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Editorial Board of JIDMR 2011 Assoc. Prof. Dr. Izzet YAVUZ Editor-in-Chief and General Director Assist. Prof. Dr. Ozkan ADIGUZEL Associate Editor and Director Assoc. Prof. Dr. Refik ULKU Associate Editor for Medicine Prof. Dr. Zulkuf AKDAG, Prof. Dr. Sinerik N. AYRAPETYAN Associate Editor for Biomedical research Assist. Prof. Dr. Filiz ACUN KAYA, Assist. Prof. Dr. Sabiha Zelal ULKU Associate Editor for Dentistry Advisory Board Betul KARGUL (TURKEY) Ferranti WONG (UNITED KINGDOM) Filiz ACUN KAYA (TURKEY) Gauri LELE (INDIA)

Gulten UNLU (TURKEY) Jalen Devecioglu KAMA (TURKEY) Moschos A. PAPADOPOULOS (GREECE) Nik Noriah Nik HUSSEIN (MALAYSIA)

Sabiha Zelal ULKU (TURKEY) Sadullah KAYA (TURKEY) PhD. Dr. Ediz KALE Language Editor

Editorial Board Abdel Fattah BADAWI (EGYPT) Abdurrahman ONEN (TURKEY) Ahmet YALINKAYA (TURKEY) Ahmet DAG (TURKEY) Ali Al-ZAAG (IRAQ) Ali BUMIN (TURKEY) Ali GUR (TURKEY) Ali Kemal KADIROGLU (TURKEY) Ali Riza ALPOZ (TURKEY) Allah Bakhsh HAAFIZ (USA) Alpaslan TUZCU (TURKEY) Aziz YASAN (TURKEY) Balasubramanian MADHAN (INDIA) Benik HARUTUNYAN (ARMENIA) Betul KARGUL (TURKEY) Betul URREHMAN (UAE) Bugra OZEN (TURKEY) Carlos Menezes AGUIAR (BRAZIL) Cemil SERT (TURKEY) Chiramana SANDEEP (INDIA) Christine Bettina STAUDT (SWITZERLAND) Cihan AKGUL (TURKEY) Claudia DELLAVIA ( ITALY ) Emin Caner TUMEN (TURKEY) Ertugrul ERCAN (TURKEY) Ertunç Dayı (TURKEY) Fadel M. ALI (EGYPT) Fahinur ERTUGRUL (TURKEY) Feral OZTURK (TURKEY) Ferhan YAMAN (TURKEY) Feridun BASAK (TURKEY) Feriha CAGLAYAN (TURKEY) Ferranti WONG (UNITED KINGDOM) Figen SEYMEN (TURKEY) Filippo BATTELLI (ITALY) Filiz Acun KAYA (TURKEY) Flavio Domingues Das NEVES (BRAZIL) Gajanan Kiran KULKARNI (CANADA) Gamze AREN (TURKEY) Gauri LELE (INDIA) Gokhan KIRBAS (TURKEY) Gonul OLMEZ (TURKEY) Guliz Nigar GUNCU (TURKEY) Gulsen YILMAZ (TURKEY) Gulten UNLU (TURKEY) Gungor ATES (TURKEY) Guvenc BASARAN (TURKEY) Guven ERBIL (TURKEY)

Halimah AWANG (MALAYSIA) Heloisa Fonseca MARAO (BRAZIL) Hilal TURKER (TURKEY) Huseyin ASLAN (TURKEY) Igor BELYAEV (SWEDEN) Ilhan INCI (ZURICH) Ilker ETIKAN (TURKEY) Isil TEKMEN (TURKEY) Isin ULUKAPI (TURKEY) Izzet YAVUZ (TURKEY) Jalen DEVECIOGLU KAMA (TURKEY) Kemal CIGDEM (TURKEY) Kewal KRISHAN (INDIA) King Nigel MARTYN(HONG KONG SAR, PR CHINA) Kursat ER (TURKEY) Levent ERDINC (TURKEY) Lucianne Cople MAIA (BRAZIL) Luciane Rezende COSTA (BRAZIL) M. Ali FADEL (EGYPT) M. Sabri BATUN (TURKEY) Mahmut METE (TURKEY) Marri Sai ARCHANA (INDIA) Manoj KUMAR (INDIA) Marcelo Rodrigues AZENHA (BRAZIL) Marcia Cancado FIGUEIREDO (BRAZIL) Marco MONTANARI (ITALY) Margaret TZAPHLİDOU (GREECE) Maria Elisa Oliveira dos SANTOS (BRAZIL) Medi GANIBEGOVIC (BOSNIA and HERZEGOVINA) Mehmet DOGRU (TURKEY) Mehmet Nuri OZBEK (TURKEY) Mehmet Zulkuf AKDAG (TURKEY) Meliksah ERTEM (TURKEY) Meral ERDİNÇ (TURKEY) Mohamed TREBAK (USA) Mohammed Mustahsen URREHMAN (UAE) Moschos A. PAPADOPOULOS (GREECE) Mostaphazadeh AMROLLAH (IRAN) M.S.Rami REDDY (INDIA) Muhammad FAHIM (INDIA) Mukadder ATMACA (TURKEY) Murat AKKUS (TURKEY) Murat KIZIL (TURKEY) Murat SOKER (TURKEY) Mustafa KELLE (TURKEY) Mustafa ZORTUK (TURKEY) Muzeyyen YILDIRIM (TURKEY) Neval Berrin ARSERIM (TURKEY)

Nezahat AKPOLAT (TURKEY) Nihal HAMAMCI (TURKEY) Nik Noriah Nik HUSSEIN (MALAYSIA) Nurten AKDENIZ (TURKEY) Nurten ERDAL (TURKEY) Orhan TACAR (TURKEY) Ozant ONCAG (TURKEY) Ozgur UZUN (TURKEY) Ozkan ADIGUZEL (TURKEY) Rafat Ali SIDDIQUI (PAKISTAN) Refik ULKU (TURKEY) Remzi NIGIZ (TURKEY) S. Yavuz SANISOGLU (TURKEY) Sabiha Zelal ULKU (TURKEY) Sadullah KAYA (TURKEY) Saul Martins PAIVA (BRAZIL) Sedat AKDENIZ (TURKEY) Seher GUNDUZ ARSLAN (TURKEY) Selahattin ATMACA (TURKEY) Selahattin TEKES (TURKEY) Serdar ERDINE (TURKEY) Serdar ONAT (TURKEY) Sergio Adriane Bezerra DE MOURA (BRAZIL) Serhan Akman (TURKEY) Serhat ATILGAN (TURKEY) Shailesh LELE (INDIA) Sinerik N. AYRAPETYAN (ARMENIA) Smaragda KAVADIA (GREECE) Sossani SIDIROPOULOU (GREECE) Stephen D. SMITH (USA) Susumu TEREKAWA (JAPAN) Suha TURKASLAN (TURKEY) Suleyman DASDAG (TURKEY) Tekin YILDIZ (TURKEY) Ufuk ALUCLU (TURKEY) Ugur KEKLIKCI (TURKEY) Xiong-Li YANG (CHINA) Yasar YILDIRIM (TURKEY) Yu LEI (USA) Yuri LIMANSKI (UKRAINE) Zafer C. CEHRELI (TURKEY) Zeki AKKUS (TURKEY) Zeynep AYTEPE (TURKEY) Zuhal KIRZIOGLU (TURKEY) Zurab KOMETIANI (GEORGIA)


Journal of International Dental and Medical Research / ISSN: 1309-100X 2011; 4: (1) / TABLE OF CONTENTS DENTISTRY ARTICLE 1. EXPRESSION OF MATRIX METALLOPROTEINASE MMP-2 AND ITS TISSUE INHIBITOR TIMP-2 IN INTRAORAL PLEOMORPHIC ADENOMA AND ADENOID CYSTIC CARCINOMA Natheer Hashim AL-Rawi, Muthanna Al-Samarai, Sausan Al-Kawas, Ahlam H. Majeed Pages 1-6 ARTICLE 2. EFFECT OF COMMONLY CONSUMED FRESH FRUIT JUICES AND COMMERCIALLY AVAILABLE FRUIT JUICES ON pH OF SALIVA AT VARIOUS TIME INTERVALS Sabyasachi Saha, Gudamarlahally Venkatarayappa Jagannath, Venkatarayappa Jagannath, Sahana Shivkumar, Sumit Kumar Pal Pages 7-11 CASE REPORT 3. RIGA’S PAPILLOMA IN A 3 MONTH OLD INFANT: A CASE REPORT Sonali Saha, Firoza Samadi, Sabyasachi Saha, Fahad Mansoor Samadi Pages 12-16 CASE REPORT 4. DENTAL PHENOTYPE IN A PATIENT WITH HYPOIDROTIC ECTODERMAL DYSPLASIA AND SEVERE IMMUNODEFICIENCY M. Callea, F. Faletra, A. Maestro, F. Verzegnassi, M. Rabusin, A. Vinciguerra, F. Radovich, G. Clarich, I. Yavuz, E. C. Tumen Pages 17-20 CASE REPORT 5. DENTIGEROUS CYST WITH AN IMPACTED CANINE: CASE REPORT Kamil Serkan Agacayak, Ibrahim Kose, Nedim Gunes, Emrullah Bahsi, Ferhan Yaman, Serhat Atilgan Pages 21-24 CASE REPORT 6. EFFECT OF PARAFUNCTIONAL FORCE ON DENTAL IMPLANT TREATMENT IN BRUXISM: A CASE REPORT (TWO YEAR RESULTS) Mustafa Zortuk, Erdem Kilic, Pinar Yildiz, Ikbal Leblebicioglu Pages 25-29 CASE REPORT 7. ELEPHANTIASIS GINGIVA IN 13 YEAR OLD BOY- A CASE REPORT Srinivasa T.S., Rachana Kaushik, R.G.Shiva Manjunath Pages 30-34 REVİEW 8. DIAGNOSIS AND MANAGEMENT OF OBSTRUCTIVE SLEEP APNEA Venigalla Naga Venu Madhav Pages 35-41 BIOMEDICAL RESEAERCH ARTICLE 9. KETAMINE- INDUCED CELL DEHYDRATION AS A MECHANISM OF IT’S ANALGESIC AND ANESTHETIC EFFECTS Armenuhi Heqimyan, Anush Deghoyan, Sinerik Ayrapetyan Pages 42-49 ARTICLE 10. EFFECTS OF PROPOFOL ON EXPRESSION ICAM-1 IN RABBIT GASTRIC ENDOTHELIAL CELLS Sennur Ketani, Berna Ersoz Kanay, Hakan Sagsoz Pages 50-53

2011 Volume 4 - Number 1


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

Pleomorphic Adenoma And Adenoid Cystic Carcinoma Natheer H. AL-Rawi et al

EXPRESSION OF MATRIX METALLOPROTEINASE MMP-2 AND ITS TISSUE INHIBITOR TIMP-2 IN INTRAORAL PLEOMORPHIC ADENOMA AND ADENOID CYSTIC CARCINOMA Natheer Hashim AL-Rawi1*, Muthanna Al-Samarai2, Sausan Al-Kawas1, Ahlam H. Majeed2 1. College of Dentistry, University of Sharjah, Sharjah, UAE. 2. College of Dentistry, University of Baghdad, Baghdad, Iraq.

Abstract Matrix metalloproteinases (MMPs) are proteolytic enzymes that are capable of degrading different substrates within extracellular matrix (ECM), and are believed to be crucial for tumor invasion and metastasis. Tissue inhibitors of MMP (TIMPs) can inhibit the action of MMPs The aim of this study was to analyze protein expression of MMP-2 and TIMP-2 in intraoral pleomorphic adenoma (PLA) and adenoid cystic carcinoma (ACC). A total of 35 formalin-fixed paraffin-embedded specimens comprising 19 PLA and 16 ACC were utilized in this study. A standard immunohistochemical technique was used to determine the expression levels of MMP-2 and TIMP-2 proteins. Sections were assessed semi quantitatively .Staining was scored as 0 (< 1% positive tumor staining), 1+ (< 25% positive tumor cells), 2+ (20-50% positive tumor cells) and 3+ (> 50% positive tumor cells). For statistical analysis, tumors were divided into two groups, low expressors ( 0-1+) and high expressors (2-3+). PLA showed higher TIMP-2 expression than ACC (p<0.05). No significant difference was observed between PLA and ACC regarding MMP-2 expression. MMP-2 and TIMP-2 expressed mainly in the cytoplasm of epithelial/ myoepithelial components of PLA and neoplastic epithelial cells of ACC. Myoepithelial cells may be the primary source of gelatinases in PLA and the down regulation of TIMP-2 expression in ACC might be responsible for metastasis and recurrence. The ratio value of MMP-2/TIMP-2 is valuable parameter to demonstrate the ECM degradation/ deposition imbalance. Article (J Int Dent Med Res 2011; 4: (1), pp. 1-6 ) Keywords: Pleomorphic Adenoma, Adenoid Cystic Carcinoma. . Received date: 02.06.2010 Introduction Intraoral minor salivary gland tumors (MSGTs) constitute a heterogeneous group of neoplasm with great histomorphologic variation1. In spite of their relative infrequency in term of total number of cases in oral or hospital surgical pathology services2, minor salivary glands tumors continue to generate considerable research interest. Several studies2,3,4 have *Corresponding author: Dr. Natheer H. AL-Rawi, College of Dentistry, University of Sharjah, Sharjah, UAE. E-mail: nhabdulla@sharjah.ac.ae

Volume 4 ∙ Number ∙ 1 ∙ 2011

Accept date: 20.08.2010 documented the distribution of series of minor salivary gland tumors seen in some countries. Reports from various parts of the world indicate that there are differences in the frequency of particular histological types and in the frequency with which minor salivary glands are involved. Benign and malignant tumors have almost equal frequency. However, some studies have previously reported that pleomorphic adenomas and adenoid cystic carcinoma seems to have a higher prevalence among Japanese 5. The palate was the main site of occurrence in many studies followed by cheek, lip and gingiva 6. Most studies in the literatures indicated that minor salivary gland tumors are somewhat more common in females than in males with Male to Female ratio varying from 1:1.2 to 1:1.5 6,7.

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PLA cases 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Age/ Years 17 25 51 14 28 45 42 15 45 50 57 61 18 23 15 70 25 73 60

Gender

Site

Female Male Male Female Female Male Male Male Female Male Male Male Female Female Male Female Female Female Female

Palate Palate Palate Palate Palate Palate Palate Palate Palate Palate Palate Palate Palate Palate Palate Palate Palate Palate Palate

MMP-2

TIMP-2

2+ 0 3+ 3+ 3+ 3+ 2+ 0 1+ 0 0 0 0 0 2+ 0 0 0 0

1+ 3+ 0 3+ 3+ 3+ 3+ 3+ 0 0 3+ 1+ 0 0 1+ 1+ 1+ 0 0

ACC cases 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Pleomorphic Adenoma And Adenoid Cystic Carcinoma Natheer H. AL-Rawi et al

Age/ Years 64 60 61 51 55 49 55 59 60 68 60 71 38 44 55 65

Gender

Site

Male Male Male Male Male Male Female Female Female Male Male Male Female Female Female Female

Palate Palate Palate Palate Palate Palate Palate Palate Palate Palate Palate Palate Palate Palate Palate Palate

MMP-2

0 3+ 2+ 1+ 3+ 1+ 1+ 0 2+ 2+ 0 1+ 2+ 0 0 1+

TIMP-2

3+ 1+ 3+ 0 0 1+ 1+ 1+ 0 0 0 0 0 0 0 0

Table 1. Clinico-pathologic data of studied sample. (N.B: Histological subtype: PLA : Classic/ cellular ; ACC: Cribriform/ tubular) One Iraqi study done on 140 minor salivary gland tumors revealed Male to Female ratio of about 1:1.1, the palate was the most commonly involved site and pleomorphic adenoma was the most common tumor type followed by mucoepidermoid carcinoma and adenoid cystic carcinoma8. In addition to traditional microscopical studies, other methods have become available to help define biologic behavior of these tumors. The application of immunohistochemistry to salivary gland pathology has shown that matrix metalloproteinases (MMPs) and their inhibitors are useful markers to determine the biologic behavior of benign and malignant minor salivary gland tumors9,10,11. The matrix metalloproteinases (Matrixins) are a family of multi domain Zn-dependent proteolytic enzymes that are capable of degrading different substrates within extracellular matrix (ECM)12,13 , both in physiological and pathological conditions such as embryogenesis, wound healing, angiogenesis, inflammation and tumor metastasis14. They are classified into four groups according to their substrate specificity: Collagenases (MMP-1,-8,-13 & -18), gelatinases (MMP-2 & -9), stromelysins (MMP-3, MMP-10 &MMP-11), membrane type MMP (MMP-14,-15,16,-17,-24 & -25) and matrilysins (MMP-7 & 26)15. Volume 4 ∙ Number ∙ 1 ∙ 2011

Tissue inhibitors of matrix metalloproteinases (TIMPs) are specific inhibitors of MMPs that participate in controlling the local activities of MMPs in tissues16,17. Four TIMPs (TIMP1-4) have been identified in vertebrates 18, and their expression is regulated during development and tissue remodeling. The expression of MMPs and TIMPs is still not well documented in intraoral minor salivary gland tumors. The aim of this study was to elucidate the expression and cellular localization of MMP-2 and TIMP-2 in intraoral minor salivary gland tumors by mean of immunohistochemistry. Materials and Methods Thirty five formalin-fixed paraffin embedded blocks were retrieved from Oral Pathology Laboratory specimen file of Dental School / University of Baghdad. The sample consisted of 19 specimens of pleomorphic adenoma of the palate and other 16 specimens of adenoid cystic carcinoma of the palate. The clinico-pathological characteristics of the patients from which the specimens were taken are summarized in table 1. All the 35 blocks were cut at 3 µM thickness sections and mounted on poly-L-lysincoated slides. The sections were deparaffinized Page 2


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

and stained by means of a standard immunohistochemical technique using a high – temperature water bath for antigen enhancement. US Biological (US Biological Co, Massachusetts) reagents was used in a peroxidase – based system to identify antigen-antibody conjugates. The sections were incubated with primary antibody against MMP-2 (mouse monoclonal anti-MMP-2, clone 0.N.430,USBM2420-51). Other sections were incubated with primary antibody against TIMP-2 (mouse monoclonal anti-TIMP-2, clone ZQ672, USB T5585-27). The incubation time for both antibodies was 30 minutes at room temperature. Negative control slides were incubated with normal phosphate buffer solution instead of primary monoclonal antibodies. The immunostaining procedure was performed using a labeled streptavidin-biotin system (LSAB system-HRP, Dakocytomation, Carpinteria, California, USA). The biotinylated secondary antibody was applied for 20 minutes. The presence of antigens was detected with streptavidin conjugated to horse radish peroxidase. DAB (3,3’-diaminobenzidine) (Dakocytomation) was used as a chromogen. Mayer hemotoxylin counter stain was then applied to all sections for 2 minutes and washed with tab water for another 2 minutes, dehydrated in graduated alcohol concentrations, then mounted with DPX and slide cover. Ten more samples of non- neoplastic salivary gland tissue were included in this study and served as control group. All slide were examined and photographed with a light microscope (Olympus BX41) with digital camera at 100, 400 & 1000 magnification. All slides were scored by two investigators without knowledge of the clinical outcomes. Occasional disagreement was discussed later to reach a consensus. In case of persistent differences between them, the sections were studied by a third independent oral pathologist and the majority decision was then considered. Staining of MMP-2 and TIMP-2 was semiquantitatively assessed. Staining was graded as 0(negatively stained cells), 1+ (< 10% positive stained cells), 2+ (10-50% positive stained cells), 3+ (> 50% positive stained cells) (11). For statistical analysis of the immunoscores we used Fisher’s exact test to test the statistical association between tumor types and expression levels for each marker. For analysis we dichotomized results by comparing low expressors (0-1+) to high Volume 4 ∙ Number ∙ 1 ∙ 2011

Pleomorphic Adenoma And Adenoid Cystic Carcinoma Natheer H. AL-Rawi et al

expressors (2-3+). All analysis was two tailed with a significant level set at (0.05). Results A benign intraoral salivary gland tumor was represented by nineteen cases of pleomorphic adenoma which were selected for this study. All were located in the palatal region as slowly-growing, painless mass. They presented histologically as epithelial cells arranged in strands, sheets and duct-like structure in mucoid or myxochondroid stroma background. Epithelial cells and modified myoepithelial cells are components of cell islands in pleomorphic adenoma. Immunoexpression of MMP-2 was detected in 8 out of 19 cases (42.1%). Four cases (50%) outof these 8 cases were strongly expressed MMP-2 (grade 3+), 3 (37.5%) showed immunostaining between 10% and 50% of tumor cells (grade 2+) and only one case (12.5%) expressed less than 10% of positive cells staining (grade 1+). MMP-2 staining intensity was most prominent in epithelialmyoepithelial components (figure1). The remaining 11 cases (57.9%) were negative for MMP-2.

Figure 1. MMP-2 in PLA ( grade 3). TIMP-2 immunoexpression was detected in 12 pleomorphic adenoma (63.15%), seven cases ( 58.33%) were strongly positive ( grade 3+), five case ( 41.66%) were with( grade 1+) .The cytoplasmic TIMP-2 staining was found in the majority of specimens and epithelialmyoepithelial cells were the most prominent cells that expressed TIMP-2 reactivity (Figure 2). The remaining seven cases (36.85%) were negative for TIMP-2 (table 2). Page 3


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

Pleomorphic Adenoma And Adenoid Cystic Carcinoma Natheer H. AL-Rawi et al

cystic carcinoma (p<0.05). The median values of MMP-2 and TIMP-2 immunoscores are summarized in table (2). The MMP-2/TIMP-2 ratio was calculated for both pleomorphic adenoma and adenoid cystic carcinoma which was 1:1.6 for pleomorphic adenoma and 2:1 for adenoid cystic carcinoma. Non-neoplastic minor salivary gland control group expressed mildmoderate MMP-2 and TIMP-2 immunostaining reaction in mucus acinar cells with MMP-2/TIMP2 ratio of 1:2.

Figure 2. TIMP-2 in PLA (grade 3).

Figure 3. MMP-2 in ACC (grade 3). Table 2. Summary of results of Immunohistochemical staining.

A malignant intraoral salivary gland tumor was represented by sixteen cases of adenoid cystic carcinoma. All were located in the palate. They all presented histologically in a classical cribriform/ tubular pattern. Immunoexpression of MMP-2 was detected in 11cases out of 16 case (68.75%), of these 11 cases only 2 cases (18.18%) were strongly positive for MMP-2 (grade 3+) (figure 3), other 4 cases ( 36.36%) expressed immunreactivity between (10-50%) (Grade 2+), the remaining five cases (45.45%) expressed very mild immune reaction (grade 1+). The remaining 5 cases (31.25%) were negative for MMP-2. TIMP-2 immunostaining in adenoid cystic carcinoma was detected in 6 cases representing only (37.5%) of the total number, 2 cases (33.33%) expressed (grade 3+) and the other 4 cases (66.66%) expressed (grade 1+). The remaining 10 cases (62.5%) were negative for TIMP-2 (figure 4) (table 2). TIMP-2 expression was significantly higher in pleomorphic adenoma compared with adenoid Volume 4 ∙ Number ∙ 1 ∙ 2011

Figure 4. TIMP in ACC ( grade 0). Discussion Intraoral minor salivary gland tumors are relatively uncommon lesions in daily practice. The balance between MMPs and their tissue inhibitors (TIMPs) is involved in the morphogenesis of normal salivary glands as well as in the mechanism of tumor invasion and Page 4


Journal of International Dental And Medical Research ISSN 1309-100X http://www.ektodermaldisplazi.com/journal.htm

metastasis10. The role of MMPs and TIMPs in pleomorphic adenoma and adenoid cystic carcinoma has not been well defined yet. Imunohistochemistry allows the study of the presence of MMPs & TIMPs in tumors and surrounding tissues. In this study MMP-2 and TIMP-2 expression was compared in pleomrphic adenoma and adenoid cystic carcinoma. The immunoscores of MMP-2 detected in tumor cells were significantly increased in malignant tumors compared with benign tumors. In this study, MMP-2 and TIMP-2 were mainly expressed in epithelial/ myoepithelial cells of pleomorphic adenoma. Previous studies have demonstrated that although tumors cells may be the source of gelatinases in some patients, in the majority of patients only stroma cells express gelatinase19. Tumor stroma is one of the critical elements that promote the transition from carcinoma in situ to invasive cancer20. However, the present study , however, strongly suggest the possibility that myoepithelial cells may be the primary source of gelatinases and perhaps play a critical role in development and/or progression of pleomorphic adenoma. This finding is in accordance of other studies10. The increased expression of MMPs was followed by increased expression of TIMPs. This suggested a positive correlation between MMP-2 and TIMP-2. Zhang et al10 suggested that the increase in both MMP and TIMP levels represented a physiological attempt of cells to control MMP activity and maintain a balanced ratio between the two proteins. It is well known that the gelatinolytic activity of MMPs is antagonized by TIMPs in a stoichiometric manner 21, maintenance of this equilibrium is essential and any disturbance of this balance will probably result in tissue damage due to increased proteolysis. Hence, the high expression of MMP2 (without TIMP-2 expression) in epithelial/ myoepithelial components of pleomorphic adenoma may facilitate the local invasiveness. Careful follow up is of great importance to early detect and prevent invasion or malignant transformation. The metastatic ability of human salivary gland cancer cells was closely associated with decreased or altered TIMP-2 expression. The down regulation of TIMP-2 in malignant epithelia of adenoid cystic carcinoma might be capable of concerning acquisition of abilities of recurrence and metastasis by the effects of TIMP-2 regarding the inhibition of Volume 4 ∙ Number ∙ 1 ∙ 2011

Pleomorphic Adenoma And Adenoid Cystic Carcinoma Natheer H. AL-Rawi et al

MMPs, tumor growth and anti angiogenic activity (10). The expression of MMP-2 in tumor stromal interface of Adenoid Cystic Carcinoma in this study suggest that stroma may play some critical role than epithelia in progression of this tumor. It has also been reported that some TIMPs can directly affect cell growth and/ or cell survival independent of their actions of MMPs22. The expression of TIMP-2 has been correlated with the metastatic ability and poor prognosis of squamous cell carcinoma of tongue14 and some studies reported the positive role of TIMP-2 in tumor metastasis and decreased survival23. The finding of the current study does not support this concept since most of ACC cases (14 out of 16 case) were low TIMP-2 expressors ( 0 -1+). The ratio of MMP-2/TIMP-2 takes into account variations in the expression of enzymes and its inhibitor and is an indicator of an imbalance between MMP (degradation of ECM) and TIMP (deposition of ECM). Because the balance of these enzymes is critical to matrix destruction, this ratio may adequately reflect the net proteolytic capacity which was significantly higher in carcinoma than in adenoma in this study. Other researchers have found a significant positive correlation between lymph node metastasis and MMP-2/TIMP-2 value24, and the evaluation of MMP-2/TIMP-2 ratio has a higher prognostic value than the evaluation of MMP-2 and TIMP-2 expression alone23. Therefore we evaluated the ratio of MMP-2/TIMP-2 for all studied sample and non-neoplastic minor salivary glands. We found that the ratio in ACC were higher than that of pleomorphic adenoma and non-neoplastic minor salivary glands. The increased MMP-2/TIMP-2 ratio was ascribed to overproduction of MMP and MMP/TIMP balance apparently favors gelatinolytic activity. Conclusions In summary, the results presented here provide supporting evidence that MMP-2 expressed in epithelial/myoepithelia cells of pleomorphic adenoma may be important in the development and progression of this tumor and the high TIMP-2 expression may prevent invasion and metastasis. In Adenoid Cystic Carcinoma, the lack or low expression of TIMP-2 in malignant epithelial cells may favor ECM degradation produced by various MMPs and the ratio value of MMP-2/TIMP-2 is valuable parameter to demonstrate this imbalance. Page 5


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Declaration of Interest The authors report no conflict of interest and the article is not funded or supported by any research grant. References 1. Yih WY, Kratochvil FJ, Stewart JC. Intraoral minor salivary gland neoplasms: review of 213 cases. J Oral Maxillofac Surg 2005;63(6):805-10 2. Regezi JA, Lloyd RV, Zarbo RJ, McClatchey KD. Minor salivary gland tumors. A histologic and immunohistochemical study. Cancer 1985; 55:108-115. 3. Isacsson G, Shear M. Intraoral minor salivary gland tumors: A retrospective study of 201 cases. J Oral Pathol 1983; 12:57-62. 4. Eveson JW, Cawson RA. Tumors of the minor (Oropharyngeal) salivary glands. A demographic study of 336 cases. J Oral pathol 1985; 14:500-509. 5. Toida M, Shimokawa K, Makita H, Kato K, Kobayashi A, Kusunoki Y, et al.Intraoral minor salivary gland tumors. A clinic o-pathological study of 82 cases. Int J Oral Maxillofacial Surg 2005; 34:528-32. 6. Sugiura R, Kuyama K, Miyake M, Wakita M, Nishiyama T, Suzuki Y, Nakamura F, Fukomuto M, Yamamato H. Intraoral minor salivary gland tumors. A retrospective study of 92 cases. Nihon University J Oral Science 2001; 27(1):10-15. 7. Pires FR, Pringle GA, Almeda OP, Chen SY. Intraoral minor salivary gland tumors. A clinic-pathological study of 546 cases. Oral Oncol 2007; 43:463-70. 8. Al-Rawi NH .Intraoral Minor salivary gland tumors: A clinicpathological study of 140 cases. Iraqi Dent J 2001; 27:143-150. 9. Nagel R, Laskawi A, Wahlers B, Hemmerlein I. Expression of matrix metalloproteinases MMP-2, MMP-9 and their tissue inhibitors TIMP-1, -2 and -3 in benign and malignanat tumors of salivary gland. Histopathol 2004; 44:222-31. 10. Zhang X, Wang Y, Tamamoto G, Tachikawa T. Expression of matrix metalloproteinases MMP-2, MMP-9 and their tissue inhibitors TIMP-1 and TIMP-2 in the epithelium and stroma of salivary gland pleomorphic adenomas. Histopathology 2009; 55(3):250-260. 11. Westernoff TH, Jordan RC, Regezi JA, Ramos DM, Schmidt BL. ß-6 Integrin, tenascin-C, and MMP-1 expression in salivary gland neoplasms. Oral Oncol 2005; 41:170-174. 12. Mignatti P, Rifkin DB. Biology and biochemistry of proteases in tumor invasion. Physiol Rev 1993; 73:161-195. 13. Stettler-Stevenson WG, Aznavoorian S, Liotta LA. Tumor cell interactions with the extracellular matrix during invasion and metastasis. Ann Rev Cell Biol 1993; 9:541-73. 14. Matrisian L. The matrix degrading metalloproteinases. Bioassays 1992; 14:455-59. 15. Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases: Structure, function, and biochemistry. Cir Res 2003; 92:827-39. 16. Stolow MA, Bauzon DD, Li J, Sedjwick T, Liang VC, Sang QA , Shi YB. Identification and characterization of a novel collagenases in Xenopus laevis: Possible roles during frog development. Mol Biol Cell 1996; 7:1471-83. 17. Patterson ML, Atkinson SJ, Knauper V, Murphy G. Specific collagenolysis by gelatinase A, MMP-2, is determined by the hemopexin domain and not the fibronectin-like domain. FEB Cell 2001; 503:158-62. 18. Brew K, Dinakarpandian D, Nagase H. Tissue inhibitors of metalloproteinases: evolution, structure and function. Biochem biophys Acta 2000; 1477:267-283. 19. Pyke C, Ralfkiaer E, Tryggvason K, Dano K. Massenger RNA for two type IV collagenases is located in stromal cells in human colon cancer. AM J Pathol 1993; 142:359-65. 20. Liotta LA, Kohn EC. The microenvironment of the tumor-host interface. Nature 2001; 411:375-79.

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Pleomorphic Adenoma And Adenoid Cystic Carcinoma Natheer H. AL-Rawi et al

21. Haykawa T, Yamashita K, Ohuchi E, Shinagawa A. Cell growth-promoting activity of tissue inhibitor of metalloproteinases-2(TIMP-2). J Cell Sci 1994:107; 2373-79. 22. Kallakury BVs, Karikehalli S, Haholu A, Sheehan CE, Azumi JS, Ross JS. Increased expression of matrix metalloproteinases 2 and 9 and tissue inhibitors of metalloprotenases 1 and 2 correlate with poor prognostic variable in renal cell carcinoma. Clin Cancer Res 2001; 7:3113-19. 23. Kanayama H, Yokota K, Kurokawa Y, Murakami Y, Nishitani S, Kagawa S. Prognostic value of matrix metalloproteinase-2 expression and tissue inhibitor of metalloproteinase-2 expression in bladder cancer. Cancer 1998; 82:1359-66. 24. Gray ST, Wilkins RJ, Yun K.Interstitial collagenase gene expression in oral squamous cell carcinoma. AM J Pathol 1992; 141:301-306.

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Effect of Fruit Juices on ph of Saliva Sabyasachi Saha et al

EFFECT OF COMMONLY CONSUMED FRESH FRUIT JUICES AND COMMERCIALLY AVAILABLE FRUIT JUICES ON pH OF SALIVA AT VARIOUS TIME INTERVALS Sabyasachi Saha1*, Gudamarlahally Venkatarayappa Jagannath2, Sahana Shivkumar3, Sumit Kumar Pal4 1. Prof.Dr. & Head, Department of Public Health Dentistry, Sardar Patel Postgraduate Institute of Dental and Medical Sciences, Rai-bareilly Road, Lucknow, India. 2. Dr. Reader, Department of Public Health Dentistry, Sardar Patel Postgraduate Institute of Dental and Medical Sciences, Rai-bareilly Road, Lucknow, India. 3. Dr. Reader, Department of Public Health Dentistry, Sardar Patel Postgraduate Institute of Dental and Medical Sciences, Rai-bareilly Road, Lucknow, India. 4. Post graduate student, Department of Public Health Dentistry, Sardar Patel Postgraduate Institute of Dental and Medical Sciences, Raibareilly Road, Lucknow, India.

Abstract To find out the acidogenic potential of the commonly consumed freshly prepared fruit juices (Pomegranate, Lime) and commonly consumed commercially available fruit juices ( Guava and Apple) at room temperature on pH of saliva at various time intervals. The double-blind study was done on 40 subjects. During the study, subjects were asked to collect their stimulated saliva in a glass bottle before having juice as a baseline score. Subjects were asked to drink the juice, their saliva sample was collected after 1minute, 5minutes, 15minutes and 30 minutes of drinking juice and the pH of the samples were measured using digital pH meter. All fruit juices were acidic and reduced the salivary pH. The maximum drop in pH was found at after min of consuming fruit juices. Drop in salivary pH was greater after consumption of commercially available fruit juices than that after consumption of fresh fruit juices. Commercially available fruit juices are more acidogenic than the fresh fruit juices. Article (J Int Dent Med Res 2011; 4: (1), pp. 7-11 ) Keywords: Fruit juice, Salivary pH, Acidogenic. Received date: 25 November 2010 Introduction As the mankind is evolving there has been drastic changes occurring in the dietary pattern as well.1 The diet we are consuming has become more refined with increased access to readymade fruit juices and high frequency of snacking. Also there has been substantial increase in consumption of carbonated beverages and fruit drinks.1 Since recently there has been considerable emphasis on “healthy food and healthy eating”. Fruit juices have been widely marketed and promoted as „healthy drinks‟.

*Corresponding author: Dr. Sabyasachi Saha Department of Public Health Dentistry Sardar Patel Postgraduate Institute of Dental and Medical Sciences, Rai-bareilly Road, Lucknow, India. Pin- 226025. Mobile No: 09451248210 E-mail: drssaha28@gmail.com

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Accept date: 28 January 2011 Fruit juices are widely popular among people of all ages as they are perceived to be good for health and are sometimes preferred over carbonated beverages which are inherently highly acidic. However claims of safety of fruit juices for teeth are unsubstantiated due to inadequate report in the literature.2 The erosive effect of fruit juices have been recognized for a long time as evident in the studies of Darby (1892)3 and W. D. Miller (1907)4 who reported tooth decalcification due to excessive fruit juice consumption. Saliva plays a very important role in maintaining the integrity of teeth by way of its buffering action and controlling the demineralization and promoting remineralization occurring continuously at the enamel surface.5 There will be a drop in salivary pH whenever one consumes sugary foods or beverages. The sweeter a food or a drink is the more will be the magnitude of the drop in salivary pH. Packaged fruit juices are sweeter having higher sugar Page 7


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content to enhance their taste.6 Hence the present study has been carried out to assess the acidogenic potential of commonly consumed fresh and commercially available fruit juices at various time intervals. Material & Methods Sample selection Around one hundred first year under graduate students of Sardar Patel Post Graduate Institute of Dental Sciences, Lucknow, were invited to voluntarily participate in the study. They were screened using following inclusion and exclusion criteria. Inclusion Criteria The inclusion criteria comprised of subjects voluntarily participating in the study with Decayed Missing Filling Tooth Index (DMFT)7 score < 3 & Oral Hygiene Index-Simplified (OHIS)8 score < 1.2, depicting good oral hygiene. Exclusion criteria Exclusion criteria comprised presence of any relevant past medical history, history of any antibiotic and medication therapy two months prior to the study and any known history of allergy to any fruit / fruit juice. Procedure of this institutionally approved study was explained to all the subjects and written consent from each subject was obtained. A total of 40 volunteers with an age range of 18 to 20 years consisting of 17 males and 23 females fulfilling the above mentioned criteria were selected and randomly allocated to the groups consuming different fruit juices. A pilot survey was conducted to know which fruit juice is most commonly consumed among the general population, which showed that among commercially available fruit juices the most commonly consumed were Apple and Guava, among the freshly prepared fruit juices the most commonly consumed were Lime and Pomegranate. Therefore only these four fruit juices were considered in the study. Experimental Design Procedure of the study was explained to all the subjects and written consent from each subject was obtained. Ethical clearance was obtained from the Institutional Ethical Committee. All the 40 subjects were randomly allocated to the groups of fresh fruit juice (Group - A) and commercially available fruit juice (Group - B) each with 20 subjects. Both groups were again Volume ∙ 4 ∙ Number ∙ 1 ∙ 2011

Effect of Fruit Juices on ph of Saliva Sabyasachi Saha et al

subdivided into two groups each having 10 subjects for consuming the assigned fruit juices: A-1 for Sweet lime, A-2 for pomegranate, B-1 for Apple, B-2 for Guava. Procedure The intrinsic pH of all four fruit juices were measured prior to the consumption. These were as follows- Fresh fruit juices: Sweet lime (A-1) pH 4.59, Pomegranate (A-2) pH 4.47; Commercially available fruit juices: Apple (B-1) pH 3.33, Guava (B-2) pH 3.10. Stimulated saliva of the subjects were collected in sterile glass bottles after they chewed one gram of paraffin wax prior to the consumption of juice as a baseline score. Then the subjects were asked to consume assigned fruit juice and the salivary sample of each subject was collected in separate sterile glass bottles after 1 minute, 5 minute, 15 minute and 30 minute of fruit juice consumption. Salivary samples were collected of only 10 subjects in a single day.

Summary of experimental design. The glass bottles were coded with a specific identity number given to each subject. The pH of the samples were measured in the institutional laboratory using calibrated digital pH meter. The same procedure was repeated with the other three groups after consumption of assigned fruit juice. Whole process was supervised and carried out by a single investigator. Tools used: Glass bottles, Digital pH meter, Paraffin wax, Fruit juices. Statistical Analysis „Student t-test‟ was used to compare the effect of one type of fruit juices with other type, where as to assess the effect of one type of fruit juice at different time intervals „paired t-test‟ was used. Page 8


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Effect of Fruit Juices on ph of Saliva Sabyasachi Saha et al

Results Mean resting salivary pH of all the subjects was 7.28 ± 0.30 and this was compared with drop in pH at 1 min, 5 minute, 15 minute and 30 minute following fruit juice consumption. Table 1 shows that on the whole the drop in pH in relation to resting pH at 1 minute was 6.11 ± 0.95, at 5 minute 6.80 ± 0.66, at 15 minute 7.18 ± 0.31 and at 30 minute it was 7.24 ± 0.26 and the difference was statistically significant (p < 0.001) except for 30 minute time interval. It was found that the magnitude of overall fall in salivary pH was more (7.28 v/s 6.11) in Group B.

Table 2. Change in salivary pH after consumption of Fresh fruit juices. (*Statistically significant)

Table 1. Overall change in salivary pH after consumption of juices. (*Statistically significant) Table 2 shows that in group A, the mean resting pH was 7.39 ± 0.27 which after consuming sweet lime and pomegranate juices, dropped to 6.58 ± 0.47 after 1 minute and the difference (0.81) was statistically highly significant, (p < 0.001), similarly at 5 minute the pH observed was 7.08 ± 0.33 and the difference (0.31) was statistically highly significant ( p < 0.001).

Table 3. Change in salivary pH after consumption of commercially available fruit juices.

(*Statistically significant) Table 4 shows the intergroup comparison regarding the difference in pH drop. The difference in drop observed at 1 minute and 5 minute intervals were statistically significant.

Table 3 shows that in group B, the mean resting pH was 7.17 ± 0.29 which after consuming Apple and Guava juices, dropped to 5.65 ± 1.09 after 1 minute, and the difference (1.52) was statistically highly significant (p = 0.001), similarly at 5 minute the pH observed was 6.52 ± 0.79 and the difference (0.65) was statistically highly significant (p < 0.001). Table 4. Difference in pH value at different time intervals between group A & Group B.

(*Statistically significant) Volume ∙ 4 ∙ Number ∙ 1 ∙ 2011

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Figure 1 shows that the maximum drop in salivary pH of all the subjects was noticed after 1 minute of fruit juice consumption. After consumption of any juice, pH of saliva gradually reached back to the baseline within 30 minute.

Figure 1. Variations in salivary consumption of different fruit juices.

pH

after

A= Change in salivary pH after consumption of freshly prepared fruit juice. B= Change in salivary pH after consumption of commercially available fruit juice. A+B= Overall change after consumption of juices.

Among the groups consuming freshly prepared fruit juice and commercially available fruit juice, the latter group showed more drop in salivary pH. Among all the juices Apple juice caused the maximum fall in salivary pH. Paired t–test value showed that drop in salivary pH after consumption of commercially available fruit juices was significant at 1 minute and 5 minute. Similarly drop in salivary pH after consumption of freshly prepared pomegranate juice was significant at 1 minute and 5 minute, whereas the drop was significant at 1 minute interval after consumption of freshly prepared sweet lime juice. Discussion The study was conducted as a pre and post effect of fruit juice consumption on salivary pH among undergraduate dental students with proportionate representation for age and sex. The consumption of fruit juices though widely prevalent, varies greatly among populations. Acidified sugar containing drinks have shown to be cariogenic and erosive in rats.9 Foods and beverages, specially fruit juices, can contain variety of acids that have the potential to damage the teeth.6 Volume ∙ 4 ∙ Number ∙ 1 ∙ 2011

Effect of Fruit Juices on ph of Saliva Sabyasachi Saha et al

The fruit juices can have either of the following effect or both on teeth: a. They may be acidic enough to erode the surfaces of teeth. b. They may contain fermentable carbohydrates, which may serve as a substrate for acidogenic bacteria which might result in dental caries. It has been shown that carbonated beverages are more efficiently buffered by contact with saliva than fruit juices.10 In the present study the group consuming commercially available packed fruit juices showed maximum drop in salivary pH, even dropping to critical pH level (5.5 ± 3) at 1 minute interval, followed by a gradual recovery within 30 minutes of study. This greater drop could be attributed to the relatively lower intrinsic pH of commercially available fruit juices. Similar results have been shown by Lata Kiran et al, 2005. The probable reason for immediate drop in salivary pH could be that intrinsic acidity of packed fruit juices rendered it more able to combat salivary buffers.10 The length of time for which this low pH remains at its minimum is important- the longer the stay at critical pH value, the higher the dissolution of enamel.11 It has been reported that solubility of dental tissue increases by a factor of 7-8 with each drop of pH by 1 unit thereby significantly increasing the potential risk for demineralization. The drop in salivary pH among the groups consuming sweet lime and pomegranate was similar and minimal. For the groups consuming packed fruit juices, there was significant drop in salivary pH in Apple juice group. Appropriate comparison could not be made due to unavailability of similar study. In present study the degree of pH drop was – Apple > Guava > Pomegranate > Sweet lime. A single acidic attack is of minor importance but if repeated, the ability of saliva to deal with the acid decreases. Hence, the danger is the frequent use of these fruit juices over time. If the challenge is frequent enough and there are few or no protective factors as in caries susceptible people, this can be quite aggressive.

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Effect of Fruit Juices on ph of Saliva Sabyasachi Saha et al

Conclusions It was found that commercially available fruit juices caused greater drop in salivary pH than that of fresh fruit juices. The results provide the basic information to the general dental practitioner regarding the consumption of fruit juices and their potential role in the development of dental erosion and / or dental caries. It could also provide basis for the development of less acidic (erosive) fruit juices. It is suggested that dietary advice and preventive care is mandatory for individuals who frequently consume fruit juices. Declaration of Interest The authors report no conflict of interest and the article is not funded or supported by any research grant. References 1. Lata Kiran Banan, Amitha M Hegde. Plaque and salivary pH changes after consumption of fresh fruit juices. The Journal of Clin Ped Dent. 2005; 30(1): 9-13. 2. Jihanson AK, Johanson A, Birkhed D, Omar R, Baghdais S, Carlson GE. Dental erosion, soft drink intake, and oral health in young Saudi men, and the development of a system for assessing erosive tooth wear. Acta Odont Scand. 1996; 54: 369-78. 3. Darby ET. Dental erosion and the gouty diathesis: Are they usually associated? Dent Cosmos 1892; 34: 629-640. 4. Miller W.D. Experiments and Observations on the Wasting of Tooth Tissue Variously Designated as Erosion, Abrasion, Chemical Abrasion, Denudation. Dent Cosmos 1907; 49: 225247. 5. George K. Stookey. The effect of saliva on dental caries. J Am Dent Assoc 2008; 139; 11S-17S. 6. Birkhed D. Sugar Content, Acidity and Effect on Plaque pH of Fruit Juices, fruit Drinks, Carbonated Beverages and Sports drinks. Caries Res 1984 (18): 120-27. 7. Henry Klein, Carrole E Palmer, Knutson JW. “Studies on dental Caries, dental status and dental needs of elementary school children”. Public health report (Washington) 1938; 53: 751-65. 8. Green J.C., Vermillion J.R. The simplified oral hygiene index. Acta Odont Scand. 1963; 21: 533-51. 9. Hartles RL, Wagg BJ. Erosive effect of drinking fluids on the molar teeth of rats. Arch Oral Biol 1962; 5: 307-15. 10. W.M Edger, B G Bibby, S Mundorff, J Rowley., Acid production in plaques after eating snacks: modifying factors in foods. Journal of American Dental Association 1975; 90: 418-25. 11. Mythri H, Chandu GN, Prashant GM, Subba Reddy V. V. Effect of Four Fruit Juices on pH of Dental Plaque – A Four Period Cross-over Study. Journal of the Indian Association of Public Health Dentistry. 2008; 11: 53-58.

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Riga’s Papilloma: A case report Sonali Saha et al

RIGA’S PAPILLOMA IN A 3 MONTH OLD INFANT: A CASE REPORT Sonali Saha1*, Firoza Samadi2, Sabyasachi Saha3, Fahad Mansoor Samadi4 1. Senior Lecturer, Department of Pedodontics, Sardar Patel Postgraduate Institute of Dental and Medical Sciences, Lucknow, India. 2. Professor & Head, Department of Pedodontics, Sardar Patel Postgraduate Institute of Dental and Medical Sciences, Lucknow, India. 3. Professor & Head, Department of Public Health Dentistry, Sardar Patel Postgraduate Institute of Dental and Medical Sciences, Lucknow, India. 4. Senior Lecturer, Department of Oral & Maxillofacial Pathology, Sardar Patel Postgraduate Institute of Dental and Medical Sciences, Lucknow, India.

Abstract Riga’s papilloma or Cardarelli's apthae describes ulceration on the ventral of the tongue in neonates and infants. It is often associated with natal or neonatal teeth. It may rarely occur in older infants. Failure to diagnose and properly treat this lesion can result in inadequate nutrient intake for the infant. Treatment should begin conservatively and should focus on eliminating the source of trauma. The purpose of this study is to describe a clinical case of an infant with Riga’s papilloma on the ventral of the tongue along with the clinical management. The case is very interesting and can be very useful to Pediatric Dentists. A 3 month-old male infant reported for evaluation of a neonatal tooth that was erupting in the mandibular anterior region together with a non-healing oral ulcer. The tooth was not present at the time of his birth. Intra Oral examination revealed one firm tooth crown in the mandibular anterior central incisor region. Examination also revealed ulceration on the ventral surface of tongue. As the neonatal tooth was the cause of the ulceration and pain, extraction of the tooth was chosen as treatment of choice. Patient was re-examined after 3 weeks. Examination revealed complete healing of the ulcer and the extraction socket. With subsequent patient follow-up (at 7 months of age), an occlusal radiograph revealed erupting mandibular central incisors. Hence it was possible to conclude that the neonatal tooth had been a supernumerary. Case report (J Int Dent Med Res 2011; 4: (1), pp. 12-16 ) Keywords: Riga’s papilloma, Cardarelli's apthae, natal and neonatal teeth, traumatic ulcer. Received date: 10 September 2010 Introduction ‘Riga’s papilloma’ is a rare, benign traumatic ulceration on the ventral surface of the tongue in neonates and infants typically between 1 week and 1 year of age1,2. It is frequently associated with natal, neonatal, or primary lower incisor teeth3. The lesion was first described by Antonio Riga, an Italian physician, in 18814. Histologic studies and additional cases

*Corresponding author: Dr. Sonali Saha (Senior Lecturer) Department of Pedodontics, Sardar Patel Postgraduate Institute of Dental and Medical Sciences, Rai-bareilly Road, Lucknow, India. Pin- 226025. E-mail: sonalisaha24@yahoo.co.in

Volume ∙ 4 ∙ Number ∙ 1 ∙ 2011

Accept date: 04 March 2011 were subsequently published by F. Fede in 1890. It has been subsequently known as ‘Riga's boil’ or ‘Riga’s disease’5. Typically, the benign lesion begins as an ulcerated area on the ventral surface of the tongue exposed to repeated trauma from a natal or neonatal tooth2. With repeated trauma, it may progress to an enlarged fibrous mass and resemble a traumatic ulcerative granuloma (with stromal eosinophilia). Often, the lesion develops a rolled white hyperkeratotic border immediately adjacent to the ulceration. Occasionally, the underlying proliferative granulation tissue results in a raised exophytic lesion similar to a pyogenic granuloma6. The clinical presentation many resemble squamous cell carcinoma causing concern but are not frequently reported7,8. Page 12


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Riga’s Papilloma: A case report Sonali Saha et al

It may interfere with proper suckling and feeding and the pain associated with the lesion may put the neonate at risk for dehydration and failure to thrive. In such instances, dental intervention may be required5, 7. Treatment should begin conservatively and should focus on eliminating the source of trauma7-11. The purpose of this study was to describe a clinical case of an infant with Riga’s papilloma on the ventral of the tongue. Case Report A 3 month-old male infant was referred to the out-patient department, Department of Pedodontics, Sardar Patel Postgraduate Institute of Dental & Medical Sciences by his attending paediatrician for evaluation of a neonatal tooth that was erupting in the mandibular anterior region together with a non-healing oral ulcer of approximately 1 month duration. The tooth was not present at the time of his birth. The mother complained of child exhibiting inadequate nutrient intake together with pain during suckling. A review of his medical chart revealed that he had a birth weight of 1,665 g. Maternal and paternal medical and social histories had no evidence of heredity influence or other significant information. Intra Oral examination revealed one firm tooth crown, whitish in colour in the mandibular anterior central incisor region (Figure 1). Examination also revealed 6 mm x 11 mm ulceration on the ventral surface of tongue that extended from anterior border of the tongue to lingual frenum (Figure 2). On palpation, area elicited a pain response from the patient.

Figure 2. Intra Oral Photograph showing ‘Riga's papilloma’ on the ventral surface of the tongue. Examination of the rest of intraoral mucosa revealed no other lesions. Difficulty in obtaining a radiograph of the region, due to the child’s age, prevented immediate confirmation of whether the tooth in question belonged to the normal series or was a supernumerary. Based on clinical findings, diagnosis of ‘Riga’s Papilloma’ was made. As the neonatal tooth was the suspected cause of the ulceration and pain, extraction of the tooth was chosen as treatment of choice. Extraction was carried out under topical local anaesthesia, which the infant tolerated well (Figure 3).

Figure 3. After Extraction of Neonatal Tooth.

Figure 1. Intra Oral Photograph of 3 month old infant showing Neonatal Tooth in mandibular anterior region. Volume ∙ 4 ∙ Number ∙ 1 ∙ 2011

Clinical examination of the extracted tooth revealed pulp tissue (Figure 4) which was sent for histological examination. Histological results revealed it to be normal, underdeveloped pulp tissue with sparse innervations and cellular components. Page 13


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Histological examination of the neonatal tooth showed of thin layer of hypoplastic enamel, atubular dentin and no evidence of root formation (Figure 5).

Figure 6. Healed ulcer after 3 weeks.

Figure 4. Extracted Tooth with Extirpated Pulp Tissue.

Figure

7. Mandibular Anterior Occlusal Radiograph taken at 7 Months. Erupting Central Incisors (71, 81) visible. Discussion

Figure 5. Ground section of the neonatal tooth showing irregular arrangement of enamel rods &irregular DE junction. Patient was re-examined after 3 weeks. Examination revealed complete healing of the ulcer and the extraction socket (Figure 6). The baby appeared to be much more content and the mother reported that her baby was feeding normally and gained weight. With subsequent patient follow-up (at 7 months of age), an occlusal radiograph revealed erupting mandibular central incisors. Hence it was possible to confirm that the neonatal tooth had been a supernumerary (Figure 7). Volume ∙ 4 ∙ Number ∙ 1 ∙ 2011

Infants born with teeth or teeth erupting immediately after birth have appeared in the medical and dental literature1-5, 7. These teeth have been referred as 'Natal teeth', 'congenital teeth', fetal teeth, and predecidual teeth2, 7. If the primary teeth erupt during the third to the fifth month of life, they are termed precocious dentition1, 2. According to Massler and Savara1, natal teeth indicates teeth present in oral cavity at birth and 'neonatal teeth' are those which erupt during the neonatal period i.e., from birth to thirtieth day of life. Natal and neonatal teeth are rare in the buccal cavity3,4. Their incidence has been investigated in many studies. In a 1995 review article, Zhu and Page 14


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King3 tabled results from 10 studies dating from 1876 to 1991. For this group, the reported incidence of both natal and neonatal teeth ranged from 1:7160 to 1:30,000. Bodenhoff & Gorlin4 reported the incidence of natal and neonatal teeth to be 0.3-0.5%. According to Elzay8, natal teeth are more frequent than neonatal teeth, ratio being approximately 3: 1. Previous clinical studies and surveys suggest that more females are affected than males1-4, 9. In our case the infant is a male child. 76% to 94% of natal and neonatal teeth occur in pairs1, 2, 6-8. In the present case, there was a single neonatal tooth present in the lower central incisor region. The etiology of this anomaly remains unknown, although it has been related to a series of factors, such as superficial positioning of the tooth germ, osteoclastic activity within the tooth germ area (bone remodeling), hereditary factors, endocrine disorders, hypovitaminosis and fever states2, 7,8,10. Three syndromes have been associated with natal teeth: (a) chondroectodermal dysplasia or Ellis-van Creveld syndrome, (b) oculomandibulo-dyscephaly with hypotrichosis or Hallermann-Streiff syndrome, and (c) pachyonychia congenita or JadassohnLewandowski syndrome. Natal teeth may also be associated with cleft lip, cleft palate and cyclopia9. Clinical studies by Kates et al. 6 and Massler and Savara1 suggested that only 1 to 8 % of natal and neonatal teeth are supernumerary. However, in the present case, with subsequent patient follow-up (at 7 months of age) and eruption of mandibular central incisors, it was possible to confirm with a radiograph that the tooth had been a supernumerary. Clinically, natal and neonatal teeth can be normal in size and shape, or conical, with enamel hypoplasia and a yellow-brownish coloration2-6. Radiographically, these teeth show low radiopacity, minimal or absent root formation1-4. Ground section of natal and neonatal teeth revealed hypo mineralized enamel, irregular arrangement of enamel rods, irregular dentino-enamel junction, dentinal tubules, more cellular and numerous vascular channels with endothelial cells and large pulp chamber7,8,11,12. There was also a failure of root formation despite eruption and a failure of cementum formation6,8. This was in accordance with the histological findings in our present case. Volume ∙ 4 ∙ Number ∙ 1 ∙ 2011

Riga’s Papilloma: A case report Sonali Saha et al

Simple chronic traumatic ulcerations occur most often on the tongue, lips, and buccal mucosa (sites usually injured by the dentition). The presence of natal or neonatal teeth may lead to formation of a traumatic ulcer on the ventral surface of the tongue, which is known as Riga’s Boil or Riga’s apthae 2, 9,10,13,14. Although usually associated with natal or neonatal teeth, it may also occur in older infants after the eruption of the primary lower incisors5-7. Various terms have been applied to this lesion, including Cardarelli's aphthae, Cardarelli's disease, Fede's disease, Riga's aphthae, Riga's boil, Riga's papilloma, Riga’s disease, RigaFede’s disease, sublingual ulcer, sublingual granuloma9,12,15. This lesion was of particular concern in Italy in the late 1800s because it was frequently associated with malnourished infants and often resulted in death. In some cases, it was also thought to be diagnostic of whooping cough. Hence it was considered that the lesion was of traumatic origin7,12. Buchanan et al. 2 & Elzay et al. 8 reported these lesions to be found on the anterior ventral surface of the tongue. In our present case the ulceration extended from anterior border of the tongue to lingual frenum. According to Slayton 11 , the dorsal surface may also be affected. Ventral tongue lesions contact the mandibular anterior incisors whereas dorsal lesions contact the maxillary incisors. Zaenglein et al. 9 reported cases on the buccal mucosa, palate, and lip, but the tongue remains to be the most common site of involvement. Feeding behaviours also contribute to the trauma that causes this type of lesion13, 15. Hence, parents are advised to modify their feeding methods to try to avoid the child positioning his tongue over the teeth and to minimize the amount of sucking needed to obtain fluids. This may involve using a bottle with a larger hole in the nipple or a Sippy cup that requires less vigorous sucking7,9. According to Jariwala et al.16 the parent can also attempt feeding by spoon to minimize trauma to the tongue. Treatment should begin conservatively and should focus on eliminating the source of trauma. For traumatic ulcerations that have an obvious source of injury, the irritating cause should be removed first8-10,14. Jariwala et al. 16 suggested biopsy for lesions that remain for 2 weeks after removal of the cause. Ahmet et al.13 Page 15


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reported a rare recurrence rate. In case of mild to moderate irritation to the tongue, conservative treatment such as smoothing the incisal edge with an abrasive instrument is advocated11,14. Alternatively, a small increment of composite may be bonded to the incisal edges of the teeth13. In this case, ulcerated area was large and denuded and even a reduced incisal edge may still contact and traumatize the tongue during suckling, enough to delay healing. Extraction of the natal or neonatal tooth, like in this case was recommended. Tooth extraction had resolved the ulceration too. Although reported by Rutt12, the possibility of pyogenic granuloma in the tooth extraction place, we did not observe this kind of alteration in our present case.

Riga’s Papilloma: A case report Sonali Saha et al

8. Elzay RP: Traumatic ulcerative granuloma with stromal eosinophilia (Riga-Fede’s disease and traumatic eosinophilic granuloma). Oral Surg Oral Med Oral Pathol 1983; 55: 497-506. 9. Zaenglein AL, Chang MW, Meehan SA, Axelrod FB, Orlow SJ: Extensive Riga- Fede disease of the lip and tongue. J Am Acad Dermatol 2002; 47: 445-47. 10. Hedge R J. Sublingual traumatic ulceration due to neonatal teeth. J Indian Soc Pedod Prev Dent 2005; 23: 51–52. 11. Slayton R. Treatment alternatives for sublingual traumatic ulceration (Riga-Fede disease). Pediatr Dent 2000; 22: 413–14. 12. Ruth AA. Natal and neonatal teeth: Histologic investigation of two black females. ASDC J Dent Child 1982; 49: 300-3. 13. Ahmet T, Ferruh B, Gurcan A. Lingual traumatic ulceration (Riga-Fede disease). Br J Oral Maxillofac Surg 1982; 41: 201. 14. Rakocz M, Frand M, Brand N. Familial dysautonomia with RigaFede's disease: report of case. ASDC J Dent Child 1987; 54: 57–59. 15. Terzioglu A, Bingul F, Aslan G. Lingual traumatic ulceration (Riga-Fede disease). J Oral Maxillofac Surg 2002; 60: 478. 16. Jariwala D, Graham RM, Lewis T. Riga fede disease. Br Dent J 2008; 204: 171-72.

Conclusions Our male infant presented a single neonatal tooth in the mandibular central incisor region with hypoplastic enamel. The paediatricians’ concern over the infant’s failure to gain weight due to ulceration's interference with suckling dictated the need for rapid resolution of the lesion. So extraction of the natal tooth was chosen over more conservative treatments. With subsequent patient follow-up, an occlusal radiograph revealed erupting mandibular central incisors. Hence it was possible to confirm that the neonatal tooth had been a supernumerary. Declaration of Interest The authors report no conflict of interest and the article is not funded or supported by any research grant. References 1. Massler M, Savara BS. Natal and neonatal teeth. J Pediatr 1950; 36: 349-59. 2. Buchanan S, Jenkins CR: Riga-Fedes syndrome: natal or neonatal teeth associated with tongue ulceration: Case report. Aust Dent J 1997; 42: 225-27. 3. Zhu J, King D. Natal and neonatal teeth. ASDC J Dent Child 1995; 62(2):123–8. 4. Bodenhoff J, Gorlin RJ. Natal and neonatal teeth: folklore and fact. Pediatrics 1963; 32:1087-1093. 5. Chow MH. Natal and neonatal teeth. ASDC J Dent Child 1995; 62(2): 123–8. 6. Kates GA, Needleman HL, Holmes LB. Natal and neonatal teeth: a clinical study. JADA 1984; 109: 441–3. 7. Tsubone H, Onishi T, Hayashibara T, Sobue S, Ooshima T. Clinicopathological aspects of a residual tooth: a case report. J Oral Pathol Med 2002; 31(4): 239–41.

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Ectodermal Dysplasia and Immunodeficiency M. Callea et al

DENTAL PHENOTYPE IN A PATIENT WITH HYPOIDROTIC ECTODERMAL DYSPLASIA AND SEVERE IMMUNODEFICIENCY M. Callea1*, F. Faletra2, A. Maestro3, F. Verzegnassi4, M. Rabusin4, A. Vinciguerra5, F. Radovich1, G. Clarich1, I. Yavuz6, E. C. Tumen6 1. Maxillo-Facial Surgery and Dentistry, Hospital for Child and Maternal Health, IRCCS Burlo Garofolo, Trieste, Italy. 2. Molecular Medicine and Laboratory Department, Hospital for Child and Maternal Health, IRCCS Burlo Garofolo, Trieste, Italy. 3. Pharmacy and Parenteral Nutrition, Hospital for Child and Maternal Health, IRCCS Burlo Garofolo, Trieste, Italy. 4. Bone-Marrow Transplants Unit, Hospital for Child and Maternal Health, IRCCS Burlo Garofolo, Trieste, Italy. 5. Ophthalmology and Visual-Motor Rehabilitation, Hospital for Child and Maternal Health, IRCCS Burlo Garofolo, Trieste, Italy. 6. Dicle University Faculty of Dentistry, Department of Pediatric Dentistry. Diyarbakir, Turkey.

Abstract Ectodermal dysplasia is a rare disease which affects at least two ectoderm-derived structures such as hair, nails, skin, sweat glands and teeth. The dentition is altered in number and shape. A 14-year-old male patient with hypodontia, micrognathia, ankylosed teeth and conical shaped teeth was referred for examination, evaluation and treatment. The child exhibited the classic dental phenotype of Ectodermal Dysplasia plus a severe immunodeficiency. Radiographic examination revealed ankylosed primary molars. Ocular findings are reported. Conservative dentistry to reduce the abnormal shape was carried out , and an ultrasound scaling every 4 months, with a strong follow up established. The child fulfilled a good occlusion. Every 3 months the patient has been seen in our department for control of hard and soft tissue in the mouth and after 36 months the dental situation is very well accomplished by patient, family and dental staff. Oral rehabilitation must be carried out at the earliest age possible in order to maintain and correct the oral functions, alignment, good occlusion and a good compliance in smiling and feeding. Case report, (J Int Dent Med Res 2011; 4: (1), pp. 17-20) Keywords: Ectodermal Dysplasia, Nemo, Immunodeficiency, Ocular finding. Received date: 18 February 2011 Introduction Ectodermal dysplasias (Eds) refer to a group of inherited disorders involving dysplasia or absence of the ectodermal appendages1. The commonly known signs of nail dystrophy (onychodysplasia), alopecia or hypotrichosis (scanty, fine light hair on the scalp and eyebrows), and palmoplantar hyperkeratosis2 are usually accompanied by a lack of sweat glands (hypohidrosis) and a partial or complete absence of the primary and/ or permanent dentition3-6. *Corresponding author: Dr. Michele Callea Maxillo-Facial Surgery and Dentistry, Hospital for Child and Maternal Health, IRCCS Burlo Garofolo, Trieste, Italy. E-mail: mcallea@gmail.com

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Accept date: 23 March 2011 Orofacial characteristics of this syndrome include hypoplastic conical teeth, underdevelopment of the alveolar ridges, frontal bossing, a depressed nasal bridge and protuberant lips4-8. The most frequently reported ectodermal dysplasia syndrome is X-linked recessive hypohidrotic ectodermal dysplasia4-7,9 where males are usually more severely affected, and carrier females show a variable severity ranging from mild to severe because of X-chromosome inactivation9,10. The hypohidrotic/anhidrotic form of ectodermal dysplasia has been attributed to at least 4 genes (EDA1 [ectodysplasin]; EDAR [the EDA-A1 isoform receptor]; and EDARADD [EDAR-associated death domain]), with at least 3 modes of inheritance: X-linked recessive (OMIM 305100), autosomal dominant (OMIM 129490), and autosomal recessive (OMIM 224900). Page 17


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Recently, the syndrome X-linked ectodermal dysplasia with immunodeficiency11 (XL-ED-ID) (OMIM 300291) was described in patients with hypomorphic mutations in IKBKG (inhibitory B kinase gene), which encodes NEMO (nuclear factor B [NF- B] essential modulator) protein, the regulatory subunit of the IKK (I B kinase) complex. Mutations in the IKBKG gene NEMO can cause an heterogeneous group of disorders, including Incontinentia Pigmenti and Hypohidrotic Ectodermal Dysplasia and immunodeficiency with or without osteopetrosis and lymphoedema. Mutations of IKBKG are difficult to diagnose early in infancy, most of them presenting with manifestations of 12 immunodeficiency . We present the case of a 14-years-old boy with XL-ED-ID carrying the c.1167dupC mutation in the IKBKG gene and showing the characteristic dental features of the syndrome, jointly with a complex set of symptoms, who required a specific, non-invasive, dental treatment.

Ectodermal Dysplasia and Immunodeficiency M. Callea et al

Figure 1. Orthpantomography showing ankylosed teeth, mesialisation of permanent mandibulary molars, and conical shaped teeth in the frontal upper and lower region.

CASE REPORT A 14 years old boy came to the attention of the Pediatric Dental Department of our Institute for Infancy after a long medical history, including bone marrow transplantation (BMT), recurrent colitis, blindness, progressive hearing loss, severe immunodeficiency13. After an oral examination the patient presented a scarce level of oral hygiene, caries and visible multiple tooth agenesis with consequent atrophy of the alveolar ridges, ankylosed teeth, with abnormal shape, especially in the crown, and in the frontal region conically shaped. All these clinical findings led us to the diagnosis of HED-ID. An Orthopantomogram and Cephalometric radiographs were carried out showing a third skeletal class, the presence of permanent maxillary central incisors, primary maxillary lateral incisors conically shaped, primary maxillary first and second molars, permanent first mandibular molars, primary mandibular second molars ankylosed in the bone although asymptomatic, primary mandibular first molars, and 5 conically shaped mandibular teeth resembling primary incisors. (Figures 1,2)

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Figure 2. Cephalometry film showing a third skeletal class. After a professional cleaning of the hard and soft tissue of the mouth with mouth rinsing and scaling by ultrasound ablation, in the second appointment, aesthetic dentistry14 with multiple fillings have been carried out. In a period of 18 months we have been able to reach a satisfying aesthetic and functional result. Whether still a skeletal third-like class was persisting, that is characteristic for patients affected by HED15, a treatment therapy plan has Page 18


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been discussed with the dentistry team and the patient’s family. The final aim to achieve a perfect occlusion was selective extraction of ankylosed teeth, extraction of primary teeth with almost complete root resorption, rapid expansion of the high narrow palate, and placement of implants in the edentulous region. Ocular features The past ocular history was significant for chronic uveitis and progressive retinitis in both eyes; because of these persistent inflammations the child underwent enucleation of right eye and lensectomy with pars plana vitrectomy of the left eye. At the last ocular examination the left eye was blind due to a persistent retinal detachment, and slit lamp examination revealed transparent cornea, deep anterior chamber, iridectomy with a slight pupil deformation (Figure 3). Although various forms of ocular involvement have been reported in anhidrotic HED16, in our case ocular findings were correlated to persistent ocular inflammation following to severe immunodeficiency without the usually related clinical condition.

Ectodermal Dysplasia and Immunodeficiency M. Callea et al

reduction, labial retrusion, chin prominence, nasolabial and chin reinforcement4. However the researchers should be in attention that these measures may be unreliable because they vary according to tooth agenesis and to the severity of ectodermal dysplasia. The consequence of the dental agenesis could curb bone growth4. All clincal signs give the ectodermal dysplasia cases a prematurely aged appearance. That is in agreement with previous research4-6. Several affected infants and children may also exhibit underdevelopment (hypoplasia) or absence (aplasia) of mucous glands within the respiratory tract and, in some cases, decreased lung capacity and function, potentially causing an increased susceptibility to certain infections and/or allergic conditions. A number of affected patients experience recurrent attacks of wheezing and breathlessness (asthma), and respiratory infections4-6. Considering the long medical history of our young patient who is now 16 year old, and his personal satisfaction with the actual dental situation, and along with the consent of the parents, in order to prevent any kind of further complains due to expansion of the palate and selective extraction under general anesthesia, the decision has been agreed for a strong followup with a 3 month dental control by maintaining the actual dental status and a daily careful toothbrushing with fluoride toothpaste and mouth rinsing at a concentration of chlorhexidine below 0.05%. A multidisciplinary approach is required in modern dentistry for diagnosis and treatment ectodermal dysplasia cases. Conclusions

Figure 3. Transparent cornea, deep anterior chamber, miosis, slight pupil deformation, iridectomy, aphakia Discussion Ectodermal dysplasia is a rare, genetically transmitted, multisystem disorder. Diagnosis of ectodermal dysplasia, without any other diagnostic precision, would be difficult at best. Steiner analyses are useful for revealing a facial height reduction and concavity4, maxillar Volume ∙ 4 ∙ Number ∙ 1 ∙ 2011

Ectodermal Dysplasia represents a large group of inherited disorders, with many gene mutations involved. Clinical findings and genetic counselling can lead to an early diagnosis. This is an unique case in which conically shaped teeth, dry skin, sparse hair and an appropriate study of the Nemo gene has led us to the correct diagnosis and to the possibility of achieving a good compliance for the treatment. Acknowledgements We thank the family for allowing us the publication of images and clinical data and for full cooperation. Page 19


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Ectodermal Dysplasia and Immunodeficiency M. Callea et al

Declaration of Interest The authors report no conflict of interest. The article is not funded or supported by any research grant. References 1. Clarke A. Hypohidrotic ectodermal dysplasia. J Med Genet 1987; 24(11):659-63. 2. Bonilla ED, Guerra L, Luna O. Overdenture prosthesis for oral rehabilitation of hypohidrotic ectodermal dysplasia: a case report. Quint Int 1997;28:657-665. 3. Tumen EC, Hamamcı N, Değer Y, Süer Tümen D, Agaçkiran E. Direct composite resin application, and prosthetic management in a patient with hypohidrotic ectodermal dysplasia: A case report. J Int Dent Med Res (JIDMR) 2009; 2(1), 19-24. 4.Yavuz I, Ulku SZ, Unlu G, Devecioglu KAMA , Kaya S, Adiguzel O, Akun Kaya F,Tumen EC, Zortuk M, Bashi E, Arslanoglu Z, “Ectodermal Dysplasia: Clinical Diagnosis”, International Dental and Medical Disorders 2008; 1, 1-10. 5.Adiguzel O, Kaya S, Yavuz I, Atakul F, “Oral Findings of Ectodermal Dysplasia and Literature Review”, International Dental and Medical Disorders, 2008; 1, 43-49. 6. Yavuz, I., Z. Baskan, R. Ulku, T.C. Dulgergil, O. Dari, A. Ece, Y. Yavuz ve O. Dari, “Ectodermal Dysplasia: Retrospective Study of 15 Cases,” Archives of Medical Research, 37(3), 403-409 (2006). 7. Levin LS. Dental and oral abnormalities in selected ectodermal dysplasia syndromes. Birth Defects Orig Artic Ser 1988;24:205-227. 8. Champlin TL, Mallory SB. Hypohidrotic ectodermal dysplasia: a review. J Ark Med Soc 1989;86:115-117. 9. Freire-Maia N, Pinheiro M. Ectodermal dysplasia: a clinical and genetic study. New York: Alan R. Liss;1984: 25-31. 10. Kere J, Srivastava AK, Montonen O, et al. X-linked anhidrotic (hipohidrotic) ectodermal dysplasia is caused by mutation in a novel transmembrane protein. Nat Genet 1996;13:409-416. 11. Permaul P, Narla A, Hornick JL, Pai SY. Allogeneic hematopoietic stem cell transplantation for X-linked ectodermal dysplasia and immunodeficiency: case report and review of outcomes. Immunol Res 2009; 44(1-3): 89-98. 12. Roberts CM, Angus JE, Leach IH, McDermott EM, Walker DA, Ravenscroft JC. A novel NEMO gene mutation causing osteopetrosis, lymphoedema, hypohidrotic ectodermal dysplasia and immunodeficiency (OL-HED-ID). Eur J Pediatr 2010; 169(11): 1403-7. 13. Fish JD, Duerst RE, Gelfand EW, Orange JS, Bunin N. Challenges in the use of allogeneic hematopoietic SCT for ectodermal dysplasia with immune deficiency. Bone Marrow Transplant 2009; 43(3): 217-21. Epub 2008 Sep 15. 14. Răducanu AM, Păuna M, Feraru IV. A simple prosthetic restorative solution of a single peg-shaped upper central primary incisor in a case of ectodermal dysplasia. Rom J Morphol Embryol 2010; 51(2): 371-4. 15. Yavuz I, Kiralp S, Baskan Z. Hypohidrotic ectodermal dysplasia: a case report. Quint Int 2008; 39(1): 81-6. 16. Saw VP, Dart JK, Sitaru C, Zillikens D. Cicatrisig conjunctivitis with anti-basement membrane autoantibodies in ectodermal dysplasia Br J Ophthalmol 2008; 92(10):1403-10.

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Dentigerous Cyst Kamil Serkan Agacayak et al

DENTIGEROUS CYST WITH AN IMPACTED CANINE: CASE REPORT Kamil Serkan Agacayak1, Ibrahim Kose1, Nedim Gunes1, Emrullah Bahsi2, Ferhan Yaman1*, Serhat Atilgan1 1. Department of Oral & Maxillofacial Surgery, Faculty of Dentistry, Dicle University. Diyarbakir / TURKEY. 2. Department of Operative Dentistry Faculty of Dentistry, Dicle University. Diyarbakir / TURKEY.

Abstract Dentigerous cysts are the second most common developmental odontogenic cysts after radicular cyst. They usually present in the second or third decades of life and these cysts are rarely seen during child hood. Their frequency in the general population has been estimated at 1.44 cysts for every 100 unerupted teeth. According to frequency of impaction, the maxillary permanent canine ranks second only to the third molar, with a prevalence of approximately 2% in the general population. Impacted canines are positioned palatally 85% of the time. The frequency of impaction is three times greater in females than males. In many instances the cyst may be asymptomatic till it attains a large size. It usually presents as a slowly enlarging, sometimes painful swelling; particularly if infected. At radiography, dentigerous cysts appear as well-defined, round or ovoid, corticated, lucent lesions around the crowns of unerupted teeth. The radiographic appearance of such dentigerous cysts is comparable with that of cystic, unilocular odontogenic keratocysts. Treatment includes extraction of the associated tooth and enucleation of the cyst. In this study we present dentigerous cyst that caused by impacted canine. Case report (J Int Dent Med Res 2011; 4: (1), pp. 21-24) Keywords: Dentigerous cyst, unerupted teeth, impacted canine. Received date: 25 January 2011 Introduction After radicular cysts, the second most common are dentigerous cysts of odontogenic origin and account for about 16.6% of all such jaw lesions1,2. They are usually asymptomatic but can become extremely large and cause cortical expansion and erosion. In 75% of the cases, they are located in the mandible. The mandibular third molar and maxillary canine are involved most frequently1,2. Radiographically, the dentigerous cyst appears as a unilocular radiolucency of variable size with well-defined sclerotic borders, *Corresponding author: Dr. Ferhan Yaman Dicle University Faculty of Dentistry Department of Oral and Maxillofacial Surgery 21280 Diyarbakir,Turkey

Accept date: 16 February 2011 associated with the crown of an unerupted tooth. Histologically, the dentigerous cyst displays a thin fibrous cyst wall with a myxomatous appearance. The epithelial lining consists of 2-4 layers of fat or cuboidal cells, which in fact is the reduced enamel epithelium and is characteristically non-keratinized. Nests, islands or strands of odontogenic epithelium are often seen in the fibrous capsule. Localized proliferation of epithelial lining may occur in response to inflammation. Several treatment options existed, including removal of the cyst via enucleation; marsupialization of the cyst to the oral mucosa, with placement of a wire to allow for drainage and decompression of the cyst; decompression of the cyst via fenestration. The aim of this study is illustrates a simplified surgical treatment for large dentigerous cysts with impacted tooth. The procedure can be performed in the office and provides the best chance to preserve.

E-mail: dtferhan@hotmail.com

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Case Report A19 year-old female patient referred to our clinic with a complaint of swelling in the anterior maxilla. The patient’s medical history was insignificant. No other abnormality was detected on complete systemic examination. The patient reported that she had first noticed the swelling one year ago. Oral and radiologic examination showed the absence of left mandibular canine tooth. Panoramic radiograph showed that mandibular permanent canine was a radiolucency with a well-defined border which measured 2.0cm x 2.0cm, surrounding the left mandibular impacted canine. (Figure 1).

Figure 1. Preoperative panoramic view.

Dentigerous Cyst Kamil Serkan Agacayak et al

The patient was further prepared for enucleation of the pathology with a provisional diagnosis of dentigerous cyst arising from the impacted canine. On incision and reflection of the mucosa, a thinned mandibular cortex was evident with areas of perforation. The bone cavity was lined by a thick epithelium that was easily enucleated (Figure 2). Impacted canine was removed along with the left lateral incisor and left first premolar that lacked sufficient bone support. The mucosa was then sutured. Post operative healing was uneventful. Biopsy specimen was sent for histopathological examination. The lesion was reported as dentigerous cyst (Figure 3).

Figure 3. Histological view. The patient was follow up for six months. In routine controls there was no problem and was also no evidence of recurrence of the cyst (Fig-4).

Figure 2. Surgical specimen. In the left lower canine region a softly palpable, remarkable swelling was evident. The overlying mucosa was of normal color and appearance. Extra oral examination revealed that there is no hard bony expansion over laying the maxilla. The oral soft tissues were within normal limits, and inferior alveolar nevre function was normal. Fine needle aspiration biopsy showed a serious cyst liquid with colestine crystals. Volume ∙ 4 ∙ Number ∙ 1 ∙ 2011

Figure 4. Postoperative panoramic view. Discussion Teeth may be impacted or erupt ectopically for a variety of reasons. Hereditary factors, lack of space, persistence of primary canines, a true ectopic path of eruption, reduced root length and aplasia of lateral incisors are a few of the factors cited3. Page 22


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Failure of eruption of the mandibular canine is an unusual event4. Mandibular canine impaction is regarded as a much rarer phenomenon, and there are limited numbers of studies revealing its frequency of occurrence5. Grover and Lorton found only 11 impacted canines (0.22%) in the mandible in 5000 individuals6. Chu et al. reported five mandibular impacted canine (0.07%) teeth in 7486 patients7. A study by Rohrer examining 3,000 patients radiographically found 62 impacted maxillary canines (2.06%) and only three impacted mandibular canines (0.1%), a 20:1 ratio.8 An another study by Aydin et al. involving 4500 Turkish patients, the incidence of mandibular canine impaction was 0.44% 5. Dentigerous cysts enclos the crown of an unerrupted tooth, attaching to the neck of the tooth and grows by expansion of its follicle. It is classified as a developmental cyst by the World Health Organization9. In 75% of cases they are located in the mandible. The mandibular third molar and maxillary canine are involved most frequently.10 The first is developmental in origin and occurs in mature teeth usually as a result of impaction. The incidence of cysts and tumours around impacted third molars is 3.1% 11. According to the literatures two types of dentigerous cysts occur. These cysts usually occur in the late second and third decades, are discovered on routine radiography, and predominantly involve mandibular third molars 12,13 . The second type is inflammatory in origin and occurs in immature teeth as a result of inflammation from a nonvital deciduous tooth follicle12. These are diagnosed in the first and early part of the second decade either on routine radiographic examination or when the patient complains of swelling and pain14. Our cases classified as the first type of dentigerous cyst. Dentigerous cysts are usually single lesions. Bilateral and multiple cysts have been reported in patients with syndromes such as basal cell nevus syndrome, mucopolysaccharidosis, and cleido cranial dysplasia15,16. Bilateral mandibular dentigerous cysts have also been reported after prolonged concurrent use of cyclosporine A and calcium channel blockers. Gingival hyperplasia and impaired dentition are the most common features shared by most of these syndromes2. No Volume ∙ 4 ∙ Number ∙ 1 ∙ 2011

Dentigerous Cyst Kamil Serkan Agacayak et al

syndrome was detected in our cases. It’s known that panoramic radiography has a limited value for evaluating the margins and extension of the lesion. CT examination aid in delineating the extent of the lesion. The indications for CT examination of dentigerous cysts are not so familiar. Conventional X-ray methods /extraoral and intraoral/ give enough information for occasional findings- asymptomatic and without clinical signs dentigerous cysts. CT imaging displays bony details and gives exact information about the size, origin, content and relationships of the lesion involving the maxilla9,18. The epithelial cells lining the lumen of dentigerous cysts are able to undergo metaplastic change to other epithelial cell types. The cyst's lining may contain areas of orthokeratinization, ciliated cells or mucinsecreting cells18. Because of this inherent ability for metaplastic change, some dentigerous cysts appear to progress to more aggressive lesions such as an odontogenic keratocyst, ameloblastoma, mucoepidermoid carcinoma or squamous cell carcinoma19. We performed long term follow up of our cases (approximately one year and still goes on) and no dysplastic changes were observed. The histopathologic findings of dentigerous cysts vary depending on whether the cyst is inflamed. In the noninflamed variant, the fibrous connective tissue wall is loosely arranged with small islands of inactive odontogenic epithelial rests18. In our cases histological sections of specimens were similar, showing cyst walls composed of fibrous tissue and lined by stratified squamous, non-keratinized epithelium. Treatment of dentigerous cysts depends on size, location, and disfigurement, and often requres variable bone removal to ensure total removal of the cyst, especilly in cases of large ones20. However, cysts causing tooth displacement and involving loss of bone should be treated with marsupialization or decompression21. In this method, new bone formation is stimulated because marsupialization decreases intracystic pressure. The major disadvantage of marsupialization is that pathologic tissue is left in situ, without a thorough histologic examination. Although the tissue taken in the window can be submitted for pathologic examination, there is a possibility of a more aggressive lesion in the residual tissue22. Page 23


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Conclusions These cysts are usually slow-growing lesions and may attain a considerable size with minimal or no symptoms. Ameloblastoma, mucoepidermoid carcinoma, and squamous cell carcinoma have also been reported to arise from the lining epithelium of dentigerous cysts, indicating the pluripotentiality of their cells. Early detection and removal of such cysts is therefore important to reduce potential morbidity. As it is common to find an unerupted tooth as the only initial presenting symptom of a dentigerous cyst, it is important to undertake radiographic examinations of all such teeth that are well past their expected eruption date. Despite the rarity of bilateral occurrence, once a dentigerous cyst has been identifed, attempts must be made to rule out the presence of any co-existent lesions in other parts of the jaws. Declaration of Interest

Dentigerous Cyst Kamil Serkan Agacayak et al

11. Kaya O., Bocutoglu O. A misdiagnosed giant dentigerous cyst involving the maxillary antrum and affecting the orbit. Case report. Aust Dent J 1994; 39,165. 12. Kramer L.R, Pindborg J.J., Shear M. International histological classification of tumors. 2nd edn 1992; 34-36 13. Ko K.S.C., Dover D.G., Jordan R.C.K. Bilateral dentigerous cysts -report of an unusual case and review of the literature: Clinical Practice 1999; 68,49-51. 14. Miyawaki S., Hyomoto M., Tsubauchi J. Eruption speed and rate of angulation change of a cyst-associated mandibular second premolar after marsupialization of a dentigerous cyst. Am J Orthod Dentofac Orthop 1999; 116,578. 15. Mintz S., Alard M., Nour R. Extraoral removal of mandibular odontogenic dentigerous cysts: A report of 2 cases. J Oral Maxillofac Surg 2001; 59,1094. 16. Norris L., Piccoli P., Papageorge M.B. Multiple dentigerous cysts of the maxilla and the mandible: report of a case. J Oral Maxillofac Surg 1987; 45,694-7. 17. Peterson LJ, Ellis E, Hupp JR, Tucker MR. Contemporary Oral and Maxillofacial Surgery 4th edn: 2003; p116 18. Rohrer A. Displaced and impacted canines. Int J Orthod Oral Surg 1929; 15,1003. 19. Shivaprakash P., Rizwanulla T., Baweja D.K., Noorani H.H. Save a tooth: Conservative surgical management of dentigerous cysts; J Indian Soc Pedod Prevent Dent 2009: 1,27, 52-5. 20. Ustuner E., Fitoz S., Atasoy Ç.,Erden İ., Akyar S. Bilateral maxillary dentigerous cysts: A case report Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003; 95,632-5. 21. Vincent B.Z., Todd I.E., Ronald E.S. Using fenestration technique to treat a large dentigerous cysts J Am Dent Assoc 1997; 128,201-205. 22. Wong M. Surgical fenestration of large periapical lesions. J Endod 1991; 17,517.

The authors report no conflict of interest and the article is not funded or supported by any research grant. References 1. Aydin U., Yilmaz H.H., Yildirim D. Incidence of canine impaction and ransmigration in a patient population. Dentomaxillofac Radiol 2004; 33,164-9. 2. Baykul T., AA. Saglam, U.Aydin, Basak K. Incidence of cystic changes in radiographically normal impacted lower third molar. Oral surg Oral Med Oral Pathol Oral Radiol Endod 2005; 99,5. 542-5. 3. Camilleri S, Scerri E. Transmigration of mandibular canines-A review of the literature and a report of five cases. Angle Orthod 2003; 73,753-762. 4. Chu F.C.S., Li T.K.L., Lui V.K.B., Newsome P.R.H., Chow R.L.K., Cheung L.K. Prevalence of impacted teeth and associated pathologies - a radiographic study of the Hong Kong Chinese population. Hong Kong Med J 2003; 9,158-163. 5. Ertaş Ü., Yavuz M. S. Interesting eruption of 4 teeth associated with a large dentigerous cyst in mandible by only marsupialization J Oral Maxillofac Surg 2003; 61,728-730. 6. Grover P.S, Lorton L. The incidence of unerupted permanent teeth and related clinical cases. Oral Surg Oral Med Oral Pathol 1985; 59,420-425. 7. Guven G.O., Keskin A., Akal U. K. The incidence of cysts and tumors around impacted third molars. Int J Oral Maxillofac Surg 2000; 29,131-135. 8. Mihailova H., Nikolov V., Slavkov S. Diagnostic imaging of dentigerous cysts of the mandible. Journal of IMAB - Annual Proceeding (Scientific Papers), book 2008; 2 ,8-12. 9. Jacoby H. The etiology of maxillary canine impactions. Am J Orthod 1983; 84,125-132 . 10. Johnson L.M., Sapp J.P., Mc Intire D.N. Squamous cell carcinoma arising in a dentigerous cyst. J Oral Maxillofac Surg 1994; 52,987-90.

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Parafunctional Force on Dental Implant in Bruxism Mustafa Zortuk et al

EFFECT OF PARAFUNCTIONAL FORCE ON DENTAL IMPLANT TREATMENT IN BRUXISM: A CASE REPORT (TWO YEAR RESULTS) Mustafa Zortuk1*, Erdem Kilic2, Pinar Yildiz3, Ikbal Leblebicioglu3 1. Assistant Professor, Department of Prosthetic Dentistry, Faculty of Dentistry, Erciyes University, Kayseri- Turkey. 2. Assistant Professor, Department of Maxillofacial Surgery, Faculty of Dentistry, Erciyes University, Kayseri- Turkey. 3. Research Assistant, Department of Prosthetic Dentistry, Faculty of Dentistry, Erciyes University, Kayseri- Turkey.

Abstract An implant fracture may be one of the major causes of implant failures. The probable cause of the implant fracture was due to biomechanical overload caused by bruxism. Bruxism (teeth grinding and clenching) is generally considered a contraindication for dental implants. So far, in the dental literature, the possible cause-and-effect relationship between bruxism and implant failure do not yield consistent and specific outcomes. This is partly because of the large variation in the literature in terms of both the technical aspects and the biological aspects of the study material. Although there is still no proof for the suggestion that bruxism causes an overload of dental implants and of their suprastructures, a careful approach is recommended. This case report illustrates the importance of an extensive clinical examination and accurately occlusal arrangements of oral implant patients for the presence of severe bruxism for two year results. Case report (J Int Dent Med Res 2011; 4: (1), pp. 25-29) Keywords: Bruxism, dental implant, fixed prosthesis. Received date: 03 September 2010 Introduction Bruxism is a movement disorder of the masticatory system that is characterized, among others, by teeth grinding and clenching, during sleep as well as during wakefulness.1 Several recent review articles describe the definitions, epidemiology, (differential) diagnosis, aetiology, and treatment of this disorder. Bruxism is frequently considered an aetiological factor for temporomandibular disorders (TMD), tooth wear (e.g. attrition), loss of periodontal support, and failure of dental restorations and dental implants, although conflicting evidence for many of these purported aetiological relationships can be found in the literature.2 *Corresponding author: Dr. Mustafa Zortuk Department of Prosthetic Dentistry Faculty of Dentistry Erciyes University, Melikgazi-Kayseri 38039, Turkey.

E-mail: mzortuk@erciyes.edu.tr

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Accept date: 15 October 2010 These possible musculoskeletal and dental consequences of bruxism illustrate the clinical importance of this disorder. Importantly, it should be borne in mind that there is still a lack of agreement about, for example, the definition of bruxism, which makes it sometimes difficult to unequivocally interpret the available evidence. 3 Since the beginning of implant dentistry, implant-supported prostheses have proven to be a highly predictable treatment for completely and partially edentulous patients. However, complications affecting osseointegrated dental implants can occur in specific situations and the clinician must be aware of the treatment limitations and avoid risky situations, which can lead to implant-supported prostheses failure due to biomechanical complications.4,5 These complications can involve loosening or fracture of the prosthetic screw, loosening or fracture of the abutment screw, and also implant fracture.6 Bruxism has also been suggested to cause excessive (occlusal) load of dental implants and their suprastructures, ultimately resulting in bone loss around the implants or even in implant failure. Not surprisingly, bruxism is, therefore, often considered a cause of Page 25


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Parafunctional Force on Dental Implant in Bruxism Mustafa Zortuk et al

Nevermore she hadn’t any temporamandibularjoint (TMJ) problem like sensitive muscles, limited occlusal opening and deviation on lower jaw opening. Following the routine oral implant treatment planning protocol of the clinic, 2 implants, with lengths of 12 mm and diameters of 3,3 and 4,1 mm, respectively, were finally placed at the former tooth sites of elements 45 to 47 (Standard Plus Implant, Straumann® AG,Basel, Switzerland) by submerged approach. The patient had not used any temporary removable denture during osseointegration. After a healing phase of almost 3 months, the implants appeared to be firmly anchored, as assessed clinically and radiographically. In July 2008, the secondary surgery was performed and healing caps were placed. After a ten day period the patient recalled in order to take the impression. The impression of the implants and teeth was taken by a custom made tray by using polyvinyl siloxane material ( ExpressTM XT, 3M ESPE AG D-82229 Seefeld-Germany). The master model was casted with Type IV stone. Afterwards the abutments were chosen for each implants ( SynOcta 048.605 for 45 and SynOcta 048.602 for 47 , Straumann® AG,Basel, Switzerland). The metal-ceramic restorations were produced for both quadrants. Subsequent to the laboratory procedures abutments were placed on the implants and the trials were performed in order to examine the coherence. Before the screwing the premature contacts during the occlusal and lateral movements were removed and occlusal management was finished by bilateral balanced Case Report occlusion with tripod sentric contact. Bridges were made on the implants and teeth, to the full In March 2008, a 64 year old woman satisfaction of both the patient and the consulted the clinic of the Department of prosthodontist (figure1). Prosthetic Dentistry of Erciyes University Dentistry Faculty, with a wish for oral implants in the right lower jaw and retreat her old fixed prosthodontics to improve her aesthetics and oral function. There was not any systemic disease in her history. On her clinical examination there were improper fixed partial prostheses at her left mandible and maxilla. Also it was identified that the occlusal surface of her teeth was abraded severely. Consequently the occlusion type of the patient was bilateral balanced occlusion. Additionally the patient confirmed her Figure 1. After the loading phase, the implants grinding and clenching habit on her anamnesis. assessed radiographically. concern or even a contraindication for implant treatment, as stated in many textbooks and conference proceedings on oral implantology and prosthetic dentistry.2 According to Misch, bruxism doesn’t necessarily represent a contraindication to implants but it does dramatically influence treatment planning. In addition, many researchers use bruxism as an exclusion criterion for the selection of their participants in clinical studies concerning treatment modalities with dental implants. These authors argue that the overloading influence of bruxism on implants and their suprastructures yields a higher risk of biological and biomechanical complications than would be the case during physiological masticatory activities.7 In the presence of bruxism, most authors recommend to place more implants than would have been necessary in the absence of this movement disorder. More specifically, as to avoid free-ending situations, one implant should be placed for each missing element.8,9 This recommendation is supported by the findings of in vivo studies that indicate a reduction of the forces that are being exerted on an individual implant when the number of implants increases.10 In addition, mechanically connecting the implants leads to a better distribution of the forces and a reduction of the stresses in the bone around the implants.11 In these 2-year follow up this report, the efficacy of bruxism is described in a patient experiencing oral implant treatment and effect of bruxism on implant survival.

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Although the patient had bruxism, the occlusal splint was not performed because of not having any symptomatic complaints as TM disorder or muscle pain. She was controlled with clinically and radiologically whether her bruxism influence the implant and its periimplant tissues at 6 month, 1 and 2 year. At 6 month control there was not anything wrong. However, in the first year control, we recognized that at the other quadrant of her mandibula, the porcelain of her bridge was fractured 1mm diameter on the occlusal surface of functional cusp (Figure 2).

Figure 2. The porcelain fracture and removed aesthetic composite on screw due to bruxism at the 1 year control. A porcelain repair system was performed for repairing the fractured porcelain piece (Ultradent Porcelain Repair Kit,Ultradent Inc, South Jordan, UT) . After the measurement of the periapical radiographs, the bone loss around the implants was evaluated by the formula below:

The bone level in the mesial and distal portion of the implants in 6 month, one year and 2 year follow up were shown in the table 1.

Table 1. The bone levels that outcome measures from radiographs. Volume ∙ 4 ∙ Number ∙ 1 ∙ 2011

Parafunctional Force on Dental Implant in Bruxism Mustafa Zortuk et al

The marginal bone level was examined at 2-year recall, and the initial bone level of actual implants placed according to the guidelines determined by the manufacturer was used as a baseline reference. Periapical radiographs were obtained by using a paralleling device (Dentsply RI˙NN, Rinn Cooperation, Elgin, IL, USA). Radiographs were digitized at 2,400 dpi by using a scanner (CanonScan Lide 200 Canon Inc Vietnam), and linear distance measurements were made (Figure 3).

Figure 3. Periapical radiographs were obtained by using a paralleling device and linear distance measurements were made. The maximum bone loss was 1,2mm at the distal side of implant, located at second molar tooth localization. The observed values are encountered the early term success criteria of the implant. Discussion Stress is a particular entity that directly related to force. As a result, any dental force factor magnifies the stress. Different patient conditions place different amounts of force magnitude, duration, type and direction. In addition, several factors may multiply or increase the effect of the other conditions once the dentist has determined the prosthesis type, the dentist should evaluate and account for in the overall treatment plan the potential force levels that will be exerted on the prosthesis.7,12 Several elements observed during the dental evaluation may be the source of additional forces on implant abutments. The initial implant survival, early loading survival, early crestal bone loss, incidence of abutment or prosthetic screw Page 27


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loosening, and unretained restorations, porcelain fracture, and component fracture are influenced by the factors of the force.13 The most harmful forces of stomatognatic system are bruxism because of its repeated or sustained forces. Bruxism has also been cause excessive (occlusal) load of dental implants and their suprastructures, which may ultimately result in bone loss around the implants or even in implant failure.3 In dental literature, bruxism was used as an exclusion criterion, success rates of about 95% are found after 18–24 months14-16 , while for studies that included bruxism patients in their study sample, lower success rates are reported, i.e. about 80% after 1–2 years.17 On the other hand, some studies report high success rates despite the inclusion of bruxists in the study population. For example, the cumulative success rate after 6 years varies between 92 and 95% in a study by Quirynen et al. 18, while Lidquist et al.19 reported a success rate of almost 99% after 15 years. In other words, epidemiological data yield equivocal results with regard to the purported causal relationship between bruxism and implant failure.2 All guidelines aim to minimize the forces that are applied to the implants. A frequent advice is related to the number of implants. In the presence of bruxism, most authors recommend to place more implants than would have been necessary in the absence of this movement disorder. More specifically, as to avoid freeending situations, one implant should be placed for each missing element. A final recommendation regarding the implants themselves is related to their length and diameter: longer implants with a larger diameter help to keep the stresses in the bone as low as possible. Articulation should be characterized by flat incline planes of the cusps as to protect the implant system against the lateral components of the forces that are being exerted during, for example, teeth grinding. In addition, mechanically connecting the implants leads to a better distribution of the forces and a reduction of the stresses in the bone around the implants.2 Conspicuous finding from our case was that the porcelain fracture increase was disproportionate with bruxism habit. Comparably Kinsel and Lin were found that there was a disproportionate increase of porcelain fracture in Volume ∙ 4 ∙ Number ∙ 1 ∙ 2011

Parafunctional Force on Dental Implant in Bruxism Mustafa Zortuk et al

patients with a bruxism habit, patients not wearing a protective occlusal device, and when the restoration opposed another implantsupported metal ceramic crown or FPD in their study. And they deduced that approximately 7 times higher odds of porcelain fracture that these patients.20 Additionally the masticatory function is not clear in current literature for bruxism with dental implant treatment. The chewing efficiency of unilateral implant supported fixed partial dentures in bruxism patients must be investigated for further informations. At the end of our study we compared our data with the success criteria of implants that were approved by dental literature. 21,22 The individual unattached implant was immobile when tested clinically and also the radiograph did not demonstrate any evidence of periimplant radiolucency. In addition, the vertical bone loss was max 1,2mm following the two years. Due to absence of persistent or irreversible signs and symptoms such as pain and these reasons we mentioned above the implant success criteria were ensured. Conclusions In this study the patient who has bruxism and as two implants on one side edentulous ending dentition was followed up during 2 years. There were not any complication existed in the bruxism patient who we investigate and also the registered bone loss was between the normal limits. On the account of our study the implant therapy is applicable and sufficient therapy after making the required occlusal arrangements. Declaration of Interest The authors report no conflict of interest and the article is not funded or supported by any research grant. References 1. Okeson JP. Orofacial pain. Guidelines for assessment, diagnosis, and management. 1996; Chicago, Il: Quintessence Publishing Co, Inc. 2. Lobbezoo F, Brouwers JE, Cune MS, Naeıje M. Dental implants in patients with bruxing habits. J Oral Rehabil 2006;33:152-159. 3. Lobbezoo F, Van Der Zaag J, Naeije M. Bruxism: its multiple causes and its effects on dental implants an updated review. J Oral Rehabil 2006;33:293-300. 4. Rangert B, Krogh PH, Langer B, Van Roekel N. Bending overload and implant fracture: A retrospective clinical analysis. Int J Oral Maxillofac Implants 1995;11:431-432.

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Parafunctional Force on Dental Implant in Bruxism Mustafa Zortuk et al

5. Eckert SE, Meraw SJ, Cal E, Ow RK Analysis of incidence and associated factors with fractures implants: A retrospective study. Int J Oral Maxillofac Implants 2000;15:662-667. 6. Mendonça G, Mendonça DB, Fernandes-Neto AJ, Neves FD Management of Fractured Dental Implants:A Case Report Implant Dent. 2009;18:10-16 7. Misch CE. The effect of bruxism on treatment planning for dental implants. Dent Today 2002;21:76-81 8. El Askary AS, Meffert RM, Griffin T. Why do dental implants fail? Part I. Implant Dent 1999; 8:173–185. 9. Hurson S. Practical clinical guidelines to prevent screw loosening. Int J Dent Symp 1995;3:22–25. 10. Duyck J, Van Oosterwyck H, Vander Sloten J, De Cooman M, Puers R, Naert I. Magnitude and distribution of occlusal forces on oral implants supporting fixed prostheses: an in vivo study. Clin Oral Implants Res. 2000;11:465–475. 11. Guichet DL, Yoshinobu D, Caputo AA. Effect of splinting and interproximal contact tightness on load transfer by implantrestorations. J Prosthet Dent. 2002;87:528–535. 12. Kim Y, Oh TJ, Misch CE, Wang HL. Occlusal considerations in implant therapy: clinical guidelines with biomechanical rationale. Clin Oral Implants Res 2005;16:26-35. 13. Misch CE. Dental implant prosthetics. 2005 Elsevier Mosby. 14. Becker W, Becker BE. Replacement of maxillary and mandibularmolars with single endosseous implant restorations: a retrospective study. J Prosthet Dent 1995;74:51–55. 15. Colomina LE. Immediate loading of implant-fixed mandibular prosthesis: a prospective 18-month follow-up clinical study – preliminary report. Implant Dent 2001;10:23–29. 16. Vanden Bogaerde L, Pedretti G, Dellacasa P, Mozzati M,Rangert B. Early function of splinted implants in maxillas and posterior mandibles using Branemark system machinedsurfaceimplants: an 18-month prospective clinical multicenter study. Clin Implant Dent Relat Re. 2003;5:21–27 17. Glauser R, Re´e A, Lundgren AK, Gottlow J, Ha¨mmerle CHF, Scharer P. Immediate occlusal loading of Branemark implants applied in various jawbone regions: a prospective,1-year clinical study. Clin Implant Dent Relat Res 2001;3:204–213. 18. Quirynen M, Naert I, van Steenberghe D, Nys L. A study of 589 consecutive implants supporting complete fixed prostheses. Part I: periodontal aspects. J Prosthet Dent. 1992;68:655–663. 19. Lindquist LW, Carlsson GE, Jemt T. A prospective 15-year follow-up study of mandibular fixed prostheses supported b osseointegrated implants. Clin Oral Implant Res 1996;7:329– 336. 20. Kinsel RP, Lin D. Retrospective analysis of porcelain failures of metal ceramic crowns and fixed partial dentures supported by 729 implants in 152 patients: patient-specific and implantspecific predictors of ceramic failure. J Prosthet Dent. 2009;101:388-394. 21. Misch CE, Perel ML, Wang HL, Sammartino G, Galindo-Moreno P, Trisi P, Steigmann M, Rebaudi A, Palti A, Pikos MA, Schwartz-Arad D, Choukroun J, Gutierrez-Perez JL, Marenzi G, Valavanis DK. Implant success, survival, and failure: the International Congress of Oral Implantologists (ICOI) Pisa Consensus Conference. Implant Dent. 2008;17:5-15. 22. Porter JA, von Fraunhofer JA. Success or failure of dental implants? A literature review with treatment considerations. Gen Dent. 2005;53:423-432.

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Elephantiasis Gingiva: A Case Report Srinivasa T.S. et al

ELEPHANTIASIS GINGIVA IN 13 YEAR OLD BOY- A CASE REPORT Srinivasa T.S.1*, Rachana Kaushik2, R.G.Shiva Manjunath3 1. Dr. Reader, Department of Periodontology, Rungta College of Dental Sciences & Research, Bhilai, Chhattisgarh- 490024, INDIA. 2. Dr.Sr. Lecturer, Department of Periodontology, Rungta College of Dental Sciences & Research, Bhilai, Chhattisgarh- 490024, INDIA. 3. Professor, Department of Periodontology, Rungta College of Dental Sciences & Research, Bhilai, Chhattisgarh- 490024, INDIA.

Abstract Hereditary Gingival Fibromatosis (HGF) is the most common genetic form of gingival fibromatosis. HGF is characterized by a slowly progressive, non-hemorrhagic, fibrous enlargement of maxillary and mandibular keratinized gingiva. The enlarged gingiva is normal in color, firm in consistency, and histologically benign, the main feature being accretion of mature collagenous connective tissues. The overlying epithelium is typically normal, with occasional areas of hyperplasia, and with rete pegs extending deep into underlying connective tissues. Gingival enlargement may be generalized or localized, either unilateral or bilateral. A 13-year-old boy reported to the with massive gingival overgrowth. On clinical examination, a generalized gingival overgrowth covering almost entire clinical crowns of the teeth was found. Radiographic examination revealed evidence of anterior teeth bone loss. A provisional diagnosis of hereditary gingival fibromatosis was done after history, clinical & radiographical examination. Full mouth gingivectomy was carried out and tissue was sent for histopathological examination and final diagnosis was made. Patient was followed up for a period of 2 year and no recurrence was detected. Case report (J Int Dent Med Res 2011; 4: (1), pp. 30-34) Keywords: Elephantiasis Gingiva. Received date: 24 January 2011 Introduction Hereditary gingival fibromatosis (HGF) is a benign, idiopathic condition affecting both arches. It effects males and females equally and is usually autosomal-dominant1. The gingiva is markedly enlarged, asymptomatic, nonhemorrhagic, nonexudative. It may be an isolated finding or associated with other syndromes2. HGF develops as a slowly progressive, benign, localized or generalized enlargement of keratinized gingiva that, in severe cases, may cover the crowns of the teeth. Localized forms of HGF usually affect the maxillary tuberosities and the labial gingiva around the mandibular molars.

Accept date: 18 March 2011 Since HGF has not been reported in edentulous patients, it appears that the presence of dentition is necessary for overgrowth to develop3. A relationship with growth hormone deficiency has been suggested. The condition is either of a nodular form or, more commonly, a symmetric form. Onset of the condition usually begins with eruption of the permanent teeth. This condition predisposes one to malpositioning of the teeth, retention of deciduous teeth, esthetic and functional problems4. It is very rare cases affects 1 in750, 000 people with varying intensity and expressivity even in individuals within the same family 5,6 Case Report

*Corresponding author: Dr. SRINIVASA T.S Rungta College of Dental Sciences & Research, Bhilai, Chhattisgarh- 490024, INDIA. E-mail: seen79@rediffmail.com

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A 13 year old boy patient reported to Department of Periodontology, Rungta College of Dental sciences & Research Bhilai, with a chief complaint of slowly growing gingival growth present in the upper and lower teeth region, since many years. Page 30


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History revealed that the overgrowth began slowly, and covering entire surface of crown. The patient was in good systemic health, medical and dental history was non-contributory. Intra oral examination revealed generalized severe enlargement of gingiva involving both the mandibular and maxillary arches (Figure 1 to Figure 6).

Elephantiasis Gingiva: A Case Report Srinivasa T.S. et al

appearance of her gingiva. His medical history is noncontributory but family history was of significance, since his sister had a similar gingival condition. He reveled that his mother had had several gingival surgeries because of slowly progressing hyperplasia. He also stated that his sister not had any type disorder, nor had she taken any medications associated with gingival hyperplasia. On the basis of history and clinical presentation, a provisional diagnosis of ‘hereditary gingival overgrowth’ was made and a gingivectomy was planned.

Figure 1. Preoperative frontal view.

Figure 4. Preoperative upper occlusal view.

Figure 2. Preoperative right buccal view.

Figure 5. Preoperative lower occlusal view.

Figure 3. Preoperative left buccal view. The gingiva was pink, and its firm, dense, fibrous consistency caused considerable difficulty with plaque removal. The presence of an overgrowth covering entire surface of exposed teeth reaching occlusal surface interfering occlusion. The patient was unhappy with the Volume ∙ 4 ∙ Number ∙ 1 ∙ 2011

Figure 6. Pretreatment orthopantamogram. Page 31


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In this case, treatment was carried quadrant-by-quadrant gingivectomy. The patient was operated under 2% Xylocaine HCl with 1:80,000 Adrenaline, after securing profound anesthesia the bleeding points were created by using crane Kaplan pocket marker(Fig.7), then excessive gingival tissue was removed by using gingivectomy knives(Kirkland knife, Orbans interdental knife) (Figure 8, Figure 9), the area was thoroughly irrigated with normal saline and the surgical wound was covered with the periodontal dressing for one week, followed by 0.2% chlorhexidine oral mouthwash twice a day for a week after each surgery. Antibiotics and analgesics were prescribed and the patient was discharged after giving him post operative instructions.

Elephantiasis Gingiva: A Case Report Srinivasa T.S. et al

collagen bundles and plump fibroblasts were seen focally (Figure 10). Considering the history, clinical radiographic features and microscopic picture, a final diagnosis of ‘Hereditary Gingival Fibromatosis’ was made.

Figure 9. After gingvectomy.

Figure 7. Bleeding points marked with pocket marker.

Figure 10. The photomicrograph showing hyperkeratotic stratified squamous of variable thickness with irregular rete ridges, highly fibrous connective tissue with dense collagen bundles arranged in haphazard manner with numerous fibroblasts. (HE, X100). Figure 8. Incision being placed with Kirkland knife. The histopathological report read as “The H & E stained section shows acanthotic stratified squamous epithelium with elongated rete pegs. There is a bulbous increase the amount of a vascular connective tissue densely arranged Volume ∙ 4 ∙ Number ∙ 1 ∙ 2011

Patient reported after one week when the pack was removed and the area was thoroughly irrigated with saline and betadine. The oral hygiene was reinforced and patient was called for checkups at regular intervals. After eighteen months observation, Scaling and prophylaxis were performed every six months. So far there was no evidence of recurrence (Figure 11 to Figure15). Page 32


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Elephantiasis Gingiva: A Case Report Srinivasa T.S. et al

Figure 11. Post operative frontal view. Figure 15. Post operative lower occlusal view. Discussion

Figure 12. Post operative right buccal view.

Figure 13. Post operative left buccal view.

Figure 14. Post operative upper occlusal view. Volume ∙ 4 ∙ Number ∙ 1 ∙ 2011

HGF is a rare condition, and, therefore, most information about its characteristics is based on case reports. Generalized gingival fibromatosis can be caused by a number of factors, including inflammation, Leukemic infiltration and medication use such as phenytoin, cyclosporine or nifedipine7,8. Gingival tissue enlargement usually begins with the eruption of the permanent dentition but can develop with the eruption of the deciduous dentition; it rarely is present at birth. The most extensive enlargement appeared to occur either during loss of the deciduous teeth or in early stages of eruption of the permanent dentition. He noted that the enlargement seems to progress rapidly during "active" eruption and decrease with the end of this stage5. Gingival fibromatosis may exist as an isolated finding or as part of a more general syndrome. It is possible that isolated gingival fibromatosis may result from a single gene mutation, while syndromic forms may result from alterations of multiple genes9. Gingival fibromatosis can occur as part of a syndrome. It has been reported as a feature of Murray- Puretic Drescher syndrome (multiple hyaline fibromas), Rutherfurd's syndrome, Laband syndrome Cross syndrom. Recently Wynne and colleagues reported a new syndrome of HGF occurring with hearing deficiencies, hypertelorism and supernumerary teeth.10 Hereditary gingival fibromatosis associated with generalized aggressive 11 periodontitis. In addition to heritable forms, several classes of pharmacological agents (calcium Page 33


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channel blockers, cyclosporin, and phenytoin) have been associated with the development of gingival hypertrophy. Yet, not everyone exposed to these medications develops gingival overgrowth, suggesting that genetic factors may underlie a differential propensity to develop druginduced gingival overgrowth. Although genetic factors appear to be important in several types of gingival fibromatosis, few of the responsible genes have been identified, and none has been identified in isolated HGF12. Recent studies about the genetic characteristics of HGF have provided novel clues about the potential pathogenic mechanisms. Genetic loci for autosomal dominant forms of HGF have been localized to chromosome 2p21p22 (HGF1) and chromosome 5q13-q22 (HGF2)13, 14. AND mutation in the Son of sevenless–1 (SOS1) gene has been identified in affected individuals.15 The best time to initiate treatment to HGF is when all of the permanent dentition has erupted because the risk of recurrence is higher before it. Treatments vary according to the degree of severity of gingival enlargement. When the enlargement is minimal, thorough scaling of teeth and home care may be sufficient. However, excessive gingival tissue and esthetic and functional impairment dictate the need for surgical intervention.16,17 In this case, HGF showed moderate hyperplasia of a dense, hyperkeratotic epithelium with elongated rete ridges these findings are similar to other reports.18,19 Conclusions Hereditary gingival fibromatosis is a relatively rare entity, cannot be cured but can be controlled with varying degrees of success. When the enlargement is minimal, good scaling of teeth and home care may be all that is required to maintain good oral health. As the excess tissue increases, appearance and function indicate need for surgical intervention.

Elephantiasis Gingiva: A Case Report Srinivasa T.S. et al

References 1. Jorgenson RJ, Cocker ME. Variation in the inheritance and expression of gingival fibromatosis. J Periodontol 1974; 45: 472-477. 2. Hart TC, Pallos DL, Bozzo OP, et.al. Evidence of genetic heterogeneity for hereditary gingival fibromatosis. J Dent Res 2000; 79; 1758. 3. Flaitz CM, Coleman GC. Differential diagnosis of oral enlargement in children. Pediatr Dent 1995; 17: 294-300. 4. Raeste AM, Collan Y, Kilpinen E. Hereditary fibrous hyperplasia of the gingiva with varying penetrance and expressivity. Scand J Dent Res1978; 86:357–365. 5. Fletcher JP. Gingival abnormalities of genetic origin: preliminary communication with special reference to hereditary gingival fibromatosis. J Dent Res 1966; 45:597-612. 6. Avelar RL, De Luna Campos GJ, Falcao PG, et al. Hereditary gingival fibromatosis: a report of four cases in the same family. Quintessence Int 2010; 41(2):99-102. 7. Seymour RA, Heasman PA. Drugs and the periodontium. J Clin Periodontol 1988; 15:1-16. 8. Hakkinen L, Csiszar A. Hereditary Gingival Fibromatosis: Characteristics and Novel Putative Pathogenic Mechanisms. J Dent Res2007; 86(1):25-34. 9. Fryns JP. Gingival fibromatosis and partial duplication of the short arm of chromosome 2 (dup (2) (p13>p21)). Ann Genet1996; 39:54-55. 10. Wynne SE, Aldred MJ, Bartold PM, et al. Hereditary gingival fibromatosis with hearing loss and supernumerary teeth-a new syndrome. J Periodontol; 66(1):75-79. 11. Casavecchia P, Uzel MI, Hart TC, et al. Hereditary gingival fibromatosis associated with generalized aggressive periodontitis: a case report. J Periodontol 2004; 75:770-778. 12. Shashi V, Pallos D, Pettenati MJ, et al. Genetic heterogeneity of gingival fibromatosis on chromosome 2p. J Med Genet 1999; 36:683-686. 13. Xiao S, Bu L, Zhu L, Zheng G, et al. A new locus for hereditary gingival fibromatosis (GINGF2) maps to 5q13-q22.Genomics 2001; 74:180-185. 14. Ye X, Shi L, Yin W, et al. Further evidence of genetic heterogeneity segregating with hereditary gingival fibromatosis. J Clin Periodontol.2009; 36(8):627-33. 15. Thomas CH, Yingze Z, Michael CG, et al. A Mutation in the SOS1 Gene Causes Hereditary Gingival Fibromatosis Type -I. Am J Hum Genet 2002; 70(4): 943–954. 16. Ramakrishnan T, Kaur M. Multispeciality Approach in the Management of Patient with Hereditary Gingival Fibromatosis: 1Year Followup: A Case Report. Int Journal Dentistry 2010; 1-5. 17. Lobao DS, Silva LC, Soares RV. Idiopathic gingival fibromatosis: a case report. Quintessence Int. 2007; 38(8):699-704. 18. Doufexi A, Mina M, Ioannidou E. Gingival overgrowth in children: epidemiology, pathogenesis, and complications. A literature review. J Periodontol 2005; 76:3-10. 19. Araujo CS, Graner E, Almeida OP, et.al. Histomorphometric characteristics and expression of epidermal growth factor and its receptor by epithelial cells of normal gingiva and hereditary gingival fibromatosis. J Periodontal Res 2003; 38:237-241.

Declaration of Interest The authors report no conflict of interest and the article is not funded or supported by any research grant. Volume ∙ 4 ∙ Number ∙ 1 ∙ 2011

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Obstructive Sleep Apnea Venigalla Naga Venu Madhav

DIAGNOSIS AND MANAGEMENT OF OBSTRUCTIVE SLEEP APNEA Venigalla Naga Venu Madhav1* 1. Dr. M.D.S. Reader, Department of Prosthodontics, Bharati Vidyapeeth Dental College and Hospital, Pune.

Abstract Collapsibility of the upper airway in obstructive sleep apnea (OSA) causes repeated arousals from sleep, decreased oxygen saturation of the blood, and excessive sleepiness. Patients with OSA are at increased risk of cardiovascular and cerebrovascular disease, and experience occupational and vehicular accidents more frequently than the general population. Nasal continuous positive airway pressure (CPAP) is the current treatment of choice, but its cumbersome nature makes tolerance and compliance less than optimal. There is growing interest in the use of oral appliances to treat snoring and OSA. The rationale is that advancement of the mandible and tongue impacts positively on upper airway caliber and function. There are many such types of appliances, and they have potential advantages over CPAP in that they are unobtrusive, make no noise, do not need a power source, and are potentially less costly. There is a growing evidence base to support the use of oral appliances in the management of OSA. Review (J Int Dent Med Res 2011; 4: (1), pp. 35-41) Keywords: Obstructive sleep apnea, Continuous positive airway pressure, Polysomnography. Received date: 28 September 2010 Introduction Obstructive sleep apnea (OSA) is a disorder in which a person stops breathing during the night, perhaps hundreds of times. These gaps in breathing are called apneas. The word apnea means absence of breath. An obstructive apnea episode is defined as the absence of airflow for at least 10 seconds. Sleep apnea is usually accompanied by snoring, disturbed sleep, and daytime sleepiness. People might not even know they have the condition. Obstructive Sleep Apnea: Obstructive sleep apnea (OSA) occurs when tissues in the upper throat collapse at

*Corresponding author: Dr V.N.V.Madhav, M.D.S. (Prosthodontics) Reader, Department of Prosthodontics, Bharati Vidyapeeth Dental College and Hospital, Pune. E-mail: vnvmadhav@yahoo.co.in

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Accept date: 07 February 2011 different times during sleep, thereby blocking the passage of air. In general, OSA occurs as follows:  On its way to the lungs, air passes through the nose, mouth, and throat (the upper airway).  Under normal conditions, the back of the throat is soft and tends to collapse inward as a person breathes.  Dilator (widening) muscles work against this collapse to keep the airway open. Interference or abnormalities in this process cause air turbulence.  If the tissues at the back of the throat collapse and become momentarily blocked, apnea occurs. Breath is temporarily stopped. In most cases the person is unaware of it, although sometimes they awaken and gasp for breath.  In some cases, the interference is incomplete (called obstructive hypopnea) and causes continuous but slow and shallow breathing. In response, the throat vibrates and makes the sound of snoring. Snoring can occur whether a person breathes through the mouth or the nose. (Snoring often occurs without sleep apnea.) Page 35


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 Apnea decreases the amount of oxygen in the blood, and eventually this lack of oxygen triggers the lungs to suck in air.  At this point, the patient may make a gasping or snorting sound but does not usually fully wake up. Obstructive sleep apnea is defined as five or more episodes of apnea or hypopnea per hour of sleep (called apnea-hypopnea index or AHI) in individuals who have excessive daytime sleepiness. Patients with 15 or more episodes of apnea or hypopnea per hour of sleep are considered to have moderate- sleep apnea. Causes: General Causes of Obstructive Sleep Apnea Structural abnormalities in the face, skull, or airways that cause some obstruction or collapse in the upper airways and reduce air pressure can produce sleep apnea syndrome. People with micrognathia, retrognathia, enlarged tonsils, tongue enlargement, and acromegaly are especially predisposed to obstructive sleep apnea. Abnormalities or weakness in the muscles that surround the airway can also contribute to obstructive sleep apnea. Problems with the soft palate (the soft tissue at the back of the roof of the mouth) are also associated with many cases of sleep apnea. Obesity can contribute to sleep apnea when fat deposits clog throat tissue. Risk Factors: Gender Sleep apnea is more common in men than in women. Men tend to have larger necks and weigh more than women. However, women tend to gain weight and develop larger necks after menopause, which increases their risk of developing sleep apnea. Age Sleep apnea is most common in adults ages 40 - 60 years old. Middle age is also when symptoms are worse. Nevertheless, sleep apnea affects people of all ages. Race and Ethnicity African-Americans face a higher risk for sleep apnea than any other ethnic group in the Volume ∙ 4 ∙ Number ∙ 1 ∙ 2011

Obstructive Sleep Apnea Venigalla Naga Venu Madhav

United States. Other groups at increased risk include Pacific Islanders and Mexicans. Family History People with a family history of obstructive sleep apnea are at increased risk of developing the condition. Obesity Obesity, especially having fat around the abdomen (the so-called apple shape), is a particular risk factor for sleep apnea, even in adolescents and children. However, not all people who are obese have sleep apnea. Specific anatomical and physiological properties in the airways are more likely to be present in obese individuals with apnea. Physical Characteristics Large Neck. A large neck (17 inches or greater in men and 16 inches or greater in women) is a risk factor for sleep apnea. Facial and Skull Characteristics. Structural abnormalities in the face and skull contribute to many cases of sleep apnea. These include:  A long lower part of the face  Brachycephaly, a birth defect in which the head tends to be shorter and wider than average  A narrow upper jaw  A receding chin  An overbite  A larger tongue Soft Palate Characteristics. Some people have specific abnormalities in the soft area (palate) at the back of the mouth and throat that may lead to sleep apnea. These abnormalities include:  The soft palate is stiffer, larger than normal, or both. An enlarged soft palate may be a significant risk factor for sleep apnea.  The soft palate and the walls of the throat around it collapse easily. Symptoms: People with sleep apnea usually do not remember waking during the night. Symptoms may include:  Excessive daytime sleepiness.

Generally,

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patients risk falling asleep during the day while performing routine activities such as reading, watching TV, sitting inactively, lying down, or riding in a car while a passenger or stopped for a few minutes in traffic. Usually, these brief episodes of sleep do not seem to relieve their overall sense of sleepiness.  Morning headaches.  Irritability and impaired mental or emotional functioning. These types of symptoms are directly related to interrupted sleep.  Snoring. Bed partners may report very loud and interrupted snoring. Patients experience snoring associated with choking or gasps. This often occurs in a crescendo pattern with the loudest noises occurring at the very end. These findings are more likely to occur when lying down (supine position). Patients often suffer from frequent arousals during sleep because of snoring. Diagnosis: The symptoms of obstructive sleep apnea are not very specific. This means that many people who snore at night or who feel tired during the day probably do not have sleep apnea. Other medical reasons for daytime sleepiness should be considered by your doctor before referral to a sleep center for diagnostic sleep tests. They include:  Having to work excessive hours or varying shifts (nights, weekends)  Medications (tranquilizers, sleeping pills, antihistamines beta blockers, many others)  Alcohol abuse  Medical conditions (such as hypothyroidism, hypercalcemia, and hyponatremia / hypernatremia)  Self-imposed short sleep time  Other sleep disorders, such as narcolepsy, insomnia, or restless legs syndrome  Chronic fatigue syndrome  Depression Symptoms or findings that make the need for evaluation by a sleep specialist include:  Sleepiness is affecting patient's quality of life  Sleepiness on-the-job places the patient or others in danger  Others have observed apnea or breath holding spells while asleep Volume ∙ 4 ∙ Number ∙ 1 ∙ 2011

Obstructive Sleep Apnea Venigalla Naga Venu Madhav

 Other medical illnesses that may be worsened by obstructive sleep apnea are present. If symptoms suggest obstructive sleep apnea or other sleep disorders, further diagnostic testing will be performed. A sleep specialist or sleep disorders center will perform an in-depth medical and sleep history and physical exam. Keeping a Record of Sleep. To help answer these questions, the patient may need to keep a sleep diary. Every day for 2 weeks, the patient should record all sleep-related information, including responses to questions listed above described on a daily basis. Recording sleep behavior using an extended-play audio or videotape can be very helpful in diagnosing sleep apnea. Physical Examination To diagnose sleep apnea, the doctor will check for physical indications of sleep apnea, including:  Abnormalities in the soft palate or upper airways, including enlarged tonsils  Upper body obesity  A wide neck measurement Polysomnography and Home Sleep Studies Polysomnography is the technical term for an overnight sleep study that involves recording brain waves and other sleep-related activity. Polysomnography involves many measurements and is typically performed at a sleep center. Home Diagnostic Portable Devices A number of portable devices are available, or being developed, so that patients have the convenience of being monitored at home. Devices that can accurately diagnose sleep apnea and titrate CPAP may eventually replace the need for many overnight sleep studies or the need for attended monitoring at home. These home devices can be very different from each other. Some are able to measure all the different factors that a sleep study performed at a sleep center is able to measure. Others are only able to measure some of them and may not get the full picture or accurate diagnosis. Unattended Monitoring with Auto-CPAP. This Home monitoring method is a recent and simple technique for detecting impaired breathing. It Page 37


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uses an auto-CPAP machine, which is programmed to apply pressure through the airways via a tube that attaches to a mask that fits the nose. A monitor is attached that digitizes and records on a computer all the information on any apnea episodes during sleep. Treatment: Treatment for sleep apnea depends on the severity of the problem. Given the data on the long-term complications of sleep apnea, it is important for patients to treat the problem as they would any chronic disease. Simply trying to treat snoring will not treat sleep apnea. Because of its association with heart problems and stroke, sleep apnea that does not respond to lifestyle measures should be treated by a doctor, ideally a sleep disorders specialist. At this time, the most effective treatments for sleep apnea are devices that deliver slightly pressurized air to keep the throat open during the night. There are a number of such devices available. Continuous Positive Airflow Pressure (CPAP) The best treatment for symptomatic obstructive sleep apnea is a system known as continuous positive airflow pressure (CPAP), sometimes referred to as nasal continuous positive airflow pressure (nCPAP). It is safe and effective in sleep apnea patients of all ages, including children. CPAP may not recommended for patients who have mild-sleep apnea as identified in sleep studies but who do not have daytime sleepiness, as they generally report little or no benefit from this treatment. Those with more moderate sleep apnea are more likely to receive a trial of CPAP, but not always. When severe sleep apnea is present, most patients will receive CPAP. Overall, CPAP is considered first-line treatment for mildto-moderate, or severe obstructive sleep apnea. CPAP works in the following way:  The device itself is a machine weighing about 5 pounds that fits on a bedside table.  A mask containing a tube connects to the device and fits over just the nose.  The machine supplies a steady stream of air through a tube and applies sufficient air pressure to prevent the tissues from collapsing during sleep. Volume ∙ 4 ∙ Number ∙ 1 ∙ 2011

Obstructive Sleep Apnea Venigalla Naga Venu Madhav

Effects on Sleep and Wakefulness. CPAP improves both objective and subjective measures of sleep. After using CPAP regularly many patients report the following benefits:  Restoration of normal sleep patterns.  Greater alertness and less daytime sleepiness.  Less anxiety and depression and better mood.  Improvements in work productivity.  Better concentration and memory.  Patients' bed partners also report improvement

in their own sleep when their mates use CPAP, even though objective sleep tests showed no real difference in the partners' sleep quality. If patients comply with the CPAP regimen but do not feel less sleepy after a period of time, or their sleep apnea symptoms do not improve, the airflow pressure may not be high enough. Patients may need to be retested. Likewise, if patients have started using an oral appliance or had a surgical procedure, their doctor probably needs to reevaluate them. Surgery: Surgery is sometimes recommended, usually by ear, nose, and throat specialists, for severe obstructive sleep apnea. A patient should be sure to seek a second opinion from a specialist in sleep disorders. Few randomized clinical trials, the gold standard of medical research, have been conducted to verify the long-term efficacy of sleep apnea surgery. Uvulopalatopharyngoplasty (UPPP) The Procedure. Surgery known as uvulopalatopharyngoplasty (UPPP) removes soft tissue on the back of the throat. Such tissue includes all or part of the uvula (the soft flap of tissue that hangs down at the back of the mouth) and parts of the soft palate and the throat tissue behind it. If tonsils and adenoids are present, they are removed. The surgery typically requires a stay in the hospital. The Goal of Surgery. The goal of UPPP is threefold:  Increase the width of the airway at the throat's opening  Block some of the muscle action in order to improve the ability of the airway to remain open Page 38


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 Improve the movement and closure of the soft palate. Success Rates. Success rates for sleep apnea surgery are rarely higher than 65% and often deteriorate with time, averaging about 50% or less over the long term. Few studies have been conducted on which patients make the best candidates. Some studies suggest that surgery is best suited for patients with abnormalities in the soft palate. Results are poor if the problems involve other areas or the full palate. In such cases, CPAP is superior and should always be tried first. Many or most patients with moderate or severe sleep apnea will likely still require CPAP treatment after surgery. Laser-Assisted Uvulopalatoplasty (LAUP) A variation on UPPP called laser-assisted uvulopalatoplasty (LAUP) is being increasingly performed to reduce snoring. It removes less tissue at the back of the throat than UPPP and can be done in a doctor's office. At this time, however, long-term success rates in the treatment of obstructive sleep apnea with LAUP are very modest, particularly for reducing apneas. Some doctors, in fact, are concerned that if LAUP eliminates snoring, they may miss a diagnosis of apnea in patients who have the more serious condition. More than 50% of patients complain of throat dryness after surgery. Throat narrowing and scarring have also been reported. In a minority of patients, snoring becomes worse afterward. Pillar Palatal Implant The pillar palatal implant is a noninvasive surgical treatment for mild-to-moderate sleep apnea and snoring. However, the main focus of the procedure is a reduction in snoring. The implant helps reduce the vibration and movement of the soft palate. In this procedure, a doctor inserts 3 short pieces of polyester string into the soft palate. The procedure can be performed in a doctor ' s office and takes about 10 minutes. Unlike uvulopalatopharyngoplasty (UPPP), the pillar procedure requires only local anesthesia. Studies indicate it works as well as UPPP, with less pain and quicker recovery time.

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Obstructive Sleep Apnea Venigalla Naga Venu Madhav

Other Procedures Other surgical procedures may be appropriate to correct facial abnormalities or obstructions that cause sleep apnea. They may be used alone or combined with each other or with UPPP. Most are invasive and reserved for patients with severe sleep apnea who fail to respond to or comply with CPAP. They include:  Genioglossus (tongue advancement), in which an opening is cut where the tongue joins the jawbone and the area is pulled forward.  Temperature controlled radiofrequency ablation tongue reduction.  Genioplasty, which is plastic surgery on the chin.  Hyoid advancement surgery, in which the movable bone underneath the chin is moved forward, pulling the tongue muscle along with it.  Maxillary or maxillomandibular advancement (MMA), which moves the upper (maxilla) or lower (mandible) jawbone forward. A survey of patients who had MMA found that the surgery changed their facial appearance, but most people thought it was a change for the better. Surgery for nasal obstructions (such as a deviated septum) that contribute to snoring and other symptoms. Dental Devices The American Academy of Sleep Medicine recommends dental devices for patients with mild-to-moderate obstructive sleep apnea who are not appropriate candidates for CPAP or who have not been helped by it. (CPAP should be used for patients with severe sleep apnea whenever possible. Pretreatment dental assessment This includes dental history and an oral examination focusing on parafunctional habits, wear facets and temperomandibular joint (TMJ) status have to be recorded prior to treatment planning. Oral appliances are worn only during sleep and work to enlarge the airway by moving the tongue (anteriorly) or the mandible to enlarge the airway. Whether they change the airway shape or increase the cross-sectional area of the upper airway is not clear. It is hypothesized that these appliances may also affect upper airway muscle Page 39


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tone and thus decrease their collapsibility. Movement of the tongue or mandible anteriorly can increase the cross-sectional size of the airway 1 and hence oral appliances help in increasing the airway size. 2 Activation of the upper airway dilator muscles by the appliance could cause a decrease in airway collapsibility and this may contribute to preservation of airway patency during sleep, 3 although the increase in airway size may be the most important factor preventing airway occlusion. A tongue-retaining device is a custommade soft acrylic appliance that covers the upper and lower teeth and has an anterior plastic bulb. It uses negative suction pressure to hold the tongue in a forward position inside the bulb. By holding the tongue in a forward direction through its attachment to the genial tubercle, it stabilizes the mandible and hyoid bone, thus preventing retrolapse of the tongue. These devices, reverse pharyngeal obstruction both at the level of the oropharynx and the hypopharynx, thereby enlarging the airway and reducing snoring and the related apnea. 4 Soft palate trainers and tongue posture trainers are rarely used.5 Oral devices are basically acrylic custom made devices to, a. Repostion and recondition the mandible. e.g., Herbst appliance 6 / snoreguard 7 / silencer. 8 They function by engaging one or both of the dental arches to modify mandibular protrusion b. Tongue repositioning or retaining devices, e.g., SnorEx. 9 c. Soft-palate lifters. 5 d. Tongue trainers. 5 e. A combination of oral appliance and CPAP in the new products deliver pressurized air directly into the oral cavity and eliminates the use of head gear or nasal mask and avoids the problems of air leaks and the claustrophobia associated with CPAP treatment. 10 Patients fitted with one of these devices should have a check-up early on to see if it is working; short-term success usually predicts long-term benefits. It may need to be adjusted or replaced periodically. Benefits of Dental Devices. Dental devices seem to offer the following benefits: Volume ∙ 4 ∙ Number ∙ 1 ∙ 2011

Obstructive Sleep Apnea Venigalla Naga Venu Madhav

 Significant reduction in apneas for those with mild-to-moderate apnea, particularly if patients sleep either on their backs or stomachs. They do not work as well if patients lie on their side. The devices may also improve airflow for some patients with severe apnea.  Improvement in sleep in many patients.  Improvement and reduction in the frequency of snoring and loudness of snoring in most (but not all) patients.  Higher compliance rates than with CPAP. Dental devices have shown better longterm control of sleep apnea when compared to uvulopalatopharyngoplasty (UPPP), the standard surgical treatment. There are also few complications with a dental device. Disadvantages of Dental Devices. Dental devices are not as effective as CPAP therapy. The cost of these devices tends to be high. Side effects associated with dental devices include:  Nighttime pain, dry lips, tooth discomfort, and excessive salivation. In general, these side effects are mild, although over the long term they cause nearly half of patients to stop using dental devices. Devices made of softer materials may produce fewer side effects.  Permanent changes in the position of the teeth or jaw have occurred in some cases of longterm use. Patients should have regular visits with a health professional to check the devices and make adjustments. In a small percentage of patients, the treatment may worsen apnea. Orthodontic Treatments An orthodontic treatment called rapid maxillary expansion, in which a screw device is temporarily applied to the upper teeth and tightened regularly, may help patients with sleep apnea and a narrow upper jaw. This nonsurgical procedure helps to reduce nasal pressure and improve breathing. Declaration of Interest The authors report no conflict of interest and the article is not funded or supported by any research grant.

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References 1. Ferguson KA, Love LL, Ryan CF. Effects of a mandibular and tongue protrusion on upper airway size. Am J Respir Crit Care Med 1997;155:1748-54. 2. Ferguson KA, Love LL, Ryan CF. Effect of mandibular and tongue protrusion on upper airway size during wakefulness. Am J Respir Crit Care Med 1997;155:1748-54 3. Rogers RR. Oral appliance therapy for the management of sleep disordered breathing. Sleep Breath 2000;4:79-84. 4. Cartwright RD, Samelson CF. The effects of a nonsurgical treatment for obstructive sleep apnea. The tongue-retaining device. JAMA 1982;2248:705-9. 5. Schlosshan D, Elliott MW. Sleep, part 3: Clinical presentation and diagnosis of the obstructive sleep apnoea hypopnoea syndrome. Thorax 2004;59:347-52. 6. Clark GT, Arand D, Chung E, Tong D. Effect of anterior mandibular positioning on obstructive sleep apnea. Am Rev Respir Dis 1993;147:624-9. 7. Ferguson KA, Ono T, Lowe AA, al-Majed S, Love LL, Fleetham JA. A short term controlled trial of an adjustable oral appliance for the treatment of mild to moderate obstructive sleep apnoea. Thorax 1997;52:362-8 8. Schmidt-Nowara WW, Meade TE, Hays MB. Treatment of snoring and obstructive sleep apnea with a dental prosthesis. Chest 1991;99:1378-85. 9. Schonhofer B, Stoohs RA, Rager H, Wenzel M, Wenzel G, K φhler D. A new tongue advancement technique for sleep disordered breathing: Side effects and efficacy. Am J Respir Crit Care Med 1997;155:732-8. 10. Hart NT, Duhamel J, Guilleminault C. Oral positive airway pressure by the OPAP dental appliance reduces mild to severe OSA. Sleep Res 1997;26:371.

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Ketamine effect on tissue hydration Armenuhi Heqimyan et al

KETAMINE- INDUCED CELL DEHYDRATION AS A MECHANISM OF IT’S ANALGESIC AND ANESTHETIC EFFECTS Armenuhi Heqimyan1, Anush Deghoyan1, Sinerik Ayrapetyan1* 1. UNESCO Chair-Life Sciences International Postgraduate Educational Center, 31 Acharyan St. 0040 Yerevan, Armenia.

Abstract Effect of intraperitoneally (i.p.) injected sub-anesthetic (8x10-5-8x10-2 mg/g) and anesthetic (0.125mg/g) doses of ketamine on rats’ pain sensitivity and tissue hydration of different organs were studied. Determination of water content of tissue was performed by Adrian’s traditional “tissue drying” experimental procedure. The number of functionally active receptors were determined by counting the number of [3H]-ouabain in tissues. Latent period of pain sensitivity was defined by means of “hot plate” test. Ketamine in sub-anesthetic doses (8x10-5-8x10-2 mg/g i.p.) had depressing effect on rats’ latent period of pain sensitivity which was accompanied by dehydration of tissues and decrease of the number of [3H]-ouabain receptors in membrane of tissues of different organs. The ouabain influence on brain cell hydration was characterized by dose dependent (10-9-10-4M) three phases and this fact was accompanied by corresponding changes of number of ouabain receptors in membrane. Ketamine anesthetic dose had reversing effect on all three phases of ouabain – induced cell hydration. It was suggested that ketamine – induced cell dehydration leading to decrease of number of functional active proteins in membrane serves as a powerful mechanism through which an analgesic and anesthetic effects of ketamine on organisms were realized. Article (J Int Dent Med Res 2011; 4: (1), pp. 42-49) Keywords: rat, ketamine, [3H]-ouabain receptor, pain sensitivity, cell hydration. Received date:

Accept date:

Introduction It is assumed that ketamine administration causes "dissociative anesthetic state" characterized by profound analgesic, moderate hypnotic properties and by marked sympathomimetic reactions. Important adverse effects are hallucinations and hypersalivation. Pharmacological profile of ketamine influence can not be explained by a single mechanism. Analgesic, anesthetic and sympathomimetic effects are mediated by different sides of action. It is suggested that Nmethyl-D-aspartate (NMDA)-receptor antagonism *Corresponding author: Prof. Sinerik Ayrapetyan UNESCO Chair-Life Sciences International Educational Center 31 Acharyan St. 0040 Yerevan, Armenia E-mail: info@biophys.am

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Postgraduate

accounts for most of the amnestic, analgesic, psychotomimetic and neuroprotective effects of ketamine1,2. It is also known that ketamine blocks an NMDA receptor-mediated component of synaptic transmission in a voltage-dependent manner3-7. At the same time it is well established that ketamine has depressing effect on variety of receptors: nicotinic8,9, muscarinic10 and opioid ones10-12 as well as on voltage sensitive Na+ 9, 13, K+ 13-15 and Ca2+ channels16 of nerve cell membrane in peripheral and central nervous system. At present it is known that influence of ketamine is not limited only by nervous system but it has also relaxing effect on smooth and heart muscles17-20 and other tissues21. However, the nature of cellular mechanism underlying in the ground of above mentioned multisided effects of ketamine on different tissues is not clear yet. Earlier on the basis of experimental data performed in vitro on snail single neurons the correlation between number of functional active Page 42


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membrane receptors having channel22, receptor 23 , enzyme functions24 and active cell membrane surface (cell volume) was established. According to this data cell swelling leads to the activation while shrinkage to inhibition of neurons functional activity. Therefore, the metabolic control of cell volume- induced changes of membrane functional activity was suggested as an essential mechanism through which the regulation of 25 neuronal ionic function is realized . It is known that there are a number of nonconductive membrane mechanisms responsible for cell volume regulation such as Na+/K+ pump, Na+/Ca++ , Na+/H+ exchangers, changes of cytoskeleton contractility and membrane fluidity and others26. Previously in vitro experiments (on snail neurons and women breast tissue) it was shown that ketamine has stimulating effect on Na+/K+ pump and Na+/Ca+ exchanger which brings to cell dehydration27-29. Therefore, the working hypothesis for present work was to clarify whether the ketamine- induced cell volume changes has a crucial role in realization of analgesic and anesthetic effects as well as it’s multisided effect on different organs. For this purpose the dose-dependent effect of ketamine on rats pain sensitivity, hydration of different organs tissue and the number of [3H]-ouabain receptors was studied. Materials and methods All procedures performed on animals were carried out following the protocols approved by Animal Care and Use Committee of Life Sciences Postgraduate International Educational Centre. Animals All experiments were performed on 96 adult male Wistar albino rats weighing from 100g to 130g. Animals were kept in a specific pathogenfree animal room, under optimum conditions of 12 hours light/dark cycle and 22 ± 2 0C temperature, received sterilized commercial diet and water ad libitum. Chemicals Physiological solution (PS) with NaCl-137; KCl-5.4; CaCl2-1.8; MgCl2-1.05; C6H12O6-5; NaHCO3-11.9; NaH2PO4-0.42 in mM (Sigma Chemicals, Steinhein, Germany) and pH-7.4 was used in all experiments. The stock solution of ketamine containing 500mg ketamine solved Volume ∙ 4 ∙ Number ∙ 1 ∙ 2011

Ketamine effect on tissue hydration Armenuhi Heqimyan et al

in 10ml of PS was used (Gedeon Richter, Budapest, Hungary). Ketamine intraperitoneally (i.p.) injections were calculated individually according to animal body weight. [3H]-ouabain (PerkinElmer, Massachusetts, USA) with 10-11 10-4 M concentrations were taken as a marker for definition of tissues volume changes. Tissue preparation Tissues of different organs (lungs, heart, spleen, liver, kidney, muscle and brain) were investigated on 50 animals. Brain tissues of next 36 animals were investigated separately. Tissues of 10 animals receiving ketamine subanesthetic dose 8x10-5 did not investigate. For definition the tissue water content and counting the number of ouabain receptors ten pieces from each organ tissue weighing from 50 to 60 mg were taken from each rat. From cortex and subcortex tissues of brain eight pieces were taken while from cerebellum tissue only four pieces for every case. Definition of Tissues Water Content Water content in tissues of brain and organs were determined after animals’ decapitation. In definition of tissues water content 86 animals were used. Animals were sharply immobilized by dipping its’ noses into liquid nitrogen for 3-4 sec30 and decapitated after 15 min. ketamine or [3H]-ouabain injection. After such procedure the full absence of somatic reflexes on extra stimuli was recorded. Determination of water content of tissue was performed by traditional “tissue drying” method (Adrian, 1956). After measuring the tissue wet weight (w.w.) it was dried in thermostat (Factory of Medical Equipment, Odessa, Ukraine) during 24 hours at 1050C for determination of dry weight (d. w.). The quantity of water in 1 g of d.w. of tissue was counted by the following equation: (w.w. – d.w.) / d.w. 10. Counting the number of ouabain receptors in cell membrane Counting the number of ouabain receptors in cell membrane was made only in samples of brain tissues (cortex, subcortex and cerebellum) in one control (18 animals) and experimental (18 animals) groups of animals. The stock [3H]ouabain solution contains 10-9M concentration (12 Ci/mM specific activities). The solutions with higher concentrations (10-8-10-4M) were prepared by adding corresponding concentration of cold (none labeled) ouabain. Ouabain solutions were intraperitoneally injected in control as well as in Page 43


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experimental groups of animals. In the last groups of animals it was made 15 minute after ketamine anesthetic dose injection. Animals were decapitated in all groups after 30 minute of injections and brain tissue samples were placed in special vials and homogenized with HNO3. Finally, 5 ml of Bray's scintillation fluid was added and the mixture was counted in Wallac1450 liquid scintillation counter (PerkinElmer, Finland). After this procedure the amount of scintillate isotopes counted per minute was received and correspondingly calculated as the number of ouabain receptors. Determination of pain sensitivity latent period This test was carried out using the specific setup developed in our laboratory. It consists of org-glass chamber with the brass bottom. The bottom temperature (51oC) was controlled with the thermometer (accuracy of measurement  0.01oC) and it was completely covered by the Plexiglas box keeping the temperature constant. Rats were placed individually on brass bottom and latent period of pain sensitivity was recorded as the time elapsed to obtain one of the following responses: licking the feet, jumping or rapidly stamping the feet. The tissue damage prevention was near 10 sec. Statistic significance in latent periods of pain sensitivity was defined between data of control and experimental groups. Statistical Analysis The Microsoft Excel and Sigma-Plot (Version 8.02A) were used for data analysis. For all statistical tests (with Student’s t-test) P value of 0.05 or less taken as (*P < 0.05, **P < 0.01, ***P < 0.001). Results In first series of experiments the effect of ketamine on latent period of pain sensitivity to hot plate was investigated on four groups of animals (n = 10 in each group) injected i.p. by different subanesthetic doses (8x10-5 - 8x10-2 mg/g) solved in 0.2 ml of PS. As a control ten rats were taken and injected i.p. with equivalent quantity (0.2 ml) of PS. Latent period of pain sensitivity was measured 15 min after injections. The mobility of animals injected with above mentioned doses of ketamine was not fully inhibited and on brass bottom of hot plate setup (t = 51o) following reactions (lick the feet, jump or rapid stamp the feet) were observed in control as well as in Volume ∙ 4 ∙ Number ∙ 1 ∙ 2011

Ketamine effect on tissue hydration Armenuhi Heqimyan et al

experimental groups of animals. On Figure 1 the bars indicate in percent the value of latent period of pain sensitivity in five groups of animals. It is shown the increase of latent period of pain sensitivity more than three times after ketamine injections (8x10-4 - 8x10-2 mg/g) and it’s threshold concentration was 8x10-5 mg/g.

Figure 1. Ketamine effect on the latent period (in %) of pain sensitivity of rats to hot plate. Black bar indicates the latent period of pain sensitivity of control group animals injected with 0.2ml of PS. Control data was taken as 100%. White bars indicate the latent period of pain sensitivity of experimental groups’ animals receiving 8x10-5 mg/g - 8x10-2 mg/g of ketamine dissolved in 0.2ml of PS. The ordinate indicates the latent period of pain sensitivity in percent (%). Each bar represents the mean of latent period from 10 animals. Error bars indicate SE. Values were statistically significant (compared with data of control group *P <0.05, ***P<0.001). Recently in our experiments the close correlation between brain tissue hydration and pain sensitivity was demonstrated31,32. That is way in the second series of experiments it was interesting to clarify whether the pain relief effect of ketamine could also be due to tissue dehydration. This study was continued on previous groups of animals after determination of latent period. Experiments were performed on control and four experimental groups of animals. Ten animals for each group were chosen. Ketamine was taken i.p. in following doses: subanesthetic (8x10-4 - 8x10-2mg/g) and anesthetic (0.125 mg/g). Dose-dependent effect of ketamine on tissue hydration of different zones Page 44


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of brain (cortex, subcortex and cerebellum) and different organs were studied. Animals of control group were received 0.2 ml of PS i.p. while animals in experimental groups were injected also i.p. with different doses of ketamine: subanethetic (8x10-4 - 8x10-2 mg/g) and anesthetic one (0.125 mg/g). On Figure 2 dose-dependent effect of ketamine on water content (in %) in brain tissues is shown. As can be seen from Figure the curve of ketamine dose-dependent effect on cortex tissue hydration is different from those of subcortex and cerebellum. Ketamine at following concentrations (8x10-4- 8x10-3 mg/g) has no significant effect on all brain tissues hydration while at 8x10-2 mg/g it has strong dehydration effect (decrease of water content) on all types’ tissues. Ketamine at concentration of 0.125 mg/g (which had anesthetic effect on rats) shows overhydration effect (increase of water content) on cortex tissue (Figure 2, a) and dehydration effect on subcortex and cerebellum tissues (Figure 2, b,c). The fact that ketamine at anesthetic dose brings to the overhydration of cortex tissue corresponds with data where the exciting effect of ketamine on brain cortex explains by it’s stimulation of some groups of glutamate receptors33.

Figure 2. Dose-dependent effect of ketamine on water content in different parts of brain tissues (cortex, subcortex and cerebellum). The results are expressed in percent and control was taken as 100%. Each point is the mean value of water content for each part of brain tissue at ketamine corresponding doses (for cortex and subcortex tissues 10 samples were taken from each animal while for cerebellum tissue 4 pieces). For control and experimental groups 10 animals were chosen. Volume ∙ 4 ∙ Number ∙ 1 ∙ 2011

Ketamine effect on tissue hydration Armenuhi Heqimyan et al

Dose-dependent effect of ketamine was also carried out on tissues of other organs (lung, heart, spleen, liver, kidney and muscle) to find out whether observed tissue hydration to ketamine is a general property or it is only specific for brain tissue. On Figure 3 changes of tissues water content (in %) to different ketamine doses are shown. As can be seen from Figure at the smallest dose (8x10-4 mg/g) of ketamine there is an overhydration effect in all tissues except the spleen tissue (small, no significant dehydration effect). At ketamine 8x10-3 mg/g dose in all investigated tissues dehydration effect were observed but in muscle tissue significant overhydration effect is revealed. The dehydration effect in lung, heart, spleen, kidney tissues were also found at higher doses of ketamine (8x10-2 mg/g i.p.) but in the case of liver and muscle tissues the opposite one (overhydration effect). In the last group of animals when the anesthetic dose of ketamine (0.125 mg/g i.p.) was injected the dehydration effect on tissues of all organs except liver was observed. Thus, the presented data indicate that ketamine has modulating effect on tissue hydration of different organs and its’ sensitivity to ketamine is different which can be probably explained by different nature of mechanisms involved in cell volume regulation or by various quantity of ketamine reaching these cells by blood flow.

Figure 3. Dose-dependent effect of ketamine on water content in tissues of different organs. The results are expressed in percent and data of control group was taken as 100%. Each point is the mean value (n=50) of the water content for each organ of 10 animals at ketamine corresponding doses. The abscissa indicates ketamine doses - 8x10-4 mg/g - 8x10-2 mg/g and 0.125mg/g. The ordinate indicates the water Page 45


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content in tissues. Error bars indicate the standard error for 10 independent experiments. Significance was calculated with Student’s t test with *P < 0.05, **P< 0.01, ***P < 0.001. Previously was shown that cell volume control is connected with Na+/K+ pump24, cGMPdependent34 and cAMP-dependent Na+/Ca++ exchangers activities35. In these studies by means of [3H]-ouabain (specific inhibitor of Na+/K+ ATP-ase) cell volume changes were defined. [3H]-ouabain binds to membrane Na+/K+ ATP-ase receptors. Kinetic curve of dosedependent [3H]-ouabain binding with neuronal membrane functionally active proteins was consisted of two saturated and one linear component. There were found that only last component of curve was connected with Na+/K+ pump activity21. From this point of view it was interesting in the last series of experiments to study the possible mechanism of ketamine anesthetic dose (0.125 mg/g) influence on dosedependent ouabain-induced cell hydration in brain tissues and find out whether this process is accompanied with corresponding change of number of membrane functionally active proteins ([3H]-ouabain was taken as a marker of membrane functionally active proteins). These experiments were performed on 12 groups of animals (three animals in each). In first part of experiments (six groups) water content and number of ouabain receptors in brain tissues after different dose of ouabain (10-9M - 10-6M, 104 M) were determined. Animals of control group were injected with 0.2 ml of PS. Animals of five experimental groups were injected with different concentrations of ouabain (10-9M - 10-6M, 10-4M) solved in 0.2ml of PS. On Figure 4 points on continuous line mean values of water content in brain tissues are shown and kinetic curve of dose-dependent ouabain effects is formed by three ranges. For ouabain 10-9 - 10-8M concentrations an overhydration process is detected, at 10-7 - 10-6M dose – dehydration effect and at 10-4M (and less) doses again overhydration process is received. These changes of water content are characterized for all investigated tissues. In second part of experiments (next six groups of animals) water content and number of ouabain receptors in brain tissues after ouabain and ketamine parallel injections were investigated. In this case animals of control group Volume ∙ 4 ∙ Number ∙ 1 ∙ 2011

Ketamine effect on tissue hydration Armenuhi Heqimyan et al

were injected with 10-9M of ouabain solved in 0.2ml of PS. Animals of experimental groups were at first received ouabain and 15 minute after ketamine in anesthetic dose (0,125mg/g). On Figure 4 points on dotted line mean values for each group are indicated. As can be seen from figure in case of ketamine influence some reversing effect on ouabain-induced hydration of brain tissues at all doses of ouabain was observed.

Figure 4. Dose-dependent effect of ouabain on tissues water content (brain cortex - A, subcortex -B and cerebellum - C). Continuous lines indicate the ouabain dose - dependent effect on water content in tissues of control group experiments where animals were injected with ouabain dissolved in 0,2ml PS. Dotted lines indicate the water content in tissues of experimental groups animals injected with ouabain dissolved in 0,2ml PS and 0,125mg/g ketamine. Each point on both lines shows the mean value of water content for each part of brain. Abscissae indicate control point and [3H]-ouabain concentrations (10-9 M 10-6 M, and 10-4 M). Ordinates indicate the mean value of water content in tissues of cortex, subcortex and cerebellum. The difference between data of experimental and control groups of animals was statistically significant (*P<0.05, **P< 0. 01, ***P< 0.001) for all ouabain doses. The comparison between two kinetic curves shows the following changes in cortex tissue (Figure 4 A). After 10-9M of ouabain injection ketamine brings to the same overhydration effect. Ketamine-induced cell dehydration become more expressive after 10-8 - 10-7M ouabain injection. Then, it can be noted that 10-6M Page 46


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ouabain dehydration effect reverses into overhydration one after ketamine influence and overhydration at 10-4M ouabain also rotates into dehydration after ketamine injection. As for subcortex tissue it can be seen (Figure 4 B) that after ketamine injections meaningful dehydration effect is observed and dotted line having same three ranges is quite parallel to continuous one. In cerebellum tissue (after 10-9M of ouabain) ketamine leads to overhydration effect more expressive than at ouabain influence (Figure 4 C). At 10-8 - 10-7M ouabain injection ketamine shows the same dehydration effect as in case of cortex tissue. 10-6M ouabain dehydration effect reverses into overhydration one after ketamine influence and overhydration (at 10-4M of ouabain) to dehydration effect after ketamine injection.

Table 1 - Binding of [3H]-ouabain to brain tissues (cortex, subcortex and cerebellum). As a control the number of ouabain receptors after [3H]ouabain injections in different concentrations (10-9 M, 10-8 M, 10-7 M, 10-6 M, 10-4 M) was taken. In the experimental part (experiment) the number of ouabain receptors was measured after [3H]ouabain and ketamine injections. On table 1 the number of ouabain receptors in all investigated tissues is presented. As can be seen ketamine causes the increase of number of ouabain receptors in all three zones when injects after ouabain 10-9M concentration. It is worth to note that ketamine- induced increase of number of ouabain receptors was more pronounced in cerebellum tissue than in cortex and sub-cortex tissues. At 10-8M and 10-7M concentrations of ouabain ketamine causes the decrease of number of ouabain receptors in all brain tissues while at ouabain 10-6M concentration of this effect was observed only in subcortex and cerebellum. At 10-4M Volume ∙ 4 ∙ Number ∙ 1 ∙ 2011

Ketamine effect on tissue hydration Armenuhi Heqimyan et al

concentration of ouabain ketamine has slight increasing effect on number of ouabain receptors in cortex and cerebellum tissues and no significant effect on subcortex tissues. Discussion Investigation of ketamine influence on rats pain sensitivity latent period, tissues hydration and number of ouabain receptors was showed that its subanesthetic doses (8x10-5-8x10-2 mg/g) have statistically significant depressing effect on rats’ pain sensitivity. The analgesic effect of these ketamine doses on mammals was commonly explained by it’s non-competitive inhibitory effect on NMDA receptors in brain3-7. However, ketamine effects on tissue hydration of various organs (lung, heart, spleen, liver, kidney and skeletal muscle) were indicated that its effect was not only connected with neuronal membrane conductive functions. This conclusion is conformed to our previous data of ketamine induced dehydration effect on women breast tissue28. At present it is a proven fact that metabolic control of cell volume is dynamic cell parameter through which the regulation of cell various functions is realized25,26,36,37. Non-linear curves of dose-dependent ketamine effect on tissue hydration in brain and different organs could be explained by multisided effects of ketamine on mechanisms involved in cell volume regulation. Although there is a number of metabolic mechanisms involved in cell volume regulation the electrogenic ionic transporting mechanisms such as Na+/K+ pump and Na+/Ca++ exchanger have a crucial role in this process38-40. Earlier it was shown that ketamine has activation effect on electrogenic Na+/K+ pump and Na+/Ca++ exchanger activity in isolated neurons of mollusc through which it causes cell dehydration and decrease of membrane fluidity24,41. In study of dose-dependent ouabain binding with neuronal membrane three types of ouabain receptors was distinguished24. Among them the function of only one-dose dependent linear binding having low agonist affinity connected with Na+/K+ pump inhibition while the function of two types receptors having higher agonist affinity and characterized by dosedependent saturated curves connected with cGMP-dependent Na+/Ca++ and cAMP+ ++ dependent Na /Ca exchangers mechanisms Page 47


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correspondingly24,34,35. As these ion transporting mechanisms are electrogenic one they could modulate the level of cell hydration: activation of cGMP-dependent Na+/Ca++ exchanger and inactivation of Na+/K+ pump brings to cell swelling while activation of cAMP-dependent Na+/Ca++ exchanger brings to cell shrinkage. Considering the curves of dose-dependent ouabain-induced tissue hydration (Figure 4), it can be clearly seen three phases of ouabaininduced changes on tissue hydration: overhydration at 10-9-10-8M, dehydration at 10-710-6M and overhydration at 10-4M. The fact that the ketamine has reverse effects on all the three components of ouabain-induced hydration brings to conclusion that above mentioned ouabain receptors function which connected with cGMPdependent Na+/Ca++, cAMP-dependent Na+/Ca++ exchangers and Na+/K+ pump activity are ketamine sensitive. This conclusion confirms the previous isotope study of ketamine effects on Na+/K++ pump activity and Na+/Ca++ exchanger in snail neurons according which ketamine has activation effect on Na+/Ca++ (Ca++ efflux Na+ influx) exchange and Na+/K+ pump activity (24). The fact that ketamine has slight hydration effect on brain tissues which was accompanied by increased number of ouabain receptors at 10-9M ouabain concentration (Table 1) could be explained by the activation of cGMP dependent Na+/Ca++ exchanger which is responsible for cell hydration as it was above mentioned. Non-anesthetic dose (8x10-2mg/g i.p.) of ketamine has dehydration effect on brain tissues which is accompanied by corresponding decrease of number of ouabain receptors at 10-8-10-7 M ouabain concentrations (Table 1) explained by activation of cAMP dependent Na+/Ca++ exchanger (Ca++influx and 3Na+ efflux) responsible for cell dehydration34,35. Thus, the above mentioned results and literature data about involvement of intracellular cyclic nucleotides in brain psychological processes42 leads to the hypothesis that hallucination effect of ketamine connected with its modulation effect on cyclic nucleotides metabolism which could serve as a subject for the future investigation. The data of close correlation between cell hydration and the number of functional active protein molecules determining the cell functional activity25 and recent data on direct correlation between brain tissue hydration and rats pain Volume ∙ 4 ∙ Number ∙ 1 ∙ 2011

Ketamine effect on tissue hydration Armenuhi Heqimyan et al

sensitivity32 allows to consider ketamine dehydration effect at sub-anesthetic doses as an essential mechanism responsible for its depression effect on pain sensitivity of rats. On basis of the fact that ketamine anesthetic dose has only dehydration effect on subcortex and cerebellum tissue and over hydration effect on cortex tissue the suggestion can made that anesthetic effect of ketamine is only responsible for subcortex and cerebellum tissues. This conclusion can not be final as it needs future investigation. Conclusions Obtained data allow to decide that analgesic effect of subanesthetic dose of ketamine can be explained by brain tissue dehydration. Dehydration effect in tissues of subcortex and cerebellum has essential role in realization of ketamine anesthetic effect on organism. Ketamine induced cell volume changes accompanied by corresponding changes of number of functional active proteins on cell membrane serve as one of the main nonconductive membrane mechanisms through which the biological effect of ketamine is realized and explanation of ketamine effects only by changes of neuronal membrane conductive functions is not adequate. Acknowledgements The authors thank to Mss. Anna Nikoghosyan and Varsik Martirosyan for technical and editorial assistance. Declaration of Interest The authors report no conflict of interest and the article is not funded or supported by any research grant. References 1. Kress HG. Wirkmechanismen von Ketamin. Anaesthesist 1997; 46: S8-s19. 2. Kohrs R, Durieux ME. Ketamine: teaching and old drug new tricks. Anesth.Analg 1998; 87: 1186-1193. 3. Davies SN, Alford ST, Coan EJ, Lester RA, Collingridge GL. Ketamine blocks an NMDA receptor- mediated component of synaptic transmission in rat hippocampus in a voltage-dependent manner. Neurosci Let 1988; 92: 213-7. 4. Reich DL, Silvay G. Ketamine: an update of the first twenty-five years of clinical experience. Canad. J. of Anaesthesa 1989; 36: 186-197.

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5. Franks NP, Lieb WR. Molecular and cellular mechanisms of general anaesthesia. Nature 1994; 367: 607–614. 6. Sakai F, Amaha K. Midazolam and ketamine inhibit glutamate release via a cloned human brain glutamate transporter. Canad. J. of Anesthesia 2000; 47: 800-6. 7. Franks NP. General anesthesia: from molecular targets to neuronal pathways of sleep and arousal. Nature Reviews (Neuroscience) 2008; 9: 370-386. 8. Scheller M, Bufler J, Hertle I, et al. Ketamine blocks currents through mammalian nicotinic acetylcholine receptor channels by interaction with both the open and the closed state. Anesth. Analg 1996; 83: 830–6. 9. Sasaki T, Tomio A, Itaru W, et al. Nonstereoselective inhibition of neuronal nicotinic acetylcholine receptors by ketamine isomers. Anesth Analg 2000; 91: 741–8. 10. Hustveit O, Maurset A, Oye I. Interaction of the chiral forms of ketamine with opioid, phencyclidine, sigma and muscarinic receptors. Pharmacol. Toxicol. 1995; 77: 355–9. 11. Finck AD, Ngai SH. Opiate receptor mediation of ketamine analgesia. Anesthesiology 1982; 56: 291–297. 12. Smith DJ, Bouchal RL, Desanctis CA, et al. Properties of the interaction between ketamine and opiate binding sites in vivo and in vitro. Neuropharmacology 1987; 26: 1253–1260. 13. Schnoebel R, Wolff M, Peters SC, et al. Ketamine impairs excitability in superficial dorsal horn neurons by blocking sodium and voltage-gated potassium currents. Brit J. of Pharmacology 2005; 146: 826–833. 14. Brau ME, Sander F, Vogel W, Hempelmann G. Blocking mechanisms of ketamine and its enantiomers in enzymatically demyelinated peripheral nerve as revealed by single-channel experiments. Anesthesiology 1997; 86: 394–404. 15. Kawano TK, Oshita S, Takahashi A, et al. Molecular Mechanisms Underlying Ketamine-mediated Inhibition of Sarcolemmal Adenosine Triphosphate-sensitive Potassium Channels. Anesthesiology 2005; 102: 93-101. 16. Hirota K, Lambert DG. Ketamine: its mechanism(s) of action and unusual clinical uses. Brit. J. of Anaesthesia 1996; 77: 441-4. 17. Akbaş L., Sarioğlu Y., Utkan T. Effect of ketamine on contractile performance of isolated frog myocardium and comparison of ketamine, thiopental and droperidol. Mater Med Pol. 1992;24 (1): 32-4 18. Abdalla SS, Laravuso RB, Will JA. Mechanisms of the Inhibitory Effect of Ketamine on Guinea Pig Isolated Main Pulmonary Artery. Anesth. Analg. 1994; 78: 17-22. 19. Kohjitani A, Shirakawa J, Okada S, Obara H. The Relaxing Effect of Ketamine on Isolated Rabbit Lower Esophageal Sphincter. Anesth Analg. 1997; 84: 433-7. 20. Han J, Kim N, Joo H, Kim E. Ketamine blocks Ca2+-activated K+ channels in rabbit cerebral arterial smooth muscle cells. Am J Physiol Heart Circ Physiol. 2003; 285: 1347-1355. 21. Chang Y, Chen TL, Wu GJ, et al. Mechanisms involved in the antiplatelet activity of ketamine in human platelets. Journal of Biomedical Science 2004; 11: 764-772. 22. Ayrapetyan SN, Rychkov GY, Suleymanyan MA. Effects of water flow on transmembrane ionic currents in neurons of Helix pomatia and in Squid giant axon. Comp. Biochem. Physiol. 1988; 89: 179-186. 23. Ayrapetyan SN, Arvanov VL. On the mechanism of the electrogenic sodium pump dependence of membrane chemosensitivity. Comp Biochem Physiol 1979; 64: 601-604. 24. Ayrapetyan SN, Suleymanyan MA, Sagian AA, Dadalyan SS. Autoregulation of Electrogenic Sodium Pump. Cell Mol Neurobiol. 1984; 4: 367- 384. 25. Ayrapetyan SN. On the physiological significance of pumpinduced cell volume changes. Adv. Physiol. Sci. 1980; 23: 67-82. 26. Hoffman EK, Lambert IH, Pedersen SF. Physiology of cell volume regulation in vertebrates. Physiol. Rev. 2009; 89:193-277. 27. Ayrapetyan SN, Takenaka MA, Bakunts IS, Saghyan AA, Dadalyan SS. Reports of USSR Academy of Sciences 1986; 291:469-472.

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28. Danielyan AA, Mirakian MM, Airapetyan SN. The dehydrating action of ketamine on malignant breast tumors. Voprosi Onkol. 1998; 44: 395-7. 29. Danielyan AA, Ayrapetian SN. Changes of hydration of rats’ tissues after in vivo exposure to 0.2 Tesla Steady Magnetic Field. Bioelectromagnetics 1999; 20: 123-128. 30. Takahashi R, Aprison MH. Acetylcholine content of discrete areas of the brain obtained by a near-freezing method. J. of Neurochemistry 1964; 11:887-892. 31. Adrian RH. The effect of internal and external potassium concentration on the membrane potential of frog muscle. J Physiol 1956; 133: 631–658. 32. Ayrapetyan S, Musheghyan G, Deghoyan A. The brain tissue dehydration as a mechanism of analgesic effect of hypertonic physiological solution in rats. J Int Dent Med Res 2010; 3: 93-9. 33. Lazzaro V, Oliviero A, Profice P, et al. Ketamine increases motor cortex excitability to transcranial magnetic stimulation Physiol. 2003; 547: 485–496. 34. Azatian KV, White AR, Walker RJ, Ayrapetyan SN. Cellular and molecular mechanisms of nitric oxide-induced heart muscle relaxation. Gen. Pharmac. 1998; 30:543-553. 35. Saghyan AA, Hunanian Ash, Ayrapetyan SN. Effect of Locust Poison (LP) on Neuromembrane Functional Activity. General Pharmacology 1997; 29:587-590. 36. Tasaki I, Iwasa K. Rapid pressure changes and surface displacements in the squid giant axon associated with production of action potentials. Jpn J Physiol. 1982; 32:69-81. 37. Haussinger D. Regulation of Cell Function by Hydration, Biochem. J. 1996; 313:697-710. 38. Cooke KR. Ouabain and regulation of cellular volume in freshly prepared slices ofrabbit renal cortex. J Physiol. 1978; 279:361374. 39. Ayrapetyan SN, Suleymanyan MA. On The Pump-Induced Cell Volume Changes. Comp Biochem Physiol 1979; 64: 571-5. 40. Annunziato L, Pignataro G, Di Renzo GF. Pharmacology of Brain Na_/Ca2_ Exchanger: From Molecular Biology to Therapeutic Perspectives Pharmacol Rev 2004; 56: 633–654. 41. Saghyan AA, Dadalian SS, Takenaka T, Suleymanyan MA, Ayrapetyan SN. The effects of short-chain fatty acids on the neuronal membrane functions of helix pomatia. III 22 Na efflux from the cells. Cell. And Mol. Neourobiol. 1986; 6: 397-405. 42. Kandel ER, Schwartz JH. Molecular biology of learning: modulation of transmitter release. Science 1982; 218: 433-442.

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Effects of Propofol Şennur Ketani et al

EFFECTS OF PROPOFOL ON EXPRESSION ICAM-1 IN RABBIT GASTRIC ENDOTHELIAL CELLS Sennur Ketani1*, Berna Ersoz Kanay2, Hakan Sagsoz3 1. Department of Biology Education, Education Faculty, University of Dicle, 21280 Diyarbakır-Turkey. 2. Department of Surgery Veterinary Medicine Faculty, University of Dicle, 21280 Diyarbakır-Turkey. 3. Department of Histology&Embriyology, Veterinary Medicine Faculty, University of Dicle, 21280 Diyarbakır-Turkey.

Abstract It was examined the dose-dependent effects of propofol on expression intracellular adhesion molecule (ICAM-1) in rabbit gastric endothelial cells. Twenty adult New Zeland albino rabbits were used in this study. One control and three experimantal groups designed. In experimantal groups 0.5, 4.0, 8.0 mg/kg propofol were applied to rabbits by marginal ear vein. One hour after applying propofol, control and experimantal group rabbits were sacrificed and their gaster were removed. The sections were stained with APAAP immunohistochemical staining for evaluation using a light microscope. No inflammatoric reactions were seen in sections of gastric endothelial cells of control and experimental groups. Article (J Int Dent Med Res 2011; 4: (1), pp. 50-53) Keywords: ICAM-1; rabbit; gastric endothelium; propofol. Received date: 20 February 2010 Introduction Propofol is widely used for the induction and maintenance of anesthesia and a sedative in intensive care units, where it is given as a constant intravenous infusion for periods of many days1,2. Gastrointestinal mucosa is one of the most rapidly changing tissues in the body and the balance between cell regeneration and cell loss may lead to mucosal lesion and ulceration3. Leucocytes are pivotal component of the inflammatory cascade that results in tissue injury in a large group of disorders such as ischemia, non-steroidal antinflammatory drugs (NSAIDs) and ethanol1,4,5. The major lines of evidence that implicate leucocytes in the tissue injury associated with them include; leucocytes accumulate in the gastric mucosa prior to or during the development of tissue injury and that deplation of *Corresponding author: D r . Ş ennur KETANI Department of Biology Education, Education Faculty, University of Dicle, 21280 Diyarbakır-Turkey E-mail: ketani.sennur@gmail.com

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Accept date: 16 April 2010 leucocytes decreases the degree of injury. Free radical production and endothelial activation promote leucocyte-endothelium interaction via endothelial expression of vascular cell adhesion molecule 1 (VCAM-1) and intracellular adhesion molecule 1 (ICAM-1)1,4,5. The aim of present study was to investigate the effects of different doses of propofol on expression intracellular adhesion molecule (ICAM-1) in rabbit gastric endothelial cells. Material and Methods Twenty adult New Zeland albino rabbits were used in this study and the sex of them wasn’t remarkable, 2000-2500 g in weight were obtained from the Department of Medical Science Application and Research Centre of Dicle University (DUSAM). They were housed in invidual cages in temperature-controlled environment (22C) with a 12 :12 h light-dark cycle. All rabbits were fed standard pellet food and adlibitum tap water, which were performed according to the Declaration of Helsinki with the permission of the Governmental Animal Protection Committee.

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Group I (Control group): In this group the interval of the study nothing was done to the rabbits (n:5). Group II (0.5 mg/kg IV propofol applied): In this group 0.5 mg/kg propofol was applied to rabbits by marginal ear vein (n: 5). Group III: (4.0 mg/kg IV propofol applied): In this group 4 mg/kg propofol was applied by marginal ear vein (n: 5). Group IV: (8.0 mg/kg IV propofol applied). In this group 8 mg/kg propofol was applied by marginal ear vein (n: 5). After 1 hour applying of propofol, control and experimental group rabbits were sacrificied and their gaster were removed. Tissues were fixed for 6-8 hours in Bouin’s solution at 4 C. They were dehydrated though increasing concentrations of the ethanol series and the tissues were embedded in paraffin and cut into 4-5m transversal, dewaxed in xylene, and incubated for 20 minutes in 0.3% H2O2 to block endogenous peroxidase activity. Section then were microwaved for 4 minutes in 20 % goat serum in PBS in order to avoid undesired background staining, put into 20 minutes. Monoclonal mouse anti-Human ICAM-1 (BioGenex San Ramon USA) primary antibody (dilution: 1/200) was applied to the sections for 3 hours at 37 C in a humidified staining chamber. Sections were then incubated in anti-mouse IgG secondary antibody (Lab Vision, dilution: 1/1000) for 1 hour, and they were put into the APAAP complex for an hour. Sections were mounted with a glycerol-PBS mixture (1:1 glycerol: PBS). Following this step, sections were incubated in the fast red/TR naphtol mixture until the specific regions were stained red, and then the sections were either briefly put into Mayer’s hematoxilen in order to visualize the nuclei, or were not subjected to counterstaining. Sections were mounted with a glycerol-PBS mixture (1:1 glycerol: PBS). The control staining of some sections was performed without the primary antibody, and no ICAM-1 immunostaining was observed in these sections6. The immunohistochemical expressions were evaluated in 3 categories such as no, weak, moderate. The microphotographs were taken by Nikon 400 Eclipse light microscope.

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Effects of Propofol Şennur Ketani et al

Results Immunohistochemical Examination The aim of present study was to invastigate the effects of propofol on expression intracellular adhesion molecule (ICAM-1) in rabbit gastric endothelial cells. There was no significance changes in immunoreactivity among section evenwithin groups, the difference between control and experimental groups was not clear (Table 1).

Table1: Immunohistochemical expression ICAM-1 in control and experimental groups.

of

The control staining of some sections was performed without the primary antibody, and no ICAM-1 positive immunostaining was observerd in these sections (Figure 1).

Figure 1 Immunonohistological appearance of Control Group. The control staining was performed without the primary antibody. Immunogens show no positive staining, Lumen (L), Lamina epithelialis (le), lamina propria (lp), gastric gland (g), blood vessel (bv), (Original magnification X40, Scale Bar: 25µm). Tunica mucosa of the stomachs of the rabbits in control and experimental groups were normal in histological examination (Figure 2,3,4,5). Page 51


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Effects of Propofol Şennur Ketani et al

In group IV (which were given 8 mg/kg propofol) showed moderate staining with ICAM-1 (Figure 5).

Figure 2 Immunonohistological appearance of Group I (Control groups). ICAM-1 weak staining in endothelium (arrows head), Lumen (L), Lamina Epithelialis (le), lamina propria (lp), Immunostaining was performed using secondary antibodies (Original magnification X 40, Scale Bar: 25µm).

Figure 4 Immunonohistological appearance of Group III. ICAM-1 weak staining in endothelium (arrows head), Lumen (L), Lamina Epithelialis (le), lamina propria (lp), gastric gland (g), blood vessel (bv), Immunostaining was performed using secondary antibodies (Original magnification X 40, Scale Bar: 25µm).

Figure 3 Immunonohistological appearance of Group II. ICAM-1 weak staining in endothelium (arrows head), Lumen (L), Lamina Epithelialis (le), lamina propria (lp), Immunostaining was performed using secondary antibodies (Original magnification X 40, Scale Bar: 25µm). In control group ımmunohistochemistry of ICAM-1 expression showed weak staining in gastric endotelial cells (Figure 2). In group II (which were given 0.5 mg/kg propofol) showed weak staining with ICAM-1 (Figure 3). In group III (which were given 4 mg/kg propofol) showed weak staining with ICAM-1 (Figure 4). Volume ∙ 4 ∙ Number ∙ 1 ∙ 2011

Figure 5 Immunonohistological appearance of Group IV. ICAM-1 modarete staining in endothelium (arrows head), Lumen (L), Lamina Epithelialis (le), lamina propria (lp), Immunostaining was performed using secondary antibodies (Original magnification X 40, Scale Bar: 25µm).

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Discussion Nuclear factor kappa ß can be activated by lesion induced oxidative stress, bacterial endotoxin or cytokines and subsequently increases transcriptionally the expression of the genes for many cytokines, enzymes and adhesion molecules, which have been believed to be involved in the acute inflammatory response. Adhesion molecules can recruit inflammatory cells, such as neutrophils, eosinophils and T lymphocytes from the circulation to the site of inflammation to release inflammatory mediators responsible for the gastric mucosal damage7. Antioxidants within cell membranes protect the phospholipids from free radical mediated lipid peroxidation and oxidative stress. α-Tocopherol (Vitamine E) is used to protect lipid from oxidation. This compound contains a phenol group that donates hydrogen to free radicals, thus terminating lipid peroxidation. Propofol is an intavenous anesthetic with a chemical structure similar to phenol-based free radical scavengers such as Vitamine E1,8. Reduction of free radical may improve outcomes of patients undergoing on surgeries. Common antioxidants such as vitamine E and buthylated hydroxytoluene can not be used routinely. Propofol may be the first candidate becouse of its anesthetic properties, rapid acting and recovering. Therefore it may have a protective role in gastric disorders and surgeries where free radical mediated injury promates leucocytes-endothelium adhesive interactions1,4,8. ICAM-1 expression is strongly inducible by inflammatory cytokines9. Here no immunohystochemical staining seen in groups shows that propofol had not caused ınflammation. Ketamine is commonly used as an anesthetic agent in veterinary medicine. Kenneth et al., (2003)10 had mentioned that ketamine inhibits gastric injury. Because ketamine interacts with a number of inflammatory pathways and may be useful in inflammatory models of tissue injury.

Effects of Propofol Şennur Ketani et al

mechanisms involved may help the understanding of the inflammatory cascade and may lead to future development for the propofol in the treatment of acute abdomen surgery and pathologic inflammation in animals. Declaration of Interest The authors report no conflict of interest and the article is not funded or supported by any research grant. References 1. Ketani M.A., Kanay B.E, Ketani Ş, Ünver Ö, Kilinç M. Dosedependent effects of propofol on expression ICAM-1 in rabbit aorta endothelial cells. African Journal of Biotechnology 2009; 8: 66886693. 2. Steward A, Allott P.R, Cowles A.L., Mapleson W.W. Solubility coefficients for inhaled anesthetics for water, oil and biological media. Br. J. Anaesth. 1973; 45: 282-293. 3. Ketani Ş, Balcı K, Kanay Z, Kanay B.E., Isı H. Effects of ethanol induced gastric lesions in sialoadenectomized rat: An ultrastructural study. Biotechnol. & Biotechnol. Eq. 2005; 19: 125-129. 4. Corcoran T.B., Engel A, Shorten G.D. The influence of propofol on the expression of intercelluler adhesion molecule 1 (ICAM-1) and vasculer cell adhesion molecule (VCAM-1) in reoxygenated human umblical vein endothelial cells. Eur. J. Anaest 2006; 23: 942947. 5. Fiona J, Andrews C.M.W., Paul E.O. Expression of adhesion molecules and leukocyte recruitment into gastric mucosa following ıschemia-reperfusion. Digestive diseases and sciences 1997; 42: 326-332. 6. Seidmen J.G. In situ hybridization and immunochemistry, In: Short Protocols in Molecular Biology,. Ausubell F.M., Brent R.: John Wiley and Sons Press, Newyork, Chichester, Brisbane, Toronto, Singapore. 1993. 7. Hang C.H., Shi J.X., Li J.S., Li W.Q., Yin H.X. Up-regulation of intestinal nuclear factor kapa B and intercelluler adhesion molecule1 following traumatic brain ınjury in rats. World J. Gastroenterol 2005; 11: 1149-1154. 8. Zhang S.H., Wang S.Y., Yao S.L. Antioxidative effect of propofol during cardiopulmonary bypass in adults. Acta Pharmacol. Sin. 2004; 25: 334-340. 9. Mclaughlin F, Hayes B.P., Horgan C.M., Beesley J.E., Campbell C.J., Randı A.M. Tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 beta down-regulate intercelluler adhasion molecule (ICAM-2) expression on the endothelium. Cell Adhes Commun 1998; 6: 381400. 10. Kenneth S, Hemler Y.C., Lily C, Ashvin D, David W.M.: Effects of ketamine/xylazine on expression of tumor necrosis factor-ά, inducible nitric oxide synthase and cyclo-oxygenase-2 in rat gastric mucosa during endotoxemia. Shock 2003; 20: 63-69.

Conclusions In this study it has been shown that propofol do the same inhibition just as ketamine in gastric mucosa by the nonexpression of ICAM1. Here it also shown that the anti-inflammatory effects of antioxidants and molecular Volume ∙ 4 ∙ Number ∙ 1 ∙ 2011

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Journal of International Dental and Medical Research