CLINICS July 2012

I S S N - 1807-5932 printed version I S S N - 1980-5322 online version

Volume 67 Number 7 - July/2012

Official Scientific Journal of Faculdade de Medicina and Hospital das ClĂ­nicas Universidade de SĂŁo Paulo

CLINICS Editor Mauricio Rocha-e-Silva Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Area Editors Ana Maria de Ulhoa Escobar Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Anuar Ibrahim Mitre Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ashok Agarwal The Cleveland Clinic Foundation Cleveland, Ohio, USA Berenice Bilharinho Mendonc¸a Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Bruno Zilberstein 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 Carmen Silvia Valente Barbas Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Claudia Regina Furquim de Andrade Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Edmund Chada Baracat Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Eliete Bouskela Universidade do Estado do Rio de Janeiro Rio de Janeiro, RJ, Brazil

Gustavo Franco Carvalhal Faculdade de Medicina da Pontifı´cia Universidade Cato´lica do Rio Grande do Sul Porto Alegre, Rio Grande do Sul, Brazil Heitor Franco de Andrade Jr. Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ivete Bedin Prado 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 Ludhmila Abrahao Hajjar 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 Lydia Masako Ferreira Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Maria Cecı´lia Solimene Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Nelson Wolosker Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Newton Kara-Junior Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Emilia Inoue Sato Universidade Federal 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

Fulvio Alexandre Scorza Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Geraldo Busatto 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

Raul Coimbra University of California, San Diego La Jolla, CA, USA Renato Delascio Lopes Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Rubens Belfort Jr. Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ruth Guinsburg Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ruy Jorge Cruz Junior University of Pittsburgh Pittsburgh, PA, USA Sergio Paulo Bydlowski Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Silvia Vanessa Lourenc¸o Faculdade de Odontologia da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Simone Appenzeller Universidade Estadual de Campinas Campinas, SP, Brazil Sophie Franc¸oise Mauricette Derchain Faculdade de Cieˆncias Me´dicas, Universidade Estadual de Campinas Campinas, SP, Brazil Suely Kazue Nagahashi Marie 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 Vale´ria Aoki Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

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

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

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

Marco Martins Amatuzzi Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o 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

Daniel Romero Mun˜oz Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Irismar Reis de Oliveira Faculdade de Medicina da Universidade Federal da Bahia Salvador, BA, Brasil

Maria Aparecida Shikanai Yasuda Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Abhijit Chandra King George’s Medical College Lucknow, India

Artur Brum-Fernandes Universite´ de Sherbrooke Que´bec, Canada´

Adamastor Humberto Pereira Universidade Federal do Rio Grande do Sul Porto Alegre, RS, Brazil

Carmita Helena Najjar Abdo Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Adauto Castelo Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ademar Lopes Fundac¸a˜o Antoˆnio Prudente, Hospital do Caˆncer Sa˜o Paulo, SP, Brazil Alberto Azoubel Antunes 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

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

Andrea Schmitt University of Goettingen Goettingen, Germany

Ernest Eugene Moore University of Colorado Denver Denver, CO, USA

Arnaldo Valdir Zumiotti 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

Mauro Perretti William Harvey Research Institute London, UK

Irshad Chaudry University of Alabama Birmingham, AL, USA

Michael Gregory Sarr Mayo Clinic Rochester, MN, USA

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

Milton de Arruda Martins Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Ke-Seng Zhao Southern Medical University Guangzhou, China

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

Laura Cunha Rodrigues London School of Hygiene and Tropical Medicine - University of London London, UK

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

Navantino Alves Faculdade de Cieˆncias Me´dicas de Minas Gerais Belo Horizonte, MG, Brazil Noedir Antonio Groppo Stolf Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Pedro Puech-Lea˜o Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o 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

Samir Rasslan Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Tarcisio Eloy Pessoa de Barros Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Valentim Gentil 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 Alberto Jose´ da Silva Duarte Ana Claudia Latronico Xavier Berenice Bilharinho de Mendonc¸a Clarice Tanaka Claudia Regina Furquim de Andrade Cyro Festa Neto Dalton de Alencar Fischer Chamone Daniel Romero Mun˜oz Edmund Chada Baracat Eduardo Massad Eloisa Silva Dutra de Oliveira Bonfa´ Euripedes Constantino Miguel Fa´bio Biscegli Jatene Flair Jose´ Carrilho Francisco Vargas Suso Gerson Chadi Gilberto Luis Camanho Irene de Lourdes Noronha Irineu Tadeu Velasco Ivan Cecconello Jorge Elias Kalil

Jose´ Antonio Franchini Ramires Jose´ Antonio Sanches Jose´ Eduardo Krieger Jose´ Ota´vio Costa Auler Jose´ Ricardo de Carvalho Mesquita Ayres Lenine Garcia Branda˜o Luiz Augusto Carneiro D’Albuquerque Luiz Fernando Onuchic Magda Maria Sales Carneiro-Sampaio Manoel Jacobsen Teixeira Marcelo Zugaib Marcos Boulos Marcus Castro Ferreira Maria Aparecida Shikanai Yasuda Maria Irma Seixas Duarte Miguel Srougi Milton de Arruda Martins Nelson de Luccia Noedir Antonio Groppo Stolf Olavo Pires de Camargo Paulo Andrade Lotufo

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

Paulo Hila´rio Nascimento Saldiva Paulo Marcelo Gehm Hoff Pedro Puech-Lea˜o Remo Susanna Ricardo Ferreira Bento Ricardo Nitrini Roberto Kalil 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 Werther Brunow de Carvalho William Carlos Nahas Wilson Jacob

Editorial Assistants Nair Gomes Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Daniela Aquemi Higa Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ariane Maris Gomes 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 CEP 05403-010 Sa˜o Paulo/SP Tel.: +55-11-2661-6235 Email: clinics.office@gmail.com Website: www.scielo.br/clinics Submission: http://mc04.manuscriptcentral.com/clinics-scielo 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. Sa˜o Paulo: Scientific Journal of Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, 2005Monthly Periodical: January to December ISSN 1807-5932 printed version ISSN 1980-5322 online version Formerly Revista do Hospital das Clı´nicas da FMUSP, 1946–2004.

1. Medicine-scientific production. 2. Medical Sciences I. Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o 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, 2012 must be registered prospectively (before patient recruitment begins) in a publicly accessible registry. Trials initiated before January 1, 2012 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.

Publication Fees CLINICS uses a business model in which expenses are recovered in part by charging a publication fee to the authors or research sponsors for each published article. Our 2012 prices are as follows: fast track: R$ 2.500,00 (US$ 1,500.00); original articles: R$ 1.850,00 (US$ 1,100.00); review articles: R$ 1.850,00 (US$ 1,100.00); case reports, technical notes, and rapid communications: R$ 1.100,00 (US$ 650.00); and invited reviews, editorials and letters to the editors: no charge.

Manuscripts involving human subjects or the use of laboratory animals must clearly state adherence to appropriate guidelines and approval of protocols by their institutional review boards.

Photographs that may identify patients or other human participants of studies shall be acceptable only when a legally valid permission (form provided when necessary) is signed by the said patient or other human participant or by his/her legally constituted representative.

Manuscripts should be submitted online, in English, digitalized in a word.doc-compatible software program. Authors are strongly advised to submit the manuscript in its final form to a spell check for English (USA). Submissions with excessive spelling or syntax mistakes, as well as articles in which the meaning is not sufficiently clear shall be returned to authors for correction.

Authors are also very strongly advised to limit abbreviations to the minimum possible. Abbreviations tend to make text unreadable.

All abbreviations must be defined when first used. Only terms or expressions used at least 5 times throughput the text should be abbreviated. Never use abbreviations spelt as common English words such as, for instance FUN, PIN, SCORE, SUN, etc.

Please make sure to submit your manuscript in the exact format described below. Failure to do so will cause the submission to be returned to you during the preliminary examination by the Editorial Office.

Manuscripts are invited in the following categories: ORIGINAL STUDY: Complete original studies should be submitted in this category. Three sections are offered: Basic, Clinical, and Surgical Research. Original Studies must follow the following format:

Title page:

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Title (up to 250 characters); Running title (up to 40 characters, letters and spaces); Full address of corresponding author only; Authors’ names (without titles or graduations).

Authors should have participated sufficiently in the work to take public responsibility for appropriate portions of the content. Such participation must be declared in this section of the manuscript.

Manuscript:

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Abstract: limited to 250 words and structured into: Objectives, Method, Results, Conclusions. Citations or abbreviations (except internationally recognized such as weights, measures, physical or chemical) are not allowed. Authors are strongly recommended not to display numerical statistical information, but merely to state what is significantly different (or not) between described parameters.

Keywords: 3–6 items from the Medical Subject Headings (Mesh) should be used.

Introduction: should set the purpose of the study, give a brief summary (not a review) of previous relevant works, and state what new advance has been made in the investigation. It should not include data or conclusions from the work being reported. A final sentence summarizing the novelty to be presented is permissible.

Materials and Methods: should briefly give clear and sufficient information to permit the study to be repeated by others. Standard techniques need only be referenced. Previously published methods may be briefly described following the reference.

Ethics: when reporting experiments on human subjects, indicate whether the procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional or regional) and with the Helsinki Declaration of 1975, as revised in 1983. When reporting experiments on animals, indicate whether the institution’s or a national research council’s guide for, or any national law on the care and use of laboratory animals was followed.

Results: should be a concise account of the new information discovered, with the least personal judgment. Do not repeat in text all the data in the tables and illustrations, but briefly describe what such material contains.

Discussion: should be limited to the significance of the new information and relate the new findings to existing knowledge. Only unavoidable citations should be included. Citation to review articles are not encouraged in this section.

Acknowledgements: should be short and concise, and restricted to those absolutely necessary.

References in text: CLINICS adopts the Vancouver format. Cite references in text with Arabic numerals, in order of appearance, within parentheses (1), after previous word, with spacing as in this example: ‘‘Diabetes (2), hypertension (3,4) and alcoholism (5–9)

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are complex medical problems (10).’’ Under exceptional circumstances authors’ names may appear in text: Single author: ‘‘Einstein (11) proposed a new theory …’’ Two authors: ‘‘Watson & Crick (12) reported on the structure of …’’ Three or more authors: ‘‘Smith et al (13) described …’’

Reference List: Only citations that appear in the text should be referenced. Unpublished papers, unless accepted for publication, should not be cited. Work accepted for publication should be referred to as ‘‘in press’’ and a letter of acceptance of the journal must be provided. Unpublished data should only be cited in the text as ‘‘unpublished observations’’, and a letter of permission from the author must be provided.

Authors are responsible for the accuracy and completeness of their references and for correct text citation. CLINICS adopts the Vancouver format; references must be restricted to directly relevant published works, papers, or abstracts that have been accepted for publication. Usually the total number of references should not exceed 35. For up to six authors, list all authors; for more than 6 authors, list first six, followed by ‘‘et al’’.

Tables and Figures: The maximum number of tables plus figures is six. Tables: Do not be incorporate Tables into the Manuscript. Upload each table individually into the system. 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 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: Do not incorporate Figures into the Manuscript. 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. 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 must be of professional quality and uploaded as *.tiff files, 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 must 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.

Comments: Authors should use this space to describe the novelty contained in their original study. Only the

Editor of CLINICS has access to this section of the submission.

FAST TRACK ARTICLES: Fast track articles should follow the same format described above for original studies. Fast track articles must be complete original studies with justifiable urgency for publication. The Editorial Office undertakes to produce a first action response in the shortest possible time and to publish accepted Fast Track articles in the next available issue. Only one article may be submitted as Fast Track in any calendar year by any author or co-author. In Comments, authors must explain the reasons for the claim to Fast Track. Rejection by journals with a higher Impact Factor that ours is an acceptable reason for requesting FAST TRACK, provided that the reviewers’ reports from the previous submission be included in the present submission. Information contained in the comments is limited to the Editor and shall remain confidential. No publication fee discount is allowed for accepted Fast Track articles.

REVIEW ARTICLES: Such articles should cover themes relevant to medical practice or mammalian function. Spontaneously submitted reviews are welcome, but potential authors should bear in mind that they are expected to have expertise in the reviewed field. The sections should be arranged as follows:

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Title page: as in Original Study.

Manuscript: Abstract, keywords and text should be arranged as best suited to cover the subject being reviewed. If appropriate, the manner of reference collection should be described. The use of headings, subheadings, and paragraph titles is encouraged for sake of clarity. Abbreviations, acknowledgements, Tables and Figures as in original studies. The number of references is at the discretion of the authors. No publication fee discount is allowed for spontaneously submitted review articles accepted for publication.

RAPID COMMUNICATIONS:

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Manuscript: Rapid communications are limited to 1,500 words, except for the reference list. Authors should format such contributions as best suited to the subject at hand. No abstract or key words are required. Please copy an introductory sentence into the abstract box on Page 1 of the submission procedure.

CASE REPORTS: Case Reports will be published only if justified by the extreme rarity of the case and/or novelty of adopted procedures. Case reports should be arranged as follows:

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Manuscript: Case reports are limited to 1,500 words, except for the reference list. The text should be divided into Introduction, Case Description, and Discussion. No abstract or key words should be included, but please make sure to copy your Introduction into the abstract box of the submission form. Abbreviations, References, Tables and Figures (maximum of four, altogether) follow the format described for Original Studies. No publication fee discount is allowed for case reports accepted for publication.

LETTERS TO THE EDITOR: Letters to the Editor expressing comments or dissenting opinions concerning articles recently published in CLINICS are not submitted to peer review and are published at the discretion of the Editor. A letter is a single section untitled text, followed by references, to include the article under discussion. No publication fee is charged for this class of manuscripts.

EDITORIAL: Editorials should cover broad aspects of Medical or Biological sciences. Such manuscripts are not submitted to peer review and are published at the discretion of the Editor. A letter is a single section untitled text, followed by references, if applicable.

No publication fee is charged for this class of manuscripts.

COMMENTARY: A commentary is an invited text with respect to an article being published by Clinics. No publication fee is charged for this class of manuscripts.

INVITED REVIEW: Invited reviews follow the general pattern proposed for general reviews but are by invitation only. No publication fee is charged for this class of manuscripts.

SPECIAL ISSUE ARTICLE: Special issue articles are by invitation only and follow a specific pattern set out by the Editor in charge of the collection.

Peer Review: Manuscripts are reviewed by at least two expert consultants. Accepted manuscripts are edited to comply with the Journal’s format, to remove redundancies, and to improve clarity and understanding without altering meaning. The edited text will be presented to authors for approval.

Submission: A copyright transfer form, signed by all authors, must be submitted by fax (55-11-2661-7524) or by mail as soon as the manuscript is submitted. Any financial or other relationships that could lead to a conflict of interest must be disclosed in the copyright transfer form. If the Editor considers this conflict of interest relevant to the paper, a footnote will be added to show the equity interest in or affiliation with the identified commercial firm(s).

As soon as authors are satisfied that the manuscript complies with the Journal format, our site should be accessed through www. clinics.org.br. The system will guide authors through the manuscript submission process and prompt authors to input information into specific fields as they are submitting their manuscript. The Editorial Office will be automatically notified of the submission and will send an email confirming it as soon as the submission letter reaches the office. Progress of the manuscript through the Editorial Office procedures will be available to authors at all times.

ISSN-1807-5932

CLINICS CONTENTS Clinics 2012 67(7):697–857

CLINICAL SCIENCES

Renal macrophage infiltration is associated with a poor outcome in IgA nephropathy Gyl Eanes Barros Silva, Roberto Silva Costa, Roberto Cuan Ravinal, Leandra Naira Zambelli Ramalho, Marlene Antonia dos Reis, Miguel Moyses-Neto, Elen Almeida Romao, Terezila Machado Coimbra, Ma´rcio Dantas . . . . . . . 697

Histopathological evaluation and risk factors related to the development of pouchitis in patients with ileal pouches for ulcerative colitis Roberta Thiery de Godoy Arashiro, Magaly Gemio Teixeira, Viviane Rawet, Alina Guimara˜es Quintanilha, Henrique Moura de Paula, Adriano Zanon Silva, Se´rgio Carlos Nahas, Ivan Cecconello . . . . . . . . . . . . . . . . . . . . . . . . . . . . 705

Metformin, but not glimepiride, improves carotid artery diameter and blood flow in patients with type 2 diabetes mellitus Helena Atroch Machado, Marcelo Vieira, Maria Rosaria Cunha, Marcia Regina Soares Correia, Rosa Tsunechiro Fukui, Rosa Ferreira dos Santos, Dalva Marreiro Rocha, Bernardo Leo Wajchenberg, Silvia G. Lage, Maria Elizabeth Rossi da Silva . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 711

Association between functional alterations of senescence and senility and disorders of gait and balance Homero Teixeira-Leite, Alex C. Manha˜es . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 719

Disparities in female breast cancer mortality rates in Brazil between 1980 and 2009 Ruffo Freitas-Junior, Carolina Maciel Reis Gonzaga, Nilceana Maya Aires Freitas, Edesio Martins, Rita de Ca´ssia de Maio Dardes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 731

Frequency and predictors of symptomatic intracranial hemorrhage after intravenous thrombolysis for acute ischemic stroke in a Brazilian public hospital Pedro Telles Cougo-Pinto, Bruno Lopes dos Santos, Francisco Antunes Dias, Soraia Ramos Cabette Fabio, Ilana Vaula Werneck, Millene Rodrigues Camilo, Daniel Giansante Abud, Joa˜o Pereira Leite, Octavio Marques Pontes-Neto . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 739

T-cell large granular lymphocytic leukemia: treatment experience with fludarabine Renata Oliveira Costa, Marcelo Bellesso, Dalton Alencar Fischer Chamone, Milton Artur Ruiz, Abraha˜o Elias Hallack Neto, Vera Lucia Aldred, Juliana Pereira . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 745

Trends and characteristics of pethidine use in Taiwan: a six-year-long survey Hsueh-Hsing Pan, Chung-Yi Li, Tso-Chou Lin, Ju-O Wang, Shung-Tai Ho, Kwua-Yun Wang . . . . . . . . . . . . . . . . 749

Postural control in women with breast hypertrophy Alessandra Ferreira Barbosa, Gabriela Cristina Raggi, Cristina dos Santos Cardoso Sa´, Ma´rcio Paulino Costa, Jonas Eraldo de Lima Junior, Clarice Tanaka. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 757

Renal oxygen content is increased in healthy subjects after angiotensin-converting enzyme inhibition Anna Stein, Silvia Goldmeier, Sarah Voltolini, Enio Setogutti, Carlos Feldman, Eduardo Figueiredo, Renato Eick, Maria Irigoyen, Katya Rigatto . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 761

Noninvasive positive-pressure ventilation in clinical practice at a large university-affiliated Brazilian hospital Liria Yuri Yamauchi, Teresa Cristina Francischetto Travaglia, Sidnei Ricardo Nobre Bernardes, Maise C. Figueiroa, Clarice Tanaka, Carolina Fu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 767

Pulse pressure variation and prediction of fluid responsiveness in patients ventilated with low tidal volumes Clarice Daniele Alves de Oliveira-Costa, Gilberto Friedman, Sı´lvia Regina Rios Vieira, Le´a Fialkow . . . . . . . . . . . . 773

BASIC RESEARCHES

Protective effects of Tualang honey on bone structure in experimental postmenopausal rats Siti Sarah Mohamad Zaid, Siti Amrah Sulaiman, Nor Hayati Othman, Ima-Nirwana Soelaiman, Ahmad Nazrun Shuid, Norazlina Mohamad, Norliza Muhamad . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 779

Rat testicular impairment induced by electromagnetic radiation from a conventional cellular telephone and the protective effects of the antioxidants vitamins C and E Mona Abdullah Al-Damegh . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 785

The use of vancomycin-loaded poly-l-lactic acid and poly-ethylene oxide microspheres for bone repair: An in vivo study De´bora Cristina Corac¸a-Huber, Eliana Aparecida de Rezende Duek, Maurı´cio Etchebehere, Luis Alberto Magna, Eliane Maria Ingrid Amstalden . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 793

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CLINICS 2012;67(7):697-703

DOI:10.6061/clinics/2012(07)01

CLINICAL SCIENCE

Renal macrophage infiltration is associated with a poor outcome in IgA nephropathy Gyl Eanes Barros Silva,I Roberto Silva Costa,I Roberto Cuan Ravinal,I Leandra Naira Zambelli Ramalho,I Marlene Antonia dos Reis,II Miguel Moyses-Neto,III Elen Almeida Romao,III Terezila Machado Coimbra,IV Ma´rcio DantasIII I University of Sa˜o Paulo, Faculty of Medicine of Ribeira˜o Preto, Department of Pathology, Ribeira˜o Preto/SP, Brazil. II Faculty of Medicine of Triaˆngulo Mineiro, Department of General Pathology, Uberaba/MG, Brazil. III University of Sa˜o Paulo, Faculty of Medicine of Ribeira˜o Preto, Division of Nephrology, Ribeira˜o Preto/SP, Brazil. IV University of Sa˜o Paulo, Faculty of Medicine of Ribeira˜o Preto, Department of Physiology, Ribeira˜o Preto/SP, Brazil.

OBJECTIVES: The objectives of our study were as follows: 1) to analyze the prognostic value of macrophage infiltration in primary IgA nephropathy (IgAN) and 2) to study the relationship between macrophages and other factors associated with the development of renal fibrosis, including mast cells, TGF-b1, a-SMA and NF-kB. METHODS: We analyzed 62 patients who had been diagnosed with IgAN between 1987 and 2003.

Immunohistochemical staining was performed with monoclonal antibodies against CD68 and mast cell tryptase and polyclonal antibodies against TGF-b1, a-SMA and NF-kB p65. We also used Southwestern histochemistry for the in situ detection of activated NF-kB. RESULTS: The infiltration of macrophages into the tubulointerstitial compartment correlated with unfavorable clinical and histological parameters, and a worse clinical course of IgAN was significantly associated with the number of tubulointerstitial macrophages. Kaplan-Meier curves demonstrated that increased macrophage infiltration was associated with decreased renal survival. Moreover, the presence of macrophages was associated with mast cells, tubulointerstitial a-SMA expression and NF-kB activation (IH and Southwestern histochemistry). In the multivariate analysis, the two parameters that correlated with macrophage infiltration, proteinuria and tubulointerstitial injury, were independently associated with an unfavorable clinical course.

CONCLUSION: An increased number of macrophages in the tubulointerstitial area may serve as a predictive factor for poor prognosis in patients with IgAN, and these cells were also associated with the expression of pro-fibrotic factors.

KEYWORDS: IgA nephropathy; Macrophage; Renal fibrosis; Glomerulonephritis; Southwestern immunohistochemistry.

Silva GEB, Costa RS, Ravinal RC, Ramalho LNZ, Reis MA, Moyses-Neto M, et al. Renal macrophage infiltration is associated with a poor outcome in IgA nephropathy. Clinics. 2012;67(7):697-703.

Received for publication on February 17, 2012; First review completed on February 29, 2012; Accepted for publication on March 5, 2012 E-mail: gyleanes@fmrp.usp.br Tel.: 55 16 3602-0224

clinical and histological parameters have been implicated in the prognosis of IgAN, including hypertension, proteinuria, high levels of serum creatinine and injury to the tubulointerstitial, glomerular and vascular compartments of the kidney. The extent of tubulointerstitial fibrosis is believed to better predict a decline in renal function and poor prognosis than other known parameters (1,5), and this process has been shown to be associated with phenotypic changes in glomerular and interstitial cells and can be recognized by the expression of smooth muscle actin (a-SMA) (6). A large number of cytokines, inflammatory mediators and growth factors produced by macrophages and other cells are related to renal fibrosis (7,8), as macrophages orchestrate kidney tissue repair and the replacement of specialized renal cells following injury (9). Recently, many reports have described two different populations of macrophages that act during either the initiation or recovery phase of scarring (9,10). M1

INTRODUCTION IgA nephropathy (IgAN) is the most common type of primary glomerulonephritis (GN) worldwide and is characterized by a highly variable clinical course (1). Within 10 to 20 years of disease onset, these patients may progress to end-stage renal disease (ESRD) (2,3), and only 3.7% of IgAN patients develop long-standing spontaneous clinical remission (4). As a result of these findings, many potential prognostic factors have been extensively studied, and many

Copyright ß 2012 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.

No potential conflict of interest was reported.

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system: absent,,10% of the total area; mild, 10 to 25% of the total area; moderate, 26 to 50% of the total area; and severe, .50% of the total area. Frozen renal tissue specimens were prepared for immunofluorescence microscopy.

macrophages are recruited during the first 48 hours after an acute injury, whereas M2 macrophages predominate at later time points (11). Moreover, transcription factors such as nuclear factor-kappa B (NF-kB) have been shown to regulate many genes involved in the progression of renal disease (12).

Our objectives for this study were as follows: 1) to analyze whether the amount of macrophage infiltration in the kidney would be associated with clinical and histological parameters for the prediction of an unfavorable clinical course in patients with IgAN and 2) to study the relationship between macrophages and other factors associated with the development of renal fibrosis, such as mast cells, TGF-b1 (transforming growth factor beta 1), aSMA and NF-kB.

Immunohistochemical (IHC) Studies These assays were performed as previously described (13). We used the following primary antibodies: a polyclonal antibody for NF-kB (1:200, Santa Cruz BiotechnologyH, Santa Cruz, CA, USA); a monoclonal mouse antibody for human macrophage CD68 (1:150, DAKO CorporationH, Glostrup, Denmark); a monoclonal antibody for human mast cell tryptase (1:30, NovocastraH, Newcastle Upon Tyne, UK); a polyclonal antibody for human TGF-b1 (1:250, Santa Cruz Biotechnology, Santa Cruz, CA, USA); and a monoclonal antibody for human a-SMA (1:1000).

Chromogenic development was performed using 3,3’ diaminobenzidine (DAB) (Sigma), and the samples were counterstained with methyl green prior to dehydration and mounting. Control kidney specimens were obtained from normal kidney tissues of ten patients with renal cell carcinoma who had no history of diabetes or hypertension and no detectable histological lesions.

Quantification of mast cells and macrophages was performed, and the number of cells/mm2 was counted using a light microscope with a 10x magnification objective and an automatic image analyzer (KS 300 Kontron Imaging System, Eching bei Mu¨nchen, Germany). Extracapillary cells, such as those present in crescents, were excluded from the count of intraglomerular immune cells. Glomerular expression of a-SMA and TGF-b1 was graded semiquantitatively as follows: (13) 0, no staining; 1, trace mesangial staining; 2, weak segmental mesangial staining; 3, strong segmental mesangial staining; and 4, strong diffuse mesangial staining. The a-SMA and TGF-b1 immunoreaction in the tubulointerstitium and tubular NF-kB expression in the renal cortex were scored as follows: 0, absent staining; 1, weak staining with focal distribution; 2, moderate staining with focal distribution; 3, strong staining with focal distribution or weak and diffuse staining; and 4, strong and diffuse staining.

PATIENTS AND METHODS Patients and Clinical Evaluation We studied the renal biopsies of 62 patients who had been diagnosed with primary IgAN between 1987 and 2003. The records survey covered a four-month period during 2008, and a review was performed in 2011. Patients with a clinical history of alcoholism, liver diseases or Henoch-Scho¨nlein purpura and any specimens with fewer than six identifiable glomeruli were excluded from the study. The biopsies were obtained from the files of the Department of Pathology at the Medical School of Ribeira˜o Preto, Brazil. Clinical and laboratory data were obtained from the medical records of the patients using a previously reported protocol (13).

The following classifications of IgAN outcomes were applied during the clinical follow-up period (13): (i) clinical remission (CR), which was defined as proteinuria below 200 mg/day with normal renal function; (ii) clinical improvement (CI), which was defined as regression of nephrotic or nephritic syndrome or reduced proteinuria with stable or improved renal function; (iii) unchanged (UC), which was defined as unchanged clinical and laboratory data during the course of the disease; (iv) clinical worsening (CW), which was defined as aggravation of nephrotic or nephritic syndrome or increased proteinuria with normal renal function; and (v) chronic kidney disease (CKD), which was defined as a serum creatinine level of 1.5 mg/dL or greater, a creatinine clearance of less than 90 mL/min per 1.73 m2, or the need for dialysis or renal transplant (ESRD). We considered remission, clinical improvement and unchanged conditions to indicate a favorable clinical outcome, whereas clinical worsening and CKD indicated an unfavorable clinical outcome.

Regardless of the schedule used, all patients were assumed to have been treated for IgAN when they received corticosteroids, regardless of whether these were administered in combination with immunosuppressive drugs. At the time of biopsy collection, 49 of the 62 patients had received angiotensin-converting enzyme (ACE) inhibitor therapy, and 6 of the 62 patients had been treated for IgAN.

Southwestern histochemical (SWH) analysis The non-radioactive in situ detection of NF-kB in paraffinembedded kidney tissue preparations was performed using the SWH method, as previously described (14-16), with digoxigenin labeling and detection kits (Roche Applied Science, Indianapolis, USA). Synthetic sense DNA (Imprint Genetics Corporation, Hialeah, USA) containing the NF-kB sequence (59-AGTTGAGGGGACTTTCCCAGGC-39) was used as the probe. After annealing with the complementary sequence, the DNA probe was labeled with digoxigenin.

The sections were then incubated with the labeled probes for 12 h at 37 ˚C. Slides were then incubated with an antidigoxigenin antibody conjugated to alkaline phosphatase and detected using an NBT/BCIP solution. The slides were then mounted with glycerol. A mutant form of the probe labeled with digoxigenin was used as a negative control.

Only morphologically recognizable cells were counted, and the cells were considered positive when distinct, homogeneous, purple nuclear staining was present. NF-kB staining by SWH was scored in the same manner as the NF-kB staining by IHC.

Morphological Evaluation The samples were analyzed using standard light and immunofluorescence microscopy. The tissues obtained by renal biopsy in the 62 IgAN patients and control sections were stained with hematoxylin and eosin, Masson’s trichrome and methenamine silver. Interstitial fibrosis was assessed using the following semiquantitative scoring

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of biopsy (p = 0.02; r = 0.28) and at the end of the follow-up period (p,0.01; r = 0.46). The number of tubulointerstitial macrophages was also correlated with the magnitude of urinary protein excretion at the time of biopsy (p = 0.01; r = 0.44) and at the end of the follow-up period (p,0.01; r = 0.33). These data are shown in Figure 2.

The presence of tubulointerstitial macrophages was not correlated with the glomerular (p = 0.50) or tubulointerstitial (p = 0.70) expression of TGF-b1 or the glomerular expression of a-SMA (p = 0.52). Macrophage numbers had a tendency to correlate with NF-kB expression, as measured by SWH (p = 0.07) and shown in Figure 3. Interestingly, the numbers of tubulointerstitial macrophages showed positive correlations with tubulointerstitial a-SMA (p = 0.01) and NF-kB expression, as measured by IHC (p,0.01), as well as tubulointerstitial injury (p,0.01) and poor clinical outcome (p = 0.01). The correlation between the presence of mast cells and macrophages infiltrating the tubulointerstitium of the kidney was also statically significant (p,0.01).

As shown in Figure 4, we observed a significant correlation between the infiltration of macrophages in the tubulointerstitial compartment and the clinical disease course (p = 0.01). Infiltrating macrophages in the tubulointerstitial compartment were also correlated with the degree of tubulointerstitial lesions (p,0.01). As shown in Figure 5, a significant association was also detected between macrophages and the annual decrement of estimated glomerular filtration rate (eGFR) during the first three years following renal biopsy (r = 0.531, p,0.01). Because a minimum of three years was required for the calculation of the annual decrement of eGFR, only 47 of the 62 cases were included in this analysis. For the other 15 patients, the follow-up duration was between one and three years or there was insufficient data for the eGFR calculation.

The Kaplan-Meier curve shown in Figure 6 demonstrates the correlation between the presence of macrophages and survival over the course of several months, whereby a higher number of macrophages infiltrating the tubulointerstitium of the renal cortex predicted a lower survival rate for renal function (p,0.05). There were no correlations between macrophage infiltration into the glomerular compartment and various clinical or laboratory parameters, unfavorable clinical course or renal survival (p.0.05).

In the multivariate analysis (Table 2), the number of macrophages was not independently associated with an unfavorable clinical course (OR = 1.0, CI = 0.91-1.09, p = 0.93).

However, the degree of tubulointerstitial injury and the levels of proteinuria were independently associated with progressive disease (OR = 10.5, CI = 212-425, p = 0.01 and OR = 7.65, CI = 1.39-42.3, p = 0.02).

Statistical Analysis For normally distributed data, values were expressed as the means ¡ standard deviations. Proportions were tested using the standard chi-squared test (x2) with a Yates correction or Fisher’s exact test. Associations between continuous variables were examined by calculating Pearson’s correlation coefficient. Simple and multivariate logistic regression models were used to assess associations. A significant difference was accepted if p,0.05.

Survival analyses were performed to test the association between renal failure and the number of macrophages infiltrating the glomerulus and tubulointerstitial area. Survival times from the first clinical assessment to the last follow-up visit were obtained for each patient. Univariate comparisons of renal survival rates were made using Kaplan-Meier curves and the log-rank test. The end-point for renal survival was defined as ESRD requiring dialysis or transplant.

RESULTS Sixty-two patients were included in this study, including 32 men (51.6%) and 30 women (48.4%). Twenty-four patients were excluded from our study due to short follow-up periods (,12 months), specimens with fewer than 6 glomeruli and one case without hematuria. We found significant correlations between an unfavorable clinical outcome of IgAN and male gender, the absence of macroscopic hematuria, arterial hypertension, high levels of serum creatinine, severe proteinuria, and elevated glomerular or tubulointerstitial injury scores (p,0.05). For the purposes of statistical analysis, we classified 28 (45%) patients as having a favorable clinical course and 34 (55%) patients as having an unfavorable clinical course. The average follow-up times were very similar between groups, as patients with an unfavorable course demonstrated an average follow-up time of 76.32¡47.84 months and those with a favorable course had an average follow-up time of 73.29¡50.85 months. The clinical and laboratory data for these two groups are shown in Table 1.

Macrophages were rarely detected in the interstitial compartment of normal control samples, although these cells were frequently detected in the glomerular compartment. In contrast, macrophages were often detected in the cortical interstitium and glomeruli of patients with IgAN (Figure 1). The average number of macrophages infiltrating the tubulointerstitial compartment was 7.14 cells/mm2 for patients with IgAN and 3.0 cell/mm2 for the controls, and the average number of macrophages infiltrating the glomerular compartment was 2.6 cells/mm2 for patients with IgAN and 2.3 cell/mm2 for the controls.

The number of tubulointerstitial macrophages was positively correlated with the serum creatinine level at the time

Table 1 - Clinical parameters of IgAN patients with favorable vs. unfavorable clinical disease courses.

Age at onset (years) Gender (male - female) Urinary protein excretion at the time of biopsy (g/24 h) Urinary protein excretion at the end of follow-up (g/24 h) Serum creatinine at the time of biopsy (mg/dL) Serum creatinine at the end of follow-up (mg/dL) Time of follow-up (months) The parameters are expressed as the means¡S.D. *p,0.05.

699

Favorable course (n = 28)

Unfavorable course (n = 34)

27.7¡9.2 10:18* 1.9¡0.8* 1.1¡0.8 1.4¡0.5* 2.2¡0.7* 76¡47.32

27.2¡13.0 22:12 2.9¡1.0 1.0¡0.5 1.9¡0.4 3.8¡1.3 73¡50.85

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Figure 1 - Renal macrophage infiltration. (A) The absence of macrophages in a preserved area of the cortex, and (B) macrophages within areas of interstitial fibrosis (A and B: objective 40x).

Figure 2 - Correlations between the number of cortical macrophages and the following variables: (A) serum creatinine (mg/dL) at the time of biopsy (p = 0.02; r = 0.28); (B) serum creatinine at the end of follow-up (p,0.01; r = 0.46); (C) protein excretion (g/24h) the time of biopsy (p = 0.01; r = 0.44); and (D) protein excretion at the end of follow-up (p,0.01; r = 0.33).

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Figure 3 - Southwestern histochemistry for NF-kB expression. (A) The absence of staining and (B) moderate-to-intense staining in the tubular compartment (A and B: objective 40x).

Studies have shown that macrophages can synthesize collagen I and fibronectin in vitro and can release several fibrogenic cytokines, including TGF-b, PDGF and IL-2 (21,22). In our sample, there was no relationship between TGF-b expression and glomerular or interstitial infiltration by macrophages, which may have been due to the limited sensitivity of immunohistochemistry to detect TGF-b activity in the kidney (8).

In a study of patients with chronic cyclosporine nephropathy, Young et al. (23) reported that macrophage infiltration precedes the development of interstitial fibrosis.

Moreover, Geleilete et al. (24) observed an increase in the diffuse distribution of a-SMA and ED-1 (monocytes/ macrophages) immunoreactivity in the renal cortex of animals treated with gentamicin. In the same study, the animals that were sacrificed 30 days after treatment demonstrated a focal distribution of these proteins that was predominantly localized within damaged areas. Therefore, these authors concluded that macrophages, TGF-b, endothelin and myofibroblasts may contribute to renal injury. In our patients, there was a correlation between the interstitial expression of a-SMA, but not the glomerular expression of a-SMA, and macrophage infiltration.

DISCUSSION In the present study, high levels of serum creatinine (at the time of biopsy and at the end of the follow-up period), severe proteinuria (at the time of biopsy and at the end of the follow-up period), an elevated degree of tubulointerstitial injury and increased tubulointerstitial expression of NF-kB and a-SMA were all significantly associated with the number of macrophages in the tubulointerstitial compartment. Furthermore, macrophage infiltration served as a marker of the annual decrement of eGFR. The infiltration of macrophages into the renal cortex has been reported in patients with progressive renal disease (17,18). In a study of 204 patients with IgAN, the presence of macrophages (CD68-positive cells) was positively correlated with the following: serum creatinine at the time of biopsy, proteinuria, progression of renal disease and a worse disease outcome (19). In addition, similar results have been observed in pediatric patients (20). These findings are similar to those of the present study, as we demonstrated a correlation between macrophage infiltration and proteinuria and serum creatinine levels both at the time of biopsy and at the end of the follow-up period.

Figure 4 - A dispersion graph showing the relationship between the number of cortical macrophages and the following variables: (A) clinical outcome (1, clinical remission; 2, clinical improvement; 3, unchanged; 4, clinical worsening; 5, chronic kidney disease; 6, endstage renal disease) (p = 0.01) and (B) tubulointerstitial fibrosis score (0, absent; 1, mild; 2, moderate; 3, severe) (p,0.01).

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Figure 5 - The correlation between the number of macrophages in the tubulointerstitial area and annual decrement of eGFR calculated after three years of follow-up.

Figure 6 - The renal survival of patients with IgAN (n = 62) as a function of the number of macrophages. A low score corresponded to those cases with a number of cells below the median of 7.14 cells/mm2, and a high score corresponded to those cases with a number of cells above the median value. The end-point for renal survival was defined as ESRD (end-stage renal disease) requiring dialysis or transplant. The vertical hash marks on the survival curves indicate the end of follow-up for patients who did not develop ESRD.

One previous study found a correlation between the activation of NF-kB and macrophage interstitial infiltration in patients with lupus nephritis (25). In addition, recent studies have suggested that the NF-kB signaling pathway is involved in the activation of macrophages (15,26). In our study, we found a correlation between macrophage infiltration and NF-kB expression, as measured by IHC, as well as a trending correlation between macrophage infiltration and NF-kB expression, as measured by SWH. Although the role of NF-kB in the activation of mast cells has been demonstrated (27), no study has examined this relationship in renal disease.

NF-kB participates in the regulation of gene transcription by binding to a specific sequence of nuclear DNA known as a response element. To better understand the operation of this transcriptional factor, an analysis of expression at the individual cell level is required. In our IHC analysis, NF-kB was detected whether or not it was bound to the response element, whereas in the SWH analysis, NF-kB was only detected when it was unbound or weakly bound to the response element. Because these methods provide complementary results, they can be used together to characterize the physiological state of NF-kB (15). However, none of the techniques described in the literature provide a fully comprehensive description of NF-kB activation and function (28,29).

In the current study, the number of macrophages was not independently associated with an unfavorable clinical course, although the degree of tubulointerstitial injury was found to be the variable most significantly associated with poor clinical course. In addition, the multivariate analyses identified a significant interaction between macrophage infiltration and levels of proteinuria and tubulointerstitial injury. In agreement with these findings, other reports have consistently shown a relationship between interstitial macrophage infiltration and the progression of renal fibrosis (11,16).

Recently, several studies have suggested that there are functionally distinct populations of macrophages that exist within the same tissues and have opposing pro- and antifibrotic actions (9,10). Despite the potential protective role of

macrophages in renal fibrosis, the number of macrophages in the tubulointerstitial area may serve as a predictive factor for poor prognosis in patients with IgAN, and their presence was associated with the expression of pro-fibrotic factors in the current study. Therefore, renal function and its prognosis are closely correlated with the extent of tubulointerstitial damage and, consequently, with the extent of mononuclear infiltration into this compartment. However, we observed no correlation between macrophage infiltration into the glomerular compartment and any of the examined clinical or histological parameters. This finding could likely be explained by the very high percentage of patients with non-proliferative histological forms (77.5% with focal and segmental sclerosis and mesangiopathic forms; 6.5% with mesangiopathic forms) in our study. In addition, the study

by Nagata et al. demonstrated that the number of macrophages in the glomerular compartment was significantly higher in patients who received pathological diagnoses of focal and diffuse proliferative glomerulonephritis than in patients with minor glomerular abnormalities or sclerosis (30).

However, certain limitations of our study should be noted. First, the investigation was performed casuistically using single-center, heterogeneous data regarding the use of angiotensin-converting enzyme (ACE) inhibitor therapy, the levels of renal function and immunosuppressive protocols.

In addition, the study used retrospective data collection analysis.

In conclusion, an increased number of macrophages in the tubulointerstitial area may serve as a predictive factor for poor prognosis in patients with IgAN, and we found the presence of these cells to also be associated with the expression of pro-fibrotic factors.

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Table 2 - Risk factors for unfavorable clinical course according to the multivariate analysis.

Gross Logistic Regression

Risk Factors

IC 95%

Odds Ratio

Macrophages Serum Creatinine at onset Proteinuria at onset (261) Proteinuria at onset (361) Interstitial Fibrosis (260/1) Interstitial Fibrosis (360/1)

1.10 6.94 4.05 5.36 4.07 40.70

Adjusted Logistic Regression p-value

LI

LS

1.01 1.94 1.21 1.24 1.17 4.50

1.19 24.82 13.54 23.21 14.15 367.91

0.04 ,0.01 0.02 0.02 0.03 ,0.01

IC 95%

Adjusted Odds Ratio*

1.00 3.17 7.65 6.98 3.52 30.05

Adjusted p-value*

LI

LS

0.91 0.81 1.39 0.88 0.67 2.12

1.09 12.44 42.03 55.61 18.53 425.69

0.93 0.10 0.02 0.07 0.14 0.01

Legend: Proteinuria (1-#1 g/24 h; 2- .1 and #3.5 g/24 h; 3 .3.5); Interstitial Fibrosis (0- absent; 1- mild; 2- moderate; 3- severe); IC- confidence intervals; LI- Lower 95% confidence limits; LS- Upper 95% confidence limits.

14. Ashizawa M, Miyazaki M, Abe K, Furusu A, Isomoto H, Harada T, et al.

Detection of nuclear factor-kappaB in IgA nephropathy using Southwestern histochemistry. Am J Kidney Dis. 2003;42(1):76-86, http://dx.doi.org/10.1016/S0272-6386(03)00411-6.

15. Koji T, Komuta K, Nozawa M, Yamada S, Nakane PK. Localization of cyclic adenosine 3’,5’-monophosphate-responsive element (CRE)-binding proteins by southwestern histochemistry. J Histochem Cytochem.

1994;42(10):1399-405, http://dx.doi.org/10.1177/42.10.7930523.

16. Donadelli R, Abbate M, Zanchi C, Corna D, Tomasoni S, Benigni A, et al.

Protein traffic activates NF-kB gene signaling and promotes MCP-1dependent interstitial inflammation. Am J Kidney Dis. 2000;36(6):122641, http://dx.doi.org/10.1053/ajkd.2000.19838.

17. Eddy A. Role of cellular infiltrates in response to proteinuria.

Am J Kidney Dis. 2001;37(1 Suppl 2):S25-9, http://dx.doi.org/10.1053/ ajkd.2001.20735.

18. Forbes JM, Hewitson TD, Becker GJ, Jones CL. Ischemic acute renal failure: long-term histology of cell and matrix changes in the rat. Kidney Int. 2000;57(6):2375-85, http://dx.doi.org/10.1046/j.1523-1755.2000.00097.x.

19. Myllymaki JM, Honkanen TT, Syrjanen JT, Helin HJ, Rantala IS, Pasternack AI, et al. Severity of tubulointerstitial inflammation and prognosis in immunoglobulin A nephropathy. Kidney Int. 2007;71(4): 343-8, http://dx.doi.org/10.1038/sj.ki.5002046.

20. Maruhashi Y, Nakajima M, Akazawa H, Shimoyama H, Nishiguchi M, Yamoto Y, et al. Analysis of macrophages in urine sediments in children with IgA nephropathy. Clin Nephrol. 2004;62(5):336-43.

21. Vaage J, Lindblad WJ. Production of collagen type I by mouse peritoneal macrophages. J Leukoc Biol. 1990;48(3):274-80.

22. Kliem V, Johnson RJ, Alpers CE, Yoshimura A, Couser WG, Koch KM, et al. Mechanisms involved in the pathogenesis of tubulointerstitial fibrosis in 5/6-nephrectomized rats. Kidney Int. 1996;49(3):666-78, http://dx.doi.org/10.1038/ki.1996.95.

23. Young BA, Burdmann EA, Johnson RJ, Andoh T, Bennett WM, Couser WG, et al. Kidney Int. 1995;48(2):431-8.

24. Geleilete TJ, Melo GC, Costa RS, Volpini RA, Soares TJ, Coimbra TM. Role of myofibroblasts, macrophages, transforming growth factor-beta endothelin, angiotensin-II, and fibronectin in the progression of tubulointerstitial nephritis induced by gentamicin. J Nephrol.

2002;15(6):633-42.

25. Zheng L, Sinniah R, Hsu SI. Renal cell apoptosis and proliferation may be linked to nuclear factor-kappaB activation and expression of inducible nitric oxide synthase in patients with lupus nephritis. Hum Pathol.

2006;37(6):637-47, http://dx.doi.org/10.1016/j.humpath.2006.01.002.

26. Wilson HM, Chettibi S, Jobin C, Walbaum D, Rees AJ, Kluth DC. Inhibition of macrophage nuclear factor-kappaB leads to a dominant anti-inflammatory phenotype that attenuates glomerular inflammation in vivo. Am J Pathol. 2005;167(1):27-37, http://dx.doi.org/10.1016/ S0002-9440(10)62950-1.

27. Miyata N, Gon Y, Nunomura S, Yamashita K, Matsumoto K, Hashimoto S, et al. Inhibitory effects of parthenolide on antigen-induced microtubule formation and degranulation in mast cells. Int Immunopharmacol.

2008;8(6):874-80, http://dx.doi.org/10.1016/j.intimp.2008.02.002.

28. Panzer U, Steinmetz OM, Turner JE, Meyer-Schwesinger C, von Ruffer C, Meyer TN, et al. Resolution of renal inflammation: a new role for NFkappaB1 (p50) in inflammatory kidney diseases. Am J Physiol Renal Physiol. 2009;297(2):F429-39, http://dx.doi.org/10.1152/ajprenal.

90435.2008.

29. Sanz AB, Sanchez-Nino MD, Ramos AM, Moreno JA, Santamaria B, Ruiz-Ortega M, et al. NF-kappaB in renal inflammation. J Am Soc Nephrol. 21(8):1254-62.

30. Nagata M, Akioka Y, Tsunoda Y, Komatsu Y, Kawaguchi H, Yamaguchi Y, et al. Macrophages in childhood IgA nephropathy. Kidney Int. 1995;48(2):527-35, http://dx.doi.org/10.1038/ki.1995.323.

AUTHOR CONTRIBUTIONS Silva GEB participated in the conception and drafting of the manuscript as well as the revisions for critically important intellectual content. Costa RS was involved in the histopathological studies of the biopsies from selected patients as well as the revision of the manuscript for critically important intellectual content. Ravinal RC participated in the immunohistochemical studies. Reis MA participated in the statistical analysis. Ramalho LZ participated in the Southwestern histochemical studies. Moyses-Neto M participated in the morphometric analyses. Romao EA participated in the translation and editing of the manuscript. Coimbra TM participated in the conception, analysis of data, and in the revision of the manuscript for critically important intellectual content. Dantas M participated in the selection of patients, collection of biopsies, analysis of data, laboratory and radiological exams, and in the revision of the manuscript for critically important intellectual content.

REFERENCES 1. Berthoux FC, Mohey H, Afiani A. Natural history of primary IgA nephropathy. Semin Nephrol. 2008;28(1):4-9, http://dx.doi.org/10.1016/ j.semnephrol.2007.10.001.

2. Coppo R, D’Amico G. Factors predicting progression of IgA nephropathies. J Nephrol. 2005;18(5):503-12.

3. Manno C, Strippoli GF, D’Altri C, Torres D, Rossini M, Schena FP. A novel simpler histological classification for renal survival in IgA nephropathy: a retrospective study. Am J Kidney Dis. 2007;49(6):76375, http://dx.doi.org/10.1053/j.ajkd.2007.03.013.

4. Costa RS, Droz D, Noel LH. Long-standing spontaneous clinical remission and glomerular improvement in primary IgA nephropathy (Berger’s disease). Am J Nephrol. 1987;7(6):440-4, http://dx.doi.org/ 10.1159/000167516.

5. D’Amico G. Natural history of idiopathic IgA nephropathy and factors predictive of disease outcome. Semin Nephrol. 2004;24(3):179-96, http:// dx.doi.org/10.1016/j.semnephrol.2004.01.001.

6. Alpers CE, Hudkins KL, Gown AM, Johnson RJ. Enhanced expression of ‘‘muscle-specific’’ actin in glomerulonephritis. Kidney Int. 1992;41(5):1134-42, http://dx.doi.org/10.1038/ki.1992.173.

7. Ehara T, Shigematsu H. Mast cells in the kidney. Nephrology (Carlton).

2003;8(3):130-8, http://dx.doi.org/10.1046/j.1440-1797.2003.00153.x.

8. Goumenos DS, Tsamandas AC, Oldroyd S, Sotsiou F, Tsakas S, Petropoulou C, et al. Transforming growth factor-beta(1) and myofibroblasts: a potential pathway towards renal scarring in human glomerular disease. Nephron. 2001;87(3):240-8, http://dx.doi.org/10.1159/ 000045921.

9. Ricardo SD, van Goor H, Eddy AA. Macrophage diversity in renal injury and repair. J Clin Invest. 2008;118(11):3522-30, http://dx.doi.org/ 10.1172/JCI36150.

10. Nishida M, Hamaoka K. Macrophage phenotype and renal fibrosis in obstructive nephropathy. Nephron Exp Nephrol. 2008;110(1):e31-6, http://dx.doi.org/10.1159/000151561.

11. Lee S, Huen S, Nishio H, Nishio S, Lee HK, Choi BS, et al. Distinct macrophage phenotypes contribute to kidney injury and repair. J Am Soc Nephrol. 22(2):317-26.

12. Harris RC, Neilson EG. Toward a unified theory of renal progression.

Annu Rev Med. 2006;57:365-80, http://dx.doi.org/10.1146/annurev. med.57.121304.131342.

13. Ravinal RC, Costa RS, Coimbra TM, Dantas M, dos Reis MA. Mast cells, TGF-beta1 and myofibroblasts expression in lupus nephritis outcome.

Lupus. 2005;14(10):814-21, http://dx.doi.org/10.1191/0961203305lu 2188oa.

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DOI:10.6061/clinics/2012(07)02

CLINICAL SCIENCE

Histopathological evaluation and risk factors related to the development of pouchitis in patients with ileal pouches for ulcerative colitis Roberta Thiery de Godoy Arashiro,I Magaly Gemio Teixeira,I Viviane Rawet,II Alina Guimara˜es Quintanilha,I Henrique Moura de Paula,I Adriano Zanon Silva,I Se´rgio Carlos Nahas,I Ivan CecconelloI I Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Departamento de Gastroenterologia, Servic¸o de Cirurgia do Co´lon e Reto, Sa˜o Paulo/SP, Brazil. II Rawet Patologia Especializada Ltda, Sa˜o Paulo/SP, Brazil.

OBJECTIVE: Many changes in mucosal morphology are observed following ileal pouch construction, including colonic metaplasia and dysplasia. Additionally, one rare but potential complication is the development of adenocarcinoma of the reservoir. The aim of this study was to evaluate the most frequently observed histopathological changes in ileal pouches and to correlate these changes with potential risk factors for complications.

METHODS: A total of 41 patients were enrolled in the study and divided into the following three groups: a nonpouchitis group (group 1) (n = 20; 8 males; mean age: 47.5 years) demonstrating optimal outcome; a pouchitis without antibiotics group (group 2) (n = 14; 4 males; mean age: 47 years), containing individuals with pouchitis who did not receive treatment with antibiotics; and a pouchitis plus antibiotics group (group 3) (n = 7; 3 males; mean age: 41 years), containing those patients with pouchitis who were administered antibiotics. Ileal pouch endoscopy was performed, and tissue biopsy samples were collected for histopathological analysis.

RESULTS: Colonic metaplasia was found in 15 (36.6%) of the 41 patients evaluated; of these, five (25%) were from group 1, eight (57.1%) were from group 2, and two (28.6%) were from group 3. However, no correlation was established between the presence of metaplasia and pouchitis (p = 0.17). and no differences in mucosal atrophy or the degree of chronic or acute inflammation were observed between groups 1, 2, and 3 (p.0.45). Moreover, no dysplasia or neoplastic changes were detected. However, the degree of mucosal atrophy correlated well with the time of postoperative follow-up (p = 0.05).

CONCLUSIONS: The degree of mucosal atrophy, the presence of colonic metaplasia, and the degree of acute or chronic inflammation do not appear to constitute risk factors for the development of pouchitis. Moreover, we observed that longer postoperative follow-up times were associated with greater degrees of mucosal atrophy.

KEYWORDS: Colonic metaplasia; Atrophy; Ileal Pouch; Pouchitis; Proctocolectomy.

Arashiro RTG, Teixeira MG, Rawet V, Quintanilha AG, De Paula HM, Silva AZ, et al. Histopathological evaluation and risk factors related to the development of pouchitis in patients with ileal pouches for ulcerative colitis. Clinics. 2012;67(7):705-710.

Received for publication on February 13, 2012; First review completed on March 3, 2012; Accepted for publication on March 3, 2012 E-mail: robertathiery@gmail.com Tel.: 55 11 3726 5544

technique has become the most widely used elective surgical procedure (1).

However, IPAA is not free of complications; nonspecific inflammation of the ileal pouch, or pouchitis, is the most common long-term complication, with an incidence ranging from 14 to 59% (2-12).

Although the physiopathology of pouchitis remains controversial, there is strong evidence that this condition represents a reactivation of UC. In particular, pouchitis occurs almost exclusively in patients who underwent IPAA for UC, rather than for FAP (7,12,13), which suggests an autoimmune etiology for this condition in genetically predisposed individuals. Other factors in support of this theory include the existence of extra-intestinal events associated with an increased risk of developing pouchitis (3,9,14) and the endoscopic and histopathological similarities observed between patients with pouchitis and those with UC (15,16).

INTRODUCTION Restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) has become the gold standard procedure for the treatment of familial adenomatous polyposis (FAP), as well as ulcerative colitis (UC) that is refractory to clinical treatment. This procedure reduces the inconvenience of the diarrhea associated with direct ileoanal anastomosis while preserving sphincter function. In the case of the UC, this

Copyright ß 2012 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.

No potential conflict of interest was reported.

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There is also an increased risk for the development of pouchitis in female patients (6) and in those with extensive or severe UC (7), extra-intestinal manifestations (3,9,14), early disease onset (3), the use of non-steroidal anti-inflammatory drugs (10), gene polymorphisms in the interleukin-1 receptor and TNF-1 antagonists (11) and the presence of perinuclear anti-neutrophil cytoplasmic antibodies (pANCA) (17). Additionally, colonic metaplasia is a common histopathological finding for the ileal pouch and is observed in approximately 50% of patients with pouchitis and approximately 18% of patients undergoing IPAA without pouchitis (18).

In addition, it was observed that the occurrence of pouchitis, particularly in its chronic course, appears to increase the risk of the development of adenocarcinoma of the ileal pouch according to the sequence of atrophydysplasia-carcinoma (19-21). Furthermore, it was found that up to 71% of ileal pouches with severe atrophy develop dysplasia, which is in contrast to cases of discrete atrophy that generally do not develop dysplastic changes (22).

This study sought to characterize the most frequent histopathological changes of ileal pouches and to correlate these changes with potential risk factors for the development of pouchitis.

Table 1 - Graduation and criteria adopted for the histopathological characterization of ileal pouches.

Histopathological Change Degree of chronic inflammation (eosinophilic and lymphoplasmacytic) Degree of acute inflammation (polymorphonuclear granulocytes)

Degree of activity

Presence of colonic metaplasia Degree of mucosal atrophy

Degree of malignancy (cytoarchitectural atypia)

Graduation Mild (0) Moderate (1) Intense (2) Absent (0) Mild (1) Moderate (2) Intense (3) Absent (0) Acute cryptitis (1) Crypt microabscesses (2) Erosion or ulceration (3) Absent (0) Present (1) Absent (0) Distortion of crypts (1) Atrophy of crypts (2) Mild (0) Low grade (1) High degree (2) Intramucosal Carcinoma (3) Invasive Carcinoma (4)

PATIENTS AND METHODS Patients

with no prior bowel preparation. The endoscopic assessment of the ileal pouch was performed and analyzed according to the PDAI (24).

This study included 41 patients who underwent IPAA for UC between 1985 and 2006 at the Hospital of the Faculty of Medicine at the University of SaËœo Paulo (HC-FMUSP) in the Division of Colon and Rectal Surgery. The ileal J-pouch technique was used according to the description provided

by Utsunomiya et al. (23).

A total of 15 men and 26 women with a mean age of 46.2 (range: 23 to 66) years were included in the study. The mean time-course of the disease was 190 (range: 15 to 312) months, and the mean duration of the postoperative follow-up period was 141.7 (range: 24 to 276) months. A total of 33 patients retained a temporary ileostomy for an average period of 22.3 (range: 1 to 168) months, whereas eight patients underwent a single surgery with primary anastomosis.

The pouchitis disease activity index (PDAI), which was previously used by Sandborn et al. (24), was adopted to define the presence or absence of pouchitis in patients based on clinical, endoscopic, and histopathological criteria.

Thus, the patients were divided into the following three groups: the non-pouchitis group (NP), containing individuals without pouchitis at the time of evaluation (n = 20); the pouchitis/no antibiotics group (PNA), containing individuals with pouchitis who were not administered antibiotics (n = 14); and the pouchitis plus antibiotics group (PA), including patients who had pouchitis and were administered antibiotics (n = 7).

The research project was approved by the CAPPESQ (Commission for Ethics Review of Research Projects) of the HCFMUSP under protocol number 1162/07. All included patients signed an informed consent form prior to participation.

Histopathological assessment After the luminal contents were aspirated, two biopsies were taken from areas of major inflammation, if present, and care was taken to avoid suture lines and stapling. The fragments were then sent for histopathological assessment.

Three pathologists participated in the study, and in addition to the criteria of the PDAI previously cited (24), these individuals described the histological patterns observed in accordance with the following criteria: the degree of acute inflammation, the degree of chronic inflammation, the degree of activity, the presence of colonic metaplasia, the degree of atrophy of the mucosa and the degree of malignancy. These data are shown in Table 1.

Statistics analysis An un-paired T-test was used for the histopathological analysis of the three groups. Furthermore, the Shapiro-Wilk normality test was applied to the NP, PNA, and PA groups to evaluate both the duration of the disease (p = 0.661, 0.589, and 0.442, respectively) and the postoperative follow-up period (p = 0.389, 0.051, and 0.218, respectively). Next, an ANOVA was performed on the same groups, which led us to conclude that these three groups were homogeneous in terms of both the duration of the disease (p = 0.222) and the postoperative follow-up period (p = 0.139). Additionally, p,0.05 was defined as the threshold of significance.

RESULTS Histopathological evaluation of biopsies

METHODS

According to the histopathological biopsy results, we observed that longer post-operative follow-up times were associated with a greater degree of mucosal atrophy (p = 0.055). However, there was no correlation between this

Endoscopic evaluation of the ileal pouch All patients underwent an endoscopic evaluation of the ileal pouch (using a sterile rigid rectoscope for children)

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Table 2 - Correlation between elapsed times of disease and post-operative follow-up with the degree of mucosal atrophy.

Degree of mucosal atrophy

0 1

Elapsed time of disease

Elapsed time of post-operative follow-up

Average

Standard error

p-value

Average

Standard error

p-value

184.6154 199.6875

13.28076 22.04535

0.5632

126.0000 162.0000

12.75617 12.96148

0.0552

complicated diseases, sphincter dysfunction, malnutrition or coexistence of cancer of the middle or distal portion of the rectum or anus), this technique has become the most widely used elective surgical procedure for patients with UC (1).

Prior to the introduction of the ileal pouch, patients underwent either the permanent ileostomy of Brooke (26) or ileoanal anastomosis and experienced a consequently high number of daily bowel movements. The first pouch technique, described by Parks and Nicholls, uses three folds of small intestine that are formed into an ‘‘S’’ shape.

Subsequently, other ileal pouch techniques have been described, such as the isoperistaltic loop technique of Fonkalsrud and Ament (27), which uses four loops shaped into a ‘‘W’’, that described by Lubowski and Nicholls (28), and the ‘‘J’’ pouch described by Utsunomiya (23), which was the technique employed in the current study.

Clinically, pouchitis is characterized by watery diarrhea, an increased number of bowel movements, abdominal pain, fecal urgency, incontinence, fever, malaise, and in certain cases, intestinal bleeding. This disorder can also be accompanied by extra-intestinal manifestations of UC. Acute or chronic forms can be observed, although the acute forms are more common (29). Additionally, recurrent episodes are reported at variable rates, ranging from 24.6 (7) to 66% (12).

We used the PDAI criteria in our study because we believed that appropriate diagnostic tests for pouchitis should be performed, including endoscopic examination, biopsy, and histopathological analysis. It has previously been shown that the diagnoses of as many as 25% of patients with the typical symptoms of pouchitis are not confirmed endoscopically or histopathologically (8). Differential diagnoses are

histopathological change and the elapsed disease time (p = 0.5632). These data are presented in Table 2.

The histopathological pattern observed in healthy ileal pouches is shown below in Figure 1, whereas atrophy of the mucosa is illustrated in Figure 2.

Colonic metaplasia was observed in 15 (36.6%) of the 41 patients evaluated; of these, five (25%) were from the NP group, eight (57.1%) were from the PNA group, and two (28.6%) were from the PA group. No dysplasias or neoplastic changes were observed.

In addition, there were no statistically significant differences between the three groups with regard to the degree of mucosal atrophy (p = 0.5203) (Figure 2); the presence of colonic metaplasia (p = 0.1697) (Figure 3); the degree of acute inflammation (p = 0.4434) (Figure 4); and the degree of chronic inflammation (p = 0.9999).

There were also no significant correlations between colonic metaplasia and elapsed disease time (p = 0.4670) or elapsed post-operative follow-up time (p = 0.2041) and between the degree of chronic inflammation and the degree of mucosal atrophy (p = 0.2396), the presence of colonic metaplasia (p = 0.8275), elapsed disease time (p = 0.2031) or elapsed post-operative follow-up time (p = 0.4980).

DISCUSSION Since its first description in 1978 by Parks and Nicholls (25), the ileal pouch technique has been widely used to achieve sphincter preservation and a reduction in the number of bowel movements in patients undergoing total proctocolectomy. Apart from its contraindications (acute

Figure 1 - Healthy ileal pouch mucosa, where villous: crypt ratio is 3 : 1. Presence of Paneth cells, ‘‘brush border’’ and less amount of goblet cells compared to colon.

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Figure 2 - Mucosal atrophy, where villous: crypt ratio is 1:1. Enlargement of the villi, Lymphocytic predominance and lamina propria’s fibroplasia.

both the basic disease, as well as to adaptive responses to anatomical conformation (31).

However, our study did not confirm an association between colonic metaplasia and the presence of pouchitis.

However, we did observe a correlation between mucosal atrophy and the duration of post-operative follow-up, regardless of the presence of pouchitis, and this finding is contrary to that of Apel et al., which suggested that the presence of villous atrophy does not increase with elapsed disease time (32). Thus, additional factors besides fecal stasis and bacterial proliferation are likely involved in the adaptive process of the ileal pouch mucosa, as preliminary studies have suggested (33).

important to rule out other inflammatory (e.g., cuffitis, Crohn’s disease, certain infections of the pouch) and noninflammatory conditions (e.g., decreased complacency, irritable pouch syndrome, stenosis, a long efferent loop, decreased emptying, pelvic floor dysfunction or adhesions) (10,30).

A previous study correlated the presence of colonic metaplasia with inflammation of the ileal pouch and suggested that villous atrophy and crypt hyperplasia predominantly represent a reparative response to the construction of the ileal pouch. In addition, the absence of metaplasia in pouches constructed by FAP would suggest that such a transformation of the mucosa is attributable to

Figure 3 - Colonic Metaplasia. Only crypts are observed. Absence of Paneth cells or villi.

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Figure 4 - Acute inflammation with erosions. There are erosions in the muscular layer of the mucosa, a fibrin-leukocyte buffer and adjacent granulation tissue.

Trovato et al. (34) assessed ileal pouches using either confocal laser endomicroscopy in vivo or conventional histopathological analysis and found incidences of colonic metaplasia of 67.7% and 83.3%, respectively, as well as an incidence of villous atrophy of 83.3% using both techniques.

In our study, both colonic metaplasia and villous atrophy were observed in 36.6% of patients, and no dysplasia was detected in either study.

Although the neoplastic transformation process was not the focus of our study, we considered the complete histopathological characterization of the ileal pouch to be relevant, including the detection of pre-neoplastic changes.

Although rare, adenocarcinoma of the reservoir has been described and is thought to be a consequence of atrophic chronic pouchitis. It has also been assumed that persistent inflammation of the pouch may result in malignant degeneration to resemble what occurs in the inflamed colon. Additionally, there are reports of the development of adenocarcinomas in the ileal pouch mucosa that exhibit intense chronic inflammation and atrophy without a prior history of neoplasms or other risk factors (19,35,36).

No dysplasia or ileal pouch neoplasia was detected in our patients, which is consistent with the results of most previous studies (37-41). The study by Kariv et al. (42) evaluated 3,203 patients with ileal pouches and found dysplasia and adenocarcinoma incidences of 0.72% and 0.36%, respectively. However, the only parameters that these authors considered to represent risk factors for ileal pouch neoplasia were a preoperative diagnosis of dysplasias or adenocarcinomas of the colon or rectum.

The study by MKoma et al. (43) reviewed 12 studies from retrospective case series and 15 case reports and observed the following risk factors for the neoplastic transformation of ileal pouches: pouchitis, preoperative dysplasia or cancer, an interval greater than 10 years from the onset of UC, type C mucosal changes of the pouch mucosa, extra-intestinal manifestations and prior treatment with the stapled anastomotic

technique. Pouch-related cancer did not occur in any of these patients within 10 years of the diagnosis of UC. Thus, early postoperative endoscopic surveillance is generally not justified for these patients. However, for pouch patients with longer postoperative times, it would be interesting to perform routine endoscopic examinations, even in asymptomatic patients, to pursue mucosal atrophy due to the typical sequence of atrophic pouchitis - dysplasia – adenocarcinoma (19-21).

The degree of mucosal atrophy, the presence of colonic metaplasia and the degree of acute or chronic inflammation do not appear to constitute risk factors for the development of pouchitis. Moreover, we found that longer postoperative follow-up times were associated with greater degrees of mucosal atrophy.

AUTHOR CONTRIBUTIONS Arashiro RTG designed the project, collected data and wrote the paper.

Teixeira MG designed the project and wrote and revised the paper. Rawet V designed the project, examined the slides and revised the paper. Quintanilha AG designed the project and revised the paper. De Paula HM and Silva ZA examined the slides. Nahas SC and Cecconello I revised the paper.

REFERENCES 1. Habr-Gama A, Teixeira MG. Tratamento ciru´ rgico da Retocolite Ulcerativa / Proctocolectomia restauradora com bolsa ileal. In: HabrGama A (ed). Doenc¸a Inflamato´ria Intestinal, Clı´nica Brasileira de Cirurgia, Ano III, Volume III. Atheneu, Brasil, 1997.

2. Rauh SM, Schoetz DJ Jr, Roberts PL, Murray JJ, Coller JA, Veidenheimer MC. Pouchitis – is it a wastebasket diagnosis? Dis Colon Rectum.

1991;34(8):685-9, http://dx.doi.org/10.1007/BF02050351.

3. Stahlberg D, Gullberg K, Liljeqvist L, Hellers G, Lo¨fberg R. Pouchitis following pelvic pouch operation for ulcerative colitis – Incidence, cumulative risk and risk factors. Dis Colon Rectum. 1996;39(9):1012-8, http://dx.doi.org/10.1007/BF02054692.

4. Lohmuller JL, Pemberton JH, Dozois RR, Ilstrup DM, van Heerden J. Pouchitis and extraintestinal manifestations of inflammatory bowel disease after ileal pouch-anal anastomosis. Ann Surg. 1990;211(5):622-9. ¨ resland T, Hulte´n L. Incidence and 5. Svaninger G, Nordgren S, O characteristics of pouchitis in the Koch continent ileostomy and the

709

Histopathological evaluation of pouchitis Arashiro RTG et al.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

CLINICS 2012;67(7):705-710

24. Sandborn WJ, Tremaine WJ, Batts KP, Pemberton JH, Phillips S. Pouchitis after ileal pouch-anal anastomosis: a pouchitis disease activity index. Mayo Clin Proc. 1994;69(5):409-15.

25. Parks AG, Nicholls RJ. Proctocolectomy without Ileostomy for Ulcerative-Colitis. Brit Med J. 1978;2(6130):85-8, http://dx.doi.org/ 10.1136/bmj.2.6130.85.

26. Brooke BN. The outcome of surgery for ulcerative colitis. Lancet. 1956; 271(6942):532-6, http://dx.doi.org/10.1016/S0140-6736(56)92021-9.

27. Fonkalsrud EW, Ament ME. Endorectal mucosal resection without proctocolectomy as an adjunct to abdominoperineal resection for non malignant conditions: clinical experience with five patients. Ann Surg.

1978;188(2):245-8, http://dx.doi.org/10.1097/00000658-197808000-00019.

28. Nicholls RJ, Lubowski DZ. Restorative proctocolectomy: the four loop W reservoir. Br J Surg. 1987;74(7):564-6, http://dx.doi.org/10.1002/ bjs.1800740705.

29. Teixeira MG, Ponte ACA, Sousa M, Almeida MG, Silva Filho E, Calache JE, et al. Short- and long-term outcomes of ileal pouch-anal anastomosis for ulcerative colitis. Rev Hosp Clin Fac Med S Paulo. 2003;58(4):193-8.

30. Pardi DS, Sandborn WJ. Systematic review: the management of pouchitis. Aliment Pharmacol Ther. 2006;23(8):1087-96, http:// dx.doi.org/10.1111/j.1365-2036.2006.02884.x.

31. Fruin AB, El-Zammer O, Stucchi AF, OBrien M, Becker JM. Colonic metaplasia in the ileal pouch is associated with inflammation and is not the result of long-term adaptation. J Gastrointest Surg. 2003;7(2):246-54, http://dx.doi.org/10.1016/S1091-255X(02)00191-9.

32. Apel R, Choen Z, Andrews CW Jr, McLeod R, Steinhart H, Odze R. Prospective evaluation of early morphological changes in pelvic ileal pouches. Gastroenterology. 1994;107(2):435-43.

33. De Silva HJ, Millard PR, Kettlewell M, Mortensen NJ, Prince C, Jewell DP. Mucosal characteristics of pelvic ileal pouches. Gut. 1991;32(1):61-5, http://dx.doi.org/10.1136/gut.32.1.61.

34. Trovato C, Sonzogni A, Fiori G, Ravizza D, Tamayo D, Botti F, et al.

Confocal laser endomicroscopy for the detection of mucosal changes in ileal pouch after restorative proctocolectomy. Dig Liver Dis. 2009;41(8):578-85, http://dx.doi.org/10.1016/j.dld.2008.09.019.

35. Heuschen UA, Heuschen G, Autschbach F, Allemeyer EH, Herfarth C. Adenocarcinoma in the ileal pouch: late risk of cancer after restorative proctocolectomy. Int J Colorectal Dis. 2001;16(2):126-30, http:// dx.doi.org/10.1007/s003840000276.

36. Hassan C, Zullo A, Speziale G, Stella F, Lorenzetti R, Morini S. Adenocarcinoma of the ileoanal pouch anastomosis: an emerging complication? Int J Colorectal Dis. 2003;18(3):276-8.

37. Nilubol N, Scherl E, Bub DS, Gorfine SR, Marion J, Harris MT, et al.

Mucosal dysplasia in ileal pelvic pouches after restorative proctocolectomy. Dis Colon Rectum. 2007;50(6):825-31, http://dx.doi.org/10.1007/ s10350-007-0217-6.

38. Herline AJ, Meisinger LL, Rusin LC, Roberts PL, Murray JJ, Coller JA, et al. Is routine pouch surveillance for dysplasia indicated for ileoanal pouches? Dis Colon Rectum. 2003;46(2):156-9, http://dx.doi.org/ 10.1007/s10350-004-6517-1.

39. Hulte´n L, Wille´n R, Nilsson O, Safarani N, Haboubi N. Mucosal assessment for dysplasia and caˆncer in the ileal pouch mucosa in patients operated on for ulcerative colitis- a 30-year follow-up study. Dis Colon Rectum. 2002;45(4):448-52, http://dx.doi.org/10.1007/s10350-004-62189.

40. Thompson-Fawcett MW, Marcus V, Redston M, Cohen Z, McLeod RS. Risk of dysplasia in long-term ileal pouches and pouches with chronic pouchitis. Gastroenterology. 2001;121(2):275-81, http://dx.doi.org/ 10.1053/gast.2001.26442.

41. Ettore GM, Pescatori M, Panis Y, Nemeth J, Crescenzi A, Valleur P. Mucosal changes in ileal pouches after restorative proctocolectomy for ulcerative and Crohns colitis. Dis Colon Rectum. 2000;43(12):1743-8, http://dx.doi.org/10.1007/BF02236861.

42. Kariv R, Remzi FH, Lian L, Bennett AE, Kiran RP, Kariv Y, et al.

Preoperative colorectal neoplasia increases risk for pouch neoplasia in patients with restorative proctocolectomy. Gastroenterology. 2010;139(3): 806-12, http://dx.doi.org/10.1053/j.gastro.2010.05.085.

43. MKoma AE, Moses HL, Adunyah SE. Inflammatory bowel diseaseassociated colorectal cancer: proctocolectomy and mucosectomy do not necessarily eliminate pouch-related cancer incidences. Int J Colorectal Dis. 2011;26(5):533-52, http://dx.doi.org/10.1007/s00384-011-1137-4.

pelvic pouch. Scand J Gastroenterol. 1993;28(8):695-700, http:// dx.doi.org/10.3109/00365529309098275.

Simchuk EJ, Thirlby RC. Risk factors and true incidence of pouchitis in patients after ileal pouch-anal anastomosis. World J Surg. 2000;24(7):8516, http://dx.doi.org/10.1007/s002680010136.

Fazio VW, Ziv Y, Church JM, Oakley JR, Lavery IC, Milson JW, et al. Ileal pouch-anal anastomosis complications and function in 1005 patients.

Ann Surg. 1995;222(2):120-7, http://dx.doi.org/10.1097/00000658199508000-00003.

Shen B, Achkar JP, Lashner BA, Ormsby AH, Remzi FH, Bevins CL, et al.

Endoscopic and histologic evaluation together with symptom assessment are required to diagnose pouchitis. Gastroenterology. 2001;121(2): 261-7, http://dx.doi.org/10.1053/gast.2001.26290.

Hata K, Watanabe M, Shinozaki M, Nagawa H. Patients with extraintestinal manifestations have a higher risk of developing pouchitis in ulcerative colitis: multivariate analysis. Scand J Gastroenterol.

2003;10:1055-8, http://dx.doi.org/10.1080/00365520310005938.

Shen B, Fazio VW, Remzi FH, Delaney CP, Bennett AE, Achkar JP, et al.

Comprehensive evaluation of inflammatory and noninflammatory sequelae of ileal pouch-anal anastomoses. Am J Gastroenterol.

2005;100(1):93-101, http://dx.doi.org/10.1111/j.1572-0241.2005.40778.x.

Aisenberg J, Legnani PE, Nilubol N, Cobrin GM, Ellozy SH, Hegazi RAF, et al. Are pANCA, ASCA or cytokine gene polymorphisms associated with pouchitis? Long-term follow-up in 102 ulcerative colitis patients.

Am J Gastroenterol. 2004;99(3):432-41.

Hurst RD, Molinari M, Chung P, Rubin M, Michelassi F. Prospective study of the incidence, timing, and treatment of pouchitis in 104 consecutive patients after restorative proctocolectomy. Arch Surg. 1996;131(5):497-500, http://dx.doi.org/10.1001/archsurg.1996.01430170043007.

Dozois RR, Kelly KA, Welling DR, Gordon H, Beart RW, Wolff BG, et al.

Ileal pouch-anal anastomosis: comparison of results in familial adenomatous polyposis and chronic ulcerative colitis. Ann Surg.

1989;210(3):268-71, http://dx.doi.org/10.1097/00000658-19890900000002.

Teixeira WGJ, Silva JH, Teixeira MG, Almeida MG, Calache JE, HabrGama A. Pouchitis: extracolonic manifestation of ulcerative colitis? Rev Hosp Clin Fac Med S Paulo. 1999;54(5):155-8.

Stallmach A, Moser C, Hero-Gross R, Muller-Molaian I, Ecker KW, Feifel G, et al. Pattern of mucosal adaptation in acute and chronic pouchitis.

Dis Colon Rectum. 1999;42(10):1311-7, http://dx.doi.org/10.1007/ BF02234221.

Kmiot WA, Youngs D, Tudor R, Thompson H, Keighley MRB. Mucosal morphology, cell proliferation and faecal bacteriology in acute pouchitis.

Br J Surg. 1993;80(11):1445-9, http://dx.doi.org/10.1002/bjs.1800801132.

Sandborn WJ, Landers CJ, Tremaine WJ, Targan SR. Antineutrophil cytoplasmic antibody correlates with chronic pouchitis after ileal pouchanal anastomosis. Am J Gastroenterol. 1995;90(5):740-7.

Kuisma J, Mentula S, Luukkonen P, Jarvinen H, Kahri A, Farkkila M. Factors associated with ileal mucosal morphology and inflammation in patients with ileal pouch-anal anastomosis for ulcerative colitis. Dis Colon Rectum. 2003;46(11):1476-83, http://dx.doi.org/10.1007/s10350004-6796-6.

Knupper N, Straub E, Terpe HJ, Vestweber KH. Adenocarcinoma of the ileoanal pouch for ulcerative colitis–a complication of severe chronic atrophic pouchitis? Int J Colorectal Dis. 2006;21(5):478-82, http:// dx.doi.org/10.1007/s00384-005-0063-8.

Stein RB, Lichtenstein GR. Complications after ileal pouch-anal anastomosis. Semin Gastrointest Dis. 2000;11(1):2-9.

Gullberg K, Lindforss U, Zetterquist H, Sta˚lberg D, Reinholt FP, Veress B, et al. Cancer risk assessment in long-standing pouchitis. DNA aberrations are rare in transformed neoplastic pelvic pouch mucosa.

Int J Colorectal Dis. 2002;17(2):92-7.

Gullberg K, Sta˚hlberg D, Liljeqvist L, Tribukait B, Reinholt FP, Veress B, et al. Neoplastic transformation of the pelvic pouch mucosa in patients with ulcerative colitis. Gastroenterology. 1997;112(5):1487-92, http:// dx.doi.org/10.1016/S0016-5085(97)70029-5.

Utsunomiya J, Iwana I, Imajo M, Matsuo S, Sawai S, Yaegashi K, et al.

Total colectomy mucosal proctectomy and ileoanal anastomosis. Dis Colon Rectum. 1980;23(7):459-66, http://dx.doi.org/10.1007/ BF02987076.

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DOI:10.6061/clinics/2012(07)03

CLINICAL SCIENCE

Metformin, but not glimepiride, improves carotid artery diameter and blood flow in patients with type 2 diabetes mellitus Helena Atroch Machado,I Marcelo Vieira,I Maria Rosaria Cunha,I Marcia Regina Soares Correia,I Rosa Tsunechiro Fukui,II Rosa Ferreira dos Santos,I Dalva Marreiro Rocha,I Bernardo Leo Wajchenberg,II Silvia G. Lage,II Maria Elizabeth Rossi da SilvaI I

Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Laboratory of Medical Investigation (LIM-18), Sa˜o Paulo/SP, Brazil.

Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Heart Institute (InCor) Sa˜o Paulo/SP, Brazil.

II

OBJECTIVE: To compare the effects of glimepiride and metformin on vascular reactivity, hemostatic factors and glucose and lipid profiles in patients with type 2 diabetes.

METHODS: A prospective study was performed in 16 uncontrolled patients with diabetes previously treated with dietary intervention. The participants were randomized into metformin or glimepiride therapy groups. After four months, the patients were crossed over with no washout period to the alternative treatment for an additional fourmonth period on similar dosage schedules. The following variables were assessed before and after four months of each treatment: 1) fasting glycemia, insulin, catecholamines, lipid profiles and HbA1 levels; 2) t-PA and PAI-1 (antigen and activity), platelet aggregation and fibrinogen and plasminogen levels; and 3) the flow indices of the carotid and brachial arteries. In addition, at the end of each period, a 12-hour metabolic profile was obtained after fasting and every 2 hours thereafter.

RESULTS: Both therapies resulted in similar decreases in fasting glucose, triglyceride and norepinephrine levels, and they increased the fibrinolytic factor plasminogen but decreased t-PA activity. Metformin caused lower insulin and pro-insulin levels and higher glucagon levels and increased systolic carotid diameter and blood flow. Neither metformin nor glimepiride affected endothelial-dependent or endothelial-independent vasodilation of the brachial artery.

CONCLUSIONS: Glimepiride and metformin were effective in improving glucose and lipid profiles and norepinephrine levels. Metformin afforded more protection against macrovascular diabetes complications, increased systolic carotid artery diameter and total and systolic blood flow, and decreased insulin levels. As both therapies increased plasminogen levels but reduced t-PA activity, a coagulation process was likely still ongoing.

KEYWORDS: Treatment; Metabolic profile; Vascular reactivity; Hemostatic factors; Type 2 diabetes.

Machado HA, Vieira M, Cunha MR, Correia MRS, Fukui RT, Santos RF et al. Metformin, but not glimepiride, improves carotid artery diameter and blood flow in patients with type 2 diabetes mellitus. Clinics. 2012;67(7):711-717.

Received for publication on November 28, 2011; First review completed on December 28, 2011; Accepted for publication on March 5, 2012 E-mail: maria_cunha@uol.com.br Tel.: 55 11 3061 7259

hypertension, oxidative stress, accelerated aging, hyperinsulinemia, disturbances in coagulation and fibrinolysis (1).

Biguanides and sulfonylureas remain the principal oral therapeutic options for treating patients with DM2 (2). The biguanide metformin has been established as a first-line drug for the management of type 2 diabetes. Its indications are supported by its potency, lack of weight gain, low risk of hypoglycemia and mode of action in countering insulin resistance (3). The drug’s anti-atherosclerotic and cardio-protective effects appear to reflect a combination of glucose-independent effects on the vascular endothelium, suppressant effects on glycation, oxidative stress and the formation of adhesion molecules, and anti-inflammatory properties, in addition to stimulating fibrinolysis and favorable effects on lipid profiles (4).

INTRODUCTION Cardiovascular disease is the leading cause of mortality in patients with type 2 diabetes mellitus (DM2). The effective control of glycemia can delay but not prevent vascular complications, which are likely related to many other poorly controlled atherogenic factors, such as hyperlipidemia,

Copyright ß 2012 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.

No potential conflict of interest was reported.

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In contrast, sulfonylureas enhance insulin release primarily by closing the ATP-sensitive K+ channels of pancreatic b-cells. The glucose-lowering potency of sulfonylureas is similar to that of metformin. However, by stimulating insulin secretion, sulfonylureas are believed to favor the development of hypoglycemia and weight gain, accelerate beta-cell apoptosis and beta-cell exhaustion and impair endothelial function, thereby increasing the risk for ischemic complications. The blocking of potassium channels in the heart by sulfonylureas has raised concern regarding the drugs’ potential adverse effects in cases of ischemic heart episodes (5).

Glimepiride is a long-acting and low potency insulin secretagogue sulfonylurea that has an insulin-sensitizer effect on the muscles and liver (6) and rarely causes hypoglycemia (5). Data from studies in humans are scarce but have suggested that the risks of developing coronary artery disease and mortality do not appear to be increased by glimepiride (7-8). In addition, no deleterious effects of glimepiride on brachial vasodilatation — an acute effect (9) — or ischemic preconditioning — acute (10) and chronic (11) effects — have been observed Only two studies have investigated the chronic vascular effects of glimepiride measured by forearm arterial blood flow, and the results were similar to those of glibenclamide (12-13) and metformin (12). There have been no reports comparing carotid artery blood flow during glimepiride and metformin therapy, and only a few have measured hemostatic factors (14-19), specifically plasminogen activator inhibitor (PAI-1), fibrinogen and adhesion molecules. In our study, the effects of glimepiride in patients with type 2 diabetes on vascular reactivity, hemostatic factors, and fat and carbohydrate metabolism were compared with those of metformin, the first-line therapy. This comparison is valuable in that these different classes of oral anti-diabetic agents — metformin and glimepiride — target the two main pathophysiological defects of type 2 diabetes, and their safety is fundamental.

Table 1 - Anthropometric data.

Pretreatment Age (yr) Sex (F:M) Dose (mg/day) Weight (kg) BMI (kg/m2) W/H DBP (mm Hg) SBP (mm Hg)

Metformin Group

Glimepiride Group

1907¡558 68.9¡12.5 26.7¡3.4 0.9¡0.1 79.4¡7.5 129.2¡16.4

4¡2 70.5¡14.2 27.2¡3.7 0.9¡0.1 81.1¡8.5 134.0¡14.0

51.8¡6.5 10:06 71.1¡13.4 27.6¡3.6 0.9¡0.1 83.3¡9.3 141.5¡14.0

Ns Ns Ns Ns Ns

Values are expressed as means¡SDs. DBP = diastolic blood pressure; SBP = systolic blood pressure; BMI = body mass index; W/H = waist-to-hip ratio.

Seven of the ten women were postmenopausal, and none had received hormone replacement therapy, while the premenopausal women were tested up to the 8th day of the follicular phase of their cycles. At the time of enrollment, a complete medical history, physical examination, and laboratory evaluation, including urinalysis, renal, hepatic, and thyroid function tests and serum lipid and electrolyte levels, were obtained for all of the subjects. An ECG and echocardiogram were performed, and subjects with left ventricular systolic dysfunction, valve abnormalities, arrhythmias and ischemic heart disease were not enrolled.

None of the patients exercised on a regular basis. Other exclusion criteria included any severe concomitant illness, nephropathy (serum creatinine .1.6 mg/dL or microalbuminuria), uncontrolled hypertension (BP .190x120 mm Hg), stroke, peripheral vascular disease, marked dyslipidemia (total cholesterol .6.5 mmol/L and triglyceride levels .2.8 mmol/L), coagulopathy, proliferative diabetic retinopathy and use of hypolipidemic and anticoagulant medications. None of the subjects demonstrated clinical evidence of autonomic neuropathy as assessed by blood pressure response to standing, beat-to-beat heart rate variation, the Valsalva maneuver and the handgrip test. The Medical Ethics Committee of Hospital das Clı´nicas and Heart Institute (INCOR) approved the study protocol, and all of the subjects provided written informed consent.

METHODS Patients A prospective study was performed in 16 uncontrolled patients with type 2 diabetes according to the ADA criteria (2); the cohort included ten women and six men with a mean age of 51.8¡6.5 years (mean¡SD) previously treated with dietary intervention. Subjects who had fasting blood glucose values .7.78 mmol/L and/or glycated hemoglobin exceeding the normal range (4-8.5%) by 1.0% or more after two or more months of a diet therapy program without medications (basal values) were included. The participants were randomly assigned to receive either metformin (M group) or glimepiride (G group). The drug dosage was titrated to achieve fasting glucose levels lower than 7.0 mmol/L using domiciliary capillary glucose measurements.

After four months, the patients were crossed over with no washout period to the alternative treatment for an additional four-month period on a similar dosage schedule. The subjects were followed on an outpatient basis every 1-2 weeks throughout the study period for drug and weightmaintaining diet adjustments. The clinical characteristics of the patients are depicted in Table 1. Three of the sixteen subjects smoked, and six had systemic arterial hypertension, which was treated with converting-enzyme inhibitors.

Study protocol The patients were instructed to continue a similar food intake and abstain from the use of tobacco, alcohol, coffee, salty food, and any physical activity for 24 h before test days and discontinue converting-enzyme inhibitors 72 h before the evaluation.

The following procedures were performed before (basal values) and after each four-month treatment period (M and G groups): 1) hormonal and metabolic evaluations: fasting plasma glucose, insulin and catecholamine levels, lipid profiles and HbA1; 2) hemostatic factor determination: tissue plasminogen activator (t-PA) antigen and activity, plasminogen activator inhibitor (PAI-1) antigen and activity, platelet aggregability, fibrinogen and plasminogen levels; and 3) a cardiovascular evaluation: by high-resolution ultrasound of the carotid and brachial arteries.

At the end of each treatment period, a 12-h metabolic profile, including measurements of glucose, insulin, glucagon, proinsulin, and triglyceride levels at fasting and every 2 h (from 7 am to 7 pm), was obtained. The meals offered to patients (breakfast, lunch, and dinner) contained 50% of

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Metabolic and vascular effects of antidiabetic drugs Machado HA et al.

intra-assay CV was 8%. Platelet aggregation was performed using a method described by Born (27). The activities of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI-1) were determined by quantitative assays (ChromolizeTM t-PA and ChromolizeTM PAI-1, respectively, Biopool, Umea, Sweden); the intra-assay CVs were 3.9% for tPA and 3.7% for PAI-1. PAI-1 and t-PA antigens were determined by Imulyse and Tint-Elize (Biopool, Umea, Sweden), respectively. The intra-assay CVs were 5% for PAI-1 and 5.5% for t-PA. All of the analyses were performed in duplicate.

their total calories as carbohydrates, 20% as protein, and 30% as fat, and the meals were provided by the Metabolic Unit of the Hospital das Clı´nicas.

Cardiovascular evaluation The study was performed in a laboratory setting at a temperature of 23 ˚C and in low luminosity, with the patients in the supine position. A catheter was inserted into the forearm vein 30 minutes before blood sample collection for catecholamine, insulin, sodium, and potassium determinations and maintained with a saline infusion.

During the study, the heart rate and systolic, diastolic, and mean blood pressure were registered using a noninvasive, automatic oscillometric device (Dinamap 1486, Critikon, Inc., Tampa, FL, USA). The images for flow velocity and diameters of the arteries were first established after a 10-minute equilibration period using a transducer (Apogee-800 Plus, ATL Inc., Bothell, WA, USA). Intimamedia thickness (IMT), the compliance and distensibility of the carotid artery, and the total and systolic blood flow indices (TFI and SFI) were obtained. Flow-mediated vasodilation of the brachial artery was measured using a method reported elsewhere (20). A cuff placed on the left forearm was inflated to 200 mm Hg for 5 minutes. Imaging of the artery was performed before cuff inflation (baseline; B) and at 60 seconds after cuff deflation (reactive hyperemia; RH) to obtain measurements of flow velocity and the diameter of the artery. Fifteen minutes after acquisition of the post-occlusion image, the baseline image was reobtained (rebase; RE), after which nitroglycerin was administered sublingually; 3 and 5 minutes later, another image was acquired (N3 and N5, respectively) to establish endothelium-independent vasodilation. The total and systolic flow indices (TFI and SFI) of the brachial artery were also calculated based on these images.

Statistical methods Numerical data are reported as means and standard deviations, and nominal data are reported as proportions.

Differences (95% CI) between the treatment groups were initially tested for treatment-time interaction (28) and then compared by an analysis of variance for repeated measurements and a Tukey’s post-test or Student’s two-tailed test, with p,0.05 considered statistically significant.

RESULTS A significant treatment period interaction effect was demonstrated for triglycerides, VLDL cholesterol, plasminogen and norepinephrine levels; hence, only the values from the first treatment period were analyzed.

Anthropometric, biochemical, hormonal and hemostatic factor measurements Weight, waist-to-hip ratio, and systolic and diastolic blood pressure did not change after glimepiride or metformin treatment (Table 1). Fasting plasma HbA1 (p = 0.000009) and glucose levels (p = 0.00009) decreased by equal amounts in both treatment groups (Table 2). Fasting insulin levels were greater in the G group (p = 0.009) compared with the M group. Similar decreases in VLDL cholesterol (p = 0.007), triglycerides (p = 0.023) and norepinephrine (p = 0.042) levels were obtained in both groups. There was an increase in plasminogen levels (p = 0.025) after the initial four months of M (118.2¡8.26142.4¡32.0) or G (128.4¡8.66130.2¡8.1) therapy, which although persistent did not remain significant when the groups switched to the alternative drugs. LDL and HDL cholesterol and epinephrine levels were unchanged.

Both therapies decreased t-PA activity (p = 0.024). There was no significant effect of either of the therapies on the other hemostatic factors measured (PAI-1 antigen and activity, fibrinogen levels and platelet aggregation — data not shown).

Biochemical and hormonal analyses Glucose was determined by the glucose oxidase method (Labtest, Sa˜o Paulo, Brazil) (21), and HbA1 (normal values: 4 to 8.5%) was determined by ionic chromatography (Labtest, Sa˜o Paulo, Brazil) (22). Total cholesterol was measured by employing the cholesterol oxidase/peroxidase method; HDL cholesterol was separated using the phosphotungstic acid/ Mg2+ method and measured using the oxidase/peroxidase method, while triglycerides were measured by the lipase/ glycerol kinase method (Labtest, Sao Paulo, Brazil) (23). LDL was estimated using the Friedewald equation (LDL cholesterol = total cholesterol minus HDL cholesterol minus 0.2 6 triglycerides). The intra-assay and inter-assay coefficients of variation (CVs) for the glucose and lipid determinations were ,3% and 0%, respectively. Insulin, proinsulin and glucagon were quantified by a double-antibody radioimmunoassay (Linco Research, St. Louis, MO, USA) (24). Catecholamines were measured by high-performance liquid chromatography (25). The intra-assay and inter-assay CVs for the hormonal analyses were 6.8% and 9.6% for insulin, 4.4% and 6.5% for glucagon, 5.5% and 6.8% for catecholamines, and 5% and 5.3% for proinsulin, respectively.

Hemostatic factors were measured using the same assay. Plasminogen was measured by chromogenic assay (Plasminogen Accucolor TM Sigma Diagnostic, ST Louis, MO, USA), and the intra-assay coefficient of variation was ,3.0%.

Fibrinogen was determined by the CLAUSS method (26) with Fibriquick Assay (Sigma Diagnostics, ST Louis, USA). The

12-h metabolic profile Only the areas under the curve during glimepiride and metformin therapy were analyzed (Figure 1). The 12-h integrated areas for glucose (M:87.7¡11.03 vs. G:104.61¡ 36.63 mmol/L/h) and triglycerides (M:20.47¡9.68 vs. G:22.4 ¡11,03 mmol/L/h) were similar for both therapies.

Treatment with metformin was associated with higher glucagon (M:1361.69¡473.25 vs. G:1044.22¡326.90 ng/L/h; p = 0.0046) and lower insulin-integrated (M:1076.61¡389.02 vs. G:1718.69¡837.03 pmol/L/h — p = 0.02) and proinsulinintegrated (M:565.38¡279.11 vs. G:834.71¡299.96 pmol/L/ h — p = 0.0016) areas compared with the G group (Figure 1).

The proinsulin-to-insulin molar ratios during the M (0.58¡0.32) and G (0.60¡0.37) therapies did not differ.

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Table 2 - Glucose, glycated hemoglobin, insulin, total and fractional cholesterol, triglycerides and catecholamine levels.

Pre-treatment Glucose (mmol/L) HbA1 (%) Insulin (pmol/L) Total cholesterol (mmol/L) HDL cholesterol (mmol/L) LDL cholesterol (mmol/L) VLDL cholesterol (mmol/L) Triglyceride (mmol/L) Norepinephrine (nmol/dL) Epinephrine (nmol/dL) t-PA activity (IU/mL) a

Metformin Group

15.1¡5.12 10.8¡2.3 59.5¡22.9 5.41¡1.29 0.99¡0.21 3.45¡1.16 0.92¡0.54 2.01¡1.19 1481.0¡986.4 104.7¡176.8 1.1¡0.5

7.10¡1.34 8.2¡1.4 55.9¡29.4 5.05¡0.93 1.01¡0.18 3.32¡0.80 0.66¡0.31 1.38¡0.67 913.3¡497.5 26.1¡72 0.7¡0.3

Glimepiride Group 8.3¡1.7 7.8¡1.3 79.6¡25.8 5.23¡1.09 0.95¡0.22 3.64¡0.90 0.70¡0.25 1.46¡0.60 958.7¡414.7 16.3¡49.6 0.8¡0.3

p-value a

0.00011 b0.037 a 0.009 b0.001 c 0.009 Ns Ns Ns d 0.007 d 0.023 d 0.042 Ns d 0.024

(pre-treatment vs. metformin); b(pre-treatment vs. glimepiride); c(metformin vs. glimepiride); d(pre. vs. post-treatment). Values are expressed as means ¡ SDs.

Figure 1 - 12-h metabolic profiles of glucose, triglyceride, insulin, proinsulin and glucagon levels. Mean levels are indicated.

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Table 3 - Total (TFI) and systolic (SFI) flow indices and percentage changes in the systolic diameter of the carotid artery.

Pre-treatment 2

TFI (L/min/m ) SFI (L/min/m2)

0.319¡0.079 0.599¡0.155

% Systolic Diameter a

Metformin Group

Glimepiride Group

0.382¡0.094 0.738¡0.167

0.285¡0.091 0.563¡0.152

2.75¡9.93

-3.50¡11.46

p-value a

0.024; c0.003 a 0.006 c 0.003 ,0.05

c

(pre-treatment vs. metformin); (metformin vs. glimepiride). Values are expressed as means ¡ SDs.

Despite these findings, the proinsulin-to-insulin ratio areas under the curve during the 12-h metabolic profile were similar for both therapies, suggesting that glimepiride did not worsen the previous secretor dysfunction of beta cells, as reported with other sulfonylureas (35).

The overall effects on plasma lipids were small, with similar lowering of fasting VLDL cholesterol and triglyceride levels after the initial four months of both therapies. TG levels during the 12-h metabolic profile were also similar between the two drugs. LDL and HDL cholesterol levels were unaffected by treatment. Metformin. and glimepiride-associated improvements in lipid metabolism were expected, but the reported changes have been small (3,16,30). The near normal triglyceride and cholesterol levels of our patients prior to both therapies were likely factors that influenced these modest results.

Despite causing lower insulin and higher glucagon secretion, metformin kept glucose and lipid profiles at similar levels compared with glimepiride. The blood glucose-lowering actions of metformin result primarily from an amelioration of insulin resistance, increase in peripheral glucose disposal, decrease in fatty acid oxidation and activation of the enzyme adenosine monophosphate (AMP) kinase to increase glucose transporter 4 (GLUT4) translocation to plasma membrane cells in the muscles and fat and reduce gluconeogenesis in the liver (3,36). The higher glucagon levels are unlikely to have interfered with metformin’s action, as glucagon appears to have little effect on the presence of insulin, suggesting that its diabetogenic action occurs only under conditions of high insulin deficiency (37). In addition, glucagon’s effects on hepatic glucose production could have been strongly counteracted by metformin.

Vascular reactivity The elastic arterial properties were evaluated.

Carotid artery (Table 3): Both the total (p = 0.004) and systolic (p = 0.002) carotid flow indices increased with metformin therapy compared with baseline values and glimepiride therapy. Moreover, the carotid systolic diameter changed in opposite directions during the therapies; its percentage change (from basal levels) increased with metformin and decreased with glimepiride, and these different behaviors reached significance (p = 0.028). The compliance, distensibility and intima-media thickness of the carotid artery did not change significantly with any therapy (data not shown).

Brachial artery: There was a similar increase in flowmediated vasodilatation of the brachial artery after reactive hyperemia (endothelium-dependent) and sublingual nitroglycerin (endothelium-independent) stimuli across the groups. There were no significant effects of medications on systolic diameter or the total and systolic flow indices of the brachial artery (data not shown).

DISCUSSION This study compared the actions of two different classes of drugs (the biguanide metformin, an insulin sensitizer, and the sulfonylurea glimepiride, an insulin secretagogue) on carbohydrate and lipid metabolism, hemostatic factors and vascular reactivity. We evaluated the same patients with type 2 diabetes before and after four months of treatment with metformin or glimepiride. The aim of this strategy was to minimize the influence of metabolic control on specific drug effects other than glucose control. The data in the literature remain poor and contradictory regarding the direct actions of these drugs on the cardiovascular risk-related factors analyzed in the present study (1519,30-33).

Hemostatic factors Glimepiride and metformin had similar effects on hemostatic factors; both increased plasminogen levels (significant after four months on each therapy) and decreased t-PA activity. These findings observed in the G group have not been reported previously.

The improvements in fibrinolysis after metformin and glimepiride therapy suggested by the increases in plasminogen levels occurred along with an unexpected decrease in another marker of fibrinolysis — t-PA activity. In contrast to our results, an increase in t-PA activity during metformin therapy has been described (38). However, as active t-PA declines as a function of increasing concentrations of PAI-1 and considering that PAI-1 antigen and activity did not change in our experiment, we can speculate that the formed complexes of PAI-1 and t-PA cleared in an accelerated fashion by the liver (39) contributed to the decrease in t-PA activity and that an impairment of fibrinolysis continued to occur. These data are likely implicated in the high frequency of cardiovascular disease in type 2 diabetes. No additional effects of any of the therapies were observed on the other

Carbohydrate and lipid metabolism Both treatment groups achieved similar and significant mean decreases from baseline in fasting plasma glucose and HbA1 levels. The lower insulin and proinsulin levels (at fasting and during the 12-h metabolic profile) observed during metformin therapy compared with glimepiride were in agreement with reports of metformin’s sparing effects on beta cell function, thus lowering the basal and postprandial insulin requirements for the same metabolic control (3,17).

These differences in insulin secretion could not be accounted for by changes in body weight, which were unaffected in both groups. Although some authors have found no increases in insulin levels (18) and others have reported that glimepiride’s insulin trophic effect might diminish in the presence of normoglycemia (34), the insulin levels in this study were found to be higher during therapy with glimepiride than metformin.

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activity, the coagulation process continued, which can worsen cardiovascular disease. Only metformin countered insulin resistance and induced an increase in carotid artery diameter and blood flow indices. Neither drug affected small brachial artery vasodilation.

hemostatic factors analyzed (fibrinogen levels and platelet aggregation — data not shown).

Vascular reactivity Increased carotid artery systolic diameter and blood flow were demonstrated only with metformin therapy, whereas an opposite trend in carotid diameter was observed with glimepiride use. Similar trends were observed even after patients treated for hypertension were excluded. No changes were observed for the other properties of the carotid (compliance, distensibility, and intima-media thickness) or brachial arteries (flow and diameter measurements after stimulus with reactive hyperemia and nitroglycerin).

Metformin’s effects on carotid artery flow, independent of glucose decay or changes in systolic and diastolic blood pressure, likely afford more protection against cerebral diabetes complications. Its countering of insulin resistance action is likely implicated. Furthermore, we observed a negative correlation between fasting serum insulin levels and carotid compliance in the M group (r = -0.5; p = 0.04).

Several observational analyses have suggested cardioprotective benefits with metformin use in patients with cardiovascular disease (3-4,40). Patients on metformin exhibited reduced nitroxidant metabolites and increased nitric oxide levels (30). As the carotid artery is more elastic and more proximal to the heart than the brachial artery, it might be more amenable to these improvements.

As both therapies improved glucose control in very similar manners, their beneficial effects on lipid profiles, hemostatic factors and norepinephrine levels can likely be ascribed to improvement in the metabolic milieu and not to a specific drug effect. Our study is the first to demonstrate decreases in norepinephrine levels after the first four months of glimepiride therapy and similar trends for epinephrine levels. The same results were obtained for metformin.

Although metformin has been confirmed as the first-line option for treating diabetes, troublesome gastrointestinal intolerance occasionally precludes its use (3). Thus, sulfonylureas remain important adjuvants for patients with intolerance to metformin or limited insulin secretion (5).

When compared with metformin, glimepiride achieved similar efficacy in controlling weight and improving metabolic and hemostatic factors. Although it led to greater insulin secretion, glimepiride did not worsen beta cell function, as measured by the proinsulin-to-insulin ratio or vascular reactivity. Long-term studies are needed to ascertain whether glimepiride can reduce beta cell exhaustion or apoptosis.

Our study has some limitations. The four-month treatment duration could have not been sufficient to demonstrate all of the effects of these medications. Additionally, as a crossover study with no washout period, a treatment period interaction effect was demonstrated for some variables (triglyceride, VLDL cholesterol, plasminogen and norepinephrine levels). To minimize this problem, only the values from the first treatment period were analyzed.

In patients with type 2 diabetes inadequately controlled by dietary therapy, M and G resulted in similar overall improvements in glycemic control, lipid profiles, norepinephrine levels, and levels of the fibrinolytic factor plasminogen. All of these beneficial effects were likely due to improvement of the metabolic environment and were not drug-specific. However, as both therapies reduced t-PA

ACKNOWLEDGMENTS We are grateful to Greci de Paula Souza for assistance with procedures.

This study was supported by the Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo-FAPESP.

AUTHOR CONTRIBUTIONS Machado HA and Cunha MR participated in designing the studies, conducting the clinical and experimental procedure and writing the paper.

Vieira M provided the cardiovascular evaluation. Correia MRS performed the biochemical analysis. Fukui RT performed the hormonal analysis.

Santos RF, Wajchenberg BL and Rocha DM collaborated on the clinical study. Lage SG collaborated on the clinical study and the cardiovascular evaluation. Silva MER collaborated on the clinical study and wrote, edited and finalized the manuscript for publication.

REFERENCES 1. Stolar M. Glycemic control and complications in type 2 diabetes mellitus.

Am J Med. 2010;123(Suppl. 3):S3-S11, http://dx.doi.org/10.1016/ j.amjmed.2009.12.004.

2. American Diabetes Association - Standards of medical care in diabetes.

Diabetes Care. 2009;32(Suppl. 1):S13–S61.

3. Scarpello JHB, Howlett HCS. Metformin therapy and clinical uses.

Diabetes and Vascular Disease Research 2008;5:157-67.

4. Bailey CJ. Metformin: Effects on micro and macro vascular complications in type 2 diabetes. Cardiovascular Drugs Ther. 2008; 22(3):215-24, http:// dx.doi.org/10.1007/s10557-008-6092-0.

5. Draeger KE, Wernicke-Panten K, Lomp HJ, Schuler E, Rosskamp R. Long-term treatment of type 2 diabetic patients with the new oral antidiabetic agent glimepiride (Amaryl): a double-blind comparison with glibenclamide. Horm Metab Res. 1996;28(9):419–25, http://dx.doi.org/ 10.1055/s-2007-979830.

6. Mori RC, Hirabara SM, Hirata AE, Okamoto MM, Machado UF. Glimepiride as insulin sensitizer: increased liver and muscle responses to insulin Diabetes Obes Metab. 2008l;10(7):596-600.

7. Sadikot SM, Mogensen CE. Risk of coronary artery disease associated with initial sulphonylurea treatment of patients with type 2 diabetes: a matched case-control study. Diabetes Res Clin Pract. 2008;82(3):391-5, http://dx.doi.org/10.1016/j.diabres.2008.09.004.

8. Monami M, Luzzi C, Lamanna C, Chiasserini V, Addante F, Desideri CM, et al. Three-year mortality in diabetic patients treated with different combinations of insulin secretagogues and metformin. Diabetes Metab Res Rev. 2006;22(6):477-82, http://dx.doi.org/10.1002/dmrr.642.

9. Bijlstra PJ, Lutterman JA, Russel FGM. Interaction of sulfonylurea derivate with vascular ATP-sensitive potassium channels in humans. Diabetologia.

1996;39(9):1083-90, http://dx.doi.org/10.1007/BF00400658.

10. Klepzig H, Kober G, Matter C, Luus H, Schneider H, Boedeker KH, et al.

Sulfonylureas and ischemic preconditioning. A double-blind, placebo controlled evaluation of glimepiride and glibenclamide. European Heart Journal. 1999;20(6):439-46.

11. Lee TM, Chou TF. Impairment of myocardial protection in type 2 diabetic patients. J Clin Endocrinol Metab. 2003;88(2):531-7, http:// dx.doi.org/10.1210/jc.2002-020904.

12. Abbink EJ, Pickkers P, Jansen van RA, Lutterman JA, Tack CJ, Russel FG, et al. Vascular effects of glibenclamide vs. glimepiride and metformin in Type 2 diabetic patients. Diabet Med. 2002;19(2):136-43.

13. Spallarossa P, Schiavo M, Rossettin P, Cordone S, Olivotti L, Cordera R, et al. Sulfonylurea treatment of type 2 diabetic patients does not reduce the vasodilator response to ischemia. Diabetes Care. 2001;24(4):738-42, http://dx.doi.org/10.2337/diacare.24.4.738.

14. Pfu¨tzner A, Marx N, Lu¨bben G, Langenfeld M, Walcher D, Konrad T, et al. Improvement of cardiovascular risk markers by pioglitazone is independent from glycemic control: results from the pioneer study. J Am Coll Cardiol. 2005;45(12):1925-31, http://dx.doi.org/10.1016/j.jacc.2005.

03.041.

15. Yener S, Comlekci A, Akinci B, Demir T, Yuksel F, Ozcan MA, et al.

Soluble CD40 ligand, plasminogen activator inhibitor-1 and thrombinactivatable fibrinolysis inhibitor-1-antigen in normotensive type 2 diabetic subjects without diabetic complications. Effects of metformin and rosiglitazone. Med Princ Pract. 2009;18(4):266-71, http://dx.doi.org/ 10.1159/000215722.

716

CLINICS 2012;67(7):711-717

Metabolic and vascular effects of antidiabetic drugs Machado HA et al.

16. Rizzo MR, Barbieri M, Grella R, Passariello N, Paolisso G. Repaglinide has more beneficial effect on cardiovascular risk factors than glimepiride: data from meal-test study. Diabetes Metab. 2005;31(3 Pt 1):255-60, http://dx.doi.org/10.1016/S1262-3636(07)70192-1.

17. Derosa G, Franzetti I, Gadaleta G, Ciccarelli L, Fogari R. Metabolic variations with oral antidiabetic drugs in patients with Type 2 diabetes: comparison between glimepiride and metformin. Diabetes Nutr Metab.

2004;17:143-50.

18. Derosa G, Mugellini A, Ciccarelli L, Crescenzi G, Fogari R. Comparison between repaglinide and glimepiride in patients with type 2 diabetes mellitus: a one-year, randomized, double-blind assessment of metabolic parameters and cardiovascular risk factors. Clin Ther. 2003;25(2):472-84, http://dx.doi.org/10.1016/S0149-2918(03)80090-5.

19. Luis BJ, Bugos C, Dirnberger G, Atherton T. Efficacy and safety profile of glimepiride in Mexican American Patients with type 2 diabetes mellitus: a randomized, placebo-controlled study. Clin Ther. 2003;25(1):194-209, http://dx.doi.org/10.1016/S0149-2918(03)90025-7.

20. Corretti MC, Anderson TJ, Benjamin EJ, Celermajer D, Charbonneau F, Creager MA, et al. Guidelines for the ultrasound assessment of endothelialdependent flow-mediated vasodilation of the brachial artery: a report of the International Brachial Artery Reactivity Task Force. J Am Coll Cardiol.

2002;39(2):257-65, http://dx.doi.org/10.1016/S0735-1097(01)01746-6.

21. Latt JA, Turner K. Evaluations of Trinder’s glucose oxidase method for measuring glucose in serum and urine. Clin Chem. 1975;21(12):1754-60.

22. Trivelli LA, Ranney HM, Lai HT. Hemoglobin components in patients with diabetes mellitus. New Engl J Med. 1971 284:353-57.

23. Warnick GR. Enzymatic methods for quantification of lipoprotein lipids.

Methods Enzymol. 1986;129:101-23, http://dx.doi.org/10.1016/00766879(86)29064-3.

24. Warnick GR, Knopp RH, Fitzpatrick V, Brauson L. Estimating lowdensity lipoprotein cholesterol by the Friedewald equation is adequate for classifying patients on the basis of nationally recommended cutpoints. Clin Chem. 1990;36(1):15-9.

25. Bouloux P, Perret D, Besser GM. Methodological considerations in the determination of plasma cathecolamines by high-performance liquid chromatography with electrochemical detection. Ann Clin Biochem 1985;22(Pt 2):194-203.

26. Clauss A. Rapid physiological coagulation method for determination of fibrinogen. Acta Haematol. 1957;17(4):237-46, http://dx.doi.org/ 10.1159/000205234.

27. Born GVR. Quantitative investigation into the aggregation of blood platelets. J Physiol. 1962;162:67-8.

28. Singer JM, Andrade DF. Analysis of longitudinal data. In Handbook of Statistics. Volume 18: Bio-Environmental and Public Health Statistics. eds. P.K. Sen and C.R. Rao. Amsterdam: North Holland.2000;115-60.

29. Neter J, Kutner MH, Nachtsheim CJ, Wasserman W. In Richard D. Irwing ed. Applied Linear Statistical Models, 4. Ed. Ilinois. 1996:1408p.

30. Meaney E, Vela A, Samaniego V, Meaney A, Asbu´n J, Zempoalteca JC, et al. Metformin, arterial function, intima-media thickness and nitroxidation in metabolic syndrome: the mefisto study. Clin Exp Pharmacol Physiol. 2008;35(8):895-903, http://dx.doi.org/10.1111/j.1440-1681.2008.

04920.x.

31. Stocker DJ, Taylor AJ, Langley RW, Jezior MR, Vigersky RA. A randomized trial of the effects of rosiglitazone and metformin on inflammation and subclinical atherosclerosis in patients with type 2 diabetes. Am Heart J. 2007;153(3):445.e1-6, http://dx.doi.org/10.1016/ j.ahj.2006.11.005.

32. Huang Z, Lei MX, Liu L, Tang QB. Effects of rosiglitazone on the IMTc and serum MMP-9 levels in newly diagnosed type 2 diabetic patients.

Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2006;31(3):367-72.

33. Stakos DA, Schuster DP, Sparks EA, Wooley CF, Osei K, Boudoulas H. Long term cardiovascular effects of oral antidiabetic agents in nondiabetic patients with insulin resistance: double blind, prospective, randomized study. Heart. 2005;91(5):589-94, http://dx.doi.org/10.1136/ hrt.2003.027722.

34. Clark HE, Matthews DR. The effect of glimepiride on pancreatic beta – cell function under hyperglycaemic clamp and hyperinsulinaemic, euglycaemic clamp conditions in non. insulin. dependent diabetes mellitus. Horm Metab Res. 1996;28(9):445-60.

35. Inoguchi T, Umeda F, Kakimoto M, Sako Y, Ishii H, Noda K, et al.

Chronic sulfonylurea treatment and hyperglycemia aggravate disproportionately elevated plasma proinsulin levels in patients with 2 diabetes. Endocr J. 2000;47(6):763-70, http://dx.doi.org/10.1507/ endocrj.47.763.

36. Reitman ML, Schadt EE. Pharmacogenetics of metformin response: a step in the path toward personalized medicine. J Clin Invest. 2007; 117(5):1226-9, http://dx.doi.org/10.1172/JCI32133.

37. Bansal P, Wang Q. Insulin as a physiological modulator of glucagon secretion. Am J Physiol Endocrinol Metab. 2008;295(4):E751-E761, http://dx.doi.org/10.1152/ajpendo.90295.2008.

38. Grant PJ. Beneficial effects of metformin on haemostasis and vascular function in man. Diabetes Metab. 2003;29(4 Pt):6S44-52, http:// dx.doi.org/10.1016/S1262-3636(03)72787-6.

39. Wing LR, Hawksworth GM, Bennett B, Booth NA. Clearance of t-PA, PAI-1, and t-PA-PAI-1 complex in an isolated perfused rat liver system. J Lab Clin Med. 1991;117(2):109-14.

40. Roussel R, Travert F, Pasquet B, Wilson PW, Smith SC Jr, Goto S, et al.

For the Reduction of Atherothrombosis for Continued Health (REACH) Registry Investigators. Metformin Use and Mortality Among Patients With Diabetes and Atherothrombosis. Arch Intern Med. 2010;170(21): 1892-9.

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DOI:10.6061/clinics/2012(07)04

CLINICAL SCIENCE

Association between functional alterations of senescence and senility and disorders of gait and balance Homero Teixeira-Leite,I,II Alex C. Manha˜esI I Universidade do Estado do Rio de Janeiro, Instituto de Biologia Roberto Alcantara Gomes, Departamento de Cieˆncias Fisiolo´gicas, Centro Biome´dico, Laborato´rio de Neurofisiologia, Rio de Janeiro/RJ, Brazil. II Hospital Adventista Silvestre do Rio de Janeiro, Unidade Integrada de Prevenc¸a˜o, Rio de Janeiro/RJ, Brazil.

OBJECTIVES: Declines in cognition and mobility are frequently observed in the elderly, and it has been suggested that the appearance of gait disorders in older individuals may constitute a marker of cognitive decline that precedes significant findings in functional performance screening tests. This study sought to evaluate the relationship between functional capacities and gait and balance in an elderly community monitored by the Preventive and Integrated Care Unit of the Hospital Adventista Silvestre in Rio de Janeiro, RJ, Brazil.

METHODS: Elderly individuals (193 females and 90 males) were submitted to a broad geriatric evaluation, which included the following tests: 1) a performance-oriented mobility assessment (POMA) to evaluate gait; 2) a minimental state examination (MMSE); 3) the use of Katz and Lawton scales to assess functional capacity; 4) the application of the geriatric depression scale (GDS); and 5) a mini-nutritional assessment (MNA) scale.

RESULTS: Reductions in MMSE, Katz and Lawton scores were associated with reductions in POMA scores, and we also observed that significant reductions in POMA scores were present in persons for whom the MMSE and Katz scores did not clearly indicate cognitive dysfunction. We also demonstrated that a decline in the scores obtained with the GDS and MNA scales was associated with a decline in the POMA scores.

CONCLUSIONS: Considering that significant alterations in the POMA scores were observed prior to the identification of significant alterations in cognitive capacity using either the MMSE or the Katz systems, a prospective study seems warranted to assess the predictive capacity of POMA scores regarding the associated decline in functional capacity.

KEYWORDS: Aging; Cognition disorders; Dementia; Locomotion; Postural balance.

Teixeira-Leite H, Manha˜es AC. Association between functional alterations of senescence and senility and disorders of gait and balance. Clinics.

2012;67(7):719-729.

Received for publication on February 25, 2012; First review completed on March 3, 2012; Accepted for publication on March 5, 2012 E-mail: amanhaes@uerj.br Tel.: 55 21 2868-8195

million elderly people (3). Moreover, this aging population trend will undoubtedly continue, as most of the world’s population lives in countries with low to moderate socioeconomic status. Therefore, delivering proper care to this increasingly older population is and will continue to be a serious challenge, and the development of strategies to preserve the functional capacity of the geriatric population is therefore of paramount importance. Early identification of cognitive decline may constitute one such strategy, although to date, no effective and long-lasting methods for averting senility have been devised (4). However, early medical, behavioral and social intervention does improve clinical outcomes, which indirectly justifies the need for better screening methods (4). Improvements in clinical outcomes may offer the opportunity for a better quality of life, both for the elderly population and their caregivers (relatives and/or hired professionals).

The loss of functional capacity is closely linked to dementia, which is defined as a persistent and progressive loss of cognitive function in two or more domains (attention,

INTRODUCTION The percentage of elderly people in the general population has steadily increased throughout the last century (1).

At the start of the 20th century, this increase occurred mainly in countries that today are considered socio-economically developed. As a result, most of these countries currently have a considerable proportion of their population above the age of 65 (1). More recently, this increase has been associated with an increase in the socio-economic status of developing countries (2). For instance, it is expected that Brazil will have a geriatric population of more than 14% of the general population by 2025, with approximately 33.4

Copyright ß 2012 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.

No potential conflict of interest was reported.

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memory, language, visuospatial skills and executive function) that interferes with the ability to perform the basic and instrumental activities of daily living (5). The main causes of dementia are related to Alzheimer’s (55%–77%) and vascular diseases (15%–25%) (6). Dementia can have a deleterious impact on other aspects of health in the elderly; for instance, mood disorders and malnutrition are more frequent in dementia patients than in cognitively unimpaired individuals (7). In older individuals, falls are not only considerably more frequent than in younger people but also constitute a major health problem with a significant psychological impact and a higher risk of serious injury (7,8). Accidents are the fifth most common cause of death in the elderly, of which falls are responsible for 2/3 of all deaths (9). Approximately 1/3 of the elderly population 65 years of age or older living in nonresidential care facilities will suffer at least one fall per year, and this number doubles for those over 80 years of age (8).

Gait and balance disorders associated with normal and pathological aging increase the frequency of falls and dependency (10). In fact, such disorders are found in 2040% of elderly individuals aged 65-84 years and in 40-50% of those aged 85 or older (11).

Motor control disorders may share relevant pathophysiological aspects with cognitive disorders (11), which may be due to the fact that human motor control is intimately associated with the functioning of the CNS (12). This association warrants further investigation into how a reduction in the functional capacity of the elderly, whether the result of normal or pathological aging, would reflect a reduction in motor control and as a consequence, an increased risk of falling.

It is often difficult to differentiate normal aging (senescence), which is characterized by natural physiological alterations, from pathological aging (senility), which is associated with illnesses that can affect quality and length of life (13). The need to differentiate senescence from senility has led to the creation of guidelines for clinical decisions, such as diagnoses, treatment and follow-up, which differ considerably between young patients and the elderly (14).

Motor control related to gait and balance is highly dependent on the integrity of the CNS, and gait and balance disorders may be significantly associated with declining functional capacity in the elderly. Therefore, in the present study, we compared the results obtained with the use of the performance-oriented assessment of mobility problems (POMA) test in elderly patients to several parameters used for the assessment of functional capacity in elderly subjects.

The objective of the current study was to examine the potential association between gait and balance disorders and functional alterations of senescence and senility.

to participate in the tests used in the study; and 5) scores $22 on the mini-mental state examination (MMSE) test.

The exclusion criterion consisted of the presence of any illness that could represent a risk to the subject’s health during the functional assessments, especially those associated with walking and standing upright. In particular, subjects reporting dizziness or altered vestibular function at the time of the initial physical examination or immediately prior to the functional assessment were not tested with the performance-oriented assessment of mobility problems in elderly patients (POMA) and therefore were not included in the final sample.

All participants were subjected to a comprehensive geriatric assessment, which consisted of two home visits.

During the first visit, the patient’s history was recorded, a physical examination was performed, and the patient signed the ICF. During the second visit one week later, the functional assessment was performed using POMA. This instrument, which is specifically used to evaluate gait and balance problems in the elderly, was originally developed by Mary E. Tinetti in 1986 (15). The patient assessment was divided into two parts; the first part contained nine items related to balance, and the second part was composed of seven items related to gait. The scores ranged between 0 and 28, and lower scores indicated greater deficiencies. Scores below 26 were suggestive of deficiency, and scores less than 19 indicated a fivefold increase in the risk of falling. The functional assessment of the patients was also performed using the mini-mental state examination (MMSE) (16), which is an instrument commonly used as a screening test in the assessment of cognitive deficiencies. The potential scores ranged between 0 and 30, and a score less than 26 indicated the possibility of dementia. Moreover, the Katz scale (17) was used to evaluate the capacity of the elderly to undertake basic activities in daily life. This scale measured scores between 0 and 6; scores of 5 or 6 indicated independence in these activities, whereas scores of 3 or 4 indicated partial dependency and scores less than 3 indicated dependency. In addition, the Lawton scale (18) was used to measure the capacity of the elderly to undertake the instrumental activities of daily living. These scores ranged from 9 to 27, and lower scores indicated greater dependency. The geriatric depression scale (GDS) (19) was used to measure depression in the elderly. These scores range from 0 to 15; scores from 0 to 5 were considered normal, whereas scores from 6 to 10 suggested a slight disruption in mood, and any result above 11 indicated significant alteration. Last, the mini-nutritional assessment scale (MNA) (20) was used to evaluate nutritional alterations in the elderly. These scores ranged from 0 to 14, and a result of less than 12 indicated potential nutritional deficiencies.

For the statistical analyses, Kolmogorov-Smirnov onesample (K-S) tests were initially used to assess the normality of the distributions. Kruskal-Wallis H (K-W) tests and MannWhitney U (M-W) tests were used to assess differences between the groups. Kendall tau-b (t) tests were used to assess the degree of association between two variables, and chisquared (x2) tests were used to compare proportions between the groups. Significance was assigned at the level of p,0.05.

MATERIALS AND METHODS The initial sample consisted of 353 elderly patients who were routinely followed up at the Preventive and Integrated Care Unit (UIP) of the Hospital Adventista Silvestre (HAS) in Rio de Janeiro, Brazil. The study was approved by the Ethics Committee of HAS (protocol number: 20307/2007), and all procedures were carried out in accordance with the Declaration of Helsinki. Prior to participation in the study, all patients signed an informed consent form (ICF). The inclusion criteria were as follows: 1) patients followed at the UIP; 2) agreement to participate in the study and completion of the ICF; 3) patients between 65 and 95 years of age; 4) ability

RESULTS The final patient sample consisted of 90 (31.8%) men and 193 (68.2%) women with the following age distribution: 16

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Functional alterations in the elderly Teixeira-Leite H and Manha˜es AC.

men and 54 women between 65 and 74 years of age; 50 men and 102 women between 75 and 84 years of age; and 24 men and 37 women between 85 and 95 years of age. No significant differences were observed between the men (79.6¡0.6 years) and women (78.9¡0.5 years) regarding mean age (t = 0.87, p.0.10) or the percentage of individuals with more than eight years of schooling (x2 = 1.0, gl = 1, p.0.05; H = 88.8%, M = 84.2%). In addition, no significant differences were found between the men and women concerning the results obtained with the MMSE, Lawton and MNA scales (M-W: p.0.05 in all comparisons) (Figure 1). However, male patients demonstrated better scores than female patients regarding the POMA (M-W: Z = 2.1, p = 0.038), KATZ (M-W: Z = 4.4, p,0.001) and GDS (M-W: Z = 2.2, p = 0.029) scales (Figure 1).

Analyses of the Kendall’s correlation coefficients indicated significant associations between age and the scores obtained with the POMA (t = -0.19, p,0.001), MMSE (t = -0.13, p = 0.004), KATZ (t = -0.13, p = 0.008), LAWTON (t = -0.28, p,0.001) and MNA (t = -0.09, p = 0.038). However, these coefficients were very low, and age was not considered to be a factor in the subsequent analyses. As shown in Figure 2, significant reductions in the scores were identified with increased age for the following variables: POMA (K-W: x2 = 19.4, gl = 2, p,0.001), MMSE (K-W: x2 = 9.0, gl = 2, p = 0.011), KATZ (K-W: x2 = 9.0, gl = 2, p = 0.011) and LAWTON (K-W: x2 = 22.1, gl = 2, p,0.001). In addition, the pairwise comparisons and the stratification of individuals according to age and functional capacity according to the various assessment instruments are shown in Table 1.

The analysis of the correlation coefficients obtained for the various assessment instruments used in the functional evaluation of the elderly indicated that the POMA scores presented significant correlations with the scores obtained using all other instruments. Particularly, the POMA-KATZ correlation (t = 0.38, p,0.001) and the POMA-LAWTON correlation (t = 0.44, p,0.001) were significantly larger than the others. In general, these correlations indicated that declines in the results obtained with the POMA were associated with declines in the results obtained with the other instruments (Figure 3).

As shown in Figure 4A, the percentage of elderly individuals with a normal POMA score ($26) as well as a normal MMSE score ($26) was significantly greater (x2 = 12.3, p = 0.002) than the percentage of elderly individuals with a normal MMSE score and a POMA score #19.

When the analysis was performed considering only the normal MMSE scores (between 26 and 30), the percentage of elderly persons with a result indicating an abnormality increased significantly (x2 = 17.4, p = 0.026) as the MMSE score decreased (Figure 5A).

The percentage of elderly individuals with a normal POMA score as well as a normal KATZ score ($5) was significantly larger (x2 = 91.4, p,0.001) than the percentage of elderly persons with a normal KATZ score and a POMA score #19 (Figure 4B). When the analysis was performed only among individuals with normal KATZ scores (5 and 6), the percentage of elderly individuals with abnormal POMA scores was not significantly affected by decreases in the KATZ score (Figure 5B) (x2 = 4.7, p = 0.097).

Figure 4C shows that the percentage of elderly persons with normal POMA scores and normal GDS scores (#5) was significantly greater (x2 = 18.0, p = 0.001) than the percentage of elderly persons with normal GDS scores and POMA

scores #19. As shown in Figure 5C, when the analysis was performed only among individuals with normal GDS results (between 0 and 5), the percentage of elderly individuals with abnormal POMA scores increased significantly (x2 = 9, p = 0.049) as the GDS scores increased.

Finally, as shown in Figure 4D, the percentage of elderly persons with a normal POMA score as well as a normal MNA score ($12) was significantly greater (x2 = 18.9, p,0.001) than the percentage of elderly persons with a normal MNA score and a POMA score #19. When the analysis was performed only among individuals with normal MNA scores (between 12 and 14), the percentage of elderly persons with an abnormal POMA score significantly increased (Figure 5D) (x2 = 14.5, p = 0.006) as the MNA scores decreased.

DISCUSSION Methodological aspects One of the prominent characteristics of our sample was the inclusion of more women than men. Interestingly, the proportion of men per age group increased as the total age increased, and the higher overall number of females in our sample may reflect the fact that women tend to live longer than men (21). However, given the increase in the proportion of males with increasing age in our sample, the most likely explanation for the observed differences is behavioral. Females are accustomed to being routinely examined by physicians (such as gynecologists), and women have been shown to pay more attention to their health than men (21). As a result, women would be expected to be more actively engaged in the activities of the Preventive and Integrated Care Unit than men, especially at younger ages. In contrast, men typically become more interested in the advantages of preventive activities later in life when health problems become more prevalent; as a result, the proportion of men in our patient sample would be expected to increase with increasing age.

Our sample had a high socioeconomic and educational level (data not shown), as more than 80% of our sample completed high school. Although studies using Brazilian subjects have suggested a MMSE cutoff score of 24 (22), we used a score of 26 as a cutoff point, as this value is frequently used in the USA and Europe. We also excluded elderly patients with a MMSE score of less than 22 due to the reduced number of subjects with such scores in our initial sample and because these scores would have compromised the quantitative analyses.

Gender and age effects In the present study, we did not observe significant gender effects regarding the scores obtained with the MMSE, Lawton and MNA instruments. Previously, educational level was shown to be a factor in the definition of cutoff scores for instruments such as the MMSE (16,20).

Elderly Brazilian men tend to have higher levels of education than women, and this trend is often accentuated in lower income classes, which comprise most of the Brazilian population (23). As previously indicated, however, our sample was comprised of individuals with higher educational and socioeconomic levels, which may explain the absence of gender differences. On the other hand, gender effects were observed in regards to the results of the POMA, Katz and GDS instruments, and these findings are

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Figure 1 - Graphs depicting the effects of gender on the POMA (A), MMSE (B), Katz (C), Lawton (D), GDS (E) and MNA (F) scores. Note that women performed more poorly than men, as measured using the POMA, Katz and GDS assessment instruments. F: females; M: males; AS: 0.10.p$0.05; * p,0.05; *** p,0.001.

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Functional alterations in the elderly Teixeira-Leite H and ManhaËœes AC.

Figure 2 - Graphs depicting the effects of age on the POMA (A), MMSE (B), Katz (C), Lawton (D), GDS (E) and MNA (F) scores. Note that performance declined as a function of increasing age when the POMA, MMSE, Katz and Lawton assessment instruments are used. 65: from 65 to 74 years old; 75: 75 to 84 years old; 85: from 85 to 95 years old; * p,0.05; *** p,0.001.

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Table 1 - Age effects on functional scores.

Assessment

K-S

instruments

POMA

MMSE

KATZ

LAWTON

EDG

MAN

Pairwise

M-W

comparisons

***

*

*

***

65 vs. 75 65 vs. 85 75 vs. 85

65 vs. 75 65 vs. 85 75 vs. 85

Age

% of subjects

Group

per score range

#

*** ***

* **

65 vs. 75 65 vs. 85 75 vs. 85

** *

65 vs. 75 65 vs. 85 75 vs. 85

** *** **

x2

65-74 75-84 .84

7-19 9% 9% 25%

20-25 11% 20% 30%

65-74 75-84 .84

22-25 3% 12% 16%

26-30 97% 88% 84%

65-74 75-84 .84

0-2 1% 3% 13%

3-4 1% 6% 7%

5-6 97% 91% 80%

65-74 75-84 .84

11-15 4% 3% 3%

6-10 28% 23% 27%

0-5 68% 74% 70%

65-74 75-84 .84

0-11 31% 27% 36%

12-14 69% 73% 64%

ns

ns

K-S: Kruskal-Wallis test; M-W: Mann-Whitney test; x2: Chi-Squared test; ns: non-significant;

#

26-28 80% 72% 46%

***

*

**

ns

ns

0.10.p$0.05; * p,0.05; ** p,0.01; *** p,0.001.

depression and undernourishment (27,28), and both health problems can usually be corrected. Conversely, there are currently no treatments that can fully reverse the cognitive decline associated with senility (5). In this sense, the number of elderly individuals with cognitive deficits (which directly affects the POMA, MMSE, Katz and Lawton scores) tends to accumulate with age. However, the same trend is not necessarily true regarding the number of individuals with depression and/or undernourishment, which affects the GDS and MNA score, respectively.

in agreement with the current literature. For instance, the higher POMA scores among men in our sample likely reflected the fact that men of all ages engage in more physical exercise, have higher muscle mass and suffer fewer falls than women (24). Moreover, the lower average Katz scores observed among women were likely due to the increased prevalence of urinary incontinence (a significant component of the Katz score) that has been reported for elderly women (17). Finally, the higher GDS scores observed for women likely reflect the fact that females are more prone to depression than males, as the European prevalence of depression in the elderly is 14.1% for women and 8.6% for men, according to the EURODEP (25).

Regarding age effects, our results indicate that the scores derived from the POMA, MMSE, Katz and Lawton instruments show a decline with increased age, which is also in agreement with the literature. Several studies have shown that aging is associated with a progressive reduction in functional capacity that eventually leads to death (26).

Interestingly, the GDS and MNA scores were not significantly affected by aging. Although there is currently no clear explanation for these last two findings, it is possible that the higher socioeconomic level of our sample explains, at least in part, the absence of age effects regarding these instruments. Generally, our subjects were closely monitored by their families and physicians, which suggests that depressive states, particularly moderate to severe ones, would likely have been identified and treated, and the same reasoning could be applied regarding undernourishment. In addition, there are well-established treatment protocols for

POMA in the context of a comprehensive geriatric assessment Aging leads to the loss of functional reserves for dealing with situations that demand additional resources, such as those involving cognition, motor performance, immune response and endocrine function (13). Under normal conditions, these reserves are not used, and a reduction or even the absence of these reserves should not have a relevant impact on an individual’s daily life. However, reductions in these functional reserves indicate an increase in fragility, given that these resources will not be available in situations of increased demand (29). As a result of the absence of significant effects on routine tasks, it is difficult to identify the loss of functional reserves. This is unfortunate because an identifiable loss of reserves could constitute advanced warning for impending health problems that could be better dealt with during early stages, especially in the elderly population (29). Therefore, the development and use of assessment instruments to identify subtle changes in

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Functional alterations in the elderly Teixeira-Leite H and ManhaËœes AC.

Figure 3 - Graphs depicting the correlations between the POMA score and the MMSE (A), Katz (B), Lawton (C), GDS (D) and MNA (E) scores. Note that the POMA score was positively correlated with all but the GDS score, for which higher values indicate a higher risk of depression.

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CLINICS 2012;67(7):719-729

Figure 4 - Graphs depicting the relative frequency of individuals in a given MMSE (A), Katz (B), GDS (C) and MNA (D) score range per POMA score range. ** p,0.01; *** p,0.001.

the functional capacity of elderly patients is of great importance. Walking involves complex and continuous strategic planning by the central nervous system and involves aspects pertaining to the individual as well as the environment (30). Walking competently and safely is a task that demands adequate sensory-motor function, an efficient interaction between executive control (integration and decision making) and cognition (navigation, visual-spatial perception and attention) and an affective dimension (mood, preoccupation and risk-taking) (30). Given the demands that walking poses on the nervous system, it is likely that patients with cognitive impairments also present problems in their ability to walk. In fact, one initial sign of neurologic disorder is when individuals with cognitive impairments have difficulties in maintaining a conversation while walking (‘‘stop walking while talking’’) (31). This sign has also been used as a predictor of future falls, which further demonstrates the relationship between cognition and gait (31). Furthermore, executive frontal function is particularly important in maintaining a stable gait, and executive dysfunction may actually explain falls from idiopathic causes in elderly subjects (32). Moreover, there is a significant correlation between executive function,

working memory and gait velocity in the elderly, particularly those with mild cognitive impairment (33). These associations suggest that cognitive and motor functions share a neural substrate, and this hypothesis has been corroborated by neuroimaging studies showing that tasks demanding these functions lead to increases in the activity of brain areas such as the prefrontal and parietal cortices, the cingulate gyrus and premotor areas (34).

In addition, our results corroborate these previously documented observations. By comparing the results obtained with the POMA to those obtained with the MMSE, Katz and Lawton scales, our results strongly suggest that a decline in functional capacity related to gait and balance is associated with a decline in cognitive capacity. In particular, it was observed that significant reductions in the POMA scores were present even for MMSE and Katz scores that would not be considered significantly altered. The POMA could thus serve as an early indicator of a cognitive decline that is unable to be detected by the most commonly employed screening instruments, particularly in populations with higher levels of education. Despite this promising and relevant possibility, a longitudinal prospective study is warranted to unequivocally demonstrate under what circumstances the use of the POMA

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Functional alterations in the elderly Teixeira-Leite H and ManhaËœes AC.

Figure 5 - Graphs depicting the relative frequency of individuals in a given POMA score range per MMSE (A), Katz (B), GDS (C) and MNA (D) normal score range. AS: 0.10.p$0.05; * p,0.05; ** p,0.01.

which in turn may have been the true cause for the decline in the POMA results. Both severe and mild depression can alter the functional capacity of the elderly and aggravate associated chronic illnesses (36). Patients with mild depressive disorders present an elevated risk of developing disabilities, which may lead to more severe depression (37). Furthermore, elderly subjects with normal cognition and mild to moderate depressive symptoms present a twofold increase in the risk of presenting mild cognitive decline over a period of six years, and this change is independent of associated vascular illness, which suggests that depressive symptoms could be considered a risk factor for the onset of cognitive impairment (38).

Our results for the mini-nutritional assessment scale (MNA) were also associated with changes in POMA scores.

Understandably, the loss of functional reserves brought about by aging becomes even more evident in the presence of an inadequate diet (28,29). In addition to the direct effects on muscular action, malnutrition interferes directly with the activity of the nervous system (12). Therefore, malnutrition in the elderly may affect both gait and balance as a result of both muscle weakness and the inability of the brain to deal with the associated sensory-motor and cognitive demands.

Furthermore, nutritional status becomes particularly critical

instrument is valid as an indicator of cognitive integrity in the elderly. For example, patients suffering from severe arthritis or those who have significant lesions in the spinal cord or peripheral nerves may have normal cognitive capacity in addition to a significantly altered POMA score.

In the present study, we evaluated two potential confounding factors regarding the use of the POMA score as an additional source of information on cognitive integrity in the elderly. Affective disorders are commonly associated with gait impairment in patients belonging to this age group.

Depression, anxiety and, in particular, a fear of falling are common among elderly individuals with an insecure gait and a previous history of falling (12). Furthermore, our results show that declines in the GDS scores are associated with reduced POMA scores. Depression is frequently observed in cases of dementia, as 25% of dementia patients present clear symptoms of depression, and dementia is an independent risk factor for early institutionalization (35). Moreover, it is relatively common for dementia to be initially identified as depression (36). Considering the relationship between cognitive function and the POMA and GDS scores, we cannot disregard the possibility that depressive state, as identified using the GDS, may serve as a marker of cognitive impairment,

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2. Kinsella K, Velkoff VA. US Census Bureau, Series P95/01-1. An Aging World: 2001. Washington, DC: US Government Printing Office; 2001.

3. Chaimowicz F, Gonc¸alves L. Population Ageing in Brazil: the transitional model in a developing country. In: Demographic Aspects of Population Ageing: East European/Latin American Perspectives, 1997. Final Report.

Valleta - Malta: International Institute of Aging - United Nations; 1997.v.

1.p.21-4.

4. Fries JF. Successful aging – an emerging paradigm of gerontology. Clin Geriatr Med. 2002;18(3):371-82, http://dx.doi.org/10.1016/S07490690(02)00021-6.

5. Cummings JL, Cole G. Alzheimer disease. JAMA. 2002;287(18):2335-8, http://dx.doi.org/10.1001/jama.287.18.2335.

6. Clarfield AM. The decreasing prevalence of reversible dementias: An updated meta-analysis. Arch Intern Med. 2003;163(18):2219-29, http:// dx.doi.org/10.1001/archinte.163.18.2219.

7. Campbell AJ, Borrie MJ, Spears GF, Jacson SL, Brown JS, Fitzgerald JL. Circumstances and consequences of falls experienced by a community populations 70 years and over during a prospective study. Age Ageing.

1990;19(2):136-41, http://dx.doi.org/10.1093/ageing/19.2.136.

8. Tinetti ME, Speechley M, Ginter SF. Risk factors for falls among elderly persons living in the community. N Engl J Med. 1988;319(16):1701-07, http://dx.doi.org/10.1056/NEJM198812293192604.

9. Rubenstein LZ, Josephson KR, Roggins AG. Falls in the nursing home.

Ann Intern Med. 1994;121(6):442-51.

10. Downton JH. Falls. In: Tallis RC, Fillit SHM, editors. Brocklehurst’s Textbook of Geriatric Medicine and Gerontology. Amsterdam: W.B. Saunders; 1998.p.789-828.

11. Alexander NB. Gait disorders in older adults. J Am Geriatr Soc. 1996;44(4):434-51.

12. Snijders AH, van de Warrenburg BP, Giladi N, Bloem BR. Neurological gait disorders in elderly people: clinical approach and classification. Lancet Neurol. 2007;6(1):63-74, http://dx.doi.org/10.1016/S1474-4422(06)70678-0.

13. Canineu PR, Diniz CMC. Envelhecimento Normal: Aspectos fı´sicos, psı´quicos e cognitivos. In: Forlenza OV, editor. Psiquiatria Geria´trica do Diagno´stico Precoce a` Reabilitac¸a˜o. Sa˜o Paulo: Atheneu; 2007.p.17-21.

14. Hazzard WR, Halter JB, Ouslander JG, Tinetti ME, Studenski S, High KP, Asthana S, editors. Hazzard’s geriatric medicine and gerontology.

Principles of Geriatric Medicine & Gerontology. 6th ed. McGraw-Hill Medical; 2009.

15. Tinetti M.E. Performance-oriented assessment of mobility problems in elderly patients. J Am Geriatr Soc. 1986;34:119-26.

16. Bertolucci PH, Brucki SM, Campacci SR, Juliano Y. The Mini-Mental State Examination in a general population: impact of educational status.

Arq Neuropsiquiatr. 1994;52(1):1-7, http://dx.doi.org/10.1590/S0004282X1994000100001.

17. Katz S, Downs TD, Cash HR, Grotz RC. Progress in development of the index of ADL. Gerontologist. 1970;10(1):20-30, http://dx.doi.org/ 10.1093/geront/10.1_Part_1.20.

18. Lawton MP, Brody EM. Assessment of older people: self-maintaining and instrumental activities of daily living. Gerontologist. 1969;9(3):17986, http://dx.doi.org/10.1093/geront/9.3_Part_1.179.

19. Yesavage JA, Brink TL, Rose TL, Lum O, Huang V, Adey M, et al.

Development and validation of a geriatric depression screening scale: a preliminary report. J Psychiatr Res. 1982;17(1):37-49, http://dx.doi.org/ 10.1016/0022-3956(82)90033-4.

20. Guigoz Y, Vellas B, Garry PJ. Mini Nutritional Assessment: a practical assessment tool for grading the nutritional state of elderly patients. In: Albarede JL, editor. Nutrition in the elderly. The Mini Nutritional Assessment (MNA). Paris: Serdi Publishing Company; 1994.p.15-59.

21. Wingard DL. The sex differential in morbidity, mortality, and lifestyle.

Annu Rev Public Health. 1984;5:433-58, http://dx.doi.org/10.1146/ annurev.pu.05.050184.002245.

22. Lourenc¸o RA, Veras RP. Mini-Mental State Examination: psychometric characteristics in elderly outpatients. Rev. Sau´de Pu´blica. 2006;40:712-19, http://dx.doi.org/10.1590/S0034-89102006000500023.

23. Camarano AA. In:Muito ale´m dos 60: os novos idosos brasileiros. Rio de Janeiro. IPEA. 1999.p.43.

24. Brach J, Rosano C, Studenski S. Mobility. In: Hazzard WR, Halter JB, Ouslander JG, Tinetti ME, Studenski S, High KP, Asthana S, editors.

Hazzard’s geriatric Medicine and Gerontology. 6th ed. McGraw-Hill Medical; 2009.p.1397-409.

25. Copeland JR, Beekman AT, Braam AW, Dewey ME, Delespaul P, Fuhrer R, et al. Depression among older people in Europe: the EURODEP studies. World Psychiatry. 2004;3(1):45-9.

26. Rowe JW, Khan RL. Human aging: usual and successful. Science, 1987;237(4811):143-9.

27. Nakajima GA, Wenger NS. Quality indicators for the care of depression in vulnerable elders. J Am Geriatr Soc. 2007;55(Suppl 2):S302-11, http:// dx.doi.org/10.1111/j.1532-5415.2007.01336.x.

28. Reuben DB. Quality indicators for the care of undernutrition in vulnerable elders. J Am Geriatr Soc. 2007;55(Suppl 2):S438-42, http:// dx.doi.org/10.1111/j.1532-5415.2007.01353.x.

in patients with cognitive impairments, given that these individuals become progressively more dependent on others to perform the basic activities of daily life, such as feeding oneself (28). Two of the six items in the MNA assessment are not related to the somatic or nutritional characteristics of the individual but are instead related to their capacity for maintaining independence and intact cognition (20).

Therefore, future studies must differentiate the direct effects of nutritional status on the POMA scores from the effects of the cognitive impairments that are relevant to the MNA assessment score and that could negatively affect both POMA and an individual’s nutritional status.

It remains unclear what the effect of normal aging (senescence) is on gait and whether all normal aging elderly individuals experience gait disorders. Although it was once assumed that difficulties related to gait would be unavoidable with the passing of time, in has been demonstrated in recent years that more than 20% of very old individuals (older than 88 years of age) present a normal gait, which suggests that gait impairments are not a consequence of normal aging (senescence) (39). This assumption is further supported by the fact that individuals with senile gait have an increased risk of developing dementia and a lower survival rate when compared to elderly people who walk normally (40). We therefore have reason to believe that gait impairments may represent early manifestations of subclinical diseases affecting the central nervous system in elderly patients.

Gait and balance are intimately associated with the functioning of the nervous system. Although gait and balance are directly linked to the motor nervous system, disorders that affect components of the nervous system involved with cognition can also have negative repercussions on motor control. In fact, we observed that a reduction in the MMSE, Katz and Lawton scores (the three scales of functional evaluation in the elderly that involve cognitive aspects) was associated with a decline in the POMA scores.

In particular, we demonstrated that significant reductions in POMA scores could be identified prior to the detection of cognitive impairment using the MMSE and Katz scores, and we also found that increased susceptibility to depression and malnutrition were associated with reduced POMA scores. Given these observations, the performance-oriented assessment of mobility problems in elderly patients (POMA) should be used in conjunction with other instruments that assess the functional status of the elderly, not only as a measure of motor performance, but also as a potential marker of cognitive status. In fact, the presence of alterations in POMA scores may be considered an early indication of cognitive decline.

ACKNOWLEDGMENTS The authors would like to thank Mirtes Ester Ferreira, Nilza Veiga Ferreira, Tereza Cristina Martins, Luciana Mescolin, Marta Gomes, Esther Bando, Dr. Roge´rio Gusma˜o and all the patients who participated in this study.

AUTHOR CONTRIBUTIONS Both authors contributed equally to the study.

REFERENCES 1. World Health Organization (WHO). Population ageing - A public health challenge. Geneva: WHO; 1998. Fact sheet n.135.

728

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Functional alterations in the elderly Teixeira-Leite H and Manha˜es AC. 35. Dorenlot P, Harboun M, Bige V, Henrard JC, Ankri J. Major depression as a risk factor for early institutionalization of dementia patients living in the community. Int J Geriatr Psychiatry. 2005;20(5):471-8, http:// dx.doi.org/10.1002/gps.1238.

36. Hybels CF, Blazer DG, Pieper CF. Toward a threshold for subthreshold depression: an analysis of correlates of depression by severity of symptoms using data from an elderly community sample. Gerontologist.

2001;41(3):357-65, http://dx.doi.org/10.1093/geront/41.3.357.

37. Lyness JM, Heo M, Datto CJ, Ten Have TR, Katz IR, Drayer R, et al.

Outcomes of minor and subsyndromal depression among elderly patients in primary care settings. Ann Intern Med. 2006;144(7): 496-504.

38. Barnes DE, Alexopoulos GS, Lopez OL, Williamson JD, Yaffe K. Depressive symptoms, vascular disease, and mild cognitive impairment: findings from the Cardiovascular Health Study. Arch Gen Psychiatry.

2006;63(3):273-79, http://dx.doi.org/10.1001/archpsyc.63.3.273.

39. Bloem Br, Haan J, Lagaay AM, van Beek W, Wintzen AR, Roos RA. Investigation of gait in elderly subjects over 88 years of age. J Geriatr Psychiatry Neurol. 1992;5(2):78-84.

40. Bloem BR, Gussekloo J, Lagaay AM, Remarque EJ, Haan J, Westendorp RG. Idiopathic senile gait disorders are signs of subclinical disease. J Am Geriatr Soc. 2000;48(9):1098-101.

29. Tas¸ U, Steyerberg EW, Bierma-Zeinstra SM, Hofman A, Koes BW, Verhagen AP. Age, gender and disability predict future disability in older people: the Rotterdam Study. BMC geriatrics. 2011;11:22.

30. Woollacott M, Shumway-Cook A. Attention and the control of posture and gait: a review of an emerging area of research. Gait Posture.

2002;16(1):1-14, http://dx.doi.org/10.1016/S0966-6362(01)00156-4.

31. Lundin-Olsson L, Nyberg L, Gustafson Y. ‘‘Stops walking when talking’’as a predictor of falls in elderly people. Lancet. 1997;349(9052): 617, http://dx.doi.org/10.1016/S0140-6736(97)24009-2.

32. Springer S, Giladi N, Peretz C, Yogev G, Simon ES, Hausdorff JM. Dualtasking effects on gait variability: the role of aging, falls, and executive function. Mov Disord. 2006;21(7):950-7, http://dx.doi.org/10.1002/ mds.20848.

33. Ble A, Volpato S, Zuliani G, Guralnik JM, Bandinelli S, Lauretani F, et al.

Executive function correlates with walking speed in older persons: the InCHIANTI study. J Am Geriatr Soc. 2005;53(3):410-15, http:// dx.doi.org/10.1111/j.1532-5415.2005.53157.x.

34. Klingberg T. Limitations in information processing in the human brain: neuroimaging of dual task performance and working memory tasks.

Prog Brain Res. 2000;126:95-102, http://dx.doi.org/10.1016/S00796123(00)26009-3.

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DOI:10.6061/clinics/2012(07)05

CLINICAL SCIENCE

Disparities in female breast cancer mortality rates in Brazil between 1980 and 2009 Ruffo Freitas-Junior,I Carolina Maciel Reis Gonzaga,I Nilceana Maya Aires Freitas,II Edesio Martins,III Rita de Ca´ssia de Maio DardesIV I

Federal University of Goias, Program of Mastology, Goiania/GO, Brazil. II Araujo Jorge Hospital, Goias Anticancer Association (ACCG), Radiotherapy Department, Goiania/GO, Brazil. III Goias Anticancer Association (ACCG), Goiania Population-Based Cancer Registry, Goiania/GO, Brazil. IV Department of Gynecology, Universidade de Sa˜o Paulo, Sa˜o Paulo/SP, Brazil.

OBJECTIVE: To describe the temporal trends in female breast cancer mortality rates in Brazil in its macro-regions and states between 1980 and 2009.

METHODS: This was an ecological time-series study using data on breast cancer deaths registered in the Mortality Data System (SIM/WHO) and census data on the resident population collected by the Brazilian Institute of Geography and Statistics (IBGE/WHO). Joinpoint regression analyses were used to identify the significant changes in trends and to estimate the annual percentage change (APC) in mortality rates.

RESULTS: Female breast cancer mortality rates in Brazil tended to stabilize from 1994 onward (APC = 0.4%).

Considering the Brazilian macro-regions, the annual mortality rates decreased in the Southeast, stabilized in the South and increased in the Northeast, North, and Midwest. Only the states of Sao Paulo (APC = -1.9%), Rio Grande do Sul (APC = -0.8%) and Rio de Janeiro (APC = -0.6%) presented a significant decline in mortality rates. The greatest increases were found in Maranhao (APC = 12%), Paraiba (APC = 11.9%), and Piaui (APC = 10.9%).

CONCLUSION: Although there has been a trend toward stabilization in female breast cancer mortality rates in Brazil, when the mortality rate of each macro-region and state is analyzed individually, considerable inequalities are found, with rate decline or stabilization in states with higher socioeconomic levels and a substantial increase in those with lower socioeconomic levels.

KEYWORDS: Breast Cancer; Mortality; Trends; Brazil.

Freitas-Junior R, Gonzaga CM, Freitas NM, Martins E, Dardes RC. Disparities in female breast cancer mortality rates in Brazil between 1980 and 2009. Clinics. 2012;67(7):731-737.

Received for publication on January 22, 2012; First review completed on January 31, 2012; Accepted for publication on March 9, 2012 E-mail: ruffojr@terra.com.br Tel.: 55 62 32437260

undergoing an accelerated aging process, with changes in the epidemiological profile of the population reflecting the slower growth rate in the number of children and adolescents together with an increase in the economically active population and the elderly (9).

As a developing country with an emerging economy, the pattern of breast cancer in Brazil has been changing, and an increase in incidence has been reported (10). The estimated number of new cases for 2012 is 52,680, with a risk of 52 cases for every 100,000 women (10). This transition, however, is occurring in an extremely unbalanced manner, which is, to a large extent, associated with the different social conditions present within the country (11). In the Southeast region, breast cancer is the most common type of malignancy in women, with an estimated risk of 69 new cases per 100,000 women, followed by the South (65/ 100,000), the Midwest (47/100,000), and the Northeast (32/ 100,000). In the North region, it is the second most common form of cancer in women, with an incidence of 19/100,000 (10).

In the last decade, opportunistic breast cancer screening in some areas of Brazil and the use of mammography (12,13)

INTRODUCTION Breast cancer is the principal cause of cancer deaths in women worldwide (1-2). The patterns of mortality rates generally follow the incidence patterns of the disease (3).

The trend in breast cancer incidence and mortality rates has remained stable or declined in some developed countries (1,4,5), largely as a result of a reduction in the use of menopausal hormone therapy (6), early detection through mammography screening, and advances in the treatment of the disease (1,2). However, breast cancer incidence and mortality have been increasing rapidly among women in many of the countries of Eastern Europe, Asia, and Latin America and some African countries (1,2).

The incidence of breast cancer is directly associated with older age groups (7,8). The Brazilian population is currently

Copyright ß 2012 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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have allowed the reduction of local advanced diseased and an increase in early breast cancer detection (14). However, as of 2004, this modification in the pattern of diagnosis had not affected breast cancer mortality (15,16), morbidity, or quality of life, considering different types of surgical treatment (17).

The continental dimensions of this vast country (9) and large social and economic disparities might play a role in breast cancer mortality. However, to date, this question has not been addressed, limiting the understanding of the impact of the disease and not allowing for a better use and allocation of resources according to the needs of each individual state. Therefore, the objectives of the present study were to analyze female breast cancer mortality trends in Brazil considering individual macro-regions and states for the 1980-2009 period.

the International Classification of Diseases (ICD) underwent two revisions; therefore, data up to 1996 were based on the ICD-9, and data from 1996 onward were based on the ICD-10. The consolidated mortality data from 1980 to 2008 and preliminary data for 2009 were included in this study.

Data on the resident population, which were obtained from the national censuses conducted in 1980, 1991, and 2000, were extracted from DATASUS. For the intermediate years, population estimates were derived from the National Household Sample Surveys (PNAD), which are conducted annually by the Brazilian Institute of Geography and Statistics IBGE/WHO (18).

Statistical analysis Once the data on deaths and the resident population were obtained, age-specific mortality rates, which were classified into seven different age groups (20-29, 30-39, 40-49, 50-59, 60-69, 70-79 and $80 years of age), were calculated, and the age-standardized mortality rates were estimated using an indirect method with the hypothetical world population proposed by Segi (1960) and modified by Doll et al. (1966) as a reference (19).

To identify significant changes in trends, we performed a joinpoint regression analysis with the software provided by the Surveillance Research Program of the US National Cancer Institute (20). With this analysis, it is possible to identify the years in which a significant change in the linear slope of the trend (on a log scale) was detected during the study period (21). The best-fitting points, called ‘‘joinpoints’’, are chosen when the rate changes significantly. The analysis starts with the minimum number of joinpoints (e.g., 0 joinpoints, which is a straight line) and tests whether one joinpoint is significant and must be added to the model.

To describe linear trends by period, the estimated annual percentage change (APC) is then computed for each of those trends by fitting a regression line to the natural logarithm of the rates using calendar year as a regressor variable [i.e., given y = a+bx, where y = ln(rate) and x = calendar year, the APC is estimated as 100 6 (eb – 1)] (20,21).

An increase in mortality rates was considered to have occurred when the trend was toward growth and the minimum value of the confidence interval was .0.

Conversely, a reduction was considered to have occurred when there was a decline in the trend and the maximum value of the confidence interval was ,0. Stability was defined when, regardless of the trend, the confidence interval included zero.

METHODS Study design This is an ecological time-series study conducted by collecting data on the number of deaths due to female breast cancer in Brazil between 1980 and 2009. Because this study was carried out using secondary open data, there was no need for Ethics Committee approval.

Brazilian macro-regions and states Brazil encompasses a total area of 8,514,876 km2 in South America and consists of 27 states in five geographical macro-regions: the South, Southeast, Midwest, North, and Northeast (9). The total population in 2009 was approximately 191 million inhabitants, and the population density was 22.5 people/km2.

The South region, consisting of the states of Parana, Santa Catarina and Rio Grande do Sul, comprises 15% of the country’s population and has the highest human development index (HDI). The Southeast region is composed of the states of Sao Paulo, Minas Gerais, Rio de Janeiro, and Espirito Santo and is the most populous (42%) and most affluent region of Brazil. The states of Mato Grosso, Mato Grosso do Sul, and Goias and the Federal District, where Brasilia, the capital of the country, is situated, constitute the Midwest. This is the least populated region (7%), with the second lowest population density in the country. The North region, which is the least densely populated, consists of the states of Acre, Amapa, Amazonas, Para, Rondonia, Roraima, and Tocantins. This region includes 45% of the total area of Brazil and 8% of its total population. The Northeast region is made up of the states of Bahia, Alagoas, Ceara, Maranhao, Paraiba, Pernambuco, Piaui, Rio Grande do Norte, and Sergipe. The population of this region represents 29% of the total population of the country, and it is the region with the lowest HDI in Brazil (9).

RESULTS Between 1980 and 2009, 213,486 cases of death from female breast cancer were registered in Brazil. The standardized mortality rate increased from 9.2 per 100,000 women in 1980 to 11.3 per 100,000 in 2009. A change occurred in the mortality trend in Brazil. Initially, between 1980 and 1994, an annual increase of 1.6% was registered; however, from 1994 onward, the rates tended to stabilize, with an annual increase of 0.4%, as shown in Table 1. The highest mortality rates were found in the South and Southeast regions of the country, with maximum values of 14.6/100,000 women in 1997 and 14.4/ 100,000 in 1994, respectively. Nevertheless, these were the regions in which the female breast cancer mortality rates

Data sources The number of deaths from female breast cancer in the different Brazilian states and macro-regions and for the country as a whole was obtained from the reports of deaths registered in the Mortality Data System (SIM/WHO) and made available by the DATASUS database (18). The states of Acre, Roraima, and Amapa were excluded from the study because data for the period to be analyzed were missing, while Tocantins was excluded because it only became an independent state in 1989 (9). During this study,

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Table 1 - Female breast cancer mortality trends between 1980 and 2009 in Brazil and the macro-regions of the country.

Trend 1

Brazil and its Period

macro-regions

Brazil South Southeast Midwest North Northeast

Trend 2

Mortality Rate

Period

Mortality Rate

Initial

Final

Initial

Final

APC

(95% CI; p-value)

Initial

Final

Initial

Final

APC

(95% CI; p-value)

1980 1980 1980 1980 1980 1980

1994 1994 1997 2009 2009 2000

9.2 10.9 12.3 5.3 4.1 4.2

11.7 14.4 14.6 10.4 6.6 6.1

1.6* 2.2* 1.2* 1.9* 2.4* 2.1*

(1.1–2.1; (1.5–3.0; (0.8–1.6; (1.5–2.4; (1.9–3.0; (1.7–2.5;

1994 1994 1997

2009 2009 2009

11.7 14.4 14.6

11.3 12.7 12.6

0.4 -0.3 -0.9*

(-0.1–0.8; p = 0.08) (-1.0–0.3; p = 0.31) (-1.6–-0.2; p,0.01)

2000

2009

6.1

9.0

5.3*

(3.9–6.7; p,0.01)

p,0.01) p,0.01) p,0.01) p,0.01) p,0.01) p,0.01)

Standardized mortality rate per 100,000 women. * Statistically significant; p,0.05.

and Rio de Janeiro (-0.6% from 1994 onward). In all of these states and in the Federal District, the capital of the country, the breast cancer mortality rates for women were higher.

The maximum value for Rio de Janeiro was 18.8/100,000 women in 1994, followed by the Federal District (18.4/ 100,000 in 2006), Rio Grande do Sul (17.1/100,000 in 1993), and Sa˜o Paulo (16.7/100,000 in 1999) (Figure 1).

In the states of Parana and Santa Catarina and the Federal District, the breast cancer mortality rates stabilized, as shown in Table 2. An increase occurred in the breast cancer mortality rates in women in 17 states, primarily those situated in the Northeast region of the country, particularly the states of Maranhao (APC of 12% from 2002 onward), Paraiba (APC of 11.9% from 2000 onward), and Piaui (APC of 10.9% from 1997 onward).

tended to decline or stabilize (Table 1). In the South, stabilization occurred from 1994 onwards. In the Southeast, a significant change occurred in the mortality rates in 1997, from an increase of 1.2% between 1980 and 1997 to a decrease of -0.9% between 1997 and 2009.

In the Midwest and North regions, increases in the breast cancer mortality rates in women of 1.9% and 2.4%, respectively, were found during the period analyzed. An increase in mortality was also registered in the Northeast region of the country, with an increase of 2.1% between 1980 and 2000 and 5.3% from 2000 onward.

When female breast cancer mortality trends were analyzed in the different states in Brazil (Table 2), a significant decrease was found in three states: Sao Paulo (-1.9% from 1999 onward), Rio Grande do Sul (-0.8% from 1993 onward),

Table 2 - Trends in female breast cancer mortality rates between 1980 and 2009 in the states of Brazil.

Trend 1

States Period

Parana Santa Catarina Rio Grande do Sul Espirito Santo Rio de Janeiro Sa˜o Paulo Minas Gerais Goias Mato Grosso do Sul Mato Grosso Federal District Amazonas Para Rondonia Bahia Rio Grande do Norte Maranhao Piaui Paraiba Alagoas Sergipe Ceara Pernambuco

Trend 2

Mortality Rate

Period

Mortality Rate

Initial

Final

Initial

Final

APC

(95% CI; p-value)

Initial

Final

Initial

Final

APC

(95% CI; p-value)

1980 1980 1980

2002 1998 1993

8.3 8.9 13.4

13.1 12.5 17.1

2.2* 2.1* 2.1*

(1.7–2.8; p,0.01) (1.4–2.8; p,0.01) (1.1–3.1; p,0.01)

2002 1998 1993

2009 2009 2009

13.1 12.5 17.1

11.8 11.3 13.9

-1.3 -0.6 -0.8*

(-4.0–1.6; p = 0.35) (-2.0–0.9; p = 0.43) (-1.4– -0.1; p = 0.03)

1980 1980 1980 1980 1980 1980

2009 1994 1999 2009 2009 2009

6.5 17.0 12.5 7.7 4.1 6.3

10.3 18.8 16.7 9.2 9.8 11.8

2.1* 1.2* 1.2* 0.7* 2.0* 2.2*

(1.5–2.7; (0.6–1.8; (0.9–1.6; (0.4–1.0; (1.3–2.6; (1.4–3.1;

1994 1999

2009 2009

18.8 16.7

16.3 12.7

-0.6* -1.9*

(-1.1– -0.1; p = 0.03) (-2.9– -0.9; p,0.01)

1980 1980 1980 1980 1980 1980 1980

2009 2006 2009 2009 2009 2001 1986

2.5 11.5 4.3 4.6 1.3 4.7 3.1

8.9 18.4 6.4 6.4 9.5 5.7 5.4

4.3* 1.1* 2.1* 1.6* 4.5* 1.3* 9.7*

(3.4–5.1; p,0.01) (0.4–1.8; p,0.01) (1.0–3.2; p,0.01) (0.9–2.4; p,0.01) (2.9–6.2; p,0.01) (0.7–1.8; p,0.01) (0.9–19.2; p = 0.03)

2006

2009

18.4

12.1

-11.0

(-25.9–7.0; p = 0.20)

2001 1986

2009 2009

5.7 5.4

7.7 9.7

4.4* 2.3*

(2.0–6.1; p,0.01) (1.2–3.4; p,0.01)

1980 1980 1980 1980 1980 1980 1980

2002 1997 2000 1996 1983 2009 2009

1.2 2.2 3.5 5.1 5.5 3.6 6.2

2.5 2.7 3.3 2.6 2.7 9.9 11.1

3.0* 0.3 1.6* -1.1 -17.4 3.8* 2.7*

(1.4–4.7; p,0.01) (-2.2–2.8; p = 0.80) (0.4–2.9; p = 0.01) (-2.4–0.2; p = 0.08) (-39.7–12.9; p = 0.21) (3.2–4.3; p,0.01) (2.4–3.1; p,0.01)

2002 1997 2000 1996 1983

2009 2009 2009 2009 2009

2.5 2.7 3.3 2.6 2.7

5.8 8.5 9.5 8.7 12.5

12.0* 10.9* 11.9* 6.1* 4.2*

(2.5–22.3; p = 0.01) (6.3–15.7; p,0.01) (7.3–16.7; p,0.01) (4.2–8.0; p,0.01) (3.0–5.4; p,0.01)

p,0.01) p,0.01) p,0.01) p,0.01) p,0.01) p,0.01)

Standardized mortality rate per 100,000 women. * Statistically significant; p,0.05.

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Figure 1 - Female breast cancer mortality rates in the states of Brazil from 1980 to 2009 (5-year average annual mortality rates). The states are grouped in accordance with their macro-regions.

of the strong association between the risk of cancer and age (23).

The exact number of breast cancer deaths registered in the country as a whole and the North region in particular is unknown due to incomplete notification in some states.

Irregularities are known to have occurred in the registration of deaths in the majority of the states in the North region and in some states in the Northeast in 1980 (24). However, a significant improvement was achieved in the quality of reports in 1990, with the coverage of deaths increasing, thus enabling reliable mortality indicators to be produced.

Currently, there are 19 hospital population-based cancer registries in Brazil. Over time, a significant improvement in the quality of mortality data in the country has been noted, which is indicated by the reduction in the deaths classified as ‘‘ill-defined causes’’, mostly from 2005. However, there is still a certain degree of underreporting (10). The database quality in a cancer registry depends on the quality of its

DISCUSSION This analysis of the trends in female breast cancer mortality in Brazil reveals a positive pattern in recent years, reflecting a leveling of the rates. These findings contradict the expected patterns for developing, lowincome countries (1,2) and differ from some studies that reported an increase in mortality in other periods (4,22).

However, the present results correlate with other studies (15,16) in which a general trend toward stabilization was found in breast cancer rates up to 1994, indicating that advances were being achieved in the treatment of breast cancer and the availability of mammography screening programs, resulting in earlier detection (1,5). Despite this supposedly favorable scenario and although breast cancer mortality rates remain constant, considerable increases will occur in the absolute number of deaths as a result of population growth and increased life expectancy because

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in women in the South, principally in Rio Grande do Sul, where a decline of 0.8% has occurred since 1993.

In the Southeast, the mortality rates are higher in Rio de Janeiro and Sao Paulo, confirming data published by INCA (10). However, there was a significant decline in breast cancer mortality in women in the states of Rio de Janeiro and Sao Paulo. The 1.9% decline found in Sao Paulo from 1999 onward is particularly noteworthy and suggests advances in treatment. The Sao Paulo State Health Department, in collaboration with the Women’s Health Referral Center, the Department of Breast Diseases of the Federal University of Sao Paulo (UNIFESP) and the Oncocenter Foundation, is developing a strategic plan to reduce breast cancer mortality.

This type of action in association with a center for efficient case resolution (CARE) may help provide immediate and individualized treatment (29).

In the Midwest region, the Federal District, the capital of the country, has the highest mortality rates from breast cancer in women compared with Mato Grosso do Sul, Goias, and Mato Grosso, following patterns similar to those found in Sao Paulo, Rio de Janeiro, and Rio Grande do Sul. Nevertheless, the 11% decline in mortality registered over the past four years in the Federal District may be due to advances in the Brazilian National Health System (SUS) (30,31) in addition to regulations and guidelines within the SUS network that prevent the migration of residents from other states. This would explain the steep decline in mortality in the Federal District and justify the increase in mortality rates in recent years in the northeastern states because patients with breast cancer would migrate from the states with poorer healthcare conditions to the Federal District, where the public healthcare network is well established and well developed.

In the North, breast cancer is the second cause of mortality from cancer in women, preceded only by cervical cancer (10). These data show an annual increase of 2.4% in the mortality rate. The profile of the individual states is similar, with increases that range from 4.5% in Rondonia to 1.6% in Para. This vast region is composed of extensive areas of unpopulated territory such as that occupied by the Amazon forest (11); however, it is a region in which the population is increasing rapidly (11). Considering these facts and the unfavorable trend in mortality, breast cancer continues to be of fundamental importance to public health in this region.

Socioeconomic indicators in the Northeast region are precarious (9). In parallel, the greatest increase in female breast cancer mortality was found in this region. An increase of 5.3% occurred between 2000 and 2009, with variation between the states that ranged from a 1.1% decline in Alagoas between 1980 and 1996 (thereafter increasing by 6.1%) to a 12% increase in Maranhao between 2002 and 2009. It is possible that some of these fluctuations may be the result of changes in the practices of codification and registration and modifications to regulations concerning migration within the SUS network because the inverse occurred in the Federal District (30). Nevertheless, in these states, breast cancer mortality was found to follow the patterns expected for developing and less developed regions (1,2). The maximum mortality rates ranged from 5.8 per 100,000 women in Maranhao to 12.5/100,000 in Sergipe.

In 2004, a consensus document was drawn up to promote the early detection of breast cancer in Brazil, with the development of public policies adapted to the reality of the infrastructure in the country (32). The current major

sources and its registration criteria. These methods should follow the international standard so that the worldwide population can be compared consistently (25).

It is crucial that cancer mortality data are standardized, updated, properly recorded, representative of the population, and disseminated in a timely manner as a tool for the epidemiologic surveillance of cancer in the country. In this way, careful interpretation and usage of the estimates are necessary when analyzing time trends. Such an approach is important due to changes in the methodology and the best quality of data over time (10).

As shown, significant regional differences exist in Brazil with respect to female breast cancer mortality rates, reproducing a similar pattern to those observed around the world (1-6). Although the Southeast (the most developed region) generally has the highest mortality rates, when temporal trends are analyzed, a pattern of decline is found, which is in line with trends identified in a large proportion of developed countries over the past 25 years (1,2,5). In the United States, an annual decrease of 2.5% has been observed, with similar findings in various European countries, such as France (-1.4%), Italy (-1.6%), and Germany (21.5%) (1,2,5).

However, the greatest increase (5.3%) was found in the Northeast, a region of medium and low development, in agreement with trends observed in some African and Asian countries, including South Korea and India (2).

Breast cancer is associated with industrialization, and the risk of the disease is related to higher socioeconomic levels, older age groups, low parity, early menarche and late menopause, obesity and alcohol consumption (10,26).

Accordingly, the results of the present study show that the risk of dying from female breast cancer in Brazil is at least twofold higher in states such as Sao Paulo, Rio de Janeiro, Rio Grande do Sul and the Federal District, where HDIs are higher, compared with the states of Alagoas, Maranhao and Piaui. Social inequalities regarding the access to and use of healthcare services are also present in this country, as reflected in the different breast cancer mortality trends found within Brazil.

Mammography screening is known to reduce breast cancer mortality rates through early diagnosis (27). In Brazil, mammography coverage is lower in the North and in some states of the Northeast, with coverage levels that range from 64% to 84% (28). According to the National Cancer Institute (INCA), mammography coverage ranges from 37% to 67% (29) in 15 state capital cities and the Federal District, which partially explains the different trends in breast cancer mortality shown in this paper, ranging from a decrease of 0.9% in the Southeast to an increase of 5.3% in the Northeast. These inequalities are even greater when the different states of Brazil are analyzed separately, with values ranging from a decline of 1.9% in Sao Paulo to a substantial increase of 12% in Maranhao.

Due to the immense inequalities between regions in Brazil, the specifications of each individual region need to be considered because they indicate different patterns within the demographic transition. In the South, mortality from breast cancer is higher in Rio Grande do Sul compared with the states of Santa Catarina and Parana, as has also been shown in other studies (10,30), suggesting heterogeneity in the profiles of exposure to risk factors and in lifestyle and genetic factors (30). However, contrary to the results of these previous studies, the present findings suggest stabilization and a decline in breast cancer mortality

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challenge facing Brazil is related to the difficulty encountered by patients in accessing the few centers in the country that specialize in breast cancer treatment. Furthermore, even these centers are often inadequately equipped to provide speedy diagnosis and treatment, resulting in patients migrating from areas in which healthcare is deficient to areas where access is easier, consequently overloading and burdening existing centers (29). In many regions of the country, the distribution of consultations was found to be poor, and access to treatment was difficult for women of lower socioeconomic levels (28,33,34).

The socioeconomic inequalities present in this country are most likely affecting mortality patterns despite the increased knowledge of risk factors for breast cancer, the improvement in mammography screening programs, resulting in the early detection of cases, and the advances achieved in the treatment of the disease (26,35,36). Therefore, although the risk of breast cancer is higher among women living in areas in which socioeconomic conditions are better (35,36), economically deprived women are at an increased risk of dying from breast cancer (36).

REFERENCES 1. Jemal A, Center MM, DeSantis C, Ward EM. Global patterns of cancer incidence and mortality rates and trends. Cancer Epidemiol Biomarkers Prev. 2010;19(8):1893–907, http://dx.doi.org/10.1158/1055-9965.EPI-100437.

2. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. Ca Cancer J Clin. 2011;61(2):69–90, http://dx.doi.org/10.3322/ caac.20107.

3. Parkin DM, Ferna´ndez LM. Use of statistics to assess the global burden of breast cancer. Breast J. 2006;2(Suppl 1):S70–S80, http://dx.doi.org/ 10.1111/j.1075-122X.2006.00205.x.

4. Bosetti C, Malvezzi M, Chatenoud L, Negri E, Levi F, La Vecchia C. Trends in cancer mortality in the Americas, 1970-2000. Ann Oncol.

2005;16(3):489–511, http://dx.doi.org/10.1093/annonc/mdi086.

5. Autier P, Boniol M, La Vecchia C, Vatten L, Gavin A, He´ry C, et al.

Disparities in breast cancer mortality trends between 30 European countries: retrospective trend analysis of WHO mortality database. BMJ. 2010;341:c3620, http://dx.doi.org/10.1136/bmj.c3620.

6. Ravdin PM, Cronin KA, Howlader N, Berg CD, Chlebowski RT, Feuer EJ, et al. The decrease in breast-cancer incidence in 2003 in the United States. N Engl J Med. 2007;356(16):1670-1674, http://dx.doi.org/ 10.1056/NEJMsr070105.

7. Freitas-Junior R, Freitas NM, Curado MP, Martins E, Moreira MA, Silva CM. Variations in breast cancer incidence per decade of life (Goiaˆnia, GO, Brazil): 16-year analysis. Cancer Causes Control. 2008;19(7):681–7, http://dx.doi.org/10.1007/s10552-008-9131-z.

8. Freitas-Junior R, Freitas NMA, Curado MP, Martins E, Silva CM, Rahal RM, et al. Incidence trend for breast cancer among young women in Goiaˆnia, Brazil. Sa˜o Paulo Med J. 2010;128(2):81–4.

9. Instituto Brasileiro de Geografia e Estatı´stica. [internet homepage]. Rio de Janeiro. http://www.ibge.gov.br. Accessed 26 June 2011.

10. Brasil. Ministe´rio da Sau´de. Estimativa 2012: incideˆncia de caˆncer no Brasil. Instituto Nacional de Caˆncer Jose´ Alencar Gomes da Silva, Rio de Janeiro. http://www.inca.gov.br/estimativa/2012/estimativa20122111. pdf. Accessed 02 Dez 2011.

11. Instituto Brasileiro de Geografia e Estatı´stica. Indicadores sociodemogra´ficos e de sau´de no Brasil: 2009. Estudos e Pesquisas: Informac¸a˜o Demogra´fica e Socioeconoˆmica, 25. Rio de Janeiro. http://www.ibge. gov.br/home/estatistica/populacao/indic_sociosaude/2009/indicsaude. pdf. Accessed 26 June 2011.

12. Caleffi M, Ribeiro RA, Duarte Filho DL, Ashton-Prolla P, Bedin AJ, Skonieski GP, et al. A model to optimize public health care and downstage breast cancer in limited-resource populations in southern Brazil. (Porto Alegre Breast Health Intervention Cohort). BMC Public Health. 2009;9:83.

13. Correa RS, Freitas-Ju´nior R, Peixoto JE, Rodrigues DCN, Lemos MEF, Marins LAP, et al. Estimativas da cobertura mamogra´fica no Estado de Goia´s, Brasil. Cad. Sau´de Pu´blica. 2011;27(9):1757-67, http://dx.doi.org/ 10.1590/S0102-311X2011000900009.

14. Nunes RD, Martins E, Freitas-Junior R, Curado MP, Freitas NMA, Oliveira JC. Descriptive study of breast cancer cases in Goiania between 1989 and 2003. Rev. Col. Bras. Cir. 2011;38(4):212-6, http://dx.doi.org/ 10.1590/S0100-69912011000400002.

15. Chatenoud L, Bertuccio P, Bosetti C, Levi F, Curado MP, Malvezzi M, et al. Trends in cancer mortality in Brazil, 1980-2004. Eur J Cancer Prev.

2010;19(2):79–86, http://dx.doi.org/10.1097/CEJ.0b013e32833233be.

16. Fonseca LAM, Eluf-Neto J, Wunsch Filho V. Trends of cancer mortality in Brazilian state capitals, 1980-2004. Rev Assoc Med Bras. 2010;56(3): 309–12, http://dx.doi.org/10.1590/S0104-42302010000300015.

17. Freitas-Silva R, Conde DM, de Freitas-Ju´nior R, Martinez EZ. Comparison of quality of life, satisfaction with surgery and shoulder-arm morbidity in breast cancer survivors submitted to breast-conserving therapy or mastectomy followed by immediate breast reconstruction. Clinics.

2010;65(8):781-7, http://dx.doi.org/10.1590/S1807-59322010000800007.

18. Brasil. Ministe´rio da Sau´de. Departamento de Informac¸a˜o e Informa´tica do SUS. DATASUS [internet homepage]. Brası´lia, DF. http://www.datasus. gov.br. Accessed 26 June 2011.

19. Jensen OM, Parkin DM, MacLennan R, Muir CS, Skeet RG. (1991). Cancer registration: principles and methods. International Agency for Research on Cancer, Lyon. (IARC Scientific Publications, no. 95).

20. National Cancer Institute [internet homepage]. Joinpoint Regression Program. Version 3.4.3. Bethesda. http://surveillance.cancer.gov/ joinpoint/. Accessed 12 June 2011.

21. Kim HJ, Fay MP, Feuer EJ, Midthune DN. Permutation tests for joinpoint regression with applications to cancer rates. Stat Med. 2000;19(3):335–51, http://dx.doi.org/10.1002/(SICI)1097-0258(20000215)19:3,335::AIDSIM336.3.0.CO;2-Z. 22. Wunsch Filho V, Moncau JE. Mortalidade por caˆncer no Brasil 1980-1995: padro˜ es regionais e tendeˆ ncias temporais. Rev Assoc Med Bras.

2002;48(3): 250–7, http://dx.doi.org/10.1590/S0104-42302002000300040.

23. Boyle P, Ferlay J. Cancer incidence and mortality in Europe, 2004. Ann Oncol. 2005;16(3):481–488, http://dx.doi.org/10.1093/annonc/mdi098

Conclusions and policy implications Although there has been a trend toward stabilization in the female breast cancer mortality rates in Brazil, when the mortality rate of each state is analyzed individually, considerable inequalities are found, with rate decline or stabilization in states with higher socioeconomic levels and a substantial rate increase in those with lower socioeconomic levels. In the majority of developing states, the mortality rates are increasing. Therefore, female breast cancer mortality should be studied in greater detail in view of the significant changes in the annual percentages, particularly in regions where the level of development is low.

The epidemiological data presented in this study provide further scientific knowledge on the temporal trends of female breast cancer in Brazil in each macro-region of the country and in each state. These data highlight important differences in the pattern of breast cancer mortality in Brazil and, for the first time, show a decline in female breast cancer mortality in some states and macro-regions of the country.

The relevance of these findings lies in the fact that they provide scientific data on which to base future decision making, primarily with respect to the northeast of the country, where the greatest increases in mortality were found. Therefore, although Brazil must be considered as a whole, the priority in terms of primary and secondary preventive actions should be established for the less developed regions of the country.

Conflicts of Interest and Source of Funding: Freitas-Junior R received a grant from Instituto AVON. Gonzaga CM received a scholarship from CAPES (Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior). For the remaining authors, no funding was declared.

AUTHOR CONTRIBUTIONS Freitas-Junior R was responsible for manuscript writing and the intellectual and scientific content of the study. Gonzaga CM was responsible for manuscript writing, the methodology applied, and revisions. Freitas NM was responsible for the methodology applied and critical revisions. Martins E was responsible for the statistical analysis. Dardes RC was responsible for the critical revisions.

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31. Brasil. Ministe´rio da Sau´de. O SUS de A a Z: garantindo sau´de nos municı´pios, 2nd ed. Conselho Nacional dos Secreta´rios Municipais de Sau´de, Brası´lia, DF, 2006.

32. Brasil. Ministe´rio da Sau´de. Controle do caˆncer de mama. Documento de consenso. Rev Bras Cancerol. 2004;50(2):77–90.

33. Brito C, Portela MC, Vasconcellos MTL. Assisteˆncia oncolo´gica pelo SUS a mulheres com caˆncer de mama no Estado do Rio de Janeiro. Rev Sau´de Pu´blica. 2005;39(6):874–81, http://dx.doi.org/10.1590/S0034-89102005 000600002.

34. Sclowitz ML, Menezes AMB, Gigante DP, Tessaro S. Condutas na prevenc¸a˜o secunda´ria do caˆncer de mama e fatores associados. Rev Sau´de Pu´blica. 2005;39(3):340–49, http://dx.doi.org/10.1590/S003489102005000300003.

35. Robert SA, Strombom I, Trentham-Dietz A, Hampton JM, McElroy JA, Newcomb PA, et al. Socioeconomic risk factors for breast cancer: distinguishing individual- and community-level effects. Epidemiology.

2004;15(4):442–50, http://dx.doi.org/10.1097/01.ede.0000129512.61698.

03.

36. Vona-Davis L, Rose DP. The influence of socioeconomic disparities on breast cancer tumor biology and prognosis: a review. J Womens Health.

2009;18(6):883–93, http://dx.doi.org/10.1089/jwh.2008.1127.

24. Paes NA, Albuquerque MEE. Avaliac¸a˜o da qualidade dos dados populacionais e cobertura dos registros de o´bitos para as regio˜es brasileiras. Rev Sau´de Pu´blica. 1999;33(1):33–43.

25. Curado MP. Techniques of registration. In: Curado MP, Edwards B, Shin HR, Storm H, Ferlay J, Heanue M, et al., eds. Cancer Incidence in Five Continents, Vol. IX. Lyon: IARC, 2009:73-101.

26. McPherson K, Steel CM, Dixon JM. ABC of breast diseases. Breast cancerepidemiology, risk factors, and genetics. BMJ. 2000;321(7261):624–8.

27. Autier P, Boniol M, Middleton R, Dore´ JF, He´ry C, Zheng T, et al.

Advanced breast cancer incidence following population-based mammographic screening. Ann Oncol. 2011;22(8):1726–1735, http://dx.doi.org/ 10.1093/annonc/mdq633.

28. Viacava F, Souza-Junior PRB, Moreira RS. Estimativas da cobertura de mamografia segundo inque´ritos de sau´de no Brasil. Rev Sau´de Pu´blica.

2009;43(Supl. 2):117–25, http://dx.doi.org/10.1590/S0034-891020090 00900015.

29. Gebrim LH, Quadros LGA. Rastreamento do caˆncer de mama no Brasil.

Rev Bras Ginecol Obstet. 2006;28(6):319–23.

30. Santos NR. Desenvolvimento do SUS, rumos estrate´gicos e estrate´gias para visualizac¸a˜o dos rumos. Cieˆnc. Sau´de Col. 2007;12(2):429–35, http://dx.doi.org/10.1590/S1413-81232007000200019.

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DOI:10.6061/clinics/2012(07)06

CLINICAL SCIENCE

Frequency and predictors of symptomatic intracranial hemorrhage after intravenous thrombolysis for acute ischemic stroke in a Brazilian public hospital Pedro Telles Cougo-Pinto,I Bruno Lopes dos Santos,I Francisco Antunes Dias,I Soraia Ramos Cabette Fabio,I Ilana Vaula Werneck,I Millene Rodrigues Camilo,I Daniel Giansante Abud,II Joa˜o Pereira Leite,I Octavio Marques Pontes-NetoI I

University of Sa˜o Paulo, Ribeira˜o Preto School of Medicine, Department of Neuroscience and Behavioral Sciences, Ribeira˜o Preto/SP, Brazil. II University of Sa˜o Paulo, Ribeira˜o Preto School of Medicine, Radiology Division of the Department of Internal Medicine, Ribeira˜o Preto/SP, Brazil.

OBJECTIVE: Scarce data are available on the occurrence of symptomatic intracranial hemorrhage related to intravenous thrombolysis for acute stroke in South America. We aimed to address the frequency and clinical predictors of symptomatic intracranial hemorrhage after stroke thrombolysis at our tertiary emergency unit in Brazil.

METHOD: We reviewed the clinical and radiological data of 117 consecutive acute ischemic stroke patients treated with intravenous thrombolysis in our hospital between May 2001 and April 2010. We compared our results with those of the Safe Implementation of Thrombolysis in Stroke registry. Univariate and multiple regression analyses were performed to identify factors associated with symptomatic intracranial transformation.

RESULTS: In total, 113 cases from the initial sample were analyzed. The median National Institutes of Health Stroke Scale score was 16 (interquartile range: 10-20). The median onset-to-treatment time was 188 minutes (interquartile range: 155-227). There were seven symptomatic intracranial hemorrhages (6.2%; Safe Implementation of Thrombolysis in Stroke registry: 4.9%; p = 0.505). In the univariate analysis, current statin treatment and elevated National Institute of Health Stroke Scale scores were related to symptomatic intracranial hemorrhage. After the multivariate analysis, current statin treatment was the only factor independently associated with symptomatic intracranial hemorrhage.

CONCLUSIONS: In this series of Brazilian patients with severe strokes treated with intravenous thrombolysis in a public university hospital at a late treatment window, we found no increase in the rate of symptomatic intracranial hemorrhage. Additional studies are necessary to clarify the possible association between statins and the risk of symptomatic intracranial hemorrhage after stroke thrombolysis.

KEYWORDS: Acute Stroke; Thrombolytic Therapy; Brain Hemorrhage; Statins; Tissue Plasminogen Activator.

Cougo-Pinto PT, Santos BL, Dias FA, Fabio SR, Werneck VI, Camilo MR et al. Frequency and predictors of symptomatic intracranial hemorrhage after intravenous thrombolysis for acute ischemic stroke in a Brazilian public hospital. Clinics. 2012;67(7):739-743.

Received for publication on January 27, 2012; First review completed on February 28, 2012; Accepted for publication on March 9, 2012 E-mail: opontesneto@fmrp.usp.br Tel.: 55 16 3602 2548

(TPA) is the only approved medical therapy for acute ischemic stroke aimed at arterial recanalization. However, the use of such therapy has been relatively low, partially due to its narrow therapeutic window but also because of concerns about the risk of symptomatic intracranial hemorrhagic transformation (SIH), which may be associated with clinical deterioration and death (2-4).

Reliable predictors of SIH are still under debate. In a systematic review that included 12 studies, there was considerable variance between results, and no predictor of SIH was common to all studies (5). Defining the accurate risk factors for SIH could increase the safety of TPA treatment for stroke and should eventually reduce the unnecessary concerns about its risks.

INTRODUCTION Acute ischemic stroke is one of the leading causes of mortality and disability worldwide and represents a lifechanging and ominous event for many patients (1). Currently, intravenous thrombolysis with tissue plasminogen activator

Copyright ß 2012 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.

No potential conflict of interest was reported.

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intracranial hemorrhages were categorized according to the ECASS classification system (hemorrhagic infarction (HI) type 1, HI type 2, parenchymal hematoma (PH) type 1, and PH type 2) (21). In the event of disagreement between investigators, images were further reviewed by an experienced stroke neurologist (OMP) for consensus. SIH was defined as any intracranial hemorrhage associated with an increase of 4 points or more on the NIHSS score or death, according to the criteria applied in the ECASS-2 trial (22).

Stroke is the main cause of death in Brazil, which has a higher age-adjusted mortality rate related to this condition than other South American countries (6-8). Little information exists on the safety of thrombolysis and the occurrence of SIH in South America (9-11). Recent studies found alarming results on public stroke awareness, delays in hospital admission and high stroke-related in-hospital mortality in Brazil (12,13). Nevertheless, there are few data about the safety of IV TPA for stroke in the country. SIH may be more common among more severe stroke patients (14), and the impact of late admission and, consequently, of late onset-toneedle time on the safety of IV TPA is also a matter of concern.

In this study, we aimed to determine the frequency of SIH and the factors associated with its occurrence among patients with an acute ischemic stroke treated with IV TPA at our public tertiary emergency unit in Brazil.

Statistical analysis In the univariate analysis, Fisher’s exact test was used for categorical data, and Wilcoxon’s rank-sum test was used for quantitative data. A forward stepwise logistic regression analysis was used to identify the independent predictors of SIH among the variables with a p-value,0.1 in the univariate analysis. The Spearman correlation coefficient (rs) was used to evaluate the correlations between quantitative variables. All of the statistical analyses were performed with the SPSS package (version 15.0 for Windows; Chicago, Illinois, USA).

PATIENTS AND METHODS We reviewed our institutional prospective stroke registry to identify all patients treated with IV TPA at the emergency unit of our hospital from May 2001 to April 2010. This prospective registry and this study were approved by the local institutional review board. Demographic data, cerebrovascular risk factors, prior medical history and medical therapy, clinical data, and relevant laboratory data were collected from this prospective registry by the investigators.

Stroke severity was measured using the National Institutes of Health Stroke Scale (NIHSS) and assessed by certified medical staff from the institution (15). The exclusion criteria were predefined as the lack of data for onset-to-treatment time, admission NIHSS score or neuroimaging scans.

Intravenous thrombolysis was administered in accordance with an institutional protocol, which was based on the National Institute of Neurological Disorders and Stroke (NINDS) trial and the European Cooperative Acute Stroke Study-3 (ECASS-3) trial (16,17). Informed consent was obtained from all patients or their proxy. NIHSS was assessed every 15 minutes during infusion, every hour during the first 6 hours, and every 6 hours during the first 48 hours after treatment. The monitoring and control of blood pressure were performed according to the Brazilian Consensus for the Thrombolysis in Acute Ischemic Stroke, which uses the same monitoring strategy and blood pressure thresholds for intervention as the NINDS trial protocol and the American Stroke Association guidelines for the early management of adults with acute stroke (18,16,19). The Brazilian protocol includes sodium nitroprusside and metoprolol as optional drugs for blood pressure control because labetalol, intravenous nicardipine, and nitropaste are not widely available in Brazil. In our hospital, sodium nitroprusside and metoprolol were used for blood pressure control. A control brain computed tomography scan was obtained 24 to 48 hours after treatment or immediately for neurological deterioration. All protocol violations were reviewed and registered by the main investigators. The results from our institution were compared with those of the Safe Implementation of Thrombolysis in Stroke – International Stroke Thrombolysis Registry (SITSISTR), which was published after the ECASS-3 trial and included patients within the 4.5-hour window of treatment (20).

RESULTS Between May 2001 and April 2010, 117 consecutive patients with acute ischemic stroke were treated with IV TPA in the emergency department of our institution. Four patients were excluded from the current analysis because relevant data regarding treatment were unavailable. None of those patients had SIH. The mean age in the remaining sample was 63¡12.8 years old. The median onset-totreatment time was 188 minutes [interquartile range (IR) = 155-227]. This median onset-to-treatment time was higher than the SITS-ISTR ,3 h cohort [140 (IR = 114-165); p,0.001] and lower than the 3-4.5 h cohort [205 (IR = 190229); p = 0.048]. Five (4.4%) patients received IV TPA within 90 minutes, 47 (41.6%) from 91-180 minutes, 51 (45.1%) from 181-270 minutes, and 10 (8.8%) after 270 minutes. The median admission NIHSS score was 16 (IR = 10-20). Our sample had more severe strokes than the SITS-ISTR in both treatment cohorts [,3 h cohort: 12 (IR = 7-17), p,0.001; 34.5 h cohort: 10 (IR = 6-15), p,0.001]. Twenty-eight (24.8%) patients had an NIHSS score equal to or less than 10, 60 (53.1%) from 11-20, 19 (16.8%) from 21-30, and six (5.3%) scored greater than 30. The demographic characteristics and clinical and laboratory data at the time of admission are shown in Table 1. Our sample had more prior stroke events and concurrent comorbidities and higher admission blood pressure than the SITS-ISTR. SIH occurred in 7 patients (6.2%), which were categorized as 2 PH-1 and 5 PH-2. SIH was related to in-hospital death (p = 0.005). Using the same ECASS-2 definition for SIH, we did not observe more hemorrhages compared with the SITS-ISTR (6.2% vs. 4.9%; p = 0.505).

Some patients received IV TPA despite having protocol violations: nine patients (8.0%) were older than 80 years old; ten (8.8%) patients received IV TPA after 270 minutes; and six (5.3%) patients had glycemia levels equal to or greater than 400 mg/dL. None of the patients above 80 years old suffered an SIH. One of the six patients with glycemia levels above 400 mg/dL suffered an SIH (16.6%; SIH in patients with glycemia #400 mg/dL: 5.4%; p = 0.325). Five patients with NIHSS scores between 11-20, two patients with scores between 21 and 30, and none of the patients with scores

Image review All of the images were independently reviewed by the main investigators (PTC, BLS, and FAD). When present,

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Table 1 - A comparative analysis of patient demographic, medical history, and clinical and laboratory data.

Present study Female sex 47.8% Hypertension 80.5% Diabetes mellitus 31.9% Hyperlipidemia 21.2% Obesity 23.9% Smoking 28.3% Alcohol abuse 17.7% Atrial fibrillation 16.8% Congestive heart 15.0% failure Ischemic heart disease 16.8% Prior stroke 18.6% Chagas’ disease 5.3% Current aspirin use 29.2% Current warfarin use 6.2% Current statin use 8.8% SBP (mmHg) 156 (140-180) DBP (mmHg)

90 (80-104)

NIHSS

16 (10-20)

Glycemia (mg/dL)

114 (101-162)

SITS-ISTR

p-value

39.7% 59.2% 16.0% 35.1% NA 25.6% NA 22.9% 7.6%

0.083 0.007 ,0.001 0.002 0.517 0.144 0.007

NA 10.3% NA 29.4% NA NA ,3 h = 150 (136-165) 3-4.5 h = 150 (136-166) ,3 h = 81 (74-90) 3-4.5 h = 80 (74-90) ,3 h = 12 (7-17) 3-4.5 h = 10 (6-15) ,3 h = 117 (102-140) 3-4.5 h = 117 (103-142)

0.003 0.999 p = 0.007

Table 2 - Univariate analysis.

Male sex Age NIHSS* Onset-to-treatment* Hypertension Diabetes High cholesterol Smoking Alcohol abuse Prior stroke Atrial fibrillation Chagas’ disease Congestive heart failure Ischemic heart disease Obesity Aspirin use Warfarin use Statin use SBP (mmHg) DBP (mmHg) Glycemia (mg/dL)

p,0.001 p,0.001 p,0.001 p,0.001 p = 0.011 p = 0.011

No SIH (n = 106)

SIH (n = 7)

p-value

55 (51.9%) 63.0¡13.1 15 (10-20) 185 (155-227) 85 (80.2%) 35 (33.0%) 21 (19.8%) 31 (29.2%) 20 (18.9%) 20 (18.9%) 18 (17.0%) 6 (5.7%) 16 (15.1%) 19 (17.9%) 26 (24.5%) 32 (30.2%) 7 (6.6%) 7 (6.6%) 160.5¡34.2 91.2¡22.8 142.9¡64.3

4 (57.1%) 62.9¡7.6 19 (16-22) 190 (100-227) 6 (85.7%) 1 (14.3%) 3 (42.9%) 1 (14.3%) Zero 1 (14.9%) 1 (14.9%) Zero 1 (14.9%) Zero 1 (14.9%) 1 (14.9%) Zero 3 (42.9%) 157.1¡27.3 97.0¡18.0 101.3¡35.3

0.999 0.979 0.083 0.560 0.999 0.428 0.164 0.671 0.350 0.999 0.999 0.999 0.999 0.599 0.999 0.671 0.999 0.015 0.800 0.543 0.121

SIH: symptomatic intracranial hemorrhage. NIHSS: National Institutes of Health Stroke Scale. SBP: systolic blood pressure at the time of admission.

DBP: diastolic blood pressure at the time of admission. Categorical variables are expressed as events (percentage). Numerical variables are expressed as the mean¡standard deviation. *Expressed as median (interquartile range).

SITS-ISTR: Safe Implementation of Thrombolysis in Stroke-International Stroke Thrombolysis Registry. NA: not available. SBP: systolic blood pressure at the time of admission. DBP: diastolic blood pressure at the time of admission. NIHSS: National Institutes of Health Stroke Scale.

Categorical variables are shown as relative frequencies (%). Numerical variables are shown as medians (interquartile range).

onset-to-needle time and SIH occurrence (23). In the NINDS trial, elevated NIHSS scores predicted SIH (14), but the ECASS-2 results did not confirm this association (4).

Although IV TPA seems to remain equally safe for ischemic stroke treatment throughout the 4.5-hour treatment window, its functional benefit decreases with prolonged onsetto-needle time (23-25). More than half of our sample was treated after 3 hours of symptom onset, and few patients received IV TPA in the first ‘golden hour’. In the last decade, a hierarchical network between emergency units has been implemented in the Brazilian emergency care systems.

However, this referral network was not specifically designed for acute stroke care, and there were no unified multi-institutional protocols. We believe that this lack may have contributed to delays in acute stroke recognition and referral to our hospital in the first years of implementation of this emergency network. More recently, there has been an important endeavor (with major input from stroke neurologists) to improve pre-hospital care and organize regional stroke networks in Brazil (26).

Although we may conclude that acute stroke thrombolysis can be safely performed, even in severe patients treated within a rather late treatment window, the true efficacy of IV TPA in this setting is most likely less beneficial. There are striking demographic disparities among Brazilian regions, and nationwide clinical registries are necessary to address this issue (27). Fortunately, the Safe Implementation of Thrombolysis in Stroke – Iberoamerican Cerebrovascular Diseases Society (SITS-SIECV) Stroke Registry has been recently created and will show broader results about the safety and efficacy of IV TPA use for stroke treatment in Latin America.

We observed a high frequency of baseline comorbidities in our sample. Indeed, our patients were more likely to have arterial hypertension, diabetes, dyslipidemia, heart disease

greater than 30 or less than 11 experienced an SIH. Three patients with an onset-to-needle time between 91 and 180 minutes and four patients with an onset-to-needle time between 181 and 270 minutes had an SIH. SIH did not occur among patients treated within 90 minutes or after 270 minutes.

Ten patients were taking statins at the time of admission, and three of the ten patients suffered an SIH. Patients currently using statins were more likely to have hyperlipidemia (p,0.001) and congestive heart failure (p = 0.042). In the univariate analysis, prior statin use was associated with SIH, and there was a trend toward an association between greater stroke severity and SIH (Table 2). After the multivariate analysis, the prior use of statins was the only factor independently associated with SIH (OR = 3.8; 95% confidence interval: 2.5-8.2; p,0.001).

DISCUSSION In this series of acute ischemic stroke patients treated with IV TPA in a single public university hospital of Brazil, we observed no increase in the SIH occurrence compared with the more recent data from the SITS-ISTR. Our patients had severe neurological deficits and a late onset-to-needle time.

Patients with SIH had greater NIHSS scores at the time of admission, although this difference did not reach statistical significance. We found no association between SIH and age greater than 80 years old or onset-to-treatment time. Prior use of statins was the only factor independently associated with SIH. A large pooled analysis of the main trials of IV TPA in acute ischemic stroke did not show a correlation between

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Nevertheless, important efforts for the promotion of public stroke awareness and the organization of national and regional stroke networks in Brazil have already shown positive and promising results, with a reduction in stroke rate and mortality (38).

In this study, we found that, despite the increased severity of strokes and delayed admissions, acute stroke treatment with IV TPA can be safely performed in Brazil without an increased risk of SIH. In severe, late-treated patients with acute stroke, statin use may be associated with the occurrence of SIH. However, further studies are necessary to clarify this association.

and higher blood pressure at the time of admission compared with the patients in the SITS-ISTR. Nevertheless, this profile was not related to a more frequent SIH occurrence in this study. Prior diabetes and congestive heart failure have been associated with SIH in some studies, but this association has not been consistently confirmed by others (5). The presence of severe comorbidities may be a surrogate marker for more critical underlying strokes and also suggests an important opportunity to reduce stroke recurrence.

The effect of prior statin use on SIH occurrence in acute ischemic stroke patients treated with recanalization therapies is controversial. The prolonged use of statins and low cholesterol levels have been associated with an increased risk of spontaneous hemorrhagic stroke, although a clear explanation for such an association has not been fully established (28). This relationship may be due to non-lipidrelated effects. In clinical and experimental studies, statins have been observed to have anti-thrombotic and profibrinolytic effects (29,30). HMG-CoA reductase inhibitors decrease platelet aggregation and reduce fibrinogen and prothrombin levels and their conversion to active forms (31,32). A positive relationship between prior statin use and SIH in intra-arterial thrombolysis-treated patients has been reported (33). However, there was no relationship between prior statin use and SIH or good functional outcome in a large prospective cohort of acute ischemic stroke patients treated with IV TPA (34). One study including patients treated with IV TPA, intra-arterial thrombolysis and endovascular embolectomy found that lower admission low-density lipoprotein levels but not prior statin use were related to SIH (35).

Recently, a large pooled analysis found that statin users had more ECASS2-defined SIH, although this positive association was no longer significant after a multivariate analysis and adjustment for imbalances between the groups of statin users and non-statin users (36). These conflicting results could be related to ethnic disparities between the populations of these studies. In fact, it has been shown that ethnicity may have striking effects on statin pharmacokinetics (37). We believe that further studies would be necessary to answer this controversy.

The main limitations of our study include its small sample size and its single-center and retrospective design. The fact that variables such as age and stroke severity were not significantly related to SIH may be due to low study power because there were only seven hemorrhages. Larger studies are required before conclusions on the relationships between clinical variables and SIH can be drawn. Additionally, the blood pressure values during the first 48 hours after treatment were not assessed, and an imbalance of adequate blood pressure control between the subgroups is possible. We were also not able to retrieve cholesterol levels before or during the acute phase of stroke in many patients, and this precluded the analysis of the relationship between cholesterol levels and SIH. Furthermore, it is probable that our results may not reflect the acute stroke treatment conditions in other regions of Brazil.

Intravenous thrombolysis remains one of the few medical therapies for acute ischemic stroke. Nevertheless, few patients admitted to emergency departments receive thrombolytic treatment in developing countries. Scarce lay knowledge about stroke symptoms, ineffective health coverage and non-organized stroke care are contributing to this scenario (2,7,12). In fact, only recently has alteplase been reimbursed in the public health system in Brazil.

AUTHOR CONTRIBUTIONS Cougo-Pinto PT contributed to the methodological planning, data collection and analysis, image review, critical review of the results, and manuscript development and review. Santos BL and Dias FA contributed to the data collection, image review, and manuscript review. Fabio SR, Werneck IV and Camilo MR contributed to the data collection and manuscript review. Abud DG and Leite JP participated in the critical review of the results and manuscript development and review. Pontes-Neto OM contributed to the methodological planning, image review, data analysis, critical review of the results, and manuscript development and review.

REFRENCES 1. World Health Organization: The World Health Report 2003: shaping the future. World Health Organization, Geneva (2003).

2. Pontes-Neto OM, Silva GS, Feitosa MR, de Figueiredo NL, Fiorot JA Jr, Rocha TN, et al. Stroke awareness in Brazil: alarming results in a community-based study. Stroke. 2008;39(2):292-6, http://dx.doi.org/ 10.1161/STROKEAHA.107.493908.

3. Caplan LR: Stroke thrombolysis: slow progress. Circulation 2006;114(3): 187–90.

4. Larrue V, von Kummer R, Mueller A, Bluhmki E. Risk Factors for Severe Hemorrhagic Transformation in Ischemic Stroke Patients Treated With Recombinant Tissue Plasminogen Activator: A Secondary Analysis of the European-Australasian Acute Stroke Study (ECASS II). Stroke.

2001;32(2):438-41, http://dx.doi.org/10.1161/01.STR.32.2.438.

5. Lansberg MG, Albers GW, Wijman CA. Symptomatic intracerebral hemorrhage following thrombolytic therapy for acute ischemic stroke: a review of the risk factors. Cerebrovasc Dis. 2007;24(1):1-10, http:// dx.doi.org/10.1159/000103110.

6. Lotufo PA, De Lolio CA. Trends of mortality from cerebrovascular disease in the state of Sa˜o Paulo: 1970 to 1989. Arq Neuropsiquiatr.

1993;51(4):441-6, http://dx.doi.org/10.1590/S0004-282X1993000400003.

7. Lavados PM, Hennis AJM, Fernandes JG, Medina MT, Legetic B, Hoppe A, et al. Stroke epidemiology, prevention, and management strategies at a regional level: Latin America and the Caribbean. Lancet Neurol.

2007;6(4):362-72, http://dx.doi.org/10.1016/S1474-4422(07)70003-0.

8. Mansur AP, do Souza MFM, Favarato D, Avakian SD, Ce´sar LAM, Aldrigui JM et al. Stroke and ischemic heart disease mortality trends in Brazil from 1979 to 1996. Neuroepidemiology. 2003;22(3):179-83, http:// dx.doi.org/10.1159/000069893.

9. Cabral NL, Goncalves AR, Longo AL, Moro CH, Costa G, Amaral CH, et al. Incidence of stroke subtypes, prognosis and prevalence of risk factors in Joinville, Brazil: a 2 year community based study. J Neurol Neurosurg Psychiatry. 2009;80(7):755–61, http://dx.doi.org/10.1136/ jnnp.2009.172098.

10. Leopoldino JF, Fukujima MM, Silva GS, do Prado GF. Time of presentation of stroke patients in Sao Paulo Hospital. Arq Neuropsiquiatr. 2003;61(2A):186-7, http://dx.doi.org/10.1590/S0004282X2003000200005.

11. Conforto AB, Paulo RB, Patroclo CB, Pereira SL, Miyahara Hde S, Fonseca CB, et al. Stroke management in a university hospital in the largest South American city. Arq Neuropsiquiatr. 2008;66(2B):308-11, http://dx.doi.org/10.1590/S0004-282X2008000300004 ´ , Machado CB, Dos Santos BF, 12. De Carvalho JJ, Alves MB, Viana GA Kanamura AH, et al. Stroke epidemiology, patterns of management, and outcomes in Fortaleza, Brazil: a hospital-based multicenter prospective study. Stroke. 2011;42(12):3341-6, http://dx.doi.org/10.1161/STROKE AHA.111.626523.

13. Pontes-Neto OM, Silva GS, Feitosa MR, de Figueiredo NL, Fiorot JA Jr, Rocha TN, et al. Stroke awareness in Brazil: alarming results in a community-based study. Stroke. 2008;39(2):292-6, http://dx.doi.org/ 10.1161/STROKEAHA.107.493908.

742

CLINICS 2012;67(7):739-743

Brain hemorrhage after IV TPA Cougo-Pinto PT et al.

14. Intracerebral hemorrhage after intravenous t-PA therapy for ischemic stroke. The NINDS t-PA Stroke Study Group. Stroke. 1997;28(11):2109-18.

15. Cincura C, Pontes-Neto OM, Neville IS, Mendes HF, Menezes DF, Mariano DC, et al. Validation of the National Institutes of Health Stroke Scale, modified Rankin Scale and Barthel Index in Brazil: the role of cultural adaptation and structured interviewing. Cerebrovasc Dis. 2009;27(2):119-122, http://dx.doi.org/10.1159/000177918.

16. Tissue plasminogen activator for acute ischemic stroke. The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group. N Engl J Med. 1995;333(24):1581–7.

17. Hacke W, Kaste M, Bluhmki E, Brozman M, Da´valos A, Guidetti D, et al.

Thrombolysis with alteplase 3 to 4.5 hours after acute ischemic stroke.N Engl J Med. 2008;359(13):1317-29.

18. Brazilian consensus for the thrombolysis in acute ischemic stroke. Arq Neuropsiquiatr. 2002;60(3-A):675-80.

19. Adams HP, del Zoppo G, Alberts MJ, Bhatt DL, Brass L, Furlan A et al.

Guidelines for the early management of adults with ischemic stroke: a guideline from the American Heart Association/American Stroke Association Stroke Council, Clinical Cardiology Council, Cardiovascular Radiology and Intervention Council, and the Atherosclerotic Peripheral Vascular Disease and Quality of Care Outcomes in Research Interdisciplinary Working Groups: the American Academy of Neurology affirms the value of this guideline as an educational tool for neurologists.

Stroke. 2007;38(5):1655-711, http://dx.doi.org/10.1161/STROKEAHA. 107.181486.

20. Ahmed N, Wahlgren N, Grond M, Hennerici M, Lees KR, Mikulik R, et al.

Implementation and outcome of thrombolysis with alteplase 3–4?5 h after an acute stroke: an updated analysis from SITS-ISTR. Lancet Neurol.

2010;9(9):866–874, http://dx.doi.org/10.1016/S1474-4422(10)70165-4.

21. Hacke W, Kaste M, Fieschi C, Toni D, Lesaffre E, von Kummer R, et al.

Intravenous thrombolysis with recombinant tissue plasminogen activator for acute hemispheric stroke: the European Cooperative Acute Stroke Study (ECASS). JAMA. 1995;274(13):1017-25, http://dx.doi.org/ 10.1001/jama.1995.03530130023023.

22. Hacke W, Kaste M, Fieschi C, von Kummer R, Davalos A, Meier D, et al.

Randomised double-blind placebo-controlled trial of thrombolytic therapy with intravenous alteplase in acute ischaemic stroke (ECASS II). Second European-Australasian Acute Stroke Study Investigators.

Lancet. 1998;352(9136):1245-51, http://dx.doi.org/10.1016/S01406736(98)08020-9.

23. Lees KR, Bluhmki E, von Kummer R, Brott TG, Toni D, Grotta JC, et al.

Time to treatment with intravenous alteplase and outcome in stroke: an updated pooled analysis of ECASS, ATLANTIS, NINDS, and EPITHET trials. Lancet. 2010;375(9727):1695-703, http://dx.doi.org/10.1016/ S0140-6736(10)60491-6.

24. Marler JR, Tilley BC, Lu M, Brott TG, Lyden PC, Grotta JC, et al. Early stroke treatment associated with better outcome: the NINDS rt-PA stroke study. Neurology. 2000;55(11):1649-55, http://dx.doi.org/10.1212/ WNL.55.11.1649.

25. Lansberg MG, Schrooten M, Bluhmki E, Thijs VN, Saver JL. Treatment time-specific number needed to treat estimates for tissue plasminogen

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

743

activator therapy in acute stroke based on shifts over the entire range of the modified Rankin Scale. Stroke. 2009;40(6):2079-84, http:// dx.doi.org/10.1161/STROKEAHA.108.540708.

Hachinski V, Donnan GA, Gorelick PB, Hacke W, Cramer SC, Kaste M, et al. Working Toward a Prioritized World Agenda. Stroke.

2010;41(6):1084-99, http://dx.doi.org/10.1161/STROKEAHA.110.586156. ´ , Machado CB, dos Santos BFC, de Carvalho JJ, Alves MB, Viana GA Kanamura AH, et al. Stroke epidemiology, patterns of management, and outcomes in Fortaleza, Brazil: a hospital-based multicenter prospective study. Stroke. 2011;42(12):3341-6, http://dx.doi.org/10.1161/STROK EAHA.111.626523.

Amarenco P, Bogousslavsky J, Callahan A, Goldstein LB, Hennerici M, Rudolph AE, et al. High-dose atorvastatin after stroke or transient ischemic attack. N Engl J Med. 2006;355(6): 549–59.

Undas A, Brummel KEMann KG. Statins and blood coagulationArterioscler Thromb Vasc Biol. 2005;25(2):287-94.

Krysiak R, Okopien´ BHerman Z,Effects of HMG-CoA reductase inhibitors on coagulation and fibrinolysis processes. Drugs. 2003;63(170:1821-4, http://dx.doi.org/10.2165/00003495-200363170-00005.

Ma LP, Nie DN, Hsu SX, Yin SM, Xu LZ, Nunes JV. Inhibition of platelet aggregation and expression of alpha granule membrane protein 140 and thromboxane B2 with pravastatin therapy for hypercholesterolemia. J Assoc Acad Minor Phys. 2002;13:23-26.

Undas A, Brummel KE, Musial J, Mann KG, Szczeklik A. Simvastatin depresses blood clotting by inhibiting activation of prothrombin, factor V, and factor XIII and by enhancing factor Va inactivation. Circulation.

2001;103(18):2248-53, http://dx.doi.org/10.1161/01.CIR.103.18.2248.

Meier N, Nedeltchev K, Brekenfeld C, Galimanis A, Fischer U, Findling O, et al. Prior statin use, intracranial hemorrhage, and outcome after intraarterial thrombolysis for acute ischemic stroke. Stroke. 2009;40(5):1729-37, http://dx.doi.org/10.1161/STROKEAHA.108.532473.

Miedema I, Uyttenboogaart M, Koopman K, De Keyser J, Luijckx GJ. Statin use and functional outcome after tissue plasminogen activator treatment in acute ischaemic stroke. Cerebrovasc Dis. 2010;29(3):263-7, http://dx.doi.org/10.1159/000275500.

Bang OY, Saver JL, Liebeskind DS, Starkman S, Villablanca P, Salamon N, et al. Cholesterol level and symptomatic hemorrhagic transformation after ischemic stroke thrombolysis. Neurology. 2007;68(10):737-42, http://dx.doi.org/10.1212/01.wnl.0000252799.64165.d5.

Engelter ST, Soinne L, Ringleb P, Sarikaya H, Bordet R, Berrouschot J, et al. IV thrombolysis and statins. Neurology. 2011;77(9):888-95, http:// dx.doi.org/10.1212/WNL.0b013e31822c9135.

Lee E, Ryan S, Birmingham B, Zalikowski J, March R, Ambrose H, et al.

Rosuvastatin pharmacokinetics and pharmacogenetics in white and Asian subjects residing in the same environment. Clin Pharmacol Ther.

2005;78(4):330-41, http://dx.doi.org/10.1016/j.clpt.2005.06.013.

Cabral NL, Gonc¸alves ARR, Longo AL, Moro CHC, Costa G, Amaral CH, et al. Trends in stroke incidence, mortality and case fatality rates in Joinville, Brazil: 1995–2006. J Neurol Neurosurg Psychiatry. 2009;80(7):749– 54, http://dx.doi.org/10.1136/jnnp.2008.164475.

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CLINICAL SCIENCE

T-cell large granular lymphocytic leukemia: treatment experience with fludarabine Renata Oliveira Costa,II,III Marcelo Bellesso,I,II Dalton Alencar Fischer Chamone,I,II Milton Artur Ruiz,I Abraha˜o Elias Hallack Neto,IV Vera Lucia Aldred,V Juliana PereiraI,II I

Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Hematology Department, Sa˜o Paulo/SP, Brazil. II Instituto do Caˆncer do Estado de Sa˜o Paulo da Faculdade de Medicina da Universidade de Sa˜o Paulo Hematology Department, Sa˜o Paulo/SP, Brazil. III Lusiadas University School of Medicine, Internal Medicine Department, Santos/SP, Brazil. IV Faculdade de Medicina da Universidade Federal de Juiz de Fora, Internal Medicine Department, Juiz de Fora/MG, Brazil. V Faculdade de Medicina da Universidade de Sa˜o Paulo, Pathology Department, Sa˜o Paulo/SP, Brazil.

OBJECTIVES: The aim of this retrospective study was to investigate the results of T-cell large granular lymphocytic leukemia treatment with fludarabine by assessing the complete hematologic response, the complete molecular response, progression-free survival, and overall survival.

METHODS: We evaluated the records of six patients with T-cell large granular lymphocytic leukemia who were treated with fludarabine as a first-, second-, or third-line therapy, at a dose of 40 mg/m2, for three to five days per month and 6 to 8 cycles.

RESULTS: Of the six patients investigated with T-cell large granular lymphocytic leukemia who were treated with fludarabine, five (83.3%) were female, and their median age was 36.5 years (range 18 to 73). The median lymphocyte level was 3.46109/L (0.5 to 8.9). All patients exhibited a monoclonal T-cell receptor gamma gene rearrangement at diagnosis. Two (33.3%) patients received fludarabine as first-line treatment, two (33.3%) for refractory disease, one (16.6%) for relapsed disease after the suspension of methotrexate treatment due to liver toxicity, and one (16.6%) due to dyspesia. A complete hematologic response was achieved in all cases, and a complete molecular response was achieved in five out six cases (83.3%). During a mean follow-up period of 12 months, both the progression-free survival and overall survival rates were 100%.

CONCLUSION: T-cell large granular lymphocytic leukemia demonstrated a high rate of complete hematologic and molecular response to fludarabine, with excellent compliance and tolerability rates. To confirm our results in this rare disease, we believe that fludarabine should be tested in clinical trials as a first-line treatment for T-cell large granular lymphocytic leukemia.

KEYWORDS: Fludarabine; Treatment; Large Granular Lymphocyte Leukemia.

Costa RO, Bellesso M, Chamone DAF, Ruiz MA, Hallack Neto AE, Aldred VL, Pereira J. T-cell large granular lymphocytic leukemia: treatment experience with fludarabine. Clinics. 2012;67(7):745-748.

Received for publication on March 10, 2012; First review completed on March 16, 2012; Accepted for publication on March 18, 2012 E-mail: dr.marcelobellesso@gmail.com Tel.: 55 11 3061-5544

CD4-CD8-, CD4+CD8- or CD4+CD8+ in rare variants of the disorder. The associated natural killer cell antigens (NKa) CD16, CD56 and CD57 are variably expressed; CD57 is the most commonly expressed cell antigen and CD56 is the least commonly expressed cell antigen. A total or partial lack of other T-cell antigens, such as CD2, CD5 and CD7, can be observed (3-4).

T-LGL leukemia is associated with autoimmune disorders, such as rheumatoid factor with or without rheumatoid arthritis, Felty’s syndrome, Coombs-positive hemolytic anemia, idiopathic thrombocytopenic purpura, pure red cell aplasia, the presence of positive anti-nuclear antibodies, the presence of anti-neutrophil cytoplasmic antibodies, hypogammaglobulinemia, and polyclonal hypergammaglobulinemia. Anemia and neutropenia are frequent (5). At diagnosis, 70% of patients require treatment, but a watchand-wait approach may be appropriate for asymptomatic patients (2). The most common indications for treatment are

INTRODUCTION T-cell large granular lymphocytic (T-LGL) leukemia is characterized by a monoclonal expansion of CD3-positive TLGL cells, as described in 1975 (1). This rare and indolent disorder represents 2% to 3% of chronic lymphoid leukemia cases, with a median age at diagnosis of 60 years and an equal male to female ratio (2). The diagnostic criterion is a persistent increase in circulating monoclonal large granular lymphocytes lasting at least six months. The neoplastic cells are CD3, CD8 and TCRab in 80% of cases, but they may be

Copyright ß 2012 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.

No potential conflict of interest was reported.

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MgCl2, 0.2 nmol/L of dNTP, and 0.5 mmol/L of each primer (IDT Technologies, Illinois, USA). PCR was performed in a PTC-100 DNA Engine Tetrad (MJ Research, Inc., Waltham, MA) with one cycle at 94 ˚C for three min, followed by 40 cycles at 95 ˚C for 60 sec, 61.8 ˚C for 30 sec, and 72 ˚C for 30 sec, with a final extension of 10 min at 72 ˚C and then held at 4 ˚C. A 0.1-mL aliquot of the PCR product was mixed with 12 mL of deionized formamide and 0.5 mL of GeneScan 500 HD Rox size standard (Applied Biosystems, Foster City, CA). Then, the mixture was injected into an ABI Prism 3130 Genetic Analyzer, and the resulting data were analyzed using the GeneMapper V 3.2 software (Applied Biosystems, Foster City, CA). A monoclonality spike was determined by visual examination of the electropherograms (12).

cytopenia, recurrent infection and pure red cell aplasia, progressive splenomegaly and B symptoms (5).

There is currently no gold standard treatment for T-LGL leukemia. Most patients are treated with low doses of methotrexate, cyclophosphamide, and cyclosporine-A. As a rule, the treatment should be continuously monitored and, if necessary, adjusted to maintain the desired response.

However, treatment compliance is frequently low, with a consequent relapse of the disease. Moreover, side effects are common and may limit the treatment effectiveness (6-7). We therefore changed our approach to treating T-LGL leukemia in an attempt to improve the hematological and molecular response rates and to reduce the treatment duration.

Previous studies have shown promising results with fludarabine (8), 29-deoxycoformycin and alemtuzumab in T-LGL leukemia, with a response rate of 40% to 60% when used as second-line therapy (9). Based on our long-term, positive experience with fludarabine in other hematological disorders and in view of the high cost and toxicity of alemtuzumab, we changed our treatment approach to TLGL leukemia by adopting fludarabine as the drug of choice. Herein, we report our experience using fludarabine in the treatment of six patients with T-LGL leukemia.

Treatment Patients with T-LGL were treated with fludarabine if they presented with B symptoms, progressive splenomegaly, recurrent infection or anemia. Fludarabine was used as a first-line treatment or salvage therapy. HIV-positive patients were excluded from the study.

Fludarabine was used at a dosage of 40 mg/m2/day pathway oral for three to five days monthly for 6/8 cycles.

Granulocyte colony-stimulating factor was given to all patients beginning from the 10th day of each cycle until the end of the cycle, and it was given for five days if the neutrophil count was less than 1.06109/L because it was not possible to differentiate between neutropenia secondary to the disease or due to drug toxicity. Prophylaxis for Pneumocystis jiroveci with trimethoprim-sulfamethoxazole was also indicated for all patients.

PATIENTS AND METHODS Patients and end points This study was a retrospective analysis of data obtained by reviewing the medical records of six patients with T-LGL leukemia. They were treated with fludarabine by the same Hematology Service of the Clinical Hospital of Sa˜o Paulo and Cancer Institute (Sa˜o Paulo, Brazil) from January 2007 to January 2009 and February 2009 to October 2010, respectively.

The primary and secondary end points were complete hematologic (CHR) and complete molecular (CMR) response and progression-free (PFS) and overall survival (OS), respectively.

Assessment of treatment response The treatment response was evaluated by periodic clinical assessments, complete blood cell counts and phenotype and molecular analysis after cycles four, six and eight. CHR was defined as the complete normalization of blood counts (i.e., hemoglobin .120 g/L, platelets .1506109/L, and neutrophils .1.56109/L) and the absence of T-LGL cells. Partial hematologic response (PHR) was defined as an improvement of more than 50% in the complete blood cell count.

CMR was defined as the absence of a monoclonal population as assessed by PCR (13).

Diagnostic criteria for T-LGL leukemia The diagnostic criterion for T-LGL leukemia was a persistent (at least six months) increase in circulating TLGL cells, as evaluated by cell morphology and phenotype and confirmed by a monoclonal T-cell receptor (TCR) gamma gene rearrangement. A complete blood cell count, peripheral blood (PB) slide examination, and phenotype determination by flow cytometry were carried out using a standard multiparameter methodology to assess the antigens CD2, CD3, CD4, CD5, CD7, CD8, CD56, CD16, CD58, TCRb, and TCRcd, as previously described (10). The equipment used was a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA) and the CellQuestPro software. For analysis, the lymphocyte region was delimitated by an electronic gate in the forward side scatter versus side scatter display.

Populations of T-LGL cells were defined by the co-expression of the T-cell CD3 antigen and at least one NKa CD57, CD16 or CD56 antigen. The normal range of T-LGL cell numbers assumed in the analysis was 0.1-0.36109/L (11).

RESULTS Clinical features Five (83.3%) patients were female, and the median patient age was 36.5 years (18 to 73) (Table 1). The median lymphocyte level was 3.46109/L (0.5 to 8.9), with a median T-LGL cell number of 2.26109/L (0.4-5.3). The median neutrophil count was 0.356109/L (0.2-0.8). Five (83.3%) patients had anemia, with a median hemoglobin level of 105 g/L (38-143); two patients (33.3%) presented with pure red cell aplasia, and the disease was associated with Felt’s syndrome in two patients (33.3%) (Table 1). Four (66.6%) patients were CD3+CD8+TCRab+, one (16.6%) was CD4+ CD8-TCRab+, and one (16.6%) was CD4-CD8-TCRcd+. Five (83.3%) patients were CD57+CD16+, and none of them were CD56+. All patients were positive for the monoclonal TCR gamma gene rearrangement.

Two (33.3%) patients received fludarabine as first-line treatment, another two (33.3%) for refractory disease, one (16.6%) for relapse disease after discontinuing methotrexate treatment due to liver toxicity, and one (16.6%) due to

T-cell monoclonality assessment The monoclonal rearrangement of the TCR gamma gene was assessed by polymerase chain reaction (PCR) using primer sequences, as described previously by Shadrach B and Warshawsky I (12). Briefly, each 25-ml PCR reaction contained 200 ng of DNA, 1.5 U of Taq, 1.5 nmol/L of

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Table 1 - Clinical Characteristics of Patients.

Patient 1 2 3 4 5 6

Age/gender

Presentation

Hemoglobin (g/L)

Lymphocytes (x 109/L)

Neutrophils (x 109/L)

Platelets (x 109/L)

54/F 45/M 73/F 25/F 28/F 18/F

Felty’s syndrome Infection Felty’s syndrome Infection PRCA PRCA

143 117 129 105 38 69

1.8 3.9 0.5 8.9 1.6 3.4

0.3 0.4 0.2 0.8 0.8 0.3

147 319 245 202 147 172

F = Female; M = Male; PRCA = pure red cell aplasia.

dyspesia. The most common sign of toxicity was grade 4 neutropenia without infection.

CHR was achieved in all cases, and CMR was achieved in five out of the six patients (83.3%) (Figure 1). No case of hematological relapse occurred within a median follow-up time of 12 months. All patients are still alive. Bone marrow transplantation was not performed after the treatment with fludarabine because T-LGL is an indolent disease and is associated with a high rate of mortality and morbidity.

showed that with cyclosporine-A and methotrexate, only a minority of patients were able to achieve better results, due to low compliance rates and drug toxicity.

Fludarabine has been used to treat malignant lymphoid disorders since the 1990s (16) and has shown high efficacy in indolent diseases, such as chronic lymphocyte leukemia and non-Hodgkin lymphoma. In 1994, Witzig et al. (17) described a case of T-LGL leukemia with transfusion-dependent anemia that was successfully treated with four cycles of fludarabine after a seven-year history of partial response to several drugs. This patient became transfusion-free, with a partial molecular response lasting more than 15 months.

Another study showed CHR in four symptomatic patients treated with fludarabine but with no CMR (8). In another report including 26 patients (14), 19 (73%) were treated with different drugs, either alone or in combination, such as cyclosporine-A, erythropoietin, granulocyte colony-stimulating factor, prednisone, alemtuzumab, pentostatin, fludarabine, cyclophosphamide and infliximab, in addition to splenectomy and hematopoietic stem cell transplantation.

Cyclosporine-A was the most commonly used drug; six patients achieved CHR, but none of them achieved immunophenotype CR or CMR. Although the authors reported an absence of response in two patients treated with fludarabine, they had been previously treated at another institution and laboratory data were not available to confirm the diagnosis.

In contrast, the best results concerning CMR in T-LGL leukemia were reported in patients treated with fludarabine, mitoxantrone and dexamethasone (18). Based on this study, we decided to use fludarabine to treat our T-LGL leukemia patients. Our results demonstrated a high rate of CHR and

DISCUSSION Herein, we describe our experience concerning T-LGL leukemia treatment with fludarabine. We observed a high rate of CHR and CMR, with good compliance and minimal side effects. Because T-LGL leukemia is a rare disease, prospective, randomized studies to evaluate different treatment protocols are not feasible. Therefore, the treatment of TLGL leukemia is based on the findings of case reports and retrospective studies (14-15). Thus, until 2006, cyclosporine and methotrexate were the only first-line therapy options for T-LGL leukemia. Although effective, maintenance therapy is required to achieve sustained responses with these drugs.

However, maintenance therapy results in significant side effects, especially in younger patients (7,14,15). In fact, in our institutions, the median age of T-LGL leukemia patients was less than described in the literature. Therefore, a short time of therapy could certainly produce better results because it avoids adverse events. Although indolent diseases, such as TLGL leukemia, require a long follow-up period for the treatment to lead to a better overall survival, our experience

Figure 1 - Peripheral blood of patient 4: (A) at diagnosis, showing T-cell receptor gamma gene monoclonal rearrangement; and (B) after eight cycles of fludarabine, showing that the monoclonal cells were completely replaced by polyclonal T-cells, which is characteristic of a complete molecular response (CMR).

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7. Sood R, Stewart CC, Aplan PD, Murai H, Ward P, Barcos M, Baer MR. Neutropenia associated with T-cell large granular lymphocyte leukemia: long-term response to cyclosporine therapy despite persistence of abnormal cells. Blood. 1998;91(9):3372-8.

8. Sternberg A, Eagleton H, Pillai N, Leyden K, Turner S, Pearson D, et al.

Neutropenia and anaemia associated with T-cell large granular lymphocyte leukaemia responds to fludarabine with minimal toxicity.

Br J Haematol. 2003;120(4):699-701, http://dx.doi.org/10.1046/j.13652141.2003.04148.x.

9. Fortune AF, Kelly K, Sargent J, O’Brien D, Quinn F, Chadwick N, et al.

Large granular lymphocyte leukemia: natural history and response to treatment. Leuk Lymphoma. 2010;51(5):839-45, http://dx.doi.org/ 10.3109/10428191003706947.

10. Stewart CC. Clinical applications of flow cytometry: Immunologic methods for measuring cell membrane and cytoplasmic antigens.

Cancer. 1992;15;69(6 Suppl):1543-52, http://dx.doi.org/10.1002/10970142(19920315)69:6+,1543::AID-CNCR2820691307.3.0.CO;2-O. 11. O’Malley DP. T-cell large granular leukemia and related proliferations.

Am J Clin Pathol. 2007;127(6):850-9, http://dx.doi.org/10.1309/ A8FHDA0VVRJ05GJP. 12. Shadrach B, Warshawsky I. A comparison of multiplex and monoplex T cell receptor gamma PCR. Diagn Mol Pathol. 2004;13(3):127-34, http:// dx.doi.org/10.1097/01.pdm.0000126419.92931.a3.

13. Dhopapkar M, Chin-Yang, Lust L, Tefferi A, Phyliky R. Clinical spectrum of clonal proliferations of T-large granular lymphocytes: a T-cell clonopathy of undetermined significance. Blood. 1994;84(5): 1620-7.

14. Aribi A, Huh Y, Keating M, Brin S, Ferrajoli A, Faderl S, et al. T-cell large granular lymphocytic (T-LGL) leukemia: Experience in a single institution over 8 years. Leuk Res. 2007;31(7):939-45, http://dx.doi.org/ 10.1016/j.leukres.2006.09.003.

15. Lamy T, Loughran TP. How I treat LGL leukemia. Blood. 2011;117:276474, http://dx.doi.org/10.1182/blood-2010-07-296962.

16. Kantarjian HM, Childs C, O’Brien S, Huh Y, Beran M, Schachner J, et al.

Efficacy of fludarabine, a new adenine nucleoside analogue in patients with prolymphocytic leukemia and the prolymphocytoid variant of chronic lymphocytic leukemia. Am J Med. 1991;90(2):223-8.

17. Witzig TE, Weitz JJ, Lundberg JH, Tefferi A. Treatment of refractory Tcell chronic lymphocytic leukemia with purine nucleoside analogues.

Leuk Lymphoma. 1994;14(1-2):137-9, http://dx.doi.org/10.3109/ 10428199409049659.

18. Tse E, Chan JCW, Pang A, Au W-Y, Leung AYH, Lam CCK, et al.

Fludarabine, mitoxantrone and dexamethasone as first-line treatment for T-cell large granular lymphocyte leukemia. Leukemia. 2007;21(10):22256, http://dx.doi.org/10.1038/sj.leu.2404767.

19. Bareau B, Rey J, Hamidou M, Donadieu J, Morcet J, Reman O, et al.

Analysis of French cohort of patients with large granular lymphocyte leukemia: a report on 229 cases. Haematologica 2010;95(9):1534-41.

CMR, along with excellent treatment compliance and drug tolerance. In accordance with the literature, our main indication for treatment was cytopenia and recurrent infection (19).

In our series of cases, we found a high rate of CHR and CMR with fludarabine, along with excellent compliance and tolerability. Even though our number of cases was small, we suggest that fludarabine should be further tested as a firstline treatment option for T-LGL to confirm these preliminary data.

AUTHOR CONTRIBUTIONS Bellesso M and Pereira J were responsible for the data review, manuscript writing and review. Oliveira RC was responsible for the data collection and review, manuscript writing and review. Chamone DAF was responsible for the manuscript review. Ruiz MA was responsible for the manuscript writing. Hallack AE was responsible for the manuscript writing and review.

Aldred VL was responsible for the diagnosis review.

REFERENCES 1. Brouet JC, Sasportes M, Flandrin G, Preud’Home JL, Seligman M. Chronic lymphocytic leukaemia of T-cell origin. Immunological and clinical evaluation in eleven patients. Lancet. 1975(7941);2:890-3.

2. Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, et al.

World Health Organization classification of tumours of Haematopoietic and Lymphoid Tissues. 4th ed. International Agency for Research on Cancer Press, Lyon, 2008.

3. Morice WG, Kurt PJ, Leibson PJ, Teferi A, Hanson CA. Demonstration of aberrant T-cell and natural killer cell antigen expression in all cases of granular lymphocytic leukemia. Br J Haematol. 2003;120(6):1026-36, http://dx.doi.org/10.1046/j.1365-2141.2003.04201.x.

4. Lundell R, Hartung L, Hill S, Perkins SL, Bahler DW. T-cell large granular lymphocyte leukemias have multiple phenotypic abnormalities involving pan-T cell antigens and receptors for MHC molecules.

Am J Clin Pathol. 2005;124(6):937-46, http://dx.doi.org/10.1309/ PH7X78HF4FW4PRKW. 5. Lamy T, Loughran TP. Clinical features of large granular lymphocyte leukemia. Semin Hematol. 2003;40(3):185-95, http://dx.doi.org/10.1016/ S0037-1963(03)00133-1.

6. Loughran TP, Kidd PG, Starkebaum G. Treatment of large granular lymphocyte leukemia with oral low-dose methotrexate. Blood. 1994; 84(7):2164-70.

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DOI:10.6061/clinics/2012(07)08

CLINICAL SCIENCE

Trends and characteristics of pethidine use in Taiwan: a six-year-long survey Hsueh-Hsing Pan,I,II Chung-Yi Li,III,IV Tso-Chou Lin,V Ju-O Wang,VI Shung-Tai Ho,V,VII Kwua-Yun WangVIII,IX I Graduate Institute of Medical Science, National Defense Medical Center, Taipei, Taiwan. II Department of Nursing, Tri-Service General Hospital, Taipei, Taiwan. III Department of Public Health, National Cheng Kung University, Tainan, Taiwan. IV Department of Public Health, China Medical University, Taichung, Taiwan. V Department of Anesthesiology, Tri-Service General Hospital, Taipei, Taiwan. VI Graduate Institute of Life Science, National Defense Medical Center, Taipei, Taiwan. VII Department of Anesthesiology, Taipei Veterans General Hospital, Taipei, Taiwan. VIII Department of Nursing, Taipei Veterans General Hospital, Taipei, Taiwan. IX School of Nursing, National Defense Medical Center, Taipei, Taiwan.

OBJECTIVES: To investigate the trends and characteristics of pethidine prescriptions and users in Taiwan from 2002 to 2007.

METHOD: All pethidine users (n = 3,301,136) in Taiwan from 2002 to 2007 were linked to National Health Insurance claims to identify pethidine prescriptions. We examined the trends in pethidine user prevalence and the proportion of pethidine prescriptions according to health care characteristics. A logistic regression model was used to compare patient demographics and health care characteristics associated with pethidine prescriptions between 2002 and 2007.

RESULTS: Despite the decline in the number of pethidine users and prescriptions over the six-year period, more than half a million people were prescribed pethidine annually. In fact, an increasing proportion of pethidine prescriptions were observed in clinics, outpatient settings, and patients who had both operations and cancer diagnoses. Pethidine prescriptions were mostly associated with a non-operation status without a cancer diagnosis (.60%). However, approximately 10% of the total pethidine prescriptions were found in patients with a cancer diagnosis but no operation. Compared to those in 2002, pethidine prescriptions in 2007 were more likely to be found in people 80 years or older, rural residents, patients from clinics, outpatient settings and operation patients with cancer diagnoses.

CONCLUSIONS: A population-based survey in Taiwan demonstrated decreasing consumption of pethidine from 2002 to 2007; however, an increased proportion of prescriptions in certain health care settings was observed. In addition, 10% of the pethidine prescriptions were for cancer patients without operations. These cases need further evaluation for the determination of appropriate pethidine use.

KEYWORDS: Pethidine; Prescription; Consumption; Defined Daily Dose; Population-Based.

Pan HH, Li CY, Lin TC, Wang JO, Ho ST, Wang KY. Trends and characteristics of pethidine use in Taiwan: a six-year-long survey. Clinics. 2012;67(7):749755.

Received for publication on January 16, 2012; First review completed on February 23, 2012; Accepted for publication on March 18, 2012 E-mail: w6688@mail.ndmctsgh.edu.tw Tel.: 886-2-28757233

mood changes) and even seizures, and interacts with monoamine oxidase inhibitor (MAOI) drugs causing complications such as serotonergic crisis (3-6). These adverse effects become evident if multiple doses are given over a period of time (3-5). In addition, adverse reactions to normeperidine can occur in patients with normal renal function if the administered doses are sufficiently high (4).

In addition, the once-purported theoretical advantages of pethidine over morphine for patients with cholecystitis or pancreatitis have also been challenged (7). Consequently, as awareness of pethidine’s adverse effects has increased, updated pain management protocols have called for its cautious use (3). The Agency for Health Care Policy and Research’s recommendation suggests that oral pethidine should not be used for pain management and strongly recommends that parenteral pethidine be restricted to patients who have a true allergy or intolerance to other

INTRODUCTION Pethidine (meperidine), which has a short duration of action, has been widely and routinely prescribed for moderate-to-severe pain in medical and surgical patients (1,2). Pethidine is a synthetic opioid that was introduced in 1939 for clinical use as an anticholinergic when its analgesic properties were discovered. It has an active metabolite, normeperidine (3), which accumulates in the organism causing stimulation (anxiety, hyperreflexia, myoclonus and

Copyright ß 2012 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.

No potential conflict of interest was reported.

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injections (drug code: A005874209) and tablets (drug code: A005858100), respectively, were written for 3,301,136 patients. From each prescription claim, we retrieved information on the patient’s date of birth, gender, the urbanization level of the patient’s residential area, the medical institution’s accreditation level (clinic, local, regional hospital, or medical center), the setting (hospital, outpatient, or emergency department), and the patient’s operation status and cancer diagnosis status. The age of each patient was calculated as the difference between the date of the prescription and the date of birth. The urbanization level for each of the 365 townships in Taiwan was categorized (i.e., urban, suburban, or rural) according to the National Statistics of Regional Standard Classification (19). Outpatient claims provided one procedure code and up to three diagnostic codes for each ambulatory care visit.

Inpatient claims included up to five codes for procedures and diagnoses separately. All codes are based on the International Classification of Diseases codes, 9th version Clinical Modification (ICD-9-CM). Operation status was determined by the entry of any procedure code of ICD-9CM except for obstetrical procedures [72-75], miscellaneous diagnostic and therapeutic procedures [87-99], and diagnostic procedures (Appendix). Any claims accompanied by primary or secondary diagnoses of malignancy (140-209, 230-239) were classified as a cancer diagnosis.

The amount of pethidine prescriptions was expressed as the defined daily dose (DDD) per 1,000 inhabitants per day (20). The DDD is a technical unit of measurement, established by an expert panel as the assumed average maintenance dose when the drug is used for its main indication by an adult, and it is based on the Anatomical Therapeutic Chemical (ATC) classification index (20). The following formula was used in this analysis: Number of DDD/1,000 inhabitants/day = (number of packages dispensed 6 number of doses per package 6 number of mg per dose) / (DDD in mg 6 number of 1,000 inhabitants in the mid-year population 6 365 days).

The DDD value of pethidine is 400 milligrams (20). The mid-year population for each year of the study period was obtained from the Ministry of the Interior of Taiwan (21).

opioids, in which case it should only be used in acute pain situations for brief courses (8).

Despite the clinical guidelines that have been issued against pethidine use in many nations (9), it continues to be used in many parts of the world, including Taiwan (10-13).

One of the arguments supporting the use of pethidine is that concurrent analgesics given with pethidine can achieve adequate analgesia without causing adverse effects in patients with renal failure (14). Moreover, pethidine was found to produce fewer side effects than morphine when used for short-term periods (15). Therefore, pethidine is still a frequently ordered opioid analgesic, especially for patients who receive shorter courses of narcotics (2,6).

Due to its habit-forming potential, serotonergic crisis risk and normeperidine toxicity, the consumption of pethidine is decreasing worldwide. A population-based study was conducted in Taiwan to describe the trends in the consumption of opioid analgesics from 2002 to 2007, and the report indicated that the consumption of transdermal fentanyl and oral morphine increased markedly over the study period (16).

Although pethidine use decreased gradually, it was predominantly prescribed to patients without cancer diagnoses (16). Taiwan recently launched the ‘‘Physician Guidelines on Clinical Use of Pethidine’’ in September 2011 (17) to limit the clinical use of pethidine; however, there is no populationbased evidence of pethidine prescriptions available for Taiwan and other nations. In addition, pethidine is currently the only available strong opioid in Taiwan for those with intolerable side effects from morphine or fentanyl. We therefore conducted this population-based study using data from National Health Insurance (NHI) claims to investigate the trends and characteristics of pethidine prescriptions and users in Taiwan from 2002 to 2007.

MATERIALS AND METHODS Source of data The data analyzed in this study were retrieved from the National Health Insurance Research Database (NHIRD) provided by the Bureau of National Health Insurance (BNHI) (18). Taiwan reformed its insurance programs into the universal National Health Insurance (NHI) system in 1995, and more than 99% of residents in Taiwan were enrolled in the NHI program in 2007 (18). This NHI research database contains registration files and original claim data for reimbursements and offers information on each patient’s characteristics, diagnosis, treatments, prescriptions, and associated ambulatory and inpatient medical benefit claims.

The database also protects the privacy and confidentiality of all beneficiaries and only transfers health insurance data to health researchers after ethical approval has been obtained.

For the current analysis, we used all pethidine prescriptions issued in outpatient and inpatient claims from 2002 to 2007. All NHI datasets can be interlinked with each individual’s personal identification number (PIN). The research protocol for this analysis was ethically approved by the Review Committee of the National Health Research Institutes.

Data analysis We first described the secular trends for the annual numbers of pethidine users, total prescriptions, total amounts, and DDD/1,000 inhabitants/day according to the forms of pethidine (i.e., injections or tablets). We then calculated the prevalence rate of pethidine users for each year. We also calculated the proportion of total pethidine prescriptions each year according to selected health care characteristics. To further compare patient demographics and health care characteristics for all pethidine prescriptions between 2002 and 2007, we performed simple and multiple logistic regression models, with a generalized estimation equation accounting for clustering of prescriptions administered to the same patient. The odds ratio (OR) of receiving a pethidine prescription in 2007 in relation to the selected patient demographics and health care characteristics was estimated. In addition, we calculated the distribution of the proportion of pethidine prescriptions across primary diagnoses based on ICD-9-CM diagnostic codes in non-operation patients over the six-year period.

All statistical analyses were performed with SAS (version

Study subjects and measurements This is a descriptive study that depicts secular trends and characteristics of pethidine prescriptions and users in Taiwan from 2002 to 2007. During the study period, a total of 3,380,884 prescriptions, of which 99.1% and 0.9% were

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Table 1 - Numbers of pethidine users, total prescriptions, total amounts, and defined daily dose, 2002-2007.

Percent change 2002 vs. 2007

Calendar year

Injections No. of users Total prescriptions Total amount (mg6106) DDD/1,000/day

2002

2003

2004

2005

2006

2007

553,364 568,389 79

527,054 543,070 77

560,044 571,415 77

550,979 563,129 76

546,515 559,779 74

533,776 545,581 69

-3.5 -4.0 -12.7

0.024

0.023

0.023

0.023

0.022

0.021

-12.5

6,283 6,317 8

6,106 6,141 9

6,461 6,479 7

4,772 4,781 3

2,870 2,883 3

2,912 2,920 4

-53.7 -53.8 -50.0

0.003

0.003

0.002

0.001

0.001

0.001

-66.7

559,647 574,706 87

533,160 549,211 86

566,505 577,894 84

555,751 567,910 79

549,385 562,662 77

536,688 548,501 73

-4.1 -4.6 -16.1

0.027

0.026

0.025

0.024

0.023

0.022

-18.5

Tablets No. of users Total prescriptions Total amount (mg6106) DDD/1,000/day Total No. of users Total prescriptions Total amount (mg6106) DDD/1,000/day

DDD/1,000/day = defined daily dose (DDD) per 1,000 inhabitants per day.

9.2; SAS Institute, Cary, NC). P values ,0.05 were considered statistically significant.

those over 80 years old. Males had a higher prevalence than females. Additionally, there was an apparent geographic variation in the prevalence of pethidine use; people living in rural areas had a higher prevalence rate of pethidine use than in urban areas. We also noted that the subgroups with higher prevalence rates of pethidine users tended to have a smaller percentage of reduction in the prevalence over the study period. People over 80 years old, males and those from rural areas all experienced small reductions in prevalence at 2.9%, 2.1% and 1.3%, respectively. However, the largest reduction in the prevalence of pethidine users was among people under 40 years old (15.0%-16.0%); the corresponding figures for females and urban residents were 8.0% and 9.6%, respectively.

Table 3 illustrates the distributions of pethidine prescriptions across various health care characteristics. Medical centers accounted for the largest proportion of prescriptions; however, these prescriptions decreased by 9.6% from

RESULTS Pethidine was prescribed to more than 500,000 people annually for pain relief from 2002 to 2007 (Table 1); the numbers of pethidine users and total prescriptions decreased slightly, by 4.1% and 4.6%, respectively. The DDD/1,000 inhabitants/day decreased from 0.027 to 0.022, representing a reduction of 18.5%; this decreasing trend was more obvious for tablets than for injections.

Table 2 shows the prevalence rate of pethidine users during the study period. The overall prevalence rate of pethidine users was 24.9 per 1,000 people in 2002; it increased slightly in 2004, and then declined gradually in 2007, representing a 6.4% decrease over the study period.

However, pethidine use increased with age, especially in

Table 2 - Prevalence rate of pethidine users, 2002-2007.

Variables Age ,18 18-40 41-64 65-79 $80 Gender Male Female Urbanization Urban Satellite Rural Total

2002 (n = 559,647)

2003 (n = 533,160)

2004 (n = 566,505)

2005 (n = 555,751)

2006 (n = 549,385)

2007 (n = 536,688)

Percent change 2002 vs. 2007

5.6 22.7 33.3 58.5 66.3

4.9 22.1 31.3 54.9 64.4

5.3 23.3 33.4 58.9 68.3

5.2 21.3 32.0 56.4 65.9

5.0 20.3 31.3 55.7 66.4

4.7 19.3 30.0 53.8 64.4

-16.0 -15.0 -9.9 -8.1 -2.9

26.2 22.9

26.0 21.0

28.0 23.2

26.9 22.2

26.5 21.7

25.6 21.1

-2.1 -8.0

22.7 24.2 30.0 24.9

21.5 22.6 29.0 23.9

23.4 24.2 31.3 25.8

21.8 23.0 30.6 24.5

21.4 22.7 30.1 24.1

20.5 21.8 29.7 23.3

-9.6 -9.9 -1.3 -6.4

Incidence density: Per 1,000 people.

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Table 3 - Proportion of total pethidine prescriptions according to health care characteristics, 2002-2007.

Variables Accreditation level Medical center Regional hospital Local hospital Clinic Patient setting Inpatient Outpatient Emergency department Operation/Cancer status Operation With cancer diagnosis Without cancer diagnosis Non-operation With cancer diagnosis Without cancer diagnosis

2002 (n = 574,706)

2003 (n = 549,211)

2004 (n = 577,894)

2005 (n = 567,910)

2006 (n = 562,662)

2007 (n = 548,501)

Percent change 2002 vs. 2007

47.4 36.0 15.7 1.0

45.6 36.6 16.7 1.1

45.3 36.8 16.3 1.6

43.9 38.4 15.9 1.8

45.0 38.5 15.2 1.4

42.8 41.4 14.7 1.2

-9.7 15.1 -6.6 19.7

73.6 10.9 15.5

71.8 11.8 16.4

73.2 12.6 14.2

71.7 13.5 14.8

71.0 13.1 15.9

70.6 13.1 16.3

-4.1 20.1 5.1

4.4 22.0

4.7 21.6

5.0 22.6

5.2 23.0

5.4 22.7

5.4 22.4

22.7 2.2

10.2 63.4

10.5 63.3

10.2 62.3

9.8 62.0

10.0 61.9

9.7 62.5

-5.4 -1.5

bronchus and lung, malignant neoplasm of the rectum, recto sigmoid junction, and anus, and malignant neoplasm of the female breast (Table 5).

2002 to 2007. Although the proportion of pethidine prescriptions for clinics was small (,2%), it showed a substantial increase (19.7%). More than 70% of pethidine prescriptions were given in inpatient settings; and 61.9% to 63.4% of pethidine was prescribed to non-operation patients without cancer diagnoses. Despite that, the proportion of prescriptions for inpatient settings and for those nonoperation patients without cancer diagnoses decreased by 4.1% and 1.5%, respectively. However, pethidine prescriptions from outpatient settings that were administrated to operation patients with cancer diagnoses demonstrated notable increases in proportion, at 20.1% and 22.7%, respectively.

The patient demographics and health care characteristics for pethidine prescriptions between 2002 and 2007 are shown in Table 4. Pethidine prescriptions tended to be administered more to older people in 2007. Compared to those prescribed in 2002, the pethidine prescribed in 2007 was 1.27 times and 1.77 times more likely to be administered to patients aged 65 to 79 years and greater than 80 years, respectively. Additionally, pethidine was more likely to be prescribed to patients living in rural areas (OR = 1.09, 95% confidence interval (CI) = 1.08-1.10) in 2007. With respect to the health care characteristics, compared to the 2002 prescriptions, the prescriptions in 2007 were more likely to be obtained in clinics (OR = 1.48, 95% CI = 1.43-1.54) and outpatient settings (OR = 1.27, 95% CI = 1.26-1.29), as well as in operation patients with a cancer diagnosis (OR = 1.24, 95% CI = 1.22-1.27).

Because non-operation patients without cancer diagnoses accounted for more than 60% of the total prescriptions, we further examined the leading diagnosis associated with these prescriptions. We noted that the top five leading diagnoses of disease included kidney and ureter calculus and other symptoms involving the abdomen and pelvis, such as abdominal pain, pancreatic diseases, hemorrhoids, and acute appendicitis; these causes accounted for 23.48% of all prescriptions (Table 5). In addition, we also noted that approximately 10% of pethidine prescriptions were administered to non-operation cancer patients, which is usually considered to be inappropriate. The top five cancer diagnoses with non-operation status included malignant neoplasm of the liver and intrahepatic bile ducts, malignant neoplasm of the colon, malignant neoplasm of the trachea,

DISCUSSION This is the first national study to analyze the trends and characteristics of pethidine use in Taiwan. Although there were declining trends in the prevalence of pethidine users, total prescriptions and DDD/1,000 inhabitants/day in Taiwan over the study years, there were still more than 500,000 people who received pethidine prescriptions for pain each year, representing 2.4% of the total population of Taiwan. Additionally, older people, males and people living in rural areas showed a smaller reduction in the prevalence rate of pethidine users than the national average (6.4%).

Moreover, there was an increasing proportion of pethidine prescriptions from clinics, outpatient settings and operation patients with cancer diagnoses. We noted that pethidine prescriptions were mostly found in non-cancer patients (85%). However, 10% of the total pethidine prescriptions were found in non-operation patients with a cancer diagnosis, and these prescriptions should be evaluated for their appropriateness.

The decreasing trend in pethidine use noted in Taiwan is consistent with previous reports from other nations (11,12,22-24). Joranson et al. (23) retrospectively surveyed the U.S. medical record database, which revealed a 35% decrease (5.2 to 3.4 million grams) in the medical use of pethidine from 1990 to 1996. Gilson et al. (22) also used the same database to demonstrate that there was a further decline in medical use of pethidine by over 6% (5.8 to 5.4 million grams) from 1997 through 2002. Additionally, in Israel from 2000 to 2008, pethidine consumption fell by 65%, from 0.07 to 0.02 DDD/1,000 inhabitants/day (12). However, two population-based surveys found that pethidine consumption was less than 0.01 DDD/1,000 inhabitants/day and remained constant in both Spain between 1992 and 2006 (11) and in the Slovak Republic between 1998 and 2002 (24). The fall in pethidine consumption in recent years may reflect the campaign by major authorities towards a decrease in regular and long-term use of pethidine. This decrease may also signify the growing awareness of pethidine’s shortcomings, including its short duration of action and conversion to a long-lasting toxic metabolite (4). Moreover, multifaceted

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Table 4 - Comparisons of the demographics and health care characteristics of patients who received pethidine prescriptions between 2002 and 2007.

Pethidine prescriptions

Variables

in 2002 (Controls)

Demographics Age (years) ,18 18-40 41-64 65-79 $80 Gender Male Female Urbanization Urban Satellite Rural Health care characteristics Accreditation level Medical center Regional hospital Local Clinic Patient setting Inpatient Outpatient Emergency department Operation/Cancer status Operation With cancer diagnosis Without cancer diagnosis Non-operation With cancer diagnosis Without cancer diagnosis Total

Crude estimate

Adjusted estimate

in 2007 (Cases)

n

n

OR

95% CI

OR

95% CI

30,849 209,079 206,909 102,193 25,676

23,685 169,413 217,571 102,205 35,627

reference 1.06 1.37 1.30 1.81

1.04-1.08 1.35-1.39 1.28-1.33 1.77-1.85

reference 1.04 1.32 1.27 1.77

1.02-1.06 1.29-1.34 1.24-1.29 1.73-1.81

307,001 259,357

299,759 248,741

1.02 reference

228,234 161,113 185,359

211,054 151,828 185,619

reference 1.02 1.08

1.01-1.03 1.07-1.09

reference 1.02 1.09

1.01-1.03 1.08-1.10

272,180 206,631 90,224 5,671

234,568 227,008 80,448 6,477

reference 1.28 1.03 1.33

1.26-1.29 1.02-1.05 1.28-1.37

reference 1.28 1.05 1.48

1.27-1.29 1.03-1.06 1.43-1.54

422,710 62,727 89,269

387,078 71,907 89,516

reference 1.25 1.10

1.24-1.27 1.08-1.11

reference 1.27 1.12

1.26-1.29 1.11-1.13

25,322 126,202

29,647 123,102

1.25 1.04

1.22-1.27 1.03-1.05

1.24 1.08

1.22-1.27 1.07-1.09

58,706 364,476 574,706

53,020 342,732 548,501

0.96 reference

0.95-0.97

0.90 reference

0.89-0.91

education, combining a pethidine formulary restriction and a computerized provider order entry regulation initiative, has also minimized pethidine prescriptions (25-27). Furthermore, restricting the use of pethidine to patients who are allergic to or unable to tolerate all other opioids has been advocated in Taiwan as well as in many other countries worldwide (3,12).

1.01-1.03

1.00 reference

0.99-1.01

Despite a decrease in the prevalence of pethidine use, our study reveals an increased number and proportion of pethidine users aged 80 years and older. Similar increases in number and proportion were also noted in pethidine prescriptions in operation patients with cancer diagnoses, as well as in those from rural areas, regional hospitals, clinics,

Table 5 - Leading diagnoses associated with pethidine prescriptions for non-operation patients with and without cancer diagnoses, 2002-2007.

Rank

ICD-9-CM

Prescription for nonoperation without cancer diagnosis (n = 2,115,366)

ICD-9-CM

% 1

592-Calculus of kidney and ureter

8.99

2

4.26

3

789-Other symptoms involving abdomen and pelvis 577-Diseases of pancreas

4

455-Hemorrhoids

3.28

5 Total

540-Acute appendicitis

3.07 23.48

Prescription for nonoperation with cancer diagnosis (n = 339,816) %

3.88

155-Malignant neoplasm of liver and intrahepatic bile ducts 153-Malignant neoplasm of colon

16.37

162-Malignant neoplasm of trachea, bronchus and lung 154-Malignant neoplasm of rectum, rectosigmoid junction, and anus 174- Malignant neoplasm of female breast

5.18

ICD-9-CM: International Classification of Diseases codes, 9th version Clinical Modification.

753

5.97

4.70 4.18 36.40

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CLINICS 2012;67(7):749-755

alive two years after their first opioid prescription increased from 38% to 55%. The results also indicate that 14% of the population’s opioid users were cancer patients and that they consumed 23% of the total opioids from 1993 to 1997.

Our findings demonstrate that a large proportion of pethidine was prescribed to non-operation patients without cancer diagnoses. The indications for pethidine use documented by emergency departments included abdominal pain, back pain, biliary pain, chest pain, migraine/headache, pancreatitis, renal colic, trauma/fracture, and morphine allergy (26), which were all non-cancer diagnoses.

Nevertheless, our data showed that 10% of the total pethidine prescriptions were found in cancer diagnoses without operations. Therefore, there was a potential for improper use of pethidine in non-operation cancer patients in Taiwan, which affirms the need for further evaluation of the appropriateness of pethidine prescriptions in non-operation cancer patients to avoid adverse events from such use.

This study has the following strengths. First, it is population based and includes all eligible pethidine users in Taiwan during the study period. Therefore, the data are highly representative and less likely to have selection bias.

Second, the study sample was collected from the NHI database, which makes it possible to cover all pethidine claims of individual patients with little likelihood of nonresponse from the study subjects. Finally, the advantage of using insurance claim datasets in clinical research is the ease of access to longitudinal records of pethidine use in a large sample of geographically dispersed patients (35).

Despite the above strengths, there are several limitations in our study. First, the data contain no information on compliance with prescription drugs in outpatient settings, which could entail a degree of pethidine use misclassification in this study. However, there is no evidence showing that medication compliance varies with secular years, which provides reassurance that the potential pethidine use misclassification should not have a great effect on the secular trends reported in this study. Second, although we used the ICD-9-CM procedure codes to determine whether a pethidine user had an operation, we were unable to differentiate between acute and chronic pain associated with the operation.

In conclusion, the prevalence of pethidine users and prescriptions steadily decreased from 2002 to 2007 and so did the DDD/1,000 inhabitants/day. Nevertheless, our data show that the number and proportion of pethidine prescriptions increased in certain subgroups, including elderly patients, rural residents, patients with cancer diagnoses who had operations and patients from regional hospitals/clinics and outpatient/emergency settings. We suggest further investigation of the appropriateness of prescribing pethidine to these patients.

outpatient settings and emergency departments. Elderly patients usually have higher risks of suffering from both acute and chronic pain (28). Valano et al. (29) conducted a cross-sectional study and reported that the majority of patients treated with opioids were aged 41 to 65 years, which is similar to our findings. A population-based study indicated that patients aged 80 years and older who received care in clinics tended to be prescribed potentially inappropriate medications (30). Previous studies also reported that pethidine was administered to only approximately one in eigth older surgical patients (10), mainly due to the greater likelihood that the elderly would experience side effects from pethidine, which may have affected physician prescribing behavior (31). The increased number and proportion of elderly pethidine users in recent years noted in our study warrants further intervention to effectively reduce the use of pethidine in this vulnerable population.

The geographic and medical institutional variations in pethidine use might be due to varying barriers of access to healthcare. Sadowski et al. (32) conducted a cross-sectional population-based study to describe accessibility and intensity of analgesic use among older Manitobans by region. The age- and sex-standardized accessibility and intensity measures revealed that the highest overall analgesic use occurred in the most rural/remote regions of the province.

Valano et al. (29) used a cross-sectional study design to demonstrate that adherence to analgesic treatment guidelines was higher in large hospitals than in medium- and small-sized hospitals. A recent study also reported that pethidine was prescribed to 7.6% and 4.3% of all study patients at a large private tertiary care teaching hospital and a smaller academically affiliated Veteran’s Affairs medical center, respectively, but the number of doses administered was similar at both sites (10). The variation in the number of pethidine doses prescribed to patients treated in hospitals with different accreditation levels may reflect the different severity of diseases that required different strategies to manage pain. Alternatively, ordering pethidine was also found to be associated with physician specialty, hospital location, patient race, insurance coverage, and physician gender (2), which might also account for the observed variations in pethidine use among geographic and medical institutions in Taiwan.

We were not surprised by the increased proportion of pethidine prescriptions in patients with cancer diagnoses together with operations, which is also consistent with previous findings (29). Previous studies reported that after surgery, patients with cancer were more frequently treated with analgesics than other patients (29). Furthermore, a cancer diagnosis was found to be associated with greater access to and use intensity of all analgesics classes (32).

Physicians do not appear hesitant to prescribe higher amounts of opioid analgesics to individuals with cancer diagnoses (33). Jarlbaek et al. (34) analyzed the changes in opioid use from 1994 to 1998 in an entire cohort of cancer patients (n = 24,190) in a Danish county. The results indicated that the overall consumption of opioids increased from 20 kg to 37 kg oral morphine equivalents (omeq) per year, the average consumption increased from 7.6 to 10.7 g omeq/ opioid user/year, the annual proportion of users increased from 17% to 20%, and the proportion of patients who were

ACKNOWLEDGMENTS This study was supported by grants from the National Bureau of Controlled Drugs, the Department of Health, Taiwan (DOH98-NNB1031), and the National Science Council (NSC100-2314-B-006 -052).

AUTHOR CONTRIBUTIONS Pan HH, Li CY, Wang JO and Lin TC conceived and designed the study and were responsible for the data acquisition, analysis and interpretation.

Ho ST and Wang KY contributed equally to this work and were

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19. Directorate-General Budget, Accounting and Statistics. National Statistics of Regional Standard Classification Data. Taipei: Accounting and Statistics; 1993.

20. WHO. WHO Collaborating Centre for Drug Statistics Methodology.

Complete ATC index 2010. Available at: http://www.whocc.no/ atcddd/.

21. Department of Statistics. Statistical yearbook of interior. 2009. Available at: http://sowf.moi.gov.tw/stat/year/list.htm.

22. Gilson AM, Ryan KM, Joranson DE, Dahl JL. A reassessment of trends in the medical use and abuse of opioid analgesics and implications for diversion control: 1997-2002. J Pain Symptom Manage. 2004;28(2):176-88, http://dx.doi.org/10.1016/j.jpainsymman.2004.01.003.

23. Joranson DE, Ryan KM, Gilson AM, Dahl JL. Trends in medical use and abuse of opioid analgesics. JAMA. 2000;283(13):1710-4, http:// dx.doi.org/10.1001/jama.283.13.1710.

24. Hudec R, Tisonova J, Bozekova L, Foltan V. Trends in consumption of opioid analgesics in Slovak Republic during 1998-2002. Eur J Clin Pharmacol. 2004;60(6):445-8, http://dx.doi.org/10.1007/s00228-0040793-5.

25. Taylor SE, Braitberg G, Lugt J. Multifaceted education initiative minimizes pethidine prescribing in the emergency department. Emerg Med Australas. 2007;19(1):25-30, http://dx.doi.org/10.1111/j.17426723.2006.00911.x.

26. Kaye KI, Welch SA, Graudins LV, Graudins A, Rotem T, Davis SR, et al.

Pethidine in emergency departments: promoting evidence-based prescribing. Med J Aust. 2005;183(3):129-33.

27. O’Connor AB, Lang VJ, Quill TE. Eliminating analgesic meperidine use with a supported formulary restriction. Am J Med. 2005;118(8):885-9, http://dx.doi.org/10.1016/j.amjmed.2005.01.061.

28. Scudds RJ, Ostbye T. Pain and pain-related interference with function in older Canadians: the Canadian Study of Health and Aging. Disabil Rehabil. 2001;23(15):654-64, http://dx.doi.org/10.1080/0963828011 0043942.

29. Vallano A, Malouf J, Payrulet P, Banos JE. Analgesic use and pain in the hospital settings. Eur J Clin Pharmacol. 2007;63(6):619-26, http://dx. doi.org/10.1007/s00228-007-0303-7.

30. Lai HY, Hwang SJ, Chen YC, Chen TJ, Lin MH, Chen LK. Prevalence of the prescribing of potentially inappropriate medications at ambulatory care visits by elderly patients covered by the Taiwanese National Health Insurance program. Clin Ther. 2009;31(8):1859-70, http://dx.doi.org/ 10.1016/j.clinthera.2009.08.023.

31. Freye E, Levy JV. Use of opioids in the elderly – pharmacokinetic and pharmacodynamic considerations. Anasthesiol Intensivmed Notfallmed Schmerzther. 2004;39(9):527-37, http://dx.doi.org/10.1055/s-2004825883.

32. Sadowski CA, Carrie AG, Grymonpre RE, Metge CJ, St John P. Access and intensity of use of prescription analgesics among older Manitobans.

Can J Clin Pharmacol. 2009;16(2):e322-30.

33. Morley-Forster PK, Clark AJ, Speechley M, Moulin DE. Attitudes toward opioid use for chronic pain: a Canadian physician survey. Pain Res Manag. 2003;8(4):189-94.

34. Jarlbaek L, Andersen M, Hallas J, Engholm G, Kragstrup J. Use of opioids in a Danish population-based cohort of cancer patients. J Pain Symptom Manage. 2005;29(4):336-43, http://dx.doi.org/10.1016/ j.jpainsymman.2004.07.010.

35. Jollis JG, Ancukiewicz M, DeLong ER, Pryor DB, Muhlbaier LH, Mark DB. Discordance of databases designed for claims payment versus clinical information systems. Implications for outcomes research. Ann Intern Med. 1993;119(8):844-50.

responsible for drafting the article and revising it critically for important intellectual content. All authors approved the final version of the manuscript.

REFERENCES 1. Costantini R, Affaitati G, Fabrizio A, Giamberardino MA. Controlling pain in the post-operative setting. Int J Clin Pharmacol Ther.

2011;49(2):116-27.

2. Panda M, Desbiens N, Doshi N, Sheldon S. Determinants of prescribing meperidine compared to morphine in hospitalized patients. Pain. 2004; 110(1-2):337-42, http://dx.doi.org/10.1016/j.pain.2004.04.016.

3. Latta KS, Ginsberg B, Barkin RL. Meperidine: a critical review. Am J Ther.

2002;9(1):53-68, http://dx.doi.org/10.1097/00045391-200201000-00010.

4. Todd M. Meperidine and the management of pain: what you need to know. Lippincotts Case Manag. 2004;9(5):241-2.

5. Vermeulen LC, Bollinger KA, Antonopoulos J, Meek PD, Goshman LM, Ploetz PA. Multifaceted approach to medication use policy development: the restriction of meperidine. Pharm Pract Manag Q. 1997;16(4):66-75.

6. Kredo T, Onia R. Pethidine–does familiarity or evidence perpetuate its use? S Afr Med J. 2005;95(2):100-1.

7. Thompson DR. Narcotic analgesic effects on the sphincter of Oddi: a review of the data and therapeutic implications in treating pancreatitis.

Am J Gastroenterol. 2001;96(4):1266-72, http://dx.doi.org/10.1111/j.

1572-0241.2001.03536.x.

8. American Pain Society Quality of Care Committee. Quality improvement guidelines for the treatment of acute pain and cancer pain. American Pain Society Quality of Care Committee. JAMA. 1995;274(22):1874-80.

9. O’Callaghan JP. Evolution of a rational use of opioids in chronic pain.

Eur J Pain. 2001;5(Suppl A):21-6, http://dx.doi.org/10.1053/eujp.2001.0275.

10. Kornitzer BS, Manace LC, Fischberg DJ, Leipzig RM. Prevalence of meperidine use in older surgical patients. Arch Surg. 2006;141(1):76-81, http://dx.doi.org/10.1001/archsurg.141.1.76.

11. Garcia del Pozo J, Carvajal A, Viloria JM, Velasco A, Garcia del Pozo V. Trends in the consumption of opioid analgesics in Spain. Higher increases as fentanyl replaces morphine. Eur J Clin Pharmacol. 2008;64(4):411-5.

12. Ponizovsky AM, Marom E, Zeldin A, Cherny NI. Trends in opioid analgesics consumption, Israel, 2000-2008. Eur J Clin Pharmacol. 2011; 67(2):165-8, http://dx.doi.org/10.1007/s00228-010-0932-0.

13. Hsu CC, Li JH. The trends of requirements in medical opioid analgesics from 1987 through 1996 in Taiwan. Chinese Journal of Public Health.

1998;17:495-503.

14. Pellegrini JE, Paice J, Faut-Callahan M. Meperidine utilization and compliance with Agency for Health Care Policy and Research guidelines in a tertiary care hospital. CRNA. 1999;10(4):174-80.

15. Cepeda MS, Farrar JT, Baumgarten M, Boston R, Carr DB, Strom BL. Side effects of opioids during short-term administration: effect of age, gender, and race. Clin Pharmacol Ther. 2003;74(2):102-12, http://dx.doi.org/ 10.1016/S0009-9236(03)00152-8.

16. Pan HH, Ho ST, Lu CC, Wang JO, Lin TC, Wang KY. Trends in the consumption of opioid analgesics in Taiwan from 2002 to 2007: a population-based study. J Pain Symptom Manage. 2012. (In press) 17. Food and Drug Administration, Department of Health, Taiwan.

Physician Guidelines on Clinical Use of Pethidine. 2011. Available at: http://www.fda.gov.tw/people_laws_list.aspx?classifysn = 183.

18. Lu JFR, Hsiao WC. Does universal health insurance make health care unaffordable? Lessons from Taiwan. Health Affairs (Millwood).

2003;22(3):77-88, http://dx.doi.org/10.1377/hlthaff.22.3.77.

Appendix- ICD-9-CM codes of diagnosis procedures classifications.

Diagnosis procedures classifications

ICD-9-CM codes

Nervous system Endocrine system Eye Ear Nose, mouth, and pharynx Respiratory system Cardiovascular system Hemic and lymphatic system Digestive system Urinary system Male genital organs Female genital organs Musculoskeletal system Integumentary system

01.1, 06.1, 08.1, 18.1, 21.2, 31.4, 37.2, 40.1, 42.2, 55.2, 60.1, 65.1, 76.1, 85.1,

03.3, 07.1 09.1, 20.3 22.1, 33.2, 38.2 41.3 44.1, 56.3, 61.1, 66.1, 78.8, 86.1

ICD-9-CM: International Classification of Diseases codes, 9th version Clinical Modification.

755

05.1 10.2, 11.2, 12.2, 14.1,15.0, 16.2 24.1, 25.0, 26.1, 27.2, 28.1, 29.1 34.2

45.1, 45.2, 48.2, 49.2, 50.1, 51.1, 52.1, 54.2 57.3, 58.2, 59.2 62.1, 63.0, 64.1 67.1,68.1,70.2,71.1 80.2, 80.3, 83.2

CLINICS 2012;67(7):757-760

DOI:10.6061/clinics/2012(07)09

CLINICAL SCIENCE

Postural control in women with breast hypertrophy Alessandra Ferreira Barbosa,I Gabriela Cristina Raggi,I Cristina dos Santos Cardoso Sa´,III Ma´rcio Paulino Costa,II Jonas Eraldo de Lima Jr.,II Clarice TanakaI I Faculdade de Medicina da Universidade de Sa˜o Paulo, Physical Therapy, Speech and Occupational Therapy Department, Servic¸o de Fisioterapia, Sa˜o Paulo/SP, Brazil. II Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Plastic Surgery Department, Sa˜o Paulo/SP, Brazil.

III Universidade Federal de Sa˜o Paulo, Departamento de Cieˆncias do Movimento Humano, Sa˜o Paulo/SP, Brazil.

OBJECTIVES: The consequences of breast hypertrophy have been described based on the alteration of body mass distribution, leading to an impact on psychological and physical aspects. The principles of motor control suggest that breast hypertrophy can lead to sensorimotor alterations and the impairment of body balance due to postural misalignment. The aim of this study is to evaluate the postural control of women with breast hypertrophy under different sensory information conditions.

METHOD: This cross-sectional study included 14 women with breast hypertrophy and 14 without breast hypertrophy, and the mean ages of the groups were 39¡15 years and 39¡16 years, respectively. A force platform was used to assess the sensory systems that contribute to postural control: somatosensory, visual and vestibular. Four postural conditions were sequentially tested: eyes open and fixed platform, eyes closed and fixed platform, eyes open and mobile platform, and eyes closed and mobile platform. The data were processed, and variables related to the center of pressure were analyzed for each condition. The Kruskal-Wallis test was used to compare the conditions between the groups for the area of center of pressure displacement and the velocity of center of pressure displacement in the anterior-posterior and medial-lateral directions. The alpha level error was set at 0.05.

RESULTS: Women with breast hypertrophy presented an area that was significantly higher for three out of four conditions and a higher velocity of center of pressure displacement in the anterior-posterior direction under two conditions: eyes open and mobile platform and eyes closed and mobile platform.

CONCLUSIONS: Women with breast hypertrophy have altered postural control, which was demonstrated by the higher area and velocity of center of pressure displacement.

KEYWORDS: Hypertrophy; Breast; Postural Balance.

Barbosa AF, Raggi GC, Sa´ CS, Costa MP, Lima Jr JE, Tanaka C. Postural control in women with breast hypertrophy. Clinics. 2012;67(7):757-760.

Received for publication on January 20, 2012; First review completed on February 17, 2012; Accepted for publication on March 18, 2012 E-mail: cltanaka@usp.br / secretaria.fisioic@hcnet.usp.br Tel.: 55 11 2661-7969

the cervical extensor muscles, resulting in frequent neck pain (6). Back, shoulder and breast pain (7,8), in addition to protraction of the scapula, ptotic breast, paresthesia of the ulnar nerve, and maceration in the infra-mammary groove due to the excessive weight of the breast and in the shoulder secondary to bra strap pressure are frequent complaints (9,10). The effects on quality of life can be evaluated by reports of limited choice of clothing, discomfort during sleep, embarrassment and difficulty performing physical exercises given the size of the breasts (7,11).

Currently, changes caused by breast hypertrophy have been described based on the alteration of body mass distribution, leading to an impact on psychological and physical aspects. However, we hypothesize that sensorimotor inputs are also impaired due to alterations in postural alignment and that postural and/or motor control are ultimately compromised.

Postural alignment itself does not answer the needs of daily life activities because the relationship between body segments is only a quick picture of the body (12). The body responds constantly with postural adjustments to both internal (physiological) and external (environmental) demands when

INTRODUCTION Breast hypertrophy is defined as an increase in glandular tissue exceeding physiological limits in the absence of pregnancy and excluding causes such as accidents, tumors, hemorrhage and inflammatory processes. Beyond the breast enlargement itself, breast hypertrophy can be associated with serious psychological and physical changes (1,2). The most common psychological changes are dissatisfaction with body image, low self-esteem, loss of sexual relations (3,4) and other factors that can considerably inhibit social activities (3), leading to varied degrees of depression (4).

With respect to the physical symptoms, the weight disparity between the upper and lower trunk enhances the physiological curvature of the spine (5) and increases the tension in

Copyright ß 2012 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.

No potential conflict of interest was reported.

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CLINICS 2012;67(7):757-760

another with a greater demand (17). In EO/FP, all of the sensory afferents are available, and it is thus the least challenging task, whereas in EC/MP, only one of three sensory inputs remains completely available, so the central nervous system has to reweight sensory integration to control posture, making it the most challenging of the test conditions.

an individual performs a simple task (13). Motor and sensory changes may interfere with other body functions. For example, the shoulder stabilizer muscles of subjects with scapular protraction may be excessively recruited in a standing position, impairing the ability to reach for or catch an object with the hands (14). Changes in alignment and trunk mobility may be related to modifications in rib cage mechanics and possibly interfere with respiratory function (8,15). These principles of motor control suggest that breast hypertrophy can lead to sensorimotor alterations and body balance impairment due to postural misalignment. We conducted this study to evaluate postural control under different sensory information conditions in women with breast hypertrophy.

Data Processing Data were processed using Matlab 6.5 software (The Mathworks, USA). Variables related to center of pressure (CoP) displacement, including the area and velocity in the anterior-posterior (MVy) and medial-lateral (MVx) directions, were analyzed for each condition.

MATERIALS AND METHODS

STATISTICAL ANALYSIS

Participants

Statistical analyses were conducted using Minitab 15.1 software (Minitab, State College, PA, USA). Student’s t test was used to compare the demographic data. The mean values of the three trials for area, MVy and MVx for each condition were compared between groups using the Kruskal-Wallis test. A Pearson product-moment correlation coefficient was applied to verify the association between variables and age when a difference was found between groups. The alpha level error was set at 0.05 for all analyses.

A cross-sectional study was conducted with 14 women with breast hypertrophy (BHG) and 14 women without breast hypertrophy for control group (CG) (Table 1).

The Sacchini index was used to classify the participants by breast size as normal or hypertrophic. The Sacchini index uses the average of the distances x (the distance between the lateral border of the sternum and mammary papilla) and y (the distance between the inframammary fold and mammary papilla). In this classification, normal-sized breasts measure between 9 and 11 cm, and hypertrophic breasts are larger than 11 cm (16).

Women in the BH group (BHG) were recruited from the Department of Plastic Surgery and the Department of Gynecology of the Clinics Hospital, School of Medicine, University of Sa˜o Paulo. The CG contained women from the local community. The exclusion criteria for both groups included rheumatic, neurological, respiratory and vestibular conditions due to possible balance alterations. None of the participants were engaged in regular physical activity. The Ethics Committee of the Clinics Hospital in Sa˜o Paulo, Brazil, approved (0102/10) the study, and all of the participants signed the informed consent form.

RESULTS Table 1 presents the subjects’ age and BMI and a comparison of the values between groups. Note that there were no differences between groups, although four women in the CG and two in the BHG were considered obese.

An analysis of postural control showed that the area is significantly higher in women with breast hypertrophy under EO/FP, EO/MP and EC/MP conditions. Furthermore, the MVy in the EO/MP and EC/MP conditions was also higher in the BHG. A significant Pearson’s correlation between age and these variables/conditions was only found for area in the BHG in the EO/FP condition. Therefore, the median age was used as a reference to divide the subjects into two age subgroups: (1) lower and equal to the median age and (2) higher than the median age. An analysis of variance with two factors (group and age) showed an effect in the group factor (p = 0.009), but age did not differ (p = 0.274), and no interaction was identified between group and age (p = 0.420). The median values and the ranges for area, MVy and MVx for each condition in each group and the p-values for the intergroup comparisons are shown in Table 2.

Procedure Evaluation of postural control. The participants were requested to stand still, barefoot, on the platform PRO Balance Master (NeuroComH Inc., Oregon, USA) with their arms alongside the body and the feet placed according to the standard indicated on the support base, remaining as relaxed and stable as possible and breathing normally.

Data were collected in three trials of 20 seconds each under four postural conditions: i) eyes open and fixed platform (EO/FP, integrity of the somatosensory, visual and vestibular information); ii) eyes closed and fixed platform (EC/FP, absence of visual information and integrity of the somatosensory and vestibular information); iii) eyes open and mobile platform (EO/MP, disturbance of somatosensory information and integrity of the visual and vestibular information); and iv) eyes closed and mobile platform (EC/MP, disturbance of somatosensory information, absence of visual and vestibular information integrity).

The postural conditions were always tested in the above described sequence, with a progressive demand of control to avoid the effect of adaptation of the postural response, which is frequent when a task with a lower demand follows

Table 1 - Subject characteristics (age and BMI) with a pvalue comparison between BH (n = 14) and CG (n = 14).

Variable Age p-value BMI p-value

Group

Mean (standard deviation)

Median (range)

CG BH

39¡15 39¡16 0.84 27.29¡3.22 27.24¡3.21 0.66

32 (24-73) 31 (24-71)

CG BH

27.49 (20.00-31.22) 26.73 (21.41-32.46)

BH = breast hypertrophy, CG = control group, BMI = body mass index.

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CLINICS 2012;67(7):757-760

Postural control in breast hypertrophy Barbosa AF et al.

the two lower limbs, anterior-posterior sway reflects variations in the activities of the ankle flexor muscles, which control CoP displacement in the sagittal plane (19).

Analogous to obese individuals (18), the increased body mass in the thoracic area of women with breast hypertrophy brings the center of mass forward, requiring higher torque in both ankle flexion and extension to promote recurrent postural adjustments for the extra anterior body mass.

Table 2 - The median values and range of area, MVy and MVx for each condition between groups.

Condition

Group

Area

MVy

MVx

Median (range) Median (range) Median (range) EO/FP BH CG

0.4 (0.1-1.7) 0.2 (0.0-0.7) 0.004

0.9 (0.7-1.2) 0.9 (0.7-1.9) 0.927

0.8 (0.7-1.1) 0.7 (0.6-1.3) 0.462

BH CG

0.9 (0.2-3.6) 0.3 (0.1-0.9) 0.073

1.1 (0.9-2.2) 1.0 (0.8-1.9) 0.215

0.8 (0.8-1.3) 0.8 (0.6-1.3) 0.383

BH CG

3.9 (0.9-14.8) 1.4 (0.8-3.8) 0.003

2.0 (1.3-4.5) 1.6 (1.4-1.9) 0.004

1.1 (1.0-2.1) 1.0 (0.8-1.3) 0.168

BH CG

10.2 (4.3-40.4) 4.7 (2.5-8.8) 0.001

4.7 (2.5-8.3) 3.3 (1.9-6.3) 0.015

1.6 (1.1-4.2) 1.6 (1.0-1.9) 0.183

p-value EC/FP

p-value EO/MP

p-value EC/MP

p-value

Sensorial manipulation changes postural control Spatial orientation in postural control is based on the interpretation of convergent information from the somatosensory, visual and vestibular systems. When one of the sensorial inputs is disturbed, the central nervous system has to reweight the integration from the remaining inputs to promote postural control. According to Horak (20), healthy individuals rely most on somatosensory information when standing on a firm support with opened eyes. Our study proved that women with breast hypertrophy presented altered postural control when somatosensory inputs were disturbed with or without visual information.

A limitation of the present study was the age range of the sample (young adults to elderly subjects). Although both groups had a similar age range, older people could possibly present changes in postural control due to breast weight and sensorimotor changes arising from the aging process. To control this limitation a correlation with age was verified and a second analysis on subgroups of age showed differences between groups; age and the interaction between age and group did not have significant effects.

The present study provides new insight into the physical changes in women with breast hypertrophy. Although aesthetics can be an issue, breast hypertrophy is associated with alterations in postural control and may have functional consequences. Therefore, breast hypertrophy should be considered for assistance by health care services.

Women with breast hypertrophy presented altered postural control with a greater area of center of pressure displacement under three out of four sensorial information conditions. Only a marginal difference was observed for the condition in which visual information was absent and somatosensory information was available. Women with breast hypertrophy also showed a higher velocity of center of pressure displacement in the sagittal plane of the body when somatosensory information was disturbed, with or without visual integrity.

Area = area of center of pressure displacement, MVy = velocity in anterior posterior direction, MVx = velocity in the medial lateral direction, BH = breast hypertrophy, CG = control group, EO/FP = eyes open and fixed platform, EC/FP = eyes closed and fixed platform, EO/MP = eyes open and mobile platform, EC/MP = eyes closed and mobile platform.

DISCUSSION This study was conducted to evaluate the effect of the sensory integration in postural control in women with breast hypertrophy and our results contribute with three important findings. First, the results provide evidence that postural control is altered in BH because body sway is higher in this group. Second, postural control is altered, with a higher CoP velocity in the anterior-posterior direction. Finally, sensorial manipulation changes postural control in women with BH.

Postural control is altered in women with breast hypertrophy with a higher CoP area Women with breast hypertrophy present a higher CoP displacement area, which is independent of the sensory input. In this study, body balance, which was measured by CoP displacement, was correlated with muscle activity. Both very large and very small amounts of muscle activity lead to postural instability (18). When standing upright, the human body is often compared to an inverted pendulum system rotating around the ankle joint (19), which determines the area of CoP displacement. Similar to obese individuals (18), we suggest that the increased tissue concentration present in the thoracic area in women with breast hypertrophy tends to dislocate the center of mass close to the anterior boundaries of the base of support. As a result, the ground reaction force is amplified to induce higher torque at the ankle level and to increase muscle activity and postural adjustments, which justifies the increase in CoP displacement.

AUTHOR CONTRIBUTIONS Barbosa AF was responsible for the data acquision and analysis. Raggi GC was responsible for the manuscript preparation. Sa´ CS was responsible for data acquisition. Costa MP was responsible for the patient selection and data acquisition. Lima Junior JE was responsible for the patient selection and data acquisition. Tanaka C was responsible for the data analysis and manuscript preparation.

REFERENCES

Postural control is altered with a higher CoP velocity in the anterior-posterior direction

1. Foged J. Symptomatologien ved mamma hypertrofi. Ugeskr Laeg.

1953;115(12):439-51.

2. Sigurdson L, Mykhalovskiy E, Kirkland SA, Pallen A. Symptoms and related severity experienced by women with breast hypertrophy. Plast Reconstr Surg. 2007;119(2):481-6, http://dx.doi.org/10.1097/01.prs.

0000246407.87267.46.

3. Freire M, Neto MS, Garcia EB, Quaresma MR, Ferreira LM. Quality of life after reduction mammaplasty. Scand J Plast Reconstr Surg Hand Surg.

2004;38(6):335-9, http://dx.doi.org/10.1080/02844310410034295.

Our results showed that women with breast hypertrophy present a higher velocity of CoP displacement in the anterior-posterior direction under conditions in which somatosensory input is disturbed. While medial-lateral sway reflects the ability to distribute body weight between

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12. Shumway-Cook A, Woollacott M. Controle Postural Normal in. Controle Motor: Teoria e Aplicaco˜es Pra´ticas Motor. 2 ed. Barueri, SP: Manole; 2003.p.153-78.

13. Gautier G, Thouvarecq R, Larue J. Influence of experience on postural control: effect of expertise in gymnastics. J Mot Behav. 2008;40(5):400-8, http://dx.doi.org/10.3200/JMBR.40.5.400-408.

14. Kaminski TR, Bock C, Gentile AM. The coordination between trunk and arm motion during pointing movements. Exp Brain Res. 1995;106(3):45766.

15. Starley IF, Bryden DC, Tagari S, Mohammed P, Jones BP. An investigation into changes in lung function and the subjective medical benefits from breast reduction surgery. Br J Plast Surg. 1998;51(7):531-4, http://dx.doi.org/10.1054/bjps.1997.0260.

16. Sacchini V, Luini A, Tana S, Lozza L, Galimbert V, Merson M, et al.

Quantitative and qualitative cosmetic evaluation after conservative treatment for breast cancer. Eur J Cancer. 1991;27(11):1395-400.

17. Horak FB, Diener HC, Nashner LM. Influence of central set on human postural responses. J Neurophysiol. 1989;62(4):841-53.

18. Menegoni F, Tacchini E, Bigoni M, Vismara L, Priano L, Galli M, et al.

Mechanisms underlying center of pressure displacements in obese subjects during quiet stance. J Neuroeng Rehabil. 2011;8:20, http:// dx.doi.org/10.1186/1743-0003-8-20.

19. Winter DA, Prince F, Frank JS, Powell C, Zabjek KF. Unified theory regarding A/P and M/L balance in quiet stance. J Neurophysiol 1996;75(6):2334–43.

20. Horak FB. Postural orientation and equilibrium: what do we need to know about neural control of balance to prevent falls? Age and Ageing.

2006;35(Suppl 2):ii7-ii11, http://dx.doi.org/10.1093/ageing/afl077.

4. Iwuagwu OC, Stanley PW, Platt AJ, Drew PJ, Walker LG. Effects of bilateral breast reduction on anxiety and depression: Results of a prospective randomised trial. Scand J Plast Reconstr Surg Hand Surg.

2006;40(1):19-23, http://dx.doi.org/10.1080/02844310500415335.

5. Kemal F, Fulya F, Selahattin O, Tuba G. The impact of breast size on vertebral column: A radiologic study. Aesthetic Plast. Surg. 2007;31(1): 23-7.

6. Evans GR, Ryan JJ. Reduction mammaplasty for teenage patient: A critical analysis. Aesthetic Plast Surg. 1994;18(3):291-7, http:// dx.doi.org/10.1007/BF00449797.

7. Sabino Neto M, Dematteˆ MF, Freire M, Garcia EB, Quaresma M, Ferreira LM. Self-esteem and functional capacity outcomes following reduction mammaplasty. Aesthet Surg J. 2008;28(4):417-20, http://dx.doi.org/ 10.1016/j.asj.2008.04.006.

8. Sood R, Mount DL, Coleman JJ, Ranieri J, Sauter S, Mathur P, Thurston B. Effects of reduction mammaplasty on pulmonary function and symptoms of macromastia. Plast Reconstr Surg. 2003;111(2):688-94, http:// dx.doi.org/10.1097/01.PRS.0000041395.02699.B7.

9. Pernia LR, Ronel DN, Leeper JD, Miller HL. Carpal tunnel syndrome in women undergoing reduction mammaplasty. Plast Reconstr Surg.

2000;105(4):1314-9.

10. Gonzalez F, Walton RL, Shafer B, Matory Jr WE, Borah GL. Reduction mammaplasty improves symptoms of macromastia. Plast Reconstr Surg.

1993;91(7):1270-6.

11. Glatt BS, Sarwer DB, O’Hara DE, Hamori C, Bucky LP, LaRossa D. A retrospective study of changes in physical symptoms and body image after reduction mammaplasty. Plast Reconstr Surg. 1999;103(1):7682.

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DOI:10.6061/clinics/2012(07)10

CLINICAL SCIENCE

Renal oxygen content is increased in healthy subjects after angiotensin-converting enzyme inhibition Anna Stein,I,II Silvia Goldmeier,I Sarah Voltolini,I Enio Setogutti,III Carlos Feldman,I,III Eduardo Figueiredo,IV Renato Eick,I Maria Irigoyen,I,VI Katya RigattoV I

Fundac¸a˜o Universita´ria de Cardiologia (IC/FUC), Instituto de Cardiologia do Rio Grande do Sul, Porto Alegre/RS, Brazil. II Universidade Federal de Sa˜o Paulo (UNIFESP), Sa˜o Paulo, SP/Brazil. III Servic¸o de Investigac¸a˜o Diagno´stica (SIDI), Porto Alegre, RS/Brazil. IV MR Res Latin America GE Healthcare, Sa˜o Paulo, SP/Brazil. V Universidade Federal de Cieˆncias da Sau´de de Porto Alegre (UFCSPA), Departamento de Cieˆncias Ba´sicas da Sau´de, Porto Alegre, RS/ Brazil. VI Hospital das Clinicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Instituto do Corac¸a˜o (InCor - USP), Sa˜o Paulo, SP/Brazil.

OBJECTIVE: The association between renal hypoxia and the development of renal injury is well established.

However, no adequate method currently exists to non-invasively measure functional changes in renal oxygenation in normal and injured patients.

METHOD: R2* quantification was performed using renal blood oxygen level-dependent properties. Five healthy normotensive women (50¡5.3 years) underwent magnetic resonance imaging in a 1.5T Signa Excite HDx scanner (GE Healthcare, Waukesha, WI). A multiple fast gradient-echo sequence was used to acquire R2*/T2* images (sixteen echoes from 2.1 ms/slice to 49.6 ms/slice in a single breath hold per location). The images were post-processed to generate R2* maps for quantification. Data were recorded before and at 30 minutes after the oral administration of an angiotensin II-converting enzyme inhibitor (captopril, 25 mg). The results were compared using an ANOVA for repeated measurements (mean ¡ standard deviation) followed by the Tukey test. ClinicalTrials.gov: NCT01545479.

RESULTS: A significant difference (p,0.001) in renal oxygenation (R2*) was observed in the cortex and medulla before and after captopril administration: right kidney, cortex = 11.08¡0.56ms, medulla = 17.21¡1.47ms and cortex = 10.30¡0.44ms, medulla = 16.06¡1.74ms, respectively; and left kidney, cortex = 11.79¡1.85ms, medulla = 17.03¡0.88ms and cortex = 10.89¡0.91ms, medulla = 16.43¡1.49ms, respectively.

CONCLUSIONS: This result suggests that the technique efficiently measured alterations in renal blood oxygenation after angiotensin II-converting enzyme inhibition and that it may provide a new strategy for identifying the early stages of renal disease and perhaps new therapeutic targets.

KEYWORDS: BOLD; Renal Oxygenation; Healthy Subjects; Captopril; Renin-Angiotensin System.

Stein A, Goldmeier S, Voltolini S, Setogutti E, Feldman C, Figueiredo E, et al. Renal oxygen content is increased in healthy subjects after angiotensinconverting enzyme inhibition. Clinics. 2012;67(7):761-765.

Received for publication on February 27, 2012; First review completed on March 20, 2012; Accepted for publication on March 20, 2012 E-mail: steinanna87@gmail.com Tel.: 55 51 3230-3757

(3). In addition, the strong association between chronic kidney disease and cardiovascular disease has led the National Kidney Foundation and the American Heart Association to recommend that all patients with cardiovascular disease also be screened for kidney disease (4).

In cardiovascular disease, it is possible to precisely measure blood pressure, cholesterol or glucose concentration. However, it is still very difficult to identify early-stage renal injury, and current laboratory measurements are insufficient for making an early diagnosis. In addition, traditional imaging methods, although excellent for morphologic assessments, are limited in detecting premature renal functional alterations (5). The sequence of events that reduces renal oxygenation leading to ischemia and/or kidney injury is still unknown (6). Glomerular hyperfiltration (7) or tubulointerstitial damage (8) can cause progressive and irreversible renal failure, regardless of the initial insult. In fact, histological studies in human kidneys showed that tubulointerstitial oxygenation is modified before structural

INTRODUCTION Despite advances in renal dialysis, there has been no substantial change in the mortality rate of acute renal failure over the past three decades. In contrast, over the same period, acute myocardial infarction mortality has fallen dramatically, from approximately 50% to 6% (1). In patients with end-stage renal disease, the mortality rate is growing because of the greater prevalence of hypertension and diabetes and the increased mean age of the population (2).

Indeed, in the United States and Australia, approximately 11% of adults have some degree of chronic kidney disease

Copyright ß 2012 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.

No potential conflict of interest was reported.

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microvascular damage takes place. This observation indicates that morphological changes begin in a very early stage of kidney disease (9). At this stage, it is not possible to diagnose renal pathology using the methods available. Thus, a method for detecting the early stages of renal functional changes would be very useful for the early treatment of renal disease. Unfortunately, renal biopsy is the only current method of confirm functional alteration (5).

From the late 1980s, when Brezis (1991) (10) inserted microelectrodes into the Sprague-Dawley rats renal parenchyma to demonstrate hypoxic changes during acute kidney injury, to the 1990s, when Prasad introduced blood oxygen level-dependent (BOLD) magnetic resonance imaging (MRI) to the study of renal blood flow (11), several different factors have been blamed for the slow progress of the prevention and treatment of acute or chronic renal failure.

There is no doubt that the renin angiotensin system (RAS) is useful to preserve peritubular capillary perfusion under normal situations (12). However, when it is excessively stimulated, the RAS can induce renal hypoxia, reducing the efficiency of oxygen diffusion in renal tissue and provoking functional (13) and structural changes, damage to the renal vasculature and fibrosis (14).

In the present study, we measured the intra-renal effect of ACE inhibition on healthy subjects using a sensitive BOLDMRI method that uses deoxygenated hemoglobin as an endogenous contrast agent. The ratio of oxyhemoglobin (diamagnetic) to deoxyhemoglobin (paramagnetic) is proportional to the partial oxygen pressure (pO2). The BOLD signal is estimated using the transverse relaxation rate R2* (1/T2*) and can be considered a sensitive indicator of tissue pO2. However, an inverse ratio also exists; when this signal is increased, renal oxygenation is decreased, and vice versa (11,15).

Therefore, considering that angiotensin II (Ang II) reduces renal blood flow, (16-17) we hypothesized that partial ACE inhibition would increase the BOLD-MRI signal in renal tissue. The study of the mechanism underlying this putative process and the responses to ACE inhibition is of considerable importance. Understanding the mechanisms by which the kidney adapts to the effects of the RAS in healthy subjects will be useful for the early detection of renal disease and for the development of new therapies to decrease the progression of the disease and its consequences.

MR Imaging Technique Images were acquired using a 1.5T HDx instrument (Sigma, GE Healthcare, Waukesha, WI) and an 8-channel body coil.

Localizer images were performed in the axial and sagittal planes following the long axis of both kidneys, using the fast spoiled gradient echo (FSPGR) technique while the patients held their breath.

Oblique axial and coronal reference images were acquired to construct color maps following approximately the short and long axes of both kidneys using FSPGR with the following parameters: field of view (FOV) = 40 cm, matrix 2566128, slice thickness = 5 cm, slice gap 6 cm, 3 slices per plane in one breath hold, repetition time (RT) = 150 ms, echo time (ET = min full, angle degrees (FLIP) = 90, band width (rBW) = 62KHz, 1NEX. This sequence provides an excellent contrast between the cortex and the medulla and avoids the unnecessary use of contrast agents.

BOLD-sensitive images were acquired using the multiple echo FGRE sequence with the same geometrical parameters.

The reference images considered the following parameters: TR = 60 ms, FLIP = 30, rBW = 60 KHz, 16 echoes from TE = 2.1 ms until 49.6 ms, an echo interval of 3.2 ms, 1 NEX.

BOLD imaging Data Processing Bold image processing was performed using the Functool R2* module for R2*/T2* fitting, assuming single exponential decay without constant offset at AW4.3 (GE Healthcare).

For visualization and quantification, R2* parametric maps were generated for each slice (11,18-19). A Puh-Thallium color table was used because the data had a dynamic range with a minimum value set at 7.0/sec and a maximum value at 23/sec. The color ranged from blue to red from the minimum R2* to the maximum R2*, respectively. Green/yellow coloring represents intermediate R2*. When a position mismatch occurred due to a different breath hold state, anatomical fatsuppressed FSPGR images were used as transparent references over a parametric map or the original BOLD images.

Three slices were acquired, and the slice that provided the best differentiation between the cortex and the medulla and the minimum partial volume was selected for quantification. A total of six regions of interest (ROIs) with a pixel size of nine were used for each plane; three regions were positioned at the cortex and three were positioned at the medulla.

The ROIs were positioned at the cortex according to the anatomical reference image (light gray) and blue on the parametric map. The ROIs were positioned at the medulla using either the anatomical reference image (dark gray) or the parametric map that showed a green, yellow and red gradient (Figure 1). Areas with susceptibility artifacts, such as bowel gas, and areas with renal hilum vessels were avoided.

METHODS Subjects The study was conducted under protocol (n˚ 4111/08), approved by the Ethics Committee of the Instituto de Cardiologia do Rio Grande do Sul-Fundac¸a˜o Universita´ria de Cardiologia/Brazil. After providing informed consent, five healthy female volunteers (50¡5.3 years old) participated in the study. None of the subjects were taking any medications, and they were asked to abstain from food and water overnight. All measurements were performed in the late morning.

After a baseline BOLD-MRI acquisition, the subjects were given an oral ACE inhibitor (captopril 25 mg). Thirty minutes after captopril administration, without moving from the scanner, blood pressure was measured and a second BOLD acquisition was performed to detect possible changes in renal oxygenation. Data were collected by two radiologists and interpreted by a physician and a physiologist.

Data analysis The statistical significance was determined using an analysis of variance for three repeated measures (ANOVA) followed by the Tukey test (SPSS 12.0). The data are presented as the mean ¡ SD. P,0.05 was considered statistically significant.

RESULTS Table 1 summarizes the R2* measurements of the left and right kidney in the cortex and medulla before and after ACE

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DISCUSSION The main finding of this study is that the oral administration of a low dose of an ACE inhibitor allowed the early detection of an increase in renal cortical and medullar oxygenation using BOLD-MRI. This result is consistent with the very well-established property of Ang II to decrease renal perfusion and renal oxygenation (16-17). Ang II is known to be responsible for the majority of the physiological and pathological effects of RAS, and ACE inhibition reduces Ang II formation. Ang II inhibition has been highly successful in hypertension management and for reducing renal disease progression (20-21).

Renal hypoxia has long been considered important in the development of renal disease due to diabetes mellitus (22).

Ries et al. used the BOLD-MRI technique in an animal model and observed a significant decrease in oxygenation in the kidneys of diabetic patients compared to controls (23).

Several studies on the pathogenesis of essential hypertension have also shown that medullar blood flow reduction is one of the most important factors in the development of the renal disease (24). However, a chronic infusion of a subpressor Ang II dose induces a long-term increase in BP. This effect could be associated with the afferent arteriolar reactivity to Ang II and might be present in the early stages of hypertension (25). These findings and the fact that Ang II decreases renal oxygenation support the utility of BOLDMRI for the study of the pathophysiology of hypertension (26-27).

Thus, efforts to measure functional renal oxygenation with the use of captopril, a renin Ang II axis inhibitor, provide data that is relevant to the prevention and treatment of renal diseases (26-27).

ACE inhibition and the subsequent reduction in Ang II plasma concentration (28) most likely provoked renal arteriolar vasodilatation and increased renal blood flow and oxygenation. Thus, the decrease in R2* signal observed in the cortex and medulla likely occurred due to the increase in tissue pO2 (11,15) induced by moderate ACE inhibition.

These findings support the idea that kidney oxygen content depends on both tubular sodium reabsorption and renal blood flow (29) and reinforce the idea that R2* has an inverse correlation with oxygen content and blood flow (11,30).

The results obtained using renal BOLD-MRI in the presence of Ang II are most likely a consequence of reduced perfusion rather than of increased renal oxygen consumption (31). Thus, the mechanism for improving oxygenation by Ang II blockade includes both hemodynamic changes and efficient oxygen usage (14). These findings are consistent with our results, which indicated that the effects of the RAS were not exclusively associated with BP, although they were principally associated with renal perfusion.

Figure 1 - An R2* map superimposed on the anatomic image of the 9th echo. The lowest R2* was set to 7 Hz (blue), and the maximum R2* was set to 23 Hz (red). The intermediate values range from green and yellow to orange according to the color scale on the left.

inhibitor administration. Our results consistently indicate that moderate ACE inhibition reduced the R2* BOLD-MRI signal in the cortex and medulla and, therefore, increased renal blood flow. For all subjects, the R2* values were significantly higher in the medulla than in the cortex, and there was a significant difference between the baseline values and those obtained thirty minutes after ACE inhibitor administration (Table 1). No significant differences were observed for the values within subjects or between subjects for the cortex and medulla in the three measurements (data not shown).

The volunteers had an initial medial systolic blood pressure of 113¡12 mmHg and a diastolic pressure of 76¡5 mmHg. Thirty minutes after captopril administration, systolic but not diastolic blood pressure showed a modest but significant decrease. However, the systolic blood pressure did not significantly differ between 30 and 60 minutes after captopril administration (Table 2).

Hematocrit (37¡3.6%) and hemoglobin (12¡1.2 mEq/L) and the plasma concentrations of sodium (139¡3.9 mEq/L), potassium (3.8¡0.6 mEq/L) and creatinine (0.6¡0.1 mEq/ L) (n = 4) were measured in the clinical laboratory of the Instituto de Cardiologia do Rio Grande do Sul.

Table 1 - R2* signal (ms) in the renal cortex and medulla in five healthy kidneys before (control) and at 30 minutes after the oral administration of an ACE inhibitor.

Cortex Control Right kidney Left kidney *

11.08 ¡ 0.56 11.79 ¡ 1.85

Medulla After captopril {

10.30 ¡ 0.44 10.89 ¡ 0.91{

Control

After captopril *

17.21 ¡ 1.47 17.03 ¡ 0.88*

p,0.001 vs. cortex (control and captopril), {p,0.001 vs. control (medulla and cortex), ACE = angiotensin-converting enzyme.

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16.06 ¡ 1.74*{ 16.43 ¡ 1.49*{

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Table 2 - Blood pressure measurements before and at 15, 30 and 60 minutes after the oral administration of an ACE inhibitor (n = 5).

Time (minutes) BP (mmHg)

Before ACEi Systolic 113¡12

15 Diastolic 76¡5

Systolic 108¡13

30 Diastolic 70¡6

Systolic 105¡8*

60 Diastolic 69¡3

Systolic 103¡12**

Diastolic 69¡5

ACEi = angiotensin-converting enzyme inhibition; BP = Blood Pressure; * p,0.05 and **p,0.01 vs. systolic BP before ACEi

The right and left kidneys did not have significantly different R2* values. It should also be emphasized that our control R2* values are very close to those found in the literature (11,15,32-35) for baseline measurements.

We recorded the BOLD signal after thirty minutes instead of 60 minutes because 25 mg of captopril decreased the systolic blood pressure (BP) equally at both time points.

Thus, our protocol predicted the second BOLD signal acquisition after 30 minutes to avoid maintaining the subjects in a static position inside the scanner for an unnecessarily long period of time.

We believe that the decrease in systolic blood pressure observed in the first 30 minutes did not interfere with renal oxygenation. In support of this assumption, Schachinger recently demonstrated that vasoactive substances, such as noradrenalin and sodium nitroprusside, did not alter the renal BOLD signal, although they were able to provoke BP changes (31). Ang II infusion is also known to decrease the renal BOLD signal as quickly as 10 s after the peripheral intravenous administration of an Ang II bolus. This is clearly faster than the arterial blood pressure response, suggesting that it may be related to reduced renal perfusion rather than to metabolic adjustment (31).

Several studies have also demonstrated that, at lower concentrations, Ang II may cause greater vasoconstriction in the medulla than in the cortex. The authors hypothesize that this effect is likely related to the greater Ang II receptor density in the outer medulla (36-37). In fact, we also found that, under normal conditions, the oxygen content was significantly higher in the cortex than in the medulla. This result was maintained after ACE inhibition, indicating that the physiological balance between cortical and medullar circulation did not change after a moderate reduction of the Ang II in the blood stream.

Our results are consistent with those of many investigators who showed that BOLD detected a higher oxygen content in the cortex than in the medulla (11,19,34,38).

Thus, BOLD-MRI is a useful, non-invasive tool for defining basic physiological mechanisms, and it will contribute to a better understanding of regional oxygenation within the kidney (39).

BOLD-MRI has opened an exciting new area for studying renal physiopathology and has created new possibilities for identifying the early stages of renal disease and perhaps new therapeutic targets (40).

However, this study has several important limitations: 1we did not include a placebo group; 2- the subject number was small; and 3- the study was conducted only in females.

It is also important to note that subjects were not removed from the scanner prior to the second BOLD acquisition.

Moreover, the conditions were the same during the data

collection for all subjects, indicating that renal oxygenation was increased after captopril administration.

There is considerable interest in developing techniques that will enable physicians to identify and evaluate early changes in tissue function. BOLD-MRI may be a very useful technique for the early assessment of renal injuries in clinical practice. More studies should be conducted to confirm this hypothesis.

ACKNOWLEDGMENTS We wish to thank the volunteers for their contribution to science and ˆ ngelo de Souza for their assistance.

Sergio Kakuta Kato and A

AUTHOR CONTRIBUTIONS Stein A recorded data, participated in the analysis and interpretation of data, statistical analysis, and manuscript writing. Goldmeier S, Voltolini S, Setogutti E, Feldman C, and Figueiredo E recorded and interpreted data.

Eick R analyzed and interpreted data and participated in manuscript writing. Rigatto K interpreted data and drafted and critically revised the manuscript. Irigoyen MC participated in the conception and design of the study, analyzed and interpreted data and drafted and critically revised the manuscript. All authors read and approved the final manuscript.

REFERENCES 1. Mehta RL, Chertow GM. Acute renal failure definitions and classification: time for change? J Am Soc Nephrol. 2003;14(8):2178-87, http:// dx.doi.org/10.1097/01.ASN.0000079042.13465.1A. 2. Alcazar JM, Rodicio JL. Ischemic nephropathy: clinical characteristics and treatment. Am J Kidney Dis. 2000;36(5):883-93, http://dx.doi.org/ 10.1053/ajkd.2000.19077.

3. Bello AK, Nwankwo E, El Nahas AM. Prevention of chronic kidney disease: a global challenge. Kidney Int Suppl. 2005(98):S11-7.

4. Brosius FC 3rd, Hostetter TH, Kelepouris E, Mitsnefes MM, Moe SM, Moore MA, et al. Detection of chronic kidney disease in patients with or at increased risk of cardiovascular disease: a science advisory from the American Heart Association Kidney and Cardiovascular Disease Council; the Councils on High Blood Pressure Research, Cardiovascular Disease in the Young, and Epidemiology and Prevention; and the Quality of Care and Outcomes Research Interdisciplinary Working Group: Developed in Collaboration With the National Kidney Foundation. Hypertension.

2006;48(4):751-5.

5. Wang TM, Chang PL, Kao PF, Hsieh ML, Huang ST, Tsui KH. The role of diuretic renography in the evaluation of obstructed hydronephrosis after pediatric pyeloplasty. Chang Gung Med J. 2004;27(5):344-50.

6. Textor SC, Glockner JF, Lerman LO, Misra S, McKusick MA, Riederer SJ, et al. The use of magnetic resonance to evaluate tissue oxygenation in renal artery stenosis. J Am Soc Nephrol. 2008;19(4):780-8, http:// dx.doi.org/10.1681/ASN.2007040420.

7. Brenner BM, Goldszer RC, Hostetter TH. Glomerular response to renal injury. Contrib Nephrol. 1982;33:48-66.

8. Nangaku M. Mechanisms of tubulointerstitial injury in the kidney: final common pathways to end-stage renal failure. Intern Med. 2004;43(1):917, http://dx.doi.org/10.2169/internalmedicine.43.9.

9. Nangaku M. Chronic hypoxia and tubulointerstitial injury: a final common pathway to end-stage renal failure. J Am Soc Nephrol.

2006;17(1):17-25.

10. Heyman SN, Brezis M, Epstein FH, Spokes K, Silva P, Rosen S. Early renal medullary hypoxic injury from radiocontrast and indomethacin.

Kidney Int. 1991;40(4):632-42, http://dx.doi.org/10.1038/ki.1991.255.

764

CLINICS 2012;67(7):761-765

Renal Oxygenation in Healthy Subjects Stein A et al.

27. Chen SX, Song T, Zhou SH, Liu YH, Wu SJ, Liu LY. Protective effects of ACE inhibitors on vascular endothelial dysfunction induced by exogenous advanced oxidation protein products in rats. European journal of pharmacology. 2008;584(2-3):368-75, http://dx.doi.org/ 10.1016/j.ejphar.2008.02.020.

28. Badzynska B, Sadowski J. Opposed effects of prostaglandin E2 on perfusion of rat renal cortex and medulla: interactions with the renin-angiotensin system. Experimental physiology. 2008;93(12):1292302, http://dx.doi.org/10.1113/expphysiol.2008.043604.

29. Kellerman PS. Cellular and metabolic consequences of chronic ischemia on kidney function. Semin Nephrol. 1996;16(1):33-42.

30. Juillard L, Lerman LO, Kruger DG, Haas JA, Rucker BC, Polzin JA, et al.

Blood oxygen level-dependent measurement of acute intra-renal ischemia. Kidney Int. 2004;65(3):944-50, http://dx.doi.org/10.1111/ j.1523-1755.2004.00469.x.

31. Schachinger H, Klarhofer M, Linder L, Drewe J, Scheffler K. Angiotensin II decreases the renal MRI blood oxygenation level-dependent signal.

Hypertension. 2006;47(6):1062-6, http://dx.doi.org/10.1161/01.HYP.000 0220109.98142.a3.

32. Simon-Zoula SC, Hofmann L, Giger A, Vogt B, Vock P, Frey FJ, et al.

Non-invasive monitoring of renal oxygenation using BOLD-MRI: a reproducibility study. NMR Biomed. 2006;19(1):84-9, http://dx.doi.org/ 10.1002/nbm.1004.

33. Hofmann L, Simon-Zoula S, Nowak A, Giger A, Vock P, Boesch C, et al.

BOLD-MRI for the assessment of renal oxygenation in humans: acute effect of nephrotoxic xenobiotics. Kidney Int. 2006;70(1):144-50, http:// dx.doi.org/10.1038/sj.ki.5000418.

34. Epstein FH, Veves A, Prasad PV. Effect of diabetes on renal medullary oxygenation during water diuresis. Diabetes Care. 2002;25(3):575-8, http://dx.doi.org/10.2337/diacare.25.3.575.

35. Zuo CS, Rofsky NM, Mahallati H, Yu J, Zhang M, Gilbert S, et al.

Visualization and quantification of renal R2* changes during water diuresis. J Magn Reson Imaging. 2003;17(6):676-82, http://dx.doi.org/ 10.1002/jmri.10314.

36. Blantz RC, Konnen KS, Tucker BJ. Angiotensin II effects upon the glomerular microcirculation and ultrafiltration coefficient of the rat. J Clin Invest. 1976;57(2):419-34, http://dx.doi.org/10.1172/JCI108293.

37. Navar LG, Inscho EW, Majid SA, Imig JD, Harrison-Bernard LM, Mitchell KD. Paracrine regulation of the renal microcirculation. Physiol Rev. 1996;76(2):425-536.

38. Prasad PV, Epstein FH. Changes in renal medullary pO2 during water diuresis as evaluated by blood oxygenation level-dependent magnetic resonance imaging: effects of aging and cyclooxygenase inhibition.

Kidney Int. 1999;55(1):294-8, http://dx.doi.org/10.1046/j.1523-1755.

1999.00237.x.

39. Gloviczki ML, Lerman LO, Textor SC. Blood Oxygen Level-Dependent (BOLD) MRI in Renovascular Hypertension. Current hypertension reports.13(5):370-7.

40. Chrysochou C, Mendichovszky IA, Buckley DL, Cheung CM, Jackson A, Kalra PA. BOLD imaging: a potential predictive biomarker of renal functional outcome following revascularization in atheromatous renovascular disease. Nephrol Dial Transplant. 2012;27(3):1013-9, http:// dx.doi.org/10.1093/ndt/gfr392.

11. Prasad PV, Edelman RR, Epstein FH. Noninvasive evaluation of intrarenal oxygenation with BOLD MRI. Circulation. 1996;94(12):3271-5.

12. Nangaku M, Inagi R, Miyata T, Fujita T. Angiotensin-induced hypoxia in the kidney: functional and structural changes of the renal circulation.

Adv Exp Med Biol. 2007;618:85-99, http://dx.doi.org/10.1007/978-0387-75434-5_7.

13. Kondo N, Kiyomoto H, Yamamoto T, Miyatake A, Sun GP, Rahman M, et al. Effects of calcium channel blockade on angiotensin II-induced peritubular ischemia in rats. J Pharmacol Exp Ther. 2006;316(3):1047-52.

14. Nangaku M, Fujita T. Activation of the renin-angiotensin system and chronic hypoxia of the kidney. Hypertens Res. 2008;31(2):175-84, http:// dx.doi.org/10.1291/hypres.31.175.

15. Prasad PV, Chen Q, Goldfarb JW, Epstein FH, Edelman RR. Breath-hold R2* mapping with a multiple gradient-recalled echo sequence: application to the evaluation of intrarenal oxygenation. J Magn Reson Imaging.

1997;7(6):1163-5, http://dx.doi.org/10.1002/jmri.1880070633.

16. Schachinger H, Dieterle T, Martina B, Haberthur C, Huber PR, Bock A, et al. Increased renovascular response to angiotensin II in persons genetically predisposed to arterial hypertension disappears after chronic angiotensin-converting enzyme inhibition. J Hypertens. 2004;22(1):17580, http://dx.doi.org/10.1097/00004872-200401000-00027.

17. Hall JE, Granger JP. Renal hemodynamic actions of angiotensin II: interaction with tubuloglomerular feedback. Am J Physiol.

1983;245(2):R166-73.

18. Sadowski EA, Fain SB, Alford SK, Korosec FR, Fine J, Muehrer R, et al.

Assessment of acute renal transplant rejection with blood oxygen leveldependent MR imaging: initial experience. Radiology. 2005;236(3):911-9, http://dx.doi.org/10.1148/radiol.2363041080.

19. Epstein FH, Prasad P. Effects of furosemide on medullary oxygenation in younger and older subjects. Kidney Int. 2000;57(5):2080-3, http:// dx.doi.org/10.1046/j.1523-1755.2000.00057.x.

20. Johnston CI. Tissue angiotensin converting enzyme in cardiac and vascular hypertrophy, repair, and remodeling. Hypertension.

1994;23(2):258-68.

21. Dzau VJ. Theodore Cooper Lecture: Tissue angiotensin and pathobiology of vascular disease: a unifying hypothesis. Hypertension. 2001;37(4): 1047-52.

22. Prasad P, Li LP, Halter S, Cabray J, Ye M, Batlle D. Evaluation of Renal Hypoxia in Diabetic Mice by BOLD MRI. Investigative radiology. 2010; 45(12):819-22, http://dx.doi.org/10.1097/RLI.0b013e3181ec9b02.

23. Ries M, Basseau F, Tyndal B, Jones R, Deminiere C, Catargi B, et al. Renal diffusion and BOLD MRI in experimental diabetic nephropathy. Blood oxygen level-dependent. J Magn Reson Imaging. 2003;17(1):104-13, http://dx.doi.org/10.1002/jmri.10224.

24. Mattson DL, Roman RJ, Cowley AW Jr. Role of nitric oxide in renal papillary blood flow and sodium excretion. Hypertension. 1992;19(6 Pt 2):766-9.

25. Imig JD. Afferent arteriolar reactivity to angiotensin II is enhanced during the early phase of angiotensin II hypertension. Am J Hypertens.

2000;13(7):810-8, http://dx.doi.org/10.1016/S0895-7061(00)00264-8.

26. Solski LV, Longyhore DS. Prevention of type 2 diabetes mellitus with angiotensin-converting-enzyme inhibitors. Am J Health Syst Pharm.

2008;65(10):935-40, http://dx.doi.org/10.2146/ajhp070388.

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DOI:10.6061/clinics/2012(07)11

CLINICAL SCIENCE

Noninvasive positive-pressure ventilation in clinical practice at a large university-affiliated Brazilian hospital Liria Yuri Yamauchi,I Teresa Cristina Francischetto Travaglia,II Sidnei Ricardo Nobre Bernardes,II Maise C. Figueiroa,II Clarice Tanaka,II Carolina FuII I Federal University of Sa˜o Paulo, Department of Human Movement Sciences, Santos/SP, Brazil. II Faculdade de Medicina da Universidade de Sa˜o Paulo, Department of Physiotherapy, Communication Science & Disorders, Occupational Therapy, Sa˜o Paulo/SP, Brazil.

OBJECTIVES: To describe noninvasive positive-pressure ventilation use in intensive care unit clinical practice, factors associated with NPPV failure and the associated prognosis.

METHODS: A prospective cohort study.

RESULTS: Medical disorders (59%) and elective surgery (21%) were the main causes for admission to the intensive care unit. The main indications for the initiation of noninvasive positive-pressure ventilation were the following: post-extubation, acute respiratory failure and use as an adjunctive technique to chest physiotherapy. The noninvasive positive-pressure ventilation failure group was older and had a higher Simplified Acute Physiology Score II score. The noninvasive positive-pressure ventilation failure rate was 35%. The main reasons for intubation were acute respiratory failure (55%) and a decreased level of consciousness (20%). The noninvasive positive-pressure ventilation failure group presented a shorter period of noninvasive positive-pressure ventilation use than the successful group [three (2-5) versus four (3-7) days]; they had lower levels of pH, HCO3 and base excess, and the FiO2 level was higher. These patients also presented lower PaO2:FiO2 ratios; on the last day of support, the inspiratory positive airway pressure and expiratory positive airway pressure were higher. The failure group also had a longer average duration of stay in the intensive care unit [17 (10-26) days vs. 8 (5-14) days], as well as a higher mortality rate (9 vs. 51%). There was an association between failure and mortality, which had an odds ratio (95% CI) of 10.6 (5.93 – 19.07). The multiple logistic regression analysis using noninvasive positive pressure ventilation failure as a dependent variable found that treatment tended to fail in patients with a Simplified Acute Physiology Score II$34, an inspiratory positive airway pressure level$15 cmH2O and pH,7.40.

CONCLUSION: The indications for noninvasive positive-pressure ventilation were quite varied. The failure group had a longer intensive care unit stay and higher mortality. Simplified Acute Physiology Score II$34, pH,7.40 and higher inspiratory positive airway pressure levels were associated with failure.

KEYWORDS: Artificial ventilation; Noninvasive Ventilation; Intensive Care Unit; Cohort Study.

Yamauchi LY, Travaglia TC, Bernardes SR, Figueiroa MC, Tanaka C, Fu C. Noninvasive positive-pressure ventilation in clinical practice at a large university-affiliated Brazilian hospital. Clinics. 2012;67(7):767-772.

Received for publication on March 8, 2012; First review completed on March 21, 2012; Accepted for publication on March 22, 2012 E-mail: liria_yamauchi@yahoo.com.br / liria.yuri@unifesp.br Tel.: 55-11-2661-6867

been used to treat chronic respiratory failure, but in more recent years it has increasingly been used to treat patients with various forms of acute respiratory failure (1). A recently published guideline has suggested the following guidelines: bilevel NPPV should be the first option for patients with either a severe exacerbation of chronic obstructive pulmonary disease (COPD) or cardiogenic pulmonary edema; continuous positive airway pressure delivered by mask appears to be as effective as bilevel NPPV for patients with cardiogenic pulmonary edema; patients with acute respiratory distress or hypoxemia, either in the postoperative setting or in the presence of immunosuppression, can be considered for a trial of bilevel NPPV; patients with COPD can be considered for a trial of early extubation to bilevel NPPV in centers with experience in the

INTRODUCTION Mechanical ventilation without the use of an invasive artificial airway is defined as noninvasive ventilation (NIV).

Noninvasive positive-pressure ventilation (NPPV) using a mask (or interface) that conducts gas from a positivepressure ventilator into the airways has become the predominant means of administering NIV. NPPV has long

Copyright ß 2012 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.

No potential conflict of interest was reported.

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use of this technique (2). All of these suggestions were based on an extensive systematic review of published randomized controlled trials.

Clinical NPPV experience has been reported by some authors (3-6,9,10). This information can elucidate the feasibility of NPPV in all of the indications mentioned in the guidelines as well as identify any issues that arise during clinical practice. This type of research may inform the identification of issues related to technical or even local characteristics that could interfere with patient outcomes.

Our aim was to describe the clinical, demographic and technical characteristics related to noninvasive positivepressure ventilation (NPPV) use in clinical practice, as well as factors associated with NPPV failure and the prognosis of critically ill adult patients at a large university-affiliated hospital. We then compared our findings with the current evidence.

face, total face); arterial blood gas levels at NPPV onset and at the time when support was discontinued; tolerance to NPPV or the lack thereof; and NPPV-related complications (skin lesions, gastric distension due to aerophagia, eye irritation, vomiting, hypotension, others); cough characteristics; the need for nasotracheal suctioning; the incidence of nosocomial pneumonia, as clinically defined according to the CDC (Centers for Disease Control and prevention) criteria (12). The study end-points were the following: NIV outcome, ICU length of stay and mortality rate. NIV success was defined as the avoidance of orotracheal intubation and independence from NPPV within 72 hours after its discontinuation, and failure was defined as a need for intubation.

Data analysis A descriptive analysis of the overall study population was carried out. The quantitative variables were expressed as the mean (standard deviation) or median (interquartile range) when more appropriate. Categorical variables were presented as proportions. Associations between categorical variables were analyzed with the chi-square test. Fisher’s exact test was applied when the expected value was not sufficient for use of the chi-square test. Continuous data with a normal distribution were compared using Student’s t-test; otherwise, the non-parametric equivalent was applied. NPPV outcome was considered as the primary endpoint. All the tests were two-tailed with a significance level of 5% (p#0.05). Factors independently associated with NPPV outcome were identified using a logistic regression model. A univariate analysis was initially performed, and all variables considered clinically relevant with a p value less than 0.20 were included in the model. Continuous variables (age, SAPS II score, IPAP, EPAP and pH) were dichotomized based on the median values of the distribution. In the preliminary stage of the analysis, we studied the possible associations between the explanatory variables examined in this study. The goodness-of-fit was assessed using the Hosmer-Lemeshow test. Statistical analyses were performed with SPSSH version 16.0

MATERIALS AND METHODS The institutional ethics committee approved the study protocol. This observational, non-interventional, prospective cohort study was performed over the course of eight months (May - December 2007) in all consecutive adult patients who underwent NPPV in 11 intensive care units at a university-affiliated hospital with a total of 140 ICU beds.

Patients meeting the inclusion criteria were observed daily until ICU discharge or death. Patients with do-not-resuscitate or do-not-intubate orders were excluded.

The researchers carried out the data collection through a form specially developed for this study. These researchers had been trained for three months before gathering the data.

The form was tested and revised during the same period.

The units were visited daily by the researchers. The prospective data were obtained from the patient’s chart and from the ICU staff at the moment of data collection. All of the decisions about the use of NPPV were made by the ICU healthcare teams. Patients whose data were incomplete during collection were excluded from the analysis.

The variables studied were selected through review of the relevant literature. These included the following information: demographic data, Simplified Acute Physiology Score (SAPS II) for the first 24 hours of ICU hospitalization (7); NPPV features; reason for installing NPPV (COPD exacerbation, asthma, neuromuscular disease, acute respiratory failure [acute lung injury or acute respiratory distress syndrome, acute cardiogenic pulmonary edema, congestive heart failure, pneumonia, trauma, upper airway obstruction]); post-extubation, preventive NPPV application (defined as the use of NPPV in patients without signs of respiratory failure with a risk of worsening, for example during the process of fluid balance adjustment, or any patient considered to be at risk of respiratory distress by physicians, such as burn patients (8), or as an adjunctive technique associated with chest physiotherapy treatment (to assist in resolving atelectasis or airway clearance in patients not responding to routine deep breathing exercises and incentive spirometry)) (9-11); days of NPPV use; time between ICU admission and NPPV initiation; time between extubation and NPPV initiation for post-extubation NPPV; type of equipment (CPAP-flow generator, Bipap VisionH, Bipap ST/D-30H, ICU conventional ventilators) at the onset and on the final day of application; NPPV parameters at the onset and on the last day of use; type of mask (nasal, full

RESULTS Between May 1st and December 31st, 2007, all of the ICU patients over 18 years old who underwent NIV treatment were studied. During the study, 407 patients were treated with NIV, and 392 of them were included in the analysis.

The patient characteristics are presented in Table 1. The patient age and SAPS II score were higher in the NPPV failure group. At the time of ICU admission, 4% of the patients came from other hospitals, and most patients came from the ward (41%) and emergency room (28%). Medical disorders (59%) and elective surgery (21%) were the most common reasons for admission. Previous ICU admissions had occurred in 15% of the patients. The previous use of invasive mechanical ventilation was observed in 15% of patients, and 9% had already been treated with NPPV on another occasion. The main indications for NPPV initiation were the following: post-extubation, acute respiratory failure and use as an adjunct to chest physiotherapy.

The NPPV failure rate in this population was estimated to be 35%. The main reasons for intubation (Table 1) were acute respiratory failure (55%) and a decreased level of consciousness (20%).

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Table 1 - Baseline characteristics of the study group according to the outcome of noninvasive positivepressure ventilation.

Characteristics Age, mean (SD), years Male gender, n (%) SAPS II, mean (SD) Types of admission, n (%) Medical Elective surgery Emergency surgery Other* Reasons for installing NPPV Post-extubation Acute respiratory failure COPD* Asthma* Neuromuscular disease* Other chronic respiratory disease* Chest physiotherapy Preventive NPPV use Unknown*

All patients N = 392

Success N = 254

Failure N = 138

p-value, failure vs. success

56 (20) 218 (56) 36 (14)

55 (19) 134 (53) 34 (14)

61 (16) 84 (61) 38 (14)

0.003 0.150 0.010

234 (60) 83 (21) 74 (19) 1 (0.2) n (%) 173 (44) 106 (27) 9 (2) 1 (0,3) 4 (1) 3 (1)

156 (61) 47 (19) 50 (20) 01 (0.4)

78 36 24 0

(57) (26) (17) (0)

0.403 0.104 0.675

113 (45) 65 (26) 7 (3) 1 (0,4) 1 (0,4) 1 (0,4)

60 41 2 0 3 2

(44) (30) (1) (0) (2) (1)

0.931 0.448

73 (19) 16 (4) 5 (1)

53 (21) 11 (4) 0 (0)

20 (15) 5 (4) 5 (4)

0.157 0.943

Table 2 - Arterial blood gas analysis at NPPV onset and on the last noninvasive positive-pressure ventilation day.

Characteristics ABG at NPPV onset, n = 336 pH PCO2 HCO3 Base excess PaO2 /FiO2 ratioa ABG on the last day of NPPV, n = 223 pH PCO2 HCO3 Base excess PaO2 /FiO2 ratiob

p-value, failure vs. success

Success

Failure

N = 221 7.37 (0.08) 38.7 (10.3) 23.6 (18.5) - 2.5 (5.6) 283.9 (139) N = 120

N = 115 7.37 (0.06) 38.4 (10) 21.9 (6.3) - 2.18 (8.1) 226 (89) N = 103

0.612 0.815 0.319 0.670 0.034

7.40 (0.07) 40 (9.7) 24.3 (6.3) - 0.57 (6.7) 312 (137)

7.35 (0.09) 41 (11.9) 21.7 (5.5) - 3.18 (5.6) 245 (123)

,0.001 0.536 0.001 0.002 0.020

NPPV = noninvasive positive-pressure ventilation.

N = 97 (success 65, failure 32), b N = 86 (success 44, failure 42).

a

Initially, the CPAP flow generator was used most commonly for NPPV, followed by the BIPAP Vision and ICU ventilator. At onset, the equipment used to perform NPPV was not associated with success or failure. On the last day of NIV use, the prevalence of CPAP flow generator use was lower, with an increase in BIPAP VisionH use and a reduction in the use of ICU ventilators. Patients examined with a BIPAP Vision presented a higher NPPV failure rate.

The NPPV parameters were also analyzed according to two sets of results collected at onset and on the last day of NPPV application. The initial IPAP and EPAP and CPAP levels were not associated with the NPPV outcome, but the FiO2 levels were significantly higher in the failure group (p = 0.006). On the last day of support, the IPAP, EPAP and FiO2 levels were significantly higher in the NPPV failure group (Table 3).

The rate of complications related to NPPV use was estimated during the study. Vomiting, either associated with aspiration or not, was the most common occurrence. There was a significant association between the occurrence of complications and NPPV failure. Another event associated

NPPV = noninvasive positive-pressure ventilation, COPD = chronic obstructive pulmonary disease. * not analyzed due to an insufficient number of cases.

The prevalence of smoking was 15.5% in the group in which treatment was successful and 20.7% in the group in which NPPV failed, but this association was not significant (p = 0.259).

The time between ICU admission and NPPV initiation was estimated. The overall mean (SD) time was 1.4 (2.4) days. In the successful NPPV group, this mean (SD) period was 1.6 (2.7) days. In the NPPV failure group, it was 1.1 (1.9) days. This difference was not statistically significant (p = 0.201).

The overall median (interquartile range) for the time between extubation and NPPV initiation for post-extubation respiratory failure cases was zero (0-1) days. No difference was found between groups (p = 0.390).

The median (interquartile range) overall length of NPPV use was four (2-6) days. The NPPV failure group presented a shorter period of use than the group in which NPPV was successful [three (2-5) versus four (3-7) days, p = 0.034, respectively].

The data related to arterial blood gas analysis (ABG) at the time of NPPV onset and on the last NPPV day were also collected (Table 2). Some patients had no ABG on these specific days. At the time of NPPV onset, 336 patients had ABG; on the final day of NPPV, this value was 223 patients.

The PaO2/FiO2 ratio was not available in all charts, and all recorded data are presented below. There were no differences in ABG values between the groups studied at the time of NPPV initiation, but on the last day, the failure group presented lower levels of pH, bicarbonate and base excess.

Patients with NPPV failure presented a lower PaO2:FiO2 ratio from the time of NPPV onset onward (Table 2).

The data related to the equipment and NPPV parameters were compared according to outcome at NPPV onset (Table 3) and on the last day of application (Table 4). The full-face mask was the used the most frequently. There was no association between the NPPV interfaces and outcome.

Table 3 - Technical characteristics of noninvasive ventilation: final equipment and parameters applied, according to the noninvasive ventilation results.

Characteristics

All patients N = 392

Success N = 254

NPPV equipment, n (%) CPAP- flow generator 187 (48) 130 (51) BIPAP VisionH 150 (38) 87 (34) BIPAP ST/D-30H 29 (7) 17 (7) ICU ventilator 17 (4) 11 (4) Others* 9 (2) 9 (4) NPPV final parameters, median (IR) IPAP cmH2O 14 (12-16) 14 (12-15) 10 (8-10) 9 (8-10) EPAP cmH2O CPAP cmH2O 10 (10-10) 10 (10-10) FiO2 (%) 35 (30-40) 30 (25-35)

Failure N = 138

p-value, failure vs. success

57 (41) 63 (45) 12 (9) 6 (4) 0 (0)

0.078 0.035 0.602 0.801

14 (14-16) 10 (8-10) 10 (10-10) 40 (35-50)

0.002 0.001 0.114 ,0.001

NPPV = noninvasive positive pressure ventilation, CPAP = continuous positive airway pressure, ICU = intensive care unit, IR = interquartile range, IPAP = inspiratory positive airway pressure, EPAP = expiratory positive airway pressure, FiO2 = inspired oxygen fraction. * not analyzed due to an insufficient number of cases.

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Table 4 - Events associated with NPPV use.

Events associated with NPPV use, n(%) NPPV complications Complications* Vomiting and aspiration Skin lesion Gastric distension Arterial hypotension Intolerance to NIV Air leakage Productive cough Ineffective cough Nasotracheal suctioning Hypersecretion Nosocomial pneumonia

Total N = 392

Success N = 254

Failure N = 138

p-value, failure vs. success

21 (5)

9 (4)

12 (9)

0.050

10 (3) 3 (1) 2 (1) 1 (0) 69 (18) 8 (2) 109 (28) 162 (41) 115 (29) 65 (17) 22 (6)

5 (2) 2 (1) 0 (0) 0 (0) 47 (18) 6 (2) 62 (24) 108 (42) 64 (25) 36 (14) 15 (6)

5 1 2 1 22 2 47 54 51 29 7

0.619 0.718 0.055 0.587 0.020 0.247 0.910

(4) (1) (1) (2) (16) (1) (34) (39) (37) (21) (5)

NPPV = noninvasive positive-pressure ventilation. * Not analyzed due to insufficient data.

with failure was the need for nasotracheal suctioning (Table 5).

The incidence of clinically defined nosocomial pneumonia in the overall study population was 6%. There was no difference between the groups.

The patients with NPPV failure had a higher length of stay in the ICU when compared to the NPPV success group [17 (10-26) days vs. 8(5-14) days]. The overall mortality rate was 24%, and the NPPV failure group also presented a higher mortality rate (9 vs. 51%). There was an association between NPPV failure and mortality, with an estimated odds ratio (95% CI) of 10.6 (5.93 – 19.07).

The multiple logistic regression analysis model using NPPV failure as the dependent variable found that patients with a SAPS II $ 34, IPAP level$15 cmH2O and pH,7.40 were independently associated with NPPV failure and were kept in the final logistic model (Table 6). The goodness-of-fit was confirmed by the Hosmer-Lemeshow test (p = 0.991).

Another indication that stood out was the use of NPPV in association with chest physiotherapy (19%). In this case, the objectives of positive pressure use were to assist in resolving atelectasis and normalizing pulmonary function in patients not responding to routine incentive spirometry and deep breathing. The use of NPPV to assist patients in improving ventilation and recruiting additional gas exchange units to maintain functional residual capacity and vital capacity with relatively little discomfort is instrumental to its success (8). Although most epidemiological studies do not highlight that NPPV indication, this technique is routine in our country and is part of the procedures used in routine hospital practice. The studies that report this type of NPPV application were published as long as 30 years ago (9-11).

The multiple logistic regression analysis model using NPPV failure as the dependent variable identified that patients with IPAP levels$15 cmH2O, SAPS II$34, and pH,7.40 were independently associated with NPPV failure.

The severity of illness, as evaluated by the SAPS II score at the time of ICU admission, was high in our population and independently associated with NPPV failure. Our study also found that a SAPS II score$34 at the time of ICU admission was independently associated with NIV failure. This finding was similar to that reported by Antonelli et al. (23), which recommended avoiding NPPV in ARDS patients with SAPS II$34 due to the high levels of mortality observed in those in whom NPPV had failed. Although our study population was not comprised entirely of ARDS patients, we observed that even in patients without ARDS, SAPS II$34 was

DISCUSSION Aside from the classic indications of NPPV use, such as post-extubation and acute respiratory failure, we have identified two distinct indications for NPPV: prophylactic application and use as an adjunct to chest physiotherapy.

The prophylactic application was defined as the use of NPPV in cases where the indication was based on clinical criteria, such as patients who needed additional support but did not require ventilatory support immediately, as in cases of fluid replacement. This indication rate was only 4%.

Table 6 - Factors associated with noninvasive ventilation failure – multivariate analysis.

Table 5 - Factors associated with noninvasive ventilation failure – multiple logistic regression, final model.

Variables Variables Age EPAP IPAP FiO2 EPAP by FiO2 EPAP by age Age by IPAP

OR

Lower

Upper

p-value

1.094 0.089 4.452 0.829 1.029 1.026 0.979

0.887 0.013 1.529 0.640 1.000 1.001 0.962

1.349 0.617 12.968 1.073 1.059 1.051 0.995

0.400 0.014 0.006 0.155 0.051 0.040 0.011

SAPS II, points ,34 $34 IPAP, cm H2O ,15 $15 pH $7.4 ,7.4

OR = odds ratio, EPAP = expiratory positive airway pressure, IPAP = inspiratory positive airway pressure, FiO2 = inspired oxygen concentration.

OR

95% CI

p-value

1.0 2.97

1.31-6.99

0.010

1.0 3.13

1.34-7.71

0.010

1.0 2.72

1.21-6.28

0.017

OR = odds ratio, IPAP = inspiratory positive airway pressure.

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reported by Schettino et al. (6) is that our study population included only a small proportion of those with chronic respiratory disease.

The availability of a CPAP flow generator in the ICU contributed to the frequent use of this type of equipment at the time of NIV onset. We observed that this device was shifted to the bilevel setting during treatment. At that time, NIV was performed using a CPAP flow generator, Bipap STD 30, Bipap Vision or conventional ICU ventilator. We did not find any association with NIV outcome. A recently published guideline by Keenan et al. (2) did not describe any influence of the ventilatory mode or equipment on the NIV outcome.

Univariate analysis showed that NIV was likely to fail in patients requiring nasotracheal suctioning. This procedure was required due to a cough that occurred in 29% of the study population. It seems important to evaluate cough severity, even in patients with NIV support. According to the literature, patients with weak coughs after planned extubation may benefit from NIV, but this procedure requires staff with expertise in invasive airway management (2). In a prospective multicenter survey, Carlucci et al. (14) estimated that 52 of the 108 patients (48%) submitted to NPPV discontinued the treatment prematurely. This was due to an inability to manage copious secretions in 32% of the patients. Our study estimated that 37% of the patients in the NPPV failure group required nasotracheal suctioning.

The overall incidence of nosocomial pneumonia was 6%.

Despite differences in the diagnostic criteria, we observed a similar rate in the literature (15). A matched case-control study compared the rates of nosocomial infections among patients submitted to NPPV and those submitted to invasive mechanical ventilation. The study concluded that the use of NPPV was associated with a lower risk of nosocomial infections and better outcomes.

Patients with NIV failure had a poor prognosis as well as a high mortality rate (51%). The overall mortality rate was 24%. Alsous et al. (13), in a retrospective study at a community teaching hospital, also found a higher mortality among those in whom BIPAP had failed. They considered some factors that contributed to this fact: 1) a ‘‘do-notintubate’’ order from 7 of 18 patients, 2) less physiological derangement among the subcohort of BIPAP success patients, and 3) the attempt at a trial of BIPAP rather than intubation may prolong cardiorespiratory decompensation and worsen patient outcomes. This study concluded that BIPAP success was associated with significantly better outcomes (ICU length of stay and mortality). This finding may indicate that NIV failure must be avoided through appropriate approaches, such as staff education. Two studies carried out at a medical ICU where there was a protocol and a well-trained staff presented lower mortality rates. The study by Girault et al. (3) conducted at a 22-bed medical ICU presented an overall mortality rate of 11%; this rate was 16% in another study by Meduri et al. (5).

An important point with respect to interpretation of our results is that the patients were not transferred to chronic respiratory units because this type of facility is rare in our country. Almost all patients remain in the ICU when they are dependent on ventilatory support. This factor may also have affected the observed mortality rates. We consider that educational approaches and the use of protocols can improve NIV outcomes, as observed in the studies mentioned above.

associated with treatment failure. Another study by Carlucci et al. (14) also found that SAPS II was an independent risk factor for secondary intubation. The authors maintain that the SAPS II score indicates that the most severely ill patients are poor candidates for NPPV. However, they recommended that the severity score cannot be used as such on an individual basis.

In relation to the NPPV pressure levels used, in the multivariate analysis, the value of IPAP$15 cmH2O was independently associated with NPPV failure. Among the studies that evaluated NPPV parameters, only the study by Antonelli et al. (23) reported higher IPAP values in the failure group, but this association was not maintained in the multivariate analysis. Other studies (6,20) did not report such a relationship, and we observed lower levels of IPAP and EPAP in both studies. Our findings suggest that the use of higher values of IPAP ($15 cmH2O) indicate a higher probability of NPPV failure.

The failure group presented lower levels of pH, bicarbonate and base excess, and the PaCO2 did not differ between groups. According to Rana et al. (18), the presence of metabolic acidosis was predictive of NPPV failure, even when shock was not present at the time NPPV was initiated.

Although patients who failed NPPV tended to have a higher level of serum lactate, this did not reach a statistically significant value, suggesting that not only tissue hypoperfusion but also metabolic acidosis per se, may be associated with poor NPPV outcomes. Their finding are consistent our results.

We also suggest that lower levels of pH may be a good marker for a poor outcome after NPPV, as shown in Tables 2 and 6.

The overall time between extubation and NPPV initiation for post-extubation respiratory failure cases was estimated.

Half of the patients used NPPV on the same day of extubation, and only 25% used NPPV after the first postoperative day. No difference was found between the groups. We observed that clinical practice reflects the findings of the literature, which recommends the early use of NPPV after extubation (19-22).

The time of NPPV use in patients with failure was shorter.

This finding is similar to that reported in the literature (13,24). Our hypothesis is that this is most likely due to the negative impact of delayed intubation patients on the prognosis (25).

In this unselected patient sample, we observed that the NIV failure rate was similar to that reported in other cohort studies (3,5). We considered the overall failure rate because our study did not classify patients according to arterial blood gas values. During the study period, there was no standardized NPPV utilization protocol, and some patients did not have arterial blood gas samples. Rather, both studies mentioned were based on standardized protocols. The study by Meduri et al. (5) applied a protocol in a large group of medical patients, and Girault et al. (3) presented two years of NIV experience at a specialized medical ICU with 22 beds. Another study by Schettino et al. (6) reported data related to NIV use over a period of one year at a university-affiliated hospital. All patients submitted to NIV at this hospital were included. The authors estimated the rate of NIV failure in the ICU setting to be 49%. They considered that ICU-managed patients were the sickest and that this population was composed of patients that could not be managed in the general ward. Patients maintained on the ward were able to breathe spontaneously for at least one hour. One important difference between our study and that

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The present study provides information about NIV as part of the clinical practice at a large university-affiliated hospital. Our intention was to analyze the main features of this procedure in a non-controlled environment, as occurs during a clinical trial. During the study period, there was no standardized NIV protocol in the observed units, but we also consider that the activities of research and teaching may influence the results at a university-affiliated hospital.

This study presented the demographic, clinical and technical features of NPPV use in clinical practice among the unselected ICU patients. Our results showed that patients with NPPV failure have a poor prognosis, with a high mortality rate and a prolonged length of stay in the ICU. The more severe patients (higher SAPS II score and lower pH) had higher failure rates. High levels of IPAP also indicate a higher risk of failure. In our study, we found that physiotherapy uses the NPPV for therapeutic purposes and not only in cases of acute respiratory failure.

A major objective of this study is to present the main features of the use of NPPV in patients admitted to the intensive care unit. We understand that the study design weakens some of our conclusions because it is an uncontrolled study without an intervention. However, our results are not discordant with those observed in most previously published studies.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

ACKNOWLEDGMENTS We are grateful to Felipe Granado de Souza and Fabio Montesano, statisticians at the Federal University of Sao Paulo, for their help in the analysis of these data.

16.

17.

AUTHOR CONTRIBUTIONS Yamauchi LY participated in the study design, performed the statistical analyses, contributed to data analysis and interpretation, and drafted the manuscript. Travaglia TC participated in the study design, data collection, and drafted the manuscript. Bernardes SR and Figueiroa MC participated in the study design and data collection. Tanaka C contributed to the study analysis and data interpretation. Fu C participated in the study design, made substantial contributions to data analysis and interpretation, and drafted the manuscript. All authors have read and approved the final version of the manuscript.

18.

19.

20.

REFERENCES

21.

1. Hill NS. Noninvasive positive-pressure ventilation. In: Principles and practice of mechanical ventilation. Mc Graw-Hill; 2006.p.433-71.

2. Keenan SP, Sinuff T, Burns KEA, Muscedere J, Kutsogiannis J, Mehta S, et al. Clinical practice guidelines for the use of noninvasive positivepressure ventilation and noninvasive continuous positive airway pressure in the acute care setting. CMAJ. 2011;183(3):E195-E214, http://dx.doi.org/10.1503/cmaj.100071.

3. Girault C, Briel A, Hellot MF, Tamion F, Woinet D, Leroy J, Bonmarchand G. Noninvasive mechanical ventilation in clinical practice: a 2-year experience in a medical intensive care unit. Crit Care Med. 2003; 31(2):552–559, http://dx.doi.org/10.1097/01.CCM.0000050288.49328.F0.

4. Sinuff T, Cook D, Randall J, Allen C. Noninvasive positive-pressure ventilation: a utilization review of use in a teaching hospital CMAJ 2000;163(8):969-73.

5. Meduri GU, Turner RE, Abou-Shaia N, Wunderink R, Tolley E. Noninvasive positive pressure ventilation via face mask: first-line intervention in patients with acute hypercapnic and hypoxemic

22.

23.

24.

25.

772

respiratory failure. CHEST. 1996;109(1):179-93, http://dx.doi.org/ 10.1378/chest.109.1.179.

Schettino G, Altobelli N, Kacmarek RM. Noninvasive positive-pressure ventilation in acute respiratory failure outside clinical trials: experience at the Massachusetts General Hospital. Crit Care Med. 2008;36(2):441– 447, http://dx.doi.org/10.1097/01.CCM.0000300084.67277.90.

Le Gall JR, Lemeshow S, Saulnier F. A New Simplified Acute Physiology Score (SAPS II) based on a European/North American multicenter study. JAMA. 1993;270(24):2957-63. Erratum in: JAMA 1994;271(17):1321.

Endorf FW, Dries DJ. Noninvasive Ventilation in the Burned Patient.

J Burn Care Res. 2010;31(2):217-28, http://dx.doi.org/10.1097/BCR. 0b013e3181d0f62c Holland AE, Denehy L, Ntoumenopoulos G, Naughton MT, Wilson JW. Non-invasive ventilation assists chest physiotherapy in adults with acute exacerbations of cystic fibrosis. Thorax. 2003;58(10):880–4, http:// dx.doi.org/10.1136/thorax.58.10.880.

Ricksten SE, Bengtsson A, Soderberg C, Thorden M, Kvist H. Effects of periodic positive airway pressure bymask on postoperative pulmonary function. Chest. 1986(6);89;774-81, http://dx.doi.org/10.1378/ chest.89.6.774.

Stock MC, Downs J B, Gauer PK, Alster JM, Imrey PB. Prevention of postoperative pulmonarycomplications with CPAP, incentive spirometry,and conservative therapy. Chest. 1985;87(2):151-7, http://dx.doi.org/ 10.1378/chest.87.2.151.

CDC definitions for nosocomial infections, 1988. Am Rev Respir Dis. 1989;139(4):1058–9.

Alsous F, Amoateng-Adjepong Y, Manthous CA. Noninvasive ventilation: experience at community hospital. Intensive Care Med. 1999;25(5):458-63, http://dx.doi.org/10.1007/s001340050880.

Carlucci A, Richard JC, Wysocki M, Lepage E, Brochard L, and the SRLF Collaborative Group on Mechanical Ventilation. Noninvasive versus conventional mechanical ventilation an epidemiologic survey.

Am J Respir Crit Care Med. 2001;163(4): 874–80.

Girou E, Schortgen F, Delclaux C, Brun-Buisson C, Blot F, Lefort Y, et al.

Association of noninvasive ventilation with nosocomial infections and survival in critically ill patients. JAMA. 2000;284(18):2361-7, http:// dx.doi.org/10.1001/jama.284.18.2361.

III Consenso Brasileiro de Ventilac¸a˜o Mecaˆnica: Ventilac¸a˜o Na˜o Invasiva com Pressa˜o Positiva. Jornal Brasileiro de Pneumologia. 2007;33(supl 2):S54-S70.

Paus-Jenssen ES, Reid JK, Cockcroft DW, Laframboise K, Ward HA. The Use of Noninvasive Ventilation in Acute Respiratory Failure at a Tertiary Care Center. Chest. 2004;126(1):165–72, http://dx.doi.org/10.1378/ chest.126.1.165.

Rana S, Jenad H, Gay PC, Buck CF, Hubmayr RD, Gajic O. Failure of non-invasive ventilation in patients with acute lung injury: observational cohort study. Crit Care. 2006;10(3):R79, http://dx.doi.org/10.1186/ cc4923.

Ferrer M, Valencia M, Nicolas J M, Bernadich O, Badia J R, Torres A. Early noninvasive ventilation averts extubation failure in patients at risk.

A randomized trial. Am J Respir Crit Care Med. 2006;173(2):164–70.

Nava S, Gregoretti C, Fanfulla F, Squadrone E, Grassi M,Carlucci A, et al.

Noninvasive ventilation to prevent respiratory failure after extubation in high-risk patients. Crit Care Med. 2005;33(11):2465-70, http:// dx.doi.org/10.1097/01.CCM.0000186416.44752.72.

Trevisan C, Vieira S. Noninvasive mechanical ventilation may be useful in treating patients who fail weaning from invasive mechanical ventilation: a randomized clinical trial. Crit Care. 2008;12(2):R51, http://dx.doi.org/10.1186/cc6870.

Nery P, Pastore L, Carvalho CRR, Schettino G. Shortening ventilatory support with a protocol based on daily extubation screening and noninvasive ventilation in selected patients Clinics. 2011;66(5):759-66.

Antonelli M, Conti G, Esquinas A, Montini L, Maggiore SM, Bello G, et al.

A multiple-center survey on the use in clinical practice of noninvasive ventilation as a first-line intervention for acute respiratory distress syndrome. Crit Care Med. 2007;35(1):18-25, http://dx.doi.org/10.1097/ 01.CCM.0000251821.44259.F3.

Antonelli M, Conti G, Moro ML, Esquinas A, Gonzalez-Diaz G, Confalonieri M, et al. Predictors of failure of noninvasive positive pressure ventilation in patients with acute hypoxemic respiratory failure: a multicenter study. Intensive Care Med. 2001;27(11):1718-28.

Epstein SK, Ciubotaru RL, Wong JB. Effect of failed extubation on the outcome of mechanical ventilation. Chest. 1997;112(1):186-92, http:// dx.doi.org/10.1378/chest.112.1.186.

CLINICS 2012;67(7):773-778

DOI:10.6061/clinics/2012(07)12

CLINICAL SCIENCE

Pulse pressure variation and prediction of fluid responsiveness in patients ventilated with low tidal volumes Clarice Daniele Alves de Oliveira-Costa,I,II Gilberto Friedman,I,II,III Sı´lvia Regina Rios Vieira,I Le´a FialkowI I

Universidade Federal do Rio Grande do Sul, Hospital de Clı´nicas de Porto Alegre, Department of Intensive Care Medicine, Porto Alegre/RS, Brazil.

Complexo Hospitalar Santa Casa de Porto Alegre, Porto Alegre/RS, Brazil. III Hospital de Clı´nicas de Porto Alegre, Porto Alegre/RS, Brazil.

II

OBJECTIVE: To determine the utility of pulse pressure variation (DRESPPP) in predicting fluid responsiveness in patients ventilated with low tidal volumes (VT) and to investigate whether a lower DRESPPP cut-off value should be used when patients are ventilated with low tidal volumes.

METHOD: This cross-sectional observational study included 37 critically ill patients with acute circulatory failure who required fluid challenge. The patients were sedated and mechanically ventilated with a VT of 6-7 ml/kg ideal body weight, which was monitored with a pulmonary artery catheter and an arterial line. The mechanical ventilation and hemodynamic parameters, including DRESPPP, were measured before and after fluid challenge with 1,000 ml crystalloids or 500 ml colloids. Fluid responsiveness was defined as an increase in the cardiac index of at least 15%.

ClinicalTrial.gov: NCT01569308.

RESULTS: A total of 17 patients were classified as responders. Analysis of the area under the ROC curve (AUC) showed that the optimal cut-off point for DRESPPP to predict fluid responsiveness was 10% (AUC = 0.74). Adjustment of the DRESPPP to account for driving pressure did not improve the accuracy (AUC = 0.76). A DRESPPP$10% was a better predictor of fluid responsiveness than central venous pressure (AUC = 0.57) or pulmonary wedge pressure (AUC = 051). Of the 37 patients, 25 were in septic shock. The AUC for DRESPPP$10% to predict responsiveness in patients with septic shock was 0.84 (sensitivity, 78%; specificity, 93%).

CONCLUSION: The parameter DRESPPP has limited value in predicting fluid responsiveness in patients who are ventilated with low tidal volumes, but a DRESPPP.10% is a significant improvement over static parameters. A DRESPPP$10% may be particularly useful for identifying responders in patients with septic shock.

KEYWORDS: Cardiac Output; Pulse Pressure Variation; Fluid Responsiveness; Low Tidal Volume.

Oliveira-Costa CD, Friedman G, Vieira SRR, Fialkow L. Pulse pressure variation and prediction of fluid responsiveness in patients ventilated with low tidal volumes. Clinics. 2012;67(7):773-778.

Received for publication on January 12, 2012; First review completed on February 5, 2012; Accepted for publication on March 25, 2012 E-mail: beto_gremio@hotmail.com Tel.: 55-51-3359-8223

(4). Avoiding unnecessary fluid loading has been shown to have a positive effect on patient outcomes (5,6).

Pulse pressure variation (DRESPPP) is one of the most accurate dynamic parameters used at the bedside to identify fluid responsiveness in patients with acute circulatory failure who are undergoing invasive mechanical ventilation (3,7).

However, most studies have evaluated patients ventilated with large tidal volumes ($8 ml/kg). The validity of DRESPPP in identifying fluid responsiveness is still under debate when using lower tidal volumes (7-12).

In preload-dependent patients on mechanical ventilation, DRESPPP is primarily the result of an inspiratory decrease in the right ventricular (RV) preload secondary to an increase in the pleural pressure, which is affected by the tidal volume (13). Cyclic changes in the stroke volume are due to pleural and intrathoracic pressure variations in this group of patients (8,14). In patients ventilated with low tidal volumes, the variation in lung volume and airway pressure may not be sufficient to significantly change the pleural

INTRODUCTION Volume expansion is frequently used to treat critically ill patients with acute circulatory failure. The goal of volume expansion is to increase the left ventricular stroke volume, which consequently increases the cardiac output (1,2) However, only approximately 50% of patients with acute circulatory failure will respond to a fluid challenge (preloaddependent patients) (3). Therefore, the ability to predict fluid responsiveness in critically ill patients is crucial, particularly in patients with acute respiratory distress syndrome (ARDS) due to increased alveolar-capillary membrane permeability

Copyright ß 2012 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.

No potential conflict of interest was reported.

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pressure, venous return or ventricular filling (15,16).

Adjusting the DRESPPP to account for the driving pressure (DP, the difference between the plateau pressure and positive end expiratory pressures) could be useful in identifying responders with a DRESPPP,13% (9). However, the adjustment of DRESPPP based on the DP was shown to be as inaccurate as DRESPPP alone in patients who were ventilated with a VT,8 ml/kg IBW (8). In lungs with normal compliance values, low tidal volumes induce small variations in the DP, particularly when the DP#20 cm H2O (9).

The parameter DRESPPP may be useful in guiding fluid therapy following lung injuries, but several physiological mechanisms may limit its validity. The current literature regarding the effects of DRESPPP during ventilation with low tidal volumes is unclear, and conflicting conclusions have been reported (10,12). The present study was designed to determine the value of DRESPPP in predicting fluid responsiveness in patients ventilated with low tidal volumes and investigate whether a lower DRESPPP cut-off point should be used when patients are ventilated with low tidal volumes.

Hemodynamic Parameters Variations in the arterial pulse pressure were visualized on bedside monitors (HP S66 and PHILIPS IntelliVue, MP60, Germany) and measured with the cursor for five breathing cycles. The DRESPPP was calculated using the following equation: DRESP PPð%Þ ~100xðPPmax PPminÞ=½ðPPmaxzPPminÞ=2 where PPmax and PPmin are the maximal pulse pressure at inspiration and expiration, respectively (1).

A pulmonary arterial catheter (Edwards Healthcare, Irvine, CA) was used to measure the cardiac output according to the thermal dilution method (three injections of a 10 ml 0.9% saline solution); the systolic, diastolic and mean pulmonary arterial pressures; the pulmonary artery occlusion pressure (PAOP, mmHg); the central venous pressure (CVP, mmHg); and the mixed venous saturation (SvO2). The mean arterial pressure (MAP, mmHg), which was measured using the arterial line and heart rate (HR, bpm), was also recorded. All of the measurements were recorded at the end of expiration before and after FC. Patients were defined as fluid responders if the cardiac index increased by at least 15% relative to the baseline.

MATERIALS AND METHODS This cross-sectional observational study included patients admitted to the Intensive Care Unit at the Hospital das Clı´nicas de Porto Alegre (HCPA) who required fluid challenge (FC). The study was approved by the HCPA Research Ethics Committee. Informed consent was waived because no interventions were performed on the patients.

Ventilation Parameters The following ventilatory parameters were measured: inspiratory and expiratory tidal volumes, respiratory rate (RR), plateau pressure (Pplat, cmH2O), peak pressure (Ppeak, cmH2O), total positive end-expiratory pressure (PEEPtot), static compliance (Cst) and driving pressure (DP = Pplat-PEEP). All of the measurements were recorded before and after FC.

Patients A total of 38 patients admitted to the HCPA ICU who received invasive mechanical ventilation between May 2006 and October 2009 were included. The following inclusion criteria were used: i) age$16 years; ii) hemodynamic instability, defined as the need for norepinephrine infusion and/or intravascular fluid administration to maintain systolic arterial blood pressure .90 mmHg; iii) arterial line in place (radial or femoral); and iv) pulmonary arterial catheter in place. The exclusion criteria were the presence of cardiac arrhythmia, pneumothorax, heart valve disease or intracardiac shunt and previously diagnosed right ventricular insufficiency. The patients were scheduled to undergo FC with colloid or crystalloid solutions as prescribed by the attending physician.

Statistical Analysis The sample size was defined as 38 patients to estimate the correlation between CI and DRESPPP 0.5 (moderate to high magnitude), with a level of significance of 0.05 and a power of 90%.

The effects of FC on the hemodynamic parameters were assessed using the paired Student’s t-test for normally distributed variables or the nonparametric Wilcoxon Signed Rank test for non-normally distributed variables. The hemodynamic parameters between both groups at baseline Table 1 - Patient Characteristics.

Study Protocol The patients were sedated with midazolan and fentanil (scores of -4 to -5 in the Richmond Agitation Sedation Scale) (17) and ventilated in a controlled pressure or controlled volume mode (Servo I system v.12 or Servo 900 C, Siemens, Sweden) with a VT,8 ml/kg IBW (51+0.9 [height in cm152.9] for men and 45.5 + 0.91 [height in cm- 152.9] for women) (7). The ventilatory and hemodynamic variables were measured before and after FC with the patients in a supine position. Zero pressure was measured at the midaxillary line. The correct position of the pulmonary artery catheter in West’s zone 3 was confirmed as described in the literature (18).

Fluid challenge was performed with 1000 ml 0.9% saline solution or lactated Ringer’s solution (n = 36) or 500 ml 6% hydroxy-ethyl-starch solution 130/0.4 for 30 minutes (n = 2).

Variables Number Age, years Male sex (%) APACHE II Septic Shock (%) Liver Transplantation* (%) Acute Pancreatitis (%) Cardiogenic Shock (%) Aortic Surgery* (%) ARDS (%) Crystalloids (%) Colloids (%)

Statistics 37 54¡17 (19-81) 20 (54%) 28¡ 8 (13-51) 25 (67.5%) 7 (19%) 3 (8%) 1 (2.7%) 1 (2.7%) 10 (27%) 35 (94.5%) 2 (5.5%)

Mean ¡ standard deviation (minimum and maximum) or percentage. * Immediate post-operative state.

APACHE II-The Acute Physiology and Chronic Health Score II; ARDS- Adult Respiratory Distress Syndrome.

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Table 2 - The hemodynamic and ventilatory data of responders and nonresponders.

Baseline (n = 37)

Responders (n = 17) Baseline

VT exp ml/kg PEEP, cmH2O Ppl, cmH2O DP PPV/DP Cst, cmH2O HR, bpm RR HR/RR MAP, mmHg MPAP, mmHg CVP, mmHg PAOP, mmHg PPV, % CI, L/min/m2 SVI (ml/m2) SvO2 (%) Lactate, mEq/L Norepinephrine, mcg/kg/min Dobutamine, mcg/kg/min

6.5 [6.0-6.5] 21.6¡6.4 12.6¡4 0.56¡0.56 34¡15 100¡25

70¡12 28¡8 11¡5 14¡5 6.5¡6 3¡0.9 64.5¡14 4¡3.7 0.37¡0.52 0.75¡1.4

Nonresponders (n = 20) Fluids

6.5 [6.2-6.5] 7 (6-12) 20.4¡6.0 11.1¡3.2 0.83¡0.63 38¡14 105¡28 19.9¡2.6 5.35¡1.37 68¡9 28¡10 12¡6 13¡6 8.8¡6.2# 2.9¡1.3 28¡12 67¡15 3.0[1.9-4.0] 0.4 [0.2-0.6] (n = 15) 2.3¡1.6 (n = 4)

103¡22 5.19¡1.08 76¡11 32¡10 16¡7 18¡6 5.1¡4.6# 4.1¡2.1 40¡16 71¡8

Baseline 6.5 [6.0-6.5] 8 (6-10) 22.6¡6.6 12.9¡3.8 0.32¡0.35 31¡16 94¡21 20.5¡2.0 4.6¡1.12 70¡14 28¡5 11¡5 14¡5 4.4¡5.1 3.4¡1.3 37¡14 62¡13 2.0[1.3-4.2] 0.2 [0.1-0. 6] (n = 13) 3.2¡0.9 (n = 6)

p-value Fluids 0.75 0.65 0.30 0.15 0.005 0.18

90¡19 4.48¡1.03 73¡17 32¡7 16¡6 18¡6 2.6¡3.1 4.1¡2.1 37¡15 68¡11

PEEP: end expiratory pressure; VT exp: expiratory tidal volume; Ppl: plateau pressure; PPV/DP: PPV/driving pressure index; Cst: static complacence; HR: heart rate; MAP: mean arterial pressure; MPAP: mean pulmonary artery pressure; CVP: central venous pressure; PAOP: pulmonary artery occluded pressure; PPV: pulse pressure variation; CI: cardiac index; SvO2: O2 mixed venous saturation; VP: vasopressor; IN: inotropic.

and after FC were compared using the two-sample Student’s t-test or the nonparametric Mann-Whitney U test.

The results were expressed as the means¡SD or the medians (25-75th percentiles).

Receiver operating characteristic (ROC) curves were constructed to evaluate the ability of DRESPPP, DRESPPP/DP, CVP and PAOP to predict fluid responsiveness. The optimal cut-off value for the DRESPPP ROC curve was determined for the study cohort. The following measures of diagnostic performance were calculated: sensitivity, specificity, predictive values and likelihood ratios. Linear correlations were tested using the Spearman rank method. The data were analyzed using SPSS 15.0. A p-value,0.05 was considered to be significant.

DRESPPP for DP, similar results were obtained: AUC equal to 0.76; 95% 0.60-0.90; sensitivity, 47%; specificity, 95%; PPV, 89%; NPV, 68%; and positive and negative likelihood ratios

RESULTS One of the 38 patients was excluded due to cardiorespiratory arrest that occurred during the study. The general characteristics of the 37 patients are summarized in Table 1.

Twenty-five of the patients (68%) were in septic shock, and 11 patients were in distributive shock as a result of various causes. None of the patients were in cardiogenic shock.

Table 2 shows the baseline hemodynamic and ventilation parameters and data for the responders (17 patients) and nonresponders (20 patients). There were no statistically significant differences between the responders and nonresponders in terms of age (57¡16 vs. 53¡18 years) or APACHE II scores (28¡05 vs. 28¡10).

Analysis of the study cohort revealed that the optimal DRESPPP cut-off value for identifying responders was 10%, with an area under the ROC curve (AUC) equal to 0.74 (95% CI: 0.56-0.9; sensitivity, 53%; specificity, 95%; PPV, 90%; NPV, 70.4%; and positive and negative likelihood ratios of 9.4 and 0.34, respectively) (Figure 1). After adjusting the

Figure 1 - Receiver operating characteristic (ROC) curves comparing pulse pressure variation (DRESPPP), DRESPPP adjusted to driving pressure (DRESPPP/DP), central venous pressure (ROC area: 0.57 [0.38-0.76]) and pulmonary artery occlusion pressure (ROC area 0.51 [0.32-0.70]) to determine patient responses to volume expansion. The area under the curve for DRESPPP or DRESPPP/DP is greater than that for the central venous pressure (CVP) or pulmonary artery occlusion pressure (PAOP). PPV: positive predictive value; NPV: negative predictive value; LR +: positive likelihood ratio; LR: negative likelihood ratio.

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Figure 2 - Linear correlation between the pulse pressure variation (DRESPPP) and cardiac index (CI) variation immediately after a fluid challenge.

static parameters. The primary finding of this study is that although the DRESPPP had a low sensitivity, almost half of the responders had a DRESPPP ,10%. With the exception of one patient, all of the patients with a DRESPPP$10% responded to FC, which indicates that this parameter is a useful variable for evaluating fluid responsiveness.

The mean baseline DRESPPP in this study was low (6.5%).

There are several explanations for this observation. First, the patients had already been resuscitated. Second, the patients were ventilated with a low VT (6.5 ml/kg). The use of low VTs in patients with normal lung compliance scores or ARDS induces less pronounced changes in blood pressure waveforms because cyclic variations in the pleural pressure are influenced by the magnitude of the tidal volume and, to a lesser extent, DP (1,7-10). As in previous studies, this phenomenon explains the lower cut-off value for DRESPPP in patients ventilated with reduced tidal volumes, particularly when the DP#20 cmH2O (9). The mean DP in this study was low (12.6ยก4), and our attempts to adjust for DP (8) did not increase the accuracy. We observed a greater DRESPPP/DP in the subgroup of responders and a DRESPPP.10% compared with the patients with a DRESPPP,10%. In patients with decreased lung compliance (acute lung injury/ARDS), the impact of the alveolar pressure on pleural pressure is even less pronounced and is not linear. The association between low tidal volumes and low alveolar compliance (high driving pressure) decreases the effect of positive

of 9.4 and 0.56, respectively. The DRESPPP and DRESPPP/DP were more accurate than CVP and PAOP for identifying fluid responsiveness (Figure 1).

Figure 2 shows the relationship between the DRESPPP and cardiac index variation. Of the ten patients with a DRESPPP$10%, nine were true responders. Twenty-seven patients had a DRESPPP,10%, but only nineteen were nonresponders. The other eight responders showed cardiac index variations of up to 75%. Of the responders, the DRESPPP/DP index was significantly lower in eight patients with a DRESPPP ,10% compared with nine patients with a DRESPPP.10% (0.33ยก0.24 vs. 1.31ยก0.5; p,0.05).

Twenty-five patients were in septic shock (15 nonresponders with a DRESPPP,10%, one nonresponder with a DRESPPP$10%, seven responders with a DRESPPP$10%, and two responders with a DRESPPP,10%). For the 10% cut-off point, the AUC was 0.84, the sensitivity was 77.8%, the specificity was 93.3%, the PPV was 87.5%, and the NPV was 88.2%. The positive and negative likelihood ratios were 0.13 and 0.23, respectively (Figure 3).

DISCUSSION The data in the present study support previous studies (710,12) by demonstrating that DRESPPP has limitations in predicting fluid responsiveness in patients ventilated with low tidal volumes but is an improvement over conventional

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Figure 3 - Receiver operating characteristic curve for pulse pressure variation (DRESPPP) in patients with septic shock ventilated with low tidal volumes. A cut-off value of 10% improved the negative predictive value (NPV) and positive predictive value (PPV).

Predicting fluid responsiveness is particularly important in patients with ARDS. There are few studies that have properly addressed this issue. Two studies reported AUCs similar to the results in this study with different DRESPPP values. In 22 patients with ARDS, Huang et al. reported a cut-off point of 11.8% with a low sensitivity (68%), a high specificity (100%), and an AUC of 0.77 (10) using similar methods. Lakhal et al. recently evaluated 65 patients with ARDS after a fluid challenge to determine whether the CI increased by 10% (12) and reported a cut-off point of 5% with a sensitivity of 73% and specificity of 85%. The authors criticized Huang et al. interpretation that DRESPPP is a reliable predictor of fluid responsiveness because they considered the lower bound of the AUC 95% confident interval to limit accuracy. The lower bound in the present study was only 0.51. Although we agree that the accuracy may be limited, we also agree with Huang et al. in that a high specificity is sufficient to justify the use of DRESPPP at the bedside. However, this study is different because we only studied ten patients with ARDS, and six were nonresponders with a DRESPPP,10%.

There were no significant differences in the hemodynamic parameters between the groups. It should be noted that 28 of the patients (75%) were being treated with norepinephrine, with similar proportions in both groups. The use of vasopressors does not appear to mask the hemodynamic impacts of mechanical ventilation (23).

pressure on venous return and myocardial contractility (19).

Third, the DRESPPP depends on the RR and HR/RR ratios.

The DRESPPP is insignificant in responders when this ratio is below 3.6, as shown in De Backer et al. (20). Although the mean HR/RR ratio was lower in the nonresponders (p = NS), it was much higher than 3.6.

PEEP induces a decrease in the cardiac output due to the negative effect of increased pleural pressure on right ventricular filling and increased transpulmonary pressure on the right ventricular afterload in a hypovolemic state, which increases the pulse pressure variation (21). However, the pulse pressure variation results from a complex interaction between hemodynamic and ventilatory mechanisms, including VT, PEEP levels, lung volume and lung compliance (8). Cyclic changes in the pleural pressure are mostly determined by the magnitude of VT (13). In this study, the effects of PEEP on DRESPPP appeared to be less important because the mean PEEP levels were low (,10 cmH20). PEEP levels were similar between responders and nonresponders.

Pulmonary artery hypertension and/or right ventricular dysfunction may also contribute to false positive cases when identifying responders (22,23). We did not evaluate signs of right ventricular failure, which can compromise the flow response to an FC. However, the mean pulmonary artery pressure was comparable to values observed in other studies in patients with ARDS (10,12).

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3. Michard F, Teboul JL. Predicting fluid responsiveness in ICU patients: a critical analysis of the evidence. Chest. 2002;121(6):2000-8, http:// dx.doi.org/10.1378/chest.121.6.2000.

4. Reuter DA, Bayerlein J, Goepfert MS, Weis FC, Kilger E, Lamm P, et al.

Influence of tidal volume on left ventricular stroke volume variation measured by pulse contour analysis in mechanically ventilated patients.

Intensive Care Med. 2003;29(3):476-80.

5. Wiedemann HP, Wheeler AP, Bernard GR, Thompson BT, deBoisblanc B, Connors AF Jr, et al. Comparison of two fluid-management strategies in acute lung injury. N Engl J Med. 2006;354(24):2564-75.

6. Westphal GA, Silva E, Goncalves AR, Caldeira FM, Poli-de-Figueiredo LF. Pulse oximetry wave variation as a noninvasive tool to assess volume status in cardiac surgery. Clinics. 2009;64(4):337-43.

7. De Backer D, Heenen S, Piagnerelli M, Koch M, Vincent JL. Pulse pressure variations to predict fluid responsiveness: influence of tidal volume. Intensive Care Med. 2005;31(4):517-23, http://dx.doi.org/ 10.1007/s00134-005-2586-4.

8. Vallee F, Richard JC, Mari A, Gallas T, Arsac E, Verlaan OS, et al. Pulse pressure variations adjusted by alveolar driving pressure to assess fluid responsiveness. Intensive Care Med. 2009;35(6):1004-10, http:// dx.doi.org/10.1007/s00134-009-1478-4.

9. Muller L, Louart G, Bousquet PJ, Candela D, Zoric L, de La Coussaye JE, et al. The influence of the airway driving pressure on pulsed pressure variation as a predictor of fluid responsiveness. Intensive Care Med. 2010;36(3):496-503, http://dx.doi.org/10.1007/s00134-009-1686-y.

10. Huang CC, Fu JY, Hu HC, Kao KC, Chen NH, Hsieh MJ, et al. Prediction of fluid responsiveness in acute respiratory distress syndrome patients ventilated with low tidal volume and high positive end-expiratory pressure. Crit Care Med. 2008;36(10):2810-6, http://dx.doi.org/10.1097/ CCM.0b013e318186b74e.

11. Kim HK, Pinsky MR. Effect of tidal volume, sampling duration, and cardiac contractility on pulse pressure and stroke volume variation during positive-pressure ventilation. Crit Care Med. 2008;36(10):2858-62, http://dx.doi.org/10.1097/CCM.0b013e3181865aea.

12. Lakhal K, Ehrmann S, Benzekri-Lefevre D, Runge I, Legras A, Dequin PF, et al. Respiratory pulse pressure variation fails to predict fluid responsiveness in acute respiratory distress syndrome. Crit Care.

2011;15(2):R85, http://dx.doi.org/10.1186/cc10083.

13. Romand JA, Shi W, Pinsky MR. Cardiopulmonary effects of positive pressure ventilation during acute lung injury. Chest. 1995;108(4):1041-8, http://dx.doi.org/10.1378/chest.108.4.1041.

14. Jardin F, Genevray B, Brun-Ney D, Bourdarias JP. Influence of lung and chest wall compliances on transmission of airway pressure to the pleural space in critically ill patients. Chest. 1985;88(5):653-8, http://dx.doi.org/ 10.1378/chest.88.5.653.

15. Feihl F, Broccard AF. Interactions between respiration and systemic hemodynamics. Part I: basic concepts. Intensive Care Med. 2009;35(1):45-54.

16. Feihl F, Broccard AF. Interactions between respiration and systemic hemodynamics. Part II: practical implications in critical care. Intensive Care Med. 2009;35(2):198-205.

17. Sessler CN, Gosnell MS, Grap MJ, Brophy GM, O’Neal PV, Keane KA, et al. The Richmond Agitation-Sedation Scale: validity and reliability in adult intensive care unit patients. Am J Respir Crit Care Med. 2002;166(1):1338-44, http://dx.doi.org/10.1164/rccm.2107138.

18. Teboul JL, Zapol WM, Brun-Buisson C, Abrouk F, Rauss A, Lemaire F. A comparison of pulmonary artery occlusion pressure and left ventricular end-diastolic pressure during mechanical ventilation with PEEP in patients with severe ARDS. Anesthesiology. 1989;70(2):261-6, http:// dx.doi.org/10.1097/00000542-198902000-00014.

19. Talmor D, Sarge T, Malhotra A, O’Donnell CR, Ritz R, Lisbon A, et al.

Mechanical ventilation guided by esophageal pressure in acute lung injury. N Engl J Med. 2008;359(20):2095-104, http://dx.doi.org/10.1056/ NEJMoa0708638.

20. De Backer D, Taccone FS, Holsten R, Ibrahimi F, Vincent JL. Influence of respiratory rate on stroke volume variation in mechanically ventilated patients. Anesthesiology. 2009;110(5):1092-7, http://dx.doi.org/10.1097/ ALN.0b013e31819db2a1.

21. Michard F, Chemla D, Richard C, Wysocki M, Pinsky MR, Lecarpentier Y, et al. Clinical use of respiratory changes in arterial pulse pressure to monitor the hemodynamic effects of PEEP. Am J Respir Crit Care Med. 1999;159(3):935-9.

22. Wyler vB, Takala J, Roeck M, Porta F, Tueller D, Ganter CC, et al. Pulsepressure variation and hemodynamic response in patients with elevated pulmonary artery pressure: a clinical study. Crit Care. 2010;14(3):R111, http://dx.doi.org/10.1186/cc9060.

23. Nouira S, Elatrous S, Dimassi S, Besbes L, Boukef R, Mohamed B, et al.

Effects of norepinephrine on static and dynamic preload indicators in experimental hemorrhagic shock. Crit Care Med. 2005;33(10):2339-43, http://dx.doi.org/10.1097/01.CCM.0000182801.48137.13 24. Mahjoub Y, Pila C, Friggeri A, Zogheib E, Lobjoie E, Tinturier F, et al.

Assessing fluid responsiveness in critically ill patients: False-positive pulse pressure variation is detected by Doppler echocardiographic evaluation of the right ventricle. Crit Care Med. 2009;37(9):2570-5, http://dx.doi.org/10.1097/CCM.0b013e3181a380a3.

In the 25 patients with septic shock, the accuracy of DRESPPP to predict fluid responsiveness was even higher than in patients without septic shock. Seven responders (78%) with a DRESPPP$10% were in septic shock, which was similar to the proportion of nonresponders with DRESPPP,10% (80%).

Although the disease heterogeneity in the group of responders with a DRESPPP,10% may partially explain why DRESPPP did not indicate an increase in stroke volume, the design of the present study precludes any conclusions regarding the role of physiological changes related to underlying diseases in DRESPPP and fluid responsiveness.

As suggested by this and previous studies (7-12), it is not possible to establish a single cut-off point for DRESPPP. The interaction between the pleural and intrathoracic pressures and the cardiovascular system is highly complex and has not been completely elucidated. In addition, this interaction involves physiological aspects related to the underlying disease. Low DRESPPP in patients ventilated with low tidal volumes is not a contraindication to fluid challenge, although the probability of response is greater when the DRESPPP is increased (with the exception of patients with right ventricle insufficiency) (24).

This study has several limitations. We analyzed data from 37 patients instead of 38 as calculated. The heterogeneity of diagnoses precludes the generalization of our findings. Only ten of the patients had ARDS, and their mean plateau pressure was low and static compliance was preserved.

Thus, the application of these results to the entire population of patients with ARDS is limited. Although the analysis of the subgroup of patients with septic shock may be an issue, this group of patients was less heterogeneous than the study cohort, and the use of DRESPPP might be particularly useful because these patients frequently present with acute lung injuries. Manual measurement of pulse pressure variation using a cursor has been previously employed (1), and there is not sufficient evidence that automatic measurement of pulse pressure variation is advantageous.

In conclusion, pulse pressure variation had limited value as a predictor of fluid responsiveness in patients ventilated with low tidal volumes. The most accurate DRESPPP cut-off point to identify fluid responsiveness was $10%. Although a universal cut-off point may not be determined, a DRESPPP$10% can assist in identifying fluid responsiveness, particularly in patients with septic shock.

ACKNOWLEDGMENTS We would like to thank the Fundo de Incentivo a Pesquisa do HCPA (FIPE/HCPA) for financial support.

AUTHOR CONTRIBUTIONS Oliveira-Costa CD was responsible for the project design, data collection and manuscript writing. Friedman G contributed to the project design, statistics, and manuscript writing. Fialkow L and Vieira SR contributed to the project design.

REFERENCES 1. Michard F, Boussat S, Chemla D, Anguel N, Mercat A, Lecarpentier Y, et al. Relation between respiratory changes in arterial pulse pressure and fluid responsiveness in septic patients with acute circulatory failure.

Am J Respir Crit Care Med. 2000;162(1):134-8.

2. Michard F, Teboul JL. Using heart-lung interactions to assess fluid responsiveness during mechanical ventilation. Crit Care. 2000;4(5):282-9, http://dx.doi.org/10.1186/cc710.

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DOI:10.6061/clinics/2012(07)13

BASIC RESEARCH

Protective effects of Tualang honey on bone structure in experimental postmenopausal rats Siti Sarah Mohamad Zaid,I Siti Amrah Sulaiman,II Nor Hayati Othman,III Ima-Nirwana Soelaiman,IV Ahmad Nazrun Shuid,IV Norazlina Mohamad,IV Norliza MuhamadIV I

Universiti Putra Malaysia, Faculty of Environmental Studies, Department of Environmental Sciences, Selangor, Malaysia. II Universiti Sains Malaysia, School of Medical Sciences, Health Campus, Department of Pharmacology, Kelantan, Malaysia. III Universiti Sains Malaysia, School of Medical Sciences, Department of Pathology, Health Campus, Kubang Kerian, Kelantan, Malaysia. IV Universiti Kebangsaan Malaysia, Faculty of Medicine, Department of Pharmacology, Kuala Lumpur, Malaysia.

OBJECTIVE: The objective of this study was to evaluate the effects of Tualang honey on trabecular structure and compare these effects with those of calcium supplementation in ovariectomized rats.

METHODS: Forty female, Sprague-Dawley rats were randomly divided into five groups (n = 8): four controls and one test arm. The control arm comprised a baseline control, sham-operated control, ovariectomized control, and ovariectomized calcium-treated rats (receiving 1% calcium in drinking water ad libitum). The test arm was composed of ovariectomized, Tualang honey-treated rats (received 0.2 g/kg body weight of Tualang honey). Both the sham-operated control and ovariectomized control groups received vehicle treatment (deionized water), and the baseline control group was sacrificed without treatment.

RESULTS: All rats were orally gavaged daily for six weeks after day one post-surgery. The bone structural analysis of rats in the test arm group showed a significant increase in the bone volume per tissue volume (BV/TV), trabecular thickness (Tb.Th) and trabecular number (Tb.N) and a significant decrease in inter-trabecular space (Tb.Sp) compared with the ovariectomized control group. The trabecular thickness (Tb.Th) in the test arm group was significantly higher compared with the ovariectomized-calcium treated group, and the inter-trabecular space (Tb.Sp) in the test arm group was significantly narrower compared with the ovariectomized-calcium treated group.

CONCLUSION: In conclusion, ovariectomized rats that received Tualang honey showed more improvements in trabecular bone structure than the rats that received calcium.

KEYWORDS: Tualang honey; Ovariectomy; Osteoporosis; Trabecular; Structural.

Zaid SSM, Sulaiman SA, Othman NH, Soelaiman IN, Shuid AN, Mohamed N, et al. Protective effects of Tualang honey on bone structure in experimental postmenopausal rats. Clinics. 2012;67(7):779-784.

Received for publication on November 7, 2011; First review completed on February 14, 2012; Accepted for publication on March 2, 2012 E-mail: hayati@kb.usm.my Tel.: 609-7672350

Following ovariectomy, bone perforations are primarily seen at the trabecular plates of cancellous bone (4,5), and its strength depends on bone volume and bone structures, which consist of connected bony plates and bone matrix (6).

After ovariectomy, the area that is initially affected is the secondary spongiosa (central metaphyseal area) of long bones (7). One of the agents used for osteoporosis treatment is calcium supplementation (8). Other alternative treatments use natural products. Polyphenols in fruits and vegetables have demonstrated beneficial effects on bone in rats (9,10).

Natural antioxidant vitamins, such as palm tocotrienols, have been shown to prevent bone loss in many osteoporosisinduced rat models (11). Tualang honey (AgroMas, Malaysia) is a wild, multi-floral honey produced by bees (Apis dorsata) that form hives on the branches of giant trees named Tualang in the Malaysian rainforest. Tualang honey has higher phenolic content and greater radical scavenging activity compared with other honey sources (12). Honey contains approximately 200 substances, including a mixture of sugars (fructose, glucose, maltose, and sucrose) and small amounts

INTRODUCTION In women, bone loss progresses much more rapidly after menopause due to estrogen deficiency (1). Declining serum estradiol levels can increase the lifespan of osteoclasts, leading to bone resorption; however, the lifespan of osteoblasts decreases, leading to less bone being formed.

Ovariectomized rats are widely used as an animal model for the menopausal state because of the similarities in bone changes (2). In postmenopausal women and ovariectomized rats, there is negative bone balance, greater loss of cancellous than the cortical regions of long bones and reductions in bone mineral density due to declining estrogen levels (3).

Copyright Ă&#x; 2012 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.

No potential conflict of interest was reported.

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of other constituents, such as minerals, proteins, vitamins, organic acids, flavonoids, phenolic acids, enzymes, and other phytochemicals (13). A study on postmenopausal women taking Tualang honey at 20 mg/day for four months was found to have similar bone densitometry findings when compared with hormone replacement therapy (14). Our preliminary study showed the positive effects of Tualang honey on the reproductive system and bones (15). The present study was conducted to further evaluate the effects of Tualang honey on trabecular structure and to compare these effects with those of calcium supplementation in ovariectomized rats.

MATERIALS AND METHODS The study was conducted on 40 three-month-old, virgin, female, Sprague-Dawley rats weighing between 220 and 240 g and obtained from the Laboratory Animal Research Unit (LARU), Health Campus, Universiti Sains Malaysia.

After acclimatization for two weeks, the rats were divided into five groups (n = 8 per group): four controls and one test arm. The four control arms were 1) baseline control (BC), 2) sham-operated (SHAM group), 3) ovariectomized-control (OVX group), and 4) ovariectomized-calcium treated (PC).

The ovariectomy and sham operation were conducted under ketamine anesthesia. The rats were anesthetized using 90 mg/kg ketamine (Troy Laboratories, Pty Ltd, Australia) and 10 mg/kg xylazil (Troy Laboratories, Pty Ltd, Australia). The BC group did not undergo ovariectomy or a sham operation but were sacrificed at the start of the experiment to obtain the baseline histomorphometric parameters. The PC group was given 1% calcium in drinking water, which was supplied ad libitum. The calcium supplement was prepared according to previous methods (16) by adding 1 g of lactic acid hemicalcium salt (Sigma, St. Louis, Missouri, USA) to 99 ml of deionized water to make a concentration of 1%. Calcium supplementation was given to the positive control group to compare the effects of honey administration with calcium supplementation. The control rats (SHAM and OVX) were given 0.5 ml of distilled water.

The rats in the test arm (TH group) also had bilateral ovariectomy under ketamine anesthesia. The rats in the TH group were given 0.2 g/kg body weight of Tualang honey.

Tualang honey was freshly prepared every morning by dissolving the honey in deionized water. The administration of test and control materials was started at day 14 after postsurgery day 1 by oral gavage (9:00-10:00 am daily for six weeks). The Tualang honey used was supplied by the Federal Agricultural Marketing Authority (FAMA), Ministry of Agriculture and Agro-Based Industry, Malaysia. The honey was filtered to remove solid particles, concentrated in an oven at 40 ËšC and subjected to c irradiation at 25 kGy at Sterilgamma (M) Sdn. Bhd. (Selangor, Malaysia). Throughout the treatment period, a daily vaginal smear was taken, while body weights and total food intake were recorded weekly. The vaginal smear cytology was performed by a licensed cytologist according to a previously described method (17).

After six weeks, the rats were sacrificed 24 hours after the last dose of treatment under excess ketamine anesthesia (100 mg/kg). The right femur was harvested, and the distal section was longitudinally cut and then fixed in 10% formalin. The bone was processed without decalcification in methyl methacrylate (BDH, Poole, England) medium. The

Figure 1 - Metaphyseal region of longitudinal section of distal femur. All histomorphometric parameter measurements were performed randomly at the metaphyseal region, which was located 3-7 mm from the lowest point of the growth plate and 1 mm from the lateral cortex, excluding the endocortical region.

bone was dehydrated with a series of ethanol solutions (70%, 90%, and 100%) and then infiltrated in methyl methacrylate. The bone was sectioned at 9 mm thickness using a microtome (Leica RM2155, Nussloch, Germany).

The sections were stained with von Kossa and subjected to histomorphometry examination using a light microscope (Leica DMRXA2, Wetzlar, Germany) attached to an image analyzer with VideoTest-Master software (VT, St. Petersburg, Russia). All histomorphometric parameter measurements were performed randomly at the metaphyseal region, which was located 3-7 mm from the lowest point of the growth plate and 1 mm from the lateral cortex, excluding the endocortical region (Figure 1). All measurements and calculations were performed according to the American Society for Bone and Mineral Research (ASBMR) nomenclature and guidelines (18). Structural parameters were analyzed using an image analyzer (Leica DMRXA2, Wetzlar, Germany) with VideoTest-Master software (VT, St. Petersburg, Russia). First, total trabecular bone area, total tissue area and bone perimeters (primary data) were measured using this software. Then, these primary data were expressed in three-dimensional form as follows: total trabecular bone volume (bone volume, BV), total tissue volume (tissue volume, TV) and total bone surface (bone surface, BS). Finally, the structural parameters were calculated based on these primary data, as shown in Table 1.

The sample size was calculated using PS Power and Sample Size Calculation version 2.1.31 and based on a

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Table 1 - Formula for calculating the structural parameter values.

Parameter 1.

2.

3.

4.

Trabecular Trabecular Trabecular Trabecular

bone bone bone bone

volume thickness number separation

Symbol

Unit

Formula

BV/TV Tb.Th Tb.N Tb.S

% mm mm-1 mm

Bone Volume (BV)/ Tissue Volume (TV) 2/[(Bone Surface (BS)/Bone Volume (BV)] Trabecular bone volume (BV/TV)/Trabecular thickness (Tb.Th) 1/Trabecular number (Tb.N) – Trabecular thickness (Tb.Th)

previous study conducted by Hermizi et al. (19). The power of the study was set at 80%, with a 95% confident interval, while the standard deviation (s) observed was 1.4 of trabecular thickness, and the difference in population means (d) was set at 3.0 of the trabecular thickness. The calculated sample size was eight per group. The data analysis was completed using the Statistical Package for Social Sciences (SPSS version 12.0.1) software. The Kolmogorov-Smirnov test was used to test for normality. For the non-parametric statistics, the Kruskal-Wallis test for multiple comparisons was used due to the lack of a normal distribution for all data, followed by the Mann-Whitney U test to compare differences between the two groups. The results are expressed as the median and interquartile range (IR: the difference between the 75th and 25th percentile). P-values of less than 0.05 were considered statistically significant.

The protocol used in this study was approved by the Animal Ethics Committee, Universiti Sains Malaysia (approval no. PPSG/07(A)/044).

control (BC) groups (Table 2 and Figure 2). Only the intertrabecular space (Tb.Sp) was significantly increased in the OVX group compared with the SHAM and BC groups. The SHAM group showed significantly lower changes in body weight compared with the OVX, TH, and PC groups (Table 3). Rats treated with Tualang honey showed the same body weight changes as rats in the OVX control groups. No changes in femur weight were observed in the TH and PC groups compared with the OVX group (Table 4).

However, there was a significant decrease in femur weight for the OVX, TH, and PC groups compared with the SHAM group.

DISCUSSION Our study shows that Tualang honey improved the trabecular structure of the femur bone in ovariectomized rats, an animal model for the menopausal state. The effect of Tualang honey was better than that observed in the rats that received calcium, indicating that honey supplementation could be used to prevent osteoporosis during menopause.

The histomorphometrical analysis of bone structures at the secondary spongiosa in the present study showed that the trabecular bone volume (BV/TV), trabecular thickness (Tb.Th) and trabecular number (Tb.N) were decreased, while the inter-trabecular space (Tb.Sp) was increased in the OVX control group compared with the SHAM group. The changes in trabecular bone structure parameters in the OVX control group were due to an imbalance in the normal remodeling process. We observed that Tualang honey prevented this change. The improvements observed in all structural parameters indicate that Tualang honey is effective in the prevention of ovariectomy-induced bone loss due to bone resorption. Tualang honey appears to be more efficacious than calcium supplementation.

Honey is a natural beehive product well known for its high phytochemical content (phenolic compounds or polyphenols that contribute to positive physiological effects). Honey is rich in antioxidants, such as flavonoids and phenolic acids (20-23). Flavonoids constitute the most important class of

RESULTS The results show that providing Tualang honey had positive effects on bone structures (Table 2) but not on the body weight of ovariectomized rats (Table 3). The ovariectomized rats treated with Tualang honey (TH group) had a significant increase in bone volume per tissue volume (BV/TV); the trabecular thickness (Tb.Th), trabecular number (Tb.N), and inter-trabecular space (Tb.Sp) were significantly lower in the TH group compared with the OVX group (Table 2). In contrast, 1% calcium treatment in the PC group did not significantly improve these parameters when compared with the OVX group. The trabecular thickness (Tb.Th) in the TH group was higher compared with the PC group, and the inter-trabecular space (Tb.Sp) was narrower compared with the PC group. The histomorphometrical analysis of bone structures showed that the bone volume per tissue volume (BV/TV), trabecular thickness (Tb.Th) and trabecular number (Tb.N) were significantly decreased in the OVX group compared with the SHAM and baseline

Table 2 - Effects of Tualang honey on bone histomorphometry.

Control groups BC BV/TV (%) Tb.Th (mm) Tb.N (mm-1) Tb.Sp (mm)

56.15 68.79 13.01 90.96

(2.34)**, #,ˆ ˆ ˆ (7.01)***, ##,ˆ ˆ ˆ (7.02)** (18.77)**, #,ˆ ˆ ˆ

SHAM 38.24 54.75 13.52 117.35

Test group OVX

(5.62)**, ˆ ˆ (9.30)***,ˆ ˆ ˆ (4.11)**, ˆ (4.46)*, ˆ ˆ ˆ

21.41 27.29 7.50 220.27

(16.72) (8.66) (4.31) (63.92)

PC 25.31 30.66 9.00 230.45

(14.81) ## (7.75)### (6.01) # (40.75)###

TH 29.13 38.64 12.51 157.78

(9.22)*, # (7.35)**, ###,ˆ ˆ (5.10)** (32.85)*, ###,ˆ ˆ ˆ

BC = Baseline control group; SHAM = Sham-operated control group; OVX = Ovariectomized control group; TH = Tualang honey- treated group at 0.2 g/ kg/bw; PC = Positive control group (1% calcium).

All values are expressed as the median (IR). a Kruskal–Wallis test. * p,0.05, **p,0.01 and ***p,0.001 vs. OVX control group; #p,0.05, ##p,0.01 and ###p,0.001 vs. SHAM operated group; ˆp,0.05; ˆˆp,0.01 and ˆˆˆp,0.001 vs. PC control group (Mann-Whitney U test).

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Table 3 - Effects of Tualang honey on body weight and food intake during six-week administration.

Control groups SHAM Body weight on D1b (g) Body weight at sacrifice (g) Changes in body weight (%) Total food intake (g)

234 237.5 4.9 702.5

(25) (30) (4.77)* (80)

Test group

OVX 262.5 277.5 12.3 797.0

PC

(12) (24) (8.19) (230.5)

259.5 276.0 14.3 853.0

(29) (27) (7.63) (331.25)

TH 258.5 272.5 14.1 811.5

(23) (19) (7.87) (138.5)

SHAM = Sham-operated control group; OVX = Ovariectomized control group; PC = Positive control group (1% calcium); TH = Tualang honey-treated group at 0.2 g/kg/bw.

All values are expressed as the median (IR). a Kruskal–Wallis test. b D1: First day of treatment. * p,0.05 vs. OVX control group (Mann-Whitney U test).

phenolic compounds in honey and can be categorized as flavonols, flavavones, flavones, anthocyanidins and isoflavones. Flavonoids found in honey include pinocembrin, pinobanksin, chrysin, galangin, luteolin, quercetin, and kaempferol (24). The use of flavonoids to improve postmenopausal osteoporosis is well documented. Quercetin and kaempferol are a subfamily of flavonoids (flavonols) reported to have a potential role in protection against postmenopausal osteoporosis in ovariectomized rats (25). Honey and other plants share similar phenolic compounds or polyphenols.

Flavonols, such as quercetin and kaempferol, have been shown to directly induce apoptosis of mature osteoclasts, thus inhibiting bone resorption and decreasing intracellular reactive oxygen species (ROS) in osteoclasts by a mechanism involving estrogen receptors (ER) (25). A study on kaempferol alone indicates that it prevents ovariectomized-induced bone loss by promoting osteoblast function (26).

The mechanisms of action of both flavonols on bone are still not completely understood. Flavanols may interact

directly with estrogen receptors ER-ß and ER-a (25). ER-ß is more abundant than ER-a in trabecular bone compared with cortical bone (27); thus, a greater proportion of flavonols might bind to trabecular bone, resulting in a greater preservation of trabecular bone than cortical bone. Studies on postmenopausal animals have shown that oxidative stress has an important impact on the differentiation and functioning of osteoclasts (31,32); oxidative stress also induces lipid peroxidation and H2O2 production and a decrease in enzymatic antioxidants, such as Superoxide Dismutase (SOD) and Glutathione Peroxidase (GPx) (31). Phenolic glycosides have potential antioxidant activity (32) and immunostimulatory effects (33). The composition and interaction of polyphenols in honey make it a unique and excellent source of stable, free radical scavengers among polyphenol-containing foods (34). Another component of honey that has been shown to increase calcium absorption is gluconic acid. Gluconic acid is a major organic acid in honey and one of the products of the enzymatic glucose oxidase

Figure 2 - Effects of Tualang honey on structure of femur bone. All pictures are stained with Von Kossa and examined under X20 magnification: BC = Baseline control group; SHAM = Sham- operated control group; OVX = Ovariectomised control group; TH = Tualang honey treated group at 0.2 g/kg/bw; PC = Positive control group (1% calcium).

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Table 4 - Effects of Tualang honey on weight of femur reported as relative organ weight (mg/g body weight).

Control groups

Absolute weight (mg) Relative weight (mg/g body weight)

Test group

BC

SHAM

OVX

PC

TH

808.15 (48.10)

884.91 (86.40)

876.51 (108.80)

919.51 (72.51)

877.50 (155.20)

3.38 (0.11) *,#

3.64 (0.23)

3.05 (0.22)

##

3.13 (0.21)

###

3.04 (0.55)

##

Absolute weight = Wet weight of tissue.

Relative weight = Wet weight of tissue normalized to final body weight.

BC = Baseline control group; SHAM = Sham-operated control group; OVX = Ovariectomized control group; PC = Positive control group (1% calcium); TH = Tualang honey-treated group at 0.2 g/kg body weight.

All values are expressed as the median (IR). a Kruskal–Wallis test. * p,0.05 vs. OVX control group; #p,0.05, ##p,0.01 and ###p,0.001 vs. SHAM-operated group (Mann-Whitney U test).

the University Kebangsaan Malaysia for the use of lab facilities for this study.

reaction (35). It is significantly useful in the modulation of intestinal calcium absorption calcium (36). When ingested in food, gluconic acid is fermented by lactic acid bacteria (Lactobacillus reuteri and L. mucosae) to produce lactate and acetate. Acid-utilizing bacteria (Megasphaera elsdenii and Mitsuokella multiacida) then convert these products to form butyrate, a type of short-chain fatty acid (SCFA) that is rapidly absorbed by the mucosa of the large intestine (37).

The greater efficacy of Tualang honey compared with calcium supplementation is likely due to the enhancement in intestinal calcium absorption caused by honey.

Tualang honey did not produce any adverse effects on body weight or food intake. Although not statistically significant, we observed that the body weight of the ovariectomized rats treated with Tualang honey was lower than that of the ovariectomized rats not given honey. This observation, if translated to humans, indicates that honey has the potential to reduce health problems related to overweight and obesity seen in the menopausal state, such as chronic osteoarthritis, cardiovascular diseases and some cancers. The limitation in the interpretation of this finding is the short study duration, which was only six weeks. It would be interesting to observe the effect of honey over a longer study period. Our findings were similar to the results of several previous studies on natural products. Treatment with kaempferol, which is highly abundant in Ginkgo biloba extracts, did not suppress increases in body weights in ovariectomized rats (27). Although the study period was longer [10 weeks], the duration of the study was similar to ours, as treatment was started at week 4 after ovariectomy.

In conclusion, Tualang honey improved the trabecular structure of bone in ovariectomized rats. The effect of Tualang honey in rats is better than the effects observed in rats that received calcium. These findings suggest that Tualang honey is better than calcium supplementation in preventing osteoporosis in the menopausal state. Therefore, Tualang honey could potentially be used as an alternative to calcium supplementation in osteoporosis treatment.

AUTHOR CONTRIBUTIONS Zaid SM conducted all the lab work and data analysis and drafted the manuscript. Sulaiman SB led the design of the study, served as the main supervisor to Zaid SM, was the principal investigator of the grant study, provided guidance in the data analysis and revised the manuscript.

Mohamad N contributed in the study design and provided guidance in the histomorphometry part of the study. Soelaiman IN, Shuid AN and Muhamad N provided guidance in the histomorphometry part of the study. Othman NH is the pathologist who evaluated the bone histology and finalized the revised manuscript.

REFERENCES 1. Pacifici R. Estrogen deficiency, T cells and bone loss. Cellular Immunology.

2008;252(1-2):68-80, http://dx.doi.org/10.1016/j.cellimm.2007.06.008.

2. Goss PE, Qi S, Josse RE, Pritzker KPH, Mendes M, Hu H, et al. The steroidal aromatase inhibitor exemestane prevents bone loss in ovariectomized rats. Bone. 2004;34(3):384-92, http://dx.doi.org/10.1016/ j.bone.2003.11.006.

3. Kalu DN. The ovariectomized rat model of postmenopausal bone loss.

Bone Miner. 1991;15(3):175-91.

4. Wronski TJ, Dann LM, Qi H, Yen CF. Skeletal effects of withdrawal of estrogen and diphosphonate treatment in ovariectomized rats. Calcif Tissue Int. 1993;53(3):210-6, http://dx.doi.org/10.1007/BF01321840.

5. Yamaura M, Nakamura T, Kanou A, Miura T, Ohara H, Suzuki K. The effect of 17 beta-estradiol treatment on the mass and the turnover of bone in ovariectomized rats taking a mild dose of thyroxin. Bone Miner.

1994;24(1):33-42, http://dx.doi.org/10.1016/S0169-6009(08)80129-8.

6. Mellish RW, Ferguson Pell MW, Cochran GV, Lindsay R, Dempster DW. A new manual method for assessing two-dimensional cancellous bone structure: comparison between iliac crest and lumbar vertebra. Bone Miner Res. 1991;6(7):689-96.

7. Bagi CM, Miller SC. Comparison of osteopenic changes in cancellous bone induced by ovariectomy and/or immobilization in adult rats. Anat Rec. 1994;239(3):243-54, http://dx.doi.org/10.1002/ar.1092390303.

8. Mackerras D, Lumley T. First- and second-year effects in trials of calcium supplementation on the loss of bone density in postmenopausal women.

Bone. 1997;21(6):527-33, http://dx.doi.org/10.1016/S8756-3282(97)00181-6.

9. Muhlbauer RC, Lozano A, Reinli A. Onion and a mixture of vegetables, salads, and herbs affect bone resorption in the rat by a mechanism independent of their base excess. J Bone Miner Res. 2002;17(7):1230-6, http://dx.doi.org/10.1359/jbmr.2002.17.7.1230.

10. Devareddy L, Hoosmand S, Collins JK, Lucas EA, Chai SC, Arjmandi BH. Blueberry prevents bone loss in ovariectomized rat model of postmenopausal osteoporosis. The Journal of Nutritional Biochemistry.

2008;19(10):694-9, http://dx.doi.org/10.1016/j.jnutbio.2007.09.004.

11. Ahmad NS, Khalid BA, Luke DA, Ima Nirwana S. Tocotrienol offers better protection than tocopherol from free radical-induced damage of rat bone. Clin Exp Pharmacol Physiol. 2005;32(9):761-70, http:// dx.doi.org/10.1111/j.1440-1681.2005.04264.x.

12. Krishna Kihsore R, Halim AS, Nurul Syazna MS, Sirajudeen KNS. Tualang honey has higher phenolic content and greater radical scavenging activity with other honey sources. Nutrition Research.

2011;31(4):322-5.

ACKNOWLEDGMENTS The authors would like to acknowledge the Federal Agriculture Marketing Authority (FAMA), the Ministry of Agriculture and Agro-Based Industry Malaysia for supplying the Tualang honey (Agromas, Malaysia) for the present research, the Universiti Sains Malaysia for providing the research grant (No: 304/PPSP/6131505), the University Putra Malaysia for financial assistance to the first author, Siti Sarah Mohamad Zaid, and

783

Honey on bone structure Zaid SSM et al.

CLINICS 2012;67(7):779-784

13. Aljadi AM and Kamaruddin MY. Evaluation of the phenolic contents and antioxidant capacities of two Malaysian floral honeys. Food Chemistry.

2004;85:513-8, http://dx.doi.org/10.1016/S0308-8146(02)00596-4.

14. Lily Husniata Y. The effects of Tualang honey on postmenopausal women. In 2nd International conference on the medicinal use of honey.

2010; Renaissance Hotel, Kota Bharu, Kelantan, Malaysia.

15. Zaid SSM, Sulaiman AS, Sirajudeen KNM, Othman NH. The effects of Tualang honey on female reproductive organs, tibia bone and hormonal profile in ovariectomised rats-animal model for menopause. BMC Complement Altern Med. 2010;10:82-8, http://dx.doi.org/10.1186/ 1472-6882-10-82.

16. Norazlina M, Ima Nirwana S, Khalid BAK. Calcium supplementation improves bone and lean mass in vitamin E deficient rats. JAFES. 2001;19(1-2):23-9.

17. Marcondes FK, Bianchi FJ, Tanno AP. Determination of the estrous cycle phases of rats: some helpful considerations. Brazil Journal of Biology.

2002;62(4A):609-14, http://dx.doi.org/10.1590/S1519-69842002000400008.

18. Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, et al. Bone histomorphometry: standardization of nomenclature, symbols and units. Reports of the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner Res. 1987;2(6):595-610.

19. Hermizi H, Faizah O, Ima-Nirwana S, Ahmad Nazrun S, Norazlina M. Beneficial effects of tocotrienol and tocopherol on bone histomorphometric parameters in sprague-dawley male rats after nicotine cessation.

Calcif Tissue Int. 2009;84(1):65-74, http://dx.doi.org/10.1007/s00223008-9190-x.

20. Aljadi AM, Kamaruddin MY. Evaluation of the phenolic contents and antioxidant capacities of two Malaysian floral honeys. Food Chemistry.

2004;85:513-518, http://dx.doi.org/10.1016/S0308-8146(02)00596-4.

21. Al-Mamary M,Al-Meeri A, Al-Habori M. Antioxidant activities and total phenolics of different types of honey. Nutrition Research. 2002;22(9):10417, http://dx.doi.org/10.1016/S0271-5317(02)00406-2.

22. Phrzynska K, Biesaga M. Analysis of phenolic acids and flavonoids in honey. Analytical Chemistry. 2008;28(7):893-902.

23. Gheldof N, Wang XH, Engeseth NJ. Identification and quantification of antioxidant components of honeys from various floral sources. J Agric Food Chem. 2007;50(21):5870-7.

24. Gheldof N, Wang XH, Engeseth NJ. Buckwheat honey increases serum antioxidant capacity in humans. J Agric Food Chem. 2003;51(5):1500-5, http://dx.doi.org/10.1021/jf025897t.

25. Wattel A, Kamel S, Mentaverri R, Lorget F, Prouillet C, Petit JP, et al.

Potent inhibitory effect of naturally occurring flavonoids quercetin and

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

784

kaempferol on in vitro osteoclastic bone resorption. Biochem Pharmacol.

2003.65(1):35-42, http://dx.doi.org/10.1016/S0006-2952(02)01445-4.

Trivedi R, Kumar S, Kumar K, Siddiqui JA, Swarnkar G, Gupta V, et al.

Kaempferol has osteogenic effect in ovariectomized adult Sprague-Dawley rats. Mol Cell Endocrinol. 2008;289(1-2):85-93, http://dx.doi.org/10.1016/ j.mce.2008.02.027.

Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S, et al. Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta. Endocrinology 1997;138(3):863-70.

Onoe Y, Miyaura C, Ohta H, Nozawa S, Suda T. Expression of estrogen receptor beta in rat bone. Endocrinology. 1997;138(10):4509-12, http:// dx.doi.org/10.1210/en.138.10.4509.

Zhang J, Munger RG, West NA, Cutler DR, Wengreen HJ, Corcoran CD. Antioxidant intake and risk of osteoporotic hip fracture in Utah: an effect modified by smoking status. Am J Epidemiol. 2006;163(1):9-17.

Ha BJ, Lee SH, Kim HJ, Lee JY. The role of Salicornia herbacea in ovariectomy-induced oxidative stress. Biol Pharm Bul. 2006;29(7):1305-9, http://dx.doi.org/10.1248/bpb.29.1305.

Ozgocmen S, Kaya H, Fadillioglu E, Aydogan R, Yilmaz Z. Role of antioxidant systems, lipid peroxidation, and nitric oxide in postmenopausal osteoporosis. Mol Cell Biochem. 2007;295(1-2):45-52, http:// dx.doi.org/10.1007/s11010-006-9270-z.

Wu Q, Fu DX, Hou AJ, Lei GQ, Liu ZJ, Chen JK, et al. Antioxidative phenols and phenolic glycosides from Curculigo orchioides. Chem Pharm Bull. 2005;53(8):1065-7, http://dx.doi.org/10.1248/cpb.53.1065.

Lakshmi V, Pandey K, Puri A, Saxena RP, Saxena KC. Immunostimulant principles from Curculigo orchioides. J Ethnopharmacol. 2003;89(23):181-4, http://dx.doi.org/10.1016/S0378-8741(03)00160-0.

Prior R, Martin A, Sofic E, McEwan J, O’Brien C, Lischner N. Antioxidant capacity as influenced by total phenolic and anthocyanin content, maturity and variety of Vaccinium species. Journal Agriculture Food Chemistry. 1998;46(7):2686-93, http://dx.doi.org/10.1021/jf980145d.

Jeffrey AE, Echazareta CM. Medicinal uses of honey. Revista Biomedica.

1996;7:43-9.

Fournier P, Dupius Y. Modulation of intestinal absorption of calcium.

J Physiol (Paris). 1975;70:479–1.

Tsukahara T, Koyama H, Okada M, Ushida K. Stimulation of butyrate production by gluconic acid in batch culture of pig cecal digesta and identification of butyrate-producing bacteria. J Nutr. 2002;132(8):222934.

CLINICS 2012;67(7):785-792

DOI:10.6061/clinics/2012(07)14

BASIC RESEARCH

Rat testicular impairment induced by electromagnetic radiation from a conventional cellular telephone and the protective effects of the antioxidants vitamins C and E Mona Abdullah Al-Damegh Department of Biology, College of Science and Arts, Onaizah, Qassim University, Kingdom of Saudi Arabia.

OBJECTIVE: The aim of this study was to investigate the possible effects of electromagnetic radiation from conventional cellular phone use on the oxidant and antioxidant status in rat blood and testicular tissue and determine the possible protective role of vitamins C and E in preventing the detrimental effects of electromagnetic radiation on the testes.

MATERIALS AND METHODS: The treatment groups were exposed to an electromagnetic field, electromagnetic field plus vitamin C (40 mg/kg/day) or electromagnetic field plus vitamin E (2.7 mg/kg/day). All groups were exposed to the same electromagnetic frequency for 15, 30, and 60 min daily for two weeks.

RESULTS: There was a significant increase in the diameter of the seminiferous tubules with a disorganized seminiferous tubule sperm cycle interruption in the electromagnetism-exposed group. The serum and testicular tissue conjugated diene, lipid hydroperoxide, and catalase activities increased 3-fold, whereas the total serum and testicular tissue glutathione and glutathione peroxidase levels decreased 3-5 fold in the electromagnetism-exposed animals.

CONCLUSION: Our results indicate that the adverse effect of the generated electromagnetic frequency had a negative impact on testicular architecture and enzymatic activity. This finding also indicated the possible role of vitamins C and E in mitigating the oxidative stress imposed on the testes and restoring normality to the testes.

KEYWORDS: Electromagnetic Radiation; Testes; Infertility; Vitamins C and E. Al-Damegh MA. Rat testicular impairment induced by electromagnetic radiation from a conventional cellular telephone and the protective effects of the antioxidants vitamins C and E. Clinics. 2012;67(7):785-792.

Received for publication on January 30, 2012; First review completed on February 16, 2012; Accepted for publication on March 7, 2012 E-mail: dr_mona_aldamegh@yahoo.com Tel.: 063 610651-1024

studies on the potential health effects of electromagnetic fields (EMFs) have been performed in Europe, North America, and elsewhere. Studies to assess the potential long-term effects of mobile phone use are ongoing. The exact mechanism of the EMF interactions is not well understood, although a few studies have suggested the involvement of lipid peroxidation and free radical formation (1), as well as biochemically induced oxidative stress.

There have been many scientific studies warning about the adverse biological effects of this type of electromagnetic radiation (EMR) on the health of humans, animals, and birds (2-4). Although the radio waves of cellular phones do not have enough energy to cause the ionization of atoms and molecules, the recent concerns over long-term exposure to the electromagnetic radiation emitted by mobile phones should be taken more seriously given the growing trend toward deterioration of the male germ line (spermatogenesis and sperm maturation) (5). A high-quality, more recent epidemiological animal study was conducted by the Technical Working Group (TWG) on the health effects of EMFs (6).

INTRODUCTION Radiofrequency (RF) fields are part of the electromagnetic spectrum and are generally defined as covering the range of frequencies from 100 kHz to 300 GHz. Mobile phones communicate by transmitting radiofrequency waves, which are electromagnetic fields and, unlike ionizing radiation (e.g., X-rays and gamma rays) cannot break chemical bonds or cause ionization in the human body.

Given the large number of mobile phone users, investigating, understanding, and monitoring any potential public health impacts of mobile phone use are important. To date, the adverse health effects of mobile phone use are debatable.

Partly in response to public concerns, a number of research

Copyright Ă&#x; 2012 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.

No potential conflict of interest was reported.

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any EMR-emitting device (such as an extra cellular phone, centrifuge, fluorescent light ballasts, and computers) was not allowed so that the EMR generated by any of this equipment would not interfere with the experimental environment (7).

The groups were separated from each other, with the control group isolated far from the source of the EMR. The EMRexposed animal group was taken to the exposure room and then exposed to the EMR emitted by a commercially available GSM cellular phone (900/1800/1900 MHz, 2-W peak power, average power density of 0.02 mW/cm2 at a specific absorption rate of approximately 0.9 W/kg). The distance between the phone and the rats was 50 cm. The rats were placed in Plexiglas cages with drilled ventilation holes of cm Ø that were attached with a mobile phone hand set (8). The control animals were exposed to a mobile phone without a battery in similar cages for the same period in a separate but similar room.

Electromagnetic radiation is one of the environmental toxicants that are capable of compromising male fertility by inducing a state of oxidative stress in the testes.

Consequently, there is an urgent need to identify antioxidants that can supplement the tissue’s own antioxidant strategies to rescue the testes from the consequences of a ROS attack. Therefore, this study has been performed to clarify the activities of some free radical scavenger enzymes in the blood and testicular tissue and the possible protective role of vitamins C and E after mobile phone-induced testicular impairment.

MATERIALS AND METHODS Animals The study population comprised 120 male Wister albino rats (body weight, 200¡20 grams) that were obtained from the Experimental Animal Center of the College of Pharmacy, King Saud University in Riyadh, Saudi Arabia.

The procedures used for animal care and housing were in accordance with the U.S. Department of Agriculture through the Animal Welfare Act (7USC 2131) of 1985 and the Animal Welfare Standards incorporated in 9 CFR Part 3, 1991. The rats were maintained under standard laboratory conditions in an air-conditioned room, where the temperature was maintained at 25 ˚C with constant humidity (4050%), and kept on a 12/12-hour light/dark cycle throughout the experiment. They were provided with standard food pellets and water ad libitum. They were randomly divided into two main groups, the control and exposure groups, as follows:

Biochemical assays to determine oxidative stress Several oxidation-related analytes were assayed to investigate a range of oxidative changes potentially associated with EMR exposure. To estimate the different oxidative stress parameters and the anti-oxidant enzymes, a portion of the testes (0.25 g) was freeze-clamped, crushed at liquid nitrogen temperature, ice-cooled, homogenized in 2.5 ml of phosphate buffer (pH 7.4), and then centrifuged at 30,000 g for 15 min at 4 ˚C. The supernatant was collected and preserved at 220 ˚C until use. Total glutathione (GSH; CS0260 Sigma-Aldrich, St. Louis, MO, USA), glutathione peroxidase (c-GPx; CGP1-1KT Sigma-Aldrich), and catalase (CAT; Cat100-1KT Sigma-Aldrich) levels were measured. In the course of our investigation of radiation-induced oxidation, we measured two indexes of peroxidation, namely conjugated dienes (CD; performed according to the method of Girotti et al. (9)) and hydroperoxides (LIP Di; PeroxiDetect kit [PD1, Sigma-Aldrich]).

Control groups Control group (without stress and with the EMR exposure device turned off) (n = 10) Standard food pellets + vitamin C (40 mg/kg/day)treated group (n = 10) Standard food pellets + vitamin E (2.7 mg/kg/day)treated group (n = 10)

Conjugated diene determination Conjugated dienes (CD) were assayed as a measure of early lipid oxidation, and thiobarbituric acid-reactive substances (TBARS) were assayed as an indicator of later aldehyde formation. The conjugated diene concentrations were determined with a modified assay (9).

Experimental EMR-exposed groups EMR-exposed (n = 30) EMR-exposed + vitamin C (40 mg/kg/day)-treated group (n = 30) EMR-exposed + vitamin E (2.7 mg/kg/day)-treated group (n = 30) Every ten rats were placed in a separate cage to avoid the stress of isolation or overcrowding. The duration of the exposure was 15, 30, and 60 min/day for 14 consecutive days for each of the six groups. At the end of each experimental period, ten animals from each group were sacrificed under mild diethyl ether anesthesia. Blood samples were taken from the retro-orbital venous plexus and centrifuged at 700 g for 30 min. The clear, nonhemolyzed supernatant sera were quickly removed and kept at 220 ˚C for further biochemical analysis. In addition, the testes were homogenized in an isotonic solution and kept at 220 ˚C for further biochemical investigations conducted to estimate the anti-oxidant parameters.

Histopathology Testis tissues were submitted for histological and morphologic examinations using conventional methods and were stained with hematoxylin and eosin.

Statistical analysis The data were analyzed using analysis of variance (ANOVA; 30), followed by LSD analysis to evaluate the variations between groups and for multiple comparisons among different groups. The results are expressed as the means¡SD. Values of p.0.05 were considered statistically insignificant, whereas values of p,0.05 were considered statistically significant.

RESULTS

Whole-body exposure to electromagnetic radiation

Histopathological alterations

In the EMR exposure room, there were no other metal or ferromagnetic materials around the clean benches that would change the structure of the electromagnetic field. The use of

The testis sections in the three control groups revealed that the seminiferous tubules consist of several layers of epithelial cells (Figure 1). The cuboidal spermatogonia cells

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Mobile phone, testes and vitamins C and E Al-Damegh MA

Figure 3 - Photomicrograph of the marked regenerative effect of protective treatment with vitamin C showing normal seminiferous tubules, spermatocytes and spermatids. H&E 6100.

Figure 1 - Section of rat testis showing the normal seminiferous tubules, interstitium, spermatids and spermatogenic cells at different stages of development. H&E 6400.

the germinal epithelium was significantly decreased. A reduction in the spermatid numbers within the lumen of the seminiferous tubules, marked mitotic divisions and pyknosis of some spermatogonic nuclei were observed in the testis sections after a two-week exposure to EMR. However, supplemental treatment with vitamin C or E for two weeks had a marked regenerative effect on the histopathological features of the seminiferous tubules (Figures 3 and 4), causing a significant decrease in the effects of EMR.

have clear cytoplasm and rounded nuclei, which may show division. Sertoli cells are large epithelial cells that have a vesicular nucleus with a large nucleolus. Next to the spermatogonia cells is a zone of spermatocytes, the nuclei of which are usually in mitotic division. A large number of small cells, the spermatids, are seen external to the spermatocytes. The spermatids then further develop into spermatozoa. The latter usually lie in groups with their heads projecting between the deeper cells and are connected with one of the Sertoli cells of the lining epithelium.

In contrast, in the sections of testis exposed to EMR, the histopathological changes in the testis exhibited degenerative changes and high levels of mitotic division after 14 days of exposure (Figures 2 and 3). The sections show degeneration in the seminiferous tubules with the complete absence of spermatozoa, and some spermatogonic nuclei also show karyomegaly and a high incidence of mitotic divisions. In the EMF group, the diameter of the seminiferous tubules was significantly increased compared with the normal unexposed EMR rat testicular architecture, whereas the mean height of

Biochemical changes The concentrations of the oxidative markers in the blood and testicular tissue at the end of each treatment period are shown in Tables 1 and 2. The changes in the levels of blood GSH, c-GPx, CD, LIP Di, and CAT are reported in Table 1.

The levels of GSH and GPx were significantly elevated in all of the EMR-exposed groups compared with the control group, whereas the levels of CD, LIP Di, and CAT were decreased. In contrast, the levels of GSH, c-GPx, CD, LIP Di, and CAT in the vitamin C- and E-supplemented groups were significantly (p,0.05) lower than those in the EMRexposed groups. Vitamins C and E seemed to provide

Figure 2 - Photograph of a section of rat testis exposed to EMR for 60 minutes showing the damaged spermatocytes and spermatids, marked mitotic divisions and pyknotic nuclei of some spermatogonia, widening of the seminiferous tubular lumen and absence of spermatozoa within the lumen. H&E 6100.

Figure 4 - Magnified photograph of a section of testis after the protective treatment with vitamin E for two weeks showing remarkable regenerative features. H&E 6400.

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3.90 ¡0.3 3.55 ¡0.3 3.48 ¡0.3 2.11 * ¡0.1 3.11 ¡0.2 2.96 ¡0.2

GSH (mmol/g)

15.80 ¡1.1 15.58 ¡1.1 15.01 ¡0.8 11.38 * ¡1.0 14.25 * ¡1.1 12.91* ¡1.0

LIP Di (nmol/ml)

CAT (mmol/ml)

GSH (mmol/g)

2.18 ¡ 0.2 2.06 ¡0.2 1.82 ¡0.2 3.40 ¡0.4 2.40 ¡0.2 2.25 ¡0.2

3.76 0.5 3.70 ¡0.4 3.70 ¡0.5 5.25 ¡0.7 5.03 ¡0.4 4.63 ¡0.4

0.49 ¡0.1 0.54 ¡0.1 0.56 ¡0.1 1.01 ¡0.1 0.72 ¡0.6 0.74 ¡0.0

3.90 ¡0.3 3.55 ¡0.3 3.48 ¡0.3 1.45 ¡0.1 3.03 * ¡0.2 3.01 * ¡0.2

15.80 ¡1.1 15.58 ¡1.1 15.01 ¡0.8 9.00 * ¡0.7 12.88 * ¡0.8 13.30 * ¡0.8

2.18 ¡0.2 2.06 ¡0.2 1.82 ¡0.2 4.60 ¡0.5 2.66 ¡0.3 3.08 ¡0.3

c-GPx CD (Units/ml) (mmol/mg)

Enzymes

Enzymes

c-GPx CD (Units/ml) (mmol/mg)

30 min/day

3.76 ¡0.5 3.70 ¡0.4 3.70 ¡0.5 5.70 * ¡0.6 5.05 ¡0.7 4.96 ¡0.6

LIP Di (nmol/ml)

Duration of EMR exposure

15 min/day

* Significant at the level of p,0.05.

1 = without stress and EMR (control).

2 = without stress and EMR + vitamin C (40 mg/kg/day) (control).

3 = without stress and EMR + vitamin E (2.7 mg/kg/day) (control).

4 = EMR-exposed.

5 = EMR-exposed + vitamin C (40 mg/kg/day).

6 = EMR-exposed + vitamin E (2.7 mg/kg/day).

GSH = Total glutathione. c-GPx = Cellular glutathione peroxidase.

CD = Conjugated dienes.

CAT = Catalase.

6

5

4

3

2

1

Treatments

0.49 ¡0.1 0.54 ¡0.1 0.56 ¡0.1 2.13 * ¡0.2 0.71 * ¡0.1 0.79 * ¡0.0

CAT (mmol/ml) 3.90 ¡0.3 3.55 ¡0.3 3.48 ¡0.3 1.08 * ¡0.1 2.76 * ¡0.2 2.76 * ¡0.3

GSH (mmol/g)

15.80 ¡1.1 15.58 ¡1.1 15.01 ¡0.8 6.60 * ¡0.5 10.63 * ¡0.5 10.60 * ¡0.5

c-GPx (Units/ml)

2.18 ¡0.2 2.06 ¡0.2 1.82 ¡0.2 6.00 * ¡0.6 3.50 * ¡0.1 3.71* ¡0.0

CD (mmol/mg)

Enzymes

60 min/day

3.76 ¡0.5 3.70 ¡0.4 3.70 ¡0.5 6.63 * ¡0.7 5.51* ¡0.7 5.28 * ¡0.5

LIP Di (nmol/ml)

0.49 ¡0.1 0.54 ¡0.1 0.56 ¡0.1 3.39 * ¡0.3 0.94 * ¡0.1 1.38 * ¡0.1

CAT (mmol/ml)

Table 1 - Effects of vitamin C and vitamin E pretreatment on the biochemical parameters in the serum of control and EMR-exposed rats (2-week exposure time).

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4.58 ¡0.4 4.20 ¡0.4 4.49 ¡0.4 2.96 * ¡0.3 3.59 ¡0.4 3.81 ¡0.5

GSH (mmol/g)

9.00 ¡0.6 9.43 ¡0.6 9.51 ¡0.5 5.81* ¡0.5 7.75 * ¡0.5 9.08 * ¡0.6

LIP Di (nmol/ml)

CAT (mmol/ml)

GSH (mmol/g)

4.58 ¡0.5 5.61 ¡0.5 4.90 ¡0.6 6.33 * ¡0.7 5.25 ¡0.7 5.20 ¡0.6

5.38 ¡0.4 4.55 ¡0.4 4.31 ¡0.4 8.93 * ¡0.7 6.36 * ¡0.5 5.80 * ¡0.7

3.70 ¡0.4 3.86 ¡0.4 3.86 ¡0.4 5.40 * ¡0.4 4.25 ¡0.5 4.36 ¡0.6

4.58 ¡0.4 4.20 ¡0.4 4.49 ¡0.4 1.71* ¡0.2 4.19 * ¡0.5 3.91* ¡0.4

9.00 ¡0.6 9.43 ¡0.6 9.51 ¡0.5 4.40 * ¡0.5 7.28 * ¡0.6 7.68 * ¡0.5

4.58 ¡0.5 5.61 ¡0.5 4.90 ¡0.6 9.21 * ¡0.5 6.68 * ¡0.6 5.75 * ¡0.6

c-GPx CD (Units/ml) (mmol/mg)

Enzymes

Enzymes

c-GPx CD (Units/ml) (mmol/mg)

30 min/day

5.38 ¡0.4 4.55 ¡0.4 4.31 ¡0.4 12.03 * ¡0.7 7.56 * ¡0.4 6.95 * ¡0.4

LIP Di (nmol/ml)

Duration of EMR exposure 15 min/day

* Significant at the level of p,0.05.

1 = without stress and EMR (control).

2 = without stress and EMR + vitamin C (40 mg/kg/day) (control).

3 = without stress and EMR + vitamin E (2.7 mg/kg/day) (control).

4 = EMR-exposed.

5 = EMR-exposed + vitamin C (40 mg/kg/day).

6 = EMR-exposed + vitamin E (2.7 mg/kg/day).

GSH = Total glutathione. c-GPx = Cellular glutathione peroxidase.

CD = Conjugated dienes.

CAT = Catalase.

6

5

4

3

2

1

Treatments

3.70 ¡0.4 3.86 ¡0.4 3.86 ¡0.4 7.48 * ¡0.4 4.65 * ¡0.4 5.21* ¡0.5

CAT (mmol/ml) 4.58 ¡0.4 4.20 ¡0.4 4.49 ¡0.4 1.03 * ¡0.3 2.79 * ¡0.4 3.36 * ¡0.5

GSH (mmol/g)

9.00 ¡0.6 9.43 ¡0.6 9.51 ¡0.5 2.34 * ¡0.4 7.66 * ¡0.5 7.34 * ¡0.6

c-GPx (Units/ml)

4.58 ¡0.5 5.61 ¡0.5 4.90 ¡0.6 12.20 * ¡0.6 7.72 * ¡0. 8 6.94 * ¡0.7

CD (mmol/mg)

Enzymes

60 min/day

5.38 ¡0.4 4.55 ¡0.4 4.31 ¡0.4 15.18 * ¡0.9 8.74 * ¡0.4 7.78 * ¡0.5

LIP Di (nmol/ml)

3.70 ¡0.4 3.86 ¡0.4 3.86 ¡0.4 10.34 * ¡0.4 5.96 * ¡0.5 5.74 * ¡0.6

CAT (mmol/ml)

Table 2 - Effects of vitamin C and vitamin E pretreatment on the antioxidant levels in the testicular tissue of control and EMR exposed rats for two weeks.

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CLINICS 2012;67(7):785-792

significant protection against the oxidative stress induced by the exposure of the rats to EMR. The changes in the levels of GSH, c-GPx, CD, LIP Di, and CAT in the testicular tissue homogenates are provided in Table 2. The GSH level and GPx activity decreased significantly (p,0.05) in the EMR-exposed rats compared with the control group. The percentage decrease ranged from 64.6 to 26.0%. In contrast, the levels of CD, LIP Di and CAT increased significantly in the EMR-exposed rats compared with the control group. However, the protective effect of vitamins C and E was evident from the increased activities of GSH and c-GPx in the irradiated testicular tissue, which reached normal levels. Moreover, the levels of CAT and the two markers of lipid peroxidation, CD and LIP Di, were significantly decreased in the irradiated testicular tissue of the supplemented vitamin groups, which reached normal levels. The protective effect of vitamins C and E was evident from the increased activity of GSH and c-GPx and decreased levels of CD, LIP Di, and CAT to approximately normal values in the testicular tissue.

reproductive system and sperm parameters and cause significant changes in the sperm cell cycle (15-24).

ROS are continuously neutralized by antioxidants present in body tissues (25). Whenever the production of ROS exceeds the scavenging capacity of antioxidants, oxidative stress (OS) will result (26). In 1992, researchers found that electromagnetic fields increase the free radical activity in cells (27). Within the last decade, in vivo animal studies have shown that OS develops in response to cell phone radiation (28-32). EMR may disturb ROS metabolism by increasing the production of ROS or by decreasing the activity of antioxidant enzymes. The capability of EMR to induce oxidative stress in the testes strongly suggests that the testis is a vulnerable tissue that is highly dependent on oxygen to drive spermatogenesis and yet highly susceptible to the toxic effects of reactive oxygen metabolites; in this context, the testis is very similar to the brain.

In an analysis of antioxidant enzymes, Kesari et al. (15) found a decrease in GSH and an increase in catalase. The group exposed to EMR exhibited an increased level of malondialdehyde, a result that was also evident in our study.

However, studies designed to measure malonaldehyde (MDA) levels have shown conflicting results (16,18,29,31,33).

Our findings demonstrate that the exposure of male rats to EMR emitted by mobile phones alters the enzymatic activity in the blood and testicular tissue. Bediz et al. (34) suggested that long-term exposure to low-frequency EMR increases lipid peroxidation in the brain and other body organs. Our results are in partial agreement with the findings of other studies (34-36).

Some authors have shown that electromagnetic fields (EMFs) penetrate living organisms and alter the cell membrane potential (37). This alteration may affect free-radical processes within the cell and alter the activities of antioxidant enzymes, particularly CAT and c-GPx, in different organs. At the level of the testes, oxidative stress is capable of disrupting the steroidogenic capacity of Leydig cells (38) and the capacity of the germinal epithelium to differentiate normal spermatozoa (39). Many studies have indicated that EMR decreases the size of the testicular organs and the diameter of the seminiferous tubules (16,33,40-42). However, a study by Ribeiro et al. (18) and a follow-up study by Dasdag et al.

(43) could not find any significant adverse effects of cellular phones (1835-1850 MHz) on the rat testis. Wang et al. (44) suggested that RF-EMR may change the permeability of the blood-testis barrier. RF-EMR-mediated ROS formation can lead to heat shock protein (hsp) production and phosphorylation, which can alter the secretion of growth factors and mediate an increase in the blood-testis barrier permeability, as suggested by Desai et al. (26).

Although the testes clearly do possess highly specialized antioxidant defensive enzymes, there are clear benefits to be gained by treating susceptible individuals with exogenous antioxidants. A variety of antioxidants have been assessed for their ability to counteract EMR-induced oxidative stress in the testes. To determine the relative potential of different antioxidants to counteract oxidative stress in the testes, the exposure of the rats to EMR for different periods and the use of vitamins C and E as defensive agents involved the application of antioxidant therapy prior to or in conjunction with the creation of a brief period of oxidative stress and the subsequent comparison of various testicular attributes (lipid peroxidation, histopathology, and antioxidant enzyme status) with those of the controls. In the present study, vitamins

DISCUSSION Mobile phones create an EMF around them when in use, thus increasing the electromagnetic contamination, also known as ‘‘electrosmog’’, in the vicinity. In response to public and governmental concern, the WHO established the International Electromagnetic Fields (EMF) Project in 1996 to assess the scientific evidence of the possible adverse health effects of the electromagnetic fields and to conduct a formal health risk assessment of radiofrequency field exposure by 2012 to fill these knowledge gaps (10). Biological systems are affected by microwave radiation primarily due to the resulting increase in temperature, i.e., thermal damage (11), although non-thermal effects have also been studied (12). The cellular target of EMF is still controversial. Recent studies on human semen have suggested an increased production of ROS in human semen due to cell phone radiation (13,14). An analysis of antioxidant enzymes glutathione peroxidase and superoxide dismutase showed a decrease, while an increase in catalase was observed. Malondialdehyde showed an increase and histone kinase showed a significant decrease in the exposed group. Micronuclei also show a significant decrease in the exposed group. A significant change in sperm cell cycle and were recorded. Generation of free radicals was recorded to be significantly increased. The findings of Kesari et al. (15) on antioxidants, malondialdehyde, histone kinase, micronuclei, and the sperm cell cycle are clear indications of an infertility pattern that is initiated due to an overproduction of reactive oxygen species. They also concluded that radiofrequency electromagnetic waves from commercially available cell phones may affect the fertilizing potential of spermatozoa.

There is evidence from several high-quality studies published before 2000 suggesting a lack of effects on testicular function in experimental animals provided that the exposures do not induce hyperthermia. The responses to RF fields are identical to those induced by heating using conventional means. Very few recent studies have addressed this issue, and these studies mostly support the conclusion that male fertility and testicular function are not affected in the absence of hyperthermia (16-19).

In contrast, several authors stated that electromagnetic waves have a wide range of damaging effects on the male

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C and E, when given as a supplement in conjunction with the EMR exposure, were shown to effectively protect the testes from the oxidative damage created by the exposure to EMR and significantly suppress the oxidative DNA damage induced in the rat testicular cells.

Vitamins C and E are powerful antioxidants that are vital for the maintenance of mammalian spermatogenesis and that counteract the testicular oxidative stress induced by pro-oxidant exposure (45-51). Vitamin E has also been shown to suppress lipid peroxidation in testicular microsomes and mitochondria (52,53) and to reverse the detrimental effects of oxidative stress on testicular function (45,49,54-59). Studies have demonstrated that antioxidants, such as melatonin, caffeic acid phenyl ester, vitamin C and vitamin E, prevent the oxidative stress and apoptosis caused

by EMR in animal tissues (28,29,41,60,61). This prophylactic treatment could thus be used to counteract the observations recorded by Mailankot et al. (62), who found that rats exposed to mobile phone emissions for 1 hour a day for 28 days had impaired semen quality that may impair male fertility because the RF-EMR exposure resulted in a significant increase in lipid peroxidation and a low GSH content in the testis and epididymis. Notably, pulsed electromagnetic field (PEMF) therapy was recently used as a scavenging agent to combat oxidative stress (63). PEMF is generated by many medical devices used in medical therapies. Therefore, the issue of whether vitamins C and E could be used along with PEMF to relieve the adverse effects of EMR remains to be elucidated.

In summary, EMR has a negative effect on testicular function through the induction of oxidative stress and the concomitant disruption of the testicular antioxidant status.

Given the importance of vitamins C and E in this defensive strategy, the ability of antioxidants, such as vitamins C and E, to ameliorate this pathology confirms their importance in overcoming oxidative stress in this context. Vitamins C and E ameliorate the EMR-induced oxidative stress in the testes, thus facilitating the restoration of testicular tissue morphology and function by suppressing testicular lipid peroxidation and restoring the levels of GST and GSH to normal physiological levels.

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AUTHOR CONTRIBUTIONS

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The author was solely responsible for the entire experiment, which included planning and performing the experiments and writing and revising the manuscript.

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REFERENCES

24.

1. Watanabe Y, Nakagawa M, Miyakoshi Y. Enhancement of lipid peroxidation in the liver of mice exposed to magnetic fields. Ind. Health. 1997;35(2):285–90, http://dx.doi.org/10.2486/indhealth.35.285.

2. Marks TA, Ratke CC, English WO. Stray voltage and developmental, reproductive and other toxicology problems in dogs, cats and cows: a discussion. Vet. Hum. Toxicol. 1995;37(2):163–72.

3. Lo¨scher W, Ka¨s G. Conspicuous behavioural abnormalities in a dairy cow herd near a TV and radio transmitting antenna. Prakt. Tierarzt.

1998;79(5):437–44.

4. Hyland GJ. Physics and biology of mobile telephony. The Lancet.

2000;356(9244):1-8.

5. Aitken RJ, Koopman P, Lewis SEM. Seeds of concern. Nature.

2004;432:48e52, http://dx.doi.org/10.1038/432048a.

6. Marino C. EMF-ET. NEffects of the exposure to electromagnetic fields: From science to public health and safer work place. Project no. SSPE-CT2004-502173 Instrument: Co-ordination action Thematic Priority: Priority 8, Policy Oriented Research – Area 2.3, Call Identifier FP6-2002-SSP-1 WP2.2 Deliverable report D4bis: Effects on reproduction and development.

Organisation name of lead contractor for this deliverable: ENEA - Ente per

25.

26.

27.

28.

29.

791

le Nuove tecnologie, l’Energia e l’Ambiente (ENEA), Section of Toxicology and Biomedical Sciences, Roma, Italy 2007.

Erogul O, Oztas E, Yildirim I, Kir T, Aydur E, Komesli G, et al. Effects of Electromagnetic Radiation from a Cellular Phone on Human Sperm Motility: An In Vitro Study. Arch. Med. Res. 2006;37(7):840-3, http:// dx.doi.org/10.1016/j.arcmed.2006.05.003.

Kavindra K, Kesari JB. Whole body 900 MHz radiation exposure effect on enzyme activity in male wistar rats. Available at: http://wifiinschools. org.uk/resources/Kesari+and+Behari+.pdf (cited date: July 15 2010).

Girotti MJ, Khan N, McLellan BA. Early measurement of systemic lipid peroxidation products in the plasma of major blunt trauma patients. J. Trauma 1991;31(1):32–5.

WHO. Electromagnetic fields and public health: mobile phones. Fact sheet No. 193. World Health Organization, Geneva, Switzerland 2010.

Stuchley MA. Biological effects of radiofrequency fields in M.H. Repacholi (Ed) Non-Ionizing Radiations, Physical characterization, Biological effects and Health Hazard Assessment. Proceeding for the International Non-Ionizing Radiation Workshop, Melbourne, 1988,pp.

197-217.

Kunjilwar KK, Behari J. Effect of amplitude-modulated radio frequency radiation on cholinergic system of developing rats. Brain Res. 1993;601(12):321-4, http://dx.doi.org/10.1016/0006-8993(93)91729-C. Agarwal A, Fnu Deepinder HCLD, Sharma RK, Ranga G, Li J. Effect of cell phone usage on semen analysis in men attending infertility clinic: an observational study. Fertil Steril. 2008;89(1):124-8, http://dx.doi.org/ 10.1016/j.fertnstert.2007.01.166.

De Iuliis GN, Newey RJ, King BV, Aitken RJ. Mobile phone radiation induces reactive oxygen species production and DNA damage in human spermatozoa in vitro. PLoS One. 2009;4(7):e6446, http://dx.doi.org/ 10.1371/journal.pone.0006446.

Kesari KK, Kumar S, Behari J. Effects of Radiofrequency Electromagnetic Wave Exposure from Cellular Phones on the Reproductive Pattern in Male Wistar Rats. Appl. Biochem. Biotechnol. 2011;164(4):546-59, http:// dx.doi.org/10.1007/s12010-010-9156-0.

Dasdag S, Akdag MZ, Aksen F, Yilmaz F, Bashan M, Dasdag MM, et al.

Whole body exposure of rats to microwaves emitted from a cell phone does not affect the testes. Bioelectromagnetics. 2003;24(3):182-8, http:// dx.doi.org/10.1002/bem.10083.

Forgacs Z, Somosy Z, Kubinyi G, Bakos J, Hudak A, Surjan A, et al. Effect of whole-body 1800MHz GSM-like microwave exposure on testicular steroidogenesis and histology in mice. Reprod. Toxicol. 2006;22(1):111-7, http://dx.doi.org/10.1016/j.reprotox.2005.12.003.

Ribeiro EP, Rhoden EL, Horn MM, Rhoden C, Lima LP, Toniolo L. Effects of subchronic exposure to Radio Frequency from a conventional cellular telephone on testicular function in adult rats. J. Urol.

2007;177(1):395-9, http://dx.doi.org/10.1016/j.juro.2006.08.083.

Sommer AM, Grote K, Reinhardt T, Streckert J, Hansen V, Lerchl A. Effects of Radiofrequency Electromagnetic Fields (UMTS) on Reproduction and Development of Mice: A Multi-generation Study.

Radiat. Res. 2009;171(1):89-95, http://dx.doi.org/10.1667/RR1460.1.

Zavaczki FZ, Szollosi J, Koloszar S, Daru J, Kovacs L, Pal A. Is there a relationship between cell phone use and semen quality? Arch Androl.

2005;51(5):385-93.

Derias EMB, Stefanis P, Drakeley A, Gazvani R, Lewis-Jones DI. Growing Concern Over the Safety of Using Mobile Phones and Male Fertility.

Arch. Androl. 2006;52(1):9-14.

Ji-Geng Y, Michael A, Bruce T, Hui YY, Amy G, Hani SM. Effects of cellular phone emissions on sperm motility in rats. Fertil Steril.

2007;88(4):957-64.

Agarwal A, Desai NR, Makker K, Varghese A, Mouradi R, Sabanegh E, et al. Effects of radiofrequency electromagnetic waves (RF-EMW) from cellular phones on human ejaculated semen: an in vitro pilot study. Fertil Steril. 2008;92(4):1318-25.

Gutschi T, Al-Ali BM, Shamloul R, Pummer K, Trummer H. Impact of cell phone use on men’s semen parameters. Andrologia. 2011; http:// dx.doi.org/10.1111/j.1439-0272.2010.01075.x Desai N, Sharma R, Makker K, Sabanegh E, Agarwal A. Physiologic and pathologic levels of reactive oxygen species in neat semen of infertile men.

Fertil Steril. 2009;92(5):1626-31, http://dx.doi.org/10.1016/j.fertnstert.

2008.08.109.

Desai NR, Kesari KK, Agarwal A. Pathophysiology of cell phone radiation: oxidative stress and carcinogenesis with focus on male reproductive system. Review Reprod Biol Endocrinol. 2009;7:114. http://dx.doi.org/10.1186/1477-7827-7-114.

Grundler W, Kaiser F, Keilmann F, Walleczek J. Mechanisms of electromagnetic interaction with cellular systems. Naturwissenschaften.

1992;79(12):551-9, http://dx.doi.org/10.1007/BF01131411.

Oktem F, Ozguner F, Mollaoglu H, Koyu A, Uz E. Oxidative damage in the kidney induced by 900-MHz-emitted mobile phone: protection by melatonin. Arch Med Res. 2005;36(4):350-5, http://dx.doi.org/10.1016/ j.arcmed.2005.03.021.

Oral B, Guney M, Ozguner F, Karahan N, Mungan T, Comlekci S, et al.

Endometrial apoptosis induced by a 900-MHz mobile phone: preventive effects of vitamins E and C. Adv. Ther. 2006;23(6):957-73.

Mobile phone, testes and vitamins C and E Al-Damegh MA

CLINICS 2012;67(7):785-792

48. Rao M, Narayana K, Benjamin S, Bairy KL. L-ascorbic acid ameliorates postnatal endosulfan induced testicular damage in rats. Indian J Physiol Pharmacol. 2005;49(3):331-6.

49. Sonmez M, Turk G, Yuce A. The effect of ascorbic acid supplementation on sperm quality, lipid peroxidation and testosterone levels of male Wistar rats. Theriogenology. 2005;63(7):2063-72, http://dx.doi.org/ 10.1016/j.theriogenology.2004.10.003.

50. Chang SI, Jin B, Youn P, Park C, Park JD, Ryu DY. Arsenic-induced toxicity and the protective role of ascorbic acid in mouse testis. Toxicol Appl Pharmacol. 2007;218(2):196-203, http://dx.doi.org/10.1016/ j.taap.2006.11.009.

51. Aitken RJ, Romanm SD. Antioxidant systems and oxidative stress in the testes. Review. Oxidative Med. Cell Longevity. 2008;1:15-24, http:// dx.doi.org/10.4161/oxim.1.1.6843 52. Lucesoli F, Fraga CG. Oxidative stress in testes of rats subjected to chronic iron intoxication and alpha-tocopherol supplementation.

Toxicology. 1999;132(2-3):179-86, http://dx.doi.org/10.1016/S0300-483X (98)00152-8.

53. Gavazza MB, Catala A. The effect of alpha-tocopherol on lipid peroxidation of microsomes and mitochondria from rat testis.

Prostaglandins Leukot Essent Fatty Acids. 2006;74(4):247-54, http:// dx.doi.org/10.1016/j.plefa.2006.01.007.

54. Verma RJ, Nair A. Ameliorative effect of vitamin E on aflatoxin-induced lipid peroxidation in the testis of mice. Asian J. Androl. 2001;3(3):217-21.

55. Ghosh D, Das UB, Misro M. Protective role of alpha-tocopherol-succinate (provitamin-E) in cyclophosphamide induced testicular gametogenic and steroidogenic disorders: A correlative approach to oxidative stress.

Free Radic. Res. 2002;36(11):1209-18, http://dx.doi.org/10.1080/107157 6021000016472.

56. Latchoumycandane C, Mathur PP. Effects of vitamin E on reactive oxygen species-mediated 2,3,7,8-tetrachlorodi-benzo-p-dioxin toxicity in rat testis. J Appl Toxicol. 2002;22(5):345-51, http://dx.doi.org/10.1002/ jat.866.

57. Manna I, Jana K, Samanta PK. Intensive swimming exercise-induced oxidative stress and reproductive dysfunction in male wistar rats: Protective role of alpha-tocopherol succinate. Can J Appl Physiol.

2004;29(2):172-85, http://dx.doi.org/10.1139/h04-013.

58. Jedlinska-Krakowska M, Bomba G, Jakubowski K, Rotkiewicz T, Jana B, Penkowski A. Impact of oxidative stress and supplementation with vitamins E and C on testes morphology in rats. J Reprod Dev. 2006;52(2):203-9, http://dx.doi.org/10.1262/jrd.17028.

59. Zhou DX, Qiu SD, Zhang J, Tian H, Wang HX. The protective effect of vitamin E against oxidative damage caused by formaldehyde in the testes of adult rats. Asian J Androl. 2006;8(5):584-8, http://dx.doi.org/ 10.1111/j.1745-7262.2006.00198.x.

60. Guney M, Ozguner F, Oral B, Karahan N, Mungan T. 900 MHz radiofrequency-induced histopathologic changes and oxidative stress in rat endometrium: protection by vitamins E and C. Toxicol Ind Health.

2007;23(7):411-20, http://dx.doi.org/10.1177/0748233707080906.

61. Devrim E, Ergu¨der IB, Kılıc¸og˘lu B, Yaykas¸lı E, C ¸ etin R, Durak J. Effects of Electromagnetic Radiation Use on Oxidant/Antioxidant Status and DNA Turn-over Enzyme Activities in Erythrocytes and Heart, Kidney, Liver, and Ovary Tissues From Rats: Possible Protective Role of Vitamin C. Toxicol Mech Methods.2008;18(9):679-83, http://dx.doi.org/10.1080/ 15376510701380182.

62. Mailankot M, Kunnath AP, I Jayalekshmi H, Koduru B, Valsalan R. Radio frequency electromagnetic radiation (RF-EMR) from gsm (0.9/ 1.8ghz) mobile phones induces oxidative stress and reduces sperm motility in rats. Clinics. 2009;64(6):561-5, http://dx.doi.org/10.1590/ S1807-59322009000600011.

63. Kumar S, Kesari KK, Behari J. The therapeutic effect of a pulsed electromagnetic field on the reproductive patterns of male Wistar rats exposed to a 2.45-GHz microwave field. Clinics. 2011;66(7):1237-45, http://dx.doi.org/10.1590/S1807-59322011000700020.

30. Ozguner F, Bardak Y, Comlekci S. Protective effects of melatonin and caffeic acid phenethyl ester against retinal oxidative stress in long-term use of mobile phone: a comparative study. Mol Cell Biochem. 2006;282(12):83-8, http://dx.doi.org/10.1007/s11010-006-1267-0.

31. Balci M, Devrim E, Durak I. Effects of mobile phones on oxidant/ antioxidant balance in cornea and lens of rats. Curr. Eye Res. 2007;32(1):21-5, http://dx.doi.org/10.1080/02713680601114948.

32. Meral I, Mert H, Mert N, Deger Y, Yoruk I, Yetkin A, et al. Effects of 900MHz electromagnetic field emitted from cellular phone on brain oxidative stress and some vitamin levels of guinea pigs. Brain Res. 2007;1169:120-4, http://dx.doi.org/10.1016/j.brainres.2007.07.015.

33. Dasdag S, Ketani MA, Akdag Z, Ersay AR, Sari I, Demirtas OC, et al.

Whole-body microwave exposure emitted by cellular phones and testicular function of rats. Urol Res. 1999;27(3):219-23, http://dx.doi. org/10.1007/s002400050113.

34. Bediz CS, Baltacy AK, Mogulkoc R, Oztekin E. Zinc supplementation ameliorates electromagnetic field-induced lipid peroxidation in the rat brain. Tohuko J Exp Med. 2006;208(2):133-40, http://dx.doi.org/ 10.1620/tjem.208.133.

35. Kula B, Sobczak A, Kuska R. Effect of static and ELF magnetic fields on free radical processes in rat liver and kidne. ElectroMagnetobiol.

2000;19(1):99-105.

36. Zecca L, Mantegazza C, Margonato V, Cerretelli P, Caniatti M, Piva F, et al. Biological effects of prolonged exposure to ELF electromagnetic fields in rats: III.50 Hz electromagnetic fields. Bioelectromagnetics.

2006;19(1):57-66.

37. Naziroglu M, Karaoglu A, Aksoy AO. Selenium and high dose vitamin E administration protects cisplatin-induced oxidative damage to renal, liver and lens tissues in rats. Toxicology. 2004;195(2-3):221–30, http:// dx.doi.org/10.1016/j.tox.2003.10.012.

38. Hales DB, Allen JA, Shankara T, Janus P, Buck S, Diemer T, et al.

Mitochondrial function in Leydig cell steroidogenesis. Ann NY Acad.

Sci. 2005;1061:120-34, http://dx.doi.org/10.1196/annals.1336.014.

39. Naughton CK, Nangia AK, Agarwal A. Pathophysiology of varicoceles in male infertility. Hum. Reprod. Update 2001;7(5):473-81, http:// dx.doi.org/10.1093/humupd/7.5.473.

40. Saunders RD, Kowalczuk CI. Effects of 2.45 GHz microwave radiation and heat on mouse spermatogenic epithelium. Int J Radiat Biol Relat Stud Phys Chem Med. 1981;40(6):623-32, http://dx.doi.org/10.1080/ 09553008114551611.

41. Ozguner M, Koyu A, Cesur G, Ural M, Ozguner F, Gokcimen A, et al.

Biological and morphological effects on the reproductive organ of rats after exposure to electromagnetic field. Saudi Med. J. 2005;26(3):405-10.

42. Salama N, Kishimoto T, Kanayama HO. Effects of exposure to a mobile phone on testicular function and structure in adult rabbit. Int. J. Androl.

2010;33(1):88-94, http://dx.doi.org/10.1111/j.1365-2605.2008.00940.x.

43. Dasdag S, Akdag MZ, Ulukaya E, Uzunlar AK, Yegin D. Mobile phone exposure does not induce apoptosis on spermatogenesis in rats. Arch. Med. Res. 2008;39(1):40-4, http://dx.doi.org/10.1016/j.arcmed.2007.06.013.

44. Wang XW, Ding GR, Shi CH, Zhao T, Zhang J, Zeng LH, et al. Effect of electromagnetic pulse exposure on permeability of blood-testicle barrier in mice. Biomed. Environ. Sci. 2008;21(3):218-21, http://dx.doi.org/ 10.1016/S0895-3988(08)60032-X. 45. Sen Gupta R, Sen Gupta E, Dhakal BK, Thakur AR, Ahnn J. Vitamin C and vitamin E protect the rat testes from cadmium-induced reactive oxygen species. Mol. Cells. 2004;17(1):132-9.

46. Senthil kumar J, Banudevi S, Sharmila M, Murugesan P, Srinivasan N, Balasubramanian K, et al. Effects of Vitamin C and E on PCB (Aroclor 1254) induced oxidative stress, androgen binding protein and lactate in rat Sertoli cells. Reprod. Toxicol. 2004;19(2):201-8, http://dx.doi.org/ 10.1016/j.reprotox.2004.08.001.

47. Mailankot M, Jayalakshmi H, Dutta S, Chakrabarti A, Vasudevan DM. Experimental therapeutic intervention with ascorbic acid in ethanol induced testicular injuries in rats. Indian J Exp Biol. 2005;43(2):172-6.

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DOI:10.6061/clinics/2012(07)15

BASIC RESEARCH

The use of vancomycin-loaded poly-l-lactic acid and poly-ethylene oxide microspheres for bone repair: An in vivo study De´bora Cristina Corac¸a-Huber,I Eliana Aparecida de Rezende Duek,III Maurı´cio Etchebehere,IV Luis Alberto Magna,V Eliane Maria Ingrid AmstaldenII I Experimental Orthopaedics, Department of Orthopaedic Surgery, Medical University Innsbruck, Austria. II Pathological Anatomy Department, Medical Sciences Faculty, Campinas State University, Sa˜o Paulo, Brazil. III Mechanical Engineering Faculty, Campinas State University, Sa˜o Paulo, Brazil.

IV Orthopaedic and Traumatology Department, Medical Sciences Faculty, Campinas State University, Sa˜o Paulo, Brazil. V Medical Genetics, Medical Sciences Faculty, Campinas State University, Sa˜o Paulo, Brazil.

OBJECTIVE: The aim of this study was to investigate bone repair after the implantation of vancomycin-loaded polyL-lactic acid/poly-ethylene oxide microspheres compared with vancomycin-unloaded poly-L-lactic acid/poly-ethylene oxide microspheres.

METHODS: Poly-L-lactic acid/poly-ethylene oxide microspheres were implanted in rat tibiae and evaluated for periods of 2, 4, 8, and 12 days and 4, 8, 16, and 32 weeks. The groups implanted with vancomycin-loaded and vancomycin-unloaded microspheres were compared. Histopathologic (semi-quantitative) and histomorphometric analyses were performed to evaluate the bone formation process.

RESULTS: During the first period (second day), fibrin and hemorrhaging areas were observed to be replaced by granulation tissue around the microspheres. Woven bone formation with progressive maturation was observed. All of the histopathological findings, evaluated by a semi-quantitative assay and a quantitative analysis (percentage of bone formation), were similar between the two groups.

CONCLUSION: Vancomycin-loaded poly-L-lactic acid/poly-ethylene oxide microspheres are a good bone substitute candidate for bone repair. Local antibiotic therapy using vancomycin-loaded poly-L-lactic acid/poly-ethylene oxide microspheres should be considered after the microbiological evaluation of its efficacy.

KEYWORDS: Poly-L-lactic acid; Poly-ethylene oxide; Vancomycin; Bone grafting.

Corac¸a-Huber DC, Duek EAR, Etchebehere M, Magna LA, Amstalden EMI. The use of vancomycin-loaded poly-l-lactic acid and poly-ethylene oxide microspheres for bone repair: An in vivo study. Clinics. 2012;67(7):793-798.

Received for publication on November 4, 2011; First review completed on January 16, 2012; Accepted for publication on March 12, 2012 E-mail: debora.coraca-huber@i-med.ac.at Tel.: 0043 512 9033-71697

Bioabsorbable polymeric materials are potential candidates for replacing metallic, ceramic, and polymeric nonbioabsorbable materials. Additionally, when manufactured together with those materials, they can enhance the osteointegration processes (3-10). Poly-L-lactic acid (PLLA) is a bioabsorbable polymer that is widely investigated for its use in drug delivery systems, surgical sutures, and bone fracture fixation materials (11). However, due to a very slow degradation rate, this polymer may be undesirable for some applications (12). Thus, polymeric blends, such as the blend of PLLA with poly-ethylene oxide (PEO), have been used to control the PLLA degradation rate. PEO is a biomaterial known for its biocompatibility and low toxicity (4). Corac¸a et al. (13) observed the crack formation in PLLA/PEO pins after implantation in rat tibia, which allowed tissue growth into the implant and improved the bone interaction compared with pure PLLA implants. PLLA is a Food and Drug Administration (FDA)-approved material that can be implanted or administered by injection (14,15).

INTRODUCTION Due to the limitations of bone grafts (1,2), synthetic materials for orthopedic use have become a focus of scientific studies. These products can replace or aid bone grafting for bone healing. Thus, many polymers have been studied. Polymethylmethacrylate (PMMA) is one of the most commonly used polymers as a bone cement. It is also sometimes applied as a substitute for bone grafts; however, this type of polymer is mechanically weak and requires high temperatures for synthesis, which can therefore cause tissue necrosis (2).

Copyright ß 2012 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.

No potential conflict of interest was reported.

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PLLA/PEO Microspheres as a Bone Substitute Corac¸a-Huber DC et al.

CLINICS 2012;67(7):793-798

the experimental period, the animals remained in plastic cages under controlled light and ventilation, receiving solid food (Nuvilab CR-1 from Nuvital) and water ad libitum. The study was carried out with the approval of the Ethical Committee of Animal Research (CEEA/Unicamp Protocol n. 339-1) of the Campinas State University, Sa˜o Paulo, Brazil.

In addition to the positive results with PLLA and PEO, infections associated with metal and polymeric surfaces or dead tissues, such as bone grafts, can still be fatal for the patient and represent a major financial burden for the economy. The adhering bacteria in these cases can evade host defenses by forming biofilms. Therefore, the prevention of bacterial colonization is of great interest. Injury to the surrounding tissue is unavoidable during implantation, resulting in impaired blood circulation in this area and triggering an inflammatory reaction. Bacteria are more apt to colonize the surface of the implant, where the host’s defense is weakened and the blood supply is insufficient.

When antibiotics are administered systemically, they do not reach a high therapeutic level in the area directly surrounding the implant, rendering long-term high doses of antibiotics unsuccessful in most cases (16). When examining the relationship among the implant, the host and bacteria, competition for the colonization of the implant surface can be observed. If the body’s own cells are the first to colonize the surface, they form a cell ‘‘lawn’’ of connective tissue, which makes adhering to the surface difficult for the bacteria and thus effectively protects the implant from infections. If, however, the bacteria colonize the surface first, they can, similar to the body’s own cells, form a biofilm that protects itself from host immune cells. This phenomenon is called the ‘‘race for surface’’ (17).

The aim of this study was to investigate bone repair using implants of vancomycin-loaded and vancomycin-unloaded PLLA/PEO microspheres as a bone graft substitute and possible carriers of antibiotics for local therapy. The microspheres containing vancomycin should not impair the process of bone healing.

Production of the microspheres Pure PLLA and pure PEO granulates (Sigma Aldrich) were mixed in a ratio of 80:20 and melted in a Mini Max Molder LMM-2017 injector (Custom Scientific Instruments, Inc., Whippany, NJ). The polymer was heated inside a mold and stirred for gravity collection. The resulting stick blend was cooled at room temperature. Half a gram of the 80:20 PLLA/PEO blend was dissolved in 10 ml of dichloromethane (DCM). Vancomycin was added to the polymeric solution (0.75 ml) and mixed to create an emulsion. To gather the microspheres, the emulsion was placed in a 2% isopropanol solution. The solution was vortexed until total DCM evaporation occurred, and the microspheres were collected from the supernatant and vacuum dried. The microspheres, sized 100-200 mm in diameter, were compressed to facilitate their use during surgery.

Surgical procedures The animals were anesthetized before the surgical procedure to create the defects. The anesthesia was induced interperitoneally with 0.1 mg/100 mg body weight ketamine (KetalarH, Pfizer, Guarulhos, Brazil) and 0.08 mg/ 100 mg body weight xylazine (RompunH, Bayer, Sao Paulo, Brazil). Under anesthesia, a longitudinal incision was made in the skin above the proximal portion of the left tibia. The muscle tissue was retracted, and the bone was exposed. An odontological drill was used to create a cavity (3-mm diameter) in the tibia. During this process, the area was continuously irrigated with a saline solution to avoid overheating. The cavity was filled with the microspheres, and the muscle and skin were sutured. In each group, 40 animals were divided into subgroups of five animals for each experimental period (2, 4, 8, and 12 days and 4, 8, 16, and 32 weeks). No animals died during these periods. At the end of each period, the animals belonging to each subgroup were euthanized by an overdose of anesthesia, and the implanted tibia was removed for histological evaluation.

MATERIALS AND METHODS Animals A total of 80 adult (eight weeks old) male Wistar rats were divided into two groups as follows: 40 animals were implanted with vancomycin-loaded PLLA/PEO microspheres and 40 animals were implanted with vancomycinunloaded PLLA/PEO microspheres as a control. Although the objective of this study was the evaluation of bone healing in the presence of vancomycin-loaded implants, no tests for spontaneous bone healing were performed. During

Table 1 - Estimated histopathological features used in the semi-quantitative analysis.

Items

Intensity Absence (0)

Light (1)

Moderate (2)

Intense (3)

-

Occupying up to 1/3 of the microscopic field Occupying up to 1/3 of the microscopic field Around the microspheres

Occupying up to 2/3 of the microscopic field Occupying up to 2/3 of the microscopic field Around and partially filling the center of the microspheres

Occupying from 2/3 to all of the microscopic field

Level 1

Level 2

Level 3

Level 4

Loose connective tissue

Dense connective tissue

Connective tissue with partial ossification

Total ossification

Granulation Tissue (HE 106) Acute Inflammation (HE 406) Bone Formation

-

Item

Intensity

Connective Septae

-

794

Occupying from 2/3 to all of the microscopic field Around and completely filling the center of the microspheres

CLINICS 2012;67(7):793-798

PLLA/PEO Microspheres as a Bone Substitute Corac¸a-Huber DC et al.

Figure 1 - Histology of a tibia implanted with vancomycin-loaded microspheres composed of PLLA/PEO: A) implant on the 4th day, characterized by exuberant granulation tissue around the microspheres (Original H&E 206); B) implant on the 12th day, showing remodeling into woven bone characterized by osteoblastic (arrow right) and osteoclastic (arrow left) activity (Original TM 406); and C and D) implants on the 8th day, characterized by woven bone surrounding the microspheres that becomes mature when composed of lamellar bone at 32 weeks (Original TM 106).

Figure 2 - Histology of tibias implanted with PLLA/PEO that were A) vancomycin-loaded or B) vancomycin-unloaded on the 4th day, showing evident granulation tissue around the microspheres (arrow) with the initial replacement by bone formation (arrow).

This figure also shows the histology of the tibias of animals implanted after 12 days with PLLA/PEO that were C) vancomycinloaded or D) vancomycin-unloaded, showing trabecular bone encasing the microspheres (Original H&E 106).

medullary cavity and were replaced by granulation tissue on approximately the fourth day (Figure 1A).

In addition, a loose interstitial connective mesh was observed wrapped around the microspheres. Septa of a connective-like tissue were observed invading the spaces between the microspheres. Those septa were comprised of fibroblast-like mesenchymal elements with osteoblast differentiation and endothelial-like cells that created vascular lumens. Discrete inflammatory cell foci with neutrophilic predomination were observed only during the initial period (two and four days). On the fourth day, the proliferation of initial immature bone formation (woven bone) was observed around the cluster of microspheres,

Microscopic evaluation and morphometric analysis The removed tibiae were fixed in 37% formalin, decalcified in ethylenediaminetetraacetic acid (EDTA) solution and submitted to histological analysis. The sections were stained with hematoxylin/eosin and Masson’s trichrome stain.

Conventional light microscopy was used for the histopathological evaluation. Qualitative (descriptive) and semiquantitative analyses were used according to the following criteria (Table 1).

For the histomorphometric analysis, the Masson’s trichromestained sections were photographed, and the images were analyzed with the software Image LabH (ImageLab, Sao Paulo, Brazil). The results were used to measure the new bone formed (%) around the implant (microspheres).

Statistical analysis The Mantel-Haenszel test was used to compare the different levels of intensity obtained by the morphometric (semi-quantitative) assessments. The co-variance analysis and Mann-Whitney test were used to compare the differences between the percentages of bone formation around the microspheres during the experimental periods, as obtained by the histomorphometric analysis. p-values of 0.05 or lower were considered statistically significant. SPSS 17.0 (SPSS Inc., Chicago, IL, USA) was used for the statistical analysis.

The software GraphPadH Prism 5.01 (GraphPad Software Inc., La Jolla, CA, USA) was used to create the graphs.

RESULTS Microscopic findings Figure 3 - Histology of tibias implanted with PLLA/PEO that were A) vancomycin-loaded or B) vancomycin-unloaded after 4 weeks, showing thin trabecular bone (arrow); and PLLA/PEO that were C) vancomycin-loaded or D) vancomycin-unloaded during a 32week period, showing massive bone formation surrounding the microspheres with evident maturation and the presence of lamellar bone (arrow) (Original H&E 106).

The microscopic findings were similar in both groups throughout the experimental periods. The vancomycin-loaded and vancomycin-unloaded PLLA/PEO microspheres were easily visible inside the medullary cavity in both groups during all evaluated periods (Figures 1, 2, and 3). After two days, fibrin and hemorrhaging were visibly close to the implant in the

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the thickness of trabecular bone. In the final periods, a completely mature ossification of the septum inside the implant and around each microsphere was observed (Figures 3C and 3D). This ossification formed a capsule encasing the implant. Tissue rejection signals, such as thick fibrous capsules or necrosis, were not observed.

gradually replacing the granulation tissue and the interstitial connective septa (Figures 2A and 2B) and becoming abundant in the eight-week implants (Figure 1C). On approximately the 12th day, obvious trabecular bone was observed around the microspheres (Figures 2C and 2D). This period was also characterized by bone remodeling with intense osteoblast and osteoclast activity (Figure 1B). Thin trabecular bone was observed in the fourth week (Figures 3A and 3B). After this event, gradual bone maturation was observed with lamellarlike bone centripetally in the microsphere group, which had

Histopathological (semi-quantitative) analysis The level of intensity for each item evaluated by the histopathological (semi-quantitative) analysis (Figure 4 and

Figure 4 - Histopathological (semi-quantitative) analysis: 0 = absent, 1 = light, 2 = moderate and 3 = intense. The periods that showed no values are equivalent to 0 = absent.

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A mild inflammatory reaction with giant cells was observed without edema, and, therefore, the bone regeneration was not affected. Beginning on the fourth day after the implantation, the areas of fibrin and hemorrhage, organized in the granulation and fibrous connective tissue, were gradually replaced by bone. The bone grew between the individual microspheres, encasing the implant. This finding demonstrated the characteristic osteointegration capacity of this material. Ueng et al. (20) detected this reaction in rabbits loaded with vancomycin-loaded poly (L-lysine) (PLL) beads, which were encased by bone after eight weeks.

The osteointegration process in the present study was better for PLLA/PEO microspheres compared with PLLA/ PEO molded as pins (13). This result can likely be attributed to the sphere morphology, which permits more tissue interaction with the bone formation that occurs not only around but also inside the implant and in between the microspheres. Another advantage of the microsphere morphology is that it facilitates the use of beads in surgical bone cavities. Furthermore, the microspheres do not need to be cast in advance or be polymerized under high temperatures during surgery. Ueng et al. (20) described this advantage for bone repair after tumor removal or infectious material curettage, such as in osteomyelitis. In addition to the morphology, the microsphere-shaped implants, in association with antibiotic drugs, could be candidates for local antibiotic therapy to prevent and treat bone infections.

In this study, the gradual bone formation was morphologically and morphometrically assessed. The observed bone was of a ‘‘membranous’’ type formed independent of cartilage.

Arrote´ia et al. (21) have confirmed that a local vessel supply and granulation tissue formed by hemorrhagic clots and fibrin nets are basic requisites for the formation of bone of this type.

The bone healing process involves a series of highly regulated steps that depend on the interactions of two cell lineages: the mesenchymal osteoblasts and hematopoietic osteoclasts.

During this process, the bone matrix is deposited in an irregular manner and with a definitive form characterized by a trabecular bone structure that is sculptured or remodeled by the reabsorptive action of the osteoclasts. This process is followed by the deposition of new bone via the action of osteoblastic cells during the following periods until reaching the thickness of the trabeculae of the local pre-existing bone bars (21,22). This study demonstrated similar aspects in the bone healing process. Gradual bone growth was morphologically and morphometrically proven during the experimental

Figure 5 - Level of maturation of the connective septae formed around the microspheres (1 = loose connective tissue, 2 = dense connective tissue, 3 = connective tissue with partial ossification and 4 = total ossification). The periods that showed no values are equivalent to 0 = absent.

5) did not exhibit any significant differences between the vancomycin-loaded and vancomycin-unloaded PLLA/PEO microsphere groups.

Histomorphometric analysis The percentage of bone formation was similar between the vancomycin-loaded and vancomycin-unloaded PLLA/PEO microsphere groups. However, a significant difference in the bone formation was noted over the course of the entire experimental period (p#0.05). The bone formation increased gradually during the four earliest experimental periods and stabilized in approximately the fourth week. Subsequently, the bone formation process underwent a maturation phase that continued into the last period (Figure 6).

DISCUSSION The PLLA/PEO blend was selected for this study due to its well-known biocompatibility (5,13). Vancomycin was added to the PLLA/PEO blend because of its common use as a local therapy for bone infection (18).

After implantation, the microspheres remained inside the medullar cavity in all animals. This result may be explained first by the connective and bone tissue proliferation encasing the microspheres and, second, by the long-term degradation of the PLLA (12). The inflammatory process was mild and present only during the initial periods (from 2 to 4 days) with neutrophilic predominance, a characteristic process of tissue repair.

Figure 6 - Percentage of bone formation during the different experimental periods for the vancomycin-loaded PLLA/PEO microspheres and vancomycin-unloaded PLLA/PEO. The periods that showed no values are equivalent to 0 = absent.

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periods. Thin trabeculae were observed after four weeks, followed by a stabilization of the bone formation. The presence of thin trabeculae can be explained by the osteoclastic activity in the bone remodeling cycle. This phenomenon was clearly verified in the previous period analyzed (12 day), which was followed by osteoblastic activity that was responsible for the new bone deposition and the progressive trabecular thickening observed in the successive periods.

According to Raisz (22), after the reabsorption activity by the osteoclasts during the bone remodeling cycle, there is a subsequent reversal phase in which mononuclear cells are present. These cells may complete the reabsorption process and produce the signals that initiate the bone formation.

Finally, successive waves of mesenchymal cells differentiate into functional osteoblasts, which lay down the matrix during the bone formation phase. In our study, this reversal phase was observed from the eight week until the last experimental periods in both groups. Complete bone maturation, characterized by lamination of the trabeculae and a return to its original thickness, was observed in the last period (32 weeks).

Ribeiro et al. (23) describe this bone architecture change during the evaluation of cavities obtained in dogs in an experimental study of spontaneous bone regeneration. In this case, from six to ten weeks after the bone formation, an osteoporotic appearance was observed in the trabecular bone compared with the appearance in the other periods.

This phenomenon could be related to the osteoclastic activity. However, the trabecular thickness and distribution returned to the initial thickness and distribution after 12 weeks. The transition reported in both the study by Ribeiro et al. and our own study did not impair the lesion healing.

The statistical analyses showed progressive bone formation during all of the experimental periods. The new bone growth curve revealed the characteristics of the bone healing process. These findings were also observed by Ribeiro et al. (23), who described bone maturation and lamination with trabecular thickening on the implants after 12 weeks during spontaneous bone regeneration in dogs.

In conclusion, our results demonstrated efficient bone regeneration using PLLA/PEO blends mixed with vancomycin. In this case, the vancomycin did not impair the bone healing process when associated with PLLA/PEO microspheres. Further investigation is needed with respect to the delivery rate of vancomycin from the PLLA/PEO microspheres and its efficacy against microorganisms usually involved in implant infections. Overall, the vancomycinloaded PLLA/PEO microspheres appear to be good candidates as a bone substitute or as an adjuvant for bone repair.

performance of the statistical analysis, analysis of the data and writing of the manuscript.

REFERENCES 1. Boyce T, Edwards J, Scarborough N. Allograft bone. The influence of processing on safety and performance. Orthop Clin North Am. 1999;30(4):571-81.

2. Cook S, Barrack R, Skinner H. Basic science in orthopedic surgery. In: Skinner HB, editor. Current diagnosis and treatment in orthopedics.

Singapore: McGraw-Hill; 2000.p.1-46.

3. Gourlay S, Rice R, Hegyeli A, Wade C, Dillon J, Jaffe H, et al.

Biocompatibility testing of polymers: in vivo implantation studies.

J Biomed Mater Res. 1978;12(2):219-32, http://dx.doi.org/10.1002/ jbm.820120207.

4. Desai NP, Hubbell JA. Solution technique to incorporate polyethylene oxide and other water-soluble polymers into surfaces of polymeric biomaterials. Biomaterials. 1991;12(2):144-53, http://dx.doi.org/ 10.1016/0142-9612(91)90193-E. 5. Meikle M, Papaioannou S, Ratledge T, Speight P, Watt-Smith S, Hill P, et al. Effect of poly DL-lactide—co-glycolide implants and xenogeneic bone matrix-derived growth factors on calvarial bone repair in the rabbit.

Biomaterials. 1994;15(7):513-21, http://dx.doi.org/10.1016/01429612(94)90017-5.

6. Huatan H, Collett JH, Attwood D, Booth C. Preparation and characterization of poly([var epsilon]-caprolactone) polymer blends for the delivery of proteins. Biomaterials. 1995;16(17):1297-303, http:// dx.doi.org/10.1016/0142-9612(95)91044-Y. 7. Bergsma JE, de Bruijn WC, Rozema FR, Bos RRM, Boering G. Late degradation tissue response to poly(-lactide) bone plates and screws.

Biomaterials. 1995;16(1):25-31, http://dx.doi.org/10.1016/0142-9612(95) 91092-D. 8. Nijenhuis AJ, Colstee E, Grijpma DW, Pennings AJ. High molecular weight poly(-lactide) and poly(ethylene oxide) blends: thermal characterization and physical properties. Polymer. 1996;37(17):5849-57, http://dx.doi.org/10.1016/S0032-3861(96)00455-7.

9. Mainil-Varlet P, Gogolewski S, Nieuwenhuis P. Long-term soft tissue reaction to various polylactides and their in vivo degradation. J Mater Sci Mater Med. 1996;7:713-21. http://dx.doi.org/10.1007/BF00121406.

10. Lowry KJ, Hamson KR, Bear L, Peng YB, Calaluce R, Evans ML, et al.

Polycaprolactone/glass bioabsorbable implant in a rabbit humerus fracture model. J Biomed Mater Res. 1997;36(4):536-41, http://dx.doi.org/10.1002/ (SICI)1097-4636(19970915)36:4,536::AID-JBM12.3.0.CO;2-8.

11. Andreopoulos AG, Hatzi EC, Doxastakis M. Controlled release systems based on poly(lactic acid). An in vitro and in vivo study. J Mater Sci Mater Med. 2000;11(6):393-7.

12. Rokkanem P, Bo¨stman O, Hirvensalo E. Bioabsorbable implants in orthopaedics. Current Orthopaedics. 1999;13:223-8, http://dx.doi.org/ 10.1016/S0268-0890(99)90007-5.

13. Coraca DC, Duek EA, Padovani CA, Camilli JA. Osteointegration of poly(L: -lactic acid)PLLA and poly(L: -lactic acid)PLLA/poly(ethylene oxide)PEO implants in rat tibiae. J Mater Sci Mater Med. 2008;19(7):2699704, http://dx.doi.org/10.1007/s10856-008-3397-2.

14. Dunn RL, Ottenbrite RM. Polymeric drugs and drug delivery system.

Washington: ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

15. Simamora P, Chern W. Poly-L-lactic acid: an overview. J. Drugs Dermatol. 2006;5(5):436-40.

16. Stemberger A, Schwabe J, Ibrahim K, Matl F, Ro¨ssner M, Vogt S, et al.

New antibiotic carriers and coatings in surgery. In: Walenkamp G, editor. Local Antibiotics in Arthroplasty. Stuttgart/New York: Georg Thieme Verlag; 2007.p.13-21.

17. Gristina AG. Implant failure and the immuno-incompetent fibroinflammatory zone. Clin Orthop Relat Res. 1994;298:106-18.

18. Mader JT, Shirtliff ME, Bergquist SC, Calhoun J. Antimicrobial Treatment of Chronic Osteomyelitis. Clin Orthop Relat Res. 1999;360:47-65, http://dx.doi.org/10.1097/00003086-199903000-00008.

19. Itokazu M, Ohno T, Tanemori T, Wada E, Kato N, Watanabe K. Antibiotic-loaded hydroxyapatite blocks in the treatment of experimental osteomyelitis in rats. J Med Microbiol. 1997;46(9):779-83, http:// dx.doi.org/10.1099/00222615-46-9-779.

20. Ueng SW, Yuan LJ, Lee N, Lin SS, Chan EC, Weng JH. In vivo study of biodegradable alginate antibiotic beads in rabbits. J Orthop Res. 2004;22(3):592-9, http://dx.doi.org/10.1016/j.orthres.2003.09.001.

21. Arrote´ia KF, Pereira LAV. Osteoblastos. In: Carvalho HF & CollaresBuzato CB, editors. Ce´lulas - Uma abordagem multidisciplinar. Manole.

Barueri, SP. Brasil. 2005.p.34-49.

22. Raisz LG. ‘‘Physiology and pathophysiology of bone remodeling’’. Clin Chem. 1999;45: (8 Pt 2):1353-8.

23. Ribeiro R, Amstalden E, Izatto I. Modelo experimental de regenerac¸a˜o o´ssea espontaˆnea. Rev Bras Orthop. 1996;31(11):931-5.

ACKNOWLEDGMENTS This work was supported by the Coordination for the Improvement of Higher Level Personnel (CAPES) and the Foundation of Support to Education and Research (FAEP), Sao Paulo, Brazil. We thank Dennis Huber for his assistance with the statistics analysis and manuscript edition.

AUTHOR CONTRIBUTIONS Corac¸a-Huber DC participated in the development of the study plan, performance of the laboratory experiments and animal surgery, analysis of the data and writing of the manuscript. Amstalden EMI participated in the development of the study plan, analysis of the data and writing of the manuscript. Duek EAR participated in the development of the study plan, performance of the laboratory experiments, production of the biomaterials and analysis of the data. Etchebehere M participated in the development of the study plan and analysis of the data. Magna LA participated in the

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DOI:10.6061/clinics/2012(07)16

BASIC RESEARCH

Time-related effects of general functional training in spinal cord-injured rats Taisa Amoroso Bortolato Miranda,I Juliana Mendes Yule Vicente,II Raphael Martus Marcon,III Alexandre Fogac¸a Cristante,III Edgard Morya,IV Angela Cristina do ValleI I

Faculdade de Medicina da Universidade de Sa˜o Paulo, Program of Experimental Physiopathology, Sa˜o Paulo/SP, Brazil. II Faculdade de Medicina da Universidade de Sa˜o Paulo, Program of Experimental Physiopathology, Sa˜o Paulo/SP, Brazil. III Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Laboratory of Spinal Cord Injury, Orthopedic and Traumatology Institute, Sa˜o Paulo/SP, Brazil. IV Sı´rio-Libaneˆs Hospital, Alberto Santos Dumont Research Support Association, Teaching and Research Institute, Sa˜o Paulo/SP, Brazil.

OBJECTIVES: This prospective, randomized, experimental study with rats aimed to investigate the influence of general treatment strategies on the motor recovery of Wistar rats with moderate contusive spinal cord injury.

METHODS: A total of 51 Wistar rats were randomized into five groups: control, maze, ramp, runway, and sham (laminectomy only). The rats underwent spinal cord injury at the T9-T10 levels using the NYU-Impactor. Each group was trained for 12 minutes twice a week for two weeks before and five weeks after the spinal cord injury, except for the control group. Functional motor recovery was assessed with the Basso, Beattie, and Bresnahan Scale on the first postoperative day and then once a week for five weeks. The animals were euthanized, and the spinal cords were collected for histological analysis.

RESULTS: Ramp and maze groups showed an earlier and greater functional improvement effect than the control and runway groups. However, over time, unexpectedly, all of the groups showed similar effects as the control group, with spontaneous recovery. There were no histological differences in the injured area between the trained and control groups.

CONCLUSION: Short-term benefits can be associated with a specific training regime; however, the same training was ineffective at maintaining superior long-term recovery. These results might support new considerations before hospital discharge of patients with spinal cord injuries.

KEYWORDS: Spinal cord injury; Rehabilitation; Exercise movement techniques.

Miranda TA, Vicente JM, Marcon RM, Cristante AF, Morya E, Valle AC. Time-related effects of general functional training in spinal cord-injured rats. Clinics. 2012;67(7):799-804.

Received for publication on December 22, 2011; First review completed on February 2, 2012; Accepted for publication on March 9, 2012 E-mail: taisaabm@hotmail.com Tel.: 55 11 82773336

studies with injured animals undergoing a range of training strategies in different environments may facilitate an understanding of SCI recovery mechanisms and may promote the development of improved human rehabilitation programs.

This study aimed to assess the therapeutic effects of three types of locomotor training in Wistar rats with moderate contusive spinal cord injury.

INTRODUCTION Spinal cord injury (SCI) either completely or partially damages the ascending and descending spinal pathways, leading to a loss of adequate stimulus perception and imprecision in the reorganization of motor and sensory behaviors (1). In such cases, physical therapy and training are considered to be the most efficient strategies for inducing neuroplasticity and motor recovery (2). Therefore, recent functional recovery studies in human and animal models focus on new methods of optimizing the sensory information associated with locomotor training (3,4). However, the effect of certain physical therapy and rehabilitation strategies used in human functional recovery remains unclear. Experimental

MATERIALS AND METHODS Animals A total of 51 male adult Wistar rats (150–300 g) were divided into five groups according to the type of exercise they underwent: control (n = 11), maze (n = 10), ramp (n = 9), runway (n = 10) and sham (n = 11). The sample size was estimated according to the Multicenter Animal Spinal Cord Injury Study (MASCIS) using the New York University (NYU) protocol, which was previously used in studies published by our laboratory (5-7). The runway, maze, ramp, and control groups were subjected to contusive SCI, whereas the sham group was subjected only to the surgical

Copyright ß 2012 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.

No potential conflict of interest was reported.

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procedure without SCI. The project and all of the procedures were approved by the University of Sa˜o Paulo Ethical Committee (CAPPesq).

apparatuses (9). The rats were food-deprived for 24 hours before the first two trainings, and water with sucrose was used as a reward during the trainings to stimulate the animals to accomplish the tasks. After the SCI, twice-aweek training sessions were initiated on the third postoperative day (POd) without food or water deprivation; these sessions also lasted 12 minutes and were continued for five weeks.

Surgical procedure The rats were anesthetized with diazepam (intraperitoneal (ip), 0.1 ml/100 g) and ketamine hydrochloride (ip 0.2 ml/100 g). Lidocaine hydrochloride was administered as a local anesthetic. The spinal cord contusion surgeries were performed by the biologist in charge of the laboratory (who was blind to the other procedures planned for each rat) and in accordance with MASCIS using the NYUImpactor. Laminectomy at the T9-T10 thoracic level exposed the spinal cord, and a 10-g rod dropped from a 25-mm height produced a moderate injury (5,8). The antibiotic cefazoline (0.1 ml/100 g) was administered immediately after surgery and once a day for seven days.

Behavioral assessment The Basso, Beattie, and Bresnahan (BBB) Open Field Locomotor Scale was used for the motor recovery assessment (10). The BBB test has a 21-point rating scale: complete hindlimb paralysis is scored 0, and normal locomotion is scored 21 (1,8). This scale assesses hindlimb movement and position, dorsal or plantar stepping, forelimb-hindlimb coordination, toe clearance and trunk and tail position, among other features of motor functional recovery. An open field (90 cm 6 90 cm) with a non-slippery floor and two video cameras were used for the locomotor assessment. All of the animals were evaluated for four minutes on the 1st, 8th, 15th, 22nd, 29th, and 36th POd by three observers who were blinded to the allocation of the animals. The videos were recorded for further analysis in case of divergence in the BBB scores assigned by the observers. If there was any divergence, the evaluators discussed the case until they reached a consensus.

Apparatuses Three locomotor training apparatuses, a runway, a maze, and a ramp (Figure 1), were constructed for this study.

Runway: the animals walked on a horizontal plane surface.

Maze: the animals walked on a horizontal surface with different textures at every 20 cm (flat, rugose, rough, smooth, pointed, and undulate) and with blockages that lead to deviations from the path. Ramp: the animals walked on an inclined surface (15 ˚, 20 ˚, 25 ˚, or 30 ˚), with the slope depending on the functional recovery.

Histology On the 45th POd, all of the animals were anesthetized with ketamine overdose and were then perfused transcardially with saline and 4% buffered paraformaldehyde. The spinal cords were dissected, and a 2-cm segment (centered on the lesion epicenter) was removed. The tissue was post-fixed overnight in the same fixative, immersed for one day in PBS and then embedded in paraffin.

Training All of the animals underwent a 10-minute handling to gain familiarity with the researcher two days before the experimental procedures. The maze, ramp, runway, and sham groups were subjected to a 12-minute training procedure twice a week during the two weeks before the SCI to learn the task that they needed to perform in the

Figure 1 - Apparatuses where the animals were trained. (A) Runway apparatus with measures. (B) Maze apparatus with measures and indication of different textures. (C) Ramp apparatus with measures and indication of four levels.

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hindlimb joints (mean locomotor BBB scale ranging between 0 to 0.4).

The results showed a significant main effect for postoperative day (F5,150 = 95.32, p,0.001), a significant POd 6 group interaction (F15,150 = 2.57, p = 0.002), and a marginally significant main effect for group (F3,30 = 2.85, p = 0.054). On the 1st POd, there were no significant differences (e.g., the rats had similar spinal cord lesions), and soon after the training intervention, on the 8th POd, the maze and ramp groups achieved better motor control performances (as verified by BBB scores) compared with the control and runway groups (F3,30 = 7.468, p = 0.001). Whereas the control and runway groups achieved BBB scores of 1.8 and 2.1, respectively, the maze and ramp groups achieved BBB scores of 5.8 and 7.4, respectively. Functionally, there is a significant difference between these scores because up to 2 points, the animals have extensive movement in one joint and slight movement in another one, whereas from 3 to 7 points, animals exhibit extensive movement of all three joints. Thus, there were practically no improvements in the control and runway groups, which resulted in a delay of functional recovery among these animals compared with the others.

By the end of the experiment, the rats subjected to training on the ramp apparatus showed 11.4 BBB punctuation, which means that these animals had frequent to consistent weight-supported plantar steps (10). This motor behavior was not achieved by any other group, even occasionally. However, there was no significant difference among the groups at the 36th POd. Notably, the control group showed motor recovery similar to that of the runway group from the beginning of the experiment. This result might be due to the lower effort required by the runway task compared with the training in the maze and ramp apparatuses. However, it should be remarked that these rats could move freely in their cages as well as the other groups. There was no significant functional difference between the control and runway groups, even in the first week; thus, exercising on the runway or moving freely in the cage result in the same, functionally. The highest BBB score obtained by these groups was 7.5, which corresponds to the capacity to move all three joints of the hindlimbs extensively but without any body weight support. This score was the same as that achieved by the ramp group on the 8th POd. Although these differences could be noted, at the end of the experiment, there were no significant differences among any group.

Transverse 7-mm serial sections were cut using a Leica RM 2155 microtome. Slices were selected every 0.5 cm rostral and caudal from the injury epicenter (slices from levels + 1.0 cm; + 0.5 cm; epicenter; -0.5 cm; -1.0 cm) and were stained with cresyl violet.

Spinal cord morphometry was conducted in five randomly chosen animals from each group (ten slices per animal, two slices for each level). The sections were examined at low magnification (5X), and the images were captured with a Canon Power Shot A620 digital camera coupled to an Axio Scope 40 microscope (Carl Zeiss, Jena, Germany). The outer borders of the white and gray matter were spared, and the entire spinal cord section (Figure 2) was manually traced using AxioVision 4.5 software to determine its area (mm2).

The accuracy of the tracing was verified by viewing the delineations at a higher magnification.

Statistical analysis The locomotor scores for the hindlimbs were averaged together for each rat. All of the parametric data were analyzed using Statistical Package for Social Sciences (SPSS) software. The open field locomotor scores were compared using an analysis of variance (ANOVA) with the factors group (between-subjects) and POd (within-subjects and repeated measure factor). A one-way analysis was used to examine the differences in the between-subjects factor. A post hoc Bonferroni test was used when necessary to examine specific differences between the groups.

One-way ANOVA was used for between-group comparisons of the spared tissue. Pairwise post hoc comparisons were made with Bonferroni-corrected t tests.

RESULTS Behavioral assessment As expected, the sham group achieved the highest BBB score (21 points) from the first day after the surgical procedure without spinal cord injury. The other groups showed no significant differences on the 1st POd (F3,30 = 0.34, p = 0.790, Figure 3), indicating a contusive injury consistency with no movements or slight movement of one or two

Histological assessment Histological analysis showed that spinal cord weight-drop contusion resulted in cavities and loss of gross tissue structure, which could be observed as alterations in both white and gray matter at the injury epicenter and throughout several millimeters rostrally and caudally in all of the injured animals.

Large cystic cavities were observed in regions previously occupied by the gray matter, as illustrated in Figure 4. The spared tissue was lightly stained and appeared similar to partial demyelination, and a microcystic zone was observed. It could be noted in many spinal cord slices that a small region of gray matter was spared in the superficial dorsal horns (Figure 4). Otherwise, as expected, the sham group histology revealed intact spinal cords, as demonstrated in Figure 2.

The results showed a significant main effect for distance (F4,80 = 12.357, p,0.001), a non-significant distance 6 group interaction (F12,80 = 0.816, p = 0.634) and a non-significant

Figure 2 - Sham spinal cord transverse slice, 7 mm thick, with indication of total spinal cord and gray matter areas (mm2). The white matter area was calculated by subtracting gray matter area of the total spinal cord area.

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Figure 3 - The time course of locomotor recovery as measured by BBB open field scores for ramp, maze, runway control and sham groups. Since the 1st POd the sham group punctuated 21, showing no motor deficits. Maze and ramp groups showed greatest recovery when compared to runway and control groups on the 8th POd (*p = 0.001), but this difference did not persist till the 36th POd, although the ramp group tended to have a better performance.

main effect for group (F3,20 = 0.913, p = 0.452). A post hoc test showed that the lesion was more severe at the epicenter than at the extremities (Figure 5).

between generation of the lesion and intervention could be more favorable for axonal regeneration and behavioral recovery. More recently, Smith et al. (13) also indicated that swimming training initiated acutely might be detrimental to functional recovery due to the exacerbation of the acute phase inflammatory processes.

Many authors agree that early intervention is an efficient strategy for rehabilitation (14-16). They support the idea that the regeneration capacity decreases over time in not only humans but also experimental models due to many factors, such as axonal demyelination, increase in the lesion area, loss of neurons and glial scar formation, among others.

The training sessions on the ramp and maze apparatuses were more effective for performance improvement soon after the SCI. This fact is critical when considering that it is desirable for injured persons to acquire functional independency as soon as possible. According to Norrie et al. (15), the early training session facilitates the synapses that are more plastic before the development of aberrant reflex connections. Thus, rehabilitation programs should initiate as rapidly as possible in an attempt to minimize secondary damage caused by injuries to achieve functional recovery through the motor features that remain after SCI. However, the beneficial aspects of maze and ramp training observed in the early stage after the contusion did not persist in the late phase. The stimulus provided by these apparatuses was not sufficient to generate a better recovery than that achieved by the runway or control groups. It is possible that the training load should vary over time for the acquisition of new motor features rather than merely maintaining those already gained.

Although 24 minutes of training per week could appear insufficient to cause motor control improvement, recently, Smith et al. (13) reported that only eight minutes of swimming per week is sufficient to induce a degree of functional recovery of hindlimb movement.

DISCUSSION In this study, training sessions in the apparatuses were initiated on the third day after injury. This early intervention appears to have led to more efficient motor recovery of the maze and ramp animals, as these groups exhibited performance improvement from the beginning compared to the control and runway groups. Certain authors (11,12) suggest that a small interval (two weeks, subchronic phase)

Figure 4 - Injured spinal cord transverse slice, 7 mm thick, with the arrows showing structural/morphological changes after injury, spared tissue, large cystic cavities in both white such as microcysts, region of gray matter and gray matters, spared in the dorsal horn.

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Figure 5 - Injured spinal cord transverse slices, 7 mm thick. The epicenter showed a significant difference compared to the damaged extremities.

Notably, the control group displayed motor recovery without being subjected to any type of training. These data indicate that there is spontaneous recovery after experimental spinal cord injury, supporting Leung and Wrathall (17), who claimed that a level of functional recovery could be reached without intervention. This recovery is mediated by a structural reorganization of the residual motor system, which may develop over weeks in animal models of SCI and over many months in humans (18). Although there is a spontaneous recovery, this recovery is delayed by the limited capacity of the mammalian central nervous system to reestablish functional neuronal connections (16). This result is in accordance with our data: until the 8th POd, the control motor recovery was less than that of the maze and ramp groups. In a study of rats with spinal injuries, Siegenthaler et al. (19) also verified that animals trained on a running wheel showed greater improvement until the second week after injury, whereas the peak improvement of sedentary animals was delayed by one more week.

Although there was earlier improvement in the animals subjected to sensorimotor training, more long-term outcomes were not different between the trained and control groups. This result was also observed by Sandrow-Feinberg et al. (20), who used forced exercise as a rehabilitation program after unilateral cervical SCI and verified an early recovery of the trained rats that was not significant compared with that of the control group at the end of the experiment. Andrade et al. (21) also observed a better evolution of the BBB score in injured treadmill-trained rats during the acute period after the injury, but not in the chronic phase.

Soon after SCI, animals develop a flaccid pattern below the lesion level, which means that there are no observable hindlimb joint movements. This flaccidity is the reason that an injured animal’s pattern of stepping remains dorsal. The ramp appeared to be the most efficient in promoting plantar stepping, which can be attributed to the task specificity required by this apparatus. It is believed that the improvement occurred due to the demand imposed by the ramp, in which the animals had to coordinate their steps while going up and down the ramp, actions which require different types of muscle recruitment against a greater load. Multon et al.

(22) have also shown that animals subjected to treadmill training after acute incomplete spinal cord compression are able to support their body weight and perform plantar steps.

The animals did not exhibit forelimb-hindlimb coordination, which further corroborates our outcomes.

There were no histological differences in the injured area between the trained and control groups. Nevertheless, the animals exhibited functional improvement. These data are in accordance with studies by Siegenthaler et al. (19), who demonstrated no significant difference in the cord area between sedentary and exercised (voluntary wheel running) groups, and Hutchinson et al. (23), who showed no significant differences in the percentages of white matter spared at lesion epicenters among the exercise (treadmill, swimming or standing) and no-exercise groups. Ahmed and Wieraszko (24) verified that the total area of the lesion epicenter and the total spared area were very similar in all of the experimental groups (acrobatic exercised plus magnetic stimulation, acrobatic exercised, magnetic stimulation and control). However, these authors also verified significant improvement in the trained animals’ motor behavior compared to that of the control animals, as in our findings. The absence of histological differences among groups shows that our study groups were homogenous.

The histological analysis revealed that different training types do not significantly change the injured spinal cord area, demonstrating that the nervous system is still able to promote functional recovery when stimulated correctly.

Additionally, the histological homogeneity indicates that the functional recovery was likely due to exercising and not to a difference in the severity of the lesions. All in all, the type of training appears to be an important factor in the functional recovery of animals with contusive spinal cord injury.

It is possible that if the training in the apparatus were conducted more frequently (i.e., five times a week instead of two), the results that we observed of greater improvements in the ramp and maze groups in the acute stage after injury could have persisted until the end of the experiment. In addition, other histological analyses could have been performed to identify more specific changes in the spinal cord after rehabilitation procedures and training, along with quantification of neurons or the verification of glial scarring.

Because the training assisted functional recovery in spinal cord injury, this study shows that specific training protocols are crucial to reduce the necessary time for motor behavior recovery in the early period. This observation is extremely relevant to therapy, as the independence of an injured person is one of the primary factors that leads to a better quality of life. However, the training sessions on the ramp and maze apparatuses failed to induce better motor improvement at the end of the experiment. These data

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8. Basso DM, Beattie MS, Bresnahan JC. Graded histological and locomotor outcomes after spinal cord contusion using the NYU weight-drop device versus transection. Exp Neurol. 1996;139(2):244-56, http://dx.doi.org/ 10.1006/exnr.1996.0098.

9. Ferreira JC, Bacurau AV, Bueno CR Jr, Cunha TC, Tanaka LY, Jardim MA, et al. Aerobic exercise training improves Ca2+ handling and redox status of skeletal muscle in mice. Exp Biol Med (Maywood).

2010;235(4):497-505, http://dx.doi.org/10.1258/ebm.2009.009165.

10. Basso DM, Beattie MS, Bresnahan JC. A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma. 1995;12(1):1-21, http://dx.doi.org/10.1089/neu.1995.12.1.

11. Reier PJ, Stokes BT, Thompson FJ, Anderson DK. Fetal cell grafts into resection and contusion/compression injuries of the rat and cat spinal cord. Exp Neurol. 1992;115(1):177-88, http://dx.doi.org/10.1016/00144886(92)90245-L. 12. Coumans JV, Lin TT, Dai HN, MacArthur L, MacAtee M, Nash C, et al.

Axonal regeneration and functional recovery after complete spinal cord transection in rats by delayed treatment with transplants and neurotrophins. J Neurosci. 2001;21(23):9334-44.

13. Smith RR, Brown EH, Shum-Siu A, Whelan A, Burke DA, Benton RL, et al. Swim training initiated acutely after spinal cord injury is ineffective and induces extravasation in and around the epicenter. J Neurotrauma.

2009;26(7):1017-27, http://dx.doi.org/10.1089/neu.2008.0829.

14. Houle JD, Tessler A. Repair of chronic spinal cord injury. Exp Neurol.

2003;182(2):247-60, http://dx.doi.org/10.1016/S0014-4886(03)00029-3.

15. Norrie BA, Nevett-Duchcherer JM, Gorassini MA. Reduced functional recovery by delaying motor training after spinal cord injury. J Neurophysiol.

2005;94(1):255-64, http://dx.doi.org/10.1152/jn.00970.2004.

16. Dietz V, Curt A. Neurological aspects of spinal-cord repair: promises and challenges. Lancet Neurol. 2006;5(8):688-94, http://dx.doi.org/ 10.1016/S1474-4422(06)70522-1.

17. Leung PY, Wrathall JR. Local and distal responses to injury in the rapid functional recovery from spinal cord contusion in rat pups. Exp Neurol.

2006;202(1):225-37, http://dx.doi.org/10.1016/j.expneurol.2006.06.007.

18. Fouad K, Tse A. Adaptive changes in the injured spinal cord and their role in promoting functional recovery. Neurol Res. 2008;30(1):17-27, http://dx.doi.org/10.1179/016164107X251781.

19. Siegenthaler MM, Berchtold NC, Cotman CW, Keirstead HS. Voluntary running attenuates age-related deficits following SCI. Exp Neurol.

2008;210(1):207-16, http://dx.doi.org/10.1016/j.expneurol.2007.10.019.

20. Sandrow-Feinberg HR, Izzi J, Shumsky JS, Zhukareva V, Houle JD. Forced exercise as a rehabilitation strategy after unilateral cervical spinal cord contusion injury. J Neurotrauma. 2009;26(5):721-31, http:// dx.doi.org/10.1089/neu.2008.0750.

21. Andrade MS, Mendonc¸a LM, Chadi G. Treadmill running protects spinal cord contusion from secondary degeneration. Brain Res. 2010;1346:26678, http://dx.doi.org/10.1016/j.brainres.2010.05.070.

22. Multon S, Franzen R, Poirrier AL, Scholtes F, Schoenen J. The effect of treadmill training on motor recovery after a partial spinal cord compression-injury in the adult rat. J Neurotrauma. 2003;20(8):699-706, http://dx.doi.org/10.1089/089771503767869935.

23. Hutchinson KJ, Go´mez-Pinilla F, Crowe MJ, Ying Z, Basso DM. Three exercise paradigms differentially improve sensory recovery after spinal cord contusion in rats. Brain. 2004;127(Pt 6):1403-14.

24. Ahmed Z, Wieraszko A. Combined effects of acrobatic exercise and magnetic stimulation on the functional recovery after spinal cord lesions.

J Neurotrauma. 2008;25(10):1257-69, http://dx.doi.org/10.1089/ neu.2008.0626.

suggest that different training protocols should be used at different rehabilitation stages, especially when discharging patients from care units, considering their capacity to perform simple daily activities.

ACKNOWLEDGMENTS The authors wish to thank the financial support of Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES), Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) and Hospital das Clı´nicas, Faculdade de Medicina, Universidade de Sa˜o Paulo (HCFMUSP). The authors also wish to thank Gustavo Bispo, the Spinal Cord Injury Laboratory technician.

AUTHOR CONTRIBUTIONS Miranda TA was responsible for the study design, experimental study planning and data collection, data analysis and interpretation, manuscript writing and final reviewing. Vicente JM was responsible for the study design, data collection, data analysis and interpretation, and final reviewing of the manuscript. Marcon RM was responsible for data analysis and interpretation and final reviewing of the manuscript. Cristante AF and Morya E were responsible for data analysis and interpretation, manuscript writing and final reviewing. Valle AC was responsible for the study design, experimental study planning, manuscript writing and final reviewing.

REFERENCES 1. Zai LJ, Wrathall JR. Cell proliferation and replacement following contusive spinal cord injury. Glia. 2005;50(3):247-57, http://dx.doi.org/ 10.1002/glia.20176.

2. Basso DM. Neuroanatomical substrates of functional recovery after experimental spinal cord injury: implications of basic science research for human spinal cord injury. Phys Ther. 2000;80(8):808-17.

3. Bigbee AJ, Crown ED, Ferguson AR, Roy RR, Tillakaratne NJ, Grau JW, et al. Two chronic motor training paradigms differentially influence acute instrumental learning in spinally transected rats. Behav Brain Res. 2007;180(1):95-101, http://dx.doi.org/10.1016/j.bbr.2007.02.029.

4. Dietz V. Body weight supported gait training: from laboratory to clinical setting. Brain Res Bull. 2008;76(5):459-63, http://dx.doi.org/10.1016/ j.brainresbull.2008.02.034.

5. Marcon RM, Cristante AF, de Barros Filho TE, de Oliveira RP, dos Santos GB. Potentializing the effects of GM1 by hyperbaric oxygen therapy in acute experimental spinal cord lesion in rats. Spinal Cord.

2010;48(11):808-13, http://dx.doi.org/10.1038/sc.2010.37.

6. Cristante AF, Barros Filho TE, Oliveira RP, Marcon RM, Rocha ID, Hanania FR, et al. Antioxidative therapy in contusion spinal cord injury.

Spinal Cord. 2009;47(6):458-63, http://dx.doi.org/10.1038/sc.2008.155.

7. Santos GB, Cristante AF, Marcon RM, Souza FI, Barros Filho TEP, Damasceno ML. Modelo experimental de lesa˜o medular e protocolo de avaliac¸a˜o motora em ratos wistar [Spinal cord injury experimental model and motion evaluation protocol in wistar rats]. Acta Ortop Bras.

2011;19(2):87-91.

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DOI:10.6061/clinics/2012(07)17

BASIC RESEARCH

The imbalance between Treg and Th17 cells caused

by FTY720 treatment in skin allograft rejection Alessandra Gonc¸alves Commodaro,I,II Juliana Figueredo Pedregosa,III Jean Pierre Peron,IV Wesley Branda˜o,IV Luiz Vicente Rizzo,II Valquiria BuenoV I

Vision Institute, Federal University of Sa˜o Paulo (UNIFESP), Sa˜o Paulo/SP, Brazil. II Hospital Israelita Albert Einstein, Sa˜o Paulo/SP, Brazil. III Nephrology Division, Federal University of Sa˜o Paulo (UNIFESP), Sa˜o Paulo/SP, Brazil. IV Faculdade de Medicina da Universidade de Sa˜o Paulo, Department of Immunology, Sa˜o Paulo/SP, Brazil. V Immunology Division, Federal University of Sa˜o Paulo (UNIFESP), Sa˜o Paulo/SP, Brazil.

OBJECTIVES: FTY720 modulates CD4+T cells by the augmentation of regulatory T cell activity, secretion of suppressive cytokines and suppression of IL-17 secretion by Th17 cells. To further understand the process of graft rejection/acceptance, we evaluated skin allograft survival and associated events after FTY720 treatment.

METHODS: F1 mice (C57BL/6xBALB/c) and C57BL/6 mice were used as donors for and recipients of skin transplantation, respectively. The recipients were transplanted and either not treated or treated with FTY720 by gavage for 21 days to evaluate the allograft survival. In another set of experiments, the immunological evaluation was performed five days post-transplantation. The spleens, axillary lymph nodes and skin allografts of the recipient mice were harvested for phenotyping (flow cytometry), gene expression (real-time PCR) and cytokine (Bio-Plex) analysis.

RESULTS: The FTY720 treatment significantly increased skin allograft survival, reduced the number of cells in the lymph nodes and decreased the percentage of Tregs at this site in the C57BL/6 recipients. Moreover, the treatment reduced the number of graft-infiltrating cells and the percentage of CD4+ graft-infiltrating cells. The cytokine analysis (splenocytes) showed decreased levels of IL-10, IL-6 and IL-17 in the FTY720-treated mice. We also observed a decrease in the IL-10, IL-6 and IL-23 mRNA levels, as well as an increase in the IL-27 mRNA levels, in the splenocytes of the treated group. The FTY720-treated mice exhibited increased mRNA levels of IL-10, IL-27 and IL-23 in the skin graft.

CONCLUSIONS: Our results demonstrated prolonged but not indefinite skin allograft survival by FTY720 treatment.

This finding indicates that the drug did not prevent the imbalance between Tr1 and Th17 cells in the graft that led to rejection.

KEYWORDS: Transplantation; Rejection; Tolerance; FTY720; Foxp3; Th17.

Commodaro AG, Pedregosa JF, Peron JP, Branda˜o W, Rizzo LV, Bueno V. The imbalance between Treg and Th17 cells caused by FTY720 treatment in skin allograft rejection. Clinics. 2012;67(7):805-813.

Received for publication on November 23, 2011; First review completed on February 20, 2012; Accepted for publication on March 14, 2012 E-mail: valquiria@nefro.epm.br Tel.: 55 11 5549 6073

the peripheral CD4+ T cells in adult rodents and humans (6).

These cells play an important role in the maintenance of self-tolerance, prevention of autoimmune diseases (7), and regulation of allogeneic immune responses (8,9).

Regulatory natural T cells (nTregs) are generated in the thymus, defined by their high expression of the IL-2 receptor a chain (CD25) and thought to be controlled by the forkhead box P3 (Foxp3) transcription factor. The population of thymus-derived Treg cells is supplemented with a population of inducible Tregs (iTregs) generated from naı¨ve T cells activated in the presence of TGF-b. iTregs are believed to be functionally similar to thymus-derived nTregs (10).

In contrast with Treg-generated transplantation tolerance, the Th17 cell population has been associated with allograft rejection (11). Th17s are described as T cells producing interleukin-17 that are dependent on signals from cytokines, such as IL-6 (12). Moreover, the Th17 response is propagated by the cytokines IL-23 and IL-21 (13,14).

INTRODUCTION The rejection responses to allografts are initiated by a specific T-cell recognition of antigens expressed on the cells within the graft (1-3). T-cell activation after allograft transplantation requires both antigen presentation and costimulatory signals (4,5), and, once they occur, several other factors are involved, leading to effector or regulatory immune responses. Recently, the immune responses to allografts have been reported to be modulated by regulatory T cells, such as CD4+CD25+ T cells, which comprise 5-10% of

Copyright ß 2012 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.

No potential conflict of interest was reported.

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monotherapy was not sufficient to prevent rejection. However, when MR1 (anti-CD154 mAb) was administered in combination with FTY720, skin allograft survival increased.

Interestingly, the expression of CD4CD25Foxp3 was similar when FTY720 monotherapy was compared with FTY720+ MR1. These findings suggest that mechanisms other than CD4CD25Foxp3 expression occur during allograft acceptance. To further understand the process of graft acceptance/rejection, we evaluated skin allograft survival and associated events following treatment with FTY720.

A member of the IL-12 family of cytokines, namely IL-27, has been shown to cause the immune-specific suppression of IL-17 secretion by down-regulating retinoid orphan nuclear receptor C expression. This inhibition is associated with the induction of the intracellular signaling factor STAT1 and the suppression of cytokine signaling protein 1 (15).

Investigations of the Treg-Th17 axis aim to identify potential pharmacological targets to promote graft acceptance.

The new compound FTY720 binds to S1P (sphingosine-1 phosphate) receptors, causing lymphocytes to be sequestered in secondary lymphoid organs. In addition, FTY720 inhibits the egress of lymphocytes from the thymus and secondary lymphoid tissues into the periphery, thereby preventing the migration of lymphocytes to inflammatory sites (16,17). Some additional mechanisms of action have been proposed for FTY720, such as its anti-proliferative effect on T cells through the augmentation of regulatory T cell activity and secretion of suppressive cytokines (18-20).

FTY720 has also been shown to cause the suppression of IL17 secretion by Th17 cells in vitro (15).

Jin et al. (21) showed that, although FTY720 improved CD4CD25Foxp3 expression after skin transplantation, the

MATERIALS AND METHODS Experimental groups Mice were cared for in accordance with the Principles of Laboratory Animal Care (NIH publication 86-23, revised 1985) and the regulations of the Brazilian Committee on Animal Experimentation. The project was submitted to and approved by the Animal Ethic Committee of UNIFESP Federal University of SaËœo Paulo (protocol 0125/08).

Eight- to ten-week-old male (C57BL/6xBALB/c) F1 and C57BL/6 mice were used as donors for and recipients of

Figure 1 - FTY720 treatment prolongs mouse skin allograft survival. F1 (BALB/c x C57BL/6) mice were donors to C57BL/6 recipients, and skin allograft rejection was determined macroscopically when the graft reached a necrosis level of 90%. A - Kaplan-Meier curve of skin allograft survival showing that FTY720 treatment significantly increased the survival of F1 (BALB/c x C57BL/6) skin allografts in the C57BL/6 recipients. B - The macroscopic signs of rejection were observed as early as day 7 in transplanted, non-treated (Tx) mice. C Rejection signs occurred later in the transplanted, FTY720-treated (Tx+FTY) mice.

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Figure 2 - Evaluation of the effect of transplantation and FTY720 treatment on draining lymph nodes (axillary) five days after F1 skin graft transplantation in the C57BL/6 mice. A - FTY720 treatment reduces the number of lymph node cells (p = 0.022). B - FTY720 treatment reduces the percentage of cells (p = 0.028) with the Treg phenotype (CD4+Foxp3+). The results are expressed as the means ÂĄ SD.

15 min with 1% paraformaldehyde, permeabilized for 6 min with 0.2% Triton X-100 and stained with rat anti-mouse Foxp3 FITC for 30 min (eBioscience, San Diego, CA, USA). The stained cells were acquired in a FACSCalibur Flow Cytometer (BD Biosciences, San Jose, CA, USA), and the data were analyzed using Cell Quest Pro software (BD Biosciences). At least 30,000 events were acquired for each analysis.

skin transplantation, respectively. Skin from the donor tail (1.0 cm2) was placed on the C57BL/6 mice and fixed with 60 nylon sutures (Brasuture, Brazil) at each corner. The recipient mice were either transplanted only (Tx, n = 5) or transplanted and submitted to the daily administration of FTY720 (Novartis, Switzerland; 1 mg/kg/day diluted in distilled water) by gavage for 21 days, starting three days before the transplantation (Tx + FTY, n = 5). The grafts were observed daily to establish skin allograft survival (rejection = 90% skin necrosis). In another set of experiments, the immunological evaluation was performed five days posttransplantation.

Quantitative analysis of gene expression by realtime PCR The total RNA from the skin graft and spleen cells was extracted with Trizol (Invitrogen Life Technologies) according to the manufacturer’s instructions and reverse-transcribed into complementary DNA with Superscript III (Invitrogen Life Technologies). For the real-time PCR of the IL-10, IL-6, FOXP3, IL-23 and IL-27 genes, 0.5 mL of primer, 1 mL of cDNA, 3.5 mL of RNAse/DNAse-free water and 5 mL of Taqman Fast Universal Master Mix (Applied BiosystemsH) were used. The PCR was performed in an Applied Biosystems 7900HT Fast Real-Time PCR SystemH. The mRNA level was determined following normalization to b-actin. We performed qPCR in the spleen for IL-10, IL-6, FOXP3, IL-23 and IL-27. In the skin graft, we performed qPCR for IL-10, FOXP3, IL-23 and IL-27.

Experimental design 1) To evaluate skin allograft survival, the following steps were completed: FTY720 treatment, skin transplantation, and graft follow-up.

2) An immunological evaluation of C57BL/6-transplanted mice either treated or not treated with FTY720 was performed.

Immunological evaluation Five days post-transplantation, the spleens, axillary lymph nodes (ALNs) and skin allografts were harvested from the recipient mice. Single-cell suspensions from the spleen, ALNs and skin were counted in a Neubauer chamber. The spleen cells were used for in vitro stimulation and qPCR analysis, whereas the ALNs and graft-infiltrating cells were evaluated by flow cytometry.

Bio-Plex The spleen cells were harvested at day five post-skin transplantation and stimulated in vitro with anti-CD3. A Bio-Plex mouse cytokine assay kit (Bio-Rad Laboratories, Hercules, CA, USA) was used to test the samples for the presence of the specific cytokines IL-10, IL-6 and IL-17. The assay was performed in the Bio-Plex suspension array system, and the data were analyzed using Bio-Plex Manager software version 4.0. The standard curves ranged from 1.95 to 32 000 pg/ml.

Flow cytometry Cell suspensions (16106) from the ALNs and grafts were incubated for 30 min with rat anti-mouse monoclonal antibody CD4PerCP-Cy5.5 (eBioscience, San Diego, CA, USA).

After this period, the cells were washed with PBS, fixed for

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Figure 3 - Evaluation of graft-infiltrating cells five days after F1 skin graft transplantation in the C57BL/6 mice. A - A representative flow cytometry dot plot showing the graft-infiltrating cells (FSC x SSC) and Treg cells (CD4+Foxp3+). B - FTY720 treatment reduces the number of graft-infiltrating cells in the F1 transplanted skin (p = 0.02). C - FTY720 treatment reduces the percentage of CD4+ (p = 0.014) graft-infiltrating cells in the F1 transplanted skin. D - FTY720 reduces but does not prevent the accumulation of Foxp3+ graft-infiltrating cells in the F1 transplanted skin (p = 0.011). The results are expressed as the means ¡ SD.

Statistical analysis

RESULTS

The skin allograft survival curve was obtained using the Kaplan-Meier method. Other data were expressed as the means¡SD. The statistical analyses were performed using Graph Pad Prism version 5.00 for Windows (Graph Pad Software, San Diego, CA, USA). The parametric Student’s t test was employed, and p-values ,0.05 were considered significant.

Treatment with FTY720 increases skin allograft survival To evaluate the effect of FTY720 on the progression of skin allograft rejection, the recipient mice were either transplanted only (Tx, n = 5) or transplanted and submitted to the daily

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administration of FTY720 (Tx + FTY, n = 5) by gavage for 21 days, starting three days before the transplantation. We observed a significant (p-value = 0.0047) increase in the survival of allografts from F1 mice (BALB/c x C57BL/6) when the recipient C57BL/6 mice were treated with FTY720 (Figure 1A). The macroscopic examination shows that the first rejection (90% of skin necrosis) occurred on day 13 for the FTY720-treated mice, whereas the first rejection occurred as early as 9 days after transplantation in the non-treated mice (Figure 1B).

Decrease in the cytokine levels in mice treated with FTY720 We evaluated the cytokine levels in the spleen cells submitted to in vitro stimulation with anti-CD3. The analysis of the 72-h culture supernatants by Bio-Plex showed decreased levels of IL-10, IL-6 and IL-17 secretion in the FTY720-treated mice compared with the non-treated mice (Figure 4). The IL-6 secretion was significantly decreased in the Tx+FTY group (p,0.05).

qPCR analysis FTY720 reduced the number of cells in the lymph nodes

To confirm our results regarding cytokine levels, we evaluated the IL-10, IL-6, FOXP3, IL-23 and IL-27 gene expression in the spleen cells by real-time PCR. In agreement with our previous observation, there was a decrease in the IL-10 and IL-6 mRNA levels in the splenocytes of the FTY720-treated mice. In this group, we also observed a decrease in the IL-23 mRNA level but an increase in the IL-27 mRNA level. The FOXP3 expression was decreased, but this decrease did not reach statistical significance (Figure 5).

In the skin, we found increased mRNA levels of IL-10, IL27, and IL-23 in mice treated with FTY720 (Figure 6), but this increase was not statistically significant. The FOXP3 expression was decreased in this group (p = 0.0948).

The total number of cells and percentage of regulatory T cells were analyzed in the lymph nodes from the Tx and Tx + FTY mice five days post-transplantation. Our results showed that the FTY720 treatment was able to significantly reduce both the number of cells and percentage of CD4+Foxp3+ T cells in the lymph nodes (Figure 2).

FTY720 reduced the number of graft-infiltrating cells Five days after the skin transplantation, we analyzed the graft-infiltrating cells and observed that the FTY720 treatment reduced the number of graft-infiltrating cells and diminished the percentage of CD4+ T cells in the skin allograft. The FTY720 did not prevent the accumulation of Foxp3+ cells in the graft but did cause a decrease in the percentage of these cells (Figure 3).

DISCUSSION In the present study, we investigated the events associated with FTY720 treatment in mice submitted to skin

Figure 4 - FTY720 decreases the cytokine production by splenocytes. Analysis of the IL-10, IL-6 and IL-17 secretion levels by the splenocytes. The spleen cells from the Tx non-treated and Tx + FTY720-treated mice were harvested at day 5 post-skin transplantation and stimulated in vitro with anti-CD3. The supernatants were collected 72 h later, and the IL-10, IL-6, and IL-17 levels were determined by multiplex ELISA. The results are expressed as the means ยก SD (*p,0.05).

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Figure 5 - FTY720-treated mice exhibit a decrease in the relative gene expression (qPCR analysis) of IL-10, IL-6, and IL-23 in the spleen cells, whereas IL-27 is increased. The relative gene expression in the spleen cells was evaluated in the Tx non-treated and Tx + FTY720treated mice. The results are expressed as the means ยก SD.

In 2008, we demonstrated that the increased skin allograft survival in FTY720-treated mice was associated with diminished cell numbers in the spleen, blood and axillary lymph nodes (24). Our present results showed a reduction in the number of cells and percentage of CD4+Foxp3+ cells in the lymph nodes of mice treated with FTY720.

Recently, we demonstrated that FTY720 was effective in suppressing experimental autoimmune uveitis (EAU), and this finding was associated with a reduction in the eyeinfiltrating cells with either Treg or Th17 phenotypes (26). In the present study, at the early time points (day +5) after transplantation, FOXP3 gene expression in the spleen and

allograft transplantation. We confirmed the previous data showing increased skin allograft survival and delayed macroscopic signs of tissue necrosis in the mice treated with FTY720. In the same model, we showed previously that FTY720 increases skin allograft survival when administered as monotherapy, as well as when combined with cyclosporine, tacrolimus and sirolimus (22-25).

The prevention of T lymphocytes from migrating to inflammatory sites is an effect already described for FTY720.

Accordingly, our present results demonstrate a reduction in the number of graft-infiltrating cells and in the percentage of CD4+ T and Foxp3+ cells.

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Figure 6 - FTY720-treated mice present an increase in the relative gene expression (qPCR analysis) of IL-10, IL-23, and IL-27 in the skin grafts. The relative gene expression (qPCR analysis) of FOXP3, IL-10, IL-23, and IL-27 in the F1 skin grafts was evaluated in the Tx nontreated and Tx + FTY720-treated mice. The results are expressed as the means ¡ SD.

skin graft, as well as Foxp3 protein expression in the lymph nodes and graft-infiltrating cells, was decreased in mice treated with FTY720. These findings show that FTY720 does not inhibit but rather decreases the expression of Foxp3 systemically. The increase in skin allograft survival due to FTY720 without reaching indefinite tolerance could be justified by the following: the increased skin graft survival induced by FTY720 is mediated by other factors in addition to Foxp3 cells; and the decreased Foxp3 expression contributes to the rejection, even if over the long-term.

In our model, the in vitro anti-CD3 stimulation of splenocytes from the mice submitted to skin transplantation and treated with FTY720 exhibited decreased secretion of IL-10 and IL-6, which was confirmed by qPCR analysis.

In addition, the splenocytes from FTY720-treated mice secreted significantly less IL-17 after in vitro anti-CD3 stimulation, and the evaluation of the mRNA in the spleen cells showed a decreased level of IL-23. Chen et al. showed an enhanced expression of Th17-related cytokines at the early stage of cornea rejection either in the draining lymph nodes (LNs) or in the allograft. They also found that responder cells in the draining LNs of the transplanted recipients secreted high levels of IL-6, TGF-b and IL-21 compared with the controls and suggested that these factors could drive naı¨ve T cells to differentiate into Th17 cells (27). Because Th17 cells have been implicated in renal allograft rejection (28) and IL-17 production has been shown to be dependent on IL-6 (29) and IL-23 levels (30),

the decreased expression of these cytokines in our model suggests that the prolonged skin allograft survival obtained

by FTY720 treatment was at least in part due to Th17 phenotype impairment in the periphery rather than Foxp3 enhancement. In agreement, Mehling et al. showed a decrease in the number of Th17 cells from the blood of patients with multiple sclerosis treated with FTY720 and of patients in disease remission (31). However, whether FTY720 increases the Treg cell population is still uncertain.

Wolf et al. showed in vitro and in vivo that FTY720 inhibits the proliferation of Treg cells without affecting their viability, phenotype or in vitro immunosuppression (32).

Zhou et al. showed that the addition of FTY720 to the mixed lymphocyte reaction (MLR) in mouse (BALB/c and C57BL/6) splenocytes increased the proportion of CD4+CD25+ T regulatory cells. Additionally, FTY720 added to the MLR caused the suppression of lymphocyte proliferation, decreased the IL-2 levels in the supernatant, and induced lymphocyte apoptosis (33).

Allograft acceptance/rejection is associated with multiple suppressor/effector mechanisms, and immunosuppressive drugs administered during transplantation have been shown to affect T cell populations.

In agreement, Hester et al. observed, in the follow-up of kidney recipients induced with alemtuzumab and treated post-transplantation with several drugs such as sirolimus or sirolimus + mycophenolate mofetil, that rejection and the long-term maintenance of immunosuppression influence the number of circulating Treg and Th17 cells (34).

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Sewgobind et al. (35) showed that the relative mRNA expression of FOXP3 decreased in the blood of kidneytransplanted patients treated with rabbit anti-thymocyte globulin (rATG) or rATG + tacrolimus, whereas the levels of IL-10 and IL-27p28 increased compared with the levels in non-transplanted blood donors.

Battaglia et al. (36) showed that the administration of IL10 in combination with the immunosuppressive drug rapamycin promoted tolerance in type 1 diabetic mice after pancreatic islet transplantation by the induction of Tr1 cells.

In addition, IL-27 secretion from dendritic cells has been shown to enhance the development of Tr1 cells and, as observed by Yoshimoto et al. (37), inhibit not only Th2 commitment but also Th2 cytokine production from already polarized Th2 cells (down-regulation of GATA-3 and upregulation of T-bet). Moreover, IL-27 was shown by Stumhofer et al. (38) to directly antagonize the development of the Th17-cell responses in experimental autoimmune models.

In our model, even though statistical significance could not be reached among groups, the prolonged skin allograft survival promoted by FTY720 treatment could be explained by the increased expression of IL-10 and IL-27 in the skin graft, suggesting the development of the Tr1 cells. However, we also found an increase in the IL-23 expression in the skin graft, confirming the presence of Th17 cells. In agreement with our data, Li et al. (39) found both Treg and Th17 cells in mouse liver transplants. They showed a time-dependent and significant increase in Th17-related cytokines (IL-17, IL6 and IL-23), whereas the expression of Treg-related cytokines (IL-10, TGF-b1) was markedly low in the liver of transplanted mice prior to graft rejection.

Our results show that FTY720 treatment causes a different modulation of IL-10 and IL-23 in the spleen and skin graft; namely, the IL-10 and IL-23 expression decreased in the spleen but increased in the graft. In contrast, IL-27 and Foxp3 exhibited the same modulation in the spleen and skin, with an increase in IL-27 and decrease in Foxp3. These findings suggest that, at early time points (day +5) after transplantation, FTY720 controls the balance between Tr1 (IL-10 and IL-27) and Th17 (IL-23) in the graft, which prevents rejection for a definite period of time.

In conclusion, we demonstrated that skin allograft survival was prolonged but not indefinite in FTY720-treated mice, indicating that the drug did not prevent the long-term imbalance between the Treg and Th17 cells in the graft, which thus led to rejection.

REFERENCES 1. Rosenberg AS, Singer A. Evidence that the effector mechanism of skin allograft rejection is antigen-specific. Proc Natl Acad Sci USA. 1988;85(20):7739-42, http://dx.doi.org/10.1073/pnas.85.20.7739.

2. Lee RS, Grusby MJ, Glimcher LH, WinnHJ, Auchincloss H Jr. Indirect recognition by helper cells can induce donor-specific cytotoxic T lymphocytes in vivo. J Exp Med. 1994;179(3):865-72, http://dx.doi.org/ 10.1084/jem.179.3.865.

3. Chen W, Murphy B, Waaga AM, Willett TA, Russell ME, Khoury SJ, et al.

Mechanisms of indirect allorecognition in graft rejection: class II MHC allopeptide-specific T cells clones transfer delayed-type hypersensitivity responses in vivo. Transplantation. 1996;62(6):705-10, http://dx.doi.org/ 10.1097/00007890-199609270-00001.

4. Sayegh MH, Turka LA. The role of T-cell co stimulatory activation pathways in transplant rejection. N Engl J Med. 1998;338(25):1813-21.

5. Chattopadhyay K, Bhatia S, Fiser A, Almo SC, Nathenson SG. Structural basis of inducible costimulator ligand costimulatory function: determination of the cell surface oligomeric state and functional mapping of the receptor binding site of the protein. J Immunol, 2006;177(6):3920-9.

6. Itoh M, Takahashi T, Sakaguchi N, Kuniyasu Y, Shimizu J, Otsuka F, et al.

Thymus and autoimmunity: production of CD25+CD4+ naturally anergic and suppressive T cells as a key function of the thymus in maintaining immunologic self-tolerance. J Immunol. 1999;162(9):5317-26.

7. Jee Y, Piao WH, Liu R, Bai XF, Rhodes S, Rodebaugh R, et al.

CD4(+)CD25(+) regulatory T cells contribute to the therapeutic effects of glatiramer acetate in experimental autoimmune encephalomyelitis.

Clin Immunol. 2007;125(1):34-42, http://dx.doi.org/10.1016/ j.clim.2007.05.020.

8. Walsh PT, Taylor DK, Turka LA. Tregs and transplantation tolerance.

J Clin Invest. 2004;114(10):1398-403.

9. Karim M, Feng G,Wood KJ, Bushell AR. CD25+CD4+ regulatory T cells generated by exposure to a model protein antigen prevent allograft rejection: antigen-specific reactivation in vivo is critical for bystander regulation. Blood. 2005;105(12):4871-7, http://dx.doi.org/10.1182/ blood-2004-10-3888.

10. Mitchell P, Afzali B, Lombardi G, Lechler RI. The T helper 17-regulatory T cell axis in transplant rejection and tolerance. Curr Opin Organ Transplant.

2009;14(4):326-31, http://dx.doi.org/10.1097/MOT.0b013e32832ce88e.

11. Afzali B, Lombardi G, Lechler RI, Lord GM. The role of T helper 17 (Th17) and regulatory T cells (Treg) in human organ transplantation and autoimmune disease. Clin Exp Immunol. 2007;148(1):32-46, http:// dx.doi.org/10.1111/j.1365-2249.2007.03356.x.

12. Weaver CT, Hatton RD, Mangan PR, et al. IL-17 family cytokines and the expanding diversity of effector T cell lineages. Annu Rev Immunol.

2007;25:821-52, http://dx.doi.org/10.1146/annurev.immunol.25.022106.

141557.

13. de Jong E, Suddason T, Lord GM. Translational mini-reviews series on Th17 cells: Development of mouse and human T helper 17 cells. Clin Exp Immunol. 2010;159(2):148-58, http://dx.doi.org/10.1111/j.13652249.2009.04041.x.

14. Ouyang W, Kolls JK, Zheng Y. The biological functions of T helper 17 cells effector cytokines in inflammation. Immunity. 2008;28(4):454-67, http://dx.doi.org/10.1016/j.immuni.2008.03.004.

15. Liao JJ, Huang MC, Goetzl EJ. Cutting Edge: Alternative Signaling of Th17 Cell Development by Sphingosine 1-Phosphate. J Immunol.

2007;178(9):5425-28.

16. Isoyama N, Takai K, Tsuchida M, Matsumura M, Naito K. Evidence that FTY720 induces rat thymocyte apoptosis. Transpl Immunol.

2006;15(4):265-71, http://dx.doi.org/10.1016/j.trim.2006.02.004.

17. Xie JH, Nomura N, Koprak SL, Quackenbush EJ, Forrest MJ, Rosen H. Sphingosine-1-phosphate receptor agonist impairs the efficiency of the local immune response by altering trafficking of naive and antigenactivated CD4+ T cells. J Immunol. 2003;170(7):3662-70.

18. Sawicka E, Dubois G, Jarai G, Edwards M, Thomas M, Nicholls A, et al.

The sphingosine1-phosphate receptor agonist FTY720 differentially affects the sequestration of CD4+CD25+ T-regulatory cells and enhances their functional activity. J Immunol. 2005;175(12):7973-80.

19. Fujiwara I, Nakajima H, Matsuda T, Mizuta N, Yamagishi H, Oka T. Prolongation of concordant xenograft survival by a newly developed drug, FTY720. Transplant Proc. 1999;31(7):2831-33, http://dx.doi.org/ 10.1016/S0041-1345(99)00582-5.

20. Wang W, Graeler MH, Goetzl EJ. Type 4 sphingosine 1-phosphate G protein coupled receptor (S1P4) transduces S1P effects on T cell proliferation and cytokine secretion without signaling migration.

FASEB J. 2005;19(12):1731-33.

21. Jin J, Yang SH, Park J, Lee C, Lee J, Kim SM, Kim YS. Fragile maintenance of allograft tolerance induced by lymphocyte sequestration and costimulation blockade. Transpl Immunol. 2009;21(4):221-7, http:// dx.doi.org/10.1016/j.trim.2009.06.001.

22. Silva FR, Silva LB, Cury PM, Burdmann EA, Bueno V. FTY720 in combination with cyclosporine - an analysis of skin allograft survival and renal function. Int Immunopharmacol. 2006;6(13-14):1911-8, http:// dx.doi.org/10.1016/j.intimp.2006.07.014.

ACKNOWLEDGMENTS This study was supported by grants from Fundac¸a˜o de Amparo a Pesquisa do Estado de Sa˜ o Paulo (FAPESP), Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) and CAPES.

AUTHOR CONTRIBUTIONS Commodaro AG performed the skin transplantation, real-time PCR, BioPlex cytokine assay, FTY720 treatment, tissue harvesting and flow cytometry; conducted the statistical analyses; made the figures; and wrote the manuscript. Pedregosa JF performed the skin transplantation, FTY720 treatment, tissue harvesting and flow cytometry. Bueno V performed the skin transplantation, FTY720 treatment, tissue harvesting and flow cytometry; conducted the statistical analyses; made the figures; and wrote the manuscript. Peron JP and Branda˜o WN performed the real-time PCR and the Bio-Plex cytokine assay. Rizzo LV conducted the statistical analyses, made the figures, and wrote the manuscript.

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23. Gallo AP, Silva LB, Franco M, Burdmann EA, Bueno V. Tacrolimus in combination with FTY720 – an analysis of renal and blood parameters.

Int Immunopharmacol. 2006;6(13-14):1919-24, http://dx.doi.org/ 10.1016/j.intimp.2006.07.012.

24. Lopes CT, Gallo AP, Palma PV, Cury PM, Bueno V. Skin allograft survival and analysis of renal parameters after FTY720 + Tacrolimus treatment in mice. Transplant Proc. 2008;40(3):856-60, http://dx.doi.org/ 10.1016/j.transproceed.2008.02.051.

25. Lopes CT, Rosin EC, Cordeiro JA, Bueno V. Cell phenotype evaluation at various sites after skin transplantation and FTY720 plus Sirolimus therapy. Transplant Proc. 2010;42(2):573-7, http://dx.doi.org/10.1016/ j.transproceed.2010.01.027.

26. Commodaro AG, Peron JP, Lopes CT, Arslanian C, Belfort R Jr., Rizzo LV, Bueno V. Evaluation of experimental autoimmune uveitis in mice treated with FTY720. Invest Ophtalmol Vis Sci. 2010;51(5):2568-74, http://dx.doi.org/10.1167/iovs.09-4769.

27. Chen H, Wang W, Xie H, Xu X, Wu J, Jiang Z, Zhang M, Zhou L, Zheng S. A pathogenic role of IL- 17 at the early stage of corneal allograft rejection. Transpl Immunol. 2009;21(3):155-61, http://dx.doi.org/ 10.1016/j.trim.2009.03.006.

28. Loong CC, Hsieh HG, Lui WY, Chen A, Lin CY. Evidence for the early involvement of interleukin 17 in human and experimental renal allograft rejection. J Pathol. 2002;197(3):322-32, http://dx.doi.org/10.1002/path.1117.

29. Veldhoen M, Hocking RJ, Atkins CJ, Locksley RM, Stockinger B. TGF beta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity. 2006;24(2):179-89, http://dx.doi.org/10.1016/j.immuni.2006.01.001.

30. Aggarwal S, Ghilardi N, Xie MH, de Sauvage FJ, Gurney AL. Interleukin23 promotes a distinct CD4 T cell activation state characterized by the production of interleukin-17. J Biol Chem. 2003;278(3):1910-4, http:// dx.doi.org/10.1074/jbc.M207577200.

31. Mehling M, Lindberg R, Raul F, Kuhle J, Hess C, Kappos L, et al. Th17 central memory T cells are reduced by FTY720 in patients with multiple sclerosis. Neurology. 2010;75(5)403-10, http://dx.doi.org/10.1212/ WNL.0b013e3181ebdd64.

32. Wolf AM, Eller K, Zeiser R, Du¨rr C, Gerlach UV, Sixt M, Markut L, Gastl G, Rosenkranz AR, Wolf D. The sphingosine 1-phosphate receptor agonist FTY720 potently inhibits regulatory T cell proliferation in vitro and in vivo. J Immunol. 2009;183(6):3751-60, http://dx.doi.org/10.4049/ jimmunol.0901011.

33. Zhou PJ, Wang H, Shi GH, Wang XH, Shen ZJ, Xu D. Immunomodulatory drug FTY720 induces regulatory CD4(+)CD25(+) T cells in vitro. Clin Exp Immunol. 2009;157(1):40-7, http://dx.doi.org/10.1111/j.1365-2249.

2009.03942.x.

34. Hester J, Mills N, Shankar S, Carvalho-Gaspar M, Friend P, Wood KJ. Th17 cells in Alemtuzumab-treated patients: the effect of long-term maintenance immunosuppressive therapy. Transplantation. 2011;91(7): 744-50.

35. Sewgobind VDKD, van der Laan LJW, Kho MML, Kraaijeveld R, Korevaar SS, Mol W, Weimar W, Baam CC. The calcineurin inhibitor tacrolimus allows the induction of functional CD4+CD25+ regulatory T cells by rabbit anti-thymocyte globulins. Clin Exp Immunol. 2010;161(2):364-77.

36. Battaglia M, Stabilini A, Draghici E, Gregori S, Mocchetti C, Bonifacio E. Rapamycin and interleukin-10 treatment induces T regulatory type 1 cells that mediate antigen-specific transplantation tolerance. Diabetes.

2006;55(1):40-9, http://dx.doi.org/10.2337/diabetes.55.01.06.db05-0613.

37. Yoshimoto T, Yoshimoto T, Yasuda K. IL-27 suppresses Th2 cell development and Th2 cytokines production from polarized Th2 cells: a novel therapeutic way for Th2-mediated allergic inflammation.

J Immunol. 2007;179(7):4415-23.

38. Stumhofer JS, Laurence A, Wilson EH, Huang E, Tato CM, Johnson LM, et al. Interleukin 27 negatively regulates the development of interleukin 17-producing T helper cells during chronic inflammation of the central nervous system. Nat Immunol. 2006;7(9):937-45, http://dx.doi.org/ 10.1038/ni1376.

39. Li J, Lai X, Liao W, He Y, Liu Y, Gong J. The dynamic changes of Th17/ Treg cytokines in rat liver transplant rejection and tolerance. Int Immunopharmacol. 2011;11(8):962-7, http://dx.doi.org/10.1016/j. intimp.2011.02.010.

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DOI:10.6061/clinics/2012(07)18

BASIC RESEARCH

Exercise training prevents diastolic dysfunction induced by metabolic syndrome in rats Cristiano Mostarda,I Ivana Cinthya Moraes-Silva,I Vera Maria Cury Salemi,II Jacqueline Freire Machi,I Bruno Rodrigues,I,III Ka´tia De Angelis,IV Vera de Moura Azevedo Farah,V Maria Claudia IrigoyenI I

Hospital das Clinicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Hypertension Unit, Heart Institute (InCor), Sao Paulo/SP, Brazil. II Hospital das Clinicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Cardiomiopathy Unit, Heart Institute (InCor), Sao Paulo/SP, Brazil. III Human Movement Laboratory, Sao Judas Tadeu University, Sao Paulo/SP, Brazil. IV Laboratory of Translational Physiology, Nove de Julho University, Sao Paulo Sao Paulo/SP, Brazil. V Center of Biological Sciences and Health, Mackenzie Presbyterian University, Sao Paulo/SP, Brazil.

OBJECTIVE: High fructose consumption contributes to the incidence of metabolic syndrome and, consequently, to cardiovascular outcomes. We investigated whether exercise training prevents high fructose diet-induced metabolic and cardiac morphofunctional alterations.

METHODS: Wistar rats receiving fructose overload (F) in drinking water (100 g/l) were concomitantly trained on a treadmill (FT) for 10 weeks or kept sedentary. These rats were compared with a control group (C). Obesity was evaluated by the Lee index, and glycemia and insulin tolerance tests constituted the metabolic evaluation. Blood pressure was measured directly (Windaq, 2 kHz), and echocardiography was performed to determine left ventricular morphology and function. Statistical significance was determined by one-way ANOVA, with significance set at p,0.05.

RESULTS: Fructose overload induced a metabolic syndrome state, as confirmed by insulin resistance (F: 3.6¡0.2 vs.

C: 4.5¡0.2 mg/dl/min), hypertension (mean blood pressure, F: 118¡3 vs. C: 104¡4 mmHg) and obesity (F: 0.31¡0.001 vs. C: 0.29¡0.001 g/mm). Interestingly, fructose overload rats also exhibited diastolic dysfunction.

Exercise training performed during the period of high fructose intake eliminated all of these derangements. The improvements in metabolic parameters were correlated with the maintenance of diastolic function.

CONCLUSION: The role of exercise training in the prevention of metabolic and hemodynamic parameter alterations is of great importance in decreasing the cardiac morbidity and mortality related to metabolic syndrome.

KEYWORDS: Metabolic Syndrome; Diastolic function; Exercise Training; Insulin resistance; Cardiac hypertrophy.

Mostarda C, Moraes-Silva IC, Salemi VM, Machi JF, Rodrigues B, De Angelis K, et al. Exercise training prevents diastolic dysfunction induced by metabolic syndrome in rats. Clinics. 2012;67(7):815-820.

Received for publication on December 20, 2011; First review completed on February 5, 2012; Accepted for publication on March 14, 2012 E-mail: hipirigoyen@incor.usp.br Tel.: 55 11 2661-5327

habits, including increased blood pressure, hyperlipidemia, obesity, glucose intolerance, insulin resistance and hyperinsulinemia.

Insulin resistance may precede and is a pathogenic factor of diabetes. Furthermore, insulin resistance is associated with left ventricular diastolic dysfunction in pre-diabetic adults, independent of blood pressure levels (6). Studies have shown that MS patients also present with left ventricular remodeling and diastolic dysfunction.

Experimentally, fructose overload in drinking water or chow has been used to study MS and promote similar metabolic and autonomic derangements. Our research group has shown that, in female rats, eight weeks of fructose overload promoted impairment in metabolic parameters and cardiac autonomic control by reducing vagal tone and increasing sympathetic modulation with additional insulin resistance in the absence of diabetes (7). However, morphometric and left ventricular diastolic functions in this model are seldom reported.

INTRODUCTION Metabolic syndrome (MS) has proven to be a major clinical challenge in this century. The presence of this syndrome increases mortality, especially due to cardiovascular disease (1). It is worth noting that the association of MS with cardiovascular disease increases overall and cardiovascular mortalities by 1.5- to 2.5-fold (2,3).

One of the factors that contributes to the increase in MS incidence is poor eating habits, which are mainly characterized by a large increase in fructose consumption (4,5).

Several changes are observed after chronic unhealthy eating

Copyright ß 2012 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.

No potential conflict of interest was reported.

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As a non-pharmacological treatment, aerobic physical training at moderate intensity can effectively mitigate fructose-induced hypertension in rats (8), and it has been considered a significant component in the treatment and prevention of human cardiovascular disease (9). However, little is known about the mechanisms by which exercise acts in models of high-fructose diets. Moreover, it is not known whether physical exercise of moderate intensity, performed concomitantly with a high-fructose diet, can minimize the metabolic changes, left ventricular hypertrophy and diastolic dysfunction that occur due to MS. Therefore, the aim of this study was to investigate whether moderate physical exercise performed during 10 weeks of a high-fructose diet can prevent metabolic changes and morphometric and left ventricular diastolic dysfunction in Wistar rats.

group, according to the guidelines of the American Society of Echocardiography. Rats were anesthetized with ketamine (50 mg/kg) and xylazine (10 mg/kg), and images were obtained using a Sequoia 512 ultrasound system (ACUSON, Mountain View, CA, USA) with a 10-14 MHz linear transducer for the measurement of morphometric parameters (left ventricular (LV) mass and relative wall thickness (RWT)), systolic function (ejection fraction (EF), fractional shortening (FS) and velocity of circumferential fiber shortening (VCF)) and diastolic function (absolute and normalized LV isovolumetric relaxation time; IVRT and nIVRT, respectively), and the ratio of maximal early diastolic peak velocity (E) and late peak velocity (A) of mitral flow (E/A ratio). The global index was quantified by the myocardial performance index (MPI).

Echocardiographic parameters were measured as previously described (13,14).

MATERIALS AND METHODS

Cardiovascular assessments After the last training session, two catheters were implanted into the femoral artery and vein (PE-10) while the animals were anesthetized (ketamine 50 mg/kg+xylazine 10 mg/kg) for the direct measurement of arterial pressure (AP) and drug administration, respectively.

Rats were studied one day after catheter placement; the rats were conscious and allowed to move freely during the experiments. The arterial cannula was connected to a straingauge transducer (P23Db, Gould-Statham, Oxnard, CA, USA), and AP signals were recorded over a 30-minute period by a microcomputer equipped with an analog-todigital converter board (Windaq, 2-kHz sampling frequency; Dataq Instruments, Inc., Akron, OH). The recorded data were analyzed on a beat-to-beat basis to quantify changes in mean AP and heart rate (HR).

Animals Experiments were performed on 21 male Wistar rats (251¡10 g) from the Animal Shelter at the University of Sao Paulo, Sao Paulo, Brazil. The rats received standard laboratory chow and water ad libitum. The animals were housed in individual cages in a temperature-controlled room (22˚C) with a 12-h dark-light cycle. All surgical procedures and protocols used were in accordance with the Guidelines for Ethical Care of Experimental Animals from the International Animal Care and Use Committee. This study was approved by the University of Sao Paulo Medical School Ethical Committee (360/11). The rats were randomly assigned to one of three groups: control, kept sedentary (C, n = 7); sedentary, receiving a high-fructose diet (F, n = 7); and trained, receiving a high-fructose diet (FT, n = 7).

The rate constant for blood glucose disappearance

High-fructose diet

One day after blood pressure recording, the blood glucose of all animals was measured after a 4-h fast with a glucosimeter (ACCUCHEK Advantage, Roche, Brazil). The rats were also submitted to an intravenous insulin tolerance test (ITT) after a 2-h fast. Animals were anesthetized with thiopental (40 mg/kg body weight, i.p.), and a drop of blood from the tail was collected to measure blood glucose.

This procedure was performed at baseline and 4, 8, 12 and 16 min after insulin administration (0.75 U/kg). The rate constant for blood glucose disappearance (KITT) was calculated using the formula 0.693/t1/2, and the blood glucose half-time (t1/2) was calculated from the leastsquares regression slope of the blood glucose concentration during the linear phase of decline (7).

The F and FT groups received an overload of D-fructose (100 g/l) in drinking water for 10 weeks. Control animals received only water during this period (7).

Exercise training Moderate-intensity exercise training (50-70% of the maximum running speed) was performed on a treadmill once per day, five days per week for 10 weeks, as described in detail elsewhere (10).

All animals were adapted to the procedure (10 min/day; 0.3 km/h) for one week before the beginning of the exercise training protocol. Sedentary and trained groups underwent a maximal treadmill test (10,11). The tests were performed in the first, fifth and tenth weeks of exercise training to determine aerobic capacity and adequate exercise training intensity.

Statistical analysis Data are reported as the means ¡ SEM, and one-way ANOVA was used to compare the groups, followed by the Student-Newman-Keuls post-hoc test. The Pearson correlation was used to study the association between variables.

Significance was established at p,0.05.

Obesity parameter in rats The Lee index for each animal was calculated after 10 weeks of fructose overload to obtain the obesity parameter. This index was calculated as the cube root of body weight (g)610/ naso-anal length (mm), and a value equal to or less than 0.300 was classified as normal at the third month of life. Rats with values higher than 0.300 were classified as obese (12).

RESULTS Hemodynamic and metabolic evaluations The body weight in the F group was higher than that in the C and FT groups (F = 515¡8 vs. C = 412¡14 and FT = 416¡ 10 g). The naso-anal length was similar among the groups (F = 26.49¡0.13, C = 26.19¡0.21 and FT = 26.04¡0.25 cm).

Echocardiographic evaluation One day after the last training session, echocardiography was performed by an observer blinded to the treatment

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left ventricular mass was higher in the F and FT groups compared with the control group. Furthermore, the RWT was increased in the F group compared with the FT and control groups. The FT and C groups exhibited similar RWT values.

Systolic function, expressed by fractional shortening, ejection fraction and VCF, was similar among the three groups. However, in the evaluation of diastolic function, lower ratios were found for IVRT, nIVRT and E/A in the F group than in the C and FT groups. The C and FT groups exhibited similar diastolic function. The myocardial performance index (MPI) was increased in the F group compared with the C and FT groups, whereas the FT and C groups exhibited similar values.

Consequently, the Lee index was increased in the F group compared with the C and FT groups (F = 0.31¡0.001 vs.

C = 0.29¡0.001 and FT = 0.29¡0.001 g/mm). The Lee index was similar between the C and FT groups (Figure 1).

Glycemia was also increased in the F group after fructose overload compared with the control rats, but there was no difference with respect to the F and FT groups (F = 100¡4, FT 99¡1 vs. C = 88¡1 mg/dl).

In the intravenous insulin tolerance test, the rate constant for blood glucose disappearance (KITT) was reduced in F rats compared with C and FT rats (3.6¡0.2 vs. 4.5¡0.2 and 4.6¡0.2 %/min, respectively). Additionally, when the area under the curve was calculated, a higher area was obtained in the F group compared with the C group (1300¡48 vs.

1044¡30 mg/dl/min). No difference was observed between the C and FT groups for this parameter (1044¡30 vs. 1208¡38 mg/dl/min) (Figure 1).

The systolic (SBP), diastolic (DBP) and mean (MBP) blood pressure were increased in the F group compared with the C and FT groups. These parameters were similar between the C and FT groups (SBP = 142¡2 vs. 122¡4 and 119¡2 mmHg, DBP = 101¡2 vs. 83¡4 and 87¡4 mmHg and MAP = 118¡3 vs. 104¡4 and 101¡2 mmHg, respectively). No difference was observed in heart rate among the studied groups (C = 330¡7, FT = 320¡6 and F = 335¡9 bpm).

Correlation Analyses Correlation analyses were carried out to associate metabolic parameters with diastolic dysfunction variables in all three groups (Figure 2). A negative correlation was found between IVRT and KITT (r = -0.67; p = 0.002). Similarly, a negative correlation between nIVRT and KITT (r = -0.70; p = 0.0016) was demonstrated. We observed a negative correlation between the E/A ratio and KITT (r = -0.63; p = 0.004). A negative correlation was also found between the global index MPI and KITT (r = -0.74; p = 0.004).

A positive correlation was found between IVRT, nIVRT and AUC (r = 0.67; p = 0.002 and r = 0.68; p = 0.0036, respectively). Furthermore, the E/A ratio and AUC were positively correlated (r = 0.66; p = 0.003), as were the MPI and AUC (r = 0.67; p = 0.003).

Echocardiographic evaluation The results of echocardiographic evaluations performed 10 weeks after fructose overload are shown in Table 1. The left ventricular morphometric evaluations indicated that the

Figure 1 - Lee Index, systolic arterial pressure, the rate constant for blood glucose disappearance and area under the curve in control (C) high fructose-fed (F) and trained high fructose-fed (FT) groups. One-way ANOVA, *p,0.05 vs. C and # p,0.05 vs. C and FT.

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weight gain, dyslipidemia, reduced insulin sensitivity and high blood pressure (15,16).

Interestingly, fructose overload promoted changes in left ventricular morphometry, diastolic dysfunction and increased cardiac effort, as evidenced by the increase in the myocardial performance index (MPI). Other authors found similar results after submitting Wistar rats to 10% fructose overload for eight weeks (17).

Left ventricular diastolic dysfunction and morphometric alterations can result from several factors, such as increased collagen concentration and fibrosis and decreased compliance, which are influenced by the autonomic nervous system and the renin-angiotensin system (17). In fact, several authors showed increased blood pressure, decreased vagal tonus and increased angiotensin II in a high fructose-fed rat model (7,18,19). Furthermore, insulin resistance is a pathogenic factor and has been associated with abnormal myocardial performance in pre-diabetic adults. Additionally, as demonstrated in Figure 1, this relationship between resistance and myocardial performance reinforces the fact that the heart is a target organ in metabolic syndrome, and its evaluation should not be neglected in clinical practice.

Table 1 - Cardiac morphometric and functional evaluations by echocardiography in control (C), fructosefed (F) and trained fructose-fed (FT) groups.

Variable morphometric LV mass (g) RWT systolic function LVFS (%) LVEF (%) VCF (circ/sec) diastolic function IVRT (ms) nIVRT (ms) E/A ratio (ms) global index MPI

C (7)

F (7)

FT (7)

0.73¡0.03 0.33¡0.007

1.3¡0.01* 0.38¡0.005*

1.2¡0.01* 0.35¡0.004

40¡1.5 36¡1 77¡1.6 72¡1.1 0.0053¡0.0003 0.0046¡0.0001

40¡1.6 76¡1.5 0.0052¡0.0002

22¡0.4 1.6¡0.03 1.72¡0.08

27¡0.9# 2.05¡0.07# 2.24¡0.12#

0.22¡0.6 1.73¡0.03 1.72¡0.15

0.32¡0.02

0.41¡0.02#

0.33¡0.01

Values are the mean ¡ SEM. The morphometric evaluation consisted of the assessment of left ventricular mass (LV mass) and relative wall thickness (RWT). Systolic function was evaluated by the left ventricular fractional shortening (LVFS), left ventricular ejection fraction (LVEF), and velocity of circumferential fiber shortening (VCF). Diastolic function was evaluated by the absolute and normalized LV isovolumetric relaxation time (IVRT and nIVRT, respectively) and the ratio of the maximal early diastolic peak velocity (E) and late peak velocity (A) of mitral flow (E/A ratio). Cardiac global function was evaluated by the myocardial performance index (MPI). * p,0.05 vs. C and # p,0.05 vs. C and FT (one-way ANOVA).

Preventive effect of exercise training during high fructose intake Physical exercise has been shown to be an effective, non-pharmacological treatment for cardiovascular disease (20-27). However, in the present study, it was used as a preventive tool when performed during the same period in which the rats received fructose overload in drinking water.

Interestingly, the FT group presented similar values to those of the C group for several parameters, such as the Lee index, insulin resistance, and systolic and diastolic blood pressure, which confirms that exercise training was effective in the prevention of these changes induced by a high-fructose diet.

The caloric expenditure due to the exercise training during the high-fructose diet period probably enhanced the energetic demand and prevented changes such as weight and fat gain, which contributed to improved insulin sensitivity and the maintenance of blood pressure in the normal range. In fact, the reduction of weight, intraabdominal adiposity and insulin resistance by exercise training may interfere with other mechanisms of action in the treatment of hypertension, as hypertension has been associated with all of these variables (28). Indeed, regular physical exercise can promote chronic metabolic and cardiovascular adaptations. It is known that regular exercise improves glycemia, the lipid profile (29) and insulin sensitivity; promotes weight reduction; and improves muscle glucose uptake.

Additionally, the morphometric and functional alterations demonstrated by echocardiography in the F rats were not observed in the C and FT groups. In diabetic and hypertensive rats, physical training has been important in the treatment of metabolic and autonomic disorders by contributing to increased baroreflex sensitivity and decreased blood pressure (10,30,31). Moreover, exercise training can modulate the renin-angiotensin system and prevent the development of cardiac hypertrophy in SHR rats (32). Exercise training can increase the antioxidant capacity, increase vascular compliance and promote atheroprotective effects (33).

Altogether, these improvements in several systems resulting from exercise training were important in the prevention of cardiovascular derangements promoted by MS. It is

DISCUSSION The purpose of this study was to investigate whether moderate physical exercise performed during 10 weeks of a high-fructose diet in Wistar rats can prevent metabolic changes, cardiac morphofunctional alterations and left ventricular diastolic dysfunction.

Interestingly, we found that metabolic and cardiac morphofunctional alterations promoted by a high-fructose diet can be attenuated by physical exercise when both occur in the same time period. This is the first study to demonstrate the preventive effect of physical training on the diastolic dysfunction induced by fructose overload in rats.

Metabolic and echocardiographic changes induced by fructose overload In our study, although high fructose consumption promoted an increase in glycemia compared with control rats, the values were still within the normal range. Other studies, even those utilizing high-fructose diets over longer periods, did not find significant alterations in glycemia (7).

However, we observed several changes that were characteristic of metabolic syndrome in the rats that received a highfructose diet. For instance, obesity was defined as an increased Lee index and was associated with insulin resistance, as demonstrated by the KITT and the area under the curve. In fact, this model characterizes an increase in insulin resistance and not hyperglycemia (7). In fact, this model is characterized by an increase in systolic and diastolic blood pressure.

In humans with normal beta cell function, the secretion of insulin may be altered to accommodate different degrees of insulin sensitivity and, thus, to maintain blood glucose within the normal range. Likewise, clinical studies have also demonstrated that the consumption of fructose can induce

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Exercise training in fructose rats Mostarda C et al.

Figure 2 - Correlation analyses between insulin resistance index, diastolic index and global index. A: normalized isovolumetric relaxation time (nIVRT), B: ratio of maximal early diastolic peak velocity (E) and late peak velocity (A) of mitral flow (E/A ratio), C: myocardial performance index (MPI) vs. area under the curve of the rate constant for blood glucose disappearance (AUC) and D: nIVRT, E: E/A ratio and F: MPI vs. the rate constant for blood glucose disappearance (KITT), involving control, fructose-fed and trained fructosefed rats.

syndrome or in patients at risk for metabolic syndrome development. Nevertheless, more clinical studies that assess cardiac function in these patients should be performed.

notable that the cardiac changes observed in the F rats and absent in the FT rats due to exercise training were correlated with the indexes of insulin resistance, suggesting that these alterations play a significant role in the process of cardiac damage due to MS or diabetes.

Among the derangements caused by metabolic syndrome, cardiac morphofunctional alterations were also observed.

Diastolic function seemed to be the cardiac parameter most influenced by the metabolic alterations, especially the insulin resistance.

Chronic high fructose intake favored the development of metabolic syndrome in rats. When performed concomitantly with a high-fructose diet, exercise training proved to be a vital tool for the prevention of not only metabolic but also cardiac changes promoted by fructose overload in rats. Additionally, it seems that the prevention of diastolic dysfunction was related to improved insulin resistance. Considering our results, we speculate that cardiac morphofunctional evaluations should also be performed in patients with metabolic

AUTHOR CONTRIBUTIONS Mostarda C performed and coordinated the experiments. Moraes-Silva IC and Machi JF participated in the data discussion and helped draft the manuscript. Salemi VM performed the echocardiography evaluation.

Rodrigues B, De Angelis K and Farah VM participated in the design and data discussion and helped draft the manuscript. Irigoyen MC conceived the study and participated in its design and coordination.

REFERENCES 1. First Brazilian Guideline for Diagnosis and Treatment of Metabolic Syndrome. 2005.

2. Lakka HM, Laaksonen DE, Lakka TA, Niskanen LK, Kumpusalo E, Tuomilehto J, et al. The metabolic syndrome and total and cardiovascular disease mortality in middle-aged men. JAMA. 2002;288(21):2709-16, http://dx.doi.org/10.1001/jama.288.21.2709.

819

Exercise training in fructose rats Mostarda C et al.

CLINICS 2012;67(7):815-820

3. Girman CJ, Rhodes T, Mercuri M, Pyorala K, Kjekshus J, Pedersen TR, et al. The metabolic syndrome and risk of major coronary events in the Scandinavian Simvastatin Survival Study (4S) and the Air Force/Texas Coronary Atherosclerosis Prevention Study (AFCAPS/TexCAPS).

Am J Cardiol. 2004;93(2):136-41, http://dx.doi.org/10.1016/j.amjcard.

2003.09.028.

4. Bray GA, Nielsen SJ, Popkin BM. Consumption of high-fructose corn syrup in beverages may play a role in the epidemic of obesity. Am J Clin Nutr. 2004;79(4):537-43.

5. Basciano H, Federico L, Adeli K. Fructose, insulin resistance, and metabolic dyslipidemia. Nutr Metab (Lond). 2005;2(1):5, http://dx. doi.org/10.1186/1743-7075-2-5.

6. Sliem H, Nasr G. Left ventricular structure and function in prediabetic adults: Relationship with insulin resistance. J Cardiovasc Dis Res. 2011;2(1):23-8, http://dx.doi.org/10.4103/0975-3583.78583.

7. Brito JO, Ponciano K, Figueroa D, Bernardes N, Sanches IC, Irigoyen MC, et al. Parasympathetic dysfunction is associated with insulin resistance in fructose-fed female rats. Braz J Med Biol Res. 2008;41(9):804-8, http:// dx.doi.org/10.1590/S0100-879X2008005000030.

8. Reaven GM, Ho H, Hoffman BB. Attenuation of fructose-induced hypertension in rats by exercise training. Hypertension. 198812(2):12932, http://dx.doi.org/10.1161/01.HYP.12.2.129.

9. Carroll S, Dudfield M. What is the relationship between exercise and metabolic abnormalities?A review of the metabolic syndrome. Sports Med. 2004;34(6):371-418, http://dx.doi.org/10.2165/00007256-20043 4060-00004.

10. Moraes-Silva IC, De La Fuente RN, Mostarda C, Rosa K, Flues K, Damaceno-Rodrigues NR, et al. Baroreflex deficit blunts exercise training-induced cardiovascular and autonomic adaptations in hypertensive rats. Clin Exp Pharmacol Physiol. 2010,37(3):e114-20.

11. Rodrigues B, Figueroa DM, Mostarda CT, Heeren MV, Irigoyen MC, De Angelis K. Maximal exercise test is a useful method for physical capacity and oxygen consumption determination in streptozotocin-diabetic rats.

Cardiovasc Diabetol. 2007;6:38, http://dx.doi.org/10.1186/1475-2840-638.

12. Bernardis LL, Patterson BD. Correlation between ’Lee index’ and carcass fat content in weanling and adult female rats with hypothalamic lesions.

J Endocrinol. 1968;40(4):527-8, http://dx.doi.org/10.1677/joe.0.0400527.

13. Mostarda C, Rodrigues B, Vane M, Moreira ED, Rosa KT, Moraes-Silva IC, et al. Autonomic impairment after myocardial infarction: role in cardiac remodelling and mortality. Clin Exp Pharmacol Physiol.

2010;37(4):447-52, http://dx.doi.org/10.1111/j.1440-1681.2009.05327.x.

14. 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;6:14, http:// dx.doi.org/10.1186/1475-2840-6-14.

15. Elliott SS, Keim NL, Stern JS, Teff K, Havel PJ. Fructose, weight gain, and the insulin resistance syndrome. Am J Clin Nutr. 2002;76(5):911-22.

16. Ferder L, Ferder MD, Inserra F. The role of high-fructose corn syrup in metabolic syndrome and hypertension. Curr Hypertens Rep. 2010;12(2):105-12, http://dx.doi.org/10.1007/s11906-010-0097-3.

17. Xing SS, Bi XP, Tan HW, Zhang Y, Xing QC, Zhang W. Overexpression of interleukin-18 aggravates cardiac fibrosis and diastolic dysfunction in fructose-fed rats. Mol Med. 2010;16(11-12):465-70.

18. Farah V, Elased KM, Chen Y, Key MP, Cunha TS, Irigoyen MC, et al.

Nocturnal hypertension in mice consuming a high fructose diet. Auton

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

820

Neurosci. 2006;130(1-2):41-50, http://dx.doi.org/10.1016/j.autneu.

2006.05.006.

Farah V, Elased KM, Morris M. Genetic and dietary interactions: role of angiotensin AT1a receptors in response to a high-fructose diet. Am J Physiol Heart Circ Physiol. 2007;293(2):H1083-9, http://dx.doi.org/ 10.1152/ajpheart.00106.2006.

Bacchi E, Negri C, Zanolin ME, Milanese C, Faccioli N, Trombetta M, et al. Metabolic Effects of Aerobic Training and Resistance Training in Type 2 Diabetic Subjects: A randomized controlled trial (the RAED2 study). Diabetes Care. 2012;35(4):676-82, http://dx.doi.org/10.2337/ dc11-1655.

Krieger EM, Da Silva GJ, Negrao CE. Effects of exercise training on baroreflex control of the cardiovascular system. Ann N Y Acad Sci. 2001;940:338-47, http://dx.doi.org/10.1111/j.1749-6632.2001.tb03689.x.

La Rovere MT, Bersano C, Gnemmi M, Specchia G, Schwartz PJ. Exercise-induced increase in baroreflex sensitivity predicts improved prognosis after myocardial infarction. Circulation. 2002;106(8):945-9, http://dx.doi.org/10.1161/01.CIR.0000027565.12764.e1",-1,"xxx/ 12764.e1.

Ignarro LJ, Balestrieri ML, Napoli C. Nutrition, physical activity, and cardiovascular disease: an update. Cardiovasc Res. 2007;73(2):326-40, http://dx.doi.org/10.1016/j.cardiores.2006.06.030.

Laterza MC, de Matos LD, Trombetta IC, Braga AM, Roveda F, Alves MJ, et al. Exercise training restores baroreflex sensitivity in never-treated hypertensive patients. Hypertension. 2007;49(6):1298-306, http:// dx.doi.org/10.1161/HYPERTENSIONAHA.106.085548.

Legramante JM, Iellamo F, Massaro M, Sacco S, Galante A. Effects of residential exercise training on heart rate recovery in coronary artery patients. Am J Physiol Heart Circ Physiol. 2007;292(1):H510-5.

Senechal M, Bouchard DR, Dionne IJ, Brochu M. Lifestyle Habits and Physical Capacity in Patients with Moderate or Severe Metabolic Syndrome. Metab Syndr Relat Disord. 2012,[Epub ahead of print].

Alves AJ, Ribeiro F, Goldhammer E, Rivlin Y, Rosenschein U, Viana JL, et al. Exercise Training Improves Diastolic Function in Heart Failure Patients. Med Sci Sports Exerc. 2011, [Epub ahead of print].

Jakicic JM, Clark K, Coleman E, Donnelly JE, Foreyt J, Melanson E, et al.

American College of Sports Medicine position stand. Appropriate intervention strategies for weight loss and prevention of weight regain for adults. Med Sci Sports Exerc. 2001;33(12):2145-56.

Durstine JL, Haskell WL. Effects of exercise training on plasma lipids and lipoproteins. Exerc Sport Sci Rev. 1994;22:477-521.

Bertagnolli M, Schenkel PC, Campos C, Mostarda CT, Casarini DE, BelloKlein A, et al. Exercise training reduces sympathetic modulation on cardiovascular system and cardiac oxidative stress in spontaneously hypertensive rats. Am J Hypertens. 2008;21(11):1188-93, http:// dx.doi.org/10.1038/ajh.2008.270.

Mostarda C, Rogow A, Silva IC, De La Fuente RN, Jorge L, Rodrigues B, et al. Benefits of exercise training in diabetic rats persist after three weeks of detraining. Auton Neurosci. 2009;28 145(1-2):11-6, http://dx.doi.org/ 10.1016/j.autneu.2008.10.010.

Zamo FS, Barauna VG, Chiavegatto S, Irigoyen MC, Oliveira EM. The renin-angiotensin system is modulated by swimming training depending on the age of spontaneously hypertensive rats. Life Sci. 2011;18 89(34):93-9, http://dx.doi.org/10.1016/j.lfs.2011.05.004.

Hagg U, Andersson I, Naylor AS, Gronros J, Jonsdottir IH, Bergstrom G, et al. Voluntary physical exercise-induced vascular effects in spontaneously hypertensive rats. Clin Sci (Lond). 2004;107(6):571-81.

CLINICS 2012;67(7):821-826

DOI:10.6061/clinics/2012(07)19

BASIC RESEARCH

S100B protein expression in the heart of deceased individuals by overdose: a new forensic marker?

Armando Faa, Giancarlo Senes, Annalisa Locci, Pietro Pampaloni, Maria Elena Pais, Bruno Piras, Ernesto d’Aloja, Gavino Faa University of Cagliari, Department of Pathology, Cagliari, Italy.

OBJECTIVE: The evaluation of S100B protein expression in the human heart and its correlation with drug-related death.

METHOD: Left ventricular samples were collected from 74 serial forensic autopsies (15 overdose-related deaths; 59 non-overdose-related deaths) from 2007 to 2010. Tissue sections from each sample were immunostained for S100B protein by a commercial antibody.

RESULTS: The S100B protein was detected in the heart samples of all 15 cases of drug-related deaths; S100B immunoreactivity was mainly observed in the cytoplasm of cardiomyocytes and as globular deposits in the interstitial spaces. No reactivity or weak reactivity was found in the cardiomyocytes of the 59 subjects who died of other causes.

CONCLUSION: Our preliminary data show that the S100B protein accumulates in injured cardiomyocytes during drug-related sudden death. Given the near absence of S100B protein in the heart of subjects who died from causes other than drug overdose, S100B immunopositivity may be used as a new ancillary screening tool for the postmortem diagnosis of overdose-related cardiac death.

KEYWORDS: Chronic myocardial ischemia; Overdose, Immunohistochemistry; S100B. Faa A, Senes G, Locci A, Pampaloni P, Pais ME, Piras B, et al. S100B protein expression in the heart of deceased individuals by overdose: a new forensic marker? Clinics. 2012;67(7):821-826.

Received for publication on February 19, 2012; First review completed on March 5, 2012; Accepted for publication on March 22, 2012 E-mail: armando.faa@hotmail.it Tel.: 0039-0706092370

arrhythmias, myocardial infarction, and sudden cocainerelated death have been reported in humans (3). In the same study, cocaine use was shown to precipitate a cardiac event with myocardial infarct even after recreational use.

Additionally, clinical evidence has shown that adverse cardiac effects are frequent during medicinal cocaine use, in which the drug is applied intranasally and intratracheally as a local anesthetic (4-6). Cocaine use has also been associated with other cardiac disorders, such as systemic hypertension, aortic aneurysms, torsades de pointes, arrhythmia, sudden coronary death (7), ischemia, ventricular tachycardia, and ventricular fibrillation (8-12). Cocaine may change cardiac rhythm by inhibiting sodium transport across membranes during depolarization and thus inhibiting the generation and conduction of action potentials (13,14). Cocaine-related myocardial ischemia or infarction may occur immediately after cocaine use: cocaine increases the risk of acute myocardial infarct (AMI) by up to 24-fold in the first hours after use (15). The incidence of AMI is much higher in younger patients (aged 18-45 years) (16), particularly in those with other cardiac risk factors (17).

The principal mechanism of cocaine-related heart damage appears to be myocardial ischemia. Three pharmacological mechanisms, likely acting in combination, are involved in the pathogenesis of cocaine-related ischemia: first, cocaine produces an increase in heart rate and blood pressure as a consequence of adrenergic stimulation; second, it causes

INTRODUCTION Cocaine, a crystalline tropane alkaloid obtained from the leaves of the coca plant, is one of the most commonly used illicit drugs and acts as a powerful sympathomimetic agent, with local and systemic effects. Local effects are the consequence of the inhibition of membrane permeability to sodium during depolarization and the inhibition of the transmission of electrical impulses. Systemic effects are attributed to the inhibition of presynaptic reuptake of norepinephrine and dopamine and the secondary excess production of these neurotransmitters at receptor sites on the postganglionic neuron (1).

The effects of cocaine on the cardiovascular system have been extensively documented in both animal models and humans. Cocaine administered to rats produces atrial and ventricular arrhythmias and myocardial ischemia with coronary vasoconstriction (2). High-grade ventricular

Copyright ß 2012 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.

No potential conflict of interest was reported.

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coronary vasoconstriction due to the stimulation of coronary a-adrenergic receptors; and third, it leads to an increase in platelet aggregability. Furthermore, the mismatch between myocardial oxygen supply and the demand from cocaineinduced vasoconstriction and increased myocardial workload are often invoked as the major mechanisms by which cocaine might induce myocardial ischemia.

In contrast, there is limited information available on the acute effects of heroin on the heart; to the best of our knowledge, only a few cases of AMI after heroin injections have been reported (18). Experimental studies have demonstrated that heroin may cause systemic and direct effects on the heart (19,20), most likely mediated by a local coronary spasm or inflammation (21). Heroin could act directly on the vasomotor center by increasing parasympathetic activity, producing bradycardia and hypotension and stimulating the release of histamine from mast cells (22,23). Heroininduced coronary vasospasm after heroin sniffing has also been reported (24-26). Taken together, these reports indicate that heroin may have a direct toxic effect on coronary arteries and induce acute coronary occlusion.

The S100 protein family comprises 16 low-molecularweight (21 KDa) calcium-binding proteins that contain two calcium-binding sites. S100B, one of the two founding members of this family, is concentrated in oligodendrocytes, astrocytes, ependymocytes, Schwann cells and neurons (27).

In clinical practice, it is a reliable biochemical marker of cerebral damage and is also utilized in postmortem diagnosis in medico-legal autopsy cases (28). Because S100B protein plasma levels have been shown to increase during ischemic stroke, S100 protein serum levels are used in clinical practice to monitor neurovascular status and functional outcome in subjects affected by cerebral damage (29) or subarachnoid hemorrhage (30,31). Elevated serum S100B levels have been reported in patients affected by obstructive sleep apnea syndrome (32). Moreover, S100B urine levels have been proposed as a useful tool to identify newborns that are at a higher risk of neonatal death (33) or full-term infants at risk of hypoxic-ischemic encephalopathy (34).

S100B is not restricted to the nervous system; chondrocytes, adipocytes, melanocytes, and Langerhans cells may also release S100B (35,36). In addition, recent investigations showed that elevated S100B serum levels may also be detected in patients without head injury, suggesting that it may originate from other damaged tissues (37,38), such as skeletal myofibers, muscle satellite cells and myoblasts.

Isolated rat heart cells were shown to release S100B (39), and the myocardium was indicated as the putative main source for the increase in S100B serum levels in patients affected by dilated cardiomyopathy (40). Recently, S100B protein expression was shown to be induced in the heart of human subjects after myocardial infarction (41), and it has been proposed as a marker of poor prognosis in patients undergoing cardiac surgery (42).

Based on these data, the current study was aimed at verifying, with immunohistochemical data, whether the S100B protein is expressed in the hearts of subjects who underwent sudden cardiac death, including drug addicts and patients without a history of drug abuse.

study. The subjects tested were subdivided into two main groups: the overdose group (15 subjects; cocaine, heroin or polypharmacological admixtures, including ecstasy) and the non-overdose group (59 subjects; traumatic and non-traumatic deaths). The traumatic group was subdivided into three subgroups (sidearms, firearms, and drowning). The victims ranged in age from 9 to 85 years old. In each case, a full autopsy and toxicological analysis were performed within 24-72 hours after death. No macroscopic sign of congenital cardiomyopathy was found in any case. The macroscopic examination of the heart did not show any sign of severe coronary atherosclerosis. Multiple tissue samples collected from the left ventricular wall were fixed in 10% formalin, routinely processed and paraffin-embedded. The initial block was cut into 6-7 blocks approximately 2-3 mm wide. At least two histological sections were prepared from each block. The tissue sections were stained with hematoxylin-eosin or immunostained with a polyclonal rabbit antiS100B antibody (DakoCytomation A/S, Glostrup, diluted 1/ 2000) followed by incubation for three hours at 37 ËšC in with universal streptavidin/biotin immunoperoxidase detection reagents. Endogenous peroxidase was inactivated by incubation with 3% hydrogen peroxide for 5 min. To confirm the specificity of immunostaining, phosphate-buffered saline was substituted for the primary antibody in control sections.

The peripheral nerves present in each section served as a positive internal control. Stained sections were read by two pathologists blinded to the clinical diagnosis.

MATERIALS AND METHODS

DISCUSSION

Seventy-four serial autopsies conducted from 2007 to 2010 (40 males and 34 females) were included in the present

The current study investigated whether the S100B protein is expressed and immunodetectable in the hearts of patients

RESULTS Immunoreactivity for the S100B protein was mainly detected in the overdose group; less reactivity was detected in the 59 subjects in the non-overdose-related group (Figure 1).

The pattern of S100B reactivity varied between cases, with different localization and different intensities. S100B staining was mainly observed inside the cytoplasm of scattered cardiac cells (Figure 2). The signals appeared as differently sized globular deposits and granules, often located near the nucleus. Similar globular deposits were also found in the interstitial spaces and were unevenly distributed throughout the cardiac tissue (Figure 3). Immunoreactive signals were also occasionally found in the perivascular spaces in the cytoplasm of stromal cells surrounding the wall of intracardiac arteries. No significant differences were found among the different subgroups in which the overdose patients were subdivided: S100B protein deposits were found in the heart in cocaine-, heroin- and other drug-related deaths. We observed the highest levels of immunoreactivity for S100B in 3 patients with a clinical history of combined cocaine and heroin abuse (Figure 4). In these cases, a large number of cardiomyocytes showed very large globular deposits of the protein.

No reactivity for the S100B protein was detected in the subpericardial fatty tissue or other soft tissues, with the exclusion of nerve fibers, which represented the internal positive control.

Finally, hematoxylin-eosin staining was performed to investigate the pathological status of these hearts; edema, and a wavy arrangement of cardiomyocytes were observed in all S100B-positive cases (Figure 5).

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Cardiac changes following overdose Faa A et al.

Figure 1 - S100B positivity and cause of death.

who died due to drug overdose and whether different drugs (heroin, cocaine, and polypharmacological admixtures, including ecstasy) could affect S100B protein retention in cardiomyocytes.

Our preliminary data clearly show that the S100B protein is significantly more expressed in the hearts of drug users, regardless of the type of drug employed, compared with patients who died from traumatic or other pathological events. From a practical point of view, this finding suggests that S100B immunostaining could be performed in all cases of cardiac death possibly related to drug abuse to confirm the role of drug use as a cause of death.

The presence of the S100B protein inside the cytoplasm of cardiomyocytes and in the interstitial spaces around cardiac cells, and the presence of myocytolysis clearly suggest cardiomyocytes as the putative main source of S100B production and release. The absence of immunoreactivity for the protein in the subepicardial fat tissue in this study is inconsistent with the previous hypothesis that cardiac adipocytes are the main source of S100B protein in the heart.

Our histological data confirm the typical histological findings previously reported in cocaine-related death; i.e., contraction band necrosis (CBN), also referred to as coagulative myocytolysis, and an anomalous hypereosinophilic crossband formed by segments of hypercontracted sarcomeres with extremely thickened Z lines (43).

Another interesting finding in our study is that a similar S100B protein expression pattern is observed in both cocaine and heroin abusers. One possible explanation for this finding

could be the acute stress caused by both drugs on the central nervous system, followed by the induction of S100B protein overproduction in cardiomyocytes (44).

Particularly strong reactivity for S100B protein was observed in some cases in which toxicological analyses suggested concomitant cocaine and heroin abuse. This finding supports the assumption that both drugs may act on the same pathways, cooperating in S100B protein overproduction, storage and release. Because both of these substances are metabolized by the same enzymes, human carboxylesterases (hCEs), we might postulate that heroin and cocaine could interfere with each other at a metabolic level when taken together, giving rise to the overproduction of S100B and leading to significant cardiac storage of the protein (45).

However, to the best of our knowledge, the exact mechanism by which S100B protein is overexpressed at the cardiac level in overdose-related deaths remains unclear. The over-expression of S100B protein might depend on environmental factors; it has been shown that several substances, including norepinephrine, phenylephrine, interleukin 1b (IL-1b), bamyloid, antidepressants and pro-inflammatory cytokines, may up- or down-regulate S100B expression. Moreover, S100B expression has been attributed to catecholamines in cardiomyocytes surviving infarction (46). In fact, the S100B protein, which is undetectable in normal cardiomyocytes, is induced in the ischemic or post-infarction myocardium. These observations suggest that S100B gene expression is under complex transcriptional regulation. S100B gene expression appears to be constitutively repressed in all cell types, implying that the expression of S100B in a given cell type

Figure 2 - Diffuse S100B intermyocite positivity with some positivity in perivessels spaces (arrow).

Figure 3 - S100B positivity in inter-myocytes spaces with accentuation of Schwann cells in a nerve twig X 400.

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Figure 4 - S100B positivity and type of substance.

mediated via RAGE activation. The S100B protein could participate in this process.

Our data confirm the hypothesized existence of a common pathway through which multiple drugs (heroin and cocaine) might cause S100B protein cardiac overload and release. The reported increase in S100B urine levels in newborns at higher risk of neonatal death (33) due to hypoxic-ischemic encephalopathy or asphyxia (34) suggest that severe hypoxia is the common mechanism of druginduced S100B protein accumulation at the cardiac level.

Finally, our results, though limited by the small number of cases available and the numerous uncontrolled variables involved (e.g., doses, survival time, individual tolerance, post-mortem redistribution, drug stability, etc.), support the hypothesis that the immunohistochemical detection of significant S100B protein in the heart supports a suspicion of drug addiction and could distinguish overdose-related death from traumatic or ischemia-related death. Based on our preliminary data, we suggest that the immunodetection of S100B protein in the heart could be useful to not only help determine the cause of death in complicated or atypical cases but also provide evidence in individual cases to be shown in court.

Further studies are needed to clarify the metabolic link between overdose and S100B protein overload in the heart of drug abuse-related sudden deaths.

requires induction of an appropriate factor to counter the action of some negative regulatory elements (47).

In this study, the S100B protein was also detectable in extracellular spaces, in which it might exert multiple important effects. Previous research has shown that astrocytes may be stimulated to excrete S100B by relatively high levels of cytosolic Ca (48). A similar enhancing action of calcium ions could also be hypothesized in cardiomyocytes under hypoxic stress. A secondary hypothesis to explain the presence of S100B outside of cells is leakage from damaged cells (49). Our observation of coagulative myocytolysis in all cases of drug abuse might confirm this hypothesis.

Whatever the source of extracellular S100B and the mechanism underlying its release, extracellular S100B might attain local concentrations sufficient to affect cellular activities by paracrine, autocrine and endocrine manners.

The extracellular effects of S100B protein on cardiomyocytes and inflammatory cells could be mediated by the receptor for advanced glycation end-products (RAGE), a member of the immunoglobulin superfamily that is generally considered a central mediator of ischemia/reperfusion injury in the heart (50-53).

Dissecting the complex pattern of intracellular signaling resulting from hypoxic insults to ischemia and reperfusion (I/R) may be a considerable challenge.

In the complex environmental interactions in the injured heart, several ‘danger signals’ are commonly liberated from ischemic cells (52). These signals signal the ischemic insult to other cells and mediate an inflammatory response but also promote tissue growth and remodeling that is partially

ACKNOWLEDGMENTS The authors wish to thank Mr. Ignazio Ferru for secretarial assistance and Mrs. Sandra Serra for technical work.

AUTHOR CONTRIBUTIONS Faa A analyzed and interpreted the data and prepared the manuscript.

Senes G processed the heart tissues (hematoxylin–eosin and S100B immunostaining). Locci A processed the heart tissues (hematoxylin–eosin and S100B immunostaining). Pampaloni P provided assistance to the manuscript preparation and assembled images. Pais ME performed the experiments. Piras B performed the experiments and critically revised the manuscript. D’Aloja E critically revised the final version of the manuscript.

Faa G contributed to the conception and design of the study.

REFERENCES 1. Hollander JE, Hollander RS. In. Goldfrank LR, ed. Goldfrank’s Toxicologic Emergencies. New York: McGraw-Hill Medical Pub Division. 2002;1004-19.

2. Hale SL, Lehmann MH, Kloner RA. Electrocardiographic abnormalities after acute administration of cocaine in the rat. Am J Cardiol.

1989;63(20):1529-30, http://dx.doi.org/10.1016/0002-9149(89)90024-6.

Figure 5 - Oedema, wavy arrangement of cardiomyocytes.

824

CLINICS 2012;67(7):821-826

Cardiac changes following overdose Faa A et al.

3. Hale SL, Alker KJ, Rezkalla S, Figures G, Kloner RA. Adverse effect of cocaine on cardiovascular dynamics, myocardial blood flow, and coronary artery diameter in an experimental model. Am Hearth J. 1989;118(5 Pt 1):927-33.

4. Aslibekyan S, Levitan EB, Mittleman MA. Prevalent Cocaine Use and Myocardial Infarction. Am J Cardiol. 2008;102(8):966-9, http:// dx.doi.org/10.1016/j.amjcard.2008.06.016.

5. Ross GS, Bell J. Myocardial infarction associated with inappropriate use of topical cocaine as treatment for epistaxis. Am J Emerg Med. 1992;10(3):219-22, http://dx.doi.org/10.1016/0735-6757(92)90213-H. 6. Osula S, Stockton P, Abdelaziz MM, Walshaw MJ. Intratracheal cocaine induced myocardial infarction: an unusual complication of fibreoptic bronchoscopy. Thorax. 2003;58(8):733-4, http://dx.doi.org/10.1136/ thorax.58.8.733.

7. Makaryus JN, Makaryus AN, Johnson M. Acute myocardial infarction following the use of intranasal anesthetic cocaine. South Med J. 2006;99(7):759-61, http://dx.doi.org/10.1097/01.smj.0000223951.68627.32.

8. Montisci M. Cannabis and cocaine: a lethal cocktail triggering coronary sudden death. Cardiovascular Pathology. 2007;17(5):344-6.

9. Krantz MJ, Rowan SB, Mehler PS. Cocaine-related torsade de pointes in a methadone maintenance patient. J Addict Dis. 2005;24(1):53-60, http:// dx.doi.org/10.1300/J069v24n01_05.

10. Geggel RL, McInerny J, Estes NAM. Tansient neonatal ventricular tachycardia with maternal cocaine use. Am J Cardiol. 1989;63(5):383-4, http://dx.doi.org/10.1016/0002-9149(89)90359-7.

11. McCann B, Hunter M, Mccann J. Cocaine/heroin induced rabdomyolisis and ventricular fibrillation. Emerg Med J. 2002;19(3):264-5, http:// dx.doi.org/10.1136/emj.19.3.264.

12. Shereif H, Rezkalla MD, Kloner RA. Cocaine-induced acute myocardial infarction. Clin Med and Research. 2007;5(3):172-6.

13. Mittlemann MA, Mintzer D, Maclure M, Tofler GH, Sherwood JB, Muller JE. Triggering of myocardial infarction by cocaine. Circulation. 1999; 99(21):2737-41.

14. Kerns W II, Garvey L, Owens J. Cocaine- induced wide complex dysrythmia. J Emerg Med. 1995;15(3):321-9.

15. Kanami PM, Guse PA, Smith WM, Barnett A, Ellinwood EHJr. Acute deleterious effects of cocaine on cardiac conduction, hemodynamics and ventricular fibrillation thresholds: effects on interaction with a selective dopamine D1 antagonist SCH39166. J Cardiovasc Pharmacol.

1998;32(1):42-8, http://dx.doi.org/10.1097/00005344-199807000-00007.

16. Mitlemann MA, Lewis RA, Maclure M, Sherwood JB, Muller JE. Triggering myocardial infarction by Marijuana. Circulation. 2001; 103(23):2805-9.

17. Qureshi Al, Suri MF, Guterman LR, Hopkins LN. Cocaine se and the likelihood of nonfatal myocardial infarction and stroke: data from the Third National Health and Nutrition Examination Survey. Circulation.

2001;103(4):502-6.

18. Hollander JE, Shih RD, Hoffmann RS, Harchelroad FP, Philips S, Brent J. Predictor of coronary artery disease in patients with cocaine-associated myocardial infarction. Cocaine-Associated Myocardial Infarction (CAMI) Study Group. Am J Med. 1997;102(2):158-63, http://dx.doi.org/ 10.1016/S0002-9343(96)00406-8.

19. Yu SL, LIU CP, Lo YK, Shoa-Lin S. Acute Myocardial infarction after heroin injections. Jpn Heart J. 2004;45(6):1021-8.

20. Paterna S, Di Pasquale P, Montaina G, Procaccianti P, Antona A, Scaglione R, et al. Effect of heroin and morphine on cardiac performance in isolated and perfused rabbit hearth: valuation of cardiac hemodynamics, myocardial enzyme activity and ultrastructure features.

Cardiologia. 1991;36(10):811-5.

21. Schwartzfarb L, Singh G, Marcus D. Heroin- associated rhabdomyolisis with cardiac involvement. Arch Intern Med. 1977;137(9):1255-7, http:// dx.doi.org/10.1001/archinte.1977.03630210121035.

22. Sztajzel J, Karpuz H, Rutishauser W. Heroin abuse and myocardial infarction. Int J Cardiol. 1994;47(2):180-2, http://dx.doi.org/10.1016/ 0167-5273(94)90187-2.

23. Schwartz RH. Adolescent heroin use: a review. Pediatrics. 1998; 102:1461-6, http://dx.doi.org/10.1542/peds.102.6.1461.

24. Lipski J, Stimmel B, Donoso E. The effect of heroin and multiple drug abuse on the electrocardiogram. Am Heart J. 1973;86(6):663-8, http:// dx.doi.org/10.1016/0002-8703(73)90344-X. 25. Silverma SH, Turner WW Jr. Intraarterial drug abuse: new treatment options. J Vac Surg. 1991;14(1):111-6, http://dx.doi.org/10.1016/07415214(91)90162-N. 26. Bartolomei F, Nicoli F, Swiader L, Gastaut JL. Ischemic cerebral vascular stroke after heroin sniffing. A new case. Press Med. 1992;21(21):983-6.

27. Adle-Biassette H, Marc B, Benhaiem-Sigaux N, Durigon M, Gray F. Cerebral infarctions in a drug addict inhaling heroin. Arch Anat Cytol Pathol. 1996;44(1):7-12.

28. Leclerc E, Fritz G, Vetter SW, Heizmann CW. Binding of S100 proteins to RAGE: an update. Biochim Biophys Acta. 2009;1793(6):993-1007, http:// dx.doi.org/10.1016/j.bbamcr.2008.11.016.

29. Li DR, Ishikawa T, Zhao D, Michiue T, Maeda H. Postmortem serum protein S100B levels with regard to the cause of death involving brain

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

825

damage in medicolegal autopsy cases. Legal Medicine. 2006;8(2):71-7, http://dx.doi.org/10.1016/j.legalmed.2005.07.004.

Wunderlich MT, Wallesch CW, Goertler M. Release of neurobiochemical markers of brain damage is related to the neurovascular status on admission and the site of arterial occlusion in acute ischemic stroke. J of the Neurological sciences. 2004;227(1):49-53, http://dx.doi.org/10.1016/ j.jns.2004.08.005.

Weiss N, Sanchez Pena P, Roche S, Beaudeux JL, Colonne C, Coriat P, et al. Prognosis value of plasma S100B protein levels after subarachnoid aneurysmal hemorrhage. Anesthesiology. 2006;104(4):658-66, http://dx. doi.org/10.1097/00000542-200604000-00008.

Gazzolo D, Marinoni E, Di Iorio R, Bruschettini M, Kornacka M, Lituania M, et al. Urinary S100B protein measurements: a tool for the early identification of hypoxic-ischemic encefalopathy in asphyxiated fullterm infants. Crit Care Med. 2004;32(1):131-6, http://dx.doi.org/ 10.1097/01.CCM.0000104116.91462.CD. Johnsonn P, Backstrom M, Bergh C, Jonsonn H, Luhrs C, Alling C. Ann Thorac Surg. 2003;75(1):162-8.

Gazzolo D, Florio P, Ciotti S, Marinoni E, di Iorio R, Bruschettini M, et al.

S100B protein in urine of preterm newborns with ominous out come.

Pediatrich Research. 2005;58(6):1170-5, http://dx.doi.org/10.1203/ 01.pdr.0000185131.22985.30.

Braga CW, Martinez D, Wofchuk S, Portela SV, Souza DO. S100B and NSE serum levels in obstructive sleep apnea syndrome. Sleep Medicine.

2006;7(5):431-35, http://dx.doi.org/10.1016/j.sleep.2005.12.012.

Haimoto H, Hosoda S, Kato K. Differential distribution of immunoreactive S100A and S100 B proteins in normal non nervous human tissues.

Laboratory Investigations. 1987;57(5):489-97.

O Kleine T, Benes L, Zofel P. Studies of the brain specificity of S 100B and neuron-specific enolase (NSE) in blood serum of acute care patients.

Brain Research Bulletin. 2003;61(3):265-79, http://dx.doi.org/10.1016/ S0361-9230(03)00090-X. Savola O, Pyhtinen J, Leino TK, Siitonen S, Niemela O, Hilbom M. Effects of head and extracranial injuries on serum protein S100B level in trauma patients. J Trauma. 2004;56:1229-1234, http://dx.doi.org/10.1097/ 01.TA.0000096644.08735.72.

Anderson RE, Hansson LO, Nilsson O, Dijlai-Merzong R, Settergren G. High serum S100B levels for trauma patients without head injuries.

Neurosurgery. 2001;48(6):1255-8; discussion 1258-60.

Mazzini GS, Schaf DV, Oliveira A, Goncalves CA, Bello-Klein A, Bordignon S et al. The ischemic rat heart releases S100B. Life Sciences.

2005;77(8):882-9, http://dx.doi.org/10.1016/j.lfs.2004.12.035.

Ambrozic J, Bunc M, Osredkar J, Brvar M. S100b protein in benzodiazepine overdose. Emergency Medical Journal. 2008;25(2):90-2.

Mussack T, Biberthaler P, Kanz KG, Wiedemann E, Gippner-Steppert C, Jochum M. S-100B, SE-selectin and P-selectin for avaluation of hypoxic brain damage in patients after cardiopulmonary resuscitation: pilot study. World J Surg. 2001;25(5):539-43; discussion 544.

Tsoporis JN, Marks A, Kahn HJ, Butany JW, Liu PP, O’Hanlon D, et al.

Inhibition of norepinephrine-induced cardiac hypertrophy in S100B transgenic mice. J Clin Invest. 1998;102(8):1609-16, http://dx.doi.org/ 10.1172/JCI3077.

Tramontina AC, Tramontina F, Bobermina LD, Zanotto C, Souza DF, Leite MC, et al. Secretion of S100B, an astrocyte-derived neurotrophic protein, is stimulated by fluoxetine via a mechanism independent of serotonin.

Progress in Neuro-Psychopharmacology and Biological Psychiatry.

2008;32(6):1580-3, http://dx.doi.org/10.1016/j.pnpbp.2008.06.001.

Polettini A, Poloni V, Groppi A, Stramesi C, Vignali C, Politti L. The role of cocaine in heroin-related deaths Hypothesis on the interaction between heroin and cocaine. Forensic Science International.

2005;153(1):23–8, http://dx.doi.org/10.1016/j.forsciint.2005.04.017.

Hsieh CH, Davis ME, Lisowsky LK, Lee LT. Endothelial-cardiomyocytes interactions in cardiac development and repair. Ann. Rev. Phisiol. 2006; 68:51-66, http://dx.doi.org/10.1146/annurev.physiol.68.040104.124629.

Tsoporis JN, Marks A, Haddad A, Dawood F, Liu PP, Parker TG. S100b expression modulates left ventricular remodeling after myocardial infarction in mice. Circulation. 2005;111(5):598-606, http://dx.doi.org/ 10.1161/01.CIR.0000154554.65287.F5.

Deloulme JC, Raponi E, Gentil BJ, Bertacchi N, Marks A, Labourdette G, et al. Nuclear expression of S100B in oligodendrocytes progenitor cells correlates with differentiation toward the oligodendroglial lineage and modulates oligodendrocytes maturation. Mol Cell Neurosc. 2004;27(4): 453-65, http://dx.doi.org/10.1016/j.mcn.2004.07.008.

Bloomfield SM., McKinney J, Smith L, Brisman J. Reliability of S100b in predicting severity of central nervous system injury. Neurocrit Care.

2007;6(2):121-38, http://dx.doi.org/10.1007/s12028-007-0008-x.

Schmidt AM, Yan SD, Yan SF, Stern DM. The multiligand receptor RAGE as a progression factor amplifying immune and inflammatory response.

J Clin Invest. 2001;108(7):949-55.

Bierhaus A, Humpert PM, Morcos M, Wendt T, Chavakis T, Arnold B et al. Understanding Rage, the receptor for advanced glycation end products. J Mol Med (Berl). 2005;83(11):876-86, http://dx.doi.org/ 10.1007/s00109-005-0688-7.

Cardiac changes following overdose Faa A et al.

CLINICS 2012;67(7):821-826

53. Aleshin A, Ananathakrishnan R, Li Q, Rosario R, Lu Y, Qu W, et al. Rage modulates myocardial injury consequent to LAD infarction via impact on JNK and STAT signaling in a murine model. Am J Physiol Heart Circ Physiol. 2008;294(4):H1823-32, http://dx.doi.org/10.1152/ajpheart.01210.2007.

52. Andrassy M, Volz HC, Igwe JC, Funke B, Eichberger SN, Kaya Z, et al.

High mobility group box-1 in ischemia-reperfusion injury of the heart.

Circulation. 2008;117(25):3216-26, http://dx.doi.org/10.1161/CIRCUL ATIONAHA.108.769331.

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DOI:10.6061/clinics/2012(07)20

REVIEW

Wound-healing and benzodiazepines: does sleep play a role in this relationship?

Flavia Egydio, Gabriel Natan Pires, Sergio Tufik, Monica Levy Andersen Universidade Federal de Sa˜o Paulo (UNIFESP), Departamento de Psicobiologia, Sa˜o Paulo/SP, Brazil.

Patients who have suffered burns frequently experience psychological consequences, among which anxiety disorders are prominent. Benzodiazepines are commonly administered to treat these symptoms. The effects of benzodiazepines on healing may not be direct but rather are modulated by alterations of the sleep architecture.

This hypothesis is supported by studies that demonstrate the effects of benzodiazepines on the immune system and the inflammatory profile under both normal sleep conditions and during sleep deprivation, particularly rapid eye movement sleep deprivation.

KEYWORDS: Benzodiazepines; Sleep Deprivation; Wound Healing; Burns; Immune System.

Egydio F, Pires GN, Tufik S, Andersen ML. Wound-healing and benzodiazepines: does sleep play a role in this relationship? Clinics. 2012;67(7):827-830.

Received for publication on January 10, 2012; First review completed on January 31, 2012; Accepted for publication on February 22, 2012 E-mail: mandersen@psicobio.epm.br / ml.andersen12@gmail.com Tel.: 55-11 21490155

the wound were increased significantly by midazolam.

Another study by this group, using the same treatment with midazolam, investigated whether psychological stress (e.g., predator exposure) could alter survival following Pseudomonas aeruginosa infection. The results showed that midazolam had a protective effect in mice (8). Together, these studies suggest that BDZ can modulate the immune system and a host of inflammatory mediators, leading to a detrimental effect on healing and tissue regeneration.

Based on this evidence, we hypothesize that inflammation of wounded tissue may not be a direct effect of midazolam but rather a consequence of sleep alterations caused by the administration of BZD.

INTRODUCTION Damage to the skin triggers mechanisms of repair that are collectively called healing. This process commences immediately after injury and is composed of the following three main phases: inflammatory, proliferative, and maturation.

The wound-healing process is not linear, and these phases depend on various intrinsic and extrinsic factors. Several factors may influence the healing process, including age, nutritional status, diabetes, atherosclerosis, stress, lack of sleep, and the integrity of the immune system (1-3). The integrity of the immune system is indispensable during the healing process, particularly during the recovery of tissue after burns, when defense cells and inflammatory mediators act in unison to promote tissue regeneration, cytokine production, and bacterial clearance.

Among patients who have suffered burns, a common sequela is the psychological consequences, among which anxiety disorders are the most prevalent (4). A therapeutic option for anxiety is the prescription of benzodiazepines (BDZ) to deal with the ensuing stress caused by the injury.

The choice of this drug for burn patients has grown considerably in the past years and 77% of burn patients received this drug in 2001 (5,6).

Recently, some studies examined the effects of midazolam, a commonly used BZD, upon the burn wound. Babcock et al. treated mice with midazolam (1 mg/kg) daily after a burn injury and observed a decrease in IL-1b, TNF-a, IL-6, IL-10, and TGF-b levels compared to saline-treated mice (7).

Moreover, the chemokines (MIP-1a, MIP-1b, and MIP-2) in

STRESS, ANXIETY, AND THE IMMUNE SYSTEM An important factor during the treatment of wounded patients is the stress resulting from the injury, which may affect the immune system. Another consideration is the interaction between stress and the immune system. As demonstrated by McGhee et al. (9), lesions caused by burns or other wounds are potentially stressful events and are one reason why the use of BZD among such patients has seen a rise. However, stress, anxiety, and the immune system are intimately associated, especially when dealing with inflammation and healing. Specifically, dermatologic lesions can trigger psychological alterations such as stress (10), and stressful situations may trigger alterations in the immune system as a consequence of the activation of the hypothalamic-pituitary adrenal axis (HPA) and increased glucocorticoids (11; see (16) for review), which may then impact healing.

Increased psychological stress is associated with a higher incidence of skin dermatoses (such as psoriasis and dermatitis) (12-14). This abnormality in the skin may interfere in the healing process and compromise the skin’s ability to act as an immune barrier, which is one of its main functions (15).

Padgett et al. (17) observed retardation in the healing process

Copyright ß 2012 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.

No potential conflict of interest was reported.

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in an animal model submitted to immobilization stress. In addition, immobilized mice presented increased corticosterone when compared to controls. Thus, delayed healing was correlated with higher concentrations of corticosterone, suggesting that a perturbation of neuroendocrine homeostasis may modulate the repair process of the lesion. Taken as a whole, these results suggest that alterations in the immune function are associated with stress and may exert further complications that hamper the regeneration and integrity of the skin.

The wound healing process consists in a perfectly coordinated cascade of cellular and molecular events that interact to produce the reconstitution of the tissue. There are several studies in the literature showing the adverse effects, particularly stress, in wound healing in animal models and humans. It is known that sleep deprivation is an event inherent to stress and we can hypothesize that the stress caused by sleep deprivation results in negative consequences in the healing process. However, it is a challenge for researchers to investigate the effects of sleep deprivation on the healing process due to the complexity of this process.

EFFECTS OF SLEEP RESTRICTION ON THE IMMUNE SYSTEM

INTEGRATION OF CONCEPTS Recovery from skin lesions, such as burns, is intimately associated with specific inflammatory mediators, which entails a direct association with the immune system. An example of such an association can be found in the formation of granulation tissue as macrophages release a variety of pro-inflammatory cytokines (interleukin 1 and 6) and growth factors (fibroblast growth factor [FGF], epidermal growth factor EGF, transforming growth factor-beta [TGF-b], and platelet-derived growth factor [PDGF]). The formation of granulation tissue is an essential process for the synthesis, deposition, and organization of new extracellular matrix [see review, (34)]. Thus, the efficacy of the healing process is directly dependent upon the integrity of the immune system. However, particularly when the wound is of great extent, depth, or severity, symptoms such as anxiety and stress can be observed, making the relationship between stress and the immune system bidirectional; the patient is stressed by the wound, and stress can compromise the immune system, slowing healing time. Thus, stress itself is responsible for the retardation of healing time (35).

Given the detrimental effects of stress in these situations, BZDs are commonly administered as a means of attenuating anxiety and stress symptoms (36). However, chronic consumption of BZD also provokes significant immunological disorders in humans (37) and alterations in sleep that may also affect immune function.

Therefore, we propose that the effects of BZD on healing are not direct, but rather are modulated by alterations of sleep, which then affect the immune system (Figure 1). This hypothesis is supported by studies that demonstrate the effects of BZD on the characteristics of the immune system and the inflammatory profile in normal sleep and sleep deprivation, particularly in REM sleep deprivation.

Recently, Kronholm and collaborators (18) showed that average sleep time has fallen approximately two hours per night over the past 50 years. This reduction in sleep time, which is characteristic of a modern lifestyle, may lead to repercussions upon one’s health and well-being. In particular, several studies have shown that sleep deprivation (SD) causes marked and adverse alterations in the immune system (19-24). One study demonstrated that forced wakefulness in the first half of the night (from 22:00 to 3:00) and in the second half of the night (from 3:00 to 7:00) promoted a reduction in the activity of natural killer (NK) cells (25). These results suggest that a few hours of sleep deprivation is sufficient to cause adverse effects on the integrity of the immune system. Recently, Ruiz et al. (24) reported that a total of two nights of SD resulted in increased leukocytes and neutrophils in healthy men compared to their baseline values. After 24 hours of sleep recovery, those figures returned to the values observed at baseline. However, the concentrations of monocytes, eosinophils, basophiles, and cytokines (IL-1b, IL-2, IL-4, IL-6, IL10, TNF-a, and IFN-c) remained unaltered after the SD protocol. Thus, SD may alter the immune profile. BZD also influence the immune system, affecting cytokines, neutrophils, and macrophages (26-28).

RELATIONSHIPS BETWEEN THE USE OF BZD AND SLEEP When first administered, hypnotics and anxiolytics of the BZD class improve sleep efficiency by reducing the latency of sleep onset and arousals during the night (29). However, after chronic use, BZDs inhibit the desynchronization process, reducing REM sleep due to a decrease of the neurotransmitter GABA (30). Additionally, BZDs increase slow-wave sleep and, in the long term, lead to a condition of REM-sleep restriction (31).

The relationship between sleep deprivation, immune parameters, and skin integrity remains under investigation.

Mostaghimi et al. (32) showed that rats subjected to five days of sleep deprivation by the multiple-platform method did not present a slower healing process when compared to controls. Similarly, Landis and Whitney (33) did not observe any detrimental effects on healing at the cellular level in rats that were sleep-deprived for 72 h using the single-platform method. Although these data cannot be interpreted as that the lack of sleep jeopardizes the healing process, nevertheless it is possible that those effects are mediated by other molecular mechanisms that are currently unidentified.

PRACTICAL IMPLICATIONS The potential damage caused by BZD to the healing process in patients who have skin wounds contradicts the use of these compounds in such cases (6). BZDs are used in such cases to improve the mental state of the patient and, as a consequence, promote better tissue regeneration; however, that advantage is offset by these potentially detrimental effects.

There are two important considerations that follow from our hypothesis. The first is that the hypothesis presents a novel manner of approaching the clinical condition in a patient with skin lesions. In this scenario, sleep becomes an important factor for that patient’s improvement. The second is that this investigation opens a debate on the use of BZD for patients with skin lesions. BZDs are not the only drugs

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Burns and benzodiazepines Egydio F et al.

Figure 1 - The proposed model for the interplay between the use of BZD and healing in patients with skin lesions. In this case, the immune system and healing continue to be mutually dependent and are presented in relation to the bidirectional immune profile and psychological profile of the patient because large lesions cause anxiety and stress, and these effects directly affect healing and inflammatory capacity. Finally, BZD compounds are ultimately responsible for alterations in healing and tissue regeneration.

3. Wild T, Rahbarnia A, Kellner M, Sobotka L, Eberlein T. Basics in nutrition and wound healing. Nutrition. 2010;26(9):862-6, http:// dx.doi.org/10.1016/j.nut.2010.05.008.

4. Ter Smitten MH, de Graaf R, Van Loey NE. Prevalence and co-morbidity of psychiatric disorders 1-4 years after burn. Burns. 2011;37(5):753-61, http://dx.doi.org/10.1016/j.burns.2010.12.018.

5. Tcheung WJ, Robert R, Rosenberg L, Rosenberg M, Villarreal C, Thomas C, et al. Early treatment of acute stress disorder in children with major burn injury. Pediatr Crit Care Med. 2005;6(6):676-81, http://dx.doi.org/ 10.1097/01.PCC.0000165562.04157.DA. 6. Ratcliff SL, Brown A, Rosenberg L, Rosenberg M, Robert RS, Cuervo LJ, et al. The effectiveness of a pain and anxiety protocol to treat the acute pediatric burn patient. Burns 2006;32(5):554-62, http://dx.doi.org/ 10.1016/j.burns.2005.12.006.

7. Babcock GF, Hernandez L, Yadav E, Schwemberger S, Dugan A. The burn wound inflammatory response is influenced by midazolam. Inflammation.

2012;35(1):259-70, http://dx.doi.org/10.1007/s10753-011-9313-9.

8. Dugan AL, Gregerson KA, Neely A, Gardner J, Noel GJ, Babcock GF, et al. Mice treated with a benzodiazepine had an improved survival rate following Pseudomonas aeruginosa infection. J Burn Care Res. 2010;31(1):1-12, http://dx.doi.org/10.1097/BCR.0b013e3181cb8e82.

9. McGhee LL, Maani CV, Garza TH, DeSocio PA, Gaylord KM, Black IH. The relationship of intravenous midazolam and posttraumatic stress disorder development in burned soldiers. The Journal of trauma.

2009;66(4 Suppl):186-90.

10. Gouin JP, Kiecolt-Glaser JK. The impact of psychological stress on wound healing: methods and mechanisms. Immunol Allergy Clin North Am. 2011;31(1):81-93, http://dx.doi.org/10.1016/j.iac.2010.09.010.

11. Heijnen CJ, Kavelaars A. The contribution of neuroendocrine substances to the immune response. Neth J Med. 1991;39(3-4):281-94.

12. Rostenberg A Jr. The role of psychogenic factors in skin diseases. Arch Dermatol. 1960;81:81-6, http://dx.doi.org/10.1001/archderm.1960.03730 010085010.

13. Tausk FA, Nousari H. Stress and the skin. Arch Dermatol. 2001; 137(1):78-82.

14. Sugarman JL, Fluhr JW, Fowler AJ, Bruckner T, Diepgen TL, Williams ML. The objective severity assessment of atopic dermatitis score: an objective measure using permeability barrier function and stratum corneum hydration with computer-assisted estimates for extent of disease. Arch Dermatol. 2003;139(11):1417-22, http://dx.doi.org/ 10.1001/archderm.139.11.1417.

15. Altemus M, Rao B, Dhabhar FS, Ding W, Granstein RD. Stress-induced changes in skin barrier function in healthy women. J Invest Dermatol.

2001;117(2):309-17, http://dx.doi.org/10.1046/j.1523-1747.2001.01373.x.

16. Stahn C, Buttgereit F. Genomic and nongenomic effects of glucocorticoids. Nat Clin Pract Rheumatol. 2008;4:525-33, http://dx.doi.org/ 10.1038/ncprheum0898.

17. Padgett DA, Marucha PT, Sheridan JF. Restraint stress slows cutaneous wound healing in mice. Brain Behav Immun. 1998;12(1):64-73, http:// dx.doi.org/10.1006/brbi.1997.0512.

18. Kronholm E, Partonen T, Laatikainen T, Peltonen M, Ha¨rma¨ M, Hublin C, et al. Trends in self-reported sleep duration and insomnia-related

available as a therapeutic option for patients with anxiety.

Medications such as buspirone, tricyclic antidepressants, and beta-blockers may be employed. Additionally, the effects of these medications on sleep differ from the effects of BZD; thus, they may reduce the negative effects on healing. This difference is particularly important when considering sleep deprivation and especially REM sleep deprivation. In these cases, the use of non-BZD hypnotics may be an option.

The physiological integrity of the skin is essential for the health of living creatures. Sleep is also a biological process that is fundamental for the quality of an individual’s mental, emotional and physical life. Thus, our hypothesis combines the interaction of BZD with sleep and the possible influence on the healing process. However, additional studies, conducted in animals and humans, are necessary to test this hypothesis. This hypothesis opens opportunities for new discussions regarding this theme.

ACKNOWLEDGEMENTS This work was supported by grants from Associac¸a˜o Fundo de Incentivo a` Pesquisa (AFIP), CNPq, and Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sao Paulo-FAPESP (No. 2011/14462-0 to F.E., No. #2010/14768-0 to G.N.P and CEPID #98/14303-3 to S.T.). M.L.A. and S.T. are recipients of CNPq fellowships.

AUTHOR CONTRIBUTIONS Egydio F contributed to the idea conception, the bibliographic review, the writing, and the final approval. Pires GN contributed to the idea conception, the writing, and the final approval. Tufik S contributed to the idea conception and the final approval. Andersen ML contributed to the idea conception, the writing, and the final approval.

REFERENCES 1. Guo S, Dipietro LA. Factors affecting wound healing. J Dent Res. 2010; 89(3):219-29.

2. Kahan V, Andersen ML, Tomimori J, Tufik S. Stress, immunity and skin collagen integrity: evidence from animal models and clinical conditions.

Brain Behav Immun. 2009;23(8):1089-95, http://dx.doi.org/10.1016/ j.bbi.2009.06.002.

829

Burns and benzodiazepines Egydio F et al.

19.

20.

21.

22.

23.

24.

25.

26.

CLINICS 2012;67(7):827-830

symptoms in Finland from 1972 to 2005: a comparative review and reanalysis of Finnish population samples. J Sleep Res. 2008;17(1):54-62, http://dx.doi.org/10.1111/j.1365-2869.2008.00627.x.

Palma BD, Gabriel A Jr, Colugnati FA, Tufik S. Effects of sleep deprivation on the development of autoimmune disease in an experimental model of systemic lupus erythematosus. Am J Physiol Regul Integr Comp Physiol. 2006;291(5):1527-32, http://dx.doi.org/10.1152/ ajpregu.00186.2006.

Perry JC, D’Almeida V, Souza FG, Schoorlemmer GH, Colombari E, Tufik S. Consequences of subchronic and chronic exposure to intermittent hypoxia and sleep deprivation on cardiovascular risk factors in rats. Respir Physiol Neurobiol. 2007;156(3):250-8, http://dx.doi.org/ 10.1016/j.resp.2006.10.004.

Zager A, Andersen ML, Ruiz FS, Antunes IB, Tufik S. Effects of acute and chronic sleep loss on immune modulation of rats. Am J Physiol Regul Integr Comp Physiol. 2007;293(1):R504-9, http://dx.doi.org/10.1152/ ajpregu.00105.2007.

Zager A, Andersen ML, Lima MM, Reksidler AB, Machado RB, Tufik S. Modulation of sickness behavior by sleep: the role of neurochemical and neuroinflammatory pathways in mice. Eur Neuropsychopharmacol.

2009;19(8):589-602, http://dx.doi.org/10.1016/j.euroneuro.2009.03.005.

Ruiz FS, Andersen ML, Zager A, Martins RC, Tufik S. Sleep deprivation reduces the lymphocyte count in a non-obese mouse model of type 1 diabetes mellitus. Braz J Med Biol Res. 2007;40(5):633-7, http:// dx.doi.org/10.1590/S0100-879X2006005000086.

Ruiz FS, Andersen ML, Martins RC, Zager A, Lopes JD, Tufik S. Immune alterations after selective rapid eye movement or total sleep deprivation in healthy male volunteers. Innate Immun. 2012;18(1):44-54, http:// dx.doi.org/10.1177/1753425910385962.

Irwin M, Mascovich A, Gillin JC, Willoughby R, Pike J, Smith TL. Partial sleep deprivation reduces natural killer cell activity in humans.

Psychosomatic medicine. 1994;56(6):493-8.

Zavala F, Taupin V, Descamps-Latscha B. In vivo treatment with benzodiazepines inhibits murine phagocyte oxidative metabolism and production of interleukin 1, tumor necrosis factor and interleukin-6.

J Pharmacol Exp Ther. 1990;255(2):442-50.

27. Schreiber AA, Frei K, Lichtensteiger W, Schlumpf M. The effect of prenatal diazepam exposure on TNF-alpha production by rat splenocytes. Agents and actions. 1993;38(3-4):265-72, http://dx.doi.org/10.1007/BF01976219.

28. Sakai M, Fonseca ES, Dagli ML, Palermo-Neto J. Diazepam effects on Ehrlich tumor growth and macrophage activity in mice. Life sciences.

2006;78(16):1777-83, http://dx.doi.org/10.1016/j.lfs.2005.08.015.

29. Roehrs R RT. Hypnotics: Efficacy and adverse events. In: Kryger TR MH, Dement eWC, ed. Principles and practices of sleep medicine. New York: 3rd ed; 2000:pp.414-8.

30. Xi MC, Morales FR, Chase MH. Evidence that wakefulness and REM sleep are controlled by a GABAergic pontine mechanism. J Neurophysiol.

1999;82(4):2015-9.

31. Marks GA, Sachs OW, Birabil CG. Blockade of GABA, type A, receptors in the rat pontine reticular formation induces rapid eye movement sleep that is dependent upon the cholinergic system. Neuroscience.

2008;156(1):1-10, http://dx.doi.org/10.1016/j.neuroscience.2008.06.067.

32. Mostaghimi L, Obermeyer WH, Ballamudi B, Martinez-Gonzalez D, Benca RM. Effects of sleep deprivation on wound healing. J Sleep Res. 2005;14(3):213-9, http://dx.doi.org/10.1111/j.1365-2869.2005.00455.x.

33. Landis CA, Whitney JD. Effects of 72 hours sleep deprivation on wound healing in the rat. Res Nurs Health. 1997;20(3):259-67, http://dx.doi.org/ 10.1002/(SICI)1098-240X(199706)20:3,259::AID-NUR8.3.0.CO;2-H. 34. Barrientos S, Stojadinovic O, Golinko MS, Brem H, Tomic-Canic M. Growth factors and cytokines in wound healing. Wound Repair Regen.

2008;16(5):585-601, http://dx.doi.org/10.1111/j.1524-475X.2008.00410.x.

35. Kiecolt-Glaser JK, Glaser R. Psychological stress and wound healing: Kiecolt-Glaser, et al. (1995). Adv Mind Body Med. 2001;17(1):15-6, http://dx.doi.org/10.1054/ambm.2000.0248.

36. Goyal R, Anil K. Protective effect of alprazolam in acute immobilization stress-induced certain behavioral and biochemical alterations in mice.

Pharmacological reports. 2007;59(3):284-90.

37. Lechin F, van der Dijs B, Vitelli-Flores G, Ba´ez S, Lechin ME, Lechin AE, et al. Peripheral blood immunological parameters in long-term benzodiazepine users. Clin Neuropharmacol. 1994;17(1):63-72, http:// dx.doi.org/10.1097/00002826-199402000-00007.

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DOI:10.6061/clinics/2012(07)21

REVIEW

Clinical pharmacokinetics of vancomycin in the neonate: a review Gian Maria Pacifici,I Karel AllegaertII I University of Pisa, Medical School, Department of Neurosciences, Section of Pharmacology, Pisa/IT. II Neonatal Intensive Care Unit, University Hospitals Leuven, Herestraat 49, 3000 Leuven, Belgium.

Neonatal sepsis is common and is a major cause of morbidity and mortality. Vancomycin is the preferred treatment of several neonatal staphylococcal infections. The aim of this study was to review published data on vancomycin pharmacokinetics in neonates and to provide a critical analysis of the literature. A bibliographic search was performed using PubMed and Embase, and articles with a publication date of August 2011 or earlier were included in the analysis. Vancomycin pharmacokinetic estimates, which are different in neonates compared with adults, also exhibit extensive inter-neonatal variability. In neonates, several vancomycin dosing schedules have been proposed, mainly based on age (i.e., postmenstrual and postnatal), body weight or serum creatinine level. Other covariates [e.g., extracorporeal membrane oxygenation (ECMO), indomethacin or ibuprofen, and growth restriction] of vancomycin pharmacokinetics have been reported in neonates. Finally, vancomycin penetrates cerebrospinal fluid (range = 7-42%). Renal function drives vancomycin pharmacokinetics. Because either age or weight is the most relevant covariate of renal maturation, these covariates should be considered first in neonatal vancomycin dosing guidelines and further adjusted by renal dysfunction indicators (e.g., ECMO and ibuprofen/indomethacin). In addition to the prospective validation of available dosing guidelines, future studies should focus on the relevance of therapeutic drug monitoring and on the value of continuous vancomycin administration in neonates.

KEYWORDS: Pharmacokinetics; Vancomycin; Neonate; Developmental pharmacology; Covariates.

Pacifici GM, Allegaert K. Clinical pharmacokinetics of vancomycin in the neonate: a review. Clinics. 2012;67(7):831-837.

Received for publication on November 22, 2011; First review completed on January 11, 2012; Accepted for publication on March 5, 2012 E-mail: pacifici@biomed.unipi.it Tel.: 39 050-2218721

and ,10 mg/ml, respectively, to both be effective and avoid oto- or nephrotoxicity in adults (6,7,8). Because vancomycin activity is primarily time-dependent, the 24-h area under the curve (AUC0-24h) divided by the minimum inhibitor concentration (MIC) value (AUC0-24h/MIC) is a better predictor of efficacy. In adults with MIC values less than 1 mg/ml, trough concentrations greater than 10 mg/ml result in AUC0-24h/MIC values .400.

In subsequent analyses addressing clinical pharmacology, it is important to note that vancomycin is water-soluble, has a limited plasma protein binding capacity (i.e., IgA and albumin) and is mainly eliminated renally by glomerular filtration, although its elimination is further modulated by renal tubular transport (4). Compared with adults, neonates have a higher extracellular fluid volume and a limited renal elimination capacity (4,9). Consequently, vancomycin pharmacokinetics are different in neonates and undergo significant alterations as neonates mature. We aimed to provide a summary of published observations on vancomycin pharmacokinetics in neonates and a subsequent discussion on areas of further research.

INTRODUCTION Neonates have a higher risk of nosocomial infections due to humoral, cellular and myeloid cell functions combined with the use of invasive techniques (e.g., central venous catheter and endotracheal intubation) (1). Because neonatal bacterial sepsis has mortality rates of 10-20% and causes morbidity in 25-30% of survivors (2,3), effective and tolerated antimicrobiologic treatment regimens are required. Coagulasenegative Staphylococci and Staphylococcus aureus are common nosocomial bacterial pathogens, and vancomycin is the first-choice antibiotic to treat these pathogens, especially in methicillin-resistance settings (4). Vancomycin is active against gram-positive bacteria. Staphylococcus epidermis, including methicillin-resistant strains, are inhibited by vancomycin concentrations of 1-4 mg/ml. Staphylococcus pyogenes, Streptococcus pneumonia, and Streptococcus viridans are susceptible to 2 mg/ml vancomycin. Bacillus spp. are inhibited by 2 mg/ml, Corynebacterium spp. by 0.04-3.1 mg/ml and Clostridium spp. by 0.39-6 mg/ml vancomycin (5). Peak and trough concentrations of vancomycin should be ,40 mg/ml

BIBLIOGRAPHIC SEARCH

Copyright ß 2012 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.

The bibliographic search was performed electronically using PubMed and Embase. Searches were performed with the following keywords: "pharmacokinetics vancomycin neonate’’; ‘‘continuous infusion vancomycin neonate’’;

No potential conflict of interest was reported.

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CLINICS 2012;67(7):831-837

Covariates that reflect maturation: creatinine, age and/or weight

Table 1 - Vancomycin dosage guidelines developed by Grimsley and Thomson (10).

Serum creatinine concentration (mmol/l) 20-29 30-39 40-49 50-59 60-79 80-100 .100

Vancomycin (mg/kg) 20 20 15 12 15 15 15

Vancomycin is almost exclusively cleared by renal glomerular filtration because renal tubular functions are still immature in neonates (4,9). Either postmenstrual age or weight is a covariate of vancomycin clearance, presumably because these covariates serve as indicators of glomerular filtration rate maturation (28). Vancomycin clearance is positively affected by weight, gestational age, postnatal age and postmenstrual age and negatively affected by creatinine levels (10-28). However, these covariates are interrelated because postmenstrual age is the sum of gestational and postnatal ages, whereas the correlation between weight and age is self-evident. Finally, a progressive decline in creatinine levels is associated with an increase in age (Tables 1-4).

Interval (h) 8 12 12 12 18 24 Check trough level at 24 h

‘‘ECMO vancomycin neonate’’; ‘‘indomethacin vancomycin neonate’’; ‘‘ibuprofen vancomycin neonate’’; ‘‘vancomycin penetration CSF neonate’’ and ‘‘vancomycin toxicity neonate’’ with the limit of ‘‘human’’. All studies published in August 2011 or earlier were included in the analysis. The bibliography of each article was examined, and selected articles were read carefully. In addition, the book Neofax: a Manual of Drugs Used in the Neonatal Care, by Young and Mangum (8), was consulted. The bibliographic search resulted in 60 original articles, 10 review articles, and two book chapters; the publication year range was from 1961 to 2011.

Covariates reflect comorbidity characteristics The level of creatinine does not only reflect maturational changes, but it may also in part reflect renal impairment. At present, the use of neonatal creatinine levels to predict renal function is controversial due to residual maternal creatinine and bio-analytical issues (see the discussion section). Other comorbidity characteristics that affect vancomycin clearance are related to renal impairment, which are either primarily caused by a drug-related reduction in renal clearance or a lower endogenous renal clearance capacity or secondarily caused by a disease-related decrease in renal function (Table 4).

As documented by Frattarelli et al., growth restriction affects vancomycin clearance capacity (17) in neonates (,28 days old), but this reduction in vancomycin clearance is limited (16-20%) and related to the reduced renal elimination capacity of these neonates (19,29).

The association between patent ductus arteriosus (PDA) and reduced vancomycin clearance is likely related to pharmacological treatment with either ibuprofen or indomethacin. Either indomethacin or ibuprofen is administered to induce PDA closure. Asbury et al. (26, Table 4) quantified the impact of indomethacin on vancomycin clearance.

Basically, indomethacin administration for PDA closure reduced vancomycin clearance by 50% (26). Allegaert (30) aimed to quantify the impact of indomethacin or ibuprofen on neonatal vancomycin clearance. Allegaert et al. confirmed the effect of indomethacin on vancomycin clearance (-40%), whereas ibuprofen reduced clearance by 28%.

Therefore, indomethacin has a much higher effect on vancomycin clearance compared with ibuprofen (30,31).

Seay et al. (21) reported on the population pharmacokinetics of vancomycin in 192 patients and documented that dopamine reduced vancomycin clearance (Table 4). In contrast, when dopamine was evaluated as one of the covariates of

RESULTS Seventy-two studies were considered in this review.

Table 1 summarizes vancomycin dose guidelines based on serum creatinine levels (10). Table 2 provides two neonatal vancomycin-dosing strategies that are based on either postmenstrual age or serum creatinine levels (11). Table 3 provides dosage guidelines based on postmenstrual age and weight (12). Finally, Table 4 summarizes demographic and pharmacokinetic parameters of vancomycin in neonates as reported in the literature (12-27).

Median estimates of vancomycin pharmacokinetics in neonates Compared with pharmacokinetic estimates in adults, the median distribution volume is higher in neonates, and the highest estimates are in the most preterm neonates (Table 4).

In contrast, vancomycin clearance is significantly lower in neonates compared with adults after correcting for body surface area (27). When expressed in ml/min/kg, the clearance estimates are lowest in the most preterm neonates (0.98 ml/min/kg) (15). In addition, Table 4 illustrates that there is at least a 2- to 3-fold difference in vancomycin clearance within the neonatal age range, which in part reflects maturation and other renal functions that are related to co-morbidity characteristics in neonates (12-27).

Table 2 - Two neonatal vancomycin dosing strategies (postnatal age ,60 days) developed by Capparelli et al. (11).

Postmenstrual age (PMA) PMA (weeks) #29 30-33 34-37 38-44 $45

Serum creatinine concentration (Scr) Dose (mg/kg)

Interval (h)

Scr (mg/dl)

Dose (mg/kg)

Interval (h)

20 20 20 15 10

24 18 12 8 6

$1.7 1.3-1.6 1.0-1.2 0.7-0.9 #0.6

15 10 15 20 15

48 24 24 24 12

832

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Pharmacokinetics of vancomycin in the neonate Pacifici GM and Allegaert K

4 mg/day intraventricularly for 30 days. The vancomycin CSF concentration range was 9.5-55.3 mg/ml.

Table 3 - Initial vancomycin dosage guidelines in neonates based on postmenstrual age (PMA) or weight, developed by McDougal et al. (12).

PMA (weeks)

Weight (g)

Dose (mg/kg)

Interval (h)

,27 27-30 31-36 $37

,800 800-1200 1200-2000 .2000

18 16 18 15

36 24 18 12

DISCUSSION Neonatal morbidity and mortality due to late-onset sepsis caused by coagulase-negative staphylococci or methicillinresistant staphylococci has resulted in an increased necessity for the effective and safe administration of vancomycin in neonates. In the 1950s, Staphylococcus strains developed resistance to treatment with penicillin. Vancomycin arose as the treatment of choice for staphylococcal infections (34), and its use declined in the 1960s when methicillin was preferred because of its safer toxicity profile compared with vancomycin (35). In 1978, the use of vancomycin resurfaced as Streptococcus species developed further resistance to cephalosporins and other penicillins (35).

At present, vancomycin is the current treatment of choice for many neonatal staphylococcal infections. There are, however, no reports yet on the association between indices (i.e., peak, trough, and AUC0-24h) of vancomycin exposure and either bactericidal effects or nephro- or ototoxicity in neonates. Consequently, the target AUC0-24h/MIC index ($400) of vancomycin, which is documented in adults, is used (36-39). Therefore, similar to a trough concentration of 10 mg/ml of vancomycin, the AUC0-24h/MIC target should be considered for any dosage recommendation to minimize treatment failure in neonates. Lustar and Metsvaht (38) suggested that for mid-moderate neonatal infections, the AUC0-24h/MIC ratio should be $400, and for severe infections in immunocompromised hosts, such as cases of ventilatorassociated pneumonia sustained by Staphylococcus aureus, this ratio should be $850. Staphylococcus warneri (40), which has reduced glycopeptide susceptibility (MIC.2 mg/ml), also requires a higher vancomycin exposure.

Obviously, this target necessitates the integration of the most relevant vancomycin pharmacokinetic covariates in neonatal dosing strategies. Van den Anker (39) stated that the most important dosing strategies in neonates are based on a combination of postmenstrual and postnatal ages, which considers the known changes in body composition and renal function with a higher dose for the treatment of neonatal meningitis. Such strategies have been suggested or evaluated by different groups.

vancomycin clearance in a data set of 214 preterm neonates, size, renal function and PMA, but neither dopamine nor respiratory support, were major contributors to clearance variability in premature neonates, which caused 18% of the variability to be unexplained (28). Extra-corporeal membrane oxygenation (ECMO) provides cardiopulmonary support for patients with potentially reversible respiratory and cardiac failure. Vancomycin clearance is decreased and its distribution volume is increased in neonates on ECMO (Table 4) (24,25). Finally, an interaction at the level of competitive binding or inhibition of renal tubular transport processes likely explains the increase in vancomycin clearance during amoxicillin-clavulanic acid co-administration in a cohort of 70 neonates (22).

Cerebrospinal fluid (CSF) pharmacokinetics of vancomycin Little is known concerning the penetration of vancomycin in neonatal CSF. Reiter and Doron (32) reported CSF concentrations in three vancomycin-treated neonates. The case 1 subject was a 1264-g male who was delivered at 28 weeks of gestation, the case 2 subject was a 670-g male who was delivered at 31 weeks of gestation, and the case 3 subject was a 1150-g female who was delivered at 26 weeks of gestation. These three case subjects were treated with vancomycin at a daily dose of 20 mg/kg for 14 days. The article by Reiter and Doron (32) includes additional clinical details. In case 1, the vancomycin CSF concentration was 5.6 mg/ml at 17.25 h after infusion on day 8 of therapy, and the CSF penetration rate, which was measured by the CSF to serum vancomycin concentration ratio, was 32%. In case 2, the vancomycin CSF concentration was 2.2 mg/ml at 10.7 h after intravenous administration on day 2 of the vancomycin therapy, and the CSF penetration rate of vancomycin was 26%. Patient 3 had a vancomycin CSF concentration of 5.5 mg/ml at 23 h after infusion on day 9 of the vancomycin therapy, and the CSF penetration rate was 42%. Schaad et al.

(27) (Table 4) administered 10-15 mg of vancomycin intravenously to three neonates whose gestational age and body weight were unreported. The vancomycin CSF concentration range was 1.2-4.8 mg/ml (mean = 3.1 mg/ml), and the CSF penetration rate of vancomycin ranged from 721% (mean = 14%). Finally, Pau et al. (33) reported the cases of two newborns who had received vancomycin intravenously and intra-ventricularly. The case 1 subject was a 1,280-g male who was born at 29 weeks of gestation and suffered from several diseases. This patient received the following vancomycin treatments: 50 mg/kg/day intravenously and 5-10 mg intra-ventricularly. The duration of vancomycin therapy was 26 days, and the vancomycin CSF concentration range was 12.7-92.1 mg/ml. Patient 2 was a female who was born at 31 weeks of gestation with multiple congenital anomalies. This neonate received the following vancomycin treatments: 60 mg/kg/day intravenously and

Dosing guideline performance Young and Mangum (8) suggested a vancomycin dosage of 15 mg/kg every 12 h in meningitis patients and 10 mg/ kg every 12 h for the treatment of bacteremia. When the postmenstrual age is #29 weeks, the vancomycin dose should be administered every 12 or 18 h according to the postnatal age (.14 days postnatal age or younger). When the postmenstrual age range is 30-44 weeks, vancomycin should be administered every 8 or 12 h according to the postnatal age. When the postmenstrual age is $45 weeks, vancomycin should be administered every 6 h. This dosing guideline aims for a trough concentration between 5-10 mg/ ml. However, Badran et al. (41) recently illustrated that 51% of the patients in their neonatal unit attained the desired therapeutic trough concentration, and the trough concentration was ,5 mg/ml in 33% of the neonates when these guidelines were utilized.

To further illustrate the difficulties of effective dosing guideline implementation, we refer to the work of de Hoog

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CLINICS 2012;67(7):831-837

Table 4 - Neonatal demographic data and pharmacokinetic parameters of vancomycin therapy. The results are presented as the mean¡standard deviation unless otherwise stated.

Comments First dose Steady state Group 1 Group 2 Group 3 Preterm Group 1 Group 2 Preterm SGA AGA GA 29 wks

GA weeks

PNA days

Weight g

28.4 20.5 1069 SD 2.6 SD 10.4 SD 435 The values refer to the 26-case population 26.6 18 908 SD 1.7 SD 8 SD 254 29.4 23 1194 SD 2.0 SD 14 SD 412 35.9 24 2405 SD 3.7 SD 18 SD 956 25.9 21a 769 SD 1.3 SD 151 29.5 15.3 1232 SD 1.1 SD 5.9 SD 263 30.1 26.0 1289 SD 1.9 SD 14.2 SD 265 a 26.5 71 880 SD 2.6 SD 340 27.3 48 1140 SD 2.5 SD 38 SD 590 26.5 38 1535 SD 1.9 SD 47 SD 986 29.3 29 1297 SD 3.4 SD 20 SD 640 30 .90% 1000 SD 2.4 , 2 wks SD 840 28.9c 14c 1045c

Population kinetics Population kinetics Population 29.5 kinetics SD 5.1 Population 32.2 kinetics SD 5.0 Preterm and full- 31.0 term SD 7.3 ECMO 38.8 SD 2.2 Controls 39.7 SD 1.3 ECMO 39 SD 1.4 Controls 29.3 SD 4.2 PDA 26.0 (indo) SD 1.2 Preterm 32a

Dose mg/kg

Cl ml/min/kg

Vd l/kg

1.22 SD 0.7 1.16 SD 0.6 1.1 SD 0.07 1.19 SD 0.08 1.36 SD 0.85 0.8b 0.98a

0.53 SD 0.13 0.52 SD 0.10 0.55 SD 0.02 0.56 SD 0.02 0.57 SD 0.02 0.63 SD 0.025 0.85a

6.0 SD 2.0 6.6 SD 2.1 6.6 SD 0.35 5.6 SD 0.36 4.9 SD 0.4 10 hb

13

12.6 q24 h 12.6 q24 h 16 q24 h 18 q18 h 15 q12 h 14.2 SD 3.2 Note A

12

Note A

0.89a

20

Note B

1.2b

20

n.a.

1.17a

123

n.a.

a

1.84

15

Note C

116 108

n 15 +11 16 15 13 12

16.5 SD 19.6 16.9 SD 10.9 9.1 SD 4.9 12.7 SD 5.1 7.7 SD 2.3 1.6 SD 1.7 33.9 SD 19.9 17.6 SD 7.1 3.3a

1492 1053 1700 SD 800 1620 SD 550 3100 SD 600 3400 SD 400 3300 SD 430 1780 SD 1080 810 SD 160 1530a

192 70 11 15

Peak mg/ml

Trough mg/ml Reference

11.3a

31.2 SD 12 46.4 SD 15 28.1 SD 1.2 27.9 SD 1.2 26.1 SD 1.3 32.6 SD 9.3 23.6a

9.5 SD 3.5 19.4 SD 9.2 3.0 SD 0.6 3.9 SD 0.6 5.5 SD 0.7 5.7 SD 4.5 10.5a

0.56a

8.7a

2 0.6a

9.9a

6.66b

n.a.

n.a.

10

7.7 SD 4.9 8.

0SD 6.4 5.6 SD 1.6 8.5a

31.5 SD 8.3 30.6 SD 10.7 22.6 SD 3.6 n.a.

6.8 SD 4.2 8.3 SD 6.4 7.6 SD 2.0 n.a.

17

1.07 SD 0.34 2.6a

0.68 SD 0.15 0.52 SD 0.26 0.72 SD 0.45 0.48 SD 0.09 0.52a

15 q12 h Note G

3.4 SD 0.11 3.5a

0.43 SD .013 0.76a

34.3 SD 7.7 n.a.

8.2 SD 2.2 n.a.

20

15 q8-24 Note F

3.96a

0.57a

6.0 SD0.27 13.4-33.7 (range) 6.3a

7.5a

,20

22

0.74 SD 0.20 0.65 SD 0.28 0.79 SD 0.41 0.78 SD 0.19 1.22 SD 0.54 0.61 SD 0.17 15 ml/min/ 1.73 m2 27 ml/min/ 1.73 m2 30 ml/min/ 1.73 m2

0.51 SD 0.03 0.45 SD 0.18 0.39 SD 0.12 1.1 SD 0.5 0.52 SD 0.08 0.57 SD 0.06 0.74a

7.2 SD 2.9 8.3 SD 2.2 6.5 SD 2.1 16.9 SD 9.5 5.6 SD 1.2 11.9 SD 3.3 9.8a

28.0 SD 5.4 27 SD 4.3 23.0 SD 5.4 30a

4.9 SD 2.0 13.7 SD 2.7 13.2 SD 4.5 5- 10

23

27.8 SD 0.7 27.9 SD 0.4 16.8a

7.5 SD 1.2 8.5 SD 0.7 5.7a

0.71a

5.9a

25.2a

n.a.

0.69a

6.7a

29.8a

n.a.

12

10 q8 h 10 q8 h 15-20

19

Note D

4

Note E

7

10 q24 h

15

Preterm

34a

4.7a

1570a

7

15 q24 h

Term

40a

2.6a

3070a

7

15 q24 h

tK h

13

12

14 15 16

18 19

21

24

25 26

27

GA = gestational age; PNA = postnatal age; n = number of cases; SGA = small for gestational age; AGA = appropriate for gestational age; ECMO = extracorporeal membrane oxygenation; PDA = patent ductus arteriosus; n.a. = not available. a Mean, SD is not available; bExtracted figure; cMedian.

Note A: The vancomycin doses were 10, 15, or 20 mg/kg.

Note B: The vancomycin dose range was 9.2-18 mg/kg.

Note C: The loading dose was 15¡0.2 mg/kg, and the maintenance dose was 14.8¡4.3 mg/kg.

Note D: The loading dose was 15¡0.2 mg/kg, and the maintenance dose was 29.6¡13.1 mg/kg.

Note E: The loading dose was 15 mg/kg, and the maintenance dose was 14.8¡4. mg/kg.

Note F: The dose range was 12.1-13.8 mg/kg (12.6¡0.9 mg/kg), and the dosing interval range was 13-40 h (22.0¡7.5 h).

Note G: The dosing was initiated at 7.5, 10, 12, or 15 mg/kg, and the interval range was from q8 h to q24 h.

et al. (20). This group aimed to determine the best therapeutic dose for the treatment of vancomycin-sensitive bacterial infections in 108 neonates. The median gestational and

postnatal ages were 28.9 weeks and 14 days, respectively, and the median body weight was 1,045 g (Table 4). The vancomycin dose was 15 mg/kg every 12 h. Clearance was

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currently observed. After eliminating impurities from the early preparations of vancomycin, the incidence of primary adverse effects, such as nephrotoxicity, ototoxicity and ‘‘red man syndrome’’, was reduced (46). The most important risk factors for developing nephrotoxicity are the following: trough concentrations .10 mg/ml, concomitant treatment with aminoglycosides and/or prolonged therapy (.21 days) (47,48). Other risk factors include high peak concentrations, high total dose, preexisting renal failure, and concurrent treatment with amphotericin and/or furosemide. However, the role of these factors in the neonatal population is not well-established (49). Proper vancomycin TDM minimized both glomerular and tubular nephrotoxicity in two studies in children and neonates (8,39,50,51). In most cases, nephrotoxicity is reversible, even after high doses (52). In contrast, there is no proven association between TDM and ototoxicity prevention (39).

0.057¡0.0018 l/h/kg (variability = 31%); the distribution volume was 0.43¡0.013 l/kg (variability = 25%), and tK was 6¡0.27 h (variability = 34%). Using this dosage (i.e., 15 mg/kg every 12 h), 95.5% of the initial trough concentration measurements were in the desired therapeutic range.

Vancomycin trough concentrations before the second dose were 8.2¡2.2 mg/ml. Trough concentrations before the fifth dose were 12.3¡4.1 mg/ml. Only one trough concentration measurement was below 5 mg/ml. The vancomycin trough concentration range before the fifth dose was 15.3-20.6 mg/ ml. Vancomycin accumulates in serum; accordingly, after the fifth dose, the peak concentration range was 16.6-34.5 mg/ml (mean = 25.8¡5.0 mg/ml). The vancomycin trough concentration range was 5-15 mg/ml, and peak concentrations were ,40 mg/ml (39) (Table 4). The authors suggested that a vancomycin dosage of 3610 mg/kg per day leads to a minimized high peak serum concentration and a trough serum concentration that is too low. However, this assertion was derived from a prospective validation study of 22 neonates in the same unit. However, these results are concomitant with the internal (same unit) prospective validation efforts of Seay et al. (n = 20) and MarquesMinana et al. (n = 41) (21,22).

Improving neonatal vancomycin effectiveness and tolerance Despite the number of reported pharmacokinetic studies on vancomycin in neonates, there are still relevant issues that require further consideration. First, covariates differ between the different studies, and the prospective validation of dosing guidelines is limited but urgently required to improve the predictability and extrapolation of dosing guidelines. This may be partially related to analytical issues specific to quantifying vancomycin or creatinine concentrations. Immunoassays that are routinely used to quantify vancomycin may overestimate vancomycin concentrations because of the cross-reactivity with some of the vancomycin degradation products or endogenous compounds, such as bilirubin (53). The introduction of a more precise analytical method, such as liquid chromatography-tandem mass spectroscopy, should be considered. Creatinine levels initially increase and are most pronounced in the most immature neonates (54). Postnatal observations also depend on techniques used to quantify creatinine levels (i.e., Jaffe colorimetry, compensated Jaffe quantification or enzymatic quantification). It is generally accepted that the Jaffe quantification method overestimates creatinine concentrations because of bilirubin and cephalosporin interference.

This observation also indicates that the age- or weightdependent creatinemia reference values will differ based on the quantification method used (55). Until recently, none of the studies in the literature explicitly discussed creatinine quantification methods.

Second, the external prospective validation of vancomycin dosing guidelines is only a first step to improving the efficacy and safety/tolerance of vancomycin. The AUC0-24h/ MIC ratio is derived from extrapolated data in adults and necessitates prospective validation in neonates that includes an analysis of the potential add-on value of continuous administration with or without a loading dose (39). Finally, data in specific settings, such as ECMO or meningitis, are limited and warrant a focused approach (56). Until these data emerge, there are no well-validated dosing guidelines that can be used routinely throughout the world. Therefore, we strongly recommend that neonatal units prospectively validate one of the dosing guidelines provided (Tables 1-4).

Local validation efforts also enable caregivers to consider the local MIC values of the isolated pathogens.

In conclusion, neonatal vancomycin pharmacokinetic data were reviewed, which was followed by a critical analysis of

Intermittent vs. continuous vancomycin administration Because the AUC concept aims to maintain the vancomycin concentration above a given threshold, it has been suggested that vancomycin can be administered as a continuous infusion (42-45). Plan et al. (42) administered vancomycin as a continuous infusion of 15-25 mg/kg/day or 20-30 mg/kg/day to 145 neonates. The body weight range of the neonates was 500-1,160 g, and their gestational age range was 26-29 weeks. Using the abovementioned dosage, bactericidal efficacy was maintained, and most subjects had serum vancomycin concentrations within the therapeutic range. Pawlotsky et al. (43) administered vancomycin as a 24-h constant infusion to two neonatal groups. Group 1 (n = 24) had a postmenstrual age range of 27-41 weeks and received 10-30 mg/kg/day according to the postmenstrual age. Group 2 (n = 29) had a postmenstrual age range of 28-51.5 weeks and received a loading dose of 7 mg/kg, followed by a continuous infusion of 10-40 mg/ kg/day according to the postmenstrual age. The mean vancomycin serum concentrations at steady state were 11.0¡3.1 and 15.4¡6.2 mg/ml in Groups 1 and 2, respectively. Both regimens were well tolerated. Oudin et al. (44) confirmed the feasibility of this approach (i.e., a 7-mg/kg loading dose and constant infusion of 30 mg/kg/day) but suggested increasing the initial loading dose to 20 mg/kg, which resulted in an exposure similar to that observed by Pawlotsky et al. (43).

The use and limitations of therapeutic drug monitoring All pharmacokinetic studies demonstrate variability, which is only in part explained by weight, age, or creatinine level. This variability explains the use of therapeutic drug monitoring (TDM) of trough concentrations to ensure effectiveness and avoid nephrotoxicity. In contrast, the quantification of peak concentrations provides no additional monitoring value (4,8,39). Vancomycin-induced nephrotoxicity and ototoxicity were previously more common than is

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15. Machado JK, Feferbaum R, Kobayashi CE, Sanches C, Santos SR. Vancomycin pharmacokinetics in preterm infants. Clinics. 2007;62(4):40510, http://dx.doi.org/10.1590/S1807-59322007000400006.

16. James A, Koren G, Milliken J, Soldin S, Prober C. Vancomycin pharmacokinetics and dose recommendations for preterm infants.

Antimicrob Agents Chemother. 1987;31(1):52-4.

17. Frattarelli DA, Ergun H, Lulic-Botica M, Lehr VT, Aranda JV. Vancomycin elimination in human infants with intrauterine growth retardation. Pediatr Infect Dis J. 2005;24(11):979-83.

18. Kildoo CW, Lin LM, Gabriel MH, Folli HL, Modanlou HD. Vancomycin pharmacokinetics in infants: relationship to postconceptional age and serum creatinine. Dev Pharmacol Ther. 1990;14(2):77-83.

19. Lo YL, van Hasselt JG, Heng SC, Lim CT, Lee TC, Charles BG. Population pharmacokinetics of vancomycin in premature Malaysian neonates: identification of predictors for dosing determination.

Antimicrob Agents Chemother. 2010;54(6):2626-32, http://dx.doi.org/ 10.1128/AAC.01370-09.

20. de Hoog M, Schoemaker RC, Mouton JW, van den Anker JN. Vancomycin population pharmacokinetics in neonates. Clin Pharmacol Ther 2000;67(4):360-7, http://dx.doi.org/10.1067/mcp.2000.105353.

21. Seay RE, Brundage RC, Jensen PD, Schilling CG, Edgren BE. Population pharmacokinetics of vancomycin in neonates. Clin Pharmacol Ther.

1994;56(2):169-75, http://dx.doi.org/10.1038/clpt.1994.120.

22. Marque´s-Min˜ana MR, Saadeddin A, Peris JE. Population pharmacokinetic analysis of vancomycin in neonates. A new proposal of initial dosage guideline. Br J Clin Pharmacol. 2010;70(5):713-20.

23. Jarrett RV, Marinkovich GA, Gayle EL, Bass JW. Individualized pharmacokinetic profiles to compute vancomycin dosage and dosing interval in preterm infants. Pediatr Infect Dis J. 1993;12(2):156-7.

24. Buck ML. Vancomycin pharmacokinetics in neonates receiving extracorporeal membrane oxygenation. Pharmacotherapy. 1998;18(5):1082-6.

25. Amaker RD, DiPiro JT, Bhatia J. Pharmacokinetics of vancomycin in critically ill infants undergoing extracorporeal membrane oxygenation.

Antimicrob Agents Chemother. 1996;40(5):1139-42.

26. Asbury WH, Darsey EH, Rose WB, Murphy JE, Buffington DE, Capers CC. Vancomycin pharmacokinetics in neonates and infants: a retrospective evaluation. Ann Pharmacother. 1993;27(4):490-6.

27. Schaad UB, McCracken GH Jr, Nelson JD. Clinical pharmacology and efficacy of vancomycin in pediatric patients. J Pediatr. 1980;96(1):119-26.

28. Anderson BJ, Allegaert K, Van den Anker JN, Cossey V, Holford NH. Vancomycin pharmacokinetics in preterm neonates and the prediction of adult clearance. Br J Clin Pharmacol. 2006;63(1):75-84.

29. Allegaert K, Anderson BJ, van den Anker JN, Vanhaesebrouck S, de Zegher F. Renal drug clearance in preterm neonates: relation to prenatal growth. Ther Drug Monit. 2007;29(3):284-91, http://dx.doi.org/10.1097/ FTD.0b013e31806db3f5.

30. Silva R, Reis E, Bispo MA, Almeida AM, Costa IM, Falca˜o F, et al. The kinetic profile of vancomycin in neonates. J Pharm Pharmacol.

1998;50(11):1255-60, http://dx.doi.org/10.1111/j.2042-7158.1998.tb03342.x.

31. Allegaert K. The impact of ibuprofen or indomethacin on renal drug clearance in neonates. J Matern Fetal Neonatal Med. 2009;22(Suppl 3):8891, http://dx.doi.org/10.1080/14767050903180965.

32. Reiter PD, Doron MW. Vancomycin cerebrospinal fluid concentrations after intravenous administration in premature infants. J Perinatol.

1996;16(5):331-5.

33. Pau AK, Smego RA Jr, Fisher MA. Intraventricular vancomycin: observations of tolerance and pharmacokinetics in two infants with ventricular shunt infections. Pediatr Infect Dis. 1986;5(1):93-6.

34. Dawson PM. Vancomycin and gentamicin in neonates: hindsight, current controversies, and forethought. J Perinat Neonatal Nurs.

2002;16(2):54-72.

35. Isaacs D. Rationing antibiotic use in neonatal units. Arch Dis Child Fetal Neonatal Ed. 2000;82(1):F1-2, http://dx.doi.org/10.1136/fn.82.1.F1.

36. Moise-Broder PA, Forrest A, Birmingham MC, Schentag JJ. Pharmacodynamics of vancomycin and other antimicrobials in patients with Staphylococcus aureus lower respiratory tract infections. Clin Pharmacokinet. 2004;43(13):925-42, http://dx.doi.org/10.2165/00003088-20044313000005.

37. Frymoyer A, Hersh AL, Benet LZ, Guglielmo BJ. Current recommended dosing of vancomycin for children with invasive methicillin-resistant Staphylococcus aureus infections is inadequate. Pediatr Infect Dis J. 2009;28(5):398-402.

38. Lutsar I, Metsvaht T. Understanding pharmacokinetics/pharmacodynamics in managing neonatal sepsis. Curr Opin Infect Dis. 2010;23(3):2017, http://dx.doi.org/10.1097/QCO.0b013e328337bb42.

39. van den Anker JN. Getting the dose of vancomycin right in the neonate.

Int J Clin Pharmacol Ther. 2011;49(4):247-9.

40. Center KJ, Reboli AC, Hubler R, Rodgers GL, Long SS. Decreased vancomycin susceptibility of coagulase-negative staphylococci in a neonatal intensive care unit: evidence of spread of Staphylococcus warneri. J Clin Microbiol. 2003;41(10):4660-5, http://dx.doi.org/10.1128/ JCM.41.10.4660-4665.2003.

the literature. Renal function drives vancomycin pharmacokinetics. Consequently, age or weight are the most relevant covariates of vancomycin clearance and should be considered first in neonatal vancomycin dosing guidelines and further adjusted by indicators of renal dysfunction (e.g., ECMO and ibuprofen/indomethacin). In addition to the prospective validation of vancomycin dosing guidelines that includes an analysis of its effectiveness and tolerance, studies should focus on the relevance and the methods applied for therapeutic drug monitoring and on the value of continuous vancomycin administration in neonates, whereas specific settings (i.e., ECMO and ibuprofen/indomethacin) warrant focused studies.

ACKNOWLEDGMENTS This work was supported by the Ministry of the University and Scientific and Technologic Research (Rome, Italy). Karel Allegaert is supported by the Fund for Scientific Research, Flanders (Belgium) (FWO Vlaanderen, Fundamental Clinical investigatorship 1800209N).

AUTHOR CONTRIBUTIONS Pacifici GM performed the systematic review and is the principal and corresponding author of the paper. Allegaert K is the second reviewer and verified the results. Both authors wrote the paper and approved the final version.

REFERENCES 1. Schelonka RL, Infante AJ. Neonatal immunology. Semin Perinatol.

1998;22(1):2-14, http://dx.doi.org/10.1016/S0146-0005(98)80003-7.

2. Manzoni P, Rizzollo S, Decmebrino L, Ruffinazzi G, Rossi Ricci A, Gallo E, et al. Recent advances in prevention of sepsis in the premature neonates in NICU. Early Human Dev. 2011;87(suppl 1):S1-3, http:// dx.doi.org/10.1016/j.earlhumdev.2011.01.026.

3. Black RE, Cousens S, Johnson HL, Lawn JE, Rudan I, Bassani DG, et al.

Child Health Epidemiology Reference Group of WHO and UNICEF. Global, regional, and national causes of child mortality in 2008: a systematic analysis. Lancet. 2010;375(9730):1969-87, http://dx.doi.org/10.1016/ S0140-6736(10)60549-1.

4. de Hoog M, Mouton JW, van den Anker JN. Vancomycin pharmacokinetics and administration regimens in neonates. Clin Pharmacokinet.

2004;43(7):417-40, http://dx.doi.org/10.2165/00003088-200443070-00001.

5. Chambers HF. Protein synthesis inhibitors and miscellaneous and antibacterial agents. In the Goodman and Gilman’s the Pharmacological Basis of Therapeutics. 11th Edition. Brunton LL Lazo JS Lazo and Parker KL eds. pp 1194-1197. McGraw-Hill, New York 2006.

6. Gabriel MH, Kildoo GC 3rd, Gennrich JL, Modanlou HD, Collins SR. Prospective evaluation of a vancomycin dosage guideline for neonates.

Clin Pharm. 1991;10(2)129-32.

7. Sato Y. Pharmacokinetics of antibiotics in neonates. Acta Paediatr Jpn. 1997;39(1):124-131, http://dx.doi.org/10.1111/j.1442-200X.1997.tb03569.x.

8. Young TE, Mangum B. Antimicrobials. Neofax: a manual of drugs used in neonatal care, 23rd edition, 2010, pages 96-97. Thomson Reuters, Montvale, NJ, USA. 9. Allegaert K, Verbesselt R, Naulaers G, van den Anker JN, Rayyan M, Debeer A, et al. Developmental pharmacology: neonates are not just small adults. Acta Clin Belg. 2008;63(1)16-24.

10. Grimsley C, Thomson AH. Pharmacokinetics and dose requirements of vancomycin in neonates. Arch Dis Child Fetal Neonatal Ed. 1999;81(3):F221-7.

11. Capparelli EV, Lane JR, Romanowski GL, McFeely EJ, Murray W, Sousa P, et al. The influences of renal function and maturation on vancomycin elimination in newborns and infants. J Clin Pharmacol. 2001;41(9):92734.

12. McDougal A, Ling EW, Levine M. Vancomycin pharmacokinetics and dosing in premature neonates. Ther Drug Monit. 1995;17(4):319-26, http://dx.doi.org/10.1097/00007691-199508000-00001.

13. Reed MD, Kliegman RM, Weiner JS, Huang M, Yamashita TS, Blumer JL. The clinical pharmacology of vancomycin in seriously ill preterm infants.

Pediatr Res. 1987;22(3):360-3, http://dx.doi.org/10.1203/00006450198709000-00024.

14. Leonard MB, Koren G, Stevenson DK, Prober CG. Vancomycin pharmacokinetics in very low birth weight neonates. Pediatr Infect Dis J. 1989;8(5):282-6.

836

CLINICS 2012;67(7):831-837

Pharmacokinetics of vancomycin in the neonate Pacifici GM and Allegaert K

41. Badran EF, Shamayleh A, Irshaid YM. Pharmacokinetics of vancomycin in neonates admitted to the neonatology unit at the Jordan University Hospital. Int J Clin Pharmacol Ther. 2011;49(4):252-7.

42. Plan O, Cambonie G, Barbotte E, Meyer P, Devine C, Milesi C, et al.

Continuous-infusion vancomycin therapy for preterm neonates with suspected or documented Gram-positive infections: a new dosage schedule. Arch Dis Child Fetal Neonatal Ed. 2008;93(6):F418-21, http://dx.doi.org/10.1136/adc.2007.128280.

43. Pawlotsky F, Thomas A, Kergueris MF, Debillon T, Roze JC. Constant rate infusion of vancomycin in premature neonates: a new dosage schedule. Br J Clin Pharmacol. 1998;46(2):163-7.

44. Oudin C, Vialet R, Boulamery A, Martin C, Simon N. Vancomycin prescription in neonates and young infants: towards a simplified dosage.

Arch Dis Child Fetal Neonatal Ed. 2011;96(5):F365-70, http://dx.doi.org/ 10.1136/adc.2010.196402.

45. Embleton ND, Berrington J. Giving vancomycin as a continuous infusion. Arch Dis Child Fetal Neonatal Ed. 2009;94(3):F233-4.

46. Fanos V, Cuzzolin L, Atzei A, Testa M. Antibiotics and antifungals in neonatal intensive care units: a review. J Chemother. 2007;19(1):5-20.

47. Goetz MB, Sayers J. Nephrotoxicity of vancomycin and aminoglycoside therapy separately and in combination. J Antimicrob Chemother.

1993;32(2):325-34, http://dx.doi.org/10.1093/jac/32.2.325.

48. Rybak MJ, Albrecht LM, Boike SC, Chandrasekar PH. Nephrotoxicity of vancomycin, alone and with an aminoglycoside. J Antimicrob Chemother. 1990;25(4):679-87, http://dx.doi.org/10.1093/jac/25.4.679.

49. Rodvold KA, Everett JA, Pryka RD, Kraus DM. Pharmacokinetics and administration regimens of vancomycin in neonates, infants and

50.

51.

52.

53.

54.

55.

56.

837

children. Clin Pharmacokinet. 1997;33(1):32-51, http://dx.doi.org/ 10.2165/00003088-199733010-00004.

Nahata MC. Lack of nephrotoxicity in pediatric patients receiving concurrent vancomycin and aminoglycoside therapy. Chemotherapy.

1987;33(4):302-4, http://dx.doi.org/10.1159/000238512.

Goren MP, Baker DK Jr, Shenep JL. Vancomycin does not enhance amikacin-induced tubular nephrotoxicity in children. Pediatr Infect Dis J. 1989;8(5):278-82.

Duffull SB, Begg EJ. Vancomycin toxicity. What is the evidence for dose dependency? Adverse Drug React Toxicol Rev. 1994;13(2):103-14.

Iwamoto T, Kagawa Y, Kojima M. Factors influencing the overestimation of plasma vancomycin concentrations measured by the Abbott TDx technique. Ther Drug Monit. 2005;27(1):58-62, http://dx.doi.org/ 10.1097/00007691-200502000-00012.

George I, Mekahli D, Rayyan M, Levtchenko E, Allegaert K. Postnatal trends in creatinemia and its covariates in extremely low birth weight (ELBW) neonates. Pediatr Nephrol. 2011;26(10):1843-9, http://dx.doi.org/ 10.1007/s00467-011-1883-0.

Kuppens M, George I, Lewi L, Levtchenko E, Allegaert K. Creatinaemia at birth is equal to maternal creatinaemia at delivery: does this paradigm still hold? J Matern Fetal Neonatal Med. 2011; Aug 25, http://dx.doi. org/10.3109/14767058.2011.602144.

Mulla H, Pooboni S. Population pharmacokinetics of vancomycin in patients receiving extracorporeal membrane oxygenation. Br J Clin Pharmacol. 2005;60(3):265-75, http://dx.doi.org/10.1111/j.1365-2125.

2005.02432.x.

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DOI:10.6061/clinics/2012(07)22

REVIEW

Does calcium intake affect cardiovascular risk factors and/or events?

Ma´rcia Regina Simas Gonc¸alves Torres,I Antonio Felipe SanjulianiII I Rio de Janeiro State University, Department of Applied Nutrition, Nutrition Institute, Rio de Janeiro/RJ, Brazil. II Rio de Janeiro State University, Discipline of Clinical and Experimental Pathophysiology, Rio de Janeiro/RJ, Brazil.

Dietary intervention is an important approach in the prevention of cardiovascular disease. Over the last decade, some studies have suggested that a calcium-rich diet could help to control body weight, with anti-obesity effects.

The potential mechanism underlying the impact of calcium on body fat has been investigated, but it is not fully understood. Recent evidence has also suggested that a calcium-rich diet could have beneficial effects on other cardiovascular risk factors, such as insulin resistance, dyslipidemia, hypertension and inflammatory states. In a series of studies, it was observed that a high intake of milk and/or dairy products (the main sources of dietary calcium) is associated with a reduction in the relative risk of cardiovascular disease. However, a few studies suggest that supplemental calcium (mainly calcium carbonate or citrate) may be associated with an increased risk of cardiovascular events. This review will discuss the available evidence regarding the relationship between calcium intake (dietary and supplemental) and different cardiovascular risk factors and/or events.

KEYWORDS: Dietary calcium; Supplemental calcium; Obesity; Cardiovascular risk factors; Cardiovascular events.

Torres MR, Sanjuliani AF. Does calcium intake affect cardiovascular risk factors and/or events? Clinics. 2012;67(7):839-844.

Received for publication on November 8, 2011; First review completed January 13, 2012; Accepted for publication on March 14, 2012 E-mail: marciarsimas@gmail.com Tel.: 00 55 21 2334-2063

fewer participants, even when a specific group of individuals was evaluated (27-30). Two studies conducted by our group observed this inverse association (28-29). In the first study (28), a higher dietary calcium intake was associated with lower total body adiposity in hypertensive patients, whereas in the second study (29), a higher dietary calcium intake was associated with lower abdominal adiposity in a group of renal transplant patients.

A global evaluation of different observational studies points to a modest negative association between dietary calcium (or dairy) and body weight. Dougkas et al. (3), based on a linear regression analysis of 18 studies, estimated that at 400 mg of calcium/day (low dietary calcium intake), a body mass index (BMI) of 25.6 kg/m2 was predicted, while for 1,200 mg of calcium/day (adequate calcium intake), a BMI of 24.7 kg/m2 was predicted. Thus, an increase in calcium intake of 800 mg/day was associated with a decrease in BMI of 1.1 kg/m2.

INTRODUCTION Considerable evidence indicates the role of dietary interventions in the prevention of cardiovascular disease (CVD) (1). Existing research has focused mainly on the role of macronutrients. However, micronutrients might also be important in the prevention of CVD. The possibility that a calcium-rich diet could affect energy balance, with antiobesity effects, has attracted considerable attention (2-3).

Several studies have also suggested that dietary calcium could have beneficial effects on insulin resistance (4-8), dyslipidemia (9-12), hypertension (13-18), inflammatory stress (19-20) and cardiovascular events (21-24). In this review, we discuss the evidence regarding calcium intake and cardiovascular risk factors and/or events.

CALCIUM AND OBESITY Observational studies The initial evidence for an inverse association between the intake of dietary calcium and/or dairy products (which are the main sources of dietary calcium) and the parameters of abdominal or total adiposity was derived from epidemiological studies in both adults and children (9,25-26). Similar results have been obtained from observational studies with

Interventional studies The effect of calcium supplementation on weight loss is uncertain. The results of randomized clinical trials designed to examine the effects of calcium supplementation on parameters of body fat have been ambiguous (31-36). One factor that likely contributes to the conflicting results is the lack of uniformity in the design of the studies. For example, some trials used supplemental calcium (mainly calcium carbonate or citrate) (37-40), while others used dietary calcium (derived from dairy products) (34-36). In some trials, calcium was supplemented during energy restriction (37,40), while in others, energy intake was not restricted (38-39).

Copyright ß 2012 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.

No potential conflict of interest was reported.

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intake raises serum levels of calcitriol (1,25(OH)2 vitamin D), which can stimulate adipocyte calcium influx by membrane vitamin D receptors, identified as the membrane-associated rapid response to steroids (1,25D MARRS). This increase in intracellular calcium levels ([Ca2+]i) might promote lipogenesis and inhibit lipolysis by increasing fatty acid synthase activity and expression inhibiting hormone-sensitive lipase (2,47). Calcitriol also acts via the classical nuclear vitamin D receptors in adipocytes to inhibit the expression of uncoupling protein-2 and thus increase energy efficiency.

Calcitriol regulation of uncoupling protein-2 and [Ca2+]i appears to exert an additional effect on energy metabolism by affecting adipocyte apoptosis (2). In contrast, an increase in calcium intake can decrease serum levels of calcitriol, consequently reducing lipogenesis and stimulating lipolysis (46).

Among the studies without concomitant energy restriction that evaluated the effects of supplemental calcium, the majority did not observe any benefits to body weight and/ or composition (38-39,41). In contrast, some studies that supplemented dietary calcium reported beneficial effects (33,42). Zemel et al. (43) compared the effects of low dairy (,1 dairy serving/day) versus recommended dairy (.3 servings/day) on weight maintenance and body composition subsequent to weight loss. Weight maintenance was similar in both groups. However, the recommended dairy group exhibited evidence of greater fat oxidation and was able to consume greater amounts of energy without greater weight gain compared to the low dairy group.

Studies that have evaluated the use of supplemental calcium during energy restriction have failed to find beneficial effects on weight loss (37,40). However, the supplementation of dietary calcium associated with energy restriction has induced significantly higher decreases in total and/or abdominal adiposity in some studies (31-33,36) but not in others (34-35). Recently, our group conducted a randomized clinical trial (36) with 50 obese subjects of both sexes, 22–55 years of age, with stable body weight and low habitual calcium intake. The participants were randomized into the following outpatient dietary regimens: (i) a lowcalcium diet (,500 mg/day) or (ii) a high-calcium diet (1,200–1,300 mg/day, supplemented with non-fat powdered milk [60 g/day]). Both groups followed an energy-restricted diet (-800 kcal/day) throughout the study (16 weeks). After 16 weeks of energy restriction, the subjects on the highcalcium diet exhibited greater reductions in waist circumference (p = 0.002) and waist-to-hip ratio (p = 0.0001) than the subjects on the low-calcium diet (36).

Studies comparing the effects of supplemental versus dietary calcium during energy restriction have observed that calcium from dairy products has a greater effect on the reduction of body fat (31,44). According to Van Loan (45), three important elements have been present in studies showing a favorable effect of calcium and/or dairy product intake on weight loss. First, all of the individuals participating in these studies were overweight and/or obese. Second, the subjects’ calcium intake levels were habitually inadequate (,600 mg/day), and an appropriate control group (,600 mg of calcium/day) was used. Third, moderate energy restriction was maintained throughout the study.

Interventional trials assessing the effects of calcium supplementation on body adiposity present inconsistent results, and further studies regarding this issue are needed.

Effect of calcium on fecal fat excretion Although it has already been shown that both dietary and supplemental calcium can increase fecal fat excretion through the formation of insoluble complexes (soaps), there is disagreement regarding the magnitude of this effect (2,4748). In general, the effect is relatively small (especially with supplemental calcium), and studies have used amounts of calcium that are higher than the usual intakes of different populations and also higher than the amounts of calcium used in studies evaluating the effects of calcium on adiposity (48-49).

In a recent meta-analysis (49), an increase in dairy calcium intake of approximately 1,200 mg/day resulted in an increase in fecal fat excretion of 5.2 g/day. Astrup et al.

(46), assuming that increased fat excretion of 3,500 kcal/year produces weight loss of 0.45 kg/year, estimated that this would correspond to a weight change of -2.2 kg/year.

According to these authors (46), this effect is sufficient to explain the differences found in observational studies but not in some interventional trials. In summary, this mechanism probably contributes to the anti-obesity effect of calcium but cannot explain it completely (2,47-48).

Effect of calcium on appetite regulation Recently, it was suggested that calcium intake could interfere in the regulation of appetite. However, this effect was evaluated in a very small number of studies, and this hypothesis is not confirmed (3,46). In humans, only two studies have been conducted (50-51). Major et al. (50) found that daily supplementation of 1,200 mg of calcium + 10 mcg of vitamin D for 15 weeks in subjects with low habitual calcium intake (,600 mg/day) resulted in decreased ad libitum lipid intake. These authors speculated that the cooccurrence of fats and calcium in an abundance of food, especially dairy foods, could influence subjects with habitual low calcium intake toward the selection of highfat foods (50). In contrast, Lorenzen et al. (51) found that acute ingestion of dietary and/or supplemental calcium has no effect on postprandial appetite, the levels of hormones involved in the regulation of appetite or energy intake at subsequent meals.

Possible mechanisms underlying the impact of calcium on adiposity The mechanisms underlying the possible relationship between calcium and/or dairy intake and BMI are unclear.

However, a reduction in body weight can only occur if fecal energy loss, energy expenditure and/or energy intake are affected. High calcium intake has been suggested to affect all of these factors (46).

Effect of calcium on fat metabolism in adipocytes Effect of calcium on abdominal obesity

There is evidence from in vitro and animal studies that increased calcium intake inhibits lipogenesis and stimulates lipolysis and thermogenesis, thereby increasing energy expenditure and lipid oxidation (46). One of the suggested mechanisms that explains these effects is that low calcium

Observational studies (27,29) and randomized clinical trials (31-33,36) have indicated that a high-calcium diet is associated with a significantly greater decrease in abdominal obesity, suggesting body fat redistribution. The exact

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fecal excretion and thus favoring the conversion of cholesterol into bile acids (56).

mechanism by which dietary calcium intake induces abdominal obesity reduction is still not clear, although recent studies describing the role of adipose tissue autocrine cortisol production could provide a plausible explanation (2). Human adipose tissue expresses 11b-hydroxysteroid dehydrogenase-1, which catalyzes the conversion of cortisone to cortisol. Recently, it was demonstrated that calcitriol stimulates the expression of 11b-hydroxysteroid dehydrogenase-1 and cortisol production by human adipocytes.

Because high-calcium diets suppress calcitriol levels, it has been suggested that the decrease in central obesity seen with these diets could be partly explained by the suppression of calcitriol levels, leading to a reduction of adipocyte cortisol production (52).

CALCIUM AND HYPERTENSION

The association is relatively consistent between low calcium (and/or dairy) intake and the increased prevalence of type 2 diabetes and/or insulin resistance (4) and has been found in several observational cohort studies (5-8). Pittas et al. (5) followed 83,779 women participating in the Nurses’ Health Study for 20 years and identified a multivariate relative risk of type 2 diabetes of 0.79 (95% CI 0.70 – 0.90; p,0.001) when comparing the highest category of calcium intake with the lowest category (dietary + supplemental). In prospective data from 41,254 men taking part in the Health Professionals Follow-up Study who were followed for 12 years, intake of dairy products was associated with a modestly lower risk of type 2 diabetes (6).

Despite the epidemiological findings, there is only limited evidence regarding the effects of calcium supplementation on insulin sensitivity (4). The results of studies with supplemental calcium appear to have been influenced by the level of insulin sensitivity at baseline. In a study by Pittas et al. (53), combined daily supplementation of vitamin D (700 IU) + calcium (500 mg) for three years only produced a significant improvement in insulin sensitivity in subjects with impaired glucose tolerance at baseline.

The exact mechanism by which calcium modulates insulin resistance is not known (4). There is evidence that an increase in [Ca2+]i in adipocytes and other insulin target cells, induced by a low-calcium diet, can result in insulin resistance via phosphorylation of glucose transporter type 4 (GLUT-4) and other substrates sensitive to insulin, rendering glucose uptake mediated by insulin less efficient (54).

An inverse relationship between calcium and/or dairy intake and blood pressure has been reported in various epidemiological studies, typically showing that reduced intake of calcium is associated with higher blood pressure and/or an increased risk of developing hypertension (1316).

The randomized clinical trials that have evaluated the effects of calcium supplementation have identified modest reductions in blood pressure (17-18). vanMierlo et al. (17) performed a meta-analysis of 40 randomized controlled trials evaluating the effects of calcium supplementation (mean daily dose: 1,200 mg) and found significant reductions in systolic (-1.86 mm Hg) and diastolic (-0.99 mm Hg) blood pressure. Calcium supplementation has a greater hypotensive effect on: (i) subjects regularly consuming small amounts of calcium (17); (ii) hypertensive individuals or groups at a higher risk of developing hypertension, such as individuals with sub-Saharan African ancestry, salt-sensitive persons and pregnant women (57); and (iii) studies using dietary calcium (18).

Reid et al. (39) conducted a randomized controlled trial of calcium supplementation in 323 generally healthy men over a period of two years. The subjects were assigned to groups taking a placebo, 600 mg of calcium/day, or 1,200 mg of calcium/day. There were downward trends in systolic and diastolic blood pressures within the calcium-supplemented groups, but there were no significant treatment effects over the entire trial period (p.0.60). In a post hoc analysis of those subjects with baseline calcium intakes less than the median value (785 mg/day), their blood pressures exhibited borderline treatment effects (p = 0.05–0.06 for changes at two years in those subjects who received 1,200 mg of calcium/day compared with those taking a placebo: systolic, -4.2 mm Hg; diastolic, -3.3 mm Hg).

To date, the evidence available provides the basis for the argument that a diet containing the recommended amounts of dairy products can reduce the risk of hypertension (57).

The protective effect of calcium on blood pressure can be partly explained by the influence of calcitriol on [Ca2+]i. An increase of [Ca2+]i in vascular smooth muscle cells can result in vasoconstriction and a consequent rise in blood pressure (58-59).

CALCIUM AND DYSLIPIDEMIA

CALCIUM AND INFLAMMATORY STRESS

The effects of calcium on lipid profiles have not yet been elucidated. Some studies using dietary or supplemental calcium have found significant reductions in total cholesterol and low-density lipoprotein (LDL) cholesterol or increases in high-density lipoprotein (HDL) cholesterol (10-12), while other studies have not found such benefits (39,55).

In the gut, saponification of free fatty acids by calcium reduces the intestinal absorption of lipids; this mechanism can explain the effects of calcium on lipid profiles (10,12).

This reduction in the absorption of fatty acids, especially saturated fatty acids, could reduce serum cholesterol levels by decreasing the production of very low-density lipoprotein (VLDL) and by increasing the uptake of LDL cholesterol by the liver. Calcium can also bind to bile acids, increasing

Some studies have suggested that calcium intake can facilitate the suppression of the inflammatory state associated with obesity (19-20). Few studies have been conducted in humans (20,60), and these studies have evaluated only the effects of dietary calcium. Zemel et al. (20) conducted a study to assess the acute effects of a diet rich in dairy products on inflammatory stress in obese and overweight subjects in the absence of changes in adiposity. In this randomized, blinded, crossover trial, normocaloric diets were supplemented with dairy or soy over two periods of 28 days, separated by a washout period of 28 days. The diet supplemented with dairy products resulted in significantly lower levels of inflammatory markers (tumor necrosis factor-a, interleukin-6 and monocyte chemotactic protein-1) and significantly increased levels of adiponectin (20%) (20).

CALCIUM AND INSULIN RESISTANCE

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The modulation of inflammatory stress by calcium is likely mediated, in part, by a reduction in body fat.

However, it has been shown that calcium could have additional effects via the suppression of calcitriol (20).

Elevated serum levels of calcitriol, in addition to increasing [Ca2+]i, appear to increase the production of reactive oxygen species (ROS) through the modulation of mitochondrial uncoupling. These two mechanisms modulate the production and release of cytokines (19-20).

Lewis et al. (65) found no increased risk of atherosclerotic CVD with daily calcium supplementation of 1,200 mg.

Mursu et al. (70) assessed the use of vitamin and mineral supplements in relation to total mortality in 38,772 older women in the Iowa Women’s Health Study; the mean age was 61.6 years old at baseline in 1986. Supplement use was self-reported in 1986, 1997, and 2004. Through December 31, 2008, a total of 15,594 deaths (40.2%) were identified.

Supplemental calcium was inversely correlated with total mortality rate (hazard ratio, 0.91; 95% CI, 0.88-0.94; absolute risk reduction, 3.8%); however, no clear dose-response relationship was observed.

Shah et al. (71) investigated the long-term cumulative effects of calcium and vitamin D supplementation on cardiovascular outcomes and death in older women. An observational cohort study using a UK electronic primary care records database was referenced; 9,910 women aged 60–89 years old were studied who began calcium and vitamin D supplementation between 2000 and 2005, with no heart disease or stroke history and who survived diseasefree for two years after supplement initiation. Two years after initiation, the women who consistently received supplementation with calcium and vitamin D did not experience more cardiovascular events or deaths than the women who received minimal supplementation.

The proposed mechanism for the increased risk of atherosclerotic disease is the acute rise in serum calcium that can be induced with supplemental calcium (but not with dietary calcium). There is epidemiological evidence that serum levels of calcium in the upper limit of the normal range are a risk factor for vascular disease (62).

ADDITIONAL BIOACTIVE COMPONENTS OF DAIRY PRODUCTS Dairy products have other bioactive components (in addition to calcium) that can have beneficial effects on adiposity and/or cardiovascular risk factors, such as leucine and the peptides that inhibit angiotensin-converting enzyme (ACE) (2). The high concentrations of leucine present in dairy products can contribute to the anti-obesity effects of these foods by stimulating muscle protein synthesis, inhibiting muscle degradation, and suppressing the energy storage of adipose tissue (61). The peptides that inhibit ACE, found in dairy whey, could contribute to the beneficial effects of dairy products on adiposity, blood pressure and inflammatory stress (2,20).

CALCIUM AND CARDIOVASCULAR EVENTS The relationship between calcium intake and the risk of cardiovascular events has been explored in some studies (21-24,62-65). The results of studies evaluating dietary calcium differ from studies evaluating supplemental calcium. Thus, these studies will be discussed separately.

Dietary calcium and cardiovascular events In a series of studies, it has been observed that high intake of milk and/or dairy products is associated with a reduction in the relative risk of CVD (21-24). In a recent meta-analysis, Elwood et al. (22) observed that the relative risks for stroke and coronary heart disease in individuals with high intake levels of milk and dairy products were 0.84 (95% CI 0.760.93) and 0.79 (95% CI 0.75-0.82), respectively, compared with subjects with low intakes.

Supplemental calcium and cardiovascular events The results of studies assessing the effects of supplemental calcium on cardiovascular events are inconsistent.

Supplemental calcium is widely recommended for postmenopausal women to prevent osteoporosis. The effects of this supplementation on bone mineral density and fracture risk have been investigated in numerous studies. Secondary analyses of the data from a few of these studies suggest that supplemental calcium (with or without co-administered vitamin D) might be associated with an increased risk of cardiovascular events, especially acute myocardial infarction (63-64,66). Bolland et al. (63) conducted a randomized clinical trial to evaluate the effects of calcium supplementation (1 g of elemental calcium daily, as citrate) on myocardial infarction, stroke, and sudden death in healthy postmenopausal women. The study included 1471 women (mean age of 74 years old): 732 were randomized to calcium supplementation and 739 to placebo. The composite end points of myocardial infarction, stroke, and sudden death were more common in the calcium group (p = 0.008). The authors concluded that calcium supplementation in postmenopausal women is associated with upward trends in cardiovascular risk.

Despite the potential harmful effects of supplemental calcium observed in the studies cited above, other authors do not agree with this point of view (67-69). Several studies have found no increased risk of CVD with calcium supplementation (with or without vitamin D) in postmenopausal women (65,70-74).

FINAL CONSIDERATIONS Epidemiological studies indicate an inverse association between calcium intake and total or abdominal adiposity, insulin resistance, and blood pressure. In contrast, the effects of calcium supplementation (either dietary or supplemental) on obesity and cardiovascular risk factors have not been proven. The most consistent findings indicate modest benefits of dietary calcium (especially in individuals with low habitual intake of this mineral) in the reduction of adiposity during periods of energy restriction and in the reduction of blood pressure.

The relationship between calcium intake and cardiovascular events is uncertain. High levels of intake of milk and/ or dairy products have been associated with a reduction in the relative risk of CVD in a series of studies. However, supplemental calcium has been associated with increased risk of cardiovascular events in some trials. Further studies are needed to define the effects of calcium intake on cardiovascular risk factors and/or events.

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AUTHOR CONTRIBUTIONS 22.

Torres MR and Sanjuliani AF conceived and designed the study and were also responsible for the references, analysis and interpretation, manuscript drafting and approval of the final version of the manuscript.

23.

REFERENCES 1. Artinian NT, Fletcher GF, Mozaffarian D, Kris-Etherton P, Van Horn L, Lichtenstein AH, et al. Interventions to promote physical activity and dietary lifestyle changes for cardiovascular risk factor reduction in adults. A Scientific Statement from the American Heart Association.

Circulation. 2010;122(4):406-41.

2. Zemel MB. The role of dairy foods in weight management. J Am Coll Nutr. 2005;24(6 supl):537S-46S. 3. Dougkas A, Reynolds CK, Givens ID, Elwood PC, Minihane AM. Associations between dairy consumption and body weight: a review of the evidence and underlying mechanisms. Nutr Res Rev. 2011;15:1-24.

4. Pittas AG, Lau J, Hu FB, Dawson-Hughes B. The role of vitamin D and calcium in type 2 diabetes. A systematic review and meta-analysis. J Clin Endocrinol Metab. 2007;92(6):2017-29, http://dx.doi.org/10.1210/ jc.2007-0298.

5. Pittas AG, Dawson-Hughes B, Li Tricia, Van Dam RM, Willett WC, Manson JE, et al. Vitamin D and calcium intake in relation to type 2 diabetes in women. Diabetes Care. 2006;29(3):650-6, http://dx.doi.org/ 10.2337/diacare.29.03.06.dc05-1961.

6. Choi HK, Willett WC, Stampfer MJ, Rimm E, Hu FB. Dairy consumption and risk of type 2 diabetes mellitus in men. Arch Intern Med. 2005;165:997-1003, http://dx.doi.org/10.1001/archinte.165.9.997.

7. Villegas R, Gao YT, Dai Q, Yang G, Cai H, Li H, et al. Dietary calcium and magnesium intakes and the risk of type 2 diabetes: the Shanghai Women’s Health Study. Am J Clin Nutr. 2009;89(4):1059–67, http:// dx.doi.org/10.3945/ajcn.2008.27182.

8. Fumeron F, Lamri A, Khalil AC, Jaziri R, Porchay-Balde´relli I, Lantieri O, et al. Dairy consumption and the incidence of hyperglycemia and the metabolic syndrome. Diabetes Care. 2011;34(4):813-7, http://dx.doi.org/ 10.2337/dc10-1772.

9. Jacqmain M, Doucet E, Despre´s JP, Bouchard C, Tremblay A. Calcium intake, body composition, and lipoprotein-lipid concentrations in adults.

Am J Clin Nutr. 2003;77(6):1448-52.

10. Ditscheid B, Keller S, Jahreis G. Cholesterol metabolism is affected by calcium phosphate supplementation in humans. J Nutr. 2005;135(7):167882.

11. Major GC, Alarie F, Dore´ J, Phouttama S, Tremblay A. Supplementation with calcium + vitamin D enhanced the beneficial effect of weight loss on plasma lipid and lipoprotein concentrations. Am J Clin Nutr.

2007;85(1):54-9.

12. Lorenzen JK, Astrup A. Dairy calcium intake modifies responsiveness of fat metabolism and blood lipids to a high-fat diet. Br J Nutr. 2011 Jan 31:1-10, http://dx.doi.org/10.1017/S0007114510005581.

13. Ruidavets J-B, Bongard V, Simon C, Dallongeville J, Ducimetie`re P, Arveiler D, et al. Independent contribution of dairy products and calcium intake to blood pressure variations at a population level.

J Hypertens. 2006;24(4):671-81, http://dx.doi.org/10.1097/01.hjh.000 0217849.10831.16.

14. Snijder MB, van der Heijden AAWA, van Dam RM, Stehouwer CDA, Hiddink GJ, Nijpels G, et al. Is higher dairy consumption associated with lower body weight and fewer metabolic disturbances? The Horn Study.

Am J Clin Nutr. 2007;85(4):989-95.

15. Wang L, Manson JE, Buring JE, Lee IM, Sesso HD. Dietary intake of dairy products, calcium, and vitamin D and the risk of hypertension in middleaged and older women. Hypertension. 2008;51(1):1-7.

16. Engberink MF, Hendriksen MAH, Schouten EG, van Rooij FJA, Hofman A, Witteman JCM, et al. Inverse association between dairy intake and hypertension: the Rotterdam Study.Am J Clin Nutr. 2009;89(6):1877-83.

17. vanMierlo LA, Arends LR, Streppel MT, Zeegers MP, Kok FJ, Grobbee DE, et al. Blood pressure response to calcium supplementation: a metaanalysis of randomized controlled trials. J Hum Hypertens.

2006;20(8):571-80, http://dx.doi.org/10.1038/sj.jhh.1002038.

18. Griffith LE, Guyatt GH, Cook RJ, Bucher HC, Cook DJ. The influence of dietary and nondietary calcium supplementation on blood pressure: an updated metaanalysis of randomized controlled trials. Am J Hypertens.

1999;12 (1 Pt 1):84-92, http://dx.doi.org/10.1016/S0895-7061(98)00224-6.

19. Sun X, Zemel MB. Calcium and 1,25-Dihydroxyvitamin D3 Regulation of Adipokine Expression. Obesity. 2007;15(2):340-8, http://dx.doi.org/ 10.1038/oby.2007.540.

20. Zemel MB, Sun X, Sobhani T, Wilson B. Effects of dairy compared with soy on oxidative and inflammatory stress in overweight and obese subjects. Am J Clin Nutr. 2010;91(1):16-22, http://dx.doi.org/10.3945/ ajcn.2009.28468.

21. Elwood PC, Pickering JE, Givens DI, Gallacher JE. The Consumption of milk and dairy foods and the incidence of vascular disease and diabetes:

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

843

An overview of the evidence. Lipids. 2010;45(10):925-39, http:// dx.doi.org/10.1007/s11745-010-3412-5.

Elwood PC, Givens DI, Beswick AD, Fehily AM, Pickering JE, Gallacher J. The survival advantage of milk and dairy consumption: an overview of evidence from cohort studies of vascular diseases, diabetes and cancer.

J Am Coll Nutr. 2008;27(6):723S-34S. Elwood PC, Pickering JE, Fehily AM. Milk and dairy consumption, diabetes and the metabolic syndrome: the Caerphilly prospective study.

J Epidemiol Community Health. 2007;61(8):695-8, http://dx.doi.org/ 10.1136/jech.2006.053157.

Elwood PC, Strain JJ, Robson PJ, Fehily AM, Hughes J, Pickering J, et al.

Milk consumption, stroke, and heart attack risk: evidence from the Caerphilly cohort of older men. J Epidemiol Community Health.

2005;59(6):502-5, http://dx.doi.org/10.1136/jech.2004.027904.

Pereira MA, Jacobs DR, Horn LV, Slattery ML, Kartashov AI, Ludwig DS. Dairy consumption, obesity, and the insulin resistance syndrome in young adults. The CARDIA study. JAMA. 2002;287(16):2081-9, http:// dx.doi.org/10.1001/jama.287.16.2081.

Hanks LJ, Casazza K, Willig AL, Cardel MI, Beasley TM, Fernandez JR. Associations among calcium intake, resting energy expenditure, and body fat in a multiethnic sample of children. J Pediatr. 2010;157(3):473-8.

Bush NC, Alvarez JA, Choquette SS, Hunter GR, Oster RA, Darnell BE, et al. Dietary calcium intake is associated with less gain in intraabdominal adipose tissue over 1 year. Obesity. 2010;18(11):2101-4, http://dx.doi.org/10.1038/oby.2010.39.

Torres MRSG, Ferreira TS, Carvalho DC, Sanjuliani AF. Dietary calcium intake and its relationship with adiposity and metabolic profile in hypertensive patients. Nutrition. 2011;27(6):666–71, http://dx.doi.org/ 10.1016/j.nut.2010.07.012.

Torres MRSG, Gioseffi G, Guimara˜es SSMS, Cardoso LG, Barroso SG, Sanjuliani AF, et al. A Pilot study on the relation between dietary calcium and clinical parameters in renal transplant recipients. J Ren Nutr.

2011;21(5):418-25, http://dx.doi.org/10.1053/j.jrn.2010.08.007.

Shahar DR, Schwarzfuchs D, Fraser D, Vardi H, Thiery J, Fiedler GM, et al. Dairy calcium intake, serum vitamin D, and successful weight loss.

Am J Clin Nutr. 2010;92(5):1017–22, http://dx.doi.org/10.3945/ ajcn.2010.29355.

Zemel MB, Thompson W, Milstead A, Morris K, Campbell P. Calcium and dairy acceleration of weight and fat loss during energy restriction in obese adults. Obes Res. 2004;12(4):582-90, http://dx.doi.org/10.1038/ oby.2004.67.

Zemel MB, Richards J, Mathis S, Milstead A, Gebhardt L, Silva E. Dairy augmentation of total and central fat loss in obese subjects. Int J Obes Relat Metab Disord. 2005;29(4):391-7, http://dx.doi.org/10.1038/ sj.ijo.0802880.

Zemel MB, Richards J, Milstead A, Campbell P. Effects of calcium and dairy on body composition and weight loss in African-American adults.

Obes Res. 2005;13(7):1218-25, http://dx.doi.org/10.1038/oby.2005.144.

Harvey-Berino J, Gold BC, Lauber R, Starinski A. The impact of calcium and dairy product consumption on weight loss. Obes Res. 2005;13(10):1720-6, http://dx.doi.org/10.1038/oby.2005.210.

Thompson WG, Holdman NR, Janzow DJ, Slezak JM, Morris KL, Zemel MB. Effect of energy-reduced diets high in dairy products and fiber on weight loss in obese adults. Obes Res. 2005;13(8):1344-53, http:// dx.doi.org/10.1038/oby.2005.163.

Torres MRSG, Francischetti EA, Genelhu V, Sanjuliani AF. Effect of a high-calcium energy-reduced diet on abdominal obesity and cardiometabolic risk factors in obese Brazilian subjects. Int J Clin Pract.

2010;64(8):1076-83, http://dx.doi.org/10.1111/j.1742-1241.2009.02312.x.

Shapses SA, Heshka S, Heymsfield SB. Effect of calcium supplementation on weight and fat loss in women. J Clin Endocrinol Metab.

2004;89(2):632-7, http://dx.doi.org/10.1210/jc.2002-021136.

Reid IR, Horne A, Mason B, Ames R, Bava U, Gamble GD. Effects of calcium supplementation on body weight and blood pressure in normal older women. J Clin Endocrinol Metab. 2005;90(7):3824-9, http:// dx.doi.org/10.1210/jc.2004-2205.

Reid IR, Ames R, Mason B, Bolland MJ, Bacon CJ, Reid HE, et al. Effects of calcium supplementation on lipids, blood pressure, and body composition in healthy older men: a randomized controlled trial.

Am J Clin Nutr. 2010;91(1):131-9, http://dx.doi.org/10.3945/ ajcn.2009.28097.

Shalileh M, Shidfar F, Haghani H, Eghtesadi S, Heydari I. The influence of calcium supplement on body composition, weight loss and insulin resistance in obese adults receiving low calorie diet. J Res Med Sci. 2010;15(4):191-201.

Palacios C, Bertra´n JJ, Rı´os RE, Soltero S. No effects of low and high consumption of dairy products and calcium supplements on body composition and serum lipids in Puerto Rican obese adults. Nutrition.

2011;27(5):520-5, http://dx.doi.org/10.1016/j.nut.2010.02.011.

Eagan MS, Lyle RM, Gunther CW, Peacock M, Teegarden D. Effect of 1year dairy product intervention on fat mass in young women: 6-month follow-up. Obesity. 2006;14(12):2242-8, http://dx.doi.org/10.1038/ oby.2006.263.

Calcium and cardiovascular disease Torres MR and Sanjuliani AF

CLINICS 2012;67(7):839-844

43. Zemel MB, Donnelly JE, Smith BK, Sullivan DK, Richards J, MorganHanusa D, et al. Effects of dairy intake on weight maintenance. Nutr Metab (Lond). 2008;5:28, http://dx.doi.org/10.1186/1743-7075-5-28.

44. Faghih SH, Abadi AR, Hedayati M, Kimiagar SM. Comparison of the effects of cows’ milk, fortified soy milk, and calcium supplement on weight and fat loss in premenopausal overweight and obese women.

Nutr Metab Cardiovasc Dis. 2011;21(7):499-503, http://dx.doi.org/ 10.1016/j.numecd.2009.11.013.

45. Van Loan M. The role of dairy foods and dietary calcium in weight management. J Am Coll Nutr. 2009;28(Suppl1):120S-9S. 46. Astrup A, Kristensen M, Gregersen NT, Belza A, Lorenzen JK, Due A, et al. Can bioactive foods affect obesity? Ann N Y Acad Sci. 2010;1190:2541, http://dx.doi.org/10.1111/j.1749-6632.2009.05272.x.

47. Major GC, Chaput JP, Ledoux M, St-Pierre S, Anderson GH, Zemel MB, et al.

Recent developments in calcium-related obesity research. Obes Reviews.

2008;9(5):428-45, http://dx.doi.org/10.1111/j.1467-789X.2007.00465.x.

48. Jacobsen R, Lorenzen JK, Toubro S, Krog-Mikkelsen I, Astrup A. Effects of short-term high dietary calcium intake on 24-h energy expenditure, fat oxidation, and fecal fat excretion. Int J Obes (Lond). 2005;29(3):292-301, http://dx.doi.org/10.1038/sj.ijo.0802785.

49. Christensen R, Lorenzen JK, Svith CR, Bartels EM, Melanson EL, Saris WH, et al. Effect of calcium from dairy and dietary supplements on faecal fat excretion: a meta-analysis of randomized controlled trials. Obes Rev. 2009;10(4):475-86, http://dx.doi.org/10.1111/j.1467-789X.2009.00599.x.

50. Major GC, Alarie FP, Tremblay A. Calcium plus vitamin D supplementation and fat mass loss in female very low-calcium consumers: potential link with a calcium-specific appetite control. Br J Nutr. 2009;101(5):65963, http://dx.doi.org/10.1017/S0007114508030808.

51. Lorenzen JK, Nielsen S, Holst JJ, Tetens I, Rehfeld JF, Astrup A. Effect of dairy calcium or supplementary calcium intake on postprandial fat metabolism, appetite, and subsequent energy intake. Am J Clin Nutr.

2007;85(3):678-87.

52. Morris KL, Zemel MB. 1,25-dihydroxyvitmin D3 modulation of adipocyte glucocorticoid function. Obes Res. 2005;13(4):670-7, http:// dx.doi.org/10.1038/oby.2005.75.

53. Pittas AG, Harris SS, Stark PC, Dawson-Hughes B. The Effects of calcium and vitamin D supplementation on blood glucose and markers of inflammation in nondiabetic adults. Diabetes Care. 2007;30(4):980-6, http://dx.doi.org/10.2337/dc06-1994.

54. Zemel MB. Insulin resistance vs. hyperinsulinemia in hypertension: insulin regulation of Ca2+ transport and Ca(2+)-regulation of insulin sensitivity. J Nutr. 1995;125(6 Suppl):1738S-43S. 55. Rajpathak SN, Xue X, Wassertheil-Smoller S, Horn LV, Robinson JG, Liu S, et al. Effect of 5 y of calcium plus vitamin D supplementation on change in circulating lipids. Am J Clin Nutr. 2010;91(4):894-9, http:// dx.doi.org/10.3945/ajcn.2009.28579.

56. Vaskonen T. Dietary minerals and modification of cardiovascular risk factors. J Nutr Biochem. 2003;14(9):492-506, http://dx.doi.org/10.1016/ S0955-2863(03)00074-3.

57. Miller GD, DiRienzo DD, Reusser ME, McCarron DA. Benefits of dairy product consumption on blood pressure in humans: a summary of the biomedical literature. J Am Coll Nutr. 2000;19(suppl):147S-64S. 58. Zemel MB. Calcium modulation of hypertension and obesity: Mechanisms and implications. J Am Coll Nutr. 2001;20(suppl):428S-35S. 59. Hoe SZ, Lee CN, Mok SL, Kamaruddin MY, Lami SK. Gynura procumbens Merr. decreases blood pressure in rats by vasodilatation via inhibition of calcium channels. CLINICS. 2011;66(1):143-50.

60. van Meijl LEC, Mensink RP. Effects of low-fat dairy consumption on markers of low-grade systemic inflammation and endothelial function in overweight and obese subjects: an intervention study. Br J Nutr. 2010;104(10):1523-7, http://dx.doi.org/10.1017/S0007114510002 515.

61. Sun X, Zemel MB. Leucine and calcium regulate fat metabolism and energy partitioning in murine adipocytes and muscle cells. Lipids.

2007;42(4):297-305, http://dx.doi.org/10.1007/s11745-007-3029-5.

62. Reid IR, Bolland MJ, Grey A. Does calcium supplementation increase cardiovascular risk? Clinical Endocrinology. 2010;73(6):689-95, http:// dx.doi.org/10.1111/j.1365-2265.2010.03792.x.

63. Bolland MJ, Barber PA, Doughty RN, Mason B, Horne A, Ames R, et al.

Vascular events in healthy older women receiving calcium supplementation: randomised controlled trial. BMJ. 2008;336(7638):262-6, http:// dx.doi.org/10.1136/bmj.39440.525752.BE. 64. Bolland MJ, Grey A, Avenell A, Gamble GD, Reid IR. Calcium supplements with or without vitamin D and risk of cardiovascular events. BMJ. 2011;342:d2040, http://dx.doi.org/10.1136/bmj.d2040.

65. Lewis JR, Calver J, Zhu K, Flicker L, Prince RL. Calcium supplementation and risk of atherosclerotic vascular disease in older women. J Bone Miner Res. 2011;26(1):35-41, http://dx.doi.org/10.1002/jbmr.176.

66. Bolland MJ, Avenell A, Baron JA, Grey A, MacLennan GS, Gamble GD, et al. Effect of calcium supplements on risk of myocardial infarction and cardiovascular events: meta-analysis. BMJ. 2010;341:doi:10.1136/ bmj.c3691.

67. Jackson G. Calcium supplements and cardiovascular disease – A response. Int J Clin Pract. 2011;65(2):227–8, http://dx.doi.org/10.1111/ j.1742-1241.2010.02605.x.

68. Nordin BE, Daly RM, Horowitz J, Metcalfe AV. Calcium and heart attacks. Making too much of a weak case. BMJ. 2010;341:c4997, http:// dx.doi.org/10.1136/bmj.c4997.

69. Grove ML, Cook D. Calcium and heart attacks. Doesnt apply to most calcium prescriptions. BMJ. 2010;341:c5003, http://dx.doi.org/10.1136/ bmj.c5003.

70. Mursu J, Robien K, Harnack LJ, Park K, Jacobs DR. Dietary supplements and mortality rate in older women. The Iowa Women’s Health Study.

Arch Intern Med. 2011;171(18):1625-33.

71. Shah SM, Carey IM, Harris T, DeWilde S, Cook DG. Calcium supplementation, cardiovascular disease and mortality in older women.

Pharmacoepidemiol Drug Saf. 2010;19(1):59–64, http://dx.doi.org/ 10.1002/pds.1859.

72. LaCroix AZ, Kotchen J, Anderson G, Brzyski R, Cauley JA, Cummings SR, et al. Calcium plus vitamin D supplementation and mortality in postmenopausal women: The Women’s Health Initiative Calcium – Vitamin D Randomized Controlled Trial. J Gerontol A Biol Sci Med Sci. 2009;64(5):559–67, http://dx.doi.org/10.1093/gerona/glp006.

73. Neuhouser ML, Wassertheil-Smoller S, Thomson C, Aragaki A, Anderson GL, Manson JE, et al. Multivitamin use and risk of cancer and cardiovascular disease in the Women’s Health Initiative Cohorts.

Arch Intern Med. 2009;169(3):294-304, http://dx.doi.org/10.1001/ archinternmed.2008.540.

74. Hsia J, Heiss G, Ren H, Allison M, Dolan NC, Greenland P, et al.

Calcium/vitamin D supplementation and cardiovascular events.

Circulation. 2007;115(7):846-54, http://dx.doi.org/10.1161/CIRCUL ATIONAHA.106.673491.

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DOI:10.6061/clinics/2012(07)23

RAPID COMMUNICATION

Anaplastic lymphoma kinase gene rearrangement in non-small-cell lung cancer in a Brazilian population Lisandro F. Lopes, Carlos E. Bacchi Consultoria em Patologia, Botucatu/SP, Brazil.

Email: bacchi@conspat.com.br Tel.: 55 14 3112-5900

Tissues from small biopsies and lung resection specimens were used. The ages and genders of the patients were registered.

INTRODUCTION Lung cancer is the leading cause of cancer deaths worldwide (1). Non-small-cell lung carcinoma (NSCLC), which represents approximately 80% of all lung cancer cases, is frequently diagnosed at advanced stages of the disease and is associated with short survival time. Although the prognosis of this disease remains poor, significant advances in the genetics and treatment of NSCLC have been made in recent years.

In 2007, Soda et al. (2) identified the transforming EML4ALK fusion gene in 6.7% of NSCLC patients. This fusion transcript, which presents potent oncogenic activity both in vitro and in vivo (2,3), is formed by the translocation of the echinoderm microtubule-associated protein-like 4 (EML4) gene (2p21) and anaplastic lymphoma kinase (ALK) gene (2p23). Subsequent studies have shown that between two and eight percent of all NSCLC cases may harbor ALK rearrangements, which are almost always mutually exclusive with EGFR and KRAS mutations. Moreover, these patients are frequently younger, non- or light smokers and have adenocarcinomas (2,4-23). Recently, crizotinib, an orally available small-molecule inhibitor of the ALK tyrosine kinase, was approved for the treatment of ALKrearranged NSCLC patients, with successful results (24-29).

The two most widely used methods for the diagnosis of ALK-rearranged NSCLC are fluorescence in situ hybridization (FISH) and immunohistochemistry.

Because there is no information about the prevalence and clinical characteristics of ALK-rearranged NSCLC in Latin America in the literature, the aim of this study was to evaluate NSCLC with ALK rearrangement in Brazilian patients.

Fluorescence in situ hybridization (FISH) FISH was performed on formalin-fixed, paraffin-embedded tumor samples using a probe specific to the ALK locus (Vysis LSI ALK dual color, break apart rearrangement probe; Abbott Molecular, Des Plaines, IL, USA) according to the manufacturer’s instructions. The FISH results were analyzed under a fluorescence microscope (Zeiss Axio Imager M1, Carl Zeiss AG, Oberkochen, Germany) with the appropriate filters (Chroma Technology GmbH, Fuerstenfeldbruck, Germany) and Metafer 4 software (MetaSystems, Altlussheim, Germany). FISH-positive cases were defined as those in which more than 15% of the cells (at least 40 neoplastic cells were counted) presented with split orange and green signals or an isolated orange signal, as previously described (31,32).

Immunohistochemistry For comparison with FISH results, ALK protein expression was determined by immunohistochemistry using the mouse monoclonal antibody 5A4 (code ab17127, ABCAM, Cambridge, MA, USA), which was found to show high concordance with ALK gene status, as determined by molecular tests (13,14,23,33-36). Briefly, 3-mm-thick unstained histologic sections of lung carcinoma tissue were used in all cases. The sections were deparaffinized in xylene and rehydrated in a graded series of alcohol in phosphatebuffered saline. The sections then were submitted to antigen retrieval in a microwave. Subsequently, slides were incubated overnight with the 5A4 antibody at a pre-standardized concentration (1:200), washed with phosphate-buffered saline, and incubated with Novocastra Novolink Polymer Detection System (Leica Biosystems Newcastle Ltd., Newcastle upon Tyne, UK). Diaminobenzidine was used as the chromogen, and sections were counterstained with hematoxylin and coverslipped. A case was considered positive for ALK if $10% of the tumor cells showed intense cytoplasmic immunostaining.

MATERIALS AND METHODS Case Selection This study included 62 consecutive NSCLC cases received from August 2010 to September 2011 for ALK rearrangement testing using FISH (30) by Consultoria em Patologia, a large Anatomic Pathology and Molecular Pathology reference laboratory located in Botucatu, Sa˜o Paulo State, Brazil.

RESULTS Of the 62 NSCLC cases included in this study, two (3.2%) showed ALK rearrangement by FISH: one case presented an isolated orange signal along with one fusion signal in tumor cells, and the other showed split orange and green signals along with one fusion signal. These two FISH-positive cases were also positive for ALK by immunohistochemistry, with

Copyright ß 2012 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.

No potential conflict of interest was reported.

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CLINICS 2012;67(7):845-847

strong cytoplasmic immunostaining in virtually all tumor cells. All of the other sixty FISH-negative cases were also negative for ALK using immunohistochemistry.

Both of the patients with FISH- and ALK immunohistochemistry-positive NSCLC were men (36 years old and 45 years old; mean: 40 years). One of them had no history of tobacco smoking, and the other was a light smoker who had stopped smoking some months prior to the diagnosis. The group of patients (n = 60) presenting ALK-negative tumors

by FISH varied in age from 32 to 87 years (mean: 58 years; median: 59 years), and most of these patients were male (33 patients).

According to the new international multidisciplinary classification of lung adenocarcinomas proposed by the International Association for the Study of Lung Cancer, the American Thoracic Society and the European Respiratory Society (37), tumors from the two patients with ALK-positive FISH were classified as invasive adenocarcinoma, acinar predominant. One tumor had papillary areas, and the other was rich in mucinous areas. The 36-year-old patient with invasive adenocarcinoma, acinar predominant with papillary areas, was also tested for EGFR and KRAS mutations; the tumor was negative for both mutations. Figure 1 presents the morphologic characteristics of the two ALK-positive cases, including FISH and immunohistochemical findings.

Table 1 - Prevalence of ALK-rearranged NSCLC in different countries.

Authors, year of publication Soda et al., 2007 (2) Rodig et al., 2009 (17) Wong et al., 2009 (4) Sakairi et al., 2010 (19) Takahashi et al., 2010 (16) Sun et al., 2010 (15) Paik et al., 2011 (11) Li et al., 2011 (12) Kim et al., 2011 (13) Paik et al., 2011 (14) Yang et al., 2012 (23) Lopes et al. (present study)

Country Japan USA China Japan Japan China Korea China Korea Korea USA Brazil

ALK-rearranged tumors 6.7% 5.6% 4.9% 6.4% 2.4% 5.7% 3.8% 5% 3.7% 4.2% 8.2% 3.2%

(n=75) (n=358) (n=266) (n=109) (n=211) (n=52) (n=735) (n=202) (n=449) (n=453) (n=300) (n=62)

(17,23). These findings show that ethnicity likely does not strongly influence the prevalence of ALK-rearranged lung carcinomas, which is in contrast to EGFR- and KRASmutated tumors (38-41).

To the best of our knowledge, however, no information about the prevalence and clinical characteristics of these tumors in Latin America, including Brazil, is available in the literature.

In the present study, 3.2% (2 out of 62) of NSCLC cases in Brazilian patients were ALK fusion positive by FISH. These two male patients were younger than those with ALKnegative FISH, had a history of no or light smoking, and their tumors presented invasive adenocarcinoma histology similar to that described in the literature. Both tumors were histologically classified as acinar predominant: one tumor had papillary areas, and the other was rich in mucinous areas.

Consistent with the results of other studies (13,14,23,3335), a high concordance between FISH and immunohistochemistry results for the detection of ALK-positive NSCLCs was found in the present study. Although FISH remains the gold standard technique for the diagnosis of ALK-rearranged lung carcinoma, immunohistochemistry may play an important role in the determination of ALK status in NSCLC in the near future. Because data regarding the role of immunohistochemistry in the detection of ALK-positive lung cancer are limited in the literature, additional studies are necessary.

The use of crizotinib has changed the history of NSCLC treatment, with dramatic results in patients with ALKrearranged tumors. A recent phase I/II clinical trial showed that 47 of 82 (57%) patients with ALK fusion-positive lung tumors confirmed by FISH had objective responses to crizotinib, and an additional 27 (33%) patients displayed stable disease (9). These data highlight that the diagnosis of ALK-rearranged NSCLC is crucial.

As previously stated, the present study is the first in Latin America to present clinicopathologic information regarding ALK-rearranged NSCLC in patients from this geographic area. Further studies are encouraged to expand our knowledge on ALK-positive NSCLC in Latin America and Brazil.

DISCUSSION Clinical characteristics associated with ALK rearrangement in NSCLC include adenocarcinoma histology, younger age at diagnosis, and non- or light smoker status, similar to those described for EGFR mutations. The prevalence of NSCLCs with ALK fusion varies from 2% to 8% in the literature, and most studies have been conducted in Asian countries or in the United States of America. Table 1 presents the frequency of ALK-rearranged NSCLC in several countries. In Japan, the prevalence of lung cancer with ALK rearrangement varied widely, from 2.4% to 6.7% (2,16,19). In Korea, the frequency of ALK-positive NSCLC ranged from 3.7% to 4.2% (11,13,14). In China, it was approximately 5% (4,12,15). In the USA, the prevalence varied from 5.6 to 8.2%

Figure 1 - ALK-rearranged NSCLCs. A. Invasive adenocarcinoma, acinar predominant with papillary areas, in a 36-year-old male patient (case 1). B. FISH of case 1 showing one isolated orange signal along with one fusion signal (positive for ALK rearrangement). C. ALK expression in case 1: diffuse and intense cytoplasmic immunostaining. D. Invasive adenocarcinoma, acinar predominant with mucinous areas, in a 45-year-old male patient (case 2). E. FISH of case 2 showing split orange and green signals along with one fusion signal (positive for ALK rearrangement). F. ALK expression in case 2: intense cytoplasmic immunostaining in virtually all tumor cells.

AUTHOR CONTRIBUTIONS Bacchi CE was responsible for designing and supervising the project, and writing and revising the manuscript. Lopes LF was responsible for data collection, data analysis, and writing the manuscript. All authors approved the final version of the manuscript.

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REFERENCES 21.

1. Siegel R, Ward E, Brawley O, Jemal A. Cancer statistics, 2011: the impact of eliminating socioeconomic and racial disparities on premature cancer deaths. CA Cancer J Clin. 2011;61(4):212-36, http://dx.doi.org/10.3322/ caac.20121.

2. Soda M, Choi YL, Enomoto M, Takada S, Yamashita Y, Ishikawa S, et al.

Identification of the transforming EML4-ALK fusion gene in non-smallcell lung cancer. Nature. 2007;448(7153):561-6, http://dx.doi.org/ 10.1038/nature05945.

3. Soda M, Takada S, Takeuchi K, Choi YL, Enomoto M, Ueno T, et al. A mouse model for EML4-ALK-positive lung cancer. Proc Natl Acad Sci U S A. 2008;105(50):19893-7, http://dx.doi.org/10.1073/pnas.0805381105.

4. Wong DW, Leung EL, So KK, Tam IY, Sihoe AD, Cheng LC, et al. The EML4-ALK fusion gene is involved in various histologic types of lung cancers from nonsmokers with wild-type EGFR and KRAS. Cancer.

2009;115(8):1723-33, http://dx.doi.org/10.1002/cncr.24181.

5. Inamura K, Takeuchi K, Togashi Y, Hatano S, Ninomiya H, Motoi N, et al.

EML4-ALK lung cancers are characterized by rare other mutations, a TTF-1 cell lineage, an acinar histology, and young onset. Mod Pathol.

2009;22(4):508-15, http://dx.doi.org/10.1038/modpathol.2009.2.

6. Boland JM, Erdogan S, Vasmatzis G, Yang P, Tillmans LS, Johnson MR, et al. Anaplastic lymphoma kinase immunoreactivity correlates with ALK gene rearrangement and transcriptional up-regulation in nonsmall-cell lung carcinomas. Hum Pathol. 2009;40(8):1152-8, http:// dx.doi.org/10.1016/j.humpath.2009.01.012.

7. Takeuchi K, Choi YL, Soda M, Inamura K, Togashi Y, Hatano S, et al.

Multiplex reverse transcription-PCR screening for EML4-ALK fusion transcripts. Clin Cancer Res. 2008;14(20):6618-24, http://dx.doi.org/ 10.1158/1078-0432.CCR-08-1018.

8. Solomon B, Varella-Garcia M, Camidge DR. ALK gene rearrangements: a new therapeutic target in a molecularly defined subset of non-small-cell lung cancer. J Thorac Oncol. 2009;4(12):1450-4, http://dx.doi.org/ 10.1097/JTO.0b013e3181c4dedb.

9. Kwak EL, Bang YJ, Camidge DR, Shaw AT, Solomon B, Maki RG, et al.

Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer.

N Engl J Med. 2010;363(18):1693-703, http://dx.doi.org/10.1056/ NEJMoa1006448.

10. Mino-Kenudson M, Mark EJ. Reflex testing for epidermal growth factor receptor mutation and anaplastic lymphoma kinase fluorescence in situ hybridization in non-small-cell lung cancer. Arch Pathol Lab Med. 2011;135(5):655-64.

11. Paik JH, Choi CM, Kim H, Jang SJ, Choe G, Kim DK, et al.

Clinicopathologic implication of ALK rearrangement in surgically resected lung cancer: a proposal of diagnostic algorithm for ALKrearranged adenocarcinoma. Lung Cancer. 2012;76(3):403-9, http:// dx.doi.org/10.1016/j.lungcan.2011.11.008.

12. Li C, Fang R, Sun Y, Han X, Li F, Gao B, et al. Spectrum of oncogenic driver mutations in lung adenocarcinomas from East Asian never smokers. PLoS One. 2011;6(11):e28204, http://dx.doi.org/10.1371/ journal.pone.0028204.

13. Kim H, Yoo SB, Choe JY, Paik JH, Xu X, Nitta H, et al. Detection of ALK gene rearrangement in non-small- cell lung cancer: a comparison of fluorescence in situ hybridization and chromogenic in situ hybridization with correlation of ALK protein expression. J Thorac Oncol.

2011;6(8):1359-66, http://dx.doi.org/10.1097/JTO.0b013e31821cfc73.

14. Paik JH, Choe G, Kim H, Choe JY, Lee HJ, Lee CT, et al. Screening of anaplastic lymphoma kinase rearrangement by immunohistochemistry in non-small-cell lung cancer: correlation with fluorescence in situ hybridization. J Thorac Oncol. 2011;6(3):466-72, http://dx.doi.org/ 10.1097/JTO.0b013e31820b82e8.

15. Sun Y, Ren Y, Fang Z, Li C, Fang R, Gao B, et al. Lung adenocarcinoma from East Asian never-smokers is a disease largely defined by targetable oncogenic mutant kinases. J Clin Oncol. 2010;28(30):4616-20, http:// dx.doi.org/10.1200/JCO.2010.29.6038.

16. Takahashi T, Sonobe M, Kobayashi M, Yoshizawa A, Menju T, Nakayama E, et al. Clinicopathologic features of non-small-cell lung cancer with EML4-ALK fusion gene. Ann Surg Oncol. 2010;17(3):889-97, http://dx.doi.org/10.1245/s10434-009-0808-7.

17. Rodig SJ, Mino-Kenudson M, Dacic S, Yeap BY, Shaw A, Barletta JA, et al.

Unique clinicopathologic features characterize ALK-rearranged lung adenocarcinoma in the western population. Clin Cancer Res. 2009;15(16):5216-23, http://dx.doi.org/10.1158/1078-0432.CCR-09-0802.

18. Solomon B, Shaw AT. Are Anaplastic Lymphoma Kinase Gene Rearrangements in Non-small-cell lung Cancer Prognostic, Predictive, or Both? J Thorac Oncol. 2012;7(1):5-7, http://dx.doi.org/10.1097/JTO. 0b013e31823f1289.

19. Sakairi Y, Nakajima T, Yasufuku K, Ikebe D, Kageyama H, Soda M, et al.

EML4-ALK fusion gene assessment using metastatic lymph node samples obtained by endobronchial ultrasound-guided transbronchial needle aspiration. Clin Cancer Res. 2010;16(20):4938-45, http:// dx.doi.org/10.1158/1078-0432.CCR-10-0099.

20. Yoshida A, Tsuta K, Nakamura H, Kohno T, Takahashi F, Asamura H, et al. Comprehensive histologic analysis of ALK-rearranged lung

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

847

carcinomas. Am J Surg Pathol. 2011;35(8):1226-34, http://dx.doi.org/ 10.1097/PAS.0b013e3182233e06.

Zhang X, Zhang S, Yang X, Yang J, Zhou Q, Yin L, et al. Fusion of EML4 and ALK is associated with development of lung adenocarcinomas lacking EGFR and KRAS mutations and is correlated with ALK expression. Mol Cancer. 2010;13(9):188, http://dx.doi.org/10.1186/ 1476-4598-9-188.

Sasaki T, Rodig SJ, Chirieac LR, Janne PA. The biology and treatment of EML4-ALK non-small-cell lung cancer. Eur J Cancer. 2010;46(10):1773-80, http://dx.doi.org/10.1016/j.ejca.2010.04.002.

Yang P, Kulig K, Boland JM, Erickson-Johnson MR, Oliveira AM, Wampfler J, et al. Worse disease-free survival in never-smokers with ALK+ lung adenocarcinoma. J Thorac Oncol. 2012;7(1):90-7, http:// dx.doi.org/10.1097/JTO.0b013e31823c5c32.

Koivunen JP, Mermel C, Zejnullahu K, Murphy C, Lifshits E, Holmes AJ, et al. EML4-ALK fusion gene and efficacy of an ALK kinase inhibitor in lung cancer. Clin Cancer Res. 2008;14(13):4275-83, http://dx.doi.org/ 10.1158/1078-0432.CCR-08-0168.

Hallberg B, Palmer RH. Crizotinib--latest champion in the cancer wars?

N Engl J Med. 2010;363(18):1760-2, http://dx.doi.org/10.1056/ NEJMe1010404.

Trial watch: success for crizotinib in ALK-driven cancer. Nat Rev Drug Discov. 2010;9(12):908.

Ou SH. Crizotinib: a novel and first-in-class multitargeted tyrosine kinase inhibitor for the treatment of anaplastic lymphoma kinase rearranged non-small-cell lung cancer and beyond. Drug Des Devel Ther. 2011;5:471-85, http://dx.doi.org/10.2147/DDDT.S19045.

Shaw AT, Solomon B, Kenudson MM. Crizotinib and testing for ALK. J Natl Compr Canc Netw, 2011;9(12):1335-41.

Ou SH, Bazhenova L, Camidge DR, Solomon BJ, Herman J, Kain T, et al.

Rapid and dramatic radiographic and clinical response to an ALK inhibitor (crizotinib, PF02341066) in an ALK translocation-positive patient with non-small-cell lung cancer. J Thorac Oncol. 2010;5(12): 2044-6, http://dx.doi.org/10.1097/JTO.0b013e318200f9ff.

Freeman A, Geddes N, Munson P, Joseph J, Ramani P, Sandison A, et al.

Anaplastic lymphoma kinase (ALK 1) staining and molecular analysis in inflammatory myofibroblastic tumours of the bladder: a preliminary clinicopathological study of nine cases and review of the literature.

Mod Pathol. 2004;17(7):765-71, http://dx.doi.org/10.1038/modpathol.

3800078.

Shaw AT, Yeap BY, Mino-Kenudson M, Digumarthy SR, Costa DB, Heist RS, et al. Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK. J Clin Oncol. 2009;27(26):4247-53, http://dx.doi.org/10.1200/JCO.2009.22.6993.

Martelli MP, Sozzi G, Hernandez L, Pettirossi V, Navarro A, Conte D, et al. EML4-ALK rearrangement in non-small-cell lung cancer and nontumor lung tissues. Am J Pathol. 2009;174(2):661-70, http://dx.doi.org/ 10.2353/ajpath.2009.080755.

Yi ES, Boland JM, Maleszewski JJ, Roden AC, Oliveira AM, Aubry MC, et al. Correlation of IHC and FISH for ALK gene rearrangement in nonsmall-cell lung carcinoma: IHC score algorithm for FISH. J Thorac Oncol.

2011;6(3):459-65, http://dx.doi.org/10.1097/JTO.0b013e318209edb9.

McLeer-Florin A, Moro-Sibilot D, Melis A, Salameire D, Lefebvre C, Ceccaldi F, et al. Dual IHC and FISH testing for ALK gene rearrangement in lung adenocarcinomas in a routine practice: A French study. J Thorac Oncol. 2012;7(2):348-54, http://dx.doi.org/10.1097/JTO.0b013e318238 1535.

Hofman P, Ilie M, Hofman V, Roux S, Valent A, Bernheim A, et al.

Immunohistochemistry to identify EGFR mutations or ALK rearrangements in patients with lung adenocarcinoma. Ann Oncol. 2011 Nov 18.

[Epub ahead of print] McLeer-Florin A, Moro-Sibilot D, Melis A, Salameire D, Lefebvre C, Ceccaldi F, de Fraipont F, Brambilla E, Lantuejoul S. Dual IHC and FISH testing for ALK gene rearrangement in lung adenocarcinomas in a routine practice: a French study. J Thorac Oncol. 2012;7(2):348-54, http:// dx.doi.org/10.1097/JTO.0b013e3182381535.

Travis WD, Brambilla E, Noguchi M, Nicholson AG, Geisinger KR, Yatabe Y, et al. International association for the study of lung cancer/ american thoracic society/european respiratory society international multidisciplinary classification of lung adenocarcinoma. J Thorac Oncol.

2011;6(2):244-85, http://dx.doi.org/10.1097/JTO.0b013e318206a221.

Tsao AS, Tang XM, Sabloff B, Xiao L, Shigematsu H, Roth J, et al.

Clinicopathologic characteristics of the EGFR gene mutation in nonsmall-cell lung cancer. J Thorac Oncol. 2006;1(3):231-9.

Shigematsu H, Lin L, Takahashi T, Nomura M, Suzuki M, Wistuba II, et al. Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers. J Natl Cancer Inst.

2005;97(5):339-46, http://dx.doi.org/10.1093/jnci/dji055.

Riely GJ, Marks J, Pao W. KRAS mutations in non-small-cell lung cancer.

Proc Am Thorac Soc. 2009;6(2):201-5, http://dx.doi.org/10.1513/ pats.200809-107LC. Inamura K, Ninomiya H, Ishikawa Y, Matsubara O. Is the epidermal growth factor receptor status in lung cancers reflected in clinicopathologic features? Arch Pathol Lab Med. 2010;134(1):66-72.

CLINICS 2012;67(7):849-851

DOI:10.6061/clinics/2012(07)24

CASE REPORT

The surgical removal of a live tapeworm with an interesting pathologic finding most likely representing the migration path: a case report of cerebral sparganosis Peng Wang,I,II Xueying Su,III Qing Mao,I Yanhui LiuI I Sichuan University, West China Hospital, Department of Neurosurgery, Chengdu, China. II The Fifth People’s Hospital of Chengdu, Department of Neurosurgery, Chengdu, China. III Sichuan University, West China Hospital, Department of Pathology, Chengdu, China.

Email: liuyanhui9@gmail.com Tel.: 8618980601509

Therefore, we hypothesized that the lesion was a live tapeworm. An operation was performed, and a light yellow lesion was identified in the posterior portion of the superior temporal gyrus with a live worm inside it. The worm was milky white, approximately 12cm long and could twist its body under stimulus (Figure 2).

The histology of the larval cestode showed a brush border and eosinophilic smooth muscle fibers (Figure 3A). Some portions of the parasite body contained calcareous bodies (Figure 3B). A new tunnel-like structure with infiltrating plasma cells and eosinophils was identified in the tissue (Figure 3C). Under higher power, calcareous bodies were also observed within the tunnel (Figure 3D). Based on the pathological findings, the patient history and serum test results, a final diagnosis of cerebral sparganosis was made.

During a follow-up period of more than two years, the patient’s symptoms disappeared. No enhanced lesion was found on MRI at 26 months after the operation (Figure 1 C1 and C2).

INTRODUCTION Cerebral sparganosis is a rare parasitic disease of the central nervous system that is caused by sparganum.

Cerebral sparganosis occurs worldwide, but it is found more frequently in China, Japan, and Southeastern Asia (12). Sparganum, the second-stage larva of Spirometra mansoni, can produce chronic active inflammation through slow migration in the brain (1). They can also parasitize other sites of the human body, such as the subcutaneous tissue, body cavities and eyes, and survive for 5-20 years (3).

In this paper, we report a case of cerebral sparganosis, including the magnetic resonance imaging (MRI) characteristics of local migration and the surgical finding of a live worm. A new pathological finding, a tunnel-like structure, was observed and most likely represented a sparganum migration path.

CASE DESCRIPTION A fifteen-year-old girl suffered from facial and upper limb numbness on her right side one year ago. She was previously diagnosed with encephalitis at the Department of Neurology until she suffered a generalized tonic–clonic seizure six months later. MRI showed a contrast-enhanced lesion with an irregular shape located in the left frontal lobe (Figure 1 A1 and A2). The patient was advised to undergo further treatment in the Department of Neurosurgery.

However, because the seizure was acutely controlled, she was noncompliant with treatment. One week before admission, the seizure became uncontrollable. An MRI revealed a contrast-enhanced lesion with an irregular shape (Figure 1 B1 and B2); however, local migration of the lesion was observed.

After admission, the patient’s serum was tested by ELISA, which was positive for sparganum. The patient mentioned that she had eaten inadequately cooked frog flesh.

Figure 1 - A series of pre-operational MR images demonstrates the local migration of an irregular, enhanced lesion (arrow) in the left hemisphere (A1-A2) six months before the operation; (B1-B2) three days before the operation. The lesion disappears in the post-operation MR images over the two-year follow-up period (C1-C2) 26 months after the operation.

Copyright ß 2012 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.

No potential conflict of interest was reported.

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An interesting pathologic finding in cerebral sparganosis Wang P et al.

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Figure 2 - A live tapeworm with the scolex, measuring approximately 12 cm in length, extracted from the patient (arrow).

DISCUSSION The clinical manifestations of cerebral sparganosis include seizure, hemiparesis, and headache with a chronic course, in which seizure is the most common symptom (1,4).

In our case, the patient first presented with sensation disorders on her right side and later with seizure. Serum ELISA is sensitive against sparganum and was essential evidence in our patient’s diagnosis.

The characteristics of cerebral sparganosis on MRI include the following: A) degeneration of the cerebral white matter with edema and ventricle dilation; B) a contrast-enhanced lesion with an irregular shape, a string-of-beads shape or tunneling signs; and C) local or contralateral migration of the lesion or a change of the shape in follow-up MRI studies (1-2,5-6). Migration may suggest a live tapeworm (1,7-8), which was observed and confirmed in this case. Other infectious granulomas, primary or secondary neoplasms, chronic infarctions, and degenerative diseases should be excluded from the diagnosis (4). Rengarajan et al. (9) indicated that cerebral sparganosis mimicked a tuberculoma on imaging, which should be included in the differential diagnosis. The patient in this case was previously diagnosed with encephalitis, which should also be included in the differential diagnosis.

The main pathologic findings in cerebral sparganosis include larval cestodes with a brush border and eosinophilic smooth muscle fibers and granulomas with calcareous bodies surrounded by plasma cells and eosinophils (1,6-8,10). In our case, a new tunnel-like structure with infiltrating plasma cells and eosinophils was found in addition to the previously mentioned structures. Calcareous bodies were also observed within the tunnel under higher power. This tunnel-like structure was most likely the migration path of the sparganum, suggesting that migration phenomena may be significant histological evidence.

Neurocysticercosis is the most common parasitic disease of the human central nervous system, and it should be considered as a very important differential diagnosis in cases such as the present one. Similar to cerebral sparganosis, neurocysticercosis typically develops following oral ingestion of the parasite, and seizures and headache are the most commonly encountered symptoms (11). However, several factors can differentiate between the two infections: A) there are some highly suggestive radiological findings for neurocysticercosis, such as cyst structures (containing a scolex), enhancing lesions, and parenchymal calcifications (12); B) the detection of Taenia solium antigens or anti-T. solium antibodies in serum or cerebrospinal fluid can suggest the presence of neurocysticercosis (13); and C) cysticerci in different stages that are associated with inflammatory responses can be found upon microscopic examination.

Figure 3 - Histological findings of the larval cestode and lesion. (A) Photomicrograph of the live larval showing a brush border and eosinophilic smooth muscle fibers (H&E 4X). (B) Photomicrograph showing calcareous bodies (H&E 40X). (C) A tunnel-like structure surrounded by plasma cells and eosinophils. (H&E 10X). (D) Calcareous bodies were observed within the tunnel. (H&E 20X).

Surgical resection of the lesion, including the tapeworm and the granuloma, is considered to be an effective treatment for cerebral sparganosis. Patients with total lesion excision can achieve good long-term outcomes (1). Many researchers (1-2,7-8) recommend surgery for patients with a live worm and signs of migration on MR images because this can prevent further damage caused by disease progression. Drug therapy, such as praziquantel, has a limited effect because live tapeworms are not affected (1). In this case, the operation was performed because local migration that was suggestive of a live worm was observed on serial MR images. The patient’s symptoms disappeared, and recurrence was not observed over a follow-up period of more than two years.

In conclusion, the observed migration was an important radiological sign for preoperative diagnosis, identification of tapeworm survival, and judgment of surgical indication.

The tunnel-like structure demonstrated by pathology might be an important diagnostic clue for cerebral sparganosis.

AUTHOR CONTRIBUTIONS Wang P drafted the manuscript, reviewed the literature, and participated in the operation. Su XY made the histopathologic diagnosis. Mao Q and Liu YH performed the operation and revised the manuscript.

REFERENCES 1. Kim DG, Paek SH, Chang KH, Wang KC, Jung HW, Kim HJ, et al.

Cerebral sparganosis: clinical manifestations, treatment, and outcome.

J Neurosurg. 1996;85(6):1066-71.

2. Moon WK, Chang KH, Cho SY, Han MH, Cha SH, Chi JG, et al. Cerebral sparganosis: MR imaging versus CT features. Radiology. 1993;188(3):7517.

3. Cho SY, Bae JH, Seo BS. Some Aspects Of Human Sparganosis In Korea.

Kisaengchunghak Chapchi. 1975;13(1):60-77.

4. Chang KH, Chi JG, Cho SY, Han MH, Han DH, Han MC. Cerebral sparganosis: analysis of 34 cases with emphasis on CT features.

Neuroradiology. 1992;34(1):1-8, http://dx.doi.org/10.1007/BF00588423.

5. Song T, Wang WS, Zhou BR, Mai WW, Li ZZ, Guo HC, et al. CT and MR characteristics of cerebral sparganosis. AJNR Am J Neuroradiol.

2007;28(9):1700-5, http://dx.doi.org/10.3174/ajnr.A0659.

6. Kim IY, Jung S, Jung TY, Kang SS, Chung TW. Contralateral migration of cerebral sparganosis through the splenium. Clin Neurol Neurosurg.

2007;109(8):720-4, http://dx.doi.org/10.1016/j.clineuro.2007.05.014.

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An interesting pathologic finding in cerebral sparganosis Wang P et al.

11. Carabin H, Ndimubanzi PC, Budke CM, Nguyen H, Qian Y, Cowan LD, et al. Clinical manifestations associated with neurocysticercosis: a systematic review. PLoS Negl Trop Dis. 2011;5(5):e1152, http:// dx.doi.org/10.1371/journal.pntd.0001152.

12. Moskowitz J, Mendelsohn G. Neurocysticercosis. Arch Pathol Lab Med. 2010;134(10):1560-3.

13. Rodriguez S, Dorny P, Tsang VC, Pretell EJ, Brandt J, Lescano AG, et al.

Detection of Taenia solium antigens and anti-T. solium antibodies in paired serum and cerebrospinal fluid samples from patients with intraparenchymal or extraparenchymal neurocysticercosis. J Infect Dis. 2009;199(9):1345-52.

7. Murata K, Abe T, Gohda M, Inoue R, Ishii K, Wakabayashi Y, et al. Difficulty in diagnosing a case with apparent sequel cerebral sparganosis. Surg Neurol. 2007;67(4):409-11, http://dx.doi.org/10.1016/j.surneu.2006.06.056.

8. Bo G, Xuejian W. Neuroimaging and pathological findings in a child with cerebral sparganosis. Case report. J Neurosurg. 2006;105(6 Suppl):470-2.

9. Rengarajan S, Nanjegowda N, Bhat D, Mahadevan A, Sampath S, Krishna S. Cerebral sparganosis: a diagnostic challenge. Br J Neurosurg.

2008;22(6):784-6, http://dx.doi.org/10.1080/02688690802088073.

10. Cummings TJ, Madden JF, Gray L, Friedman AH, McLendon RE. Parasitic lesion of the insula suggesting cerebral sparganosis: case report. Neuroradiology. 2000;42(3):206-8, http://dx.doi.org/10.1007/s002340050047.

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DOI:10.6061/clinics/2012(07)25

CASE REPORT

Giant obstructive left atrial myxoma resembling mitral valve stenosis Solange Desire´e Avakian, Julio Yoshio Takada, Antonio de Padua Mansur Hospital das Clinicas da Faculdade de Medicina da Universidade de Sa˜o Paulo Medical, Heart Institute (InCor), Sa˜o Paulo/SP, Brazil.

Email: apmansur@usp.br Tel.: 55 11 2661-5387

flow, and an atrium-ventricular pressure gradient of 11 mmHg (Figure 1). This mass was attached to the inferior edge of the fossa ovalis. Left ventricular function was normal, and the right ventricular diameter was 3.2 cm. His systolic pulmonary artery pressure was 100 mmHg. The patient was referred for cardiac surgery. The adopted technique was a biatrial approach. The intraoperative findings included a mass, and anatomopathologic analysis confirmed that it was an atrial myxoma. Two days after surgery, an echodopplercardiogram showed an important reduction in systolic pulmonary artery pressure to 30 mmHg. Five years later, an echocardiogram showed no mass in the left atrial chamber.

INTRODUCTION Primary tumors of the heart are rare, with an incidence between 0.0017% and 0.19% in unselected patients at autopsy. Approximately 75% of the tumors are benign, and nearly half of them are myxomas (1). Atrial myxoma is the most common benign cardiac neoplasm, and its origin has been ascribed to a multipotential mesenchymal cell (2).

Myxomas commonly occur between the third and sixth decades of life and are three times more frequent in women (3). There are two distinctive forms of the tumors: solid and ovoid myxomas and soft and papillary myxomas. Solid tumors are more likely to present with symptoms of congestive heart failure (CHF), while papillary tumors are more likely to embolize to the cerebral and other peripheral vessels (4). At the time of diagnosis, 92% of the patients have symptoms of CHF (5). Atrial myxoma may mimic valvular heart disease, cardiac insufficiency, cardiomegaly, bacterial endocarditis, disturbances of ventricular and supraventricular rhythm, syncope, and systemic or pulmonary embolism (3). The purpose of this paper is to present a case report of atrial myxoma mimicking severe mitral valve stenosis at cardiac auscultation and the importance of echocardiograpy in the diagnosis.

DISCUSSION This case report showed an atrial myxoma mimicking mitral valve stenosis. The diagnosis was made by echocardiogram, which disclosed a huge mass obstructing the left atrial blood flow. Heart valve disease is one of the differential diagnoses of atrial myxomas. The clinical findings of cardiac tumors are characterized by the triad of embolism, intracardiac obstruction, and constitutional findings. Congestive heart failure is the most common clinical presentation, followed by neurologic and systemic embolic events. Embolic events more frequently affect the cerebral arteries, including the retinal arteries.

Embolization into visceral, renal, or coronary arteries has also been reported and can affect nearly 29% of patients (3).

Although the large majority of myxomatous emboli are cerebral, it is possible for atrial myxomas to embolize and implant in other locations in the visceral and peripheral arterial circulation. Ectopic myxoma implants have been described in other unusual locations, including the skin, the skeleton, and the small bowel. Intracardiac obstruction with impaired filling of the left or right ventricle causes subsequent dyspnea, recurrent pulmonary edema, rightheart failure or even syncope or sudden death (6). Three fourths of myxomas are located in the left atrium (7).

General or constitutional manifestations, such as fatigue, fever, weight loss, night sweats, arthralgia, and laboratory abnormalities, such as elevation of C-reactive protein, anemia, and the erythrocyte sedimentation rate, can also occur.

Mitral valve obstruction caused by atrial myxoma represents an important hemodynamic consequence leading to symptoms of congestive heart failure, pulmonary hypertension, syncope, and sudden death. Clinicopathologic correlations showed that mitral stenotic effects occurred when the tumor diameter exceeded 5 cm (8). Echodopplercardiography

CASE DESCRIPTION A 77-year-old Caucasian man presented at the Emergency Care Unit with a six-month history of congestive heart failure and in funcional class III, according to the New York Heart Association (NYHA) criteria. He had no previous history of diabetes, arterial hypertension, or ischemic coronary disease. The physical examination disclosed distended neck veins, rales at the lung base, and hepatomegaly.

Cardiac auscultation revealed a loud first heart sound and diastolic rumble resembling mitral valve stenosis. Chest radiography showed a normal cardiac silhouette image, enlarged hilar shadows and moderate right-sided pleural effusion. The electrocardiogram showed a regular sinus rhythm with a nonspecific repolarization alteration. The transthoracic echocardiogram showed an image of a large pediculate mass of 5.5 mm64.0 mm in the left atrium in his greater diameters, restricting transmitral diastolic blood

Copyright ß 2012 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.

No potential conflict of interest was reported.

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Figure 1 - Giant obstructive left atrial mixoma (arrows) in systole and diastole.

is the most important method for the differential diagnosis with primary valve disease. Moreover, it can estimate the transvalve gradient. It has been reported that patients with left atrial myxomas have reduced transmitral blood flow.

Nevertheless, only large myxomas can resemble severe mitral stenosis. The differential diagnosis of an intracardiac mass encompasses benign and malignant primary heart tumors, metastatic tumors and thrombi. Eletrocardiographic findings are unspecific. The cardiac rhythm is usually normal. Chest Xray may reveal an enlargement of the left atrium and some signs of pulmonary hypertension and congestion. Calcifications, which make the tumor visible on routine radiographic examination, are unusual. Computed tomography and magnetic resonance imaging can differentiate tissue composition, making it possible to identify the nature of the mass. The preoperative diagnosis of cardiac tumors could be performed correctly utilizing angiography, echocardiography, or both. Coronary angiography is reserved for older patients who may have coexisting coronary artery disease (9).

Controversy remains regarding the optimal operative approach to atrial myxomas. The biatrial approach has been advocated to achieve the most complete tumor excision and assess the possibility of tumors in the right atrium (10).

Other options include minimally invasive thoracoscopic surgery and the use of robotic technology. Surgical excision of the tumor appears to be curative, and recurrence is rare, as shown five years later in an echocardiogram of this patient.

AUTHOR CONTRIBUTIONS All of the authors participated in manuscript preparation, data collection and data interpretation. All of the authors read and approved the final manuscript.

REFERENCES 1. Silverman NA. Primary cardiac tumors. Ann Surg. 1980;191(2):127-138, http://dx.doi.org/10.1097/00000658-198002000-00001.

2. Johansson L. Histogenesis of cardiac myxomas. An immunohistochemical study of 19 cases including one with glandular structures and review of the literature. Arch Pathol Lab Med. 1989;113(7):735-41.

3. Reynen K. Cardiac myxomas. N Eng J Med. 1995;333(24):1610–7, http:// dx.doi.org/10.1056/NEJM199512143332407.

4. Shimono T, Makino S, Kanamori Y, Kinoshita T, Yada I. Left atrial myxomas: Using gross anatomic tumor types to determine clinical features and coronary angiographic findings. Chest. 1995;107(3):674-679, http:// dx.doi.org/10.1378/chest.107.3.674.

5. Swartz MF, Lutz CJ, Chandan VS, Landas S, Fink GW. Atrial Myxomas: Pathologic Types, Tumor Location, and Presenting Symptoms. J Card Surg.

2006;21(4):435-40, http://dx.doi.org/10.1111/j.1540-8191.2006.00265.x.

6. Jung R, Ghahramani A, Mallon S, Richter S, Sommer L, Gottlieb S, Myerburg R. Hemodynamic features of prolapsing and nonprolapsing left atrial myxoma. Circulation. 1975;51(5):342–9.

7. Schaff HV, Mullany CJ. Surgery for cardiac myxomas. Semin Thorac Cardiovasc Surg. 2000;12(2):77-88.

8. Pucci A, Gagliardotto P, Zanini C, Pansini S, di Summa M, Mollo F. Histopathologic and clinical characterization of cardiac myxoma: review of 53 cases from a single institution. Am Heart J. 2000;140(1):134–8, http://dx.doi.org/10.1067/mhj.2000.107176.

9. Larsson S, Lepore V, Kennergren C. Atrial myxomas: Results of 25 years’ experience and review of the literature. Surgery. 1989;105(6):695-8.

10. Jones DR, Warden HE, Murray GF, Hill RC, Graeber GM, Cruzzavala JL, et al. Biatrial approach to cardiac myxomas: A 30-year clinical experience.

Ann Thorac Surg. 1995;59(4):851-6, http://dx.doi.org/10.1016/00034975(95)00064-R.

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DOI:10.6061/clinics/2012(07)26

CASE REPORT

Spinal hematoma complicating a Churg-Strauss syndrome patient: a previously unreported association Camila Cristina Kukita,I Andre´ Luiz Nunes Gobatto,I Alice Zoghbi Lobo,II Leandro Utino TaniguchiI,III I Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Discipline of Emergency Medicine, Sa˜o Paulo/SP, Brazil. II Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Department of Dermatology, Sa˜o Paulo/SP, Brazil. III Hospital Sı´rio Libaneˆs, Intensive Care Unit, Sa˜o Paulo/SP, Brazil.

Email: leandro.taniguchi@gmail.com Tel.: 55 11 2661 6336

thromboplastin time of 30 s). Perinuclear anti-neutrophil cytoplasmic antibodies and low titers of FAN-HEP2 were detected in her serum. A computed tomography (CT) scan exhibited bilateral maxillary, ethmoid and sphenoid sinus opacification and diffuse lung infiltrates. A skin biopsy demonstrated predominant extravascular eosinophils and necrotizing vasculitis (Figure 1). A diagnosis of ChurgStrauss syndrome was confirmed, and corticosteroid treatment was initiated.

The next morning, the patient awoke with urinary incontinence and an inability to move her legs. She maintained consciousness and had no pain or respiratory or cardiac distress. Her vital signs were normal. A neurological examination revealed complete paraplegia.

Neurological and neurosurgical consultants were contacted, and emergency magnetic resonance imaging (MRI) was ordered. MRI of the thoracic spine revealed a hyperintense mass on the posterior topography, causing spinal cord compression from T5-T6 to T7-T8 (Figure 2).

The compressed spinal cord already demonstrated signal alteration on T2-weighted images. Based on her physical examination and her image findings, a hematoma of the thoracic spine was suspected, and she was taken to the operating room for an emergency decompression laminectomy. During the operation, a subdural hematoma was discovered and removed. The pathologic report revealed a hematoma, but no blood vessels were present in the specimen to analyze whether vasculitis was present. After the surgery, she was admitted to the Intensive Care Unit (ICU). However, the patient was unable to move or feel her legs, and the urinary incontinence persisted. The patient continued on 1 g of methylprednisolone intravenously for three days.

Her postoperative course was further complicated on the 6th day, when she developed surgical site infection and meningitis. She received appropriate antibiotic therapy.

No microorganisms were identified. No new neurological symptoms appeared, and she was discharged without paraplegia improvement. After six months, she still had not recovered from the paraplegia or recovered from urinary retention. The asthma symptoms, eosinophilia, visual complaints, renal function (0.65 mg/dl) and paresthesia in her arms improved.

INTRODUCTION Churg-Strauss syndrome (CSS) is a systemic disease characterized by an eosinophil-rich and granulomatous inflammation involving the respiratory tract, necrotizing eosinophilic vasculitis affecting small- to medium-sized vessels, and an association with asthma and eosinophilia (1,2). Neurological involvement in CSS occurs in approximately 62% of patients, usually as peripheral neuropathy (3). Nevertheless, central nervous system (CNS) involvement is rare. Cerebral infarctions, subarachnoid hemorrhage, intracerebral hemorrhage and optic neuritis are reported events (4-8).

Spinal hematoma is a rare pathology [Kreppel et al. identified only 613 cases over 170 years of medical literature (9)]. The classic presentation is an acute onset of severe, radiating back pain followed by the signs and symptoms of spinal cord compression, which develop minutes to days later (9-11). The true etiology remains unknown, but associations with predisposing conditions, such as blood dyscrasias and arteriovenous malformation, have been reported (9,12). No previous spinal hemorrhage has been described in association with CSS. In this case report, we describe a 40-year-old woman with CSS presenting with a spinal hematoma.

CASE DESCRIPTION A previously healthy 40-year-old woman was admitted to the general ward due to recent asthma symptoms, purpuric skin lesions, and intermittent paresthesia and paresis in both arms and legs, which were suggestive of multiplex mononeuritis. She also complained of diminished visual acuity. The relevant initial laboratory tests revealed a slightly increased creatinine value (1.55 mg/dl) and elevated leukocytes (24,520/mm3) due to eosinophilia (15,000/ mm3). A urinary sediment evaluation demonstrated microscopic hematuria (.100 red cells per high-power field) and leukocyturia (30 leukocytes per high-power field). A coagulation evaluation revealed no remarkable contribution (platelets 459,000/mm3, prothrombin activity 75%, partial

Copyright ß 2012 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.

DISCUSSION The American College of Rheumatology has established six criteria for the classification of CSS in a patient with docu-

No potential conflict of interest was reported.

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Spinal hematoma in Churg-Strauss syndrome Kukita CC et al.

CLINICS 2012;67(7):855-857

Figure 1 - Panel displaying the thoracic computed tomography findings and the skin histopathology. (A) The thoracic computed tomography scan demonstrates small nodular lesions with discrete ground-glass attenuation. (B) The skin biopsy (hematoxylin-eosin) demonstrates small-vessel vasculitis with eosinophils. (C) Magnification of the inflammatory infiltrate demonstrates the presence of numerous eosinophils (small arrow).

mented vasculitis: asthma, .10% eosinophilia in a differential white blood cell count, mononeuropathy (including multiplex) or polyneuropathy, non-fixed pulmonary infiltrates on radiography, paranasal sinus abnormality, and a biopsy showing blood vessels with extravascular eosinophils. The presence of four or more of these criteria reportedly yields a sensitivity of 85% and a specificity of 99.7% (13). Our patient could not be evaluated with a nerve conduction study to further characterize the symptoms clinically attributed to mononeuritis multiplex before the surgery and the corticosteroid treatment.

Nevertheless, she displayed at least five criteria (excluding the neuropathy). The visual alterations might be related to optic neuritis, which improved after corticosteroid treatment, as did the other CSS-related symptoms. Unfortunately, she also could not undergo any ophthalmologic diagnostic study to conclude that she indeed had optic neuritis before treatment. The overall prognosis for CSS is good, and treatment with prednisone is usually successful (1).

Although neurological involvement in CSS is common, with peripheral neuropathy present in 53–78% of the patients (1,3), central nervous system involvement is uncommon (6–10%) and usually presents as cerebral infarction and rarely with subarachnoid hemorrhage, intracerebral hemorrhage, optic neuritis, convulsions, coma or psychosis (1,2). To the best of our knowledge, no previous case of CSS associated

with spinal hematoma has been documented. Only three cases of subdural hematoma with ANCA-associated vasculitis (Wegener’s Granulomatosis) have been reported, and only one presented with a spinal subdural hematoma (14-16).

The majority of spinal hematomas (29.7%) have no etiological factor identified (9). After idiopathic spinal hematoma, cases related to anticoagulant therapy and vascular malformations represent the second and third most common categories (9). The present case had no history of trauma or other precipitating factors and no coagulation abnormality. Additionally, no vascular malformations were observed using MRI or CT. Unfortunately, the absence of vessels in the spinal specimen sent for pathologic evaluation precluded adequate study for evidence of vasculitis or eosinophilic infiltration. Nevertheless, because both CSS, characterized by an incidence of 0.11 to 2.66 new cases per million population per year, and spontaneous spinal hematoma are rare disorders [an estimated incidence of approximately one case per million patients per year (18)], we believe that a coincident association between these two conditions is unlikely.

In conclusion, we described a CSS case presenting with a novel neurological association, a spinal subdural hematoma, which, at the time of publication, had never been documented.

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Spinal hematoma in Churg-Strauss syndrome Kukita CC et al.

Figure 2 - Magnetic resonance imaging of the thoracic spine. Sagittal T2-weighted imaging reveals a hyperintense posterior mass extending from T5-T6 to T7-T8 (white arrows), which compresses the spinal cord anteriorly.

9. Kreppel D, Antoniadis G, Seeling W. Spinal hematoma: a literature survey with meta-analysis of 613 patients. Neurosurg Rev. 2003;26(1):149, http://dx.doi.org/10.1007/s10143-002-0224-y.

10. Liu WH, Hsieh CT, Chiang YH, Chen GJ. Spontaneous spinal epidural hematoma of thoracic spine: a rare case report and review of literature.

Am J Emerg Med. 2008;26(3):384.e1-2, http://dx.doi.org/10.1016/ j.ajem.2007.05.036.

11. Taniguchi LU, Pahl FH, Lu´cio JED, Brock RS, Gomes MQT, Adoni T, et al. Complete motor recovery after acute paraparesis caused by spontaneous spinal epidural hematoma: case reportBMC Emerg Med. 2011;11:10.

12. Groen RJ, Ponssen H. The spontaneous spinal epidural hematoma.

A study of the etiology. Journal of Neurological Sciences. 1990;98:12138.

13. Masi AT, Hunder GG, Lie JT, Michel BA, Bloch DA, Arend WP, et al. The American College of Rheumatology 1990 criteria for the classification of Churg-Strauss syndrome (allergic granulomatosis and angiitis). Arthritis Rheum. 1990;33(8):1094-100.

14. Guilfoyle MR, Khan S, Helmy A, Jalloh I, Trivedi S, Trivedi R, et al. Spinal intradural haemorrhage in a patient with Wegener’s Granulomatosis. Clin Neurol Neurosurg. 2010;112(4):341-3, http://dx.doi.org/10.1016/j.clineuro.

2009.12.004.

15. Yokote H, Terada T, Nakai K, Itakura T. Subdural and meningeal involvement related to Wegener’s Granulomatosis: Case Report.

Neurosurgery. 1997;40(5):1071-3; discussion 1073-4.

16. Shiotani A, Mukobayashi C, Oohata H, Yamanishi T, Hara T, Itoh H, et al.

Wegener’s Granulomatosis with dural involvement as the initial clinical manifestation. Internal Medicine. 1997;36(8):514-8, http://dx.doi.org/ 10.2169/internalmedicine.36.514.

17. Baldini C, Talarico R, Rossa AD, Bombardieri S. Clinical manifestations and treatment of Churg-Strauss syndrome. Rheum Dis Clin North Am. 2010;36(3):527-43, http://dx.doi.org/10.1016/j.rdc.2010.05.

003.

18. Holtas S, Heiling M, Lo¨ nntoft M. Spontaneous spinal epidural hematoma: findings at MR imaging and clinical correlation. Radiology. 1996;199:409-13.

AUTHOR CONTRIBUTIONS All of the authors participated in manuscript preparation, the literature search, data collection and data interpretation. All of the authors read and approved the final manuscript. This study was performed at the Emergency Department and Dermatology Department, Hospital das Clı´nicas, University of Sa˜o Paulo Medical School, Sa˜o Paulo, Brazil.

REFERENCES 1. Noth I, Strek ME, Leff AR. Churg-Strauss syndrome. Lancet. 2003; 361(9357):587–94, http://dx.doi.org/10.1016/S0140-6736(03)12518-4.

2. Sinico RA, Bottero P. Churg–Strauss angiitis. Best Pract Res Clin Rheumatol.

2009;23(3):355–66, http://dx.doi.org/10.1016/j.berh.2009.02.004.

3. Sehgal M, Swanson JW, DeRemee RA, Colby TV. Neurologic manifestations of Churg-Strauss syndrome. Mayo Clin Proc. 1995;70(4):337-41, http://dx.doi.org/10.4065/70.4.337.

4. Sakamoto S, Ohba S, Eguchi K, Shibukawa M, Kiura Y, Okazaki T, et al.

Churg-Strauss syndrome presenting with subarachnoid hemorrhage from ruptured dissecting aneurysm of the intracranial vertebral artertery. Clin Neurol Neurosurg. 2005;107(5):428-31, http:// dx.doi.org/10.1016/j.clineuro.2004.09.020.

5. Mencacci NE, Bersano A, Cinnante CM, Ciammola A, Corti S, Meroni PL, et al. Intracerebral haemorrhage, a possible presentation in ChurgStrauss syndrome: Case report and review of the literature. J Neurol Sci. 2011;301(1-2):107-11, http://dx.doi.org/10.1016/j.jns.2010.10.016.

6. Takanashi T, Uchida S, Arita M, Okada M, Kashii S. Orbital Inflammatory Pseudotumor and Ischemic Vasculitis in Churg-Strauss Syndrome. Ophthalmology 2001;108(6):1129-33, http://dx.doi.org/ 10.1016/S0161-6420(01)00557-7.

7. Kumar N, Vaish AK. Hemiplegia due to Churg Strauss syndrome in a young boy. J Assoc Physicians India. 2011;59:172-3.

8. Sacco S, Casalena A, Gallucci M, Carolei A. Showered cortical infarctions and brain atrophy in Churg-Strauss syndrome. Eur Neurol. 2011;65(2): 112, http://dx.doi.org/10.1159/000324006.

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