Clinics E-Book August 2011 by eBook Clinics

CLINICS Editor Mauricio Rocha-e-Silva Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Area Editors Anuar Ibrahim Mitre Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil

Heitor Franco de Andrade Jr. Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Rubens Belfort Jr. Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Berenice Bilharinho Mendonça Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil

Ivete Bedin Prado Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Ruy Jorge Cruz Junior University of Pittsburgh Pittsburgh, PA, USA

Bruno Zilberstein Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Joaquim Prado Moraes-Filho Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Sergio Paulo Bydlowski Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Carlos Serrano Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Luı´z Eugeˆnio Garcez-Leme Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Silvia Vanessa Lourenço Faculdade de Odontologia da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil

Maria Cecı´lia Solimene Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Sophie Franc¸oise Mauricette Derchain Faculdade de Cieˆncias Me´dicas, Universidade Estadual de Campinas Campinas, SP, Brazil

Newton Kara-Junior Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Suely Kazue Nagahashi Marie Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Olavo Pires de Camargo Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Thelma Suely Okay Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Paulo Hoff Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Vale´ria Aoki Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Edmund Chada Baracat Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Eliete Bouskela Universidade do Estado do Rio de Janeiro Rio de Janeiro, RJ, Brazil Eloisa Silva Dutra de Oliveira Bonfa´ Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Geraldo Busatto Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Gustavo Franco Carvalhal Faculdade de Medicina da Pontifıćia Universidade Cato´lica do Rio Grande do Sul Porto Alegre, Rio Grande do Sul, Brazil

Renato Delascio Lopes Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Editorial Board Abhijit Chandra King George’s Medical College Lucknow, India Adamastor Humberto Pereira Universidade Federal do Rio Grande do Sul Porto Alegre, RS, Brazil Adauto Castelo Universidade Federal de Saõ Paulo Saõ Paulo, SP, Brazil Ademar Lopes Fundaçaõ Antoˆnio Prudente, Hospital do Caˆncer Saõ Paulo, SP, Brazil

Daniel Romero Munõz Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Edmund Neugebauer Witten/Herdecke University Witten, North Rhine - Westphalia, Germany Egberto Gaspar de Moura Jr. Universidade do Estado do Rio de Janeiro Rio de Janeiro, RJ, Brazil Ernest Eugene Moore University of Colorado Denver Denver, CO, USA

Alberto Azoubel Antunes Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Euclides Ayres Castilho Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Alexandre Roberto Precioso Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Fa´bio Biscegli Jatene Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Andrea Schmitt University of Goettingen Goettingen, Germany

Francisco Laurindo Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Arnaldo Valdir Zumiotti Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Artur Brum-Fernandes Universite´ de Sherbrooke Que´bec, Canada´ Ashok Agarwal The Cleveland Clinic Foundation Cleveland, Ohio, USA Carmita Helena Najjar Abdo Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Cesar Gomes Victora Faculdade de Medicina da Universidade Federal de Pelotas Pelotas, RS, Brasil

Hiroyuki Hirasawa Chiba University School of Medicine Chiba, Japan Irismar Reis de Oliveira Faculdade de Medicina da Universidade Federal da Bahia Salvador, BA, Brasil Irshad Chaudry University of Alabama Birmingham, AL, USA Ivan Cecconello Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ke-Seng Zhao Southern Medical University Guangzhou, China

Laura Cunha Rodrigues London School of Hygiene and Tropical Medicine - University of London London, UK Marcelo Zugaib Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Marco Martins Amatuzzi Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Maria Aparecida Shikanai Yasuda Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Mauro Perretti William Harvey Research Institute London, UK Michael Gregory Sarr Mayo Clinic Rochester, MN, USA Milton de Arruda Martins Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil

Olavo Pires de Camargo Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Pedro Puech-Leaõ Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Peter Libby Brigham and Women’s Hospital Boston, Boston, MA, USA Philip Cohen University of Houston Health Center Houston, Texas, USA Rafael Andrade-Alegre Santo Toma´s Hospital Republic of Panama´, Panama´ Ricardo Antonio Refinetti Faculdade de Medicina da Universidade Federal do Rio de Janeiro Rio de Janeiro, RJ, Brazil Roberto Chiesa San Raffaele Hospital Milan, Italy Ronald A. Asherson Netcare Rosebank Hospital Rosebank, Johannesburg, South A´frica

Mitchell C. Posner The University of Chicago Medical Center Chicago, IL, USA

Samir Rasslan Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Moyses Szklo Johns Hopkins Bloomberg School of Public Health Baltimore, USA

Tarcisio Eloy Pessoa de Barros Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Navantino Alves Faculdade de Cieˆncias Me´dicas de Minas Gerais Belo Horizonte, MG, Brazil

Valentim Gentil Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Noedir Antonio Groppo Stolf Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Wagner Farid Gattaz Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Board of Governors Antonio Alci Barone Arnaldo Valdir Zumiotti Berenice Bilharinho de Mendonça Clarice Tanaka Claudia Regina Furquim de Andrade Dalton de Alencar Fischer Chamone Daniel Romero Munõz Edmund Chada Baracat Eduardo Massad Eloisa Silva Dutra de Oliveira Bonfa´ Euclides Ayres de Castilho Euripedes Constantino Miguel Evandro Ararigboía Rivitti Fa´bio Biscegli Jatene Flair Jose´ Carrilho Francisco Vargas Suso Gerson Chadi Giovanni Guido Cerri Irineu Tadeu Velasco Ivan Cecconello

Jorge Elias Kalil Jose´ Antonio Franchini Ramires Jose´ Eduardo Krieger Jose´ Eluf Jose´ Ota´vio Costa Auler Jose´ Ricardo de Carvalho Mesquita Ayres Luiz Augusto Carneiro D’Albuquerque Luiz Francisco Poli de Figueiredo Magda Maria Sales Carneiro-Sampaio Manoel Jacobsen Teixeira Marcelo Zugaib Marcos Boulos Marcus Castro Ferreira Maria Aparecida Shikanai Yasuda Maria Irma Seixas Duarte Miguel Lorenzo Barbero Marcial Miguel Srougi Milberto Scaff Mı´lton de Arruda Martins Noedir Antonio Groppo Stolf

Editorial Director Kavita Kirankumar Patel-Rolim Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Editorial Office: Rua Dr. Ovı´dio Pires de Campos, 225 - 6 ˚ Andar Email: clinics@hcnet.usp.br Website: www.scielo.br/clinics

Olavo Pires de Camargo Paulo Andrade Lotufo Paulo Hila´rio Nascimento Saldiva Paulo Marcelo Gehm Hoff Pedro Puech-Lea˜o Ricardo Ferreira Bento Roberto Zatz Roger Chammas Samir Rasslan Sandra Josefina Ferraz Ellero Grisi Selma Lancman Tarcı´sio Eloy Pessoa de Barros Uenis Tannuri Umbertina Conti Reed Valentim Gentil Venaˆncio Avancini Ferreira Alves Vicente Odone Wagner Farid Gattaz Wilson Jacob

Editorial Assistant Nair Gomes Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

CEP 05403-010 Sa˜o Paulo/SP Tel.: +55-11-3069-6235 Submission: http://mc.manuscriptcentral.com/clinics

Indexations: LILACS; MEDLINE; PubMed; PubMed Central; SciELO; Science Citation Index Expanded (ISI Web of Knowledge); Scopus; Ulrich’s Periodical Directory; Qualis/Capes - Classified as an International Circulation Journal in Medicine. Clinics. Saõ Paulo: Scientific Journal of Hospital das Clıńicas da Faculdade de Medicina da Universidade de Saõ Paulo, 2005Monthly Periodical: January to December ISSN 1678-3379 Formerly Revista do Hospital das Clıńicas da FMUSP, 1946–2004. 1. Medicine-scientific production. 2. Medical Sciences I. Hospital das Clıńicas da Faculdade de Medicina da Universidade de Saõ Paulo. CDD 610

PUBLICATION INFORMATION AND EDITORIAL POLICIES CLINICS publishes peer-reviewed articles of interest to clinicians and researchers in the medical sciences. CLINICS is registered with PubMed Central and SciELO. This complies with the policies of funding agencies, such as, the Wellcome Trust, the Research Councils UK - RCUK, the National Institutes of Health (NIH), and the DFG, German Research Foundation, which request or require deposition of the published articles that they fund into such publicly available databases. CLINICS supports the position of the International Committee of Medical Journal Editors (http://www.icmje.org/) on trial registration. All trials initiated after January 1, 2010 must be registered prospectively (before patient recruitment begins) in a publicly accessible registry. Trials initiated before January 1, 2007 must be registered before submission to our journals. See the ICMJE faq on trial registration for further details. Visit http://www.who.int/ ictrp/network/list_registers/en/index.html for the WHO’s list of approved registries. CLINICS suggests: http://www.clinicaltrials. gov/, a user friendly site.

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Journal articles: Wolosker N, Nakano L, D’Hippolito G, Rosoky RA, Borri ML, Wolosker AM. Gadolinium magnetic angioresonance in the study of aortoiliac disease. Angiology. 2003;54:163–8. Lee JT, Ling D, Heiken JP, Glazer HS, Sicard GA, Totty WG, et al. Magnetic resonance imaging of abdominal aortic aneurysms. Am J Roentgenol. 1984;143:1197–202. Citation of unpublished material or personal communication. should be noted parenthetically in the text and not added to the reference list. TABLES: Should be incorporated into the Main Document, after the end of the reference list. Tables should be constructed using the Table feature in your word processor or using a spreadsheet program such as Excel. Tables should be numbered in order of appearance in the text with Arabic numerals. Each table should have a title and, if necessary, an explanatory legend. All tables must be referred to and succinctly described in the text. Under no circumstances should a table repeat data that are presented in an illustration. Statistical measures of variation (i.e., standard deviation, standard error) should be identified, and decimal places in tabular data should be restricted to those with mathematical and statistical significance. FIGURES: Photographs, illustrations, charts, drawings, line graphs, etc are all defined as Figures. We do not publish Pictures Graphics, Photos, etc. Number your figures consecutively in Arabic numerals in order of appearance. Upload each Figure individually into the system. Do not incorporate Figures into the Main Document. Legend(s) should be descriptive and allow examination of the figure without reference to text. Legends should be incorporated into the Main Document, after the Tables (if any) or after the references. Photographic illustrations should should be of professional quality and uploaded as *.jpeg files, and. They must be clear even after reduction in size for publication. Typewritten or hand lettering is unacceptable, as are figures generated by dot matrix printers. Generally, figures will be reduced to fit one column of text. Actual magnification of all photomicrographs should be provided, preferably by placing a length scale on the print. Line graphs and charts should never be sent as jpeg illustrations. It is usually best to prepare these as ExcelH files. When ready copy the line graph or chart to a word.doc sheet. For each submitted line graph or chart, upload the corresponding ExcelH data matrix as a supplementary file. Identify it as ‘‘Fig_N_excel’’ and upload as supplementary file. TITLE PAGE: Please submit a separate file containing: (A) the full title; (B) a running title of not more than 45 characters; (C) Scientific address where project was executed; (D) authors in the order in which they are to appear if the article if accepted opposite the name of each author specify his her scientific address; (C) his/her contribution for the project and production of the article. Authors should have participated sufficiently in the work to take public responsibility for appropriate portions of the content; (E) Acknowledgments: Contributors who do not meet the criteria for authorship should be listed in the acknowledgments section. All

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ISSN-1807-5932

CLINICS CONTENTS Clinics 2011 66(8):1307–1511

CLINICAL SCIENCES

High prevalence of chronic pelvic pain in women in Ribeira˜o Preto, Brazil and direct association with abdominal surgery Gabriela Pagano de Oliveira Gonc¸alves da Silva, Anderson Luı´s do Nascimento, Daniela Michelazzo, Fernando Filardi Alves Junior, Marcelo Gondim Rocha, Ju´lio Ce´sar Rosa-e-Silva, Francisco Jose´ Candidodos-Reis, Antonio Alberto Nogueira, Omero Benedicto Poli-Neto . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1307

Expression of Hypoxia-inducible factor 1-a and Vascular endothelial growth factor-C in locally advanced breast cancer patients Luiz Gustavo Oliveira Brito, Viviane Fernandes Schiavon, Jurandyr Moreira de Andrade, Daniel Guimara˜es Tiezzi, Fernanda Maris Peria, Heitor Ricardo Cosiski Marana . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1313

Effects of thyroxine replacement on endothelial function and carotid artery intima-media thickness in female patients with mild subclinical hypothyroidism Monica Dias Cabral, Patricia Teixeira, Debora Soares, Sandra Leite, Elizabeth Salles, Mario Waisman . . . . . . . . . 1321

Color Doppler imaging of the superior ophthalmic vein in patients with Graves’ orbitopathy before and after treatment of congestive disease Ma´rio L. R. Monteiro, Rodrigo B. S. Moritz, He´lio Angotti-Neto, Joseph E. Benabou . . . . . . . . . . . . . . . . . . . . . 1329

Improving the outcomes of elderly patients with acute myeloid leukemia in a Brazilian University Hospital Alex Freire Sandes, Juliana Correa da Costa Ribeiro, Rodrigo S. Barroso, Maria R. R. Silva, Maria L. L. F. Chauffaille . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1335

Urinary incontinence and vaginal squeeze pressure two years post-cesarean delivery in primiparous women with previous gestational diabetes mellitus Ange´lica Me´rcia Pascon Barbosa, Adriano Dias, Gabriela Marini, Iracema Mattos Paranhos Calderon, Steven Witkin, Marilza Vieira Cunha Rudge. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1341

Tonsil volume, tonsil grade and obstructive sleep apnea: is there any meaningful correlation? Michel Burihan Cahali, Carolina Ferraz de Paula Soares, Danielle Andrade da Silva Dantas, Gilberto Guanaes Simo˜es Formigoni . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1347

Attenuation of neuropsychiatric symptoms and caregiver burden in Alzheimer’s disease by motor intervention: a controlled trial Florindo Stella, Ana Paula Canonici, Sebastiaõ Gobbi, Ruth Ferreira Santos-Galduroz, Joaõ de Castilho Caçaõ, Lı´lian Teresa Bucken Gobbi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1353

Pharmacogenetics of glucocorticoid replacement could optimize the treatment of congenital adrenal hyperplasia due to 21-hydroxylase deficiency Ricardo P. P. Moreira, Alexander A. L. Jorge, Larissa G. Gomes, Laura C. Kaupert, Joa˜o Massud Filho, Berenice B. Mendonca, Taˆnia A. S. S. Bachega. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1361

Subsequent reproductive outcome in women who have experienced a potentially life-threatening condition or a maternal near-miss during pregnancy Rodrigo S. Camargo, Rodolfo C. Pacagnella, Jose´ G. Cecatti, Mary A. Parpinelli, Joa˜o P. Souza, Maria H. Sousa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1367

The TNF-a -308 polymorphism may affect the severity of Crohn’s disease Genoile Santana, Maria Teresita Bendicho, Tamara Celi Santana, Lidiane Bianca dos Reis, Denise Lemaire, Andre´ Castro Lyra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1373

Detection of cognitive impairment in the elderly by general internists in Brazil Alessandro Ferrari Jacinto, Sonia Brucki, Cla´udia Sellitto Porto, Milton de Arruda Martins, Ricardo Nitrini . . . . . 1379

The relationship between the oral and pharyngeal phases of swallowing Rachel Aguiar Cassiani, Carla Manfredi Santos, Luana Casari Parreira, Roberto Oliveira Dantas . . . . . . . . . . . . . 1385

Comparison of pelvic floor muscle strength evaluations in nulliparous and primiparous women: a prospective study Moˆnica Orsi Gameiro, Vanessa Oliveira Sousa, Luiz Felipe Gameiro, Rosana Carneiro Muchailh, Carlos Roberto Padovani, Joa˜o Luiz Amaro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1389

Effects of a multidisciplinar cognitive rehabilitation program for patients with mild Alzheimer’s disease Luciane F. Viola, Paula V. Nunes, Monica S. Yassuda, Ivan Aprahamian, Franklin S. Santos, Glenda D. Santos, Paula S. Brum, Sheila M. Borges, Alexandra M. Oliveira, Gisele F. S. Chaves, Eliane C. Ciasca, Rita C. R. Ferreira, Vanessa J. R. de Paula, Oswaldo H. Takeda, Roberta M. Mirandez, Ricky Watari, Deusivania V. S. Falca˜o, Meire Cachioni, Orestes V. Forlenza . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1395

Association analysis of anti-Epstein-Barr nuclear antigen-1 antibodies, anti-cyclic citrullinated peptide antibodies, the shared epitope and smoking status in Brazilian patients with rheumatoid arthritis Michel Alexandre Yazbek, Silvia de Barros-Mazon, Cla´udio Lu´cio Rossi, Ana Carolina Londe, Lilian Tereza Lavras Costallat, Manoel Barros Be´rtolo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1401

BASIC RESEARCHES

Role of cGMP and cAMP in the hemodynamic response to intrathecal sildenafil administration Gabriela Bombarda, Joa˜o Paulo J. Sabino, Carlos Alberto A. da Silva, Rubens Fazan Jr, Maria Cristina O. Salgado, Helio C. Salgado . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1407

The impact of previous para-areolar incision in the upper outer quadrant of the breast on the localization of the sentinel lymph node in a canine model Paulo Henrique Dio´genes Vasques, Luiz Gonzaga Porto Pinheiro, Joa˜o Marcos de Meneses e Silva, Jose´ Ricardo de Moura Torres-de-Melo, Karine Bessa Porto Pinheiro, Joa˜o Ivo Xavier Rocha . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1413

Effects of diabetes on myocardial capillary density and serum angiogenesis biomarkers in male rats Majid Khazaei, Ali Reza Fallahzadeh, Mohammad Reza Sharifi, Noushin Afsharmoghaddam, Shaghayegh Haghjooy Javanmard, Ensieh Salehi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1419

Endovascular treatment of peripheral arterial injury with covered stents: an experimental study in pigs Sergio Belczak, Erasmo Sima˜o da Silva, Ricardo Aun, Igor Rafael Sincos, Alessandro Rodrigo Belon, Ivan Benaduce Casella, Vitor Gornati, the late Luiz Francisco Poli de Figueiredo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1425

The metabolic dynamics of cartilage explants over a long-term culture period E. K Moo, N.A Abu Osman, B. Pingguan-Murphy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1431

Short-term diabetes attenuates left ventricular dysfunction and mortality rates after myocardial infarction in rodents Bruno Rodrigues, Diego Mendrot Taboas Figueroa, Jiao Fang, Kaleizu Teodoro Rosa, Suzana Llesuy, Ka´tia De Angelis, Maria Cla´udia Irigoyen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1437

The effect of subconjunctival bevacizumab on corneal neovascularization, inflammation and re-epithelization in a rabbit model Glauco Reggiani Mello, Marcos Longo Pizzolatti, Daniel Wasilewski, Marcony R. Santhiago, Vinı´cius Budel, Hamilton Moreira . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1443

Effects of mycophenolate sodium on mucociliary clearance using a bronchial section and anastomosis rodent model Viviane Ferreira Paes e Silva, Rogerio Pazetti, Sonia de Fatima Soto, Mariana Moreira Quinhones Siqueira, Aristides Tadeu Correia, Fabio Biscegli Jatene, Paulo Manuel Peˆgo-Fernandes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1451

Rosuvastatin prevents proteinuria and renal inflammation in nitric oxide–deficient rats Jose´ Marcos Girardi, Roge´rio Estevan Farias, Ana Paula Ferreira, Na´dia Rezende Barbosa Raposo. . . . . . . . . . . . 1457

REVIEW

Surgical treatment of male infertility in the era of intracytoplasmic sperm injection – new insights Sandro C. Esteves, Ricardo Miyaoka, Ashok Agarwal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1463

LETTER TO THE EDITORS

Caveats in the interpretation of natriuretic peptide levels Chia-Ter Chao . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1479

Alopecia areata incognita: a comment Alfredo Rebora . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1481

LETTER TO EDITOR

Diastolic dysfunction in end-stage renal disease patient: what the ticking clock has told us? Mustafa Duran, Aydin Unal, Mehmet Tugru Inanc, Fatma Esin, Yucel Yilmaz, Ender Ornek . . . . . . . . . . . . . . . . 1483

RAPID COMMUNICATION

Frequency of HLA B*5701 allele carriers in abacavir treated-HIV infected patients and controls from northeastern Brazil Sergio Crovella, Lara Biller, Sergio Santos, Ana Salustiano, Lucas Brandao, Rafael Guimaraes, Ludovica Segat, Jose´ Luiz de Lima Filho, Luiz Claudio Arraes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1485

TECHNICAL NOTE

Polyvinyl alcohol as a viable membrane in artificial tissue design and development Nahrizul Adib Kadri, Mat Ghazali Raha, Belinda Pingguan-Murphy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1489

CASE REPORTS

Gastropleural fistula from gastric perforation due to renal cell carcinoma after bevacizumab chemotherapy: a case report Olı´via Meira Dias, Caroline Chaul de Lima Barbosa, Lisete Ribeiro Teixeira, Francisco S. Vargas . . . . . . . . . . . . . 1495

Clinical and cytogenetic aspects of giant cell tumor of the sternum Edgard Eduard Engel, Marcello Henrique Nogueira-Barbosa, Maurı´cio Eiji de Almeida Santos Yamashita, Federico Enrique Garcia Cipriano, Elvis Terci Valera, Maria Sol Brassesco . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1501

Primary progressive aphasia beginning with a psychiatric disorder Leonardo Caixeta, Marcelo Caixeta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1505

Contact-lens-related corneal ulcer caused by klebsiella pneumoniae ¨ zsoy, Mufide Cavdar, Yusuf Yakupogullari . . . . . . . . . . . . . . . . . . . 1509 Tongabay Cumurcu, Pembegul Firat, Ercan O

ERRATA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1511

CLINICS 2011;66(8):1335-1339

DOI:10.1590/S1807-59322011000800005

CLINICAL SCIENCE

Improving the outcomes of elderly patients with acute myeloid leukemia in a Brazilian University Hospital Alex Freire Sandes,I Juliana Correa da Costa Ribeiro,I Rodrigo S. Barroso,I Maria R.R. Silva,II Maria L.L.F. ChauffailleI I

Disciplina de Hematologia e Hemoterapia, UNIFESP - Escola Paulista de Medicina, Universidade Federal de Saõ Paulo, Saõ Paulo/SP, Brazil. Departamento de Patologia, UNIFESP - Escola Paulista de Medicina, Universidade Federal de Saõ Paulo, Saõ Paulo/SP, Brazil.

OBJECTIVE: To evaluate the outcomes of acute myeloid leukemia patients who were older than 60 years of age at the time of diagnosis following the implementation of a treatment algorithm based on age, performance status, and cytogenetic results. METHODS: We retrospectively compared the results of 31 elderly acute myeloid leukemia patients (median age of 74 years) who were treated according to the new algorithm. RESULTS: Fifteen patients with a good performance status and no unfavorable karyotypes were treated with either intensive cytotoxic chemotherapy (,70 years, nine cases) or adapted etoposide, 6-thioguanine and idarubicine (.70 years, six cases); 16 cases with a poor performance status or unfavorable cytogenetics received supportive care only. Six patients achieved a complete remission and two achieved a partial remission after chemotherapy. There were three toxic deaths during induction, two in the adapted etoposide, 6thioguanine and idarubicine group and one in the intensive cytotoxic chemotherapy group. The overall median survival time was 2.96 months, 1.3 months in the supportive care group, and 4.6 months in the treatment group. CONCLUSIONS: Our results illustrate the importance of treatment guidelines adapted to local resources in an attempt to improve the survival of elderly acute myeloid leukemia patients in developing countries. KEYWORDS: Acute Myeloid Leukemia; Elderly; Intensive Chemotherapy; Adapted ETI; Prognosis. Sandes AF, Ribeiro JCC, Barroso RS, Silva MRR, Chauffaille MLLF Improving the outcomes of elderly patients with acute myeloid leukemia in a Brazilian University Hospital. Clinics. 2011;66(8):1335-1339. Received for publication on January 17, 2011; First review completed on February 24, 2011; Accepted for publication on April 24, 2011 E-mail: alex.sandes@bol.com.br Tel.: 55 11 55764240

mortality rates following intensive chemotherapy.3 A previous study from our group4 had similar findings, reporting an adverse outcome in AML patients older than 60 with no impact of intensive treatment on overall survival time. The last two decades have brought few advances in the outcomes and survival of elderly patients with AML despite the introduction of new therapy modalities, including granulocyte colony-stimulating factors, modulating drugs of the multidrug resistance status and FLT3 inhibitors. Although several centers recommend that the best treatment for this group of patients is inclusion in new clinical trials, some elderly patients may benefit from intensive chemotherapy regimens. The correct selection of treatment remains fundamental to the management of this disease.1,2 Consistent with the above evidence and to improve our results, we retrospectively evaluated the outcomes of elderly AML patients treated at the Escola Paulista de Medicina, Universidade Federal de Sa˜o Paulo after the implementation of an adapted treatment algorithm based on age, performance status, and cytogenetic results that benefited from the

INTRODUCTION Acute myeloid leukemia (AML) comprises a heterogeneous group of clonal myeloid precursor cell disorders with distinct clinical, morphologic, immunophenotypic, cytogenetic, and molecular features. Age is one of the most important predictors of outcomes in adult AML, and 20-30% of patients are older than 60 years of age at the time of diagnosis.1 The unfavorable prognosis of elderly patients may be related to their poor performance status, an increased incidence of high-risk cytogenetic abnormalities, the common expression of multidrug resistance phenotypes, and the presence of previous hematologic disorders.2 The treatment of elderly AML patients is challenging, with poor response rates and high

Copyright ß 2011 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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participation of a multidisciplinary team and the continuous training of medical and nursing staff.

For cytogenetic analysis, 5 ml of each BM samples was collected in a syringe containing sodium heparin. Karyotyping was performed using standard techniques, and the results were reported according to the International System for Human Cytogenetic Nomenclature guidelines.5 At least 20 G-banded metaphases were analyzed. A clone was defined either as two BM cells showing the same gain of chromosomal material or the same structural aberration or as at least three BM cells showing loss of the same chromosome. A complex aberrant karyotype was defined as three or more independent cytogenetic abnormalities in at least two BM cells. Cytogenetic risk was defined according to the National Comprehensive Cancer Network guidelines.6 AML was diagnosed and classified according to the World Health Organization (WHO) criteria, which define AML as the presence of at least 20% of blasts in a BM sample or the presence of a recurrent cytogenetic abnormality.7

MATERIALS AND METHODS Patients The sample comprised 31 patients greater than 60 years of age with a de novo AML diagnosis or with a previous history of myelodysplastic syndrome. All the participants had been diagnosed at the Escola Paulista de Medicina, Universidade Federal de SaË&#x153;o Paulo (UNIFESP) between January 1, 2003 and July 31, 2008, and the sample represented 26.27% (31/118) of all the adult AML cases diagnosed in the institution during this period. The outcomes of all the patients were followed until death. The clinical features at presentation of all 31 patients are shown in Table 1. This study was conducted according to the requirements of the Institutional Review Board of UNIFESP.

Cytomorphologic, immunophenotypic, and cytogenetic studies

Treatment protocols Patients were treated according to their age, Eastern Cooperative Oncology Group (ECOG) performance status, comorbidities, and cytogenetic risks and were divided into two groups: treatment and supportive care. In the treatment group, patients younger than 70 years of age with a good performance status (ECOG index ,II) and no unfavorable karyotypes were treated with intensive cytotoxic induction chemotherapy comprising intravenous (IV) cytarabine (200 mg/m2 per day for seven days) and IV daunorubicin (45 mg/m2 per day on days 1-3). Patients who were older than 70 years of age but had a good performance status and no unfavorable cytogenetics were treated with an alternative chemotherapy regimen adapted from the Finnish Leukemia Group (ETI protocol),8-10 which comprised IV

Peripheral blood (PB) and bone marrow (BM) smears collected using BM first-pull aspiration were stained with May-GruÂ¨nwald-Giemsa (MGG), and cell morphology was analyzed using conventional bright-field microscopy. Immunophenotypic studies were performed in BM samples using a four-color combination of monoclonal antibodies (MAbs). Immunofluorescence staining was performed using a standardized direct stain-and-then-lyse-and-wash technique combined with fixation and permeabilization for the detection of intracellular antigens. Immediately after staining, samples were acquired using a FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA, USA) using the CellQuest software program (Becton-Dickinson, San Jose, CA, USA).

Table 1 - Patient characteristics at the time of diagnosis in the two groups. Treatment (n = 15)

Supportive care (n = 16)

Median age (range) Gender (male/female) ECOG performance status 0 I II III IV Karyotype Low risk t(8;21) inv(16) Standard risk Normal del(20 q) +8 t(8;16) Unfavorable risk complex karyotype del(7) t(1;3) del(11 q)(23) Unknown Hb (g/dL) WBC (6109/L) Platelets (6109/L) Bone marrow blast cells (%) Splenomegaly

Adapted ETI (n = 6)

Intensive treatment (n = 9)

79 (60-89) 6/10

77 (68-80) 3/3

68 (60-75) 6/3

1 8 3 4 0

1 5 0 0 0

1 8 0 0 0

0 0 0 8 3 0 3 2 4 1 1 1 1 4 8.53 56.5 58 57.5 (21-97) 3/16

2 1 1 1 1 0 0 0 1 1 0 0 0 2 6.5 21.6 38.7 56.5 (20-92) 1/6

1 1 0 3 2 1 0 0 1 0 1 0 0 4 6.5 20.1 30 46 (20-79) 1/9

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Improving the survival of elderly leukemia patients Sandes AF et al.

etoposide (80 mg/m2 twice daily on days 1-5); IV thioguanine (100 mg/m2 twice daily on days 1-5) and IV mitoxantrone (12 mg/m2 once daily on days 1-3). The original ETI protocol contained idarubicine, which was replaced by mitoxantrone in our study because of the unavailability of this chemotherapeutic agent in our hospital and the similar pharmacodynamic features of both drugs. The response assessment criteria were defined according to the guidelines reported by Cheson et al.11 Complete remission was defined by the following criteria: the presence of ,5% blast cells; the absence of blasts with Auer rods; the absence of extramedullary disease; an absolute neutrophil count .1.06109/l and a platelet count .1006109/l. Partial remission was defined as a decrease in BM blast percentage to 5-25% and a decrease in the pretreatment BM blast percentage by at least 50%. The overall survival time was the time measured from the date of leukemia diagnosis to the date of death from any cause. After the achievement of complete remission, consolidation therapy was identical to induction therapy in both treatment schedules. Granulocyte colony-stimulating factor was administrated to all patients who presented with a neutrophil count ,0.56109 cells/l after chemotherapy administration. Patients with a poor performance status and those with unfavorable cytogenetics were treated with supportive care, regardless of age. Monotherapy with hydroxycarbamide 500-1500 mg/day was initiated when the white blood cell count was .206109/l, and blood cell transfusions and antibiotic therapy were administrated when necessary.

(two with t(8;21) and one with inv(16)); 11 cases of AML with myelodysplasia-related changes; one case of therapyrelated AML and 16 cases of AML not otherwise specified (one case of AML with minimal difference, four of AML without maturation, four of AML with maturation, two of acute myelomonocytic leukemia, three of acute monoblastic leukemia and two of erythroleukemia). At diagnosis, the patientsâ&#x20AC;&#x2122; hemoglobin levels ranged from 2.9 to 15.5 g/dl (median 7.5 g/dl), and the white blood cell counts ranged from 1.1 to 1286109 cells/l (median 39.56109 cells/l). The ECOG performance status, the BM blast cell percentage, and the presence of splenomegaly are listed in Table 1. Cytogenetic studies were performed in 29 patients, and 21 (72.4%) presented the following karyotype results: three had low-risk abnormalities (14.3%), twelve had intermediaterisk abnormalities (57.1%) and six had high-risk abnormalities (28.6%).

Response and survival time Table 2 shows the overall responses of the AML patients by patient group. In the intention-to-treat analysis used, the overall response (complete remission + partial remission) of the patients who received treatment (8 of 15 patients, 53.3%) was superior to the response of the patients in the supportive care group (0 of 16 patients) (p,0.05). Of the six patients who achieved complete remission following induction, five received intensive therapy, and one received adapted ETI. In all, two patients achieved a partial response, and both were in the ETI group. One patient presented with treatment failure after ETI, and three patients presented with treatment failure after intensive chemotherapy. Three toxic deaths occurred during induction: two in the adapted ETI group and one in the intensive chemotherapy group. All the toxic deaths were due to severe infection. The overall median survival time was 2.96 months (range 0.07 to 9.5): 1.3 months in the supportive care group and 4.6 months in the treatment group. The p-value produced by the log-rank test was p = 0.001 for the comparison between the two groups. The treatment comparisons in terms of overall survival time are shown in Figure 1. Patients older than 70 years of age exhibited a median survival time of 2.8 months, and no significant difference was observed between them and the patients less than 70 years old (3.4 months, p = 0.86).

Statistical analyses Analyses were performed on an intention-to-treat basis. The response rates of the different patient groups were compared using the chi-squared (x2) test of proportions. Overall survival time was estimated with the Kaplan-Meier method and compared using the log-rank test. A p-value less than 0.05 was considered statistically significant. Statistical analyses were performed using SPSS 12.0 software (Chicago, USA).

RESULTS Patient characteristics The median age was 74 years (range 60 to 89 years), and the male:female ratio was 151. Fifteen patients received treatment [nine with intensive chemotherapy and six with adapted etoposide, 6-thioguanine and idarubicine (ETI)], and 16 received supportive care. The median ages in the treatment and supportive care groups were 68 and 79 years old, respectively. According to the WHO classification criteria, the cases were subdivided into the following groups: three cases of AML with recurrent translocations

Time of hospitalization The median duration of hospitalization was 1.6 months overall (range 0.07 to 5.2) and 0.56 and 2.8 months in the supportive care and treatment groups, respectively. The rate of hospitalization during the follow-up period did not vary significantly by patient group (75.7% for the supportive care group and 77.6% for the treatment group).

Table 2 - Response rates and overall survival times by patient group.

Response rate Complete remission Partial remission Resistant disease Death during induction Overall survival time in months (range)

Supportive care (n = 16)

Adapted ETI (n = 6)

Intensive treatment (n = 9)

ETI + intensive treatment (n = 15)

0 0 16 0 1.3 (0.07-9.2)

1 2 1 2 4.4 (1.05-7.25)

5 0 3 1 4.6 (0.92-9.5)

6 2 4 3 4.6 (0.92-9.5)

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CLINICS 2011;66(8):1335-1339

Figure 1 - Overall survival time of elderly AML patients according to patient group, 2003-2008.

idarubicine that displays good response rates and an acceptable toxicity profile in advanced age AML patients.8-10 The adapted ETI evaluated herein was associated with a significant increase in AML survival time and may be an alternative approach for the treatment of older patients who cannot participate in clinical trials. The prevalence and survival times of AML in advanced age have not been adequately studied in Latin American countries. Beyr覺織es et al. reported that Cuban AML patients older than 60 years of age experienced an overall median survival time of less than two months, and those selected to receive high-dose chemotherapy presented high rates of resistance and mortality.15 A previous study conducted by our group showed that 33% of all AML cases occurred in patients who were older than 60 years of age, and the overall survival time was two months.4 Moreover, no difference in overall survival time was noted between elderly AML patients who received intensive cytotoxic chemotherapy and those who received supportive care, suggesting that this group of patients should receive only palliative treatment. Five years later, our results indicate a historical improvement in the treatment of elderly patients with AML. This change can be attributed to a paradigm shift in the selection of therapeutic modalities coupled with improved medical and nursing training and better medical support. Despite the improvement in the overall survival of these patients, there was no difference in the hospitalization rate between the patient groups. The impact of treatment guidelines adapted to local resources is exemplified by the initiative of the International Consortium on Acute Promyelocytic Leukemia (APL)16 to improve the response and cure rates and reduce the mortality associated with APL treatment in developing countries.

DISCUSSION The results confirm the unfavorable prognosis of AML patients older than 60 years of age. The overall survival time was 2.9 months, and patients selected to receive intensive cytotoxic treatment or an alternative treatment (adapted ETI) experienced significant increases in overall survival time compared with patients who received supportive care only. However, the survival times described in this study are inferior to those reported in developed countries.1,2 This phenomenon may be attributed to both socioeconomic differences and characteristics of the Brazilian national public health system. Brazil was one of the first countries in Latin America to make universal access to free health care a constitutional right, and the available services range from primary care to highly technologically complex treatments.12 Nevertheless, this system has not been fully implemented, and patients wait a long time for access to specialized services.13 This delay is particularly experienced by patients waiting to access the limited number of specialized hematology centers. The optimal management of AML and other hematological malignancies14 necessitates a prompt diagnosis and the initiation of treatment without delay. A number of new chemotherapy agents are under investigation in older patients with AML, such as DNA methyltransferase inhibitors (e.g., decitabine) and the purine nucleoside analog clofarabine. However, such drugs are not available via the public health services of developing countries, and alternative treatments are necessary for the management of elderly patients not suitable for intensive chemotherapy. The Finnish Leukemia Group has proposed an alternative oral treatment composed of etoposide, 6-thioguanine and

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Improving the survival of elderly leukemia patients Sandes AF et al. 5. Shaffer LG, Slovak ML, Campbell LJ. ISCN 2009: An International System for Human Cytogenetic Nomenclature (2009). Karger; 2009. 6. Appelbaum FR, Baer MR, Carabasi MH, Coutre SE, Erba HP, Estey E, et al. NCCN Practice Guidelines for Acute Myelogenous Leukemia. Oncology (Williston Park). 2000;14:53-61. 7. Swerdlow SH, Campo E, Harris NL, Pileri SA, Stein H, Thiele J, et al. WHO Classification of tumours of haematopoietic and lymphoid tissues. IARC: Lyon; 2008. 8. Ruutu T, Elonen E. Etoposide, 6-thioguanine and idarubicin, an oral combination regimen (ETI) for the induction treatment of acute leukemia. Hematol Oncol. 1991;9:87-92, doi: 10.1002/hon.2900090204. 9. Ruutu T, Almqvist A, Hallman H, Honkanen T, Ja¨rvenpaä¨ E, Ja¨rventie G, et al. Oral induction and consolidation of acute myeloid leukemia with etoposide, 6-thioguanine, and idarubicin (ETI) in elderly patients: a randomized comparison with 5-day TAD. Finnish Leukemia Group. Leukemia. 1994;8:11-5. 10. Ruutu T, Koivunen E, Nousiainen T, Pelliniemi TT, Almqvist A, Anttila P, et al. Oral treatment of acute myeloid leukaemia with etoposide, thioguanine, and idarubicin (ETI) in elderly patients: a prospective randomised comparison with intravenous cytarabine, idarubicin, and thioguanine in the second and third treatment cycle. Eur J Haematol. 2004;72:38-44, doi: 10.1046/j.0902-4441.2003.00182.x. 11. Cheson BD, Bennett JM, Kopecky KJ, Bu¨chner T, Willman CL, Estey EH, et al. Revised recommendations of the International Working Group for Diagnosis, Standardization of Response Criteria, Treatment Outcomes, and Reporting Standards for Therapeutic Trials in Acute Myeloid Leukemia. J Clin Oncol. 2003;21:4642-9, doi: 10.1200/JCO.2003.04.036. 12. Cornwall A, Shankland A. Engaging citizens: lessons from building Brazil’s national health system. Social Science & Medicine. 2008;66:217384, doi: 10.1016/j.socscimed.2008.01.038. 13. Ocke´-Reis CO, Marmor TR. The Brazilian national health system: an unfulfilled promise? Int J Health Plann Mgmt. 2010. 14. Scerni AC, Alvares LA, Beltraõ AC, Bentes IR, Azevedo TC, Bentes AQ, et al. Influence of late treatment on how chronic myeloid leukemia responds to imatinib. Clinics. 2009;64:731-4, doi: 10.1590/S180759322009000800004. 15. Beyrıés LS, Urıá JC, Arenas RC, Perurena JM, Hernańdez CM, Sua´rez GL, et al. Leucemias agudas en pacientes mayores de 60 anõs. Rev Cubana Med. 2003;42:18-26. 16. Ribeiro RC, Rego E. Management of APL in developing countries: epidemiology, challenges and opportunities for international collaboration. Hematology Am Soc Hematol Educ Program. 2006;162-8

The low number of patients enrolled in our study could be interpreted as a limitation, and a larger number of AML cases are currently being followed by our hospital to confirm our findings. In conclusion, as part of the ongoing search to enhance the quality of health care and with all limitations of a single institution in the Brazilian public health system, we implemented a medical assistance approach that showed benefits for the management of AML patients. The treatment of AML in advanced age is challenging, and some patients may benefit from treatment with intensive or alternative chemotherapy that is appropriate for their age, performance status, and cytogenetics. We believe that outcomes in elderly AML patients can be improved by better and faster access to specialized health services, consistent training of medical and nursing staff in all spheres of care and the development of therapeutic algorithms and planned solutions that are directed at and customized for different regional realities.

REFERENCES 1. Erba HP. Prognostic Factors in Elderly Patients with AML and the Implications for Treatment. Hematology Am Soc Hematol Educ Program. 2007;2007:420-8. 2. Appelbaum FR, Gundacker H, Head DR, Slovak ML, Willman CL, Godwin JE, et al. Age and acute myeloid leukemia. Blood. 2006;107:34815, doi: 10.1182/blood-2005-09-3724. 3. Do¨hner H, Estey EH, Amadori S, Appelbaum FR, Bu¨chner T, Burnett AK, et al. Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel, on behalf of the European LeukemiaNet. Blood. 2010;115:453-74, doi: 10.1182/blood2009-07-235358. 4. Rodrigues CA, Chauffaille ML, Pelloso LA, Ghaname FS, Kerbauy DM, Campos MG, et al. Acute myeloid leukemia in elderly patients: experience of a single center. Braz J Med Biol Res. 2003;36:703-8.

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CLINICS 2011;66(8):1341-1345

DOI:10.1590/S1807-59322011000800006

CLINICAL SCIENCE

Urinary incontinence and vaginal squeeze pressure two years post-cesarean delivery in primiparous women with previous gestational diabetes mellitus Ange´lica Me´rcia Pascon Barbosa,I Adriano Dias, Gabriela Marini,I Iracema Mattos Paranhos Calderon,I Steven Witkin,II Marilza Vieira Cunha RudgeI I Department of Gynecology and Obstetrics, Botucatu Medical School, Universidade Estadual Paulista (UNESP), Botucatu/SP, Brazil. II Cornell University, New York, United States.

OBJECTIVE: To assess the prevalence of urinary incontinence and associated vaginal squeeze pressure in primiparous women with and without previous gestational diabetes mellitus two years post-cesarean delivery. METHODS: Primiparous women who delivered by cesarean two years previously were interviewed about the delivery and the occurrence of incontinence. Incontinence was reported by the women and vaginal pressure evaluated by a Perina perineometer. Sixty-three women with gestational diabetes and 98 women without the disease were screened for incontinence and vaginal pressure. Multiple logistic regression models were used to evaluate the independent effects of gestational diabetes. RESULTS: The prevalence of gestational incontinence was higher among women with gestational diabetes during their pregnancies (50.8% vs. 31.6%) and two years after a cesarean (44.8% vs. 18.4%). Decreased vaginal pressure was also significantly higher among women with gestational diabetes (53.9% vs. 37.8%). Maternal weight gain and newborn weight were risk factors for decreased vaginal pressure. Maternal age, gestational incontinence and decreased vaginal pressure were risk factors for incontinence two years after a cesarean. In a multivariate logistic model, gestational diabetes was an independent risk factor for gestational incontinence. CONCLUSIONS: The prevalence of incontinence and decreased vaginal pressure two years post-cesarean were elevated among women with gestational diabetes compared to women who were normoglycemic during pregnancy. We confirmed an association between gestational diabetes mellitus and a subsequent decrease of vaginal pressure two years post-cesarean. These results may warrant more comprehensive prospective and translational studies. KEYWORDS: Cesarean delivery; Gestational diabetes mellitus; Vaginal squeeze pressure; Pregnant; Urinary incontinence. Barbosa AMP, Dias A, Marini G, Calderon IMP, Witkin S, Rudge MVC. Urinary incontinence and vaginal squeeze pressure two years post-cesarean delivery in primiparous women with previous gestational diabetes mellitus. Clinics. 2011;66(8):1341-1345. Received for publication on April 25, 2011; First review completed on April 26, 2011; Accepted for publication on April 26, 2011 E-mail: marilzarudge@ig.com.br Tel.: 55 14 38116181

Gestational diabetes mellitus (GDM) is a metabolic disorder that is considered to be similar to type 2 DM. In GDM, pregnancy acts as a ‘‘stress factor,’’ revealing an individual’s tendency to become diabetic.7 GDM is associated with macrosomia, shoulder dystocia, cesarean section (CS), intrauterine fetal growth alterations and perinatal mortality.8,9 Fetal macrosomia increases the risk for the development of GDM-independent UI,10 and one of the confounding variables in the development of UI is delivery method. Chawla et al. demonstrated an association between multiparity and vaginal delivery with an increased risk for UI and decreased vaginal squeeze pressure (VSP).11 Diabetes and vaginal deliveries are the most significant risk factors implicated in the incidence of UI,3 although little is known about whether GDM and the length of time elapsed after GDM are connected to a loss of bladder control. Kim et al. demonstrated that UI is common among

INTRODUCTION Diabetes increases the risk of urinary incontinence (UI) 2.5 fold,1-3 although the mechanisms by which type 2 Diabetes Mellitus (type 2 DM) contributes to UI development and severity are not well understood.4 Diabetes is associated with an impairment of muscle strength and physical function.5 There is a temporal relationship between the time of diabetes diagnosis and subsequent development of muscle weakness and other related complications, such as diabetic amyotrophy.6

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women with a history of GDM but does not appear to be associated with physical activity level or body mass index (BMI).12 The Kim study neither quantified pelvic floor muscle function nor controlled for the mode and number of deliveries and the time from delivery to evaluation. More detailed information would allow for quantification of UI risk of UI within the population affected by GDM, such as women who will be receiving a CS for their current pregnancy or those who have received a CS during previous pregnancies. The aim of this study was to evaluate the prevalence of UI and VSP two years post-CS in primiparous women with or without previous Gestational diabetes mellitus (pGDM) and to investigate the association between UI, VSP and pGDM.

MATERIALS AND METHODS The Institutional Review Board of Botucatu Medical School, UNESP-Univ Estadual Paulista, Sa˜o Paulo, Brazil, granted approval for this prospective study, and all subjects gave informed written consent. This study consisted of a cross-sectional survey conducted at the Department of Gynecology and Obstetrics General Hospital, a university hospital. All women who participated in this study, when pregnant, were patients of this hospital and were routinely screened for GDM at 24-28 weeks of gestation. The cutoff values for the diagnosis of GDM were those proposed by Carpenter & Coustan,13 i.e., 95 mg/dl, 180 mg/dl, 155 mg/dl, and 140 mg/dl, at fasting, one, two and three hours after oral glucose load, respectively. All women in this perinatal database who had delivered two years earlier (between January 1st, 2004, and September 30th, 2004), a total of 529 subjects, were eligible for inclusion in the study. They were contacted by phone and invited to participate. The women answered questions based on the Questionnaire for Urinary Incontinence Diagnosis14 and the International Consultation on Incontinence Questionnaire Short Form (ICIQ-SF) translated into Portuguese and validated with Brazilian female patients complaining of urinary incontinence.15 The patients were questioned about the following aspects of pregnancy and childbirth: maternal weight gain, the newborn weight, if they entered partum labor, postpartum complications, involuntary urine loss (for efforts such as coughing, sneezing, laughing, catching weight, squatting, walking and/or involuntary urine lost when feeling a strong urge to urinate, contact with water, sound of running water or if exposed to low temperatures) during pregnancy and postpartum. Of the 529 possible participants, 161 eligible primiparous women with singleton pregnancies delivered by CS who met the inclusion criteria were identified. An overview of the study design is outlined in Figure 1. Power analysis was performed based on a positive diagnosis of hyperglycemia during pregnancy with a type I error of 0.05 and a type II error of 0.20. The analysis indicated that the minimum number of patients enrolled in each group should be 62. A trained physician who was blinded to the results of the oral glucose tolerance test obtained obstetric and maternal data relating to the index pregnancy from the hospital records. Demographic data collected included maternal age, gestational age at delivery, body mass index (BMI-weight in kilograms divided by height in meters squared), weight gain during pregnancy, delivery method and birth weight.

Figure 1 - Overview of the study design.

Two years postpartum, all patients were interviewed regarding their UI symptoms and the prevalence of these symptoms before, during and two years after the index pregnancy. The same author conducted all interviews. UI was as defined by Abrams et al.12 as an involuntary leakage of urine. After the interview, bidigital palpation and perineometry were performed. First, vaginal bidigital palpation was performed with the woman’s knees semiflexed. The subjects were asked to squeeze the vagina three times.16 While the subjects were in the same position, a vaginal latex sensor called a Perina perineometer was employed to measure the strength of the squeezes (Quark, Sa˜o Paulo, Brazil). Based on a previous study of nulliparous women, perineometric values higher than 33.6 mmHg were considered to be normal. The perineometer used was not a validated device. It was selected based on the results of a pilot study of Barbosa.17 Statistical analyses were performed using the PASW Statistics v.17.0.2 and SAS 9.03 software programs. The statistical significance level was set at p,0.05. Categorical variables were analyzed using descriptive procedures and are expressed as percentages. The impacts of maternal age, gestational age at delivery, BMI, weight gain during pregnancy, birth weight, GDM, VSP and UI two years postpartum were evaluated in univariate analyses. Multiple logistic regression models were then used to evaluate the independent effects of GDM and maternal age on gestational UI after controlling for other significant factors. Odds ratios were computed to express the magnitude of the association between UI, VSP and GDM with 95%

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Incontinence and vaginal squeeze pressure following gestational diabetes Barbosa AMP et al.

confidence intervals to express statistical significance. The adjusted odds ratios (OR) and estimates of 95% confidence intervals (95% CI) were based on the Mantel-Haenszel x2 test. The adjusted odds ratios were calculated to express the magnitude of the association between prior GDM and UI/ VSP and were controlled for other confounding variables. For logistic regression analyses, UI and VSP were used as the response variables.

DISCUSSION GDM is considered to be an early stage in the natural history of type 2 DM. Women with prior GDM are at a higher risk of developing type 2 DM or some degree of glucose intolerance two to twelve years after the index pregnancy.18 In the present study, the presence of pGDM increased the incidence rate of UI not only during pregnancy but also after a CS. Approximately half of the women with pGDM reported having UI during their pregnancy (50.8%) and two years post-CS (44.4%). These results confirm an association between pGDM, UI and decreased VSP. It is interesting to note that blood glucose levels do not become sufficiently elevated to cause osmotic diuresis or increase involuntary urinary loss during pregnancy.9 A history of at least one vaginal delivery, family history of prolapsed and a macrosomatic fetus have been shown to be independent risk factors for urinary incontinence and prolapse.10 The greater weight gain for fetuses in women with GDM, resulting in increased intra-abdominal pressure, could also contribute to UI. It is important to take into consideration that delivery at an earlier gestational age could decrease the rate of UI perinatal complications because of the smaller expected size of prematurely delivered newborns.19 An increased prevalence of UI among women with diabetes has been reported in cross-sectional analyses.20 In the NHANES 2001-2002 study, one in three women with impaired fasting glucose or diabetes reported weekly or more frequent UI.21 However, no previous studies have examined whether women with pGDM also have an increased prevalence of UI two years after a CS. The results presented here suggest that the cesarean delivery route does not protect against UI two years after delivery in women with pGDM. Instead, our results suggest that the complications of pGDM, including possible microvascular disease, seem to be a more relevant factor than the mode of delivery. The potential mechanisms by which diabetes causes incontinence include microvascular damage to the innervation of the bladder and urethral sphincter, detrusor muscle dysfunction, sphincter dysfunction, overactive bladder, urinary retention and elevated post-void residual urine volume, which contributes to overflow incontinence, chronic bacterial colonization, urinary tract infections and hyperglycemia.22 Moreover, the microvascular complications

RESULTS Clinical data for pGDM patients and control subjects at the time of pregnancy and two years after CS are shown in Table 1. Among the 161 patients enrolled, 63 (39.1%) had pGDM and 98 (60.9%) did not have pGDM. The length of time since the index pregnancy was similar for both groups (25.8¡3.2 months for pGDM and 24.8¡2.1 months for controls). The overall prevalence of gestational UI and twoyear postpartum UI was significantly higher among women with pGDM (50.8% and 44.4%, respectively) than among normoglycemic pregnant women (31.6% and 18.4%, respectively, p,0.01). In addition, decreased VSP was more frequently found in women with pGDM two years after receipt of CS than in the control group (53.9% and 31.6%, respectively, p = 0.04). Subject skin color, black, was homogeneous for both groups (14.2% for pGDM and 13.3% for controls, p = 0.08). Women with pGDM were older (29.3¡6.1 years vs. 25.4¡3.2 years) and experienced greater weight gain during pregnancy (17.3¡6.1 vs. 14.6¡4.1) compared to controls. Women with pGDM were more likely to have a lower gestational age at delivery (38.7¡0.8 weeks vs. 39.7¡1.3 weeks) and a higher newborn weight (3546.2¡440.6 grams vs. 3143.1¡472.9 grams, Table 1). In the multivariate logistic model, newborn weight (OR 1.002; 95% CI: 1.001-1.002) and maternal weight gain (OR 1.295; 95% CI: 1.176-1.425) was associated with a decrease in VSP in cases of pGDM (Table 2). We constructed a separate model for two-year post-CS UI in women with pGDM and found that maternal age (OR 1.181; 95% CI: 1.022-1.365), gestational UI (OR 4.992; 95% CI: 1.383-18.023) and a decrease in VSP incidence (OR 20.416; 95% CI: 3.548117.479) were associated factors (Table 3). In the multivariate logistic models, pGDM was an independent risk factor (OR 2.26; 95% CI: 1.116-4.579) for gestational UI (Table 4).

Table 1 - Characteristics of women with and without GDM during their pregnancies and two years post- cesarean section. Control group n = 98 n (%) Maternal age two years post-cesarean Gestational age at delivery (w) Weight gain during index gestation (kg) Newborn weight (g) Length of time since index pregnancy (y) BMI (kg/m2) Decresead of VSP Pre-gestational UI Gestational UI UI two years postpartum Black skin color

37 7 31 18 13

— — — — — — (37.8) (7.1) (31.6) (18.4) (13,3)

GDM n = 63

m ¡ sd

n (%)

m ¡ sd

p-value

25.4¡3.2 39.7¡1.3 14.6¡4.1 3143.1¡472.9 24.8¡2.1 25.5¡4.3 — — — — —

— — — — — — 34 (53.9) 10 (15.9) 32 (50.8) 28 (44.4) 9 (14,2)

29.3¡6.15 38.7¡0.8 17.3¡6.1 3546.2¡440.6 25.0¡0.8 25.8¡3.2 — — — — —

0.0001 0.0001 0.0003 0.0001 0.4621 0.5748 0.0431 0.0785 0.0151 0.0004 0.0823

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Table 2 - Estimated ß parameter, odds ratio (OR), 95% confidence interval (95% CI) and p-value obtained by adjusting the multivariate logistic model for investigating decresead of VSP two years after GDM.

CLINICS 2011;66(8):1341-1345

Table 4 - Estimated ß parameter, odds ratio (OR), 95% confidence interval (95% CI) and p-value obtained by adjusting the multivariate logistic model for investigating gestational UI.

VSP – Perineometer Variables Maternal age two years postcesarean Gestational age (w) Newborn weight (g) Weight gain during gestation (kg) Two groups (GDM)

Gestational UI

95% CI

p-value

Variable

0.994

0.910 - 1.087

0.8425

Maternal age at time pregnancy GDM

0.759 1.002 1.295 0.516

0.533 1.001 1.176 0.191

1.081 1.002 1.425 1.394

0.1265 0.0006 ,0.0001 0.1917

UI two-year postpartum OR

95% CI

p-value

Maternal age two years post- cesarean Pre-gestational UI Gestational UI Abnormal VSP

1.181

1.022 - 1.365

0.024

4.498 4.992 20.416

0.437 - 46.313 1.383 - 18.023 3.548 - 117.479

0.206 0.014 0.001

95% CI

p-value

0.997 2.260

0.930-1.068 1.116-4.579

0.9250 0.0236

mobility, which could lead to the association seen between BMI and UI.22 Excessive body weight could also affect bladder pressure and could also be a factor in the development of UI.27 Therefore, decreased VSP two years post-CS in women with pGDM may be a consequence of the interaction between pregnancy and GDM. Our findings that advanced maternal age, UI during pregnancy and decreased VSP are risk factors for UI suggest that UI may also be an indirect consequence of GDM. We are aware that our study sample and findings may be biased by the facts that the incontinence outcomes were selfreported and that patients were from a prenatal care service with routine screening for GDM. Patients who are symptomatic may be more willing to undergo an evaluation and receive treatment. However, our findings are in agreement with a previous study.12 Our data confirm the interaction between pGDM, UI and decreased VSP. Our data also extend this association to women who delivered by CS. These observations strongly suggest that further more comprehensive, prospective controlled and translational studies are warranted.

Table 3 - Estimated ß parameter, odds ratio (OR), 95% confidence interval (95% CI) and p-value obtained by adjusting the multivariate logistic model for investigating UI two years after GDM.

Variable

associated with type 2 DM may damage the innervation of the bladder and/or alter detrusor muscle function.23 The present study has also demonstrated that patients with pGDM have a larger VSP decrease (50.8%) two years post-CS than the controls (37.8%). The results of a study by Casey et al. indicated that the increased prevalence of decreased VSP could be related to high rates of obesity and macrosomia pregnancies. Our results partially confirm these results, as there remains an association between newborn weight and VSP (OR 1.295 CI95% 1.176-1.425). Diabetes is associated with impaired muscle strength and physical function and may provide a link between the metabolic and mechanical functions of muscles.5 Motor dysfunction is considered an end-stage manifestation of severe polyneuropathy with an annual decline in muscle strength of approximately 3%, although motor function in type 2 DM is still unknown.24 Muscle weakness is considered a progressive late complication in diabetic distal symmetric polyneuropathy and has been associated with atrophy of striated muscle, probably because of insufficient re-innervation.24 Almost all muscle weakness in diabetes relates to distal weakness and the atrophy of the muscles of the lower leg and foot.24,25 In a multivariate logistic model, maternal weight gain during pregnancy and newborn weight were associated with decreased VSP, and GDM itself was not a significant risk factor (Table 2). This finding suggests that decreased VSP two years post-CS may be a consequence of either maternal or perinatal adverse effects related to GDM, maternal weight gain during pregnancy and newborn weight. GDM has previously been associated with both an increased risk of fetal and neonatal macrosomia26 and high maternal BMI.27 A higher BMI could theoretically enhance abdominal pressure, thereby increasing bladder pressure and urethral

CONCLUSIONS The prevalence of UI and decreased VSP two years postCS were elevated among women with pGDM compared to normoglycemic controls during pregnancy. We confirmed an association between pGDM and a subsequent VSP decrease two years post-CS. These results may warrant the beginning of more comprehensive prospective and translational studies.

ACKNOWLEDGMENTS This research was supported in part by the Research Support Foundation of the State of Sa˜o Paulo and Coordination for the Improvement of Higher Education Personnel. The authors are grateful to Tatiana Casagrande and Carlos Negrato and to the Research Support Center of Botucatu Medical School, UNESP - Univ Estadual Paulista for their valuable contributions to the study design and statistical analysis.

REFERENCES 1. Sarma AV, Kanaya AM, Nyberg LM, Kusek JW, Vittinghoff E, Rutledge B, et al. Urinary incontinence among women with type 1 diabetes-how common is it? J Urol. 2009;181:1224-30, doi: 10.1016/j.juro.2008.11.024. 2. Izci Y, Topsever P, Filiz TM, Cinar ND, Uludag C, Lagro-Janssen T. The association between diabetes mellitus and urinary incontinence in adult women. Int Urogynecol J Pelvic Floor Dysfunct. 2009;20:947-52, doi: 10. 1007/s00192-009-0888-8. 3. Alvaro R, Araco F, Gravante G, Sorge R, Overton J, Vellone E, et al. Epidemiological aspects of urinary incontinence in a female population of an Italian region. Int Urogynecol J Pelvic Floor Dysfunct. 2010;21:87383, doi: 10.1007/s00192-010-1112-6. 4. Brown JS, Wing R, Barrett-Connor E, Nyberg LM, Kusek JW, Orchard TJ, et al. Lifestyle intervention is associated with lower prevalence of urinary

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incontinence: the Diabetes Prevention Program. Diabetes Care. 2006;29: 385-90, doi: 10.2337/diacare.29.02.06.dc05-1781. Sayer AA, Dennison EM, Syddall HE, Gilboy HJ, Phillips DIW, Cooper C. Type 2 Diabetes, Muscle Strength, and Impaired Physical Function. Diabetes Care. 2005;28:2541-2, doi: 10.2337/diacare.28.10.2541. Helander I, Westerblad H, Katz A. Effects of glucose on contractile function, Ca2+, and glycogen in isolated mouse skeletal muscle. Am J Physiol Cell Physiol. 2002;282:1306-12. Buchanan TA, Xiang AH. Gestational diabetes mellitus. J Clin Invest. 2005;115:485-91. Lucas M J. Diabetes complicating pregnancy. Obstet Gynecol Clin North Am. 2001;28:513-36, doi: 10.1016/S0889-8545(05)70215-1. Rudge MVC, Calderon IMP, Ramos MD, Abbade JF, Rugolo LM. Perinatal outcome of pregnancies complicated by diabetes and by maternal daily hyperglycemia not related to diabetes. A retrospective 10-year analysis. Gynecol Obstet Invest. 2000;50:108-12, doi: 10.1159/ 000010293. Rodrigues AM., Gira˜o MJB, Sartori C, Ferreira MG, Martins KF, Castro RA. COL1A1 Sp1-binding site polymorphism as a risk factor for genital prolapse. Int Urogynecol J. 2008;19:1471–5, doi: 10.1007/s00192-008-06623. Chawla A, Reddy S, Thomas J. Risk factors for persistent stress urinary incontinence after mid-urethral procedures. Indian J Urol. 2008;24:130-1, doi: 10.4103/0970-1591.44272. Kim C, McEwen LN, Sarma AV, Piette JD, Herman WH. Stress urinary incontinence in women with a history of gestational diabetes mellitus. J Womens Health (Larchmt). 2008;17:783-92, doi: 10.1089/jwh.2007.0616. Carpenter MW, Coustan DR. Criteria for screening tests for gestational diabetes. Am J Obstet Gynecol. 1982;144:768-73. Bradley CS, Rovner ES, Morgan MA, Berlin M, Novi JM, Shea JA, et al. A new questionnaire for urinary incontinence diagnosis in women: development and testing. Am J Obstet Gynecol. 2005;192:66-73, doi: 10. 1016/j.ajog.2004.07.037. Tamanini JT, Dambros M, D’Ancona CA, Palma PC, Rodrigues Netto N, Jr. [Validation of the ‘‘International Consultation on Incontinence Questionnaire - Short Form’’ (ICIQ-SF) for Portuguese]. Rev Saude Publica. 2004;38:438-44, doi: 10.1590/S0034-89102004000300015. Barbosa PB, Franco MM, Souza Fde O, Antonio FI, Montezuma T, Ferreira CH. Comparison between measurements obtained with three

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different perineometers. Clinics. 2009;64:527-33, doi: 10.1590/S180759322009000600007. Barbosa AMP, Carvalho LR, Martins AMVC, Calderon IMP, Rudge MVC. Efeito da via de parto sobre a forc¸a muscular do assoalho pe´lvico. Rev Bras Ginecol Obstet. 2005;27:677-82, doi: 10.1590/S010072032005001100008. Seely EW. Does treatment of gestational diabetes mellitus affect pregnancy outcome? Nat Clin Pract Endocrinol Metab. 2006;2:72-3, doi: 10.1038/ncpendmet0081. Pimenta WP, Calderon IMP, Cruz NS, Santos ML, Aragon FF, Padovani CR. Subclinical anormalities o glucose metabolism in Brazilian women with a history of gestational diabetes mellitus. Acta Obstet Gynecol Scand. 2004;83:1152-8. Jackson SL, Scholes D, Boyko EJ, Abraham L, Fihn SD. Urinary incontinence and diabetes in postmenopausal women. Diabetes Care. 2005;28:1730-8, doi: 10.2337/diacare.28.7.1730. Brown JS, Vittinghoff E, Lin F, Nyberg LM, Kusek JW, Kanaya AM. Prevalence and risk factors for urinary incontinence in women with type 2 diabetes and impaired fasting glucose: findings from the National Health and Nutrition Examination Survey (NHANES) 2001-2002. Diabetes Care. 2006;29:1307-12, doi: 10.2337/dc05-2463. Jackson RA, Vittinghoff E, Kanaya AM, Miles TP, Resnick HE, Kritchevsky SB, et al. Urinary incontinence in elderly women: findings from the Health, Aging, and Body Composition Study. Obstet Gynecol. 2004;104:301-7, doi: 10.1097/01.AOG.0000133482.20685.d1. Ellenberg M. Development of urinary bladder dysfunction in diabetes mellitus. Am Intern Med 1980;92:321-3. Andreassen CS, Jakobsen J, Andersen H. Muscle weakness. Diabetes. 2006;55:806-12, doi: 10.2337/diabetes.55.03.06.db05-1237. Andersen H, Nielsen S, Mogensen CE, Jakobsen J. Muscle strength in type 2 diabetes. Diabetes. 2004;53:1543-8, doi: 10.2337/diabetes.53.6.1543. Oh W. Neonatal outcome and care (2004) In Diabetes in women. Adolescence, Pregnancy and Menopause. 3rd ed. Reece EA, Coustan DR, Gabbe SG, Eds. Lippincott, Williams & Wilkins, pp. 451-9. Mestman JH. Interaction between pregnancy, gestational diabetes, and long-term maternal outcome (2004).In Diabetes in women. Adolescence, Pregnancy and Menopause. 3rd ed. Reece EA, Coustan DR, Gabbe SG, Eds. Lippincott, Williams & Wilkins, pp. 233-41.

CLINICS 2011;66(8):1347-1351

DOI:10.1590/S1807-59322011000800007

CLINICAL SCIENCE

Tonsil volume, tonsil grade and obstructive sleep apnea: is there any meaningful correlation? Michel Burihan Cahali,I,II Carolina Ferraz de Paula Soares,I Danielle Andrade da Silva Dantas,II Gilberto Guanaes Simo˜es FormigoniII I

Department of Otolaryngology. Hospital do Servidor Pu´blico Estadual de Saõ Paulo, Saõ Paulo/SP, Brazil. II Department of Otolaryngology. Hospital das Clıńicas da Faculdade de Medicina da Universidade de Saõ Paulo, Saõ Paulo/SP, Brazil.

OBJECTIVES: The aims of this study were to evaluate the correlation between oropharyngeal examination and objective palatine tonsil volume in snoring adults and verify the influence of the oropharyngeal anatomy, body mass index, age, and severity of obstructive sleep apnea on actual tonsil volume. In addition, we aimed to assess the influence of tonsil size on obstructive sleep apnea in adults. INTRODUCTION: Pharyngeal wall geometry is often altered in adults who have obstructive sleep apnea, and this might influence the findings of the oropharyngeal examination that, in turn, are the key factors when considering surgical management for this condition. Furthermore, the correlation between the actual tonsil volume and the severity of obstructive sleep apnea in adults is currently unknown. METHODS: We prospectively studied 130 patients with obstructive sleep apnea or primary snoring who underwent pharyngeal surgery with intraoperative measurement of tonsil volume. We compared tonsil volume with preoperative polysomnography, oropharyngeal examination, and anthropometric data. RESULTS: We found a significant correlation between actual tonsil volume and subjective tonsil grade. We also found a significant correlation between tonsil volume and the apnea-hypopnea index. Using a multivariate linear regression model, tonsil volume was found to be significantly correlated with age, body mass index, and oropharyngeal examination, but not with polysomnography. Clinically, only the rare tonsil grade IV was indicative of more severe obstructive sleep apnea. CONCLUSIONS: There is a strong correlation between clinical tonsil grade and objective tonsil volume in snoring adults, and this correlation exists regardless of the presence or severity of obstructive sleep apnea. Pharyngeal tissue volume likely reflects the body mass index rather than obstructive sleep apnea severity. KEYWORDS: Oropharyngeal Examination; Sleep Apnea; Tonsil. Cahali MB, Soares CFP, Dantas DAS, Formigoni GGS. Tonsil volume, tonsil grade and obstructive sleep apnea: is there any meaningful correlation? Clinics. 2011;66(8):1347-1351. Received for publication on March 9, 2011; First review completed on April 22, 2011; Accepted for publication on April 28, 2011 E-mail: mcahali@gmail.com / cahalimd@terra.com.br Tel.: 55 11 5055-0312

examinations; however, it can be difficult to adequately distinguish between lymphoid tissue and muscle. An oropharyngeal examination, including a subjective evaluation of tonsil size, is a simple task for a general physician. However, tonsil evaluation is based on the free pharyngeal airway space rather than the tonsil’s volume itself, and theoretically, this evaluation might be influenced by structural variations in the components of the lateral pharyngeal wall. In a recent study of children, a good correlation was observed between subjective tonsil size and objective tonsil volume.4 To our knowledge, this association has never been investigated in adults. The purpose of this study was to evaluate the correlation between subjective measurements and objective palatine tonsil volume in adults and to verify the influence of the oropharyngeal anatomy, body mass index, age, and the severity of OSA on actual tonsil volume. In addition, we assessed the influence of tonsil size on OSA in adults.

INTRODUCTION The role of thickened and exceedingly collapsible lateral pharyngeal walls in the pathophysiology of obstructive sleep apnea (OSA) is well recognized in adults.1-3 The tonsils notwithstanding, narrowing of the lateral pharyngeal walls is associated with OSA in males.2 In the pathogenesis of OSA, the influence of each component of the lateral pharyngeal wall, including fat tissue, muscles, tonsils, and vessels, is currently under investigation. Volumetric analysis of these components is usually performed with imaging

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least 10 seconds) by more than 50% of the basal ventilatory value or a reduction by 50% or less associated with either a decrease in oxyhemoglobin saturation of more than 3% or an arousal. The variables that were evaluated were the apnea-hypopnea index (AHI) and the lowest oxyhemoglobin saturation (LSaO2). With respect to the severity of the disease, the patients were classified into four groups: simple snoring (AHI#5) or mild (5,AHI#15), moderate (15, AHI#30) or severe (AHI.30) OSA.

PATIENTS AND METHODS Data were collected prospectively for 130 consecutive patients who underwent pharyngeal surgery to treat simple snoring or OSA from 2001 through 2008 at two institutions. The study was approved by the Ethics Committees of both institutions and registered at the Brazilian National Committee of Ethics (CONEP) with the number NCT 01022320. We included patients who had both palatine tonsils removed during surgery and for whom the following information was available: intraoperative measurement of tonsil volume, preoperative oropharyngeal examination, full-night polysomnography, Epworth sleepiness scale, body mass index (BMI, in km/m2) and a subjective evaluation of snoring on a scale from 1 (irrelevant) to 10 (worst possible). All of the histories, physical examinations and surgeries were performed by the same examiner. The surgery performed was either a lateral pharyngoplasty5 (n = 119) or uvulopalatopharyngoplasty (n = 11), always with cold dissection tonsillectomy. The volume of the removed tonsils was assessed by liquid displacement.

Statistical Analysis To compare the groups with different OSA severities with respect to the variables studied, we used an analysis of variance (ANOVA) and the Fisher’s exact test. Correlations between tonsil volume and the other variables were determined using the Spearman’s correlation test and an ANOVA. We performed post-hoc comparisons using the Tukey and Dunnett’s tests. The influence of the variables that were studied on the volume of the tonsils was analyzed using a model of multivariate linear regression. An evaluation of a possible outlier influence was also performed. Differences with a p-value of less than 0.05 were considered significant. Statistical analyses were performed using the Statistical Package for Social Sciences (SPSS) software version 11.0 for Windows.

Otorhinolaryngologic Physical Examination The tonsils were subjectively measured using a grading system. In grade I, the tonsils were hidden in the tonsillar fossa and were barely visible behind the anterior pillars. In grade II, the tonsils were visible behind the anterior pillars and occupied up to 50% of the pharyngeal space (the distance between the medial borders of the anterior pillars). In grade III, the tonsils occupied between 50 and 75% of the pharyngeal space. In grade IV, the tonsils occupied more than 75% of the pharyngeal space. To evaluate the palate-tongue position, we used a modification of Mallampati’s technique,6 with the tongue kept in place without the use of a tongue depressor. In grade 1, the tonsils, pillars, pharynx, and soft palate were clearly visible. In grade 2, the uvula and only the upper part of the pillars and tonsils were visible between the palate and the tongue. In grade 3, only the soft and hard palate were visible, whereas the tonsils, pillars, pharynx and base of the uvula were hidden behind the tongue. In grade 4, only the hard palate was visible. The combination of the palate-tongue position and tonsil size forms the Friedman staging system,7 which is an anatomical classification that is used for selecting cases for uvulopalatopharyngoplasty. Stage 1 corresponds to palate grade 1 or 2 with tonsil grade III or IV. In stage 2, the palate grade is 1 or 2 with a tonsil grade I or II or a palate grade 3 or 4 with a tonsil grade III or IV. Stage 3 corresponds to palate grade 3 or 4 with tonsil grade I or II. The success of uvulopalatopharyngoplasty markedly decreases with an increase in Friedman stage from 1 to 3.7,8 Stage 4 corresponds to cases with a BMI that is higher than 40 kg/m2 or with gross craniofacial abnormalities and is usually not suitable for pharyngeal surgeries.

RESULTS The clinical characteristics of the 130 patients in this study are presented in Table 1. The cohort included 92 men (70.8%) and 38 women (29.2%) with a combined mean ¡ SD age of 44.5¡11.4 (men, 42.1; women, 50.5) (range 19 – 70) years and a mean BMI of 29.0¡3.1 (range 22.5 – 37.0) kg/ m2. With respect to OSA severity, 6.2% had simple snoring, 22.3% had mild OSA, 31.5% had moderate OSA, and 40% had severe OSA. There were significant differences between the severity groups with respect to BMI (severe.mild; severe.moderate). However, there were no significant differences between the groups with respect to tonsil grade (p = 0.56, Figure 1), Mallampati grade (p = 0.36), Friedman stage (p = 0.59), or gender (p = 0.18). The oropharyngeal examination of the tonsils revealed that 39.2% of the subjects had grade I, 43.1% had grade II, 16.2% had grade III and 1.5% had grade IV tonsils. A Mallampati grade of 1, 2, 3 or 4 occurred in 13.8, 32.3, 48.5, and 5.4% of the subjects, respectively. According to the Friedman staging system, 10.8% of the cases were classified as Friedman stage 1, 41.5% as stage 2 and 47.7% as stage 3. A univariate analysis of tonsil volume revealed that this volume was significantly associated with tonsil grade (grade I,II,III, p = 0.005, Figure 2) and Friedman stage (volume in stage 1.2.3, p,0.001); however, there was no association with the Mallampati grade (p = 0.11). There was also a positive correlation between tonsil volume and both AHI (s = 0.18, p = 0.04) and the Epworth scale (s = 0.19, p = 0.04) as well as a negative correlation with age (s = 20.45, p,0.001). Tonsil volume was not significantly correlated with any other variables, including BMI (s = 0.15, p = 0.09), snoring scale (s = 0.09; p = 0.34) or LSaO2 (s = 20.07, p = 0.44). Tonsil volume (p = 0.053, Figure 3), but not tonsil grade (p = 0.56), had a tendency to correlate with OSA rank of severity. There were no significant differences in mean AHI between patients with grade I (30.5), II (29.6) or III (38.2) tonsils. Patients with grade IV tonsils had a higher

Polysomnography Each subject underwent an overnight polysomnography in a sleep center in a standard fashion with a test time of seven to eight hours. The polysomnograms were scored according to Rechtschaffen and Kales.9 Respiratory event scoring was based on the American Academy of Sleep Medicine 1999 criteria10 as follows: apnea, an absence of airflow for at least 10 seconds; hypopnea, a reduction (for at

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Tonsil volume, tonsil grade and sleep apnea Cahali MB et al.

Table 1 - Clinical data of the studied groups (mean). Parameter Age (years) BMI (kg/m2) AHI LSaO2 (%) Epworth sleepiness scale Snoring scale Volume of the tonsils (ml)

Simple snoring (n = 8)

Mild OSA (n = 29)

Moderate OSA (n = 41)

Severe OSA (n = 52)

p-value

43.1 28.9 3.1 87.3 14.0 9.6 5.4

41.8 27.0 10.2 87.3 11.8 9.4 5.6

47.4 28.4 22.1 82.7 13.1 9.2 5.3

44.0 30.5 57.6 74.7 13.7 9.3 6.8

.21 ,0.001* ,0.001* ,0.001* 0.58 0.82 0.053

*Intergroup significant difference (p,0.05). OSA = obstructive sleep apnea; BMI = body mass index; AHI = apnea-hypopnea index; LSaO2 = lowest oxyhemoglobin saturation.

AHI (mean, 103.2) than those with grade I (p = 0.01), II (p = 0.01) or III (p = 0.03). Using the multiple linear regression model, a multivariate analysis of the relationship between tonsil volume and the other variables revealed that tonsil volume was significantly correlated with age (p,0.001), BMI (p = 0.004), tonsil grade (p,0.001), Mallampati grade (p = 0.006) and Friedman stage (p = 0.028), but not with polysomnography. According to the model, tonsil volume can be expressed by the equation: Volume = 1.31 2 (0.07*Age) + (0.17*BMI) + (1.92*Tonsil grade) + (0.99*Mallampati) 2 (1.2*Friedman)

system for palatine tonsils that has been correlated with actual tonsil volume in adults. What is the relevance of this correlation between subjective and objective tonsil volume in patients with OSA? The grade of the palatine tonsils is usually defined by the volume of space that is claimed by the tonsils in the pharyngeal airway. Among several anatomic factors that are involved in the pathogenesis of OSA, most patients present with either an excess amount of soft tissue or an underdeveloped bony skeleton that leads to the narrowing of the pharynx.11 The narrow lateral pharyngeal wall with which these patients present is either the result of larger lateral fat pads, muscle thickening, or tissue edema, which may be due to cervical vascular congestion.1,3,12,13 Each of these factors may displace the palatine tonsils towards the pharyngeal lumen, leading to an overestimation of their real size. Our data revealed that this is not the case: following a major increase of more than 11 kg/m2 in BMI, the same tonsil volume would be clinically graded as smaller, not bigger. Therefore, the clinical tonsil grade is a simple, reliable and independent predictor of actual tonsil volume in patients with OSA. Our multivariate equation demonstrates that tonsil volume decreases as age advances, which is expected given the tissueâ&#x20AC;&#x2122;s natural involution, and with increasing Friedman stage, which is also expected because this parameter usually implies a lower tonsil stage and therefore less tonsil volume. In contrast, tonsil volume increases as BMI and the Mallampati score increase, which implies a more crowded

DISCUSSION Measuring tonsil volume in surgically removed specimens is likely the most accurate method for quantifying that volume. In this study, we found that in adults with snoring and OSA, the clinical tonsil grade correlated well with the actual volume of this tissue, despite large variations in age and/or BMI. For example, according to our equation, an increase of one grade in the tonsils of a patient would yield an increase of 1.92 ml in tonsil volume, whereas a relatively large difference in BMI of 10 kg/m2 between two patients of the same age, palate position and tonsil grade would yield a difference of 1.70 ml in tonsil volume. Additionally, our measurements show that grade III tonsils are significantly larger than grade II tonsils, which are larger than grade I tonsils. We only had two cases of the rare tonsil grade IV. To our knowledge, this is the most discriminative grading

Figure 1 - Distribution of the observed tonsil grade (I, II, III or IV) among the various obstructive sleep apnea (OSA) severities (p = 0.56).

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significant relationship between these parameters.6 More important, these studies showed that a combination of increased oropharyngeal tissues served as a much stronger predictor of OSA than isolated upper airway findings. Because our data show that a higher BMI is associated with increased pharyngeal tissues, we suspect that such combinations simply predict the BMI, which is the strongest predictor of the presence of OSA. Thus, one should weight other anthropometric features before defining the role of large tonsils in OSA. In addition, we believe that future research in this field should investigate correlations between anthropometric features and the severity rank of OSA rather than solely with AHI because that rank would be more informative for the clinician. Clearly, the removal of extremely large tonsils (grade IV) is indicated in the management of adult OSA and produces a highly positive impact on the patientâ&#x20AC;&#x2122;s quality of life.16-18 Our rare cases with tonsil grade IV had the highest AHI. These cases aside, the impact of tonsils that extend beyond the tonsillar bed on OSA is controversial. Some authors classify all of these cases as large tonsils (grade III or IV), which implies possible surgical intervention;8 however, our grading system, which is easy to learn and implement, may identify tonsils that extend beyond the tonsillar bed as grade II, III and IV, and only grade IV tonsils have a definite impact on OSA severity. There was a potential bias in our study because all of our cases underwent pharyngeal surgery for snoring or OSA and because we excluded cases with a BMI over 35 kg/m2 or with a gross maxillary or mandible deformity. In theory, this bias would have restricted the oropharyngeal features of our subjects to a particular subtype, because the success of traditional uvulopalatopharyngoplasty or tonsillectomy in OSA treatment is largely related to the presence of large tonsils.7,8,16-18 In contrast, the majority of our patients (91.5%) underwent a lateral pharyngoplasty procedure5, which is based on the reconstruction of the lateral pharyngeal wall, and has an improved outcome over uvulopalatopharyngoplasty regardless of tonsil volume.19 Thus, we were able to include the 82.3% of cases with tonsil grades I or II, which appear to be similar to the general OSA population.15 We also recognize that we cannot use our data as a screen for OSA because we did not include a control group of non-snoring, non-OSA subjects. All of our patients complained of heavy snoring that significantly impacted their social life. Even our simple snoring group had excess daytime somnolence (with a mean Epworth scale score of 14), which we attributed to increased upper airway resistance because it improved after surgery. Consistent with reports by other groups, we believe that heavy snoring and OSA represent a progressive disease, and this progression depends more heavily on functional factors than anatomical factors and may also be influenced by the use of some drugs.20,21 The mere presence of an obstacle within the upper airway is not sufficient to produce OSA,22 and even in children, for whom the influence of anatomical factors is more definitive for OSA, tonsil volume does not correlate with AHI.4

Figure 2 - Box plot of tonsil volume versus tonsil grade (I,II,III, p = 0.005). Open circles indicate the outliers.

upper airway. Therefore, as with tongue volume,14 pharyngeal tissues also appear to be proportional to body size among both heavy snorers and OSA patients, thereby suggesting a secondary role of anatomy in OSA. It is interesting to note that the effect of uvulopalatopharyngoplasty would be noticeable in patients who do not follow this trend and have a disproportionate anatomy, for example, in patients with large tonsils and a low Mallampati score, as opposed to patients with large tonsils and a high Mallampati score.7,8 The second issue we investigated was the relationship between tonsil size and OSA severity. None of the clinical oropharyngeal data (i.e., tonsil grade, Mallampati grade or Friedman stage) was correlated with the severity rank of OSA. In contrast, tonsil volume was correlated with AHI and had a tendency to correlate with the severity rank of OSA (p = 0.053). Other groups have studied the relationship between clinical tonsil evaluation and AHI and reported a tendency approaching significance15 or a

CONCLUSIONS In adult snorers and OSA patients, there is a good correlation between clinical tonsil grade and objective tonsil volume. The clinical ability to infer tonsil volume is not

Figure 3 - Box plot of tonsil volume versus the severity of obstructive sleep apnea (OSA) (p = 0.053). Open circles indicate the outliers.

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Tonsil volume, tonsil grade and sleep apnea Cahali MB et al. 10. American Academy of Sleep Medicine. Sleep-related breathing disorders in adults: recommendations for syndrome definition and measurement techniques in clinical research. Sleep. 1999;22:667-89. 11. Ryan CM, Bradley TD. Pathogenesis of obstructive sleep apnea. J Appl Physiol. 2005;99:2440-50, doi: 10.1152/japplphysiol.00772.2005. 12. Anastassov GE, Trieger N. Edema in the upper airway in patients with obstrutictive sleep apnea syndrome. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 1998;86:644-7, doi: 10.1016/S1079-2104(98)90197-4. 13. Bradley TD, Rutherford R, Grossman RF, Lue F, Zamel N, Moldofsky H, et al. Role of daytime hypoxemia in the pathogenesis of right heart failure in the obstructive sleep apnea syndrome. Am Rev Respir Dis. 1985;131:835–9. 14. Do KL, Ferreyra H, Healy JF, Davidson TM. Does tongue size differ between patients with and without sleep-disordered breathing? Laryngoscope. 2000;110:1552-5, doi: 10.1097/00005537-200009000-00027. 15. Zonato AI, BIttencourt LR, Martinho FL, Ju´nior JFS, Grego´rio LC, Tufik S. Association of systematic head and neck physical examination with obstructive sleep apnea syndrome. Laryngoscope. 2003;113:973-80, doi: 10.1097/00005537-200306000-00011. 16. Nakata S, Noda A, Yanagi E, Suzuki K, Yamamoto H, Nakashima T. Tonsil size and body mass index are important factors for efficacy of simple tonsillectomy in obstructive sleep apnea. Clin Otolaryngol. 2006;31:41-5, doi: 10.1111/j.1749-4486.2006.01130.x. 17. Martinho FL, Zonato AI, Bittencourt LR, Soares MCM, Silva RFN, Grego´rio LC, et al. Obese obstructive sleep apnea patients with tonsil hypertrophy submitted to tonsillectomy. Braz J Med Biol Res. 2006;39:1137-42, doi: 10.1590/S0100-879X2006000800017. 18. Verse T, Kroker BA, Pirsig W, Brosch S. Tonsillectomy as a treatment of obstructive sleep apnea in adults with tonsillar hypertrophy. Laryngoscope. 2000;110:1556–9, doi: 10.1097/00005537-200009000-00029. 19. Cahali MB, Formigoni GGS, Gebrim EMMS, Miziara ID. Lateral pharyngoplasty versus uvulopalatopharyngoplasty: a clinical, polysomnographic and computed tomography measurement comparison. Sleep. 2004;27:942-50. 20. Friberg D. Heavy snorer’s disease: a progressive local neuropathy. Acta Otolaryngol. 1999;119:925-33, doi: 10.1080/00016489950180306. 21. Neves C, Tufik S, Chediek F, Poyares D, Cintra F, Roizenblatt M, et al. Effects of sildenafil on autonomic nervous function during sleep in obstructive sleep apnea. Clinics. 2010;65:393-400, doi: 10.1590/S180759322010000400008. 22. Strohl K, Redline S. State-of-the-art: recognition of sleep apnea. Am J Respir Crit Care Med. 1996;154:279-89.

impaired by possible changes in pharyngeal geometry that are related to OSA. Although tonsil volume correlates with AHI, from a clinical perspective, only tonsil grade IV predicts severe OSA. Pharyngeal tissue volume likely reflects body mass index rather than OSA.

REFERENCES 1. Schwab RJ, Pasirstein M, Pierson R, Mackley A, Hachadoorian R, Arens R, et al. Identification of upper airway anatomic risk factors for obstructive sleep apnea with volumetric magnetic resonance imaging. Am J Respir Crit Care Med. 2003;168:522-30, doi: 10.1164/rccm.200208866OC. 2. Schellenberg JB, Maislin G, Schwab RJ. Physical findings and the risk for obstructive sleep apnea. Am J Respir Crit Care Med. 2000;162:740-8. 3. Schwab RJ, Gupta KB, Gefter WB, Metzger LJ, Hoffman EA, Pack AI. Upper airway and soft tissue anatomy in normal subjects and patients with sleep-disorders breathing: significance of the lateral pharyngeal walls. Am J Respir Crit Care Med. 1995;152:1673-89. 4. Howard NS, Brietzke SE. Pediatric tonsil size: Objective vs subjective measurements correlated to overnight polysomnogram. Otolaryngol Head Neck Surg. 2009;140:675-81, doi: 10.1016/j.otohns.2009.01.008. 5. Cahali MB. Lateral pharyngoplasty: a new treatment for obstructive sleep apnea hypopnea syndrome. Laryngoscope. 2003;113:1961-8, doi: 10.1097/00005537-200311000-00020. 6. Friedman M, Tanyeri H, La Rosa M, Landsberg R, Vaidyanathan KMS, Pieri S, et al. Clinical predictors of obstructive sleep apnea. Laryngoscope. 1999;109:1901-7, doi: 10.1097/00005537-19991200000002. 7. Friedman M, Ibrahim H, Bass L. Clinical staging for sleep disordered breathing. Otolaryngol Head Neck Surg. 2002;127:13–21, doi: 10.1067/ mhn.2002.126477. 8. Friedman M, Ibrahim H, Joseph NJ. Staging of obstructive sleep apnea/ hypopnea syndrome: a guide to appropriate treatment. Laryngoscope. 2004;114:454-9, doi: 10.1097/00005537-200403000-00013. 9. Rechtschaffen A, Kales A. A manual of standardized terminology, techniques and scoring system of sleep stages of human subjects. Los Angeles: Brain Research Institute UCLA, 1968.

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DOI:10.1590/S1807-59322011000800008

CLINICAL SCIENCE

Attenuation of neuropsychiatric symptoms and caregiver burden in Alzheimer’s disease by motor intervention: a controlled trial Florindo Stella,I Ana Paula Canonici,I,II Sebastiaõ Gobbi,I Ruth Ferreira Santos-Galduroz,I,III Joaõ de Castilho Caçaõ,IV Lı´lian Teresa Bucken GobbiV I UNESP - Universidade Estadual Paulista, Biosciences Institute, Aging and Physical Activity Laboratory, Campus of Rio Claro/SP, Brazil. II Physiotherapy Unity, University of Araras/SP, Brazil. III Federal University of ABC - Mathematics, Computation & Cognition Center, Saõ Paulo/SP, Brazil. IV FAMERP Geriatric Unity, Faculty of Medicine at Saõ Jose´ do Rio Preto/SP, Brazil. V UNESP – Universidade Estadual Paulista, Biosciences Institute, Posture and Gait Studies Laboratory, Campus of Rio Claro/SP, Brazil.

OBJECTIVE: To analyze the effects of motor intervention on the neuropsychiatric symptoms of Alzheimer’s disease and on the caregivers’ burden. DESIGN: This is a controlled trial evaluating the effects of a motor intervention program on the neuropsychiatric symptoms. SETTING: The intervention was performed on community patients from two university centers specializing in physical exercise for the elderly. SUBJECTS: Patients with Alzheimer’s disease were divided into two groups: sixteen received the motor intervention and sixteen controls (five controls were excluded because of clinical intercurrences). INTERVENTIONS: Aerobic exercises (flexibility, strength, and agility) and functional balance exercises were conducted over six months for 60 minutes three times per week. MAIN MEASURES: Psychopathological features of patients were evaluated with the Neuropsychiatric Inventory and Cornell Scale for Depression in Dementia. Caregivers were evaluated using the Neuropsychiatric Inventory-Distress and Burden Interview. A two-way analysis of variance (ANOVA) was applied to observe interactions (pre- vs. postintervention; participants vs. controls). RESULTS: Patients from the intervention presented a significant reduction in neuropsychiatric conditions when compared to controls (Neuropsychiatric Inventory: F511.12; p = 0.01; Cornell Depression scale: F511.97; p = 0.01). The burden and stress of caregivers responsible for patients who participated in the intervention significantly decreased when compared to caregivers responsible for controls (Neuropsychiatric Inventory-Distress: F: 9.37; p = 0.01; Burden Interview: F: 11.28; p = 0.01). CONCLUSIONS: Aerobic exercise was associated with a reduction in the neuropsychiatric symptoms and contributed to attenuate the caregivers’ burden. However, the researchers were not blinded to the patient’s intervention status, which constitutes an important limitation of this study. KEYWORDS: Alzheimer’s disease; Aerobic exercise; Neuropsychiatric symptoms; Caregiver’s burden. Stella F, Canonici AP, Gobbi S, Santos-Galduroz RF, Cac¸a˜o JC, Gobbi LTB. Attenuation of neuropsychiatric symptoms and caregiver burden in Alzheimer’s disease by motor intervention: a controlled trial. Clinics. 2011;66(8):1353-1360. Received for publication on March 14, 2011; First review completed on March 28, 2011; Accepted for publication on April 28, 2011 E-mail: fstella@rc.unesp.br Tel.: 55 19 35264245

INTRODUCTION Neuropsychiatric symptoms are important clinical manifestations of Alzheimer’s disease (AD), which is a neurodegenerative disorder that causes a decrease in cognition, functionality, and behavior.1,2 Although the major etiological agent is the neurodegenerative process causing

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an appropriate motor intervention program could contribute to a decrease in the psychopathological disturbances of AD and attenuate the caregiver’s burden. The purpose of this study was to examine the effects of a six-month aerobic exercise program on the neuropsychiatric symptoms of patients with AD and the impact of exercise on alleviating the caregiver’s burden.

the dementia, there is a complex interplay between the biological, social, and psychological factors that are involved in the pathogenesis of these symptoms.3 General clinical conditions, environmental stressors, and demand surpassing the caregiver’s capacity may result in psychopathological manifestations.1 These symptoms are highly prevalent and represent a source of burden for the caregiver, resulting in a shorter time to institutionalization4 and an increase in the suffering of the caregivers or relatives.5 In this context, caregivers of AD patients display important burdens associated with their daily caregiving responsibilities because of the frequency of and the stress related to the provision of assistance.6 At present, pharmacological interventions for AD patients with behavioral disturbances are limited to the treatment of symptoms, although treated patients have shown significant benefits compared to those without treatment.7 Improving the management of AD patients with neuropsychiatric symptoms such as agitation, aggression, depression, apathy, and other psychopathological phenomena requires the attenuation of the frequency and severity of these disturbances. Whereas studies have increasingly targeted the retardation of cognitive decline through pharmacological or non-pharmacological strategies, few studies have focused on the use of aerobic exercise to alleviate neuropsychiatric symptoms and reduce the caregiver’s burden in AD. Research concerning the effects of exercise on the risk of dementia in the elderly has produced inconsistent results, with some studies, but not others,10,11 reporting a lower risk of all-cause dementia, including AD.8,9 However, in addition to traditional medical approaches to prevent disease progression, regular exercise may significantly improve well-being and reduce the risk of dementia later in life.9 Consequently, there is currently a significant, increasing effort to clarify the impact of physical exercises on the risk of dementia in this neurodegenerative condition. Recently, Hamer & Chida12 used a meta-analysis to conclude that physical exercise is protective against the future risk of dementia and reduces the risk of AD. Aiming to reduce disability in patients with AD, Teri et al.13 performed a randomized controlled trial that demonstrated the favorable effects of exercise on behavioral management and on depressive symptoms among patients with AD in comparison to those under routine medical care. Landi et al.14 similarly reported that aerobic exercise reduced neuropsychiatric symptoms, such as wandering, aggression, and sleep disturbances, in nursing home residents who were cognitively impaired. In this same context, Heyn et al.15 demonstrated by a meta-analysis that regular and systematic aerobic exercise, such as walking, dancing, and cycling, provided favorable effects on dementia, including AD. The authors attributed these benefits to a reduction in cognitive decrease and to an improvement in daily living activities. However, in another meta-analysis, Forbes et al.16 found no similar benefits of global physical activity programs for persons with dementia. After a one-year exercise program, Rolland et al.17 similarly failed to observe a significant decrease in the behavioral symptoms of AD. There is emerging but insufficient evidence that exercise prescription could be an established treatment for neuropsychiatric symptoms in AD.18 Furthermore, more scientific support is needed to clarify whether exercises targeted at patients can reduce the caregiver’s burden and the suffering of relatives. To address this question, we hypothesized that

METHODS This controlled trial involved 32 community patients and their respective caregivers recruited from two centers: the Aging and Physical Activity Laboratory (UNESP – Univ Estadual Paulista) and the Alzheimer’s Brazilian Association (Regional-Araras, SP). Several strategies were implemented for the recruitment of subjects to the physical exercise program: a) explanations to clinicians about the importance of our research project; b) community-wide advertisements on television, newspaper and radio; and c) community meetings to present the objectives and development stages of our project. After these disclosures, families and clinicians began to refer their patients to our services. The physical exercise program for AD was implemented at UNESP – Univ Estadual Paulista in 2003, and the patients from the present study were assessed at the beginning of 2008. Inclusion criteria were as follows: (a) diagnosis of probable AD by a trained clinician; (b) mild or moderate stage of the disease; (c) locomotion ability, preserved vision and hearing (eyeglasses and/or hearing aid were permissible) sufficient for compliance with testing procedures; and (d) presence of a respective caregiver. Patients with motor disorders who had difficulty with the motor performance required for physical tasks were excluded. The clinicians responsible for the patients established the diagnosis of Alzheimer’s disease according to the consensus criteria of the National Institute for Neurological and Communicative Disorders/Association (NINCDS/ADRDA).19 They referred the patients to our services for participation in the present investigation. The assessment of dementia was made according to the Diagnostic and Statistical Manual of Mental Disorders.20 The dementia severity was measured according to the Clinical Dementia Rating scale.21 This scale assesses the patient’s cognitive and functional state, and it is graded on a scale from 0 (normal) to 0.5 (questionable dementia), 1 (mild dementia), 2 (moderate dementia), and 3 (severe dementia). A physician trained in geriatric psychiatry confirmed the diagnosis to include only patients with mild or moderate AD. Furthermore, he supervised all cognitive and neuropsychiatric assessments while blind to the patient’s assignment to the motor intervention or control groups. The main outcome measures concerned the impact of aerobic exercise on neuropsychiatric symptoms in AD. To this end, the interviewers completed a cognitive assessment at each time point (baseline and final application) using the Mini-Mental State Examination22 to characterize global cognitive functioning. This instrument is a brief, cognitive screen that assesses the patient’s time and place orientation, immediate and delayed recall, concentrated attention and calculation, naming, repetition, comprehension, and constructional praxis. The interviewers additionally applied the Neuropsychiatric Inventory23 to identify psychopathological features including delusions, hallucinations, agitation, dysphoria, anxiety, euphoria, apathy, dis-inhibition, irritability,

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addition, after the end of the exercise program, the patients from the control group were invited to engage in other physical activity studies performed at the same sites. The aerobic exercise included five phases suggested by Gobbi et al.27 for the elderly in Brazil. Each exercise session was comprised of a warm-up period, pre-exercise stretching, the main exercise procedures, a cool-down period, and post-exercise stretching. At the end of each phase, there was a progressive increase of load. The main exercise program involved aerobic procedures such as walking, dancing, and exploring upper and lower limb mobility with the goal of enhancing flexibility, strength, and agility as well as improving functional balance. There is evidence to suggest that these motor components are important for maintaining the functional capacity of patients with AD. Sixteen patients participated in the program of aerobic exercise with moderate intensity and on a regular basis over six months. The aerobic exercises (moderate intensity over a long duration) encompassed activities that simultaneously benefit several components of functional capacity such as flexibility (stretching), muscular resistance (specific exercises for the large muscle groups), motor coordination (rhythmic activities), and balance (recreational motor activities). The exercise program was administered to two subgroups of eight participants simultaneously, each under the supervision of at least one expert on physical education. The respective caregivers did not participate in this program. They observed the activities of their patients (exercise program plus medical routine vs. medical routine only) during the six-month period from a distance as spectators. In addition, the caregivers did not engage in the interventions themselves. This strategy aimed to assess effects on the caregivers’ burden that could be attributed to the hypothesized reduction in neuropsychiatric symptoms of the respective participant patients. The caregivers were evaluated with respect to their burden and the occurrence of stress at the beginning and the end of this period using the Neuropsychiatric InventoryDistress23 and Burden Interview questionnaires.24 Data processing and statistical analysis were performed using the ‘Statistica 5.0’ software. The descriptive analyses displayed mean values and standard deviations. The ShapiroWilk test was performed to assess the normal distribution of the data, and because the confirmed data were normal, a parametric analysis was considered. First, the t-test was applied to independent samples to verify group homogeneity for baseline values. The demographic and clinical variables of the groups were compared using the two-way analysis of variance (ANOVA) for repeated variables. The two-way ANOVA was similarly applied to verify interactions between time points (pre- and post-intervention) and groups (with motor intervention and controls). Because five patients left the motor intervention group before the completion of the trial, a complete case analysis was made. Statistical significance was defined at a level of p,0.05. The local ethics committee approved the study, and patients and/or relatives (directly responsible for patients) provided written informed consent.

aberrant motor behavior, sleep disorders, and appetite disorders. This instrument consists of a structural interview for measuring the frequency and severity of symptoms, with higher scores related to greater severity. Moreover, the Neuropsychiatric Inventory allows the assessment of the caregiver’s distress related to patient disturbances. To determine the caregiver’s mental suffering and burden, the interviewers completed the Burden Interview,24 and to identify depressive symptoms in the patients, they applied the Cornell Scale for Depression in Dementia,25 with higher scores representing greater severity. Two experts on neuropsychiatric disorders in the elderly supervised all cognitive and psychopathological procedures. The interviewers administered the scales to patients (e.g., the Mini-Mental State Examination) and caregivers (e.g., the Neuropsychiatric Inventory-Distress and Burden Interview) either together or separately when appropriate, taking into account the patient’s behavior or emotional condition. In general, the interviewers questioned the caregiver while observing the patient when they were together, and the interviewers requested the patient’s attention for clarifying some items when necessary. In conducting these procedures, the interviewers made an effort to limit the emotional influence of the patient or the caregiver on the results. At baseline, the raters were unaware as to how the groups would be divided. In addition, the raters themselves did not know the group to which the patient and the caregiver belonged. Although the raters had planned to be blinded in completing the scales, this strategy was not implemented because of the insufficient number of experts. At baseline, prior to the start of the study, no patients had been engaged in a systematic physical exercise program. The patients were divided into two groups to generate groups with overall similar physical activity levels, as measured by the Modified Baecke Questionnaire for the Elderly,26 and similar psychopathological conditions, as measured by the Neuropsychiatric Inventory (total score) and the Cornell Scale for Depression in Dementia. Following group assignment, the 16 patients in the treatment group participated in the exercise program plus a medical routine, whereas the 16 patients in the control group abstained from systematic physical exercise and adhered to their standard medical routine (based on prescribed medicines and general health orientation established by their respective clinicians, excluding participation in physical exercise programs). Neither the patients nor the caregivers were informed of their allocation to one group or the other prior to the start of the trial. For the treatment group, exercises were performed over a six-month period for 60 minutes three times per week on nonconsecutive days. Exercises were conducted in locations and buildings with appropriate conditions that were, in general, close to the homes of the participants. Patients came to the sites with their caregivers by automobile or by special public transport, and this travel did not involve systematic physical exercise. Patients in the control group traveled to the exercise sites only on the days of the assessment: once at the beginning and once at the end of the six-month period. During the intervention, patients from the control group stayed at home or performed the typical activities of daily life. Researchers ensured that these patients refrained from systematic motor intervention during this period and instead followed only their regular medical routine. In

RESULTS The sample comprised 32 community patients with AD and their respective caregivers. At baseline, 34% of all patients were receiving anticholinesterases, 17% were receiving antidepressants, and 12% were receiving benzodiazepines. The

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caregivers of patients with dementia.1,28 However, caregivers whose patients participated in the aerobic program presented a significant reduction in Neuropsychiatric InventoryDistress (18.3 to 3.3). Conversely, caregivers whose patients belonged to the control group maintained similar scores at the end of the program (19.6 to 20.4), with a significant difference between groups (two-way ANOVA: F59.37; p = 0.01). In the Burden Interview, the scores of caregivers whose patients participated in the aerobic program similarly significantly decreased (32.3 to 14.6), whereas the scores of caregivers whose patients belonged to the control group remained the same (35.6 to 35.5). The difference between the groups was significant (two-way ANOVA: F:11.28; p = 0.01) (Table 3). The improvement in the caregivers’ burden was attributed to a reduction in the behavioral disturbances by patients who performed the exercise program. For instance, agitation/aggression, anxiety, apathy/indifference, disinhibition, and appetite alterations, as well as depression, were reduced in this group. However, caregivers whose patients belonged to the control group did not experience the same benefit (Figure 3).

severity of dementia was rated from 1 (mild) to 2 (moderate) according to the Clinical Dementia Rating scale.21 The sociodemographic characteristics of the patients, together with selected clinical aspects, are displayed in Table 1. During the exercise program, five patients from the control group suffered severe clinical occurrences such as stroke or falls with fractures and sudden cognitive decline. Because of these events, these patients and their respective caregivers were excluded from the sample. This group was composed of 11 patients and 11 caregivers at the end of the study (Figure 1). The five patients who were lost from the control group received appropriate medical care. Participants from the exercise group attended at least 70% of the motor sessions. At baseline, both groups had similar levels of psychopathological manifestations, as measured by their Neuropsychiatric Inventory scores (participants vs. controls: 40.3 vs. 39.6; t-test: -0.4; p = 0.69), which suggests these manifestations were clinically relevant. However, after the six-month period of the aerobic program, the Neuropsychiatric Inventory scores of the participants had decreased and were lower than those of the controls (40.3 to 16.9 vs. 39.6 to 43.3) with significant differences between the groups (two-way ANOVA: F:11.12; p = 0.01). Similarly, when examining the Cornell Scale for Depression in Dementia, the participants’ scores decreased over the six-month period, and they presented significantly lower values than the controls (11.8 to 6.4 vs. 12 to 17.1; t-test: 0.25; p = 0.81), with significant differences between groups (two-way ANOVA: F:11.97; p = 0.01). Here, the main differences were based on a reduction in neuropsychiatric symptoms among participants (agitation/aggression, depression, anxiety, apathy/indifference, dis-inhibition, irritability, and appetite alterations) and an increase in these symptoms among patients in the control group (Table 2; Figure 2). With regard to the burden and stress of the caregivers, at baseline, the scores on the Neuropsychiatric InventoryDistress and Burden Interview were high in both groups. However, there were no significant differences between these caregiver groups (Neuropsychiatric Inventory – total burden score: 18.3 vs. 20.1; t-test: -0.24; p = 0.81; Burden Interview: 32.3 vs. 36.4; t-test: -0.57; p = 0.57). These scales measure the burden and stress, respectively, experienced by the

DISCUSSION The results of this analysis indicate that aerobic exercise over a six-month period contributed to a decrease in the neuropsychiatric symptoms of patients with AD and to a reduction in caregiver burden. In the control group, these symptoms slightly increased, and the burden of the respective caregivers remained unchanged. It should be noted that during the intervention, five patients were excluded from the control group. The primary reasons for exclusion were clinical intercurrences, such as stroke or falls, which were associated with a severe cognitive decrease. Because it is possible that these patients differed from those who experienced no intercurrences, they were excluded to maintain the original features of the control group. For this reason, a complete case analysis was performed. Patients who participated in aerobic exercise experienced improvement in several domains on the Neuropsychiatric Inventory, including irritability, anxiety, apathy/indifference, and appetite alterations, when compared to controls. Differences between the groups could be attributed to the reciprocal influence of two factors, i.e., the reduction in symptoms among patients who participated in aerobic exercise and the increase of these symptoms in the control group. This interaction was significant but was simultaneously based on a decrease in psychopathological manifestations in the exercise group and an increase of these symptoms in the control group. Therefore, because AD is a neurodegenerative process with progressive impairment of cognition and behavior, aerobic exercise is a useful strategy for alleviating patient suffering and contributed to a reduction in caregiver stress by allowing the patients to better manage their own daily responsibilities.29-31 In addition, apathy/indifference, anxiety, and appetite alterations represented the primary sources of caregiver burden. Unsurprisingly, behavioral disturbances rather than cognitive impairment have been previously reported to be important factors linked to patient and caregiver burden.1,3 Two meta-analyses15,16 that examined the effect of physical activity on cognition, function and behavior in patients with dementia arrived at distinct conclusions.

Table 1 - Socio-demographic and clinical aspects of 32 patients with Alzheimer’s disease and their respective caregivers at baseline (percentage, mean and standard deviation).

Characteristics Gender - Male - Female Age (years) Mini-Mental (points) Education (years) CDR (points) Diagnosis duration (years) Care duration (years)

Patients (n = 32)

Caregivers (n = 32)

(%, mean and SD)

12 (37.5%) 20 (62.5%)

2 (6.3%) 30 (93.7%)

77.8 (5.8) 15.4 (6.0) 4.5 (4.1) 1.5 2.8 (1.3) -----

54.2 (11.7) ----9.8 (4) --------2.7 (1.4)

CDR: Clinical Dementia Rating; Mini-Mental: Mini-Mental State Examination. SD: Standard deviation.

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Figure 1 - Flow diagram of disclosure, patient recruitment, intervention, and assessments.

Whereas Heyn et al.15 verified that individuals with cognitive impairment or dementia who took part in an exercise program experienced benefits to cognition, daily life activities, and behavioral disturbances, Forbes et al.16 found no sufficient evidence to support similar conclusions. Additionally, Steinberg et al.32 recently verified the benefits of physical exercise for functionality in basic daily activities, but not for behavioral disturbances. To measure psychopathological symptoms, these authors32 applied the Neuropsychiatric Inventory, which is the same scale used in our investigation. Despite a growing number of studies that emphasize the effects of aerobic exercise on cognitive function in AD, there are few reports in the literature aimed at analyzing the impact of such exercise on behavioral disturbances and functionality. Our findings are in agreement with studies reporting that physical exercise may be associated with the attenuation of neuropsychiatric symptoms in AD,13,14,17,30,31 but diverge from the conclusions of others.16,32,33 Alternative methodological strategies that could be implemented may produce different results compared with our data. For instance, reductions in neuropsychiatric

symptoms may have been improved if the caregivers were performing the same aerobic program as the patients. Furthermore, this type of research is impacted by the difficulty of measuring both the effects of physical exercise itself and the psychosocial stimulation that may influence the patientâ&#x20AC;&#x2122;s motivation. In addition, if the control group had been provided with joint activities, such as recreational games with their caregivers during the same period of time, their neuropsychiatric symptoms may have been improved. These questions remain to be answered. It should be noted that the Neuropsychiatric InventoryClinician rating scale is a uniform assessment system for accurately measuring behavioral disturbances in patients with dementia and caregiversâ&#x20AC;&#x2122; burden.34 This scale includes a clinician-rating judgment to provide an overall severity rating for neuropsychiatric symptoms in dementia. In Brazil, Fialho et al.35 recently reported a significant association between the behavioral manifestations of patients with dementia, as assessed by the Neuropsychiatry Inventory, and a high level of burden among caregivers. The similarly favorable scores from both the Neuropsychiatric InventoryDistress and Burden Interview questionnaires suggests that

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Table 2 - Neuropsychiatric symptoms according to Neuropsychiatric Inventory domains and the Cornell Scale for Depression in Dementia for patients with Alzheimer’s disease (significant values: mean and SD) at baseline and at the time of final evaluation, after six months of exercise. Controls (n = 11) Neuropsychiatry Inventory

Delusions Hallucinations Agitation/aggression Depression Anxiety Euphoria Apathy/indifference Dis-inhibition Irritability Motor behaviors Sleep disturbances Appetite alterations NPI-Total Cornell Depression

First evaluation

Patients with motor intervention (n = 16) Final evaluation

First evaluation

Final evaluation

(mean, SD)

4.3 3.7 2.7 4.5 5.4 2.4 4.5 0.5 5.6 2.5 4.4 1.1 39.6 12

2.5 1.7 5 6.2 6.8 0.2 6 1.4 6.9 1.1 2.7 2 43.3 17.1

1.9 1.4 2.4 5.3 6.6 1.5 6.4 2.7 6.6 2.9 2.8 3.6 40.3 11.8

0.4 (1.1) 0.1 (0.3) 0.3 (1) 1.4 (3.1) 1.3 (3.2) 0 1.4 (3.2) 0.1 (0.3) 0.8 (3) 0.5 (1.4) 0.3 (0.7) 0.9 (3) 16.9 (17.6) 6.4 (4.8)

(4.7) (5) (3.9) (4.1) (4.9) (4.8) (4.5) (1.2) (5.4) (4) (5.6) (2) (25) (7.9)

(3.2) (3.6) (5.4) (5) (5.7) (0.6) (4.6) (2.8) (5.3) (2.4) (4.7) (2.5) (18.4) (6.3)

(3.7) (3.1) (3.3) (5.1) (4.7) (4.1) (5.2) (4.5) (4.4) (4.6) (4.2) (4.3) (34) (6.5)

0.01 0.11 5.52 6.4 9.14 0.15 8.87 4.95 10.95 0.29 0.27 8.03 11.12 11.97

0.90 0.73 0.02 0.01 0.01 0.69 0.01 0.03 0.01 0.59 0.60 0.01 0.01 0.01

Values of F interaction between group and motor intervention and ANOVA two-way for repeated measures. SD: Standard deviation.

early Alzheimer’s disease, Burns et al.40 found that high fitness levels were associated with less brain atrophy, resulting in preserved brain volume. In a comprehensive review, Kramer & Erickson38 reported that BDNF is an important molecule in the effects of physical exercise on the human brain and cognition. Based on experimental models, voluntary exercise increases both mRNA and protein levels of BDNF, with a favorable impact on the hippocampus, the frontal cortex, and other brain regions38-40 and increased matter integrity in select cerebral structures41, probably with a favorable impact on cognition and behavior.42 However, the neurobiological mechanisms connecting exercise and neuro-protection in patients with AD remain unclear. In the present study, interviewers were not blinded when made data collection. The non-blinded assessment of outcome can result in an overestimation of the effects of an intervention, with the risk of apparent benefit or expectation bias. Although we recognize that blinding during data collection is a critical aspect of any controlled trial to eliminate subjective bias and improve accuracy and agree that blinding should be routinely incorporated into the design of such research, in this study, this procedure was not operationally possible. The primary reason for this was the limited availability of volunteer interviewers for the assessments. However, this problem was partially controlled for by a careful and appropriate orientation and training of the interviewers by experts on psychometric assessment and motor procedures to avoid subjective interference. Regarding the clinical approach, physical exercise is already recommended to improve neuropsychiatric symptoms in patients with dementia and to reduce the suffering of caregivers in conducting their daily responsibilities. Furthermore, evidence supporting the favorable effects of physical exercise on neuropsychiatric symptoms has provided sufficient reason to encourage participation in motor intervention programs among patients with AD. Our results must be interpreted with caution because there are several noteworthy methodological limitations. First, the use of non-blinded interviewers constitutes a

regular aerobic exercise should help to attenuate the caregivers’ suffering. In addition, given the limited efficacy of pharmacological strategies for AD, exercise intervention could represent a useful resource to delay the need for placement of patients under a complex level of care.32 Concerning the neurobiological approach, exercise has been related to enhance neuronal survival36, vascularization, and growth factors in the brain regions linked to cognitive functions such as memory.37 In experimental models evaluating the effects of motor activity, neurogenesis, synaptogenesis, decreasing beta-amyloid, angiogenesis, and increasing brain-derived neurotropic factor (BDNF) have been observed, and these phenomena are likely to be present in the non-model, human brain.38 In addition, exercise promotes vascular health by lowering blood pressure, lipids, obesity and inflammatory markers, which are associated with risk of cognitive aggravation in AD.38 Furthermore, enlargement of the hippocampus was observed in subjects who performed systematic aerobic exercise.39 In a study with

Figure 2 - Impact of motor intervention program on psychopathological features according to Neuropsychiatric Inventory and Cornell Scale for Depression in Dementia among patients with Alzheimer’s disease.

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Table 3 - Caregivers’ burden and stress (significant values: mean and SD) related to Neuropsychiatric Inventory domains and Burden Interview among patients with Alzheimer’s disease at baseline and at the time of the final evaluation, after six months of exercise. Caregivers of Controls (N = 11)

Caregivers of Participants (N = 16)

Neuropsychiatric Inventory-Distress

First Evaluation (mean, SD)

Final Evaluation (mean, SD)

First Evaluation (mean, SD)

Agitation/aggression Anxiety Apathy/indifference Dis-inhibition Appetite alterations NPI-Distress (total) Burden Interview

1.6 2.5 2.2 0.4 0.8 19.6 35.6

2.2 2.8 2.8 0.9 1.5 20.4 35.5

1.6 2.5 2.8 1 1.6 18.3 32.3

(2) (2) (1.9) (0.8) (1.3) (14.3) (14.9)

(2.2) (2.4) (1.9) (1.6) (2) (9.6) (13.7)

(2.1) (1.9) (2) (1.7) (1.9) (13.8) (14.7)

Final evaluation (mean, SD) 0.1 0.4 0.6 0.1 0.4 3.3 14.6

(0.5) (1) (1.3) (0.3) (1.2) (6.5) (8.1)

4.98 8.83 10.23 5.00 6.74 9.37 11.28

0.03 0.01 0.01 0.03 0.01 0.01 0.01

Values of F interaction between group and motor intervention and ANOVA two-way for repeated measures. SD: Standard deviation.

patients in the control group, the scores in several domains on this scale slightly decreased. Whether this reduction was related to a placebo response or to natural fluctuations in the course of the disease is not clear. Another limitation consists of the absence of psychotropic controls. The effect of these drugs was not investigated in either group in the first or second evaluation. It is possible that this could represent a bias, and our results may not be readily transferable to a clinical population. Regarding the number of patients, there was a small sample size, particularly in the control group, which substantially reduces the statistical power and capacity for the generalization of our results. In conclusion, our results suggest that six months of aerobic exercise is associated with a reduction in the neuropsychiatric symptoms of patients with AD and contributes to the attenuation of the caregiver’s burden. Irritability, anxiety, apathy, and appetite alterations were the primary improved psychopathological manifestations. The positive effects should be interpreted with caution in view of our limitations. Whether these benefits are maintained for longer periods will require further investigation.

limitation, as this methodological strategy could have weakened the reliability of the data. Furthermore, patients and caregivers were not blinded; they may have been more positive in describing their symptoms because of the effort they had put into the physical exercise during the intervention program. In addition, psychological states might have influenced the assessment of the caregivers. It is possible that their emotional and cognitive conditions have an uncontrolled impact on the interpretation of a patient’s behavior. In Brazil, family members frequently provide daily care for elderly people, and the presence of professional caregivers, who would avoid psychological influences, is a not common occurrence. The absence from this study of a group of patients with severe dementia represents another limitation. Because of this deficit, it was impossible to assess the effects of physical exercise on the neuropsychiatric symptoms of severe dementia or to compare the results with mild or moderate dementia. Concerning the procedures described in the methods section, the data collection for the patient and the caregiver were completed together or separately, according to specific situations, such as emotional condition or behavior control. Although these procedures could interfere with the accuracy and quality of the measurements, the interviewers made an effort to limit the emotional influences of the patient or caregiver on the results. Although the total score on the Neuropsychiatric Inventory increased among

Clinical Message

N N

Aerobic exercise represents a favorable, non-pharmacological strategy for reducing behavioral disturbances of patients with AD. Caregivers whose patients participated in the exercise program presented an attenuation of burden and stress.

ACKNOWLEDGMENTS The authors acknowledge Etna Maca´rio for English revision.

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Figure 3 - Impact of motor intervention performed by patients on caregivers burden and stress according to Neuropsychiatric Inventory and Burden Interview.

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23. Cummings JL, Mega M, Gary K, Rosenberg-Thompson S, Carusi DA, Gornbein J. The Neuropsychiatric Inventory: comprehensive assessment of psychopathology in dementia. Neurology. 1994;44:2308-14. 24. Zarit SH, Reever KE, Bach-Peterson J. Relatives of the impaired elderly: correlates of feeling of burden. Gerontologist. 1980;20:649-55, doi: 10. 1093/geront/20.6.649. 25. Alexopoulos GS, Abrans RC, Young RC, Shamoian CA. Cornell Scale for Depression in Dementia. Biol Psychiatr. 1998;23:271-84, doi: 10.1016/ 0006-3223(88)90038-8. 26. Voorrips LE, Ravelli AC, Dongelmans PC, Deurenberg P. Van Staveren WA: A physical activity questionnaire for the elderly. Med Sci Sports Exerc. 1991;23:974-9. 27. Gobbi S, Villar R, Zago AS. Bases Teo´rico-Pra´ticas do Condicionamento Fı´sico. Rio de Janeiro: Editora Guanabara Koogan, 2005. 28. Taub A, Andreoli SB, Bertolucci PH. Dementia Caregiver Burden: reliability of the Brazilian version of the Zarit caregiver burden interview. Cad Sau´ de Pu´ blica. 2004;20:372-6, doi: 10.1590/S0102311X2004000200004. 29. Cohen GD. Alzheimer’s disease: managing behavioral problems in patients with progressive dementia. Geriatrics. 2002;57:53-4. 30. Castro CM, Wilcox S, O’Sullivan P, Baumann K, King AC. An exercise program for women who are caring for relatives with dementia. Psychosom Med. 2002;64:458-68. 31. Williams CL, Tappen RM. Exercise training for depressed older adults with Alzheimer’s disease. Aging Mental Health. 2008;12:72-80, doi: 10. 1080/13607860701529932. 32. Steinberg M, Leoutsakos JMS, Podewils LJ, Lyketsos CG. Evaluation of a home-based exercise program in the treatment of Alzheimer’s disease: the maximizing independence in dementia (MIND) study. Int Psychogeriatr. 2009;24:680-5. 33. Rolland Y, Pillard F, Klapouszczak A, Reynish E, Thomas D, Andrieu S, et al. Exercise program for nursing home residents with Alzheimer’s disease: a 1-year randomized, controlled trial. Journal of Am Geriatr Soc. 2007;55:158-65, doi: 10.1111/j.1532-5415.2007.01035.x. 34. De Medeiros K, Robert P, Gauthier S, Stella F, Politis A, Leoutsakos J, et al. The Neuropsychiatric Inventory-Clinician rating scale (NPI-C): reliability and validity of a revised assessment of neuropsychiatric symptoms in dementia. Int Psychogeriatr. 2010;22:984-94, doi: 10.1017/ S1041610210000876. 35. Fialho PPA, Koenig AM, Santos EL, Guimara˜es HC, Beato RG, Carvalho VA, et al. Dementia caregiver burden in a Brazilian sample: association to neuropsychiatric symptoms. Dement Neuropsychologia. 2009;3:132-5. 36. Carro E, Trejo JL, Busiguina S, Torres-Aleman I. Circulating insulin-like growth factor I mediates the protective effects of physical exercise against brain insults of different etiology and anatomy. J Neurosci. 2001;21:5678-84. 37. Cotman CW, Berchtold NC. Exercise: a behavioural intervention to enhance brain health and plasticity. Trends Neurosci. 2002;25:295-301, doi: 10.1016/S0166-2236(02)02143-4. 38. Kramer A, Erickson KI. Capitalizing on cortical plasticity: influence of physical activity on cognition and brain function. Trends Cognit Sci. 2007;11:342-8, doi: 10.1016/j.tics.2007.06.009. 39. Erickson KI, Prakash RS, Voss MW, Chaddock L, Hu L, Morris KS, et al. Aerobic fitness is associated with hippocampal volume in elderly humans. Hippocampus. 2009;19:1030-9, doi: 10.1002/hipo.20547. 40. Burns JM, Cronk BB, Anderson HS, Donelly JE, et al. Cardiorespiratory fitness and brain atrophy in early Alzheimer disease. Neurology. 2008;71:210-6, doi: 10.1212/01.wnl.0000317094.86209.cb. 41. Marks BL, Madden DJ, Bucur B, Provenzale JM, White LE, Cabeza R, Hueteel SA. Role of aerobic fitness and aging on cerebral white matter integrity. An N York Acad Sci. 2007;1097:171-4, doi: 10.1196/annals.1379. 022. 42. Torta R, Badino E, Scalabrino A. Therapeutic strategies for behavioral and psychological symptoms (BPSD) in dementia patients. Arch Gerontol Geriatr Suppl. 2004;9:443-54, doi: 10.1016/j.archger.2004.04.057.

4. Cummings JL, Mackell J, Kaufer D. Behavioral effects of current Alzheimer’s disease treatments: a descriptive review. Alzheimer Dement. 2008;4:49-60, doi: 10.1016/j.jalz.2007.10.011. 5. Lovheim H, Sandman PO, Karlsson S, Gusafson Y. Behavioral and psychological symptoms of dementia in relation to level of cognitive impairment. Int Psychogeriatr. 2008;20:777-89, doi: 10.1017/S1041610208006777. 6. Cotter EM, Burgio LD, Stevens AB, Roth DL, Gitlin LN. Correspondence of the Functional Independences Measure (FIM) self-care subscale with real-time observations of dementia patients’ ADL performance in the home. Clin Rehabil. 2002;16:36-45, doi: 10.1191/0269215502cr465oa. 7. Gauthier S, Loft H, Cummings J. Improvement in behavioural symptoms in patients with moderate to severe Alzheimer’s disease by memantine: a pooled data analysis. Int J Geriatr Psychiatry. 2008;23:537-45, doi: 10. 1002/gps.1949. 8. Colcombe S, Kramer AF. Fitness effects on the cognitive function of older adult: a meta-analytic study. Psychol Sci. 2003;14:125-30, doi: 10.1111/ 1467-9280.t01-1-01430. 9. Larson EB, Wang L, Bowen JD, McCormick WC, Teri L, Crane P, et al. Exercise is associated with reduced risk for incidence dementia among persons 65 years of age and older. An Int Med. 2006;144:73-81. 10. Verghese J, Lipton RB, Katz MJ, Hall CB, Derby CA, Kuslansky G, et al. Leisure activities and the risk of dementia in the elderly. N Engl J Med. 2003;348:2508-16, doi: 10.1056/NEJMoa022252. 11. Podewils LJ, Guallar E, Kuller LH, Fried LP, Lopez OL, Carlson M, et al. Physical activity, APOE genotype, and dementia risk: findings from the cardiovascular health cognition study. Am J Epidemiol. 2005;161:639-51, doi: 10.1093/aje/kwi092. 12. Hamer M, Chida Y. Physical activity and risk of neurodegenerative disease: a systematic review of prospective evidence. Psychol Med. 2009;39:3-11, doi: 10.1017/S0033291708003681. 13. Teri L, Gibbons LE, McCurry SM, Logsdon RG, Buchner DM, Barlow WE, et al. Exercise plus behavioral management in patients with Alzheimer disease: a randomized controlled trial. J Am Med Assoc. 2003;290:2015-22, doi: 10.1001/jama.290.15.2015. 14. Landi F, Russo A, Bernabei R. Physical activity and behavior in the elderly: a pilot study. Arch Gerontol Geriatr. 2004;9:235-41, doi: 10.1016/ j.archger.2004.04.033. 15. Heyn P, Abreu BC, Ottenbacher KJ. The effects of exercise training on elderly persons with cognitive impairment and dementia: a metaanalysis. Arch Phys Med Rehabil. 2004;85:1694-1704, doi: 10.1016/j.apmr. 2004.03.019. 16. Forbes D, Forbes S, Morgan DG, Markle-Reid M, Wood J, Culum I. Physical activity programs for persons with dementia [review]. Cochrane Database Systemat Rev. 2009;3:CD006489. 17. Rolland Y, Rival L, Pillard F, Lafont C, Rive´re D, Albare´de J, et al. Feasibility of regular physical exercise for patients with moderate to severe Alzheimer disease. J Nutr Health Aging. 2000;4:109-13. 18. Yu F, Kolanowski A. Facilitating aerobic exercise training in older adults with Alzheimer’s disease. Geriatr Nurs. 2009;30:250-9, doi: 10.1016/j. gerinurse.2008.11.001. 19. McKhann G, Drachman D, Folstein M, Katzman R, Price D, Stadlan EM. Clinical diagnosis of Alzheimer’s disease: report of the NICNCDSADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer’s Disease. Neurology. 1984;34:939-74. 20. APA – American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders. 4th ed., Test Revised. American Psychiatric Association, Washington DC., 2000. 21. Morris J. The Clinical Dementia Rating (CDR): current version and scoring rules. Neurology. 1993;43:2412-4. 22. Folstein MF, Folstein SE, McHugh PR. ‘‘Mini-Mental State’’: A practical method for grading the cognitive state of patients for the clinician. J Psychiatr Res. 1975;12:189-98, doi: 10.1016/0022-3956(75)90026-6.

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DOI:10.1590/S1807-59322011000800009

CLINICAL SCIENCE

Pharmacogenetics of glucocorticoid replacement could optimize the treatment of congenital adrenal hyperplasia due to 21-hydroxylase deficiency Ricardo P. P. Moreira,I Alexander A. L. Jorge,II Larissa G. Gomes,I Laura C. Kaupert,I Joa˜o Massud Filho,III Berenice B. Mendonca,I Taˆnia A. S. S. BachegaI I

Unidade de Endocrinologia do Desenvolvimento, Laborato´rio de Hormoˆnios e Gene´tica Molecular LIM 42, Disciplina de Endocrinologia da Faculdade de Medicina da Universidade de Saõ Paulo, Saõ Paulo/SP, Brazil. II Unidade de Endocrinologia Gene´tica - LIM 25, Disciplina de Endocrinologia da Faculdade de Medicina da Universidade de Saõ Paulo, Saõ Paulo/SP, Brazil. III Curso de Especializaçaõ em Medicina Farmaceûtica da Universidade Federal de Saõ Paulo – UNIFESP, Saõ Paulo/SP, Brazil.

INTRODUCTION: 21-hydroxylase deficiency is an autosomal recessive disorder that causes glucocorticoid deficiency and increased androgen production. Treatment is based on glucocorticoid replacement; however, interindividual variability in the glucocorticoid dose required to achieve adequate hormonal control has been observed. OBJECTIVE: The present study aimed to evaluate the association between polymorphic variants involved in glucocorticoid action and/or metabolism and the mean daily glucocorticoid dose in 21-hydroxylase deficiency patients. METHODS: We evaluated 53 patients with classical forms of 21-hydroxylase deficiency who were receiving cortisone acetate. All patients were between four and six years of age and had normal androgen levels. RESULTS: The P450 oxidoreductase A503V, HSD11B1 rs12086634, and CYP3A7*1C variants were found in 19%, 11.3% and 3.8% of the patients, respectively. The mean¡SD glucocorticoid dose in patients with the CYP3A7*1C and wild-type alleles was 13.9¡0.8 and 19.5¡3.2 mg/m2/d, respectively. We did not identify an association between the P450 oxidoreductase or HSD11B1 allelic variants and the mean glucocorticoid dose. CONCLUSION: Patients carrying the CYP3A7*1C variant required a significantly lower mean glucocorticoid dose. Indeed, the CYP3A7*1C allele accounted for 20% of the variability in the cortisone acetate dose. The analysis of genes involved in glucocorticoid metabolism may be useful in the optimization of treatment of 21-hydroxylase deficiency. KEYWORDS: 21-hydroxylase deficiency; Glucocorticoid replacement therapy; Pharmacogenetics; Polymorphism; CYP3A7*1C allele. Moreira RPP, Jorge AAL, Gomes LG, Kaupert LC, Massud Filho J, Mendonca BB, et al. Pharmacogenetics of glucocorticoid replacement could optimize the treatment of congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Clinics. 2011;66(8):1361-1365. Received for publication on February 18, 2011; First review completed on March 16, 2011; Accepted for publication on May 2, 2011 E-mail: tbachega@usp.br Tel.: 55 11 3069-7512

to restore the levels of the steroid hormones to the normal range and to decrease adrenocorticotrophic stimulation, which would suppress the effects of androgen overproduction. The aims of GC replacement are to avert adrenal crisis and to allow normal growth and puberty development. Similarly, mineralocorticoid replacement restores serum electrolyte levels and water balance without excessive salt retention. During growth, preference is given to short half-life glucocorticoids (e.g., hydrocortisone acetate) that avoid growth suppression and weight gain, which are commonly observed when long half-life GCs (e.g., prednisone or dexamethasone) are used.1 Among the GCs with a short half-life, cortisone acetate (CA) is one therapeutic option for the treatment of children with CAH.3 Cortisone acetate is converted to cortisol by the action of 11b-hydroxysteroid

INTRODUCTION Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is a common autosomal recessive disorder, caused by mutations in the 21-hydroxylase gene (CYP21A2). These mutations result in decreased glucocorticoid (GC) secretion with or without mineralocorticoid deficiency and excess androgens.1,2 GC replacement therapy significantly improves the prognosis of CAH. The goal of GC replacement therapy is

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The medical records of 53 patients with CAH were reviewed from a group of 213 classical CAH patients who underwent glucocorticoid replacement therapy in our hospital. The selection criteria included the use of CA, good compliance and hormonal control between the ages of 4 and 6 years (before starting pubertal development). Adequate control was defined by normal auxological parameters and biochemical assessments according to age during the two years of this study. The assessment comprised eight laboratory measurements. Testosterone and androstenedione levels were maintained at levels lower than 0.48 nmol/l (14 ng/dl) and 69.9 pmol/L (2 ng/ml), respectively. Bone age was evaluated yearly using the Greulich-Pyle system. No patient presented with adrenal crisis during this period or clinical/ hormonal parameters suggestive of overtreatment (e.g., Cushing features, decreased growth rate or 17-hydroxyprogesterone levels less than 36 nmol/l). Thirty-eight patients (27 females) had the salt-wasting (SW) form, which was defined as the development of dehydration, a sodium level less than 130 mmol/l and high plasmatic renin activity in the neonatal period. Fifteen patients (11 females) had the simple virilizing (SV) form, which was characterized by ambiguous genitalia in girls and signs of postnatal virilization in both sexes without dehydration or salt waste. At diagnosis, all patients had basal 17hydroxyprogesterone levels that were greater than 50 ng/ml. Daily CA doses (mg/m2/day) were evaluated retrospectively. The CA doses were administered three times a day, and the highest dose was administered at bedtime. Patients with the SW form also received fludrocortisone 50¡25 mg/day in the morning. No patient received gonadotropin-releasing hormone analogs, aromatase inhibitors or anticonvulsants. Body mass index (BMI) was calculated as the weight (in kilograms) divided by the height (in meters) squared, and the BMI Z-score was determined according to the curves of the Centers for Disease Control and Prevention. Of the 53 patients, 13 achieved final height (FH). The relationship between the achieved FH and target height (TH) is expressed as FH.SDS - TH.SDS, and this value was individually calculated for each patient.

dehydrogenase type 1 (11bHSD1); however, variability in 11bHSD1 activity has been described.4 Despite great advances in our understanding of the pathophysiology of CAH, most studies still report mean final heights in CAH patients under 21.5 Standard Deviation (SD).5 These shorter heights also reflect the difficulties in treatment compliance and the complexity of GC dose adjustments to balance cortisol insufficiency and androgen overproduction. Furthermore, the interindividual variability in daily glucocorticoid dose requirements poses an additional challenge in the treatment of CAH8 and suggests that genetic factors may modulate the glucocorticoid response. In the treatment of Addison’s disease, genetic factors play a major role in the differential response to glucocorticoid replacement regimens and/or the incidence of side effects.9 However, genetic factors have never been evaluated in CAH, which led us to hypothesize that interindividual variability in glucocorticoid metabolism could be involved. The CYP3A family of enzymes is the most important family of enzymes for drug metabolism in humans because they metabolize the majority of commercially available drugs. Many studies have shown that polymorphisms in CYP3A genes account for interindividual variability in the metabolism of many clinically used drugs.10 The CYP3A7 gene, which encodes P4503A7, is abundantly expressed in the fetal liver, and its expression declines rapidly after birth. The polymorphic variant CYP3A7*1C, which results from the replacement of an approximately 60-bp sequence within the promoter of CYP3A7 with an analogous region of the CYP3A4 promoter, leads to persistent P4503A7 expression in the adult liver11 and has been associated with lower serum dehydroepiandrosterone sulfate (DHEAS) and testosterone levels compared with the levels in individuals lacking this variant.12 Because of the presence of lower androgen levels, we speculated that the CYP3A7*1C allele may decrease the glucocorticoid requirement in CAH patients. The 11bHSD1 enzyme is involved in glucocorticoid metabolism by converting inactive cortisone into active cortisol.13 A polymorphism located in intron 3 (rs12086634) has been associated with decreased conversion of cortisone to cortisol and may function as an intronic enhancer.14 Therefore, CAH patients with this polymorphism could require higher CA doses compared with patients lacking this polymorphism. Finally, the P450 oxidoreductase (POR) gene encodes a flavoprotein that donates electrons to cytochrome P450 type II enzymes, including P450c21 and P450c17. A frequent POR variant (A503V) has been associated with reduced P450c17 activity and, consequently, with lower androgen secretion.15 Because of the critical role of this polymorphism in steroid hormone production, we hypothesized that it could reduce the glucocorticoid requirement in CAH patients. Therefore, the aim of the present study was to investigate whether interindividual variability in glucocorticoid requirements amongst treated CAH patients is influenced by the presence of polymorphisms in CYP3A7, POR or HSD11B1.

Methods

SUBJECTS AND METHODS

CYP21A2 mutations were screened by Southern blotting to identify large gene rearrangements and by allele-specific polymerase chain reaction (PCR) to determine the 15 common microconversions.16 If mutations were not identified in both alleles, CYP21A2 sequencing was performed. The identified mutations were segregated in parent DNA samples. CYP21A2 genotypes were divided according to predicted impairment of enzymatic activity as previously described: null and A and B groups, prediciting 0%, 2% and 3–7% residual enzymatic activity, respectively .1 Selected regions of the CYP3A7, POR, and HSD11B1 genes were amplified and sequenced as previously described.11,15,17 One hundred healthy volunteers were selected to determine the allelic frequency of the identified polymorphisms. None of the control subjects had a history of malignancy.

Subjects

Statistical Analyses

The present study was approved by the Ethic Committee of the Faculdade de Medicina da Universidade de Sa˜o Paulo, and written consent was obtained from patients and controls.

Qualitative variables are listed as frequencies and percentages, whereas quantitative variables are presented as the mean¡SD. Group comparisons based on genotype were

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performed using an unpaired Student t-test or a MannWhitney rank sum test for numerical variables and a Chisquare or Fisher exact test for nominal variables, as appropriate. To assess whether the genetic variants had independent prognostic significance for outcome, we performed single regression analysis followed by multiple regression analysis adjusting for established influential factors. A p-value less than 0.05 was considered statistically significant.

Table 1 - Clinical, auxological and hormonal data of 53 patients with CAH.

Female/Male Age at diagnosis (y) Mean bone age (y) at diagnosis Basal 17OHP (mmol/L) levels at diagnosis Mean daily CA doses (mg/m2/day) Fludrocortisone ( mg/day) Mean BMI at 4 y (kg/m2) Mean BMI at 6 y (kg/m2) Mean BMI at 4 y (SDU)1 Mean BMI at 6 y (SDU)1 Height SDS for bone age at 4 y Height SDS for bone age at 6 y Final height SDS** Target height SDS

RESULTS The POR A503V, HSD11B1 rs12086634, and CYP3A7*1C polymorphic variants were found in 19%, 11.3%, and 3.8% of the CAH patients and in 26.4%, 17.4%, and 2.3% of the control subjects, respectively. The CYP3A7*1C and POR variants were identified only in heterozygous state in the studied patients. In contrast, 1% of the CAH patients and 6% of the controls were homozygous for the HSD11B1 rs12086634 allele, whereas 10.3% of the CAH patients and 12% of the controls were heterozygous. The frequency of these genetic variants did not differ between children with CAH and the controls (p.0.05). All polymorphisms were in HardyWeinberg equilibrium. In patients between 4 and 6 years of age with the SW and SV forms, the mean CA dose was 18.5¡3.4 and 20.1¡3.3 mg/m2/day, respectively (p = 0.086). In SW patients between 4 and 6 years of age, the mean fludrocortisone dose was 50¡25 mg/day. There were no differences in the mean CA doses among patients with the null, A or B CYP21A2 genotypes (p.0.05). In the period of this study, the mean bone age advancement in SW and SV patients was 1.5¡0.7 and 1.2¡0.6 years, respectively (p.0.05). The mean BMI Z-scores at 4 and 6 years of age were 0.8¡0.003 and 0.7¡¡0.02, respectively. In addition, the mean 17-hydroxyprogesterone levels during these two years were 194.8¡157.6 nmol/l (6,283¡5,063 ng/ dl) and 202.7¡132.8 nmol/l (6,540¡4,285 ng/dl) in patients with SW and SV forms, respectively. Of the 53 patients, 13 achieved FH, and the corrected FH ranged from 20.87 to +0.86 SD. The clinical data are described in Table 1. There were no differences in the mean daily CA doses between the patients with the wild-type POR allele and the patients heterozygous for the POR A503V allele or between the patients with the wild-type HSD11B1 allele and the patients heterozygous or homozygous for the HSD11B1 rs12086634 allele (Table 2). Interestingly, the mean daily CA dose was lower in patients with the CYP3A7*1C allele compared with patients with the wild-type allele: 13.9¡0.8 vs. 19.5¡3.2 mg/m2/d, respectively (95% confidence interval for difference of means: 2.4 to 8.8 mg/m2/d; p = 0.001, power of calculation 96.2%). Moreover, single regression analysis followed by multiple linear regression analysis revealed that the influence of this polymorphism on CA dose did not vary according to clinical form, sex, CYP21A2 genotype or concomitant use of fludrocortisone. The CYP3A7*1C variant alone accounted for 20% of the CA dose variability (p = 0.001), and 33% of the observed variability was explained by the presence of CYP3A7*1C in association with the age at diagnosis (p = 0.004).

SW patients

SV patients

27/11 0.2¡0.8 303¡341 18.5¡3.4 50¡25 17.1¡2.1 16.2¡1.6 1.0¡0.9 1.0¡1.1 0.02 0.16 21.4¡1.4 20.9¡1.0

11/4 2.9¡1.7 5.8¡4.5 199¡166 20.1¡3.3 16.2¡1.3 17.6¡2.7 0.6¡0.8 0.4¡0.8 0.9 1.3 21.0¡0.9 20.6¡1.2

SW: salt-wasting form, SV: simple virilizing form, CA: cortisone acetate dose used from 4–6 y of age. Values are presented as the mean¡SD. **13/53 patients achieved final height. 1 SDU scores are the number of SD units relative to the mean values for normal children of the same age and sex.

hormonal control. Several approaches have been proposed to improve treatment, such as the addition of flutamide and testolactone to glucocorticoid replacement therapy18 and therapy optimization strategies based on CYP21A2 genotypes.19 Although a good correlation has been observed between genotype and the clinical form, no such correlation has been identified between genotype and glucocorticoid requirements in classical forms.19 Similarly, in our cohort, there were no differences in the mean glucocorticoid dose among patients with null, A, or B CYP21A2 genotypes, which suggests that other factors influence the glucocorticoid requirement. To investigate these factors, we chose to evaluate the glucocorticoid dose in patients between 4 and 6 years of age, which appears to be a period of steady linear growth. Several studies have shown a tendency to overtreat in the first three years of life to avoid SW crises. During puberty, however, the glucocorticoid requirement generally increases because of alterations in cortisol pharmacokinetics.20 Despite suppressed androgen levels, we ruled out overtreatment in our cohort because the serum 17-hydroxyprogesterone levels were greater than 36 nmol/l, and the mean BMI Z-score and mean growth rate of all patients were both in the normal range. Genetic factors have been shown to contribute to interindividual variability in the response to different drugs.10 Recently, genetic polymorphisms have been associated with Table 2 - Mean¡SD daily cortisone acetate doses (mg/m2/ day) according to genetic polymorphisms in patients with classical 21-hydroxylase deficiency.

DISCUSSION A short FH and adverse effects have been described during CAH treatment despite the achievement of adequate

Allelic Variant

Genotype

CA dose (mg/m2/d)

p-value

HSD11B1 (rs12086634) CYP3A7 (CYP3A7*1C) POR (A503V)

C/C C/T Wild type CYP3A7*1C C/C C/T

19.1¡3.6 19.3¡2.7 19.5¡3.2 13.9¡0.8 19.2¡3.5 18.7¡3.4

0.852

CA: cortisone acetate.

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This research was supported by grants from FAPESP (05/04726-0 and 09/54238-2), RPPM by FAPESP (# 09/54394-4), TASSB by CNPq (# 305117/2009-2), AALJ by CNPq (# 301477/2009-4) and BBM by CNPq (# 301339/2008-9).

variability in glucocorticoid doses and susceptibility to osteoporosis in Addisonâ&#x20AC;&#x2122;s disease.9 Among treated CAH patients, there is great interindividual variability in the daily glucocorticoid dose. To the best of our knowledge, however, there are no data evaluating the effect of allelic variants other than CYP21A2 gene variants. The frequency of the POR A503V, HSD11B1 rs12086634, and CYP3A7*1C allelic variants was similar among patients and controls in our study. Although POR A503V is associated with decreased P450c17 activity and decreased androgen synthesis, our data suggest that this enzymatic impairment, at least in the heterozygous individuals seen in our cohort, does not influence the daily CA requirement. Interestingly, the HSD11B1 rs12086634 variant has been shown to reduce gene transcription in vitro, which was consistent with reduced cortisol generation and probably higher CA requirements.14 In our cohort, however, HSD11B1 rs12086634 carriers required a CA dose similar to that required by individuals lacking this variant. The CYP3A7 gene is predominantly expressed in the fetal liver, and its expression is sharply downregulated after birth.11 However, the CYP3A7*1C allele contains a modified promoter that causes constitutive CYP3A7 expression in the adult liver.21 P4503A7 catalyzes the 16a-hydroxylation of DHEA and DHEAS22, and the CYP3A7*1C allele has been associated with lower DHEAS levels in women with polycystic ovary syndrome.12 Although the allelic frequency of this genetic variant did not differ between the children with CAH and the controls, the glucocorticoid replacement doses were significantly lower in children with the CYP3A7*1C allele. Because CAH treatment is designed to normalize androgens, we hypothesized that the CYP3A7*1C allele results in less severe hyperandrogenism and, consequently, decreased glucocorticoid requirements. However, the CYP3A7*1C allele was a rare variant in our Brazilian cohort and probably only affects the treatment of a minority of patients. There is no doubt that other variants in different genes, such as those related to gastrointestinal absorption, could contribute to the interindividual variability in glucocorticoid doses. We cannot exclude a sample-size effect to explain the absence of a correlation between the POR and HSD11B1 variants and the mean daily glucocorticoid dose. This was a pilot study involving a large series of Brazilian patients followed at a single center, and we were able to select 53 patients who were treated with the same glucocorticoid (i.e., CA) since their diagnosis. Despite the limitations of this study design, other retrospective studies have also highlighted the influence of pharmacogenetics on individual responses to drugs.23,24 Indeed, pharmacogenetic testing has become an important tool to guide therapy with drugs such as warfarin, dasatinib and trastuzumab.25 The present study is the first to raise the potential advantage of genetic screening to obtain better CAH treatment. Future studies involving both larger cohorts and other candidate genes are certainly warranted to consolidate this approach.

REFERENCES 1. Speiser PW, White PC. Congenital adrenal hyperplasia. N Engl J Med. 2003;9:776-88, doi: 10.1056/NEJMra021561. 2. Demirci C, Witchel SF. Congenital adrenal hyperplasia. Dermatol Ther. 2008;21:340-53, doi: 10.1111/j.1529-8019.2008.00216.x. 3. Gasparini N, Di Maio S, Salerno M, Argenziano A, Franzese A, Tenore A. Growth pattern during the first 36 months of life in congenital adrenal hyperplasia (21-hydroxylase deficiency). Horm Res.1997;47:17-22, doi: 10.1159/000185361. 4. Nordenstrom A, Marcus C, Axelson M, Wedell A, Ritzen EM. Failure of cortisone acetate treatment in congenital adrenal hyperplasia because of defective 11beta-hydroxysteroid dehydrogenase reductase activity. J Clin Endocrinol Metab.1999;84:1210-3, doi: 10.1210/jc.84.4.1210. 5. Balsamo A, Cicognani A, Baldazzi L, Barbaro M, Baronio F, Gennari M, et al. CYP21 genotype, adult height, and pubertal development in 55 patients treated for 21-hydroxylase deficiency. J Clin Endocrinol Metab.2003;88:5680-8, doi: 10.1210/jc.2003-030123. 6. Bonfig W, Bechtold S, Schmidt H, Knorr D, Schwarz HP. Reduced final height outcome in congenital adrenal hyperplasia under prednisone treatment: deceleration of growth velocity during puberty. J Clin Endocrinol Metab.2007;92:1635-9, doi: 10.1210/jc.2006-2109. 7. Muthusamy K, Elamin MB, Smushkin G, Murad MH, Lampropulos JF, Elamin KB, et al. Clinical review: Adult height in patients with congenital adrenal hyperplasia: a systematic review and metaanalysis. J Clin Endocrinol Metab.2010;95:4161-72, doi: 10.1210/jc.2009-2616. 8. Bryan SM, Honour JW, Hindmarsh PC. Management of altered hydrocortisone pharmacokinetics in a boy with congenital adrenal hyperplasia using a continuous subcutaneous hydrocortisone infusion. J Clin Endocrinol Metab.2009;94:3477-80, doi: 10.1210/jc.2009-0630. 9. Lovas K, Gjesdal CG, Christensen M, Wolff AB, Almas B, Svartberg J, et al. Glucocorticoid replacement therapy and pharmacogenetics in Addisonâ&#x20AC;&#x2122;s disease: effects on bone. Eur J Endocrinol.2009;160:993-1002, doi: 10.1530/EJE-08-0880. 10. Ingelman-Sundberg M, Sim SC, Gomez A, Rodriguez-Antona C. Influence of cytochrome P450 polymorphisms on drug therapies: pharmacogenetic, pharmacoepigenetic and clinical aspects. Pharmacol Ther.2007;116:496-526, doi: 10.1016/j.pharmthera.2007.09.004. 11. Burk O, Tegude H, Koch I, Hustert E, Wolbold R, Glaeser H, et al. Molecular mechanisms of polymorphic CYP3A7 expression in adult human liver and intestine. J Biol Chem.2002;277:24280-8, doi: 10.1074/ jbc.M202345200. 12. Goodarzi MO, Xu N, Azziz R. Association of CYP3A7*1C and serum dehydroepiandrosterone sulfate levels in women with polycystic ovary syndrome. J Clin Endocrinol Metab.2008;93:2909-12, doi: 10.1210/jc.20080403. 13. Tomlinson JW, Walker EA, Bujalska IJ, Draper N, Lavery GG, Cooper MS, et al. 11beta-hydroxysteroid dehydrogenase type 1: a tissue-specific regulator of glucocorticoid response. Endocr Rev.2004;25:831-66, doi: 10. 1210/er.2003-0031. 14. Draper N, Walker EA, Bujalska IJ, Tomlinson JW, Chalder SM, Arlt W, et al. Mutations in the genes encoding 11beta-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase interact to cause cortisone reductase deficiency. Nat Genet.2003;34:434-9, doi: 10.1038/ ng1214. 15. Miller WL, Huang N, Agrawal V, Giacomini KM. Genetic variation in human P450 oxidoreductase. Mol Cell Endocrinol.2009;300:180-4, doi: 10. 1016/j.mce.2008.09.017. 16. Bachega TA, Billerbeck AE, Madureira G, Arnhold IJ, Medeiros MA, Marcondes JA, et al. Low frequency of CYP2B deletions in Brazilian patients with congenital adrenal hyperplasia due to 21-hydroxylas deficiency. Hum Hered. 1999;49:9-14, doi: 10.1159/000022833. 17. Robitaille J, Brouillette C, Houde A, Despres JP, Tchernof A, Vohl MC. Molecular screening of the 11beta-hsd1 gene in men characterized by the metabolic syndrome. Obes Res. 2004;12:1570-75, doi: 10.1038/oby.2004. 196. 18. Merke DP, Keil MF, Jones JV, Fields J, Hill S, Cutler GB Jr. Flutamide, testolactone, and reduced hydrocortisone dose maintain normal growth velocity and bone maturation despite elevated androgen levels in children with congenital adrenal hyperplasia. J Clin Endocrinol Metab. 2000;85:1114-20, doi: 10.1210/jc.85.3.1114. 19. Pinto G, Tardy V, Trivin C, Thalassinos C, Lortat-Jacob S, Nihoul-Fekete C, et al. Follow-up of 68 children with congenital adrenal hyperplasia due to 21-hydroxylase deficiency: Relevance of genotype for management. J Clin Endocrinol Metab.2003;88:2624-33, doi: 10.1210/jc.2002021433. 20. Charmandari E, Hindmarsh PC, Johnston A, Brook CG. Congenital adrenal hyperplasia due to 21-hydroxylase deficiency: Alterations in

ACKNOWLEDGEMENTS The authors gratefully acknowledge Dr. Bruno Ferraz de Souza for English language review.

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Pharmacogenetics in CAH treatment Moreira RPP et al. study of the variability in response to oral anticoagulant therapy.Blood Coagul Fibrinolysis. 2010;21:558-63, doi: 10.1097/MBC.0b013e32833c 2988. 24. Anglicheau D, Legendre C, Thervet E. Pharmacogenetics of tacrolimus and sirolimus in renal transplant patients: from retrospective analyses to prospective studies. Transplant Proc. 2007;39:2142-4, doi: 10.1016/j. transproceed.2007.06.018. 25. Gervasini G, Ben覺織tez J, Carrillo JA. Pharmacogenetic testing and therapeutic drug monitoring are complementary tools for optimal individualization of drug therapy. Eur J Clin Pharmacol.2010;66:755-74, doi: 10.1007/s00228-010-0857-7.

cortisol pharmacokinetics at puberty. J Clin Endocrinol Metab. 2001; 86:2701-08, doi: 10.1210/jc.86.6.2701. 21. Schuetz JD, Beach DL, Guzelian PS. Selective expression of cytochrome P450 CYP3A mRNAs in embryonic and adult human liver. Pharmacogenetics.1994;4:11-20, doi: 10.1097/00008571-199402000-00002. 22. Miller KK, Cai J, Ripp SL, Pierce WM Jr., Rushmore TH, Prough RA. Stereoand regioselectivity account for the diversity of dehydroepiandrosterone (DHEA) metabolites produced by liver microsomal cytochromes P450. Drug Metab Dispos.2004;32:305-13, doi: 10.1124/dmd.32.3.305. 23. Kovac MK, Maslac AR, Rakicevic LB, Radojkovic DP. The c.-1639G.A polymorphism of the VKORC1 gene in Serbian population: retrospective

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DOI:10.1590/S1807-59322011000800001

CLINICAL SCIENCE

High prevalence of chronic pelvic pain in women in Ribeira˜o Preto, Brazil and direct association with abdominal surgery Gabriela Pagano de Oliveira Gonc¸alves da Silva,I Anderson Luı´s do Nascimento,I Daniela Michelazzo,I Fernando Filardi Alves Junior,I Marcelo Gondim Rocha,I,III Ju´lio Ce´sar Rosa-e-Silva,I Francisco Jose´ Candidodos-Reis,I Antonio Alberto Nogueira,I Omero Benedicto Poli-NetoII I

Obstetrics and Gynecology Department, Hospital das Clıńicas - Medical School From Ribeiraõ Preto - University of Saõ Paulo (HCFMRP-USP) Ribeiraõ Preto/SP, Brazil. II Surgery and Anatomy and Obstetrics and Gynecology Departments, Hospital das Clıńicas - Medical School From Ribeiraõ Preto University of Saõ Paulo (HCFMRP-USP), Ribeiraõ Preto/SP, Brazil. III Discipline of Gynecology - Faculty of Medicine - University of Fortaleza (UNIFOR), Fortaleza/CE, Brazil.

INTRODUCTION: Chronic pelvic pain is a disease that directly affects the social and professional lives of women. OBJECTIVE: To estimate the prevalence of this clinical condition and to identify independent factors associated with it in women living in Ribeiraõ Preto, Brazil. METHODS: A one-year cross-sectional study was conducted in a population sample of 1,278 women over the age of 14 years. The target population was predominantly composed of women who are treated by the public health system. The questionnaire was administered by interviewers who were not linked to the city health care programs. The prevalence of the morbidity was estimated. First, we identified the significant variables associated with pelvic pain (p,0.10) and then we attributed values of 0 or 1 to the absence or presence of these variables. Logistic regression analysis was used to identify and estimate the simultaneous impact of the independent variables. The results were expressed by odds ratio and their 95% confidence interval with p,0.05. RESULTS: The disease was found in 11.5% (147/1,278) of the sample. The independent predictors were dyspareunia, previous abdominal surgery, depression, dysmenorrhea, anxiety, current sexual activity, low back pain, constipation, urinary symptoms, and low educational level. CONCLUSION: The prevalence of chronic pelvic pain in Ribeiraõ Preto is high and is associated with conditions that can usually be prevented, controlled, or resolved by improvement of public health policies and public education. KEYWORDS: Chronic Pelvic Pain; Prevalence; Public Health, Abdominal Surgery; Cesarean Section. Silva GPOG, Nascimento AL, Michelazzo D, Alves Jr FF, Rocha MG, Ju´lio Rosa-e-Silva C, et al. High prevalence of chronic pelvic pain in women in Ribeiraõ Preto, Brazil and direct association with abdominal surgery. Clinics. 2011;66(8):1307-1312. Received for publication on January 17, 2011; First review completed on February 15, 2011; Accepted for publication on April 18, 2011 E-mail: polineto@fmrp.usp.br Tel.: 55 16 36022583

The prevalence may vary from country to country. In primary care, the prevalence of CPP is found to be comparable to that of asthma and back pain, with values of 3.7%, 3.8%, and 4.1%, respectively.4 In a US study, the prevalence among women of reproductive age reached 24%,4-8 whereas in New Zealand, Grace, and Zondervan identified a prevalence of 25.4% when considering a threemonth duration of symptoms.9 This elevated prevalence was confirmed by Latthe et al. in a recent review of this condition.10 The direct and indirect costs of this condition amount to over two billion dollars per year, and the condition is responsible for 10% of all gynecologic visits, 40 to 50% of all gynecologic laparoscopies and 12% of all hysterectomies.5 CPP has a direct impact on marital status as well as on the social and professional lives of women, and it thus constitutes an important public health problem. In

INTRODUCTION Chronic pelvic pain (CPP) in women is commonly described as continuous or intermittent pain in the anatomic pelvis (anterior abdominal wall at or below the umbilicus) that lasts for at least six months, is not exclusively related to menstruation or sexual intercourse, and is sufficiently severe to cause functional disability or to lead to medical care.1-2 CPP may originate from one or more organ systems or pathologies and may have multiple contributing factors.3

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responsible for the subjects) gave written informed consent to participate in the study.

Brazil, the prevalence of CPP is not well known. International studies have demonstrated a high prevalence of persistent pain in women from Brazil, including approximately 36% in Rio de Janeiro.11 According to the Health Ministry of Brazil, in 1997 there were 1.8 million gynecologic visits and approximately 300,000 hospital admissions of women aged 15 to 69 with complaints associated with CPP.12 In a survey of diseases and/or health problems among working women in Saõ Paulo, Gomes & Tanaka13 reported that 13% of them mentioned abdominal and pelvic pain among their major complaints. Recently, some studies have concluded that drug or alcohol abuse, abortions, inflammatory pelvic disease, cesarean sections, and psychological diseases are associated with CPP.14 The etiology of CPP in women is not clear, and CPP usually involves a complex interaction between the gastrointestinal, urinary, gynecologic, musculoskeletal, and neurologic and endocrine systems. It is also influenced by psychological and sociocultural factors.6 Several other factors may be associated with the condition, including (1) neuroplastic changes occurring in the posterior horn of the spinal cord as a consequence of electrophysiological, biochemical, and metabolic changes promoted by the initial noxious stimulus, (2) cross-sensitivity between viscera that share the same innervation, and (3) development of a visceromuscular reflex that may culminate not only in dysfunctional repercussions but also in the development of myofascial syndrome and the generation of new pain points.15-16 Although there is a consensus that CPP has a high prevalence among women, there are a limited number of studies in our country, which can be attributed to the lack of a precise diagnosis, the heterogeneity of the group of diseases that result in CPP, and the variation of primary diseases in different populations.17 Considering that knowledge about the magnitude and the demographic and socioeconomic characteristics of CPP contributes to its prevention, control, and treatment, the aim of the present study was to estimate the community prevalence of CPP in women living in Ribeiraõ Preto (a southeastern city in the state of Saõ Paulo, Brazil with a population of about 700,000 people), as well as to identify independent factors associated with it.

Questionnaire We chose a questionnaire as the instrument for data collection because it could be applied to all segments of our population, which is very heterogeneous, consisting of both illiterate and literate people, and also because it allowed us to explain the objectives of the research and to answer any questions. The variables investigated were age; body mass index (BMI); number of pregnancies; parity; having been subjected to episiotomy, to forceps delivery, or to cesareansection delivery; abortions; abdominal surgery; umbilical and laparoscopy incisions; oblique, longitudinal. and transverse incisions; perineal surgery; sedentary lifestyle (women who engaged in activities such as running, walking, pedaling, dancing, or other sport activity for at least three hours a week were considered active); previous sexual activity; current sexual activity; contraception; sexual desire; lubrication; orgasm; per capita income; employment status; dysmenorrhea; dyspareunia; having been a victim of violence (physical and sexual); stable marital relationship; educational level (schooling was stratified as low when women had completed the eighth grade in primary school); mood (depression or anxiety based on previous medical diagnosis or criteria for the detection and diagnosis of psychiatric disorders in primary medical care settings)18 and comorbid health conditions (e.g., migraine and low back pain); constipation (Roma III criteria);19 urinary symptoms (bladder irritation: increased voiding episodes per day, urgency, pain); nocturia; alcoholism (excessive intake during the weekend with frequent drunkenness and/ or daily consumption with or without drunkenness); smoking (current or former daily smoker); excessive coffee intake (more than 6 cups a day); illicit drug use; menstrual status; pain intensity (visual analogue scale); pain duration (in months) and pain frequency (at least once a week); and self-medication for pain relief. Because our population is racially diverse, it is difficult to determine the ethnicity accurately, and therefore, we did not perform an ethnic analysis because of the high probability of misclassification. Interviewers who were not linked to the city health care programs were trained and selected by the researcher responsible for the study. The data collected by the interviewers were entered during the interview and sent to an electronic database. The personal identification data were coded and kept confidential. All women with CPP were evaluated by experts, and their diagnosis was confirmed before inclusion in the database. Five percent of the subjects were selected at random and re-interviewed to check data consistency.

MATERIALS AND METHODS Study design and participants A one-year cross-sectional study was conducted. The study was approved by the Research Ethics Committee of the University Hospital, Faculty of Medicine of Ribeiraõ Preto, University of Saõ Paulo (HCFMRP-USP). Women were recruited from the Western district of Ribeiraõ Preto between April 2008 and March 2009 for two particular reasons: first, the region presents the same social and demographic patterns as those of the major parts of the city (socioeconomic level, distribution of age, sex, and ethnicity), and second, the university hospital provides medical evaluation and treatment for women with a diagnosis of CPP. The target population consisted of working-class women treated by the public health system. A total of 1,306 women over 14 years of age were contacted, but 38 of them refused to participate. Therefore, 1,278 women were included in the present study. The women who were contacted were selected randomly from the population using their residential address. All subjects (or those

Definitions and grouping CPP in women has been described as continuous or intermittent pain in the anatomic pelvis (anterior abdominal wall at or below the umbilicus) that lasts for at least six months, is not exclusively related to menstruation or sexual intercourse, and is sufficiently severe to cause functional disability or lead to medical care. For this study, we defined CPP as having at least one weekly episode with an intensity higher than 3 cm on a 10-cm visual analog scale. Women who had been pregnant in the previous 12 months were excluded from the analyses. Dysmenorrhea was defined as pelvic pain during or shortly before/after menstruation. The

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intensity of dysmenorrhea was classified according to its effect on the ability to work, the coexistence of systemic symptoms, and the need to use any kind of analgesic medication. Classifications included absence (absence of pain during the periods); mild (occasional pelvic discomfort that does not impair daily activity, occasional need for medication), moderate (pain lasting almost throughout the menstrual period that impairs daily activity and is responsive to the use of medication), and intense (pain lasting throughout the menstrual period, with significant limitation of daily activity, frequent use of potent analgesics, and without effective improvement). For analysis, we considered only moderate and intense dysmenorrhea to be positive. Dyspareunia was defined as pelvic pain during sexual intercourse or during a period of 24 hours after sexual intercourse. The intensity of dyspareunia was classified according to the disruption of sexual activity during intercourse, as follows: absence (absence of pain during sexual contact), mild (tolerable pain, does not lead to the interruption of sexual contact), moderate (intense pain sufficient to lead to the interruption of sexual contact), and intense (pain that hinders sexual contact). For analysis, we considered only moderate and intense dyspareunia to be positive. The clinical measurement of pain severity was made using a 10-cm visual analog pain scale ranging from ‘least possible pain’ to ‘worst possible pain’.

(127/841). The characterization of associated history elements is presented in Table 1. In total, 82.4% of healthy women (932/1,131) and 90.5% of women with CPP (133/147) stated that they make regular clinic visits at basic health centers (at least two appointments per year). Only 4.1% of women with the disease, however, knew their diagnosis before the interview (n = 6/147). The average duration of the pain was 51.8¡61.2 [6-360] months: 6-12 months in 34.0% of the women (50/147), 13-36 months in 28.6% (42/147), and over 36 months in 37.4% (55/147). The mean intensity of pain obtained with a visual analogue scale (VAS) was 58.5¡23.4 mm. The intensity was 30-50 mm in 52.4% of the women (77/147), 51-70 mm in 17.7% (26/147), and 71100 mm in 29.9% (44/147). In total, 44.9% of the women reported a spontaneous onset of pain (66/147); in 3.4% (5/ 147), the pain was related to food intake; in 8.5% (9/106 – excluding those who were not sexually active), the pain was related to intercourse; in 21.8% (32/147), the pain was related to physical activity; in 30.6% (30/98 – excluding those who were menopausal), the pain was related to menstruation; in 2.0% (2/98), the pain was related to ovulation; in 0.7% (1/ 147), the pain was related to stress; and in 1.4%, it was related to other factors (2/147). The frequency of self-medication was 1.4% (16/1131) and 24.5% (111/147) among healthy and CPP women, respectively. The common occupations were maidservant, hairdresser, and seamstress, with no possibility of grouping the women according to their occupations. A univariate analysis of the variables investigated is presented in Table 2. In the logistic regression, the factors independently associated with CPP were dyspareunia, previous abdominal surgery, depression, dysmenorrhea, anxiety, current sexual activity, low back pain, constipation, urinary symptoms, and low educational level. These results are detailed in Table 3.

Statistical analysis The D9Agostino and Pearson test was used to determine whether data followed a Gaussian distribution. Data with and without a normal distribution were represented respectively by the mean (standard deviation) and median (range). Data were analyzed using the Fisher exact test or Chi-square test (qualitative variable) and the unpaired t test after confirmation of normal distribution (quantitative variables). We first selected only the significant variables identified (p,.10) and then assigned values of 0 or 1 to the absence or presence of these variables in each case. Logistic regression was used to identify the significant independent variables and to estimate the simultaneous impact of these factors on the assessment of CPP. The results were expressed as the odds ratio (OR) and 95% confidence interval (95% CI), with the level of significance set at p,.05.

DISCUSSION In the present study, we detected a one-year prevalence of 11.5% for CPP among women from Ribeira˜o Preto and a prevalence of 15.1% in women of reproductive age. To our knowledge, this is the first time that a high prevalence of this disease has been reported in Brazil. The presence of chronic pain was found to be 48.4% in women from Salvador, Brazil.20 Although that study described pain at various sites, it made no reference to abdominal or pelvic pain. A recent review published by Latthe et al.10 has shown that the worldwide prevalence of this condition ranges from 2% to 24%, which places Brazil among the countries with a higher prevalence of CPP. Zondervan et al., for example, observed a community prevalence of 24.0% in women between 18 and 49 years of age.7 In that study, the questionnaire response rate was 74%. A higher response rate is natural among women with the disease, which may explain the difference in prevalence compared with our study, which had a response rate of 97.9% (1,278/1,306). We believe that our data are representative of the entire Ribeira˜o Preto community because the social-demographic indicators of the covered area are similar to those of the general population of the city (socioeconomic level, age distribution, sex, and ethnicity). A notable result was that only 4% of the women with CPP stated that they had previously received a specific diagnosis, even though 90% of them attended a basic health center. Three different factors may have contributed to this

RESULTS The prevalence of CPP was 11.5% (147/1,278). Considering only women of reproductive age, the prevalence was 15.1% Table 1 - Characteristics of the subjects included in the present study. Parameters Age (mean¡SD) BMI (mean¡SD) Parity (median [range]) Age at menarche (mean ¡SD) Menopause (n [%]) First sexual intercourse (mean ¡SD) Duration of pain (mean ¡SD [range]) VAS (mean ¡SD)

Controls (n = 1,131)

Cases (n = 147)

43.0¡15.6 40.4¡15.0 24.7¡4.5 24.5¡5.0 1 [0–15] 1 [0–9] 12.7¡1.7 12.5¡2.0 388 [34.3] 49 [33.3] 19.3¡6.7 18.3¡3.8 --51.8¡61.2 [6-360] ---

58.5¡23.4

p 0.25 0.84 0.71 0.23 0.85 0.11 -----

Age in years; BMI: body mass index; Duration of pain in months; VAS: visual analogue scale, in millimeters; SD: standard deviation.

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Table 2 - Variables related to chronic pelvic pain (univariate analysis).

Number of subjects Stable marital status % (n) Single % (n) Separated % (n) Married % (n) Occupation (paid work) % (n) Per capita income ,R$450.00짜 % (n) Low educational level Sedentary lifestyle1 % (n) Prior sexual intercourse % (n) Current sexual activity % (n) Contraception % (n) Sexual appetite % (n) Vaginal lubrication % (n) Orgasm % (n) Victim of violence % (n) Alcoholism % (n) Smoking % (n) Coffee intake % (n) Illicit drugs % (n) Depression % (n) Anxiety % (n) Migraine % (n) Low back pain % (n) Episiotomy % (n) Forceps delivery % (n) Abortion % (n) .2 Abortions % (n) Abdominal surgery % (n) .2 Abdominal surgeries % (n) Longitudinal incision % (n) Transverse incision % (n) Oblique incision % (n) Umbilical incision/laparoscopic % (n) Cesarean section % (n) .2 Cesarean sections % (n) Perineal surgery # % (n) Dysmenorrhea % (n) Dyspareunia % (n) Constipation % (n) Urinary symptom (bladder irritation) % (n) Nocturia % (n)

Control

CPP

1,131 44.6 (499/1,120) 35.5 (398/1120) 19.9 (223/1120) 44.6 (499/1120) 43.3 (490/1,131) 43.6 (491/1,126) 27.0 (302/1,118) 56.7 (642/1,131) 91.4 (1,002/1,096) 60.1 (602/1,002) 47.1 (533/1,131) 49.2 (557/1,131) 34.2 (387/1,131) 54.4 (514/1,131) 1.6 (18/1,131) 5.2 (59/1,131) 4.7 (53/1,131) 9.3 (105/1,131) 1.9 (21/1,131) 16.9 (191/1,131) 15.1 (171/1,131) 18.0 (204/1,131) 15.3 (173/1,131) 28.7 (325/1,131) 5.6 (63/1,131) 16.4 (186/1,131) 5.6 (63/1,131) 37.0 (418/1,131) 20.8 (235/1,131) 7.6 (86/1,131) 24.8 (281/1,131) 7.1 (80/1,131) 1.6 (18/1,131) 23.1 (261/1,131) 13.8 (156/1,131) 5.2 (59/1,131) 21.5 (160/743) 2.8 (17/602) 22.7 (249/1,099) 19.0 (211/1,108) 9.4 (106/1,131)

147 56.9 (82/144) 27.1 (39/144) 16.0 (23/144) 56.9 (82/144) 48.3 (71/147) 54.8 (80/146) 37.1(53/143) 68.0 (100/147) 97.8 (135/138) 76.3 (103/135) 49.0 (72/147) 43.5 (64/147) 35.4 (52/147) 47.6 (70/147) 2.7 (4/147) 5.4 (8/147) 8.8 (13/147) 17.7 (26/147) 3.4 (5/147) 46.9 (69/147) 30.6 (45/147) 31.3 (46/147) 29.2 (43/147) 38.8 (57/147) 10.9 (16/147) 27.9 (41/147) 14.3 (21/147) 66.0 (97/147) 37.4 (55/147) 17.0 (25/147) 47.6 (70/147) 10.2 (15/147) 2.7 (4/147) 43.5 (64/147) 23.8 (35/147) 6.1 (9/147) 38.8 (38/98) 19.4 (20/103) 34.0 (50/147) 39.0 (57/146) 19.0 (28/147)

--0.01* 0.05 0.31 ,0.01* 0.29 0.01* 0.01* 0.01* 0.01* ,0.01* 0.73 0.22 0.78 0.66 0.31 0.85 0.04* ,0.01* 0.21 ,0.01* ,0.01* ,0.01* ,0.01* 0.02* 0.03* ,0.01* ,0.01 ,0.01* ,0.01 ,0.01 ,0.01 0.18 0.31 ,0.01 ,0.01 0.70 ,0.01* ,0.01* ,0.01* ,0.01* ,0.01*

not including episiotomy; minimum wage in the country during the study (approximately USD $ 276.07); 1 ,300 minutes of physical activity per week. 짜

on21); and whether the women complain about their clinical conditions to physicians. Studies have shown that gender is an important determining factor of pain complaints in women and might be the reason for diagnosis underestimation at primary care centers.22 The frequency of selfmedication was also a notable finding. Approximately one quarter of the women used some kind of medication (especially analgesics and anti-inflammatory drugs) for the relief of their symptoms in an indiscriminate way. This selfmedication predisposes them to side effects and requires financial expenditures that do not guarantee clinical improvement, especially over the long term.23 We believe that this percentage may be underestimated because people responding to an interviewer may not want to report the use of their pain medication. Additionally, more than one third of the women had symptoms consistent with a diagnosis of CPP for over three years, and the same proportion had symptoms that are considered severe (VAS.70 mm). Although we have no data to support this speculation, it is plausible that the high prevalence of CPP is associated

finding: whether the physician is able to provide the correct diagnosis; whether the relationship between the patient and the physician enables effective communication and explanation of the diagnosis (which other studies have commented Table 3 - Variables independently related to chronic pelvic pain identified by logistic regression. OR Dyspareunia Abdominal surgery Depression Dysmenorrhea Anxiety Current sexual activity Low back pain Constipation Urinary symptoms Low educational level

11.0 2.9 2.8 2.6 2.1 1.9 1.7 1.6 1.6 1.6

95% CI 5.1 1.9 1.9 1.6 1.3 1.2 1.1 1.0 1.0 1.0

to to to to to to to to to to

23.8 4.5 4.4 4.2 3.3 3.0 2.7 2.5 2.4 2.4

p ,0.01 ,0.01 ,0.01 ,0.01 ,0.01 ,0.01 0.02 0.03 0.04 0.05

OR: odds ratio; CI: confidence interval.

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High prevalence of chronic pelvic pain in women Silva GPOG et al.

with high rates of absenteeism (about half of these women have paid employment outside the home), which has a direct impact on social and economic life. We identified the following as factors independently associated with chronic pelvic pain: dyspareunia, previous abdominal surgery, depression, dysmenorrhea, anxiety, current sexual activity, low back pain, constipation, urinary symptoms, and low educational level. Dyspareunia is an important element of sexual dysfunction that ranges in prevalence from 7% to 75% depending on the diagnostic criteria and the associated clinical conditions, such as CPP.24-29 In a recent study, we observed that dyspareunia was associated with pelvic muscle tenderness.30 Although pelvic muscle tenderness may be a primary cause of CPP,31 we hypothesize that it is more often secondary to cross-talk communication between the viscera and muscles32 through neurogenic inflammation caused by the release of inflammatory mediators at the periphery in response to the antidromic stimulus over time.33 Similarly, current sexual activity may be linked in some way to dyspareunia, although this relationship is still unclear. Dysmenorrhea is a condition that is frequently concomitant with CPP. Its association with endometriosis or adenomyosis may explain the relationship to CPP. Another hypothesis is that women with dysmenorrhea have a lower pain threshold, which may favor the onset of illness.34 Moreover, mechanisms of viscero-visceral hyperalgesia between organs probably involve the sensitization of viscero-viscero-somatic convergent neurons.35 The most notable factor observed in the present study is the association of CPP with abdominal surgery, particularly because we observed that two-thirds of the women studied had undergone a previous abdominal surgery, and more than 40% of the women had previously undergone a cesarean section surgery. Considering the high rates of cesarean section surgery in our country36-37 (including this sample of our community), and the previous detection of an association with CPP by our group38 and by Latthe et al. in a recent systematic review,14 we stress the need for detailed studies regarding this possible causal relationship between CPP and cesarean section. Some studies suggest the possibility of an association between CPP and adhesions.39 However, the correlation between pelvic pain and adhesions is uncertain because adhesiolysis has not been shown to be effective in achieving pain control.40 Thus, we emphasize the importance of recognizing abdominal myofascial syndrome as a differential diagnosis.41 Anxiety and depression disorders are frequently concomitant with chronic pain, particularly in women, in both developed and developing countries.42-43 We have also observed a direct relationship between depression, anxiety, chronic pelvic pain, and quality of life.44 In addition to being a risk factor for CPP, mood disorders may make it more difficult for a women to engage in cognitively or emotionally demanding rehabilitation.45 Because the individual experience of pain is personal and subjective, it is probably affected by emotional states and, therefore, by psychosocial factors. There is some evidence that it is the stress of living with chronic pain, and not personal or family predisposition, that causes depression in these patients.46 The idea that pain, particularly chronic pain, can lead to feelings of frustration, worry, anxiety, and depression seems obvious. There is also evidence, however, for reverse causality, in which negative moods and emotions can lead to or exacerbate pain.47 It

therefore remains uncertain whether depression/anxiety precedes or is a consequence of chronic pain.48 Low back pain is usually comorbid with CPP. This association probably reflects the same pathophysiological mechanisms,49 although each can reduce the thresholds and thus contribute to the development of the other. We believe that both are the consequence of some other factors. Urinary and intestinal symptoms are common in women with CPP. Painful bladder syndrome and constipation are nongynecologic conditions that may cause or exacerbate CPP (level of evidence A).2 The prevalence of constipation identified in our sample of healthy women (22.7%) was similar to that observed by Oliveira et al.50 Because the women reported that constipation was present before CPP, we may infer that this condition may at least contribute to the development of CPP. Though this was beyond the scope of our study, some studies indicate that constipation may be secondary to the dysfunction of the levator ani muscle,51-52 which is a common condition in CPP. Several studies in the literature have shown that a lower educational level is linked to a higher prevalence of chronic pain.20,53 Because CPP increases with age and most education takes place in early life, it is likely that a lower educational level increase the risk for the development of CPP, or that both are the consequences of some other undescribed factors. Our study has identified several factors as independent predictors for CPP. We have not, however, reached final conclusions about the causal relationship between these factors and CPP. A thorough investigation and other different types of studies are necessary to corroborate our results.

CONCLUSIONS We conclude that the prevalence of CPP in women from RibeiraË&#x153;o Preto is very high, even though only 4% sufferers are aware of their diagnosis. This clinical condition is related to several independent predictors. Specifically designed studies are necessary to confirm our hypothesis. This confirmation is crucial for the prevention, early diagnosis, control, and resolution of CPP.

ACKNOWLEDGMENT The authors are grateful to CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico) for financial support.

REFERENCES 1. Williams RE, Hartmann KE, Sandler RS, Miller WC, Steege JF. Prevalence and characteristics of irritable bowel syndrome among women with chronic pelvic pain. Obstet Gynecol. 2004;104:452-8, doi: 10.1097/01.AOG.0000135275.63494.3d. 2. ACOG. ACOG Practice Bulletin No. 51. Chronic pelvic pain. Obstet Gynecol. 2004;103:589-605. 3. Weijenborg PT, Greeven A, Dekker FW, Peters AA, Ter Kuile MM. Clinical course of chronic pelvic pain in women. Pain. 2007;132 Suppl 1:S117-23, doi: 10.1016/j.pain.2007.06.020. 4. Zondervan KT, Yudkin PL, Vessey MP, Dawes MG, Barlow DH, Kennedy SH. Prevalence and incidence of chronic pelvic pain in primary care: evidence from a national general practice database. Br J Obstet Gynaecol. 1999;106:1149-55, doi: 10.1111/j.1471-0528.1999.tb08140.x. 5. Mathias SD, Kuppermann M, Liberman RF, Lipschutz RC, Steege JF. Chronic pelvic pain: prevalence, health-related quality of life, and economic correlates. Obstet Gynecol. 1996;87:321-7, doi: 10.1016/00297844(95)00458-0. 6. Howard FM. Chronic pelvic pain. Obstet Gynecol. 2003;101:594-611, doi: 10.1016/S0029-7844(02)02723-0.

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7. Zondervan KT, Yudkin PL, Vessey MP, Jenkinson CP, Dawes MG, Barlow DH, et al. The community prevalence of chronic pelvic pain in women and associated illness behaviour. Br J Gen Pract. 2001;51:541-7. 8. Zondervan KT, Yudkin PL, Vessey MP, Dawes MG, Barlow DH, Kennedy SH. The prevalence of chronic pelvic pain in women in the United Kingdom: a systematic review. Br J Obstet Gynaecol. 1998;105:939, doi: 10.1111/j.1471-0528.1998.tb09357.x. 9. Grace VM, Zondervan KT. Chronic pelvic pain in New Zealand: prevalence, pain severity, diagnoses and use of the health services. Aust N Z J Public Health. 2004;28:369-75, doi: 10.1111/j.1467-842X.2004. tb00446.x. 10. Latthe P, Latthe M, Say L, Gulmezoglu M, Khan KS. WHO systematic review of prevalence of chronic pelvic pain: a neglected reproductive health morbidity. BMC public health. 2006;6:177, doi: 10.1186/1471-24586-177. 11. Gureje O, Von Korff M, Simon GE, Gater R. Persistent pain and wellbeing: a World Health Organization Study in Primary Care. JAMA. 1998;280:147-51, doi: 10.1001/jama.280.2.147. 12. Ravsky A. Dor pe´lvica croˆnica. In: Coopmed E, editor. Ginecologia ambulatorial. Belo Horizonte: Camargos, A. F., Melo, V. H., Carneiro, M. M., Reis, F. M. 2001;293-301 13. Gomes KR, Tanaka AC. Reported morbidity and use of health services by working women, Brazil. Rev Saude Publica. 2003;37:75-82, doi: 10. 1590/S0034-89102003000100012. 14. Latthe P, Mignini L, Gray R, Hills R, Khan K. Factors predisposing women to chronic pelvic pain: systematic review. Bmj. 2006;332:749-55, doi: 10.1136/bmj.38748.697465.55. 15. Butrick CW. Interstitial cystitis and chronic pelvic pain: new insights in neuropathology, diagnosis, and treatment. Clin Obstet Gynecol. 2003;46:811-23, doi: 10.1097/00003081-200312000-00011. 16. Chen HS, He X, Wang Y, Wen WW, You HJ, Arendt-Nielsen L. Roles of capsaicin-sensitive primary afferents in differential rat models of inflammatory pain: a systematic comparative study in conscious rats. Experimental neurology. 2007;204:244-51, doi: 10.1016/j.expneurol.2006. 10.011. 17. Zondervan K, Barlow DH. Epidemiology of chronic pelvic pain. Baillieres Best Pract Res Clin Obstet Gynaecol. 2000;14:403-14, doi: 10. 1053/beog.1999.0083. 18. Staab JP, Datto CJ, Weinrieb RM, Gariti P, Rynn M, Evans DL. Detection and diagnosis of psychiatric disorders in primary medical care settings. The Medical clinics of North America. 2001;85:579-96, doi: 10.1016/ S0025-7125(05)70330-8. 19. Drossman DA, Dumitrascu DL. Rome III: New standard for functional gastrointestinal disorders. J Gastrointestin Liver Dis. 2006;15:237-41. 20. Sa KN, Baptista AF, Matos MA, Lessa I. Chronic pain and gender in Salvador population, Brazil. Pain. 2008;139:498-506, doi: 10.1016/j.pain. 2008.06.008. 21. Grace VM. Problems of communication, diagnosis, and treatment experienced by women using the New Zealand health services for chronic pelvic pain: a quantitative analysis. Health care for women international. 1995;16:521-35, doi: 10.1080/07399339509516207. 22. Grace VM, MacBride-Stewart S. ’Women get this’: gendered meanings of chronic pelvic pain. Health (London). 2007;11:47-67, doi: 10.1177/ 1363459307070803. 23. Peterson GM. Selecting nonprescription analgesics. American journal of therapeutics. 2005;12:67-79, doi: 10.1097/00045391-200501000-00010. 24. Aydin G, Basar MM, Keles I, Ergun G, Orkun S, Batislam E, et al. Relationship between sexual dysfunction and psychiatric status in premenopausal women with fibromyalgia. Urology. 2006;67:156-61, doi: 10.1016/j.urology.2005.08.007. 25. Heim LJ. Evaluation and differential diagnosis of dyspareunia. Am Fam Physician. 2001;63:1535-44. 26. Kwan KS, Roberts LJ, Swalm DM. Sexual dysfunction and chronic pain: the role of psychological variables and impact on quality of life. Eur J Pain. 2005;9:643-52, doi: 10.1016/j.ejpain.2004.12.008. 27. Ryan L, Hawton K. Female dyspareunia. Bmj. 2004;328:1357, doi: 10. 1136/bmj.328.7452.1357. 28. Verit FF, Verit A, Yeni E. The prevalence of sexual dysfunction and associated risk factors in women with chronic pelvic pain: a crosssectional study. Arch Gynecol Obstet. 2006;274:297-302, doi: 10.1007/ s00404-006-0178-3. 29. Danielsson I, Sjoberg I, Stenlund H, Wikman M. Prevalence and incidence of prolonged and severe dyspareunia in women: results from a population study. Scand J Public Health. 2003;31:113-8, doi: 10.1080/ 14034940210134040. 30. Montenegro ML, Mateus-Vasconcelos EC, Rosa e Silva JC, Nogueira AA, Dos Reis FJ, Poli Neto OB. Importance of pelvic muscle tenderness evaluation in women with chronic pelvic pain. Pain medicine (Malden, Mass. 2010;11:224-8, doi: 10.1111/j.1526-4637.2009.00758.x. 31. Montenegro ML, Mateus-Vasconcelos EC, Candido dos Reis FJ, Rosa e Silva JC, Nogueira AA, Poli Neto OB. Thiele massage as a therapeutic option for women with chronic pelvic pain caused by tenderness of

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pelvic floor muscles. J Eval Clin Pract. 2010;16:981-2, doi: 10.1111/j.13652753.2009.01202.x. Pezzone MA, Liang R, Fraser MO. A model of neural cross-talk and irritation in the pelvis: implications for the overlap of chronic pelvic pain disorders. Gastroenterology. 2005;128:1953-64, doi: 10.1053/j.gastro.2005. 03.008. Wesselmann U. Neurogenic inflammation and chronic pelvic pain. World J Urol. 2001;19:180-5, doi: 10.1007/s003450100201. Granot M, Yarnitsky D, Itskovitz-Eldor J, Granovsky Y, Peer E, Zimmer EZ. Pain perception in women with dysmenorrhea. Obstet Gynecol. 2001;98:407-11, doi: 10.1016/S0029-7844(01)01465-X. Giamberardino MA, Costantini R, Affaitati G, Fabrizio A, Lapenna D, Tafuri E, et al. Viscero-visceral hyperalgesia: characterization in different clinical models. Pain. 2010;151:307-22, doi: 10.1016/j.pain.2010.06.023. Freitas PF, Drachler Mde L, Leite JC, Marshall T. Inequalities in cesarean delivery rates by ethnicity and hospital accessibility in Brazil. International journal of gynaecology and obstetrics: the official organ of the International Federation of Gynaecology and Obstetrics. 2009;107:198-201. Potter JE, Hopkins K, Faundes A, Perpetuo I. Women’s autonomy and scheduled cesarean sections in Brazil: a cautionary tale. Birth (Berkeley, Calif. 2008;35:33-40. Almeida EC, Nogueira AA, Candido dos Reis FJ, Rosa e Silva JC. Cesarean section as a cause of chronic pelvic pain. International journal of gynaecology and obstetrics: the official organ of the International Federation of Gynaecology and Obstetrics. 2002;79:101-4. Mettler L, Alhujeily M. Role of laparoscopy in identifying the clinical significance and cause of adhesions and chronic pelvic pain: a retrospective review at the Kiel School of Gynecological Endoscopy. Jsls. 2007;11:303-8. Hammoud A, Gago LA, Diamond MP. Adhesions in patients with chronic pelvic pain: a role for adhesiolysis? Fertility and sterility. 2004;82:1483-91, doi: 10.1016/j.fertnstert.2004.07.948. Montenegro ML, Gomide LB, Mateus-Vasconcelos EL, Rosa-e-Silva JC, Candido-dos-Reis FJ, Nogueira AA, et al. Abdominal myofascial pain syndrome must be considered in the differential diagnosis of chronic pelvic pain. European journal of obstetrics, gynecology, and reproductive biology. 2009;147:21-4, doi: 10.1016/j.ejogrb.2009.06.025. Castro M, Kraychete D, Daltro C, Lopes J, Menezes R, Oliveira I. Comorbid anxiety and depression disorders in patients with chronic pain. Arq Neuropsiquiatr. 2009;67:982-5, doi: 10.1590/S0004282X2009000600004. Tsang A, Von Korff M, Lee S, Alonso J, Karam E, Angermeyer MC, et al. Common chronic pain conditions in developed and developing countries: gender and age differences and comorbidity with depression-anxiety disorders. J Pain. 2008;9:883-91, doi: 10.1016/j.jpain.2008.05. 005. Romao AP, Gorayeb R, Romao GS, Poli-Neto OB, dos Reis FJ, Rosa-eSilva JC, et al. High levels of anxiety and depression have a negative effect on quality of life of women with chronic pelvic pain. International journal of clinical practice. 2009;63:707-11, doi: 10.1111/j.1742-1241.2009. 02034.x. Kerns RD, Haythornthwaite JA. Depression among chronic pain patients: cognitive-behavioral analysis and effect on rehabilitation outcome. J Consult Clin Psychol. 1988;56:870-6, doi: 10.1037/0022-006X. 56.6.870. Gallagher RM, Verma S. Managing pain and comorbid depression: A public health challenge. Semin Clin Neuropsychiatry. 1999;4:203-20. Wiech K, Tracey I. The influence of negative emotions on pain: behavioral effects and neural mechanisms. NeuroImage. 2009;47:98794, doi: 10.1016/j.neuroimage.2009.05.059. Fishbain DA, Cutler R, Rosomoff HL, Rosomoff RS. Chronic painassociated depression: antecedent or consequence of chronic pain? A review Clin J Pain. 1997;13:116-37, doi: 10.1097/00002508-19970600000006. Biyani A, Andersson GB. Low back pain: pathophysiology and management. J Am Acad Orthop Surg. 2004;12:106-15. Oliveira JN, Tahan S, Goshima S, Fagundes-Neto U, Morais MB. [Prevalence of constipation in adolescents enrolled in Sao Jose dos Campos, SP, Brazil, school’s and in their parents]. Arquivos de gastroenterologia. 2006;43:50-4, doi: 10.1590/S0004-28032006000100013. Shafik A, Haugstad GK, Haugstad TS, Kirste UM, Leganger S, Wojniusz S, et al. The role of the levator ani muscle in evacuation, sexual performance and pelvic floor disorders Posture, movement patterns, and body awareness in women with chronic pelvic pain. Int Urogynecol J Pelvic Floor Dysfunct. 2000;11:361-76, doi: 10.1007/ PL00004028. Shafik A, El-Sibai O. Study of the levator ani muscle in the multipara: role of levator dysfunction in defecation disorders. J Obstet Gynaecol. 2002;22:187-92, doi: 10.1080/01443610120113391. Rashiq S, Dick BD. Factors associated with chronic noncancer pain in the Canadian population. Pain Res Manag. 2009;14:454-60.

CLINICS 2011;66(8):1313-1319

DOI:10.1590/S1807-59322011000800002

CLINICAL SCIENCE

Expression of Hypoxia-inducible factor 1-a and Vascular endothelial growth factor–C in locally advanced breast cancer patients Luiz Gustavo Oliveira Brito,I Viviane Fernandes Schiavon,I Jurandyr Moreira de Andrade,I Daniel Guimara˜es Tiezzi,I Fernanda Maris Peria,II Heitor Ricardo Cosiski MaranaI I Department of Gynecology and Obstetrics School of Medicine of Ribeiraõ Preto, Saõ Paulo University, Ribeiraõ Preto/SP, Brazil. II Internal Medicine, School of Medicine of Ribeiraõ Preto, Saõ Paulo University, Ribeiraõ Preto/SP, Brazil.

BACKGROUND: Locally advanced breast cancers are more prevalent in underdeveloped countries. Targeted therapy has been improved to identify hallmarks that are specific to these subtypes of tumors. OBJECTIVES: We aimed to prospectively assess the expression of Hypoxia inducible factor-1 a and vascular endothelial growth factor-C in locally advanced breast cancer patients. METHODS: Thirty women underwent incisional biopsies for the histopathological diagnosis of breast carcinoma and participated in neoadjuvant chemotherapy. The association of Hypoxia inducible factor-1 a and vascular endothelial growth factor-C with age, tumor size, histological grade, clinical staging, hormonal and axillary status, clinical and pathological response after neoadjuvant chemotherapy, expression of estrogen and progesterone receptors, and the presence of c-erbB-2 antigen was studied. RESULTS: Hypoxia inducible factor-1 a expression and Vascular endothelial growth factor-C expression were observed in 66.7% and 63.3% of all patients, respectively, and were marginally associated with each other (p = 0.06). Among the studied variables, only positive axillary status was associated with the presence of HIF-1a (p = 0.02). Complete pathological response was significantly associated (p = 0.04) with the expression of vascular endothelial growth factor-C prior to neoadjuvant chemotherapy. CONCLUSION: We concluded that Hypoxia inducible factor-1 a was associated with a poor prognosis and that vascular endothelial growth factor-C could be used as a predictive factor in locally advanced breast cancer patients with complete pathological response after neoadjuvant chemotherapy. KEYWORDS: Locally advanced breast cancer; HIF-1a; VEGF; Axillary lymph nodes; Immunohistochemistry. Brito LGO, Schiavon VF, Andrade JM, Tiezzi DG, Peria FM, Marana HRC. Expression of Hypoxia-inducible factor 1-a and Vascular endothelial growth factor–C in locally advanced breast cancer patients. Clinics. 2011;66(8):1313-1319. Received for publication on April 6, 2011; First review completed on April 18, 2011; Accepted for publication April 18, 2011 E-mail: lgobrito@gmail.com Tel.: 55 16 3602-2804

government provides incentives for mammography, approximately 10% of breast tumors are diagnosed as locally advanced tumors (LABC), which have a greater risk of metastasis and a reserved prognosis.3 Unlike the USA and European countries, where the incidence of breast cancer is increasing and mortality is decreasing, the mortality rate of breast cancer in Brazil is high due to the relatively high percentage (50%) of LABC cases.4 Surgery is not the primary recommended method of treatment for LABC. Neoadjuvant therapy reduces the tumor’s primary volume and transforms inoperable breasts into operable ones, increasing the conservative surgery rate. By identifying patients who present an optimal response to certain treatments, mechanisms that support tumor growth can be discovered, and novel targeted therapies can be developed. All solid tumors require a microenvironment that promotes angiogenesis, which either maintains tumor viability and its growth or contributes to the spread of the

INTRODUCTION Breast cancer is one of the main causes of death in occidental women. Statistics have indicated that the frequency of breast cancer has recently increased in developed and developing countries.1 In the USA, 192,370 women were diagnosed with breast cancer in 2009, and 40,170 deaths occurred.1 Randomized trials performed between 1976 and 1990 have shown that early detection through mammographic examination reduced mortality from breast cancer by 25% in women between 50 and 69 years old.2 Although the

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HIF-1a and VEGF-C were prevalent in locally advanced breast cancer patients Brito LGO et al.

disease.5 Vascular endothelial growth factors (VEGFs) constitute a family of potent angiogenic peptides that act on the development of hematopoietic stem cells, remodel the extracellular matrix (ECM) and regenerate inflammatory cytokines. The VEGF family is classified into various subtypes (A to D). VEGF-C and -D are produced by tumor-associated macrophages (TAMs), which express VEGFR-3 (the VEGF-3 receptor). Microvascular lymphatic density and the abovementioned hallmarks promote the lymphatic dissemination of breast tumor cells, which is directly related to the axillary status and prognosis of the patient.6 Another important mechanism that leads to angiogenesis is tissue hypoxia. Hypoxia is present in many solid tumors and is caused by abnormal neoplastic vascularization and rapid cell production, which results in apoptosis and areas of necrosis.7 Based on the results of several studies on the clinical applicability of hallmarks as a targeted therapy, biochemical hallmarks produced in hypoxic environments are related to several cancers (especially breast tumors) and have been considered as prognostic factors for highly undifferentiated tumors.8,9 However, it is not known if hypoxia is the cause or the effect of the formation of aggressive tumors. Hypoxia inducible factor-1 (HIF-1) is a heterodimeric nuclear transcription factor that is divided into two subunits (alpha [a] and beta [b]). Subtype 1a is overexpressed in breast tumors 10 and is of functional importance. Under normoxic conditions, HIF-1a is recognized by von HippelLindau (pVHL) proteins, hydroxylated by proline hydroxylases (PHDs) and factor-inhibiting HIF (FIH), and is ubiquitinated by the proteasome.10 However, the PHD and FIH levels is low during hypoxia. Thus, HIF-1 migrates from the cytoplasm to the nucleus and binds to hypoxia response elements (HREs), leading to the expression of targeted genes.10 Specifically, increases in the activation of genes that control glucose transportation, glycolysis, growth factor production, high-energy phosphate metabolism, erythropoiesis, heme metabolism, iron transport and nitric oxide synthesis are observed. The role of HIF-1 in breast carcinogenesis is related to the induction of VEGF transcription, which leads to a greater rate of tumor angiogenesis.11,12 In preclinical studies with metastatic patients, monoclonal antibodies against VEGF, such as bevacizumab, were used to control tumors.13 HIF-1a can also be overactivated by vascular growth factors, such as PDGF (platelet-derived growth factor), EGF (epidermal growth factor), FGF-2 (fibroblast growth factor type 2), TGF1b (transforming growth factor type 1 beta), IGF (insulin growth factor) and cytokines, such as TNF-a (tumor necrosis factor alpha) and interleukin (IL) 1b. Overactivation occurs through two important signaling pathways, the RAS/MEK/ MAPK and PI3K/AKt/mTOR cascades.10 Several investigations on the prognostic value of HIF-1a in breast cancer have been conducted. Schindl et al.14 showed that HIF-1a overexpression was associated with poor overall survival rates in LABC patients. However, Bos et al.5 identified an association between HIF-1a expression and survival in negative lymph node patients. In our previous study, significant differences between the serum levels of HIF-1a and VEGF were not observed before or after neoadjuvant chemotherapy (NACT).16 Studies on the modifications of the immunohistochemical and serum expression of HIF-1a and VEGF after NACT are scarce.

CLINICS 2011;66(8):1313-1319

Although VEGF is a classical prognostic and predictive factor for breast cancer, the relationship between VEGF and HIF-1a has not yet been established. Therefore, we analyzed the expression of HIF-1a and VEGF in LABC patients with the goal of identifying associations between the expression of these two genes and other prognostic factors.

METHODS AND MATERIALS Patients The present investigation was a prospective study. Consecutive sampling was conducted in the outpatient clinic of the Breast Division of the Hospital das Clıńicas, Ribeiraõ Preto School of Medicine – University of Saõ Paulo (HC-FMRP-USP) from March to November of 2009, and 30 locally advanced breast cancer patients were evaluated. All the patients signed informed consent forms before participating in the study. The Ethics Research Committee of HCFMRP-USP approved this research. The following inclusion criteria were applied: no previous treatment for breast cancer, between 25 and 80 years of age, and with an indication for NACT (the presence of stage II and III tumors, where the tumor volume/breast volume ratio did not allow for conservative surgery), as assessed by our clinic. The exclusion criteria were as follows: patients with a reduced Karnofsky performance status scale, previous malignant neoplasm, pregnancy, and confirmed metastatic disease. Prior to chemotherapy, an incisional biopsy was performed for histopathological diagnosis. During the procedure, a 1-cm3 tissue block was removed and stored in Tissue-Tek O.C.T. (Qiagen, USA). Except for one patient, who was diagnosed with lobular carcinoma, all of the patients were diagnosed with invasive ductal carcinoma (WHO criteria).

Baseline characteristics The following clinical variables were studied: age (years), tumor size (in centimeters), histological grade,17 clinical staging,18 and menopausal status (pre- and post-menopause). Clinical and pathologic responses were evaluated in all of the patients. The clinical assessment of tumor features was based on RECIST criteria;19 however, the following modified definitions were employed: complete response: no existence of clinical disease; partial response: less than a 50% reduction in lesion measurements; minor response/ progression: less than 25% reduction or an increase in tumor volume. Pathologic response was determined according to an identical rubric. The histopathological results of lymph node dissection after NACT were considered when the axilla was clinically negative. Alternatively, if the physical examination indicated that the cancer had metastasized to the lymph nodes, NACT was immediately conducted. NACT consisted of docetaxel (75 mg/m2) and epirubicin (60 mg/m2) on the first day, preceded by hydration with isotonic fluid, dexamethasone and antiemetic (ondansentron). The procedure was repeated every three weeks. The mean/standard deviation and median age of all 30 patients were 51.96¡12.65 and 52 years, respectively. Thus, most (66.7%) of the patients were more than 50 years old. The mean/standard deviation and median size of breast lumps were 5.85¡4.19 (range 2.7 to 25) and 5 cm, respectively. Table 1 shows the baseline features of the

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HIF-1a and VEGF-C were prevalent in locally advanced breast cancer patients Brito LGO et al.

hematoxylin counterstain (Zymed Laboratories Inc., CA, USA). HercepTest (Dako System) was employed to immunohistochemically quantify the HER-2 antigen. A score of +3 (10% of the tumor cells showed strong and complete nuclear membrane staining) was considered positive for HER-2. The test results were considered negative when membrane staining did not occur or was absent in less than 10% of the tumor cells.20 When intermediate tumors were identified (2+), CISH (chromogenic in situ hybridization) was conducted. Estrogen and progesterone receptor (ER and PR) status was determined when nuclear staining was visualized and was considered positive when more than 10% of the cells were stained.21 Mouse MAb was applied to analyze HIF-1a (Abcam Company), and quantification of darkly stained epithelial nuclei was performed.22 Cytoplasmic stainings were ignored, and the scoring rubric applied to ER/PR was employed. An anti-VEGF-C antibody was used (BD Biosciences) to analyze VEGF-C. To score VEGF-C stains, the percentage of strongly stained tumor cells was assessed.12 Staining was scored by three observers (L.G.O.B, V.F.S, and H.R.C.M), who were blinded to the clinical outcomes.

Table 1 - Baseline characteristics of the studied patients (Ribeira˜o Preto, Brazil, 2010). Variables Age (years) ,50 50+ Tumor size (cm) #5 5.1-10 .10 Menopausal status Pre-menopause Post-menopause Histological subtype Ductal Lobular Histological grade I II III Clinical staging (TNM) IIA IIB IIIA IIIB IIIC Estrogen receptor (ER) Positive Negative Progesterone receptor (PR) Positive Negative c-erbB-2 Positive Negative HIF-1a Positive Negative VEGF-C Positive Negative Total

10 20

33.3 66.7

15 13 2

50 43.3 6.7

14 16

46.7 53.3

28 2

93.3 6.7

3 16 11

10 53.3 36.7

4 4 10 7 5

13.3 13.3 33.4 23.3 16.7

19 11

63.3 36.7

14 16

46.7 53.3

10 20

33.3 66.7

20 10

66.7 33.3

19 11 30

63.3 36.7 100.0

Statistical analysis The intercooled Stata statistical package version 8.0 (Stata Corporation, Texas, USA) was used to interpret the data. A Fisher’s test was performed to assess the link between ER, PR, c-erbB-2 and the presence or absence of HIF-1a and VEGF-C. The mean, median and standard deviation of the continuous variables were also calculated. A significance level of 5% was used in two-tailed tests.

RESULTS Table 2 displays the positivity/negativity ratio of the immunohistochemical variables. Approximately 30% of all patients were positive for c-erbB-2 antigen. HIF-1a expression was observed in 66.7% of the patients; however, the number of patients with HIF-1a expression was reduced to 25.9% when only strong and diffuse staining was considered (Figure 1). VEGF-C was expressed in two-thirds of all cases, primarily in the cytoplasm of tumor cells (Figure 2). A trend between HIF-1a expression and age less than 50 years (p = 0.08) was observed, as was a statistically significant association with positive axillary status (p = 0.02) (Table 2). A marginally significant trend between elevated HIF-1a levels and the following variables was also observed: pre-menopausal women (p = 0.10), reduced pathologic response (p = 0.12) and higher clinical response (p = 0.09). A statistically significant association between HIF-1a expression and immunohistochemical markers (ER, PR and c-erbB-2) was not observed (Table 2). When VEGF-C expression was correlated with other variables, only the complete pathological response was associated with it (p = 0.04). Histological grading, axillary status, tumor size, age, menopausal status, ER, PR and cerbB-2 were not associated with VEGF-C. HIF-1a expression was marginally associated with VEGF staining (p = 0.06).

patients. Most of the women were postmenopausal and possessed aggressive histological tumors in advanced clinical stages (IIIA: 33%). In total, 54.2% displayed complete clinical response, and 28.6% displayed complete pathological response. Half of the patients possessed a positive axillary status.

Immunohistochemistry and quantification of ER, PR, C-erbB-2, VEGF-C and HIF-1-a Tissue sections with a diameter of 4 micrometers were obtained with a cryostat, and immunohistochemistry was performed according to the avidin-biotin-peroxidase complex (ABC) method. The tissue sections were fixed in 100% acetone for 10 minutes at 20 ˚C. Endogenous peroxidase activity was blocked by incubating the slides in a solution of hydrogen peroxide (0.3%) and PBS for 30 minutes. After antigen retrieval, the sections were cooled for 20 minutes and then incubated with the primary antibody for two hours at 37 ˚C. The specimens were washed and incubated for 45 minutes with biotinylated secondary antibodies. When the incubation was complete, the signal was amplified by the formation of an avidin-biotin complex and was developed with diaminobenzidine and Mayer’s

DISCUSSION The objectives of the present study were to assess the expression of HIF-1a and VEGF-C in LABC patients,

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Table 2 - The association between HIF-1a/VEGF-C expression and clinical variables (RibeiraË&#x153;o Preto, Brazil, 2010). Variables

HIF-1a expression (%) Negative

Age (years) ,50 50+ Tumor size (cm) ,5 5+ Clinical staging (TNM) II III Histological grade* I II III Menopausal status Menopause Pre-menopause Clinical response Complete Incomplete/no response Pathological response Complete Incomplete/no response Axillary status Positive Negative ER Positive Negative PR Positive Negative c-erbB-2 Positive Negative Total

Positive

VEGF-C expression (%) Negative

Positive

4 (40) 7 (35)

6 (60) 13 (65)

4 (33.3) 7 (38.8)

8 (66.7) 11 (61.2)

4 (50) 7 (31.8)

4 (50) 15 (68.2)

1 (33.3) 7 (43.8) 3 (27.3)

2 (66.7) 9 (56.2) 8 (72.7)

6 (37.5) 5 (35.7)

10 (62.5) 9 (64.3)

4 (30.8) 5 (45.5)

9 (69.2) 6 (54.5)

0 (0) 7 (46.6)

6 (100) 8 (53.4)

5 (33.3) 6 (40)

10 (66.7) 9 (60)

7 (36.8) 4 (36.4)

12 (63.2) 7 (63.6)

5 (35.7) 6 (37.5)

9 (64.3) 10 (62.5)

7 (35) 4 (40) 11 (36.3)

13 (65) 6 (60) 19 (63.3)

0.06 6 (60) 4 (20)

4 (40) 16 (80)

4 (33.3) 6 (33.3)

8 (66.7) 12 (66.7)

2 (25) 8 (36.4)

6 (75) 14 (63.6)

0 (0) 8 (50) 2 (18.2)

3 (100) 8 (50) 9 (81.8)

3 (18.7) 7 (50)

13 (81.3) 7 (50)

2 (15.4) 5 (45.5)

11 (84.6) 6 (54.5)

0 (0) 5 (33.3)

6 (100) 10 (66.7)

8 (53.3) 2 (13.3)

7 (46.7) 13 (86.7)

7 (36.8) 3 (27.3)

12 (63.2) 8 (72.7)

6 (42.9) 4 (25)

8 (57.1) 12 (75)

3 (30) 7 (35) 10 (33.3)

7 (70) 13 (65) 20 (66.7)

0.79

1.0

0.76

0.56

0.74

0.18

0.68

0.07

0.91

0.10

0.46

0.10

0.04

0.02

0.70

0.59

0.98

0.30

0.92

0.78

0.79

*The second and third histological grades were clustered into one category for the Fisherâ&#x20AC;&#x2122;s test.

determine possible prognostic and/or predictive functions, and identify associations with clinical and immunohistochemical markers. The expression level of HIF-1a (66.7%) was similar to those observed in other studies. For instance, Gruber23 and Bos12 observed an expression rate of 56%, and Kronblad24

obtained an expression level of 67%. However, some deviations were observed due to the diversity of cut-off points used in the aforementioned studies.10 Subjectively, a higher concentration of HIF-1a was detected in perinecrotic areas, which likely corresponds to activation by hypoxia. However, Vleugel et al.25 showed that 44% of their samples

Figure 1 - The presence (a) and absence (b) of HIF-1a expression in breast cancer cells (DAB antibody, 200x).

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HIF-1a and VEGF-C were prevalent in locally advanced breast cancer patients Brito LGO et al.

Figure 2 - The presence (a) and absence (b) of VEGF-C expression in breast cancer cells (Anti-VEGF-C antibody mouse isotype IgG2b,k, 200x).

Similar to previous reports,15,23 a significant association between HIF-1a and large tumors and more advanced clinical staging was not observed in the present study. Conversely, Kronblad et al.24 observed a significant positive correlation, especially in tumors with diameters greater than 5 centimeters. However, this association did not remain after multivariate analysis. Logistic regression could not be performed in our study due to limited sampling. Nevertheless, we concur with Gruberâ&#x20AC;&#x2122;s hypothesis,23 which states that the impact of HIF-1a is minimized in more advanced tumors due to repression by adaptive mechanisms. Bos et al.15 conducted a study on 81 negative axilla and 69 positive axilla patients and concluded that high HIF-1a levels had a profound impact on the overall survival and disease-free survival of patients with negative axilla. Alternatively, high HIF-1a levels did not have an effect on positive axillary patients. In the abovementioned study, patients did not receive NACT because all locally advanced tumors were excluded; however, NACT would likely affect patients in more advanced stages. Tumor volume has been associated with a determined factor. For example, larger breast tumors are positively related to VEGF levels.22 Furstenberger et al.31 analyzed patients in diverse clinical stages and found that the VEGF values of subjects in the control group and patients with tumors were 92 pg/ml and 132 pg/ml, respectively. However, Marana et al.16 did not observe any significant relationship between tumor volume and plasmatic VEGF level (p = 0.736). This result may be attributed to the occurrence of locally advanced breast cancers, not to the presence of tumors with different sizes. Similar results were observed in the present study. Histological grade was not associated with HIF-1a, even when this variable was divided into low and high grades. This result may be attributed to the sample used in the current investigation, which was different from that of other studies.15, 24 Another possibility is that the patients in our sample had a high percentage of positive lymph nodes (50%). This result could also explain the results obtained from previous studies conducted on patients with positive axillary status.14,23 However, Kronblad et al.24 evaluated 564

were positive for HIF-1a, and 13.5% of HIF-1a was distributed perinecrotically. By contrast, 30.5% of HIF-1a had a more diffuse expression, which was indicative of alternative activation (of p53 mutation or HER-2 overexpression, for example). The effect of HER-2 on HIF-1a activation has been determined by identifying significant associations (with the expression of the c-erbB-2 antigen) and has been demonstrated using plasma analyses on locally advanced tumors conducted by our institution.16 Moreover, the effect of HER-2 on HIF-1a activation has been extensively described.10, 15, 22, 24 However, in the present study, an association between HER-2 and HIF-1a activation was not observed due to limited sampling. The expression level of VEGF-C (63.3%) in the present investigation was similar to the results obtained in previous studies15, 26-28 on breast ductal carcinomas. In our study, staining was localized to the cytoplasm, which is in accordance with the results of previous studies.29 Although VEGF-C expression could not be correlated with clinical parameters (except for complete pathological response), angiogenesis is extremely important for tumor growth. The level of VEGF-C expression was expected to be reduced, leading to rapid cell apoptosis. Singh et al.27 observed reduced levels of VEGF expression in the tissues of patients who displayed excellent clinical responses after NACT. A significant association between positive axillary status and strong/diffuse HIF-1a staining was observed in locally advanced tumors. This finding is in accordance with the results of a previous study conducted by Gruber et al., who evaluated 77 patients.23 In the abovementioned investigation, HIF-1a was expressed in the majority of patients with positive axilla and served as a poor prognostic factor, especially for T1/T2 tumors. In particular, upon association with HIF-1a, no variables had significant association in the multivariate analysis. Schindl and colleagues14 obtained similar results in patients with positive axilla or T1/T2 tumors (192/206 patients) and demonstrated that HIF-1a expression was highly significant. Moreover, Schoppmann et al.30 found a significant association between HIF-1a expression and the amount of peritumoral lymphangiogenesis in breast cancer patients.

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CLINICS 2011;66(8):1313-1319

expression was higher in patients with complete pathological response, which confirms its ability to act as a predictive factor.

patients and observed a positive correlation between higher histological grade and HIF-1a expression (p = 0.003). In the present study, age less than 50 years and premenopausal status were slightly associated with increased HIF-1a expression; however, the crude odds ratio did not corroborate this association, which is similar to the results of previous studies.15, 23 A similar correlation with VEGF was not observed. HIF-1a expression did not have an effect on the clinical and pathological responses of patients who underwent NACT; thus, HIF-1a is not a predictive factor. Few studies aiming to establish a predictive value of HIF-1a in neoadjuvant treatments have been conducted; however, further investigations would stimulate research on targeted therapies. Neither a positive nor a negative association between HIF-1a expression and the estrogen receptor concentration was observed in the present study. The expression of estrogen receptors in mammary cancer cells is reduced under hypoxic conditions (MCF-7 and CAMA-1). The observed reduction in protein production in these cells and the oxygen level are directly related.10 Compared to normoxic conditions, tamoxifen response in breast cancer cells of premenopausal patients is reduced under hypoxic conditions.32 Thus, proteosomic degradation occurs at these receptors, reducing the concentration of activated forms. Interestingly, Generali et al.33 found that HIF-1-a was an independent predictor of poor response, especially in ERpositive patients. Similar to the results obtained from other authors, VEGF expression was not correlated with ER and PR status in the present study;27 however, VEGF production in breast cancer cells was stimulated by estrogen and progestin. Foekens et al.29 studied breast cancer cell lines and suggested that this discrepancy might be due to the constitutive expression of high levels of VEGF by ER-negative breast cancer cells. Alternatively, the expression of VEGF is well-controlled in better-differentiated ER-positive breast cancer cells. The expression of c-erbB-2 antigen in patients with HIF1a expression has been well documented. Giatromanolaki et al.34 showed that patients with high HIF-1a expression and HER-2 overexpression had a lower overall survival rate. In our study, no correlation with HIF-1a or VEGF-C expression was observed. Assuming that HER2/neu protein expression could affect cell migration and proliferation, as well as the spread of lymphangiogenic tumors via VEGF-C upregulation, Schoppmann et al.35 demonstrated that HER2/neu expression was related to significantly stronger VEGF-C expression and lymphangiogenesis in lymph nodepositive breast cancer patients. The results of the present study emphasized the role of HIF-1a as a poor prognostic factor and demonstrated that increased levels of this protein are associated with positive axillary lymph nodes. A connection between the expression of hallmarks related to hypoxia and poor outcomes was also observed, which reinforces the need for studies concerning the role of HIF-1a as a predictive factor, the creation of new HIF-1a inhibitors and their genic products, the identification of a subpopulation of patients who will benefit from this therapy (especially previously treated patients), and the attainment of knowledge about a hallmark that may be used in targeted therapies. The VEGF-C expression results obtained in the present investigation were in accordance with those of previous studies. In particular, VEGF-C

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predictive marker for tamoxifen response. Int J Cancer. 2006;118:2609-16, doi: 10.1002/ijc.21676. Vleugel MM, Greijer AE, Shvarts A, van der Groep P, van Berkel M, Aarbodem Y, et al. Differential prognostic impact of hypoxia induced and diffuse HIF-1a expression in invasive breast cancer. J Clin Pathol. 2005;58:172-7, doi: 10.1136/jcp.2004.019885. Nakamura Y, Yasuoka H, Tsujimoto M, Yang Q, Tsukiyama A, Imabun S, et al. Clinicopathological significance of vascular endothelial growth factor-C in breast carcinoma with long-term follow-up. Mod Pathol. 2003;16:309-14, doi: 10.1097/01.MP.0000062858.98295.9F. Singh M, Capocelli KE, Marks JL, Shleicher RB, Finlayson CA, Seligman PA. Expression of vascular endothelial growth factor and proliferation marker MIB1 are influenced by neoadjuvant chemotherapy in locally advanced breast cancer. Appl Immunohistochem Mol Morphol. 2005;13:147-56, doi: 10.1097/01.pai.0000137364.36091.b0. Gisterek I, Matkowaski R, Koslak J, Dus D, Lacko A, Szelachowska J, et al. Evaluation of prognostic value of VEGF-C and VEGF-D in breast cancer â&#x20AC;&#x201C; 10 years follow-up analysis. Anticancer Research. 2007;27:2797-802. Foekens JA, Peters HA, Grebenchtchikov N, Look MP, Meijer-van Gelder ME, Geurts-Moespot A, et al. High tumor levels of vascular endothelial growth factor predict poor response to systemic therapy in advanced breast cancer. Cancer Res. 2001;61:5407-14. Schoppmann SF, Fenzl A, Schindl M, Bachleitner-Hofman T, Nagy K, Gnant M, et al. Hypoxia inducible factor-1-alpha correlates with VEGF-C

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CLINICS 2011;66(8):1321-1327

DOI:10.1590/S1807-59322011000800003

CLINICAL SCIENCE

Effects of thyroxine replacement on endothelial function and carotid artery intima-media thickness in female patients with mild subclinical hypothyroidism Monica Dias Cabral,I Patricia Teixeira,I Debora Soares,I Sandra Leite,II Elizabeth Salles,I Mario WaismanI I

Universidade Federal do Rio de Janeiro, Rio de Janeiro/RJ, Brazil. II Centro de Estudos e Pesquisas da Mulher, Rio de Janeiro/RJ, Brazil.

BACKGROUND: Previous studies have suggested an association between subclinical hypothyroidism and coronary artery disease that could be related to changes in serum lipids or endothelial dysfunction. METHODS: Thirty-two female subclinical hypothyroidism patients were randomly assigned to 12 months of Lthyroxine replacement or no treatment. Endothelial function was measured by the flow-mediated vasodilatation of the brachial artery, as well as mean carotid artery intima-media thickness, and lipid profiles were studied at baseline and after 12 months of follow-up. RESULTS: The mean (¡SD) serum thyroid-stimulating hormone levels in the L-thyroxine replacement and control groups were 6.09¡1.32 and 6.27¡1.39 mUI/ml, respectively. No relationship between carotid artery intima-media thickness or brachial flow-mediated vasodilatation and free T4 and serum thyroid-stimulating hormone was found. The median L-T4 dose was 44.23¡18.13 mg/day. After 12 months, there was a significant decrease in the flowmediated vasodilatation in the subclinical hypothyroidism control group (before: 17.33¡7.88 to after: 13.1¡4.75%, p = 0.03), but there were no significant differences in flow-mediated vasodilatation in the L-thyroxine treated group (before: 16.81¡7.0 to after: 18.52¡7.44%, p = 0.39). We did not find any significant change in mean carotid intimamedia thickness after 12 months of L-thyroxine treatment. CONCLUSION: Replacement therapy prevents a decline in flow-mediated vasodilatation with continuation of the subclinical hypothyroidism state. Large prospective multicenter placebo-controlled trials are necessary to investigate endothelial physiology further in subclinical hypothyroidism patients and to define the role of L-thyroxine therapy in improving endothelial function in these patients. KEYWORDS: Subclinical hypothyroidism; Endothelial function; Flow-mediated vasodilatation; Carotid artery intima-media thickness; Levothyroxine treatment. Cabral MD, Teixeira P, Soares D, Leite S, Salles E, Waisman M. Effects of thyroxine replacement on endothelial function and carotid artery intimamedia thickness in female patients with mild subclinical hypothyroidism. Clinics. 2011;66(8):1321-1327. Received for publication on January 6, 2011; First review completed on January 26, 2011; Accepted for publication on April 19, 2011 E-mail: mdcabral@globo.com Tel.: 55 21 32825071

apply to all patients with SHT, especially when the serum TSH is only slightly elevated and there are no other associated cardiovascular risks.7 It is unclear whether the association between SHT and coronary artery disease is related to SHT-induced changes in serum lipid levels.8-10 In a population-based survey, SHT emerged as a significant risk factor for aortic atherosclerosis and myocardial infarction in elderly women independently of serum cholesterol level.11 Therefore, further studies should clarify the role of SHT in cardiovascular disease. The endothelium plays a major role in the maintenance of vascular function and integrity through the production of vasodilator and vasoconstrictor substances.12,13 Endothelial dysfunction that results in reduced availability of nitric oxide is an early marker of atherosclerosis and can be used to predict coronary artery disease before the development of atherosclerotic changes.14 Recent studies have shown that hypothyroidism and SHT may have adverse effects on endothelial function independently of other well-known

INTRODUCTION Subclinical hypothyroidism (SHT) is a disorder characterized by elevated serum thyroid-stimulating hormone (TSH) levels despite normal free thyroid hormone values.1 The prevalence of SHT ranges from 5 to 10%, and the condition affects 6 to 10% of women (approximately 15% of women over 60 years of age) and 2.4 to 3% of the male population.2,3 A recent meta-analysis demonstrated that SHT can lead to an increased risk of developing cardiovascular disease and, consequently, elevate cardiovascular-associated mortality, especially in patients under 65 years of age.4-6 However, different authors propose that this higher risk does not

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atherosclerotic risk factors.15-22 Considerable controversy surrounds the hypothesis that SHT has a significant effect on the risk profile of cardiovascular disease. Boekholdt et al.23 provided important information that addressed this issue. Although higher elevated LDL cholesterol levels were found in the SHT group, thyroid dysfunction was not linked with a significantly increased risk of coronary heart disease. Despite the association between thyroid hormone levels and cardiovascular risk factors, thyroid status was not statistically significantly associated with the risk of future coronary heart disease or all-cause mortality in that large cohort over a follow-up of 10.6 years. The endothelial physiology in SHT and the role of levothyroxine (L-T4) therapy in improving endothelial function are still uncertain. We previously reported no differences in flow-mediated vasodilatation (FMD) of the brachial artery and carotid artery intima-media thickness (IMT) in a group of women with mild SHT without previously diagnosed cardiovascular disease compared with euthyroid subjects.24,25 The aims of the present study were to assess the effect of L-T4 replacement on endothelial function measured by FMD of the brachial artery, as well as carotid artery IMT in a group of female patients with mild SHT.

another half corresponding to observation. For those patients who were treated, the initial L-T4 dose was 0.75 mg/kg/day. Patients were re-evaluated every four weeks based on the clinical evaluation (including diet habits and physical activity) and TSH and FT4 levels to adjust the dose. The dose that achieved a normal serum TSH was subsequently maintained. After a normal TSH level was obtained, patients were seen every three months to assess the presence of adverse effects, disease progression, and whether the L-T4 dose needed adjustment. The patients in the observation group (OG) were re-evaluated every four weeks, and blood tests were used to measure TSH and FT4 levels. All patients were instructed to reduce the fat in their daily diet. The patients received general recommendations of a healthy diet, including the following: consume fish, especially oily fish, at least twice a week; limit the intake of saturated fat and trans fat by choosing lean meats and vegetable alternatives, as well as fat-free or low-fat dairy products and minimizing the intake of partially hydrogenated fats; minimize the intake of beverages and foods with added sugars; and choose and prepare foods with little or no salt. These recommendations were written and provided to all patients. A re-evaluation of serum lipids, FMD and carotid IMT was performed one year after the restoration of the euthyroidism state or one year after inclusion in the group of patients with SHT who did not receive L-T4 treatment. The subjects did not use any lipid-lowering medications during the study. The study was approved by the UFRJ Institutional Ethics Committee, and all subjects provided written informed consent to participate. The number of the protocol for the Ethics Committee in Research was 12-01. A general physical examination was performed, including assessment of height (without shoes), weight, and waist circumference (the minimum value between the iliac crest and the lateral costal margin). BMI was calculated as the weight (kg) divided by the height squared (m2). Systolic and diastolic blood pressures were measured from the right brachial artery of the subjects in a supine position after 10 min of rest using a pneumatic sphygmomanometer. Venous blood samples were drawn between 8:00 and 9:00 am after a 12-hour overnight fast. Serum was centrifuged and stored at -80 Ë&#x161;C until assayed. Serum TSH, FT4 and thyroid peroxidase antibody (TPO-Ab), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-c), triglyceride (TG), apoprotein B (apoB), apoprotein A (apoA), and lipoprotein a [Lp(a)] levels were determined in both groups. Serum TSH, FT4 and TPO-Ab were measured by immunochemiluminescence (Immulite 2000; DPC, Diagnostic Products Corporation, USA). Reference ranges for TSH and FT4 were 0.4-4.0 mIU/ml and 0.8-1.9 ng/dl, respectively. TPO-Ab levels of .35 IU/ml were considered positive. The intra-assay coefficients of variation were 3.8-12.5, 4.4-7.5, and 4.3-5.6% for TSH, FT4, and TPO-Ab, respectively. The interassay coefficients of variation were 4.6-12.5, 4.8-9.0, and 7.810.5%, respectively. TC and TG (Vitros Chemistry Products, USA), as well as HDL-c (Boehringer Mannheim Systems, Germany), were determined enzymatically. Low-density lipoprotein cholesterol (LDL-c) was calculated by the Friedewald equation: LDL-c = (TC â&#x20AC;&#x201C; HDL-c) - (TG/5). The reference ranges for TC, HDL-c, TG, and LDL-c were based on the Adult Treatment Panel III of the National Cholesterol

PATIENTS AND METHODS The study was a randomized open-label prospective trial comparing the use of L-T4 in female patients with mild SHT. Thirty-two women with mild SHT were recruited from the Outpatient Clinic of the Clementino Fraga Filho University Hospital (HUCFF), Federal University of Rio de Janeiro (UFRJ). The inclusion criteria were at least two documented laboratory determinations of SHT conducted at least six weeks apart, as defined by both elevated TSH (.4.0 mIU/ml) and normal free thyroxine (FT4) levels. The patients had no previous history of thyroid disease. The maximum TSH value accepted was 12 mIU/ml. This cutoff represents three times the superior acceptable limit of the TSH level, which is 4 mIU/ml. Individuals were excluded from the study if they had a history of alcohol use or were suffering from any cardiovascular disease or concomitant non-thyroid illnesses (e.g., obesity, diabetes mellitus, arterial hypertension, liver, or renal diseases). We examined their personal histories, physical examinations, and laboratory tests. The routine laboratory test included kidney function tests (urea, creatinine), liver function tests (ALT, AST, and GGT), and mean glycemia. The routine laboratory chemistry was normal in all patients, and no patient was taking any drug that could interfere with thyroid, lipoprotein, or endothelial function. The drugs that could interfere included amiodarone, corticosteroids, estrogens, lithium, statins, diuretics, and fibrates; additional drugs were used to treat diabetes, arterial hypertension, or obesity. The patients had no history of recent hospitalization. Patients with SHT were randomly allocated to treatment with L-T4 (levothyroxine group) or no treatment (observation group). Fixed-block numbers were used to generate the random allocation sequence of the treatment group. Each patient received a number in a block of four or six random numbers. Each number corresponded to one of the follow-up groups (levothyroxine group or observation group). Blocks of six and four numbers were continuously distributed, and each block had half of the numbers corresponding to treatment and

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Education Program (NCEP).26 The Lp(a) concentration was determined by an immunoradiometric assay (Diagnosis System International, Novatech, USA) and apoA and apoB were measured by a rate nephelometric immunoassay (Beckman Coulter, USA), and their reference ranges were lower than 30, 90-200, and 30-100 mg/dl, respectively. The subjects were investigated by high-resolution colorDoppler ultrasound imaging (Toshiba Nemium, Japan; 14mHz linear probe) of the brachial artery in the dominant arm. The study was performed in a temperature-controlled room (25 ˚C) with subjects resting in the supine position. The blood pressure and heart rate were recorded on the opposite arm every three min using an automatic sphygmomanometer. The subjects’ dominant arm was comfortably immobilized in the extended position to allow consistent access to the brachial artery. Doppler ultrasound measurements were performed before and 60 seconds after reactive hyperemia (RH). To avoid interobserver variability, all measurements were performed by the same examiner, who was blinded to the subjects’ clinical status. Brachial artery vasodilation in response to RH was determined by a previously validated technique.27,28 The intraclass correlation coefficient of this technique has been reported previously by our laboratory and ranges from R = 0.7001 to R = 0.8420 (p,0.05).29,30 The scans were recorded on S-VHS videotape. The internal diameter of the brachial artery was assessed at the end of diastole, and the arterial flow was measured using the pulse Doppler sample volume at an angle of 60 ˚ or less in the center of the artery. For each subject, optimal brachial artery images were obtained approximately 5 cm above the antecubital fossa. Arm pressure was generating by inflating a pneumatic arm band above the elbow cuff to a pressure up to 30 mmHg higher than the subject’s systolic arterial pressure for 5 min. The cuff was then deflated, the arterial flow was immediately recorded and the diameter was measured 60 to 90 seconds after deflation. For both diameters, one measurement was recorded. FMD was calculated according to the following formula: FMD = (post-occlusion diameter - baseline diameter) 6100/baseline diameter. The carotid images were obtained with the patient in the supine position with the neck mildly extended and the head rotated contralaterally to the side. The imaging protocol involved obtaining a minimum of four longitudinal B-mode images of the distal 10 mm of the right and left common arteries and the carotid bifurcation. The whole image session was recorded on videotape. The IMT of the common carotid artery (CCA) and carotid bifurcation were calculated by the same examiner with high-resolution ultrasound imaging (Acuson Aspen Advanced, 10 mHz linear probe, USA), as previously described.31 The final analysis was performed with IMT images of both the near and far wall of the right and left common artery and the carotid bifurcation. The recordings were evaluated by the same reader, who was blinded to the clinical data. The diastolic frames of each carotid segment were digitized. Lines were drawn along the lumen-intimal and medial adventitial interfaces in all analyzed segments, and the IMT of each segment was automatically computed as an average of several measurements. For statistical analysis, IMT readings in all the carotid segments were averaged (mean IMT). Plaque was considered as a focal structure if it encroached into the arterial lumen by at least 0.5 mm or 50% of the surrounding IMT value or demonstrated a thickness .2.0 mm, as

measured from the media-adventitia interface to the intima-lumen interface. The reproducibility of the IMT measurement was acceptable, as demonstrated by coefficients of variation of 7.7¡4.3%.31

Statistical analysis A statistical analysis was performed using SPSS for Windows, version 10.0. The mean values of the numerical variables were compared between groups by the MannWhitney test if they were not normally distributed, as detected by the Kolmogorov-Smirnov Liliefors test. Parametric variables were compared by the Student t-test. Data were expressed as means ¡ SD values unless otherwise stated. We also determined the frequencies of qualitative variables. For multi-group comparisons, oneway analysis of variance (ANOVA) or the Kruskal-Wallis test was applied, as appropriate. Analysis of frequencies was performed with the use of contingency tables (chisquare test). Correlations between parameters were tested by Pearson or Spearman correlation coefficients. The variation in the studied variables for each SHT patient who completed the follow-up protocol was calculated. The effect of LT-4 was assessed by comparing the mean variations between the two SHT groups (treated and observational), based on a per-protocol approach. The different effects of L-T4 replacement were also assessed by comparing the means before and after intervention by the Wilcoxon Signed Rank Test or Student’s t-test. A p value of ,0.05 was considered to be significant.

RESULTS Thirty-two female patients with mild SHT without previously treated thyroid or vascular disease were randomly assigned to 12 months of L-T4 replacement or no treatment and were evaluated. 14 patients were randomly assigned to the L-T4 treatment group (LtG), and 18 patients were randomly assigned to the observational group (OG). During the study, no patient was excluded. Both groups were similar with respect to age, BMI, waist circumference, activity level, smoking behavior, menopausal status, and laboratory and vascular parameters. Baseline data: Table 1 shows the baseline information of the patients in the different studied groups. All patients had spontaneous and primary hypothyroidism, and the majority had positive circulating TPOAb (Table 1). No relationship between carotid IMT or brachial FMD and free T4 and TSH was found. Follow-up data: TSH and FT4 levels at the end of the study (12 months) are shown in Table 2. No significant changes were observed in BMI and waist circumference (data not shown) or in other laboratory variables (Table 2). The median dose of L-T4 was 44.23¡18.13 mg/day. As shown in Figure 1, there was a significant decrease in the brachial artery FMD after 12 months in the OG (p = 0.03) and no significant difference in FMD in the LtG (p = 0.39). We did not find any correlation between the carotid IMT and the studied variables or a reduction of carotid IMT after one year of LT4 treatment (Table 2). We found plaques in 2 patients in each group, and there was no difference after one year of treatment.

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triglycerides, elevated LDL-c and/or decreased HDL-c. There was a non-significant decrease in FMD in the OG in both subgroups (dyslipidemia present or absent) and a nonsignificant increase in FMD in the LtG in both subgroups. Changes in brachial FMD and carotid artery IMT after one year of follow-up in the LtG and OG based on the presence or absence of dyslipidemia are shown in Table 3.

Table 1 - Baseline data of levothyroxine-treated and observation groups.

Age (yr) BMI (kg/m2) Waist (cm) Sedentary behavior (%) Smokers (%) Menopause (%) HRT (%) Familial dyslipidemia (%) TSH (mUI/ml) FT4 (ng/dl) TPO-Ab (%) TC (mg/dl) HDL-c (mg/dl) LDL-c (mg/dl) TG (mg/dl) Lipoprotein(a) (mg/dl) ApoB (mg/dl) ApoA (mg/dl) Mean IMT (mm) Flow-mediated dilatation (%)

LtG

47.59¡8.4 26.24¡2.71 84.06¡8 94.4 11.1 55.6 0 33.3 6.77¡1.96 0.95¡0.15 61.1 226.11¡43.55 51.83¡10.28 147.5¡37.58 132.5¡50.82 53.83¡62.87 128.61¡34.16 148.83¡24.73 0.65¡0.23 17.33¡7.88

43.36¡9.8 25.89¡2.29 83.17¡6.5 85.7 14.3 50 0 34.4 6.79¡2.0 0.97¡0.13 64.3 213.43¡53.17 56.78¡11.15 137.92¡47.94 136.07¡119.18 61.78¡62.71 109.28¡44.15 150.21¡24.73 0.66¡0.11 16.81¡7.0

DISCUSSION LT-4 treatment did not significantly improve endothelial function or reduce the carotid IMT in subjects with mild SHT who were similar with respect to age, BMI, smoking, menopausal status, and endothelial function modifiers. After one year, there was a significant decrease in brachial artery FMD in the OG and no difference in LtG, which suggests that thyroid hormone replacement could prevent a decline in FMD in patients with SHT. The exclusion of participants with concomitant hormonal, renal, or liver diseases allowed us to control our analysis for confounding variables and yielded a homogeneous population. In addition, by excluding co-morbid conditions, the mean age was reduced, allowing a smaller sample size for the study. Few studies have evaluated carotid IMT and the effect of L-T4 replacement in subclinical thyroid dysfunction. Monzani et al.15 were the first to show higher mean IMT values in SHT patients compared with age- and sexmatched controls. The maximal thickness in any particular segment was also measured (maximal IMT) and was higher in the SHT group. We previously reported no significant differences in the mean carotid IMT in a group of subclinical hypothyroid patients compared with a euthyroid group, which suggests that mild SHT is not associated with an increase in cardiovascular risk when assessed by carotid IMT.24,25 Monzani et al.25 were also the first to evaluate the benefit of six months of L-T4 replacement therapy on IMT in patients with SHT. L-T4 significantly reduced both LDL-c and the mean IMT, suggesting that lipid infiltration of the arterial wall may represent the main mechanism underlying the increase in IMT in patients with SHT. In the present work, we did not find any association between carotid IMT

Notes: Values are expressed as means ¡ SD or frequency. The p values were .0.05. OG, observation group; LtG, levothyroxine group; HRT, hormone replacement treatment; TSH, thyroid-stimulating hormone; FT4, free thyroxine; TPO-Ab, antiperoxidase antibody; TG, triglycerides; HDL-c, high-density lipoprotein cholesterol; TC, total cholesterol; LDL-c, lowdensity lipoprotein cholesterol; apoA, apoprotein A; apo B, apoprotein B; IMT, intima-media thickness; FMD, flow-mediated vasodilatation.

When comparing the mean changes during the study in the two TPO-Ab patients within the prospective SHT groups, no significant differences in brachial artery FMD were found (LtG, 1.13¡9.5; OG -5.58¡9.59; p = 0.12). When the patients in each group were subdivided into low ($4 but ,8 mIU/ml) or high ($8 but ,12 mIU/ml) TSH levels, no differences were observed in metabolic or vascular parameters (data not shown). A stratified analysis that considered the presence of any type of dyslipidemia based on NCEP criteria was performed. Dyslipidemia was defined as elevated TC, elevated

Table 2 - Initial (baseline) and final (after one year) results of the levothyroxine-treated and observation groups. Observation

TSH (mUI/ml) FT4 (ng/dl) TC (mg/dl) HDL-c (mg/dl) LDL-c (mg/dl) TG (mg/dl) Lipoprotein (a) (mg/dl) ApoB (mg/dl) ApoA (mg/dl) Mean IMT (mm) FMD (%)

group

Levothyroxine

Group

Baseline

1 year

Baseline

1 year

6.77¡1.96b (4.2-11.0) 0.95¡0.15 226.11¡43.55 51.83¡10.28 147.5¡37.58 132.5¡50.82 53.83¡62.87 128.61¡34.16 148.83¡24.73 0.65¡0.23 17.33¡7.88a,b

6.69¡3,51b, (3.27-13.5) 1.06¡0.15 227.6¡36.9 49.61¡9.74 150.6¡34.74 137.0¡56.06 48.21¡47.22 125.94¡27.47 136.78¡24.04 0,67¡0,18 13.10¡4.75a,b

6.79¡2.0a,b (4.04-10.65) 0.97¡0.13 213.43¡53.1 56.78¡11.15 137.92¡47.94 136.07¡119.18 61.78¡62.71 109.28¡44.15 150.21¡24.73 0.66¡0.11 16.81¡7.0 b

3.02¡0.89a,b (1.54-4.0) 1.16¡0.18 208.4¡36.7 54.36¡12.3 132.8¡37.5 106.0¡36.7 58.87¡64.09 105.1¡27.17 144.14¡25.57 0.66¡0.15 18.52¡7.44 b

Values are expressed as means ¡ SD; TSH values are expressed as the mean ¡ SD (interquartile range). a p,0.05 comparing before and after values within each group. b p,0.05 comparing the mean changes between the two prospective SHT groups during the study. For all other values, p.0.05. TSH, thyroid-stimulating hormone; FT4, free thyroxine; TG, triglycerides; HDL-c, high-density lipoprotein cholesterol; TC, total cholesterol; LDL-c, lowdensity lipoprotein cholesterol; apoA, apoprotein A; apoB, apoprotein B; IMT, intima-media thickness; FMD, flow-mediated vasodilatation.

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Figure 1 - Changes in FMD% in the levothyroxine-treated and observation groups.

that higher cholesterol levels may be associated with endothelial dysfunction. Dagre et al.36 noninvasively assessed the NO-dependent endothelial function of resistance arteries in subjects with hypothyroidism of varying severity by measuring the forearm blood flow response during reactive hyperemia via utilizing venous occlusion strain-gauge plethysmography. Endothelial dysfunction was only detected in the microvasculature of patients with overt hypothyroidism. We previously reported no significant differences in brachial FMD in a group of subclinical hypothyroid patients compared with a euthyroid group, which suggests that mild thyroid dysfunction had no adverse effects on endothelial function in the population studied.24 Taddei et al.22 showed in a group of subclinical hypothyroid patients that 6 months of euthyroidism by L-T4 replacement increased acetylcholine-induced vasodilation. They also observed a significant correlation between the maximal response to acetylcholine and changes in LDL-c. In a randomized, double-blind, crossover study of L-T4 or placebo for 12 weeks, brachial FMD was significantly improved in the group of patients who took a daily dose of 100 mg of L-T4 compared with the placebo group. Multivariate analysis showed that increased serum FT4 levels were the most significant variable predicting a reduction in TC or improvement in FMD.23 Xiang et al. showed that TSH, FT3 (free T3), LDL-c and Lp(a) levels were

and the variables studied. Furthermore, no reduction of carotid IMT was appreciated after one year of L-T4 therapy. The cardiovascular system is a specific target of thyroid hormones (THs); thus, thyroid dysfunction is accompanied by profound changes in cardiovascular hemodynamics.26,27 Both the vasculature and the endothelium play pivotal roles in modulating vascular tone, and both are potential targets of THs.28 Several studies have demonstrated the presence of thyroid receptors in endothelial cells.32,33 Some studies have suggested a role for vascular smooth muscle (VSM) cells in mediating the vasodilating effect of T3 infusion in the endothelium in human euthyroid subjects.34,35 Little is known about the interaction of TSH with endothelial cells. The TSH receptor is present in the thyroid gland and, also, in many other sites, such as VSM cells.34 Dardano et al. demonstrated that an acute systemic increase of serum TSH levels by rhTSH injection induced a significant impairment of endothelial vasodilatation in conduit arteries. Few studies have evaluated the relationship between SHT and endothelial dysfunction and the effect of L-T4 replacement in the endothelial function of subclinical hypothyroid patients. Lekakis et al.17 were the first to describe the negative association between borderline and mild hypothyroidism and FMD. In their study, cholesterol did not differ significantly among groups but tended to be higher in the hypothyroidism and SHT groups, and the authors concluded

Table 3 - Changes in brachial FMD and mean EIM after one year of follow-up in the L-T4 group and the observation group based on the presence or absence of dyslipidemia. Dyslipidemia present Group Mean IMT (mm) Flow-mediated dilatation (%)

Dyslipidemia absent

LtG

0.00¡0.01 -3.84¡5.97

0.00¡0.01 1.15¡7.95

0.00¡0.01 -5.0¡11.7

0.00¡0.01 2.46¡8.62

Values are expressed as the mean changes between the two prospective SHT groups through the study. All other p.0.05. OG, observation group; LtG, levothyroxine group; FMD, flow-mediated vasodilatation; IMT, intima-media thickness.

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of the SHT state in comparison with untreated subjects. Large, prospective, multicenter placebo-controlled trials are necessary to further investigate the endothelial physiology of patients with SHT and to define the role of L-T4 therapy in improving endothelial function in patients with SHT.

significant factors associated with endothelium-dependent arterial dilation in Hashimoto’s thyroiditis patients with euthyroidism.35 The lack of FT3 measurement is a limitation of our study and is also important for ruling out the possible inclusion of subjects with non-thyroidal illness syndrome. The interpretation of the results strongly depends on the effectiveness of substitutive treatment. According to the literature,7 in this case, the optimal values for TSH in L-T4 treated patients are unclear. As shown in some studies, it is not uncommon for patients in clinical trials to be outside of the therapeutic target at the end of the study. Ravzi et al.6 demonstrated a significant improvement in cardiovascular risk factors in a group of patients with SHT treated by LT-4, and 27 patients had discordant thyroid function tests during the course of the study. This study was a randomized open-labeled prospective trial. A double-blind, placebo-controlled study would be a more suitable approach. Another limitation of this study was the small sample size, which makes it difficult to obtain a clear decision about the benefit of L-T4 treatment in SHT patients. More studies with larger patient populations are needed to elucidate this subject more fully. We did not measure the maximal IMT during the internal carotid artery measurements, which could be considered a limitation of the study. An important issue is which IMT measure (e.g., mean IMT value; maximal IMT value; internal carotid artery from the anterior, lateral, and posterior angle; and composite measures from the left and right side or from different arterial sides) should be used. There is no definite answer to this question, but, for study purposes, all segments should be analyzed. The brachial artery vasodilation in response to RH was determined by FMD of the brachial artery. FMD is an indirect way of measuring the endothelial function using vascular reactivity. We did not assess endothelium-independent vasodilatation, which could be assessed with a dose-response curve to sodium nitroprusside. While it is important to perform nitrate dilatation to confirm that the findings were not due to media disease, as opposed to endothelial dysfunction, we were unable to perform this evaluation and suggest that it be performed in future works. Some studies that assessed the response to sodium nitroprusside (which specifically acts on smooth muscle cells) have shown similar responses in both groups (control and SHT groups) and a reduced vasodilatory effect of acetylcholine in SHT patients; these results indicate that SHT is characterized by the presence of endothelial dysfunction.17,22 Another limitation of this study is that we did not measure estradiol levels or the duration of menopause. This finding is important because abnormal FMD values can be obtained in older women and women who experienced a long menopausal period. Although both groups were similar in relation to age, the control group was more than four years older than the treated one; thus, the changes in FMD could be related to the development of climacterium. In the present study, the reported mean values for TSH after treatment were in the upper limit of the normal range. Some patients were under-treated, and this finding could explain why we did not find a significant benefit after LT-4 treatment.

REFERENCES 1. Ayala A, Wartofsky L. Minimally symptomatic (subclinical) hypothyroidism. The Endocrinologist. 1997;7:44-50. 2. Canaris GJ, Manowitz NR, Mayor G, Ridgway EC. The Colorado thyroid disease prevalence study. Arch Intern Med. 2000;160:526-34. 3. Cooper DS. Clinical practice. Subclinical hypothyroidism. N Engl J Med. 2001;345:260-5. 4. Rodondi NR, Aujesky D, Vittinghoff A, Cornus J, Bauer D. Subclinical hypothyroidism and the risk of coronary heart disease: a meta-analysis. Am J Med. 2006;119:541-51 5. Ochs N, Auer R, Bauer D, Nanchen D, Gussekloo J, Cornuz J, et al. Metaanalysis: subclinical thyroid dysfunction and the risk for coronary heart disease and mortality. Ann Intern Med. 2008;148:832-45. 6. Razvi S, Ingle L, Keeka G, Oates C. The beneficial effect of L-thyroxine on cardiovascular risk factors, endothelial function, and quality of life in subclinical hypothyroidism: randomized, crossover trial. The Journal of Clin Endoc Metab. 2007;92:1715-23. 7. Biondi B, Cooper D. The clinical significance of subclinical thyroid dysfunction. Endoc Rev. 2008;29:76-131. 8. Caraccio N, Ferrannini E, Monzani F. Lipoprotein profile in subclinical hypothyroidism: response to levothyroxine replacement, a randomized placebo-controlled study. The Journal of Clin Endoc Metab. 2002;87: 1533-8. 9. Teixeira PF, Reuters VS, Ferreira MM, Almeida CP, Reis FA, Melo BA, et al. Treatment of subclinical hypothyroidism reduces atherogenic lipid levels in a placebo-controlled double-blind clinical trial. Horm Metab Res 2008;40:50-5. 10. Rodondi N, Newman AB, Vittinghoff E, Rekeneire N, Satterfield S, Harris T, et al. Subclinical hypothyroidism and risk of heart failure, other cardiovascular events and death. Arch Inter Med. 2005;165:2460-6. 11. Hak AE, Pols HA, Visser TJ, Drexhage H, Hofman A, and Witteman JCM. Subclinical hypothyroidism is an independent risk factor for atherosclerosis and myocardial infarction in elderly women: the Rotterdam Study. Ann Intern Med. 2000;132:270-8. 12. Vane JR, Anggard EE, Botting RM. Regulatory functions of the vascular endothelium. N Engl J Med. 1990;323:27-36. 13. Healy B. Endothelial cell dysfunction: an emerging endocrinopathy linked to coronary disease. J Am Coll Cardiol. 1990;16:357-8. 14. Bonetti PO, Lerman LO, Lerman A. Endothelial dysfunction: a marker of atherosclerotic risk. Arterioscler Thromb Vasc Biol. 2003;23:168-75. 15. Monzani F, Caraccio N, Kozakowa M, Dardano F, Vittone A, Virdis S, et al. Effect of levothyroxine replacement on lipid profile and intimamedia thickness in subclinical hypothyroidism: a doubleblind, placebocontrolled study. The Journal of Clin Endoc Metab. 2004;89:2099-106. 16. Nagasaki T, Inaba M, Henmi Y, Kumeda Y, Ueda M, Tahara H, et al. Decrease in carotid intima-media thickness in hypothyroid patients after normalization of thyroid function. Clin Endocrinol. 2003;59:607-12. 17. Lekakis J, Papamichael C, Alevizaki M, Piperingos G, Marafelia P, Mantzos J, et al. Flow-mediated, endotheliumdependent vasodilation is impaired in subjects with hypothyroidism, borderline hypothyroidism, and high-normal serum thyrotropin (TSH) values. Thyroid. 1997;7:411-4. 18. Papaioannou GI, Lagasse M, Mather JF, Thompson PD. Treating hypothyroidism improves endothelial function. Metabolism. 2004;53: 278-9. 19. Cikim AS, Oflaz H, Ozbey N, Cikim K, Umman S, Meric M, et al. Evaluation of endothelial function in subclinical hypothyroidism and subclinical hyperthyroidism. Thyroid. 2004;14:605-9. 20. Volzke H, Robinson DM, Schminke U, Lu¨demann J, Rettig R, Felix S, et al. Thyroid function and carotid wall thickness. The Journal of Clin Endoc Metab. 2004;89:2145-9. ¨ zarmag˘an S, Tezelman S. 21. Erbil Y, Ozbey N, Giris M, Salmaslıog˘lu S, O Effects of thyroxine replacement on lipid profile and endothelial function after thyroidectomy. Br J Surg. 2007;94:1485-90. 22. Taddei S, Caraccio N, Virdis A, Dardano A, Versari D, Ghiadoni L, et al. Impaired endothelium-dependent vasodilatation in subclinical hypothyroidism: beneficial effect of levothyroxine therapy. The Journal of Clin Endoc Metab. 2003;88:3731-7. 23. Boekholdt MS, Titan SM, Wiersinga WM. Initial thyroid status and cardiovascular risk factors: The EPIC-Norfolk prospective population study. Clin Endocrinol (Oxf). 2009;21:6-9. 24. Cabral MD, Teixeira PFT, Silva NA, Morais FC, Soares DV, Salles E, et al. Normal flow-mediated vasodilation of the brachial artery and carotid artery intima-media thickness in subclinical hypothyroidism. Braz J Med Biol Res. 2009;42:426-33. 25. Almeida CA, Teixeira PF, Soares DV, Cabral MD, Costa SM, Salles E, et al. Intima-media thickness measurement as cardiovascular risk marker

CONCLUSION Our data suggest that thyroid hormone replacement therapy could prevent a decline in FMD with the continuation

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26. 27. 28.

29. 30. 31.

Effects of thyroxine replacement Cabral MD et al.

in patients with subclinical hypothyroidism. Arq Bras Endocrinol Metabol. 2007;51:472-7. Klein I, Ojaama K. Thyroid hormone and the cardiovascular system: from theory to practice. The Journal of Clin Endoc Metab. 1994; 78:1026-7. Kahaly GJ, Dillmann WH. Thyroid hormone action in the heart. Endocr Rev. 2005;26:704-28. Corretti MC, Anderson TJ, Benjamin EJ, Celermajer D, Charbonneau F, Creager MA, et al. Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery: a report of the International Brachial Artery Reactivity Task Force. J Am Coll Cardiol. 2002;39:257-65. Meirelles CM, Leite SP, Montenegro CA, Gomes PS. Reliability of brachial artery flow-mediated dilatation measurement using ultrasound. Arq Bras Cardiol. 2007;89:180-3. Garrido KU, Leite S, Montenegro C, Koch H, Soares A. Artery flowmediated dilatation measurement: study of endothelial function in women in menopause. Rev Bras Ecocardiog. 2008;21:22-6. Soares DV, Spina LD, Brasil RR, Silva EC, Lobo PM, Salles E, et al. Carotid artery intimamedia thickness and lipid profile in adults with

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growth hormone deficiency after longterm growth hormone replacement. Metabolism. 2005;54:321-9. Dietrich JB, Kuchler-Bopp S, Boutillier S. Expression of thyroid hormone receptors alpha and beta-1 messengers RNA in human endothelial cells. The T3 hormone stimulates the synthesis of the messenger RNA of the intercellular adhesion molecule-1. Cellular and Molecular Biology. 1997;43:1205-12. Baumgartner-Parzer SM, Wagner L, Reining G. Increase by triiodothyronine of endothelin-1, fibrobectin and von Willebrand factor in cultured endothelial cells. The Journal of Endocrinology. 1997;154: 231-9. Ojamara K, Klemperer JD, Klein I. Acute effects of thyroid hormone on vascular smooth muscle. Thyroid. 1996;6:505-12. Yoneda K, Takasa N, Hija S. Direct effects of thyroid hormones on rat coronary artery: nongenomic effects of triiodothyronine and thyroxine. Thyroid. 1998;8:609-13. Xiang G, He Y, Zhao L, Hou J, Yue L and Xiang H. Impairment of endothelium-dependent arterial dilation in Hashimotoâ&#x20AC;&#x2122;s thyroiditis patients with euthyroidism. Clin Endocrinol. 2006;64:698-72.

CLINICS 2011;66(8):1329-1333

DOI:10.1590/S1807-59322011000800004

CLINICAL SCIENCE

Color Doppler imaging of the superior ophthalmic vein in patients with Graves’ orbitopathy before and after treatment of congestive disease Ma´rio L. R. Monteiro,I Rodrigo B. S. Moritz,I He´lio Angotti-Neto,I Joseph E. BenabouII I

Division of Ophthalmology, Hospital das Clıńicas of the University of Saõ Paulo Medical School, Saõ Paulo, Brazil. II Department of Radiology, Hospital das Clıńicas of the University of Saõ Paulo Medical School, Saõ Paulo, Brazil.

OBJECTIVE: To compare superior ophthalmic vein blood flow parameters measured with color Doppler imaging in patients with congestive Graves’ orbitopathy before and after treatment and in normal controls. METHODS: Twenty-two orbits from 12 patients with Graves’ orbitopathy in the congestive stage and 32 orbits from 16 normal controls underwent color Doppler imaging studies. Color Doppler imaging was repeated after treatment in the group of patients with Graves’ orbitopathy, which included orbital decompression in 16 orbits and corticosteroids in six orbits. The findings for each group were compared. RESULTS: In the group of orbits with congestive disease, superior ophthalmic vein flow was detected in 17 orbits (anteroposteriorally in 13 and in the opposite direction in four) and was undetectable in five. After treatment, superior ophthalmic vein flow was detected and anteroposterior in 21 and undetected in one orbit. In normals, superior ophthalmic vein flow was detected anteroposterior in 29 orbits and undetectable in three orbits, indicating a significant difference between groups. There was also a significant difference between controls and congestive Graves’ orbits and between congestive orbits before and after treatment, but not between controls and patients after treatment. A comparison of superior ophthalmic vein flow parameters revealed a significant difference between the groups. The superior ophthalmic vein flow was significantly reduced in the congestive stage compared with the flow parameters following treatment and in the untreated controls. CONCLUSIONS: Superior ophthalmic vein flow was significantly reduced in the orbits affected with congestive Graves’ orbitopathy and returned to normal following treatment. Congestion appears to be a contributing pathogenic factor in the active inflammatory stage of Graves’ orbitopathy. KEYWORDS: Color Doppler imaging; Graves’ orbitopathy; Superior ophthalmic vein; Congestive orbitopathy; Endocrine exophthalmos. Monteiro MLR, Moritz RBS, Angotti-Neto H, Benabou JE. Color Doppler imaging of the superior ophthalmic vein in patients with Graves’ orbitopathy before and after treatment of congestive disease. Clinics. 2011;66(8):1329-1333. Received for publication on February 3, 2011; First review completed on March 15, 2011; Accepted for publication on April 20, 2011 E-mail: mlrmonteiro@terra.com.br Tel.: 55 11 3661-7582

of hyperthyroidism and, less frequently, in euthyroid or hypothyroid patients. GO is divided into a congestive stage and a fibrotic stage. In the congestive or inflammatory stage, autoimmunity is believed to play a key role in the process leading to inflammatory cellular infiltration of the muscles and transformation of fibroblasts into adipose tissue. The fibrotic stage is characterized by fibrosis in the orbital tissues with residual manifestations of the disease such as proptosis and strabismus.3,4 The pathogenesis of the acute inflammatory stage has been attributed to autoimmunity, and treatment of patients in this stage has traditionally been based on corticosteroids and radiotherapy. Superior orbital vein (SOV) congestion also plays an important role in the inflammatory stage, as demonstrated by computed tomography5-7 and color Doppler imaging (CDI) studies.8-11 In a recent study using

INTRODUCTION Graves’ orbitopathy (GO) is an autoimmune inflammatory process that affects the periorbital and orbital tissues, mainly the extraocular muscles. The enlargement of these muscles is responsible for most of the manifestations of the disease, including lid retraction, proptosis, extraocular muscle restriction, dysthyroid optic neuropathy, and congestive signs such as conjunctival hyperemia, chemosis, and lid swelling.1,2 GO occurs before, during, or after the onset

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least two examinations.3,13 Patients with active congestive disease had a clinical activity score of four or more points, whereas patients classified with fibrotic disease, not included in this study, had a clinical activity score equal to or lower than two. To avoid any possible doubt regarding disease activity in this study, patients with a score of three were also excluded. Eye movement restrictions in the field of action of the superior rectus muscle (elevation) due to inferior rectus restriction and of the lateral rectus muscle (abduction) due to medial rectus restriction were graded from 0 (no limitation) to 4 (absence of eye movement from primary position in the muscle’s field of action). Grade 1, 2 and 3 restrictions indicated 75%, 50%, and 25% excursion, respectively, from the primary position, either by elevation (restriction caused by the inferior rectus) or by abduction (restriction of the medial rectus). A combined restriction index ranging from 0 to 8 was calculated by adding the two scores. Orbits were also classified according to the presence or absence of dysthyroid optic neuropathy. The diagnostic criteria for dysthyroid optic neuropathy were diminished best corrected visual acuity not caused by changes in transparency, presence of a relative afferent pupillary defect and presence of visual field defect on automated or Goldmann perimetry. To qualify as an abnormal visual field on standard automated perimetry, at least three adjacent abnormal points at the p,0.05 level or two adjacent points with one abnormal at the p,0.01 level on the pattern deviation plot were required.14 On the Goldmann perimetry, visual field was considered abnormal in the presence of central or paracentral scotoma, localized depression of the isopters greater than 10 degrees or generalized constriction of the isopters.15,16 When visual acuity was normal and visual field abnormalities were present on standard automated perimetry, a repeat examination using automated or the Goldmann manual perimetry was performed in order to rule out false-positive results. All clinical assessments were performed within one week of the CDI study. After the ophthalmic examination, the patients were transferred to the Radiology Department for CDI with a 6-12 megahertz linear-array transducer coupled to a Logiq 9 Doppler scanner from General Electric (Santa Clara, California, USA). Maximum and minimum blood flow in the SOV was determined in both eyes with the patient resting on a bed with 30 degrees head elevation. During the examination, the patients were asked to remain still with both eyes closed and to fixate straight ahead. The transducer was gently placed over the closed eyes (right eye first), and care was taken to avoid applying pressure to the eye. Blood flow velocity was measured in the superior nasal part of the SOV, anterior to the point where it crosses the optic nerve. Velocity was measured in each vessel several times until obtaining at least two good readings, maintaining the angle between the sound beam and the blood flow direction less than 30 degrees. Only the highest values were used for statistical analysis. All CDI measurements were performed by the same experienced professional (JEB) who was blinded to the clinical status of the patients with GO. Maximum and minimum SOV blood flow velocities were registered, and the mean values and standard deviations were calculated. All of the patients underwent treatment of congestive disease. Sixteen orbits were treated with two-wall orbital decompression, and six were treated with corticosteroids. CDI studies were repeated from 6-36 months after treatment

CDI, we demonstrated that SOV flow was significantly reduced in orbits with congestive GO and with myogenic fibrotic GO, but not in orbits with fibrotic lipogenic orbitopathy.11 This finding was consistent with previous studies using CDI in patients with GO at different stages.8-10,12 Our previous study lends support to the notion that venous congestion plays a significant role in the pathogenesis of the active stage of the orbitopathy and suggests that patients can benefit from relief of SOV congestion by clinical or surgical treatment.11 To our knowledge, however, no previous study has evaluated CDI flow parameters in patients with congestive orbitopathy before and after clinical and/or surgical treatment of the disease. The purpose of this study was to evaluate SOV blood flow parameters using CDI in patients with active GO before and after treatment of the disease. We investigated the effect of treatments such as orbital decompression and/ or corticosteroids in CDI-measured SOV flow parameters.

METHODS AND MATERIALS This interventional, prospective, and cross-sectional study was conducted between October 2005 and September 2010. The study followed the principles of the Declaration of Helsinki. Approval from the Institutional Review Board Ethics Committee was obtained, and all of the participants gave their informed consent. A total of 12 patients (seven men, five women) with congestive GO were examined. The mean age of the patients was 53.2 (SD 9.5) years. The diagnosis of GO was established according to previously published criteria.1 All of the patients had Graves’ disease but became euthyroid under treatment. Sixteen normal subjects (four men, twelve women), with a mean age of 48.1 (SD 10.9) years, were included in the study as a control group. The normal subjects were defined as healthy euthyroid volunteers without ocular diseases. The patients underwent a complete ophthalmic examination that included best corrected visual acuity, applanation tonometry, pupillary reactions, extraocular motility evaluation, slit lamp examination, evaluation of eyelid and soft tissue inflammation, measurement of the lid fissure, Hertel exophthalmometry, fundoscopy, and visual field evaluation with standard automated perimetry using the 24-2 SITAStandard strategy (Humphrey Field Analyzer, Carl-Zeiss Meditec, Dublin, CA). Goldmann manual perimetry was also used when necessary to confirm optic nerve involvement. Both orbits of the patients were scanned with a 16-slice multidetector computed tomography scanner (Brilliance 16; Philips Medical Systems, Nederland B.V., the Netherlands). GO was classified as active (congestive) or fibrotic according to a 10-item clinical activity score based on four of five well-known classical signs of inflammation (pain, redness, swelling, and impaired function). The clinical activity score included orbital pain, redness of the eyelid or conjunctiva, swelling of the conjunctiva (chemosis), caruncle, or eyelid and impaired function represented by decreased visual function or decreased eye movement.3 One point was given per eye for each item present. The evaluated items corresponded to symptoms (orbital ache and gaze-evoked pain), signs (conjunctival redness, eyelid erythema, eyelid edema, chemosis, and swelling of the plica or caruncle) and indications of progressive disease in at

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between the GO orbits after treatment and the control orbits (p = 0.638). Table 2 compares SOV flow values for the two GO groups and for normal controls. The results of the unpaired t-test revealed a significant difference between GO patients before treatment and controls with regard to the maximum (p = 0.009), minimum (p = 0.013), and differential (p = 0.005) SOV flow. There was no significant difference between the GO patients after treatment and the controls with regard to the maximum, minimum, and differential flow (p = 0.703, 0.599 and 0.969, respectively). The paired t-test revealed a significant difference in the maximum (p = 0.007), minimum (p = 0.012) and differential (p = 0.006) SOV flow in GO patients before and after treatment (Table 2).

of congestive disease. After treatment, GO was inactive in all orbits according to the clinical activity score classification (a score of two or less).

Statistical analysis The descriptive statistics included mean values ¡ SD for normally distributed variables after the analysis of histograms and the Shapiro-Wilk test confirmed the normality assumption. We used an unpaired t-test for comparisons of the three groups (normal controls, active GO before treatment and GO after treatment) and a paired t-test for comparison of active GO before and after treatment. Each group of orbits was initially classified according to whether the flow in the SOV was anteroposterior, absent (not detected), or posteroanterior (reverse). The observed proportions were compared using Fisher’s exact test. For further statistical analyses, the anteroposterior flow was expressed in positive numbers, undetected flow was assigned a value of zero, and the posteroanterior flow was expressed in negative numbers. A p value of less than .05 was considered statistically significant.

DISCUSSION Autoimmunity is likely the primary mechanism involved in the pathogenesis of Graves’ orbitopathy. It is, however, suspected that orbital venous blood flow congestion contributes to the development of clinical signs and symptoms (e.g., proptosis, muscle restriction, periorbital swelling, and chemosis) during the active stage of the disease,17 as suggested by a number of clinical and experimental findings. Previous studies have reported prompt improvement in congestive signs after orbital decompression, suggesting that venous congestion is an important factor in the pathogenesis of the disease.18 In another study, venous drainage impairment and subsequent resolution was visible on fluorescein angiographs of the optic discs from patients with dysthyroid optic neuropathy.19 Hudson et al.6 suggested that the orbital SOV enlargement observed on computed tomography scans of patients with dysthyroid optic neuropathy may be a contributing factor in the development of optic nerve impairment. A previous study indicated that experimentally induced orbital venous stasis can closely mimic many of the clinical changes that occur in GO.17 CDI allows simultaneous imaging of the anatomic structures by B-mode ultrasonography with superimposed color-coded vascular flow. The current study confirmed a significant difference in CDI parameters between patients with GO in the active stage and controls or orbits with GO following treatment. Reverse and absent blood flow were observed in four and five out of 22 orbits with active GO, respectively. Flow may be undetectable in eyes of normal controls for several reasons, including insufficient sensitivity of the device for measuring very small venous flow rates, or anatomic flow variations. In abnormal orbits, absent flow is most often related to the impairment of venous return at the orbital apex, leading to a venous flow of zero or close to

RESULTS A total of 76 CDI studies were performed: 22 orbits from 12 patients (seven men and five women, mean age ¡ SD: 53.1¡9.5 years) with active GO (clinical activity score index of four or higher), the same 22 orbits after treatment (activity score index of two or less), and 32 orbits from 16 control subjects (twelve women and four men; mean age ¡ SD: 48.3¡10.9). There was no significant age difference between the patients and controls (p = 0.231). Before treatment, the exophthalmometry measurements ranged from 22 to 28 mm (mean ¡ SD: 25.52¡1.96 mm) in GO patients, and the restriction index ranged from 0 to 8 (mean ¡ SD: 5.0¡1.93). Before treatment, 21 orbits of patients with GO were categorized as having predominantly myogenic disease (with a restriction grade of 3-8). One orbit was categorized as having predominantly lipogenic disease (with muscle restriction graded as 0). In addition, 15 orbits presented with dysthyroid optic neuropathy before treatment. In the normal controls, SOV was detected in 29 orbits and was undetectable in three. Reverse blood flow was not observed. In orbits with active congestive disease, SOV flow was absent in five, present in 13 and reversed in four orbits before treatment. After treatment, SOV flow in the same 22 orbits was anteroposterior (n = 21) or undetectable (n = 1) (Table 1). A significant difference was found between GO orbits before treatment and the control orbits (p = 0.007, Fisher’s exact test). No significant difference was found

Table 1 - Detection and direction of blood flow in the superior ophthalmic vein using color Doppler imaging in patients with Graves’ orbitopathy and control subjects. Superior orbital vein blood flow Anteroposterior Orbitopathy before treatment * Orbitopathy after treatment { Controls *{ Total

13 21 29 63

Posteroanterior (reverse)

Not detected

4 (18.2%) 0 (0.0%) 0 (0.0%) 4 (5.3%)

5 (22.7%) 1 (4.5%) 3 (9.4%) 9 (11.8%)

(59.1%) (95.4%) (90.6%) (82.9%)

Fisher’s exact test (p = 0.007, controls compared with orbitopathy before treatment). Fisher’s exact test (p = 0.638, controls compared with orbitopathy after treatment).

{

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Number of orbits 22 22 32 76

(100%) (100%) (100%) (100%)

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CLINICS 2011;66(8):1329-1333

Table 2 - Comparison of blood flow parameters in the superior ophthalmic vein (SOV) in patients with Graves’ orbitopathy and controls. Mean (range) velocity (cm/s)

Congestive GO before treatment (22 orbits) GO after treatment (22 orbits) Normals (32 orbits)

SOV maximum velocity *

SOV minimum velocity *

Differential flow velocity *

3.07*{ (-13.16 – 9.55) 7.80 * (0 – 11.96) 7.49 { (0 – 11.68)

2.11*{ (-10.0 – 7.26) 5.31 * (0 – 8.75) 5.02 { (0 – 8.91)

0.96*{ (-4.22 – 6.66) 2.48 * (0 – 4.43) 2.47 { (0 – 5.48)

* {

p,0.05, paired t-test, comparing GO orbits before and after treatment. p,0.05, unpaired t-test, active GO compared with controls.

zero. Although flow may occasionally be absent in normal subjects, we believe the absence of flow is most likely an indication of venous congestion. In our study, a direct comparison revealed the absence of flow in five of 22 congestive GO orbits but only in three out of 32 normal controls and one of 22 GO orbits after treatment. Conversely, reverse blood flow has not been reported in normal subjects20 and may indicate more severely impaired orbital venous drainage. While reverse blood flow occurred in four of 22 active GO orbits, it did not occur in any of the 32 control orbits or in the 22 GO orbits following treatment. A comparative analysis of our groups also indicated that SOV was significantly reduced in orbits with congestive GO compared with control orbits or the GO orbits following treatment, suggesting that venous congestion is most likely a contributing factor to the pathogenesis of this condition. Our findings of reduced SOV flow in patients with active GO are consistent with results from several previous studies. Nakase et al.8 found reversed SOV flow in 15% of 39 orbits with GO, but not in controls (n = 22). Reversed SOV flow was present in 36% of 14 orbits with GO and apical crowding. Nakase et al. suggested that these findings were a strong indication of severe venous stasis in the orbits, possibly related to the development of dysthyroid optic neuropathy. No data were provided, however, regarding the disease activity status of each orbit. Benning et al.12 found a significantly smaller maximum SOV blood flow velocity in patients with GO than in normal subjects and a slight correlation between reduced venous outflow in the SOV and disease severity. Somer et al.10 used CDI in 48 orbits of 24 patients with GO and in 20 orbits of 10 healthy volunteers. The authors found the mean venous blood flow velocity to be significantly decreased in patients with GO. No SOV flow, however, was detected in 18 orbits with GO. Alp et al.9 performed CDI on 111 patients with Graves’ disease and 46 healthy controls. Patients with Graves’ disease were divided into two groups: 69 with GO and 42 with no orbitopathy. Reverse blood flow in the SOV was observed in 13% of patients with GO. The authors found blood flow velocities in the ophthalmic artery and the central retinal artery to be significantly higher in patients with GO than in orbitopathy-free patients with Graves’ disease or healthy controls. Blood flow was significantly reduced in the SOV. The study included only patients who were not receiving local or systemic treatment for GO, and no disease activity criteria were provided. Despite reporting significant changes in orbital blood flow in patients with GO, none of the aforementioned studies were designed to directly compare findings for

different forms of GO. Only a few studies have attempted to correlate disease activity, based on the clinical activity score,13 with CDI blood flow parameters. Yanik et al.21 studied 118 patients with Graves’ disease, 25 without orbitopathy and 93 with activity-scored GO.13 The authors found a negative correlation between orbital blood flow parameters and their clinical activity scores.21 They excluded patients requiring urgent orbital decompression or receiving systemic medication, thereby likely excluding subjects with more severe forms of orbitopathy. The study did not separate patients in predominantly myogenic or lipogenic forms of the disease. In a recent study, we evaluated patients with GO after clearly distinguishing between active and inactive as well as between myogenic (restrictive) and lipogenic (non-restrictive) GO.14 Our findings indicated that most patients with congestive GO had reduced or reversed maximum SOV flow. Patients with fibrotic GO in the myogenic form also displayed a significant reduction in SOV flow compared with normal controls. Patients with fibrotic lipogenic disease did not differ significantly from the controls. In the current study, we compared the same group of active GO patients before and after treatment. The results indicated dramatic differences in the SOV flow parameters between the active stage of the disease and post-treatment GO or normal controls, indicating that venous congestion is an important element in the active stage of the disease. The CDI data provided by this study and by previous studies may be useful for the management of GO. Treatment of GO in the acute stage is traditionally immunosuppressive (using corticosteroids or immunosuppressants), but congestive signs sometimes remain despite adequate treatment and may require orbital decompression. Persistently reduced or reversed SOV blood flow despite adequate treatment may be an indication for decompressive surgery in select patients. The current data appear to confirm this assumption, as most of our patients were in fact treated with orbital decompression. In conclusion, the fact that SOV flow was significantly reduced in the congestive stage of GO and normalized after treatment of active orbitopathy suggests that venous congestion plays a significant role in the pathogenesis of the disease. Our data lends support to the notion that clinical improvement with treatment is due to the control of not only the autoimmune process but also orbital venous congestion. On the basis of these findings, we believe that if diminished SOV flow persists despite adequate clinical treatment, orbital decompression should be considered to improve congestive signs. Further studies with more

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Color Doppler imaging in patients with Graves’ orbitopathy Monteiro MLR et al. 9. Alp MN, Ozgen A, Can I, Cakar P, Gunalp I. Colour Doppler imaging of the orbital vasculature in Graves’ disease with computed tomographic correlation. Br J Ophthalmol. 2000;84:1027-30, doi: 10.1136/bjo.84.9.1027. 10. Somer D, Ozkan SB, Ozdemir H, Atilla S, Soylev MF, Duman S. Colour Doppler imaging of superior ophthalmic vein in thyroid-associated eye disease. Jpn J Ophthalmol. 2002;46:341-5, doi: 10.1016/S00215155(02)00485-9. 11. Monteiro ML, Angotti-Neto H, Benabou JE, Betinjane AJ. Color Doppler imaging of the superior ophthalmic vein in different clinical forms of Graves’ orbitopathy. Jpn J Ophthalmol. 2008;52:483-8, doi: 10.1007/ s10384-008-0594-y. 12. Benning H, Lieb W, Kahaly G, Grehn F. [Color duplex ultrasound findings in patients with endocrine orbitopathy]. Ophthalmologe. 1994;91:20-5. 13. Mourits MP, Prummel MF, Wiersinga WM, Koornneef L. Clinical activity score as a guide in the management of patients with Graves’ ophthalmopathy. Clin Endocrinol (Oxf). 1997;47:9-14, doi: 10.1046/j. 1365-2265.1997.2331047.x. 14. Monteiro ML, Portes AL, Moura FC, Regensteiner DB. Using frequencydoubling perimetry to detect optic neuropathy in patients with Graves’ orbitopathy. Jpn J Ophthalmol. 2008;52:475-82, doi: 10.1007/s10384-008-0579-x. 15. Neigel JM, Rootman J, Belkin RI, Nugent RA, Drance SM, Beattie CW, et al. Dysthyroid optic neuropathy. The crowded orbital apex syndrome. Ophthalmology. 1988;95:1515-21. 16. Panzo GJ, Tomsak RL. A retrospective review of 26 cases of dysthyroid optic neuropathy. Am J Ophthalmol. 1983;96:190-4. 17. Saber E, McDonnell J, Zimmermann KM, Yugar JE, Feldon SE. Extraocular muscle changes in experimental orbital venous stasis: some similarities to Graves’ orbitopathy. Graefes Arch Clin Exp Ophthalmol. 1996;234:331-6, doi: 10.1007/BF00220709. 18. Trobe JD, Glaser JS, Laflamme P. Dysthyroid optic neuropathy. Clinical profile and rationale for management. Arch Ophthalmol. 1978;96:1199209. 19. Yoshikawa K, Higashide T, Inoue T, Inoue Y. Fluorescein angiographic findings in optic discs with dysthyroid optic neuropathy. Orbit. 1991;10:89-96, doi: 10.3109/01676839109023088. 20. Erickson SJ, Hendrix LE, Massaro BM, Harris GJ, Lewandowski MF, Foley WD, et al. Color Doppler flow imaging of the normal and abnormal orbit. Radiology. 1989;173:511-6. 21. Yanik B, Conkbayir I, Acaroglu G, Hekimoglu B. Graves’ ophthalmopathy: comparison of the Doppler sonography parameters with the clinical activity score. J Clin Ultrasound. 2005;33:375-80, doi: 10.1002/jcu.20154.

patients are necessary confirm these findings and to better evaluate potential differences in congestion relief between clinical and surgical treatment of the congestive stage of GO.

ACKNOWLEDGMENTS This work was supported by a grant from Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico, CNPq (No 309709/2007-5), Brası´lia, Brazil. Study registered on ClinicalTrial.gov number: NCT00697528

REFERENCES 1. Bartley GB, Gorman CA. Diagnostic criteria for Graves’ ophthalmopathy. Am J Ophthalmol. 1995;119:792-5. 2. Bartley GB, Fatourechi V, Kadrmas EF, Jacobsen SJ, Ilstrup DM, Garrity JA, et al. Clinical features of Graves’ ophthalmopathy in an incidence cohort. Am J Ophthalmol. 1996;121:284-90. 3. Mourits MP, Koornneef L, Wiersinga WM, Prummel MF, Berghout A, van der Gaag R. Clinical criteria for the assessment of disease activity in Graves’ ophthalmopathy: a novel approach. Br J Ophthalmol. 1989;73:639-44, doi: 10.1136/bjo.73.8.639. 4. Bartalena L, Pinchera A, Marcocci C. Management of Graves’ ophthalmopathy: reality and perspectives. Endocr Rev. 2000;21:168-99, doi: 10. 1210/er.21.2.168. 5. Monteiro ML, Goncalves AC, Silva CT, Moura JP, Ribeiro CS, Gebrim EM. Diagnostic ability of Barrett’s index to detect dysthyroid optic neuropathy using multidetector computed tomography. Clinics. 2008;63:301-6, doi: 10.1590/S1807-59322008000300003. 6. Hudson HL, Levin L, Feldon SE. Graves exophthalmos unrelated to extraocular muscle enlargement. Superior rectus muscle inflammation may induce venous obstruction. Ophthalmology. 1991;98:1495-9. 7. Nugent RA, Belkin RI, Neigel JM, Rootman J, Robertson WD, Spinelli J, et al. Graves orbitopathy: correlation of CT and clinical findings. Radiology. 1990;177:675-82. 8. Nakase Y, Osanai T, Yoshikawa K, Inoue Y. Color Doppler imaging of orbital venous flow in dysthyroid optic neuropathy. Jpn J Ophthalmol. 1994;38:80-6.

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DOI:10.1590/S1807-59322011000800010

CLINICAL SCIENCE

Subsequent reproductive outcome in women who have experienced a potentially life-threatening condition or a maternal near-miss during pregnancy Rodrigo S. Camargo,I Rodolfo C. Pacagnella,I Jose´ G. Cecatti,I,II Mary A. Parpinelli,I Joa˜o P. Souza,I Maria H. SousaII I Department of Obstetrics and Gynecology, School of Medical Sciences, University of Campinas - UNICAMP, Campinas, SP, Brazil. II Campinas Center for Studies in Reproductive Health (CEMICAMP), Campinas, SP, Brazil.

OBJECTIVE: To evaluate the long-term reproductive consequences that affect women who have experienced potentially life-threatening or life-threatening (near-miss) maternal complications. INTRODUCTION: Although advances have been made in reducing maternal death, few studies have investigated the long-term repercussions of significant events such as severe maternal morbidity and maternal near-misses. These repercussions may be long-lasting and negatively affect quality of life. METHODS: A total of 382 women who had experienced a potentially life-threatening pregnancy-related condition within the last five years were analyzed in this retrospective cohort study. A control group of 188 women who gave birth without complications was also included. Trained interviewers contacted the subjects by telephone and completed a pre-coded, structured questionnaire on reproductive health. Data were analyzed using odds ratios adjusted for age. The main outcome measures were occurrence and outcome of subsequent pregnancies. RESULTS: The estimated risk of becoming infertile as a result of tubal ligation or hysterectomy was 3.5 times higher in women who experienced a maternal near-miss or severe maternal morbidity during the index pregnancy as compared to controls. Likewise, the risk of complications in subsequent pregnancies was five times greater in women who had experienced severe maternal morbidity. However, no differences were found in the occurrence or number of subsequent pregnancies or perinatal outcome. CONCLUSION: The occurrence of a life-threatening or potentially life-threatening maternal condition reduces future reproductive potential and increases the risk of complications in subsequent pregnancies. KEYWORDS: Maternal near-miss; Maternal morbidity; Reproductive Outcome; Maternal death; Subsequent pregnancy. Camargo RS, Pacagnella RC, Cecatti JG, Parpinelli MA, Souza JP, Sousa MH. Subsequent reproductive outcome in women who have experienced a potentially life-threatening condition or a maternal near-miss during pregnancy. Clinics. 2011;66(8):1367-1372. Received for publication on March 18, 2011; Published review completed on April 18, 2011; Accepted for publication on May 2, 2011 E-mail: cecatti@unicamp.br Tel.: 55 19 3521-9482

Nevertheless, the number of cases of severe maternal morbidity remains substantial, even in regions in which human development is progressing. Depending on the management strategy, pregnancy complications may pass through several stages and advance from being potentially life-threatening to actually life-threatening. These complications are classified retrospectively, according to the outcome, as maternal near-misses or maternal death.3-5 For this reason, rates of severe maternal morbidity and near-misses are now being used together with mortality as important indicators of maternal health.6 Women who experience a near-miss have survived a severe clinical condition leading to organ dysfunction or failure (e.g., eclampsia, sepsis) during pregnancy, childbirth or the postpartum period.7,8 Recently, the prevalence of maternal near-misses was estimated at around 21 cases per 1,000 liveborn infants or a total of about 70,000 cases

INTRODUCTION Although advances have been made towards achieving the fifth Millennium Development Goal, which advocates reducing the maternal mortality ratio by 75% by 2015, more than 300,000 avoidable maternal deaths still occur annually worldwide, especially in less developed nations.1,2 In developed countries, maternal mortality is a rare event because social development, better care in cases of severe complications, and family planning all contribute to its reduction.1

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annually in Brazil.9 In the Netherlands, a nationwide survey using different criteria for case definitions estimated a lower rate of severe maternal morbidity of 7.1 per 1,000 births.10 However, little work has been done on the long-term repercussions of significant events such as severe maternal morbidity and maternal near-misses. The possibility that both the health and social condition of women who suffer complications in pregnancy may deteriorate over time has been increasingly recognized.11,12 Nevertheless, although the risk of death remains high following childbirth in women who suffered a life-threatening condition during pregnancy,13-15 in practice, the repercussions of pregnancy complications have not been thoroughly explored. These repercussions may be long-lasting, negatively affect quality of life and/or have adverse effects on the women and their children.14,16,17 However, few studies have been conducted on postpartum maternal health, especially in middle income countries and particularly in women who almost died from pregnancy-related complications.15,17-20 With respect to the subsequent reproductive health of these women, even less information is available. In 2002, Murphy & Charlett evaluated reproductive outcomes in pregnancies subsequent to an episode of maternal nearmiss.21 In 50 women who had required intensive care during the index pregnancy, just 32 women conserved their potential fertility. Among them, 16 reported a subsequent pregnancy with live birth, suggesting that success in terms of subsequent reproductive outcome signifies a good recovery following adverse events. However, the high rate of lost reproductive potential reflects the severity of the situation and probable physical and emotional consequences.21 In such cases and especially when perinatal death occurs, women frequently experience emotions such as frustration and alienation, described as maternal near-miss syndrome.18 A more comprehensive understanding of the long-term reproductive health performance of this group of women will help identify the characteristics and consequences that could assist providers in determining the optimal management of these cases. Therefore, the objective of the present analysis was to evaluate the subsequent reproductive outcomes of women who suffered a potentially lifethreatening or life-threatening condition during a pregnancy that had occurred up to five years prior to the interview.

pregnancy, childbirth, and/or the postpartum period. The questionnaire was pre-tested in an independent sample and adapted accordingly. Eligible women were identified retrospectively in accordance with a list supplied by the hospital’s data system. Following approval by the institutional review board, we obtained the information required to trace and contact all women admitted to the intensive care unit (ICU) within the previous five-year period (20022007) as well as low-risk women admitted to the rooming-in ward during the same period. Detailed information on this questionnaire, its validation and the selection of women for the study was published previously.22 Sample size estimates were performed to validate the questionnaire. The current analysis therefore encompassed the women who experienced pregnancy complications during a five-year period and were able to be traced. For the purpose of selection, cases were defined as all of the women admitted to the institution’s ICU between October 2002 and September 2007 and who i) suffered at least one severe maternal complication such as severe preeclampsia, eclampsia, hemorrhage, infection or jaundice; ii) underwent any procedure for the treatment of a maternal complication, including hysterectomy, blood transfusion, laparotomy, inter-hospital transfer or mechanical ventilation; or iii) were hospitalized for longer than a week. Controls were defined as women who did not experience any severe complication and were not submitted to any of the aforementioned procedures. The control group was selected from the women who were hospitalized in the rooming-in ward following childbirth in the same time period as the cases. We arbitrarily decided to enroll two cases for each control; therefore, the selection process established that the first woman to be discharged from the rooming-in ward on the day that the second case was discharged would be selected for inclusion in the study. Data on the medical condition of cases and controls were obtained from the patients’ medical charts and recorded on forms specifically designed for this purpose. Because the women were interviewed at different periods post-delivery and because contact telephone numbers (a landline, mobile, or telephone number at which a message could be left) were registered in the hospital admission records and thus available for all of the eligible women, the interviews were conducted using the CATI system (Computer Assisted Telephone Interview). Between July and October 2007, the eligible women were contacted by telephone to obtain their verbal consent and schedule the interview. Three specially trained interviewers contacted the women by telephone under the supervision of a research coordinator experienced in telephone interviews related to reproductive health. The interviewers were unaware of the group to which the women belonged (maternal morbidity or control). The women who could not be traced through the registered telephone numbers were excluded from the study after five unsuccessful attempts. Successful contacts continued until a complete interview was obtained or until the occurrence of an unsuccessful later contact. To maintain the desired proportion of cases and controls, a mixed list of cases and controls containing only the name, registration number and contact information of eligible women was made available to the interviewers on a weekly basis. After the interviewer had read the informed consent form and the woman had verbally agreed to participate in the

MATERIALS AND METHODS This study was performed at the Campinas Center for Studies in Reproductive Health (CEMICAMP) and at the Center for Women’s Integrated Healthcare (CAISM) at the University of Campinas (UNICAMP), a teaching hospital that offers tertiary care to women in a catchment area of approximately three million inhabitants in the state of Sa˜o Paulo, Brazil. Women who survived a potentially lifethreatening or life-threatening condition as recently defined by the World Health Organization7,8 (severe maternal morbidity) between 2002 and 2007 participated in the study, together with a random control sample of women who experienced childbirth with no complications during the same period. A structured, pre-coded questionnaire on reproductive health was written in Brazilian Portuguese and focused on the occurrence of episodes of maternal morbidity during

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study, the interviews were recorded, and the data were concomitantly entered into a database within the SPSS program by the interviewers, who were seated in front of a computer with access to the database and were using a headset while they conducted the interview. As a quality control procedure, 5% of the data collected were reviewed by the research coordinator, who compared the forms to the voice recordings, provided feedback to each interviewer with respect to errors and later checked that all corrections to the database had been made. In addition, the coordinator conferred with the interviewers whenever the latter considered that a specific question required a more indepth approach. Quality control was performed concomitantly with the interviews throughout the entire duration of the study. The analytical approach corresponded to a retrospective cohort with a limited, fixed size, control group. Pearson’s Chi-square test was used to compare the women’s characteristics between groups. To compare the risks of certain reproductive events that occurred after the index pregnancy, odds ratios (ORs), adjusted for maternal age, were calculated using multiple logistic regression analysis, together with their respective 95% confidence intervals. SPSS (v. 11.5) and Epi Info (v. 6.04d) software were used for statistical analysis.

Table 1 - Characteristics of the women who experienced severe maternal morbidity (SMM) and the controls.

RESULTS

difficulties related to the woman’s lack of availability (all cases) and 14 interviews could not be conducted because the women had died following the hospital discharge. Four cases were excluded because the women failed to supply information on any subsequent tubal ligation and/or pregnancies, thus leaving 382 cases for the present analysis. All recorded deaths occurred in the group of women who suffered an episode of severe maternal morbidity and were recorded at different intervals following the episode (0-5 years). The number of deaths corresponded to a mortality rate of 33 per 1000 (14/419). The causes of death were reported as cancer (3), infection with respiratory complications (3), cardiac complications (2), renal failure (1), multiple organ failure (1), and undetermined cause (1); the other

Characteristic Age at delivery (years) #19 20 – 24 25 – 29 30 – 34 $35 Years of schooling Up to 4th grade 5th to 8th grade High school University Length of interval between delivery and interview (months) #12 13-24 25-36 .36 Total

SMM* (%)

Control (%)

6.5 18.3 23.6 23.8 27.7

4.3 28.2 31.4 19.1 17.0

14.9 32.5 48.7 3.9

10.1 34.0 48.4 7.4

p-value

0.002

0.147

0.602

26.7 29.8 16.0 27.5 (382)

23.9 26.6 18.6 30.9 (188)

*Cases of severe maternal morbidity with at least one related diagnosis and/or procedure. & Pearson’s Chi-square test.

Between 2002 and 2007, 655 women were admitted to the obstetric ICU and survived a pregnancy-related complication. In the same period, 12,198 women were admitted to the rooming-in ward. From this latter group, 343 women were selected for telephone interviews. Of the 998 women considered eligible for this study, 602 women were successfully contacted, and 574 were interviewed, including 188 controls and 386 cases in which an episode of severe maternal morbidity had occurred. Twenty-eight women were contacted but not interviewed: seven women refused to answer the questionnaire (three controls and four cases), seven interviews were not carried out because of practical

Table 2 - Reproductive outcomes following the index pregnancy in patients who experienced severe maternal morbidity (SMM) and controls. Group * Reproductive outcomes Tubal ligation or hysterectomy during index pregnancy Yes No Rate (/1000 women months) Subsequent pregnancy Yes No & Rate (/1000 women months) Number of subsequent pregnancies 0 1 2 Rate (/1000 women months) Total #

SMM

Control

ORadj (95% CI)@

104 (27.2) 278 (72.8) 11.1

16 (8.5) 172 (91.5) 3.3

3.41 (1.89-6.16)

21 (7.5) 257 (92.5) 2.2

16 (9.3) 156 (90.7) 3.3

0.91 (0.68-1.22)

361 (94.5) 20 (5.2) 1 (0.3)

172 (91.5) 15 (8.0) 1 (0.5)

1.44 (0.73-2.85)

(382)

(188)

*Cases of severe maternal morbidity with at least one related diagnosis and/or procedure. & Excluded cases with tubal ligation or hysterectomy. @ Controlled for age. #Four women failed to provide information on tubal ligation or hysterectomy.

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Table 3 describes the outcome of the pregnancy following the index pregnancy. No statistically significant differences were found between the groups with respect to the outcome of the pregnancy, with the majority delivering a live newborn. Furthermore, there was no difference in the period of time elapsing before the subsequent pregnancy or the interview. However, the risk of developing some form of complication was more than five times higher in the group of women who had suffered an episode of severe morbidity in the index pregnancy than in the control women. Of those women who had complications during a subsequent pregnancy, 12 women were from the group of women who had experienced morbidity, and four women were from the control group (table 4). Although these numbers are low, there seems to be a trend towards a greater prevalence of complications during subsequent pregnancies in women who had experienced an episode of severe maternal morbidity in the index pregnancy.

Table 3 - Outcomes of pregnancies subsequent to the index pregnancy and the occurrence of severe maternal morbidity (SMM). Group Outcome of subsequent pregnancy

SMM

Pregnancy outcome Delivery of a liveborn infant 16 (a) Delivery of a stillborn infant 1 Abortion 4 Pregnancy-related complication Yes 12 No 9 Interval between pregnancies (months)* ,24 10 $24 9 Total (21)

Control

ORadj (95% CI)

1.21 (0.25-5.88)

1 2

4 12

5.15 (1.12-23.64)

6 8 (16)

1.59 (0.38-6.64)

DISCUSSION

@Controlled for age (a) One woman in the case group and one woman in the control group had two pregnancies each. *Data missing for two women in the case group and two women in the control group.

This study suggests that a womanâ&#x20AC;&#x2122;s reproductive potential decreases following an episode of severe maternal morbidity. Furthermore, the analysis indicates that such women experience a higher risk of developing additional episodes of severe morbidity in subsequent pregnancies, although this conclusion was limited by the relatively small sample size. The marked loss of fertility as a surgical consequence of childbirth complications (tubal ligation or hysterectomy) found in women who experienced an episode of severe maternal morbidity was expected according to previous reports. Evaluating a cohort with no comparison group, Murphy & Charlett21 reported a high percentage of fertility loss in women who were admitted to an ICU during pregnancy, childbirth, or the postpartum period. Potential fertility was preserved in 64% of these women, 62% of whom had subsequent pregnancies resulting in liveborn infants. However, the authors did not investigate the prevalence of complications in the subsequent pregnancy. In the present study, 72.8% of the women who had an episode of severe maternal morbidity maintained their

three cases of maternal death occurred during the index pregnancy while the women were giving birth in another institution. The only statistically significant difference between the two groups with respect to patient characteristics was maternal age: the women who had experienced an episode of severe maternal morbidity were significantly older than the women who did not (Table 1). As shown in Table 2, the number of women who became sterile as a consequence of a tubal ligation or hysterectomy was 3.5 times greater in the group of women who had experienced morbidity compared to the control group. No statistically significant differences were found between the two groups in the number or the occurrence of subsequent pregnancies. Only one woman in each group had more than one pregnancy after the index pregnancy.

Table 4 - Absolute frequencies of complications and procedures in the pregnancy following the index pregnancy associated with severe maternal morbidity (SMM). Group Complication Hypertension: the woman had seizures with increased blood pressure, edema and/or visual symptoms during pregnancy, delivery or the postpartum period. Hemorrhage: the woman had severe bleeding during pregnancy or increased bleeding during childbirth or the postpartum period. Infection: the woman had high fever during childbirth or the postpartum period, with chills and/or a foul-smelling vaginal discharge. Jaundice: the woman became jaundiced during childbirth or the postpartum period. Procedures Admission to an intensive care unit Inter-hospital transfer Laparotomy Length of hospital stay .1 week postpartum Blood transfusion Total * *Complications and procedures are not mutually exclusive.

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SMM

Control

2 2 1 3 1 (12)

2 (4)

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findings that would permit us to categorically affirm this hypothesis, studies conducted with larger sample sizes may show significant differences. The present study suggests that there is a reduction in future reproductive capacity associated with severe maternal morbidity and a greater risk of further episodes of severe morbidity in subsequent pregnancies. Our group is committed to investigating the reproductive consequences of episodes of severe maternal morbidity and hopes to be able to report additional findings in the near future. An increasing understanding of the adverse effects of an episode of severe maternal morbidity, particularly on women’s reproductive health, should open up new research perspectives that may positively affect the practical management of severe cases and prevent women who suffer a maternal morbidity condition from losing their reproductive potential.

reproductive capacity (compared to 91.5% in the control group). Nevertheless, only 7.5% of these women became pregnant again during the five-year period of the study (compared to 9.3% of the controls). There was no statistically significant difference between the groups with respect to the occurrence of subsequent pregnancies. A similar finding was reported by other authors;17 here, while women with a history of near-miss appeared less likely to become pregnant again compared to women in the control group, this finding did not prove to be statistically significant. The small number of women who had another pregnancy following the index pregnancy may represent an argument in support of halting a woman’s reproductive capacity following a morbidity event. Furthermore, this study revealed that women with a history of severe maternal morbidity experience a greater occurrence of complications and need for procedures in subsequent pregnancies. This is a novel finding that, although intuitively expected, had not yet been formally ascertained. As reported by some investigators,14,17-19,23 the repercussions of an event requiring ICU admission do not end with the pregnancy. In this respect, the present study provides further information on how such events may affect the future reproductive health of affected women. Although not evaluated in this study, loss of reproductive capacity as a consequence of hysterectomy or tubal ligation may represent a negative health-related emotional factor.18 In fact, undesired tubal ligations or hysterectomies should be treated as sequelae and may contribute to an even more negative situation when associated with the loss of a child, as reported in another study.17 Nonetheless, studies with larger sample sizes, specifically designed for this purpose, should be conducted to evaluate this possible additional component of maternal near-miss syndrome. Although not a formal part of the present study, the finding that 14 women who suffered an episode of severe maternal morbidity in the institution died at some point between this episode and the attempt to contact them by telephone is very important. Other authors have also reported a higher postpartum mortality rate in women with a history of a near-miss. A prospective cohort study showed that both women who had severe obstetrical complications and their babies were significantly more likely to die within one year of hospital discharge compared to the respective controls.19 In another study, a mortality rate of around 10% was found in women during the follow-up period (18 months to 12 years) after an episode of pregnancy-related hospitalization in an ICU.21 These data suggest that risk, both in terms of reproduction and general health, increases substantially after an episode of severe maternal morbidity. The implications of these findings may be substantial and are certainly important in terms of defining the specialized follow-up care required by these women during their future reproductive lives from reproductive health providers and other specialists.

ACKNOWLEDGMENTS We gratefully acknowledge the financial support of the Foundation of Support to Research of the State of Sao Paulo (FAPESP), Brazil, grant 2007/00290-8. Another grant of FAPESP (2011/09701-6) was responsible for sponsoring the current publication.

REFERENCES 1. World Health Organization. Maternal mortality in 2005: estimates developed by WHO, UNICEF, and UNFPA and the World Bank. Geneva: World Health Organization. 2007. 2. Hogan MC, Foreman KJ, Naghavi M, Ahn SY, Wang M, Makela SM, et al. Maternal mortality for 181 countries, 1980-2008: a systematic analysis of progress towards Millennium Development Goal 5. Lancet. 2010;375:160923, doi: 10.1016/S0140-6736(10)60518-1. 3. Say L, Pattinson RC, Gu¨lmezoglu AM. WHO systematic review of maternal morbidity and mortality: the prevalence of severe acute maternal morbidity (near miss). Reprod Health. 2004;1:3, doi: 10.1186/ 1742-4755-1-3. 4. Geller SE, Rosenberg D, Cox SM, Brown ML, Simonson L, Driscoll CA, et al. The continuum of maternal morbidity and mortality: factors associated with severity. Am J Obstet Gynecol. 2004;191:939-44, doi: 10. 1016/j.ajog.2004.05.099. 5. Pattinson R. Near miss audit in obstetrics. Best Pract Res Clin Obstet Gynaecol. 2009;23:285-6, doi: 10.1016/j.bpobgyn.2009.01.013. 6. Penney G, Brace V. Near miss audit in obstetrics. Curr Opin Obstet Gynecol. 2007;19:145-50, doi: 10.1097/GCO.0b013e328014a860. 7. Say L, Souza JP, , Pattinson RC; WHO working group on Maternal Mortality and Morbidity classifications. Maternal near miss - towards a standard tool for monitoring quality of maternal health care. Best Pract Res Clin Obstet Gynaecol. 2009;23:287-96, doi: 10.1016/j.bpobgyn.2009. 01.007. 8. Pattinson R, Say L, Souza JP, Broek N, Rooney C; WHO Working Group on Maternal Mortality and Morbidity Classifications. WHO maternal death and near-miss classifications. Bull World Health Organ. 2009;87:734, doi: 10.2471/BLT.09.071001. 9. Souza JP, Cecatti JG, Parpinelli MA, Sousa MH, Lago TG, Pacagnella RC, et al. Maternal morbidity and near miss in the community: findings of the 2006 Brazilian Demographic Health Survey. Br J Obstet Gynaecol. 2010;117:1586-92, doi: 10.1111/j.1471-0528.2010.02746.x. 10. Zwart JJ, Richters JM, Ory F, de Vries JI, Bloemenkamp KW, van Roosmalen J. Severe maternal morbidity during pregnancy, delivery and puerperium in the Netherlands: a nationwide population-based study of 371,000 pregnancies. BJOG. 2008;115:842-50, doi: 10.1111/j.1471-0528. 2008.01713.x. 11. Pacagnella RC, Cecatti JG, Camargo RS, Silveira C, Zanardi DT, Souza JP, et al. Rationale for a long term evaluation of the consequences of potentially life threatening maternal conditions and maternal near miss incidents using a multidimensional approach. J Obstet Gynaecol Can. 2010;32:730-8. 12. Gill K, Pande R, Malhotra A. Women deliver for development. Lancet. 2007;370:1347-57, doi: 10.1016/S0140-6736(07)61577-3. 13. Hurt LS, Alam N, Dieltiens G, Aktar N, Ronsmans C. Duration and magnitude of mortality after pregnancy in rural Bangladesh. Int J Epidemiol. 2008;37:397-404, doi: 10.1093/ije/dym274. 14. Glazener CM, Abdalla M, Stroud P, Naji S, Templeton A, Russell IT. Postnatal maternal morbidity: extent, causes, prevention and treatment.

CONCLUSIONS This study did not detect any significant differences between groups in relation to the total number of subsequent pregnancies and their outcomes. Although, to the best of our knowledge, no other studies have produced

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15.

16.

17.

18.

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Br J Obstet Gynaecol. 1995;102:282-7, doi: 10.1111/j.1471-0528.1995. tb09132.x. Bang RA, Bang AT, Reddy MH, Deshmukh MD, Baitule SB, Filippi V. Maternal morbidity during labour and the puerperium in rural homes and the need for medical attention: A prospective observational study in Gadchiroli, India. BJOG. 2004;111:231-8, doi: 10.1111/j.1471-0528.2004. 00063.x. Seng JS, Oakley DJ, Sampselle CM, Killion C, Graham-Bermann S, Liberzon I. Posttraumatic stress disorder and pregnancy complications. Obstet Gynecol. 2001;97:17-22, doi: 10.1016/S0029-7844(00) 01097-8. Filippi V, Goufodji S, Sismanidis C, Kanhonou L, Fottrell E, Ronsmans C, et al. Effects of severe obstetric complications on women’s health and infant mortality in Benin. Trop Med Int Health. 2010; 15:733-42, doi: 10. 1111/j.1365-3156.2010.02534.x. Souza JP, Cecatti JG, Parpinelli MA, Krupa F, Osis MJ. An emerging ‘‘maternal near-miss syndrome’’: narratives of women who almost died during pregnancy and childbirth. Birth. 2009;36:149-58, doi: 10.1111/j. 1523-536X.2009.00313.x.

19. Filippi V, Ganaba R, Baggaley RF, Marshall T, Storeng KT, Sombie´ I, et al. Health of women after severe obstetric complications in Burkina Faso: a longitudinal study. Lancet. 2007;370:1329-37, doi: 10.1016/S01406736(07)61574-8. 20. Cecatti JG, Souza JP, Parpinelli MA, Haddad SM, Camargo RS, Pacagnella RC, et al. Brazilian network for the surveillance of maternal potentially life threatening morbidity and maternal near-miss and a multidimensional evaluation of their long term consequences. Reprod Health. 2009;6:15, doi: 10.1186/1742-4755-6-15. 21. Murphy DJ, Charlett P. Cohort study of near-miss maternal mortality and subsequent reproductive outcome. Eur J Obstet Gynecol Reprod Biol. 2002;102:173-8, doi: 10.1016/S0301-2115(01)00320-7. 22. Souza JP, Cecatti JG, Pacagnella RC, Giavarotti TM, Parpinelli MA, Camargo RS, et al. Development and validation of a questionnaire to identify severe maternal morbidity in epidemiological surveys. Reprod Health. 2010;7:16, doi: 10.1186/1742-4755-7-16. 23. Waterstone M, Wolfe C, Hooper R, Bewley S. Postnatal morbidity after childbirth and severe obstetric morbidity. BJOG. 2003;110:128-33.

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DOI:10.1590/S1807-59322011000800011

CLINICAL SCIENCE

The TNF-a -308 polymorphism may affect the severity of Crohn’s disease Genoile Santana,I,IV Maria Teresita Bendicho,II Tamara Celi Santana,III Lidiane Bianca dos Reis,III Denise Lemaire,II Andre´ Castro LyraIII I

Federal University of Bahia/Prof. Edgard Santos University Hospital – Gastroenterology, Salvador/BA, Brazil. II Federal University of Bahia/Health Science Institute – Immunology, Salvador/BA, Brazil. III Federal University of Bahia - Department of Medicine, Salvador/BA, Brazil. IV Roberto Santos General Hospital - Gastroenterology, Salvador/BA, Brazil.

OBJECTIVE: The goal of this project was to analyze the association between Crohn’s disease, its clinical features, and the tumor necrosis factor alpha (TNF-a) -308 polymorphism. METHODS: This is a case-control and cross-sectional study that enrolled 91 patients with Crohn’s disease and 91 controls. Patients with Crohn’s disease were characterized according to the Montreal Classification, along with their clinical and surgical treatment history. Analysis of the TNF-a -308 polymorphism was performed using a commercial kit. A stratified analysis was applied using an OR (odds ratio) with a 95% confidence interval. The chi-square and Fisher’s exact tests were utilized for analysis of the association between the polymorphism and the clinical features of Crohn’s disease. RESULTS: The low producer predicted phenotype was present in 76.9% of Crohn’s disease cases and 75.8% of controls (OR 0.94 [0.45-1.97]). The TNF2 allele and the high producer predicted phenotype were more frequent among patients with Crohn’s disease penetrating behavior (p = 0.004). The TNF2 allele and the high producer predicted phenotype were also associated with a history of colectomy (p = 0.02), and the TNF2 allele was associated with small bowel resection (p = 0.03). CONCLUSIONS: The TNF-a -308 polymorphism appears to affect the severity of the disease. However, TNF-a -308 polymorphism does not appear to be important for the susceptibility in the development of Crohn’s disease. KEYWORDS: Crohn’s disease; Inflammatory bowel disease; Tumor necrosis factor alpha; Genetic polymorphism; Mixed-race. Santana G, Bendicho MT, Santana TC, Reis LB, Lemaire D, Lyra A. The TNF-a -308 polymorphism may affect the severity of Crohn’s disease. Clinics. 2011;66(8):1373-1377. Received for publication on March 27, 2011; First review completed on April 7, 2011; Accepted for publication on May 4, 2011 E-mail: genoile@uol.com.br Tel.: 55 71 99572121

ethnic backgrounds. Overall, there is a slight predominance of CD in females.3 Additionally, smoking is a potential risk factor for development of CD.4 TNF-a is mainly secreted by cells of the immune system, including monocytes, macrophages, neutrophils, NK cells, and T lymphocytes. The TNF-a gene is located on the short arm of chromosome 6 in the HLA region. Several polymorphisms in its promoter region have been described.5 The most important of these polymorphisms is a GRA substitution at position -308, where the TNF1 (G) and TNF2 (A) alleles originate.6 The GG genotype is associated with a low TNF-a producing predicted phenotype, and the other two genotypes (GA+AA) are associated with a high TNF-a producing predicted phenotype.7,8 The association between the TNF-a -308 polymorphism and the susceptibility to IBD has been previously evaluated, with conflicting results.9,10 A European study found that the frequency of the TNF2 allele was lower in patients with CD than in healthy controls.11 Another study found a lower frequency of the TNF2 allele in ulcerative colitis patients compared with controls.12 However, a Japanese study

INTRODUCTION Crohn’s disease (CD) is a chronic illness of unknown etiology. Current studies suggest that a combination of environmental and genetic factors contribute to its pathogenesis. The inflammatory process in CD is partially due to the dysregulation of an immune reaction to gut bacteria. Monocytes and macrophages in the gastrointestinal tract of CD patients often secrete high amounts of tumor necrosis factor alpha (TNF-a), which has an essential role in the disease activity.1 Crohn’s disease appears to be more common among whites than individuals of African descent.2 Some studies suggest that different racial groups present different characteristics of CD, which could be due to unique pathogenetic mechanisms in patients of different

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observed a fourfold increase in the frequency of the TNF2 allele among patients with ulcerative colitis compared with controls.13 Another study detected a similar frequency of the TNF2 allele between IBD and control patients.14 Polymorphisms at the -308 site of TNF-a are also associated with the clinical presentation of CD.15 A study of European CD patients detected a connection between the frequency of the TNF2 allele and fistulizing, steroid-dependent and colonic disease.11 The aim of this study was to analyze the association between the incidence of CD and the TNF-a -308 polymorphism and to examine the association between this polymorphism, severity criteria and the clinical features of Crohn’s disease, such as age at diagnosis, behavior, and location of the disease.

was designed to have a power of 80%, with a 5% probability of an alpha standard error. Using these parameters, it was determined that each group should contain 91 individuals. To analyze the results, databases were generated and analyzed with the SPSS software, version 11.0, and Epi Info, version 6.04. The study groups were evaluated to determine whether the TNF alleles were in Hardy-Weinberg Equilibrium. The main independent variable was carrying the TNF2 allele, which corresponds with a high TNF-a producing predicted phenotype. The dependent variable was the diagnosis of CD. A 262 table was constructed with the main independent and the dependent variable to obtain odds ratios (OR) and 95% confidence intervals. The co-variables of interest were gender, racial group (evaluated as whites or African descent), and smoking (evaluated as the presence or absence of a smoking history). To analyze effects of confounding factors and modifiers on the main association, we performed a stratified analysis using estimated ORs with 95% confidence intervals, MantelHaenszel adjusted ORs and the Mantel-Haenszel chi-square test. The co-variables were also evaluated for the possibility of confounding effects by verifying the difference between crude OR and Mantel-Haenszel adjusted OR. The chi-square test or Fisher’s test was used to examine the associations between the presence of the TNF2 allele and the CD characteristics. The differences were considered statistically significant when the probability of the standard error was ,0.05. Informed written consent was obtained from all subjects. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Hospital Ethics Committee.

MATERIALS AND METHODS This was a cross-sectional and case-control study. We evaluated patients from outpatient gastroenterology clinics at the Professor Edgard Santos University Hospital and at the Roberto Santos General Hospital in Salvador. Patients and controls were enrolled between March 2006 and May 2007. Patients 18 years or older were enrolled if they had an established diagnosis of CD, according to the criteria of Lennard-Jones.16 All included patients were subjected to at least one small bowel follow-through and one colonoscopy with biopsy. When a colonoscopy could not be performed because of a suspected stricture, patients underwent a radiological evaluation of the colon. In cases with symptoms of the upper GI tract, the patients were subjected to an upper endoscopy. The classification of CD was based on the Montreal criteria,18 which include age at diagnosis, behavior, and location. Race was evaluated according to the criteria of Krieger.19 Patients were considered smokers if they had smoked seven cigarettes or more per week for at least one year. Only one patient from each family was enrolled. The complete clinical history of CD, including clinical and surgical treatment, was obtained from each patient. Controls were individuals 18 or older who were evaluated at the listed clinics during the same period of time. These patients may have been diagnosed with dyspepsia or esophageal reflux disease. However, these subjects were required to not have had peptic ulcers, diarrhea, hematochezia, abdominal pain, fistulae, or a family history of inflammatory bowel disease. A patient’s family history was considered positive for IBD if it occurred in a first cousin or closer relative. To obtain genomic DNA, 10 milliliters of whole blood was collected from each patient and stored at -4 ˚C in EDTA tubes until DNA extraction was performed. Genomic DNA was purified with the commercial EZ-DNA Kit (Biological Industries, Kibbutz Beit Haemek, Israel), according to the manufacturer’s instructions. The TNF-a -308 polymorphism was genotyped with the Cytokine Genotyping Tray Kit (One Lambda, Canoga Park, CA) according to the manufacturer’s instructions. The results were interpreted using maps of the genotyping plates supplied by the manufacturer. Sample size was calculated to detect an odds ratio of 2, considering a frequency of 18% for the TNF2 allele among controls and a frequency of 36% among cases. The study

RESULTS In total, 91 patients with CD and 91 control patients were enrolled in the study. Of these 182 patients, 64 cases and 62 controls were from the Professor Edgard Santos University Hospital, while 27 cases and 29 controls were from the Roberto Santos General Hospital. The control group comprised 81 patients with gastroesophageal reflux disease and 10 patients with functional dyspepsia. The genotype frequencies of both cases and controls were found to be in Hardy-Weinberg equilibrium. The mean age of patients with CD was 38.0¡12.8 years, and that of controls was 50.3¡13.6 years. Levene’s test was used to evaluate the homogeneity of variances, and they were determined to be equal. The difference between the mean age of cases and controls was statistically significant (p,0.001). Three individuals with CD were of Jewish ancestry. Otherwise, the distribution of racial groups was similar between cases and controls. Only two patients with CD had a family history of IBD. The distribution of the allelic and genotypic frequencies, along with the predicted phenotypes, for the -308 G/A polymorphism in the TNF-a promoter region for both patients with CD and controls is shown in Table 1. The crude OR and its respective 95% confidence interval for the presence of the A allele and CD were 1.09 [0.58–2.05], while the OR for displaying the high producing predicted phenotype and CD was 0.94 [0.45–1.97]. A positive association was observed between whites (1.25 [0.45–3.50]), male gender (1.91 [0.98–3.76]) and Crohn’s disease, and a

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Table 1 - Distribution of alleles, genotypes and predicted phenotype frequencies of the TNF-a -308 polymorphism in controls and CD patients.

Allele G, n (%) A, n (%) Genotype GG, n (%) GA, n (%) AA, n (%) Predicted Phenotype Low Producer, n (%) High Producer, n (%)

Cases

Controls

n = 182 155 (85.2) 27 (14.8) n = 91 70 (76.9) 15 (16.5) 6 (6.6) n = 91 70 (76.9) 21 (23.1)

n = 182 157 (82.3) 25 (13.7) n = 91 69 (75.8) 19 (20.9) 3 (3.3) n = 91 69 (75.8) 22 (24.2)

Table 3 - Montreal classification for Crohn’s disease. Montreal Classification

Age at diagnosis, n = 91 A1 (#16) A2 (17 - 40) A3 (.40) Behavior, n = 90 B1 – Non-stricturing, non-penetrating B2 – Stricturing B3 – Penetrating Location n = 82 L1 – Terminal ileum L2 – Colon L3 – Ileocolic L4 – Upper gastrointestinal

negative association was identified between a smoking history (0.67 [0.32–1.37]) and Crohn’s disease. However, these results were not statistically significant. Table 2 summarizes the results of the crude ORs with their respective 95% confidence intervals for displaying the high producing predicted phenotype and CD and the stratified analysis for gender, racial group, and smoking. The results of the Mantel-Haenszel adjusted OR are also listed in Table 2. Notably, the adjusted OR did not differ from the crude OR for any of the variables. The mean time from the onset of initial symptoms to the establishment of a CD diagnosis was 40.2¡59.2 months (range 1–324 months), and the median time was 34 months. The mean age at diagnosis was 32.3¡12.5 years. The median age at diagnosis was 33 years old, with a range from 5 to 63 years old. The mean time since CD diagnosis was 68.8¡66.2 months. The CD description, according to the Montreal classification, is outlined in Table 3. The disease behavior was defined in 90 patients, and location was identified in 82 patients. In 81 patients, it was possible to classify both location and behavior. Most patients were diagnosed between 17 and 40 years old and displayed non-stricturing, non-penetrating behavior, and an ileocolic location. A history of perianal fistula was detected in 45.1% (41/91) of the patients, and a rectovaginal fistula occurred in 5.6% (3/54) of women. Two patients (3.3%) had an enterocutaneous fistula.

Total n (%)

12 (13.2) 55 (60.4) 24 (26.4) 31 12 7

B1+p B2+p B2+p

31 1 8

62 (68.9) 13 (14.4) 15 (16.7)

17 21 36 1

L1+L4 L2+L4 L3+L4

_ _ 7

17 21 43 1

(20.7) (25.6) (52.4) (1.3)

The alleles, genotypes, and predicted phenotype frequencies were not associated with age at diagnosis or location (data not shown). Patients with penetrating behavior showed a greater frequency of the high producing phenotype and of the A allele. Specifically, they were mainly of the AA genotype. This difference was statistically significant. A complete description of the frequencies of the alleles, genotypes and predicted phenotypes according to CD behavior is shown in Table 4. There was no association between perianal disease and the TNF-a -308 polymorphism. The analysis of the severity criteria showed associations between the TNF2 allele, colectomy (p = 0.04) and small bowel resection (p = 0.03). Furthermore, the high producing predicted phenotype was associated with colectomy (p = 0.02) (Table 5).

DISCUSSION Recent advances in studies of genetic polymorphisms and CD are evident, in that the trend for the molecular classification of this disease is based on studies of genes associated with particular clinical phenotypes. For example, the influence of HLA regions appears to be more important in determining colonic and/or perianal disease, while mutations in NOD2/CARD15 seem to establish ileal disease in people diagnosed at a younger age.20 The first study from Brazil evaluating genetic polymorphisms in IBD patients

Table 2 - Stratified analysis of association between TNF-a high producing predicted phenotypes and Crohn’s disease with regard to gender, racial group and smoking. Variable Gender Female Male Racial Group African descent Whites Smoking Yes No

Case n (%)

Control n (%)

ORS { [95% CI]

OR A { [95% CI]

p-value *

54 (59.3) 37 (40.7)

67 (73.6) 24 (26.4)

1.60 [0.63–4.06] 0.32 [0.08–1.24]

0.93 [0.44–1.96]

80 (87.9) 11 (12.1)

82 (90.1) 9 (9.9)

0.84 [0.38–1.86] 2.0 [0.19–23.4]

0.93 [0.44–1.96]

20 (22) 71 (78)

27 (29.7) 64 (70.3)

2.33 [0.55–10.14] 0.67 [0.27–1.66]

0.98 [0.46–2.05]

Note: Crude odds ratio (OR) for having high producing predicted phenotype and CD = 0.94 [0.45–1.97]. { stratified OR. { adjusted OR. * Mantel-Haenszel chi-square to test equality of the OR in strata. NS, not statistically significant.

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Table 4 - Frequencies of the alleles, genotypes and predicted phenotypes for the TNF-a -308 polymorphism according to CD behavior.

Variable

B1+p (Non-stricturing nonpenetrating +perianal)

Alleles G, n (%) A, n (%) Genotypes GG, n (%) GA, n (%) AA, n (%) Predicted Phenotypes Low Producer, n (%) High Producer, n (%)

p-value*

n = 124 112(90.3) 12(9.7) n = 62 52 (83.9) 8(12.9) 2(3.2) n = 62 52 (83.8)

0.003

B2+p (Stricturing +perianal)

p-value*

n = 26 21 (80.8) 5(19.2) n = 13 9 (69.2) 3 (23.1) 1 (7.7) n = 13 9 (69.2)

0.02

10 (16.2)

4 (30.8)

B3+p (Penetrating +perianal) n = 30 20 (66.7) 10(33.3) n = 15 8 (53.3) 4 (26.7) 3 (20.0) n = 15 8 (53.3)

p-value* 0.004**

0.02

7 (46.7)

Number of alleles = 180; number of patients = 90. Comparison with the other behaviors. ** Fisher’s exact test.

found that NOD2/CARD15 confers susceptibility to CD but does not influence the disease phenotype.21 TNF-a is a key cytokine in the inflammatory response of CD and appears to be important in the digestive and systemic manifestations of the disease. Research on the polymorphism at position -308 in the promoter region of the TNF-a gene has demonstrated its importance with regard to the clinical presentation of CD. Vatay et al.22 found that carriers of the TNF2 allele had higher C-reactive protein levels in the active phase of the disease compared with the inactive phase. In a study by Gonza´lez et al.,15 50 patients with the penetrating form of CD were evaluated, and the authors detected an association between the presence of the A allele and increased serum levels of TNF-a, interleukin 1ß and proteins of the acute phase. A more intense inflammatory response occurred in GA individuals compared with GG individuals, along with a higher frequency of arthritis. Their study included no AA patients. In an analysis stratified by gender, racial group, and smoker/non-smoker variables in the current study, none of these factors were found to modify the effect or confound the association between the studied polymorphism and CD. This result reinforces the possibility that this polymorphism does not have relevance as a risk factor for CD in the studied population, and it is also in agreement with previous studies performed in other populations.15,23 In agreement with our findings, another study of Brazilian IBD patients

showed no association between this polymorphism and susceptibility to CD or UC.24 Although our study was conducted in another state, those results are still comparable with ours because a multicentric study evaluating genetic ancestry in the Brazilian population showed a perfect genetic admixture from Portuguese, Amerindian and African genetic ancestries in all states studied, regardless of the different skin colors of the individuals.25 Some considerations are relevant in studies that evaluate the association of genotype and phenotype using a casecontrol design. Saito et al. demonstrated the necessity for a plausible biological rationale when studying cytokine polymorphisms. It is not justifiable to examine polymorphisms only because of their availability, without any suggestion for a possible association between the studied cytokine and the pathogenesis of the disease.26 This study aimed to evaluate the TNF-a polymorphism, as this cytokine is involved in the pathogenesis of CD. In addition, it is important to include a detailed characterization of the control group, which may sometimes not be comparable with the disease group.27 To better match our groups, we only included control subjects who were treated at the same centers where the study patients were evaluated and managed. Individuals under 40 years old at diagnosis presented with a lower frequency of the high producing predicted phenotype. It is possible that the TNF-a gene contributes more to the inflammatory response in older individuals.

Table 5 - Frequencies of the alleles and predicted phenotypes for the TNF-a -308 polymorphism according to severity criteria. Allele

Severity Criteria Immunosuppressive treatment Steroid treatment Fistula surgery Colectomy Small bowel resection Hospitalization in the last year { {

A n (%) 10 19 8 10 5 8

(37.0) (70.4) (29.6) (37.0) (18.5) (29.6)

TNF-a predicted phenotype G n (%) 60 101 24 30 13 48

p-value

(38.7) (65.2) (15.5) (19.4) (8.4) (31.4)

NS NS NS 0.04 0.03* NS

90 patients, with 27 A alleles, 153 G alleles, 21 high producers and 69 low producers. Fisher’s exact test. NS, not statistically significant.

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High producer n (%) 8 15 5 9 5 6

(30.1) (71.4) (23.8) (42.9) (31.3) (28.6)

Low producer n (%) 27 45 11 11 4 22

(38.6) (64.3) (15.7) (15.7) (5.7) (31.9)

p-value NS NS NS 0.02 NS N

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Other genes, such as NOD2/CARD15, might contribute to the occurrence of the disease in the youngest individuals.20 In another study, an association was detected between the penetrating form of CD and a high TNF-a producing predicted phenotype.11 We obtained similar results, showing that the frequency of a high producing predicted phenotype was greater among individuals with the penetrating behavior and that this difference was statistically significant. Reinforcing this finding, the non-stricturing, non-penetrating behavior was associated with the low producing predicted phenotype. Furthermore, the TNF2 allele was associated with the presence of colectomy and small bowel resection, both of which are associated with more severe disease. Another study of Brazilian IBD patients (43 CD patients and 42 UC patients) found no association between disease phenotype and this polymorphism, but the authors did not report the classification applied, which prevents a valid comparison with our results.24 The uniform description of this complex disease may contribute to the recognition of the real mechanisms involved in its pathogenesis. We postulate that the lack of a single classification could be the reason for the absence of an association between the studied polymorphism and the severity of CD in other studies. It is also possible that this polymorphism is in linkage disequilibrium with other genetic markers. A detailed description of the evolution and treatment of patients with CD in genetic studies would be useful in understanding many of the currently unclear pathogenetic mechanisms of CD.

7. 8.

10.

11.

12.

13.

14.

15.

16. 17.

CONCLUSIONS In conclusion, the TNF-a -308 polymorphism does not appear to predispose individuals to the development of CD. This observation is in agreement with research from other centers. However, this polymorphism might affect the behavior of CD. Additionally, the TNF2 allele was associated with prior colectomy and small bowel resection, reinforcing the notion that the TNF-a -308 polymorphism might affect the severity of CD.

18.

19.

20.

ACKNOWLEDGMENTS 21.

We are in debt to Dr. Robert Summers for critically reviewing this manuscript and are thankful to the following people for their assistance and/or support of this study: Maurı´cio Cardeal, Maria Victo´ria Prieto, Jorge Carvalho Guedes and Cristiane Fortes.

22.

REFERENCES 23.

1. Komatsu M, Kobayashi D, Saito K, Furuya D, Yagihashi A, Araake H, et al. Tumor necrosis factor-alpha in serum of patients with inflammatory bowel disease as measured by a highly sensitive immuno-PCR. Clin Chem. 2001;47:1297-301. 2. Kurata JH, Kantor-Fish S, Frankl H, Godby P, Vadheim CM. Crohn’s disease among ethnic groups in a large health maintenance organization. Gastroenterology. 1992;102:1940-8. 3. Loftus EV Jr. Clinical Epidemiology of Inflammatory bowel disease: incidence, prevalence, and environmental influences. Gastroenterology. 2004;126:1504-17, doi: 10.1053/j.gastro.2004.01.063. 4. Calkins BM. A meta-analysis of the role of smoking in inflammatory bowel disease. Dig Dis Sci. 1989;34:1841-54, doi: 10.1007/BF01536701. 5. Allen R. Polymorphism of human TNF-a promotor-random variation or functional diversity? Mol Immunol. 1999;36:1017-27, doi: 10.1016/S01615890(99)00127-3. 6. Wilson AG, di Giovine FS, Blakemore AI, Duff GW. Single base polymorphism in the human tumour necrosis factor alpha (TNF alpha)

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gene detectable by NcoI restriction of PCR product. Hum Mol Genet. 1992;1:353, doi: 10.1093/hmg/1.5.353. Zimmerman P, Guderian R, Nutman T. A new TNFA promoter allele identified in South American Blacks. Immunogenetics. 1996;44:485-6. Brinkman BM, Giphart MJ, Verhoef A, Kaijzel EL, Naipal AM, Daha MR, et al. Tumor necrosis factor alpha-308 gene variants in relation to major histocompatibility complex alleles and Feltys syndrome. Hum Immunol. 1994;41:259-66, doi: 10.1016/0198-8859(94)90044-2. Louis E, Satsangi J, Roussomoustakaki M, Parkes M, Fanning G, Welsh K, et al. Cytokine gene polymorphism in inflammatory bowel disease. Gut. 1996;39:705-10, doi: 10.1136/gut.39.5.705. Hampe J, Shaw SH, Saiz R, Leysens N, Lantermann A, Mascheretti S, et al. Linkage of inflammatory bowel disease to human chromosome 6p. Am J Hum Genet. 1999;65:1647-55, doi: 10.1086/302677. Louis E, Peeters M, Franchimont D, Seidel L, Fontaine F, Demolin G, et al. Tumour necrosis factor (TNF) gene polymorphism in Crohns Disease (CD): influence on disease behaviour? Clin Exp Immunol. 2000;119:64-8, doi: 10.1046/j.1365-2249.2000.01106.x. Bouma G, Xia B, Crusius JB, Bioque G, Koutroubakis I, Von Blomberg BM, et al. Distribution of four polymorphisms in the tumor necrosis factor (TNF) genes in patients with inflammatory bowel disease (IBD). Clin Exp Immunol 1996;103:391-6, doi: 10.1111/j.1365-2249.1996.tb08292.x. Sashio H, Tamura K, Ito R, Yamamoto Y, Bamba H, Kosaka T, et al. Polymorphisms of the TNF gene and the TNF receptor superfamily member 1B gene are associated with susceptibility to ulcerative colitis and Crohn’s disease, respectively. Immunogenetics. 2002; 53:1020-7, doi: 10.1007/s00251-001-0423-7. Koss K, Satsangi J, Fanning GC, Welsh KI, Jewell DP. Cytokine (TNF alpha, LT alpha and IL-10) polymorphisms in inflammatory bowel diseases and normal controls: differential effects on production and allele frequencies. Genes Immun. 2000;1:185-90, doi: 10.1038/sj.gene. 6363657. Gonza´lez S, Rodrigo L, Martinez-Borra J, Lo´pez-Va´zquez A, Fuentes D, Nin˜o P et al. TNF-a -308A promoter polymorphism is associated with enhanced TNF-a production and inflammatory activity in Crohn’s patients with fistulizing disease. Am J Gastroenterol. 2003;98:1101-6. Lennard-Jones JE. Classification of Inflammatory Bowel Disease. Scand J Gastroenterol. 1989;170:2-6, doi: 10.3109/00365528909091339. Gasche C, Scholmerich J, Brynskov J, D’Haens G, Hanauer SB, Irvine EJ, et al. A simple classification of Crohn’s Disease: report of the Working Party for the World Congresses of Gastroenterology, Vienna 1998. Inflamm Bowel Dis. 2000;6:8-15, doi: 10.1002/ibd.3780060103. Silverberg MS, Satsangi J, Ahmad T, Arnott IDR, Bernstein CN. Toward an integrated clinical, molecular and serological classification on inflammatory bowel disease: Report of a Working Party of 2005 Montreal Congress of Gastroenterology. Can J Gastroenterol. 2005; 19:5-36. Krieger H, Morton NE, Mi MP, Azeveˆdo E, Freire-Maia A, Yasuda N. Racial admixture in north-eastern Brazil. Ann Hum Genet. 1965; 29:11325, doi: 10.1111/j.1469-1809.1965.tb00507.x. Ahmad T, Armuzzi A, Bunce M, Mulcahy-Hawes K, Marshall SE, Orchard TR, et al. The molecular classification of the clinical manifestations of Crohn’s disease. Gastroenterology. 2002;122:854-66, doi: 10.1053/ gast.2002.32413. Baptista ML, Amarante H, Picheth G, Sdepanian VL, Peterson N, Babasukumar U, et al. CARD15 and IL23R influences Crohn’s disease susceptibility but not disease phenotype in a Brazilian Population. Inflamm Bowel Dis. 2008;14:674-9, doi: 10.1002/ibd.20372. ˜ ¡cs A, Proha´szka Z, Szalai C, Romics L, et al. Vatay A, Bene L, KovA Relationship between the tumor necrosis factor alpha polymorphism and the serum C-reactive protein levels in inflammatory bowel disease. Immunogenetics. 2003;55:247-52, doi: 10.1007/s00251-003-0575-8. Cantor MJ, Nickerson P, Bernstein CN. The role of cytokine gene polymorphism in determining disease susceptibility and phenotype in inflammatory bowel disease. Am J Gastroenterol. 2005; 100:1134-42, doi: 10.1111/j.1572-0241.2005.40979.x. Queiroz DMM, Oliveira AG, Saraiva IEB, Rocha GA, Rocha AMC, Sanna MGP, et al. Immune response and gene polymorphism profiles in Crohn’s disease and ulcerative colitis. Inflamm Bowel Dis. 2009;15:353-8, doi: 10.1002/ibd.20757. Parra FC, Amado RC, Lambertucci JR, Rocha J, Antunes CM, Pena SDJ. Color and genomic ancestry in Brazilians. Proc Natl Acad Sci. 2003;100:177-82, doi: 10.1073/pnas.0126614100. Saito YA, Talley NJ, Andrade M, Petersen GM. Case-control genetic association studies in gastrointestinal disease: Review and recommendations. Am J Gastroenterol. 2006;101:1379-89, doi: 10.1111/j.1572-0241. 2006.00587.x. Healy DG. Case-control studies in the genomic era: a clinicians guide. Lancet Neurol. 2006;5:701-7, doi: 10.1016/S1474-4422(06)70524-5.

CLINICS 2011;66(8):1379-1384

DOI:10.1590/S1807-59322011000800012

CLINICAL SCIENCE

BACKGROUND: Cognitive impairment, from mild forms to dementia, is an important social and health concern, principally among older individuals. Elderly patients are usually followed by general internists, who may overlook this condition. OBJECTIVE: Our aim was to determine whether cognitive impairment diagnosed by specialists had been previously detected by general internists. SUBJECTS AND METHODS: A total of 248 elderly individuals randomly selected from a list of outpatients seen by general internists in a public university hospital in Sa˜o Paulo, Brazil, were evaluated by a geriatrician. Patients were then classified as having probable cognitive impairment or not, based on their performance on the Mini-Mental State Examination and the Informant Questionnaire on Cognitive Decline in the Elderly. Cases of probable impairment were submitted to routine laboratory investigation, brain computed tomography, and neuropsychological evaluation. The final diagnoses were established by a consensus panel comprising two neurologists and the geriatrician who evaluated the patients using all available data. General internists’ files for all cognitively impaired cases and for a selected sample of individuals without cognitive impairment were checked for any record of cognitive complaints or decline. RESULTS: Forty-three patients were classified as demented (n = 21) or as cognitively impaired but not demented (n = 22). The evaluation of the general internists’ files revealed that information on cognitive complaints or decline was recorded for seven (16.3%) of the 43 patients with dementia or cognitive impairment without dementia. CONCLUSIONS: General internists seldom detected cognitive decline in elderly patients in Brazil. Further studies should be conducted to elucidate the reasons for this low rate of detection. KEYWORDS: Aged; Cognition disorders; Dementia; Diagnosis; Primary care. Jacinto AF, Brucki S, Porto CS, Martins MA, Nitrini R. Detection of cognitive impairment in the elderly by general internists in Brazil. Clinics. 2011;66(8):1379-1384. Received for publication on February 6, 2011; First review completed on February on 25, 2011; Accepted for publication on May 6, 2011 E-mail: alessandrojacinto@uol.com.br Tel.: 55 11 55752692

to be 8% in a study conducted at the neurology outpatient clinic of a university-based Brazilian tertiary hospital.5 Several studies in the medical literature have shown that in the elderly, cognitive impairment and other neuropsychiatric disorders, such as depression, are frequently undiagnosed in primary care settings in multiple countries.6-10 Other studies have also found GI expertise on dementia to be lacking.11,12 The aim of this study was to determine whether GIs are effectively diagnosing cognitive impairment and dementia in Brazil.

INTRODUCTION Cognitive impairment, which ranges from mild cognitive decline to dementia, represents an important social and health concern, principally among the elderly.1 In Brazil, most older individuals are followed by general internists (GIs) in a range of settings, including public basic healthcare units, university-based tertiary hospitals, and private practices.2 Two population-based studies performed in the state of Sa˜o Paulo, Brazil, found a prevalence of dementia from 7.1% to 8.8% in patients aged 65 years and older.3,4 Furthermore, the prevalence of potentially reversible dementia was found

SUBJECTS AND METHODS The study was conducted at two outpatient services, the General Teaching Outpatient Service (GTOS) and the Internal Medicine Outpatient Service (IMOS), both run by the Internal Medicine Department of the Hospital das Clı´nicas of the University of Sa˜o Paulo Medical School, a public university hospital in Brazil. In the GTOS, patients

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scores on both tests was randomly selected (non-probable case group) to serve as the control for comparison. The probable cases underwent neuropsychological evaluation, including the Dementia Rating Scale,22,23 laboratory tests (blood count, thyroid hormones, syphilis serology, liver function, kidney function, vitamin B12, and folic acid levels), and a brain computed tomography (CT) scan.24 Non-probable cases were not submitted to this evaluation. The final diagnoses were established in a consensus meeting of two neurologists specializing in dementia and the geriatrician who evaluated the patients using all available data. The probable cases and the control group were evaluated on the basis of clinical data, performances on neuropsychological tests and questionnaires, and the probable cases were also diagnosed using their laboratory and CT results. Patients were classified as having dementia,25 being cognitively impaired but not demented (CIND)26 or being without cognitive impairment. The GIs’ files were checked for any record of cognitive impairment in both the probable and non-probable cases. All GIs’ notes until the patients’ last appointment (prior to their evaluation by the geriatrician) were checked. Figure 1 shows the diagnosis flowchart. The Ethics Committee of the Hospital das Clı´nicas of the University of Sa˜o Paulo Medical School in Brazil approved this study.

who are referred by physicians from the Emergency Unit are seen and treated in up to three medical consultations. In the GTOS, interns and medical residents examine patients and discuss all clinical cases with experienced GIs. In the IMOS, patients are followed for longer periods because they usually harbor more chronic conditions. Experienced GIs and residents of the Internal Medicine Department care for these patients. We assumed that the prevalence of cognitive impairment in the elderly within hospital settings is approximately 20%. Thus, to evaluate 50 individuals with cognitive impairment, we estimated that 250 patients should be screened. The inclusion criteria were as follows: to have been followed at one of the two outpatient services, aged 65 or older, and contactable by telephone. Patients were contacted by telephone and invited to undergo a cognitive evaluation. To assure the randomness of the sample, a list randomizer was used. We initially invited patients selected from a list of 1,480 elderly individuals seen by GIs during a three-month period (November 1, 2003 to January 31, 2004). Of 296 patients contacted by telephone, 118 patients agreed to be evaluated by the geriatrician. These evaluations were conducted up to May 2005. To provide a shorter interval between the evaluations by the GI and the geriatrician, a second list of 1637 elderly patients seen by GIs from a different three-month period (June 1 to August 31, 2005) was used. Of the 204 contacted patients from this second list, 130 patients were evaluated up to May 2006. Thus, the final sample comprised 248 elderly patients who underwent evaluation by the geriatrician. It was not possible to compare the included subjects with the patients who refused evaluation. The patients’ informants were invited to accompany the patients during the first evaluation. When this was not possible, informants were later contacted by telephone to gather further information on the patient’s cognitive status. All of the participants signed a written consent form before taking part in the study. The assessment consisted of recording subjective memory complaints, medical antecedents, and use of medications as well as the application of the following tests and questionnaires: the Mini-Mental State Examination (MMSE),13,14 the short version of the Informant Questionnaire on Cognitive Decline in the Elderly (Short-IQCODE),15 the Brief Screening Cognitive Battery,16-18 the Functional Activities Questionnaire (FAQ),19 the Forward and Backward Digit Span, and the 15-item Geriatric Depression Scale (GDS).20 Initially, ShortIQCODE and MMSE scores were used to classify patients as having probable cognitive impairment or otherwise, as recommended by previous Brazilian studies.14,21 These two screening instruments were chosen because they are frequently used in Brazil.16 The MMSE is a brief neuropsychological screening instrument consisting of questions on temporal and spatial orientation, immediate memory, attention/concentration, delayed recall, and language as well as a task involving the copy of a drawing. The maximum score is 30. The short version of the IQCODE is a 16-item questionnaire, in which an informant compares the patient’s current versus previous (ten years ago) cognitive performance. Scores higher than three are suggestive of decline, and the worst possible score is five. Patients with either a belownormal score on the MMSE or an above-normal score on the Short-IQCODE were classified as probable cases (suspected cognitive impairment). A group of patients with normal

Statistical analysis Statistical analysis was used to compare the three groups: ‘‘dementia’’, ‘‘cognitive impairment with no dementia’’ (CIND), and ‘‘without cognitive impairment’’. Continuous variables were compared using the non-parametric KruskalWallis test followed by a multiple comparisons test (Dunn’s post-hoc test). Frequencies of categorical variables were compared using the Chi-square test. Data were analyzed using SPSS (Statistical Package for Social Sciences) version 11.5 for Windows. The accepted level of significance was set at 0.05.

RESULTS Of the 248 evaluated elderly patients, 52 were classified as probable cases of cognitive impairment and 196 as without cognitive impairment, based on the screening interview. For the control group, 53 individuals were randomly selected from the 196 subjects without cognitive impairment. Although many patients were undergoing treatment for chronic disorders at the time of the interview, in no case did these disorders hamper the cognitive evaluation. The median age and level of education of these subjects are shown in Table 1. A difference in education was found among the three groups (p = 0.02), with the dementia group that had a lower level of education than the group of healthy individuals. Of the 52 probable cases, 33 were seen by GIs at the IMOS and 19 at the GTOS unit. Eighteen individuals had scores below the cutoff on the MMSE; 12 had above-cutoff scores on the IQCODE; and 22 were impaired according to both questionnaires. The mean time interval between the GI evaluation and the geriatrician evaluation was 5.0 (¡1.7) months. Forty-three probable-case patients underwent neuropsychological evaluation, and six subjects were not submitted to further neuropsychological evaluation due to severe

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Figure 1 - Flow diagram from outpatient services to study completion. BCSB: Brief Cognitive Screening Battery. CT: Computed Tomography. FAQ: Functional Activities Questionnaire. GDS: Geriatric Depression Scale. MMSE: Mini-Mental State Exam. ShortIQCODE: Informant Questionnaire on Cognitive Decline in the Elderly (short version).

consensus meeting. Table 2 shows the nosological diagnoses of the probable and non-probable case groups. Information related to cognitive impairment was found in the outpatient service medical records of seven (16.3%) of the 43 patients diagnosed with dementia or CIND. Six of these seven cases had moderate or severe dementia based on MMSE and Short-IQCODE scores, while one case had CIND (Table 3). These seven patients had been seen at either the GTOS (4 patients) or the IMOS (3 patients). All of the information related to cognitive impairment recorded in the

cognitive impairment. Two individuals refused further evaluation after the first appointment, and one moved to another town. All 52 probable cases and the 53 control cases without cognitive impairment were evaluated at the consensus meeting. Of the 52 probable cases, 21 were given a final diagnosis of dementia, and 22 were diagnosed with CIND, producing a total prevalence of cognitive impairment of 17.3%. None of the cases without cognitive impairment were diagnosed as having dementia or CIND at the

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Table 1 - Age, education level and gender of the subjects with and without cognitive impairment.

Age, median (IQI) Years of Education Median (IQI) Gender (Female) *

NCI (n = 53)

CIND (n = 22)

Dementia (n = 21)

70 (67-74)

69.5 (67-73)

4 (3-10) 37(69.8%)

p-value

Multiple comparisons

72 (67-75)

0.28*

Dementia = CIND = NCI

4 (2-8)

2 (2-4)

0.02*

20(90.9%)

18(85.7%)

0.71

Dementia = CIND{ CIND = NCI Dementia,WCI Dementia = CIND = NCI

Kruskal-Wallis test; {Dunn’s post hoc test; IQI: interquartile interval; 1Chi-Square Test; CIND: cognitive impairment with no dementia; NCI: no cognitive impairment.

outpatient service files is shown in Table 3. No record of cognitive impairment was present in the GI files for the 53 cases without cognitive impairment.

In 2000, Renshaw et al. demonstrated that doctors in the United Kingdom believed that knowledge about the symptoms of cognitive impairment in the elderly is not important because there is no effective treatment for these conditions. The same study also revealed that these professionals felt they had received insufficient training on dementia issues in their medical graduate programs.11 In Canada and Australia, Lorentz et al. showed that GIs felt that applying cognitive tests in their work settings (a large number of patients and a short period to attend to them) was not feasible because these instruments are very complex and time consuming.7 The present study did not investigate knowledge about dementia and its respective diagnostic tools among the GIs. The medical literature shows GIs’ knowledge about dementia to be limited, and this might explain the findings of our study.11,12 In Brazil, Bertolucci et al. gave a questionnaire about dementia knowledge to specialists (geriatricians, psychiatrists, and neurologists) and GIs. The results revealed that GIs had less knowledge about dementia than did neurologists, psychiatrists, and geriatricians.12 Other factors that may be causally related to the low diagnostic rate of cognitive impairment in our study are the difficulties that GIs face in the public healthcare system, where there is limited time available for each appointment and where patients often present with multiple co-morbidities. Another factor contributing to the under-reporting of the condition could be the low educational level of the patients and caregivers in this study, who consequently reported few or

DISCUSSION GIs had previously identified 16.3% of the cases that were later diagnosed with dementia (6 cases) or CIND (1 case) by specialists. Each of these seven patients had been seen by a different GI; four were seen at one outpatient service, whereas three were seen in the other service, indicating that the lack of recognition of cognitive impairment did not come from a specific professional or outpatient service. It should be emphasized that there was no record of cognitive or functional decline in the files of 15 patients diagnosed with dementia, despite the fact that this study was conducted in a university-based tertiary hospital where professionals are highly qualified and kept abreast of recent scientific developments. Two studies adopting a similar methodology (analyzing GIs’ files after specialists had diagnosed cognitive impairment in elderly patients) have also been published. Valcour et al. reported that 65% of the dementia cases diagnosed by geriatricians in their study had previously been identified by GIs who had followed these patients routinely.6 Another study using a similar methodology was conducted in Chicago, Illinois (USA) and showed that less than 20% of the cognitively impaired cases had been previously recorded as such in the GIs’ files.8

Table 2 - Nosological diagnoses of individuals in the probable case group and in a subsample of the non-probable case group.

Diagnosis

Probable cases (%) (n = 52)

Probable AD AD+cerebrovascular disease Vascular dementia Dementia due to intracranial mass lesions Parkinson’s disease dementia Alcoholic dementia CIND Depression Not cognitively impaired (only subjective memory complaint) No subjective or objective cognitive impairment No diagnosis (insufficient data)

12 4 2 1 1 1 22

(23.0) (7.7) (3.8) (1.9) (1.9) (1.9) (42.3) 0 2 (3.8) 4 (7.7) 3 (5.8)

AD: Alzheimer’s disease; CIND: cognitive impairment with no dementia.

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Sample of non-probable cases (%) (n = 53) 0 0 0 0 0 0 0 7 (13.2) 17 (32.0) 29 (54.7) 0

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Table 3 - Patient demographic data, diagnoses, MMSE and Short-IQCODE scores, and records from general internists’ files. Case

Age

Gender

Education

Diagnosis

MMSE

Short-IQCODE

GIs’ file notes

Female

Probable AD

3.83

2 3 4

88 73 79

Female Female Female

5 0 4

Probable AD Probable AD Probable AD

11 13 17

4.84 4.37 3.9

5 6

70 85

Female Female

1 4

0 18

5.0 3.96

Female

Probable AD AD plus cerebrovascular disease CIND

3.76

Subjective memory complaint AD diagnosis AD diagnosis Subjective memory complaint and impairment in daily life activities AD diagnosis Impairment in daily life activities Subjective memory complaint

AD: Alzheimer’s disease; CIND: cognitively impaired but not demented; GI: general internist; MMSE: Mini-Mental State Exam; Short-IQCODE: short version of the Informant Questionnaire on Cognitive Decline in the Elderly.

no complaints of cognitive impairment. A limitation of this study might be the fact that it was conducted at one specific location (University Hospital in Sa˜o Paulo), so these findings cannot be generalized to all of Brazilian society. Problems associated with an aging population were faced by economically developed countries almost one century ago. Brazil is now having to deal with the rapid aging of its population and should begin developing strategies in the near future to avoid serious repercussions. In Brazil, despite the efforts of public officials, there has been little reform of the public healthcare system to cater to an aging population, and the elderly encounter difficulties when seeking public healthcare services. Access to services equipped to receive older patients remains very limited. Basic healthcare units and even some large tertiary hospitals (university-associated or otherwise) are inadequately prepared for the growing demands placed on them by the elderly population.27 Advanced medical and scientific resources allow the early detection of diseases; cancer, diabetes, arterial hypertension, osteoporosis, and dyslipidemia are examples of diseases commonly screened for in public health programs on a preventive basis. However, the different types of dementia are often excluded from such programs despite their high prevalence.28,29 Dementia is the largest contributor to disability in elderly people in countries with low and middle incomes,30 and the societal costs of dementia have been increasing in recent years.31 Early diagnosis of cognitive impairment is important for several reasons. Ruling out potentially reversible conditions is the most relevant of these reasons, but providing more detailed instructions on how to take medications is another significant reason. The safety of the patient and of others may be safeguarded by preventing car accidents and disease complications arising from patients’ inability to take care of themselves.32-34 For the patient and his/her family, a diagnosis of cognitive impairment has an impact on their plans for the future, including financial and legal matters.35,36 Mild cognitive decline that does not significantly affect patients’ daily living activities may be more frequently overlooked by GIs. In this study, only one case classified as CIND contained a report of cognitive impairment in the GI file. Considering that patients with CIND may have higher rates of progression to dementia than non-impaired subjects, this diagnosis may be relevant for the patient.37

A note of caution should be made regarding the 17.3% prevalence of cognitive impairment found in this study. In spite of the random selection of cases selected for evaluation, many of them could not participate, and only a small sample was interviewed. It is possible that those who came for the interview had greater concerns about their cognitive functions. Further studies should be conducted to elucidate the reasons underlying the low detection of cognitive impairment by GIs and to investigate the advantages and pitfalls of early detection of dementia.

REFERENCES 1. Ferri CP, Prince M, Brayne C, Brodaty H, Fratiglioni L, Ganguli M, et al. Global prevalence of dementia: a Delphi consensus study. Lancet. 2005;366:2112-7, doi: 10.1016/S0140-6736(05)67889-0. 2. Rodrigues MA, Facchini LA, Piccini RX, Tomasi E, Thume´ E, Silveira DS, et al. Use of outpatient services by the elderly in the South and Northeast of Brazil. Cad Saude Publica. 2008;24:2267-78, doi: 10.1590/S0102311X2008001000008. 3. Herrera E, Caramelli P, Silveira ASB, Nitrini R. Epidemiologic survey of dementia in a community-dwelling Brazilian population. Alzheimer Dis Assoc Disord. 2002;16:103-8, doi: 10.1097/00002093-200204000-00007. 4. Bottino CM, Azevedo D Jr, Tatsch M, Hototian SR, Moscoso MA, Folquitto J, et al. Estimate of dementia prevalence in a community sample from Sa˜ o Paulo, Brazil. Dement Geriatr Cogn Disord. 2008;26:291-9, doi: 10.1159/000161053. 5. Takada LT, Caramelli P, Radanovic M, Anghinah R, Hartmann AP, Guariglia CC, et al. Prevalence of potentially reversible dementias in a dementia outpatient clinic of tertiary university-affiliated hospital in Brazil. Arq Neuropsiquiatr. 2003;6:925-9, doi: 10.1590/S0004282X2003000600007. 6. Valcour VG, Masaki H, Curb JD, Blanchette PL. The detection of dementia in the primary care setting. Arch Int Med. 2000;160:2964-8, doi: 10.1001/archinte.160.19.2964. 7. Lorentz WJ, Scanian JM, Borson S. Brief screening tests for dementia. Can J Psychiatry. 2002;47:723-33. 8. Finkel SI. Cognitive screening in the primary care setting: the role of physicians at the first point entry. Geriatrics. 2003;58:43-4. 9. Barret JJ, Haley WE, Harrel LE, Powers RE. Knowledge about Alzheimer’s disease among primary care physicians, psychologists, nurses and social workers. Alzheimer Dis Assoc Disord. 1997;11:99-106, doi: 10.1097/00002093-199706000-00006. 10. Henriques SG, Fra´guas R, Iosifescu DV, Menezes PR, Lucia MC, Gattaz WF, Martins MA. Recognition of depressive symptoms by physicians. Clinics. 2009;64:629-35, doi: 10.1590/S1807-59322009000700004. 11. Renshaw J, Scurfield LC, Cloke L, Orrel M. General practitioner’s views on the early diagnosis of dementia. Br J Gen Pract. 2001;51:37-8. 12. Bertolucci PHF, Ferretti CEL, Naylor FGM. Knowledge of dementia among doctors in Brazil. Neurobiol Aging. 2002;23(Suppl 1):S168. 13. Folstein MF, Folstein SE, McHugh PR. ‘‘Mini-mental state’’: a practical method for grading the cognitive state of patients for the clinician. J Psychiatr Res. 1975;12:189-98, doi: 10.1016/0022-3956(75)90026-6. 14. Brucki SMD, Nitrini R, Caramelli P, Bertolucci PH, Okamoto IH. Suggestions for utilization of the mini-mental state examination in

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15. 16.

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19. 20. 21.

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25. American Psychiatry Association. Diagnostic and Statistical Manual of Mental Disorders. 4th ed. Washington DC;American Psychiatric Press; 1994. 26. Ebly EM, Hogan DB, Parhad IM. Cognitive impairment in the nondemented elderly: results from the Canadian Study of Health and Aging. Arch Neurol. 1995;52:612-9. 27. Chaimowicz F. Health of Brazilian elderly just before of the 21st century: current problems, forecasts and alternatives. Rev Saude Publica. 1997;31:184-200, doi: 10.1590/S0034-89101997000200014. 28. Larson EB. Recognition of dementia: discovering the silent epidemic. J Am Geriatr Soc. 1998;46:1576-7. 29. Borson S, Scanlan JM, Watanabe J, Tu SP, Lessig M. Improving identification of cognitive impairment in primary care. Int J Geriatr Psychiatry. 2006;21:349-55, doi: 10.1002/gps.1470. 30. Sousa RM, Ferri CP, Acosta D, Albanese E, Guerra M, Huang Y, et al. Contribution of chronic diseases to disability in elderly people in countries with low and middle incomes: a 10/66 Dementia Research Group population-based survey. Lancet. 2009;374:1821-30, doi: 10.1016/ S0140-6736(09)61829-8. 31. Wimo A, Winblad B, Jo¨nsson L. The worldwide societal costs of dementia: estimates for 2009. Alzheimers Dement. 2010;6:98-103, doi: 10. 1016/j.jalz.2010.01.010. 32. Boustani M, Peterson B, Hanson L, HarrisRLohrKN. Screening for dementia in the primary care: a summary of the evidence for the US preventive services task force. Ann Intern Med. 2003;138:927-37. 33. Clarfield AM. Reversible dementia: the implications of a fall in prevalence. Age Ageing. 2005;34:544-5, doi: 10.1093/ageing/afi203. 34. Tierney MC, Charles J, Naglie G, Jaglal S, Kiss A, Fisher RH. Risk factors for harm in cognitively impaired seniors who live alone: a prospective study. J Am Geriatr Soc. 2004;52:1435-41, doi: 10.1111/j.0002-8614.2004.52404.x. 35. Ashford JW, Borson S, O’Hara R, Dash P, Frank L, Robert P, et al. Should older adults be screened for dementia? Alzheimer’s and Dementia. 2006;2:76-85, doi: 10.1016/j.jalz.2006.02.005. 36. Raicher I, Shimizu MM, Takahashi DY, Nitrini R, Caramelli P. Alzheimer’s disease diagnosis disclosure in Brazil: a survey of specialized physicians’ current practices and attitudes. Int Psychogeriatr. 2008;20:471-81, doi: 10.1017/S1041610207005819. 37. Petersen RC, Stevens JC, Ganguli M, Tangalos EG, Cummings JL, DeKosky ST, et al. Practice parameter: early detection of dementia: mild cognitive impairment (an evidence-based review): Report of the Quality Standards Subcommittee of the American Academy of Neurology. Neurology. 2001;56:1133-42.

Brazil. Arq Neuropsiquiatr. 2003;61:777-81, doi: 10.1590/S0004282X2003000500014. Jorm AF. A short form of the Informant Questionnaire on Cognitive Decline in the Elderly (IQCODE): development and cross-validation. Psychol Med. 1994;24:145-53, doi: 10.1017/S003329170002691X. Nitrini R, Caramelli P, Bottino CM, Damasceno BP, Brucki SM, Anghinah R, Academia Brasileira de Neurologia. [Diagnosis of Alzheimer’s disease in Brazil: cognitive and functional evaluation. Recommendations of the Scientific Department of Cognitive Neurology and Aging of the Brazilian Academy of Neurology]. Arq Neuropsiquiatr. 2005;63:720-7, doi: 10. 1590/S0004-282X2005000400034. Nitrini R, Caramelli P, Herrera Ju´nior E, Porto CS, Charchat-Fichman H, Carthery MT, et al. Performance of illiterate and literate nondemented elderly subjects in two tests of long-term memory. J Int Neuropsychol Soc. 2004;10:634-8, doi: 10.1017/S1355617704104062 Takada LT, Caramelli P, Fichman HC, Porto CS, Bahia VS, Anghinah R, et al. Comparison between two tests of delayed recall for the diagnosis of dementia. Arq Neuropsiquiatr. 2006;64:35-40, doi: 10.1590/S0004282X2006000100008. Pfeffer RI, Kurosaki TT, Harrah CH Jr, Chance JM, Filos S. Measurement of functional activities in older adults in the community. J Gerontol. 1982;37:323-9. Sheikh JI, Yesavage JA. Geriatric depression scale (GDS): recent evidence and development of a shorter version. Clin Gerontol. 1986;5:165-73, doi: 10.1300/J018v05n01_09. Bustamante SE, Bottino CM, Lopes MA, Azevedo D, Hototian SR, Litvoc J, et al. Combined instruments on the evaluation of dementia in the elderly: preliminary results. Arq Neuropsiquiatr. 2003;61:601-6, doi: 10. 1590/S0004-282X2003000400014. Mattis S. Dementia Rating Scale. Professional Manual. Florida: Psychological Assessment Resources, 1998. Porto CS, Fichman HC, Caramelli P, Bahia VS, Nitrini R. Brazilian version of the Mattis dementia rating scale: diagnosis of mild dementia in Alzheimer’s disease. Arq Neuropsiquiatr. 2003;61:339-45, doi: 10.1590/ S0004-282X2003000300004. Nitrini R, Caramelli P, Bottino CM, Damasceno BP, Brucki SM, Anghinah R. Diagnosis of Alzheimer’s disease in Brazil: diagnostic criteria and auxiliary tests. Recommendations of the Scientific Department of Cognitive Neurology and Aging of the Brazilian Academy of Neurology. Arq Neuropsiquiatr. 2005;63:713-9, doi: 10.1590/S0004282X2005000400033.

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DOI:10.1590/S1807-59322011000800013

CLINICAL SCIENCE

The relationship between the oral and pharyngeal phases of swallowing Rachel Aguiar Cassiani, Carla Manfredi Santos, Luana Casari Parreira, Roberto Oliveira Dantas Clıńica Me´dica - Faculdade de Medicina de Ribeiraõ Preto, Universidade de Saõ Paulo, Ribeiraõ Preto/SP, Brazil.

OBJECTIVE: This study was designed to investigate a possible relationship between the duration of the oral and pharyngeal phases of swallowing. INTRODUCTION: The oral and pharyngeal phases of swallowing are independent from each other but may be related. METHODS: We used videofluoroscopy to evaluate 30 healthy volunteers between 29 and 77 years of age who swallowed 5- and 10-ml liquid and paste boluses in duplicate. The duration of the oral phase, pharyngeal transit, and pharyngeal clearance were measured. RESULTS: There were no differences in oral or pharyngeal transit times between the liquid and paste boluses or between the volumes of 5 and 10 ml (p.0.40). The pharyngeal clearance time for the paste bolus (0.48¡0.27 s) was longer than for the liquid bolus (0.38¡0.11 s, p = 0.03) with no difference between the volumes of 5 and 10 ml. There was no significant correlation between the oral transit time and the duration of pharyngeal transit for the liquid (5 ml, Spearman’s coefficient r: 20.14; 10 ml, r: 0.18) or the paste (5 ml, r: 0.08; 10 ml, r: 0.10). The correlation between the oral transit time and the pharyngeal clearance time was not significant for the liquid bolus (5 ml, r: 0.31; 10 ml, r: 0.18), but it was significant for both the 5 ml (r: 0.71) and 10 ml (r: 0.64) paste boluses. DISCUSSION: The relationship between the oral and pharyngeal phases of swallowing can be affected by bolus consistency. CONCLUSION: There is a correlation between the duration of oral transit and the duration of pharyngeal clearance during the swallowing of paste boluses. KEYWORDS: Swallowing; Deglutition; Oral swallowing; Pharyngeal swallowing; Bolus consistency. Cassiani RA, Santos CM, Parreira LC, Dantas RO. The relationship between the oral and pharyngeal phases of swallowing. Clinics. 2011;66(8):13851388. Received for publication on March 9, 2011; First review completed on April 4, 2011; Accepted for publication on May 9, 2011 E-mail: rodantas@fmrp.usp.br Tel.: 55 16 3602-2457

the same. The responses of the pharynx and esophagus depend on the characteristics of the bolus.1-5 Our hypothesis was that the oral phase might be able to influence the pharyngeal phase under certain conditions depending on the consistency and volume of the bolus. Our objective in this investigation was to use videofluoroscopy to evaluate the influence of oral phase duration on pharyngeal phase duration in healthy subjects after swallowing either liquid or paste boluses. Our hypothesis was that the duration of the oral phase of swallowing would influence the duration of the pharyngeal phase.

INTRODUCTION Swallowing is a complex function that is controlled by the central pattern-generating circuitry of the brain stem and the peripheral reflexes.1 The oral, pharyngeal, and esophageal phases of swallowing occur independently of one another.1,2 Although central pattern generators of the brain stem control the timing of these events, the peripheral manifestations of these phases depend on sensory feedback through pharyngeal and esophageal reflexes.2 The oral phase of swallowing is a voluntary event, and the pharyngeal phase is an involuntary, independent event.1 However, swallowing occurs in a sequence that is always

MATERIAL AND METHODS Videofluoroscopic evaluations of swallowing were performed for a group of 30 healthy volunteers that was composed of 18 men and 12 women (age: 29-77 years; mean: 58¡13 years). Four of the subjects were between 29 and 40 years of age, 14 were between 41 and 60 years old and 12 were between the ages of 61 and 77. None of the

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No correlation was observed between the oral transit time and the pharyngeal transit time for the liquid (5 ml, r: -0.14; 10 ml, r: 0.18) and paste (5 ml, r: 0.08; 10 ml, r: 0.10) boluses. For liquid boluses, the correlation between the oral transit time and the pharyngeal clearance time was also not significant (5 ml, r: 0.31; 10 ml, r: 0.18). The correlation between the oral transit time and the pharyngeal clearance time of paste boluses was significant for both 5-ml (r: 0.71; Figure 1) and 10-mL boluses (r: 0.64; Figure 2) (p,0.01).

volunteers had dysphagia, gastroesophageal reflux symptoms, previous head, neck, esophagus, or stomach surgery, neurological diseases, or any problems ingesting liquid or solid food. No volunteer was taking any medication that could affect swallowing. The study was approved by the Human Research Committee of the University Hospital of Ribeira˜o Preto, and written informed consent was provided by all volunteers. Videofluoroscopy was performed using an Arcomax Phillips Model BV 300 radiologic instrument (Veenpluis, The Netherlands), and the Ever Focus digital image processing system Model EDSR 100 V1.2 (Taipei, Taiwan), with a DVR monitor (Ever Focus) run at 60 frames/second and a digital clock that indicated the time in seconds and hundredths of a second on each video frame. The volunteers were evaluated while seated and were viewed on the lateral plane. Images included the mouth, pharynx, and proximal esophageal body. The subjects swallowed, in duplicate, 5 and 10 ml boluses of liquid barium (100% barium sulfate, Bariogel, Laboratory Crista´lia, Itapira, SP, Brazil) and 5 and 10 ml boluses of paste barium, which were prepared by adding 4.5 g of instant food thickener (Thick & Easy, Hormel Health Labs, Savannah, GA, USA) to 50 ml of liquid barium. We measured the following features: 1 - the onset of propulsive tongue-tip movement at the maxillary incisors; 2 – the passage of the bolus head through the fauces; 3 - the passage of the bolus tail through the fauces; and 4 - the offset of the upper esophageal sphincter (UES) opening. From these times, we calculated the oral transit time (the time required for the bolus to pass from the tip of the tongue at the incisors until the time that the tail of the bolus passes through the fauces), pharyngeal transit time (bolus tail at the fauces to the offset of the UES opening), and pharyngeal clearance (bolus head at the fauces to the offset of the UES opening). Statistical analyses were performed at the Quantitative Methods Center (CEMEQ) of the Medical School of Ribeira˜o Preto USP. The data were analyzed using the Spearman correlation coefficient and a mixed-effects linear model.6 The results are reported as means, standard deviations, 95% confidence intervals, and Spearman’s coefficients (r).

DISCUSSION Our results indicate that paste boluses require a longer pharyngeal clearance time than liquid boluses and demonstrate a positive correlation between the oral transit time and pharyngeal clearance time during paste-bolus swallowing. Although the oral and pharyngeal phases of swallowing are independent, we found that a relationship exists between the two phases for paste boluses. Such factors as bolus consistency and volume and the subject’s gender and age affect the temporal relationship of the onset of specific motor events.5 It has been consistently observed that paste boluses cross the pharynx more slowly than do liquid boluses.4,5,7,8 Compared to less viscous boluses, boluses with high viscosity exhibit increases in the duration of oral ejection, the duration of UES opening, the deglutitive tongue force, and the duration of the hyoid movement.4,7,9,10 The results of this investigation suggest that the oral and pharyngeal phases of swallowing paste boluses may be related to each other; i.e., as a consequence of a slower oral transit, the duration of the bolus’s transport through the pharynx increases. Pharyngeal transit times and intrabolus pressure within the hypopharynx increase with increasing bolus consistency.3 This finding is in agreement with the findings of scintigraphy studies that suggest that increased bolus consistency is associated with slower pharyngeal transit.11 There is no change in the velocity of pharyngeal propagation contraction with alterations in bolus consistency, but the pressure difference between the bolus head and the bolus tail increases sharply.12 Greater pressure is applied to viscous boluses to maintain their transfer velocity through the pharynx; thus, the normal pharynx has adequate reserves to compensate for boluses of a thicker consistency.12 A lack of velocity alterations to propagation contractions is associated with no alterations of bolus transit duration with increasing bolus consistency. Manometry studies have revealed an increase in UES relaxation duration with bolus viscosity increases in younger adults who swallowed a volume of 10 ml. This relationship was not observed in older subjects or when swallowing smaller volumes (5 ml).2

RESULTS There were no differences in the duration of oral transit or pharyngeal transit between liquid and paste boluses (p.0.16; Table 1) or between the 5- and 10-ml boluses (p.0.40). The pharyngeal clearance time for paste boluses (0.48¡0.27 s) was longer than for liquid boluses (0.38¡0.11 s, p = 0.03), but no differences were observed between the 5- and 10–ml volumes.

Table 1 - Duration, in seconds, of oral and pharyngeal transit times and pharyngeal clearance time after swallowing two volumes (5 ml and 10 ml) of liquid and paste boluses. Liquid

Oral transit time Pharyngeal transit time Pharyngeal clearance time

Paste

MEAN (SD)

95% CI

MEAN (SD)

95% CI

p-value

0.42 (0.23) 0.22 (0.09) 0.38 (0.11)

0.36-0.48 0.20-0.24 0.35-0.41

0.41 (0.28) 0.23 (0.07) 0.48 (0.27)

0.34-0.49 0.21-0.25 0.41-0.55

0.72 0.17 0.03

CI – Confidence interval. SD – Standard deviation.

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Figure 1 - The correlation between oral transit time and pharyngeal clearance time after swallowing a 5-ml paste bolus. The Spearman coefficient (r) was 0.71.

no relationship between increased bolus volume and oral and pharyngeal transit times or pharyngeal clearance for the consistencies tested. The increased UES opening duration with increased bolus volume is actually a consequence of the earlier opening of the UES to accommodate a larger bolus.2 The aging process causes prolonged oral transit, prolonged pharyngeal transit and prolonged pharyngeal clearance.16 Because we included volunteers over the age of 60 years, it is possible that some mild swallowing abnormalities were present in some subjects. However, evaluations of the correlation between the oral and pharyngeal phases of swallowing performed in the same subjects revealed a positive correlation between the durations of these phases of swallowing. Prolonged oral transit was followed by prolonged pharyngeal transit duration. All of the volunteers were asymptomatic.

On the other hand, authors have claimed that oropharyngeal swallowing represents a synergy of overlapping and interdependent events that propel the bolus through the oropharyngeal cavities, close the valves critical for airway protection, and open the valves necessary for bolus entry into the esophagus.13 The stereotypic movements of each phase of swallowing are controlled by the patterngenerating circuitry of the brain.1 The oral phase is voluntarily initiated at the subjectâ&#x20AC;&#x2122;s discretion, but the pharyngeal and esophageal phases of swallowing occur in response to stimulation of the pharynx and esophagus by the bolus.1 It has also been reported that an increase in bolus volume has no effect on oral and pharyngeal bolus transit time or on the duration of pharyngeal peristaltic waves but leads to prolonged UES opening, longer laryngeal closure duration and longer swallowing apnea.2,14,15 In this study, we found

Figure 2 - The correlation between oral transit time and pharyngeal clearance time after swallowing a 10-ml paste bolus. The Spearman coefficient (r) was 0.64.

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8. Taniguchi H, Tsukada T, Ootaki S, Yamada Y, Inoue M. Correspondence between food consistency and suprahyoid muscle activity, tongue pressure, and bolus transit times during the oropharyngeal phase of swallowing. J Appl Physiol. 2008;105:791–9, doi: 10.1152/japplphysiol. 90485.2008. 9. Pouderoux P, Kahrilas PJ. Deglutitive tongue force modulation by volition, volume, and viscosity in humans. Gastroenterology. 1995; 108:1418–26, doi: 10.1016/0016-5085(95)90690-8. 10. Lazarus CL, Logemann JA, Rademaker AW, Kahrilas PJ, Pajak T, Lazar R, et al. Effects of bolus volume, viscosity, and repeated swallows in nonstroke subjects and stroke patients. Arch Phys Med Rehabil. 1993;74:1066-70, doi: 10.1016/0003-9993(93)90063-G. 11. Okubo PCMI, Dantas RO, Troncon LEA, Moriguti JC, Ferriolli E. Clinical and scintigraphic assessment of swallowing of older patients admitted to a tertiary care geriatric ward. Dysphagia. 2008;23:1-6, doi: 10.1007/ s00455-007-9087-2. 12. Raut VV, McKeeGJ, JohnstonBT. Effect of bolus consistency on swallowing – does altering consistency help? Eur Arch Otorhinolaryngol. 2001;258:49–53, doi: 10.1007/s004050000301. 13. Martin-Harris B, Michel Y, Castell DO. Physiologic model of oropharyngeal swallowing revisited. Otolaryngology–Head and Neck Surgery. 2005;133:234-40, doi: 10.1016/j.otohns.2005.03.059. 14. Kendall KA, McKenzie S, Leonard RJ, Gonc¸alves MI, Walker A. Timing of events in normal swallowing: A videofluoroscopic study. Dysphagia. 2000;15:74–83. 15. Tsukada T, Taniguchi H, Ootaki S, YamadaY, Inoue M. Effects of food texture and head posture on oropharyngeal swallowing. J Appl Physiol 2009;106:1848–57, doi: 10.1152/japplphysiol.91295.2008. 16. Cook IJ, Weltman MD, Wallace K, Shaw DW, Mckay E, Smart RC, et al. Influence of aging on oral – pharyngeal bolus transit and clearance during swallowing: scintigraphic study. Am J Physiol. 1994;265:G972–7.

In conclusion, for paste boluses, there was a relationship between the oral transit duration during swallowing and the pharyngeal clearance of the bolus. Slower oral transit was associated with slower pharyngeal clearance.

REFERENCES 1. Lang IM. Brain stem control of the phases of swallowing. Dysphagia. 2009;24:333-48, doi: 10.1007/s00455-009-9211-6. 2. Butler SG, Stuart A, Castell DO, Russel BG, Koch K, Kemp S. Effects of age, gender, bolus condition, viscosity, and volume on pharyngeal and upper esophageal sphincter pressure and temporal measurements during swallowing. J Speech Lang Hear Res. 2009;52:240–53, doi: 10. 1044/1092-4388(2008/07-0092). 3. Castell JA, Dalton CB, Castell DO. Effects of body position and bolus consistency on the manometric parameters and coordination of the upper esophageal sphincter and pharynx. Dysphagia. 1990;15:179–86, doi: 10.1007/BF02412685. 4. Dantas RO, Kern MK, Massey BT, Dodds WJ, Kahrilas PJ, Brasseur JG, et al. Effect of swallowed bolus variables on oral and pharyngeal phases of swallowing. Am J Physiol. 1990;258:G675-81. 5. Mendell DA, Logemann JA. Temporal sequence of swallow events during the oropharyngeal swallow. J Speech Lang Hear Res. 2007; 50:1256-71, doi: 10.1044/1092-4388(2007/088). 6. Shall R. Estimation in generalized linear models with random effects. Biometrika. 1991;78:719-27, doi: 10.1093/biomet/78.4.719. 7. Perlman AL, Schultz JG, Van Daele DJ. Effects of age, gender, bolus volume, and bolus viscosity on oropharyngeal pressure during swallowing. J Appl Physiol 1993;75:33–7.

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DOI:10.1590/S1807-59322011000800014

CLINICAL SCIENCE

Comparison of pelvic floor muscle strength evaluations in nulliparous and primiparous women: a prospective study Moˆnica Orsi Gameiro,I Vanessa Oliveira Sousa,I Luiz Felipe Gameiro,I Rosana Carneiro Muchailh,I Carlos Roberto Padovani,II Joaõ Luiz AmaroIII I Physiotherapy Service, School of Medicine, Saõ Paulo State University (UNESP), Botucatu/SP, Brazil. II Department of Biostatistics, School of Medicine, Saõ Paulo State University (UNESP), Botucatu/SP, Brazil. III Department of Urology, School of Medicine, Saõ Paulo State University (UNESP), Botucatu/SP, Brazil.

OBJECTIVE: This study aimed to compare the pelvic floor muscle strength of nulliparous and primiparous women. METHODS: A total of 100 women were prospectively distributed into two groups: Group 1 (G1) (n = 50) included healthy nulliparous women, and Group 2 (G2) (n = 50) included healthy primiparous women. Pelvic floor muscle strength was subjectively evaluated using transvaginal digital palpation. Pelvic floor muscle strength was objectively assessed using a portable perineometer. All of the parameters were evaluated simultaneously in G1 and were evaluated in G2 during the 20th and 36th weeks of pregnancy and 45 days after delivery. RESULTS: In G2, 14 women were excluded because they left the study before the follow-up evaluation. The median age was 23 years in G1 and 22 years in G2; there was no significant difference between the groups. The average body mass index was 21.7 kg/m2 in G1 and 25.0 kg/m2 in G2; there was a significant difference between the groups (p = 0.0004). In G2, transvaginal digital palpation evaluation showed significant impairments of pelvic floor muscle strength at the 36th week of pregnancy (p = 0.0006) and 45 days after vaginal delivery (p = 0.0001) compared to G1. Objective evaluations of pelvic floor muscle strength in G2 revealed a significant decrease 45 days after vaginal delivery compared to nulliparous patients. CONCLUSION: Pregnancy and vaginal delivery may cause weakness of the pelvic floor muscles. KEYWORDS: Musculature Strength; Nulliparous; Pelvic Floor Muscle; Primiparous. Gameiro MO, Sousa VO, Gameiro LF, Muchailh RC, Padovani CR, Amaro JL. Comparison of pelvic floor muscle strength evaluations in nulliparous and primiparous women: a prospective study. Clinics. 2011;66(8):1389-1393. Received for publication on January 3, 2011; First review completed on February 28, 2011; Accepted for publication on May 12, 2011 E-mail: jamaro@fmb.unesp.br Tel.: 55 14 3811-6271

and 6% to 29% after delivery.3,4 The evaluation of the PFM is important to provide prophylaxis and improve treatment of PFM dysfunctions.5 The function of the PFM is evaluated using a simple, welltolerated and minimally invasive method that identifies whether there is correct muscular recruitment and predicts PFM dysfunction.6,7 However, the correlation of this method with objective evaluation methods and its reproducibility remain questionable.1,8 Several classifications for subjective evaluations of PFM strength have also been proposed.8,9 Evaluation with a perineometer is a reliable method to objectively assess the strength of the PFM.10 This evaluation is frequently performed in the supine or semi-sitting position to improve the comfort of the patient and the clinician.11 However, these positions may not be relevant to daily activities. Frawley et al.12 found that both although manometry and digital muscle testing were reliable tools for measuring the maximum voluntary contraction in lying and upright positions, manometry exhibited a greater reliability.

INTRODUCTION The pelvic floor muscles (PFM) may be exposed to alterations during different phases of a woman’s life, such as pregnancy, the postpartum period, and physiological aging (menopause). These factors can impair the integrity of the PFM and lead to urinary incontinence (UI).1 During pregnancy, the female body undergoes several adaptive modifications. Mechanical and hormonal factors due to pregnancy can predispose women to UIs, which increase the number of micturitions and worsen urinary urgency and any preexisting stress urinary incontinence (SUI).2 The effects of normal gestation on the physiology of the urinary tract have not been completely elucidated. The prevalence of UIs ranges from 23% to 67% during gestation

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The identification and standardization of PFM strength in nulliparous women of different ages can help predict urinary and fecal incontinence as well as sexual dysfunction in a woman’s first pregnancy. However, this correlation is not well established because there have been few studies describing reference values for PFM strength.13 Because there is little information on this subject in the literature, we compared the PFM strength of nulliparous and primiparous women using subjective and objective evaluation methods.

The objective measurements were obtained using a Dynamed portable perineometer (model DM 01) with the volunteers in three different positions: supine with the lower limbs straight (P1), supine with the lower limbs bent (P2) and sitting (P3). After the participants were positioned, the examiner introduced a balloon catheter, sized 1162.6 cm, into the vagina. The balloon catheter was covered with an unlubricated condom and filled with 60 ml of air using a Plastipack syringe (Becton Dickinson, Sa˜o Paulo, Brazil), which permitted contact with the vaginal wall. The equipment was immediately zeroed, and the patient was asked to hold three PFM contractions as long as possible, with approximately 30-second rest intervals between them. The maximal peak of each contraction was registered in cmH2O. The lengths of these contractions in seconds were recorded with a Casio chronometer. The average of the measurements was used to avoid biased results. The vaginal catheters were disinfected after each evaluation by washing with liquid detergent and immersion in peracetic acid for 10 minutes. This procedure was standardized by the Infection Committee of the Clinical Hospital of Botucatu. The qualitative variables were analyzed using the Goodman Test for contrasts among and within multinomial populations.16 The Mann-Whitney Test was used for comparisons between quantitative variables and time points, along with a nonparametric variance model for repeated measurements. The Turkey Test and the Kruskal-Wallis nonparametric variance model complemented with the Dunn multiple comparisons test were used to analyze multiple comparisons.17 Statistical analysis was performed at the 5% significance level.

MATERIALS AND METHODS A total of 100 healthy women aged 20-30 years were evaluated and divided into two groups. Group 1 (G1) was composed of 50 nonpregnant nulliparous women, and Group 2 (G2) was composed of 50 primiparous women. This study was approved by the Ethical Research Committee of Medical School of Botucatu - UNESP (protocol no. 375/2007). All of the participants understood and signed an informed consent form. The sample size was established by considering a confidence level of 95%, a test power of 80% (based on comparisons in the literature) and a maximum estimated error of 5 cmH2O (based on pilot studies assessing objective evaluations of PFM strength). According to these calculations, each group consisted of approximately 50 participants. The exclusion criteria for G1 (nulliparous) were a current UI, neurological diseases, previous pelvic surgeries, diabetes, smoking, and cognitive difficulties. The same criteria were used for G2 (primiparous) in addition to gestational complications, such as diabetes, hypertension, and vaginal and urinary infections. The subjects were evaluated using a clinical questionnaire. The BMI of G1 was calculated and classified according to the World Health Organization (WHO) criteria.14 In G2, Atalah’s curve15 was used to evaluate the pregnant women’s BMIs at the 20th week of pregnancy. Subjective and objective PFM evaluations were performed at the initial evaluation in G1 and at the 20th and 36th weeks of pregnancy as well as 45 days after delivery in G2. The G2 subjects were divided into two subgroups according to type of delivery: vaginal (n = 17) and cesarean section (n = 19). For the subjective evaluations, the volunteers were placed in a supine position, undressed from waist to feet, covered with a sheet with the lower limbs bent and separated and instructed about the correct way to perform PFM contractions. The patients were evaluated by a single examiner, who used bidigital vaginal palpation in the anterior and posterior areas of the vaginal introitus. The patients were also required to contract the perineal muscles and to hold this contraction as long as possible. The degree of contraction strength was classified according to the description of Amaro et al.8 (Table 1), a system that has not been used prior to the present study.

RESULTS In G2, 14 women were excluded because they left the study before the follow-up examination. The mean age was 23 years in G1 and 22 years in G2. There was no significant difference between the groups. The average BMI was 21.7 kg/m2 in G1 and 25,0 kg/m2 in G2; there was a significant difference between the groups (p = 0.0004). All of the primiparous women had been continent before the pregnancy; however, 19% reported urinary incontinence episodes at the 20th week, 27% at the 36th week, and 36% at 45 days after delivery (p,0.05), which indicated a gradual increase in urinary incontinence episodes throughout the pregnancy. The subjective evaluations of the anterior and posterior regions of the PFM (Table 2) revealed no significant differences between the G1 subjects and the G2 subjects at the 20th week. There were significant reductions in PFM strength in the G2 women at the 36th week and 45 days after delivery compared to the G1 women (Table 2). The comparison of the groups evaluated objectively in the P1, P2, and P3 positions revealed no significant differences between the nulliparous women and the primiparous women at the 20th and 36th weeks of pregnancy (p.0.05) (Table 3). However, 45 days after vaginal delivery, a significant reduction in PFM strength in all the positions was observed in the primiparous women compared to the nulliparous women (p,0.05) (Table 4). There were no significant differences in the contraction time in the P1 position between the nulliparous and primiparous women when delivery type was not considered

Table 1 - Subjective classification of different degrees of PFM contraction.8 Degree 0 1 2 3

Digital palpation Absence of Muscular Contraction Light Contraction Mild Contraction – not held longer than 5 seconds Normal Contraction – held longer than 5 seconds

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Table 2 - Results of a subjective evaluation of anterior PFM strength in the nulliparous compared to the primiparous group at different time points using the classification system of Amaro et al.8 Subjective evaluation of PFM

Groups 1 (light) % (n) Nulliparous Primiparous 20 weeks Primiparous 36 weeks* Primiparous 45 days postpartum** *

0,00 5.55 19.44 30.55

(0) (2) (7) (11)

2 (mild) % (n)

3 (normal) % (n)

48 (24) 55.55 (2) 58.33 (21) 44.44 (16)

52 (26) 38.88 (14) 22.28(8) 25 (9)

Total 100 100 100 100

(50) (36) (36) (36)

p = 0.0006. p = 0.0001.

highest in the third trimester.22 This change is due to the increased concentration of relaxin hormone, which promotes the relaxation of the pelvic floor organs and reduces the resistance to stress on the perineum muscles. Simultaneously, high levels of progesterone cause hypotonicity, which has a relaxing effect on smooth muscles and can lead to urinary frequency and incontinence.22 Although the objective evaluation of PFM strength using a perineometer has been validated by the International Continence Society (ICS),10 there are several devices available with different strength units and probe sizes, which makes concordance among studies difficult.23 Furthermore, the effects of pregnancy, body weight, and body position on PFM strength have not been totally elucidated. There were no significant differences in PFM strength in any of the three different positions among the nulliparous and pregnant primiparous women. According to Scheer,24 the support of the pelvic organs weakens significantly following the first vaginal delivery, but not during pregnancy. However, the evaluation performed 45 days after delivery in the present study showed a significant decrease in muscular strength in all of the body positions in women who had undergone vaginal delivery. Baytur et al.25 evaluated nulliparous and primiparous women and observed reduced PFM strength after vaginal compared to cesarean delivery. Baessler and Schuessler26 also reported that the strength of this musculature was not changed in women who underwent cesarean delivery. This study is in agreement with the results of most research, which have associated vaginal delivery with PFM weakness. Using digital palpation, perineometry, and electromyography, Marshall et al.27 compared the PFM strength of nulliparous and primiparous women ten months after vaginal delivery and observed significantly higher muscular strength and endurance in the nulliparous women. In terms of the different body positions and time points, there were no significant differences in the PFM contraction time between the primiparous patients after vaginal

or when vaginal and cesarean deliveries were compared (Table 5). However, the contraction time in the P2 position was significantly lower in the primiparous women after cesarean deliveries compared to the nulliparous group (6.0ยก1.8 seconds vs. 7.1ยก1.3 seconds, respectively; p = 0.021), and it was also significantly lower in the P3 position when women with vaginal and cesarean deliveries were compared (8.6ยก2.9 seconds vs. 7.1ยก1.3 seconds, respectively; p = 0.016). There were no significant differences between nulliparous and primiparous women after vaginal delivery in the P2 or P3 positions (Table 5).

DISCUSSION The average BMI was significantly higher in the primiparous group compared with the nulliparous group, which suggests that obesity influenced the participants. A previous study18 has demonstrated that overweight and obesity are important risk factors for UIs. Alterations in the genital and urinary tracts may occur during pregnancy, delivery, and the puerperium. Mechanical and hormonal factors may influence urinary symptoms, e.g., by increasing the voiding frequency and worsening urge incontinence.19,20 The primiparous women in this study were continent prior to pregnancy and showed a gradual increase in urine loss episodes throughout the pregnancy. Amaro et al.8 validated PFM evaluation methods using digital vaginal palpation with a four-degree scale with an intensity range of 0-3. Bo et al.21 concluded that vaginal palpation was not a reproducible and valid method of measuring PFM strength. Despite the controversies and its low reproducibility, the vaginal palpation method of PFM evaluation is considered easy to use and minimally invasive in daily clinical practice. By using this evaluation, we observed significant reductions in the PFM strength of primiparous women at the 36th week of pregnancy and 45 days after delivery compared to the nulliparous group. The prevalence of UIs increases as pregnancy progresses and is

Table 3 - Maximum amplitude of PFM contraction (cmH2O) (median and range) in the nulliparous compared to the primiparous group at 20 weeks and 36 weeks of gestation measured using a perineometer in different positions: supine with the lower limbs straight (P1), supine with the lower limbs bent (P2), and sitting (P3). Groups

Positions

P1 P2 P3

G1 (n = 50)

G2 (n = 36) at 20 weeks of gestation

G2 (n = 36) at 36 weeks of gestation

Statistical results

16.3 (6.6-55.3) 15.6 (7.0-59.0) 19.3 (3.6-87.3)

17.6 (3.0-55.3) 15.8 (8.0-59.0) 19.0 (6.0-87.3)

11.8 (4.0-66.7) 11.8 (5.3-43.3) 16.5 (5.3-57.3)

p.0.05 p.0.05 p.0.05

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REFERENCES

Table 4 - Maximum amplitude of PFM contractions (cmH2O) (median and range) in the nulliparous compared to the primiparous 45 days postpartum group according to the type of delivery measured using a perineometer in different positions: supine with the lower limbs straight (P1), supine with the lower limbs bent (P2), and sitting (P3). Different lower-case letters indicate significant differences between groups at the same time point. Positions

1. Amaro J, Moreira E, Gameiro M, Padovani C. Pelvic floor muscle evaluation in incontinent patients. Int Urogynecol J Pelvic Floor Dysfunct. 2005;16:352–4, doi: 10.1007/s00192-004-1256-3. 2. Morkved S, Bø K. Prevalence of urinary incontinence during pregnancy and postpartum. J Pelvic Floor Dysfunc. 1999;10:394–8, doi: 10.1007/ s001920050067. 3. Hong P, Leong M, Selzer V. Uroflowmetric observation in pregnancy. Neurourol Urodyn. 1988;7:61–70, doi: 10.1002/nau.1930070107. 4. Goldberg R, Kwon C, Gandhi S, Atkuru L, Sand P. Urinary incontinence after multiple gestation and delivery: impact on quality of life. Int Urogynecol J Pelvic Floor Dysfunct. 2005;16:334–6, doi: 10.1007/s00192004-1252-7. 5. Peschers U, Fanger G, Schaer G. Bladder neck mobility in continent nulliparous women. Br J Obstet Gynecol. 2001;108:320-4, doi: 10.1016/ S0306-5456(00)00066-8. 6. Thompson J, O’Sullivan P, Briffa N, Neumann P. Assessment of voluntary pelvic floor muscle contraction in continent and incontinent women using transperineal ultrasound, manual muscle testing and vaginal squeeze pressure measurements. Int Urogynecol J Pelvic Floor Dysfunct. 2006;17:624–30, doi: 10.1007/s00192-006-0081-2. 7. Hundley A, Wu J, Visco A. A comparison of perineometer to brink score for assessment of pelvic floor muscle strength. Am J Obstet Gynecol. 2005;192:1583-91, doi: 10.1016/j.ajog.2004.11.015. 8. Amaro J, Gameiro M, Padovani C. Treatment of urinary stress incontinence by intravaginal electrical stimulation and pelvic floor physiotherapy. Int Urogynecol J Pelvic Floor Dysfunct. 2003;14:204–8, doi: 10.1007/s00192-003-1061-4. 9. Ortiz O, Nunez F, Ibañez G. Evaluation funcional del piso feminino (Classificacion Funcional). Bol Soc Latinoam Uroginecol Cir Vag. 1996; 1:5-9. 10. Messelink B, Benson T, Berghmans B, Bø K, Corcos J, Fowler C, et al. Standardization of Terminology of Pelvic Floor muscle function and dysfunction: report from the pelvic floor clinical assessment group of the International Continence Society. Neurourol Urodyn. 2005;24:374–80, doi: 10.1002/nau.20144. 11. Bø K, Finckenhagen HB. Is there any difference in measurement of pelvic floor muscle strength in supine and standing position? Acta Obstet Gynecol Scand. 2003; 82:1120-4, doi: 10.1046/j.1600-0412.2003.00240.x. 12. Frawley HC, Galea MP, Phillips BA, Sherburn M, Bo K. Reliability of pelvic floor muscle strength assessment using different test positions and tools. Int Urogynecol J Pelvic Floor Dysfunct. 2006;25:236–42. 13. Rosenbaum T. Pelvic floor involvement in male and female sexual dysfunction and the role of pelvic floor rehabilitation in treatment: a literature review. J Sex Med. 2007;4:4-3, doi: 10.1111/j.1743-6109.2006. 00393.x. 14. World Health Organization [homepage on the Internet] (2006) BMI Classification. Geneva: WHO [cited 2008 nov1 12]. Available from: www.who.int/bmi 15. Atalah. Propuesta de un nuevo estańdar de evaluacioń nutricional en embarazadas. Rev Me´d Chile. 1997;125:1429–36. 16. Goodman LA. Simultaneus confidence intervals for multinomial proportions. Technometrics. 1965;7:247–54, doi: 10.2307/1266673. 17. Zar JH. Biostatical analysis. New Jersey : Prentice Hall, 5th Ed., 2009;1960. 18. Subak LL, Richter HE, Hunskaar S. Obesity and urinary incontinence: Epidemiological and clinical research update. J Uro Dec. 2009;182(6 Suppl):S2-7, doi: 10.1016/j.juro.2009.08.071. 19. Wesnes SL, Rortveit G, Bø K, Hunskaar S. Urinary incontinence during pregnancy. Obstet Gynecol. 2007;109:922–8, doi: 10.1097/01.AOG. 0000257120.23260.00. 20. Chin HY, Chen MC, Liu YH. Postpartum urinary incontinence: a comparison of vaginal delivery, elective, and emergent cesarean section. Int Urogynecol J Pelvic Floor Dysfunct. 2006;17:631-5, doi: 10.1007/ s00192-006-0085-y. 21. Bo K, Finckenhagen HB. Vaginal palpation of pelvic floor muscle strength: inter-test reproducibility and comparison between palpation and vaginal squeeze pressure. Acta Obstet Gynecol Scand. 2001;80: 883-7. 22. Fitzgerald MP, Graziano S. Anatomic and functional changes of the lower urinary tract during pregnancy. Urol Clin N Am. 2007;34:7-12, doi: 10.1016/j.ucl.2006.10.007. 23. Barbosa PB, Franco MM, Souza FO, Antoˆnio FI, Montezuma T, Ferreira CHJ. Comparison between measurements obtained with three different perineometers. Clinics. 2009;64:527-33, doi: 10.1590/S1807-59322009000600 007. 24. Scheer I, Thakar R, Sultan AH, Jones PW. Does pregnancy and delivery affect pelvic organ support?Pregnancy and prolapse assessement [PAPA] study. In:37thAnnual Meeting of the International Continence Society (ICS). Rotherdan, Bristol. 2007. 25. Baytur YB, Deveci A, Uyar Y, Ozcakir HT, Kizilkaya S, Caglar H. Mode of delivery and pelvic floor muscle strength and sexual function after

Groups G1 (n = 50)

16.3 (6,6-55.3)b

15.6 (7.0-59,0)b

19.3 (3.6-87.3)b

G2 (n = 36) VD 8.3 CD 11.0 VD 8.3 CD 13.7 VD 10.3 CD 16.0

(3.6-32.6)a (4.7-46.3)ab (4.6-23.7)a (5.3-39.3)ab (6.6-26.3)a (5.3-43.3)ab

Statistical results p = 0.012 p.0.05 p = 0.011 p.0.05 p = 0.002 p.0.05

VD = Vaginal delivery (n = 17). CD = Cesarean delivery (n = 19).

delivery and the nulliparous women. However, we observed a significantly shorter contraction time in the primiparous women who underwent cesarean deliveries compared to the nulliparous women. One possible explanation is that compromising the rectus abdominis muscle during cesarean delivery impairs the sustained contraction of the PFM and reduces the contraction time. Other authors28,29 have suggested that the synergistic activity of the abdominal muscles and the PFM is important for maintaining adequate urethral closing pressure. Despite all of the limitations and difficulties in performing this type of study, further research is necessary to provide more information about the PFM strength in pregnant women compared to a control group, such as nulliparous women.

CONCLUSION During pregnancy and after vaginal delivery, women demonstrated weaker PFM compared to nulliparous controls. Table 5 - Length of PFM contractions (cmH2O) (seconds) (mean and standard deviation) in the nulliparous group compared to the primiparous 45 days postpartum group according to the type of delivery, measured using a perineometer in different positions: supine with the lower limbs straight (P1), supine with the lower limbs bent (P2), and sitting (P3). Different lower-case letters indicate significant differences between the groups at the same time point. Groups

Positions G1 (n = 50) P1

7.3¡1.7a

7.1¡1.3b

7.7¡1.7ab

G2 (n = 36) VD CD VD CD VD CD

7.3¡1.6a 6.7¡2.0a 7.3¡2.0b 6.0¡1.8a* 8.6¡2.9b 7.1¡1.3a**

Statistical results p.0.05 p.0.05 p.0.05 p = 0.021 p.0.05 p.0.05

VD = Vaginal delivery (n = 17). * p = 0.045. ** p = 0.016. CD = Cesarean delivery (n = 19).

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Pelvic floor muscle strength Gameiro MO et al. 28. Neumann P, Gill V. Pelvic floor and abdominal muscle interaction: EMG activity and intra-abdominal pressure. Int Urogynecol J Pelvic Floor Dysfunct. 2002;13:125-32, doi: 10.1007/s0019202000 27. 29. Maddil SJ, Mclean L. Relationship between abdominal and pelvic floor muscle activation and intravaginal pressure during pelvic floor muscle contractions in healthy continent women. Neurourol Urodyn. 2006;25:722-30, doi: 10.1002/nau.20285.

childbirth. Int J Gynecol Obstet. 2005;88:276-80, doi: 10.1016/j.ijgo.2004. 12.019. 26. Baessler K, Schuessler B. Childbirth-induced trauma to the urethral continence mechanism: review and recommendations. Urology. 2003; 62:39-44, doi: 10.1016/j.urology.2003.08.001. 27. Marshall K, Walsh DM, Baxter GD. The effect of a first vaginal delivery on the integrity of the pelvic floor musculature. Clin Rehabil. 2002;16:795-9, doi: 10.1191/0269215502cr556oa.

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DOI:10.1590/S1807-59322011000800015

CLINICAL SCIENCE

Effects of a multidisciplinar cognitive rehabilitation program for patients with mild Alzheimer’s disease Luciane F. Viola,I Paula V. Nunes,I Monica S. Yassuda,II Ivan Aprahamian,I Franklin S. Santos,III Glenda D. Santos,I Paula S. Brum,I Sheila M. Borges,I Alexandra M. Oliveira,I Gisele F. S. Chaves,I Eliane C. Ciasca,I Rita C. R. Ferreira,I Vanessa J. R. de Paula,I Oswaldo H. Takeda,I Roberta M. Mirandez,I Ricky Watari,I Deusivania V. S. Falca˜o,I,II Meire Cachioni,I,II Orestes V. ForlenzaI I

Laboratory of Neuroscience – LIM 27, Department and Institute of Psychiatry, Faculdade de Medicina da Universidade de Saõ Paulo, SP, Brazil. II School of Arts, Sciences and Humanities, Universidade de Saõ Paulo, SP, Brazil. III Clinical Emergencies - Hospital das Clıńicas da Faculdade de Medicina da Universidade de Saõ Paulo, SP, Brazil.

OBJECTIVE: To evaluate the effects of a multidisciplinary rehabilitation program on cognition, quality of life, and neuropsychiatric symptoms in patients with mild Alzheimer’s disease. METHOD: The present study was a single-blind, controlled study that was conducted at a university-based dayhospital memory facility. The study included 25 Alzheimer’s patients and their caregivers and involved a 12-week stimulation and psychoeducational program. The comparison group consisted of 16 Alzheimer’s patients in waiting lists for future intervention. INTERVENTION: Group sessions were provided by a multiprofessional team and included memory training, computer-assisted cognitive stimulation, expressive activities (painting, verbal expression, writing), physiotherapy, and physical training. Treatment was administered twice a week during 6.5-h gatherings. MEASUREMENTS: The assessment battery comprised the following tests: Mini-Mental State Examination, Short Cognitive Test, Quality of Life in Alzheimer’s disease, Neuropsychiatric Inventory, and Geriatric Depression Scale. Test scores were evaluated at baseline and the end of the study by raters who were blinded to the group assignments. RESULTS: Measurements of global cognitive function and performance on attention tasks indicated that patients in the experimental group remained stable, whereas controls displayed mild but significant worsening. The intervention was associated with reduced depression symptoms for patients and caregivers and decreased neuropsychiatric symptoms in Alzheimer’s subjects. The treatment was also beneficial for the patients’ quality of life. CONCLUSION: This multimodal rehabilitation program was associated with cognitive stability and significant improvements in the quality of life for Alzheimer’s patients. We also observed a significant decrease in depressive symptoms and caregiver burden. These results support the notion that structured nonpharmacological interventions can yield adjunct and clinically relevant benefits in dementia treatment. KEYWORDS: Alzheimer’s disease; Treatment; Rehabilitation; Cognition; Quality of life. Viola LF, Nunes PV, Yassuda MS, Aprahamian I, Santos FS, Santos GD, et al. Effects of a multidisciplinar cognitive rehabilitation program for patients with mild Alzheimer’s disease. Clinics. 2011;66(8):1395-1400. Received for publication on May 17, 2011; First review completed on May 17, 2011; Accepted for publication on May 25, 2011 E-mail: forlenza@usp.br Tel.: 55 11 3069-7267

treatment in addition to standard outpatient care is an asset of good clinical practice. The clinical perception supports that nonpharmacological interventions of various kinds may be helpful in the long-term global management of the disease. More controlled studies are required, however, to yield evidence-based information on the effectiveness of cognitive rehabilitation. Rehabilitation is a process of active change that allows disabled people to reach an ideal level of physical, psychological, and social functioning in the presence of ongoing or previous disease or impairment.3,4 Cognitive

INTRODUCTION Given the progressive, irreversible nature of Alzheimer’s disease (AD) and the limited symptomatic benefits delivered by pharmacotherapy,1,2 the provision of nonpharmacological

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approved by the local ethics committee, and informed consent was obtained from patients and/or caregivers. Participants were referred from outpatient units dedicated to psychogeriatric care at the same institution. In addition, some patients were referred from other sources in the local community. Inclusion criteria were mild dementia, which was indicated by a score of 0.5 or 1.0 in the Clinical Dementia Rating Scale (CDR)19 and a score of 16 or more in the Minimental State Examination (MMSE),20 and concomitant standard pharmacological treatment for AD (i.e., use of cholinesterase inhibitors and/or memantine in stable therapeutic doses for at least three months).

rehabilitation refers to the use of techniques to improve performance for specific mental functions,5 whereas neuropsychological rehabilitation, in a broader sense, aims to help patients and their family members deal with the cognitive, emotional, and social burden of the disease, thereby ultimately improving the quality of life.6,7 Several methods targeting cognition and functionality have been proposed for patients with AD dementia.8-11 Most of these techniques involve multiprofessional teamwork and include restructuring of the home environment, nutritional advice, physical activities, psychological counseling, support for family members and caregivers, and neuropsychological rehabilitation.12 Because AD is a progressive neurodegenerative disorder, cognitive stimulation rather than rehabilitation per se may be a more appropriate term to refer to the possible interventions within the context of this disease.13 The comparison among distinct studies using cognitive stimulation techniques is sometimes difficult because of a wide variability in the type, complexity, and duration of interventions; lack of uniformity in target cognitive functions and training protocols; small sample sizes in the majority of studies; unavailability of comparison groups; and other methodological constraints. As a whole, however, physicians generally accept that cognitive stimulation can improve cognition and behavioral symptoms of patients with dementia.1 Loewenstein et al.14 showed that a combination of cognitive and functional rehabilitation with pharmacological treatment with cholinesterase inhibitors promoted cognitive and functional stability for patients with mild AD. Similarly, Talassi et al.15 recently found that systematic cognitive training may optimize the benefits of pharmacological treatment in patients with early-stage AD. Interestingly, no training-related improvement in memory was obtained from a 12-week multimodular cognitive rehabilitation program in older adults.16 Spector and colleagues17 published a multicenter, randomized, placebocontrolled study on psychosocial interventions in AD showing that patients who received a 14-session reality orientation and cognitive stimulation schedule had significant benefits in cognition and quality of life. In another study, Raggi et al.10 administered a multidisciplinary rehabilitation program to AD patients in a hospital setting that used reality orientation and computerized cognitive training to stimulate attention, language, numerical and spatial skills, psychomotor speed, and memory. The intervention yielded significant improvements in activities of daily living, neuropsychiatric symptoms, and cognition. The objective of the present study was to evaluate the effect of a multifunctional stimulation program on cognition, neuropsychiatric symptoms, and quality of life in patients with mild AD in a controlled, single-blind design. We also addressed the benefits of this intervention for the mental health parameters of caregivers.

Measurements All patients and controls were submitted to clinical, cognitive, and quality of life assessments by two independent raters who were blinded to the group assignments. The same instruments were used at the baseline and the endpoint. The assessment battery included the MMSE,20 which is a brief cognitive screening test that evaluates temporal and spatial orientation, memory, attention, calculation, language, and visuoconstructive capacity; the Short Cognitive Test (SKT),21 which is a broad cognitive screening battery that addresses attention, processing speed, and memory; the Neuropsychiatric Inventory (NPI),22 which evaluates the presence of 10 psychiatric symptoms (i.e., delusions, hallucinations, irritability, disinhibition, agitation, anxiety, depression, euphoria, apathy, and psychomotor abnormalities); the Geriatric Depression Scale (GDS),23 and the Quality of Life in Alzheimer’s Disease Evaluation Scale (QoL-AD).24 The caregivers were assessed with the GDS and the caregiver’s protocol of the QoL-AD, which assessed the caregivers’ perceptions of their patient’s quality of life. Although the rehabilitation program included physical training, and physiotherapy (as detailed below), the assessment protocol did not include direct measures of these intervention components because the primary goal of the study was to investigate the effects of multiprofessional intervention on cognitive, functional, psychiatric, and quality of life outcome variables. The lack of direct measures, however, can be regarded as a limitation of the study.

Intervention Four distinct intervention groups were formed (one per semester), and each group contained a maximum of 12 patients plus their respective caregivers. The first 12 patients to be referred to the service were assigned to the experimental group, and the following 12 patients were assigned to the waiting-list control group. In the second wave of intervention, subjects in the first control group were assigned to the second experimental group, and the next referrals constituted the second control group. This procedure was repeated for the subsequent waves of intervention. This recruitment strategy was chosen to avoid delays in treatment to patients formerly allocated in the control group (i.e., intervention was provided within a maximum time-lag of 6 months from referral). The final sample consisted of 25 patients in the experimental group and 16 patients in the control group. Five patients with moderate dementia (CDR = 2) were referred to our service during the recruitment phase. Although these patients did not meet inclusion criteria (their data were not

METHODS Participants and setting Forty-one AD patients, who were diagnosed according to the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer’s Disease and Related Disorders Association (NINCDS-ADRDA)18 criteria, and their respective caregivers were recruited at the memory clinic of the Psychogeriatric Unit of the Institute of Psychiatry between August 2007 and June 2009. The study was

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preventing falls through exercises administered to patients. In addition, caregivers were also coached to reinforce exercises and habits at home. Physical training was offered as a complementary program for physically able patients to improve physical conditioning. Special emphasis was placed on motor, emotional, social, and cognitive aspects. In addition to strength and balance exercises, patients were invited to group walks and stretching sessions. Speech therapy was also provided to enhance general communication and communication strategies in specific daily-life situations. The sessions also involved cognitive stimulation through logic games (e.g., simplified chess and related games), which consisted of preliminary lessons on the rules and objectives of the games and was designed to improve concentration, logical-mathematical reasoning, rapid thinking, and decision making.

included in the analysis), participation in all treatment sessions was granted for ethical reasons. From the total sample of 41 patients, five subjects failed to reach the experimental endpoint: one died, two were unable to comply with all sessions because of limited accessibility, and two dropped out for personal reasons. Compliant patients were able to attend at least 90% of the treatment sessions (i.e., 22 from a total of 24 treatment days). Caregivers were mostly represented by family members (90.2%) of AD patients. Professional caregivers were expected to spend at least 12 h a day with their patients. The intervention was administered in group sessions offered twice a week at the day-hospital facilities for 12 consecutive weeks. Sessions lasted from 9:00 AM to 3:30 PM (lunch and refreshments were provided and lasted 90 minutes), and the 24 sessions resulted in a total of 120 h of intervention (5 h daily). The program consisted of the following activities: cognitive rehabilitation, computerassisted cognitive training, speech therapy, occupational therapy, art therapy, physical training, physiotherapy, and cognitive stimulation with reading and logic games. Each activity lasted for 60-90 minutes and was offered once a week (Table 1). Psychoeducation and psychological counseling were provided in group sessions for caregivers twice a week. The purpose of these meetings was to explain the clinical aspects of the disease by focusing on its progressive course and the expected loss of autonomy of the patient. The exchange of personal accounts and experiences among participants was also encouraged. Patients on the waiting list for forthcoming intervention groups (control group) received standard outpatient care, including monthly follow-up visits to the memory clinic. The rehabilitation sessions contained a variety of components. Cognitive rehabilitation was delivered through exercises to improve attention, memory, spatial and temporal orientation, and self-adaptations to cognitive impairment. Computer-assisted cognitive training consisted of a preparatory session (in which participants were made familiar with the use of the computer) followed by the engagement in different tasks that primarily consisted of memory and attention games (user-friendly and adjustable for complexity). Art therapy aimed at stimulating cognitive, emotional, and interpersonal skills through expressive and artistic techniques, and a special emphasis was placed on nonverbal expression that was devoid of any critical judgment of the actual quality of the artistic work. Occupational therapy was intended to develop resources and strategies to improve the completion of functional goals, including training in basic (hygiene, feeding, getting dressed) and instrumental activities of daily living (paying bills, shopping, leisure, and social activities). In addition, patients and caregivers were instructed about the need of household adaptations to enhance orientation and autonomy. Physiotherapy was aimed at improving balance and

Statistical analysis The Kolmogorov-Smirnov test was used to determine whether study variables followed a normal distribution, which supported the use of parametric tests (t test for two independent samples) in the analysis of outcomes. Chisquare (when necessary, adjusted by the Monte Carlo method) and independent-sample t tests were used to assess the similarity between experimental and control groups at baseline. Paired-sample t tests were used to address changes from the baseline within each group.

RESULTS No significant differences were observed in the mean age and education level between patients in the experimental and control groups. The average age of the patients was 75 years, and the patients had an average of 10 years of schooling. In addition, there were slightly more females in each group (experimental group, 64%; control group, 62%, p = 0.9). All patients were either married or widowed, and more patients in the experimental group group were married (56%, N.S.). At baseline, no significant differences between experimental and control groups were found with respect to mean scores in the various psychometric tests pertaining to the assessment battery. Nine patients in the experimental group and 7 in the control group had CDR = 0.5, whereas 16 patients in the experimental group and 9 in the control group had CDR = 1. Fisherâ&#x20AC;&#x2122;s exact test did not indicate any significant difference between the CDR scores of experimental and control groups. Most caregivers were family members (34.1% spouses, 56.1% children, 9.8% formal caregivers). The mean age of the caregivers was 51.6 years (15.3 SD), and the mean educational level was 9.1 years (4.1 SD). There were no significant differences for age and education between caregivers in the experimental and control groups (p = 0.996 and p = 0.423, respectively).

Table 1 - Schedule of activities (experimental group). Time 9:00-10:30 AM 10:30-12:00 AM 12:00-1:00 PM 1:00-2:00 PM 2:00-2:30 PM 2:30-3:30 PM

Tuesdays

Time

Thursdays

Cognitive rehabilitation (computer-assisted) Art therapy Lunch Occupational therapy Rest Physiotherapy

9:00-10:30 AM 10:30-12:00 AM 12:00-1:00 PM 1:00-2:00 PM 2:00-2:30 PM 2:30-3:30 PM

Cognitive rehabilitation Physical training Lunch Logic games Rest Speech therapy

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the experimental group remained stable, whereas those in the comparison group had a mild but significant worsening in attention and global performance. We understand that this effect is plausible given the progressive nature of the disease, which can render untreated patients prone to cognitive deterioration over time. Although some cognitive changes may not have been detected by the MMSE in the relatively short duration of this study (i.e., three months), cognitive changes were seen in the SKT. Indeed, there were significant changes in the total score and the attention subscore, which indicated subtle worsening of patients in the control group. Studies involving combined pharmacological and nonpharmacological treatment with multimodal stimulation programs reported slight improvements in MMSE scores.8,10 Most studies that have evaluated the effect of nonpharmacological interventions in AD observed stabilization or, at most, modest improvement of cognitive functions.1 Conversely, untreated patients may show cognitive decline. Our results are in agreement with the findings of previous studies, which have suggested that robust changes in cognition are unlikely to occur as a consequence of cognitive training in patients with AD. Gains tend to be modest and may be best documented as a slight improvement in certain cognitive domains.16,25 Compared with the MMSE, the SKT may be more sensitive to subtle changes because it is a more comprehensive cognitive assessment battery and takes into account processing speed and response accuracy.21,26 In addition, the availability of five parallel versions of the test makes it less prone to learning effects upon retesting, which is an important issue in longitudinal studies.27 Although well-established outcome measures have become available in pharmaceutical trials in recent years, the effects of nonpharmacological interventions may not be properly identified by commonly used psychometric tests. Interestingly, functional or quality-of-life outcome measures may be better tools to measure nonpharmacological effects. Thus, negative data based on quantitative testing must be interpreted with caution, particularly in light of the clinical experience, which suggests that qualitative benefits to global function are observed in the close, continuous management of AD patients. Benefits associated to nonpharmacological interventions may encompass minor, nonsignificant changes in test scores, and we understand that the modification of noncognitive functions may partially explain the clinical perception of change. In a recent study conducted by our group, Machado et al.28 suggested that the impressions of changes in the quality of life in patients with AD can be better depicted by a qualitative analysis of patientsâ&#x20AC;&#x2122; reports than by objective test scores. Accordingly, a relevant outcome of the present study was the reduction of depressive symptoms in patients who received the intervention along with their caregivers. This finding is clinically relevant and is in line with similar studies in the literature1,10,29 (i.e., there is a high rate of depression in caregivers of AD patients).30-32 In addition, the incidence of neuropsychiatric symptoms was low in both groups (there was no significant difference between the groups), which seemed to be because our sample only contained patients with mild dementia (i.e., those who were less likely to present with important behavioral manifestations). Interestingly, participation in the program resulted in a mild reduction in caregiver distress. In a recent study that

Table 2 displays the mean values and standard deviations for psychometric test scores at baseline and at the end of the study. Paired-sample t tests addressing within-group differences (baseline vs. endpoint) in test scores showed that patients in the control group had a tendency for cognitive decline, which was indicated by a slight, but significant, increase in total SKT scores and in the attention SKT subscore (i.e., higher scores in the SKT mean worse performance). Conversely, patients in the experimental group remained stable with respect to the aforementioned variables. Although the MMSE scores remained unchanged in both groups (irrespective of treatment), the intervention was associated with a significant reduction in GDS scores, which indicated improvement in depressive symptoms from both the patientsâ&#x20AC;&#x2122; and caregiversâ&#x20AC;&#x2122; perspectives. In addition, there was a significant decrease in caregiver distress, which was indicated by a reduction in the NPI distress subscore. Interestingly, patients and caregivers in the experimental group also reported an improvement in their quality of life according to the QoL-AD.

DISCUSSION This was a single-blind, controlled study addressing the effects of a nonpharmacological treatment program in AD patients with mild dementia. The intervention consisted of an extensive, multimodal stimulation program that primarily focused on the rehabilitation of cognitive abilities; however, the ultimate goal was to promote well being and improve quality of life. In regards to cognition, patients in

Table 2 - Psychometric test scores at baseline and after intervention (endpoint). Variable Mini-Mental State Examination Short Cognitive Test (SKT) Total SKT score Memory subscore Attention subscore Neuropsychiatric Inventory Total score Distress subscore

Group

Baseline#

Endpoint p-value*

CG EG

23.3 (3.9) 22.6 (2.9)

22.4 (2.8) 22.5 (3.8)

0.1 0.9

CG EG CG EG CG EG

12.6 (5.4) 14.5 (5.4) 5.5 (1.9) 5.2 (2.2) 7.1 (5.0) 9.3 (4.3)

13.8 (5.5) 14.6 (6.1) 5.2 (2.2) 4.9 (2.6) 8.6 (4.8) 9.6 (4.7)

0.05 0.9 0.4 0.5 0.01 0.5

CG EG CG EG

36.5 (23.9) 28.7 (18.5) 27.5 (22.4) 25.9 (20.8) 13.5 ( 9.1) 13.6 (9.2) 11.7 (8.9) 9.9 (7.9)

0.1 0.4 0.9 0.02

Geriatric Depression Scale (GDS) Patient CG EG Caregiver CG EG Quality of Life in AD Scale Patient CG EG Caregiver CG EG

4.3 4.7 4.0 3.9

(3.2) (3.1) (3.2) (3.5)

4.7 3.4 3.9 3.1

(3.4) (3.0) (3.3) (2.9)

0.7 0.001 0.9 0.02

36.1 35.2 31.1 30.8

(5.8) (5.0) (7.4) (5.2)

35.4 37.3 32.7 33.0

(6.1) (4.4) (6.6) (6.0)

0.5 0.004 0.3 0.04

EG, experimental group; CG, control group. Values represent means and standard deviations (SD) of test scores; * p-values in the right column: paired-sample t tests addressing withingroup differences (baseline vs. endpoint) in test scores (significant differences are shown in bold). # Independent-sample t tests comparing mean scores at baseline yielded nonsignificant differences between EG and CG.

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was also conducted in Brazil, Camara et al.33 reported decreases in caregiver distress as an indirect benefit of the rehabilitation of patients with AD. Caring for patients with dementia is highly stressful because of the progressive loss of autonomy and the presence of behavioral symptoms associated with AD. Thus, psychoeducation and psychological counseling, which was offered biweekly in the present study, may have contributed to the reported benefits, even in the absence of significant changes in the presentation of the disease.34,35 Moreover, we found significant improvements in the quality of life for both patients and caregivers after the intervention. This outcome corroborated earlier studies10,28,29,36 suggesting that improvements in quality of life can be demonstrated by objective measures (to a certain extent). The relatively small sample of patients, especially in the control group, was a limitation of the present study. In addition, the current intervention program required a significant investment of time and human resources to deliver the therapeutic sessions that may not be available in most settings because of the requirement for specialized training. Future studies should investigate the contribution of individual rehabilitation components to the outcome measures because the present investigation only assessed the impact of the global rehabilitation protocol. In addition, there may have been some training redundancies in the format of the present study (e.g., in the computer assisted training and cognitive rehabilitation components and in the physical training and physiotherapy components). In future protocols, these rehabilitation modalities might be merged to guarantee the best use of financial and human resources.

7. Avila R, Bottino CMC, Carvalho IAM, Santos CB, Seral C, Miotto EC. Neuropsychological rehabilitation of memory deficits and activities of daily living patients with Alzheimer’s disease: a pilot study. Braz J Med Biol Res. 2004;37:1721-9, doi: 10.1590/S0100-879X2004001100018. 8. Bottino CMC, Carvalho IAM, Alvarez AMMA, Avila R, Zukauskas PR, Bustamante SE, et al. Cognitive rehabilitation combined with drug treatment in Alzheimer’s disease patients: a pilot study. Clin Rehabil. 2005;19:861-9, doi: 10.1191/0269215505cr911oa. 9. Acevedo A, Loewenstein DA. Nonpharmacological cognitive interventions in aging and dementia. J Geriatr Psychiatry Neurol. 2007;20(4):23949, doi: 10.1177/0891988707308808. 10. Raggi A, Iannaccone S, Marcone A, Ginex V, Ortelli P, Nonis A, et al. The effects of a comprehensive rehabilitation program of Alzheimer’s Disease in a hospital setting. Behav Neurol. 2007;18:1-6. 11. Miotto EC, Serrao VT, Guerra GB, Lu´cia MCS, Scaff M. Cognitive rehabilitation of neuropsychological deficits and mild cognitive impairment. Dement Neuropsychol. 2008;2:139-45. 12. Bottino CMC, Carvalho IAM, Alvarez AMMA, Avila R, Zukauskas PR, Bustamante SE, et al. Cognitive rehabilitation in Alzheimer’s disease patients: multidisciplinary team report. Arq Neuropsiquiatr. 2002;60:709, doi: 10.1590/S0004-282X2002000100013. 13. Grandmaison E, Simard M. A critical review of memory stimulation programs in Alzheimer’s disease. J Neuropsychiatry Clin Neurosci. 2003;15:130-44, doi: 10.1176/appi.neuropsych.15.2.130. 14. Loewenstein DA, Acevedo A, Czaja SJ, Duara R. Cognitive rehabilitation of mildly impaired Alzheimer disease patients on cholinesterase inhibitors. Am J Geriatr Psychiatry. 2004;12:395-402. 15. Talassi E, Guerrecshi M, Feriani M, Fedi V, Bianchetti A, Trabucchi M. Effectiveness of a cognitive rehabilitation program in mild dementia and mild cognitive impairment: a case control study. Arch Gerontol Geriatr. 2007;44:391-9, doi: 10.1016/j.archger.2007.01.055. 16. Craik FIM, Winocur G, Palmer H, Binns MA, Edwards M, Bridges K, et al. Cognitive rehabilitation in the elderly: effects on memory. J Int Neuropsychol Soc. 2007;13:132-42, doi: 10.1017/S1355617707070166. 17. Spector A, Woods B, Orrel M. Cognitive stimulation for the treatment of Alzheimer’s disease. Expert Rev Neurother. 2008;8(5):751-7, doi: 10. 1586/14737175.8.5.751. 18. McKhann G, Drachman D, Folstein M, Katzman R, Price D, Stadlan EM. Clinical diagnosis of Alzheimer’s disease – Report from the NINCDSADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer’s Disease. Neurology. 1984;34:939-44. 19. Morris JC. The Clinical Dementia Rating (CDR): current version and scoring rules. Neurology. 1993;43:2412-14. 20. Folstein MF, Folstein SE, McHugh PR. Mini-mental state: a practical method for grading the cognitive state of patients for the clinician. J Psychiatr Res. 1975;12:189-98, doi: 10.1016/0022-3956(75)90026-6. 21. Flaks MK, Forlenza OV, Pereira FS, Viola LF, Yassuda MS. Short Cognitive Performance Test: diagnostic accuracy and education bias in older Brazilian adults. Arch Clin Neuropsychol. 2009;24:301-6, doi: 10. 1093/arclin/acp033. 22. Cummings JL, Mega M, Gray K. The Neuropsychiatric Inventory: comprehensive assessment of psychopathology in dementia. Neurology. 1994;44:2308-14. 23. Shiekh JI, Yesavage JA. Geriatric Depression Scale (GDS): recent evidence and development of a shorter version. Clin Gerontol. 1986;5:165-73, doi: 10.1300/J018v05n01_09. 24. Novelli MM, Dal Rovere HH, Nitrini R, Caramelli P. Cross-cultural adaptation of the quality of life assessment scale on Alzheimer disease. Arq Neuropsiquiatr. 2005;63:201-6, doi: 10.1590/S0004-282X20050002 00002. ´ vila R, Carvalho IAM, Bottino CMC. Neuropsychological rehabilitation 25. A in mild and moderate Alzheimer’s disease patients. Behav Neurol. 2007;18:225-33. 26. Diniz BS, Nunes PV, Yassuda MS, Pereira FS, Flaks MK, Viola LF, et al. Mild cognitive impairment: cognitive screening or neuropsychological assessment? Rev Bras Psiquiatr. 2008;30:316-21, doi: 10.1590/S151644462008000400003. 27. Flaks MK, Yassuda MS, Regina ACB, Cid CG, Camargo CH, Gattaz WF, et al. The Short Cognitive Performance Test (SKT): a preliminary study of its psychometric properties in Brazil. Int Psychogeriatr. 2006;18:121-33, doi: 10.1017/S1041610205002577. 28. Machado F, Nunes PV, Viola LF, Santos FS, Forlenza OV, Yassuda MS. Quality of life and Alzheimer’s disease. Influence of participation at a rehabilitation center. Dement Neuropsychol. 2009;3:241-7. 29. Rozzini L, Costardi D, Chilovi BV, Franzoni S, Trabucchi M, Padovani A. Efficacy of cognitive rehabilitation in patients with mild cognitive impairment treated with cholinesterase inhibitors. Int J Geriatr Psychiatry. 2007;22:356-60, doi: 10.1002/gps.1681. 30. Engelhardt E, Brucki SMT, Cavalcanti JLS, Forlenza OV, Laks J, Vale FA. Treatment of Alzheimer’s disease: recommendations and suggestions of the Scientific Department of Cognitive Neurology and Aging of the Brazilian Academy of Neurology. Arq Neuropsiquiatr. 2005;63:1104-12, doi: 10.1590/S0004-282X2005000600035.

CONCLUSION The current multimodal stimulation program was beneficial for patients with mild AD. Future studies that are based on larger, more homogeneous samples and use more rigorous randomization methods should be pursued in this field of research.

ACKNOWLEDGEMENTS This work was supported by the Associac¸a˜o Beneficente Alzira Denise Hertzog da Silva (ABADHS) and by an institutional grant from SantanderReal Bank (Talentos da Maturidade, Cat. Programas Exemplares).

REFERENCES 1. Spector A, Orrel M, Davies S, Woods B. Can reality orientation be rehabilitated? Development and piloting of an evidence-based programme of cognition-based therapies for people with dementia. Neuropsychol Rehabil. 2001;11:377-97. 2. De Vreese LP, Iacono S, Finelli C, Gianelli MV, Neri M. Enhancement of therapeutic effects of drug treatment in DAT when combined with cognitive training? Preliminary results of a three month program. Neurobiol Aging. 1998;19:212-3. 3. Yassuda MS, Flaks MK. Revisaõ crı´tica de programas de reabilitaçaõ cognitiva para pacientes com demeˆncia. In: Forlenza OV, editor. Psiquiatria Geria´trica: do Diagno´stico a` Reabilitaçaõ. 1st edn. Atheneu: Saõ Paulo; 2007;411-22. 4. McLellan DL. Functional recovery and the principles of disability medicine. In: Swash M, Oxbury J, editors. Clinical neurology. Churchill Livingstone: London; 1991;p.768-90. 5. Nomura S, Garcia JL, Fabrıćio AM, Bolognani SAP, Camargo CHP. Reabilitaçaõ neuropsicolo´gica. In:Forlenza OV, Caramelli P, editores. Neuropsiquiatria Geria´trica. Atheneu: Saõ Paulo; 2000;p.539-47 6. Prigatano GP. Learning from our successes and failures. Reflections J Int Neuropsychol Soc. 1997;3:497-9.

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34. Rabins PV, Mace NL, Lucas MJ. The impacto of dementia on the family. JAMA. 1982;248:333-5, doi: 10.1001/jama.248.3.333. 35. Steele C, Rovner B, Chase GA, Folstein M. Psychiatric symptoms and nursing home placement of patients with Alzheimerâ&#x20AC;&#x2122;s disease. Am J Psychiatry. 1990;147:1049-51. 36. Moore S, Sandman CA, Mcgrady K, Kesslak JP. Memory training improve cognitive ability in patients with dementia. Neuropsychol Rehabil. 2001;11:245-62, doi: 10.1080/09602010042000222.

31. Garrido R, Almeida OP. Behavior problems in patients with dementia. The impact on the career life. Arq Neuropsiquiatr. 1999;57:427-34, doi: 10.1590/S0004-282X1999000300014. 32. Garrido R, Menezes PR. Brazil is aging: good and bad news from an epidemiological perspective. Rev Bras Psiquiatr. 2002;24:3-6, doi: 10. 1590/S1516-44462002000500002. 33. Camara VD, Gomes SS, Ramos F, Moura S, Duarte R, Costa AS, et al. Cognitive rehabilitation of dementia. Rev Bras Neurol. 2009;45:25-33.

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DOI:10.1590/S1807-59322011000800016

CLINICAL SCIENCE

Association analysis of anti-Epstein-Barr nuclear antigen-1 antibodies, anti-cyclic citrullinated peptide antibodies, the shared epitope and smoking status in Brazilian patients with rheumatoid arthritis Michel Alexandre Yazbek,I Silvia de Barros-Mazon,II Cla´udio Lu´cio Rossi,II Ana Carolina Londe,I Lilian Tereza Lavras Costallat,I Manoel Barros Be´rtoloI I

Department of Rheumatology, University of Campinas (UNICAMP), Campinas, SP, Brazil. (UNICAMP), Campinas, SP, Brazil.

Department of Clinical Pathology, University of Campinas

INTRODUCTION: Epstein-Barr virus exposure appears to be an environmental trigger for rheumatoid arthritis that interacts with other risk factors. Relationships among anti-cyclic citrullinated peptide antibodies, the shared epitope, and smoking status have been observed in patients with rheumatoid arthritis from different populations. OBJECTIVE: To perform an association analysis of anti-Epstein-Barr nuclear antigen-1 antibodies, anti-cyclic citrullinated peptide antibodies, the shared epitope, and smoking status in Brazilian patients with rheumatoid arthritis. METHODS: In a case-control study, 140 rheumatoid arthritis patients and 143 healthy volunteers who were matched for age, sex, and ethnicity were recruited. Anti-Epstein-Barr nuclear antigen-1 antibodies and anti-cyclic citrullinated peptide antibodies were examined using an enzyme-linked immunosorbent assay, and shared epitope alleles were identified by genotyping. Smoking information was collected from all subjects. A comparative analysis of anti-Epstein-Barr nuclear antigen-1 antibodies, anti-cyclic citrullinated peptide antibodies, the shared epitope, and smoking status was performed in the patient group. Logistic regression analysis models were used to analyze the risk of rheumatoid arthritis. RESULTS: Anti-Epstein-Barr nuclear antigen-1 antibodies were not associated with anti-cyclic citrullinated peptide antibodies, shared epitope alleles, or smoking status. Anti-cyclic citrullinated peptide antibody positivity was significantly higher in smoking patients with shared epitope alleles (OR = 3.82). In a multivariate logistic regression analysis using stepwise selection, only anti-cyclic citrullinated peptide antibodies were found to be independently associated with rheumatoid arthritis (OR = 247.9). CONCLUSION: Anti-Epstein-Barr nuclear antigen-1 antibodies did not increase the risk of rheumatoid arthritis and were not associated with the rheumatoid arthritis risk factors studied. Smoking and shared epitope alleles were correlated with anti-cyclic citrullinated peptide-antibody-positive rheumatoid arthritis. Of the risk factors, only anticyclic citrullinated peptides antibodies were independently associated with rheumatoid arthritis susceptibility. KEYWORDS: Rheumatoid arthritis; Risk factors; Epstein-Barr virus; Epstein-Barr virus nuclear antigens; Brazilians. Yazbek MA, Barros-Mazon S, Rossi CL, Londe AC, Costallat LTL, Be´rtolo MB. Association analysis of anti-Epstein-Barr nuclear antigen-1 antibodies, anti-cyclic citrullinated peptide antibodies, the shared epitope and smoking status in Brazilian patients with rheumatoid arthritis. Clinics. 2011;66(8):1401-1406. Received for publication on May 11, 2011; First review completed on May 19, 2011; Accepted for publication on May 30, 2011 E-mail: m-yazbek@uol.com.br Tel.: 55 19 3521-7106

pathophysiology of RA is complex and multifactorial and involves both genetic and environmental factors. Many studies have demonstrated the importance of certain human leukocyte antigens (HLA) molecules that contain the shared epitope (SE) as the major genetic risk factor for RA. Nevertheless, genetic factors fail to completely explain the occurrence of RA.1,2 In addition, antibodies to cyclic citrullinated peptides (antiCCP) are highly specific predictors of RA. Anti-CCP have been observed many years prior to disease onset, indicating an

INTRODUCTION Rheumatoid arthritis (RA) is an inflammatory autoimmune disease with a pathogenesis that is not fully understood. The

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System, Siemens, Marburg, Germany). All assays were performed according to the manufacturers’ instructions. HLA-DRB1 typing was undertaken by polymerase chain reaction (PCR), using specific primers and hybridization with sequence-specific oligonucleotides, as previously described.22 HLA-DRB1*01, HLA-DRB1*04, and HLADRB1*10 were the SE alleles studied.5 Patients carrying zero, one, or two alleles of SE were classified as SE2/2, SE+/2, or SE +/+, respectively.

important role for them in the development of RA. An association between the positivity of anti-CCP and the presence of the SE in RA has been reported by numerous researchers. The presence of SE alleles appears to be a risk factor related to anti-CCP production.1,3 Interactions between tobacco exposure and SE status in anti-CCP-positive patients have been demonstrated by several studies, whereas neither smoking nor SE status confers an increased risk of developing anti-CCP negative RA.4-10 Among other environmental factors, infectious agents, most notably viruses, have long been suspected of promoting the development of RA and other autoimmune diseases.11-13 Links between Epstein-Barr virus (EBV) and RA have been a focus of research for the last three decades. Studies have demonstrated that EBV is a potent B-cell stimulator, antibodies in RA cross-react with EBV nuclear antigens, and EBV glycoproteins share sequence similarities with the HLA-DRB1 SE. These viral properties support the theory that EBV is a strong risk factor for RA.12-15 It has been observed that the levels of some anti-EBV antibodies (anti-EBNA, anti-VCA, and anti-EA) are higher in RA patients than in controls.16,17 In particular, Epstein-Barr nuclear antigen-1 (EBNA-1) contains glycine/alanine sequences that constitute a dominant EBNA-1 epitope against which specific reactivity has been demonstrated.18,19 An antibody against this EBNA-1 sequence also reacted with a protein expressed by synovial lining cells in RA patients.20 Identical glycine/alanine repeat sequences of EBNA-1 are also present in collagen, cytokeratin, and actin.21 Many studies have suggested that some RA risk factors are highly associated with EBV in the complex etiopathogeny of RA. Among the antibodies against EBV, anti-EBNA1 appears to be the most prominent in the interactions with other risk factors. In the present study, we evaluated associations between anti-EBNA-1, anti-CCP, shared epitope alleles, and smoking status in Brazilian RA patients.

Statistical analysis The chi-squared and Fisher’s exact tests were used to compare categorical variables between the groups. The differences between the mean or median values of two groups were analyzed using the Mann-Whitney test. The Kruskal-Wallis test was used when the subjects were separated into three or more groups. To evaluate RA risk, we calculated odds ratios (OR) with 95% confidence intervals (CI). A logistic regression analysis with univariate and multiple models was used with stepwise variable selection. The significance level for statistical tests was set at 5% (p,0.05). All statistical analyses were performed using SAS (Statistical Analysis System) software for Windows, version 9.1.3 (SAS Institute Inc., Cary, NC, USA).

RESULTS Patient and control characteristics The demographic, clinical, and laboratory characteristics of the RA cases and matched controls are listed in Table 1.

Anti-EBNA-1, anti-CCP, SE status, and smoking status in patients and controls The rates of anti-EBNA-1 and anti-CCP positivity, as well as the mean antibody levels detected using these assays, along with the percentages related to SE status and smoking status in the two groups, are listed in Table 2. No significant differences were observed between the patient group and the control group in terms of the positivity and the mean levels of anti-EBNA-1 antibodies. The positivity and mean levels of anti-CCP antibodies were significantly higher in the patients than in the controls (p,0.001). The presence of the SE alleles (SE+/2 and SE+/+) and a smoking history of at least 15 pack-years were significantly higher in the patients than in the controls (p,0.001 and p = 0.003, respectively).

METHODS AND MATERIALS Patients and controls A total of 140 RA Brazilian patients (including 122 females) aged 25-82 years (mean = 54.24 years), comprising 74 Caucasians, 20 Blacks, and 46 Mulattoes were evaluated at the University of Campinas Teaching Hospital in Brazil. All the patients fulfilled the 1987 RA revised criteria of the American College of Rheumatology. For the control group, we screened 143 healthy individuals who were matched for age, sex, and ethnicity and had no personal or family history of autoimmune disease. Smoking exposure was measured in pack-years, and all subjects were classified as never smoker, past smoker or current smoker. Clinical information was collected using a questionnaire at the time of enrollment. This study was approved by the ethics committee of the University of Campinas, Brazil, and informed consent was obtained from all patients and controls.

Table 1 - Demographic, clinical, and laboratory characteristics. Variables Women, n (%) Ethnicity, n (%) Caucasian Mulattoes Black Age (years) Duration of disease (years) RF-positive, n (%)

Serology and genotyping Anti-EBNA1 antibodies were analyzed using an enzymelinked immunosorbent assay (ELISA) kit containing synthetic peptides (ETI-EBNA-G, DiaSorin, Saluggia, Italy). Anti-CCP antibodies were determined by ELISA (QUANTA Lite CCP3 IgG, Inova Diagnostics Inc., USA). Rheumatoid factor was evaluated by a nephelometric immunoassay (BN ProSPec

RA patients (n = 140)

Controls (n = 143)

122 (87.1) 74 (52.9)

123 (86.0) 83 (58.0)

46 (32.9) 20 (14.3) 54.2¡11.3 10.2¡6.9 111 (79.3)

39 (27.3) 21 (14.7) 55.8¡13.4 NA ND

Data are reported as numbers of subjects with percentages or Mean ¡ Standard Deviation (SD) in parentheses. RA = Rheumatoid Arthritis; RF = Rheumatoid Factor; NA = not applicable; ND = not determined.

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arthritis nuclear antigen’’ (RANA). Reactivity to RANA was not observed in uninfected B cells and was not influenced by the HLA-DR phenotype or the presence of rheumatoid factor. Subsequently, it was established that RANA was, in fact, the same molecule as EBNA-1. The QKRAA sequence present in HLA-DRB1*0401 shares sequence homology with EBV gp110 protein, a target of the immune response to infection.24 Deblon et al. demonstrated that RA patients who carried the SE had a stronger humoral response to EBNA-1 than did SEnegative RA patients.25 These data are conflicting but suggest that EBV infection is related to the presence of HLA-DR molecules that contain the SE. The anti-citrulline immune response is strongly involved in RA etiology, and anti-citrulline antibodies can precede RA development by several years. Anti-CCP antibodies are highly specific and are considered a valuable disease marker and a strong risk factor for RA. A gene-environment interaction among anti-CCP antibodies, SE status, and smoking has been observed in several studies. The presence of HLA-DR SE only confers a risk of RA to patients who are positive for anti-CCP.1,10,26,27 Smoking is associated with the increased presence of citrulline-modified proteins in genetically predisposed individuals who carry HLA-DR SE alleles.8-10 In this study, we did not perform the HLADRB1 subtyping to identify HLA-DR SE alleles, since Lundstro¨m et al. observed that interactions of SE status with anti-CCP antibodies and smoking were independent of HLA-DRB1 subtyping and occurred with *01, *04, and *10 groups, regardless of fine specificity.5 EBV antigens can also undergo citrullination, thus becoming targets of anti-CCP. Antibodies to citrullinated EBV peptides have been identified in sera from RA patients, and the viral-citrullinated-peptide antibody levels of these patients correlated with their anti-CCP levels.28 A relationship between EBV and citrullinated peptides was supported by the observation that anti-citrulline antibodies reacted with deiminated EBNA-1.29 Toussirot and Roudier suggested that EBV should be considered as an environmental factor associated with anti-CCP production, just as smoking is associated with anti-CCP development in RA patients.15 It remains unknown how EBV influences the risk of developing RA. Anti-EBNA-1 antibodies are increased in individuals who are exposed to EBV and remain increased for a lifetime. Multiple sclerosis (MS) is a good example of an autoimmune disease for which anti-EBNA-1 antibodies have been shown to be a strong risk factor. Interestingly, this risk is increased by smoking but is independent of the MS susceptibility HLA-DR15 allele.30,31 In the present study, we showed a high positivity of anti-EBNA-1 in both patients and controls, and there was no association between antiEBNA-1 levels and anti-CCP levels, SE status, or smoking status in RA patients. The relative risk for RA was not increased in patients with higher levels of anti-EBNA-1. Thus, we have demonstrated that anti-EBNA-1 serology cannot evaluate the interactions between EBV and the most well-known RA risk factors. Further studies could assess the role of EBV in RA pathogenesis. The accurate quantification of EBV by real-time polymerase chain reaction and the evaluation of antibodies to viral citrullinated peptides are promising techniques that could aid in this process.28,32,33 Brazil has a heterogeneous population with a distinct genetic profile that has been influenced by various racial and ethnic groups, predominantly Africans and Europeans. As reported in more homogeneously Caucasian populations5-8,

Table 2 - Comparative analysis of anti-EBNA-1, anti-CCP, SE status, and smoking status in RA patients and controls.

Anti-EBNA1-positive, n (%) Mean anti-EBNA1 levels (U/ml) Anti-CCP-positive, n (%) Mean anti-CCP levels (U/ml) Shared epitope status, n (%) SE 2/2 SE +/2 SE +/+ Smoking status, n (%) Non-smokers ,15 pack-years $15 pack-years

RA patients

Controls

p-value

127 (90.7) 246.1 109 (77.9) 117.0

130 (90.9) 277.1 2 (1.4) 6.8

0.955 0.111 ,0.001 ,0.001 ,0.001

52 (37.1) 67 (47.9) 21 (15.0)

81 (56.6) 55 (38.5) 7 (4.9)

82 (58.6) 24 (17.1) 34 (24.3)

107 (74.8) 22 (15.4) 14 (9.8)

0.003

Data are reported as numbers of subjects with percentages or mean levels in parentheses. RA = Rheumatoid Arthritis; Anti-EBNA-1 = Anti-EpsteinBarr Nuclear Antigen 1 Antibodies; Anti-CCP = Anti-Cyclic Citrullinated Antibodies; SE = Shared Epitope; p = significance based on Pearson’s chi-squared or Mann-Whitney test. Differences were considered to be significant at p,0.05.

Anti-EBNA-1 profile according to anti-CCP, SE status, and smoking status in patients Anti-EBNA-1 levels were standardized in quartiles (1 = ,63.71 U/ml; 2 = 63.71-125.26 U/ml; 3 = 125.27-333.44 U/ml; 4 = $333.45 U/ml). No significant differences were found in the distribution of anti-EBNA-1 antibodies according to antiCCP positivity, SE status, or smoking status (Figure 1).

Anti-CCP profile according to SE status and smoking status in patients Although it was not significant, there was a trend for patients with higher levels of anti-CCP to carry SE alleles (p = 0.059). Smokers had significantly higher levels of antiCCP (p = 0.018). SE carriers and smoking patients had a significantly higher risk of being positive for anti-CCP (Figure 2, OR = 3.82; 95% CI = 1.28211.40; p = 0.016).

Anti-EBNA1, anti-CCP, SE status, and smoking status in RA risk The univariate logistic regression analysis showed that anti-CCP positivity carried a highly significant RA risk (p,0.001; OR = 247.49). The presence of one or two SE alleles increased the risk of RA 1.90-fold (p = 0.012) and 4.67-fold (p = 0.001), respectively. A smoking history of 15 or more pack-years led to a 3.17-fold increased risk of RA (p = 0.001). Anti-EBNA-1 levels were not found to be a risk factor for RA in this model. In the multivariate logistic regression analysis with stepwise selection, anti-CCP was the only selected variable that was independently associated with RA.

DISCUSSION There is increasing evidence of the roles of a number of environmental factors, including viruses exposure and smoking, in the pathogenesis of RA. EBV has been studied for many decades; however, it remains unclear how its interactions with other RA risk factors develop. In 1976, Alpsaugh and Tan observed a higher serologic reactivity to the nuclei of EBV-infected B cells in RA patients than in the controls.23 The target B cell antigen was named ‘‘rheumatoid

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Figure 1 - Anti-EBNA-1 profiles in RA patients. A - profile according to anti-CCP (p = 0.98); B - profile according to SE status (p = 0.96); C - profile according to smoking status (p = 0.14).

RA risk. However, through a multivariate logistic regression analysis, we showed of the risk factors, only anti-CCP antibodies were independently associated with RA. AntiEBNA-1 antibodies were neither correlated with the other factors nor influenced disease risk, unlike in other autoimmune conditions, such as MS. Despite some supporting

we showed that SE alleles and tobacco exposure are related to anti-CCP antibodies in RA patients. Several previous studies observed these same relationships in Brazilian RA patients.34,35 In this study, we demonstrated the contributions of antiCCP antibodies, the presence of the SE, and smoking history to

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Figure 2 - Anti-CCP positivity according to SE status and smoking status. * statistically significant (p = 0.038); p = significance based on Pearson’s chi-squared test.

evidence, the role of EBV in the development of RA remains unclear and needs to be studied further. We emphasize the importance of establishing the role of EBV in conjunction with other factors involved in RA pathogenesis.

17.

18.

REFERENCES 1. Klareskog L, Catrina AI, Paget S. Rheumatoid Arthritis. Lancet. 2009;373:659-72, doi: 10.1016/S0140-6736(09)60008-8. 2. Plenge RM. Recent progress in rheumatoid arthritis genetics. Curr Opin Rheumatol. 2009;21:262-71, doi: 10.1097/BOR.0b013e32832a2e2d. 3. Rantapa¨a¨-Dahlqvist S. What happens before onset of rheumatoid arthritis? Curr Opin Rheumatol. 2009;21:272-8, doi: 10.1097/BOR. 0b013e32832a2e44. 4. Liao KP, Alferdsson L, Karlson EW. Environmental influences on risk for rheumatoid arthritis. Curr Opin Rheumatol. 2009;21:279-83, doi: 10.1097/ BOR.0b013e32832a2e16. 5. Lu¨ndstrom E, Ka¨llberg H, Alfredsson L, Klareskog L, Padyukov L. Geneenvironment interaction between the DRB1 shared epitope and smoking in the risk of anti-citrullinated protein antibody-positive rheumatoid arthritis- All alleles are important. Arthritis Rheum. 2009;60:1597-603, doi: 10.1002/art.24572. 6. Karlson EW, Chang S-C, Cui J, Chibnik LB, Fraser PA, De Vivo I. Geneenvironment interaction between HLA-DRB1 shared epitope and heavy cigarette smoking in predicting incident rheumatoid arthritis. Ann Rheum Dis. 2010;69:54-60, doi: 10.1136/ard.2008.102962. 7. Bang S-Y, Lee K-H, Cho S-K, Lee H-S, Lee KW, Bae S-C. Smoking increases rheumatoid arthritis susceptibility in individuals carrying the HLA-DRB1 shared epitope, regardless of rheumatoid factor or anti-cyclic citrullinated peptide antibody status. Arthritis Rheum. 2010;62:369-77. 8. Linn-Rasker SP, van der Helm-van Mil AHM, van Gaalen FA, Kloppenburg M, de Vries RRP, le Cessie S, et al. Smoking is a risk factor for anti-CCP antibodies only in rheumatoid arthritis patients who carry HLA-DRB1 shared epitope alleles. Ann rheum Dis. 2006; 65:366-71, doi: 10.1136/ard.2005.041079. 9. Baka Z, Buza´s E, Nagy G. Rheumatoid arthritis and smoking: putting the pieces together. Arthritis Res Ter. 2009;11:238-51, doi: 10.1186/ar2751. 10. Klareskog L, Stolt P, Lundberg K, Ka¨llberg H, Bengtsson C, Grunewald J, et al. A new model for an etiology of rheumatoid arthritis: smoking may trigger HLA-DR (shared epitope)- restricted immune reactions to autoantigens modified by citrullination. Arthritis Rheum. 2006;54:38-46, doi: 10.1002/art.21575. 11. Vaughan JH. Viruses and autoimmune disease. J Rheumatol. 1996;23:1831-3. 12. Vaughan JH. The Epstein-Barr virus in autoimmunity. Springer Semin Immunopathol. 1995;17:203-30, doi: 10.1007/BF00196166. 13. Toussirot E, Roudier J. Epstein-Barr virus in autoimmune diseases. Best Pract Res Clin Rheumatol. 2008;22:883-96, doi: 10.1016/j.berh.2008.09.007. 14. Costenbader KH, Karlson EW. Epstein-Barr virus and rheumatoid arthritis: is there a link? Arthritis Res Ther. 2006;8:204, doi: 10.1186/ar1893. 15. Toussirot E, Roudier J. Pathophysiological links between rheumatoid arthritis and the Epstein-Barr virus: an update. Joint Bone Spine. 2007;74:418-26, doi: 10.1016/j.jbspin.2007.05.001. 16. Inoue N, Harada S, Miyasaka N, Oya A, Yanagi K. Analysis of antibody titers to Epstein-Barr virus nuclear antigens in sera of patients with

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Sjo¨ gren’s syndrome and with rheumatoid arthritis. J Infect Dis. 1991;164:22-8, doi: 10.1093/infdis/164.1.22. Silverman SL, Schumacher HR. Antibodies to Epstein-Barr viral antigens in early rheumatoid arthritis. Arthritis Rheum. 1981; 24:1465-8, doi: 10. 1002/art.1780241202. Kouri T, Petersen J, Rhodes G, Aho K, Palusuo T, Helio¨vaara M, et al. Antibodies to synthetic peptides from Epstein-Barr nuclear antigen-1 in sera of patients with early rheumatoid arthritis and in preillness sera. J Rheumatol. 1990;17:1442-9. Petersen J, Rhodes G, Roudier J, Vaughan JH. Altered immune response to glycine-rich sequences of Epstein-Barr nuclear antigen-1 in patients with rheumatoid arthritis and systemic lupus erythematosus. Arthritis Rheum. 1990;33:993-1000, doi: 10.1002/art.1780330711. Fox R, Sportsman R, Rhodes G, Luka J, Pearson G, Vaughan J. Rheumatoid arthritis synovial membrane contains a 62,000-molecularweight protein that shares an antigenic epitope with the Epstein-Barr virus encoded associated EBNA-1 antigen. J Clin Invest. 1986;77:1539-47, doi: 10.1172/JCI112469. Baboonian C, Venables PJW, Williams DG, Williams RO, Maini RN. Cross-reaction of antibodies to a glycine/alanine repeat sequence of EpsteineBarr virus nuclear antigen-1 with collagen, cytokeratin, and actin. Ann Rheum Dis. 1991;50:772-5, doi: 10.1136/ard.50.11.772. Kimura A, Sasazuki T. Eleventh International Histocompatibility Workshop reference protocol for the HLA DNA typing technique. In: Tsuji K, Aizawa M, Sasazuki T, eds. HLA 1991. Proceedings of the Eleventh International Histocompatibility Workshop and Conference,vol 1, Oxford: Oxford University Press. 1992:397-439. Alpsaugh MA, Tan EM. Serum antibody in rheumatoid arthritis reactive with a cell- associated antigen. Demonstration by precipitation and immunofluorescence. Arthritis Rheum. 1976;19: 711-9, doi: 10.1002/15290131(197607/08)19:4,711::AID-ART1780190409.3.0.CO;2-I. Roudier J, Petersen J, Rhodes JH, Luka J, Carson DA. Susceptibility to rheumatoid arthritis maps to a T-cell epitope shared by the HLA-Dw4 DR beta-1 chain and the Epstein-Barr virus glycoprotein gp110. Proc Natl Acad Sci USA. 1989;86:5104-8, doi: 10.1073/pnas.86.13.5104. Deblon N, Toussirot E, Auger I, Roudier J, Roudier C. Les proteins EBNA-1 et gp110 du virus d’EpsteinBarr sont les cibles d’une forte re´ponse anticorps chez les patients atteints de polyarthrite rhumatoı¨de qui expriment l’e´pitope partage´. Rev Rhum. 2005;72:978-9. Szodoray P, Szabo´ Z, Kapita´ny A, Gyetvai A, Lakos G, Sza´nto´ S, et al. Anti-citrullinated protein/peptide autoantibodies in association with genetic and environmental factors as indicators of disease outcome in rheumatoid arthritis. Autoimmun Rev. 2010;9:140-3, doi: 10.1016/j. autrev.2009.04.006. van der Helm-van Mil AH, Verpoort KN, Breedveld FC, Huizinga TW, Toes RE, de Vries RR. The HLA-DRB1 shared epitope alleles are primarily a risk factor for anti-cyclic citrullinated peptide antibodies and are not an independent risk factor for development of rheumatoid arthritis. Arthritis Rheum. 2006;54:1117-21, doi: 10.1002/art.21739. Anzilotti C, Merlini G, Pratesi F, Tommasi C, Chimenti D, Migliorini P. Antibodies to viral citrullinated peptide in rheumatoid arthritis. J Rheumatol. 2006;33:647-51. Pratesi F, Tommasi C, Anzilotti C, Chimenti D, Migliorini P. Deiminated Epstein-Barr virus nuclear antigen 1 is a target of anti-citrullinated protein antibodies in rheumatoid arthritis. Arthritis Rheum. 2006;54:73341, doi: 10.1002/art.21629.

Rheumatoid Arthritis Risk Factors Yazbek MA et al.

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patients affected by rheumatoid arthritis, chronic arthritides and connective tissue disorders. Rheumatology (Oxford). 2007;46:1579-82, doi: 10.1093/rheumatology/kem193. 34. Louzada-Junior P, Freitas MV, Oliveira RD, Deghaide NH, Conde RA, Bertolo MB, et al. A majority of Brazilian patients with rheumatoid arthritis HLA-DRB1 alleles carry both the HLA-DRB1 shared epitope and anti-citrunillated peptide antibodies. Braz J Med Biol Res. 2008;41:493-9, doi: 10.1590/S0100-879X2008005000021. 35. Oliveira RD, Junta CM, Oliveira FR, Silva LM, Donadi EA, LouzadaJunior P. Share epitope, citrullinated cyclic peptide antibodies and smoking in Brazilian rheumatoid arthritis patients. Clin Rev Allergy Immunol. 2008;34:32-5, doi: 10.1007/s12016-007-8017-2.

30. Simon KC, van der Mei IA, Munger KL, Ponsonby A, Dickinson J, Dwyer T, et al. Combined effects of smoking, anti-EBNA antibodies, and HLADRB1*1501 on multiple sclerosis risk. Neurology. 2010;74:1365-71, doi: 10.1212/WNL.0b013e3181dad57e. 31. De Jager PL, Simon KC, Munger KL, Rioux JD, Hafler DA, Ascherio A. Integrating risk factors: HLA-DRB1*1501 and Epstein-Barr virus in multiple sclerosis. Neurology. 2008;25;70:1113-8. 32. Balandraud N, Meynard JB, Auger I, Sovran H, Mugneir B, Reviron D, et al. Epstein-Barr virus load in the peripheral blood of patients with rheumatoid arthritis. Arhtritis Rheum. 2003;48:1223-8, doi: 10.1002/art.10933. 33. Anzilotti C, Riente L, Pratesi F, Chimenti D, Delle Sedie A, Bombardieri S, et al. IgG, IgA, IgM antibodies to a viral citrullinated peptide in

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DOI:10.1590/S1807-59322011000800017

BASIC RESEARCH

Role of cGMP and cAMP in the hemodynamic response to intrathecal sildenafil administration Gabriela Bombarda,I Joa˜o Paulo J. Sabino,I Carlos Alberto A. da Silva,I Rubens Fazan Jr.,I Maria Cristina O. Salgado,II Helio C. SalgadoI I

Department of Physiology, School of Medicine of Ribeiraõ Preto, University of Saõ Paulo, Saõ Paulo, SP, Brazil. II Pharmacology, School of Medicine of Ribeiraõ Preto, University of Saõ Paulo, Saõ Paulo, SP, Brazil. Ribeiraõ Preto, SP, Brazil.

INTRODUCTION: Results from our laboratory have demonstrated that intracerebroventricular administration of sildenafil to conscious rats promoted a noticeable increase in both lumbar sympathetic activity and heart rate, with no change in the mean arterial pressure. The intracerebroventricular administration of sildenafil may have produced the hemodynamic effects by activating sympathetic preganglionic neurons in the supraspinal regions and spinal cord. It is well documented that sildenafil increases intracellular cGMP levels by inhibiting phosphodiesterase type 5 and increases cAMP levels by inhibiting other phosphodiesterases. OBJECTIVE: To examine and compare, in conscious rats, the hemodynamic response following the intrathecal administration of sildenafil, 8-bromo-cGMP (an analog of cGMP), forskolin (an activator of adenylate cyclase), or dibutyryl-cAMP (an analog of cAMP) in order to elucidate the possible role of the sympathetic preganglionic neurons in the observed hemodynamic response. RESULTS: The hemodynamic responses observed following intrathecal administration of the studied drugs demonstrated the following: 1) sildenafil increased the mean arterial pressure and heart rate in a dose-dependent manner, 2) increasing doses of 8-bromo-cGMP did not alter the mean arterial pressure and heart rate, 3) forskolin did not affect the mean arterial pressure but did increase the heart rate and 4) dibutyryl-cAMP increased the mean arterial pressure and heart rate, similar to the effect observed following the intrathecal injection of the highest dose of sildenafil. CONCLUSION: Overall, the findings of the current study suggest that the cardiovascular response following the intrathecal administration of sildenafil to conscious rats involves the inhibition of phosphodiesterases other than phosphodiesterase type 5 that increase the cAMP level and the activation of sympathetic preganglionic neurons. KEYWORDS: Sympathetic preganglionic neurons; Lumbar sympathetic activity; Forskolin; Phosphodiesterase 5; Spinal cord. Bombarda G, Sabino JPJ, Silva CAA, Fazan R Jr, Salgado MCO, Salgado HC. Role of cGMP and cAMP in the hemodynamic response to intrathecal sildenafil administration. Clinics. 2011;66(8):1407-1411. Received for publication on April 1, 2011; First review completed on April 20, 2011; Accepted for publication on April 20, 2011 E-mail: hcsalgado@fmrp.usp.br Tel.: 55 16 3602-3201

organum vasculosum lamina terminalis, and area postrema) and hypothalamic sites controlling the sympathetic drive.2-5 Thus, the increase in lumbar sympathetic activity and tachycardia observed in conscious rats following the administration of sildenafil into the left lateral ventricle may be explained by an effect in the spinal cord that is probably mediated by SPN activation.1 Therefore, in order to test the hypothesis that sildenafil is able to directly activate the SPNs and cause a hemodynamic response, the first goal of the present study was to examine, in conscious rats, the effects of sildenafil on the mean arterial pressure (MAP) and heart rate (HR) following intrathecal administration. Sildenafil increases intracellular cGMP levels via inhibition of phosphodiesterase type 5 (PDE5).6-11 Moreover, sildenafil can inhibit other phosphodiesterases, including those that preferentially degrade cGMP, cAMP, or both depending on the dose. The inhibition of phosphodiesterases leads to an

INTRODUCTION Previous results from our laboratory have demonstrated that the administration of sildenafil into the left lateral ventricle of conscious rats elicited a marked increase in the lumbar sympathetic activity associated with tachycardia but no change in the arterial pressure.1 The intracerebroventricular administration of sildenafil may have produced sympathetic overactivity in the spinal cord, thereby activating sympathetic preganglionic neurons (SPNs) in addition to supraspinal areas, such as the rostral ventrolateral medulla, circumventricular organs (subfornical organ,

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increase in the intracellular cAMP level.12-15 In anesthetized rats, a dose-dependent increase in the MAP following the intrathecal administration of an cGMP analog (8-bromocGMP) has been observed. A 8-bromo-cGMP-dependent increase in the MAP suggests that the mechanism underlying this increase involves cGMP.16 As the hemodynamic response caused by sildenafil may involve a lack of cGMP degradation, the second goal of this study was to evaluate the hemodynamic effects of the intrathecal administration of 8-bromo-cGMP in conscious rats and to compare the results with the sildenafil-induced hemodynamic response. Moreover, several studies have proposed that sildenafil, by itself, increases the levels of cAMP.13-15 After taking this idea into account, the final objective of the present study was to examine the cardiovascular response of conscious rats following the intrathecal administration of forskolin, a direct activator of adenylate cyclase that increases the intracellular levels of cAMP, and dibutyryl-cAMP, an analog of cAMP, and to compare the results with the sildenafil-induced response.

n = 3), 8-bromo-cGMP (0.03 mg, n = 3; 0.47 mg, n = 6; 1.4 mg, n = 7), forskolin (100 mg, n = 7), dibutyryl-cAMP (100 mg, n = 4) or vehicle (1% DMSO, n = 6). The drugs and vehicle (10 mL) were administered via the intrathecal cannula using a Hamilton microsyringe. After drug administration, an additional 10 mL of aCSF was injected to clear the catheter of the drug solution. At the end of the experiment, 10 mL of Evan’s blue was injected into the subarachnoid space. The rats were then anesthetized with tribromoethanol (250 mg/kg, i.p.) and perfused through the heart with 4% paraformaldehyde in 0.1 M phosphate buffered saline. Finally, a necropsy was performed to confirm the position of the intrathecal catheter and to determine how far the dye spread within the subarachnoid space.17,19

Drugs The drugs used in the current study were sildenafil citrate (Pfizer), 8-bromo-cGMP (8-bromoguanosine 39,59-cyclicomonophosphate sodium salt), dibutyryl-cAMP (N6,29-Odibutyryladenosine 39,59-cyclic monophosphate sodium salt), and forskolin (Sigma-Aldrich). The vehicle used was DMSO (dimethyl sulfoxide). The components of the aCSF were 127 mM NaCl, 1.9 mM KCl, 1.2 mM KH2PO4, 2.4 mM CaCl2, 1.3 mM MgSO4, 26 mM NaHCO3 and 10 mM Dglucose (Sigma-Aldrich). All drugs were dissolved in 1% DMSO immediately before administration, and the pH of the drug solutions was adjusted to 7.4.

MATERIAL AND METHODS Animals All experiments were performed on male Wistar rats (320¡30 g) housed individually with free access to food and water. The rats were maintained on a 12512 h lightdark cycle. The experimental protocols used in this research were approved by the Committee of Ethics in Animal Research of the School of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo (protocol 199/2008).

Statistical analysis The results are expressed as the mean ¡ SEM. Comparisons were performed using one-way and twoway analysis of variance (ANOVA) tests followed by a posthoc Newman-Keuls test. The statistical differences were considered significant at p,0.05.

Surgical procedures The rats were anesthetized with tribromoethanol (250 mg/kg, i.p., Sigma Aldrich) and underwent a twostage surgery. Initially, a polyethylene cannula PE-10 (,15 cm long, total volume 10 mL) filled with heparinized saline (pH 7.4) was inserted through the intervertebral space (L5-L6) into the spinal subarachnoid space in a cranial direction (2 cm). The cannula was then implanted using the method reported by Prado et al.17 with a slight modification developed by Storkson et al.18 Once the procedure was completed, the animals were allowed five days to recover. On the day before the experiment, polyethylene tubing was inserted into the left femoral artery for direct measurement of the arterial pressure. The experiments were performed in conscious rats 24 hours after femoral artery catheterization.

RESULTS Basal MAP and HR. The baseline MAP and HR in rats that received an intrathecal administration of sildenafil, 8-bromo-cGMP, forskolin, dibutyryl-cAMP, or vehicle (DMSO) were similar (Table 1).

Effects of intrathecal administration of sildenafil on the MAP and HR Intrathecal sildenafil administration elicited a dosedependent increase in the MAP and HR (Figure 1). Although the maximal pressor and tachycardic response induced by 100 and 10 mg of sildenafil were similar, there was a considerable delay in the maximal response with the lower dose.

Measurement of arterial pressure During the experiments, silence was maintained to avoid undue stress on the animals. The pulsatile arterial pressure was recorded by connecting the arterial catheter to a pressure transducer (P23Gb, Statham, Hato Hey, PR) and was continuously sampled (2 kHz) using an IBM/PC equipped with an analog-to-digital interface (DI-220 Dataq Instruments, Akron, OH). The pulsatile arterial pressure recordings were then analyzed using the CODAS software (Dataq Instruments, Akron, OH) to obtain the MAP and HR values.

Table1 - Basal hemodynamic data of conscious rats.

DMSO Sildenafil 8-Bromo-cGMP Forskolin Dibutyryl-cAMP

Experimental protocols The animals received an single intrathecal dose (10 mL) of sildenafil (100 mg, n = 7; 10 mg, n = 6; 1 mg, n = 4; 0.1 mg,

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MAP (mm Hg)

HR (bpm)

108¡3 101¡3 104¡2 106¡2 103¡7

386¡14 378¡6 381¡6 375¡6 379¡16

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Hemodynamic response to intrathecal sildenafil Bombarda G et al.

Figure 1 - Time course of the mean arterial pressure (D MAP) and heart rate (D HR) induced by the intrathecal administration of 100 mg (A), 10 mg (B), 1 mg (C), or 0.1 mg (D) sildenafil or the vehicle (DMSO). *p,0.05 compared to DMSO.

with tachycardia that is unrelated to cGMP action as the administration of a cGMP analog (8-bromo-cGMP) had no effect. In addition, a direct activator of adenylate cyclase (forskolin) increased the HR but did not affect the MAP. In contrast, a cAMP analog (dibutyryl-cAMP) increased the MAP and HR, similar to the increase observed following the administration of 100 mg sildenafil. One possible explanation for the results observed following the intrathecal administration of sildenafil is the activation of SPNs located in the intermediolateral column of the spinal cord that predominantly innervate the heart and blood vessels. The hemodynamic response obtained with 10 and 1 mg of sildenafil indirectly support this hypothesis, as the increase in the MAP and HR was delayed when compared to results observed after the administration of 100 mg of the drug. The magnitude of the hemodynamic response was comparable even though the doses differed. A possible explanation for this delay may be that lower doses

Effects of intrathecal administration of 8-bromocGMP on the MAP and HR. The intrathecal administration of different doses of the membrane-permeable analogue of cGMP, 8-bromo-cGMP or its vehicle, DMSO, did not affect the MAP or HR (Figure 2).

Effects of intrathecal administration of forskolin and dibutyryl-cAMP on the MAP and HR. The administration of forskolin did not affect the MAP but did increase the HR, while the membrane-permeable cAMP analog, dibutyryl-cAMP, induced a rapid and significant increase in both the MAP and HR (Figure 3).

DISCUSSION The major findings of this study indicate that the intrathecal administration of sildenafil in conscious rats results in a dose-dependent increase in the MAP combined

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Figure 2 - Time course of the mean arterial pressure (D MAP) and heart rate (D HR) following the intrathecal administration of different doses of the cGMP analog 8-bromo-cGMP or the vehicle (DMSO).

anesthesia and its associated detrimental effects. One possible explanation for the absence of any cardiovascular effects following the 8-bromo-cGMP injection is that this compound can act directly on high conductance potassium channels in the cell membrane, thereby promoting hyperpolarization of the cell and decreasing their excitability.22 Therefore, the possible excitatory effect of 8-bromo-cGMP could be antagonized by its direct action on potassium channels, masking any possible activation of SPNs and blunting the hemodynamic response. Finally, it is reasonable to hypothesize that the increase in the MAP and HR caused by the intrathecal administration of sildenafil are not mediated by cGMP. There is evidence that sildenafil can interfere with cAMP activity and inhibit the activity of other PDEs.12-15 It is well known that drug selectivity, dose and route of administration, distribution, pharmacokinetics and the extent of activation of the NO-cGMP pathway are important mechanisms determining the functional role of sildenafil.23 Bischoff12 observed that sildenafil has different inhibitory activity against the 11 families of PDEs. It is possible that the dose of sildenafil used in the present study inhibited not only PDE5 but also other PDEs, including those PDEs that preferentially degrade cGMP, cAMP, or both. Therefore, the additional treatments used in the current study aimed to verify a possible role of cAMP in the hemodynamic response promoted by sildenafil. To accomplish this, the rats were intrathecally injected with a direct activator of adenylate cyclase (forskolin) or a membrane-permeable analogue of cAMP (dibutyrylcAMP). The intrathecally injected forskolin had no effect on the MAP but increased the HR. However, the intrathecally injected dibutyryl-cAMP increased the MAP and the HR, an effect similar to sildenafil, suggesting that cAMP may be involved in the mechanism underlying the

of sildenafil require a longer time to achieve the effective concentration required for the activation of SPNs located deep inside the spinal cord. In fact, an increase in sympathetic activity has been observed in humans after only a single dose of sildenafil.20,21 In addition, a study recently conducted in our laboratory1 has indicated that the intracerebroventricular administration of sildenafil in rats causes a noticeable increase in lumbar sympathetic activity without an accompanying change in the MAP. To fully understand the implications for human health, the hemodynamic effects caused by the intrathecal administration of sildenafil in conscious rats observed in this study necessitate comparison with similar studies in humans. In a study by Philips et al.20 in which healthy males received oral sildenafil, sildenafil had no effect on the MAP and HR but did elicit a significant increase in muscle sympathetic nerve activity. Despite the absence of hemodynamic changes (arterial pressure and heart rate), the findings of the previous researchers20 demonstrate that sildenafil was able to promote sympathetic activation. However, a study recently conducted by our laboratory1 demonstrated that intracerebroventricular administration of sildenafil (100 mg/ 5 mL) results in a noticeable increase in lumbar sympathetic activity without an accompanying change in the MAP. This contrasts with the significant tachycardia observed in the aforementioned Philips study.20 In the current study, the MAP and HR were unaffected by the intrathecal administration of increasing doses of 8bromo-cGMP. Conversely, Malik et al.16 performed intrathecal administration of 8-bromo-cGMP in anesthetized rats, with the same doses used in the present study, and observed a dose-dependent increase in the MAP. However, it should be noted that the results of the current study were obtained using conscious rats, without the use of

Figure 3 - Time course of the mean arterial pressure (D MAP) and heart rate (D HR) following the intrathecal administration of an adenylate cyclase agonist (forskolin), a cAMP analog (dibutyryl-cAMP) or the vehicle (DMSO). *p,0.05 compared to DMSO.

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Hemodynamic response to intrathecal sildenafil Bombarda G et al.

cardiovascular effects of sildenafil. Uckert et al.14 observed that both forskolin and sildenafil administered independently caused the relaxation of isolated human corpus cavernosum tissue. However, this effect was reversed by PKA-I (a cAMP-dependent kinase inhibitor) and PKG-I (a cGMP-dependent kinase inhibitor) in both cases. These findings suggest that cross-regulation may exist between the cyclic nucleotides cAMP and cGMP. The results of the current study are in line with those of Stief et al.,24 who observed that the administration of increasing concentrations of sildenafil in isolated human corpus cavernosum tissue did not modify the cGMP levels in the tissue, although it did increase the cAMP levels. Similarly, increasing concentrations of sildenafil administered to isolated human heart muscle did not alter the cGMP levels, although an increase in the cAMP levels was evident.24 In addition, the findings of Stief et al.24 demonstrate that milrinone, a PDE3-specific inhibitor, causes an increase in the intracellular cAMP levels without altering the cGMP levels, as PDE3 preferentially degrades cAMP. In addition, Botha et al.25 verified that while the serum levels of cAMP increase after the administration of two different doses of sildenafil and milrinone, the serum levels of cGMP are not altered. Overall, the results of the current study strongly suggest that the pressor response and tachycardia observed after the intrathecal administration of sildenafil to conscious rats involves the inhibition of non-PDE5, cAMP-increasing phosphodiesterases that activate sympathetic preganglionic neurons. The hemodynamic response elicited by the intrathecal administration of sildenafil to conscious, freely moving rats may aid in understanding the cardiovascular outcomes associated with the clinical use of this drug.

5. Collister JP, Nahey DB. The cardiovascular response of normal rats to dual lesion of the subfornical organ and area postrema at rest and to chronic losartan. Brain Res.2009;1302:118-24, doi: 10.1016/j.brainres.2009. 09.021. 6. Boolell M, Allen MJ, Ballard SA, Gepi-Attee S, Muirhead GJ, Naylor AM, et al. Sildenafil: an orally active type 5 cyclic GMP-specific phosphodiesterase inhibitor for the treatment of penile erectile dysfunction. Int J Impot Res. 1996;8:47-52. 7. Zusman RM. Cardiovascular data on sildenafil citrate: introduction. Am J Cardiol.1999; 83:1C-2C, doi: 10.1016/S0002-9149(99)00041-7. 8. Zusman RM, Morales A, Glasser DB, Osterloh IH. Overall cardiovascular profile of sildenafil citrate. Am J Cardiol. 1999;83:35C-44C, doi: 10.1016/ S0002-9149(99)00046-6. 9. Kloner RA. Cardiovascular risk and sildenafil. Am J Cardiol. 2000; 86:57F-61F, doi: 10.1016/S0002-9149(00)00895-X. 10. Lin CS, Lin G, Xin ZC, Lue TF. Expression, distribution and regulation of phosphodiesterase 5. Curr Pharm Des. 2006;12:3439-57, doi: 10.2174/ 138161206778343064. 11. Uthayathas S, Karuppagounder SS, Thrash BM, Parameshwaran K, Suppiramaniam V, Dhanasekaran M. Versatile effects of sildenafil: recent pharmacological applications. Pharmacol Rep. 2007;59:150-63. 12. Bischoff E. Potency, selectivity, and consequences of nonselectivity of PDE inhibition. Int J Impot Res. 2004;16Suppl 1:S11-4., doi: 10.1038/sj.ijir. 3901208 13. Kim NN, Huang Y, Moreland RB, Kwak SS, Goldstein I, Traish A. Crossregulation of intracellular cGMP and cAMP in cultured human corpus cavernosum smooth muscle cells. Mol Cell Biol Res Commun. 2000;4:104, doi: 10.1006/mcbr.2000.0249. 14. Uckert S, Hedlund P, Waldkirch E, Sohn M, Jonas U, Andersson KE, et al. Interactions between cGMP- and cAMP-pathways are involved in the regulation of penile smooth muscle tone. World J Urol. 2004;22:261-6, doi: 10.1007/s00345-003-0394-4. 15. Kass DA, Takimoto E, Nagayama T, Champion HC. Phosphodiesterase regulation of nitric oxide signaling. Cardiovasc Res. 2007;75:303-14, doi: 10.1016/j.cardiores.2007.02.031. 16. Malik V, Holobotovskyy VV, Phillips JK, McKitrick DJ, Arnolda LF. Intrathecal cGMP elicits pressor responses and maintains mean blood pressure during haemorrhage in anaesthetized rats. J Physiol. 2007;581: 543-52, doi: 10.1113/jphysiol.2006.125690. 17. Prado WA. Antinociceptive potency of intrathecal morphine in the rat tail flick test: a comparative study using acute lumbar catheter in rats with or without a chronic atlanto - occipital catheter. J Neurosci Methods. 2003;129:33-39, doi: 10.1016/S0165-0270(03)00197-3. 18. Storkson RV, Kjorsvik A, Tjolsen A, Hole K. Lumbar catheterization of the spinal subarachnoid space in the rat. J Neurosci Methods.1996;65:16772, doi: 10.1016/0165-0270(95)00164-6. 19. Arnolda LF, McKitrick DJ, Llewellyn-Smith IJ, Minson JB. Nitric Oxide limits pressor responses to sympathetic activation in rat spinal cord. Hypertension. 2000;36:1089-92. 20. Phillips BG, Kato M, Pesek CA, Winnicki M, Narkiewicz K, Davison D, et al. Sympathetic activation by sildenafil. Circulation. 2000;102:3068-73. 21. Piccirillo G, Nocco M, Lionetti M, Moise A, Naso C, Marigliano V, et al. Effects of sildenafil citrate (viagra) on cardiac repolarization and on autonomic control in subjects with chronic heart failure. Am Heart J. 2002;143:703-10, doi: 10.1067/mhj.2002.121547. 22. Choi J, Farley JM. Effects of 8-bromo-cyclic GMP on membrane potential of single swine tracheal smooth muscle cells. J Pharmacol Exp Ther. 1998; 285:588-94. 23. Patil CS, Singh VP, Kulkarni SK. Peripheral and central activation of nitric oxide- cyclic GMP pathway by sildenafil. Inflammopharmacology. 2005;13:467-78, doi: 10.1163/156856005774649359. 24. Stief CG, Uckert S, Becker AJ, Harringer W, Truss MC, Forssmann WG, et al. Effects of sildenafil on cAMP and cGMP levels in isolated human cavernous and cardiac tissue. Urology. 2000;55:146-50, doi: 10.1016/ S0090-4295(99)00371-4. 25. Botha P, Parry G, Dark JH, Macgowan GA. Acute hemodynamic effects of intravenous sildenafil citrate in congestive heart failure: comparison of phosphodiesterase type-3 and -5 inhibition. J Heart Lung Transplant. 2009;28:676-82, doi: 10.1016/j.healun.2009.04.013.

ACKNOWLEDGMENTS Fundaçaõ de Amparo a` Pesquisa do Estado de Saõ Paulo (FAPESP), Conselho Nacional de Desenvolvimento Cientifico e Tecnolo´gico (CNPq), and Coordenadoria de Aperfeiçoamento de Pessoal de Nı´vel Superior (CAPES).

REFERENCES 1. Fazan Jr R, Huber DA, Silva CA, Dias da Silva VJ, Salgado MCO, Salgado HC. Sildenafil acts on the central nervous system increasing sympathetic activity. J Appl Physiol. 2008;104:1683-9, doi: 10.1152/japplphysiol.01142. 2007. 2. Guyenet PG. The sympathetic control of blood pressure. Nat Rev Neurosci. 2006;7:335-46, doi: 10.1038/nrn1902. 3. Simms AE, Paton JF, Pickering AE. Disinhibition of the cardiac limb of the arterial baroreflex in rat: a role for metabotropic glutamate receptors in the nucleus tractus solitarii. J Physiol. 2006;575:727-38, doi: 10.1113/ jphysiol.2006.112672. 4. Chen QH, Toney GM. Excitability of paraventricular nucleus neurones that project to the rostral ventrolateral medulla is regulated by smallconductance Ca2+-activated K+ channels. J Physiol. 2009;587:4235-47, doi: 10.1113/jphysiol.2009.175364.

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DOI:10.1590/S1807-59322011000800018

BASIC RESEARCH

The impact of previous para-areolar incision in the upper outer quadrant of the breast on the localization of the sentinel lymph node in a canine model Paulo Henrique Dio´genes Vasques,II Luiz Gonzaga Porto Pinheiro,I,II,III Joa˜o Marcos de Meneses e Silva,II Jose´ Ricardo de Moura Torres-de-Melo,II Karine Bessa Porto Pinheiro,III Joa˜o Ivo Xavier RochaII,III I

Department of Surgery, Federal University of Ceara´, Fortaleza, Ceara´, Brazil. II Saul Goldenberg Experimental Surgery Laboratory, Fortaleza, Ceara´, Brazil. Group Study and Education in Oncology, Department of Surgery, Federal University of Ceara´, Fortaleza, Ceara´, Brazil.

III

OBJECTIVES: This paper discusses the influence of a para-areolar incision in the upper outer quadrant of the breast on the location of the sentinel lymph node in a canine model. METHODS: The sentinel lymph node was marked with technetium-99, which was injected into the subareolar skin of the cranial breast. After the marker had migrated to the axilla, an arcuate para-areolar incision was performed 2 cm from the nipple in the upper outer quadrant. Patent blue dye was then injected above the upper border of the incision. At the marked site, an axillary incision was made, and the sentinel lymph node was identified by gamma probe and/or by direct visualization of the dye. The agreement between the two injection sites and the two sentinel lymph node identification methods was determined. Our sample group consisted of 40 cranial breasts of 23 adult females of the species Canis familiaris. The data were analyzed by using the McNemar test and by determining the kappa agreement coefficient. RESULT: Our findings showed that in 95% of the breasts, the sentinel lymph node was identified by the injection of technetium-99 m into the subareolar region, and in 82% of the cases, the sentinel lymph node was identified by the injection of patent blue dye above the upper border of the incision. The methods agreed 82% of the time. CONCLUSIONS: Previous para-areolar incisions in the upper outer quadrant did not interfere significantly with the biopsy when the dye was injected above the upper border of the incision. KEYWORDS: Breast cancer; Gamma probe; Sentinel node; Animal model; Oncologic surgery. Vasques PHD, Pinheiro LGP, Silva JMM, Torres-de-Melo JRM, Pinheiro KBP, Rocha JIX. The impact of previous para-areolar incision in the upper outer quadrant of the breast on the localization of the sentinel lymph node in a canine model. Clinics. 2011;66(8):1413-1418. Received for publication on February 11, 2011; First review completed on March 25, 2011; Accepted for publication on April 24, 2011 E-mail: luizgporto@uol.com.br Tel.: 55 85 3283-5700

INTRODUCTION

not yet committed to the tumor state, then the other nodes in the lymphatic network would also be healthy. They reported a 1% rate of false negatives, opening prospects for research throughout the world.5-7 In 1993, Alex et al.8 injected technetium and introduced the technique of gamma probe-guided surgery to identify the sentinel node. In the same year, Krag et al.9 published a study on the sentinel node using technetium-99 (Tc99) with probes used for the detection of gamma rays in breast cancer. They concluded that the radiolocation and selective resection of the sentinel lymph node was feasible and could be used to determine the status of the axillary lymph node. The initial techniques of SLN injection in breast cancer were intended to aid in the location of the drainage route of tumor metastasis; the dye or radiopharmaceuticals were injected into the peritumoral region. Following the increased understanding of the lymphatic drainage of the breast, superficial injection techniques have been implemented.10,11

In 1977, Cabanas reported the identification of nodal drainage pathways and a sentinel node from penile cancer. Sentinel lymph node (SLN) biopsy, introduced in the mid1990s, has revolutionized the management of breast cancer patients.2,3 The use of this method has significantly reduced the need for extensive axillary dissection to establish a reliable prognosis in breast cancer treatment. Using isosulfan blue dye, Morton et al.4 demonstrated the accuracy of sentinel node biopsy for nodal staging and the management of patients with primary cutaneous melanoma. The authors determined that if the sentinel node tumor was

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The impact of previous para-areolar incision on sentinel lymph node localization Vasques PHD et al.

Based on this knowledge, most centers have adopted the following superficial injection techniques: subdermal, intradermal, subareolar, or periareolar. These techniques have been chosen because they are technically easy, highly reproducible and provide a higher rate of identification and lower false-negative rates than does peritumoral injection.12 In daily practice, the surgeon often uses the arcuateareolar incision for access to nodes or foci of microcalcifications in the upper breast quadrant. In cases of previously diagnosed, biopsied cancer patients where the histopathological diagnosis pointed to additional surgical procedures, some authors claim that the SLN identification should be performed by injecting markers immediately above the incision.13 In this case, it is questionable if the node identified by this technique is the same as the one identified with the marker injected into the subpapilar space following a skin incision. Therefore, this study aimed to evaluate, in an experimental model, whether para-areolar incisions in the upper outer quadrant of the paired cranial thoracic breasts of bitches interfere with the identification of the SLN.

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Figure 1 - Intradermal injection of 99mTc into the subareolar region of cranial thoracic breasts using a fine-gauge insulin needle.

MATERIALS AND METHODS

promote the migration and transport of the radiocolloid particles to the lymphatic system. Five minutes after the Tc-99 subareolar injection, axillary mapping was performed using a gamma probe that was covered with a surgical glove (Figure 2) to identify the location of the point of maximum uptake of the gamma radiation in the axilla, which represents the projection of the sentinel node. The upper breast quadrants were outlined with a dermographic pen, using the nipple as the center point. The quadrants delineated segments with axes that were 2 cm distant from the center of the nipple and formed a 90-degree angle at the junction point. The arcuate incision position and extent were determined by linking the two distal ends of the axes. Therefore, the incisions were placed between 12 – 15 h (left breast) and 9 – 12 h (right breast), 2 cm from the nipple. After incision and hemostasis by compression of the wound, 0.5 ml blue dye (bleu patente´ V Guerbet 2.5%) was injected just above the midpoint of the arcuate top edge of

All studies were conducted according to the rules of the Brazilian College of Animal Experimentation (COBEA). The use of the radioactive tracer Tc99 was approved by the National Nuclear Energy Commission (CNEN) and the Institute for Energy and Nuclear Research (IPEN). Approval for the experimental use of laboratory animals was obtained from the Committee of Ethics in Animal Research of the Federal University of Ceara´. Sample group - The sample group consisted of 40 breasts obtained from 23 healthy bitches of undefined breeds of the species Canis familiaris, housed in the Zoonosis Control Center Kennel of the City of Fortaleza. Inclusion criteria - Inclusion in the study required clinically healthy bitches, who showed well-defined cranial thoracic breasts and nipples. Exclusion criteria – The animals weren’t included if they were under six months old, weighed either less than 5 kg or more than 15 kg and presented with cranial thoracic breast atrophy, previous surgery and/or scars. As a radiotracer, we used 0.2 mL Tc99 phytate (Conselho Nacional de Energia Nuclear – CNEN, Brazil).

Surgical procedure After immobilization on the operating table with the legs abducted, the animals were anesthetized subcutaneously with 0.05 mg/kg atropine (CentralvetH, Brazil) followed by 15 mg/kg ketamine hydrochloride (SyntecH, Brazil) and 1.5 mg/kg xylazine hydrochloride (SyntecH, Brazil) intramuscularly. The level of anesthesia was monitored continuously by clinical parameters, such as movements of the nostrils and other muscle groups and respiratory and cardiac rates. Additional anesthesia was provided as required. Venipuncture in an upper paw with a 9- or 21gauge ‘‘scalp’’ needle was performed for the administration of 0.9% saline to secure adequate venous access. The surgical areas of the breast and axilla were shaved to remove excess hair. Subsequently, 0.2 ml Tc99 phytate was injected intradermally using a fine-gauge insulin needle in the subareolar region of both cranial thoracic breasts (Figure 1). Two-minute bidigital massage was provided to

Figure 2 - Performing axillary mapping using a gamma probe to identify the location of the projection of the sentinel node in the axilla.

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The impact of previous para-areolar incision on sentinel lymph node localization Vasques PHD et al.

Figure 4 - Complete exposure of the identified SLN and measurement of the radiocolloid uptake in vivo before the removal of the SLN. Figure 3 - Injection of blue dye (bleu patenteÂ´ V Guerbet 2.5%) just above the midpoint of the arcuate top edge of the incision in the subdermal region.

We recorded all radiation rates at the injection site before the incision was made in the armpit, the sentinel lymph node radioactivity in vivo and ex vivo and the radioactivity from the central bed where the sentinel bed was located. The parameter used was the background radioactivity (radiation control). The tabulated data quantified the intersection between the two markings and the correlation between the methods.

the incision in the subdermal region (Figure 3). Local compression and gentle massage at the site of injection were applied to prevent the contrast from spreading farther and to allow it to be transported by the lymphatic network of the area to the sentinel node. After five minutes, the axillary point of the highest radiation uptake was identified with the gamma probe NuclearLab-DGC-8. A 3-cm-long axillary incision was made at this point, followed by a careful dissection that was guided by the visualization of a bluish afferent lymphatic system that pointed to the SLN(s). When the afferent lymphatic vessels were difficult to identify, the location of the SLN with the gamma probe was necessary to guide the incision site. A significant radioisotope uptake should be at least fivefold higher than the background radioactivity in the armpit. The background radioactivity represents the count that was obtained in four axillary equidistant points from the point of injection or the location of the sentinel node. Upon complete exposure of the identified SLN, the radiocolloid uptake was measured in vivo and ex vivo after the removal of the SLN (Figures 4-5). The values obtained were registered for subsequent analysis. The position of the sentinel lymph node, as identified by the blue dye, was compared with the location identified by the gamma probe to check whether there was agreement between the two methods. Radiation was also measured in the surgical bed to identify any remaining hot lymph nodes. If positive, the lymph node was removed, and its radiation and dye uptake characteristics were recorded. The area ratio of the greatest uptake/background radiation of the surgical bed should be greater than or equal to ten. Verification of the radioactivity of the SLN after removal from the surgical field (ex-vivo counting) and comparison with the radioactivity from the surgical bed confirmed that the SLN was actually removed and that there was no other source of radiation that could justify the continuation of a search for another node.

Figure 5 - Measurement the radiocolloid uptake ex vivo after the removal of the SLN.

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Table 1 - Kappa coefficient of concordance when comparing isolated patent blue dye with a combination of Tc99 and patent blue dye in SLN identification.

Table 3 - Identification of the SLN in the bitch axilla using blue dye injection into the dermal region of the upper edge of the arcuate incision in the left superior quadrant of the right and left breasts.

Tc99 + Patent Blue Dye Patent Blue Dye

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Yes No Total Yes 32 1 33 No 1 6 7 Total 33 7 40 Tc99: technetium99 SLN: Sentinel lymph node Kappa = 0.827/Observed agreements: 95%

SLN identification

Positive 32 Negative 7 SLN: Sentinel lymph node

% 82% 18%

Sentinel node biopsy has evolved with the publication of the many studies since Cabanasâ&#x20AC;&#x2122; original paper in 1977.1 Morton et al.4 identified the SLN in cases of melanoma using

dye, while Krag et al.9 used radiopharmaceuticals and a gamma detector during surgery and identified the SLN in 82% of cases with a 100% accuracy rate. Using the blue dye, Giuliano et al.14 obtained rates of 66% for node identification and 96% for accuracy. Albertini et al.3 used the combination of dye+Tc99, attaining positive SLN identification 92% of the time with 100% accuracy. For the past 20 years, SLN biopsy, which is considered to be a minimally invasive procedure, has replaced axillary dissection with high accuracy (97 to 99%) in predicting the axillary lymph node status. Since then, the SLN biopsy has become a standard tool for addressing the axilla in early breast cancer in many medical centers worldwide.12-16 The release of the SLN biopsy method and increased knowledge of breast anatomy have resulted in many questions about the technical peculiarities of the method, the best site of injection, the best method, the best contrast and radiotracers, injection techniques, combinations of methods, the learning curve, tumor characteristics, and the morbidity associated with the method. In short, many variables are still under evaluation. One of the important points that is being discussed concerns the role that surgery and previous breast biopsies play in identifying the SLN. Many authors exclude from their studies patients who were previously diagnosed by incisional biopsy or by formal excisional resections based on the assumption that these procedures could interfere in the identification of the SLN, increase the rates of false negatives and derail the method.3,17-25 More recently, some researchers have suggested that previous excisional biopsies are no impediment to identifying the SLN.26,27 In 2005, the American Society of Clinical Oncology released some information based on current data with limited levels of evidence, suggesting that SLN biopsy was not contraindicated in patients with previous diagnoses and excisional biopsies of the breast.12 Because of the importance of this topic, our study aimed to assess the influence of the para-areolar incision in the upper quadrant of the breasts of bitches, considering that this incision is a common practice of breast clinics when they are faced with patients with previous surgeries or biopsies. In these circumstances, the contrast has been injected into the edges and into the region overlying the scar without a previous SLN biopsy. The initial use of subareolar

Table 2 - Identification of the SLN in the bitch axilla using the sub-areolar injection of Tc99 into that region of the right and left breasts.

Table 4 - Comparison of the intersection of the methods discussed in this research to identify the SLN in the axilla (right and left breasts).

The animals were sacrificed after the experiment with the rapid intravenous injection of 10% potassium chloride, stored in a suitable plastic bag, placed in refrigeration for at least two hours, and subsequently sent to the Zoonosis Control Center Kennel.

Statistical methods The Graphpad software (http://www.graphpad.com/ quickcalcs/index.cfm) was used for computation and statistical analysis. The data were evaluated by a McNemar test and the kappa coefficient of concordance. P-values were determined using Fisherâ&#x20AC;&#x2122;s exact test. A p-value ,0.05 was considered to be statistically significant.

RESULTS From a sample group of 23 bitches (46 breasts), only 40 fulfilled the inclusion/exclusion criteria; 95% of the breasts studied had an SLN that was identified within the axillary site by using the gamma probe after an injection of Tc99 phytate into the subareolar region (Table 1). After the injection of patent blue into the dermis of the upper edge of the arcuate incision in the upper quadrant of the left breast, 82% (33/40) of the isolated lymph nodes were stained (Table 2). In the cases studied, there was agreement among the methods evaluated in 82% (28/43) of cases (Table 3). Table 4 shows the results that were obtained using Tc99 and patent blue dye (sentinel nodes from the left and right cranial thoracic bitch breasts). The McNemar test resulted in p = 0.1306 (IC: 95%). The kappa coefficient of concordance when comparing Tc99 isolated with a combination of Tc99 and patent blue in a sentinel node assay was 0.157 (82% observed agreements) (Table 5). The kappa coefficient of concordance when comparing the single use of patent blue dye with a combination of Tc99 and patent blue was 0.827 (95% observed agreements) (Table 6).

DISCUSSION

N SLN identification

Positive 38 95% Negative 2 5% Tc99: technetium99 SLN: Sentinel lymph node

SLN identification

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Positive 33 Negative 7 SLN: Sentinel lymph node

% 82% 18%

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The impact of previous para-areolar incision on sentinel lymph node localization Vasques PHD et al.

placed between the subareolar plexus and the lymphatic system that drains the upper left quadrant of the breast, in particular the section of the main collecting duct, so as not to allow part of the lymph nodes to capture the blue patent dye, as was shown in this model. In our study, due to the injection of the contrast in the upper edge of the incision, a positive SLN identification in 82% of cases (32/40) was obtained. These results corroborate some of the findings that the most recent publications have emphasized; namely, there has been an emphasis on the recommendation of, not a contraindication to, SLN biopsy in patients with a previous excisional biopsy because a significant percentage of those patients may benefit from this method.26,27 The hypothesis that the breast skin and underlying parenchyma drain into the same lymph nodes at the base of the axilla is based upon their common embryological origin28,29, justifying the injection of the tracer into the skin above the incision. Our experimental results reinforce this hypothesis.

Table 5 - Identification of the SLN in the right and left cranial thoracic breasts of bitches when using technetium and patent blue (McNemar test). Tc99 Patent Blue Dye

Yes No Total Yes 32 1 33 No 6 1 7 Total 38 2 40 Tc99: technetium99 SLN: Sentinel lymph node p = 0.1306/IC of 95% (McNemar Test)

Tc99 injection, with identification of the local projection of the SNL in the armpit after five minutes and the subsequent use of patent blue dye injected into the upper edge of the incision, as a method capable of identifying the SLN was purposely performed in that order because the distance between the areola and the axilla in the bitch is relatively small. It is known that the greater the distance between the injection of the radio tracer and the uptake area of the radioactive material by the SLN, the better the lymph node identification by radio-guided surgery because interference is avoided between the markings of the injection site and the uptake site. This phenomenon was observed in this study because the SLN was identified in 95% of cases (38/40) with a sub-areolar Tc99 injection. Based on the anatomicalphysiological similarity of the drainages of the human and bitch breast, we identified the sentinel node by injecting Tc99 into the subareolar region, and then, after identifying the presence of radiation in the projection in the axillary SLN, we performed the para-areolar incision for the left breast along with the injection of patent blue dye in the upper edge of the incision. The experiment demonstrated an SLN identification rate with blue dye and Tc99 of 95% (32/ 33) (Table 5-6). In 33 of the 40 cases, there was agreement (82%) between the two methods of the SLN identification, i.e., between the percentage of lymph nodes that captured the Tc99 radioisotope and were stained with patent blue or that did not capture the Tc99 radioisotope and were not stained with the blue dye. The lymph nodes were not identified in 5% of breasts with the Tc99 subareolar injection method and in 17.5% using blue dye on the edge of the para-areolar incision. These findings indicate that the injection of contrast at the upper edge of the incision, involving the entire left upper quadrant of the bitch breast, presents a greater failure rate in identifying the SLN compared to injection of Tc99 into the breast. This fact can be explained by injury to the lymphatic drainage pathways that was caused by the para-areolar incision. The incision was

CONCLUSION In this study, 95% (38/40) of all lymph nodes captured 82% (33/40) of the Tc99 radioisotope and were stained with the blue dye. With one exception, all nodes were labeled as sentinel nodes (95%). In 82% of the cases (33/40), concordance between the methods was verified (Table 5). These findings may explain why many researchers initially exclude patients with previous breast biopsies from their studies because there is a decrease in the rate of identification of the SLN in cases where the lymphatic network of the upper quadrant of the left breast has been damaged by previous surgery. One fact that must be considered is that the canine experimental model we created does not evaluate time interference in the mapping of the sentinel node. This study shows that lymph nodes found from injection at the upper edge of a recent incision correspond to the sentinel node in bitch breasts in 95% of cases (32/33). The rates of identification of the SLN by the subareolar injection of Tc99 and the injection of patent blue in the para-areolar incision were 95% (38/40) and 82% (32/40), respectively. A previous para-areolar incision in the upper outer breast quadrants of cranial thoracic bitch breasts does not interfere significantly in the identification of the sentinel lymph node when the dye is injected into the upper edge of a recent incision.

REFERENCES 1. Cabanas RM. An approach for the treatment of penile carcinoma. Cancer. 2000;39:456-466, doi: 10.1002/1097-0142(197702)39:2,456:: AID-CNCR2820390214.3.0.CO;2-I. 2. Giuliano AE. Sentinel lymphadenectomy in primary breast carcinoma: an alternative to routine axillary dissection. J Surg Oncol. 1996;62:75–77, doi: 10.1002/(SICI)1096-9098(199606)62:2,75::AID-JSO1.3.0.CO;2-N. 3. Albertini JJ, Lyman GH, Cox C, Yeatman T, Balducci L, Ku N, et al. Lymphatic mapping and sentinel node biopsy in the patient with breast cancer. JAMA. 1996;276:1818–22, doi: 10.1001/jama.276.22.1818. 4. Morton DL, Wen DR, Wong JH, Economou JS, Cagle LA, Storm FK, et al. Technical details of intraoperative lymphatic mapping for early stage melanoma. Arch Surg. 1992;127:392-9. 5. Oliveira Filho RS, Santos ID, Ferreira LM, de Almeida FA, Simo˜es e Silvia Enokihara MM, et al. Is intra-operative gamma probe detection really necessary for inguinal sentinel lymph node biopsy? Sao Paulo Med J. 2000;118:165-8, doi: 10.1590/S1516-31802000000600003. 6. Udi C. Axillary node Sample to Evaluate the Axilla. World J Surg. 2001;25:773-9, doi: 10.1007/s00268-001-0004-9.

Table 6 - Kappa coefficient of concordance when comparing technetium isolated with a combination of Tc99 and Patent blue dye in the identification of the SLN. Tc99 + Patent Blue Dye Tc99

Yes No Total Yes 32 6 38 No 1 1 2 Total 33 7 40 Tc99: technetium99 SLN: Sentinel lymph node Kappa = 0.157/Observed agreements: 82%

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18. Krag DN. Minimal access surgery for staging regional lymph nodes: the sentinel-node concept. Curr Probl Surg. 199835:951-1016. 19. Borgstein PJ, Pijpers R, Comans EF, van Diest PJ, Boom RP, Meijer S. Sentinel lymph node biopsy in breast cancer: guidelines and pitfalls of lymphoscintigraphy and gamma probe detection. J Am Coll Surg 1998;186:275-83, doi: 10.1016/S1072-7515(98)00011-8. 20. Veronesi U, Paganelli G, Viale G, Galimberti V, Luini A, Zurrida S, et al. Sentinel lymph node biopsy and axillary dissection in breast cancer: results in a large series. J Natl Cancer Inst. 1999;91:368-73, doi: 10.1093/ jnci/91.4.368. 21. Rodier JF, Routiot T, Mignotte H, Janser JC, Bremond A, David E. Lymphatic mapping and sentinel node biopsy of operable breast cancer. World J Surg. 2000;24:1220-25, doi: 10.1007/s002680010240. 22. Giuliano AE, Haigh PI, Brennan MB, Hansen NM, Kelley MC, Ye W. Prospective observational study of sentinel lymphadenectomy without further axillary dissection in patients with sentinel node-negative breast cancer. J Clin Oncol. 2000;18:2553-9. 23. Mariani G, Moresco L, Viale G, Villa G, Bagnasco M, Canavese G. Radioguided sentinel lymph node biopsy in breast cancer surgery. J Nucl Med. 2001;42:1198-215. 24. Dâ&#x20AC;&#x2122;Eredita G, Serio G, Mele M, Giardina C, Martellotta M, Ferrarese F. Effect of the use of vital dye, lymphoscintigraphy, or a combination for axillary lymphatic mapping and sentinel node biopsy in breast cancer. World J Surg. 2002;26:588-90, doi: 10.1007/s00268-001-0272-4. 25. Veronesi U, Paganelli G, Viale G, Luini A, Zurrida S, Galimberti V. A randomized comparison of sentinel-node biopsy with routine axillary dissection in breast cancer. N Engl J Med. 2003;349:546-53, doi: 10.1056/ NEJMoa012782. 26. Mcmasters KM, Tuttle TM, Carlson DJ, Brown CM, Noyes RD, Glaser RL. Sentinel lymph node biopsy for breast cancer: a suitable alternative to routine axillary dissection in multiinstitutional practice when optimal technique is used. J Clin Oncol. 2000;18:2560-6. 27. Tafra L, Lannin DR, Swanson MS, Van Eyk JJ, Verbanac KM, Chua AN. Multicenter trial of sentinel node biopsy for breast cancer using both technetium sulfur colloid and isosulfan blue dye. Ann Surg. 2001;233:519, doi: 10.1097/00000658-200101000-00009. 28. Grant RN, Tabah J, Adair FE. The surgical significance of the subareolar symph plexus in cancer of the breast. Surgery. 1953;33:71-78. 29. Turner-Warwick RT. The lymphatics of the breast. Br J Surg. 1959;574-82, doi: 10.1002/bjs.18004620004.

7. Oliveira Filho RS, Silva AM, Hochman B, Oliveira RL, Arcuschin L, Wagner J, et al. Vital dye is enough for inguinal sentinel lymph node biopsy in melanoma patients. Acta Cir Bras. 2006;21:12-5, doi: 10.1590/ S0102-86502006000100004. 8. Alex JC, Weaver DL, Fairbank JT, Rankin BS, Krag DN. Gamma-probeguided lymph node localization in malignant melanoma. Surg Oncol. 1993;2:303-8, doi: 10.1016/S0960-7404(06)80006-X. 9. Krag DN, Weaver DL, Alex JC, Fairbank JT. Surgical resection and radiolocalization of the sentinel lymph node in breast cancer using a gamma probe. Surg Oncol. 1993;335-9, doi: 10.1016/0960-7404(93)90064-6. 10. McMasters KM, Wong SL, Martin RC 2nd, Chao C, Tuttle TM, Noyes RD, et al. Dermal injection of radioactive colloid is superior to peritumoral injection for breast cancer sentinel lymph node biopsy: results of a multiinstitutional study. Ann Surg. 2001;233:676-87, doi: 10. 1097/00000658-200105000-00012. 11. Chagpar A, Martin RC, 3rd, Chao C, Wong SL, Edwards MJ, Tuttle T, et al. Validation of subareolar and periareolar injection techniques for breast sentinel lymph node biopsy. Arch Surg. 2004;139:614-8, doi: 10. 1001/archsurg.139.6.614. 12. Lyman GH, Giuliano AE, Somerfield MR, Benson AB3rd, Bodurka DC, Burstein HJ, et al. American Society of Clinical Oncology guideline recommendations for sentinel lymph node biopsy in early-stage breast cancer. J Clin Oncol. 2005;23:7703-20, doi: 10.1200/JCO.2005.08.001. 13. Krag DN, Harlow S, Weaver D, Ashikaga T. Radiolabeled sentinel node biopsy: collaborative trial with the National Cancer Institute. World J. Surg. 2001;25:823-8, doi: 10.1007/s00268-001-0012-9. 14. Giuliano AE, Kirgan DM, Guenther JM, Morton DL. Lymphatic mapping and sentinel lymphadenectomy for breast cancer. Ann Surg.1994;220:3918, doi: 10.1097/00000658-199409000-00015. 15. Wilke LG, Mccall LM, Posther KE, Whitworth PW, Reintgen DS, Leitch AM, et al. Surgical complications associated with sentinel lymph node biopsy: results from aprospective international cooperative group trial. Ann Surg Oncol. 2006;13:491-500, doi: 10.1245/ASO.2006.05.013. 16. Mansel RE, Fallowfield L, Kissin M, Goyal A, Newcombe RG, Dixon J, et al. Randomized multicenter trial of sentinel node biopsy versus standard axillary treatment in operable breast cancer: the ALMANAC Trial. J. Natl. Cancer Inst. 2006;98:599-609, doi: 10.1093/jnci/djj158. 17. Giuliano AE, Jones RC, Brennan M, Statman R. Sentinel lymphadenectomy in breast cancer. J Clin Oncol. 1997;15:2345-50.

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DOI:10.1590/S1807-59322011000800019

BASIC RESEARCH

Effects of diabetes on myocardial capillary density and serum angiogenesis biomarkers in male rats Majid Khazaei,I Ali Reza Fallahzadeh,I Mohammad Reza Sharifi,I Noushin Afsharmoghaddam,II Shaghayegh Haghjooy Javanmard,III Ensieh SalehiI I

Department of Physiology, Isfahan University of Medical Sciences, Isfahan, Iran. II Department of Pathology, Isfahan University of Medical Sciences, Isfahan, Iran. III Physiology Research Center, Department of Physiology, Isfahan University of Medical Sciences, Isfahan, Iran.

INTRODUCTION: Cardiovascular disease is one of the main causes of mortality and morbidity in diabetic patients. This study evaluated the effects of diabetes on myocardial capillary density and several serum angiogenic factors including nitric oxide, vascular endothelial growth factor, and soluble vascular endothelial growth factor receptors. METHODS: Twelve male rats were divided into two groups: control and diabetic (n = 6 each). Diabetes was induced with a single dose of streptozotocin (50 mg/kg). After 21 days, capillary density in the myocardial tissue was evaluated using immunohistochemical staining and is reported as capillaries per mm2. Blood samples were collected before and after the induction of diabetes. RESULTS: In the diabetic group, serum nitric oxide and soluble vascular endothelial growth factor receptor 2 concentrations were lower than the levels in the control group, while the level of soluble vascular endothelial growth factor receptor 1 was significantly higher. There was no significant change in the serum vascular endothelial growth factor concentration between the diabetic and control groups; however, the ratio of vascular endothelial growth factor to vascular endothelial growth factor receptor 1 was significantly lower in the diabetic animals. The myocardial capillary density was also lower in the diabetic group compared with the control group (1549¡161 vs. 2156¡202/mm2, respectively). CONCLUSION: Reduced serum nitric oxide and vascular endothelial growth factor receptor 2 levels, increased serum vascular endothelial growth factor receptor 1 levels and a lower vascular endothelial growth factor to vascular endothelial growth factor receptor 1 ratio may be responsible for the decreased myocardial capillary density in diabetic rats. KEYWORDS: Diabetes; Capillary Density; Vascular Endothelial Growth Factor; Nitric Oxide; Myocardium. Khazaei M, Fallahzadeh AR, Sharifi MR, Afsharmoghaddam N, Javanmard SH, Salehi E. Effects of diabetes on myocardial capillary density and serum angiogenesis biomarkers in male rats. Clinics. 2011;66(8):1419-1424. Received for publication on December 23, 2010; First review completed on February 21, 2011; Accepted for publication on April 26, 2011 E-mail: Khazaei@med.mui.ac.ir Tel.: 98 3117922417

The two VEGF receptor subtypes that are primarily involved in angiogenesis are VEGF receptor type 1 (VEGFR-1) and VEGF receptor type 2 (VEGFR-2). Soluble VEGFR-1 (sVEGFR-1) binds with a high affinity to VEGF and reduces VEGF activity and angiogenesis,6-8 whereas VEGFR-2 has proangiogenic effect in the angiogenesis process.9 Increased VEGF-mediated angiogenesis in diabetic retinopathy and nephropathy and a decreased angiogenic response in wound healing and ulcers have been documented in diabetic subjects.10,11 This study aimed to evaluate the effect of diabetes on the serum concentrations of VEGF, NO and the soluble forms of VEGFR-1 and VEGFR-2 (sVEGFR-1 and sVEGFR-2) as well as myocardial capillary density in type I diabetic rats.

INTRODUCTION Cardiovascular disease is one of the main causes of mortality and morbidity in diabetic subjects, and several long-term complications of diabetes involve impaired angiogenesis. Angiogenesis is the growth of new capillaries from pre-existing vessels1 and occurs during physiological conditions such as wound healing. However, abnormal angiogenesis may occur in some pathological conditions, including diabetic retinopathy.1 Nitric oxide (NO) and vascular endothelial growth factor (VEGF) are important factors affecting angiogenesis.2,3 NO has important roles in multiple physiological processes and pathological states,4,5 and VEGF and its receptors have essential roles in pathological and physiological angiogenesis.2,6

METHODS Copyright ß 2011 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Animals Male Wistar rats (aged 10–12 weeks, weighing 230¡30 g) were used in the present study. The animals were kept two

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the serum was kept at -70 ˚C until the serum VEGF, NO, sVEGFR-1, and sVEGFR-2 concentrations were measured. After 21 days, the animals were euthanized. The apex of the heart was removed, washed with normal saline and fixed in formalin solution for immunohistochemical evaluation. Body weight was measured before induction of diabetes and 21 days after the experiment.

Table 1 - Fasting blood glucose level (mg/dl) and body weight (g) before and after the induction of diabetes. Group

control 6 diabetic 6

Body Weight (g)

Fasting Blood Glucose Level (mg/dl)

Day 0

Day 21

Day 0

245¡4.8 240¡7.6

285.71¡7.4* 173¡9.6*

122.57¡3.5 125¡6.0

Day 21 117.43¡5.4 508.6¡25.4*

Serum VEGF, sVEGFR-1, sVEGFR-2, and NO Measurements

*p,0.05 versus day 0.

Serum VEGF, sVEGFR-1, and sVEGFR-2 concentrations were measured using the quantitative sandwich enzyme immunoassay technique with the appropriate kits (R&D Systems, Minneapolis, USA).14,15 The serum nitrite concentration, one of the main metabolites of NO, was determined using the Griess reagent method (Promega, Madison, USA).16

per cage at room temperature (20–25 ˚C) with a 12 h light/ dark cycle and ad libitum access to food and water. The experimental protocols were approved by the ethics committee of Isfahan University of Medical Sciences.

Induction of Diabetes After one week of adaptation to the animal room, the rats were intraperitoneally injected with a single 50 mg/kg dose of streptozotocin (STZ, Sigma) to induce diabetes.12,13 STZ was dissolved in cold, normal saline and administered immediately. Two days after injection, the fasting blood glucose levels were determined. Animals with blood glucose greater than 300 mg/dl were considered diabetic.12,13 In the control group, normal saline was injected.

Capillary density was measured using immunohistochemistry with a rat monoclonal antibody directed against mouse CD31 (Abcam, Cambridge UK).17 Fifteen fields of three different sections from each cardiac tissue sample were randomly examined using a light microscope (4006) by two blinded observers. The number of capillaries in each tissue sample is reported as the number of capillaries per mm2.

Experimental Design

Statistical Analysis

The rats were divided into normal and diabetic groups (n = 6 each). Blood samples were taken before and after the induction of diabetes. The samples were centrifuged, and

All values are reported as the mean ¡ SE. Student’s t-test was used to evaluate differences between the two groups. Paired data were used for the data analysis before and after

Immunohistochemistry

Figure 1 - Serum nitrite concentration in control and diabetic rats before and after the induction of diabetes. *p,0.05.

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Figure 2 - The serum VEGF (A), sVEGF-R1 (B), and sVEGF-R2 concentrations (C) and the VEGF:VEGF-R1 ratio (D) before (white column) and after (black column) the induction of diabetes. *p,0.05 compared to day 0.

the induction of diabetes. Bivariate correlations were calculated using Pearsonâ&#x20AC;&#x2122;s correlation coefficient. A p-value less than 0.05 was considered statistically significant.

Effect of diabetes on myocardial capillary density The capillary density in the myocardial tissue (expressed as the number of capillaries per mm2) was significantly reduced in the diabetic group compared with the control group (p,0.05) (Figure 3B). Representative images of the immunohistochemical staining are presented in figure 3A.

RESULTS Blood glucose and body weight Table 1 shows the fasting blood glucose levels and body weight in the two groups. Body weight significantly increased over the course of the experiment in the control group, whereas the body weight of diabetic rats decreased over time (p,0.05). The fasting blood glucose level in the diabetic animals was higher than the level in the controls and remained elevated throughout the experiment.

Correlation analysis In the correlation analysis, we found that capillary density in myocardial tissue was positively correlated with the serum nitrite level (r = 0.70), the VEGF level (r = 0.40), the sVEGFR-1 level (r = -0.69) and the VEGF:sVEGFR-1 ratio (r = 0.71) (Figure 4).

DISCUSSION

Effects of diabetes on serum angiogenic factors Figure 1 illustrates the serum NO concentration in the two groups before and after the induction of diabetes. In the diabetic group, the serum NO level was significantly decreased at the end of experiment when compared with the level prior to the induction of diabetes (p,0.05; Figure 1). While there was no significant difference in the serum VEGF concentration between the groups (Figure 2A), the serum sVEGFR-1 concentration was increased and the sVEGFR-2 level was decreased in the diabetic group compared with the control group (p,0.05; Figure 2B and C). In addition, the VEGF:sVEGFR-1 ratio in diabetic animals was lower than the ratio in the controls (p,0.05; Figure 2D).

Our results show that the capillary density in myocardial tissue, the serum NO level, and the sVEGFR-2 level in diabetic rats were lower than the levels in the controls, while the sVEGFR-1 level was higher in the diabetic animals. Moreover, the VEGF:sVEGFR-1 ratio was significantly decreased in diabetic animals. The correlation analysis demonstrated that capillary density in myocardial tissue was correlated with the above serum angiogenic markers. Angiogenesis is controlled by a number of proangiogenic and antiangiogenic factors that are released in tissues. NO and VEGF have crucial roles during angiogesis.6,18 NO not only has a direct effect on angiogenesis, but other

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Figure 3 - Effect of diabetes on the myocardial capillary density. A. Representative images of the immunohistochemical staining (4006) with an anti-CD31 monoclonal antibody. B. Myocardial capillary density in the control and diabetic groups. n = 6 per group. * p,0.05.

ischemia, diabetic mice had increased VEGF and sVEGFR-1 levels and decreased phospho-AKT/AKT and phosphoendothelial NO synthase/endothelial NO synthase levels. Lu et al. also found that the post-ischemic capillary density and revascularization in the limbs of diabetic rats was decreased, with reduced mRNA and protein expression of eNOS, VEGF, and bFGF.31 However, despite the similarity in serum VEGF levels between the groups, we found that diabetic animals had higher sVEGFR-1 and lower sVEGFR-2 levels compared with the controls. sVEGFR-1 binds with a high affinity to VEGF9 and reduces VEGF activity, endothelial cell proliferation, angiogenesis and tumor growth.6-8 Thus, sVEGFR-1 acts as an antagonist of VEGF.36 In contrast, VEGF-R2 is an effector of proangiogenic signaling in the angiogenesis process.9 Therefore, reduced sVEGFR-2 levels and increased sVEGFR-1 levels in the serum may be responsible for the lower capillary density in the myocardial tissue of diabetic rats. We also investigated the VEGF to sVEGFR-1 ratio, as some studies have used the VEGF:sVEGFR-1 ratio as an indicator of angiogenesis status.37,38 In the present study, we found that the VEGF:sVEGFR-1 ratio was reduced in diabetic rats, which suggests decreased angiogenesis in the diabetic group. In conclusion, myocardial capillary density was lower in the diabetic group compared with the control group. Furthermore, the lower serum levels of the proangiogenic factors NO and sVEGFR-2, the higher level of the antiangiogenic factor sVEGFR-1 and the reduced VEGF:sVEGFR-1 ratio may

angiogenic growth factors affect angiogenesis as well by increasing NO production.19 A decrease in NO production in endothelial NO synthase窶電eficient mice disrupts the development of the coronary vessels and angiogenesis.20 In the present study, the serum NO concentration in diabetic rats was lower than the concentration in the control group, which supports the results of previous studies.21-24 Some possible mechanisms responsible for decreased NO production under high glucose conditions include the suppression of endothelial NO synthase expression and activity,25 overproduction of superoxides,26 and activation of protein kinase C.23 VEGF has been shown to be an important inducer of angiogenesis in a variety of in vivo models.6 Several experimental and clinical reports examining the effect of diabetes on the serum or plasma VEGF level have been published, and the results are contradictory: an increase,27-29 decrease,30,31 and no change32,33 in the VEGF levels in type I and II diabetes have been reported. In the present study, although the capillary density was lower in diabetic animals, the serum VEGF concentration was similar to that in control animals. It has been suggested that even with an increase or no change in the VEGF level, the VEGF signaling pathway is defective during diabetes, and diabetes is considered VEGF resistant.29,34 In a recent study, Hazarika et al. showed that in the absence of ischemia, VEGF signaling in diabetic mice was lower than that in control mice.35 They also showed that in the absence of

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Figure 4 - Correlation between the capillary density in myocardial tissue and the serum nitrite (r = 0.70) (A), sVEGFR-1 (r = -0.69) (B), and VEGF (r = 0.40) (C) levels.

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21. Lash JM, Nase GP, Bohlen HG. Acute hyperglycemia depresses arteriolar NO formation in skeletal muscle. Am J Physiol. 1999;277:H1513-H1520. 22. Brodsky SV, Morrishow AM, Dharia N, Gross SS, Goligorsky MS. Glucose scavenging of nitric oxide. Am J Physiol Renal Physiol. 2001;280:F480-F486. 23. Bohlen HG, Nase GP. Arteriolar nitric oxide concentration is decreased during hyperglycemia-induced betaII PKC activation. Am J Physiol Heart Circ Physiol. 2001;280:H621-H627. 24. Silva AM, Penno Lde M, Bertoluci MC, Irigoyen MC, Schaan BD. Insulin therapy does not interfere with venous endothelial function evaluation in patients with type 2 diabetes mellitus. Clinics. 2010;65:1139-42, doi: 10. 1590/S1807-59322010001100015. 25. Ding Y, Vaziri ND, Coulson R, Kamanna VS, Roh DD. Effects of simulated hyperglycemia, insulin, and glucagon on endothelial nitric oxide synthase expression. Am J Physiol Endocrinol Metab. 2000; 279:E11-E17. 26. Bagi Z, Toth E, Koller A, Kaley G. Microvascular dysfunction after transient high glucose is caused by superoxide-dependent reduction in the bioavailability of NO and BH(4). Am J Physiol Heart Circ Physiol. 2004;287:H626-H633, doi: 10.1152/ajpheart.00074.2004. 27. Blann AD, Belgore FM, McCollum CN, Silverman S, Lip PL, Lip GY. Vascular endothelial growth factor and its receptor, Flt-1, in the plasma of patients with coronary or peripheral atherosclerosis, or Type II diabetes. Clin Sci (Lond). 2002;102:187-94, doi: 10.1042/CS20010178. 28. Li Y, Hazarika S, Xie D, Pippen AM, Kontos CD, Annex BH. In mice with type 2 diabetes, a vascular endothelial growth factor (VEGF)-activating transcription factor modulates VEGF signaling and induces therapeutic angiogenesis after hindlimb ischemia. Diabetes. 2007;56:656-65, doi: 10. 2337/db06-0999. 29. Sasso FC, Torella D, Carbonara O, Ellison GM, Torella M, Scardone M, et al. Increased vascular endothelial growth factor expression but impaired vascular endothelial growth factor receptor signaling in the myocardium of type 2 diabetic patients with chronic coronary heart disease. J Am Coll Cardiol. 2005;46:827-34, doi: 10.1016/j.jacc.2005.06.007. 30. Chou E, Suzuma I, Way KJ, Opland D, Clermont AC, Naruse K, et al. Decreased cardiac expression of vascular endothelial growth factor and its receptors in insulin-resistant and diabetic States: a possible explanation for impaired collateral formation in cardiac tissue. Circulation. 2002;105:373-9, doi: 10.1161/hc0302.102143. 31. Gao L, Yu DM. Molecular mechanism of limbsâ&#x20AC;&#x2122; postischemic revascularization improved by perindopril in diabetic rats. Chin Med J (Engl). 2008;121:2129-33. 32. Kivela R, Silvennoinen M, Lehti M, Jalava S, Vihko V, Kainulainen H. Exercise-induced expression of angiogenic growth factors in skeletal muscle and in capillaries of healthy and diabetic mice. Cardiovasc Diabetol. 2008;7:13, doi: 10.1186/1475-2840-7-13. 33. Larger E, Marre M, Corvol P, Gasc JM. Hyperglycemia-induced defects in angiogenesis in the chicken chorioallantoic membrane model. Diabetes. 2004;53:752-61, doi: 10.2337/diabetes.53.3.752. 34. Waltenberger J. VEGF resistance as a molecular basis to explain the angiogenesis paradox in diabetes mellitus. Biochem Soc Trans. 2009;37:1167-70. 35. Hazarika S, Dokun AO, Li Y, Popel AS, Kontos CD, Annex BH. Impaired angiogenesis after hindlimb ischemia in type 2 diabetes mellitus: differential regulation of vascular endothelial growth factor receptor 1 and soluble vascular endothelial growth factor receptor 1. Circ Res. 2007;101:948-56, doi: 10.1161/CIRCRESAHA.107.160630. 36. Ambati BK, Nozaki M, Singh N, Takeda A, Jani PD, Suthar T, et al. Corneal avascularity is due to soluble VEGF receptor-1. Nature. 2006;443:993-7, doi: 10.1038/nature05249. 37. Chang YT, Chang MC, Wei SC, Tien YW, Hsu C, Liang PC, et al. Serum vascular endothelial growth factor/soluble vascular endothelial growth factor receptor 1 ratio is an independent prognostic marker in pancreatic cancer. Pancreas. 2008;37:145-50, doi: 10.1097/MPA.0b013e318164548a. 38. Bando H, Weich HA, Brokelmann M, Horiguchi S, Funata N, Ogawa T, et al. Association between intratumoral free and total VEGF, soluble VEGFR-1, VEGFR-2 and prognosis in breast cancer. Br J Cancer. 2005;92:553-61.

explain the reduced coronary angiogenesis observed in diabetic animals.

ACKNOWLEDGMENTS This study was supported by a grant from Isfahan University of Medical Sciences (grant number: 188103).

REFERENCES 1. Carmeliet P. Angiogenesis in health and disease. Nat Med. 2003;9:653-60, doi: 10.1038/nm0603-653. 2. Folkman J. Angiogenesis. Annu Rev Med. 2006;57:1-18, doi: 10.1146/ annurev.med.57.121304.131306. 3. Milkiewicz M, Hudlicka O, Brown MD, Silgram H. Nitric oxide, VEGF, and VEGFR-2: interactions in activity-induced angiogenesis in rat skeletal muscle. Am J Physiol Heart Circ Physiol. 2005;289:H336-H343, doi: 10.1152/ajpheart.01105.2004. 4. Cengel A, Sahinarslan A. Nitric oxide and cardiovascular system. Anadolu Kardiyol Derg. 2006;6:364-8. 5. Tuck ML. Nitric oxide in diabetes mellitus. J Hypertens. 2003;21:1081-3, doi: 10.1097/00004872-200306000-00005. 6. Olsson AK, Dimberg A, Kreuger J, Claesson-Welsh L. VEGF receptor signalling - in control of vascular function. Nat Rev Mol Cell Biol. 2006;7:359-71, doi: 10.1038/nrm1911. 7. Hasumi Y, Mizukami H, Urabe M, Kohno T, Takeuchi K, Kume A, et al. Soluble FLT-1 expression suppresses carcinomatous ascites in nude mice bearing ovarian cancer. Cancer Res. 2002;62:2019-23. 8. Roberts DM, Kearney JB, Johnson JH, Rosenberg MP, Kumar R, Bautch VL. The vascular endothelial growth factor (VEGF) receptor Flt-1 (VEGFR-1) modulates Flk-1 (VEGFR-2) signaling during blood vessel formation. Am J Pathol. 2004;164:1531-5, doi: 10.1016/S0002-9440(10) 63711-X. 9. Wu FT, Stefanini MO, Mac GF, Kontos CD, Annex BH, Popel AS. A systems biology perspective on sVEGFR1: its biological function, pathogenic role and therapeutic use. J Cell Mol Med. 2010;14:528-52. 10. Brem H, Tomic-Canic M. Cellular and molecular basis of wound healing in diabetes. J Clin Invest. 2007;117:1219-22, doi: 10.1172/JCI32169. 11. Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med. 1995;1:27-31, doi: 10.1038/nm0195-27. 12. Movahedian A. Antihyperlipidemic effect of peucedanum pastinacifolium extract in streptozotocin-induced diabetic rats. Clinics. 2010;65:62933. 13. Budin SB. The effects of palm oil tocotrienol-rich fraction supplementation on biochemical parameters, oxidative stress and the vascular wall of streptozotocin-induced diabetic rats. Clinics. 2009;64:235-44, doi: 10. 1590/S1807-59322009000300015. 14. Carlstrom M. Angiogenesis inhibition causes hypertension and placental dysfunction in a rat model of preeclampsia. J.Hypertens. 2009;27:829-37, doi: 10.1097/HJH.0b013e328324f8ce. 15. Pratheeshkumar P. Vernolide-A inhibits tumour specific angiogenesis by regulating proinflammatory cytokines, VEGF, MMPs and TIMP. Eur J Pharmacol. 2011;656:10-8, doi: 10.1016/j.ejphar.2010.12.041. 16. Nematbakhsh M. The effect of estrogen on serum nitric oxide concentrations in normotensive and DOCA Salt hypertensive ovariectomized rats. Clin Chim Acta. 2004;344:53-7, doi: 10.1016/j.cccn.2004.01.019. 17. Li P. Role of bradykinin, nitric oxide, and angiotensin II type 2 receptor in imidapril-induced angiogenesis. Hypertension. 2008;51:252-58, doi: 10. 1161/HYPERTENSIONAHA.107.097394. 18. Cooke JP. NO and angiogenesis. Atheroscler Suppl. 2003;4:53-60, doi: 10. 1016/S1567-5688(03)00034-5. 19. Hood JD, Meininger CJ, Ziche M, Granger HJ. VEGF upregulates ecNOS message, protein, and NO production in human endothelial cells. Am J Physiol. 1998;274(3 Pt 2):H1054-H1058. 20. Zhao X, Lu X, Feng Q. Deficiency in endothelial nitric oxide synthase impairs myocardial angiogenesis. Am J Physiol Heart Circ Physiol. 2002;283:H2371-H2378.

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DOI:10.1590/S1807-59322011000800020

BASIC RESEARCH

Endovascular treatment of peripheral arterial injury with covered stents: an experimental study in pigs Sergio Belczak,I Erasmo Sima˜o da Silva,I Ricardo Aun,I Igor Rafael Sincos,I Alessandro Rodrigo Belon,II Ivan Benaduce Casella,I Vitor Gornati,III and the late Luiz Francisco Poli de FigueiredoIV I

Cirurgia Vascular - Faculdade de Medicina da Universidade de Saõ Paulo, Saõ Paulo/SP, Brasil. II Faculdade de Medicina da Universidade de Saõ Paulo, Saõ Paulo/SP, Brasil. III Cirurgia Geral - Faculdade de Medicina da Universidade de Saõ Paulo, Saõ Paulo/SP, Brasil. IV Disciplina de Tećnica Ciru´rgica da Faculdade de Medicina da Universidade de Saõ Paulo, Saõ Paulo/SP, Brasil.

OBJECTIVE: To evaluate the feasibility of using endovascular repair to treat penetrating arterial injuries with covered stents. Feasibility was examined according to the circumferential extent of the injury. INTRODUCTION: Surgical trauma often increases the risk of major morbidity and mortality associated with vascular injury, and endovascular repair has many advantages in such situations. METHODS: Twenty white male domestic pigs weighing 28-38 kg with controlled vascular injuries were divided into four equal groups according to the circumferential extent of their vascular lesion (i.e., no lesion, lesion ,50%, lesion .50%, and complete lesion). The left common carotid artery was dissected with proximal and distal control, and this procedure was followed by controlled sectioning of the arterial wall. Local manual compression was applied for 10 min and was followed by endovascular repair with the placement of a 5650 mm VIABHANTM covered stent using the femoral approach. We also monitored additional variables, such as the duration of the procedures (the mean was 56.3¡19.1 min), ultrasound parameters (e.g., maximum arterial diameter, peak systolic and diastolic velocity, and resistance index), arteriography findings, and fluctuations in vital signs (e.g., cardiac output, arterial pressure, and central venous pressure). RESULTS: The experimental procedure was found to be feasible and reproducible. Repairs were successful in all animals in the control (no lesion) and ,50% lesion groups. Success was also achieved in four out of five pigs in the .50% group and in one pig in the complete lesion group. DISCUSSION: The endovascular repair of an arterial injury is possible, but success depends on the circumferential extent of the arterial lesion. The present experimental model, which involved endovascular techniques, highlighted important factors that must be considered in future studies involving similar animals and materials. KEYWORDS: Vascular trauma; Cerebrovascular trauma; Penetrating carotid injuries; Stent; Endovascular procedures. Belczak S, Silva ES, Aun R, Sincos IR, Belon AR, Casella IB, et al. Endovascular treatment of peripheral arterial injury with covered stents: an experimental study in pigs. Clinics. 2011;66(8):1425-1430. Received for publication on March 8, 2011; First review completed on March 24, 2011; Accepted for publication on April 26, 2011 E-mail: belczak@gmail.com Tel.: 55 11 3257-7784

collisions.3 Other damage, such as the laceration of the outer adventitial layer of the arterial wall or the complete transection of the vessel with a high risk of exsanguinating hemorrhage, commonly results from direct-penetrating injuries.4 Both blunt and penetrating trauma forces can disrupt the vessel wall and result in the formation of a pseudoaneurysm.5 Excessive morbidity and mortality rates are associated with penetrating vascular injuries.6 Open wound exploration and appropriate surgical repair is the treatment of choice for polytrauma patients and patients showing signs of peripheral vascular trauma.1,7 Surgical trauma, however, can increase the risk of morbidity in stable patients, and endovascular treatment has been notably beneficial in such cases.8,9 Vessel embolization techniques, for example, are used to treat heavy bleeding from pelvic fractures resulting from blunt trauma.10

INTRODUCTION Trauma-related vascular injuries constitute 3% of civilian trauma casualties,1 and 5-10% of all vascular traumas are carotid artery injuries that are usually attributed to directpenetrating forces.2 The consequences of such traumarelated injuries depend on the type of traumatic mechanism. Avulsion of large vessels, intimal tears, and dissection flaps resulting in luminal thrombosis are generally caused by direct crushing and deceleration injuries from blunt

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Surgical technique: Controlled carotid injuries were created with the animals in a prone position. The right femoral artery was dissected to allow for puncture, catheterization, and invasive monitoring of arterial pressure. A longitudinal cervical incision was created through the dissection of the left common carotid artery, and the incision was controlled by means of two short incisions made distally and proximally to the cervicotomy, where the vascular clamps were placed. Xylocaine (2%) was administered locally. Next, the arterial wall was sectioned (Fig. 1), and the subcutaneous and cutaneous planes were sutured. The sutures in three planes mimicked the action of perilesional tissue in penetrating trauma when there is no access incision. After releasing the vascular clamps, the site was manually compressed for ten minutes before the endovascular procedure began. This period of manual compression and fluid administration mimicked the prehospital care received by trauma patients. The left common femoral artery was cannulated with an 8F introducer, and this process was followed by systemic heparinization (145 U/kg). A control arteriogram was carried out using a 0.0035-inch hydrophilic guidewire and a 5F VERT catheter to identify the location of the lesion (Figure 2). A 5650 mm VIABAHNH covered stent was released as soon as the guidewire passed over the lesion. At this time, papaverine (0.7 mg/kg) and heparin (15 U/kg) were administered via a catheter. The animals were sacrificed at the end of the procedures while still under general anesthesia. Assessment of the procedure’s feasibility: After the successful release of the covered stent, another arteriogram was conducted to examine the stent placement and luminal patency at the site of implantation and distal to the implantation site (Figure 3). Local and distal blood flow was registered by Doppler ultrasonography before and after the procedures, and these data were used to assess peak systolic and diastolic velocity and the resistance index. Statistical analysis: The data were analyzed using parametric (Student’s t test) and nonparametric (Wilcoxon signed-rank test) procedures to compare the quantitative parameters (means and standard deviation) observed before

Covered stents seem to be ideal for treating arterial ruptures and aneurysms and for sealing off intravascular communication between the true and false lumen of a dissection.3 The use of covered stents in the endovascular repair of trauma-related peripheral vascular injuries, however, is still under development, and few experimental studies have examined this procedure. Endovascular repair is thought to be inappropriate for complete arterial sections because the distal stump cannot be catheterized,10 but no scientific evidence has proved that this procedure is not possible. The association between the circumferential extent of damage to the vessel wall and the feasibility of endovascular management has not previously been studied. The present study presented an animal model to examine the feasibility of endovascular repair using covered stents in the treatment of penetrating peripheral vascular injuries. Feasibility was evaluated according to the circumferential extent of the injured arterial wall.

MATERIALS AND METHODS Twenty white male domestic pigs weighing 28 to 37.5 kg (the mean weight was 31.3¡2.5 kg) were divided into four groups of five pigs each. Group 1 included control pigs that were not submitted to the controlled carotid injury, which allowed the feasibility of the procedure to be analyzed in models with healthy vessels. In the other three groups, controlled vascular (carotid) lesions were carried out, and the lesions had circumferential extents of 33% (i.e., ,50%), 66% (i.e., .50%), and 100% for Groups 2, 3, and 4, respectively. The measurement of the circumferential extent of the arterial lesion was subjective and based on the opinion of two researchers. All pertinent guidelines prescribed by current Brazilian legislation (Decree 6,899/2009) on the use and protection of animals in experimental research were rigorously followed, and this study was approved by the Ethics Board in Scientific Research of the Medical School of Sa˜o Paulo University (protocol number: 0603/09). A BV Pulsera Philips RX 09301 was used to take arteriographs before and after the procedure to evaluate patency, the presence of arterial spasm, extravasation and the flow rate of the contrast (meglumine Ioxitalamato Telebrix1). A Doppler ultrasound was taken with a Sonosite Titan1 before the surgical preparation and after the control arteriogram. We evaluated the maximum arterial diameter (in centimeters), the systolic peak velocity (in centimeters per second), the diastolic peak velocity (in centimeters per second), and the resistance index. Before this study began, a pilot study was conducted with four pigs to address technical issues. Anesthesia and perioperative maneuvers: All animals were pre-anesthetized with intramuscular ketamine (5.0 mg/ kg) and midazolam (0.25 mg/kg). Anesthesia was induced with sodium thiopental (20 mg/kg) and an initial volume of NaCl (0.9%) to compensate for fasting-related volume loss. Anesthesia was maintained with inhaled halothane and fentanyl (5 mg/kg) and was followed by continuous intravenous infusion of 0.4 mg/kg/min. Breathing was maintained within physiological parameters with mechanical ventilation, and fluids (20 ml/kg) were continuously administered during surgery.

Figure 1 - Controlled section of the vessel wall in a lesion with a circumferential extent of 33%.

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Figure 2 - Lesions confirmed by arteriograms in the four groups.

and after the procedures. The groups were compared by the Kruskal-Wallis test. Differences were considered significant at the 95% confidence level (p,0.05).

In most cases, arterial spasm and the leakage of contrast were present in the preprocedure arteriograms. In the postprocedure arteriography findings, we observed cases where the procedure was not successful (Table 3).

RESULTS DISCUSSION

The control arteriography resulted in the successful placement of covered stents (introduced through the left femoral artery) to repair a perforating lesion in the left carotid artery. The procedure was successful in all five pigs in the control group (no lesion), in all five pigs in the group with lesions measuring 33% in circumference (the ,50% group), in four pigs in the group with lesions measuring 66% in circumference (the .50% group) and in one pig with a complete lesion (the 100% group). The duration of the complete procedure from the induction of anesthesia to the control arteriography varied from 38 to 118 min (the mean duration was 56.3ยก19.1 min). After the lesion was induced, the length of time from the clamp release to the control arteriography varied from 17 to 45 min (the mean time was 27.0ยก6.9 min). In 14 (70.0%) successful procedures (not including the sole successful procedure in Group 4), the mean maximal arterial diameter before the procedure decreased significantly from 0.50ยก0.04 mm to 0.45ยก0.07 mm after the procedure (t = 0.02; T = 11.5; p,0.025). Peak systolic and diastolic velocities increased significantly after the procedures in all groups, but no differences were observed in peak resistance after the procedures (Table 1). These parameters did not vary significantly when comparing Groups 1, 2, and 3 among themselves (Table 2).

Researchers continue to debate the best way to approach injuries to the proximal common carotid and distal internal carotid arteries in stable patients. Approaches vary from routine exploration to selective exploration based on either clinical findings alone or based on clinical findings combined with radiological features. This debate continues even though the feasibility and safety of endovascular management has already been well established.9 The use of covered stents has been proposed for the management of femoral artery lesions,11 injuries to the subclavian and axillary arteries,12 and even in carotid artery trauma.2,13,14 These stents can help avoid the high rates of morbidity and mortality associated with surgical exploration. Covered stents have important advantages. For example, they can be inserted with a minimally invasive procedure and deployed through a remote site of percutaneous access under local anesthesia.3 Most previous studies, however, included unhealthy arteries (i.e., those with atherosclerosis), and covered stents have been developed with a focus on diseased arteries. The endovascular management of lesions in normal arteries must be studied to establish the feasibility of this procedure in the treatment of perforating blunt trauma injuries and to reveal the longterm behavior of covered stents. This research can also help

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Figure 3 - Control arteriograms showing patency and blood flow in a successful case from each of the four groups.

determine the circumferential extent of a lesion that can be managed with an endovascular approach. All of these issues can be answered in experimental studies. The intent of the present study was to create an animal model to understand better the endovascular repair of penetrating injuries to peripheral arteries. A pilot study that included four pigs was carried out so that technical difficulties could be solved before the full experiment began. The first difficulty was the occurrence of vasospasm, which is common in experimental vascular studies with swine because their peripheral blood vessels are prone to vasospasm and rupture relatively easily.15 The local administration of xylocaine (2%) and the administration of papaverine (0.7 mg/kg) via a catheter solved this problem. Systemic heparinization (140 U/kg) after the placement of the introducer and local heparinization (15 U/kg) via a catheter after the stent was released addressed problems with intrastent thrombosis. The covered stent placement was successful in all of the animals in the control group (those without a carotid lesion) and in the group with lesions with a circumference of 33%

(,50%). Furthermore, the covered stent placement was successful in 80% of the animals with lesions with a circumference of 66% (.50%) and, surprisingly, in one pig with a complete (100%) lesion. These results point to the feasibility of the experimental procedure and show that success is associated with the circumferential extent of the lesion. These results are especially significant because pigs share important aspects of cardiovascular anatomy and physiology with humans.15 There are differences, however, in the extracranial cerebrovascular anatomy between pigs and humans, and caution should be used when extrapolating from these preliminary findings to surgical practice. It is important to clarify that the large range in the duration of the procedures (38 to 118 min) was related to the learning curve of the surgical team rather than the circumferential extent of the lesions. Indeed, the first procedures were much longer than the final ones. The resistance index did not significantly differ before and after the procedure or among Groups 1, 2, and 3. We did not analyze Doppler ultrasonography parameters in the

Table 1 - Doppler ultrasonography parameters (means ¡ standard deviation) before the lesion was created and after the placement of the covered stent and control arteriography with a perforating lesion model. Doppler ultrasonography parameters Maximum arterial diameter Peak systolic velocity Peak diastolic velocity Resistance index

Before 0.50¡0.04 56.18¡13.83 16.14¡5.53 0.73¡0.08

After

Student’s t test

0.45¡0.07 65.28¡17.48 24.40¡9.96 0.67¡0.11

p = 0.02 p = 0.05 p = 0.00 p = 0.09

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p,0.025 p,0.025 p,0.005 p.0.05

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Table 2 - Doppler ultrasonography parameters (means ¡ standard deviation) before the lesion was created and after the placement of the covered stent and control arteriography in control and experimental groups. Doppler ultra-sonography parameters Maximum arterial diameter Before After Mean difference Peak systolic velocity Before After Mean difference Peak diastolic velocity Before After Mean difference Resistance index Before After Mean difference

Group 1

Group 2

Group 3

Kruskal-Wallis

0.50¡0.03 0.44¡0.06 -0.06

0.52¡0.07 0.46¡0.08 -0.06

0.47¡0.04 0.47¡0.05 -0.01

H = 1.75; p.0.10

48.48¡7.05 61.64¡15.00 13.26

62.22¡20.18 71.18¡21.35 8.96

54.84¡10.04 62.47¡18.05 4.10

H = 0.22; p.0.30

16.36¡3.28 20.98¡3.70 4.62

16.24¡7.48 26.10¡3.51 6.66

15.75¡6.59 23.26¡19.11 10.8

H = 0.61; p.0.30

0.66¡0.06 0.70¡0.07 0.04

0.79¡0.09 0.62¡0.08 -0.16

0.73¡0.03 0.69¡0.17 -0.03

H = 4.52; p.0.08

group with complete lesions because the procedure only succeeded in one pig. Nevertheless, it is important to emphasize that the resistance index increased in the group without lesions and decreased in the groups with lesions (i.e., in both the ,50% and .50% groups) (Table 2). This difference between the lesioned groups and those without injury could have occurred as a result of cerebral vasoreactivity. In animals with lesions, insufficient cerebral vascular reactivity results in vasodilation, which leads to a decrease in vascular resistance to blood flow. This effect did not occur in the control group.16 The maximum luminal diameter decreased significantly in Groups 1, 2, and 3, and peak systolic and diastolic velocity increased significantly after the procedure; however, none of these variables differed between the three groups. The increase

in velocity was expected, due to the decreased compliance of the vessel at the site of the stent implantation.17 This effect has been documented in clinical practice by monitoring patients who received uncoated stents in the internal carotid artery to treat atherosclerotic stenosis. This observation also indicated that a spectral velocity with duplex scan can be used for clinical follow-up as long as it is recognized that the velocity changes with the insertion of stents. Because we faced difficulties with some aspects of the ultrasonographic assessment, including the measurement of hematoma and atmospheric air and intrastent and distal visualization, the analyzed parameters did not include measures taken distally to the placement of the covered stent. These parameters were only measured in three out of five control pigs, in one pig with a lesion ,50% and in one

Table 3 - Arteriography findings before and after the endovascular procedure to place the covered stent in the left common carotid artery in control and experimental groups. Angiography findings Preprocedure

G1 1

G2 2

G3 2

G4 2

Good contrast and speed flow Intense spasm, extravasation of contrast Intense spasm, extravasation of contrast Extravasation of contrast Intense spasm, extravasation of contrast, no contrast in distal stump Extravasation of contrast, no contrast in distal stump Angiography findings Postprocedure

G1 1

G2 2

Good contrast and speed flow Good contrast, moderate spasm and slowed flow Without contrast, no runoff, local thrombosis Impraticable

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8. Malley E. Results of a multicenter trial for the treatment of traumatic vascular injury with a covered stent. J Trauma. 2006;60:1189-96, doi: 10. 1097/01.ta.0000220372.85575.e2",-1,"xxx/85575.e2. 9. du Toit DF, Coolen D, Lambrechts A, de V Odendaal J, Warren BL. The endovascular management of penetrating carotid artery injuries: Longterm follow-up. Eur J Endovasc Surg. 2009;38:267-72, doi: 10.1016/j.ejvs. 2009.05.003. 10. Starnes SW, Arthurs ZM. Endovascular management of vascular trauma. Perspect Vasc Surg Endovasc Ther. 2006;18 Suppl. 2:114-29, doi: 10.1177/ 1531003506293418. 11. Alimi YS, Hakam Z, Hartung O, Boufi M, Barthe`lemy P, Aissi K, et al. Efficacy of ViabahnH in the treatment of severe superficial femoral artery lesions: which factors influence long-term patency? Eur J Vasc Endovasc Surg. 2008;35:346-52, doi: 10.1016/j.ejvs.2007.09.005. 12. Xenos ES, Freeman M, Stevens S, Cassada D, Pacanowski J, Goldman M. Covered stents for injuries of subclavian and axillary arteries. J Vasc Surg. 2003;38:451-4, doi: 10.1016/S0741-5214(03)00553-6. 13. Maras D, Lioupis C, Magoufis G, Tsamopoulos N, Moulakakis K, Andrikopoulos V. Covered stent-graft treatment of traumatic internal carotid artery pseudoaneurysms: a review. Cardiovasc Intervent Radiol. 2006;29:958-68, doi: 10.1007/s00270-005-0367-7. 14. Assadian A, Senekowitsch C, Rotter R, ZoÂ¨lss C, Strassegger J, HagmuÂ¨ller GW. Long-term results of covered stent repair of internal carotid artery dissections. J Vasc Surg. 2004;40:484-7, doi: 10.1016/j.jvs.2004.06.031. 15. Swindle MM. Swine in the laboratory: Surgery, anesthesia, imaging, and experimental techniques. 2nd ed. London/New York: CRC Press; 2007. 16. Chang TY, Liu HL, Lee TH, Kuan WC, Chang CH, Wu HC, et al. Change in cerebral perfusion after carotid angioplasty with stenting is related to cerebral vasoreactivity: a study using dynamic susceptibility-weighted contrast-enhanced MR imaging and functional MR imaging with a breath-holding paradigm. AJNR Am J Neuroradiol. 2009;7:1330-6, doi: 10.3174/ajnr.A1589. 17. Santiago C, Miller MT, Pigott JT, Whalen RC, Jones L, Camerota AJ. Duplex ultrasound velocity criteria for the stented carotid artery. J Vasc Surg. 2008;1:63-73.

pig with a lesion .50%. The measurements were similar before and after the covered stent placement. The present results can be useful in the development of endovascular treatment for victims of trauma-related vascular injuries. In addition, the present pig model can contribute to the development of more complete information that will allow an endovascular approach to be applied in humans.

REFERENCES 1. Compron C, Rhee R. Peripheral vascular trauma. Perspct Vasc Surg Endovasc Ther. 2005;17:297-307, doi: 10.1177/153100350501700404. 2. Peck MA, Rasmussen TE. Management of blunt peripheral arterial injury. Perspct Vasc Surg Endovasc Ther. 2006;18:159-73, doi: 10.1177/ 1531003506293450. 3. Katsanos K, Sabharwal T, Carrell T, Dourado R, Adam A. Peripheral endografts for the treatment of traumatic arterial injuries. Emerg Radiol. 2009;16:175-84, doi: 10.1007/s10140-008-0771-9. 4. Ruppert V, Sadeghi-Azandaryani M, Mutschler W, Steckmeier B. Vascular injuries in extremities. Chirurg. 2004;75:1229-40, doi: 10.1007/ s00104-004-0965-y. 5. Saad NE, Saad WE, Davies MG, Waldman DL, Fultz PJ, Rubens DJ. Pseudoaneurysms and the role of minimally invasive techniques in their management. Radiographics. 2005;25(Suppl 1):173-89, doi: 10.1148/rg. 25si055503. 6. Demetriades D, Murray J, Sinz B, Myles D, Chan L, Sathyaragiswaran L, et al. Epidemiology of major trauma and trauma death in Los Angeles County. J Am Coll Surg. 1998;187:373-83, doi: 10.1016/S10727515(98)00209-9. 7. Nicholosn AA. Vascular radiology in trauma. Cardiovasc Intervent Radiol. 2004;27:105-20.

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DOI:10.1590/S1807-59322011000800021

BASIC RESEARCH

The metabolic dynamics of cartilage explants over a long-term culture period E.K Moo, N.A Abu Osman, B. Pingguan-Murphy Department of Biomedical Engineering, Faculty of Engineering, University of Malaya, Kuala Lumpur, Malaysia.

INTRODUCTION: Although previous studies have been performed on cartilage explant cultures, the generalized dynamics of cartilage metabolism after extraction from the host are still poorly understood due to differences in the experimental setups across studies, which in turn prevent building a complete picture. METHODS: In this study, we investigated the response of cartilage to the trauma sustained during extraction and determined the time needed for the cartilage to stabilize. Explants were extracted aseptically from bovine metacarpal-phalangeal joints and cultured for up to 17 days. RESULTS: The cell viability, cell number, proteoglycan content, and collagen content of the harvested explants were analyzed at 0, 2, 10, and 17 days after explantation. A high percentage of the cartilage explants were found to be viable. The cell density initially increased significantly but stabilized after two days. The proteoglycan content decreased gradually over time, but it did not decrease to a significant level due to leakage through the distorted peripheral collagen network and into the bathing medium. The collagen content remained stable for most of the culture period until it dropped abruptly on day 17. CONCLUSION: Overall, the tested cartilage explants were sustainable over long-term culture. They were most stable from day 2 to day 10. The degradation of the collagen on day 17 did not reach diseased levels, but it indicated the potential of the cultures to develop into degenerated cartilage. These findings have implications for the application of cartilage explants in pathophysiological fields. KEYWORDS: Cartilage metabolism; Explants; Cell Density; Proteoglycan; Collagen. Moo EK, Abu Osman NA, Pingguan-Murphy B. The metabolic dynamics of cartilage explants over a long-term culture period. Clinics. 2011;66(8):14311436. Received for publication on March 7, 2011; First review completed on April 5, 2011; Accepted for publication on April 24, 2011 E-mail: azuan@um.edu.my Tel.: 603 79674581

extensive worldwide research on cartilage transplantation with the goal of finding a sustainable solution. Most researchers opt to study cartilage in vitro to better understand the response of cartilage to various stimuli and to eliminate unrelated disturbances that may occur in vivo. There are various models for cartilage culture, including monolayer culture,5-8 micromass/pellet culture,9,10 scaffold culture,11,12 and explant culture.7,10,13-16 Monolayer culture is a two-dimensional culture of chondrocytes on the flat surface of a plastic dish or culture flask. Micromass/pellet and scaffold cultures are three-dimensional (3D) culture systems that provide the cells with a suitable 3D environment to secrete and build a new ECM from scratch. Although 3D culture is clearly a better model than monolayer culture, it has been argued that chondrocytes in the newly-built matrix may act differently from those in naturally occurring ECM;8 therefore, on this basis, explant culture is advantageous. Through the direct culture of a small piece of tissue harvested directly from the host, explant culture provides a more controlled and physiologically relevant model, with both the morphological and biosynthetic characteristics of the chondrocytes remaining intact. Additionally, explant culture minimizes the complicated

INTRODUCTION Articular cartilage plays an integral role in the loadbearing ability of knee joints. In its healthy state, it provides excellent friction and lubrication to help sustain the rigorous dynamic loads that are placed on the knee joint during daily activities.1 Typical articular cartilage consists of four major constituents: water, cells, proteoglycans, and collagen networks. Both proteoglycans and collagen are responsible for the load-bearing properties of the cartilage. The primary constituent of proteoglycans is aggrecan, while the majority of the collagen fibers are formed from type-II collagen.2,3 Cartilage extracellular matrix (ECM) metabolism is regulated by chondrocytes, which constitute only 5% of the total content of cartilage. Due to its avascular and aneural characteristics, the healing ability of cartilage is limited to such an extent that partial articular cartilage defects rarely or never heal spontaneously.4 This finding has prompted

Copyright Ă&#x; 2011 CLINICS â&#x20AC;&#x201C; This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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systemic and environmental parameters found in vivo.14,16 Thus, explant culture is generally regarded as the superior model for the study of cartilage metabolism. Regardless of the various advantages provided by cartilage explant culture, the dynamics of cartilage metabolism after extraction from the host are still poorly understood. Admittedly, the vigorous extraction that is necessary to obtain a regularly sized cartilage explant places the adjacent cartilage under immense mechanical trauma. Thus, the primary focus of this study was to investigate the response of cartilage to the trauma sustained following explantation and to determine the time needed for stabilization. The latter consideration is particularly imperative, as the effects of the various stimuli applied to the cartilage explant can be accurately assessed only if the explant is stable. Previous research has shed some light on the dynamics of cartilage explant culture; however, our understanding of culture dynamics is incomplete and has been obstructed by differences in the anatomical location of the tissue harvests, the extraction method, the culture conditions, and the age of the tissue used in various studies.15 The aim of this study was to investigate the response of cartilage to the trauma sustained during explant extraction by observing biochemical changes (i.e., cell number, proteoglycan content, and collagen content following excision and culture of full-thickness explants with an intact subchondral bone layer). Furthermore, we aimed to determine whether chondrocytes can withstand the vigorous extraction process that is necessary for preparing an explant culture. Finally, the results of this study were compared to those of related studies.

(P4333, Sigma Aldrich, Malaysia) and 0.68 mM L-ascorbate (Ducheta Biochemie, Malaysia).17 The explants were cultured at 37 ˚C under an environment of 95% air and 5% CO2 for up to 17 days, and the medium was changed every two days. The explants harvested on day 0 were grouped according to their washing condition. The explants in the first group were washed three times with sterile PBS, while those in second group were washed once with 70% ethanol, once with PBS containing antibiotics and three times with sterile PBS. The cell viability of the explants in each group were tested using Trypan blue exclusion. The explants were harvested at days 0, 2, 10, and 17 of culture and washed with PBS. Full-thickness cartilages samples were extracted from the explants and individually weighed. The samples were then digested with papain (P3125, Sigma Aldrich, Malaysia) for 18-24 hours at 60 ˚C.18 Next, the total DNA content (cell number) of the digests was analyzed using a fluorometric Hoechst 33258 assay.19 The remaining digests were stored at -80 ˚C for future analysis and analyzed colorimetrically to measure chondroitin-6sulfate (C6S) and hydroxyproline content. C6S content was assayed using the dimethylmethylene blue (DMB) assay.20 The digests were acid-hydrolyzed for 18 hours at 110 ˚C before having their hydroxyproline content analyzed.21,22 All of the analytical data were normalized to the cartilage wet weight. The results obtained were expressed as the mean ¡ the standard error of the mean. All of the data were pooled into four groups based on the culture time. The means of two or more different groups were compared using one-way between-groups analysis of variance (ANOVA) and Tukey’s Honestly Significant Difference (HSD) test, which were calculated using Statistical Package for Social Sciences (SPSS). The mean differences between the two groups were compared using independent t-tests. The significance level was set at 0.05 (p#0.05).

MATERIALS AND METHODS Bovine metacarpal-phalangeal joints with intact synovial membranes from skeletally mature adult cows were obtained from a local abattoir. The joints were opened aseptically and sawn laterally at the joint end to extract two large bone-cartilage samples. These bone-cartilage samples were further processed in the laminar flow hood to remove most of the bone, leaving only ,2 mm of the bone with the cartilage on top. The sawing process was accompanied by constantly irrigating the exposed cartilage surface with sterile phosphate-buffered saline (PBS) solution at 4 ˚C. Approximately twenty 6-mm-diameter cartilage disks were then extracted from the processed bone-cartilage samples using an in-house-built hollow punch. The thickness of the overall cartilage-bone explant was ,3 mm. All of the explants were washed once with 70% ethanol for 30 seconds, once with sterile PBS containing antibiotics and three times with sterile PBS, with the exception of one group, which was washed with sterile PBS to assess the effect of washing with ethanol on cell viability. All of the disks were transferred to six-well TechnoPlastic-Product (TPP) plates (Trans-Techno Enterprise, Malaysia), with four explants in each well. The explants were immobilized using 2% agar (Fisher Scientific, Malaysia) and cultured in 5 ml/well of Dulbecco’s modified Eagle’s medium (D5921, Sigma Aldrich, Malaysia) supplemented with 20% (v/v) fetal bovine serum (F9665, Sigma Aldrich, Malaysia), 16 mM HEPES (H0887, Sigma Aldrich, Malaysia), 1.6 mM L-glutamine (G7513, Sigma Aldrich Malaysia), 160 U/ml penicillin-160 mg/ml Streptomycin

RESULTS The effect of the washing method on the cell viability of the explants was revealed by the significantly higher (90.33¡1.61%) mean cell viability of the explants in the first group (washed three times with sterile PBS; n = 8) compared to those in the second group (washed once with 70% ethanol, once with sterile PBS containing antibiotics, and three times with sterile PBS; n = 8), which had a cell viability of 83.56¡1.62% (p#0.01). The mean wet weight of the cartilage explants (Figure 1A) remained stable at 20¡0.7 mg throughout the culture period. The mean cell density (Figure 1B) was significantly lower at day 0 than during the remainder of the experiment (p#0.05), with a value of 36.70¡1.57 (*103 cells/mg wet weight of cartilage). However, the cell density stabilized after day 2, with an average of 54.36¡2.35 (6103 cells/mg wet weight of cartilage). The mean C6S weight (Figure 2A), which was normalized to the wet weight of cartilage, gradually decreased between day 0 (0.036¡0.002 mg C6S/mg wet weight of cartilage tissue, hereafter abbreviated as w/w) and day 17 (0.028¡0.001 [w/w]) (p#0.05). The mean C6S weight was 0.033¡0.002 (w/w) on day 2; on day 10, the weight was 0.031¡0.001 (w/w), which was also significantly lower than the value on day 0.

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Figure 2 - (A) The weight of chondroitin-6-sulfate (C6S) in the cartilage explants normalized to the cartilage wet weight. (B) The weight of C6S normalized to the DNA weight. The error bars represent SEMs. n = 22, 12, 51, and 37 for day 0, 2, 10, and 17, respectively. In (A), * indicates p#0.05 compared to the results for day 0. In (B), * indicates p#0.05 compared to the results for day 2, day 10 and day 17.

Figure 1 - (A) The wet weight of the full-thickness cartilage. (B) The cell density of the cartilage normalized to the cartilage wet weight. The error bars represent SEMs. n = 22, 12, 51, and 37 for day 0, 2, 10, and 17, respectively. In (B), * indicates p#0.05 compared to the results for day 2, day 10 and day 17.

10 but significantly decreased to 0.046¡0.002 (w/w) on day 17 (p#0.05).

Similarly, the mean C6S weight normalized to the weight of DNA (Figure 2B) was found to be significantly higher (p#0.05) on day 0 (0.128¡0.009 w/w) when compared to the values at the later time points. The values were relatively stable for these groups, with an average of 0.076¡0.004 (w/w). In contrast, the mean hydroxyproline weight, which was normalized to the cartilage wet weight (Figure 3A), remained stable from day 0 to day 10, with an average value of 0.026¡0.001 (w/w). However, this value dropped significantly (p#0.05) to 0.018¡0.0004 (w/w) on day 17. The mean hydroxyproline weight normalized to the DNA weight (Figure 3B) was significantly higher (0.096¡0.005 w/w) on day 0 (p#0.05) than at the later time points. The value stabilized at 0.063¡0.003 (w/w) between days 2 and

DISCUSSION A cell viability test was performed to verify that washing the explants with 70% ethanol would not have a negative effect on cell viability. We found that, although washing with 70% ethanol did significantly decrease cell viability, the viability remained at a high level of ,80%. The suitability of this treatment was further demonstrated by the fact that the chondrocytes responded with an increase in cell density after two days of culture. These observations suggest that only the chondrocytes in the superficial layers were killed by the ethanol, while the others remained unaffected. This tradeoff is worthwhile, as most of the explants that were not

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layer. Furthermore, previous studies23-25 have shown that co-culturing cartilage with osteochondral explants does not significantly affect the metabolism of the cartilage, despite the potential for the release of chemical factors, such as cytokines and/or free radicals. The fluorometric assay measured the DNA content of only the live cells, as most of the DNA from the dead cells was lost during DNA extraction and discarded along with the cell debris pellet.26 We found that the cell density was the lowest on day 0, with a value that was ,32% lower than the densities observed at the later time points. Due to the density of the extracellular matrix, it is unlikely that the increase in cell density after day 0 was due to cell proliferation. Instead, it is more likely that the explants studied at day 0 experienced more cell death than the explants that were given sufficient time to equilibrate. These cartilage explants were all washed once with 70% ethanol, which might have affected the chondrocytes in the superficial zone; if these cells had been given time to recover, they may have been able to survive. However, the explants on day 0 were digested immediately after the 70% ethanol wash. Therefore, those weakened cells from the superficial zone might have been unable to withstand the vigorous digestion and died, resulting in much lower cell viability. Consistent with this hypothesis, the cell density remained relatively stable after day 2, and there was no DNA accumulation throughout the culturing process, indicating that no cell outgrowth occurred.27 The chondroitin-6-sulfate (C6S) content of the explants was found to be equivalent to the proteoglycan content of healthy cartilage. It was noted that, when the C6S content was normalized to the cartilage wet weight, the value exhibited a gradual decrease from day 0. This observation is sensible if one considers that the collagen networks at the periphery of the explants were largely distorted during explant extraction. Therefore, it was inevitable that some of the proteoglycan would leak into the bathing culture medium. Although significant differences in the proteoglycan content were observed between day 0 and day 10 and between day 0 and day 17, the proteoglycan content become relatively stable after day 2, with no significant differences recorded after that time point. Additionally, when the C6S content was normalized to the DNA weight, we found that the proteoglycan content remained relatively stable after day 2. It can be argued that the high value at day 0 was due to the low cell density; furthermore, the proteoglycans bound for leakage were not given sufficient time to diffuse out of the cartilage by this assay point. Regarding the hydroxyproline weight/cartilage wet weight (w/w), we noted that this value remained very stable from day 0 to day 10 in full-thickness cartilage. This finding agrees with previous findings that the turnover rate for collagen is the slowest of all of the cartilage components.16 However, we observed an abrupt ,30% drop in collagen content at day 17, suggesting that the collagens were slowly degraded after 10 days of culture. Because a partial or total loss of proteoglycan always precedes collagen degradation in diseased cartilage,28-30 the cartilage studied here was not developing into diseased cartilage, as the proteoglycan content remained relatively stable through day 17. However, because the osteochondral explants were cultured for a prolonged period without any stimulation, it is possible that the ‘‘unstimulated’’ chondrocytes failed to maintain their normal metabolism, which led to gradual

Figure 3 - (A) The weight of hydroxyproline (hyp) in the cartilage explants normalized to the cartilage wet weight. (B) The weight of hyp normalized to the DNA weight. The error bars represent SEMs. n = 22, 12, 51, and 35, respectively. In (A), * indicates p#0.05 compared to the results for day 0, day 2 and day 10. In (B), * indicates p#0.05 compared to the results for day 2, day 10 and day 17. ** indicates p#0.05 compared to the results for day 0, day 2 and day 10.

washed with 70% ethanol became contaminated after two days of culture and had to be discarded. The wet mass of the cartilage tissue remained stable throughout the culture period. No apparent swelling was observed visually. This observation was supported by the constant wet weight of the cartilage. The cartilage tissues were co-cultured with the underlying subchondral bone to avoid further damage to the cartilage tissue (especially the deep-zone tissue), which could occur while cutting the cartilage tissue out of the bone

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collagen degradation.16 Consistent with this hypothesis, it is generally accepted that cartilage metabolism changes according to the direct mechanical loading exerted by an individualâ&#x20AC;&#x2122;s daily activities. It would be interesting to determine if the cartilage explants would eventually develop into diseased cartilage after a culture period of more than one month and with no stimulation. According to a study by Hoemann,18 the glycosaminoglycan (GAG), collagen and DNA contents in 1 mg of wet mass of typical articular cartilage samples are approximately 50 mg, 150 mg and 0.2-0.6 mg, respectively. These values are comparable to the results obtained in this study, in which contents of 31 mg of GAG, 198 mg of type-II collagen (a conversion factor of 7.6 for extrapolating hydroxyproline content to type-II collagen type II31), and 0.42 mg of DNA were observed during the stable culture period. However, it should be noted that this study was performed in vitro, and the in vivo reproducibility of the biochemical content of cartilage tissue has not been verified. The results of this study were further compared to those of previous studies by Dumont et al.14 and Brighton et al.13 The explants used in the study by Dumont et al. were similar to the explants used in this study in the sense that both were cartilage-bone explants. However, Brighton et al. cultured eight small pieces of full-thickness cartilage derived from 8-mm-diameter samples, and those eight pieces were taken as equivalent to one explant. As for the culture medium involved, Dumont et al. used serum-free DME/F12, while Brighton et al. substituted the serum for 1% insulin-transferrin-selenium G in DMEM. The results of the biochemical analyses performed in these previous studies differ slightly from those of the current study. Part of this difference could be attributed to the various anatomical harvest positions (the shoulder joint for Dumont et al. and the patella for Brighton et al.).15 Dumont et al. reported that 4 days were required for the GAG content of their cartilage explants to stabilize at 22.5 mg/ml after dropping from an initial value of ,31 mg/ml, while the cartilage explants in this study only needed two days to stabilize. The higher GAG value found in this study (31 mg/ ml) may be due to the addition of serum.7 The average number of chondrocytes in 1 mg of cartilage after equilibration was found to be 38,300 cells/mg of cartilage, which is about 70% of the value found in this study (54,360 cells/mg of cartilage). Again, this difference could be due to the addition of serum in our study. However, the type-II collagen content was similar in both studies (,187 mg/ml in the study by Dumont et al. and ,198 mg/ml in this study). The only difference was that Dumont et al. found the type-II collagen content to be constant for durations of up to 21 days, while, in this study, it decreased significantly on day 17. A comparison with the results of Brighton et al. revealed some interesting findings. Although Brighton et al. also studied the effect of electrical stimulation on cartilage explants, only the results of their control set were used for comparison. The authors found that their cartilage explants were relatively stable from day 0 of culture, and washing the explants with 70% ethanol did not appear to affect the cell number (reflected by the DNA weight). They reported a proteoglycan content normalized to DNA weight (w/w) of 65 on day 0, which was followed by an insignificant increase to 70 on day 3 and to 75 on day 10 before dropping back to 65 on day 17. In this study, we observed that the GAG content remained relatively stable, reaching an average of 76

after two days of equilibration. The results that Brighton et al. found for the hydroxyproline weight normalized to the DNA weight (w/w) also showed a stable value, with an average of ,46 from day 0 to day 2. However, their collagen content increased to 65 on day 10 and decreased back to 51 on day 17. A decrease in collagen content on day 17 was also observed in this study. Aside from the high collagen content normalized to the DNA weight (w/w) found on day 0 (caused by the low cell density on day 0), the collagen content was quite stable in this study, remaining at a value of 63 prior to its abrupt decrease to 46 on day 17. Based on the results of this study, we conclude that healthy cartilage responds well to the blunt trauma (exerted during fixation by the clamp) and the abrasive, thermal, and lacerative trauma (exerted during cutting with the saw and hollow punch) sustained by the adjacent cartilage during explantation. After the two-day equilibration period, the cartilage remained stable for up to 10 days of culture. Serum-supplemented culture medium, despite its potential for adverse effects (cell outgrowth, which did not occur in this study),27 did help the cartilage respond positively to the sustained trauma. The degradation of collagen on day 17, albeit not to diseased levels, showed that explanted cartilage could potentially develop into degenerated cartilage. This has applications in pharmacological and pathophysiological fields.15

CONCLUSIONS Healthy cartilage metabolism was assessed by studying changes in GAG, collagen, and DNA content over 17 days of culture. In general, cartilage metabolism was found to be relatively stable for a culture period of up to 10 days if the explant was given two days to equilibrate. However, the collagen fibers in the cartilage begin to degrade if the culture period is longer than ten days, which means that the cartilage could potentially develop into degenerated cartilage. It is apparent that tissue stability and remodeling dynamics are complex and influenced by many parameters. In this study, we sought to report the dynamics of cartilage metabolism in the context of a generalized culture process; future studies should aim to elucidate the factors that affect the long-term culture characteristics of bovine articular cartilage.

ACKNOWLEDGEMENTS We would like to thank Vincent Murphy for his invaluable comments and Adhli Iskandar Putera bin Hamzah for his efforts in procuring the bovine feet. This research was funded by the Institute of Research Management and Monitoring, University of Malaya, Malaysia.

REFERENCES 1. Poole AR. Cartilage in health and disease. In: Kelley WN, Harris ED, S. R, C.B. S, editors. Textbook of rheumatology. Philadelphia, Saunders. 1997;225-308 2. Goldring MB. The role of the chondrocyte in osteoarthritis. Arthritis and Rheumatism. 2000;43:1916-26, doi: 10.1002/1529-0131(200009)43:9,1916:: AID-ANR2.3.0.CO;2-I. 3. Mankin HJ, Mow VC, Buckwalter JA. Articular Cartilage Structure, Composition, and Function. In: Buckwalter JA, Einhorn TA, Simon SR, editors. Orthopaedic Basic Science. Rosemont;American Academy of Orthopaedics Surgeons. 2000;443-88 4. Breinan HA, Martin SD, Hsu HP, Spector M. Healing of canine articular cartilage defects treated with microfracture, a type-II collagen matrix, or cultured autologous chondrocytes. Journal of Orthopaedic Research: Official Publication of the Orthopaedic Research Society. 2000;18:781-9. 5. Brighton CT, Unger AS, Stambough JL. In vitro growth of bovine articular cartilage chondrocytes in various capacitively coupled electrical

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fields. Journal of Orthopaedic Research: Official Publication of the Orthopaedic Research Society. 1984;2:15-22. Chao PH, Roy R, Mauck RL, Liu W, Valhmu WB, Hung CT. Chondrocyte translocation response to direct current electric fields. Journal of Biomechanical Engineering. 2000;122:261-7, doi: 10.1115/1.429661. Hascall VC, Handley CJ, McQuillan DJ, Hascall GK, Robinson HC, Lowther DA. The effect of serum on biosynthesis of proteoglycans by bovine articular cartilage in culture. Archives of Biochemistry and Biophysics. 1983;224:206-23, doi: 10.1016/0003-9861(83)90205-9. Wang W, Wang Z, Zhang G, Clark CC, Brighton CT. Up-regulation of chondrocyte matrix genes and products by electric fields. Clinical Orthopaedics and Related Research. 2004;427(Suppl):S163-73-S-73, doi: 10.1097/01.blo.0000143837.53434.5c. Armstrong PF, Brighton CT, Star AM. Capacitively coupled electrical stimulation of bovine growth plate chondrocytes grown in pellet form. Journal of Orthopaedic Research: Official Publication of the Orthopaedic Research Society. 1988;6:265-71. Brighton CT, Wang W, Clark CC. The effect of electrical fields on gene and protein expression in human osteoarthritic cartilage explants. The Journal of Bone and Joint Surgery American Volume. 2008;90:833-48, doi: 10.2106/JBJS.F.01437. Akanji OO, Lee DA, Bader DA. The effects of direct current stimulation on isolated chondrocytes seeded in 3D agarose constructs. Biorheology. 2008;45:229-43. Hutmacher DW. Scaffolds in tissue engineering bone and cartilage. Biomaterials. 2000;21:2529-43, doi: 10.1016/S0142-9612(00)00121-6. Brighton CT, Wang W, Clark CC. Up-regulation of matrix in bovine articular cartilage explants by electric fields. Biochemical and Biophysical Research Communications. 2006;342:556-61, doi: 10.1016/j. bbrc.2006.01.171. Dumont J, Ionescu M, Reiner A, Poole AR, Tran-Khanh N, Hoemann CD, et al. Mature full-thickness articular cartilage explants attached to bone are physiologically stable over long-term culture in serum-free media. Connective Tissue Research. 1999;40:259-72, doi: 10.3109/0300820 9909000704. Sauerland K, Raiss RX, Steinmeyer J. Proteoglycan metabolism and viability of articular cartilage explants as modulated by the frequency of intermittent loading. Osteoarthritis and Cartilage / OARS, Osteoarthritis Research Society. 2003;11:343-50, doi: 10.1016/S1063-4584(03)00007-4. Wolf A, Ackermann B, Steinmeyer J. Collagen synthesis of articular cartilage explants in response to frequency of cyclic mechanical loading. Cell and Tissue Research. 2006;327:155-66, doi: 10.1007/s00441-006-0251z. Pingguan-Murphy B, El-Azzeh M, Bader DL, Knight MM. Cyclic compression of chondrocytes modulates a purinergic calcium signalling pathway in a strain rate- and frequency-dependent manner. Journal of Cellular Physiology. 2006;209:389-97, doi: 10.1002/jcp.20747. Hoemann C. Molecular and Biochemical Assays of Cartilage Components. In: Ceuninck F, Sabatini M, Pastoureau P, editors. Cartilage and Osteoarthritis (Methods in Molecular Medicine):

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Structure and in vivo Analysis New Jersey. Humana Press Inc. 2004. 127-57 Kim YJ, Sah RL, Doong JY, Grodzinsky AJ. Fluorometric assay of DNA in cartilage explants using Hoechst 33258. Analytical Biochemistry. 1988;174:168-76, doi: 10.1016/0003-2697(88)90532-5. Farndale RW, Buttle DJ, Barrett AJ. Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochimica Et Biophysica Acta. 1986;883:173-7. Stegemann H, Stalder K. Determination of hydroxyproline. Clinica Chimica Acta; International Journal of Clinical Chemistry. 1967;18:26773, doi: 10.1016/0009-8981(67)90167-2. Woessner JF. The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. Archives of Biochemistry and Biophysics. 1961;93:440-7, doi: 10.1016/00039861(61)90291-0. Chahine NO, Ateshian GA, Hung CT. The effect of finite compressive strain on chondrocyte viability in statically loaded bovine articular cartilage. Biomechanics and Modeling in Mechanobiology. 2007;6:103-11, doi: 10.1007/s10237-006-0041-2. Isaac DI, Meyer EG, Haut RC. Chondrocyte damage and contact pressures following impact on the rabbit tibiofemoral joint. J Biomech Eng. 2008;130:041018, doi: 10.1115/1.2948403. Krueger JA, Thisse P, Ewers BJ, Dvoracek-Driksna D, Orth MW, Haut RC. The extent and distribution of cell death and matrix damage in impacted chondral explants varies with the presence of underlying bone. Journal of Biomechanical Engineering. 2003;125:114-9, doi: 10.1115/1. 1536654. Nocker A, Camper AK. Selective Removal of DNA from Dead Cells of Mixed Bacterial Communities by Use of Ethidium Monoazide. Applied and Environmental Microbiology. 2006;72:1997-2004, doi: 10.1128/AEM. 72.3.1997-2004.2006. Luyten FP, Hascall VC, Nissley SP, Morales TI, Reddi AH. Insulin-like growth factors maintain steady-state metabolism of proteoglycans in bovine articular cartilage explants. Archives of Biochemistry and Biophysics. 1988;267:416-25, doi: 10.1016/0003-9861(88)90047-1. Ellis AJ, Curry VA, Powell EK, Cawston TE. The prevention of collagen breakdown in bovine nasal cartilage by TIMP, TIMP-2 and a low molecular weight synthetic inhibitor. Biochemical and Biophysical Research Communications. 1994;201:94-101, doi: 10.1006/bbrc.1994.1673. Kozaci LD, Buttle DJ, Hollander AP. Degradation of type II collagen, but not proteoglycan, correlates with matrix metalloproteinase activity in cartilage explant cultures. Arthritis and Rheumatism. 1997;40:164-74, doi: 10.1002/art.1780400121. Nagase H, Woessner JF. Role of endogenous proteinases in the degradation of cartilage matrix. In: Woessner JF, Howell DS, editors. Joint Cartilage Degradation: Basic and Clinical Aspects. Basel. Marcel Dekker. 1993;15986. Venn M, Maroudas A. Chemical composition and swelling of normal and osteoarthrotic femoral head cartilage. I. Chemical composition. Annals of the Rheumatic Diseases. 1977;36:121-9, doi: 10.1136/ard.36.2. 121.

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DOI:10.1590/S1807-59322011000800022

BASIC RESEARCH

Short-term diabetes attenuates left ventricular dysfunction and mortality rates after myocardial infarction in rodents Bruno Rodrigues,I,II Diego Mendrot Taboas Figueroa,II Jiao Fang,II Kaleizu Teodoro Rosa,II Suzana Llesuy,III Ka´tia De Angelis,IV Maria Claúdia IrigoyenII I Human Movement Laboratory, Saõ Judas Tadeu University, Saõ Paulo/SP, Brazil. II Hypertension Unit, Heart Institute (InCor), Medical School of University of Saõ Paulo, Saõ Paulo/SP, Brazil. III Ca´tedra de Quı´mica General y Inorgańica, Facultad de Farmaćia y Bioquı´mica, Universidad de Buenos Aires, Buenos Aires, Argentina. IV Nove de Julho University, Saõ Paulo/SP, Brazil.

OBJECTIVES: To investigate the effects of hyperglycemia on left ventricular dysfunction, morphometry, myocardial infarction area, hemodynamic parameters, oxidative stress profile, and mortality rate in rats that had undergone seven days of myocardial infarction. INTRODUCTION: Previous research has demonstrated that hyperglycemia may protect the heart against ischemic injury. METHODS: Male Wistar rats were divided into four groups: control-sham, diabetes-sham, myocardial infarction, and diabetes + myocardial infarction. Myocardial infarction was induced 14 days after diabetes induction. Ventricular function and morphometry, as well as oxidative stress and hemodynamic parameters, were evaluated after seven days of myocardial infarction. RESULTS: The myocardial infarction area, which was similar in the infarcted groups at the initial evaluation, was reduced in the diabetes + myocardial infarction animals (23¡3%) when compared with the myocardial infarction (42¡7%, p,0.001) animals at the final evaluation. The ejection fraction (22%, p = 0.003), velocity of circumferential fiber shortening (30%, p = 0.001), and left ventricular isovolumetric relaxation time (26%, p = 0.002) were increased in the diabetes + myocardial infarction group compared with the myocardial infarction group. The diabetes-sham and diabetes + myocardial infarction groups displayed increased catalase concentrations compared to the controlsham and myocardial infarction groups (diabetes-sham: 32¡3; diabetes + myocardial infarction: 35¡0.7; controlsham: 12¡2; myocardial infarction: 16¡0.1 pmol min-1 mg-1 protein). The levels of thiobarbituric acid-reactive substances were reduced in the diabetes-sham rats compared to the control-sham rats. These positive adaptations were reflected in a reduced mortality rate in the diabetes + myocardial infarction animals (18.5%) compared with the myocardial infarction animals (40.7%, p = 0.001). CONCLUSIONS: These data suggest that short-term hyperglycemia initiates compensatory mechanisms, as demonstrated by increased catalase levels, which culminate in improvements in the ventricular response, infarcted area, and mortality rate in diabetic rats exposed to ischemic injury. KEYWORDS: Diabetes; Myocardial Infarction; Ventricular Function; Oxidative Stress; Mortality Rate. Rodrigues B, Figueroa DMT, Fang J, Rosa KT, Llesuy S, De Angelis K, et al. Short-term diabetes attenuates left ventricular dysfunction and mortality rates after myocardial infarction in rodents. Clinics. 2011;66(8):1437-1442. Received for publication on February 9, 2011; First review completed on April 4, 2011; Accepted for publication on May 2, 2011 E-mail: bruno.rodrigues@incor.usp.br Tel.: 55 11 3069 5006

abnormalities and microvascular complications.1 In fact, clinical studies have demonstrated that diabetes, which is often associated with cardiomyopathy and cardiovascular neuropathy, is an independent risk factor for cardiovascular disease, and diabetes is associated with a two- to four-fold increased risk of coronary heart disease.2 Previous research has demonstrated that hyperglycemia in diabetic animals leads to changes in the heart that contribute to injury during and following a myocardial infarction (MI); however, the response of an uncontrolled hyperglycemic diabetic heart to ischemic injury remains controversial.3-6

INTRODUCTION The prevalence of diabetes in developed countries is approaching epidemic proportions, and this disease has been associated with an increased risk of cardiovascular

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investigated during the final seven days of the protocol in all of the rats (n = 88) beginning after the MI surgery (to exclude the influence of anesthesia or stress from the surgical procedure). After MI, the animals, divided into 4 groups (8 animals each), underwent hemodynamic and left ventricular function evaluations, as well as oxidative stress analyses.

Experimental studies using an ischemia/reperfusion protocol have shown that hearts from hyperglycemic rats7 and pigs8 undergoing a period of no-flow ischemia presented a reduced MI area and better recovery of ventricular function than those of normoglycemic animals, which indicates a possible cardioprotective role for hyperglycemic status. Accordingly, exposure to a high-glucose medium or diabetes for short periods has been found to protect the heart against a variety of pathological insults, including ischemia, hypoxia, and calcium overload.8 A recent study from our group6 demonstrated that streptozotocin (STZ)diabetic rats had a greater plasticity and cellular resistance to myocardial ischemic injury than nondiabetic rats did, as demonstrated by a reduced MI area and preserved systolic function. These results, together with the increased expression of regulatory genes involved in cardiac cellular survival and apoptosis, as well as decreased inflammatory cytokines, further suggest that hyperglycemia may play an important role after an ischemic event. Free radical-mediated oxidative stress is intimately involved in the pathogenesis of heart failure.9 Under pathological conditions, the delicate balance between free radical production and the protective antioxidant defense system may shift to favor a relative increase in free radicalmediated oxidative stress. An increased level of oxidative stress has also been shown to accompany a hyperglycemic diabetic status;10 however, little is known regarding the oxidative stress that accompanies the experimental diabetes associated with MI. Therefore, the aim of the present study was to investigate the effects of hyperglycemia induced by STZ-induced diabetes on left ventricular dysfunction and morphometry, MI area, hemodynamic parameters, oxidative stress profile, and mortality rate in rats that had undergone seven days of MI.

Myocardial Infarction Fourteen days after the STZ injection or administration of the citrate buffer, the rats in the CI and DI groups were anesthetized (80 mg/kg ketamine and 12 mg/kg xylazine, administered i.p.) and underwent MI induction by surgical occlusion of the left coronary artery, as described elsewhere.6,11 Briefly, a left thoracotomy was performed, the third intercostal space was dissected, and the heart was exposed. The left coronary artery was occluded with a single nylon (6.0) suture approximately 1 mm distal to the left atrial appendage. The chest was closed with a silk suture. The animals were maintained under ventilation until recovery. The sham rats (CS and DS) underwent the same procedures except that myocardial ischemia was not induced.

Left Ventricular Morphometry and Function Evaluations Echocardiography was performed seven days after MI induction or sham operation by a double-blinded observer according to the guidelines of the American Society of Echocardiography. The rats were anesthetized (80 mg/kg ketamine and 12 mg/kg xylazine), and images were obtained using a 10- to 14-MHz linear transducer in a Sequoia 512 ultrasound system (Acuson Corporation, Mountain View, CA, USA) for measuring (i) morphometric parameters: left ventricular mass corrected by body weight (LV mass); left ventricular end-diastolic diameter (LVEDD); parameters of systolic function: ejection fraction (EF) and velocity of circumferential fiber shortening (VCF); (ii) parameters of diastolic function: left ventricular isovolumetric relaxation time (IVRT) and peak E deceleration time (EDT); and global function: myocardial performance index (MPI). The echocardiographic parameters were measured as described in detail elsewhere.11,12

MATERIALS AND METHODS Experimental Design Experiments were performed using adult male Wistar rats (250-270 g) obtained from the animal facility at the University of Saõ Paulo (Saõ Paulo, Brazil). The rats were fed standard laboratory chow and water ad libitum. They were housed in collective polycarbonate cages in a temperature-controlled room (22 ˚C) with a 12-hour darklight cycle (light 07:00-19:00 h). The experimental protocol was approved by the Institutional Animal Care and Use Committee of the Medical School of the University of Saõ Paulo, and this investigation was conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985). The rats were randomly assigned to 4 groups: control sham (CS, n = 17), diabetic sham (DS, n = 17), control infarcted (CI, n = 27), and diabetes + myocardial infarction (DI, n = 27). Experimental diabetes was induced by an intravenous injection of 50 mg/ kg STZ (Sigma Chemical Co., St. Louis, MO) dissolved in citrate buffer (pH 4.2). The rats were fasted overnight before the STZ injection. The control rats were injected with buffer only (10 mM citrate buffer, pH 4.5). Forty-eight hours after the STZ injection, diabetes was confirmed by the measurement of blood glucose levels above 200 mg/dL. The MI and sham operations were performed in the respective groups 14 days after the STZ injection according to the procedures described below. The mortality rate was

Hemodynamic Assessment One day after the final echocardiographic evaluation, two catheters filled with 0.06 ml of saline were implanted into the femoral artery of each anesthetized rat (80 mg/kg ketamine and 12 mg/kg xylazine, i.p.). Twenty-four hours later, the arterial cannula was connected to a straingauge transducer (Blood Pressure XDCR; Kent Scientific, Torrington, CT, USA), and arterial pressure (AP) signals were recorded over a 30-min period in conscious animals using a microcomputer equipped with an analog-to-digital converter board (WinDaq, 2 kHz, DATAQ, Springfield, OH, USA). The recorded data were analyzed on a beat-to-beat basis to quantify changes in the mean AP (MAP) and heart rate (HR).11

Catalase and Thiobarbituric Acid-Reactive Substances Analyses One day after the hemodynamic evaluations, the animals were killed by decapitation, the hearts were rapidly

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removed and weighed, and the MI area of the left ventricle was identified and dissected for analysis. The remainder of the heart was placed in an ice-cold solution containing 140 mM KCl and 20 mM HEPES (pH 7.4). The hearts were homogenized using an Ultra Turrax blender and 1 g of tissue per 5 ml of a 1.15% (w/v)-KCl and 20-mM phenylmethylsulfonyl fluoride (PMSF) solution. The homogenates were then centrifuged at 6006 g for 10 min at 22 ˚C to remove nuclei and cell debris, as described elsewhere.13 The supernatants were used for catalase and thiobarbituric acidreactive substances (TBARS) assays. For the TBARS assay, trichloroacetic acid (10%, w/v) was added to the homogenate to precipitate proteins and to acidify the samples.14,15 This mixture was then centrifuged (10006 g, 3 min), the protein-free sample was extracted, and thiobarbituric acid (0.67%, w/v) was added to the reaction medium. The tubes were placed in a water bath (100 ˚C) for 15 min. The absorbances were measured at 535 nm using a spectrophotometer. Commercially available malondialdehyde (MDA) was used as a standard, and the results are expressed as nanomoles per milligram of protein. Catalase (CAT) activity was determined by measuring the decreased absorbance (240 nm) of hydrogen peroxide (H2O2). The results are expressed as picomoles of reduced H2O2 per minute per milligram of protein.15,16

(223¡8 g) groups exhibited reduced body weights at the end of the experimental protocol compared with their initial values and the final values of the nondiabetic groups (CS: 274¡4 and CI: 252¡9 g). Furthermore, the STZ-diabetic rats (DS: 290¡27 and DI: 301¡37 mg/dl) had higher plasma glucose levels compared to the normoglycemic rats (CS: 90¡3 and CI: 96¡3 mg/dl) in all measurements.

LV Morphometry and Function The echocardiographic parameters of LV morphometry and function are shown in Table 1. The evaluation performed seven days after sham or MI surgery revealed that the LV masses were similar between all the experimental animals; however, an increased LV chamber (LVEDD) was observed in the MI groups (CI and DI) compared to the noninfarcted groups (CS and DS). The MI groups (CI and DI) demonstrated systolic dysfunction, as evaluated by VCF and EF, compared to the CS and DS groups. Notably, the DI rats displayed an attenuation of systolic dysfunction, as demonstrated by increases in EF and VCF, compared with the CI rats. Similarly, diastolic function was preserved in the DI animals compared with the CI animals, as demonstrated by the IVRT echocardiographic parameter. In contrast, no differences in EDT were observed between the experimental groups. In addition, the DI group had a reduced MPI compared with the CI group, which demonstrated a lower level of global ventricular dysfunction in the diabetic rats after MI (Table 1).

Myocardial Infarction Size Determinations Seven days after MI, infarction size was delimited by subjective identification of the movement of the LV walls by observing the longitudinal, apical, and transversal echocardiographic views of the LV. Regions with a less-thannormal systolic thickness and portions with paradoxal movement were considered infarcted. Thus, the infarction size (%) was determined by the ratio of these regions to the total perimeter of the LV walls at the initial evaluation (one day) and seven days after MI surgery.6,11 To confirm the echocardiography measurement, the MI size was also evaluated by dissecting the fibrous scar from the remaining LV muscle. The outlines of both fragments were drawn on graph paper, and their areas were measured using the crosspoint method17 with the interventricular septum considered to be part of the left ventricle.

Hemodynamic Assessments The hemodynamic evaluation data are listed in Table 2. The systolic AP was reduced in all of the experimental groups compared with the CS group; however, the diabetic groups (DS and DI) displayed additional reductions compared with the CS and CI groups. Similarly, the DS and DI groups exhibited diastolic AP and MAP lower than those of the nondiabetic animals (CS and CI). HR was only reduced in the DS group compared with the other experimental groups (Table 2). Table 1 - Left ventricular morphometry and function one week after myocardial infarction and three weeks after STZ-induced diabetes.

Statistical Analyses The data are reported as means ¡ SEM. After confirming that all continuous variables were normally distributed using the Kolmogorov-Smirnov test, statistical differences between the groups were identified using two-way analysis of variance (ANOVA) or repeated-measures ANOVA followed by the Student-Newman-Keuls post-test. Pearson’s correlation was used to study the association between the measured MI areas. A survival curve was constructed using the KaplanMeier method and the survival curves of the experimental groups were compared using the log-rank test. All of the tests were 2-sided, and the significance level was established at p,0.05. Calculations were performed with Statistical Package for Social Sciences (SPSS) software, version 12.0.

Parameters Morphometric LV mass (g/kg) LVEDD (cm) Systolic Function EF (%) VCF (circ/s) (10-4) Diastolic Function IVRT (ms) EDT (ms) Global Function MPI

1.02¡0.02 0.95¡0.01 1.05¡0.04 1.04¡0.01 0.65¡0.01 0.63¡0.01 0.75¡0.01*{ 0.77¡0.03*{ 74¡2.1 52¡3

76¡0.6 57¡2

33¡2 30¡1 1.87¡0.11 1.95¡0.10

44¡2.3*{ 30¡2*{

54¡3.2*{{ 39¡3*{{

26¡1* 1.97¡0.10

33¡1{ 1.83¡0.09

0.43¡0.04 0.44¡0.04 0.50¡0.03*{ 0.40¡0.04{

Values are expressed as means ¡ SEM. LV mass – Left ventricular mass corrected by body weight; LVEDD – Left ventricular end-diastolic diameter; EF – Ejection fraction; VCF – Velocity of circumferential fiber shortening; IVRT – Left ventricular isovolumetric relaxation time; EDT – Peak E desacceleration time; MPI - Myocardial performance index. *p,0.05 vs. CS; {p,0.05 vs. DS; {p,0.05 vs. CI (n = 8 for each group).

RESULTS Animals The mean body weights were similar among all of the groups at the beginning of the study (,260¡5 g). As a result of diabetic induction, the DS (225¡9 g) and DI

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Myocardial Infarction Area

Table 2 - Hemodynamic parameters in control sham (CS), diabetic sham (DS), control infarcted (CI), and diabetic infarcted (DI) groups. CS SAP (mm Hg) DAP (mm Hg) MAP (mm Hg) HR (bpm)

128¡2 93¡2 110¡8 364¡12

The MI area, measured by echocardiography, was similar between the infarcted groups at the beginning of the protocol (CI: 40¡3 and DI: 38¡3% of the LV wall). Seven days after the MI, the infarction size was reduced in the DI animals (23¡3%) compared to the CI animals (42¡7%). In addition to echocardiography, the MI size was also evaluated using millimeter graph paper stamps. Corroborating the echocardiographic data, the DI group showed a reduced MI area (15¡6%) compared to the CI group (37¡5%) and displayed a positive correlation (r = 0.9, p = 0.0001) between these two MI size measurements.

p-values

109¡3* 117¡3*{ 106¡2*{ ,0.0001 83¡3* 90¡3 84¡2* 0.003 96¡3* 104¡2 98¡2* 0.005 302¡8* 367¡6{ 366¡10{ 0.002

Values are expressed as the means ¡ SEM. SAP – Systolic arterial pressure; DAP – Diastolic arterial pressure; MAP – Mean arterial pressure; HR – Heart rate. *p,0.05 vs. CS; {p,0.05 vs. DS; {p,0.05 vs. CI (n = 8 for each group).

Mortality Rate Figure 2 shows the total mortality rate (Kaplan-Meier survival curve) for the study groups throughout the sevenday experimental period. During the seven days after sham or MI surgery, the total mortality was similar between the CS (no deaths) and DS (1 death among 17 DS rats, 5.8%) animals. However, the mortality rate was higher (p,0.003) for the CI and DI rats compared with the CS and DS rats. Notably, the DI group (5 deaths among 27 DI rats, 18.5%) had a reduced mortality rate compared with the CI group (11 deaths among 27 rats, 40.7%; Figure 2).

Catalase and TBARS Analyses The activity of the antioxidant enzyme catalase (CAT), which catalyzes the reduction of H2O2 to H2O and O2, is shown in Figure 1A. The diabetic groups (DS and DI) displayed increased CAT activity in relation to the nondiabetic groups (CS and CI). The lipoperoxidation product, as indicated by TBARS analysis, was reduced in the DS animals compared to the CS, CI, and DI animals. Surprisingly, no difference was observed between the CI and DI rats compared to the CS rats (Figure 1B).

DISCUSSION The key finding of the present investigation is that the left ventricular dysfunction induced by MI was attenuated in diabetic rats, which is an attenuation that may be associated with the reduced myocardial infarction area and the increased concentration of catalase, an important antioxidant enzyme, observed in these animals. Furthermore, these positive adaptations were reflected in a reduction in the mortality rate in the diabetic rats following MI compared with the nondiabetic rats. Evidence has been accumulating from clinical studies with diabetic patients that an increased risk of MI and a higher rate of mortality exists in these individuals. Although it has been acknowledged that intensively controlling glucose levels in patients with diabetes reduces the risk of vascular complications, the evidence supporting the role of glycemic control in reducing cardiovascular risk is more limited. Several prospective intervention studies published in the past two years have investigated whether intensive glucose control to near normoglycemia reduces cardiovascular events and mortality in individuals with diabetes.

Figure 1 - Oxidative profiles of cardiac homogenates in control sham (CS), diabetes sham (DS), myocardial infarction (CI), and diabetes + myocardial infarction (DI) rats (n = 8 for each group). A: Catalase activity. B: Lipid peroxidation indicated by thiobarbituric acid-reactive substances (TBARS). * p,0.05 vs. CS; { p,0.05 vs. DS; { p,0.05 vs. CI.

Figure 2 - Mortality rates, as estimated by Kaplan-Meier survival curves for the control sham (CS), diabetes sham (DS), myocardial infarction (CI), and diabetes + myocardial infarction (DI) rats. * p,0.05 vs. CS; { p,0.05 vs. DS; { p,0.05 vs. CI.

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In diabetes or after myocardial infarction, the augmented production of reactive oxygen species such as superoxide (O2-), hydroxyl (OH) and non-radicals capable of generating free radicals (eg, hydrogen peroxide (H2O2), leads to reductions of most antioxidant defenses9,15 and macromolecule damage. These reactive oxygen species participate in some intracellular pathways, such as the activation of various mitogen activated protein kinases, increased activation of NF-KB, disruption of calcium cycling, alteration of myofilament responsiveness to the calcium, expression of pro-fibrotic growth factors, and inflammatory cell infiltration, lading to cardiomyocyte hypertrophy, apoptosis, interstitial fibrosis, and consequently LV dysfunction.26-28 The increased TBARS levels observed in the CI group are in agreement with findings from previous studies.9 However, the DS group exhibited a reduction in TBARS levels compared to the controls. This result may be attributed to short-term exposure to diabetes or even to the increased concentration of CAT observed in the DS and DI groups. In fact, the increased CAT activity in the LV of the diabetic groups may have blocked one pathway of lipid peroxidation, which is a mechanism likely to explain the reduced TBARS levels in these animals.29 CAT activity in the heart peroxisomes of diabetic rats may be increased in response to the augmented formation of H2O2, a subproduct of the enzymatic activity of acyl-CoA oxidase that initiates the Ă&#x;-oxidation of fatty acids in peroxisomes.15,30 Therefore, the reduced MI area and attenuated LV dysfunction observed in DI rats may be explained, at least in part, by the increase in CAT levels. In conclusion, in the present investigation, we demonstrated that the MI area and left ventricular dysfunction in diabetic animals were attenuated after seven days of MI. These findings are probably caused by compensatory mechanisms associated with metabolic and molecular adjustments of cardiac tissue, particularly the increased level of catalase. Furthermore, these compensatory mechanisms culminated in a reduction in the mortality rate of DI animals compared to CI animals. These data suggest that short-term hyperglycemia may protect the heart against an ischemic insult and thus reduce the mortality rate and preserve ventricular function. It is important to emphasize that, in the present investigation, the time courses of diabetes and MI were predetermined, in contrast to what occurs in clinical situations. Therefore, it is possible that this improved cardiac function is transitory and that cardiovascular autonomic dysfunction (not evaluated in the present study) may lead to cardiovascular damage in the long term, as observed in diabetic patients following an ischemic event.

These studies have produced conflicting results, where the use of intensive therapy to target normal glycated hemoglobin levels increased mortality and did not significantly reduce major cardiovascular events.18-20 In this sense, conflicting data have also been observed, mainly in experimental studies, and a greater resistance of hyperglycemic diabetic animals to ischemic injury has been demonstrated.6-8 Previous reports have shown that glucose supply plays a critical cardioprotective role in cardiac responses during acute ischemia. These findings suggest that a higher rate of glucose delivery to the heart may improve tolerance to ischemic stress and may lead to changes in the signaling mechanisms involved in cardioprotection.21,22 However, these protective alterations may be eliminated by preoperative treatment with insulin.23 To further investigate the mechanisms underlying the improved tolerance to ischemia in STZ-diabetic rats, we evaluated cardiac morphometry and function in the experimental groups. Myocardial remodeling after MI is typically characterized by compensatory hypertrophy of myocytes, ventricular dilatation, and increased interstitial collagen deposition.5 In our study, LVEDD was increased in the infarcted groups compared with the noninfarcted groups; however, the CI and DI groups had similar LV mass and LVEDD values, which highlights the lack of influence of STZ-induced diabetes on cardiac remodeling after MI in this study. In regard to cardiac function, although a reduction in SAP was observed in the DI group compared with the CI group, our data indicate that diabetes partially attenuated the ventricular dysfunction caused by myocardial ischemia (DI animals compared with CI animals). In fact, the DI animals displayed improvements in systolic (EF and VCF) and diastolic (IVRT) function indices compared to the CI animals. Along with reduced MPI, increases in these indices indicate global myocardial stress.12 These data corroborate previous findings from our group, which showed that, 15 days after MI, STZ-diabetic rats exhibited an attenuation of cardiac dysfunction, as evaluated by echocardiography, compared with normoglycemic rats.6 Notably, this attenuation was associated with improved net balances of inflammatory cytokines and regulatory genes that participate in cardiac cellular survival.6 Recently, Chu et al.8 provided evidence that diabetes induced by aloxan, which results in hyperglycemia, was protective against myocardial ischemic-reperfusion injury in pigs in the short term. The researchers observed pronounced reductions in MI area in diabetic animals, which was associated with the expression of cell survival proteins, including phosphorylated endothelial nitric oxide synthase and heat shock protein-27. Similarly, in the present investigation, the DI rats exhibited reductions in MI area compared with the CI rats, which was determined using echocardiography and was confirmed using millimeter graph paper stamps. These data are similar to previous findings from our group6 and may explain the attenuated cardiac dysfunction observed in diabetic animals. Some studies have demonstrated that a reduction in MI size6,8,24 may be caused by the diminished number of dead myocytes in diabetic animals;25 increased capillary density; and the expression of cardioprotective proteins, including vascular endothelial growth factor, eNOS, and phosphoAkt23 and would thus be instrumental in the higher resistance of diabetic rats to ischemic injury.

Study Limitations Some possible limitations of the present investigation deserve comment. First, because the LV was only evaluated at the end of the protocol and necropsies were not performed on the animals that died during the seven days of the protocol, it may be difficult to predict survival outcomes (or mortality) based on these parameters. Second, in the present study, we only evaluated a small sample of anti- and pro-oxidant products; however, these evaluations may provide a general concept of the profile of oxidative stress in diabetic rats, infarcted or not.

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13. Llesuy SF, Milei J, Molina H, Boveris A, Milei S. Comparison of lipid peroxidation and myocardial damage induced by adriamycin and 49epiadrimicin in mice. Tumori. 1985;71:241-9. 14. Buege JA, Aust SD. Microsomal lipid peroxidation. Methods Enzymol. 1978;52:302-10, doi: 10.1016/S0076-6879(78)52032-6. 15. De Angelis K, Cestari IA, Barp J, Dall’Ago P, Fernandes TG, de Bittencourt PI, et al. Oxidative stress in the latissimus dorsi muscle of diabetic rats. Braz J Med Biol Res. 2000;33:1363-8, doi: 10.1590/S0100879X2000001100016. 16. Aebi H. Catalase in vitro. Meth Enzymol. 1984;105:121-6, doi: 10.1016/ S0076-6879(84)05016-3. 17. Flores LJ, Figueroa D, Sanches IC, Jorge L, Irigoyen MC, Rodrigues B, et al. Effects of exercise training on autonomic dysfunction management in an experimental model of menopause and myocardial infarction. Menopause. 2010;17:712-7. 18. Action to Control Cardiovascular Risk in Diabetes Study Group, Gerstein HC, Miller ME, Byington RP, Goff DCJr, Bigger JT, et al. Effects of intensive glucose lowering in type 2 diabetes. N Engl J Med. 2008;358:2545-59, doi: 10.1056/NEJMoa0802743. 19. The ADVANCE Collaborative Group, Patel A, MacMahon S, Chalmers J, Neal B, Billot L, et al. Intensive blood glucose control and vascular outcomes in patients with type 2 diabetes. N Engl J Med. 2008;358:256072, doi: 10.1056/NEJMicm066227. 20. Duckworth W, Abraira C, Moritz T, Reda D, Emanuele N, Reaven PD, et al. VADT Investigators. Glucose control and vascular complications in veterans with type 2 diabetes. N Engl J Med. 2009;360:129-39, doi: 10. 1056/NEJMoa0808431. 21. Cave AC, Ingwall JS, Friedrich J, Liao R, Saupe KW, Apstein CS, et al. ATP synthesis during low-flow ischemia: influence of increased glycolytic substrate. Circulation. 2000;101:2090-6. 22. King LM, Opie LH. Glucose and glycogen utilisation in myocardial ischemia changes in metabolism and consequences for the myocyte. Mol Cell Biochem. 1998;180:3-26, doi: 10.1023/A:1006870419309. 23. Ma G, Al-Shabrawey M, Johnson JA, Datar R, Tawfik HE, Guo D, et al. Protection against myocardial ischemia/reperfusion injury by short term diabetes: enhancement of VEGF formation, capillary density, and activation of cell survival signaling. Naunyn Schmiedebergs Arch Pharmacol. 2006;373:415-27, doi: 10.1007/s00210-006-0102-1. 24. Ravingerova T, Neckar J, Kolar F. Ischemic tolerance of rat hearts in acute and chronic phases of experimental diabetes. Mol Cell Biochem. 2003;249:167-74, doi: 10.1023/A:1024751109196. 25. Xu G, Takashi E, Kudo M, Ishiwata T, Naito Z. Contradictory effects of short- and long-term hyperglycemias on ischemic injury of myocardium via intracellular signaling pathway. Exp Mol Pathol. 2004;76:57-65, doi: 10.1016/j.yexmp.2003.08.003. 26. Saraste A, Pulkki K, Kallajoki M, Henriksen K, Parvinen M, Voipio-Pulkki LM. Apoptosis in human acute myocardial infarction. Circulation. 1997;95:320-3. 27. Hockenbery DM, Oltvai ZN, Yin X-M, Milliman CL, Korsmeyer SJ. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell. 1993;75:241-51, doi: 10.1016/0092-8674(93)80066-N. 28. Sabri A, Hughie HH, Lucchesi PA. Regulation of hypertrophic and apoptotic signaling pathways by reactive oxygen species in cardiac myocytes. Atiox Redox Signal. 2003;5:731-40, doi: 10.1089/152308603770380034. 29. Maritim AC, Sanders RA, Watkins JB, 3rd. Diabetes, oxidative stress, and antioxidants: a review. J Biochem Mol Toxicol. 2003;17:24-38, doi: 10. 1002/jbt.10058. 30. Christie KN. Catalase in skeletal muscle fibers. J Histochem Cytochem. 1979;27:814-9, doi: 10.1177/27.4.376691.

ACKNOWLEDGMENTS This study was supported by the Fundaçaõ de Amparo a` Pesquisa do Estado de Saõ Paulo (FAPESP- 01/00009-0; 07/57595-5) and Fundaçaõ E.J. Zerbini. B.R. received doctoral and post-doctoral scholarships from the Coordenaçaõ de Aperfeiçoamento de Pessoal de Nı´vel Superior (CAPES) and the Conselho Nacional de Pesquisa e Desenvolvimento (CNPq), respectively. M.C.I and K.D.A receive financial support from Conselho Nacional de Pesquisa e Desenvolvimento (CNPq-BPQ).

REFERENCES 1. Wild S, Roglic G, Green A, Sicree R, King H. Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. Diabetes Care. 2004;27:1047-53, doi: 10.2337/diacare.27.5.1047. 2. Haffner SM, Lehto S, Ronnemaa T, Pyorala K, Laakso M. Mortality from coronary heart disease in subjects with type 2 diabetes and in nondiabetic subjects with and without prior myocardial infarction. N Engl J Med. 1998;339:229-34, doi: 10.1056/NEJM199807233390404. 3. Feuvray D, Lopaschuk GD. Controversies on the sensitivity of the diabetic heart to ischemic injury: the sensitivity of the diabetic heart to ischemic injury is decreased. Cardiovasc Res. 1997;34:113-20, doi: 10. 1016/S0008-6363(97)00037-0. 4. Ravingerova T, Neckar J, Kolar F, Stetka R, Volkovova K, Ziegelho¨ffer A, et al. Ventricular arrhythmias following coronary artery occlusion in rats: is the diabetic heart less or more sensitive to ischaemia? Basic Res Cardiol. 2001;96:160-8, doi: 10.1007/s003950170066. 5. Ba¨cklund T, Palojoki E, Saraste A, Eriksson A, Finckenberg P, Kyto¨ V, et al. Sustained cardiomyocyte apoptosis and left ventricular remodelling after myocardial infarction in experimental diabetes. Diabetologia. 2004;47:325-30, doi: 10.1007/s00125-003-1311-5. 6. Malfitano C, Alba Loureiro TC, Rodrigues B, Sirvente R, Salemi VM, Rabechi NB, et al. Hyperglycaemia protects the heart after myocardial infarction: aspects of programmed cell survival and cell death. Eur J Heart Fail. 2010;12:659-67, doi: 10.1093/eurjhf/hfq053. 7. Ravingerova T, Stetka R, Volkovova K, Pancza D, Dzurba A, Ziegelho¨ffer A, et al. Acute diabetes modulates response to ischemia in isolated rat heart. Mol Cell Biochem. 2000;210:143-51, doi: 10.1023/A:1007129708262. 8. Chu LM, Osipov RM, Robich MP, Feng J, Oyamada S, Bianchi C, et al. Is hyperglycemia bad for the heart during acute ischemia? J Thorac Cardiovasc Surg. 2010;140:1345-52, doi: 10.1016/j.jtcvs.2010.05.009. 9. Hill MF, Singal PK. Antioxidant and oxidative stress changes during heart failure subsequent to myocardial infarction in rats. Am J Pathol. 1996;148:291-300. 10. Dandona P, Thusu K, Cook S, Snyder B, Makowski J, Armstrong D, et al. Oxidative damage to DNA in diabetes mellitus. Lancet. 199617;347:444-5, doi: 10.1016/S0140-6736(96)90013-6. 11. Jorge L, Rodrigues B, Rosa KT, Malfitano C, Loureiro TC, Medeiros A, et al. Cardiac and peripheral adjustments induced by early exercise training intervention were associated with autonomic improvement in infarcted rats: role in functional capacity and mortality. Eur Heart J. 2011;32:904-12, doi: 10.1093/eurheartj/ehq244. 12. Wichi R, Malfitano C, Rosa K, De Souza SB, Salemi V, Mostarda C, et al. Noninvasive and invasive evaluation of cardiac dysfunction in experimental diabetes in rodents. Cardiovasc Diabetol. 2007;26;6:14, doi: 10. 1186/1475-2840-6-14.

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DOI:10.1590/S1807-59322011000800023

BASIC RESEARCH

The effect of subconjunctival bevacizumab on corneal neovascularization, inflammation and re-epithelization in a rabbit model Glauco Reggiani Mello,I Marcos Longo Pizzolatti,III Daniel Wasilewski,II Marcony R. Santhiago,I Vinı´cius Budel,II Hamilton MoreiraIII I Cornea and Refractive Surgery – Cole Eye Institute, Cleveland Clinic, Cleveland-OH, USA. II Cornea and External Diseases, Universidade Federal do Parana´, Curitiba, PR, Brazil. III Hospital de Olhos do Parana´, Curitiba, PR, Brazil.

PURPOSE: To evaluate the use of subconjunctival bevacizumab on corneal neovascularization in an experimental rabbit model for its effect on vessel extension, inflammation, and corneal epithelialization. METHODS: In this prospective, randomized, blinded, experimental study, 20 rabbits were submitted to a chemical trauma with sodium hydroxide and subsequently divided into two groups. The experimental group received a subconjunctival injection of bevacizumab (0.15 m; 3.75 mg), and the control group received an injection of 0.15 ml saline solution. After 14 days, two blinded digital photograph analyses were conducted to evaluate the inflammation/diameter of the vessels according to pre-established criteria. A histopathological analysis of the cornea evaluated the state of the epithelium and the number of polymorphonuclear cells. RESULTS: A concordance analysis using Kappa’s statistic showed a satisfactory level of agreement between the two blinded digital photography analyses. The neovascular vessel length was greater in the control group (p,0.01) than in the study group. However, the histopathological examination revealed no statistically significant differences between the groups in terms of the state of the epithelium and the number of polymorphonuclear cells. CONCLUSIONS: Subconjunctival bevacizumab inhibited neovascularization in the rabbit cornea. However, this drug was not effective at reducing inflammation. The drug did not induce persistent corneal epithelial defects. KEYWORDS: Antiangiogenic drugs; Corneal neovascularization; Cornea; Neovascularization; Pathology; Eye burns. Mello GR, Pizzolatti ML, Wasilewski D, Santhiago MR, Budel V, Moreira H. The effect of subconjunctival bevacizumab on corneal neovascularization, inflammation and re-epithelization in a rabbit model. Clinics. 2011;66(8):1443-1449. Received for publication on February 20, 2011; First review completed on March 16, 2011; Accepted for publication on May 2, 2011 E-mail: glaucohrm@gmail.com Tel.: 216 778-0244

INTRODUCTION

side effects related to the non-specificity of corticosteroids limits their use. Such side-effects include the increased risk of cataracts and glaucoma due to high intra-ocular pressure (IOP).6 Vascular endothelial growth factor (VEGF) and its receptors play an important role in the neovessel formation that is observed in diabetic retinopathy, venous retinal occlusion, age-related macular degeneration, and corneal neovascularization.7 High VEGF expression was observed in neovascularized corneas after penetrating keratoplasty in corneal inflammatory diseases8 and in guinea pigs’ corneas that were burned by alkalis during the healing process.9 Anti-VEGF drugs have sparked a revolution in the treatment of neovascular diseases by reducing neovascularization and also by their supposed action on fibroblasts.10 These drugs can provide beneficial effects after intra-vitreous injection in age-related macular degeneration (ARMD) neovascularization, diabetic retinopathy, and glaucoma, with minimal toxicity or side effects.11 These effects may also include the reduced formation of new vessels in other regions of the eye.

Ocular trauma, infection, inflammation, and degeneration result in corneal neovascularization.1 Neovessels cause structural changes that allow the overflow of fluid to the extravasculature, blood stasis and hemorrhage, and they can reduce corneal transparency with subsequent and progressive vision impairment.1 Corneal neovascularization is one of the greatest risk factors for corneal transplant rejection2 because it allows leukocytes access to donor tissue antigens. Corticosteroids are the first-line treatment for corneal neovascular diseases because of their ability to reduce the inflammatory process4 and vascular proliferation, both of which are initiated soon after the ocular trauma.5 However,

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The aim of this prospective study was to investigate effects of subconjunctival injections of bevacizumab experimentally induced corneal neovascularization focusing on the neovessel length, inflammation, and epithelization.

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the on by re-

MATERIALS AND METHODS This prospective, randomized, blinded study was performed at the Instituto de Pesquisas Me´dicas (IPEM) of the Faculdade Evange´lica do Parana´ (FEPAR) - Brazil and Hospital Universita´rio Evange´lico de Curitiba (HUEC). The Animal Experimentation Norms and Principles proposed by the Cole´gio Brasileiro de Experimentac¸a˜o Animal (1994) were followed. The studied variables include the vessels’ lenght, degree of inflammation/diameter, epithelium integrity, and number of polymorphonuclear cells (PMN).

Intervention Twenty corneas of twenty New Zealand rabbits were studied. All rabbits were healthy male albinos, weighing between 2.300 and 2.500 kg and were three to four months old. The rabbits were intramuscularly anesthetized with xylazine hydrochloride 0.1 ml/Kg (2.3 mg/kg) and ketamine hydrochloride 0.2 ml/Kg (10 mg/kg). The animals were divided randomly into a control group (Group 1) (n = 10) and a study group (Group 2)(n = 10). The left corneas of the animals were exposed to 1 N sodium hydroxide (NaOH), through a 5 mm diameter filter paper that was applied to the superior cornea tangential of the limbus for 20 seconds, followed by washing with 20 ml of 0.9% saline solution. Subsequently, the control group animals received a subconjunctival injection of 0.15 ml of 0.9% saline solution, whereas the study group animals received a subconjunctival injection with 0.15 ml (3.75 mg) of bevacizumab (Figure 1). The injections were performed superiorly and close to the burned area. One surgeon performed all of the procedures in a blinded fashion. The rabbits in Groups 1 and 2 were again anesthetized, examined and digitally photographed 14 days after the corneal burn and were euthanized by a 5-ml intramuscular sodium pentobarbital injection. Subsequently the corneas were removed, maintaining a scleral margin of 3 mm, fixed in formalin for 24 hours and submitted for histopathological study with hematoxylin-eosin. The corneal epithelium integrity, or lack thereof, and the number of PMN cells per field were observed at a magnification of 400x using an optical microscope. The most damaged field in the opacity area was utilized. Digital images were obtained to examine the vessel extent and the vessel inflammation/diameter in the frontal position using a Sony CybershotH (Sony Corporation – Tokyo/Japan) 7.2-megapixel camera, which was supported by a fixed tripod with an accessory ZeissH (Carl Zeiss AG – Oberkochen/Germany) lens with a magnification of 40x at a distance of 5 cm. Each animal was submitted to two digital photography evaluations for each variable (vessel extent and vessel inflammation/diameter). A cornea specialist performed the two evaluations of the digital pictures in a blinded and randomized fashion. The corneal neovascularization was determined using a score given by

Figure 1 - Animal 8 from the control group showing the vessel extent reaching 75% of the burned area and intense inflammatory reaction (Degree 3).

the cornea specialist according to the vessel extent and inflammation/diameter degree observed. The vessel extent was determined using a scale from 0 to 4 (0 – no vessels on the corneal limbus; 1 – vessels that advanced over the corneal limbus, covering 0-25% of the burned area; 2 – vessels that reached 25-50% of the burned area; 3 – vessels that reached 50-75% of the burned area; 4 – vessels that extended to the entire burned area). The inflammation and diameter degree on the vessels were determined using a scale from 0 to 3: (0 – no inflammation or vessels; 1 – little inflammation and vessels of small diameter; 2 – moderate inflammation and vessels of medium diameter; 3 – intense inflammation and vessels of large diameter).

Statistical Analysis To validate the evaluation model, the reproducibility of these measurements was evaluated using the Kappa statistic. For each evaluation, the two groups were compared with respect to the ordinal variables by using the Mann-Whitney non-parametrical test. The group comparison for dichotomous variables was executed using the Fisher exact test. p,0.05 indicated statistical significance.

RESULTS On the seventh day after the corneal burn, a rabbit from the study group presented corneal perforation and was excluded from the research. Thus, 19 animals were left in the study, including ten from the control group and nine from the study group. The digital pictures of each animal were submitted to two evaluations for each variable, as shown in Table 1 (control group) and Table 2 (study group).

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Table 1 - Blinded photographic analysis of vessel extent and inflammation/diameter in the control group, fourteen days after chemical burn. Animal

1 2 3 4 5 6 7 8 9 10

Vessel Extent

Vessel Inflammation/Diameter

Evaluation 1

Evaluation 2

Evaluation 1

Evaluation 2

2 3 2 2 3 3 4 3 3 2

3 3 2 3 3 3 4 3 3 2

1 2 2 1 2 2 1 3 3 1

1 2 1 1 2 2 1 3 3 2

Animal 8 of the control group (Figure 1) had a cornea classified as degree 3 in both evaluations (vessels reaching up to 75% of the burned area) according to the vessel extent criteria and inflammatory reaction (intense inflammation and vessels of large diameter). Animal 5 of the study group (Figure 2) had a cornea classified as degree 1 for both vessel extent criteria (vessels advancing over the corneal limbus, reaching up to 25% of the burned area) and inflammatory reaction (little inflammation and small-diameter vessels). The concordance analysis of the vessel extent estimated by the Kappa coefficient was 0.705 (95% confidence interval: 0.514 â&#x20AC;&#x201C; 0.895), which indicates a high level of agreement between the two evaluations. The estimated Kappa coefficients for each of the vessel extent classifications are presented in Table 3. The concordance was better characterized at the most extreme degrees (1 and 4). Regarding the vessel inflammation/diameter, the estimated Kappa coefficient between the two blinded evaluations was 0.500 (95% confidence interval: 0.269 â&#x20AC;&#x201C; 0.731), which again indicates a satisfactory level of agreement between the two evaluations. The estimated Kappa coefficients for each classification of vessel diameter are presented in Table 4. Although a satisfactory level of reproducibility was found in the evaluations of large diameter vessels, the concordance seemed to be weak at Degrees 1 and 2, indicating a difficulty in distinguishing between little and moderate inflammation. The comparison among the study and control groups considered the average of the two evaluations. The null hypothesis of the same results in both groups was tested

Figure 2 - Animal 5 from the study group showing the vessel extent reaching up to 75% of the burned area and minimal inflammatory reaction (Degree 1).

versus the alternative hypothesis of different values for the considered variable. The analysis of the vessel extent showed a significant difference between the control and the study group, with smaller vessels being found 14 days after the chemical burn in the study group (Table 5). Nevertheless, no meaningful difference was found among the same groups during the same follow-up when evaluating the vessel inflammation and diameter (Table 6). The microscopic evaluation of the corneal epithelial integrity revealed that the epithelium of all corneas from the study group had healed at 14 days compared with 60% in the control group (Table 7). Despite the reduced number of corneas that had epithelized in the control rabbits, no statistically significant difference was found between the groups. A lower number of polymorphonuclear cells was found in the treated group compared with the control group. Figure 3 shows the polymorphonuclear infiltration in Animal 1 of the control group, whereas Figure 4 shows Animal 3 of study group, which displayed little polymorphonuclear infiltration and full corneal epithelium integrity. In spite of the discrepancies among some rabbits (Figures 3 and 4) and an overall lower number of PMNs being found in the study group, no meaningful statistical differences among the groups were encountered in terms of PMN cell number (Figure 5).

Table 2 - Blinded photographic analysis of vessel extent and inflammation/diameter in the study group, fourteen days after the corneal burn. Animal

1 2 3 4 5 6 7 8 9

Vessel Extent

Vessel Inflammation/Diameter

Evaluation 1

Evaluation 2

Evaluation 1

Evaluation 2

1 1 2 1 1 1 1 1 1

1 1 1 2 1 1 1 1 1

2 3 2 1 1 1 1 1 2

2 3 2 2 1 2 2 2 2

DISCUSSION Previous studies used subconjunctivally administered bevacizumab at dosages varying from 1.25 mg to 3.75 mg.6,12,13 In this study, we chose a dose of 3.75 mg. Because we were only administering one injection during the study, we wanted a high dose that would suppress the initial angiogenic stimulus. No drug-related toxicity has been reported using this concentration. Hurmeric et al.13 compared the effect of injected bevacizumab when it was

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Table 3 - Concordance analysis among evaluations with respect to vessel extent. Classification

Kappa

CI 95%

Concordance

Degree 0: no vessels on the corneal lamina Degree 1: vessels that advanced over the corneal limbus, reaching up to J of the burned area Degree 2: vessels that advanced over the corneal limbus, reaching up to K of the burned area Degree 3: vessels that advanced over the corneal limbus, reaching up to 3/4 of the burned area Degree 4: vessels that extended throughout the entire burned area

0.863

0.541 – 1

Excellent

0.471

0.149 – 0.794

Good

0.644

0.322 – 0.967

Good

0.824

0.502 – 1

Excellent

CI – confidence interval.

and 4 and good for identifying vessel extension at Degrees 2 and 3. The method was also good for identifying vessel inflammation/diameter, as indicated by the good agreement between the two blinded evaluations. For this variable, Degree 3 (intense inflammation and vessels of large diameter) showed excellent agreement. The results obtained show that this method may be reproduced for the evaluation of other anti-angiogenic drugs intended to reduce corneal neovascularization. There was a statistically significant difference in the vessel extent between the two groups, with a lesser extent in the study group compared with the control group. These data indicate a positive effect on the chemical burn and agree with the data found in recent literature.12,13 Neovascularization is considered to be related to a poor prognosis in corneal transplants because it interferes with corneal transparency, which can result in low visual acuity.1 Thus, bevacizumab may improve the prognosis in cases with progressive neovessels after penetrant keratoplasty or in the case of some chemical burns, especially those that require a corneal transplant. The degree of inflammation was evaluated because although the drug does not possess direct anti-inflammatory actions, it does affect fibroblasts.15,16 Although its action in acute inflammation is not fully understood, some authors suggest that the drug decreases neutrophil invasion within the wound.17 The results for inflammation/diameter and the number of PMN cells showed that the drug does not have a clinically meaningful anti-inflammatory effect, which agrees with the theory that the inflammatory process has many pathways and the inhibition of VEGF alone does not significantly affect inflammation. Through these data, we suggest that this treatment may be more effective when administered along with corticosteroids, which would reduce the inflammation. Further studies are necessary to determine whether concomitant inhibition of inflammation and neovascularization leads to massive cellular death by acute hypoxia, which may occur because of the inhibition of all physiological mechanisms involved in such ischemia.

used immediately after the injury or at three days after the injury. Better anti-angiogenic responses were found in the group treated soon after the induced damage. Thus, we also administered the subconjunctival injection soon after the injury. Vascular growth, which is mainly analyzed using the angiogenesis parameters of vessel extent and degree of inflammation, is usually determined through photographic analysis.12,6 However, no established model for this analysis was found. Thus, an experimental model of digital photographic analysis was established, which allowed the vessel extent and inflammation/diameter to be evaluated within the indicated time period. Other photographic evaluation methods have been applied with pixel counting or measurements. Although these methods give the impression of a mathematical certainty as they appear quantitative, the data are subjective with regard to where the vessels begin and end. In addition, the delineation of the affected area may not be accurate when it is determined mathematically. Thus, we have proposed a photographic clinical analysis, in which the evaluator, who is a cornea specialist, observes not only the vessel extent but also the vessel aspect and inflammation degree. We chose to evaluate the corneas at 14 days because angiogenesis has shown a rapid growth until the 12th day in other models.14 In the previous studies, the vessel extent stabilized after this initial period. Fourteen days was sufficient to produce the highest level for each variable (vessel extent and inflammation). On the seventh day, a rabbit from the study group showed corneal perforation and was excluded from the study. As only one rabbit showed perforation and the remaining rabbits in the group showed an intact epithelium by the end of the 14-day period, the perforation was unlikely to be directly caused by the drug. The rabbit may have had a bacterial infection in the wound that was acquired early in the experiment when the corneal epithelium was not fully healed. Statistical analysis confirmed that the evaluation method was excellent for identifying vessel extension at Degrees 1

Table 4 - Concordance analysis among the evaluations with respected to vessel diameter/inflammation. Classification

Kappa

CI 95%

Concordance

Degree Degree Degree Degree

0.383 0.339 0.853

0.061 – 0.706 0.017 – 0.661 0.531 – 1

Weak Weak Excellent

0: 1: 2: 3:

no vessels or inflammation little inflammation, vessels of small diameter moderate inflammatory reaction, medium diameter vessels intense inflammatory reaction, vessels of large diameter

CI – confidence interval.

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Table 5 - Analysis of vessel extent among the groups. Group

Median

Minimum

Control Study

10 9

3 1

2 1

Maximum

p-value

4 1.5

,0.001*

*Mann-Whitney non-parametric test.

Table 6 - Vessel inflammation/diameter analysis among the groups. Group

Median

Minimum

Maximum

p-value

Control Study

10 9

1.75 1.5

1 1

3 3

= 0.968*

*Mann-Whitney non-parametric test.

Figure 3 - Histopatology of the cornea from Animal 1 of the control group with intense polymorphonuclear infiltration.

The integrity of the corneal epithelium was evaluated on the 14th day because the drug may have affected the healing process and delayed wound healing. Rapid restoration of the epithelium is fundamental to the healing process and prevents wound contamination. The comparison between the two groups did not show persistent epithelial defects in the study group at the end of the follow-up. The results showed fewer corneas with an exposed stroma in the study group compared with the control group. However, no statistical difference was observed between the groups, which suggests that the drug did not impair epithelial healing. This study suggests that the bevacizumab has potential for use during the acute healing stage, in which the epithelium is still open. Corticosteroids should be used carefully during this stage because they may impair epithelial healing.18 Delayed epithelial healing has been described previously19,20 and is believed to be caused by the reduced expression of surface integrins and collagens, which interferes with epithelial cell adhesion. However, these studies were performed with higher doses that were applied topically several times a day. Kim et al. showed a doserelated effect.20 Thus, we suggest that the subconjunctival administration is safer because in our study, it did not jeopardize the re-epithelialization. Chalam et al.21 showed that bevacizumab is not toxic to corneal cells or fibroblasts when administered in

concentrations up to 25 mg/ml. This finding is supported by the current study, in which there was no inhibition of re-epithelialization. Although the neovascularization induced by the burn was suppressed, the suppression was not as complete as has been described in previous studies.12 This is possibly because VEGF is not the only factor responsible for angiogenesis. Other pro-angiogenic factors, such as TGFbeta and fibroblast growth factor, may still be involved.16 We suggest that bevacizumab should not to be used as a monotherapy; instead, it should serve as one aspect of a multi-factorial treatment alongside other drugs, such as corticosteroids, in acute cases. This association has not been fully studied, and some conflicting results have been reported. Using a rabbit model, Kang et al.22 showed that a combined treatment of bevacizumab and triamcinolone acetonide was no more effective than the use of bevacizumab alone. Another study23 examined the inhibition of neovascularization in a rat model using bevacizumab alone and with dexamethasone. However, again there was no clear advantage when dexamethasone was included.

Table 7 - Comparative analysis among the groups regarding the epithelial integrity 14 days after the chemical burn. EPITHELIUM DEFECT

YES NO Total

GROUP Control

Study

4 40.0% 6 60.0% 10

0 0.0% 9 100.0% 9

Figure 4 - Cornea from Animal 3 of the study group with little polymorphonuclear infiltration and full corneal epithelium integrity.

p-value: 0.087 Fischerâ&#x20AC;&#x2122;s exact test.

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REFERENCES 1. Chang JH, Gabison EE, Kato T, Azar DT. Corneal neovascularization. Curr Opin Ophthalmol. 2001;12:242-9, doi: 10.1097/00055735200108000-00002. 2. Hill JC, Maske R. An animal model for corneal graft rejection in high-risk keratoplasty. Transplantation. 1988;46:26-30, doi: 10.1097/00007890198807000-00003. 3. Koay PY, Lee WH, Figueiredo FC. Opinions on risk factors and management of corneal graft rejection in the United kingdom. Cornea. 2005;24:292-6, doi: 10.1097/01.ico.0000138841.44926.f8. 4. Kuckelkorn R, Schrage N, Keller G, Redbrake C. Emergency treatment of chemical and thermal eye burns. Acta Ophthalmol Scand. 2002;80:4-10, doi: 10.1034/j.1600-0420.2002.800102.x. 5. Kvanta A. Ocular angiogenesis: the role of growth factors. Acta Ophthalmol Scand. 2006;84:282-8, doi: 10.1111/j.1600-0420.2006.00659.x. 6. Papathanassiou M, Theodossiadis PG, Liarakos VS, Rouvas A, Giamarellos-Bourboulis EJ, Vergados IA. Inhibition of corneal neovascularization by subconjunctival bevacizumab in an animal model. Am J Ophthalmol. 2008;145:424-31, doi: 10.1016/j.ajo.2007.11.003. 7. Tolentino MJ, Miller JW, Gragoudas ES, Chatzistefanou K, Ferrara N, Adamis AP. Vascular endothelial growth factor is sufficient to produce iris neovascularization and neovascular glaucoma in a nonhuman primate. Arch Ophthalmol. 1996;114:964-70. 8. Philipp W, Speicher L, Humpel C. Expression of vascular endothelial growth factor and its receptors in inflamed and vascularized human corneas. Invest Ophthalmol Vis Sci. 2000;41:2514-22. 9. Gan L, Fagerholm P, Palmblad J. Vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 in the regulation of corneal neovascularization and wound healing. Acta Ophthalmol Scand. 2004;82:557-63, doi: 10.1111/j.1600-0420.2004.00312.x. 10. Kahook MY, Lin SC. Anti-VEGF Therapy: Next Stop, Glaucoma? Review of Ophthalmology. 2008;15:47-9. 11. Zhu Q, Ziemssen F, Henke-Fahle S, Tatar O, Szurman P, Aisenbrey S, et al. Vitreous levels of bevacizumab and vascular endothelial growth factor-A in patients with choroidal neovascularization. Ophthalmology. 2008;115:1750-1755, 1755 e1751, doi: 10.1016/j.ophtha.2008.04.023. 12. Hosseini H, Nejabat M, Mehryar M, Yazdchi T, Sedaghat A, Noori F. Bevacizumab inhibits corneal neovascularization in an alkali burn induced model of corneal angiogenesis. Clin Experiment Ophthalmol. 2007;35:745-8, doi: 10.1111/j.1442-9071.2007.01572.x. 13. Hurmeric V, Mumcuoglu T, Erdurman C, Kurt B, Dagli O, Durukan AH. Effect of subconjunctival bevacizumab (Avastin) on experimental corneal neovascularization in guinea pigs. Cornea. 2008;27:357-62, doi: 10.1097/ ICO.0b013e318160d019. 14. Fechine-Jamacaru FV. Modelo de angiogeˆnese inflamato´ria em co´rnea de coelho induzida pela cauterizac¸a˜o alcalina pontual. Acta Cir. bras. 2005;20:64-73. 15. Li Z, Van Bergen T, Van de Veire S, Van de Vel I, Moreau H, Dewerchin M, et al. Inhibition of vascular endothelial growth factor reduces scar formation after glaucoma filtration surgery. Invest Ophthalmol Vis Sci. 2009;50:5217-25, doi: 10.1167/iovs.08-2662. 16. Yoeruek E, Ziemssen F, Henke-Fahle S, Tatar O, Tura A, Grisanti S, et al. Safety, penetration and efficacy of topically applied bevacizumab: evaluation of eyedrops in corneal neovascularization after chemical burn. Acta Ophthalmol. 2008;86:322-8, doi: 10.1111/j.1600-0420.2007.01049.x. 17. Saravia M, Zapata G, Ferraiolo P, Racca L, Berra A. Anti-VEGF monoclonal antibody-induced regression of corneal neovascularization and inflammation in a rabbit model of herpetic stromal keratitis. Graefes Arch Clin Exp Ophthalmol. 2009;247:1409-16, doi: 10.1007/s00417-009-1101-y. 18. Cole N, Hume EB, Jalbert I, Vijay AK, Krishnan R, Willcox MD. Effects of topical administration of 12-methyl tetradecanoic acid (12-MTA) on the development of corneal angiogenesis. Angiogenesis. 2007;10:47-54, doi: 10.1007/s10456-007-9063-3. 19. Bock F, Konig Y, Kruse F, Baier M, Cursiefen C. Bevacizumab (Avastin) eye drops inhibit corneal neovascularization. Graefes Arch Clin Exp Ophthalmol. 2008;246:281-4, doi: 10.1007/s00417-007-0684-4. 20. Kim T-im, Chung JL, Hong JP, et al. Bevacizumab application delays epithelial healing in rabbit cornea. Investigative ophthalmology & visual science. 2009;50:4653-9, doi: 10.1167/iovs.08-2805. 21. Chalam KV, Agarwal S, Brar VS, Murthy RK, Sharma RK. Evaluation of cytotoxic effects of bevacizumab on human corneal cells. Cornea. 2009;28:328-33, doi: 10.1097/ICO.0b013e31818b8be0. 22. Kang S, Chung SK. The effect of subconjuctival combined treatment of bevacizumab and triamcinolone acetonide on corneal neovascularization in rabbits. Cornea. 2010;29:192-6, doi: 10.1097/ICO.0b013e3181b1c82f. 23. Hoffart L, Matonti F, Conrath J, Daniel L, Ridings B, Masson GS, et al. Inhibition of corneal neovascularization after alkali burn: comparison of different doses of bevacizumab in monotherapy or associated with dexamethasone. Clinical & experimental ophthalmology. 2010;38:346-52, doi: 10.1111/j.1442-9071.2010.02252.x.

Figure 5 - Graphic representation of the comparison of PMN cell numbers between the groups.

Newer anti-angiogenic drugs have also been studied over the last year with promising results. For example, suramin is a non-specific anti-angiogenic drug that has a longer half-life than bevacizumb. It has been suggested that this association could provide a more potent and longer effect.24 Pe´rez-Santonja et al.25 showed a greater inhibition of the neovessels by sunitinib (an anti-VEGF and antiPDGF drug) compared with bevacizumab. It has been found that established neovessels, such as those in diabetic retinopathy, are re-established after the drug is discontinued, thereby requiring additional treatments.26 The drug’s effect has not been extensively studied in cases of acute neovascularization, such as that following burns or the initial neovessel growth following a corneal transplant. It is also possible that if the angiogenic stimulus is reduced by specific inhibitors such as bevacizumab in the acute stage, neovessel formation may decrease in the long-term because the angiogenic stimulus has a short duration and is not maintained by the injury’s physiopathology as it is in diseases such as diabetic retinopathy. Thus, this treatment could have a positive effect in the long-term for some treatments such as subsequent corneal transplants. Anti-angiogenic drugs have a great potential for beneficial effects in severely neovascularized eyes with little or no limbal damage, which generally present with very low visual acuity. Presently, there is no available treatment for this condition. Further studies with different dosages and steroids combinations with the various VEGF inhibitors that compare not only the neovascularization itself but also the inflammatory response and side effects would contribute to the search for an ideal treatment. This association may have a synergistic effect on burns or in cases with a possibility of acute neovascularization, where the neovascular stimulus occurs as a part of the acute inflammatory response. Thus, we conclude that the subconjunctival administration of bevacizumab efficiently reduces corneal neovascularization without inducing persistent epithelial defects following a chemical burn. However, this treatment does not show meaningful anti-inflammatory effects.

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Subconjunctival bevacizumab and corneal neovascularization Mello GR et al. Model. Am J ophthalmol. 2010;150:519-528.e1, doi: 10.1016/j.ajo.2010. 04.024. 26. Brown DM, Regillo CD. Anti-VEGF agents in the treatment of neovascular age-related macular degeneration: applying clinical trial results to the treatment of everyday patients. Am J Ophthalmol. 2007;144:627-37, doi: 10.1016/j.ajo.2007.06.039.

24. Lee HS, Chung SK. The effect of subconjunctival suramin on corneal neovascularization in rabbits. Cornea. 2010;29:86-92, doi: 10.1097/ICO. 0b013e3181ae91e3. 25. Pe´rez-Santonja JJ, Campos-Mollo E, Lledo´-Riquelme M, Javaloy J, Alio´ JL. Inhibition of Corneal Neovascularization by Topical Bevacizumab (Anti-VEGF) and Sunitinib (Anti-VEGF and Anti-PDGF) in an Animal

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DOI:10.1590/S1807-59322011000800024

BASIC RESEARCH

Effects of mycophenolate sodium on mucociliary clearance using a bronchial section and anastomosis rodent model Viviane Ferreira Paes e Silva, Rogerio Pazetti, Sonia de Fatima Soto, Mariana Moreira Quinhones Siqueira, Aristides Tadeu Correia, Fabio Biscegli Jatene, Paulo Manuel Peˆgo-Fernandes Hospital das Clıńicas da Faculdade de Medicina da Universidade de Saõ Paulo - Laboratory of Thoracic Surgery Research-LIM/61, Thoracic Surgery Department, Heart Institute (InCor), Saõ Paulo, Saõ Paulo/SP, Brazil.

OBJECTIVE: To study the effects of mycophenolate sodium on mucociliary clearance. INTRODUCTION: Mycophenolate is one of the most commonly used immunosuppressive drugs in lung transplantation. Although its pharmacokinetic properties are well defined, its side effects on mucociliary clearance have not yet been studied. METHODS: Sixty rats were subjected to left bronchial section and anastomosis. The right bronchus was used as a control. After surgery, the rats were assigned to two groups based on whether they received saline solution (n = 30) or mycophenolate sodium (n = 30). After 7, 15, or 30 days of treatment, 10 animals from each group were sacrificed, and in vitro mucus transportability, in situ mucociliary transport velocity and ciliary beat frequency were measured. RESULTS: The analysis of mucus transportability revealed that neither mycophenolate nor bronchial section altered any transportability related property for up to 30 days of treatment after surgery (p.0.05). With regard to ciliary beat frequency, the operated left bronchi from the mycophenolate group showed a significant decrease on postsurgical day 30 (p = 0.003). In addition, we found a significant reduction in the in situ mucociliary transport velocity in the mycophenolate-treated group (p = 0.0001). DISCUSSION: These data add important information regarding mucociliary clearance dysfunction following mycophenolate therapy and suggest that mycophenolate might contribute to the high incidence of respiratory tract infections in lung transplant patients. Further studies are needed to investigate the combined action of mycophenolate with other immunosuppressive drugs and to establish methods to protect and recover mucociliary clearance, an important airway defense mechanism. KEYWORDS: Immunosuppressive Agents; Lung Transplantation; Mucociliary Clearance; Animals. Paes e Silva VF, Pazetti R, Soto SF, Siqueira MMQ, Correia AT, Jatene FB, Peˆgo-Fernandes PM. Effects of mycophenolate sodium on mucociliary clearance using a bronchial section and anastomosis rodent model. Clinics. 2011;66(8):1451-1455. Received for publication on April 7, 2011; First review completed on May 6, 2011; Accepted for publication on May 6, 2011 E-mail: paulo.fernandes@incor.usp.br Tel.: 55-11-30695351

MCC is a first-line defense mechanism that protects the lungs from the accumulation of particles and pathogens by removing bacteria, viruses, antigens, and toxins that are trapped in the mucus on the tracheobronchial airway surface.5 An essential balance between mucus production and its removal by ciliated cells is required for optimal airway defense.6 Many studies have reported that MCC is impaired in lung transplant patients.5 Several features that are unique to LTx determine the predominance of fungal colonization and infections in the early postoperative period. Perhaps one of the most important factors is the direct communication between the graft and the outside environment. In addition, blood supply to the anastomosis is disrupted,7 causing chronic mild ischemia and placing the graft at high risk for infection.8 In previous studies, we showed that cyclosporine,9,10 azathioprine,11 and bronchial section12 can all impair MCC

INTRODUCTION Mycophenolate is currently one of the most commonly used immunosuppressive drugs in lung transplantation (LTx).1,2 Mycophenolate is a cell cycle inhibitor that produces reversible noncompetitive blockade of the purine synthesis pathway enzyme type II isoform of inosine monophosphate dehydrogenase.3,4 Although its pharmacokinetic properties are well defined, the side effects of mycophenolate on respiratory epithelium, and mucociliary clearance (MCC) in particular, have yet to be studied.

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by decreasing ciliary activity and mucus production and secretion. We hypothesize that mycophenolate can also play an important role in impaired MCC in a bronchial section and anastomosis rodent model. To test this hypothesis, we analyzed the components of bronchial ciliated epithelium in immunosuppressed rats by measuring ciliary beat frequency (CBF), mucociliary transport velocity (MCTV), and mucus transportability (MT).

main bronchus, and mucus collection was performed by inserting a small hair paintbrush into the lumen of each bronchus. The mucus that adhered to the paintbrush was then placed in a 0.6-ml microtube containing mineral oil (to prevent dehydration) and stored at -70 ˚C. MT was measured using an in vitro frog palate model.15 The mucus, which was previously defrosted at room temperature, was placed on the frog palate ciliated epithelium, and its movement was observed and timed with the aid of a stereomicroscope equipped with a reticulated eyepiece. The MT of the rat mucus was compared to that of the frog mucus itself, and the results are therefore expressed as relative velocity (rat/frog). After collection of the mucus sample, the bronchi were placed under a light microscope (Olympus, BX50, Tokyo, Japan) connected to a video camera (Sony Trinitron, 3CCD, Tokyo, Japan). A stroboscope (Machine Vision Strobe, Cedarhurst, NY) was placed in front of the bronchi, and CBF was measured by synchronization between cilia movement and a stroboscope flashlight. Finally, under the same microscope, in situ MCTV was measured by direct observation of particles deposited on the mucous layer moving across the bronchi. The movement of the particles was timed, and the velocity was registered as the distance covered over one minute.

MATERIALS AND METHODS Experimental design Sixty male Wistar rats weighting 300 g were subjected to left bronchial section and anastomosis. The right bronchus was used as an internal control. Following surgery, the rats were assigned to two groups that received either saline solution (Sal group, n = 30) or mycophenolate sodium (MPS group, n = 30). The animals were maintained according to the Guide for the Care and Use of Laboratory Animals,13 and our institution’s ethics committee approved the protocol.

Surgical procedure Anesthesia was induced with inhaled isoflurane (Isoforine, Crista´lia, Itapira, SP, Brazil) in a closed chamber, followed by orotracheal intubation with a 14-gauge catheter that was 6.5 cm in length. General anesthesia was maintained by inhalation of 2% isoflurane in pure oxygen via a nebulizer (Model 1223; Takaoka; Sa˜ o Paulo, SP, Brazil). Ventilation was achieved by a volumecycled ventilator (Harvard Apparatus, Holliston, MA, USA) with a respiratory rate of 70 breaths/min and a tidal volume of 10 ml/kg. A left thoracotomy was then performed. The left main stem bronchus was dissected, clamped, and completely transected, followed by an endto-end anastomosis with continuous running 8-0 polypropylene sutures. Finally, airflow was restored, and the atelectasis of the left lung was resolved by hyperinflation. Prior to closing the incision, a chest tube was inserted. The chest tube and tracheal tube were removed immediately after spontaneous respiration was restored. The surgical procedure was performed with the aid of a stereomicroscope at 8x the original magnification.

Statistical analysis All of the data were analyzed using the Statistic Package for Social Sciences (SPSS version 13.0). An analysis of variance was used to test the interference and interaction of the factors. Comparisons between groups were performed using the Bonferroni post-hoc test. The results are expressed as mean¡SD, and the differences were considered significance when p,0.05.

RESULTS In the MT data collected from the rat mucus samples and tested in the frog palate model, we found that neither mycophenolate sodium (MPS) nor bronchial section altered any transportability property for up to 30 days of treatment after surgery (p.0.05; Figure 1). With regard to the CBF measurements (Figure 2), the left bronchi from MPS group showed a significant decrease on postoperative day 30 (8.47¡1.12 Hz) compared to day 7 (10.72¡1.21 Hz, p,0.001) and day 15 (9.80¡1.16 Hz, p = 0.026). In addition, a significant decrease was also observed compared to the right bronchi from MPS group at 30 days (9.73¡0.98 Hz, p = 0.003). In situ MCTV (Figure 3) was significantly slower in the MPS left group (0.021¡0.009 mm/min) than in the Sal left group (0.038¡0.023 mm/min, p = 0.016) after 30 days. The Sal left group showed an increase in MCTV compared to 7 (0.018¡0.011 mm/min, p = 0.009) and 15 days (0.017¡ 0.013 mm/min, p = 0.005) after the surgery and compared to the Sal right group at 30 days (0.023¡0.018 mm/min, p = 0.003). After 15 days of therapy, MCTV in the MPS right group (0.038¡0.017 mm/min) was higher than in the Sal right (0.018¡0.016 mm/min, p = 0.014) and MPS left (0.019¡0.008 mm/min, p,0.001) groups. On postoperative day 7, only the Sal left group showed MCTV impairment (0.018¡0.011 mm/min) relative to the Sal right group (0.028¡0.024 mm/min, p = 0.034).

Therapy Following surgery, mycophenolate sodium (Myfortic, 180 mg, Novartis, Stein, Switzerland) was administered to the animals via an orogastric tube at a daily dosage of 400 mg/m2/day diluted in saline. The animals in the Sal group were subjected to the same therapeutic regimen but received only the vehicle (saline) in an equivalent volume.

Euthanasia After 7, 15, or 30 days of treatment, ten animals from each group were anesthetized with intraperitoneal sodium pentobarbital (30 mg/kg) and sacrificed by exsanguination by section of the abdominal aorta.14

Data collection Immediately after the animals were sacrificed, their lungs were removed en bloc from the thoracic cavity and placed in a petri dish. After dissection, an incision was made in each

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Effects of mycophenolate on mucociliary clearance Paes e Silva VF et al.

Figure 1 - Mucociliary transportability (MT and rat/frog) of mucus samples obtained from the left (operated) or right (intact) bronchi of rats treated with saline (Sal) or mycophenolate sodium (MPS) for 7, 15, or 30 days (p.0.05).

wealth of evidence available in the renal transplant field and the paucity, or at least a lack of consistency, of information for LTx.19 Several factors could explain this problem, including the small sample sizes and often contradictory results of original studies of LTx and patients from a heterogeneous group of pulmonary diseases who receive lung transplants, which results in widely varying patient phenotypes and individual pharmacogenomics.16 Based on these difficulties, the advantages of conducting studies using animal models are clear. We believe that our bronchial section rodent model is useful for clarifying the adverse effects of immunosuppressive drugs on the respiratory epithelium, specifically on mucociliary clearance. We previously demonstrated that cyclosporine impaired both ciliary beat frequency and mucus production/secretion for up to 90 days of treatment.9,10 Conversely, azathioprine caused a transiently perturbed on mucociliary transport on postoperative days 7 and 15, and function was fully recovered in the animals that were treated for 30 days.11 Together, these results reflect the controversy we find in the literature regarding immunosuppression therapy for LTx, even regarding drugs of the same class such as azathioprine and mycophenolate, both of which are cell cycle inhibitors. The use of mycophenolate (in place of azathioprine) has increased since its commercialization and testing in renal and

DISCUSSION In the present study, we tested a drug that is commonly used as part of the immunosuppressant triple therapy regimen that consists of a corticosteroid (prednisone, prednisolone, or methylprednisolone), a calcineurin inhibitor (cyclosporine or tacrolimus), and an antimetabolite (azathioprine or mycophenolate).1,2,16,17 We found that mycophenolate impairs MCC in the operated bronchi of animals treated for 30 days. Consistent with our previous results,10,12 MCC was also impaired by bronchial section for up to 15 days after surgery in saline-treated animals and showed significant recovery by postoperative day 30. In the immunosuppressed animals, however, this was not the case. These data partially corroborate our initial hypothesis and suggest that MPS might contribute to the high incidence of infection in the respiratory tract of lung transplant patients. Clinicians are continually searching for an adequate immunosuppressive regimen in an attempt to maximize efficacy against rejection while avoiding toxicity and infection.17 Unfortunately, the choice of the immunosuppressive regimen for an individual patientâ&#x20AC;&#x2122;s requirements is usually reactive rather than proactive.1 With respect to the optimal early and maintenance immunosuppression regimens, a recent review showed a strong contrast between the

Figure 2 - Ciliary beatfrequency (CBF) of the left (operated) or right (intact) bronchi of rats treated with saline (Sal) or mycophenolate sodium (MPS) for 7, 15, or 30 days. *The MPS left group showed the slowest frequency in 30 days (p,0.001 and p = 0.026 vs. 7 and 15 days, respectively; p = 0.003 vs. MPS right at 30 days).

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Figure 3 - Mucociliary transport velocity (MCTV) from the left (operated) or right (intact) bronchi of rats treated with saline (Sal) or mycophenolate sodium (MPS) for 7, 15, or 30 days. *Statistical differences between groups at each time point: 7 days - Sal right vs. Sal left, p = 0.034; 15 days - MPS right vs. Sal right and vs. MPS left, p = 0.014 and p,0.001, respectively; 30 days – Sal left vs. Sal right and MPS left, p = 0.003 and p = 0.016, respectively. Behavior of groups over time: Sal left 30 days vs. 7 and 15 days, p = 0.009 and 0.005, respectively.

heart transplantation in the early 1990s. Some studies suggest that mycophenolate may be more efficient than azathioprine at preventing acute rejection in LTx, whereas the single prospective, randomized study that compared these two drugs did not find any benefit.18 Korom et al.2 concluded in their review that there is currently no unambiguous evidence to support the superiority of mycophenolate over azathioprine in LTx that could justify its wide-scale use as a first-choice antimetabolite. Immunosuppression by mycophenolate occurs by virtue of its active component mycophenolic acid (MPA) through the inhibition of inosine monophosphate dehydrogenase (IMPDH). T and B cells are more dependent on this pathway than are other cells. Other potential mechanisms of immunosuppression include inducing apoptosis in activated T cells, decreasing the expression of adhesion molecules (thereby decreasing the recruitment of inflammatory cells), and decreasing the production of inducible nitric oxide and the resulting tissue damage.16 Mycophenolate has also been shown to inhibit the proliferation of smooth muscle cells, fibroblasts, and endothelial cells by blocking DNA synthesis in the S phase of the cell cycle.20 To our knowledge, this is the first study to directly test the effects of mycophenolate on the mucociliary apparatus. Furthermore, we separately analyzed each of the two principal components of this clearance mechanism, thereby showing that only cilia movement from the left (sectioned) bronchus was impaired after MPS therapy for 30 days. By itself, this treatment did not alter the viscoelastic properties of the mucus, at least in the frog palate model employed here. There are several techniques for monitoring MCC, the most common of which is the use of insoluble particles that can be directly observed, primarily in humans, through a bronchoscope, by radiography or by external monitoring of radiolabelled particles.21 In our rodent model, we used a microscope equipped with a reticulated eyepiece, which permitted the direct observation and timing of the progression of the particles trapped in the mucus layer across the bronchus. Moreover, we developed a setup consisting of an optic fiber connected to a stroboscope for analyzing CBF.10

A limiting point in this setup is its dependence on an expert researcher to analyze and register CBF according to the frequency of the stroboscopic flashlight. Finally, we used the bullfrog palate, a well-established method for analyzing the transportability of mucus samples.15 Because of its similarity with mammalian respiratory epithelium, we could test the transportability properties of the rat mucus samples in comparison with the autologous (frog) mucus. We have thus accumulated a considerable amount of information regarding the physiological variability of mucociliary clearance as a result of the effects of various immunosuppressant agents.9-11 Presently, we do not intend to analyze the intracellular mechanisms of action of MPS in the cells of the mucociliary system. We can, however, infer from our results that MPS affects ciliated cells more than secretory (goblet) cells. This is important for future studies that aim to avoid this undesirable side-effect of mycophenolate therapy on MCC. Presumably, the most informative method to study the mechanism of MPS is to use cultured respiratory epithelial cells. Given that we observed a reduction in CBF, we propose including a well-known ciliary stimulant such as extracellular adenosine triphosphate or acetylcholine22 when testing the effects of MPS.

ACKNOWLEDGEMENTS This study was performed at the Thoracic and Cardiovascular Surgery Post-Graduation Program of Heart Institute of Clinics Hospital of Saõ Paulo University Medical School, Saõ Paulo, SP, Brazil. The study was supported by grants from The State of Saõ Paulo Foundation (FAPESP), Saõ Paulo, SP, Brazil.

REFERENCES 1. Hopkins PM, McNeil K. Evidence for immunosuppression in lung transplantation. Current Opinion in Organ Transplantation. 2008;13:47783, doi: 10.1097/MOT.0b013e32831040bf. 2. Korom S, Boehler A, Weder W. Immunosuppressive therapy in lung transplantation: state of the art. European Journal of Cardio-thoracic Surgery. 2009;35:1045-55, doi: 10.1016/j.ejcts.2009.02.035. 3. Sanford M, Keating GM. Enteric-coated mycophenolate sodium. A review of its use in the prevention of renal transplant rejection. Drugs. 2008;68:2505-33.

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Effects of mycophenolate on mucociliary clearance Paes e Silva VF et al. 13. Institute of laboratory animal resources. Guide for the care and use of laboratory animals. National Research Council of the National Academy of Sciences. Washington, D.C.: National Academy Press. 1996;1-35. 14. American Veterinary Medical Association. 2000 Report of the AVMA Panel on Euthanasia. Journal of American Veterinary Medical Association. 2001;218:669-96, doi: 10.2460/javma.2001.218.669. 15. Rubin BK, Ramirez O, King M. Mucus depleted frog palate as a model for the study of mucociliary clearance. Journal of Applied Physiology. 1990;69:424-9. 16. Bhorade SM, Stern E. Immunosuppression for lung transplantation. Proceedings of the American Thoracic Society. 2009;6:47-53, doi: 10.1513/ pats.200808-096GO. 17. Snell GI, Westall GP. Immunosuppression for lung transplantation: Evidence to Date. Drugs. 2007;67:1531-9, doi: 10.2165/00003495200767110-00002. 18. Knoop C, Haverich A, Fischer S. Immunosuppressive therapy after human lung transplantation. European Respiratory Journal. 2004;23:15971, doi: 10.1183/09031936.03.00039203. 19. Dilling DF, Glanville AR. Advances in lung transplantation: The year in review. Journal of Heart and Lung Transplantation. 2011;30:247-51, doi: 10.1016/j.healun.2010.12.005. 20. Allison AC, Eugui EM. Mycophenolate mofetil and its mechanisms of action. Immunopharmacology. 2000;47:85-118, doi: 10.1016/S01623109(00)00188-0. 21. EdsbaÂ¨cker S, Wollmer P, Selroos O, BorgstroÂ¨m L, Olsson B, Ingelf J. Do airway clearance mechanisms influence the local and systemic effects of inhaled corticosteroids? Pulmonary Pharmacology & Therapeutics. 2008;21:247-58, doi: 10.1016/j.pupt.2007.08.005. 22. Teff Z, Priel Z, Gheber LA. The forces applied by cilia depend linearly on their frequency due to constant geometry of the effective stroke. Biophysical Journal. 2008;94:298-305, doi: 10.1529/biophysj.107.111724.

4. Villarroel MC, Hidalgo M, Jimeno A. Mycophenolate mofetil: an update. Drugs of Today. 2009;45:521-32. 5. Laube BL, Karmazyn YJ, Orens JB, Mogayzel PJ. Albuterol improves impaired mucociliary clearance after lung transplantation. Journal of Heart and Lung Transplantation. 2007;26:138-44, doi: 10.1016/j.healun. 2006.11.005. 6. Voynow JA, Rubin BK. Mucins, Mucus, and Sputum. Chest. 2009;135:505-12, doi: 10.1378/chest.08-0412. 7. Herold U, Jakob H, Kamler M, Thiele R, Tochtermann U, Weinmann J, et al. Interruption of bronchial circulation leads to a severe decrease in peribronchial oxygen tension in standard lung transplantation technique. European Journal of Cardio-Thoracic Surgery. 1998;13:176-83, doi: 10.1016/S1010-7940(97)00314-X. 8. Adler FR, Aurora P, Barker DH, Barr ML, Blackwell LS, Bosma OH, et al. Lung transplantation for cystic fibrosis. Proceedings of the American Thoracic Society. 2009;6:619-33, doi: 10.1513/pats.2009008-088TL. 9. Pazetti R, Pego-Fernandes PM, Ranzani OT, Parra ER, Lorenzi-Filho G, Jatene FB. Cyclosporine A reduces airway mucus secretion and mucociliary clearance in rats. Clinics. 2007;62:345-52, doi: 10.1590/ S1807-59322007000300021. 10. Pazetti R, Pego-Fernandes PM, Lorenzi-Filho G, Saldiva PHN, Moreira LFP, Jatene FB. Effects of cyclosporine A and bronchial transection on mucociliary transport in rats. Annals of Thoracic Surgery. 2008;85:1925-9, doi: 10.1016/j.athoracsur.2008.02.084. 11. Pego-Fernandes PM, Said MM, Pazetti R, Moreira LFP, Jatene FB. Effects of azathioprine on mucociliary clearance after bronchial section and anastomosis in a rat experimental model. Jornal Brasileiro de Pneumologia. 2008;34:273-9, doi: 10.1590/S1806-37132008000500005. 12. Rivero DHRF, Lorenzi-Filho G, Pazetti R, Jatene FB, Saldiva PHN. Effects of bronchial transection and reanastomosis on mucociliary system. Chest. 2001;119:1510-5, doi: 10.1378/chest.119.5.1510.

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DOI:10.1590/S1807-59322011000800025

BASIC RESEARCH

Rosuvastatin prevents proteinuria and renal inflammation in nitric oxide–deficient rats Jose´ Marcos Girardi,I,II Roge´rio Estevan Farias,III Ana Paula Ferreira,III Na´dia Rezende Barbosa RaposoI,IV I Faculdade de Farmaćia, Universidade Federal de Juiz de Fora, MG, Brazil. II Serviço de Cardiologia do Hospital Universita´rio, HU/CAS, Universidade Federal de Juiz de Fora, MG, Brazil. III Instituto de Cieˆncias Biolo´gicas, Universidade Federal de Juiz de Fora, MG, Brazil. IV Laborato´rio de Neurocieˆncias (LIM-27), Faculdade de Medicina, Departamento e Instituto de Psiquiatria, Universidade de Saõ Paulo, Brazil.

OBJECTIVE: The aim of the present study was to assess the effects of rosuvastatin on renal injury and inflammation in a model of nitric oxide deficiency. METHODS: Male Wistar rats were randomly divided into four groups (n = 10/group) and treated for 28 days with saline (CTRL); 30 mg/kg/day L-NAME (L-name); L-NAME and 20 mg/kg/day rosuvastatin (L-name+ROS-20); or L-NAME and 2 mg/kg/day rosuvastatin (L-name+ROS-2). Systolic blood pressure was measured by plethysmography in the central artery of the tail. The serum total cholesterol, triglycerides, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, creatinine, nitric oxide, interleukin-6, and tumor necrosis factor alpha levels were analyzed. Urine samples were taken to measure the albumin5urinary creatinine ratio. Kidneys were sectioned and stained with hematoxylin/eosin and Masson’s trichrome. Immunohistochemical analysis of the renal tissue was performed to detect macrophage infiltration of the glomeruli. RESULTS: The systolic blood pressure was elevated in the L-name but not the L-name+rosuvastatin-20 and L-name+rosuvastatin-2 groups. The L-name group had a significantly reduced nitric oxide level and an increased interleukin-6 and tumor necrosis factor alpha level, albumin5urinary creatinine ratio and number of macrophages in the renal glomeruli. Rosuvastatin increased the nitric oxide level in the L-name+rosuvastatin-2 group and reduced the interleukin-6 and tumor necrosis factor alpha levels, glomerular macrophage number and albumin5urinary creatinine ratio in the L-name+rosuvastatin-20 and L-name+rosuvastatin-2 groups. CONCLUSION: Rosuvastatin treatment reduced glomerular damage due to improvement in the inflammatory pattern independent of the systolic blood pressure and serum lipid level. These effects may lead to improvements in the treatment of kidney disease. KEYWORDS: L-NAME; Hypertension; HMG-CoA reductase inhibitors; Kidney; Interleukins. Girardi JM, Farias RE, Ferreira AP, Raposo NRB. Rosuvastatin prevents proteinuria and renal inflammation in nitric oxide–deficient rats. Clinics. 2011;66(8):1457-1462. Received for publication on December 10, 2010; First review completed on January 1, 2011; Accepted for publication on May 11, 2011 E-mail: jgirardi@cardiol.br Tel.: 55 32 3229 3809

pleiotropic effects, including antiinflammatory, antiproliferative, and antithrombotic effects; attenuation of NADPH oxidase–mediated superoxide generation; and improvement of endothelial vasomotor function. All of these effects may influence cardiovascular outcomes, including hypertension, in high-risk patients.4 Statins reduce vascular inflammation, suppress extracellular matrix production, exhibit inhibitory effects on the Rho/Rho kinase pathway,5,6 and block small G protein isoprenylation, which is a pivotal step in the activation of Rho. The kidney is a target organ of hypertension. Renal disease secondary to hypertension progressively develops, culminating in chronic renal failure with a loss of glomeruli and several morphological and quantitative alterations.7,8 The renal lesions caused by nitric oxide (NO) blockade are glomerulosclerosis, interstitial fibrosis, and microvascular lesions.9,10 The current chronic nephropathy treatments are limited to angiotensin-converting enzyme inhibitors and angiotensin

INTRODUCTION Statins, which are 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, are currently the most popular drugs for the management of dyslipidemia for the primary and secondary prevention of cardiovascular disease.1 Their widespread use is driven by a robust body of evidence that supports their efficacy in preventing cardiovascular events.2 Their benefits are derived both from reducing atherogenic lipoprotein levels (LDL-C) and from increasing antiatherogenic lipoproteins levels (HDL-C).3 In addition to modulating lipid levels, recent clinical and experimental studies have shown that statins have

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receptor blockers.11 However, similar to observations in the cardiovascular system, increasing clinical12 and experimental13-17 evidence indicates that statins have beneficial effects in the course of progressive renal disease. In addition, statins influence intrarenal hemodynamics by reducing angiotensin and aldosterone levels.18 Recent studies have shown that chronic administration of L-arginine analogues such as L-nitro-arginine methyl ester (L-NAME) to rats induces dose-dependent systemic arterial hypertension by blocking endothelial nitric oxide synthase (eNOS) and, therefore, NO biosynthesis.19,20 The rat hypertension model induced by chronic inhibition of NO generation has been shown to be a useful tool for studying both the development and treatment of renal lesions resembling those found in human hypertension, a disease associated with early generalized impairment of endothelial function.21 This hypertension and renal disease model seems to be a suitable model to examine the lipid lowering–independent effects of statins because the administration of statins, even at high doses, does not alter serum cholesterol levels in normal rats.22-23 The aim of this study was to assess the effects of rosuvastatin on renal injury and inflammation in a model of nitric oxide deficiency.

NO was assessed using the Griess method.24 The levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-a) were measured using enzyme-linked immunosorbent assay (ELISA) (Opt, BD, USA) according to the manufacturer’s instructions. Urine was obtained by bladder puncture to establish the urine albumin: creatinine ratio (Biotecnica kit, Cobas Mira, Roche). Analyses were performed in duplicate, and samples were stored at -80˚C until analysis.

Histological analysis After euthanasia, a fixative solution (1.27 mol/l of freshly prepared formaldehyde in 0.1 M phosphate buffer; pH 7.4) was perfused through the vascular system using a catheter placed into the left ventricle until the body was rigid. The kidneys were removed, cut into two halves, placed in 10% formaldehyde for 48 h at room temperature, dehydrated, and embedded in paraffin for histological analysis. Sections (3 mm thick) were cut and stained with hematoxylin/eosin for morphological analysis or Masson’s trichrome to highlight collagen fibers. Histological analysis was performed using a light microscope (Olympus BX51). Images were captured (Color Coolsnap-Pro, Media Cybernetics) and analyzed using Image Pro-Plus software (Version 6.0, Media Cybernetics, Silver Spring, USA). The sections were evaluated by a pathologist without prior knowledge of the groups to which the animals belonged. Tubules (to study tubular dilation), blood vessels (to identify intimal fibrosis, microthrombosis and fibrinoid necrosis), glomeruli (to identify glomerulosclerosis and glomerular atrophy), and the interstitial compartment (to detect changes in collagen and inflammatory cells (4006)) were analyzed morphologically to identify the range of changes. The glomerular surface area (mm2) was morphometrically analyzed in at least 50 glomeruli per animal.25

METHODS AND MATERIAL Animals All animals (n = 40; 6-week-old male Wistar rats) were maintained in standard cages at room temperature (22¡3 ˚C) and 60–70% humidity with a 12 h dark/12 h light cycle and ad libitum food (Nuvilab, Parana´, Brazil) and water. This diet contained 22.5% crude protein and 0.27% sodium. Twelve hours before each experiment, the animals received only water to avoid interference with absorption of the treatments. The rats were euthanized with 50 mg/kg i.p. sodium pentobarbital. Systolic blood pressure was measured weekly in conscious rats using noninvasive tail-cuff plethysmography (Letica 5001, Panlab, Spain) at baseline and at the end of each week of treatment. The experiment was conducted for four weeks. This study was conducted in accordance with guidelines set forth by the Brazilian Association for Laboratory Animal Science (COBEA) and has been approved by the Research Commission for Ethics and Animal Experimentation of UFJF.

Treatments The rats were randomly divided into four groups (n = 10/ group) and treated as follows: a control group (CTRL); 30 mg/ Kg/day L-NAME hydrochloride (Sigma, St. Louis, USA) diluted in the drinking water (L-name); L-NAME+20 mg/kg/ day rosuvastatin (Crestor, AstraZeneca, Brazil) delivered by gavage (L-name+ROS-20); and L-NAME+2 mg/kg/day rosuvastatin (L-name+ROS-2) delivered by gavage. The L-NAME in the drinking water was at a concentration of 500 mg/l, and each non-control rat received 30 mg/kg/day of L-NAME for 28 days.

Biochemical parameters

Figure 1 - Systolic blood pressure over the entire experimental period in control and treated rats. The data are expressed as the mean ¡ SEM for = 10 rats/group. After the first week, all of the treated groups (L-name, L-name+ROS-20, L-name+ROS-2) had significantly increased systolic blood pressures when compared with the CTRL group (p,0.0001).

Blood samples were drawn by cardiac puncture from the right ventricle to measure the serum levels of total cholesterol (TC), triglycerides (Tg), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and creatinine (Cr) (Cobas Mira, Roche). The serum level of

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Table 1 - Biochemical parameters of the experimental groups at the end of the experiment. Parameter n Serum creatinine, mg/dl Total cholesterol, mg/dl Triglycerides, mg/dl Alanine aminotransferase, U/l Aspartate aminotransferase, U/l Alkaline phosphatase, U/l

CTRL

L-name

L-name+ROS-20

L-name+ROS-2

10 1.1¡0.04 71.0¡6.3 73.8¡5.8 250.3¡45.1 102.3¡12.1 236.0¡14.6

10 0.9¡0.03 74.1¡4.4 89.2¡6.8 222.4¡7.8 112.5¡3.5 254.2¡10.2

10 1.0¡0.09 82.5¡7.0 78.7¡15.7 227.3¡13.1 116.0¡4.0 259.9¡23.5

10 1.0¡0.07 72.8¡3.9 72.6¡4.5 186.1¡13.2 99.2¡2.4 236.7¡15.3

Data are expressed as the mean ¡ SEM. All groups had similar biochemical parameters when compared with the CTRL group.

Immunohistochemical analysis

Biochemical analysis

Immunohistochemical analysis of renal tissues to detect the number of macrophages in 100 random glomeruli per animal was performed on paraffin-embedded sections as described by Hartner et al.26 using antibodies against macrophages/monocytes (Mouse anti-rat CD68; Serotec). The percentage of glomeruli with macrophages was determined by counting 100 random clusters from nearly 20 microscopic fields, in accordance with Pomaro et al.,27 and was determined by a pathologist without prior knowledge of the groups to which the animals belonged.

The experimental groups had similar TC, Tg, AST, ALT, ALP, and Cr levels (Table 1). Compared with the control, the L-name group had increased serum levels of IL-6 (p,0.001) and TNF-a (p,0.01). However, when compared with the L-name group, treatment with 2 or 20 mg/kg/day rosuvastatin inhibited the L-NAME-induced increase in the serum IL-6 (p,0.001 for both groups) and TNF-a levels (Lname+ROS-20, p = 0.030; L-name+ROS-2, p = 0.028). The Lname group showed a significant reduction in the NO level compared with the control group (p = 0.01). Only the L-name+ROS-2 group showed significantly higher levels of NO compared with the L-name group (p = 0.047; Table 2). The L-name group showed an increase in the urinary albumin5creatinine ratio compared with the CTRL group (p = 0.023). Rosuvastatin at both tested doses (2 and 20 mg/ kg/day) lowered the urinary albumin5creatinine ratio compared with the L-name group (p = 0.008 and p = 0.047, respectively) (Figure 2).

Statistical analysis The results are presented as the mean ¡ standard error (SEM). The data were all normally distributed in each group (Kolmogorov-Smirnov test, p.0.05). Statistical analysis between groups was performed using an analysis of variance (one-way ANOVA) followed by Tukey’s test when the data were homoscedastic. If the data were heteroscedastic, the nonparametric Kruskal-Wallis test followed by the Tamhane test was performed. A p-value less than 0.05 was considered significant. Analyses were conducted and graphs were created using GraphPad Prism Version 5.01 for Windows (GraphPad Software, San Diego, CA, USA) and Statistical Package for Social Sciences Version 13.0 (SPSS, Chicago, IL, USA).

Histological analysis Histological evaluation following staining with hematoxylin/eosin (Figure 3 A,D,G,F) or Masson’s trichrome (Figure 3 B,E,H,K) revealed no detectable renal pathology in any of the groups in all of the renal compartments. The cross-sectional glomerular areas were similar among all of the experimental groups (Table 3).

RESULTS

Immunohistochemical analysis

Systolic blood pressure analysis

The L-name group demonstrated an increased number of macrophages in the renal glomeruli compared with the CTRL group (p,0.0001). The L-name+ROS-20 and L-name+ROS-2 groups demonstrated decreased infiltration of macrophages into the glomeruli compared with the L-name group (p,0.0001) (Figure 3 C,F,I,L and Table 3).

The initial systolic blood pressure (SBP) was similar among the groups. At the end of the first week of treatment with L-NAME, all L-NAME-treated groups had an elevated SBP compared with the control group (p,0.0001 for all of the groups). This hypertension was observed for all L-NAME-treated groups throughout the experiment (Figure 1; p,0.0001 for all groups). Table 2 - Inflammatory parameters of the experimental groups at the end of the experiment. Group CTRL L-name L-name+ROS-20 L-name+ROS-2

IL-6 (pg/mL)

TNF-a (pg/mL)

NO (mM)

7.4¡0.4* 25.3¡2.0 8.7¡0.1* 10.2¡0.5*

25.5¡1.8# 35.3¡4.7 26.1¡1.4+ 25.4¡1.2+

2.1¡0.5# 0.5¡0.1 0.8¡0.1 2.3¡0.6#

The results are expressed as the mean ¡ SEM. n = 10 rats/group. *p,0.001; #p,0.01; +p,0.05 when compared with the L-name group. NO: serum nitric oxide level; IL-6: interleukin-6; TNF-a: tumor necrosis factor-alpha.

Figure 2 - Urinary albumin5creatinine ratio in experimental groups. #p,0.05, *p,0.01 when compared with the L-name group.

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Figure 3 - Histological and immunohistochemical analysis. Representative glomerular sections stained with hematoxylin/eosin (A,D,G,J; 4006), cortical sections stained with Masson’s trichrome (B,E,H,K; 206) and immunohistochemical analyses of glomerular macrophages (C,F,I,L) in each group of rats (n = 10/group). These analyses did not detect changes in the glomerular, vascular, tubular or interstitial compartments. Glomerular macrophages were most abundant in the L-name group (arrow).

and regional hemodynamics.23 At 2 mg/kg/day, rosuvastatin has been shown to significantly increase eNOS mRNA and protein expression and decrease iNOS mRNA and protein expression and nitrite production after ischemia reperfusion.30 The L-name group developed hypertension after the end of the first induction week. At the end of the experiment, there was a decrease in the serum NO level and an increase in inflammatory parameters (the serum IL-6 and TNF-a levels), macrophage infiltration into the glomeruli and proteinuria. The effects of statins on blood pressure can be dose dependent in some animal models, such as spontaneously hypertensive31 and angiotensin II–dependent rats.32 Our results did not demonstrate a dose-dependent reduction in blood pressure following treatment with rosuvastatin (2 or 20 mg/kg/day). A similar finding was reported by Susic et al.23 In rats, serum lipid levels are usually low, and statins do not change this profile. Therefore, rats are an excellent model to study the pleiotropic effects of statins.23 In our study, treatment with rosuvastatin did not alter the serum Tg, TC, ALT, AST, ALP, or Cr levels, which indicates that rosuvastatin exerted lipid lowering–independent effects. Furthermore, in our study, the serum ALT, AST, and ALP levels indicated no liver toxicity. Biochemical markers, particularly markers of vascular inflammation, such as high-sensitivity C-reactive protein (hs-CRP), have been suggested to be predictive of cardiovascular events.33 The primary proinflammatory cytokines TNF-a and IL-6 are the main inducers of hs-CRP. Furthermore, according to Navarro and Mora-Fernandez,34 TNF-a and IL-6 have been associated with significant direct renal effects.

DISCUSSION Chronic nitric oxide synthesis inhibition induced by the treatment of rats with L-NAME results in endothelial dysfunction, vascular hypertrophy, cardiac fibrosis, atherosclerosis, perivascular inflammation, renal failure, and increased vascular responses to adrenergic stimuli. Several other factors, including Renin-angiotensin system, endothelial constrictor factors, arterial remodeling and the sympathetic nervous system are involved in these effects.19,28 Additionally, this experimental model is characterized by easy handling and low animal mortality. The dose of 30 mg/kg/day of L-NAME was sufficient to induce arterial hypertension,29 and the dose of 20 mg/kg/ day of rosuvastatin used in our study is known to have beneficial effects on endothelial dysfunction and systemic

Table 3 - Glomerular macrophage infiltration and the cross-sectional glomerular area of the kidneys of animals at the end of the experiment. Group CTRL L-name L-name+ ROS-20 L-name+ ROS-2

Glomerular macrophage (%)

Cross-sectional glomerular area (mm2)

39¡3* 107¡4 56¡4*

3833¡61 3673¡64 3688¡83

45¡6*

3911¡75

The results are expressed as the mean ¡ SEM. n = 10 rats/group. The number of macrophages was analyzed in 100 random glomeruli per animal. The glomerular surface area was morphometrically analyzed in at least 50 glomeruli per animal. *p,0.0001 when compared with the L-name group.

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Rosuvastatin reduces renal inflammation in rats Girardi JM et al.

In this study, treatment with rosuvastatin (2 or 20 mg/ kg/day) reduced the serum IL-6 and TNF-a levels. Experimental and clinical studies have demonstrated the pathogenic role of TNF-a in the development of renal injury and the potential benefit of modulating TNF-a activity as a therapeutic target in diverse renal diseases.35 IL-6 has been correlated with an increased width of the glomerular basement membrane.35 It also enhances fibronectin expression, affects extracellular matrix dynamics at both the mesangial and podocyte levels, stimulates mesangial cell proliferation, and increases endothelial permeability.36,37 IL-6 plays an important role in vascular remodeling and has been reported to be a useful biomarker in predicting future cardiovascular events.38 TNF-a is cytotoxic to glomerular, mesangial and epithelial cells, and it is able to induce direct renal damage.39 Furthermore, TNF-a stimulates endothelial generation of reactive oxygen species by activating the NADPH oxidase subunits gp91 phox, NOX-1, p47phox, and p22phox. TNF-a also activates the transcription of NF-kB, which regulates the expression of genes involved in inflammation, oxidative stress and endothelial dysfunction.11,38 Animals treated simultaneously with L-NAME and rosuvastatin (2 mg/kg/day; L-name+ROS-2) had an increased serum NO level and reduced L-NAME-induced inflammatory parameters. However, treatment with 20 mg/ kg/day rosuvastatin (L-name+ROS-20) did not alter the serum NO levels compared with the L-name group. This finding could be because a lower dose (2 mg/kg/day) of rosuvastatin may increase the constitutive endothelial bioavailability of NO, which may not occur at higher doses (20 mg/kg/day). Di Napoli et al.30 evaluated the effects of rosuvastatin on reperfusion injury in rat hearts using 0.2, 2, and 20 mg/kg/day rosuvastatin. They found that 2 mg/ kg/day rosuvastatin significantly increased the mRNA expression of constitutive endothelial NOS (eNOS) compared with the untreated group and reduced the mRNA expression of inducible NOS (iNOS). These effects were more evident at 2 mg/kg/day rosuvastatin than at 0.2 mg/ kg/day rosuvastatin. However, at the highest dosage (20 mg/kg/day) there was a beneficial effects loss in the expression of eNOS and iNOS associated with increased mitochondrial damage and microcirculatory dysfunction. The current treatment of chronic nephropathy is limited to angiotensin-converting enzyme inhibitors and angiotensin AT1 receptor blockers; however, evidence indicates that statins can be effective.11 Glomerulosclerosis, glomerular ischemia, and interstitial fibrosis were observed in the kidneys of nitric oxide– deficient rats fed a high-salt diet.40 Changes in glomerular and vascular densities were also observed in this model using stereological analysis.41 The BPs achieved in the animals used in these previous studies were 212 and 200–225 mmHg, respectively. However, Lecian et al.42 found no histological abnormalities in the kidneys of rats treated with a nitric oxide synthesis inhibitor, and these animals had a BP of 175 mmHg. In our study, the BP of the hypertensive group only reached 140 mmHg, which may explain the absence of histological abnormalities. Statins inhibit several intracellular signaling pathways (Rho/RhoA and MAPK pathways) activated by angiotensin II that are involved in the regulation of profibrotic factors, connective tissue growth factor and the deposition of

extracellular matrix components, which are all modulated by redox processes.43 Our data demonstrate that there was a significant increase in the accumulation of macrophages in the glomeruli and proteinuria in L-NAME-treated rats. Rosuvastatin was effective in reducing glomerular macrophage infiltration, which is indicative of an improved inflammatory pattern; however, this effect was most evident at a dose of 2 mg/kg/day. Additionally, rosuvastatin inhibited proteinuria at both of the tested doses (2 or 20 mg/kg/day).

CONCLUSIONS In conclusion, rosuvastatin reduces the serum levels of IL6 and TNF-a, reduces glomerular macrophage infiltration and prevents proteinuria in this experimental model. All of the effects of rosuvastatin were independent of its lipidlowering effects. Future mechanistic studies will examine the molecular mechanisms involved in these effects. Our data suggest that rosuvastatin may act beneficially in preventing proteinuria and glomerular inflammation in a model of nitric oxide deficiency.

REFERENCES 1. Executive Summary of the Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). JAMA. 2001;285:2486–97. 2. Alsheikh-Ali AA, Ambrose MS, Kuvin JT, Karas RH. The Safety of Rosuvastatin as Used in Common Clinical Practice. A Postmarketing Analysis. Circulation. 2005;111:3051-7. 3. Nicholls SJ, Tuzcu EM, Sipahi I, Grasso AW, Schoenhagen P, Hu T, et al. Statins, high-density lipoprotein cholesterol, and regression of coronary atherosclerosis. JAMA. 2007;297:499–508, doi: 10.1001/jama.297.5.499. 4. Suh JW, Choi DJ, Chang HJ, Cho YS, Youn TJ, and Chae IH, et al. HMGCoA reductase inhibitor improves endothelial dysfunction in spontaneous hypertensive rats via down-regulation of caveolin-1 and activation of endothelial nitric oxide synthase. J Korean Med Sci. 2010;25:16–23, doi: 10.3346/jkms.2010.25.1.16. 5. Campese VM, Nadim MK, Epstein M. Are 3-hydroxy-3-methylglutarylCoA reductase inhibitors renoprotective? J Am Soc Nephrol. 2005;16 (suppl):S11–S17, doi: 10.1681/ASN.2004110958. 6. Gojo A, Utsunomiya K, Taniguchi K, Yokota T, Ishizawa S, Kanazawa Y, et al. The Rho-kinase inhibitor, fasudil, attenuates diabetic nephropathy in streptozotocininduced diabetic rats. Eur J Pharm. 2007;568;242–247, doi: 10.1016/j.ejphar.2007.04.011 7. Brenner BM, Garcia DL, Anderson S. Glomeruli and blood pressure. Less of one more the other? Am J Hypertens.1988;1:335-47. 8. Caetano ER, Zats R, Saldanha LB, Praxedes Jn. Hypertensive nephrosclerosis as a relevant cause of chronic renal failure. Hypertension. 2001;38:171-6. 9. Zats R, Baylis C. Chronic nitric oxide inhibition model six years on. Hypertension.1998;32:958-64. 10. Barbuto N, Almeida JR, Pereira LMM, Mandarim-de-Lacerda CA. Renal co´rtex remodeling in nitric oxide deficient rats treated with enalapril. J Cell Mol Med. 2004;8:102-8, doi: 10.1111/j.1582-4934.2004.tb00264.x. 11. Bae EH, Kim IJ, Park JW, Ma SK, Lee JU, Kim SW. Renoprotective effect of rosuvastatin in DOCA–salt hypertensive rats. Nephrol Dial Transplant. 2010;25:1051-9, doi: 10.1093/ndt/gfp604. 12. Fried LF, Orchard TJ, Kasiske BL. Effect of lipid reduction on the progression of renal disease: a meta-analysis. Kidney Int. 2001;59;260-9, doi: 10.1046/j.1523-1755.2001.00487.x 13. Wilson TW, Alonso-Galicia M, Roman RJ. Effects of lipid-lowering agents in the Dahl salt-sensitive rat. Hypertension.1998;31:225-31. 14. Park JK, Muller DN, Mervaala EM, Dechend R, Fiebeler A, Schmidt F, et al. Cerivastatin prevents angiotensin II-induced renal injury independent of blood pressure- and cholesterol-lowering effects. Kidney Int. 2000;58:1420-30, doi: 10.1046/j.1523-1755.2000.00304.x. 15. Zhou MS, Jaimes EA, Raij L. Atorvastatin prevents end-organ injury in salt-sensitive hypertension: role of eNOS and oxidant stress. Hypertension. 2004;44:186-90. 16. Li C, Yang CW, Park JH, Lim SW, Sun BK, Jung JY, et al. Pravastatin treatment attenuates interstitial inflammation and fibrosis in a rat model of chronic cyclosporine-induced nephropathy. Am J Physiol Renal Physiol. 2004; 286:46-57, doi: 10.1152/ajprenal.00428.2002.

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17. Mauro Vieira J Jr, Mantovani E, Tavares Rodrigues L, Delle H, Noronha IL, Fujihara CK, et al. Simvastatin attenuates renal inflammation, tubular transdifferentiation and interstitial fibrosis in rats with unilateral ureteral obstruction. Nephrol Dial Transplant. 2005;20:1582-91, doi: 10.1093/ndt/ gfh859. 18. Bayorh MA, Ganafa AA, Eatman D, Walton M, Feuerstein GZ. Simvastatin and losartan enhance nitric oxide and reduce oxidative stress in salt-induced hypertension, Am J Hypertens. 2005;18:1496-502. 19. Ribeiro MO, Antunes E, Nucci G, Lovisolo SM, Zatz R. Chronic inhibition of nitric oxide synthesis. A new model of arterial hypertension, Hypertension. 1992;20:298–303. 20. Rossi MA, Colombini-Netto M. Chronic inhibition of NO synthesis per se promotes structural intimal remodeling of the rat aorta. J Hypert.2001;19:1567–79, doi: 10.1097/00004872-200109000-00008. 21. Jover B, Mimran A. Nitric oxide inhibition and renal alterations. J Cardiovasc Pharmacol. 2001;38:65-70, doi: 10.1097/00005344200111002-00016. 22. Fujioka T, Nara F, Tsujita Y, Fukushige J, Fukami M, Kuroda M. The mechanism of lack of hypocholesterolemic effects of pravastatin sodium, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, in rats. Biochim Biophys Acta. 1995;1254:7–12. 23. Susic D, Varagic J, Ahn J, Slama M, Frohlich ED. Beneficial pleiotropic vascular effects of rosuvastatin in two hypertensive models. J Am Coll Cardiol. 2003;42:1091–7, doi: 10.1016/S0735-1097(03)00926-4. 24. Green LC, Wagner DA, Glogwski J, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Anal Biochem. 1982;126:131-8, doi: 10.1016/00032697(82)90118-X. 25. Tu X, Chen X, Xie Y, Shi S, Wang J, Chen Y, Li J. Anti-inflammatory Renoprotective Effect of Clopidogrel and Irbesartan in Chronic Renal Injury. J Am Soc Nephrol. 2008;19:77–83, doi: 10.1681/ASN.2007020160. 26. Hartner A, Veelken R, Wittmann M, Cordasic N. Effects os diabetes and hypertension on macrophage infiltration and matrix expansion in the rat kidney. BMC Nephrology.2005;6:6, doi: 10.1186/1471-2369-6-6. 27. Pomaro D, Fonseca FAH, Ebihara Fet al. 2006. Quinapril promotes renal protection independently of glucose serum levels in hypercholesterolemic and diabetic rabbits. Int J Atheroscler. 2006;1:156-61. 28. Paulis L, Zicha J, Kunes J, Hojna S, Behuliak M, Celec P, et al. Regression of L-NAME-induced hypertension: the role of nitric oxide and endothelium-derived constricting factor. Hypertens Res. 2008;31:793– 803, doi: 10.1291/hypres.31.793. 29. Morań A, Urbina AVO, Martıń ML, Rodrı´guez-Barbero A, Romań LS. Characterization of the contractile 5-hydroxytryptamine receptor in the autoperfused kidney of L-NAME hypertensive rats. Eur J Pharmacol. 2009;620:90-6, doi: 10.1016/j.ejphar.2009.08.026. 30. Di Napoli P, Taccardi AA, Grilli A, De Lutiis MA, Barsotti A, Felaco M, et al. Chronic treatment with rosuvastatin modulates nitric oxide synthase expression and reduces ischemia-reperfusion injury in rat

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hearts. Cardiovasc. Research. 2005;66:462-71, doi: 10.1016/j.cardiores. 2005.02.008. Zhai Y, Gao X, Wu Q. Fluvastatin decreases cardiac fibrosis possibly through regulation of TGF-beta(1)/Smad 7 expression in the spontaneously hypertensive rats. Eur J Pharmacol. 2008;587:196–203, doi: 10. 1016/j.ejphar.2008.03.006. Delbosc S, Cristol JP, Descomps B, Mimran A, Jover B. Simvastatin prevents angiotensin II-induced cardiac alteration and oxidative stress. Hypertension. 2002;40:142–47, doi: 10.1161/01.HYP.0000024348.87637.6F. Ridker PM, Danielson E, Fonseca FA, Genest J, Gotto AM Jr, Kastelein JJ, et al. JUPITER Study Group. N Engl J Med. 2008;359:2195-207, doi: 10. 1056/NEJMoa0807646. Navarro JF, Mora-Fernandez C. The role of TNF-a in diabetic nephropathy: Pathogenic and therapeutic implications Cytokine & Growth Factor Reviews. 2006;17:441–50. Bourraindeloup M, Christophe A, Candiani G, Calleret M, Bourin MC, Badoual T, et al. N-Acetylcysteine Treatment Normalizes Serum Tumor Necrosis Factor-a Level and Hinders the Progression of Cardiac Injury in Hypertensive Rats. Circulation. 2004;110:2003-9, doi: 10.1161/01.CIR. 0000143630.14515.7C. Dalla Vestra M, Mussap M, Gallina P, Bruseghin M, Cernigoi A, Saller A, et al. Acute-phase markers of inflammation and glomerular structure in patients with type 2 diabetes. J Am Soc Nephrol. 2005;16(Suppl 1): S78–82, doi: 10.1681/ASN.2004110961. Hirano T, Akira S, Taga T, Kishimoto T. Biological and clinical aspect of interleukin 6. Immunol Today. 1990;11:443–9, doi: 10.1016/01675699(90)90173-7. Dela Paz NG, Simeonidis S, Leo C, Rose DW, Collins T. Regulation of NF-kB-dependent gene expression by the POU domain transcription factor Oct-1. J Biol Chem. 2007;282:8424–34, doi: 10.1074/jbc.M606923200. Baud L, Ardaillou R. Tumor necrosis factor in renal injury. Miner Electrolyte Metab.1995;21:336–41. Vieira Jr. JM, Rodrigues LT, Mantovani E, Delleˆ H, Mattar AL, Malheiros DMACet al. Statin Monotherapy Attenuates Renal Injury in a SaltSensitive Hypertension Model of Renal Disease. Nephron Physiol. 2005;101:82–91, doi: 10.1159/000087576. Pereira LMM, Almeida JR, Mandarim-de-Lacerda CA. Kidney adaptation in nitric oxide-deficient Wistar and spontaneously hypertensive rats. Life Sciences.2004;74:1375-86, doi: 10.1016/j.lfs.2003.08.015. Lecian D, Demova H, Lodererova A, Zdychova J, Kluckova H, Teplan V, et al. Renal effects of HMG-CoA Reductase Inhibition in a rat modelo f chronic inhibition of nitric oxide synthesis. Kidney Blood Press Res. 2006;29:135-43, doi: 10.1159/000094988. Ruperez M, Rodrigues-Diez R, Blanco-Colio LM, Sanchez-Lopez E, Rodriguez-Vita J, Esteban V, et al. HMG-CoA reductase inhibitors decrease angiotensin II-induced vascular fibrosis: role of RhoA/ROCK and MAPK pathways. Hypertension. 2007;50:377–83, doi: 10.1161/ HYPERTENSIONAHA.107.091264.

CLINICS 2011;66(8):1463-1477

DOI:10.1590/S1807-59322011000800026

REVIEW

Surgical treatment of male infertility in the era of intracytoplasmic sperm injection – new insights Sandro C. Esteves,I Ricardo Miyaoka,I Ashok AgarwalII I ANDROFERT – Andrology & Human Reproduction Clinic, Campinas, Sa˜o Paulo, Brazil. Foundation, Cleveland, Ohio, USA.

Center for Reproductive Medicine, The Cleveland Clinic

Assisted reproductive technology is an evolving area, and several adjuvant procedures have been created to increase a couple’s chance of conceiving. For male infertility, the current challenges are to properly accommodate old and new techniques that are both cost-effective and evidence-based. In this context, urologists are expected to diagnose, counsel, provide medical or surgical treatment whenever possible and/or correctly refer male patients for assisted conception. Urologists are sometimes part of a multiprofessional team in an assisted reproduction unit and are responsible for the above-cited tasks as well as the surgical retrieval of sperm from either the epididymides or testicles. We present a comprehensive review of the surgical treatment options for infertile males, including the perioperative planning and prognostic aspects, with an emphasis on the role of microsurgery in the optimization of treatment results. This review also discusses current techniques for sperm retrieval that are used in association with assisted reproductive technology and includes sperm retrieval success rates according to the technique and the type of azoospermia. New insights are provided with regard to each surgical treatment option in view of the availability of assisted conception to overcome male infertility. KEYWORDS: Male; Infertility; Surgery; Sperm retrieval; Review. Esteves SC, Miyaoka R, Agarwal A. Surgical treatment of male infertility in the era of intracytoplasmic sperm injection – new insights. Clinics. 2011;66(8):1463-1477. Received for publication on April 9, 2011; First review completed on April 15, 2011; Accepted for publication on April 19, 2011 E-mail: s.esteves@androfert.com.br Tel.: 55 19 3295-8877

pregnancy.2,3 Several sperm retrieval methods were developed to collect epididymal and testicular sperm for ICSI in azoospermic men. Microsurgery facilitated sperm collection from the epididymides in men with obstructive azoospermia (OA) and from the testicles in men with nonobstructive azoospermia (NOA).2,4 As for all surgical reconstructive procedures discussed in this chapter, an evaluation of the female partner’s reproductive potential is recommended before an intervention is indicated, and options should be individually discussed with each couple.

INTRODUCTION Infertility is a common complaint at urologists’ offices and affects approximately 8% of reproductive-age men. Among these men, 1-10% present with conditions that affect their reproductive potential.1 In a group of 2,875 infertile couples who attended the first author’s tertiary center for male reproduction, conditions that could potentially be corrected with surgical procedures were identified in approximately one third of the male partners. Azoospermic males who were not candidates for surgery or who remained azoospermic after surgical reconstruction could benefit from sperm retrieval techniques and assisted conception. Thus, surgical management may be offered to more than 50% of the infertile male patient population. Two major advances have occurred in the area of male infertility surgery. One was the development of microsurgery, which increased success rates for the reconstruction of the reproductive tract with minimal morbidity. The second major advancement was the development of intracytoplasmic sperm injection (ICSI) and the demonstration that spermatozoa retrieved from either the epididymis or the testis were capable of fertilizing an egg and leading to

WHAT ARE THE AVAILABLE SURGICAL TREATMENTS? Varicocele repair Varicocele can be diagnosed in up to 35% of infertile men.1 It is currently recommended that treatment should be offered to couples with documented infertility whose male partner has a clinically palpable varicocele associated with an abnormal semen analysis. The preferred diagnostic method is a physical examination with the patient standing in a warm room.5 In the case of bilateral palpable varicocele, studies have recommended that both sides be corrected during the same procedure.6 Imaging studies should be performed when physical examination is inconclusive. When a varicocele is not palpable but a retrograde blood flow is detected by other diagnostic methods (such as venography, Doppler examination, ultrasonography, scintigraphy or thermography), the varicocele is termed subclinical.7,8 Current evidence

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identified. External spermatic veins running parallel to the spermatic cord or perforating the floor of the inguinal canal can also be identified and ligated. The use of magnification facilitates the identification and preservation of internal spermatic arteries and lymphatics; the preservation of these structures may prevent testicular atrophy and hydrocele formation, respectively.17 Microsurgical varicocelectomy can be performed via an inguinal or subinguinal approach using a testicular artery and lymphatic-sparing subinguinal microsurgical repair (Figure 1).11,18 The latter offers the advantage of sparing the aponeurosis of the external oblique muscle, which may result in less postoperative pain and a shorter time before the patient can return to work. The operation is performed under the microscope with magnification ranging from 6-16X. All dilated veins of the spermatic cord are identified, tagged, and ligated.

does not support treating infertile men who have subclinical varicocele.9,10

Preoperative planning A preoperative hormone profile that includes an analysis of follicle-stimulating hormone (FSH) and testosterone, a testicular volume assessment using a measurement instrument such as the Prader orchidometer or a pachimeter, and the results of at least two semen analyses should be obtained.11 Men with clinical varicoceles presenting with azoospermia may be candidates for surgical repair, but genetic evaluation including Giemsa karyotyping and polymerase chain reaction screening for Yq microdeletion of the AZFa, AZFb, and AZFc regions is recommended. A testis biopsy (open or percutaneous) provides testicular histology, which has been shown to be the only significant prognostic factor for the restoration of spermatogenesis in azoospermic individuals with varicocele.11,12 The benefit of varicocelectomy in azoospermic men with genetic abnormalities is doubtful and the procedure should be carefully considered. The same caution must be used for patients with atrophic testes and/or a history of cryptorchidism, testicular trauma, orchitis, or systemic or hormonal dysfunction; varicocele may be coincidental in these cases rather than the cause of infertility.13

Postoperative care Local dressing and scrotal support are maintained for 4872 h and one week, respectively. Scrotal ice packing is always recommended to control local edema for the first 48 h. Patients are counseled to refrain from physical activity and sexual intercourse for 2-3 weeks. Oral analgesics usually suffice to control postoperative pain. Follow-up aims to evaluate improvement in semen parameters, complications and spontaneous or assisted conception. Semen analysis should be performed every three months until the semen parameters stabilize or pregnancy occurs.

Operative aspects Varicocele repair may be carried out using local, regional, or general anesthesia (the choice is solely dependent on the surgeonâ&#x20AC;&#x2122;s preferences). We perform varicocele repair on an outpatient basis using short-acting intravenous propofol anesthesia along with anesthesia of the spermatic cord using lidocaine hydrochloride.11 Varicocelectomy can be performed through either open (with or without magnification) or laparoscopic approaches. The main concern is the occlusion of the dilated veins of the pampiniform plexus. The high retroperitoneal and laparoscopic approaches are used for internal spermatic vein ligation, whereas the inguinal and subinguinal approaches allow for the ligation of the internal and external spermatic and cremasteric veins that may contribute to the varicocele. High retroperitoneal open varicocele ligation is performed through an incision medial to the anterior superior iliac spine at the level of the internal inguinal ring. Exposure of the internal spermatic vessels is carried out retroperitoneally near the ureter. At this level, the internal spermatic artery may not be easy to identify. In addition, neither the parallel inguinal and retroperitoneal collateral veins that may bypass the retroperitoneal area of ligation nor the cremasteric veins can be identified. This may explain the high recurrence rate seen in retroperitoneal varicocelectomy. Laparoscopic varicocelectomy is similar to the retroperitoneal procedure, but it uses high magnification. The spermatic artery and the lymphatics are easily identified and spared. In addition, collateral veins can be clipped or coagulated. External spermatic veins, however, which are the second cause of varicocele recurrence, cannot be treated (this leads to a recurrence close to 5%).14 Laparoscopic varicocele repair is more invasive, costly and associated with higher complication rates compared with open procedures.14-16 The classical approach to the inguinal varicocelectomy involves an incision over the inguinal canal, opening of the external oblique aponeurosis and isolation of the spermatic cord. The internal spermatic veins are dissected and ligated. Testicular arteries and the lymphatics should be actively

RECONSTRUCTIVE SURGERY OF THE VAS DEFERENS AND EPIDIDYMIS Vasovasostomy (VV) and vasoepididymostomy (VE) are designed to bypass an obstruction in the male genital tract. The number of men seeking vasectomy reversal due to changes in marital status or reproductive goals has increased and varies from 2-6%.19 In Brazil, it is estimated that 200,000 vasectomies and 7,000 reversals are performed each year.20 In addition to vasectomy, epididymal or vasal obstructions may be secondary to genital infections or iatrogenic injuries related to inguinal or scrotal surgery (especially during the early childhood years).21

Preoperative planning A detailed medical history must be taken, and prognostic factors should be identified. Obstruction intervals after vasectomy play a major role in determining surgical outcomes. Vasoepididymostomy is more likely to be required after long-interval obstructions because they are associated with a higher incidence of epididymal obstruction. A computer model based on obstructive interval and patient age was created to determine the need for VE.22 Free shareware versions are available at www.uroengineering. com. A history of a previous vasectomy reversal attempt does not preclude a new attempt. Patency and pregnancy rates of 79% and 31%, respectively, have been reported for repeated reversals.23 Small and soft testes may indicate impaired spermatogenesis. Indurate, irregular epididymides, and the presence of hydrocele are often associated with epididymal obstruction and may indicate the need for VE. The presence of a granuloma in the vas deferens should be interpreted as a favorable prognostic sign because this means that sperm have leaked from the vasectomy site (i.e., this prevents too much

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Figure 1 - Microsurgical Subinguinal Varicocele Repair. A) A transverse incision is made just below the level of the external inguinal ring. B-D) Intraoperative photographs of the spermatic cord. B) Dilated cremasteric veins are identified by elevating the spermatic cord with a Babcock clamp. C) Testicular artery (blue vessel loop), lymphatics (blue cotton suture), and dilated varicose veins (red vessel loops) are demonstrated. D) Final surgical aspects of the varicose veins are transected and ligated with nonabsorbable sutures.

pressure from building up within the epididymides tubules, which can lead to rupture).19,24,25 Specific laboratory tests are not necessary before reconstructive surgeries. Serum FSH is only recommended as a marker of testicular reserve if testicular damage is suspected on physical examination. The usefulness of antibody testing remains controversial, and

evidence suggests that late failures following reversals are likely to be technical rather than immunological.25,26

Operative Aspects Vasovasostomy and vasoepididymostomy may be performed using local, regional, or general anesthesia. We

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the anastomosis, a dilated epididymal tubule must be identified immediately above the level of obstruction. The tubule must be opened, and the fluid should be inspected for the presence of motile sperm. If no sperm are identified, a more proximal site of the epididymis will be required for the anastomosis. Microsurgical techniques are clearly superior than macrosurgical or loupe-assisted anastomoses.19,36 The likelihood of sperm appearance in the semen and of pregnancy after microsurgical vasectomy reversal are inversely related to the duration of time since the vasectomy.19 Other factors that are related to success rates include the gross appearance of vas fluid at the time of surgery, the presence or absence of sperm in the vas fluid (and sperm quality), the length of the remaining segment adjacent to the epididymis, the age of the female partner, and the experience of the microsurgeon.

perform VV and VE on an outpatient basis using continuous intravenous propofol anesthesia along with spermatic cord anesthesia with a lidocaine hydrochloride solution. A longitudinal scrotal incision is made in the anterior aspect of the scrotum on each side. The incision is made onto the palpable granuloma or onto the identified vasal gap, and only the vas ends are delivered through the skin incision. In cases where the vasectomy was performed high in the scrotum or a large segment was removed or repeat reconstructions were performed with difficult vasal mobilization, the incision may be extended to the inguinal region. Microsurgical dissection is carried out in the region of the prior vasectomy site to free the vas and its vascular pedicle from the surrounding scar tissue. Hemostasis is obtained with great care using either bipolar or hand-held thermal cautery units. After the vas has been mobilized and its scarred ends excised, patency of the abdominal vas end is confirmed with the introduction of a 24-gauge blunt tipped angiocatheter into the lumen and the injection of sterile saline. Fluid from the testicular vas end is examined for the presence of sperm. Copious, clear, watery or cloudy fluid and motile sperm has been shown to be associated with an excellent patency rate of 94%, which is compared with 60% when no sperm is found in the vasal fluid.19 Thick, toothpaste-like vasal fluid is suggestive of epididymal obstruction.19,27 The most important factors in determining the type of reconstructive technique are the quality of sperm found in the intravasal fluid and the surgeonâ&#x20AC;&#x2122;s microsurgical skills. Indeed, attention to surgical details directly affects the success of reconstructive microsurgeries. Important surgical details include the accurate mucosa-to-mucosa approximation, a water-tight tension-free anastomosis, preservation of the vasal blood supply and healthy tissue (mucosa and muscularis), and an adequate microscopic atraumatic technique. Several techniques have been described for VV: (i) the modified one-layer technique, which has been described by Sharlip,28 (ii) the two-layer technique, which has been described by Belker,29 and (iii) the multilayer microdot technique, which was originally described by Goldstein.30 Recent reports have shown that it is possible to perform classical techniques using robotic assistance. Robots can offer the benefits of enhanced imaging (up to 100X magnification) and control of tremor.31,32 Vasoepididymostomy is a challenging surgical procedure that should only be attempted by experienced microsurgeons. Meticulous microsurgical technique and high magnification are required for a precise anastomosis of the vas (luminal diameter of 300-400 mm) to the epididymal tubule (150-250 mm). Intraoperative sperm harvesting and cryopreservation can be offered during VE.33 The VE procedure starts with the placement of a longitudinal incision in the upper scrotum. The testis is delivered, and the testis and epididymis are thoroughly inspected. The site of obstruction can often be grossly visible as an area where the epididymis transitions from a firm, wide caliber to a smaller, softer structure. The distal end of the vas deferens is mobilized in a similar fashion to the VV procedure, but a longer length is generally required to perform an epididymal anastomosis. Anastomosis is performed under an operating microscope. Currently, three variations of the technique have been used for precise approximation of the vas deferens lumen to a single epididymal tubule: end-to-end, end-to-side, and endto-side intussusception techniques (Figure 2).34,35 Prior to

Postoperative care Local dressing and ice packing are maintained for 4872 h, and a scrotal supporter should be used for two weeks. Patients are counseled to restrain from physical activity and sexual intercourse for one and two months in cases of VV and VE, respectively. Oral analgesics usually suffice to control postoperative pain. Postoperative follow-up should evaluate improvement in semen parameters, complications and spontaneous or assisted conception. Semen analysis should be performed every two months after surgery until the semen parameters stabilize or pregnancy occurs.

TRANSURETHRAL RESECTION OF THE EJACULATORY DUCT (TURED) Ejaculatory duct obstruction (EDO) is a cause of male infertility that can potentially be corrected by surgery. Congenital obstructions are caused by atresia or stenosis of the ejaculatory ducts as well as utricular, mullerian, and wolffian duct cysts. Acquired obstructions may be secondary to trauma or infection/inflammation. Traumatic damage to the ejaculatory ducts may occur after the removal of seminal vesicle cysts, pull-through operations for an imperforate anus or even prolonged urethral catheterization or instrumentation. Genital or urinary infection and prostatic abscess may cause stenosis or complete obstruction of the ducts.37

Preoperative planning Diagnostic assessment should include history, physical examination, semen analyses, and transrectal ultrasound. The clinical presentation may be highly variable. Indeed, in addition to a history of infertility, complaints may include painful ejaculation, hemospermia, and perineal and/or testicular pain; however, some patients may be completely asymptomatic. Unaltered physical examination is the rule. Occasionally, the seminal vesicles or a mass are palpable on rectal examination. Prostatic tenderness and/or epididymal enlargement may also exist. Hormone profiles are generally normal. Semen analyses may reveal oligozoospermia or azoospermia, decreased motility, and decreased ejaculate volume. The presence of a low volume (,1.5 mL) of acidic (pH,7.0) azoospermic ejaculate with absent fructose, palpable vasa, and epididymal thickening is virtually pathognomonic. The typical clinical picture, however, may be complicated because obstruction could be unilateral, partial, or functional.37 Postejaculate urinalyses are often performed

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Figure 2 - Microsurgical Vasoepididymostomy Techniques. A) Illustration of the end-to-end, B) end-to-side, and C) triangulation end-toside anastomoses.

to exclude retrograde ejaculation in patients with lowvolume ejaculates. High-resolution transrectal ultrasound evaluation (TRUS) using with a 5-7 MHz biplanar transducer is recommended in all cases of suspected EDO. The precise identification of obstruction on TRUS, however, is still a matter of debate because of marked variability in the size and shape of the vas deferens, seminal vesicles, and ejaculatory ducts in both fertile and infertile men. Common ultrasound findings include dilation of the seminal vesicles (defined as a cross-sectional width greater than 1.5 cm) or ejaculatory ducts (defined as an internal duct diameter greater than 2.0 mm), calcifications or calculi in the region of the ejaculatory duct or verumontanum and midline or eccentrically located prostatic cysts.38-40 Ultrasound-guided transrectal seminal vesiculography has been shown to provide excellent radiographic visualization of the ejaculatory ducts.41 In addition, TRUS-guided seminal vesicle aspiration revealing the presence of motile sperm in the aspirates seem to be an useful diagnostic tool because the seminal vesicles are not sperm reservoirs.42 Moreover, a testicular biopsy can be performed to document the presence of normal spermatogenesis. Our preference is to perform a ‘‘wet prep’’ using the percutaneous testicular sperm aspiration technique either before or at the time of surgery; the presence of motile sperm is highly indicative of ductal obstruction.

initially documented using intraoperative vasotomy and vasography. The vas is delivered using a small scrotal incision and dissected free of the associated perivasal vessels. A mixture of injectable saline, radiographic contrast material, and methylene blue dye is injected into the abdominal end of the vas by direct vas puncture with a 30-gauge lymphangiogram needle.37 Vasography is used to confirm the obstruction, and dye injection is used to confirm patency by visualization of the effluxing dye mixture during TURED. A suture is placed at the muscular layer of the vas to close the vasotomy site. Transurethral resection of the ejaculatory ducts is performed with the patient in the dorsal lithotomy position. A strip of tissue is removed from the floor of the prostate just proximal to and including a portion of the verumontanum. The ducts are confirmed to be properly opened by visualizing their dilated portion and the dye efflux.

Operative aspects

WHAT ARE THE AVAILABLE SPERM RETRIEVAL TECHNIQUES?

Postoperative care An indwelling catheter is kept in place for 24-48 h, and patients are discharged the following day. Oral quinolone antibiotics and anti-inflammatory medication are prescribed for five days. In addition, scrotal support is recommended for one week to avoid vasotomy-induced scrotal edema. Frequent ejaculation is recommended 3-4 weeks postoperatively, and patients are monitored with monthly semen analyses.

Transurethral resection of the ejaculatory ducts (TURED) is performed using regional or general anesthesia according to the procedure that was originally described by Farley and Barnes,43 with minor modifications.37 The obstruction is

Azoospermia, which is defined as the absence of spermatozoa in the ejaculate after centrifugation, is found in 1-3% of

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CFTR gene mutation is identified (approximately 4% of female partners are carriers), testing should also be offered to the male, and counseling is recommended before proceeding with sperm retrieval and ICSI because of the risk of the transmission of cystic fibrosis to the offspring.51,52 Azoospermic men with idiopathic obstruction and men with a clinical triad of chronic sinusitis, bronchiectasis, and obstructive azoospermia (Youngâ&#x20AC;&#x2122;s syndrome) may also be at higher risk for CF gene mutations. Serum FSH is a critical factor in determining whether a diagnostic testicular biopsy is needed to differentiate the type of azoospermia in men with normal semen volume. Elevated FSH and small testicles are indicative of testicular failure (i.e., NOA); therefore, a testicular biopsy is not necessary for diagnostic purposes.51 If sperm retrieval with ICSI is being considered, however, a biopsy may be performed for prognostic purposes because histology correlates with the odds of finding sperm by aspiration or dissection. The absence of sperm in a biopsy specimen taken from a man with NOA, however, does not absolutely predict whether sperm are present elsewhere within the testicle.4,52 Conversely, men with normal levels of FSH and semen volume may have either NOA or OA.53 In such cases, no noninvasive method can differentiate between the two forms, and a testicular biopsy is usually required to provide a definitive diagnosis. Testicular biopsy can be performed by a standard open-incision technique or by percutaneous methods. Histological evaluation of testicular specimens may indicate the presence of normal spermatogenesis in cases of OA, whereas hypospermatogenesis (HS), maturation arrest (MA), or Sertoli-cell-only (SCO) syndrome are seen in cases of NOA. All men with primary testicular failure of unknown origin should be offered karyotyping and Yq microdeletion testing. The frequency of karyotypic abnormalities is reported to be 10-15% in men with NOA, and Klinefelter syndrome accounts for approximately two-thirds of cases.54 Genetic testing may provide prognostic information for sperm retrieval.50 While sperm can be found within the testes of approximately 70% of patients with partial or complete AZFc deletion, it is unlikely that sperm will be found in men with complete AZFa or AZFb deletions.55,56 Genetic counseling should be offered whenever a genetic abnormality is detected in the male prior to performing ICSI with his sperm. Sperm retrieval from the epididymis is only indicated in obstructive cases. Testicular sperm retrieval can be performed in either OA or NOA cases. In OA, testicular retrievals are carried out after failed epididymal retrieval or as a primary retrieval procedure in cases of absent epididymis or intense epididymal fibrosis. In NOA, testicular sperm retrievals are the only option for the collection of sperm.

the male population and approximately 10% of infertile males.42 In obstructive azoospermia (OA), spermatogenesis is normal, but a mechanical blockage in the genital tract, somewhere between the epididymis and the ejaculatory duct, or the absence of the epididymis and vas deferens prevents spermatozoa from being expelled in semen. Acquired OA may be secondary to vasectomy or result from a failed vasectomy reversal, postinfectious diseases, surgical procedures in the scrotal, inguinal, pelvic or abdominal regions, or trauma. Congenital causes of OA include cystic fibrosis, congenital absence of the vas deferens (CAVD), ejaculatory duct or prostatic cysts, and Youngâ&#x20AC;&#x2122;s syndrome.42 Nonobstructive azoospermia (NOA) comprises a spectrum of testicular histopathology resulting from various causes, such as environmental toxins, medications, cryptorchidism, genetic and congenital abnormalities, varicocele, trauma, viral orchitis, endocrine disorders, or idiopathic causes. In both OA and NOA, pregnancy may be achieved through in vitro fertilization (IVF) associated with ICSI.2,3 Several surgical methods have been developed to retrieve epididymal and testicular sperm from azoospermic men. Either percutaneous epididymal sperm aspiration (PESA)44 or microsurgical epididymal sperm aspiration (MESA)2 can be successfully used to retrieve sperm from the epididymides in men with obstructive azoospermia. In addition, testicular sperm aspiration (TESA) can be used to retrieve sperm from the testes in men with OA who fail PESA and in men with NOA.44 Moreover, testicular sperm extraction (TESE) using single or multiple open biopsies45,46 and, more recently, microsurgery (micro-TESE) are indicated for men with NOA.4,46-49 The goals of surgical sperm retrieval are (i) to retrieve an adequate number of sperm for both immediate use and for cryopreservation, (ii) to obtain the best quality sperm possible, and (iii) to minimize damage to the reproductive tract to avoid jeopardizing future sperm retrieval attempts or testicular function. Sperm can be easily obtained from infertile men with OA, whereas individuals exhibiting NOA have historically been the most difficult to treat.

Preoperative Planning It is important to distinguish whether the lack of sperm in the ejaculate is from an obstructive or nonobstructive cause because the choice of the retrieval method is based on the type of azoospermia. History, physical examination, and hormonal analysis (FSH and testosterone) provide about a 90% ability to predict whether the cause is obstructive or nonobstructive.50 Men with OA usually have normal testes and a normal hormone profile. Occasionally, the epididymides or the seminal vesicles may be enlarged or a cyst may be palpable on rectal examination. The presence of a low volume (,1.5 ml) of acidic (pH,7.0) azoospermic ejaculate with absent or low fructose and epididymal thickening is pathognomonic of OA due to either congenital bilateral absence of the vas deferens or EDO (the differential diagnosis would be the presence of the vas in the latter). Approximately two thirds of men with CAVD have mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Failure to identify a CFTR abnormality in a man with CAVD does not rule out the presence of a mutation because some mutations are undetectable by routine testing methods. The female partner should be offered cystic fibrosis (CF) testing before proceeding with treatments that utilize the sperm from men with CAVD because of the high risk of the male being a CF carrier. If a

Operative aspects Sperm retrieval techniques may be performed using local, regional, or general anesthesia. Percutaneous sperm retrievals by needle aspiration are easily repeatable and less expensive than open macro- or microsurgical techniques. Moreover, the procedures do not require microsurgery training and causes less postoperative pain. Microsurgical techniques, however, are associated with better quality and higher numbers of sperm retrieved per attempt, which

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(at least three at each testicular pole) are performed. Testicular tissue specimens are placed in an outerwell dish containing sperm media. Specimens are washed grossly to remove blood clots, and they are sent to the laboratory for processing and to search for sperm. The albuginea and scrotal layers are closed using nonabsorbable and absorbable sutures, respectively. Conventional TESE, using single or multiple open biopsies, can also be used to obtain sperm in cases of both OA and NOA, but TESE is primarily used in cases of NOA. TESE can also be used as a diagnostic tool to obtain testicular parenchyma for histological analysis and to search for sperm prior to the ICSI cycle. Conventional TESE is carried out without magnification. A small self-retaining eyelid retractor is used to improve exposure of the tunica albuginea and facilitate incision. Gentle pressure is applied to the testis to extrude testicular parenchyma out of the small incision and assist in its removal. TESE can be repeated in a different testicular pole if the multiple biopsies approach is selected. The amount of testicular parenchyma removed in micro-TESE is low compared with conventional TESE; this low removal is particularly important because it helps preserve testicular androgen production in men with NOA who already have compromised testes.

optimize the opportunity to cryopreserve sperm for future ICSI procedures. Typically, PESA is performed on an outpatient basis using a needle attached to a syringe. The main goal is to aspirate epididymal fluid for diagnostic or therapeutic purposes. Loupe magnification may be used to avoid injuring small vessels in the scrotal skin. Negative pressure is created, and the tip of the needle is gently moved in and out of the epididymis until fluid enters the syringe. The amount of epididymal fluid obtained during aspiration is often minimal (,0.1 mL), except in cases of CAVD, when 0.31.0 ml may be aspirated. After the needle is withdrawn from the epididymis, the aspirate is flushed into sperm medium. The tube containing the epididymal aspirate is transferred to the laboratory for microscopic examination. PESA is repeated at a different site of the same epididymis (from the cauda up to the caput) and/or at the contralateral epididymis until an adequate number of motile sperm are retrieved. If PESA fails to retrieve motile sperm for ICSI, TESA is performed at the same operative time. For TESA, the epididymis is stabilized between the index finger, thumb, and forefinger while the anterior scrotal skin is stretched. A needle is inserted through the stretched scrotal skin into the anteromedial or anterolateral portion of the superior testicular pole in an oblique angle towards the medium and lower poles. When a small piece of testicular tissue is aspirated, the needle is gently withdrawn from the testis while the negative pressure is maintained. The specimen is flushed into a tube containing 0.5-1.0 ml warm sperm medium and transferred to the laboratory for microscopic examination. TESA or TESE may be performed at the contralateral testis if insufficient or no sperm are obtained. Microsurgical sperm retrieval allows direct visualization of epididymal and seminiferous tubules with high magnification. These techniques have been associated with the retrieval of higher sperm numbers and better quality sperm in MESA and higher retrieval success rates in micro-TESE. Importantly, MESA does not compromise the success of future reconstructive procedures because the damage to the epididymal tubule is minimal. In addition, sperm cryopreservation is feasible in most MESA cases. In MESA, fluid exuding from the tubule is aspirated with a silicone tube or blunted needle attached to a 1-ml tuberculin syringe (Figure 3). The aspirate is flushed into 0.5-1.0 ml of sperm medium at 37 Ë&#x161;C, and the tube containing the epididymal aspirate is transferred to the laboratory for microscopic examination. MESA is repeated at a different site of the same epididymis (from the cauda up to the caput) and/or at the contralateral epididymis until an adequate number of motile sperm are retrieved. If MESA fails to retrieve motile sperm, TESA or TESE may be performed at the same operative time. For testicular sperm extraction (micro-TESE), a single large, mid-portion incision is made in the tunica albuginea to expose the testicular parenchyma (Figure 3). Dissection of the testicular parenchyma is carried out at 16-25X magnification, and the surgeon should search for enlarged seminiferous tubules. The superficial and deep testicular regions may be examined if necessary, and microsurgical-guided testicular biopsies are performed by removing enlarged tubules, which are more likely to harbor active spermatogenesis (Figure 3). If enlarged tubules are not observed, then any tubule that is a different size than the rest of the tubules should be excised. If all tubules are identical in appearance, random microbiopsies

Postoperative care Patients are discharged on the same day and can return to normal activities one and three days after percutaneous and open techniques, respectively. Scrotal ice packing and support is recommended to control edema and alleviate pain. Patients should refrain from ejaculation and strenuous physical activity for approximately 7-10 days. In addition, oral analgesics are generally prescribed.

NEW INSIGHTS INTO THE SURGICAL TREATMENT OF MALE INFERTILITY Varicocele Repair In a recent systematic review comparing surgical modalities used to treat varicocele,14 open microsurgical inguinal or subinguinal varicocelectomy techniques resulted in higher spontaneous pregnancy rates and fewer recurrences and postoperative complications than laparoscopic, radiologic embolization, and macroscopic inguinal or retroperitoneal techniques. A study by Cayan showed that hydrocele formation was the most common complication of varicocelectomy, and the incidence ranged from 0-10%. Recurrences were reported in the range of 0%â&#x20AC;&#x201C;35%. Overall recurrence rates were lower for microsurgical varicocelectomy and higher for retroperitoneal and macrosurgical inguinal approaches.14 The rate of accidental testicular artery ligation during microsurgical varicocelectomy has been reported to be about 1%, and this may cause testicular atrophy (Table 1). Recently, a study demonstrated that the concomitant use of intraoperative vascular Doppler monitoring during microsurgical varicocelectomy allows for more arterial branches to be preserved, and more internal spermatic veins are likely to be ligated.57 Varicocelectomy studies have reported significant improvements in one or more semen parameters in approximately 65% of men.58 Higher preoperative semen parameters or varicocele repair for large varicoceles are more likely to show postoperative improvements in semen parameters.58,59 The mean time for semen improvement and spontaneous pregnancy after surgery is approximately five and seven

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Figure 3 - Microsurgical Sperm Retrieval Techniques. Operating microscope and microsurgical techniques are used throughout the procedures. The top image shows microsurgical epididymal sperm aspiration (MESA). After exposure of the testis and epididymis, a dilated epididymal tubule is dissected and opened. Fluid is aspirated, diluted with sperm medium and sent to the laboratory for examination. The bottom images show microsurgical testicular sperm extraction (micro-TESE). A) After the testicle is exteriorized, a single and large incision is made in an avascular area of the albuginea to expose the seminiferous tubules. B) Dilated tubules are identified and removed with microforceps (intraoperative photograph at 40X magnification). C) Illustration of the histopathology cross-section of a dilated seminiferous tubule with active spermatogenesis. D) Illustration of the histopathology cross-section of a thin tubule with Sertoli-cell-only syndrome.

months, respectively.60 Studies have also reported that the surgical treatment of clinical varicoceles is highly effective in decreasing seminal oxidative stress and increasing seminal concentrations of antioxidants.61,62 Conversely, reduced preoperative testicular volume, elevated serum FSH levels, diminished testosterone concentrations, subclinical varicocele, and the presence of Y chromosome microdeletions seem to be negative predictors for fertility improvement after surgery.63,64 The management of infertility in men with a unilateral clinical varicocele and a subclinical varicocele on the contralateral side is a matter of debate. For example, Zheng et al. found that bilateral varicocelectomy had no

benefit over left clinical varicocelectomy.65 Elbendary et al., however, observed that the magnitude of change in sperm count and motility and the spontaneous pregnancy rates were significantly higher in the group of men who had bilateral varicocele repair.66 Several guidelines propose that varicoceles should be treated if palpable and if abnormal semen analyses are observed.67-69 A recent meta-analysis of varicocelectomy conducted by Marmar et al. (2007) demonstrated the benefit of the surgical treatment of clinical varicoceles in infertile men with abnormal semen analyses.70 The authors showed that the chances of spontaneous conception were 2.8-times

Table 1 - Treatment Results for Varicocele Repair in Infertile Men. Postoperative Recurrence, Hydrocele Formation and Spontaneous Pregnancy Rates using Various Techniques*. Technique 14,16

Retroperitoneal High-Ligation Laparoscopic14,16 Macroscopic Inguinal14,16 Microscopic Inguinal or Subinguinal6,14,16,59 *

Recurrence Rate

Hydrocele Formation Rate

Spontaneous Pregnancy Rate

7-35% 2-7% 0-37% 0-0.3%

6-10% 0-9% 7% 0-1.6%

25-55% 14-42% 34-39% 33-56%

Values are expressed as ranges.

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oxidative stress levels show they already have altered sperm function.61 Studies have shown that men who achieved a postoperative total motile sperm count greater than 20 million and sperm DNA fragmentation decrease after varicocelectomy were more likely to initiate a pregnancy either spontaneously or via assisted reproductive technology (ART).61,62,79-81 Our group has recently demonstrated that the treatment of clinical varicoceles may improve the outcome of ICSI in couples with varicocele-related infertility.52 In our study, the chances of live birth were significantly increased by 1.9-fold, and the chance of miscarriage was reduced by 2.3-fold if the varicocele had been treated before assisted conception. Taken together, this knowledge challenges the current recommendations for varicocele treatment and highlights the importance of a continuous debate. It is still unclear why fertility potential does not always improve after surgery. The distribution of antioxidant enzyme genes in infertile men with varicocele has recently been determined. Studies have suggested that genetic polymorphisms in the glutathione S-transferase T1 gene may affect individual responses to varicocelectomy.96 In addition, doctors and researchers have also debated whether varicoceles can cause or contribute to azoospermia. A recent meta-analysis reported the appearance of sperm in the ejaculates of 39% of azoospermic individuals whose varicoceles had been treated.12 In addition, testicular histopathology results were predictive of success. Moreover, the postoperative appearance of sperm in the ejaculates increased 9.4-fold in patients with biopsy-proven HS or MA compared with SCO syndrome. Although the use of motile ejaculated sperm is preferred for ICSI82, persistent azoospermia after varicocele repair is still a potential problem, and sperm extraction before ICSI will be inevitable for many individuals. In the case of persistent azoospermia after varicocelectomy, successful sperm retrieval rates of 60% have been reported using micro-TESE sperm extraction.83 Interestingly, one study suggested that varicocele repair may maximize the chances of retrieving sperm for ICSI in azoospermic men with clinical varicoceles.84

higher in the varicocelectomy group compared with the group of patients who either did not receive treatment or received medication. Interestingly, the reference values for semen parameters normalcy are lower in the newly released edition of the World Health Organization (WHO) manual for examination of human sperm than in previous editions.71-74 According to these new reference values, several patients with clinical varicoceles who had been previously categorized as having abnormal semen parameters would be reclassified as having normal semen and would not be eligible for treatment under the current guidelines for varicocele treatment. The question is not simply whether a man should undergo repair of clinical varicoceles if he has normal semen variables. Indeed, what we are really interested in determining is what the semen parameters of the same individual would have been if varicocele had been treated. It would be very informative to reanalyze the meta-analysis studies of varicocelectomy to determine the magnitude of sperm quality improvement in the subgroup of patients that is now classified as having ‘normal’ semen. This information will certainly come, but emerging evidence seems to indicate the benefits of treatment for men with clinical varicocele and so-called ‘normal’ semen parameters according to the new WHO reference values. In one study, Agarwal et al. examined the effect of varicocelectomy on the semen parameters of adults and demonstrated a significant increase in sperm concentration, motility and morphology by 9.7 million/ml, 9.9%, and 3.1%, respectively.75 In another study, Mori et al. examined a group of 360 nonselected adolescents between the ages of 14-18 years who attended a public school in Brazil.76 They found that 27.8% presented a palpable grade II or III varicocele, but only half of the adolescents had testicular asymmetry. More importantly, semen analysis results revealed that adolescents without varicocele ejaculated a significantly higher number of progressively motile sperm (134.1 million) compared with adolescents with grade II (72.7 million) or grade III (30.3 million) varicocele. Despite the marked difference in the seminal profile between adolescents with and without varicocele, all individuals were still within the reference range for normality according to the latest editions of the WHO manual. Because these semen samples are still considered normal, and because testicular asymmetry will not be detected in $50% of adolescents, treatment would not be recommended according to the published guidelines of current professional societies, such as the American Urological Association, American Society for Reproductive Medicine, European Association of Urology, and Brazilian Society of Urology.67-69 Thus, surgical correction of the varicocele would only be offered when adolescents have already crossed into the infertile range, even though initial evaluation had already shown that their seminal profiles were significantly lower than their counterparts without varicocele. Due to the progressive nature of varicocele, it is to be expected that treatment of varicocele halts deterioration of sperm quality and prevents individuals with ‘normal’ semen analysis from crossing into the infertile range.77,78 Moreover, improvements in sperm quality after varicocele repair may also increase the male reproductive potential even if pre- and post-treatment values are within the newly proposed reference values. Adolescents and adults with palpable varicoceles may present with a normal semen analysis, but elevated DNA fragmentation rates and

Reconstructive Surgery of the Vas Deferens and Epididymis Overall patency and pregnancy rates have been shown to be 92% and 55%, respectively, following microsurgical VV and 78% and 40%, respectively, following VE (Table 2).19,24,27,34,35,85-89 Most pregnancies occur within 24 months after surgery. Pregnancy rates are related to the time elapsed between vasectomy and reversal and to female age. Although the female partner’s age does not seem to affect patency rates after vasectomy reversal (86-90% in female partners under the age of 40 years old versus 83% in those over the age of 40 years), it does affect pregnancy rates (14% in women over 40 years of age vs. 56% in those under 39 years of age).90 Pregnancy rates are also lower after a longer duration of vasal obstruction. Approximately 30-40% of couples achieve pregnancy following surgical reconstructions performed after obstruction intervals greater than 15 years; however, more than 50% achieve pregnancy after shorter intervals.34,91 Vasectomy reversal has also been shown to be feasible in patients who failed PESA. Marmar et al. showed that PESA procedures cause limited trauma to the epididymis, and pregnancy rates as high as 50% may be

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Table 2 - Treatment Results for Vasovasostomy and Vasoepididymostomy. Type of Anastomosis, Patency, and Spontaneous Pregnancy Rates using Various Techniques. Author Vasovasostomy Belker et al.19 Boorjian & Lipkin24 Chan & Goldstein85 Kolettis et al.86 Vasoepididymostomy Silber34 Thomas35 Berger87 Marmar88 Chan et al.89 Schiff et al.27

Patients (n)

Technique

Patency Rate (%)

Pregnancy Rate (%)

1,247

Modified one-layer Two-layer Two-layer Two-layer Both

89 86 95 99 76

57 51 83 54 35

End-to-end End-to-side Triangulation intussusception Modified intussusception Triangulation intussusception End-to-end End-to-side three-suture intussusception two-suture intussusception

78 79 92 78 84 73 74 84 80

56 50 NR 22 40 NR

159 1,048 34 139 137 12 9 68 153

NR = not reported.

on the possibility of enhancing physiologic static tremor correction, visual magnification (up to 100X when using a digital microscopic camera), and ergonomics.96 Animal studies have suggested that robotic-assisted vasectomy reversals are easier to perform and yield better pregnancy rates than microsurgical reversal.32 In a preliminary experience in humans, Parekattil et al. reported shorter operative time and higher postoperative sperm counts with robot-assisted vasectomy reversal compared with microsurgical techniques.94 The advantages of a robot over an experienced microsurgeon, however, are yet to be proven in larger series. A robotic system costs more than 1 million dollars and its annual maintenance surpasses one hundred thousand dollars. These cost issues will certainly represent a barrier to the wide adoption of robotics in microsurgical urologic practices.

obtained in vasectomy reversal after PESA; however, success is higher for couples whose female partners are 37 years old or younger.92 Because patency and pregnancy rates of the existing surgical procedures do not reach 100% and are technically demanding, efforts continue to be made to widen the options for reconstructive repair. Several modifications have been suggested, such as intussusception VE anastomotic techniques, the use of novel biomaterials/sealants, absorbable and nonabsorbable stents, and the use of robotics.32,34,89,93-97 Recent modifications to the conventional VE techniques simplified and fastened the anastomoses. In a prospective study, Chan et al. reported overall patency and pregnancy rates of 84% and 40%, respectively, using an intussusception technique.93 These findings were confirmed by Schiff et al., who reported patency and pregnancy rates of approximately 82% and 45%, respectively, using simplified intussusception techniques.32 Interestingly, these studies have also suggested that anastomoses are more water-tight after the use of intussusception techniques, which decreases granuloma formation. Because pregnancy rates following VE are below 50% and late failures occur in approximately 20% of the cases, it may be useful to retrieve sperm intraoperatively for cryopreservation, particularly in cases that involve difficult reconstruction. Sealants are used around the anastomotic site to decrease the operative time and to simplify the procedure without compromising success rates. Fibrin sealant can stimulate the coagulation cascade and produce a fibrin seal around the anastomosis. When mixed with thrombin and calcium, fibrinogen is converted to fibrin monomer, which is converted to a stable cross-linked fibrin polymer.94 Ho et al. achieved 85% patency rates and 23% pregnancy rates in a mean follow-up time of 6.2 months by using three transmural sutures and fibrin glue.94 There are concerns, however, about the potential contact of the glue with the vas lumen, which may result in obstruction. In addition, there are concerns about the transmission of viral disease because fibrin glue is derived from pooled plasma.93 The use of nonabsorbable polymeric stents has only been reported in animal models, but the results have been promising.95 The use of robotics for microsurgical procedures is also a novel concept. The rationale behind the addition of robotic technology to the already existing armamentarium relies

Transurethral Resection of Ejaculatory Ducts Ejaculatory duct obstruction is a treatable cause of male infertility, but it is difficult to diagnose, particularly in cases of partial obstruction. Transrectal ultrasound is valuable, but not specific. Indeed, studies have suggested that adjunctive procedures, such as magnetic resonance imaging, chromotubation, seminal vesicle aspiration, seminal vesicle scintigraphy, and ejaculatory duct manometry are more sensitive for diagnosis.98-100 Transurethral resection of ejaculatory ducts remains the treatment of choice, but less invasive approaches using balloon dilation with or without transurethral incision of the ejaculatory ducts has been proposed with similar results and fewer complications than TURED.101,102

Sperm Retrieval Techniques Currently, there is not a consensus about the best techniques for sperm retrieval in men with OA and NOA. To date, no randomized controlled trial has compared the efficiency of the various strategies; thus, current recommendations are based on cumulative evidence provided by descriptive, observational, and few controlled studies.103 For example, PESA can be performed without surgical scrotal exploration, it is easily repeatable at a low cost, does not require an operating microscope or expertise in microsurgery, can be performed under local anesthesia,

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infertile men undergoing ART is related to the type of azoospermia (i.e., obstructive or nonobstructive). Postoperative complications of sperm retrieval techniques include persistent pain, swelling, infection, hydrocele and hematoma.111-113,115,116 The development of an intratesticular hematoma has been observed in most patients undergoing TESE with single or multiple biopsies based on ultrasounds results performed after surgery, but the hematomas often resolve spontaneously without compromising testicular function.113 In a larger-volume standard testicular biopsy, the risk of transient or even permanent testicular damage (e.g., complete devascularization) can result in decreased serum testosterone levels.110,112 Less invasive techniques, such as TESA and micro-TESE, aim to reduce the incidence of complications and long-term consequences of these surgical approaches. Several studies have documented a lower incidence of complications following micro-TESE compared with conventional techniques.109,110,112,115 Using micro-TESE, proper identification of testicular vessels under the tunica albuginea is made prior to the placement of an incision into the testis. Optical magnification and microsurgery techniques allow for the preservation of intratesticular blood supply and the identification of tubules that are more likely to harbor sperm production.112 Therefore, the efficacy of sperm retrieval is improved, and the risks of large tissue removal are minimized. The small amount of tissue extracted also facilitates sperm processing.111 For certain groups of patients (e.g., patients with Klinefelter syndrome), however, who already have diminished androgen production, a temporary decrease in serum testosterone has been documented following micro-TESE.111 The pregnancy rates of ICSI using testicular sperm extracted by TESA or micro-TESE in NOA are significantly lower than those obtained with either ejaculated or epididymal/testicular sperm from men with OA.82,105,114 Testicular spermatozoa of men with severely impaired spermatogenesis have decreased fertility potential and may have a higher tendency to carry deficiencies (e.g., related to the centrioles and genetic material), which ultimately affect the capability of the male gamete to activate the egg and trigger the formation and development of a normal zygote and a viable embryo.116 Although there is limited available data, studies have suggested that the sperm retrieval technique itself has no impact on ICSI success rates.109 Nonetheless, frozen-thawed surgicallyretrieved sperm from NOA men have significantly impaired reproductive potential compared with fresh sperm.114,116 Meta-analysis results have shown that fertilization rates by ICSI remain similar, but implantation was significantly higher (by 73%) with the use of fresh compared with frozenthawed testicular sperm.114 The question of whether ICSI using sperm retrieved from men with either OA or NOA might be associated with an increased risk of birth defects is still unresolved. In general, IVF techniques are associated with multiple gestation and an increased risk of congenital abnormalities (including hypospadias).117 ICSI in particular carries an increased risk of endocrine abnormalities and epigenetic imprinting effects.117 Although the absolute risk of any of these conditions remains low,117-120 current data is limited, and study populations are heterogenic. Therefore, well-defined groups of ICSI with ejaculated sperm, ICSI with epididymal sperm, ICSI with testicular sperm, and a

and is associated with minimal postoperative discomfort. Microsurgical aspiration has the advantage of retrieving a larger number of sperm, which facilitates cryopreservation, and it is associated with a reduced risk of hematoma.104 Meta-analysis results have not demonstrated any significant differences in any outcome measures between the use of epididymal or testicular sperm in men with OA.105 The etiology of the obstruction and the use of fresh or frozenthawed epididymal/testicular sperm do not seem to affect ICSI outcomes in terms of fertilization, pregnancy, or miscarriage rates.104,106,107 In our series of 142 men with OA, the cumulative successful retrieval rate of PESA and/or TESA was 97.9%, and an adequate number of motile sperm for cryopreservation were obtained in approximately one third of the cases.104 Motile spermatozoa were obtained in approximately 73% of the cases after the first or second PESA aspirations, and TESA was carried out as a rescue procedure after failed PESA in about 14% of the individuals. In our series, success in sperm retrieval using percutaneous techniques and pregnancy outcomes by ICSI were similar in the vasectomy, CAVD, and postinfectious etiology categories.106 Either epididymal or testicular spermatozoa retrieved from these men exhibited similar reproductive potential, and overall live birth rates were 40.2%. In addition, our data indicated that ICSI outcomes using fresh epididymal and testicular spermatozoa retrieved from men with OA were comparable to those obtained with ejaculated sperm.82 In cases of NOA, the efficiency of TESA was lower than TESE,108-110 except in the favorable cases of men with previously successful TESA or testicular histopathology showing HS. In these circumstances, sperm retrieval rates (SRR) may be as high as 100%.106 In a recent systematic review the mean reported SRR for TESE was 49.5%.109 TESE with multiple biopsies resulted in a higher SRR than fineneedle aspiration, a variation of TESA, especially in cases of SCO syndrome and MA.109 In NOA, current evidence suggests that micro-TESE performs better than conventional TESE or TESA in cases of SCO, where tubules containing active foci of spermatogenesis can be positively identified using microsurgery. Sperm retrieval rates ranging from 3577% have been reported with micro-TESE.49,32,111-113 To allow for adequate healing and the resumption of spermatogenesis, the minimum recommended interval between sperm retrieval procedures in NOA is 3-6 months.97,112,113 According to our results involving approximately 200 individuals with NOA, the SRR was 55.7%, and sperm could be obtained in similar rates in multiple etiological categories, including cryptorchidism, orchitis, genetic, radio-/chemotherapy, and idiopathic causes. Testicular histopathology results were predictive of sperm collection using both TESA and micro-TESE.49,52 In our group of NOA men presenting with either HS or a history of previous successful TESA attempt, SRR by TESA was 100% and 82.3%, respectively. Using micro-TESE, SRR were significantly higher than TESA in cases of SCO and MA (39.2% vs. 22.8%).52 Both methods yielded an SRR of 100% in cases of HS. According to our data, the chances of retrieving spermatozoa (odds ratio [OR] = 43.0; 95% confidence interval [CI]: 10.3-179.5) and of achieving a live birth by ICSI (OR = 1.86; 95% CI: 1.03-2.89) were significantly increased in couples whose male partner had OA rather than NOA.114 These findings indicated that the reproductive potential of

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microsurgical vasectomy reversal are inversely related to the interval of obstruction since the vasectomy. Other factors that affect success rates include the intraoperative appearance of vasal fluid, the presence or absence of sperm in the vasal fluid (and their quality), the length of the remaining segment adjacent to the epididymis, the age of the female partner, and the experience of the surgeon. TURED is the treatment of choice for EDO. After TURED, sperm return to the ejaculate in approximately 50-75% of men, and approximately 20% of couples achieve pregnancy; however, results depend on the etiology (acquired or congenital) and type (partial or complete) of obstruction. Complications of TURED, which include hematuria, hematospermia, urinary tract infection, epididymitis, and a watery ejaculate due to a reflux of urine, occur in approximately 20% of men. In OA, sperm production is normal and gametes can be easily retrieved from the epididymides or testicles in approximately 100% of cases, irrespective of the technique. In NOA, successful sperm retrieval is approximately 50%. The use of microsurgery during TESE may improve the efficacy of sperm extraction with significantly less tissue removed, which ultimately facilitates sperm processing. Testicular histology results, if available, may be useful to predict the chances of retrieving sperm in men with NOA. Interestingly, sperm can be obtained in almost all scenarios except cases of Y chromosome infertility with complete AZFa and/or AZFb microdeletions. In both OA and NOA, the sperm retrieval technique itself does not seem to impact IVF/ICSI success rates. Nonetheless, the chances of retrieving spermatozoa and of achieving a live birth by ICSI are increased in couples whose male partner had OA rather than NOA. Children conceived using sperm retrieved from men with OA or NOA should be monitored because it is still unclear if there is an increased risk of birth defects when ICSI is carried out with nonejaculated sperm.

control group of naturally conceived children should be closely monitored.

What is the best treatment option for azoospermic males in the ICSI era? Both microsurgical reconstruction and sperm retrieval combined with IVF/ICSI can be effective treatments for infertility due to obstructive azoospermia. A choice between the two must be based not only on the needs and preferences of the individual couple but also on the couple’s clinical profile (i.e., taking into account the cause of azoospermia and any coexisting factors in the female partner). Consequently, both partners should be evaluated thoroughly before making a specific treatment recommendation. Cost issues also play a role in the decision-making process because ART is seldom reimbursed by health insurance companies in most countries. Most importantly, infertility clinics and doctors should not limit a couple’s options for treatment based on their own technical limitations (i.e., they should always provide all treatment options available for the particular case scenario). According to the most recent data, microsurgical reconstruction of the vas (when performed by an experienced microsurgeon) remains a cost-effective and reliable means of restoring fertility in the majority of men who have previously undergone vasectomy.104,121-124 Data comparing surgical reconstruction versus sperm retrieval/ICSI, however, are not randomized or homogenous. Therefore, a comprehensive understanding of the factors that can affect outcomes, overall cost, and the morbidity associated with each treatment modality is recommended.

FINAL CONSIDERATIONS Varicocele treatment based on the presence of clinically palpable varicocele and abnormal semen parameters should be reconsidered in light of the newly proposed WHO reference values for laboratory semen analysis. Open microsurgical inguinal or subinguinal techniques are currently the best treatment modalities because they result in higher spontaneous pregnancy rates and fewer recurrences and postoperative complications than laparoscopic, radiologic embolization and macroscopic inguinal or retroperitoneal varicocelectomy techniques. There are no absolute predictive factors for successful varicocele repair, and existing evidence does not support the treatment of infertile men with subclinical varicocele. Surgical repair of varicocele improves semen parameters and functional markers of oxidative stress and DNA integrity. The chance for either spontaneous or assisted conception is increased after the repair of clinical varicocele. In addition, recovery of spermatogenesis can be achieved after the repair of clinical varicocele in infertile men with NOA. Testicular histopathology is predictive of success, and men with MA and HS are more likely to ejaculate motile spermatozoa after surgery. Furthermore, the chance of retrieving testicular sperm for ICSI is optimized in nonobstructed azoospermic men with treated clinical varicocele. Men with OA may father children either by surgical correction of the obstruction, which may allow the couple to conceive naturally, or retrieval of sperm directly from the epididymis or testis, which is followed by ICSI. The return of sperm to ejaculate after microsurgical reconstructions is achieved in 70-95% of cases, and 30-75% of couples achieve unassisted pregnancy. Patency and pregnancy after

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Fibrin glue assisted 3-suture vasovasostomy. J Urol. 2005;174:1360-3, doi: 10.1097/01.ju.0000173941. 87775.35. 95. Vrijhof EJ, De Bruine A, Zwinderman AH, Lycklama a` Nijeholt AA, Koole LH. The use of newly designed nonabsorbable polymeric stent in reconstructing the vas deferens: a feasibility study in New Zealand white rabbits. BJU Int. 2005;95:1081-5, doi: 10.1111/j.1464-410X.2005. 05471.x. 96. Parekattil SJ, Atalah HN, Cohen MS. Video technique for human robotassisted microsurgical vasovasostomy. J Endourol. 2010;24:511-4, doi: 10.1089/end.2009.0235. 97. Schiff J, Li PS, Goldstein M. Robotic microsurgical vasovasostomy and vasoepididymostomy in rats. Int J Med Robot. 2005;1:122-126. 98. Eisenberg ML, Walsh TJ, Garcia MM, Shinohara K, Turek PJ. Ejaculatory duct manometry in normal men and in patients with ejaculatory duct obstruction. J Urol. 2008;180:255-60, doi: 10.1016/j.juro. 2008.03.019. 99. Orhan I, Duksal I, Onur R, Balci TA, Poyraz K, Firdolas F, et al. Technetium Tc 99m sulphur colloid seminal vesicle scintigraphy: a novel approach for the diagnosis of the ejaculatory duct obstruction. Urology. 2008;71:672-6, doi: 10.1016/j.urology.2007.11.103. 100. Onur MR, Orhan I, Firdolas F, Onur R, Kocakoc¸ E. Clinical and radiological evaluation of ejaculatory duct obstruction. Arch Androl. 2007;53:179-86, doi: 10.1080/01485010701426448. 101. Lawler LP, Cosin O, Jarow JP, Kim HS. Transrectal US-guided seminal vesiculography and ejaculatory duct recanalization and balloon dilation for treatment of chronic pelvic pain. J Vasc Interv Radiol. 2006;17:16973, doi: 10.1097/01.RVI.0000186956.00155.26. 102. Manohar T, Ganpule A, Desai M. Transrectal ultrasound- and fluoroscopic-assisted transurethral incision of ejaculatory ducts: a problem-solving approach to nonmalignant hematospermia due to ejaculatory duct obstruction. Endourol. 2008;22:1531-5, doi: 10.1089/ end.2007.0415. 103. Van Peperstraten A, Proctor ML, Johnson NP, Philipson G. Techniques for surgical retrieval of sperm prior to ICSI for azoospermia. Cochrane Database Syst Rev. 2006;3:CD002807. 104. Male Infertility Best Practice Policy Committee of the American Urological Association; Practice Committee of the American Society for Reproductive Medicine. Report on the management of infertility due to obstructive azoospermia. Fertil Steril. 2008;90(Suppl. 3):S121–24. 105. Nicopoullos JD, Gilling-Smith C, Almeida PA, Norman-Taylor J, Grace I, Ramsay JW. Use of surgical sperm retrieval in azoospermic men: a meta-analysis. Fertil Steril. 2004;82:691–701, doi: 10.1016/j.fertnstert. 2004.02.116. 106. Esteves SC, Verza S, Prudencio C, Seol B. Success of percutaneous sperm retrieval and intracytoplasmic sperm injection (ICSI) in obstructive azoospermic (OA) men according to the cause of obstruction. Fertil Steril. 2010;94(Suppl):S233, doi: 10.1016/j.fertnstert.2010.07.906. 107. Kamal A, Fahmy I, Mansour R, Serour G, Aboulghar M, Ramos L, et al. Does the outcome of ICSI in cases of obstructive azoospermia depend on the origin of the retrieved spermatozoa or the cause of obstruction? A comparative analysis. Fertil Steril. 2010;94:2135-40, doi: 10.1016/j. fertnstert.2010.01.041. 108. Hauser R, Yogev L, Paz G, Yavetz H, Azem F, Lessing JB, et al. Comparison of efficacy of two techniques for testicular sperm retrieval in nonobstructive azoospermia: multifocal testicular sperm extraction versus multifocal testicular sperm aspiration. J Androl. 2006;27:28–33, doi: 10.2164/jandrol.05055. 109. Donoso P, Tournaye H, Devroey P. Which is the best sperm retrieval technique for non-obstructive azoospermia? A systematic review. Hum Reprod Update. 2007;13:539-549, doi: 10.1093/humupd/dmm029. 110. Carpi A, Sabanegh E, Mechanick J. Controversies in the management of nonobstructive Azoospermia. Fertil Steril. 2009;91:963–970, doi: 10. 1016/j.fertnstert.2009.01.083. 111. Schiff JD, Palermo GD, Veeck LL, Goldstein M, Rosenwaks Z, Schlegel PN, et al. Success of testicular sperm injection and intracytoplasmic sperm injection in men with Klinefelter syndrome. J Clin Endocrinol Metab. 2005;90:6263–7, doi: 10.1210/jc.2004-2322.

resulted in significant DNA improvement. Syst Biol Reprod Med. 2009;55:109-15, doi: 10.1080/19396360902787944. Kondo Y, Ishikawa T, Yamaguchi K, Fujisawa M. Predictors of improved seminal characteristics by varicocele repair. Andrologia. 2009;41:20-3, doi: 10.1111/j.1439-0272.2008.00882.x. Cocuzza M, Cocuzza MA, Bragais FM, Agarwal A. The role of varicocele repair in the new era of assisted reproductive technology. Clinics. 2008;63:395-404, doi: 10.1590/S1807-59322008000300018. Zheng YQ, Gao X, Li ZJ, Yu YL, Zhang ZG, Li W. Efficacy of bilateral and left varicocelectomy in infertile men with left clinical and right subclinical varicoceles: a comparative study. Urology. 2009;73:1236-40, doi: 10.1016/j.urology.2008.11.050. Elbendary MA, Elbadry AM. Right subclinical varicocele: how to manage in infertile patients with clinical left varicocele? Fertil Steril. 2009;92:2050-3, doi: 10.1016/j.fertnstert.2009.05.069. The Male Infertility Best Practice Policy Committee of the American Urological Association; Practice Committee of the American Society for Reproductive Medicine. Report on varicocele and infertility. Fertil Steril 2004;82:S142–5. European Association of Urology. Guidelines on Male Infertility; 2010; Available from: http://www.uroweb.org/gls/pdf/Male%20Infertility% 202010.pdf (cited: February 27th, 2011). Sociedade Brasileira de Urologia & Cole´gio Brasileiro de Radiologia; Projeto Diretrizes da Associac¸ a˜ o Me´dica Brasileira. Varicocele. Available from: http://www.projetodiretrizes.org.br/8_volume/40Varicocele.pdf (cited: February 27th, 2011). Marmar JL, Agarwal A, Prabakaran S, Agarwal R, Short RA, Benoff S, et al. Reassessing the value of varicocelectomy as a treatment for male subfertility with a new meta-analysis. Fertil Steril. 2007;88:639–48, doi: 10.1016/j.fertnstert.2006.12.008. World Health Organization. WHO Laboratory Manual for the Examination of Human Semen and Sperm-cervical Mucus Interaction, 2nd ed. Cambridge: Cambridge University Press 1987;80p. World Health Organization. WHO Laboratory Manual for the Examination of Human Semen and Sperm-cervical Mucus Interaction, 3rd ed. Cambridge: Cambridge University Press 1992;107p. World Health Organization. WHO Laboratory Manual for the Examination of Human Semen and Sperm-cervical Mucus Interaction, 4th ed. Cambridge: Cambridge University Press 1999;128p. World Health Organization. WHO Laboratory Manual for the Examination and Processing of Human Semen, 5th ed. Geneva: WHO press, 2010, 287p. Agarwal A, Deepinder F, Cocuzza M, Agarwal R, Short RA, Sabanegh E, et al. Efficacy of Varicocelectomy in Improving Semen Parameters: New Meta-analytical Approach. Urology. 2007;70:532-8. Mori MM, Bertolla RP, Fraietta R, Ortiz V, Cedenho AP. Does varicocele grade determine extent of alteration to spermatogenesis in adolescents? Fertil Steril. 2008;90:1769-73, doi: 10.1016/j.fertnstert.2007.09.052. Gorelick JI, Goldstein M. Loss of fertility in men with varicocele. Fertil Steril 1993;59:613–6. Witt MA, Lipshultz LI. Varicocele: a progressive or static lesion? Urology. 1993;42:541–3, doi: 10.1016/0090-4295(93)90268-F. Matkov TG, Zenni M, Sandlow J, Levine LA. Preoperative semen analysis as a predictor of seminal improvement following varicocelectomy. Fertil Steril. 2001;75:63-68, doi: 10.1016/S0015-0282(00)01644-7. Smit M, Romijn JC, Wildhagen MF, Veldhoven JL, Weber RF, Dohle GR. Decreased sperm DNA fragmentation after surgical varicocelectomy is associated with increased pregnancy rate. J Urol. 2010;183:270-4, doi: 10. 1016/j.juro.2009.08.161. Jeng SY, Wu SM, Lee JD. Cadmium accumulation and metallothionein overexpression in internal spermatic vein of patients with varicocele. Urology. 2009;73:1231-5, doi: 10.1016/j.urology.2009.01.008. Verza S Jr, Esteves SC. Sperm defect severity rather than sperm source is associated with lower fertilization rates after intracytoplasmic sperm injection. Int Braz J Urol. 2008,34:49-56. Schlegel PN, Kaufmann J. Role of varicocelectomy in men with nonobstructive azoospermia. Fertil Steril. 2004;81:1585-8, doi: 10.1016/ j.fertnstert.2003.10.036. Inci K, Hascicek M, Kara O, Dikmen AV, Gu¨rgan T, Ergen A. Sperm retrieval and intracytoplasmic sperm injection in men with nonobstructive azoospermia, and treated and untreated varicocele. J Urol. 2009;182:1500-5, doi: 10.1016/j.juro.2009.06.028. Chan PT, Goldstein M. Superior outcomes of microsurgical vasectomy reversal in men with the same female partners. Fertil Steril. 2004; 81:1371-4, doi: 10.1016/j.fertnstert.2003.09.066. Kolletis PN, Burns JR, Nangia AK, et al. Outcomes for vasovasostomy performed when only sperm parts are present in the vasal fluid. J Androl. 2006;27:565-7, doi: 10.2164/jandrol.05190. Berger RE. Triangulation end-to-side vasoepididymostomy. J Urol. 1998;159:1951-3, doi: 10.1016/S0022-5347(01)63205-1. Marmar JL. Modified vasoepididymostomy with simultaneous double needle placement, tubulotomy and tubular invagination. J Urol. 2000;163:483-6, doi: 10.1016/S0022-5347(05)67907-4.

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112. Ramasamy R, Yagan N, Schlegel PN. Structural and functional changes to the testis after conventional versus microdissection testicular sperm extraction. Urology. 2005;65:1190–4, doi: 10.1016/j.urology.2004.12.059. 113. Carpi A, Menchini Fabris FG, Palego P, Di Coscio G, Romani R, Nardini V, et al. Fine-needle and large needle percutaneous aspiration biopsy of the testicle in men with nonobstructive azoospermia: safety and diagnostic performance. Fertil Steril. 2005;83:1029–33, doi: 10.1016/j. fertnstert.2004.09.027. 114. Prudencio C, Seol B, Esteves SC. Reproductive potential of azoospermic men undergoing intracytoplasmic sperm injection is dependent on the type of azoospermia. Fertil Steril. 2010;94(Suppl):S232-S233, doi: 10. 1016/j.fertnstert.2010.07.903. 115. Turunc T, Gul U, Haydardedeoglu B, Bal N, Kuzgunbay B, Peskircioglu L, et al. Conventional testicular sperm extraction combined with the microdissection technique in nonobstructive azoospermic patients: a prospective comparative study. Fertil Steril. 2010;94:2157-60, doi: 10. 1016/j.fertnstert.2010.01.008. 116. TesarikJ: Paternal effects on cell division in the human preimplantation embryo. Reprod Biomed Online. 2005;10:370-5, doi: 10.1016/S14726483(10)61798-1. 117. Alukal JP, Lamb DJ. Intracytoplasmic sperm injection (ICSI)—what are the risks? Urol Clin North Am. 2008;35:277–88, doi: 10.1016/j.ucl.2008. 01.004. 118. Knoester M, Helmerhorst FM, Vandenbroucke JP, van der Westerlaken LA, Walther FJ, Veen S, et al. Artificial Reproductive Techniques

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Follow-up Project. Cognitive development of singletons born after intracytoplasmic sperm injection compared with in vitro fertilization and natural conception. Fertil Steril. 2008;90:289–96, doi: 10.1016/j. fertnstert.2007.06.090. Belva F, Henriet S, Liebaers I, et al. Medical outcome of 8-year-old singleton ICSI children and a spontaneously conceived comparison group. Hum Reprod. 2007;22:506–15, doi: 10.1093/humrep/del372. Woldringh GH, Besselink DE, Tillema AH, Hendriks JC, Kremer JA. Karyotyping, congenital anomalies and follow-up of children after intracytoplasmic sperm injection with non-ejaculated sperm: a systematic review. Hum Reprod Update. 2010;16:12-9, doi: 10.1093/humupd/ dmp030. Lee R, Li PS, Goldstein M, Tanrikut C, Schattman G, Schlegel PN. A decision analysis of treatments for obstructive azoospermia. Hum Reprod. 2008;23:2043-9, doi: 10.1093/humrep/den200. Robb P, Sandlow JI. Cost-Effectiveness of Vasectomy Reversal. Urol Clin N Am. 2009;36:391-6, doi: 10.1016/j.ucl.2009.05.003. Malizia BA, Hacker MR, Penzias AS. Cumulative live-birth rates after in vitro fertilization. N Engl J Med. 2009;360:236-43, doi: 10.1056/ NEJMoa0803072. Hsieh MH, Meng MV, Turek PJ. Markov modeling of vasectomy reversal and ART for infertility: how do obstructive interval and female partner age influence cost effectiveness? Fertil Steril. 2007;88:840-6, doi: 10.1016/j.fertnstert.2006.11.199.

CLINICS 2011;66(8):1479

DOI:10.1590/S1807-59322011000800027

LETTER TO THE EDITOR

Caveats in the interpretation of natriuretic peptide levels Chia-Ter Chao Division of Nephrology, Department of Internal Medicine, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China. Email: b88401084@ntu.edu.tw. Tel.: 886-2-23123456

I read the article entitled ‘‘B type natriuretic peptide as a predictor of anterior wall location in patients with non-STelevation myocardial infarction’’ by Ramos et al.1 in the March 2011 issue of Clinics with great interest. Although the finding is instructive, I think it would be more informative if several factors were also considered: First, renal function was not evaluated in Ramos et al. ’s population. B-type natriuretic peptide (BNP) is a small polypeptide with a plasma half life of 20–30 minutes that reflects the stress sustained by the left ventricle (LV) and that correlates well with the LV filling pressure and the heart failure status.2,3 Several important studies have elucidated the fact that this miraculous myocardial stress marker can be affected by chronic kidney disease (CKD). In the PRIDE (PRoBNP Investigation of Dyspnea in the Emergency department) study, Anwaruddin et al. discovered that the N-terminal pro-BNP (NT-proBNP) level is inversely associated with the glomerular filtration rate (GFR) and that a higher cut-off value should be set for patients with poorer renal function (GFR,60 ml/min).4 Takami et al. also tested this theory in a group of CKD patients who had not undergone dialysis and found that the BNP level was significantly increased compared with hypertensive patients with normal renal function.5 They also concluded that LV overload (higher end-diastolic volume and pressure) may correlate fairly well with the BNP level, independent of the severity of renal dysfunction. McCullough et al. provided further convincing evidence of the graded relationship between BNP cut-off values and the GFR, further supporting the view that the interpretation of BNP levels should build upon the differential range of renal function.6 Theories concerning the elevation of BNP in CKD patients include poor tolerance of fluid loading and resultant subclinical volume overload in early CKD stages, a higher degree of LV hypertrophy and dysfunction in advanced CKD stages, and decreased clearance of biomarkers as renal function deteriorates.6,7 Given this understanding, it is prudent to include renal function, specifically creatinine data, in the assessment of the impact and predictive power of the BNP level. In addition, age by itself may also influence the BNP level. Fabbian et al. recently performed an elegant study

to investigate the confounding factors in decoding the NTproBNP level in elderly patients. They discovered that in a medium-sized geriatric population, with a mean age of 80 years old, elevated NT-proBNP levels were associated with lower hemoglobin levels, more atrial fibrillation and less severe pulmonary disease and were significantly influenced by the CKD stage.8 These results are indicative of the more complex situation that we might face with the elderly population, which is representing an increasing proportion of our society as time passes. In conclusion, considering the various obstacles that may hinder the elucidation of the relationship between the BNP level and patient-level outcomes, it is of vital importance that we gather other relevant information needed to determine the truth.

REFERENCES 1. Ramos RB, Strunz CM, Avakian SD, Ramires JA, Mansur AP. B-type natriuretic peptide as a predictor of anterior wall location in patients with non-ST-elevation myocardial infarction. Clinics. 2011;66:436-41, doi: 10.1590/S1807-59322011000300013. 2. Pedersen EB, Pedersen HB, Jensen KT. Pulsatile secretion of atrial natriuretic peptide and brain natriuretic peptide in healthy humans. Clin Sci. 1999;97:201-6, doi: 10.1042/CS19990103. 3. Parsonage WA, Galbraith AJ, Koerbin GL, Potter JM. Value of B-type natriuretic peptide for identifying significantly elevated pulmonary artery wedge pressure in patients treated for established chronic heart failure secondary to ischemic or idiopathic dilated cardiomyopathy. Am J Cardiol. 2005;95:883-5, doi: 10.1016/j.amjcard.2004.12.021 4. Anwaruddin S, Lloyd-Jones DM, Baggish A, Chen A, Karuser D, Tung R, et al. Renal function, congestive heart failure, and amino-terminal probrain natriuretic peptide measurement. J Am Coll Cardiol. 2006;47:91-7, doi: 10.1016/j.jacc.2005.08.051. 5. Takami Y, Horio T, Iwashima Y, Takiuchi S, Kamide K, Yoshihara F, et al. Diagnostic and prognostic value of plasma brain natriuretic peptide in non-dialysis-dependent CRF. Am J Kidney Dis. 2004;44:420-8. 6. McCullough PA, Duc P, Omland T, McCord J, Nowak RM, Hollander JE, et al. B-type natriuretic peptide and renal function in the diagnosis of heart failure: an analysis from the Breathing Not Properly Multinational study. Am J Kidney Dis. 2003;41:571-9, doi: 10.1053/ ajkd.2003.50118. 7. Mark PB, Petrie CJ, Jardine AG. Diagnostic, prognostic, and therapeutic implications of brain natriuretic peptide in dialysis and nondialysisdependent chronic renal failure. Sem Dial. 2007;20:40-9, doi: 10.1111/j. 1525-139X.2007.00240.x. 8. Fabbian F, De Giorgi A, Pala M, Tiseo R, Portaluppi F. Elevated NTproBNP levels should be interpreted in elderly patients presenting with dyspnea. Eur J Int Med. 2011;22:108-11, doi: 10.1016/j.ejim.2010.07.013.

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DOI:10.1590/S1807-59322011000800028

LETTER TO THE EDITOR

Alopecia areata incognita: a comment Alfredo Rebora Department of Endocrinologic and Medical Sciences, Section of Dermatology, University of Genova, Viale Benedetto XV, Genova, IT.

Email: rebdermo@unige.it Tel.: 00390103538414

I read the article by Dr. Molina et al. on alopecia areata incognita (AAI)1 with great interest. As the author of the original 1987 article in which the existence of AAI was hypothesized,2 I feel entitled to present my dissenting opinion with respect to some of statements made by Dr. Molina et al. The case Dr. Molina et al. described and successfully cured is not a case of AAI but rather of diffuse alopecia areata (AA). Contrary to the claims of Dr. Molina et al., AA is not ‘‘identical’’ to AAI but is a different entity. Of course, the distinction between AAI and AA is a matter of minute differences that, nonetheless, should matter to dermatologists. AAI has a typical clinical presentation: it looks like an acute telogen effluvium with an intensely positive pull test and trichodynia. There is no rarefaction of hair, unless the hair is shed for months or years, and there are rarely exclamation point hairs. More than 350 hairs are usually collected with the modified wash test (MWT),3 and, sometimes, as many as 1000 hairs are collected. Dystrophic hairs can be observed in about 3-4% of patients and represent 27% of all hairs collected at each MWT.4 This observation is possible, however, only if the hairs are collected by means of the MWT. Because the dystrophic hairs are notably rare and are scattered randomly throughout the scalp, finding them with the traditional trichogram is impossible. If patients are followed up, patches of alopecia areata are detected between the sixth and ninth week in approximately 50% of patients with dystrophic hairs at the MWT.4 The patches are small and have no tendency to enlarge.4 Diffuse AA, in contrast, presents with patches of hair rarefaction, which differ from those of classical AA because of the indistinctness of their borders and their tendency to merge. Often, other areas are affected, such as the eyebrows and limbs, an occurrence that is not observed in AAI. Diffuse AA easily results in alopecia totalis in a few weeks. Of course, AAI shares with both classical and diffuse AA some dermoscopic and histopathological features, such as

the ‘‘yellow dots’’ and the telogen germinal units reported by Tosti et al.5 The most important difference between AAI and AA, however, involves the different ways in which hair sheds. In classical AA, hairs shed in dystrophy, and the glabrous patch is formed because a group of hairs in the same early subphase of anagen is stricken simultaneously by the antimitotic insult,2 hence the neatly designed borders of the patch. In diffuse AA, hairs are shed in a mixed fashion, with both dystrophic and telogenic characteristics. In AAI, however, the hair loss is practically only telogenic, hence the absence of rarefaction or patches. Finally, I cannot agree with the statement that AAI is more common in young people. Were this the case, AAI would not differ from classical AA, which rarely occurs after the fifth decade. One of the possible explanations for this curious epidemiological behavior is the higher prevalence of androgenetic alopecia (AGA) at that age. In other words, as I postulated in 1987, the shortening of the hair cycles in AGA may be the crucial factor that prevents the formation of dystrophy and favors the ‘‘telogen escape’’ from AA.1

REFERENCES 1. Molina L, Donati A, Valente NSY, Romiti R. Alopecia areata incognita. Clinics. 2011;66:513-5, doi: 10.1590/S1807-59322011000300027. 2. Rebora A. Alopecia areata incognita: a hypothesis. Dermatologica. 1987; 174:214-8, doi: 10.1159/000249182. 3. Rebora A, Guarrera M, Baldari M, Vecchio F. Distinguishing androgenetic alopecia from chronic telogen effluvium when associated in the same patient: a simple noninvasive method. Arch Dermatol. 2005;141:1243-5, doi: 10.1001/archderm.141.10.1243. 4. Quercetani, Rebora AE, Fedi MC, Carelli G, Mei S, Chelli A, et al. Patients with profuse hair shedding may reveal anagen hair dystrophy. A diagnostic clue of alopecia areata incognita. J Eur Acad Dermatol Venereol, 2010, Oct.15. 5. Tosti A, Whiting D, Iorizzo M, Pazzaglia M, Misciali C, Vincenzi C, et al. The role of scalp dermoscopy in the diagnosis of alopecia areata incognita J Am Acad Dermatol. 2008;59:64-7.

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DOI:10.1590/S1807-59322011000800029

LETTER TO EDITOR

Diastolic dysfunction in end-stage renal disease patient: what the ticking clock has told us? Mustafa Duran,I Aydin Unal,II Mehmet Tugru Inanc,III Fatma Esin,III Yucel Yilmaz,III Ender OrnekI I Cardiology, Etlik Education Training And Research, Etlik Ihtisas Research and Educational Hospital, Ankara/Turkey. Faculty of Medicine, Kayseri/Turkey. III Department of Cardiology, Erciyes University, Kayseri/Turkey.

Nephrology, Erciyes University,

Email: mduran2@gmail.com Tel.: 0090 505 3911620

We reported an effect of maintenance hemodialysis on diastolic left ventricular function in end-stage renal disease in the October 2010 issue of Clinics Journal.1 We read with interest the letter to the editor about our article titled ‘‘Diastolic dysfunction in end-stage renal disease patient: what the ticking clock has told us?’’ by Dr. Chao.2 We thank him and reply to this interesting comments on our article. Firstly, we agree about the shortening of the follow-up period. Long term effects of hemodialysis on left ventricular diastolic functions may be due to left ventricular hypertrophy, myocardial ischemia, and heightened cardiac afterload. However, several studies have shown that a single hemodialysis session is associated with acute deterioration of diastolic and systolic parameters of myocardial function.3,4 This may be related to many factors, such as the ultrafiltration rate, the interdialysis weight gain, change of serum ionized calcium concentration, sympathetic hyperactivity, increased oxidative stress during hemodialysis treatment, and disease of smaller resistance vessels.3 Dr. Chao noted that in several studies, the average length of the follow-up period ranged from just over half a year to four years. Of course the duration of the follow-up period is very important for identifying myocardial remodeling in hemodialysis patients. But we must not ignore the acute effects of hemodialysis. In our study, the median duration of the follow-up period was approximately two months.1 This period is small for myocardial change but it is a relatively long and sufficient time to detect the acute effects of hemodialysis on left ventricular diastolic functions. Secondly, although echocardiography is inherently deficient in determining the echo window and spatial resolution,

and suffers from an operator-dependent nature, we know that echocardiographic parameters are useful markers for predicting the development of left ventricular dysfunction.5 Also, to avoid the influence of operator-dependent factors, all echocardiographic evaluations were performed by the same medically qualified operator. Dr. Chao noted that the volume-dependent effect also attenuates diagnostic accuracy. To avoid a volume-dependent effect, the second echocardiographic evaluations were performed after 6-8 hours from the last hemodyalisis session. As Dr. Chao emphasized, cardiac magnetic resonance imaging, left atrium circulation transit time, and late gadolinium enhancement sequence are the newer techniques to detailed depiction of left ventricular geometry and structural variation. However, our study design did not allow us to use these techniques.

REFERENCES 1. Duran M, Unal A, Inanc MT, Esin F, Yilmaz Y, Ornek E. Effect of maintenance hemodialysis on diastolic ventricular function in end-stage renal disease. Clinics. 2010;65:979–84, doi: 10.1590/S180759322010001000010. 2. Chao CT. Letter to the editor Diastolic dysfunction in end-stage renal disease patient: what the ticking clock has told us? Clinics. 2011; 66:913-4. 3. Galetta F, Cupisti A, Franzoni F, Carpi A, Barsotti G, Santoro G. Acute effects of hemodialysis on left ventricular function evaluated by tissue Doppler imaging. Biomed Pharmacother. 2006;60:66-70, doi: 10.1016/j. biopha.2005.10.008. ¨, 4. Dincer I, Kumbasar D, Nergisoglu G, Atmaca Y, Kutlay S, Akyurek O et al. Assessment of left ventricular diastolic function with Doppler tissue imaging: effects of preload and place of measurements. Int J Cardiovasc Imaging. 2002;18:155–60, doi: 10.1023/A:1014697208218. 5. Foley RN, Parfrey PS, Kent GM, Harnett JD, Murray DC, Barre PE. Serial change in echocardiographic parameters and cardiac failure in end-stage renal disease. J Am Soc Nephrol. 2000;11:912–6.

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DOI:10.1590/S1807-59322011000800030

RAPID COMMUNICATION

Frequency of HLA B*5701 allele carriers in abacavir treated-HIV infected patients and controls from northeastern Brazil Sergio Crovella,V Lara Biller,II Sergio Santos,I Ana Salustiano,I Lucas Brandao,II Rafael Guimaraes,II Ludovica Segat,II Jose´ Luiz de Lima Filho,II Luiz Claudio ArraesIV I

Department of Genetics, Federal University of Pernambuco, Recife, Brazil. II Laborato´rio de Imunopatologia Keizo Azami (LIKA), Federal University of Pernambuco Recife, Brazil. III Laborato´rio Central de Sau´de Pu´blica - Dr. Milton Bezerra Sobral LACEN, Recife/PE, Brazil. IV Instituto de Medicina Integrada Prof. Fernando Figueira (IMIP), Recife, Brazil. V Genetic Service, IRCCS Burlo Garofolo, Trieste, Italy. Email: crovelser@gmail.com Tel.: 39 0403785424

population resulting from contributing African (44%), Caucasian (34%), and native American (22%) genomes.5

INTRODUCTION In clinical practice, using pharmacogenetics has risen in importance since the technologies for genomic variation searches for different responses to drugs became widespread, easier, more rapid, and affordable for a specialized laboratory. The main pharmacogenetics studies have mostly involved drug resistance monitoring in oncology patients;1 however, other applications are being developed to detect genetic molecular markers associated with resistance or hypersensitivity/adverse reactions to antiretroviral drug treatment in Human Immunodeficiency Virus (HIV)infected patients.2 HIV treatment is known to be limited by adverse drug reactions and the development of resistance. One significant example is the strong association of the Abacavir hypersensitivity reaction with HLA-B*5701 in HIV-positive patients.3 Abacavir, a common drug for treating HIVinfected patients, is an efficient nucleoside reverse-transcriptase inhibitor with a beneficial long-term toxicity profile, often used with other antiretroviral agents. However, Abacavir can yield adverse effects such as immunologically-driven hypersensitivity in 5 to 8% of HIV-positive subjects during the first six weeks of use.2 Hypersensitivity symptoms immediately reverse after the interruption of Abacavir.4 The hypersensitivity reaction to Abacavir has been reportedly associated with the presence of the major histocompatibility complex class I allele HLA-B*5701 and this association has been confirmed in several replication studies in different ethnic groups of HIV-positive patients.3 In our study, we searched for the presence of HLA B*5701 in 96 HIV-positive patients treated with Abacavir and in 243 healthy subjects from Northeastern Brazil (Recife, Pernambuco) to verify the percentage of HLA B*5701 allele carriers in HIV patients and in the general population from Northeast Brazil. This area is known to harbor a tri-hybrid

MATERIAL AND METHODS Subjects Genomic DNA of 96 HIV-infected patients (50 males, mean age 29.5 years, range 19-48; 46 females, mean age 22.7, range 16-31) treated with Abacavir and 243 healthy blood donors (120 males, mean age 21.3 years, range 18-41; 123 females, mean age 24.2 years, range 19-48) from the Recife metropolitan area stored at the ‘‘Laborato´rio Central de Sau´de Pu´blica - Dr. Milton Bezerra Sobral’’ (LACEN - PE) and the ‘‘Laboratorio de Imunopatologia Keizo Azami’’ (LIKA) were used for the genetic screening. Genomic DNA was previously extracted from peripheral whole blood and stored at 220 ˚C at the LIKA laboratory of Recife using the EZ1 DNA extraction kit and EZ1 robotic device (Quiagen) following the manufacturer’s instructions. Patients and controls were stratified for ethnicity and classified as European- or African-derived according to phenotypic characteristics of individuals and ethnicity data of parents/grandparents reported by the participants in an appropriate questionnaire. The issue concerning the skin color-based classification criteria adopted in Brazil is welldocumented and has been already assessed by other research groups in previous studies.6,7 HLA B*5701 genotyping HLA B*5701 genotyping has been performed in triplicate by Melting Temperature Assay using the DUPLICaRealTime HLA-B*5701 Genotyping kit (Euroclone, Milan, Italy). Briefly, 5 ml of genomic DNA (50 ng/ml) were added to 10 ml of amplification mix (with Hot Start Taq DNA polymerase, nucleotides, MgCl2, buffer and SYBR GREEN) and 10 ml of oligo mix (with primers to amplify HLA-B*5701 allele and an internal control, human growth hormone: HGH). Real time PCR reactions were run on the Cepheid (Euroclone, Milan, Italy) platform using the following temperatures and cycles profile: 29 50 ˚C 1 hold, 109 95 ˚C 1 hold, then 40 cycles 150 95 ˚C and 600 60 ˚C. The melting protocol was as follows: initial hold 29 65 ˚C with a melting curve from 65 ˚C to 95 ˚C increasing 0.2 ˚C each time. Each specific amplicon (i.e. HLA B*5701 and HGM) was detectable by a different melting temperature.

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in the control population, eight individuals out of the 243 screened (3.4%) were carriers for HLA B*5701. The HLA B*5701 allele was heterozygous in all carriers (the results were double-checked using the PCR-SSP Dynal commercial kit). Table 1 summarizes the results for the HIV patients and the controls from Northeast Brazil and reports HLA B*5701 carriers and allele frequencies for other ethnic groups (reported in the http://www.allelefrequencies.net).8 When stratifying for ethnicity, within the 96 HIV infected patients 36 were European-derived (two HLA B*5701 carriers, 5.5%) and 60 were African-derived (one HLA B*5701 carrier, 1.6%). In the controls group, 90 individuals were classified as European-derived (five HLA B*5701 carriers, 5.5%) and 153 were classified as African-derived (three HLA B*5701 carriers, 1.9%).

RESULTS We searched for the presence of HLAB*5701 allele in 96 HIV-infected patients treated with Abacavir and 243 Northeastern Brazilian subjects from Recife (PE) using a real time allelic-specific PCR approach. The melting temperature assay, performed after real time PCR amplification, allowed us to easily identify the presence of HLA B*5701, characterized by a melting profile temperature between 91.5 and 92.5˚C, while the internal control, the human beta globin gene, showed a melting temperature between 88.8 and 89.8˚C. Figure 1 shows the melting profiles of the HLAB*5701 positive and HLAB*5701 negative samples. The real time PCR followed by melting temperature assay were performed in triplicate on three different real time PCR platforms to test the robustness of the genotyping chemistry and its adaptability to different technological platforms. The results obtained by using the ABI 7900 HT Sequence Detection System, the RotorGene 3000 and the Cepheid (Euroclone, Milan Italy) real time PCR platforms were absolutely the same and were highly reproducible, always allowing the detection of the HLA B*5701 allele in the unknown genotype samples. Within the 96 HIV infected patients treated with Abacavir, three (3.1%) were carriers of the HLA B*5701 allele and presented with hypersensitivity characterized by cutaneous rash and severe gastrointestinal tract symptoms;

DISCUSSION Recently, the presence of the HLA-B*5701 allele has been correlated with immunological hypersensitivity to Abacavir in white and black HIV-positive patients from United States.9 Moreover, HLA-B*5701 allele carriers in HIV-positive Hispanic patients showed a clinically-diagnosed hypersensitivity reaction to Abacavir.10 HLA-B*5701 allele frequency is known to depend upon the HIV-positive patients’ ethnicity; in fact, while white Caucasians have an incidence of around

Figure 1 - Melting temperature assay profiles of two subjects: one carrier of HLA B*5701 allele (A) and one non-carrier of the HLA B*5701 allele (B). The two melting profiles are easily distinguishable and allow easy detection of the HLA B*5701 allele.

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HLA-B*5701 carriers in Northeast Brazil Crovella S et al.

Table 1 - HLA B*5701 frequencies in HIV-infected patients (HIV_BRA) and the controls (BRA_NE) from Northeast Brazil in this study and in previously genotyped populations9 (http://www.allelefrequencies.net).

HLA B*5701 carriers HLA B*5701 allele frequency

HIV_BRA n = 96

BRA_NE n = 234

BRA_C n = 95

HIV_CHI n = 492

CHI_P n = 300

CHI_M n = 70

AUT_C n = 200

USA_A n = 187

USA_C n = 307

3.1%

3.4%

1.1%

2.2%

3.7%

4.0%

5.5%

2.1%

6.2%

1.5%

1.7%

0.5%

1.1%

1.8%

2.0%

2.8%

1.1%

3.1%

BRA_C: Brazil (Belo Horizonte) Caucasian; HIV_CHI: HIV infected patients from Chile: CHI_P: Chilean general population; CHI_M: Chile (Santiago) Mixed; AUT_C: Austria Caucasian; USA_A: USA (Bethesda) African-American; USA_C: USA (Philadelphia) Caucasian.

6%, only about 2.5% present in the black population.11 Thus, the screening for a genetic marker for hypersensitivity to Abacavir, such as the HLA-B*5701, is strongly dependent upon the population’s racial background. In our study performed with Northeastern Brazilian individuals, the frequency of HLA-B*5701 carriers was 3.1% in HIV-infected patients and 3.4% in the general population. These frequencies are between those reported for white Caucasian and black subjects from the United States. The difference can be explained by the ethnic composition of the Pernambuco population, known to be an admixture of Caucasian (34%), Afro-American (44%), and Amerindian (22%) genomes.5 Moreover, our findings more closely resembled those by Poggi et al.12 in HIV patients and the controls from Chile than the frequencies reported in a Caucasian population from Belo Horizonte (see Table 1 reporting HLA B*5701 frequencies in different ethnic groups for comparison).8 The importance of HLA-B*5701 screening for diminishing the frequency of hypersensitivity to Abacavir has been reported in the PREDICT-1 study13; the frequency of HLA B*5701 carriers at high risk of hypersensitivity to Abacavir was 6% in white Caucasian patients. In our study population from Northeastern Brazil, the frequency of HLA-B*5701 was between Caucasian and African ethnic groups; however, when stratifying for ethnicity, our findings show that the HLA B*5701 carriers’ frequencies are more similar to those previously reported for Caucasians and Africans. Finally, considering the clinical importance of hypersensitivity to Abacavir treatment in HIV-infected patients and the frequency of HLA B*5701 carriers reported in this study (3.1% in patients and 3.4% in the controls), we suggest the preventive use of HLA B*5701 testing in clinical practice in Abacavir treatment in Northeastern Brazil.

Fundac¸a˜o de Amparo a` Cieˆncia e Tecnologia do Estado de Pernambuco (FACEPE). LS is a recipient of a fellowship grant (APQ-0020-4.01/08) from FACEPE. SC is recipient of a grant from the TALENTS Programme (7th R&D Framework Programme, Specific Programme: PEOPLE – Marie Curie Actions – COFUND).

REFERENCES 1. Dawood S, Leyland-Jones B. Pharmacology and pharmacogenetics of chemotherapeutic agents. Cancer Invest. 2009;2:482-8, doi: 10.1080/ 07357900802574660. 2. Tozzi V. Pharmacogenetics of antiretrovirals. Antiviral Res. 2010;85:190200, doi: 10.1016/j.antiviral.2009.09.001. 3. Roca B. Pharmacogenomics of antiretrovirals. Recent Pat Antiinfect Drug Discov. 2008;3:132-5, doi: 10.2174/157489108784746650. 4. Hetherington S, McGuirk S, Powell G, Cutrell A, Naderer O, Spreen B, et al. Hypersensitivity reactions during therapy with the nucleoside reverse transcriptase inhibitor abacavir. Clin Ther. 2001;23:1603-14, doi: 10.1016/S0149-2918(01)80132-6. 5. Alves-Silva J, da Silva Santos M, Guimara˜es PE, Ferreira AC, Bandelt HJ, Pena SD, et al. The ancestry of Brazilian mtDNA lineages. Am J Hum Genet. 2000;67:444. 6. Vargas AE, Marrero AR, Salzano FM, Bortolini MC, Chies JA. Frequency of CCR5delta32 in Brazilian populations. Braz J Med Biol Res. 2006;39:321-325, doi: 10.1590/S0100-879X2006000300002. 7. Veit TD, Cordero EA, Mucenic T, Monticielo OA, Brenol JC, Xavier RM, et al. Association of the HLA-G 14 bp polymorphism with systemic lupus erythematosus. Lupus. 2009;18:424-430, doi: 10.1177/ 0961203308098187. 8. Middleton D, Menchaca L, Rood H, Komerofsky R. New Allele Frequency Database: http://www.allelefrequencies.net.TissueAntigens. 2003; 61:403-407. 9. Saag M, Balu R, Phillips E, Brachman P, Martorell C, Burman W, et al. High Sensitivity of Human Leukocyte Antigen–B*5701 as a Marker for Immunologically Confirmed Abacavir Hypersensitivity in White and Black Patients. Clin Infect Dis. 2008;46:1111-8, doi: 10.1086/529382. 10. Arrizabalaga J, Rodriguez-Alca´ ntara F, Castan˜ er JL, Ocampo A, Podzamczer D, Pulido F, et al. Prevalence of HLA-B*5701 in HIVinfected patients in Spain (results of the EPI Study). HIV Clin Trials. 2009;10:48-51, doi: 10.1310/hct1001-48. 11. Rodriguez-No´voa S, Soriano V. Current trends in screening across ethnicities for hypersensitivity to abacavir. Pharmacogenomics. 2008;9:1531-41, doi: 10.2217/14622416.9.10.1531. 12. Poggi H, Vera A, Lagos M, Solari S, Rodrı´guez P L, Pe´rez CM. HLAB*5701 frequency in Chilean HIV-infected patients and in general population. Braz J Infect Dis. 2010;14:510-2 13. Mallal S, Phillips E, Carosi G, Molina JM, Workman C, Tomazic J, et al. HLA-B*5701 screening for hypersensitivity to abacavir. N Engl J Med. 2008;358:568-79.

ACKNOWLEDGMENTS This work has been supported by the RC07/08 project from IRCCS Burlo Garofolo (Trieste Italy) and by the project APQ APQ-0757-2.02/10 from

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DOI:10.1590/S1807-59322011000800031

TECHNICAL NOTE

Polyvinyl alcohol as a viable membrane in artificial tissue design and development Nahrizul Adib Kadri, Mat Ghazali Raha, Belinda Pingguan-Murphy Department of Biomedical Engineering, Faculty of Engineering, University of Malaya, 50603 Kuala Lumpur, Malaysia. Email: nahrizuladib@um.edu.my Tel.: 603 79674485

INTRODUCTION Polyvinyl alcohol (PVA) membranes have long been used in many applications, most notably in a number of recent biomedical applications. These applications include the encapsulation of hybrid-type artificial organs,1-3 the controlled release of specific molecules,4,5 targeted drug delivery systems,6,7 enhanced wound dressings,8,9 and myriad other applications that utilize the semi-permeability and high biocompatibility of PVA. Nevertheless, prior to its designation for use as the biomaterial of choice in the design and development processes of artificial tissue fabrication applications, the exact nature of PVA’s physiological properties needs to be ascertained. In particular, the permeability and the diffusion coefficient are two important parameters that need to be analyzed. The experimental results can subsequently be used to determine the appropriate fabrication technique and design geometry for the design and development of the required tissue engineering applications. This study is based on the following presumption: if a researcher could place a semi-permeable membrane at the interface of two chambers (Chambers 1 and 2) that contain different concentrations of the same solution, then the diffusive parameters of the membrane can be measured simply by continuously measuring the changes in solute concentration in the chamber with the lower concentration. If the researcher waits until equilibrium between the two chambers is reached, both of the chambers would then have the same amount of solute (and, therefore, the same concentration of solute). A complete concentration profile will therefore provide the information that is needed to determine (using Fick’s Law) the desired parameters of the membrane. Nevertheless, this procedure is highly impractical, due to the long period of time that is required to record the observations and any related changes. On the other hand, by deriving a model of the underlying diffusion process using Fick’s Law, the researcher can predict the diffusive parameters based on the collected data over a relatively short period of time. The derivation of the model begins with constructing materials that are balanced on either side of the membrane. The material balance on the higherconcentration chamber (Chamber 1) can be expressed as

dc1 ~{PA(c1 {c2 ), dt

ð1Þ

where V1 is the chamber volume, P is the permeability, A is the exposed area of the membrane, c1 is the concentration in Chamber 1, and c2 is the concentration in Chamber 2. Similarly, the material balance in the lower-concentration chamber (Chamber 2) can be expressed as V2

dc2 ~PA(c1 {c2 ): dt

ð2Þ

Assuming the initial conditions of c1 equals c0, and c2 equals zero, the following mathematical model can be derived and used to determine the permeability, P, and thus the diffusion coefficient, D, of the hydrogel membrane. 2ct 2A Pt ~{ ln 1{ V c0

ð3Þ

If the ratio of ct/c0 is sufficiently small (as is the case if the period of the experiment is sufficiently short), then the lefthand side of the equation will be equal to –2ct/c0, according to Taylor’s series.5,7 In other words, a linear concentration profile can be expected if the experimental period is sufficiently short, and the permeability and diffusion coefficient can be easily determined thereafter. The aim of this technical report was to study the fundamental parameters in the design and development of an aqueous PVA hydrogel membrane to be used in generic artificial tissue engineering applications; these parameters included the permeability and diffusion coefficient of the membrane and were measured using dextran-fluorescein isothiocyanate (Dextran-FITC) as the solute of choice.

MATERIALS AND METHODS Various methods can be used to measure concentration changes. The method that was chosen for the current study utilizes modified dextran molecules that were tagged with a fluorescent material (Dextran-FITC). Next, the fluorescence levels were easily measured using a fluorometer, and the levels were linearly correlated with the concentration of the dextran molecules in the chamber. A standard concentration curve was constructed such that the concentrations could be calculated from the fluorescence levels based on a linear

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transfer studies was used in this study. Because DextranFITC was chosen as the solute of choice, the transparent chamber walls needed to be covered with aluminum foil to prevent degradation of the fluorescent marker. A number of openings in the top portion of the chamber were sealed using a sealant to reduce evaporation, which would have greatly affected the concentration of the samples. The device was thoroughly washed before each experimental run to ensure that no residual Dextran-FITC remained to affect the accuracy of the fluorescence readings.

equation that was derived from the curve. The derivations of the permeability and diffusion coefficient values are based on Fick’s Law, thus requiring the values of several parameters (including the chamber volume, membrane aperture, and sample times) in addition to the concentrations. The methods used in this study were divided into two stages: the preparation stage and the running stage. The preparation stage consisted of the preparation of a phosphate-buffered saline (PBS) solution, a Dextran-FITC solution, the PVA hydrogel membrane to be tested, and the experimental chamber apparatus; this stage helped to minimize any foreseeable problems during the experimental runs. In addition, apart from the actual running of the experiment, the running stage also consisted of the calculation of the permeability and diffusion coefficient for the particular hydrogel membrane that was used in the experiment.

Permeability studies Each device component (particularly the surface of the ports where the membrane was clamped) was thoroughly cleaned and dried. Next, the prepared hydrogel membrane was mounted, any folds and creases were removed, and the membrane was clamped and placed into position using four mounting bolts. Deionized water or PBS (12 ml) was subsequently placed in Chamber 2, and Chamber 1 was filled with 12 ml of Dextran-FITC solution. Both chambers were stirred continuously on the vertical axis, and the ports where the membrane was clamped were chamfered to enhance mixing near the membrane surface. The exposed membrane area was 0.385 cm2 (0.7 cm diameter), and the experiments were conducted at room temperature (22.5¡1.5 ˚C) over an average run time of seven hours. The diffusion of Dextran-FITC (or any solute, for that matter) through the polymer network of the PVA hydrogel follows Fick’s Law and can be expressed in the following form:

PBS solutions To prepare a phosphate-buffered saline solution of pH 7.4, approximately 7 g of PBS was dissolved in 1 l of deionized water with continuous stirring. The pH was then measured, and a small amount of PBS was added until the solution pH reached 7.4.

Dextran-FITC solutions Two concentrations of Dextran-FITC (100 and 200 mg/ml) were used in this study. The latter concentration was used later in the course of the study to decrease the uncertainties and errors that are associated with using a low concentration of solute. The 100 mg/ml solution was prepared by dissolving 2 mg of Dextran-FITC in 20 ml of deionized water (or PBS). The amount of Dextran-FITC that was dissolved was doubled to produce the 200 mg/ml solution. Only 12 ml of the solution was used to fill one of the permeation device chambers, and the remaining solution was used for the preparation of the standard Dextran-FITC samples. Each solution was freshly prepared prior to each run of the experiment and was used only once.

2ct 2A Pt, ~{ ln 1{ V c0

ð4Þ

where ct is the concentration of the solute in Chamber 2 at time t, c0 is the initial concentration of the solute in Chamber 1, t is the diffusion time in seconds, A is the exposed membrane area (0.385 cm2), V is the volume of the chamber (12 ml), and P is the permeability of the hydrogel membrane. The diffusion coefficient is related to the permeability based on the following relationship:

PVA hydrogel membranes A 15% w/w PVA solution was prepared from a 30% stock solution. To prepare a 1-ml solution, 0.5 ml of the stock solution was mixed with 0.5 ml of deionized water in a tube. The solution was thoroughly mixed using an electric mixer, and any air bubbles were removed using a vacuum flask in a fume cupboard. The solution was stored at 4 ˚C until use. A small volume was drawn from the tube using a disposable syringe and injected onto a microscope slide. Two 15-mm-thick cover slips were placed on each end of the slide, and another slide was placed on top of the cover slips to produce a thin membrane of fluid. A UV spot cure system was used for cross-linking, and the exposure time was set at 100 sec. The separation between the microscope slide arrangement and the tip of the UV system was kept at a distance of approximately 2530 mm.

D , kd

ð5Þ

where D is the diffusion coefficient of the membrane, k is the partition coefficient of the solute, and d is the membrane thickness at the end of the experiment. The partition coefficient of Dextran-FITC is approximately equal to 1.10 Next, the permeability and related diffusion coefficient were calculated by measuring the slope of the linear portion in Equation (1). A strong linear dependence of ln(1 – 2ct/c0) over t was observed when the dextran molecules passed through the PVA hydrogel membrane.

Experimental apparatus

Experimental protocol

A two-chamber device (Figure 1) that was made of Perspex and was previously developed for generic mass

Samples were taken from Chamber 1 only at the beginning (t0) and the end (tf) of the run, whereas samples

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between the two variables, we are of the opinion that it can be used as a measure of linearity to a certain degree. Therefore, for both water and PBS, the relationship between the two variables can be considered linear within the range of these concentrations, and hence this relationship can be used to determine the concentration of the samples. Figure 3 shows an example concentration plot using water as the solvent of choice (from Run 8). Figure 4 shows the plot of ln(1 – 2ct/c0)* versus t; this plot was used determine the permeability and diffusion coefficient of the membrane. The asterisk was included to indicate that the value was multiplied by 100,000 to yield a much more precise slope value that was then used to calculate permeability. Next, permeability and thus the diffusion coefficient was calculated using Equations (1) and (2). The calculation for determining P and D is shown below where V was 12 ml, A was 0.385 cm2, and dwater was 0.015 cm. Equating the m-part of Equation (1) to the slope of the graph in Figure 4 (removing the multiplication factor by multiplying the slope value by 10-5) yields: 2A P~3:91|10{7 V Substituting the values of V and A gives the permeability of the hydrogel membrane: Figure 1 - The two-chamber device that was used in the experiments (a) and a schematic diagram of the device (b). The dotted line between the two chambers indicates the clamped membrane.

(3:91|10{7 )(12) ~6:10|10{6 cm= sec 2|0:385

Finally, substituting the values of P and d in Equation (5) yields the diffusion coefficient of the membrane:

were taken from Chamber 2 at t0, tf, and at a number of specified intermediate times. Using a pipette and a clean tip, 1 ml of sample was taken at each specified time point and placed in a microtube; 1 ml of deionized water (or PBS) was subsequently added to the respective chamber using a clean tip to restore the volume. The microtubes were quickly placed in a rack and stored in the freezer overnight alongside the standard concentration samples. The fluorescence levels of the samples were read on the following day. Three 0.2-ml samples from each microtube was drawn and placed in a fluorescence counting plate. The frequency range of the fluorometer was set at the default setting of 485-538 nm.

D~(6:10|10{6 )(0:015)~9:1|10{8 cm2 = sec Similar calculation steps and parameter values were used to determining the permeability and diffusion coefficient when PBS was used as the solvent of choice. Table 1 summarizes the values for a select number of parameters, including the initial and final concentrations in Chamber 1, the final concentrations in Chamber 2, and the permeability and diffusion coefficient values of the hydrogel membrane for each experimental run.

RESULTS DISCUSSION

The outcome of the study was entirely dependent on the fluorescence readings of the samples. Thus, it was of the utmost importance that the construction of the standard curve from which the concentrations of the samples were determined yielded consistent and reproducible results. Figure 2 shows the standard curves for both solvents (i.e., water and PBS). The mean R-squared values when water or PBS was used as the solvent were 0.99¡0.0049 and 0.98¡0.0022, respectively. Although an R-squared value is merely a measure of the predictability of the next data points (with 0 being impossible to predict and 1 being the most predictable) and is based on the construction of the best-fit linear equation

In accordance with the objectives of this study, the calculated permeability and diffusion coefficient values of the membrane were comparable with the expected values from the literature. As part of their diffusion studies, Hickey and Peppas10 studied the membrane diffusion coefficient of 10 and 15% PVA hydrogel membranes using theophylline and Dextran-FITC that were prepared using the freezing/ thawing technique. Their studies yielded a range of values for the diffusion coefficient that was dependent on the degree of crystallinity and the equilibrium swelling ratio. Although the diffusion coefficient ranged from 1.70-5.28 6 10-8 cm2/sec, this range was still within an order of

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Figure 2 - Standard curves when using either water (open symbols) or PBS (closed symbols) as the solvent.

concentration clearly should have dropped with a corresponding increase in the concentration in Chamber 2. Moreover, even when there was a concentration drop in Chamber 1 (when using PBS), this drop did not closely correspond to the concentration increase that was observed in Chamber 2. There was also a significant difference between the two solvent types that were used with respect to the amount of fluorescence that was obtained from the Dextran-FITC samples (Figure 2). When using water as the solvent, there was a decrease of up to three times the amount of fluorescence that was obtained using PBS as the solvent. Due to time constraints, this phenomenon was not investigated, but it could play a role in explaining the inconsistencies that were found in the results. In addition, another inconsistency that is worth noting for future studies concerns the thickness of the hydrogel membrane at the conclusion of the experimental runs. On a number of occasions in which the thickness was measured post-experimentally, the thickness changed when using water as the solvent but remained constant when using PBS. When using water, the membrane thickness was almost always increased by up to twofold at the end of the experimental run relative to the start of the experiment. This phenomenon clearly had a large impact on the diffusion

magnitude, and thus the authors believed that these values (which were calculated using water as the solvent) are still relevant as reference values. As listed in Table 1, the values that were calculated in the present study differed from, but were still within an order of magnitude to, the values that were reported in previous studies,10,11 even though the solute and solvent used in the experiments are exactly the same. There are many differences in the setup of the experiments, and these differences may have affected the outcome of the experiments. For example, the degree of crystallinity and the equilibrium swelling ratio were not determined in the current study, thereby making it difficult to place the calculated value within the range of the previously determined diffusion coefficient values. These diffusion coefficient values seemed to increase non-linearly with an increase in equilibrium swelling ratio, whereas the degree of crystallinity remained relatively constant. In our opinion, one of the fundamental issues that needed to be considered before the difference in the calculated diffusion coefficient values could be interpreted concerns experimental consistency. As noted in Table 1, there was an increase in the final concentration in Chamber 1 (cf) when water was used as the solvent (Runs 6 to 10). This is counter-intuitive, as the

Figure 3 - Concentration curve of Dextran-FITC in water (from Run 8).

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Figure 4 - Plot of ln(1 – 2ct/c0)* for Dextran-FITC in water. The slope and R-squared values are shown.

ACKNOWLEDGMENTS

Table 1 - The measured and calculated values for the indicated parameters. Runs 6 through 10 used water as the solvent, and Runs 11 through 13 used PBS. Runs 6 and 8 used 100 mg/ml Dextran-FITC, and the rest of the runs used 200 mg/ml Dextran-FITC. Experimental runs

6 8 9 10 Average (¡SD) 11 12 13 Average (¡SD)

Chamber 1 (mg/ml)

Chamber 2 (mg/ml)

NAK and RMG would like to thank the University of Malaya (Malaysia) and the Malaysian Public Services Department for the financial grants that contributed to the completion of the postgraduate studies on which this report was based.

REFERENCES Permeability Diff. coefficient (10-6 cm/sec) (10-7 cm2/sec)

131.7 101.5 239.2 193.8

196.6 174.0 394.8 301.0

0.63 0.46 1.25 0.82

7.1 6.1 5.9 5.6 6.2 (¡0.65)

1.1 0.9 0.9 0.8 0.9 (¡0.98)

196.6 171.1 191.6

159.7 146.9 138.8

1.59 1.21 1.33

10.1 9.0 8.0 9.0 (¡1.08)

1.5 1.3 1.2 1.4 (¡0.16)

1. Wang Y, Hsieh Y. Immobilization of lipase enzyme in polyvinyl alcohol (PVA) nanofibrous membranes. Journal of Membrane Science. 2008;309:73-81, doi: 10.1016/j.memsci.2007.10.008. 2. Santos J, Paula A, Rocha C, Nunes G, de Castro H. Morphological and mechanical properties of hybrid matrices of polysiloxane-polyvinyl alcohol prepared by sol-gel technique and their potential for immobilizing enzyme. Journal of Non-Crystalline Solids. 2008;354:4823-6, doi: 10. 1016/j.jnoncrysol.2008.04.019. 3. Santos JC, Mijone PD, Nunes GF, Perez VH, de Castro HF. Covalent attachment of Candida rugosa lipase on chemically modified hybrid matrix of polysiloxane-polyvinyl alcohol with different activating compounds. Colloids and Surfaces B: Colloids Surf B Biointerfaces. 2008;61:229-36, doi: 10.1016/j.colsurfb.2007.08.006. 4. Koutsopoulos S, Unsworth LD, Nagai Y, Zhang S. Controlled release of functional proteins through designer self-assembling peptide nanofiber hydrogel scaffold. Proc Natl Acad Sci U S A. 2009;106:4623-8, doi: 10. 1073/pnas.0807506106. 5. Censi R, Vermonden T, Van Steenbergen M, Deschout H, Braeckmans K, De Smedt S, et al. Photopolymerized thermosensitive hydrogels for tailorable diffusion-controlled protein delivery. Journal of controlled release. 2009;140:230-6, doi: 10.1016/j.jconrel.2009.06.003. 6. Mahmoudi M, Simchi A, Imani M, Milani A, Stroeve P. Optimal Design and Characterization of Superparamagnetic Iron Oxide Nanoparticles Coated with Polyvinyl Alcohol for Targeted Delivery and Imaging’, The Journal of Physical Chemistry B. 2008;112:14470-81, doi: 10.1021/ jp803016n. 7. Taleb A, Ismail S, El-Kelesh N. Radiation Synthesis and Characterization of Polyvinyl Alcohol/Methacrylic Acid-Gelatin Hydrogel for Vitro Drug Delivery’, Journal of Macromolecular Science, Part A: Pure and Applied Chemistry. 2009;46:170-8, doi: 10.1080/10601320802594808. 8. Gwon H, Lim Y, An S, Youn M, Han S, Chang H, et al. Characterization of PVA/glycerin hydrogels made by gamma-irradiation for advanced wound dressings. Korean Journal of chemical engineering. 2009;26:16868, doi: 10.1007/s11814-009-0246-z. 9. Kim JO, Park JK, Kim JH, Jin SG, Yong CS, Li DX, et al. Development of polyvinyl alcohol-sodium alginate gel-matrix-based wound dressing system containing nitrofurazone. International journal of pharmaceutics. 2008;359:79-86, doi: 10.1016/j.ijpharm.2008.03.021. 10. Hickey A, Peppas N. Mesh size and diffusive characteristics of semicrystalline poly (vinyl alcohol) membranes prepared by freezing/ thawing techniques’, Journal of membrane science. 1995;107:229-37, doi: 10.1016/0376-7388(95)00119-0. 11. Bodugoz-Senturk H, Choi J, Oral E, Kung JH, Macias CE, Braithwaite G, et al. The effect of polyethylene glycol on the stability of pores in polyvinyl alcohol hydrogels during annealing. Biomaterials. 2008;29:1419, doi: 10.1016/j.biomaterials.2007.09.015.

dynamics and on the subsequent accuracy of the calculation that was used to determine the permeability and diffusion coefficient such that they could not be ascertained. Due to this fact, the initial membrane thickness was used for our calculations. The initial membrane thickness was similar for all of the membrane types, and we ensured a consistent initial membrane thickness by using a cover slip to separate the microscope slides while preparing the membranes.

CONCLUSIONS Although the experimental protocol that was used in these experiments was sufficiently altered to obtain the diffusion coefficient for a given membrane, there remain certain inexplicable aspects of the results that may render the calculated values less useful and relevant. Therefore, these inconsistencies require further investigation, particularly with respect to the design and fabrication of the experimental chambers, before this protocol can be used in any future permeability-related studies. Nevertheless, from these preliminary data, it can be confidently concluded that PVA is suitable for use in the design and development of artificial membranes in various tissue engineering studies.

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DOI:10.1590/S1807-59322011000800032

CASE REPORT

Gastropleural fistula from gastric perforation due to renal cell carcinoma after bevacizumab chemotherapy: a case report Olı´via Meira Dias,I Caroline Chaul de Lima Barbosa,II Lisete Ribeiro Teixeira,I Francisco S. VargasI I

Pulmonology Department, Heart Institute (INCOR) – Hospital das Clıńicas da Faculdade de Medicina da Universidade de Saõ Paulo, Saõ Paulo/SP, Brazil. Oncology Department, Saõ Paulo State Cancer Institute (ICESP) - Saõ Paulo/SP, Brazil.

Email: meiradias@yahoo.com.br Tel.: 55 11 3442-0010

lactate dehydrogenase (DHL) and amylase (16864 U/l and 873 U/l, respectively). The level of serum amylase was 23 U/l. The cytological exam was inconclusive. Because of the high levels of amylase, an esophageal perforation was initially suspected. A methylene blue test was performed at the bedside with a positive result, indicating leakage of this substance in the chest drain insertion after oral administration. Computed tomography with oral contrasted medium through the feeding tube showed a gastropleural fistula originating from the greater curvature and extending to the left subphrenic space (Figure 2). The patient was submitted to parenteral nutrition. Two days after the diagnosis, the patient presented sudden bleeding exteriorized by the chest tube (approximately 1800 ml of sanguineous pleural effusion) and hypovolemic shock. The patient was transferred again to the ICU where the patient received fluid, blood, and vasopressor support and recovered from the shock. Upper gastrointestinal endoscopy revealed a large blood clot in the greater curvature and fundus, which impeded further exploration. The patient was submitted to embolization of the splenic arteries in the interventional radiology service with success, and no further bleeding episodes occurred. After discussion with the Gastro Surgery team and considering the surgical risk due to the patient’s poor status, the patient was transferred to a palliative care facility. The patient died ten days after the embolization, due to shock and acute respiratory insufficiency.

CASE HISTORY A 39-year-old female was admitted for antibiotic treatment of sepsis of unknown origin, which had been empirically treated with piperacillin-tazobactam. The patient had previously (five months prior) been diagnosed with renal cell carcinoma with clear cell histological features and had undergone a left partial nephrectomy without excision of the entire tumor. Three months before admission, the patient was recruited to participate in a clinical trial investigating hepatic and pancreatic metastasis; the participants received interferon and bevacizumab in one of the protocols. The patient did not present any indication of pulmonary involvement from cancer in a tomography scan taken one month before admission. During her hospital stay, the patient presented a sudden onset of shortness of breath, tachypnea, and chest discomfort without hemodynamic repercussion. On physical examination, the patient exhibited decreased breath sounds in her left hemithorax and tympanic percussion. Trachea deviation was also observed to the right and the patient presented with bilateral jugular stasis. Her saturation was 91% despite oxygen given at a 10-l/min rate through an oxygen mask. A thoracocentesis with air escape in the second intercostal space was immediately performed by the thoracic surgeon. A left-sided hydropneumothorax was diagnosed in the chest roentgenogram with contralateral mediastinal shift (Figure 1). The patient was transferred to the intensive care unit (ICU) with chest discomfort despite drainage with a chest drain. A small amount of odorless, turbid brownish fluid came from the pleural space just after the drainage. After non-invasive mechanical ventilation, the chest discomfort progressively improved, and both lungs were expanded. Upon discharge from the ICU, a liquid resembling nasogastric feed started to emerge from the chest drain in increasing amounts and shortly after commencing feeding with an output of more than 2300 ml per day. Biochemical analysis of the pleural effusion revealed a neutrophilic exudate with a low pH (6.32), low protein (1.5 g/dl), normal glucose (103 mg/dl), and high levels of

DISCUSSION Gastropleural fistula is an uncommon diagnosis. It has been previously reported as a complication after pulmonary resections,1 trauma (especially due to traumatic diaphragmatic hernia), complications of peptic ulcer disease, and malignancy.2 Some case reports have indicated a predisposition caused by oral steroids or anti-inflammatory drug intake when the cause is gastric perforation.3 According to a recent literature review,4 subphrenic pathologies less frequently lead to this condition, generally due to diaphragmatic erosion. Supradiaphragmatic conditions, such as infections, intrathoracic surgeries, and fistulas due to procedures (caused by forceful intercostal tube insertion4 and after nasogastric tube placement after gastric adenocarcinoma resection)5 can also result in this condition. Gastric ulcers have been described as the most frequent cause of gastropleural fistulas. With the advent of intensive

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cessation of angiogenesis, regression of some tumor vessels, normalization of others, and improved drug delivery. Unlike the blood vessels in tumors, the normal vasculature in adults is considered to be stable and, therefore, largely independent of VEGF for survival. However, VEGF and VEGF receptors are still highly expressed in several adult organs, and endothelial fenestrations can be induced by VEGF inhibitors in vitro and in vivo.9 The inhibition of VEGF signaling can eliminate fenestrations in tumor vessels, leading to impaired wound repair and predisposing the patient to fistula formation and bleeding. This potent anti-neoplastic agent has recently been reported to be associated with an increased risk of wound suture dehiscence requiring intervention, fistulas, gastrointestinal perforation, and serious bleeding/ hemorrhage.10 Among the nongastrointestinal fistulas, tracheoesophageal, bronchopleural, biliary, vaginal, renal, and bladder fistulas have been observed, most commonly within the first six months of treatment.11 No cases of gastropleural fistulas have been reported. The diagnosis of gastropleural fistula is usually determined by contrast radiology,12 upper gastrointestinal endoscopy, and pleural fluid testing. In the present case, the cytological exam was not helpful, due to cellular lysis. Although conservative management has been suggested in a recent review of enteral fistulas after bevacizumab chemotherapy,11 this approach is often unsuccessful in patients with gastropleural fistulas, making surgery mandatory. Unfortunately, the potential bleeding caused by bevacizumab would impose unacceptable risks to this patient if surgery was attempted. In conclusion, although gastropleural fistulas are relatively uncommon, the condition requires prompt surgical intervention. Although its incidence due to gastric perforations is low, subdiaphragmatic and intrathoracic tumors will increase the likelihood of this complication. New chemotherapeutic agents, such as bevacizumab, can promote fistulas caused by tumor necrosis, and gastropleural fistulas should be considered as a possible complication.

Figure 1 - Chest roentgenogram showing left-sided tension hydropneumothorax with contralateral mediastinal shift.

treatment regimens involving proton pump inhibitors and eradication therapy for Helicobacter pylori infection, one would expect that the incidence of gastropleural fistulas would decrease.6 Unfortunately, gastropleural fistulas can also occur secondarily to intra-abdominal or thoracic malignancies, which occur increasingly frequently with aging. Published case reports include a B-cell lymphoma7 and two gastric mucosa-associated lymphoid tissue (MALT) lymphomas.6,8 To our knowledge, this is the first reported case of gastropleural fistula caused by gastric perforation due to renal cell carcinoma invasion. Bevacizumab is a recombinant, humanized monoclonal antibody that binds to vascular endothelial growth factor (VEGF) and inhibits angiogenesis. VEGF is a survival factor for blood vessels in certain types of tumors. Inhibition of VEGF signaling in tumors results in the

Figure 2 - Coronal computed tomography reconstruction with oral contrasted medium through the feeding tube. A heterogeneous solid mass with extensive necrotic areas in the left renal space invades the posterior wall of the stomach. The gastropleural fistula originates from the greater curvature extending to the left subfrenic space. Bilateral pleural effusion and partial athelectasis from the left lower lobe with air bronchograms are observed. Other small hipoattenuating hepatic lesions can also be seen due to hepatic metastasis.

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Gastropleural fistula Dias OM et al. 7. Mehran A, Ukleja A, Szomstein S, Rosenthal R. Laparoscopic partial gastrectomy for the treatment of gastropleural fistula. JSLS. 2005;9:213-5. 8. Warburton CJ, Calverly PM. Gastropleural fistula due to gastric lymphoma presenting as tension pneumothorax and empyema. Eur Resp J. 1997;10:1678-9, doi: 10.1183/09031936.97.10071678. 9. Kamba T, Tam BY, Hashizume H, Haskell A, Sennino B, Mancuso MR, et al. VEGF-dependent plasticity of fenestrated capillaries in the normal adult microvasculature. Am J Physiol Heart Circ Physiol. 2006;290: 560-76, doi: 10.1152/ajpheart.00133.2005. 10. Spigel DR, Hainsworth JD, Yardley DA, Raefsky E, Patton J, Peacock N, et al. Tracheoesophageal fistula formation in patients with lung cancer treated with chemoradiation and bevacizumab. J Clin Oncol. 2010;28:438, doi: 10.1200/JCO.2009.24.7353. 11. Badgwell BD, Camp ER, Feig B, Wolff RA, Eng C, Ellis LM et al. Management of bevacizumab-associated bowel perforation: a case series and review of the literature. Ann Oncol. 2008;19:577-82, doi: 10.1093/annonc/mdm508. 12. Chow H, Jung A, Talbott J, Lin AM, Daud AI, Coakley FV. Tumor fistulization associated with targeted therapy: computed tomographic findings and clinical consequences. J Comput Assist Tomogr. 2011;35: 86-90, doi: 10.1097/RCT.0b013e3181fce2cb.

REFERENCES 1. Takeda S, Funaki S, Yumiba T, Ohno K. Gastropleural fistula due to gastric perforation after lobectomy for lung cancer. Interact CardioVasc Thorac Surg. 2005;4:420-2, doi: 10.1510/icvts.2005.108779. 2. Schwab RJ, Jarvik JG. Tension pneumothorax secondary to a gastropleural fistula in a traumatic diaphragmatic hernia. Chest. 1991;99:247-9, doi: 10.1378/chest.99.1.247. 3. Keefe P, Goldstraw P. Gastropleural fistula following pulmonary resection. Thorax. 1993;48:1278-9, doi: 10.1136/thx.48.12.1278. 4. Darbari A, Tandon S, Singh GP. Gastropleural fistula: rare entity with unusual etiology. Ann Thorac Med. 2007;2:64-5, doi: 10.4103/1817-1737. 32233. 5. Bini A, Grazia M, Petrella F, Stella F, Bazzocchi R. Spontaneous biliopneumothorax (Thoracobilia) following gastropleural fistula due to stomach perforation by nasogastric tube. Ann Thorac Surg. 2004;78:339-41, doi: 10.1016/S0003-4975(03)01282-7. 6. Adachi Y, Sato Y, Yasui H, Nishimura S, Tanimura A, Yuasa H, et al. Gastropleural fistula derived from malignant lymphoma. J Gastroenterol. 2002;37:1052-6, doi: 10.1007/s005350200177.

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DOI:10.1590/S1807-59322011000800033

CASE REPORT

Endovascular treatment for iliac artery pseudoaneurysm with arteriovenous fistula after abdominal aortic aneurysm open repair Rodrigo Bono Fukushima, Nelson Wolosker, Daniel Augusto Benitti, Pedro Puech-Leao Department of Vascular and Endovascular Surgery, Faculdade de Medicina da Universidade de Sa˜o Paulo, Sa˜o Paulo/SP, Brazil. Email: rodrigobono@gmail.com Tel.: 55 11 30630207

INTRODUCTION

DISCUSSION

Iliac arteriovenous fistula complicating an iliac anastomotic pseudoaneurysm is a rare occurrence after abdominal aortic aneurysm open repair.1,2 We report the third case in the literature and the first case to be treated by endovascular means.

Anastomotic false aneurysm is a well-known complication after arterial revascularization procedures. The progression of atherosclerotic disease, wound infection, chronic hypertension, and weakness of the host artery are possible causes for its development.3 Arteriovenous fistula complicating an anastomotic pseudoaneurysm is also common. It has been observed following spine surgery, gynecological surgery, difficult labors, malignancies, and trauma.4,5 However, it is an extremely rare complication after abdominal aortic aneurysm open repair, which is one of the most commonly performed arterial reconstructive procedures. This complication has been reported only twice in the literature.1,2 In a patient with a past medical history of an abdominal aortic aneurysm open repair, clinical findings (such as abdominal pulsatile mass, intra-abdominal bruit, and

CASE REPORT A 71-year-old man underwent an abdominal aortic aneurysm open repair eleven years ago. The operation was performed with an aorto-bi-iliac Dacron graft. Three years later, the patient developed a right iliac artery occlusion. Correction was made with a femoral-to-femoral bypass. The patient presented in the emergency department with acute onset of orthopnea and exertional dyspnea. A physical examination indicated an abdominal thrill and murmur, jugular venous distention, cardiomegaly, and diminished distal pulses in both legs. His vital signs were within normal limits, and biochemical analysis revealed no significant abnormalities. A contrast-enhanced computed tomography angiography (CTA) scan revealed a left iliac artery anastomotic pseudoaneurysm complicated with an ilio-iliac arteriovenous fistula. The femoral-to-femoral bypass and the lower limb arteries were patent (Figures 1 and 2). An intra-operative arteriography confirmed the false aneurysm and arteriovenous fistula to the left iliac vein. The patient underwent endovascular repair with iliac extender grafts (‘‘Medtronic Talent 16612675; 146126105 and 16612675’’) in the left iliac artery, sealing the fistula. Control arteriography revealed no residual fistula, no endoleak and adequate lower limb perfusion. There were no postoperative complications, and the patient was discharged five days after surgery. His congestive heart failure symptoms disappeared. The follow-up CTA scan demonstrated no fistula, no endoleak and adequate lower limb perfusion (Figure 3).

Figure 1 - Contrast-enhanced computed tomography angiography (CTA), showing the anastomotic pseudoaneurysm and the ilio-iliac arteriovenous fistula. The inferior vena cava is fully contrasted, indicating an arteriovenous fistula.

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Figure 2 - 3D reconstruction, showing a fully contrasted aorta and inferior vena cava, in addition to a left iliac artery pseudoaneurysm with an ilio-iliac arteriovenous fistula and patency of the femoral-to-femoral bypass. Figure 3 - Postoperative contrast-enhanced computed tomography angiography reconstruction, showing complete resolution of the arteriovenous fistula and pseudoaneurysm.

murmur and acute symptoms of congestive heart failure) can lead to the diagnosis of an arteriovenous fistula complicating an anastomotic false aneurysm. Contrastenhanced CTA is the preferred noninvasive exam for confirming the diagnosis. It is also helpful for planning endovascular repair. Once diagnosed, arteriovenous fistula complicating an anastomotic false aneurysm must be treated to prevent its growth, rupture, and cardiac decompensation.6 Open techniques have been proposed for the treatment of complex aorto-iliac disease, but these cases involve high risks of bleeding, wound infection, and mortality. Endovascular techniques have been used to decrease these risks by reducing bleeding, the need for blood transfusion, operative time, and infection.7,8 There are no reports of the use of endovascular procedures for an arteriovenous fistula complicating an anastomotic pseudoaneurysm after an abdominal aortic aneurysm open repair. The two cases described in the literature1,2 were both treated with open surgical repair.

REFERENCES 1. Levy PJ, Holt JB, Close TP, Rush DS, Haynes JL. Rare presentation of anastomotic iliac artery false aneurysm: rupture with formation of ilioiliac arteriovenous fistula. Am Surg. 1993;59:713-5. 2. McCann RL, Makhoul R, Damiano R. Diagnosis of arteriovenous fistula by venous oxygen saturation: case report. J Vasc Surg. 1986;3:921-3, doi: 10.1067/mva.1986.avs0030921. 3. Aguiar ET, Langer B, Lobato AC. Risk of development of false aneurysm and graft infection after aorta-femoral bypass graft. Retrospective study. Report of 211 cases. J Mal Vasc. 1996;21:36-9. 4. Papadakos N, Wales L, Hayes K, Belli AM, Loftus I, Ray S. Posttraumatic pelvic pseudoaneurysm and arterio-venous fistula: combined endovascular and surgical approach. Eur J Vasc Endovasc Surg. 2008;36:164-6, doi: 10.1016/j.ejvs.2008.03.011. 5. Kiguchi M, Oâ&#x20AC;&#x2122;Rourke HJ, Dasyam A, Makaroun MS, Chaer RA. Endovascular repair of 2 iliac pseudoaneurysms and arteriovenous fistula following spine surgery. Vasc Endovascular Surg. 2010;44:126-30, doi: 10.1177/1538574409352809. 6. Wolosker N, Oba CM, Espirito Santo FR, Puech-Leao P. Endovascular treatment for chronic arteriovenous fistula between renal artery and inferior vena cava: image in vascular surgery. Vasc Endovascular Surg. 2010;44:489-90, doi: 10.1177/1538574410375124. 7. Puech-Leao P, Wolosker N, Zerati AE, Nascimento LD. Impact of endovascular technique in vascular surgery training at a large university hospital in Brazil. J Surg Educ. 2011;68:19-23, doi: 10.1016/j.jsurg.2010.08. 002. 8. Wolosker N, Mendes Cde A, Jacob CE, Wolosker AM, Puech-Leao P. Endovascular infrarenal aortic aneurysm repair combined with laparoscopic cholecystectomy. Clinics. 2010;65:743-4, doi: 10.1590/S180759322010000700015.

CONCLUSION Endovascular treatment appears to be an effective and minimally invasive alternative to open surgery for an arteriovenous fistula complicating an anastomotic pseudoaneurysm after an aorto-bi-iliac Dacron graft bypass.

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DOI:10.1590/S1807-59322011000800034

CASE REPORT

Clinical and cytogenetic aspects of giant cell tumor of the sternum Edgard Eduard Engel,I Marcello Henrique Nogueira-Barbosa,II Maurı´cio Eiji de Almeida Santos Yamashita,III Federico Enrique Garcia Cipriano,IV Elvis Terci Valera,V Maria Sol BrassescoV I

Faculty of Medicine of Ribeiraõ Preto, University of Saõ Paulo - Department of Orthopedics and Traumatology, Ribeiraõ Preto/SP, Brazil. II Faculty of Medicine of Ribeiraõ Preto, University of Saõ Paulo - Division of Diagnostic Imaging, Department of Internal Medicine, Ribeiraõ Preto/SP, Brazil. III Faculty of Medicine of Ribeiraõ Preto, University of Saõ Paulo - Department of Pathology, Ribeiraõ Preto/SP, Brazil. IV Faculty of Medicine of Ribeiraõ Preto, University of Saõ Paulo - Department of Surgery, Ribeiraõ Preto/SP, Brazil. V Faculty of Medicine of Ribeiraõ Preto, University of Saõ Paulo - Division of Pediatric Oncology, Department of Pediatrics, Ribeiraõ Preto/SP, Brazil. Email: engel@fmrp.usp.br Tel.: 55 16 3602-2416

and included additional areas of high signal intensity on the T2-weighted images (Figure 1D-E). Histology from a prior biopsy performed at an outside institution revealed a benign tumor composed of multinucleated giant cells compatible with a GCT or a brown

INTRODUCTION Giant cell tumor (GCT) is a benign, locally aggressive bone tumor that usually affects the metaphysis and the epiphysis of long tubular bones. It is most frequently located around the knee. It is commonly diagnosed in patients between the ages of 20 and 45 years.1 GCT metastasizes merely to the lungs in 2% of cases with unpredictable behavior. The sternum is infrequently affected by bone tumors. Although uncommon, malignant neoplasms, such as chondrosarcoma, osteosarcoma, myeloma, and lymphoma, are most likely to occur in this location.2 GCT of the sternum is an even rarer condition,3,4 and only seven cases are well documented in the literature.5-11 The present article describes the clinical and cytogenetic aspects of an additional case of GCT of the sternum occurring in a young male and includes a discussion of the surgical treatment.

Case Report A 32-year-old man presented with a sternal mass that had shown progressive growth toward the suprasternal notch over the previous ten months. He complained of local pain that radiated to the cervical region and shoulders. Physical examination showed a bony, tender, six cm large mass of the manubrium (Figure 1A). The patient’s pain was increased by palpation and elevation of his arms. Plain radiographs showed an expansile osteolytic lesion of the sternum. Computed tomography (CT) and magnetic resonance imaging (MRI) demonstrated focal cortical bone destruction, soft tissue extension and subchondral extension in relation to the sternoclavicular joints. CT images confirmed the osteolytic nature of the tumor and the absence of internal mineralization. Post-contrast CT and MRI demonstrated small foci that lacked the enhancement consistent with cystic or hemorrhagic areas. MRI showed that the lesion was of intermediate signal intensity on T1- and T2-weighted images

Figure 1 - A) Clinical aspect before and after surgery; B) intraoperative aspect after sternum resection; C) intraoperative aspect after reconstruction; and D) CT sagittal reconstruction showing anterior focal cortical bone destruction and soft tissue extension (arrow). In the post-contrast image, it is also possible to identify a small region without contrast enhancement (arrowhead). E) Axial T2-weighted MRI revealed intermediate signal intensity predominance in the sternum giant cell tumor (arrow), although regions of high signal intensity were also present (arrowhead).

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Figure 2 - A-D) Histological aspect of the lesion showing a triphasic aspect with giant, mononuclear and fusiform cells (B), bone (C) and soft tissue (D) invasion of the tumor, and cystic formations (H&E, A - 4006, B - 2006, C - 1006, D - 1006); E) Cytogenetic analysis of a GCT sample showing a 51, XY, 27, +9, del(10)(q23), add(11)(p15)62, 215, add(18)(q23), +mar1, +mar262, +mar3, +mar4, +mar5 karyotype.

tumor of hyperparathyroidism. Laboratory evaluation revealed normal levels of calcium (1.25 MMol/l) and parathyroid hormone (21 PG/ml), making the diagnosis of a brown tumor highly unlikely. The patient underwent resection of the manubrium with wide margins and post-resection reconstruction with

GOREH DUALMESHH PLUS Biomaterial (W. L. Gore & Associates, Inc., Flagstaff, USA) mesh fixed with polyester separated stitches. Both the clavicles and the first ribs were fixed to the mesh. Thoracic inspection showed no mediastinal or lung nodules. Disarticulation of the bodymanubrium joint was uneventful. A bilateral pectoralis

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Giant Cell Tumor of the Sternum Engel EE et al.

Table 1 - Reported cases of giant cell tumor (GCT) of the sternum. Reference

Gender

Age (years)

Symptom

Location

Size (cm)

Surgery

Reconstruction

Follow-up (months)

Cytogenetic study

painless

manubrium

none

pain

manubrium

3.963.2

prosthesis

Yes

pain

body

3.563.0

subtotal sternectomy subtotal sternectomy curretage

PMMA

pain

body

8.564.562.5

prosthesis

pain

body

86462.5

subtotal sternectomy curretage

PMMA

painful mass

body

6.464.364.4

prosthesis

painful mass

body

1267.564.5

fascia lata

painful mass

manubrium

7,665,164,7

subtotal sternectomy subtotal sternectomy subtotal sternectomy

Gore Dualmesh PlusH

Yes

Sundaram et al., 1982 Bay et al., 1999 Segawa et al., 2004 Imai et al., 2006 Futani et al., 2008 Abate et al., 2009 Faria et al., 2010 Current case

Abbreviations: NA, data not available; PMMA, bone cement filling.

et al. suggest that the initial treatment should involve extended curettage followed by filling with bone cement (Polimetilmetacrilate - PMMA). Because local recurrence rates reach approximately 50% due to the locally aggressive behavior of GCTs, more aggressive forms of surgery, such as marginal or wide resection, should be considered.8,9 Futani et al. suggest that a prosthetic replacement is needed after a subtotal sternectomy to protect the lungs, heart, and main vessels as well as to restore functional thoracic movement to prevent paradoxical respiration.9 As described by Gonfiotti et al., we did not use a rigid prosthetic replacement after the partial sternectomy. No respiratory distress was observed in our patient.13 Three of the eight described cases affected the manubrium and were treated by subtotal sternectomy. Reconstructions were performed with prosthetics in one case, with nothing in one case and with a mesh in our case. In the five cases that affected the body of the sternum, two were treated with curettage and PMMA filling and three with subtotal sternectomy and reconstruction (with a prosthesis in two cases and fascia lata in one) (Table 1). There are only limited data in the literature on chromosomal aberrations in GCT of the bone. Typically, this tumor is characterized by a high frequency of telomeric fusion, which has been implicated in the initiation of chromosomal instability and tumorigenesis.14 Correspondingly, GTGbanding analysis has demonstrated a more frequent involvement of chromosomes 11p, 13p, 15p, 18p, 19p, and 21p in this process.15 Cytogenetic analysis of our sample showed a complex karyotype characterized by the presence of clonal rearrangements and numerous marker chromosomes. In the literature, some clonal aberrations have been described.15-17 Most notably, Bridge et al. demonstrated clonal structural translocations involving 11p15 in more than one patient; this region was also affected in our tumor sample.15 Sawyer et al. described abnormal karyotypes with clonal telomeric fusions involving the short arm of chromosome 11.18 In three of the five cases studied, telomeric fusions of 11pter were apparently the precursor lesions to the progression of sub-clones of 11p with structural chromosomal aberrations. Sternal GCT is a very rare disease and should be treated with partial sternectomy. Reconstruction may be done with rigid or flexible materials depending on the stability of the

major flap covered the mesh and allowed skin closure (Figure 1B-C). Histological analysis of the resected specimen revealed the presence of multinucleated giant cells, mononucleated small cells and spindle cells with elongated nuclei without atypias. Occasionally, more than 100 nuclei could be perceived in the giant cells, a typical characteristic of GCT (Figure 2A). The tumor growth had induced extensive bone erosion and focally infiltrated adjacent soft tissues (Figure 2B-C). Blood-filled dilated spaces were seen, suggesting an association with an aneurysmal bone cyst (Figure 2D). Less than one typical mitotic figure was seen per ten high power fields. Atypical mitosis, vascular invasion, and tumor necrosis were not detected. To allow for cytogenetic studies, a fresh GCT sample (adjacent to areas of the tumor verified by frozen section) was aseptically collected in the operating room and processed as previously described.12 Cytogenetic analysis was performed on 30 metaphases and showed a complex complement characterized by the presence of numerous marker chromosomes. The karyotype was denoted as 51, XY, -7, +9, del(10)(q23), add(11)(p15)x2, -15, add(18)(q23), +mar1, +mar262, +mar3, +mar4, +mar5 (Figure 2E).

DISCUSSION In the seven previously presented cases and this additional case, the median age of diagnosis is 48 years (range: 28 to 74 years); this age is slightly higher than that of GCT patients in general (Table 1). Although imaging can strongly indicate GCT, a biopsy is necessary for diagnosis. Biopsy is of particular importance when the neoplasm affects an unusual location. The variety of giant cell-containing bone tumors (including osteosarcoma, chondroblastoma, and aneurysmal bone cysts) can cause biopsies with small sampling or fine-needle aspirations to be misleading.10 Even with larger tumor samples, it may be difficult to distinguish between GCT and parathyroid brown tumor, and it is necessary to examine specific hormone, calcium, and phosphate serum levels. There is no consensus regarding the optimal surgical treatment of sternal GCT; interventions ranging from simple curettage to wide resection have been proposed.9 Futani

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8. Imai K, Minamiya Y, Saito H, Kawai H, Ito M, Ogawa J. Giant cell tumor of the sternum. Jpn J Thorac Cardiovasc Surg. 2006;54:405-8, doi: 10. 1007/s11748-006-0017-2. 9. Futani H, Okumura Y, Fukuda Y, Fukunaga S, Hasegawa S, Yoshiya S. Giant cell tumor of the sternum: a case report and review of the literature. Anticancer Res. 2008;28:4117-20. 10. Abate E, Banki F, Hagen JA, Klipfel N. Giant cell tumor of the sternum. Ann Thorac Surg. 2009;88:645-7, doi: 10.1016/j.athoracsur.2008.12.059. 11. Faria RA, Silva CM, Miziara JEA, Melo FY, Silva SRM, Viana CR. Giant cell tumor of the sternum. J Bras Pneumol. 2010;36:517-20, doi: 10.1590/ S1806-37132010000400020. 12. Brassesco MS, Valera ET, Neder L, Castro-Gamero AM, Arruda D, Machado HR, et al. Polyploidy in atypical grade II choroid plexus papilloma of the posterior fossa. Neuropathology 2009;29:293-8. 13. Gonfiotti A, Santini PF, Campanacci D, Innocenti M, Ferrarello S, Caldarella A, et al. Malignant primary chest-wall tumours: techniques of reconstruction and survival. Eur J Cardiothorac Surg. 2010;38:39-45, doi: 10.1016/j.ejcts.2009.12.046. 14. Zheng MH, Siu P, Papadimitriou JM, Wood DJ, Murch AR. Telomeric fusion is a major cytogenetic aberration of giant cell tumors of bone. Pathology. 1999;31:373-8, doi: 10.1080/003130299104756. 15. Bridge JA, Neff JR, Mouron BJ. Giant cell tumor of bone. Chromosomal analysis of 48 specimens and review of the literature. Cancer Genet Cytogenet 1992;58:2â&#x20AC;&#x201C;13. 16. Molenaar WM, Van den Berg E, Dolfin AC, Zorgdrager H, Hoekstra HJ. Cytogenetics of fine needle aspiration biopsies of sarcomas. Cancer Genet Cytogenet. 1995;84:27â&#x20AC;&#x201C;31, doi: 10.1016/0165-4608(95)00068-2. 17. Sciot R, Dorfman H, Brys P, Dal Cin P, De Wever I, Fletcher CD, et al. Cytogenetic-morphologic correlations in aneurysmal bone cyst, giant cell tumor of bone and combined lesions. A report from the CHAMP study group. Mod Pathol. 2000;13:1206-10. 18. Sawyer JR, Goosen LS, Binz RL, Swanson CM, Nicholas RW. Evidence for telomeric fusions as a mechanism for recurring structural aberrations of chromosome 11 in giant cell tumor of bone. Cancer Genet Cytogenet. 2005;159:32-6, doi: 10.1016/j.cancergencyto.2004.09.001.

chest wall. Biopsy is indispensable, and the ability to differentiate between GCT and hyperparathyroidism depends on blood sampling. Although no association has been established between unfavorable outcomes and chromosomal changes other than telomeric associations, the frequent involvement of 11p15 in clonal rearrangements suggests that chromosomal changes play an important role in GCT initiation and progression. Whether this alteration is associated with this particular tumor location requires further investigation.

REFERENCES 1. Reid R, Banerjee SS, Sciot S. Giant cell tumor in: In: Fletcher CDM, Unni KK, Mertens F, editors. World health organization classification of tumors. Pathology and genetics of tumors of soft tissue and bone. 1st ed. Lyon: IARC Press 2002;p.234-6. 2. Downey RJ, Huvos AG, Martini N. Primary and secondary malignancies of the sternum. Semin Thorac Cardiovasc Surg. 1999;11:293-6. 3. Campanacci M, Baldini N, Boriani S, Sudanese A. Giant cell tumor of bone. J Bone Joint Surg Am. 1987;69:106-14. 4. Campanacci M. Giant cell tumor. In: Campanacci M, editor. Bone and soft tissue tumors. 2nd ed.Wien: Springer-Verlag. 1999;p.99 5. Sundaram M, Martin SA, Johnson FE and Wolverson MK. Case report 198. Giant cell tumor of manubrium. Skeletal Radiol. 1982;8:225-7, doi: 10.1007/BF00355512. 6. Bay JO, Bignon YJ, Gros P, Jancovici R, Desangles F, Dubayle P, et al. Giant cell tumor of the sternum. Report of a case with a 17q isochromosome. Rev Rhum Engl Ed. 1999;66:49-52. 7. Segawa T, Kanamori M, Ohmori K, Nobukiyo M, Nogami S, Kimura T, et al. Giant cell tumor of the sternum: a case report. J Orthop Sci. 2004;9:175-7, doi: 10.1007/s00776-003-0752-5.

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DOI:10.1590/S1807-59322011000800035

CASE REPORT

Primary progressive aphasia beginning with a psychiatric disorder Leonardo Caixeta, Marcelo Caixeta Neuropsychiatry, Faculdade de Medicina da Universidade Federal de Goia´s, Goiaˆnia/GO, Brazil. Email: leonardocaixeta1@gmail.com Tel.: 55 62 3204-7223

frequent pauses, phonemic paraphasias, and transpositional errors (e.g., ‘animal’ pronounced as ‘amimal’). Her insight was preserved. Two years ago, she began to show diminished interest in her daily activities. One year ago, the process of dementia became more evident, with a progressive loss of independence and an increasing need for help from caregivers. A magnetic resonance imaging (MRI) showed focal left temporal lobe atrophy (Figure 1). Ethyl cysteinate dimer (ECD) brain single-photon emission computed tomography (SPECT) showed severe hypoperfusion in the left temporal and inferior frontal regions (Figure 2). Both MRI and SPECT showed frank asymmetrical finds, and the left hemisphere more affected than the right. Neuropsychological assessment was conducted with difficulty because of the severe communication and behavioral disturbances presented by the patient. As the majority of the classical neuropsychological tests are based on language skills, we resorted to more ecologically designed tests to examine the patient’s cognitive function. She showed striking deficits in language tests, executive functions, and visuospatial processing. In addition, the patient exhibited strongly impaired verbal fluency (she was only able to name one animal in one minute in a verbal fluency task) and severe anomia (the inability to name even very easy figures, such as a house, tree, and comb in the Boston Naming Test). Her attention was impaired in many domains (visuospatial inattention, inability to maintain focus, distractibility, and working memory), and she could not recite automated sequences (e.g., counting from one to ten). Language tests revealed that both comprehension and expression of language were similarly impaired. She seemed be unable to recognize the colors and geometrical shapes presented in the Token Test and could not read (alexia) or write (agraphia). The patient’s speech output was normal; however, it was unintelligible, and she could not engage in a conversation. She did not recognize famous faces (e.g., Pele´, Roberto Carlos, and Lula). Her performance was impaired even in neuropsychological tests less dependent on language skills. She showed constructive apraxia (she could not reproduce simple geometric forms) and some aperceptive agnosia (the Boston Naming Test). Her final Mini-mental State Examination (MMSE) score was five (one point for the day of the week, two points for immediate memory, and two points for verbal command). Finally, the patient was unable to understand how to arrange numbers to form a clock. The patient was submitted to a workup for the differential diagnosis of dementia and to search for organic causes of

INTRODUCTION Primary progressive aphasia (PPA), frontotemporal dementia (FTD), and semantic dementia (SD) are clinical subtypes of frontotemporal lobar degeneration (FTLD). PPA is characterized by marked changes in language ability.1 Although anxiety is identified in 39% of FTLD subjects, agitation, irritability, and depression may also be observed.2 Frustration and anxiety have been frequently reported as early symptoms of PPA, but they are commonly construed by patients and their relatives as secondary consequences of the social embarrassment caused by the difficulty with language.3 Similarly, the avoidance of social situations by these patients is frequently interpreted as a reaction to the loss of language abilities. Here, we report a patient with PPA who initially presented with panic attacks. To the best of our knowledge, this is the first case of PPA manifesting with panic attacks as the first symptom. This case provides further evidence of the variable and circumscribed nature of the clinical presentation of focal cerebral degeneration.

CASE REPORT A 75-year-old, right-handed widowed woman with 2 years of formal education first experienced a panic attack four years ago at the age of 71. She had no previous history of psychiatric disease or panic disorder. The panic attack was characterized by a sudden onset of symptoms, including palpitations, trembling, a sensation of shortness of breath, and a feeling of choking, which reached a peak within 10 minutes. During the subsequent months, the patient suffered from recurrent and unexpected panic attacks several times a week in crowded places and became afraid of feeling bad again in such places, soon developing agoraphobia. She was therefore given a diagnosis of panic disorder with agoraphobia. During the next few months, her panic attacks reduced in frequency after being prescribed sertraline at a dose of 50 mg/d. Three years ago, the patient gradually started showing progressive isolated loss of language function (without significant impairment in other cognitive domains) characterized by a decline in linguistic fluency, word-finding difficulties, effortful speech, hesitant utterances with

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to find a panic disorder (usually a disorder of young people) beginning in old age.11 Moreover, the panic disorder presentation in this case was atypical, with fewer psychological symptoms (e.g., depersonalization, derealization, feeling of imminent death) when compared with classic (functional) panic disorder beginning at an earlier age. Thus, it is likely that the patient’s first symptom of panic attacks at the age of 70 years was a symptom of early-stage dementia. Traditionally, PPA is described primarily as a language deficit disorder. Conversely, FTD is essentially defined as a behavioral variant of FTLD. Our case demonstrates that these notions of PPA and FTD should not be viewed as rigid rules and that PPA can initially manifest with behavioral symptoms. In fact, PPA may be mistaken for a primary psychiatric disorder in many clinical situations10. Several neuropsychiatric symptoms such as depression, apathy and agitation have been reported in PPA.5 Panic attacks have only been reported as a first symptom in one case of FTLD, but this case involved FTD and not PPA.6 Therefore, this is the first report of PPA presenting with panic attacks and agoraphobia as the initial symptoms. In our case, anxiety disorder cannot be described as a secondary consequence of the social embarrassment produced by language difficulties in PPA, as commonly assumed, as the panic attacks began before any language disorder was apparent. Similarly, the social withdrawal in this case cannot be explained as a psychological reaction to the social embarrassment produced by limited language skills; rather, it is better understood as an agoraphobic response to crowded environments. The progressive language deterioration observed in this case, including the anomia, reduced spontaneous verbal output, loss of fluency, and phonemic paraphasic errors, is consistent with the clinical features of progressive aphasia as defined by Neary et al.7 The findings of both focal atrophy of the left temporal lobe on MRI and left frontotemporal hypoperfusion on the ECD SPECT support the diagnosis of PPA. In contrast, episodic and semantic memory, perception, and spatial skills were all well preserved. Therefore, the patient was diagnosed with primary progressive aphasia. A neuroanatomical hypothesis has suggested that panic disorder may result from the dysfunction of the ‘‘fear network’’ located in the center of the amygdala. A recent neuroimaging study8 indicated that subjects with panic disorder have a smaller-volume amygdala compared with normal healthy subjects. Even in the early stages of FTLD,

Figure 1 - MRI (axial T1) showing focal atrophy of the left temporal lobe.

anxiety. A complete blood count with differential; renal, liver, and thyroid function tests; determination of vitamin B12 and folic acid levels; syphilis serology; urinalysis; lipidogram; glycemic testing; hemosedimentation speed; and electrocardiogram and electroencephalogram were conducted. All of these test results were normal.

DISCUSSION This case presented with panic attacks as a symptom of the initial phase of PPA. In this phase, the patient fulfilled the DSM-IV4 diagnostic criteria for panic disorder with agoraphobia. Her language abilities gradually changed after the appearance of the panic attacks, with progressive anomia, loss of fluency, reduced verbal output, and inadequate use of generic words. Her previous medical and psychiatric histories were unremarkable, and she had no personal or familiar antecedents of panic disorder. It is rare

Figure 2 - SPECT (sequence of axial slices) showing left frontotemporal focal hypoperfusion.

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Primary progressive and psychiatric disorder Caixeta L and Caixeta M 2. Porter VR, Buxton WG, Fairbanks LA, Strickland T, O’Connor SM, Rosenberg-Thompson S, et al. Frequency and characteristics of anxiety among patients with Alzheimer’s disease and related dementias. J Neuropsychiatry Clin Neurosci. 2003;15:180–6, doi: 10.1176/appi. neuropsych.15.2.180. 3. Rosen HJ, Allison SC, Ogar JM, Amici S, Rose K, Dronkers N, et al. Behavioral features in semantic dementia vs other forms of progressive aphasias. Neurology. 2006;67:1752-6, doi: 10.1212/01.wnl.0000247630. 29222.34. 4. American Psychiatric Association. Diagnostic and statistical manual of mental disorders. Washington, APA, (4th ed., text revision), 2000. 5. Rohrer JD, Warren JD. Phenomenology and anatomy of abnormal behaviors in primary progressive aphasia. J Neurol Sci. 2010;293:35-8, doi: 10.1016/j.jns.2010.03.012. 6. Nakaaki S, Murata Y, Shinagawa Y, Hongo J, Furukawa TA, Sato J, et al. A case of frontotemporal lobar degeneration (FTLD) with panic attack as the first symptom. J Neuropsychiatry Clin Neurosci. 2007;19:485-7, doi: 10.1176/appi.neuropsych.19.4.485. 7. Neary D, Snowden JS, Gustafson L, Passant U, Stuss D, Black S, et al. Frontotemporal lobar degeneration: a consensus on clinical diagnostic criteria. Neurology. 1998;51:1546-54. 8. Massana G, Serra-Grabulosa JM, Salgado-Pineda P, Gasto´ C, Junque´ C, Massana J, et al. Amygdalar atrophy in panic disorder patients detected by volumetric magnetic resonance imaging. Neuroimage. 2003;19:80–90, doi: 10.1016/S1053-8119(03)00036-3. 9. Boccardi M, Pennanen C, Laakso MP, Testa C, Geroldi C, Soininen H, et al. Amygdaloid atrophy in frontotemporal dementia and Alzheimer’s disease. Neurosci Lett. 2002;25:139–43, doi: 10.1016/S0304-3940(02) 01169-2. 10. Philbrick KL, Rummans TA, Duffy JR, Kokmen E, Jack CR Jr. Primary progressive aphasia. An uncommon masquerader of psychiatric disorders. Psychosomatics. 1994;35:138-41. 11. Luchins DJ, Rose RP. Late-life onset of panic disorder with agoraphobia in three patients. Am J Psychiatry. 1989;146:920-1.

atrophy of both the frontal lobe and the amygdala has been shown by quantitative structural neuroimaging.9 In our case, the most affected brain area in the early phases of the disease was the temporal lobe, where the amygdala is located. Therefore, dysfunction of the amygdala might be associated with the panic attacks in this case. This finding may also be an important indicator of the precise neuroanatomical location where the neuropathological process of PPA begins. In conclusion, we must carefully observe the course of elderly subjects diagnosed with panic disorder in old age because this psychiatric phenomenon may represent the beginning of a degenerative process involving dementia. Moreover, it is important to consider that PPA can manifest first with behavioral symptoms. Thus, behavioral symptoms should no longer be used as a characteristic that distinguishes between the early clinical indicators of PPA and FTD, as this class of symptoms is not exclusive to the latter. Further study may lend support to our assertion that PPA is inaugurated by psychiatric symptoms whose phenomenology is linked to the pathology of frontotemporal regions of the left hemisphere.

REFERENCES 1. Mesulam MM. Primary progressive aphasia: a 25-year retrospective. Alzheimer Dis Assoc Disord. 2007;21:S8-S11, doi: 10.1097/WAD. 0b013e31815bf7e1.

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DOI:10.1590/S1807-59322011000800036

CASE REPORT

Contact-lens-related corneal ulcer caused by klebsiella pneumoniae ¨ zsoy,I Mufide Cavdar,I Yusuf YakupogullariII Tongabay Cumurcu,I Pembegul Firat,I Ercan O I Inonu University School of Medicine, Department of Ophthalmology, Malatya/Turkey. Microbiology, Malatya/Turkey.

Inonu University School of Medicine, Department of

Email: tongabay@superonline.com Tel.: 90-422-342 06 60/4003

After clinical improvement (four days later), topical corticosteroid drops were prescribed. The infection resolved, but residual central stromal opacification remained (Figure 2). The patient achieved a final visual acuity of 20/100 in the right eye.

INTRODUCTION The use of contact lenses (CLs) has been widely associated with corneal alterations that range in symptomatology and severity. Lesions may vary from small peripheral sterile infiltrates to infectious central ulcers with sight-threatening potential.1 Contact lens wear has been described as the most important predisposing factor to microbial keratitis worldwide.2 Infections are more frequent in soft-lens users, and the risk with gas-permeable rigid lenses is estimated to be onethird of that with daily-wear soft lenses. Extended wear is also associated with a higher risk of microbial keratitis.3 Bacterial keratitis is most commonly caused by aggressive gram-negative Pseudomonas spp.; Klebsiella spp. is rarely the causative agent of keratitis due to contact lens wear.4 We present a case of contact-lens-related bacterial keratitis caused by Klebsiella pneumoniae.

DISCUSSION Contact-lens–related corneal infections continue to present a major challenge to ophthalmologists. With the continuous improvement in lens materials and design, as well as in disinfecting and storing solutions, contact lens use is on the rise. Currently, patients can use a single solution to rinse, disinfect, and store their lenses without the need for rubbing or enzymatic cleaning.5 The patient described herein used multipurpose solution for cleaning, disinfecting, and storing daily-wear soft contact lenses. The extended wear reported may have caused this case of microbial keratitis.3 Gram-negative bacteria include opportunistic enterobac-teria (such as Serratia spp. and Klebsiella spp.) that can survive in contact lens fluid and on plastic surfaces, which explains their increased numbers in contact-lens-induced corneal infections.6 Contact-lens-related keratitis may vary from small peripheral sterile infiltrates to infectious central ulcers with sight-threatening potential, as in our case.7 Overnight wear and, to a lesser extent, improper lens care are known to be major risk factors for corneal infection.8

Case Presentation A 19-year-old man presented to our service complaining of pain and hyperemia of two days’ duration in the right eye. He had been a regular contact lens wearer for two years. The patient used multipurpose solutions for cleaning, disinfecting, and storing daily-wear soft contact lenses. Recently, the lenses had been worn for extended periods of time. His visual acuity was at the level of finger counting. Ophthalmologic examination revealed central corneal keratitis in his right eye (Figure 1) Samples from conjunctival swabbing and corneal scraping, the contact lens, the cleaning solution, and the case were all submitted for microbiological analysis. Gram staining revealed gram-negative microorganisms in the corneal scraping and in the contact lens cleaning solution. Cultures on appropriate media were positive for Klebsiella pneumoniae in these specimens. The organism was susceptible to aminoglycosides and to fourth-generation fluoroquinolones. The patient was treated topically with moxifloxacin (0.5%) and tobramycin (15 mg/ml) drops every hour and cyclopentolate 2% three times daily in the right eye. N-acetylcysteine eye drops were administered four times per day.

Figure 1 - Central corneal keratitis in the right eye.

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The majority of keratitis was caused by soft contact lens wear. One-fifth of these comparatively young patients displayed corneal scars.12 This case highlights the need for clinicians to be aware that Klebsiella spp. can induce contactlens-related corneal ulcers.

REFERENCES 1. Moriyama AS, Hofling-Lima AL. Contact lens-associated microbial keratitis. Arq Bras Oftalmol. 2008;71:32-6, doi: 10.1590/S0004-27492008000700007. 2. Bourcier T, Thomas F, Borderie V, Chaumeil C, Laroche L. Bacterial keratitis: predisposing factors, clinical and microbiological review of 300 cases. Br J Ophthalmol. 2003;87:834-8. 3. Lam DS, Houang E, Fan DS, Lyon D, Seal D, Wong E, et al. I˙ncidence and Risk Factors for Microbial Keratitis in Hong Kong: comparison with Europe and North America. Eye. 2002;16:608-18, doi: 10.1038/sj.eye. 6700151. 4. Stapleton F, Keay LJ, Sanfilippo PG, Katiyar S, Edwards KP, Naduvilath T. Relationship between climate, disease severity, and causative organism for contact lens-associated microbial keratitis in Australia. Am J Ophthalmol. 2007;144:690-8. 5. Najjar DM, Aktan SG, Rapuano CJ, Laibson PR, Cohen EJ. Contact lensrelated corneal ulcers in compliant patients. Am J Ophthalmol. 2004;137:170-2. 6. Holden BA, La Hood D, Grant T, Newton-Howes J, Baleriola-Lucas C, Willcox MD, et al. Gram-negative bacteria can induce contact lens related acute red eye (CLARE) responses. CLAO J. 1996;22:47–52. 7. Efron N, Morgan PB. Can subtypes of contact lens-associated corneal infiltrative events be clinically differentiated? Cornea. 2006;25:540-4, doi: 10.1097/01.ico.0000214219.67872.3c. 8. Cheng KH, Leung SL, Hoekman HW, Beekhuis WH, Mulder PG, Geerards AJ, et al. Incidence of contact-lens-associated microbial keratitis and its related morbidity. Lancet. 354:181–5, doi: 10.1016/S01406736(98)09385-4. 9. Yilmaz S, Ozturk I, Maden A. Microbial keratitis in West Anatolia, Turkey: aretrospective review. Int Ophthalmol. 2007;27:261-8, doi: 10. 1007/s10792-007-9069-2. 10. Palamar M, Masarogulları M, Egrilmez S, Aydemir S, Yagcı A. Our Microbiological Analysis Results in Microbial Contact Lens Keratitis. Turk J Ophthalmol. 2010;40:349-53. 11. Pinna A, Sechi LA, Zanetti S, Carta F. Detection of Virulance Factors in a corneal Isolate of Klebsiella pneumoniae. Ophthalmology. 2005;112:883-7, doi: 10.1016/j.ophtha.2004.12.024. 12. Neumaier-Ammerer B, Stolba U, Feichtinger H, Bindler S. Contact lens related corneal infiltrates and ulcers – a retrospective study of 134 eyes. Spektrum Augenheilkd. 2008;22:297–300, doi: 10.1007/s00717-008-0284-7.

Figure 2 - The infection resolved, but residual central stromal opacification remained in the right eye.

Lam et al. reported a five-fold increase in the risk of microbial keratitis among patients who wear their contact lenses overnight.3 As reported by several authors, contact-lens-induced keratitis is mainly caused by gram-negative micro-organisms.4,8 However, Klebsiella spp. are not a common cause of bacterial keratitis. One study that investigated the causes of microbial keratitis showed that 3.2% of microbial keratitis cases were due to contact lens use. The same study showed that in 2.2% of these keratitis patients, Enterobacter spp. was the causative agent.9 In another study, 33 patients with contact-lensrelated keratitis were followed. Klebsiella spp. were observed in the cultures of only two patients.10 N-acetylcysteine is a potential candidate for use as an inhibitor of Klebsiella biofilm formation. Therefore, we added N-acetylcysteine eye drops to the patient’s treatment regimen.11

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DOI:10.1590/S1807-59322011000800037

ERRATA CLINICS 2011;66(6):1101-1104 Page 1101 Replace Regina Ce´lia Gomes Teixeira,I Carina Verna,II Ricardo S Simo˜es,III Roberta B Wolff,II Edmund C. Baracat,III Jose´ Maria Soares-JrII For Regina Ce´lia Teixeira Gomes,I Carina Verna,II Ricardo S Simo˜es,III Roberta B Wolff,II Edmund C. Baracat,III Jose´ Maria Soares-JrII

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