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Editorial Board of JIDMR 2010 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 PhD. Dr. Ediz KALE Language Editor

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)

Editorial Board Abdel Fattah BADAWI (EGYPT) Ali Al-Zaag Iraq Ali GUR (TURKEY) Ali Riza ALPOZ (TURKEY) Alpaslan TUZCU (TURKEY) Ayca DENIZ IZGI (TURKEY) Aziz YASAN (TURKEY) Benik Harutunyan (ARMENIA) Betul KARGUL (TURKEY) Betul URREHMAN (UAE) Bugra OZEN (TURKEY) Cemil SERT (TURKEY) Christine Bettina STAUDT (SWITZERLAND) Claudia DELLAVIA ( ITALY ) Emin Caner TUMEN (TURKEY) Ertugrul ERCAN (TURKEY) Eylem OZDEMIR (TURKEY) Fadel M. ALI (EGYPT) Fahinur ERTUGRUL (TURKEY) Feral OZTURK (TURKEY) Feridun BASAK (TURKEY) Feriha CAGLAYAN (TURKEY) Ferranti WONG (UNITED KINGDOM) Figen SEYMEN (TURKEY) Filippo BATTELLI (ITALY) Filiz Acun KAYA (TURKEY) Gajanan Kiran KULKARNI (CANADA) Gamze AREN (TURKEY) Gauri LELE (INDIA) Gonul OLMEZ (TURKEY) Guliz Nigar GUNCU (TURKEY) Gulten UNLU (TURKEY) Halimah AWANG (MALAYSIA) Hilal TURKER (TURKEY) Igor BELYAEV (SWEDEN) Ilker ETIKAN (TURKEY)

Isin ULUKAPI (TURKEY) Izzet YAVUZ (TURKEY) Jalen Devecioglu KAMA (TURKEY) Kewal KRISHAN (INDIA) King Nigel MARTYN (HONG KONG SAR, P R CHINA) Kursat ER (TURKEY) M.Sabri BATUN (TURKEY) Mahmut METE (TURKEY) Marco MONTANARI (ITALY) Margaret TZAPHLIDOU (GREECE) Medi GANIBEGOVIC (BOSNIA and HERZEGOVINA) Mehmet Nuri OZBEK (TURKEY) Mehmet Zulkuf AKDAG (TURKEY) Meral ERDİNÇ (TURKEY) Mohammad FAHIM (DELHI) Mohamed TREBAK (USA) Moschos A. PAPADOPOULOS (GREECE) Mostaphazadeh AMROLLAH (IRAN) Muhammad FAHIM (INDIA) Muhammed Mustahsen URREHMAN (UAE) Murat AKKUS (TURKEY) Muzeyyen YILDIRIM (TURKEY) Neval Berrin ARSERIM (TURKEY) Nezahat AKPOLAT (TURKEY) Nihal HAMAMCI (TURKEY) Nik Noriah Nik HUSSEIN (MALAYSIA) Nurten ERDAL (TURKEY) Orhan TACAR (TURKEY) Ozant ONCAG (TURKEY) Ozkan ADIGUZEL (TURKEY) Rafat Ali SIDDIQUI (PAKISTAN) Refik ULKU (TURKEY) S. Yavuz SANISOGLU (TURKEY) Sabiha Zelal ULKU (TURKEY)

Sadullah KAYA (TURKEY) Sedat AKDENIZ (TURKEY) Selahattin ATMACA (TURKEY) Selahattin KATAR (TURKEY) Selahattin TEKES (TURKEY) Serdar ERDINE (TURKEY) Serdar ONAT (TURKEY) Shailesh LELE (INDIA) Sinerik N. AYRAPETYAN (ARMENIA) Smaragda KAVADIA (GREECE) Sossani SIDIROPOULOU (GREECE) Stephen D. SMITH (UNITED STATES OF AMERICA) Susumu Terekawa (JAPAN) Süleyman DASDAG (TURKEY) Ufuk ALUCLU (TURKEY) Ugur KEKLIKCI (TURKEY) Xiong-Li YANG (CHINA) Yuri LIMANSKI (UKRAINE) Zafer C. CEHRELI (TURKEY) Zeki AKKUS (TURKEY) Zeynep AYTEPE (TURKEY) Zurab KOMETIANI (GEORGIA)


Journal of International Dental and Medical Research

ISSN: 1309-100X

TABLE OF CONTENTS DENTISTRY THE EFFECT OF GALLIUM-ARSENIDE LASER IRRADIATION ON ODONTOGENESIS Eman Adnan Mustafa, Sausan Al Kawas Pages 52-56 SALIVARY LIPID PEROXIDATION AND LIPID PROFILE LEVELS IN PATIENTS WITH RECENT ISCHEMIC STROKE Natheer H Al-Rawi Pages 57-64 UNUSUAL INVERTED AND MOLARIFORM SUPERNUMERY TEETH – A CASE REPORT S. Navit, Firoza Samadi, Prashant Babaji, Rohit Anand, Anju Bansal Pages 65-68 RESTORATION OF POSTERIOR EDENTULOUS SPACES AFTER MAXILLARY MOLAR INTRUSION WITH FIXED APPLIANCES (CASE REPORT) Guvenc Basaran, Emrah Ayna, Emine Goncu Basaran, Gulten Unlu Pages 69-74 MAXILLARY CANINE-LATERAL INCISOR TRANSPOSITION: A CASE REPORT Demet Suer Tumen, Filiz Acun Kaya, Nihal Hamamci, E. Caner Tumen, Gulay Berber Pages 75-78 GINGIVAL METASTASE AND INTRACEREBRAL HAEMORRHAGE RESULTING FROM UNSUSPECTED CHORIOCARCINOMA: A CASE REPORT Mehmet Serhan Tasdemir , Ayfer Aktas, Nebahat Tasdemir, Yusuf Nergiz Pages 79-81 MEDICINE OUR DIAGNOSTIC AND THEARAPEUTIC SURGICAL APPROACHES TO MEDIASTINAL MASSES Cemil Deniz Yorgancilar, Ozgur Karakurt, Osman Korcan Tilkan, Sedat Demircan Pages 82-87 THE CRITERIA FOR CLASSIFICATION TREE METHODS IN CLINICAL RESEARCHES Zeki Akkus, S.Yavuz Sanisoglu, Mehmet Ugurlu, M. Yusuf Celik Pages 88-92 BIOMEDICAL RESEAERCH THE BRAIN TISSUE DEHYDRATION AS A MECHANISM OF ANALGESIC EFFECT OF HYPERTONIC PHYSIOLOGICAL SOLUTION IN RATS Sinerik Ayrapetyan, Gohar Musheghyan, Anush Deghoyan Pages 93-99 THE INVESTIGATION BY DOPPLER ULTRASONOGRAPY OF BLOOD FLOW DYNAMICS OF TESTES AND LIVER IN MEN EXPOSED CHRONICALLY TO TOLUENE Cemil Sert, Ocal Sirmatel, Erkan Yildiz, Ferat Oruc Pages 100-103

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The Effect of Gallium-Arsenide Laser Eman Adnan Mustafa and Sausan Al Kawas

THE EFFECT OF GALLIUM-ARSENIDE LASER IRRADIATION ON ODONTOGENESIS Eman Adnan Mustafa1*, Sausan Al Kawas2 1. BDS, MSc, lecturer, Department of Oral & Craniofacial Health Sciences, College of Dentistry, University of Sharjah, United Arab Emirate, P.O. Box: 27272 Sharjah, UAE. 2. DDS, PhD, Associate Professor, Head of Oral & Craniofacial Health Sciences Department, College of Dentistry, University of Sharjah, United Arab Emirates, P.O. Box: 27272 Sharjah, UAE.

Abstract The applications of lasers in dentistry show that dental structures react differently to various types of laser. This study explores the effect of gallium-arsenate (GaAs) laser radiation on rats’ incisor teeth. A total number of 20 rats, aged 1-21 days, are divided into five groups; one control group (4 rats) and four laser groups (a total of 16 rats). Laser groups were exposed to 60 seconds of laser radiation on alternate days from day 1 till day 19. The groups were sacrificed at different ages (15, 17, 19 and 21 days old). The specimens were prepared for processing and staining with hematoxylin and eosin and examined by light microscope. The results showed that the developing teeth of laser groups exhibit uneven thickness of dentin and multiple areas of interglobular dentin whereas pulp tissue appeared to be more cellular and vascular. However, no effect of laser can be noticed on the enamel, cementum, periodontal ligament and eruption process. It is concluded that GaAs laser irradiation has a positive effect on dentin formation. Consequently, GaAs laser can be used within pulp-capping procedures. (J Int Dent Med Res 2010; 3: (2), pp. 52-56 ) Keywords: Dentin, dentinogenesis, diode laser, histology, pulp. Received date: 01 Jauary 2010 Introduction

Accept date: 12 May 2010 other investigators to consider the use of Nd:YAG laser for caries removal 3-5. Another group of lasers termed as low level lasers (LLL) or low power lasers were applied in 1980s. Those lasers showed to be less traumatic and have more stimulating effect than other groups of lasers 2. LLL raise tissue temperature much less than high energy lasers as Nd-YAG and CO2 which can increase tissue temperature to a level high enough to vaporize the tissue 6. LLL stimulate a set of structural and functional changes in the living tissues. They have an immediate effect on cell vitality as mitochondrial production of ATP increases. Their biostimulation effect depends mainly on the wave length, power energy values, exposure time and optical features of the tissues 7, 8. The most effective irradiation is that within the red and infra-red wavelength, such as heliumneon laser (He-Ne 632.8 nm), helium-neonarsenate laser (He-Ne-As 780-870 nm) and gallium arsenate laser (GaAs 904 nm). They can penetrate deep into living tissues because the P

In the past 50 years, the use of laser in dentistry went through many dramatic changes. It is evidenced that the efficiency of the laser can be determined by the ability of the tissue to absorb and transmit laser to the surrounding tissues 1, 2. The first applications of laser to the dental tissues were reported by Goldman (1965) then followed by Stern and Sogannaes (1972). In their studies, they used ruby laser on enamel and dentin. In 1977, Adrian found that Nd:YAG laser caused no pulp necrosis, this information allowed P

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*Corresponding author:

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Dr. Eman Adnan Mustafa, Department of Oral & Craniofacial Health Sciences, College of Dentistry, University of Sharjah, United Arab Emirate, P.O. Box: 27272 Sharjah, UAE. E-mail: emustafa@sharjah.ac.ae

Volume 3 ∙ Number ∙ 2 ∙ 2010

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hemoglobin does not absorb such wavelengths 1, . A series of biological effects promote the use of LLL in the treatment of pain, wound healing, inflammation and edema, as they can stimulate fibroblasts proliferation, collagen production, enzyme activity, leukocytes activity, and angiogenesis 11-15. Different authors described the effect of LLL on erupted teeth. Their conclusions suggest that laser can alter composition and morphology of enamel 16, and can stimulate dentin formation and pulp cells 17, 18. In addition, the effect of laser was demonstrated in the induction of new periodontal ligament, cementum, and bone 19. Silvestri (2004) indicated that diode lasers can selectively result in tooth agenesis 20. There has been relatively few studies evaluation the effects of laser radiation on the developing teeth. Accordingly, the present study attempts to highlight the effect of LLL on the odontogenesis of the rat incisors teeth. 2, 9, 10

Materials and Methods Experimental animal groups: Twenty Sprague-Dawley rats aged from 1-21 days post partum were arranged into two main groups. During the time of the study, all the rats were housed in similar quarters and the environmental conditions were kept constant. The first group of rats is the control group which is consisted of four animals. They were sacrificed at different ages at 15, 17, 19 and 21 days old. For comparison purposes with the laser groups both left and right sides of the heads were used. The second group is the laser group which consisted of sixteen rats that were divided into four groups according to different doses of laser irradiation: Laser group A (4 rats): this group was utilized until 15 days old. It was exposed to multiple doses of laser irradiation for 60 sec/dose, on alternated days from day 1 to day 13 of their age. The group was sacrificed at 15 days old. Laser group B (4 rats): this group was exposed to multiple doses of laser radiation as with group A except that irradiation was continued until day 15 of their age. The group was sacrificed at 17 days old. Laser group C (4 rats): this group exposed to laser radiation as with groups A and Volume 3 ∙ Number ∙ 2 ∙ 2010

The Effect of Gallium-Arsenide Laser Eman Adnan Mustafa and Sausan Al Kawas

B except that irradiation was proceeded until day 17. The group was sacrificed at 19 days old. Laser group D (4 rats): this group is similar to the other groups except that irradiation was extended until day 19. The group was sacrificed at 21 days old. Laser device \ Optodent: Optodent is a patented dental unit for infra-red and laser therapy invented by Mario Scalvini in 1989. Specifications of Optodent are 220 Voltage supply, 50HZ power frequency, maximum power 40 Watt, dimensions 340x120x210 mm. The Optodent system is made up of two different emitting sections; infra-red emitting hand piece and optic fiber giving off Gallium-arsenate (GaAs) laser. Laser section was used to carry out this study. GaAs laser is an infra-red diode laser with 20 Watt peak power, average power of 5mw, the wave-length is 904 nm, impulse width 200 nsec, impulse frequency 3000 HZ, and the emission is continuous. Application of laser: all the groups of rats were subjected to the same lab conditions. During laser application, the optic fiber was applied to the right lower jaw below the lower border of the eye in close contact with the skin. Laser was applied according to the correct exposure time. Preparation of the specimens: The animals were killed with chloroform vapor. The samples were immediately immersed in 10% formalin, then decalcified with 10% formic acid. After that the samples were dehydrated in ethanol, cleared with xylene, and subsequently embedded in paraffin wax. Serial sagittal sections were prepared for Harris hematoxylin and eosin staining procedures. Sections were examined by Olympus & Leitz light microscopes at different powers starting with low power 4x then high powers 10x and 40 x. Results Observation of serial sagittal sections reveals the effect of laser in the development and eruption of rats’ incisor teeth as explained below: 1. Histological appearance of the rats’ incisors in the control group from 15th -21st day old: From 15th to 17th day old, the erupted incisor rat tooth appeared as an arc-shaped which is consisted of a powerful crown with no root. The labial portion is covered with enamel while the lingual and lateral portions are covered Page 53


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The Effect of Gallium-Arsenide Laser Eman Adnan Mustafa and Sausan Al Kawas

with cementum. The bulk of the tooth is formed by dentin core. At the proximal end of the tooth there was an odontogenic epithelium which is contained a mass of undifferentiated mesenchymal cells (Figures. 1 & 2).

Figure 3. Histological appearance of late developing rat incisor in the control group: The histological observation showing normal histology of enamel space =ES, labial dentin =AD, lingual dentin= LD, and the pulp = P. (H&E , original magnification 4x). Figure 1. Histological appearance of early developing rat incisor in the control group: Low power observation of developing incisor tooth in control group, show normal histological appearance. ES=enamel space, D=labial and lingual dentin, P= pulp. (H&E , original magnification 4x).

Figure 2. Histological appearance of the growing end of rat incisor in the control group: The histological observation of developing incisor tooth in different region, showing the incisor tooth growing end (GE), EM=enamel matrix, D=labial dentin, P=pulp. (H&E , original magnification 4x). The developing incisor tooth from 15 -21 days old shows three main stages from proximal to distal ends beginning respectively with growth, then calcification ended with eruption process at the distal end (Figure. 3). Volume 3 ∙ Number ∙ 2 ∙ 2010

2. Histological appearance of the rats’ incisors in the laser groups from 15th -21st day old: In all laser groups, the morphology of the incisor teeth appears similar to control groups. The developing enamel at the labial side shows no disturbances in formation. Interestingly, the dentin in laser groups is thicker than the dentin of control groups especially at the middle third of the tooth (Figure. 4).

Figure 4. Histological appearance of late developing rat incisor in laser group: Low power observation of a developing incisor tooth, shows interglobular dentin (GD) with thick layer of predentin (PD) at the labial side (A). At the lingual side (L), dentin (D), pulp (P) and acellular cementum (AC) show normal histological appearance. (H&E , original magnification 4x). Page 54


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The affected dentin represents a marked degree of interglobular dentin formation which is accompanied by increased thickness of predentin (Fig. 5). The pulp in turn shows prominent changes by increase in its blood vessels and cells mainly at the areas of increased dentin formation (Fig. 6). In all laser groups, the portion that covered with cementum showed no developmental disturbances in cells or structures. The eruption process of the incisor teeth in all groups seems normal compared to control group.

Figure 5. Histological section of early developing rat incisor in laser group: A histological changes in early developing rat incisor, showing increase thickness of predentin with interglobular (G) dentin surround by dense pulp cells (P). (H&E, original magnification 10x).

Figure 6. Histological section in the pulp region of rat incisor in laser group: A high power observation of rat incisor demonstrates increasing thickness of predentin adjacent to newly formed dentin (D). Pulp (P) shows increase cellularity near the predentin. (H&E, original magnification 40x). Volume 3 ∙ Number ∙ 2 ∙ 2010

The Effect of Gallium-Arsenide Laser Eman Adnan Mustafa and Sausan Al Kawas

Discussion Studies report that single doses of lowpower laser irradiation can stimulate dentin and pulp tissues in fully erupted human teeth 17, 21, 22. Conversely, other report that low power laser has no positive effects on hard tissues like bone or teeth 23, 24. Accordingly and due to the lack of sufficient research in the field of fractional laser irradiation, the present study investigates the effects of laser irradiation on the rats’ teeth. Rat incisors included in this present study are selected on the basis of the presence of successive developmental stages along their structure, which exhibits many similarities to human tooth formation. Furthermore rat incisors continue to grow and erupt; which in turn give a clear view of the effectiveness of lasers on the eruption pattern 25. The laser groups showed a remarkably irregularity in the distribution of dentin thickness, this can be attributed to the disturbance in the histodifferentiation of the odontoblasts. These results go in line with Godoy’s study as well as & Olivi’s 2007 which are undertaken on pulpexposed teeth 17, 21. The findings are also consistent with the results of studies carried on human teeth presented by Tate 2006 and Matsui 2007, who concluded that use of laser may induce formation of dentin, but higher energies of laser may cause irreversible changes to the pulp 26, 27. The changes in the pulp are due to the influence of laser on the cell proliferation in addition to the formation of newly formed blood vessels that are also confirmed by other studies 17, 21, 27. It is interesting to note that laser has a selective effect on the odontoblasts among all dental tissue cells. It is possible that odontoblast cells are stimulated in the same manner as the fibroblast cells that also affected by laser. Many studies stressed that there is an increase in collagen synthesis by fibroblasts after laser irradiation of wounded tissues 19, 24, 28. It can be noted that the main effect of laser is mostly on the growth and calcification stages rather than eruption stage; this is in agreement with studies carried out by Murakami 2005 and Masuda 2006 on rats’ teeth. They prove that different types of laser can provoke changes in the pulp and dentin without disturbing Page 55


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the eruption process 18, 29. The primary effects of laser in this study are at the middle third of the teeth, which was related to the area that was subjected first to the laser irradiation on day one of the experiment. This area manifested the effects of laser at these specific locations by changes in the developing tooth structures. This finding is agreed on by other investigators how could localize the influence of laser in specific areas of the pulp in the fully erupted teeth 17, 26, 29. GaAs laser directly influences the dentin and pulp rather than enamel and cementum; this can be attributed to the translucency of the latter structures which enhances laser transmission to deeper structures 24, 30, 31. It can also be attributed to their structural diversity from dentin and pulp 24, 29. Conclusion In conclusion, it can be said that GaAs laser irradiation have positive effects upon dentin formation. Consequently, GaAs lasers can be used during pulp-capping procedures. Declaration of Interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article. References 1. Sulieman M. An overview of the use of lasers in general dental practice: Laser physics and tissue interactions. Dent Update. 2005; 32:228-30. 2. Makinson OF. Soft lasers and dentistry. Aust. Dent. J. 1986; 13139. 3. Goldman I , Homby P, Meyer R, Goldman B. Effect of laser beam impacts on teeth. J. Amer Dent Assoc. 1965; 70: 599-606. 4. Stern RH, Sognnaes RF. Laser inhibition of dental caries suggested by first tests in vivo. J AM Dent Assoc. 1972;85: 108790. 5. Adrian JC. Pulp effects of neodymium laser. Oral surg. 1977; 44:301-5. 6. Pogrel MA. Application of laser and cryosurgery in oral and maxillofacial surgery. Curr Opin Dentistry. 1991; 1:263-70. 7. Kimura Y. Effect of Er:Cr:YSGG laser irradiation on canine mandibular bone. J Periodontology. 2001; 72(9):1178-82. 8. Theodoro LH. Effect of Er:YAG and diode laser irradiation on the root surface. J Periodontology. 2003; 74(6): 838-43. 9. Aoki A, Mizutani K, Takasaki A. Current status of clinical laser applications in periodontal therapy. Featured in General Dentistry. 2008 ; 674-87. 10. Vladimirov YA, Osipov AN, & Klebanov GI. Photobiolgic principles of therapeutic applications of laser radiation. Review. Biochemistry 2004; 69(1):81-90. 11.Basford JR. Low-energy laser treatment of pain and wounds; hype, hope, or hokum? Mayo Clin Proc. 1986; 61(8):671-5.

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The Effect of Gallium-Arsenide Laser Eman Adnan Mustafa and Sausan Al Kawas

12. Anneroth, G, Hall G, Ryden H, Zetterqvist L. The effect of lowenergy infra-red laser radiation on wound healing in rats. Brit Assoc of Oral and Maxillofac Surg. 1988; 26:12-17. 13. Pereira AN, Eduardo C P, Matson E, Marques MM. Effect of low-power laser irradiation on cell growth and procollagen synthesis of cultured fibroblasts. Lasers Surg Med. 2002;31:263-66. 14. Kreisler M. Effect of diode laser irradiation on the attachment rate of periodental ligament cells: an in vitro study. J Periodontology. 2001; 72(10): 1312-17. 15. Kreisler M, Christoffers AB, Willershausen B, Hoedt B. Effect of low-level GaALAs laser irradiation on the proliferation rate of human periodontal ligament fibroblasts: An in vitro study. J Clin Periodontol. 2003; 30:353-58. 16. Feuerstein O, Mayer I, Deutsch D. Physico-chemical changes of human enamel irradiated with ArF excimer laser. Lasers Surg. Med. 2005; 37:245-51. 17. Godoy BM, Arana-Chavez VE, Nunez SC, Ribeiro MS. Effects of low-power red laser on dentine-pulp interface after cavity preparation. An ultra structural study. Archives of Oral Biology. 2007; 52(9):899-903. 18. Murakami Y, Hossain M, Wang X, Okano T, Matsumoto K. Pulsed Nd:YAG laser effect on eruption of rat mandibular incisors following disturbance of the enamel organ in the pulp. Lasers Med Sci. 2005; 20(2):95-8. 19. Crespi R, Covani U, Margarone JE, Andreana S. Periodontal tissue regeneration in Beagle dogs after laser therapy. Lasers Surg Med. 1997; 21: 395-402. 20. Silvestri AR, Mirkov MG, Connolly RJ. Prevention of third molar tooth development in neonate rat with a long pulse diode laser. Lasers Surg Med. 2004; 35(5):385-91. 21. Olivi G, Genovese MD, Maturo P, Docimo R. Pulp capping: advantages of using laser technology. Eur J Paediatr Dent. 2007; 8(2):89-95. 22. Myers TD. Lasers in dentistry. JAM Dent Assoc. 1991; 122:4650. 23. Demir H, Balay H, Kirnap M. A comparative study of the effects of electrical stimulation and laser treatment on experimental wound healing in rats. J Rehabil Res Dev. 2004; 41(2):147–154. 24. Cobb CM. Lasers in periodontics: a review of the literature. J Periodontology. 2006;77: 545-64. 25. Warshawsky H, Josephsen K, Thylstrup A, Fejerskov O. The development of enamel structure in rat incisors as compared to the teeth of monkey and man. Anat Rec. 1981; 200:371-99. 26. Tate Y, Yoshiba K, Yoshiba N, Iwaku M, Okiji T, Ohshima H. Odontoblast responses to GaAlAs laser irradiation in rat molars: an experimental study using heat-shock protein-immunohistochemistry. Eur J Oral Sci. 2006; 114(1):50-7. 27. Matsui S, Tsujimoto Y, Matsushima K. Stimulatory effects of hydroxyl radical generation by Ga-Al-As laser irradiation on mineralization ability of human dental pulp cells. Biological & Pharmaceutical Bulletin. 2007; 30(1):27-31. 28. Marques M, Pereira N, Neusa A, Fernando N, Carlos P. Effect of low-power laser irradiation on protein synthesis and ultrastructure of human gingival fibroblasts. Lasers in Surgery & Medicine. 2004;34(3):260-5. 29. Masuda YM, Hossain M, Wang X, Matsuoka E, Okano T, Matsumoto K. Effect of Er,Cr:YSGG laser irradiation on eruption of rat mandibular incisor after disturbance of the enamel organ in the pulp. Lasers Med Sci. 2006;21(3):165-9. 30. Pretel H, Oliverira JA, Lizarelli RF, Ramalho LT. Evaluation of dental pulp repair using low level laser therapy (688nm& 785nm) morphologic study in capuchin monkeys. Laser Phys Lett. 2009 ; 6 (2): 149-158. 31. Dimitrov SI, Dogandzhiyska V, Ishkitiev N. Effect of laser irradiation with different wavelength on the proliferation activity of human pulp fibroblast cells, depending on irradiation parameters and hard tissue thickness. JIMAB 2009: 28-31.

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Salivary Lipid peroxidation & Ischemic Stroke Natheer H Al-Rawi

SALIVARY LIPID PEROXIDATION AND LIPID PROFILE LEVELS IN PATIENTS WITH RECENT ISCHEMIC STROKE Natheer H Al-Rawi* 1. B.D.S, M.Sc, Ph.D, Department of Oral & Craniofacial Health Sciences, College of Dentistry, University of Sharjah.

Abstract Abnormal lipid levels are an important risk factor in the development of atherosclerotic complications like stroke. Oxidative stress, lipid and lipoprotein Peroxidation are involved in neuronal damage induced by ischemia-reperfusion in stroke. Malondialdehyde (MDA) is widely utilized as a marker of lipid Peroxidation in state of elevated oxidative stress. The role of lipid in oxidative damage in saliva of patients with stroke is not yet completely elucidated. The aim of this study was evaluate the relationship of MDA as a marker of lipid Peroxidation with lipids and lipoprotein fractions and to find out the cut-off values of the studied parameters in saliva of patients with recent stroke . We studied 50 patients with ischemic stroke and other 25 ages and sex matched health control. To evaluate the oxidative status we measured the levels of Malondialdehyde in saliva and serum of all participants. Lipid profile was also estimated by the total cholesterol, triglycerides, LDL-C and HDL-C. MDA levels were significantly higher in patients with ischemic stroke than that of healthy control. Salivary critical value of triglycerides more than 0.5 mmol/L yields in highest accuracy rate (93%) to differentiate patients with ischemic from healthy control , followed by salivary MDA values which should be equal to or more than 0.38 µmol/L to be helpful in differentiation between two groups with (92% accuracy). Assessment of salivary lipid Peroxidation together with salivary lipid profile may be useful in early detection and monitoring of patients with increased risk of stroke. (J Int Dent Med Res 2010; 3: (2), pp. 57-64 ) Keywords: Dislipid, Lipid Peroxidation, Lipids, Cholesterol, Triglycerides, Saliva. Received date: 14 May 2010 Introduction Stroke (CVA) is a term that describes a clinical events caused either by occlusion or hemorrhage in the arterial blood supply to the CNS resulting in tissue infarction 1. It is potentially the most common cause of severe disability. In term of mortality, stroke is the third most common cause of death in industrialized countries, following coronary heart disease and

*Corresponding author: Dr. Natheer H AL-Rawi Department of Oral & Craniofacial Health Sciences, College of Dentistry, University of Sharjah. E-mail: nhabdulla@yahoo.com

Volume 3 ∙ Number ∙ 2 ∙ 2010

Accept date: 10 August 2010 cancer 2. As a matter of fact, cholesterol can be differently involved in stroke depending on the etiologic subtypes 3. The elevated low density lipoprotein cholesterol (LDL-C) and reduced high density lipoprotein cholesterol (HDL-C) may increase the risk of athero- thrombotic brain infarction 4. The higher levels of products of lipid and protein oxidation observed in plasma isolated from stroke patients compared to healthy subjects demonstrated that oxidative damage is involved 5-9. Oxidative stress, lipid and lipoproteins Peroxidation and inflammation are involved in neuronal damage induced by ischemiareperfusion 10. The brain contains high levels of polyunsaturated fatty acids in membrane lipids; therefore, lipid Peroxidation is one of the major consequences of free radical-mediated injury to brain 11. Malondialdehyde (MDA) level is widely Page 57


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utilized as a marker of lipid Peroxidation in state of elevated oxidative stress 12. MDA is one of many low molecular weight end products of lipid Peroxidation 13. The role of lipid in oxidative damage in saliva of patients with stroke is not yet completely elucidated .Saliva is increasingly used and well validated in diagnosing, monitoring systemic diseases status and predicting diseases progression 14. It is an important physiologic fluid that contains a highly complex mixture of substances. Salivary assays present lots of advantages when compared to blood assay; the sampling is very easy to do especially in nonmedical environment, it does not disturb intimacy when control is needed. Multiple samples could be collected providing more information than that of single blood sample 15. Karajalainen et al 16 assessed cholesterol in saliva of healthy adults; they concluded that salivary concentration levels reflect serum concentration to some extent. The present study was undertaken to evaluate the relationship of MDA as a marker of lipid Peroxidation with lipids and lipoprotein fractions and to find out the cutoff values of the studied parameters in saliva of patients with recent stroke and compare it with healthy individuals. Materials and Methods Seventy five individuals from Al-Diwaniya province in Iraq were enrolled in this observational study. They are grouped into two groups, the study group which consist of fifty patients (24 males and 26 females) having recent attack of ischemic stroke, the other twenty five (12 males and 13 females) were age and sexmatched healthy individuals and served as control group. All individuals were evaluated by full medical history and clinical examination with laboratory investigations to exclude any other systemic and /or local diseases that may affect the parameters examined in this study. Oral and periodontal examination was done for each individual and anyone with symptoms and signs of any active oral inflammation, advanced periodontitis or severe gingivitis were excluded from the study. The study was approved by the Institutional Ethical Committee of Al-Diwaniya Teaching hospital, prior signed consent was taken from all individuals participating in the study. Volume 3 ∙ Number ∙ 2 ∙ 2010

Salivary Lipid peroxidation & Ischemic Stroke Natheer H Al-Rawi

Laboratory Analysis Saliva and blood samples were taken from each participant after overnight fasting (8.00-9.00 am). For serum isolation, 10 ml of blood sample was taken from each individual, centrifuged at 3000 rpm at 4 0C for 5 minutes; the supernatant was aspirated and stored in tubes at -20 0C until analyzed. Saliva samples were always collected in restful and quite circumstances following flushing of the mouth with 100 ml of distilled water. The whole saliva was collected for 5 minutes by the individual leaning forward and spitting saliva in test tubes that were cold centrifuged at 3000 rpm at 4 0C for 5 minutes. The supernatant was aspirated and stored at 20 0C until analyzed. Salivary and serum lipid Peroxidation products, Malondialdehyde (MDA), was measured by the method outlined by Buege and Aust17 where MDA react with Thiobarbituric acid (TBA) to yield a pink color product. The absorbance of 3 ml colored layer was measured at 335 nm spectrophotometrically. Total cholesterol and triglycerides concentration in saliva and serum were measured by enzymatic methods18, 19. The HDLC concentration was measured by the method described by Warnick et al20. LDL-C concentration was then calculated from the concentration of total cholesterol, HDL-C and triglycerides by Friedwald and Levy method21. All data were analyzed with SPSS-17 (Chicago, IL-USA). The significance of difference in the mean between groups was performed by Benferoni test. To compare the diagnostic performance of each test, Receiver Operating Characteristic (ROC) curve test was used. A pvalue < 0.05 was considered statistically significant. Results The mean age of patients with ischemic stroke was 58.2 years. All of them had history of one or more of the following underlying diseases: Hypertension (82%), diabetes mellitus (68%), ischemic heart diseases (30%). Regarding smoking habit, 52% of ischemic stroke patients were heavy smokers (table 1). MDA and lipid sub fractions concentration in saliva and serum in study group did not vary with age and gender, therefore results of both gender were grouped together. All tested Page 58


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parameter in saliva of ischemic stroke group has a significant direct relation to that recorded in serum, except for total cholesterol and LDL-C (table 2). Salivary concentration of MDA, triglycerides, LDL-C was significantly higher in patients with ischemic stroke when compared with that of control group. Salivary HDL-C concentration, on the other hand, was significantly lower in study group than that in control group (table 3). Serum total cholesterol and LDL-C concentration did not show any significant differences between the study and control group (table 4). Pearson correlation was applied for different parameters used in the present study; a highly significant direct correlation was observed between MDA with triglycerides and LDL-C in both saliva and serum of study groups, whereas a highly significant negative correlation was seen between MDA and HDL-C (table 5). The atherogenic index as indicated by various risk ratios is shown in table (6). The risk ratio calculated as total cholesterol/HDL-C, LDLC/HDL-C and Triglycerides/HDL-C. All ratios were significantly elevated in patients with stroke when compared to control. Since increased lipid

Females Males Total

Hypertension 18 (44%) 23 (56%) 41/50 (82%)

Diabetes 17(50%) 17 (50%) 34/50 (68%)

Salivary Lipid peroxidation & Ischemic Stroke Natheer H Al-Rawi

Peroxidation is also risk factor for Ischemic stroke, it has been suggested that MDA values in saliva and serum multiplied by risk ratios may provide a new index which serves as better predictor of Ischemic stroke. All the ratios multiplied by MDA were significantly elevated in stroke patients when compared with controls. ROC curve equation was applied for different cut-off values of the selected parameters to differentiate individuals with ischemic stroke from healthy one. The area under ROC curve for salivary triglycerides, LDLC and MDA were significantly higher from 0.5 value of an equivocal test (table 7). Salivary critical value of triglycerides was ≥ 0.5 mmol/L yields in highest accuracy rate (93%) to differentiate patients with ischemic from healthy control , followed by salivary MDA values which should be equal to or more than 0.38 µmol/L to be helpful in differentiation between two groups with (92% accuracy). Both salivary LDL-C critical value (0.15 mmol/L) and serum HDL-C critical ratio (1.16 µg/L) can achieve 90.7% accuracy in discrimination between stroke group and control group (table 8).

Ischemic heart disease 9 (60%) 6 (40%) 15/50 (30%)

Smoking 6(23%) 20 (77%) 26 (52%)

Table 1. Distribution of underlying diseases and risk factors in ischemic stroke group.

Serum versus Salivary Estimates Total cholesterol concentration (mmol/L) Triglycerides concentration (mmol/L) HDL cholesterol concentration (mmol/L) LDL cholesterol concentration (mmol/L) MDA concentration (µmol/L)

r 0.05 0.3 0.3 0.1 0.28

p >0.05 <0.01 <0.01 >0.05 <0.01

Table 2. Pearson correlation between serum and salivary estimates.

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Salivary Lipid peroxidation & Ischemic Stroke Natheer H Al-Rawi

Control (n=25) Ischemic stroke (n=50) MDA (µmol/l) 0.23± 0.07 0.64± 022 Total cholestrol 0.45± 0.08 0.84± 0.15 Triglycerides 0.34± 0.09 0.8± 0.23 HDL-C 0.19± 0.04 0.16± 0.04 LDL-C 0.13± 0.16 0.31± 0.13 Table 3. Salivary concentration of MDA and lipid fractions.

P value < 0.001 NS < 0.001 < 0.001 < 0.001

Control (n=25) Ischemic stroke (n=50) MDA (µmol/l) 1.32± 0.35 2.51± 1.1 Total cholestrol 3.91± 0.66 4.44± 1.11 Triglycerides 1.68± 1.03 2.96± 0.19 HDL-C 1.59± 0.51 0.78± 0.26 LDL-C 1.59± 0.56 2.32± 1.19 Table 4. Serum concentration of MDA and lipid fractions.

P value < 0.001 NS < 0.001 NS < 0.001

parameters

Saliva r 0.7

P value p< 0.01

Total Cholesterol vs Triglycerides Total Cholesterol vs LDL-C 0.79 p< 0.01 Triglycerides vs MDA 0.28 p< 0.01 Triglycerides vs HDL-C -0.23 p< 0.05 HDL-C vs MDA -0.32 p< 0.01 HDL-C vs LDL-C -0.27 p< 0.01 LDL-C vs MDA 0.28 p< 0.01 Table 5. Pearson correlation between estimates.

SALIVA Total cholesterol/HDL-C LDL-C/HDL-C Triglycerides/HDL-C Total cholesterol/HDL-C * MDA LDL-C/HDL-C * MDA Triglycerides/HDL-C * MDA SERUM Total cholesterol/HDL-C LDL-C/HDL-C Triglycerides/HDL-C Total cholesterol/HDL-C * MDA

Control (n=25) 2.384± 0.552 0.710± 0.838 1.853± 0.766 0.568 ± 0.243

Serum r 0.23

P value p< 0.05

0.95 0.29 -0.22 -0.36 -0.25 0.29

p< 0.01 p< 0.01 p< 0.05 p< 0.01 p< 0.01 p< 0.01

Ischemic stroke (n=50) 5.476± 1.50 2.078±1.062 5.271± 1.933 3.417± 1.36

P value <0.01 <0.01 <0.01 <0.001

1.27±0.759 3.307±1.64

<0.001 <0.001

6.73± 4.17 3.66± 3.074 4.685± 2.96 17.81± 13.03

<0.01 <0.01 <0.01 <0.001

LDL-C/HDL-C * MDA 1.22± 0.704 9.56±9.01 Triglycerides/HDL-C * MDA 1.474±1.307 12.61±9.88 Table 6. Atherogenic index as indicated by various risk ratios

<0.001 <0.001

0.169 ± 0.182 0.437±0.230 2.595± 1.00 1.117± 0.601 1.336± 1.197 2.88± 1.23

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Parameters

ROC area

Salivary Triglycerides Salivary MDA Salivary LDL-C Serum HDL-C Serum MDA Serum triglycerides Salivary HDL-C Serum LDL-C Serum total cholesterol Table 7. ROC area for different cut-off values to control. Positive if ≥ cutoff value

Salivary Lipid peroxidation & Ischemic Stroke Natheer H Al-Rawi

Sensitivity %

Specificity %

P value

0.979 < 0.001 0.969 < 0.001 0.897 < 0.001 0.894 < 0.001 0.885 < 0.001 0.855 < 0.001 0.760 < 0.001 0.685 < 0.001 0.626 0.08 (NS) diagnose cases with ischemic stroke from healthy

Accuracy %

PPV at pretest probability =50%

PPV at pretest probability =90%

NPV at pretest probability =10%

Salivary total cholesterol concentration (mmol/L) 0.47 100.0 64.0 88.0 73.5 96.2 100.0 0.61 94.0 96.0 94.7 95.9 99.5 99.3 0.64 90.0 100.0 93.3 100.0 100.0 98.9 Salivary Triglycerides concentration (mmol/L) 0.40 100.0 72.0 90.7 78.1 97.0 100.0 0.50 92.0 96.0 93.3 95.8 99.5 99.1 0.53 86.0 100.0 90.7 100.0 100.0 98.5 Salivary MDA concentration (µmol/L) 0.23 100.0 52.0 84.0 67.6 94.9 100.0 0.38 88.0 100.0 92.0 100.0 100.0 98.7 Salivary LDL cholesterol concentration (mmol/L) 0.05 100.0 24.0 74.7 56.8 92.2 100.0 0.15 96.0 80.0 90.7 82.8 97.7 99.4 0.57 2.0 96.0 33.3 33.3 81.8 89.8 Serum HDL-C concentration (µg/L) 28.0 100.0 52.0 100.0 100.0 92.6 0.61 1.16 98.0 76.0 90.7 80.3 94.7 99.7 100.0 68.0 89.3 75.8 96.6 100.0 1.27 Serum Triglycerides concentration (mmol/L) 1.40 100.0 52.0 84.0 67.6 94.9 100.0 1.85 96.0 68.0 86.7 75.0 96.4 99.4 4.66 2.0 96.0 33.3 33.3 81.8 89.8 Serum MDA concentration (µmol/L) 0.84 100.0 20.0 73.3 55.6 91.8 100.0 1.85 72.0 100.0 81.3 100.0 100.0 97.0 Table 8. Validity of some parameters to differentiate cases with ischemic stroke from healthy control. Discussion To our knowledge, this is the first study to evaluate the salivary lipid Peroxidation and its relation to lipid fractions among patients with recent attack of ischemic stroke. Oxidative stress, Volume 3 ∙ Number ∙ 2 ∙ 2010

lipid and lipoprotein Peroxidation and inflammation are involved in neuronal damage induced by ischemia reperfusion 22. Lipid Peroxidation was measured by lipid hydroxyl peroxides 23 which are unstable and degrade to various secondary products like MDA and MDAP

P

P

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like substances which were jointly called thiobarbituric acid reactive substance (TBARS) 24. The brain contains high levels of polyunsaturated fatty acids in membrane lipid; therefore, lipid Peroxidation is one of the major consequences of free-radical mediated injury to brain. The assessment of lipid Peroxidation products (MDA) is important in measuring free radical- induced cerebral injury in patients with stroke. Lipid Peroxidation end products MDA has been observed in plasma from stroke patients compared to healthy subjects. MDA level is widely utilized as a marker of lipid peroxidation in states of elevated oxidative stress 12. In the present study, MDA levels were measured in both saliva and serum which were significantly higher in patients with ischemic stroke than that of healthy control (p< 0.001). This finding means elevated oxidative stress as a result of freeradical- induced cerebral injury in patients with stroke as reported by other studies 12, 25, 26. Salivary MDA levels are directly affected by systemic oxidative stress. These findings was also supported by the results of ROC test which revealed that salivary MDA was significantly accurate parameter in predicting patients at risk of stroke with 92% accuracy rate and 100% specificity for the optimum cut-off value ≥0.38µmol/L. The low level of salivary glutathione (GSH) recorded in stroke patients 27indicated that salivary GSH was not consumed, to a considerable extent, in scavenging or detoxification of free radicals or lipid peroxidation products, this lead to significant increase in salivary MDA levels among stroke patients. The association of blood cholesterol with the risk of stroke, a very important clinical and public health issue appears to be in dispute. Some studies found increased risk of ischemic stroke associated with increases total cholesterol levels 28-32, while other studies found no clear association 33-36. Regarding cholesterol fractions, an association between LDL-C and Ischemic stroke is less studied and inconsistent 36-42. In the present study all lipid parameters (except for total cholesterol) measured in serum and saliva of patients with stroke showed significant differences when compared with control group. Total cholesterol concentration alone in saliva and serum is considered as non sensitive parameter since it did not show any significant differences between disease and control group. Salivary and serum triglycerides Volume 3 ∙ Number ∙ 2 ∙ 2010

Salivary Lipid peroxidation & Ischemic Stroke Natheer H Al-Rawi

concentrations were 2-3 times higher in ischemic stroke group than in control group , moreover , both salivary and serum levels of triglycerides were directly correlated with MDA, LDL-C and inversely correlated with HDL-C .This finding may support the evidence that triglycerides concentration had positive risk factor-adjusted association with the risk of cerebral stroke and patients with highest levels of triglycerides were 2-7 times more likely to suffer from atherosclerotic stroke than those with lower levels43. Few prospective population-based studies have examined the association between HDL-C and stroke 44. HDL-C concentration is markedly reduced in patients with stroke in comparison to that in healthy control. HDL-C are particles with numerous athero-protective functions including facilitation of reverse cholesterol transport, improvement of endothelial function, protection of LDL-C against oxidation, limitation of hemostasis and retardation of inflammatory activity related to the vascular wall 45. There is a well established inverse relationship between serum HDL-C concentration and the risk of coronary heart diseases 31, but it is not well documented risk factor for strokes, although few case-control studies have noted an inverse relationship between HDL-C and risk of stroke or TIA 39-41. The highly significant inverse relationship between HDL-C with MDA and LDLC in saliva and serum of stroke group in the present study reflect the deficiency of atheroprotective function of HDL-C in patients with stroke . The association between LDL-C and risk of ischemic stroke has only been evaluated in few studies. A large study of over 11000 patients with coronary heart diseases showed a 14% increase in relative risk of verified ischemic stroke per 1.03 mmol/L42. In contrast, a large cohort study of over 14000 middle aged men and women found no consistent association between LDL-C and ischemic stroke during 10 years follow up 32. The present finding comes in accordance with the abovementioned study, since no significant statistical difference was found between the tested groups. On the other hand, salivary LDL-C levels were significantly higher in stroke group in comparison to healthy group. Therefore, a combination formula like MDA multiplied by atherogenic indecies was used to test their significance in determining patients at increased risk to ischemic stroke which was Page 62


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several times more sensitive than atherogenic index alone. Therefore, this index can be used as a valuable salivary marker for screening individuals who are at risk of cardio and / or cerebrovascular diseases. Total cholesterol hazard value was (3.45 mmol/L) which was greater than that recorded in other study 43 which was ((5.85 mmol/L). HDL-C hazard value in Kurth et al study 44 was (0.20 mmol/L) and was not significant. However, the hazard value (cut-off value) of HDL-C recorded in this study was significant and greater than that recorded by others 44, 45. The highest validity of salivary lipid parameters in predicting ischemic stroke was clearly seen in the present investigation. Analysis of saliva may therefore provide effective, noninvasive approach for screening large population 46. The constituents are derived from the local vasculature of salivary glands. The accuracy of salivary lipid peroxidation and lipid profile may help physicians and other health professional to pay much attention for the use of saliva as screening tool for patients at risk of cardio and /or cerebrovascular diseases.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

Conclusion Lipid peroxidation (indicated by MDA) as well as lipid fractions particularly triglycerides can be assessed in saliva and may be used alone or in combination with other lipid parameters for monitoring patients at increased risk of ischemic stroke . Declaration of Interest The author reports no conflict of interest and the article is not funded or supported by any research grant. References

17. 18.

19.

20.

21.

22. 23.

1. Buckley BM. Lipids and Stroke. Br J Diab & Vas Dis 2003;3:170-6. 2. Gariballa SE, Hutchin TP, Sidair AJ. Antioxident capacity after acute ischemic stroke. QIM 2002;95:685-90. 3. Denti L, Cechetti A, Annoni V, Franceska M, Ablondi F, et al. The role of lipid profile in determining the risk of ischemic stroke in the elderly: a case- control study. Arch GeroontolGeriatr 2003;37:51-62. 4. Tirschwell DL, Smith NL, Heckbert SR, Lamaitre RN, Longstreth WT, etal. Association of cholesterol with stroke risk varies in stroke subtypes and patients subgroups. Neurology 2004;23:1868-75. 5. Muralikrishna A, Dibhatie R, Hetcher JF. Phospholipase A2, reactive oxygen species and lipid Peroxidation in cerebral

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ischemia. Free Radic Biol Med 2006;40:376-87. Polidori MC, Frei B, Chimbini A, Nelles G, Rordorf G, Kearey JF,et al. Increased plasma levels of lipid hydroxyperoxidase in patients with ischemic stroke. Free Radic Biol Med 1998;25:561-7. Uno M, Kitazato KT, Nishi K, Itabe H, Nagahiro S. Raised plasmaoxidized LDL in acute cerebral infarction. J Neurol Neurosurg Psychiatry 2003;74:312-7. Cherubini A, Ruggiero C, Polidori MC, Mecocci P. Potential markers of oxidative stress on stroke. Free Radic Biol Med 2005;39:841-52. Leinonen JS, Ahonen JP, Loennrot K, Jenkonen M, Destider P, Moinar G, et al. Plasma antioxidant capacity is associated with high lesion volume and neurological impairment in stroke. Stroke 2000;31:33-9. Watson BD. Evaluation of the concomitance lipid Peroxidation in experimental model of cerebral ischemia and stroke. Prog Brain Res 1993;96:69-95. Ferriti G, Bacchetti T, Masciangelo S, Nanetti L, Mazzanti L, Sivestrini M, et al. Lipid Peroxidation in stroke patients Clin Chem Lab Med 2008;46(1):113-17. Yildirim A, Kotan D, Yildirim S, Aygol R, Akcay F. Increased lipid Peroxidation and decreased antioxidant response in serum and cerebrospinal fluid in acute ischemic stroke. Turk J Med Sci 2007;37(2):75-81. Bhattachaejee J, Srivastava DK. Serum Malondialdehyde (MDA) in relation to lipidemic status and atherogenic index. Ind J Clin BioChem 1993;8(1):12-15. Lawrence HP. Salivary markers of systemic disease: Non invasive diagnosis of disease and monitoring of general health. J Can Dent Assoc 2002;68(3):170-4. Lu Y, Bently G, Gann P, Hodgesk K. Salivary estradiol and progesterone levels in conception and non-conception cycle in women: Evaluation of a new assay for salivary estradiol. Fertility & Sterility 1999;71(5):863-68. Karajalainen S, Sewan L, Soderling E, Larsson B, Johansson I, et al. Salivary cholesterol of healthy adults in relation to serum cholesterol concentration and oral health. J Dent Res 1997;76:1637-43. Buege JA, Aust SD. Microsomal lipid Peroxidation. Methods Enzymol 1978;52:302-310. Richmond W. Preparation and properties of a cholesterol oxidase from Nocardia species and its application to the enzymatic assay of total cholesterol in serum. Clin Chem 1973;19:1350-6. Fossati P, Prencipe L. Serum triglycerides determined calorimetrically with an enzyme that produces hydrogen peroxide. Clin Chem 1982;28:2077-80. Warnick GR, Mayfield C, Benderson J, Chen JS, Albers JJ. HDL-cholesterol quantification by phosphotungstate/MgCl 2 and dextran sulfate Mn+2 polyethylene glycol precipitation , both with enzymatic cholesterol assay compared with the lipid research clinic method. Am J Clin Pathol 1982;78:718-23. Friedwald WT, Levy RI, Fredrickson D. Estimation of the concentration of LDL-C in plasma without the use of preoperative ultracentrifuge. Clin Chem 1972;18(6):494-515. Wang O, Tang XN, Yenari MA. The inflammatory response in stroke. J Neuroimmunol 2007;184:53-68. Hermes-Lima M, Willmore WG, Story KB. Quantification of Lipid Peroxidation in Tissue Extracts Based on Fe (III) Xylenol Orange Complex Formation. Free Radic Biol Med 1995;19: 271-280. Draper HH, Squires EJ, Mahmoodi H, Wu J, Agarwal S, Hadley M. A Comparative Evaluation of Thiobarbituric Acid Methods for the Determination of Malondialdehyde in Biological Materials. Free Radic Biol and Med 1993;15: 353-363. Sakamoto A, Ohnishi ST, Ohnishi T & Ogawa R. Relationship between free radical production and lipid peroxidation during ischemia-reperfusion injury in rat brain. Brain Res 1991;554: 186−192. Sharpe, P.C., Mulholland, C. & Trinick, T. Ascorbate and malondialdehyde in stroke patients. Ir J Med Sci 1994; 163: 488-491.

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27. Atiyah KM, Al-Rawi NH. Evaluation of Salivary and Serum Oxidative Stress and Neuronal Damage Markers as Potential Parameters in Prediction of Ischemic Stroke from StrokeRelated Risk Factors. Ph.D Thesis. University of Baghdad. 28. Sacco RL, Bensen RT, Karagman DE. High-density lipoprotein cholesterol and ischemic stroke in the elderly. The Northern Manhattan Stroke study. JAMA 2001; 285:2729-2735. 29. Benfante R, Yano K, Hwang LJ, Curb JD, Kagan A, Ross W. Elevated serum cholesterol is a risk factor for both coronary heart diseases and thrombo-embolic stroke in Hawaiian Japanese men. Implications of shared risk. Stroke 1994;25:81420. 30. Di Mascio R, Marchioli R, Vitullo F, Di Pasquale A, Cavasinni L, Tognoni G. Serum cholesterol and risk of ischemic stroke: results of a case control study. On behalf of PROGETTO3A investigators. Prev Med 1995; 24:124-133. 31. Ebrahim S, Sung J, Song YM, Ferrer RL, Lawlor DA, Davey Smith G. Serum cholesterol, hemorrhagic stroke, ischemic stroke and myocardial infarction:Korean National Health System Prospective Cohort Study. BMJ 2006:333:22. 32. Zhang X, Patel A, Horibe H et al. Cholestrol, Coronary heart disease and stroke in the Asia Pacific region. Int J Epidemiol 2003; 32:563-72. 33. Kagan A, Papper JS, Rhoods GG. Factors related to stroke incidence in Hawaii Japanese men. The Honolulu Heart study. Stroke 1980; 11:14-21. 34. Prospective studies collaboration. Cholesterol, diastolic blood pressure and stroke: 13,000 strokes in 450,000 people cohorts. Lancet 1995; 346:1647-53. 35. Bowman TS, Sesso HD, Ma J, et al. cholesterol and risk of ischemic stroke. Stroke 2003; 34:2930-34. 36. Shahar E, Chambles LE, rasmond WD, Boland LL, Ballantyne CM, et al. Plasma lipid profile and incident ischemic stroke: The Atherosclerosis Risk in Communities (ARIC) study. Stroke 2003; 34:623-30. 37. Gorelick PB, Mazzone T. Plasma lipids and stroke. J Cardiovas Risk 1999; 6:217-21. (s) 38. Gordon DJ and Rifkind BM. High-density lipoproteins: The clinical implication of recent studies. N Eng J Med 1989; 321:1311-16. 39. Oizilbash N, Jones L, Warlow C, Mann J. Fibrinogen and lipid concentrations as risk factors for transient ischemic attacks and minor ischemic stroke. BMJ 1991; 303:605-609. 40. Sridharan R. risk factors for ischemic stroke: A case control analysis. Neuroepidemiol 1992; 11:24-30. 41. Bihari-Varga M, Szekely J, Gruber E. Plasma high density lipoproteins in coronary, cerebral and peripheral vascular disease: The influence in coronary risk factors. Athersclerosis 1981; 40:337-45. 42. Koren- Morag N, Tanne D, Gralf E, Goldbourt U. low and high density lipoprotein cholesterol and ischemic cerebro vascular diseases: the Bezafibrate infarction prevention registry. Arch Intern Med 2002; 162:993-99. 43. Salonen JT and Puska P.Relation of serum cholesterol and triglycerides to the risk of acute MI, cerebral stroke and death in eastern Finnish male population. Int J Epidemiol 1982; 12(1):26-31. 44. Kurth T, Evert BM, Buring JE, Kase CS, Ridker PM, Gaziano JM. Lipid levels and the risk of ischemic in women. Neurology 2007; 68:556-62. 45. Gaziano JM, Buring JE, Breslow JL, et al. moderate alcohol intake, increased levels of high-density lipoproteins and its subfractions, and decreased risk of myocardial infarction. N Eng J Med 1993; 329:1829-34. 46. Ritschel WA, Thompson GA. Monitoring of drug concentrations in saliva: a non - invasive pharmacokinetic procedure. Methods and findings in Experimental and Clinical Pharmacology 1983; 5:511-25.

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Molariform Supernumery Teeth: A Case report S. Navit, and et al

UNUSUAL INVERTED AND MOLARIFORM SUPERNUMERY TEETH – A CASE REPORT S. Navit1, Firoza Samadi2, Prashant Babaji3*, Rohit Anand4, Anju Bansal5 1. MDS, Assoc. Prof., Department of Pedodontics & Preventive Dentistry, Sardar Patel Post Graduate Institute of Dental and Medical Sciences, Lucknow. 2. MDS, Prof. & Head, Department of Pedodontics & Preventive Dentistry, Sardar Patel Post Graduate Institute of Dental and Medical Sciences, Lucknow. 3. MDS, Reader, Department of Pedodontics & Preventive Dentistry, Sardar Patel Post Graduate Institute of Dental and Medical Sciences, Lucknow. 4. MDS, Sr Lecturer, Department of Pedodontics & Preventive Dentistry, Sardar Patel Post Graduate Institute of Dental and Medical Sciences, Lucknow. 5. Post Graduate Student, Department of Pedodontics & Preventive Dentistry,Sardar Patel Post Graduate Institute of Dental and Medical Sciences, Lucknow.

Abstract Supernumerary teeth are a relatively frequent disorder of odontogenesis, characterised by an excess number of teeth. The various forms of supernumerary teeth are, supplemental, conical, tuberculate and molariform. Molariform supernumerary teeth in the maxillary central incisor area are uncommon. This article reports an unusual presence of molariform mesiodens along with an inverted conical mesiodens in an eight year old boy. ( J Int Dent Med Res 2010; 0: (0), pp. 65-68 ) Keywords: Mesiodens, Molariform tooth, Supernumerary Teeth. Received date: 08 February 2010 Introduction Supernumerary teeth may be defined as any teeth or tooth substance in excess of the usual configuration of twenty deciduous and thirty two permanent teeth1. Supernumerary teeth may occur singly, multiply, unilaterally or bilaterally, and in one or both jaws2. Supernumerary teeth can be classified based on the time of appearance; according to the position in arch; and according to their shape3. The various forms of supernumerary teeth are, supplemental, conical, tuberculate and molariform4. Molariform type of supernumerary teeth as reported in the present case is rare and uncommon. They either appear alone or in pairs in central incisor region

*Corresponding author: Dr. PRASHANT BABAJI Department of Pedodontics & Preventive Dentistry, Sardar Patel Post Graduate Institute of Dental and Medical Sciences, Lucknow – 226025 State: Utter pradesh, India E-mail: babajipedo@rediffmail.com

Volume 3 ∙ Number ∙ 2 ∙ 2010

Accept date: 14 May 2010 with complete root formation and may cause delayed eruption of adjacent teeth4. The aetiology of supernumerary teeth is not well understood. Though many theories have been proposed to explain the anomaly based on developmental interference and heredity, environmental factors, phylogenetic process of atavism and syndromes may also play a part2, 5. CASE REPORT An eight year old boy was reported to the department of Pedodontics & Preventive Dentistry of S.P.P.G.I.D.M.S Lucknow, with the complain of a extra tooth erupting behind upper front teeth. His medical history was noncontributory. There was no history of similar anomalies (supernumerary teeth) among family members. Intra-oral examination revealed mixed dentition stage with Angle's class I molar relation. A palatally erupted abnormal shaped mesiodens was seen causing labial displacement of the upper right central incisor(Figure 1). Intra oral periapical radiograph confirmed the molariform mesiodens (Figure 2) and also revealed another well developed, conical, inverted supernumery Page 65


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tooth placed near the apex of upper left central incisor. Cone shift technique confirmed the position of supernumerary teeth.

Molariform Supernumery Teeth: A Case report S. Navit, and et al

deciduous right canine, was performed to raise a full thickness palatal envelope flap (Figure 3).Bone overlying the upper left central incisor was removed with a round bur to facilitate it’s removal and the flap sutured over the socket with four black silk sutures. Extracted mesiodens resembles molariform with complete root formation (Figure 4). Acrylic splint was fitted over the patient to prevent haematoma and to support the flap. On discharge the patient was prescribed Amoxicillin 250mg caps for five days. At one week recall for removal of sutures, healing was uneventful.

Figure 1. Photograph of upper jaw, showing a palatally erupted molariform mesiodens and labially placed right central incisor.

Figure 3. Palatally positioned supernumerary teeth seen upon rising palatal flap.

Figure 2. Intra oral periapical radiograph showing a well developed conical inverted supernumeryand molariform mesiodens. Patient was advised for routine blood investigation including, clotting time and bleeding time. Later it was decided to remove the supernumery teeth by raising palatal flap. A gingival sulcular incision, extending from the upper left deciduous canine to the upper Volume 3 ∙ Number ∙ 2 ∙ 2010

Figure 4. Occlusal supernumery tooth.

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Discussion A supernumerary tooth is one that is additional to the normal series and can be found in almost any region of the dental arch 6. The prevalence of hyperdontia in various populations is reportedly between 0.1-3.8% with male to female ratio of 2:1 7. They occur less commonly in the deciduous dentition (prevalence of 0.021.9%) when compared to permanent dentition (prevalence 0.10-3.6%) 8. The relative frequency of occurrence of different supernumerary in decreasing frequency is, upper lateral incisors, mesiodens, upper central incisor, followed by bicuspids9. A large percentage of anterior supernumerary teeth remain unerrupted7. Classification of supernumerary teeth may be on the basis of position or form. Positional variations include mesiodens, paramolars, distomolars and parapremolars. Variations in form consist of conical type, tuberculate type, supplemental teeth and odontome2. Multiple supernumerary teeth are more common when syndrome is involved. Common syndromes showing multiple supernumerary teeth along with other conditions include Gardiner’s syndrome, cleidocranial dysostosis; and cleft lip and palate2. Supernumeraries are more common in the relatives of affected children than in the general population. However, the anomaly does not follow a simple Mendalian pattern 6. A carefull check for a familial history of supernumerary teeth could point to the presence of genetically determinsed syndrome2. However supernumerary teeth can occur without any syndrome or familial history as in present case. Among the supernumerary teeth, mesiodens is the most common type. The term mesiodens refers to a supernumerary tooth present in the premaxilla between the two central incisors. Mesiodens are more common in the permanent than in primary dentition8. They can morphologically present as a cone shaped tooth, tuberculate or molariform. Very few cases has been reported with molariform mesiodens, there was three grooves in incisal/Occlusal aspect with three cusp like structures on labial half and a single cusp like structure on the palatal half 3, 7, 8. Developmentally anterior teeth develop from four lobes, three lobes on labial and one on the lingual represented by the cingulum. Hence it has been proposed that, a lack of fusion of the lobes Volume 3 ∙ Number ∙ 2 ∙ 2010

Molariform Supernumery Teeth: A Case report S. Navit, and et al

during development could be a reason for unusual morphology of mesiodens8. A radiographic examination is indicated if abnormal clinical signs are found. An anterior occlusal or periapical radiograph is useful to show the incisor region in detail. The buccolingual position of unerupted supernumeraries can be determined using the parallax radiographic principle: the horizontal tube shift method utilizes two periapical radiographs taken with different horizontal tube positions, whereas an occlusal film together with a panorex view are routinely used for vertical parallax. If the supernumerary moves in the same direction as the tube shift it lies in a palatal position, but if it moves in the opposite direction then it lies buccally. A true lateral radiograph of the incisor region assists in locating the supernumeraries that are lying deeply in the palate and enables the practitioner to decide whether a buccal rather than a palatal approach should be used to remove them6. Supernumerary teeth can create various problems to adjacent teeth like, failure of eruption, displacement, crowding, dentigerous cyst formation or resorption of roots adjacent to supernumerary tooth6. Management of supernumeraries depends on the type and position of the supernumerary tooth and on its effect or potential effect on adjacent teeth. Some authors believe that, supernumerary teeth have to be removed as soon as it has been diagnosed, to prevent any further problem on adjacent teeth2. Conclusions Early detection and management of all supernumerary teeth is a necessary part of preventive dentistry. The importance of early radiographic investigation of suspected cases cannot be underestimated. Declaration of Interest The authors report no conflict of interest and the article is not funded or supported by any research grant. References 1. Schulze C. Developmental abnormalities of the teeth and jaws. In: Gorlin R J, Goldman H M, eds Thoma’s oral pathology. St Louis: CV Mosby, 1970: 112-22.

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Molariform Supernumery Teeth: A Case report S. Navit, and et al

2. Scheiner M A, Sampson W J. Supernumerary teeth: A review of the literature and four case reports. Aust Dent J. 1997; 42(3):160-5. 3. Shashikiran ND, Reddy VV, Mandroli P. Molariform supernumerary tooth: A case report. J Indian Soc Pedo Prev Dent. 2000; 18: 18-20. 4. Primosch R K. Anterior supernumerary teeth assessement and surgical intervention in children. Pediatr Dent. 1981;3:204-215 5. Hattab FN, Yassin OM, Rewashdeh MA. Supernumerary teeth: Report of three cases and review of the literature. J Dent Child. 1994; 61:382-393. 6. Garvey MT, Barry HJ, Blake M. Supernumerary teeth –A overview of classification, diagnosis and management. J Canadian Dent Assoc. 1999; 65(11): 612-16. 7. Sharma A. Familial occurance of mesiodens- A case report. J Ind Soc Ped Prev Dent. 2003; 21(2): 84-85. 8. Srivatsan P, Babu AN. Mesiodens with an unusual morphology and multiple impacted supernumerary teeth in a non-syndromic patient. Indian J Dent Res. 2007; 18(3): 130-140. 9. Lutin JR, Jnr. The prevalence of supernumerary teeth in primary and mixed dentitions. J Dent Child. 1967; 34: 48-9.

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Restoration of Posterior Edentulous Spaces Guvenc Basaran,and et al

RESTORATION OF POSTERIOR EDENTULOUS SPACES AFTER MAXILLARY MOLAR INTRUSION WITH FIXED APPLIANCES (CASE REPORT)* Guvenc Basaran1**, Emrah Ayna2, Emine Goncu Basaran3, Gulten Unlu4 1. Assistant Professor DDS PhD Dicle University Faculty of dentistry Department Of orthodontics Diyarbakir / TURKEY. 2. Associate Professor DDS PhD Dicle University Faculty of dentistry Department Of prosthodontics Diyarbakir / TURKEY. 3. Research Assitant DDS PhD Dicle University Faculty of dentistry Department Of prosthodontics Diyarbakir / TURKEY. 4. Professor DDS PhD Dicle University Faculty of dentistry Department Of Oral Surgery Diyarbakir / TURKEY.

Abstract This case report includes the mini screw supported intrusion of the extruded teeth due to the absence of its antagonist and fixed prosthetic rehabilitation supported with osseointegrated implants. Four mini-screws with 2 mm diameter and 10 mm length were placed in buccal and palatal regions of extruded molars in both left and right sides. The 4 mm intrusion was achieved with Ni-Ti closed spring and elastomeric chain in each side. After the intrusions of the extruded antagonist molars, dental implants were placed in edentulous areas. After 3 months of healing period, fixed prosthetic restorations were made. As the benefit of orthodontic intrusion of extruded molars, no endodontic treatment was needed in order to gain enough vertical space for prosthetic restoration of antagonist edentulous area and the masticatory function was successfully given to the patient. (J Int Dent Med Res 2010; 3: (2), pp. 69-74 ) Keywords: Mini Screw, Intrusion, Molar, Fixed Prosthesis. Received date: 13 October 2009 Introduction The overeruption of maxillary molars usually results from early loss of antagonistic teeth. The elongated dentoalveolar process may cause problems of occlusal interferences and functional disturbances and may result in great difficulty during prosthetic reconstruction. To provide prosthodontic treatment of the missing teeth, these overerupted teeth need to be intruded, but molar intrusion is difficult in adults.1,2 Prosthodontic treatment replaces missing

*This case report was presented at ICOI Europe Symposium (International Congress of Oral Implantologist) at April 15-17 2010 in İstanbul / TURKEY. **Corresponding author: Assist. Prof. Dr. Guvenc BASARAN Dicle University Faculty of dentistry Department Of orthodontics Diyarbakir / TURKEY. E-mail: basaran@dicle.edu.tr

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Accept date: 28 April 2010 teeth and restores occlusal surfaces for improved masticatory function, esthetics, and phonetics. Partial edentulous jaw includes various forms and may be accompanied with displaced or deformed remaining teeth and surrounding tissues. When these deformities are severe, orthognathic correction can often facilitate prosthodontic treatment. Other surgical interventions include placement of osseointegrated dental implants and autotransplantation of teeth.3 Generally, several conventional options are available to increase occlusal clearance. Coronal reduction often requires crown restorations at the expense of tooth vitality. Another alternative raised by Schoeman and Subramanian4 is a posterior segmental osteotomy of the maxilla to impact the elongated segment, but patients must undergo the risk of general anesthesia and high cost associated with this procedure. Recent reports have demonstrated the clinical efficiency of mini-implants in providing sufficient anchorage against orthodontic forces.5,6 Page 69


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The advantages of using mini-implant as orthodontic anchorage include ease of application, minimal patient compliance needed, and the ability to immediately load after initial wound healing.7 The surgical procedure for inserting or removing the miniscrew is simple, with minimal unfavorable complications. In contrast, miniplates require flap surgery often done by oral surgeons. The mini screw implants are used for various proposes in dentistry, including space clousure or space open, open bite treatment and uprighting of posterior teeth. We aimed to place implant supported prosthodontic restorations to mandibular posterior segments by intruding upper molars extruded due to early loss of lower mandibular molars with mini screws. CASE REPORT A 36 year old women was referred from prosthodontic department because her right firstsecond molars and left second molar had overerupted. (Figures 1,2) Mandibular right-left first and second molars, maxillary right first molar and canine had been lost ten years ago. Maxillary right, left central and left lateral restorated had been fixed crown bridge restoration. The patient wanted to have the mandibular right-left posterior area restored with prosthodontic implants. However, because of the extruded maxillary left second molar and right first-second molars, less than 1 mm of vertical space was available, making proper restoration difficult. After consulting with the patient and the prosthodontic department, we planned intrusion of the maxillary left first molar and right firstsecond molars.

Figure 1. Pre treatment intraoral photograph Volume 3 â&#x2C6;&#x2122; Number â&#x2C6;&#x2122; 2 â&#x2C6;&#x2122; 2010

Restoration of Posterior Edentulous Spaces Guvenc Basaran,and et al

(Right side).

Figure 2. Pre treatment intraoral photograph (Left side). Clinical Procedure The procedure for implanting a mini-screw is as follows. First, anesthetize applied the implant side. After checking the shape and location of the roots on panoramic and periapical x-rays, the implant site marked on the gingiva by making an indentation with a periodontal probe. After checking the position of the mucogingival junction from the buccal side, implant the miniscrew (in the attached gingiva, whenever possible). When the screw is placed on the palatal side of the maxilla, determine the length needed by measuring the soft tissue thickness in the area. To ensure retention and avoid fracture, use a screw with a diameter of 2 mm (Dewimed, Medizintechnic Gmbh, Tuttingen, Germany). Use a contra-angle screwdriver and the self-tapping method to implant the screw; a steady implantation technique is important. To facilitate soft tissue healing, begin loading 5 days after the implantation. Light force (10-20 g per tooth) is recommended for the intrusion of the anterior teeth, but a heavier force (150-200 g per tooth) is needed to intrude posterior teeth. To verify the position between the mini-screw and the proximal roots, take periapical x-rays, changing the position of the cone mesiodistally. Use periodic periapical or panoramic radiographs to check for root resorption. After mini-screw operation, upper left second molar was intruded with power chains (RMO Morita Corp., Chiyoda, Tokyo, Japan).(Figure 3) The magnitude of force was measured with dynamometer. The power chains were changed per week. Maxillary right side, Page 70


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1mm thick stainless steel wire was bonded on the occlusal surfaces of upper left first and second molars. Force was applied via 7 mm closed Ni-Ti coil spring (G&H Wire Company, Greenwood, Ind) extending from the buccally placed mini-screw to the palatinally placed miniscrew.(Figures 4,5) In both sides, 3 mm intrusion was achieved before the placement of prosthetic implants. After occlusally enough space was achieved, assisted prosthetic implants were placed surgically in both mandibular posterior segments (BioHorizons Implant Systems Inc, Birmingham, AL). During the 3 months healing period, intrusion was continued and totally 4 mm intrusion was achieved at the end of treatment. Becasuse of vertical space was less than 4.5 mm, screwable prothesis was selected for mandibular right implants. Fabrication abutments for mandibular right implants, castable abutments for mandibular left implants were selected. (Figure 6) The healing abutments were removed and custom and plastic abutments were adjusted and screwed. An closed-tray impression of the abutment copings was made with vinyl polysiloxane impression material (Elite H-D, Zhermack, Italy). Individual abutments that obtained from plastic abutments were adapted.

Restoration of Posterior Edentulous Spaces Guvenc Basaran,and et al

placed and secured using 35-N cm torque. The metal-ceramic restorations of mandibular left implants were cemented, the metal-ceramic restorations of mandibular right implants screwed onto the implant. Metal-ceramic restorations were placed on to abutments to verify marginal integrity, occlusal relationships, and esthetic results. For the first year after treatment, the patient was followed for routine hygiene and assessment of long-term outcome. The patient acknowledged having improved function and esthetics, and was pleased with the results. (Figures 7,8)

Figure 4: For intrusion, Ni-Ti coil spring applied right side.

Figure 3. For intrusion, elastic chain applied left side. After metal-ceramic restorations were completed, at insertion, the healing abutments were removed and custom abutments were Volume 3 â&#x2C6;&#x2122; Number â&#x2C6;&#x2122; 2 â&#x2C6;&#x2122; 2010

Figure 5. Occlusal applications.

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Figure 6. Castable plastic abutments.

Figure 7. Post treatment intraoral photograph (Right Side).

Figure 8. Post treatment intraoral photograp (Right Side). Discussion When prosthodontic treatment of a missing molar has been delayed, the traditional treatment has been to reduce the crown length of Volume 3 â&#x2C6;&#x2122; Number â&#x2C6;&#x2122; 2 â&#x2C6;&#x2122; 2010

Restoration of Posterior Edentulous Spaces Guvenc Basaran,and et al

the tooth opposite the extruded tooth8 or to adjust the path of intrusion. Intrusion by subapical osteotomy9 or extraction of the extruded molar are more aggressive alternatives, but most patients today refuse to sacrifice a healthy tooth. Anchorage control plays an important role in orthodontic mechanics. During conventional orthodontic treatment for intruding overerupted molars, it is difficult to avoid the side effect of extrusion of the anchorage teeth. Some appliances such as high-pull headgears could be used for molar intrusion, but the patient's compliance is essential. Various implant systems have been used for orthodontic intrusion. Southard et al10 reported that molar intrusion is possible by using dental implants. Sherwood et al6 reported four cases with miniplate anchorage to close skeletal open bite. They reported that superimposition of panoramic tracings showed that a mean molar intrusion of 1.99 mm. Kanomi11 reported an adult patient with a deep bite, which was corrected with 6 mm of lower incisor intrusion by an intrusive force from a mini-implant. Umemori et al5 presented a skeletal anchorage system to correct an anterior open bite. They implanted the titanium miniplates at buccal aspects of the mandibular molars and intruded the molars about 3 to 5 mm. Daimaruya et al12 intruded the mandibular molars 3.4 mm by the intrusive force from buccal miniplate and lingual bone screw in dogs. Erverdi et al13 reported that the zygomatic area was on useful anchorage site for maxillary molar intrusion. A cephalometric study demonstrated the effectiveness of skeletal anchorage for intrusion of maxillary posterior teeth to correct anterior open-bite malocclusion.14 Our experience substantiates that successful intrusion of molars can be consistently achieved with mini-implants as anchorage. Todays mini screws are widely using for molar teeth intrusion. In contrast to traditional orthodontics, the molar intrusion facilitated with the mini-implants causes minimum extrusion of the adjacent teeth. Incorporation of mini-implants can achieve a significant amount of maxillary molar intrusion and is an excellent alternative to traditional method.6,15,16 Regarding the optimum force for intrusion, Burstone17 suggested 20 g of force for intruding anterior tooth, and Gianelly and Goldman18 recommended 15 to 50 g of force for small teeth. For molar intrusion, Umemori et al19 Page 72


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recommended an initial force of 500 g. Kalra et al20 suggested about 90 g per tooth for molar intrusion in growing children, and Melson and Fiorelli21 used about 50 g buccolingually to intrude maxillary molars in adult patients. Considering the number and the surface area of posterior tooth roots, it is reasonable to apply intrusion forces 2 or 3 times greater than those applied on anterior teeth. In our study, we used 200 to 300 g of intrusion force on maxillary posterior teeth with 3 roots and obtained 0.5 to 1 mm of continuous intrusion per month without notable root resorption or vitality problems. However, further research is needed to provide a biological basis for these figures. Fixed, removable partial, cantilever and implant supported prosthodontic restorations are frequently used for the prosthetic replacement of missing teeth.22 Bone quality, surgical procedure, the localization of implant, abutment and cementation are some of the factors affecting the success of implant supported prosthodontic restoration.23 Precise fit between an implant body and an abutment and between an implant abutment and a superstructure are important factors in determining the long-term success of implant-supported restorations. Thus, when these fits are poor, tensile, compressive, and bending forces may be introduced into an implant-supported restoration and may result with loosening of the prosthesis or abutment screws, distortion or breakage of the restoration, microfractures in the bone surrounding the implant, or fracture of the implant body. As a result, they may induce loss of osseointegration.24,25 Cement retention is well-documented in the dental literature that several factors influence the amount of retention in cement-retained restorations, whether they exist on natural teeth or implant abutments.26 These factors are (1) taper or parallelism, (2) surface area and height, (3) surface finish or roughness, and (4) type of cement. Taper greatly influences the amount of retention that can be generated in a cementretained prosthesis. Jorgensen26 established that a 6-degree taper is ideal in crown preparations. He also determined the relative amount of retention for other tapers on prepared teeth and established an inverse relationship between taper and retention. His data show that a 15degree taper provides approximately one third of the retention of the ideal 6-degree taper, and a Volume 3 ∙ Number ∙ 2 ∙ 2010

Restoration of Posterior Edentulous Spaces Guvenc Basaran,and et al

25-degree taper provides approximately 25% or one quarter of the retention generated by the ideal taper. Screw retention of implant-supported prostheses was validated by studies of the Branemark system.24,27 Screws may be used to attach abutments to implants and prostheses to abutments. It is important that all screws should be torqued to the manufacturer's specifications. Screws designed for different purposes have different mechanical properties because of their size, design, and metallurgic composition. Screws should be tightened to 50% to 75% of their yield strength to provide optimum clamping force.28 The torque that is applied to the screw is converted into tensile force in the screw (preload), and while under tension the screw holds the two components together (the prosthesis to the abutment or the abutment to the implant).28 Fulcrums or pivot points are created at the edge where the abutment or casting meets the head of the implant In a situation where there is an accurate fit between the head of the implant and the abutment, a continuum of pivot points is created around the circumference. In this stable situation, vertical occlusal forces that occur over the prosthetic head of the implant will produce vertical loading and will not stress the screw or cause screw loosening. This does not apply when inaccurate castings are screwed into implants and gaps are created. Conclusions By simply implanting mini-screws and controlling the direction and amount of force, successful molar intrusion can be obtained, satisfying both the patient and the dentist. Declaration of Interest The authors report no conflict of interest and the article is not funded or supported by any research grant. References 1. Melsen B. Limitation in adult orthodontics. In Melsen B, ed. Current Controversies in Orthodontics. Chicago, III Quintensentence; 1991:147-80. 2. Suya H. Corticotomy in orthodontics. In Hösl E, Baldauf A eds. Mecanical and Biological in Orthodontic Theraphy. Heidelberg, Germany: Hüthig; 1991:207-26. 3. Battistuzzi P. Treatment modalities for prosthetic rehabihtation in

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patients with full and partial edentulism. In: Naert I, van Steenberghe D, Worthington P. Osseointegration in oral rehabilitation. London: Quintessence Publishing Co; 1993:25-32. 4. Schoeman R, Subramanian L. The use of orthognatic surgery to faciliate implant replacement: a case report. Int J Oral Maxillofac Implants 1996;11:682-4. 5. Umemori M, Sugawara J, Mitani H, Nagasaka H, Kawamura H. Skeletal anchorage system for open-bite correction. Am J Orthod Dentofacial Orthop 1999;115:166–74. 6. Sherwood KH, Nurchg JG, Thompson WJ. Closing anterior open bites by intruding molars with titanium miniplate anchorage. Am J Orthod Dentofacial Orthop 2002;122:593–600. 7. Park HS, Bae SM, Kyung HM, Sung JH. Micro-implant anchorage for treatment of skeletal Class I bialveolar protrusion. J Clin Orthod 2001;35:417–22. 8. Norton LA, Lopes I. Specific mechanics for abutment uprighting. Aust Dent J 1980;25: 273–8. 9. Mostafa YA, Tawfik KM, El-Mangoury NH. Surgical-orthodontic treatment for overerupted maxillary molars. J Clin Orthod 1985;19:350–1. 10. Southard TE, Buckley MJ, Spivey JD, Krizan KE, Casko JS. Intrusion anchorage potential of teeth versus rigid endosseous implants: a clinical and radiographic evaluation. Am J Orthod Dentofacial Orthop 1995;107:115–20. 11. Kanomi R. Mini-implant for orthodontic anchorage. J Clin Orthod 1997;31:763–7. 12. Daimaruya T, Nagasaka H, Sugawara J, Mitani H. The influences of molar intrusion on the inferior alveolar neurovascular bundle and root using the skeletal anchorage system in dog. Angle Orthod 2001;71:60–70. 13. Erverdi N, Tosun T, Keles A. A new anchorage site for the treatment of anterior openbite: zygomatic anchorage case report. World J Orthod 2002;3:147–53. 14. Erverdi N, Keles A, Nanda R. The use of skeletal anchorage in openbite treatment: a cephalometric evaluation. Angle Orthod 2004;74:381–90. 15. Gray JB, Steen ME, King GJ, Clark AE. Studies on the efficacy of implants as orthodontic anchorage. Am J Orthod 1983;83:311-7. 16. Sugawara J. JCO interviews Dr Junji Sugawara on the skeletal anchorage system. J Clin Orthod 1999;33:689–96. 17. Burstone CR. Deep overbite correction by intrusion. Am J Orthod 1977;72:1–22. 18. Gianelly AA, Goldman HM. Biologic basis of orthodontics, Lea and Febiger, Philadelphia ;1971:136. 19. Umemori M, Sugawara J, Mitani H, Nagasaka H, Kawamura H. Skeletal anchorage system for open-bite correction. Am J Orthod Dentofacial Orthop 1999;115:166–74. 20. Kalra V, Burstone CJ, Nanda R. Effects of a fixed magnetic appliance in the dentofacial complex. Am J Orthod Dentofacial Orthop 1989;95:467–78. 21. Melsen B, Fiorelli G. Upper molar intrusion. J Clin Orthod 1996;30:91–6. 22. Nevalaınen MI, Rantanen T, Narhı T, Ainamo A. Complete dentures in the prosthetic rehabilitation of elderly persons: five different criteria to evaluate the need for replacement J Oral Rehabil 2008;24:251-8. 23. Tsolaki IN, Madianos PN, Vrotsos JA. Outcomes of dental implants in osteoporotic patients. A literature review. J Prosthodont 2009;18:309-23. 24. Adell RM, Lekholm U, Rockler B, Brånemark P-I. A 15- year study of osseointegrated implants in the treatment of the edentulous jaw. Int J Oral Surg 1981;10:387–416. 25. Albrektsson T, Jansson T, Lekholm U. Osseointegrated dental implants. Dent Clin North Am 1986;30:151–74. 26. Jorgensen KD. The relationship between retention and convergence angle in cemented veneer crowns, Acta Odontol Scand 1955;13:35–40. 27. Adell R, Eriksson B, Lekholm U, Brånemark PI, Jemt T. Longterm follow-up study of osseointegrated implants in the treatment of totally edentulous jaws, Int J Oral Maxillofac Implants 1990;5:347–9. 28. Jorneus L, Jemt T, Carlsson L. Loads and designs of screw joints for single crowns supported by osseointegrated implants. Int J Oral Maxillofac Implants 1992;7:353–9.

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Canine-Lateral Incisor Transposition: A case report Demet Suer Tumen and et al.

MAXILLARY CANINE-LATERAL INCISOR TRANSPOSITION: A CASE REPORT Demet Suer Tumen1*, Filiz Acun Kaya2, Nihal Hamamci3, E. Caner Tumen4, Gulay Berber5 1. Research Assist. MsC, DDS, Dicle University, Faculty of Dentistry Department of Orthodontics Diyarbakir/TURKEY. 2. Assoc. Prof. DDS PhD, Dicle University, Faculty of Dentistry Department of Periodontology Diyarbakir/TURKEY. 3. Assist. Prof. DDS PhD, Dicle University, Faculty of Dentistry Department of Orthodontics Diyarbakir/TURKEY. 4. Assist. Prof. DDS PhD, Dicle University, Faculty of Dentistry Department of Pediatric Dentistry Diyarbakir/TURKEY. 5. Research Assist. MsC, DDS, Dicle University, Faculty of Dentistry Department of Orthodontics Diyarbakir/TURKEY.

Abstract This case report aims to present the orthodontic treatment of a patient who referred to our clinic with a chief complaint of crowding and who had left lateral canine tranposition at maxilla. In the clinical examinations of the patient; severe crowding, retained deciduous canine tooth and dental Angle Class I relation were detected. In the radiographic examinations, impacted and transposed canine tooth was observed. Skeletal Class I relation (ANB: 2º) was observed in cephalometric analysis. Fixed appliances were used in order have the transposed and unerupted canine tooth to erupt. Initially, lateral tooth was completely moved to the place of the canine and the space was obtained for canine in the arch. Then we began to have canine tooth erupt by fixing surgically eruption appliance to canine tooth. After having had an ideal occlusion and canine eruption, canine tooth was grinded to make it look like a lateral tooth and aesthetic of the gingiva at canine was provided by applying connective tissue graft. As a result of the applied orthodontic treatment, a functional occlusion, ideal overjet and overbite relation, and aesthetic smile were provided. In the treatment planning and for the success of the transposed teeth, tooth’s position and the multidisciplinary cooperation play a key role. (J Int Dent Med Res 2010; 3: (2), pp. 75-78 ) Keywords: Transposition, impacted tooth, connective tissue graft. Received date: 29 November 2010 Introduction Tooth transposition is an anomaly of eruption characterized by the interchanged positions of two adjacent teeth1. The etiology of transposed teeth has been attributed to genetic factors related to the position of developing dental lamina, or trauma to the deciduous teeth and/or retained deciduous canines2,3. Unilateral transposition has been reported more often than bilateral transposition4,5. Also, transpositions

*Corresponding author: Dr. Demet SUER TUMEN Dicle University Faculty of Dentistry Department of Orthodontics Diyarbakır/TURKEY E-mail: demetsuer@gmail.com

Volume 3 ∙ Number ∙ 2 ∙ 2010

Accept date: 06 April 2010 occur more often the maxilla, however they can occasionally be seen in the mandible5-8. Displacement and migration of teeth is common. The maxillary canine is probably the tooth most frequently displaced. When displaced in the palatolabial plane, it may become palatally or labially impacted. When displaced distally or mesially, an ectopically erupting canine can become transposed with one of the adjacent teeth9. Additionally, maxillary tooth transposition most frequently involves the canine with the first premolar, and less frequently involves the lateral incisor9-11. The treatment of these patients frequently requires multidisciplinary treatment planning to achieve a long-term aesthetic and functional result12-16. This case report aims to present the orthodontic treatment of the patient with maxillary left canine lateral transposition. Page 75


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CASE REPORT Clinical and radiographic examination A 14-year-old girl was referred to the Dicle University Faculty of Dentistry, Department of Orthodontics with a chief complaint of crowding. The patient was in good general health, and the medical and dental history indicated no contraindications to dental treatment. A clinical and oral examination showed severe crowding, retained deciduous canine tooth and dental Angle Class I (Figures 1,2). Impacted and transposed canine tooth was observed in the radiographic examination (Figure 3). In lateral cephalometric evaluations, skeletal Class I relation (ANB: 2º) was detected.

Figure 1. Pretreatment intraoral photographs.

Canine-Lateral Incisor Transposition: A case report Demet Suer Tumen and et al.

should be considered. The first option is aligning the involved teeth in their transposed positions and the other one is moving them to their correct anatomic position in the arch. Dental and facial aesthetics of the maxillary anterior teeth should be carefully evaluated and considered in deciding which treatment option to follow. Repositioning the completely transposed anterior teeth to their normal sequence in the arch is very complex and time-consuming. Some authors suggest that it should not even be attempted as there is a risk of jeopardizing the roots and damaging the supporting structures. Despite the compromised aesthetic results, they suggest aligning the teeth in their transposed positions5,17. Therefore, we planned to align the involved teeth in their transposed positions. We began the treatment of the patient by extracting retained deciduous canine tooth. In order to make transposed and impacted canine tooth erupt, firstly 0.018 x 0.022 inch fixed Roth Edgewise appliances were applied. After the leveling, lateral tooth was moved into the space of the canine tooth and the space was obtained in the arch for canine tooth. Then, eruption appliance was placed surgically to the canine tooth. After the canine tooth erupted completely (Figure 4), it was grinded to make it look like a lateral tooth and the aesthetic of the canine was provided by applying connective tissue graft. As a consequence of the treatment, the patient has had pleasing aesthetic and smile (Figures 5,6,7). In 14-month follow-up; the patient’s occlusion was stable, oral hygiene was adequate, and the patient was satisfied with the aesthetic results (Figure 8).

Figure 2. Pretreatment facial photographs.

Figure 3. Pretreatment panoramic radiographs.

cephalometric

and

Treatment plan and procedure In the treatment planning of the maxillary caninelateral incisor transposition, at least 2 options Volume 3 ∙ Number ∙ 2 ∙ 2010

Figure 4. Canine was erupted into lateral tooth’s position. Page 76


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Figure 5. Posttreatment intraoral photographs.

Figure 6. Posttreatment facial photographs.

Figure 7. Posttreatment cephalometric and panoramic radiographs.

Figure 8. Before and after applying connective tissue graft. Discussion Canine transposition has been reported to be the most common transposition found in the human dentition18,19. Maxillary canine-lateral incisor transposition is a relatively rare anomaly, with both dental and facial aesthetic implications12. Transpositions affect both sexes, but female patients have been reported to outnumber male patients in the prevalence of this Volume 3 ∙ Number ∙ 2 ∙ 2010

Canine-Lateral Incisor Transposition: A case report Demet Suer Tumen and et al.

anomaly2,20. This approximate 2:1 ratio is in general agreement with many reports that indicate female predominance for this 2,5,6,10,20,21 . anomaly There is a distinction between a complete and an incomplete transposition22. In complete transposition, both the crowns and the entire root structures of the involved teeth are found parallel in their transposed positions. In incomplete transposition (also called “pseudo” or “partial”) the crowns may be transposed while the root apices remain in their normal positions. Alternatively, the crowns may be in the correct order while the root apices are transposed. Thus, the 2 involved teeth overlap and their long axes cross each other. In addition, the crowns and roots of the 2 involved teeth may completely superimpose each other on normally projected radiographs9. Therefore, complete radiographic analysis is crucial in the treatment planning and evaluations of transposed teeth. Transposition treatments can vary from case to case. In the treatment planning, the existence of the other tooth anomalies and the evaluation of the tooth and gingiva together at crowding region are very important. In maxillary canin-lateral transposition, there are two problems to overcome: the ability of the lateral incisor to function as a canine and the ability to disguise the canine and lateral incisor as each other. The upper lateral incisor is less favorable for “canine guidance”, as its root is usually thin and short. Hence, conversion to group function may be suggested for non-extraction cases. Camouflage of the upper canine often requires grinding of its tip, a combination of grinding and adding composite resin, or a porcelain veneer. The canine has a broader and higher gingival contour, compared with the lateral incisor, so this may make the aesthetic result pleasing in those with a high smile line23. However, the patient’s transposed canine gingival contour was very high after the orthodontic treatment. Therefore, we applied periodontically connective tissue graft in order to obtain an aesthetic smile and pleasing result. Conclusions In the treatment planning and success of the transposed teeth, tooth’s position and interdisciplinary cooperation play an important role. As a result of applied multidisiplinary Page 77


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treatment, we have achieved a functional occlusion, ideal overjet and overbite relations and aesthetic facial appearance and smile.

Canine-Lateral Incisor Transposition: A case report Demet Suer Tumen and et al.

transposition-Orthodontic management. Am J Orthod Dentofacial Orthop 1989;95:439-44. 23. Weeks EC, Power SM. The presentations and management of transposed teeth. Br Dent J 1996;181:421-4.

Declaration of Interest The authors report no conflict of interest and the article is not funded or supported by any research grant. References 1. Shapira Y, Kuftinec MM. Tooth transposition: a review of the literature and treatment considerations. Angle Orthod 1989;59: 2716. 2. Peck L, Peck S, Attia Y. Maxillary canine-first premolar transposition, associated dental anomalies and genetic basis. Angle Orthod 1993;63: 99-109. 3. Chattopadhyay A, Srinivas K. Transposition of teeth and genetic etiology. Angle Orthod 1996;66:147-52. 4. Joshi MR, Bhatt NA. Canine transposition. Oral Surg Oral Med Oral Pathol 1971;31:49-54. 5. Peck S, Peck L. Classification of maxillary tooth transpositions. Am J Orthod Dentofacial Orthop 1995;107:505-17. 6. Peck S, Peck L, Kataja M. Mandibular lateral incisor-canine transposition, concomitant dental anomalies, and genetic control. Angle Orthod 1998;68:455-66. 7. Taner T, Uzamis¸ M. Orthodontic management of mandibular lateral incisor-canine transpositions: reports of cases. ASDC J Dent Child 1999;66:110-5. 8. Peck S. On phenomenon of intraosseous migration of nonerupting teeth. Am J Orthod Dentofacial Orthop 1998;113:515-7. 9. Shapira Y, Kuftinec MM. Maxillary tooth transpositions: Characteristic features and accompanying dental anomalies. Am J Orthod Dentofacial Orthop 2001;119:127-34. 10. Plunkett DJ, Dysart PS, Kardos TB, Herbison GP. A study of transposed canines in a sample of orthodontic patients. Br J Orthod 1998;25:203-8. 11. Ranta R. Tooth germ transposition: report of cases. J Dent Child 1989;56:366-70. 12. Shapira Y, Kuftinec MM. A unique treatment approach for maxillary canine-lateral incisor transposition. Am J Orthod Dentofacial Orthop 2001;119:540-5. 13. Chaushu S, Becker A, Zalkind M. Prosthetic considerations in the restoration of orthodontically treated maxillary lateral incisors to replace missing central incisors: a clinical report. J Prosthet Dent 2001;85:335-41. 14. Beznos C. An alternative approach to replacement of a congenitally missing maxillary central incisor: a case report. Quint Int 1996;27:759-62. 15. Kokich VG, Nappen DL, Shapiro PA. Gingival contour and clinical crown length: their effect on the esthetic appearance of maxillary anterior teeth. Am J Orthod 1984;86:89-94. 16. Rabie AB, Wong RW. Bilateral transposition of maxillary canines to the incisor region. J Clin Orthod 1999;33:651-5. 17. Laptook T, Silling G. Canine transposition: approaches to treatment. J Am Dent Assoc 1983;107:746-8. 18. Mader C, Konzelman JL. Transposition of teeth. J Am Dent Assoc 1979;98:412-43. 19. Nestel E, Walsh JS. Substitution of a transposed premolar for a congentially absent lateral incisor. Am J Orthod Dentofac Orthop 1988;93:395-9. 20. Umweni AA, Ojo MA. The frequency of tooth transposition in Nigerians, its possible aetiologic factors and clinical implications. J Dent Assoc South Africa 1997;52:551-4. 21. Shapira Y. Transposition of canines. J Am Dent Assoc1980;100:710-2. 22. Shapira Y, Kuftinec MM. Maxillary canine-lateral incisor

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Gingival Metastase and Intracerebral Haemorrhage Mehmet Serhan Tasdemir and et al

GINGIVAL METASTASE AND INTRACEREBRAL HAEMORRHAGE RESULTING FROM UNSUSPECTED CHORIOCARCINOMA: A CASE REPORT Mehmet Serhan Tasdemir1 , Ayfer Aktas2*, Nebahat Tasdemir3, Yusuf Nergiz4 1. Research Assistant PhD, University of Dicle Medical Faculty, Department of Histology and Embryology, 21280Diyarbakir, Turkey. 2. Associate Professor. Dr. University of Dicle Medical Faculty, Department of Histology and Embryology, 21280Diyarbakir, Turkey. 3. Professor Dr.University of Dicle Medical Faculty Department of Neurology, 21280Diyarbakir, Turkey. 4. Professor Dr.University of Dicle Medical Faculty, Department of Histology and Embryology, 21280Diyarbakir, Turkey.

Abstract Gestational choriocarcinoma is a higly malignant epithelial tumour arising from the trophoblasts of any type of gestational event, most often a hydatidiform mole . Choriocarcinoma is for all practical purposes limited to reproductive age women but rare examples of choriocarcinoma in postmenoupausal women have been reported. In case of a 24- years- old was a female patient with left hemiparalysis. Biopsy that obtained from lower gingival ulcer indicated the availability of choriocarcinoma metastasis. Before putting her ın our intensive care unit she suddenly became unconscious. Focally haemorrhagic fragile gingival tissue (grossly, 1× 1× 0.5 cm) was processed. In the formaline fixed , paraffin embedded and hematoxylin and eosin(H&E) stained slides, beneath the stratified squamous epithelia, there was tumoral infiltration. Tumor was consisted of abnormal cytotrophoblasts and syncytiotrophoblasts cells and diffuse intercerebral haemorrhage. In this report we presented a rare case of metastatic gingival choriocarcinoma and intracranial haemorrhage resulting from an unsuspected choriocarcinoma metastasis. (J Int Dent Med Res 2010; 3: (2), pp. 79-81 ) Keywords: Intracerebral haemorrhage, gestational choriocarcinoma, gingiva. Received date: 17 November 2009 Introduction Gestational choriocarcinoma is a higly malignant epithelial tumour arising from the trophoblasts of any type of gestational event, most often a hydatidiform mole. Choriocarcinoma is for all practical purposes limited to reproductive age women but rare examples of choriocarcinoma in postmenopausal women have been reported1. Theoretically, choriocarcinoma may arise in the trophoblast of the primitive blastocyst during implantation, but most cases of choriocarcinoma appear to follow a recognizable *Corresponding author: Assist. Prof. Dr. Ayfer Aktas, University of Dicle Medical Faculty, Department of Histology and Embryology. 21280 Diyarbakir, Turkey. E-mail: aaktas@dicle.edu.tr

Volume 3 ∙ Number ∙ 2 ∙ 2010

Accept date: 21 April 2010 gestational event. The signs and symptoms of choriocarcinomas are protean. Abnormal uterinal bleeding is one of the most frequent symptoms of choriocarcinoma, but uterinal lesions may be restricted to the myometrium and remain asymptomatic. Not all patients have a demonstrable lesion in the uterus after an intrauterine gestation. Many examples of metastatic choriocarcinoma without a primary uterine tumour have been described2. It is highly likely that the neoplasm undergoes regression in the uterus. Sometimes, symptoms related to metastases are the first indication that a choriocarcinoma is present, and the lungs are the most frequent sites for metastasis2,3. Secondary involvement of the gingival metastasis by choriocarcinoma is uncommon. In this report we presented a rare case of metastatic gingival and intracranial haemorrhage resulting from unsuspected choriocarcinoma metastasis. Page 79


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Gingival Metastase and Intracerebral Haemorrhage Mehmet Serhan Tasdemir and et al

CASE REPORT We described a 24 years old female patient with left hemiparalysis. Biopsy that obtained from lower gingival ulcer indicated the availability of choriocarcinoma metastasis. Before putting her in our intensive care unit she suddenly became unconscious. She had 3 children and her last pregnanacy was 3 months ago. In her neurological examination: We found left Babinski’s sign positive and her whole deep tendon reflexes were brisk with left hemiparalysis. Her gynaecologic examination was normal. ß-hCG: 10 000( ≤ 3). We detected a haematoma at right parieto-occipital region. It was 3.5 ×5 cm size by CT scan (Figure 1). Abdominal ultrasonograpic examination of uterus and bilateral ovaries were found to be normal. Dermatological consultation: Pyogenic granuloma of the mouth. Clinical diagnosis: Malign pyogenic parenchymal tumour.

Figure 1. Patient CT-Scan.

Tissue pathologic findings: grossly, 1× 1× 0.5 focally haemorrhagic fragyl tissue processed, In the formaline fixed, paraffin embedded and H.E stained. Beneath stratified squamous epithelium, these were tumoral infiltration. Tumour was consisted of abnormal cytotrophoblasts and syncytiotrophoblasts cells (Figure 2).

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Figure 2. Biopsy tissue from gingiva.(H&E,X80). Discussion Hertig found an incidence of choriocarcinoma 1 in 160.000 normal gestations, 1 in 15.386 abortions, 1 in 5.333 ectopic pregnancies, and 1 in 40 molar pregnancies4. In that series, one–half of the cases of choriocarcinoma were preceded by hydatidiform mole, with 25% following abortion , 22.5 % following normal pregnancy , and 2.5% following ectopic pregnancy4. Other studies have generally confirmed these figures5. Metastases to the gingiva are uncommon. They can be a diagnostic challenge clinically because of their rarity and tendency to mimic benign lesions. Sometimes, symptoms related to metastases are the first indication that a choriocarcinoma is present, and the lungs are the most frequent sites for metastasis6. Large amounts of trophoblast showing atypic should be viewed suspiciously for choriocarcinoma. If the diagnosis is in doubt, a chest radiograph and careful monitoring of ßhCG levels should resolve the problem. Radioimmunoassay of beta human chorionic gonadotrophin (hCG) should be used to confirm the diagnosis. Discriminating choriocarcinoma from other carcinomas either within the uterus or at other sites usually is not a problem. Occasionally a biopsy of choriocarcinoma may show few syncytiotropblastic cells, or the entire lesion is composed of mononucleate trophoblastic cells, a pattern that can mimic a poorly differentiated carcinoma6. We also found syncytiotrophoblastic cells in her gingival biopsy tissue. Patients usually are in the reproductive Page 80


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age group ( 19-62 years with an average of 30 years) and can present with either amenorrhea or abnormal uterinal bleeding, often 7,8 and accompanied by uterine enlargement frequently are thought to be pregnant. When this differential diagnosis arises, the clinical history may reveal a previous molar pregnancy or another suspicious pregnancy event that can assist in the diagnosis. Serum hCG levels and immunohistochemical localization of hCG, hPL, and inhibin–a in syncytiotrophoblast can be useful. We found serum hCG level was over than 10 000. Choriocarcinoma has been described as a primary tumour arising in a number of different sites besides the uterus and gonads. In women of reproductive age, however , pure choriocarcinoma that appears to be an extrauterine primary tumour probably represents gestational chorocarcinoma in which the index pregnancy is undetected9. True primary choriocarcinoma at an unusual site may be derived from differentiation of an ordinary carcinoma. Our patient had her last pregnancy 3 months ago and normaly delivered. Her last gynaecologic examination was completely normal. Primary somatic tumours of the gastrointestinal tract, bladder , breast , lung , or endometrium rarely show choriocarcinomatous differentiation , and these show transitions from ordinary carcinoma to the trophoblastic component. Over the past four decades there has been a substantial improvement in survival of patients with choriocarcinoma3,9. Brain metastases are relatively uncommon and their incidence in patients with choriocarcinoma is 3-27%1,3,7,8. However, the incidence of cerebral metastasis found at autopsy on patients with choriocarcinoma is significantly higher (66.7% of patients)10. The prognosis for choriocarcinomas in the uterus is very good. Although these tumours have often spread throughout the body, chemotheraphy results in a cure or remission in at least 80-90% of cases. Women who have had choriocarcinomas often go on to have normal pregnancies and deliveries but choriocarcinomas in other sites have a worse prognosis. These tumors are a worse prognosis. These tumours tend to spread quickly and don’t always respond well to the chemotherapy. Although treatment can be effective, the outcome usually depends Volume 3 ∙ Number ∙ 2 ∙ 2010

Gingival Metastase and Intracerebral Haemorrhage Mehmet Serhan Tasdemir and et al

on

how widely the cancer is dispersed. Generally, the prognosis is worse if the cancer can be found in the liver or brain, and the original tumour developed outside the gonads. Five year survival with testicular cancers can range from 92% for tumours that have spread only to the lungs to 48% to tumours that have spread to other internal organs1,2,4,5. Conclusions In this report we presented a rare case of metastatic gingival choriocarcinoma and intracranial hemorrhage resulting from unsuspected choriocarcinoma metastasis. Declaration of Interest The authors report no conflict of interest and the article is not funded or supported by any research grant. References 1. Dougherty CM, Cunninham C, Mickal A. Choriocarcinoma with metastasis in a postmenoupausal woman . Am J Obstet Gynecol. 1978; 132:700-701. 2. Berkowitz RS, Goldstein DP. Chorionic tumours. N. Eng. J.Med. 1996;.335:1740-8. 3. Bakri Y, Berkowitz RS Goldstein DP et al . Brain metastases of gestational trophoblastic tumour. Reprod Med. 1994; 39:179-84. 4. Hertig AT. Tumours of the female sex organs . Part 1. Hydatidiform mole and choriocarcinoma , Atlas of tumour pathology , sec 9, fasc 33. Armed Forces Institute of pathology; 1956: Washington , DC. 5. Mazur MT, Lurain JR, Brewer JI. Fatal gestational choriocarcinoma .Clinicopathologic study of patients treated at a trophoblastic disease center. Cancer (Phila ) 1982; 50 :1833-1846. 6. Wurzel J, Brooks JJ. Primary gastric choriocarcinoma : İmmunohistochemistry , postmortem documentation and hormonal effects in a postmenoupausal female . Cancer (Phila) 1981; 48:2765-2761. 7. Komeichi T, Igarashi K, Takigami M et al, A case of metastatic choriocarcinoma associated with cerebral thrombosis and aneurysm formation . No Shinkel Geka. 1996; 24: 463-7. 8. Kobayashi T , Kida Y Yoshida , Shibuya N, Kageyama N, Brain metastases of choriocarcinoma . Surg .Neurol. 1982;17:395-403. 9. Lurain JR, Brewer JI , Torok EE, Halpern B. Gestational trophoblastic disease center. Obstet Gynocology. 1982; 60:354360. 10. Young RH, Scully RE . Placental –site trophoblastic tumour: current status. Clin Obstet Gynocol. 1984; 27:248-258.

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Surgical Approaches to Mediastinal Masses Cemil Deniz Yorgancilar, and et al

OUR DIAGNOSTIC AND THEARAPEUTIC SURGICAL APPROACHES TO MEDIASTINAL MASSES Cemil Deniz Yorgancilar1*, Ozgur Karakurt2, Osman Korcan Tilkan3, Sedat Demircan3 1. MD, Department of Thoracic Surgery, Balikligöl State Hospital, Sanliurfa, TURKEY. 2. MD, Department of Thoracic Surgery, Numune Training and Research Hospital, Ankara,TURKEY. 3. MD, Department of Thoracic Surgery, Gazi University Medical School, Ankara,TURKEY.

Abstract The mediastinum is defined as the thoracic space that lies between the two pleural layers. It extends from the thoracic inlet to the superior surface of the diaphragm. It is bordered anteriorly by the internal surface of the sternum and posteriorly by the longitudinal spinal ligaments. The mediastinum comprises of important anatomical structures. Surgical treatments and the results of patients who had a pre-diagnosis of mediastinal masses were examined. A total of 93 patients who underwent surgery for a pre-diagnosis of mediastinal masses between June 2003 and June 2008 were retrospectively reviewed. Preoperative evaluations, operative procedures and results of the patients were examined. Of the 93 patients, 47 were females and 46 were males. The age range was 5-76 years, while the mean age was 46.5 years. Mediastinal lesions were mostly located in the anterior mediastinum. Cough and chest pain were observed as the two main symptoms in symptomatic patients. The surgical procedures performed were mediastinoscopy, posterolateral thoracotomy, median sternotomy, and anterior mediastinotomy. Post-operative histopathological examination of collected specimens resulted in a variety of pathologic diagnoses. Two patients developed incision wound infection, whereas one patient, known to have thymoma, died on the 16th post-operative day as a result of respiratory muscle insufficiency. Two patients diagnosed with osteosarcoma and chondrosarcoma metastasis died four months after surgery due to primary pathologies. The surgical procedure used in the diagnosis and treatment of mediastinal masses is very important. The surgical approach is determined according to localization. Operative mortality is very low. Surgical management of mediastinal masses is suggested to be the most appropriate approach since histopathological diagnosis and treatment can be achieved synchronously in many patients. (J Int Dent Med Res 2010; 0: (0), pp. 82-87 ) Key words: Mediastinal mass, surgical treatment. Received date: 13 April 2010 Introduction The mediastinum is an important anatomical compartment consisting of very important structures that are vital for life. Benign or malignant masses may be found in the mediastinum. Malignant mediastinal tumors may *Corresponding author: Cemil Deniz YORGANCILAR Sanliurfa Balikligöl State Hospital, Department of Thoracic Surgery SANLIURFA / TURKEY

Accept date: 20 July 2010 be primary or secondary to lymphatic nodal spread of neoplasia. On the other hand, benign mediastinal masses may arise from primary mediastinal structures and from infectious or inflammatory diseases with lymphatic nodal involvement. This wide spectrum forms a diverse histopathology in the mediastinum1 (Table 1). Diagnosis and treatment are imperative in patients with mediastinal masses, which are generally coincidentally identified. They may lead to life threatening clinical scenarios, such as local invasion or compression on adjacent organs. Surgery plays a very important role in the diagnosis and treatment of these patients.

E-mail: denizy2000@yahoo.com

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Surgical Approaches to Mediastinal Masses Cemil Deniz Yorgancilar, and et al

Table 1. Localization of Mediastinal Tumor and Cyst.

Materials and Methods A total of 93 patients who underwent surgery for the pre-diagnosis of mediastinal masses between June 2003 and June 2008 were retrospectively reviewed. Preoperative evaluations, operative procedures and results of the patients were examined. The mediastinum is classified anatomically as the anterior, middle and posterior mediastinum. Pathologies associated with the esophagus and the diaphragms were not included in the study. Of the 93 patients, 47 were females and 46 were males. The age range was 5-76 years, while the mean age was 46.5 years. The female/male ratio was calculated as 1.1. Preoperative posterior-anterior (PA) chest X-rays, lateral X-rays and computerized tomography (CT) scans of the thorax of all the patients were performed(Figures 1,2). The localization and character of mediastinal masses and their relation with neighboring structures were evaluated. Eighteen patients suspected of having local invasion or compression of adjacent organs were also evaluated regarding resectability using magnetic resonance imaging (MRI) (Figures 3,4).

Figure 1. Thorax computed tomography image of a left paravertebral sulcus mass, histopathologic diagnosis was schwannoma.

Volume 3 â&#x2C6;&#x2122; Number â&#x2C6;&#x2122; 2 â&#x2C6;&#x2122; 2010

Figure 2. Thorax computed tomography image of a left paravertebral sulcus mass, histopathologic diagnosis was ganglioneuroma.

Figure 3. Magnetic resonance image of a left paravertebral sulcus mass, histopathologic diagnosis was schwannoma.

Figure 4. Magnetic resonance image of a left Page 83


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paravertebral sulcus mass, diagnosis was thymoma.

Surgical Approaches to Mediastinal Masses Cemil Deniz Yorgancilar, and et al

histopathologic

Evaluation with positron emission tomography (PET) were performed on three patients with suspicion of malignity following clinical and radiological examination, and on one patient previously diagnosed with malignity. Additional evaluation with bone scintigraphy in one patient and thyroid scintigraphy in three patients was performed. Fiberoptic bronchoscopy was performed on all patients before surgery. The appropriate surgical approach was determined on the basis of radiological findings. Mediastinoscopy was performed on 42 (45%) patients, right posterolateral thoracotomy on 30 (32%), median sternotomy on 12 (13%), left posterolateral thoracotomy on eight (8.5%) and anterior mediastinotomy was performed on one (1.5) patient (Table 2).

Table 3. Distribution of complaints.

Table 4. The localization distribution, number and the percentage of cases in the mediastinal compartments.

Table 2. The surgical procedure carried out, the number of operations in respect to patients, and the percentage distribution. Results Twenty-six (28%) patients were asymptomatic. Of the symptomatic patients 32 (34%) had cough, 14 (15%) had chest pain, 10 (11%) had dyspnea, seven (7.6%) had hoarseness, two (2.2%) patients had mediastinal findings, one (1.1%) had joint pain, and one patient (1.1%) had headache (Table 3). Before the operation all patients were evaluated using fiberoptic bronchoscopy. Compression of the trachea from the outside was observed in 11 patients, while bluntness of the carina was observed in five patients; no endobronchial lesion was identified. Anatomically, three compartments of the mediastinum were examined. It was demonstrated that 79% of the mediastinal masses were localized in the anterior mediastinum (n=74), 15.5% were localized in the posterior mediastinum (n=14), and 5.5% in the middle mediastinum (n=5) (Table 4). Volume 3 â&#x2C6;&#x2122; Number â&#x2C6;&#x2122; 2 â&#x2C6;&#x2122; 2010

Table 5. Histopatologic distributions.

diagnoses

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Histopathological examination of surgery specimens obtained from the patients who underwent surgery for mediastinal masses revealed 21 cases of sarcoidosis (22.5%); 11 of tuberculous lymphadenitis (11.7%); 13 of thymoma (14%); 7 of reactive lymphadenopathy (7.5%); 6 of schwannoma (6.4%); 8 of metastatic carcinoma (8.4%); 4 of nodular goiter (4.2%); 6 of Hodgkin’s lymphoma (6.4%); 3 of bronchogenic cyst (3.3%); 2 of Castleman disease (2.2%); 2 of ganglioneuroma (2.2%); 1 of Askin’s tumor (1.1%); 1 of chondrosarcoma (1.1%); 1 of tracheal adenoid cystic carcinoma (1.1%); 1 of amyloidosis (1.1%); 1 of parathyroid adenoma (1.1%); 1 of teratocarcinoma (1.1%); 1 case of usual interstitial pneumonia (1.1%); 1 of squamous hyperplasia (1.1%); 1 of paraganglioma (1.1%); 1 of neuroendocrine tumor (1.1%); 1 of lymphangiectasia (1.1%) and 1 case of pericardial cyst (1.1%) (Table 5). Incision wound infections developed in two patients during the post-operative early period. Healing was achieved through antibiotic treatment and wound care. The perioperative mortality was 1.1%. One patient who was known to have thymoma died on the 16th post-operative day as a result of respiratory muscle insufficiency. Discussion The mediastinum is bordered laterally by the pleura, superiorly by the thoracic inlet and inferiorly by the diaphragm. It is divided into three compartments based on lateral X-ray: the anterior mediastinum, middle mediastinum and posterior mediastinum. This division is not made according to a real anatomic or facial plan. The anterior mediastinum is the area covering the sternum posteriorly, the anterior border of the pericardium and bordered by the brachiocephalic vein anteriorly at the superior border. It extends from the diaphragm to the thoracic inlet, and contains the thymus gland, mediastinal adipose tissues and lymph nodes. The middle mediastinum is the area containing the heart, pericardium, aortic arc, brachiocephalic vessels, pulmonary arteries, pulmonary veins, trachea, bronchus and lymph nodes; and extending from the front of the esophagus to the anterior mediastinum. On the other hand, the posterior mediastinum is the area that extends from the anterior border of the esophagus to the vertebrae. The posterior mediastinum contains Volume 3 ∙ Number ∙ 2 ∙ 2010

Surgical Approaches to Mediastinal Masses Cemil Deniz Yorgancilar, and et al

the descending aorta, the esophagus, azygos and hemizygos veins, ganglions and nerves, thoracic dust, lymph nodes and adipose tissues. Hence, these compartments can be approached as important for the pre-diagnosis of mediastinal masses. The most encountered tumors of the anterior mediastinum are thymomas, lymphomas, and germ cell tumors. Apart from these, vascular and mesenchymal tumors, ectopic thyroid tissue, and tumors of the parathyroid gland are also seen. The most commonly encountered tumor of the middle mediastinum is lymphoma. Structures that cover cystic areas are most commonly found in the middle mediastinum. Of the tumors that are localized in the posterior mediastinum, 70% are of neurogenic origin1-4 (Table 1). Mediastinal masses can be detected in all age groups. There is a close female to male incidence rate5. In our clinical study, the female/male ratio was found to be 1.1, at various ages between 5 to 76 years. The female to male incidence rate was also similar in our study. Mediastinal masses are mostly asymptomatic. They are coincidentally detected through lung X-ray. In literature, symptoms are seen between 63-93% of the patients. Signs and symptoms depend on whether the mass is malignant or benign, the size, localization, presence of infection, presence of an accompanying systemic disease, and also on whether the tumor produces specific endocrine or chemical secretions. In symptomatic cases, there is compression or invasion of adjacent organs. Symptoms associated with compression are dyspnea, cough and dysphagia; symptoms associated with invasion of adjacent tissue are hoarseness, paralysis of the diaphragm, pain, and hemoptysis. There is a strong correlation between malignity and symptoms. Mediastinal tumors can cause syndromes such as Cushing’s syndrome and myasthenia gravis due to hormonal factors. Large anterior mediastinal masses cause airway obstruction when the patient is in the supine position. There is severe pain during invasion of the wall of the chest and thorax. There may be effusion when there is pleural and pericardial invasion. Horner syndrome associated with invasion of the stellate ganglion may be identified in the posterior mediastinum, in the presence of a neurogenic tumor. Effort dyspnea, rhythmic abnormalities and syncope may exist in the presence of cardiac Page 85


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compression6. The incidence rate of symptoms in this clinical study was 74%. Cough and chest pain were the most common complaints. Literature data is in support of these findings. The following information is investigated during preoperative diagnostic evaluation: 1) Determination of the differential diagnosis of masses, which have the same radiological image as the mediastinal mass; 2) Identification of the systemic problems, which may be detected before or after the operation; 3) Identification of compression and invasion of the tracheobronchial tree, pulmonary vascular structures, superior vena cava and other mediastinal vascular structures; 4) Identification of vertebrae invasion, if present; 5) Resectability; 6) Identification and prevention of possible medical morbidity. The first test to be performed in radiological examination of mediastinal lesions is to obtain lateral and anteroposterior X-rays. Evaluation of localization, size, density, contents of the mass, presence of calcification, structure of the mass and its relationship with neighboring tissues are performed using CT. CT scans should definitely be performed using an intravenous contrast substance when evaluating mediastinal pathologies7. MRI may be performed in cases of suspected vascular structures invasion and of masses having posterior mediastinal localization. MRI is suggested to produce good results during mediastinal evaluation. The cardiac and pericardial origin of middle mediastinal lesions can be investigated using echocardiography. Gallium scintigraphy is especially sensitive in cases of mediastinal lymphadenopathy. Aberrant thyroid tissue may be analyzed through thyroid scintigraphy, while cases suspected of having mediastinal lymphadenopathies and mediastinal mass may be evaluated with PET/CT1,8,9. In this study, all patients were first evaluated using lung roentgenogram and CT of the thorax. MRI was performed in patients with vascular involvement, patients suspected of adjacent organ invasion (n=18), while PET/CT evaluation was performed in four patients who were thought to have metastatic mediastinal masses. Those who were thought to have aberrant thyroid tissue following radiological investigation were evaluated by Volume 3 â&#x2C6;&#x2122; Number â&#x2C6;&#x2122; 2 â&#x2C6;&#x2122; 2010

Surgical Approaches to Mediastinal Masses Cemil Deniz Yorgancilar, and et al

thyroid scintigraphy. These evaluations, known to play important diagnostic roles, were very helpful before surgery10,11. Fiberoptic bronchoscopic assessment was performed on all patients before commencing the surgical operation. The relationship of the mediastinal mass with the trachea was investigated. Tracheal compression from the outside was identified in 11 patients with anterior mediastinal masses. These patients were symptomatic and were those with malignant mediastinal masses. This result strongly supports the fact that symptoms are most commonly observed in patients with anterior mediastinal masses and that there is a close relationship with malignity. Of the mediastinal masses, 50% are located in the anterior mediastinum; this is followed by posterior and middle mediastinal localization. In a study conducted with 400 patients, Davis demonstrated that masses were located, 59% in the anterior mediastinum, 27% in the posterior mediastinum and 14% in the middle mediastinum. In this study, the most common site was the anterior mediastinum, with a rate of 75%. This result was in line with literature12. In patients with mediastinal masses, the surgical procedure should be determined according to the site of localization. The surgical approach can be planned only with biopsy, or with biopsy and resection. Diagnostic mediastinoscopy performed in patients with mediastinal lymphadenopathy is considerably beneficial. In these patients, the first procedure to be carried out following imaging techniques should be mediastinoscopy1,13. A variety of histopathological diseases are seen in the mediastinum. With regards to this histopathological diversity, anatomically dividing the mediastinum into three compartments and evaluating them accordingly, plays an important role in pre-diagnotic determination. The surgical morbidity rate of mediastinal masses is 17%, while the mortality rate is between 3-6% 9, 14. In our series, the mortality rate was 1.1%, while the morbidity rate was 2.2%. One patient with osteosarcoma died four months after the operation due to primary disease, while another patient with chondrosarcoma died five months after the operation also as a result of primary disease. The mortality and morbidity rates in our study were within acceptable limits and were even at a very low level. Page 86


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Surgical Approaches to Mediastinal Masses Cemil Deniz Yorgancilar, and et al

Conclusions In conclusion, it is suggested that surgical intervention should be performed in order to make histopathological diagnoses of mediastinal masses. Apart from the beneficial role it plays in diagnosis, its role in treatment is also very important. Surgery constitutes a low mortality and morbidity rate. Surgery, for a diagnostic or treatment purpose, should be performed in all suitable patients. Declaration of Interest The author report no conflict of interest and the article is not funded or supported by any research grant. References 1.

2. 3. 4. 5.

6.

7.

8.

9.

10.

11. 12.

13.

14.

Shields TW. The Mediastinum, Its Compartments, and the Mediastinal Lymph Nodes. In Shields TW, LoCicero J, Poon RB: General Thorasic Surgery 6th ed. Vol.1. Philadelphia: Lippincott Williams&Wilkins, 2005:951-71. Crespo JD, Glassroth J, Karlinsky J. Baum’s textbook of pulmonary diseases. Philadelphia, PA:Lippincott Williams&Wilkins. 2004; 883-912. Azarow KS, Pearl RH, Zurcher R, et al. Primary mediastinal masses. J Thorac Cardiovasc Surg 1993; 106: 67. Benjamin SP, McCormack LJ, Effler DB, et al. Primary tumors of the mediastinum. Chest 1972; 106:67. Capoferri M, Furrer M, Ris HB. Surgical diagnosis and therapy in patients with mediastinal space-occupying lesions: A retrospective analysis of 223 intervention with special reference to long-term course. Swiss Surg 1998; 4: 121-128. Davis RD Jr, New Oldham H Jr, Sabiston DC Jr. Primary cysts and neoplasms of the mediastinum: recent changes in the clinical presentation, methods of diagnosis, management and results. Ann Thorac Surg 1987:44;229-237. Rendina EA, Venuta F, Ceroni L, et al. Computed tomographic staging of anterior mediastinal neoplasm. Thorax 1998; 43: 441. Cohen AJ, Thompson LN, Edwards FH, Bellamy RF. Primary cysts and tumors of the mediastinum. Ann Thorac Surg 1991; 51:378-384. Divisi D, Battaglia C, Crisci R. Diagnostic and Therapeutic approaches for masses in the posterior mediastinum. Acta Biomed Ateneo Parmense 1998; 69:123-128. Shapiro B. Summary, conclusion and future directions of 131I metiodobenzylguanidine therapy in the treatment of the neural chest tumors. J Nucl Biol Med 1991; 35:357. Park HM. Efficacy of thyroid scintigraphy in the diagnosis of intrathoracic goiter. AJR Am J Roentgenol 1987; 148: 527. Daniel PR, Thomas PD. Mediastinal Anatomy and Mediastinoscopy. In Sellke FW (ed): Surgery of The Chest. 7.Ed. Philadelphia: Sabiston&Spencer 2005; 657-666. Pattison CW, Westaby S, Wetter A, Towncard ER. Mediastinoscopy in the investigation of primary mediastinal lymphadenopathy. Scand J Thorac Cardiovasc Surg 1989; 23:177-179. Graeber GM, Shiver CD, Albus RA. The use of computed tomography in the evaluation of mediastinal masses. J Thorac Cardiovasc Surg 1986; 91: 662-666.

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Methods in Clinical Researches Zeki Akkus and et al

THE CRITERIA FOR CLASSIFICATION TREE METHODS IN CLINICAL RESEARCHES* Zeki Akkus1**, S.Yavuz Sanisoglu2, Mehmet Ugurlu3, M. Yusuf Celik4 1. Assoc. Prof, Department of Biostatistics, Medical Informatics Dicle University, Diyarbakir / TURKEY. 2. Assoc. Prof, Consultant, Turkish Ministry of Health, Ankara / TURKEY. 3. MD, Field Coordinator, Turkish Ministry of Health, Ankara / TURKEY. 4. Prof. Dr. Department of Biostatistics, Medical Informatics Dicle University, Diyarbakir / TURKEY.

Abstract This study aimed at evaluating a statistical method, classification tree, which are recently developed parallel to the improvements in computer technology. The advantages over other methods and the criterions developed for classification tree are reported in this study. Classification tree (CT) is a non-parametric statistical method using a tree algorithm for reaching diagnosis by utilizing one or more risk factors. Classifications (discriminative, logistic regression and cluster analysis etc) and regression methods are frequently employed in analysing data acquired from scientific studies. However, hypothesis in these models makes the statistical analysis limited to be performed in wide range of disciplines. As there is no need for hypothesis in analysing these data sets, classification trees are serious alternative for other statistical classification and regression techniques. Classification tree, also known as Decision tree, is a good choice for data mining classifications in respect to both understanding and explaning the some particular rules about estimating the results. These methods are evolved following the improvements in computer technology. Classification tree is becoming more important in practice as it provides reliable measures in building accurate classifications. The advantages of the method over others are the following: simplification of the results, provision of non-parametric and lineer solutions, generalization of the conclusions optained by inductive reasoning. More over the technique can utilize mixed data types and the same variable can be employed in different parts of the tree. The determination of choices, which is crucially important in accurate interpretation of the results, needs time and effort in practicing the method. In field of medicine, classification tree is one of the favorable methods particulary utilized in clinical studies. (J Int Dent Med Res 2010; 0: (0), pp. 88-92 ) Keywords: Clinical research and classification tree method. Received date: 22 March 2010 Introduction This study aimed at evaluating a statistical method, classification tree, which are recently developed parallel to the improvements *A part of the study was presented as a poster in the IX. National Biostatistics Congress 2006, Zonguldak/Turkey and its abstract was published in the congress booklet. **Corresponding author: Assoc. Prof. Zeki AKKUŞ, Dicle University Medical School, Department of Biostatistics , Medical Informatics ,21280 Diyarbakir / TURKEY. E-mail: zakkus@dicle.edu.tr

Volume 3 ∙ Number ∙ 2 ∙ 2010

Accept date: 15 july 2010 in computer technology. Classification Trees were introduced during the early 90s by Grimm and Grochtmann for the structured 1,2 representation of test cases . Classisfication Trees (CT) is a nonparametric technique that can select from among a large number of variables those and their interactions that are most important in determining the outcome variable to be explained3. Classification tree is an observational method used in order to classify explanatory variables. Common features of the classification tree methods can be listed as follows: 1- Merging: In this method, relative to the target variable, non-significant predictor categories are Page 88


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grouped with the significant categories. 2- Splitting: In this method, variable that distinguishes the split point population is chosen to be compared with all others. 3-Stopping: This method determines how far to extend the splitting of nodes. 4- Pruning: Determines the removed branches. Trees are a completely different way of partitioning. All we require is that the partition can be achieved by successive binary partitions based on the different predictors. The main questions related to creating the classification tree are: How to (1) select the splits, (2) determine the terminal nodes, and (3) assign the terminal node a class?.4 Classification tree methods known also as decision trees can be accepted as a good method in the classification of data mining, estimation of the results and easy understanding and explanation of some rules. These methods were emerged in recent years by the developments in the computer technology. Classification models can be created using various statistical approaches, including generalized linear models (GLM) such as logistic regression, generalized additive models which are semi-parametric extensions of GLMs, and fully nonparametric methods such as classification trees5. Classification tree gains more importance since it yields confidence measurements in accurate classification. Advantages of the methods against the other ones are listed as follows: Simplifies the results after the analysis, ensures non-parametric and non-linear results, gets results that may be generalized by means of induction, may use mixed data types, same variable can be reused in the different parts of a tree. Two algorithms are used in the classification tree: First one is classification and regression trees (CART) and the second one is the QUEST algorithms formed of quick, unbiased, efficient, statistical trees.6 Since the variables used in CART analysis are independent from the distributions, it ensures a great convenience to the users. Classification Trees In CT analysis there are four basic steps. First step is the structuring of the tree. Second step is stopping the tree structuring method. Third step is called as pruning and fourth step is Volume 3 ∙ Number ∙ 2 ∙ 2010

Methods in Clinical Researches Zeki Akkus and et al

called as optimal tree selection. Tree construction begins on the main node by handling all observations. Method tries to create a separate node by checking the possible subvariables and all variable values in order to find out the best variable. Possible sub groups in the categorical variables are divided into the number of categorical variables quickly. Therefore, it is beneficial to determine the maximum class numbers in each categorical variable in the program. Determining the node classes is carried out as follows: Including root node, each node is determinant in class formation. Each node is dependent on three factors in class formation. 1- Priority possibility of each class in data set, 2- Decision or cost matrix, 3- Distinction of the observations stopped in each node. In a classification model, error rate is calculated as the proportion of mis-classified events to the entire events and accuracy rate is calculated by dividing the number of the accurate events to the number of the entire events (Accuracy Rate = 1- Error Rate). Risk matrix is used in order to decide on the error rates of the models established to classify the data. In the result of the distinction, the most appropriate class to be assigned for any node is estimated as follows: Where; C (j / i) : cost of classifying i class as j class (coefficients of risk matrix), π i : former probability of class i, N i : number of trial units found in dataset N i(t ) : number of the trial units found in class i of node t, If the inequation

C ( j / i )π i N i(t ) Ni > C (i / j )π j N (j t ) N j is ensured for all values of j (j = 1, 2, …. k and j ≠ i), class i is assigned to node t as the most appropriate class.7 Classification tree is a non-parametric statistical method developed to estimate the values of a dependent variable in a categorical structure.7,8 In the classification trees, there are three alternative accuracy estimation methods. These are replacement estimation, test sample Page 89


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estimation and cross validity test.7-9 Even in the situations where dataset is very complex, CART may display the variables that affect the dependent variable and the significance of these variables in the model within a simple tree structure. If the handled dependent variable is in a categorical structure, method is called as Classification Tree, CT and if they are continuous it is called as Regression Tree, RT.7-9 Geometrical Structure of the Classification Tree For a better understanding of the geometrical structure of the classification tree, it should be examined visually how the observations are classified on a geometrical plane. Such examination will be displayed by means of a graphical distinction that is realized by the multiple linear discriminating analysis that has a strong mathematical structure. Cutoff planes on the classification of data for discriminant model are shown in the following model.

Methods in Clinical Researches Zeki Akkus and et al

Cut off planes on the classification of data for classification tree are shown in the following graphic.

Figure 2. Cutoff planes for tree model. When we examine the graphic above, the most significant difference here is that the cutoff planes are parallel with the axis. In Graphic 2, we can only see the black dotted group that is the closest subgroup. Others cannot be seen since they lay under the planes. Although such situation restricts the flexibility of the planes, tree model allows for the interaction between the variables that are not seen in the sequenced linear discriminant model.10 When the study results are examined, it is seen that the classification tree is able to distinct the complex problems into simple and comprehensible sub problems.11 Advantages and disadvantages of the Method

Figure 1. Cutoff planes for discriminant model When we examine the graphic above, we can see the explanatory variables of X, Y, Z and four subgroups of data. Four subgroups are determined as black, shadowed, white and hidden. Fourth group is not seen on the graphic. Because it lays under the plane. Although there are three explanatory and six cutoff planes for four groups, only four planes are seen in Graphic 1. Generally, if there is group k, k.( k - 1)/2 plane is formed in linear discriminant model. Volume 3 ∙ Number ∙ 2 ∙ 2010

Classification trees are computationally efficient, can handle mixed variables (continuous and discrete) easily and the rules generated by them are relatively easy to interpret and understand12. Increase in the usage rate of classification tree models is connected with the following reasons:  Since CT is a non-parametric model, its assumptions are limited.  In the model there is not any assumption and limitation regarding the types of the variables (continuous, categorical, sequential or mixed).  Since the relationship between the Page 90


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dependent and independent variables have a visual presentations, model results in form of a tree can be interpreted easily without having the necessity for a lot of statistical information.  For the defined dependent variable, CT includes all possible independent variables and combinations of the model and performs the most accurate classification possible.  It is easily applied on the complex datasets.  It is a method that remains unaffected from the lost or missing values and also from the extreme values for both dependent and independent variables.  It is an alternative for many traditional statistic techniques (multiple regression, variance analysis, logistic regression, discriminant analysis, grouping analysis).  It considers the tree methods that are not certain but based on solid grounds.  It is a practical method in ensuring objective results in complex and broad datasets. On the other hand, CT method has some disadvantages as listed below:  May have unstable decision trees.  Splits only by one variable 13  The tree-space is huge, so it may need a lot of data.  It can be hard to assess uncertainty in inference about trees.  Actual additivity becomes a mess in a binary tree.  Simple trees usually do not have a lot of predictive power.  There is a selection bias for the splits14. Conclusions CART (classification and regression trees), has been used extensively as a means for clinical risk assessments15,16. In comparison with other statistical methods, CART analysis has been shown to perform equally or better than logistic regressions17-20, discriminant function analysis21,22, and neural networks23. In this study, we aimed to give some useful information about classification trees. As a result, use of Classification Tree method is more convenient than the other methods since it is a computer based analysis method and has a non-parametric feature. Volume 3 ∙ Number ∙ 2 ∙ 2010

Methods in Clinical Researches Zeki Akkus and et al

Declaration of Interest The author report no conflict of interest and the article is not funded or supported by any research grant. References 1.Grimm, K., “Systematisches Testen von Software - Eine neue Methode und eine effektive Teststrategie (Systematic Software Testing–A new method and an effective test strategy), GMDReport-251, GMD, Oldenbourg, 1995. 2.Grochtmann, M,Grimm K. Classification Trees for Partition Testing, Software Testing, Verification and Reliability, 1993,3(2), 63–82. 3. Yohannes Y, Hoddinott J. Classification and regression trees-An Introduction, International Food Policy Research Institute, Technical Guide #3, USA,1999. 4. Creating Imputation Classes Using Classification Tree Methodology, Creel1 D V, Krotki K,1RTI International, ASA Section on Survey Research Methods,2884-2887. 5.Edwards T C, Cutler D R, Zimmermann N E, Moisen L G and G. Effects of sample survey design on the accuracy of classification tree models in species distribution odels, Ecological Modelling, 2006, 199(2), 132-141. 6. Lewis R. An introduction to classification and regression tree (CART) analysis. Academic Emergency Medicine.California, 2004; 1-14. 7. Fu CY. Combining loglinear model with classification and regression tree (CART): An application to birth data . Computational Statistics&Data Analysis. 2004;45(4):865-874. 8. Breiman L, Friedman JH, Stone CJ, Olshen RA. Classification and Regression Trees, Boca Raton, Florida: Chapman&Hall . 2003;18-23. 9. Temel GO, Çamdeviren H, Akkuş Z. Sınıflama Ağaçları Yardımıyla Restless Legs Syndrome (RLS) Hastalarına Tanı Koyma. Journal Of İnönü Üniversity School Of Medicine 2005;12(2):111-117. 10. Wilkinson L. Graphical displays.Statistical Methods in Medical Research.1992;(1):3-25. 11. Bittencourt H R, Clarke RT. Feature selection by using classification and regression trees (CART) International Archives of Photogrammetry Remote Sensing and Spatial Information Sciences. 2004;35(7):66-70. 12.Ding Y, Simonoff J S. An Investigation of Missing Data Methods for Classification Trees Applied to Binary Response Data, Journal of Machine Learning Research,2010,11: 131-170 13.Timofeev R. Classification and Regression trees Theory and applicances(Master thesis).2004;20-25 14.Breiman L. Friedman J H, Olshen R A, Stone C J.Classification and Regression Trees, Wadsworth Inc, 1984. 15. Steadman H J, Silver E, Monahan J et al. A classification tree approach to the development of actuarial violence risk assessment tools. Law Human Behav., 2000, 24:83-100. 16. Huland H. Radical prostatectomy: Options and issues,Eur. Urol.,2001, 39: 3 -9. 17. Germanson, T P, Lanzino G, Kongable G L et al.Risk classification after aneurysmal sub-arachnoid hemorrhage. Surg. Neurol.1998, 49: 155-163. 18. Rudolfer S M, Paliouras G,Peers I S. A comparison of logistic regression to decision tree induction in the diagnosis of carpal tunnel syndrome. Comput Biomed Res,1999, 32: 391-414. 19. Wietlisbach V, Vader J P, Porchet F et al. Statistical approaches in the development of clinical practice guidelines from expert panels: the case of laminectomy in sciatica patients. Med Care, 1999.37: 785-797. 20.Vayssie`res M P, Plant R E,Allen-Diaz B H,Classification trees: An alternative non-parametric approach for predicting species distributions, Veg Sci, 2000, 11: 679-694.

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Methods in Clinical Researches Zeki Akkus and et al

21. Smith S J, Iverson S J, Bowen W D,Fatty acid signatures and classification trees: new tools for investigating the foraging ecology of seals, Can J Fish Aquat Sci, 1997,54:1377-1386. 22. Kirkwood C A, Andrews B J,Mowforth P, Automatic detection of gait events: a case study using inductive learning techniques, J Biomed Eng, 1989. 11: 511-516. 23. Selker H P, Griffith J L, Patil S et al., A comparison of performance of mathematical predictive methods for medical diagnosis: identifying acute cardiac ischemia among emergency department patients, J Invest Med, 1995. 43: 468-476..

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Journal of International Dental And Medical Research ISSN 1309-100X Analgesic effect of hypertonic solution http://www.ektodermaldisplazi.com/journal.htm Sinerik Ayrapetyan, Gohar Musheghyan, and Anush Deghoyan

THE BRAIN TISSUE DEHYDRATION AS A MECHANISM OF ANALGESIC EFFECT OF HYPERTONIC PHYSIOLOGICAL SOLUTION IN RATS Sinerik Ayrapetyan1*, Gohar Musheghyan2, Anush Deghoyan3 1. Prof. UNESCO Chair-Life Sciences International Postgraduate Educational Center, 31 Acharyan St. 0040 Yerevan, Armenia. 2. PhD. UNESCO Chair-Life Sciences International Postgraduate Educational Center, 31 Acharyan St. 0040 Yerevan, Armenia. 3. MS. UNESCO Chair-Life Sciences International Postgraduate Educational Center, 31 Acharyan St. 0040 Yerevan, Armenia.

Abstract Previously were shown that neuronal shrinkage in hypertonic solution causes the decrease of the number of functional active protein molecules in membrane having channel forming, receptor and enzyme properties. On the basis of this data the correlation between rats’ brain tissue hydration and the number of ouabain receptors in membrane and pain threshold to the “hot plate” were studied. For the estimation of brain tissue hydration the differences between wet and dry weights of tissue were measured. The number of functionally active ouabain receptors was determined by counting the number of binding labeled molecules of ouabain-3H in the brain tissue. Pain threshold was determined by means of hot plate test. Hypertonic solution caused the decrease of brain tissue hydration and the number of ouabain receptors and the increase of pain threshold. Time-dependent increase of brain tissue hydration and decrease of pain threshold were observed. On the basis of the obtained data we concluded that the pain-relieving effect of hypertonic solution is due to the decrease of the number of functionally active protein molecules in cell membrane determining its excitability. (J Int Dent Med Res 2010; 0: (0), pp. 93-99 ) Keywords: Rat, cell hydration, pain threshold. Received date: 05 February 2010 Introduction The hypertonic saline (HTS) is an osmotic agent which can help patients in the acute phase of severe traumatic brain injury (TBI) and it can diminish the effects of the secondary brain injury in patients with TBI. However, the molecular and cellular mechanisms underlying the HTS-induced therapeutic effect on TBI are still unknown. The elucidation of these mechanisms could also have considerable clinical impact, especially since osmolarity disturbances are described in various

*Corresponding author: Prof. Sinerik Ayrapetyan, UNESCO Chair-Life Sciences International Postgraduate Educational Center 31 Acharyan str. Yerevan, 375040, Armenia E-mail: info@biophys.am

Volume 3 ∙ Number ∙ 2 ∙ 2010

Accept date: 07 May 2010 diseases such as diabetes 1, alcoholism 2 and aquadynia 3. In our previous works performed on snail single neurons we showed the close correlation between cell volume and membrane excitability, chemosensitivity and enzymatic activity: cell swelling leads to the increase in the number of functionally active protein molecules in the membrane, which have channel forming, receptor and enzymatic properties, while the cell shrinkage decreases its number 4-6. On the basis of these data a hypothesis was suggested, according to which the cell overhydration-induced generation of neuromembrane abnormal excitation could serve as a nociceptive signal for the central nervous system 7- 9. According to this hypothesis the pain-relieving effect of hypertonic solution is explained by the decrease of brain sensitivity (namely cortex neurons) to the input signals as a result of cell shrinkage-induced depression of neuromembrane functional activity. For testing this hypothesis the dependence of Page 93


Journal of International Dental And Medical Research ISSN 1309-100X Analgesic effect of hypertonic solution http://www.ektodermaldisplazi.com/journal.htm Sinerik Ayrapetyan, Gohar Musheghyan, and Anush Deghoyan

pain threshold to the hot plate on brain tissue hydration and the number of ouabain receptors (markers for membrane active surface) in rats interperitoneally injected to physiological solution (PS) with different tonicities as well as in rats preliminary provided with distilled water (DW) (instead of regular drinking water) ad libitum during 3 and 5 days, were studied. The obtained data in the present work allowed us to suggest that the analgesic effect of hypertonic solution on rats is determined by the decrease in the number of functionally active protein molecules in brain cells caused by cell shrinkage. The close correlation between cell hydration and the number of ouabain receptors (Na,KATPase molecules) in cell membrane was shown in our previous works6,11. The cell swelling in hypertonic and shrinkage in hypertonic mediums caused the increase and the decrease of the number of oubain receptors in the cell membrane, correspondingly. The study of the dosedependent effects of ouabain on the Na+/K+ pump activity showed that high (more than 10-7 M) concentrations of ouabain inhibited the pump activity 6, while at lower concentrations it had no effect on pump function 7. Therefore, the number of 3H-ouabain receptors in the membrane at its concentration of less than 10-7 M was suggested as a new and extrasensitive membrane marker for determining the size of the membrane active surface which correlated with the cell volume 6, 11. In these experiments the correlation between rats’ brain tissue hydration and the number of ouabain receptors in membrane and pain threshold to the “hot plate” were studied for revealing the mechanism(s) through which the pain-relieving effect of hypertonic solution is realized. Materials and Methods All the procedures performed on the animals were carried following the protocols approved by the Animal Care and Use Committee of LSIEPC. Animals All the experiments were performed on naive male unstrained albino rats (Wt 150-200 g). The animals were housed under optimum conditions of 12 hour-light/dark cycle and 22 ± 2 0C temperature with food and water access ad libitum. The brain tissue hydration was provoked Volume 3 ∙ Number ∙ 2 ∙ 2010

by providing the animals with DW ad libitum during 3 and 5 days or by single application of interperitioneal injections of 3 ml DW, isotonic and hypertonic (2 M Mannitol containing) PS. Chemicals Thyrode solution (in mM: NaCl (137); KCl (5,4); CaCl 2 (1,8); MgCl 2 (1,05); C 6 H 12 O 6 (5); NaHCO 3 (11,9); NaH 2 PO 4 (0,42); pH 7.4) served as a normal PS for the experiments. The tissue dehydration was provoked by concentrated osmoduretics mannitol [C 6 H 8 (OH) 6 , mol. Wt.=182,18], 2 M dissolved in 3 ml normal saline. In control experiments the animals were treated by 3 ml isotonic saline injection. The intraperitoneally injections of mannitol (n=56) and saline (n=11) were performed 30 min. before the experiments. Tissue preparation First of all the possible effects of emotional stress and pain sensation (arising at the ordinary technique of intraperitoneally PS injections, animal decapitation during the forcible immobilization of the awaked animal) on the baseline level of water in brain tissue were determined in non-anesthetized and anesthetized animals. Water content and the number of ouabain receptors in different organs and in different brain zones were determined in intact and testing (saline injected) rats. The decapitation of rats was performed after their anesthetization by Diphenyl ether. In order to stop the brain metabolism, the removed brain was dipped into liquid nitrogen for 15 sec. For the estimation of the saline injection-induced pain sensation on brain tissue hydration, the nonanesthetized animals (n=5) were decapitated after their sharp immobilization by dipping the head into liquid nitrogen (3-4 sec). After such procedures the full absence of somatic reflexes on extra stimuli was recorded. The testing tissue slices (thickness ~0,5mm) of heart (n=10), kidney (n=10), liver (n=10), spleen (n=10), brain cortex (n=10), stem (n=10) and cerebellum (n=10) were taken from each rat. Definition of tissue water content After measuring the wet weight (w.w.) of brain tissue samples, the latter were dried in thermostat (T-121, Russian production) at 1050C during 24 hours for determination of dry weight Page 94


Journal of International Dental And Medical Research ISSN 1309-100X Analgesic effect of hypertonic solution http://www.ektodermaldisplazi.com/journal.htm Sinerik Ayrapetyan, Gohar Musheghyan, and Anush Deghoyan

(d.w.). The quantity of water in 1 g of d.w. was counted by the following equation: (w.w. – d.w.) / d.w. (10) and expressed as water content in grams per gram of dry weight.

The statistical probability was expressed on figures with the help of asterisks (*).

Counting the number of ouabain receptors in cell membrane of different brain tissues Brain tissue slices with 1mm thickness were prepared by microtome MT-23 (Russian production). Brain tissue samples (ten from each tissue of one rat) were incubated in testing saline containing 10-8 M [3H]ouabain (12 Ci/mM activity), and then washed threefold for 10 min with the same concentration of testing saline containing unlabeled ouabain molecules to remove the tritium-labeled ouabain absorbed into the extracellular space and not connected with the receptors 6. The tissue pieces were placed in special vials and homogenized with HNO 3 . Finally, 5 ml of Bray's scintillation fluid was added, and the mixture was counted in a Wallac-1450 liquid scintillation counter (Perkin Elmer, Finland).

The level of tissue hydration of different organs of rats, including brain, can be changed by the intraperitioneally injection of DW or PS of different tonicities, or by giving the rats DW instead of regular drinking water during 3 and 5 days. To find out the emotional stress effect of the saline injection procedures on rat brain tissue hydration, the effect of 3 ml of isotonic PS injection on brain tissue hydration in anesthetized and non-anesthetized animals was studied. As can be seen in Figure 1, in anesthetized group of animals (n=10) the saline injection had no significant effect on brain tissue hydration (Figure 1A), while in non-anesthetized animals it led to the cortex hydration by 10%±1.32 and to the stem and cerebellum tissue dehydration by 15,8%±1.77 and 5,5%±2.13, correspondingly, as compared with the control (non-injected animals) group (Figure 1B). The sensitivity of brain tissue hydration to the injection of isotonic solution in non-anesthetized animals could be explained by the injection-induced pain sensation of animals. The differences between the responses of various parts of the brain to the injection-induced stress could be explained by their different functional role in stress responses of organism, which could be the subject for a special investigation.

Determination of pain threshold The hot plate test was conducted by a specific setup constructed in our laboratory and approved by the Ethic Committee of UNESCO Chair in Life Sciences. The setup consists of the org-glass chamber with the brass bottom. The temperature of the bottom (510C) was controllable and a thermometer (accuracy of measurement ±0,01oC) was placed on it. In order to keep the temperature constant, the brass bottom was completely covered by the Plexiglas box. A rat was placed in this chamber and pain latency was recorded. Latency (in sec) is defined visually as the time elapsed until one of the following responses: licking the feet, jumping or rapidly stamping the feet. For preventing the tissue damage the cut-off time was chosen 50 sec. Statistical analysis The Microsoft Excel and Sigma-Plot (Version 8.02A) were used for the data analysis. The mean value and standard error of hydration index of different tissues and pain threshold were calculated and the statistical probability was determined by Student's t-test with the help of the computer program Sigma-Plot (Version 8.02A). Volume 3 ∙ Number ∙ 2 ∙ 2010

Results

Figure 1a.

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Journal of International Dental And Medical Research ISSN 1309-100X Analgesic effect of hypertonic solution http://www.ektodermaldisplazi.com/journal.htm Sinerik Ayrapetyan, Gohar Musheghyan, and Anush Deghoyan

Figure 1b. Figure 1ab.The effect of 3 ml isotonic PS interperitoneal injection on tissue hydration of different brain zones in anesthetized (a) and nonanesthetized (b) animals, expressed in %, compared to the data of intact animals. *P<0.05. For studying of the correlation between tissue hydration and pain threshold in rats, the experimental animals were divided into six groups, per 10 animals in each group: control-1intact; Control-2 -animals intraperitoneally injected of 3 ml isotonic PS; animals intraperitoneally injected of hypertonic solution (Mannitol); and DW, as well as the intact animals whose were given DW instead of regular drinking water for 3 and 5 days. In all control and experimental groups the pain threshold to the hot plate was determined before the dissection. From the data presented in Table 1 we can see that intraperitoneally injection of 3 ml DW led to brain tissue hydration that was accompanied by the increase in the number of ouabain receptors in it. Such DW injection led to the increase of brain cortex, stem, and cerebellum hydration by 42%±2.37, 34.6%±2.75, 23%±3.075 and the number of ouabain receptors- by 45%±2.45, 38%±2.76, 23%±1.13, correspondingly, compared with the data of Control-2 (isotonic PS injected) animals. The intraperitoneal injection of hypertonic solution (Mannitol) led to the brain tissue dehydration: cortex- 26,5%±0.69, stem- 12,3%±0.69 and cerebellum- 17,5%±0.73 and the decrease in the number of ouabain receptors: cortex- 27%±1, stem- 10.2%±0.55, cerebellum- 23%±0.9, correspondingly. These data clearly show that Volume 3 ∙ Number ∙ 2 ∙ 2010

there are close correlation between brain tissue hydration and the number of functionally active ouabain receptors in the cell membrane that could serve as a marker for other membrane proteins, like as ionic channels and receptors. The fact that the brain cortex hydration is more sensitive to the osmotic stress than the stem and cerebellum tissues hydration could indicate on the crucial role of the cortex in stress sensation of the organism rather than other brain zones. To find out the correlation between brain cortex hydration and pain threshold the measurements of the pain threshold of each animal before and after 30 min of test solution injection were performed. Because of a big variation of the initial value of pain thresholds in different animals, the factor induced changes of pain threshold compared with the initial one were expressed in %.

Figure 2. The pain threshold of rats to the “hot plate” before (Control) and after interperitioneal injection with isotonic (PS), hypertonic (2 M Mannitol) physiological solutions and distilled water (DW), expressed in % compared to the initial pain threshold of the same animal (n=10). *P<0.05; **P<0.01. As shown in Figure 2, the pain thresholds (latent period) to the hot plate of rats injected to isotonic PS, and DW were decreased by 48,2%±2.34 and 17%±5.41, correspondently, while in hypertonic solution (Mannitol) injected animals it was increased by 21%±5.41 (n=10). The fact that in rats injected to isotonic PS, the pain threshold was decreased can be explained by the emotional stress effect on animals to the injection procedures, which was accompanied by the increase of brain cortex hydration (Fig 1). The DW injection had double effect on pain threshold; Page 96


Journal of International Dental And Medical Research ISSN 1309-100X Analgesic effect of hypertonic solution http://www.ektodermaldisplazi.com/journal.htm Sinerik Ayrapetyan, Gohar Musheghyan, and Anush Deghoyan

from one hand there was a dilution of extracellular Na ion concentration that caused the decrease of membrane excitability, from the other hand the dilution of extracellular medium caused the cell swelling. To avoid the injectioninduced side effects, in the next series of experiments the correlation between brain tissue hydration and pain threshold was studied on two groups of animals which were provided with DW (instead of normal drinking water) ad libitum during 3 and 5 days. As can be seen on Table 2, in animals provided with DW there are timedependent increase of brain tissue hydration and the increase on the number of ouabain receptors in it, compared to the brain tissue hydration of animals provided with normal drinking water. It is worth to note that as in previous series of experiments again the cortex tissue hydration was more sensitive to DW than brain stem and cerebellum tissues hydration. The comparative study of the pain threshold of rats before and after 3 and 5 days of providing the DW as drinking water can be seen on data presented in Figure 3, where the time-dependent decrease of pain threshold was observed. Such negative close correlation between brain tissue hydration (number of ouabain receptors) and pain threshold could serve as a strong evidence for the working hypothesis of the present work that the pain-relieving effect of hypertonic saline can be explained by dehydration–induced depression of neuromembrane excitability in brain cortex tissue.

Figure 3. The pain threshold of rats to “hot plate” before (Control) and after 3 and 5 days of providing with distilled water (DW) instead of drinking water, expressed in % compared to the initial pain threshold of the same animal. *P<0.05. Volume 3 ∙ Number ∙ 2 ∙ 2010

Discussion Hyperosmolar therapy is one of treatment interventions in the care of patients with severe head injury resulting in cerebral edema and intracranial hypertension. However, the cellular and molecular mechanisms underlying the therapeutic effect of hypertonic solution are not clear yet. Earlier studies have shown that the metabolic controlling cell hydration is a dynamic parameter determining the cell functional activity, like membrane excitability, chemo sensitivity, enzyme activity12, 13. Even during the generation of a single action potential the cell swells and shrinks: at the ascending phase of action potential (depolarization) the squid’s giant axon and crab’s nerve fibers swell 14. The cell swelling also takes place as a result of electrical and chemical substances-induced increase of membrane excitability 6 or cell poison 15-17. As the close correlation between cell hydration and membrane excitability is well documented on single isolated neurons, the cell over hydration was suggested as a messenger for nociceptive signals generation7-9. The cardiac glycoside–ouabain, which is traditionally considered as a specific inhibitor for Na+/K+ pump, has high affinity receptors in neuronal membrane (at concentration of less than 10-7 M), the function of which is not connected with the function of Na+/K+ pump 6,13. The number of these receptors as the other membrane proteins is in functionally active and inactive (reserve) states, depending on cell hydration6. Therefore the number of binding labeled molecules of 3H-ouabain to cell membrane at its concentration of less than 10-7 M was suggested as a marker for estimation of cell membrane active surface (cell hydration)11. The obtained data in the present work clearly demonstrate that there is a close correlation between tissue hydration and number of ouabain receptors (marker for membrane proteins) in the membrane: the increase of tissue hydration leads to the increase in the number of ouabain receptors, while the hypertonic PS application has opposite effect on it (Tables 1 and 2). The fact that the increase of animal brain tissue hydration and number of ouabain receptors (Table 1 and 2) decrease the pain thresholds to hot plate, while its dehydration and decrease of number of ouabain receptors hava opposite effect on pain threshold (Figure 2 and 3) Page 97


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confirms our early hypothesis according which the nerve cell over hydration serves as a messenger for pain signal generation (7-9). The data on the increase of brain cortex hydration and number of ouabain receptors, in response to the isotonic PS-injection in nonanesthetized animals (Figure 1B), which was absent in anesthetized animals (Figure 1A) can be explained by direct influence of the injectioninduced pain sensation on cortex tissue hydration. The fact that in non-anesthetized animals, after isotonic PS injection we observed also the decrease of pain threshold (Figure 1) indicates on the existence of close correlation between cell hydration in cortex and pain thresholds in rats. A comparatively higher sensitivity of the brain cortex hydration to osmotic stress induced by injection of hypertonic solution and DW, as well as to the animals, to which DW was provided as a drinking water (Tables 1 and 2), indicates the

crucial role of cortex in stress reaction of organisms. Conclusions Thus, the obtained data in the present work and our early data obtained on single snail neurons and squid axons 13, allow us to conclude that the analgesic effect of hypertonic saline on brain trauma (over-hydrated cells) is due to dehydration-induced cell shrinkage of brain cortex cells, causing the depression of membrane excitability as a result of the decrease in the number of functional active proteins in the membrane. Acknowledgements The authors thank to Ms. Hekimyan for technical assistance.

Armenuhi

Table 1. The tissue hydration and number of ouabain receptors of different brain zones in intact (Control 1), isotonic PS injected (Control 2), DW and hypertonic PS (Mannitol injected) animals. The experimental results were compared with control 2 group of animal. Each group of animal was consist of 10 animal. The difference between control 2 and experimental groups was statistically significant (***P<0.001).

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Journal of International Dental And Medical Research ISSN 1309-100X Analgesic effect of hypertonic solution http://www.ektodermaldisplazi.com/journal.htm Sinerik Ayrapetyan, Gohar Musheghyan, and Anush Deghoyan

Table 2. The tissue hydration and number of ouabain receptors of different brain zones in intact animals (Control), provided with normal drinking water and animals provided with DW during 3 and 5 days. The experimental results were compared with control group. Each group of animal was consist of 10 animal. The difference between control 2 and experimental groups was statistically significant (***P<0.001). References 1. Puliyel JM, Bhambhani V. Ketoacid levels may alter osmotonicity in diabetic ketoacidosis and precipitate cerebral edema. Archives of Disease in Childhood 2003;88:366 2. Vamvakas S, Brüning T, Thomasson B. Renal cell cancer correlated with occupational exposure to trichloroethylene. J Cancer Res Clin Oncol. 1998; 124: 374–82. 3. Misery L, Meyronet D, Pichon M, Brutin JL, Pestre P, Cambazard F. Aquadynia: a role for VIP? Ann Dermatol Venereol 2003; 130: 195–8. 4. 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(2): 179-186. 5. Ayrapetyan SN, Arvanov VL, On the mechanism of the electrogenic sodium pump dependence of membrane chemosensitivity. Comp Biochem Physiol 1979; 64(A): 601-604. 6. Ayrapetyan SN, Suleymanyan MA, Sagian AA, Dadalyan SS. Autoregulation of Electrogenic Sodium Pump. Cell Mol Neurobiol 1984; 4: 367- 384. 7. Ayrapetyan SN. Cellular Mechanism of pain. In: Kepplinger B, Ray AD, Schmid H, eds. Pain- Clinical Aspects and Therapeutical Issues, Edition Selva Verlag, Linz, Austria, 1995; 311-327. 8. Ayrapetyan SN. Theoretical aspects of magnitotherapy of pain. Abstr. of the 7th Int. Symposium "The Pain Clinic", Istanbul, Turkey, 1996: 171-2. 9. Ayrapetyan SN. The application of the theory of metabolic regulation to pain. In: Ayrapetyan SN, Apkarian AV, eds. Pain Mechanisms and Management. IOS press, Netherlands, 1998; 3 14. 10. Adrian RH. The effect of internal and external potassium concentration on the membrane potential of frog muscle. J Physiol 1956; 133: 631–658. 11. Danielian A.A., Ayrapetyan S.N. Changes of Hydration of Rats’ Tissues after in Vivo Exposure to 0.2 Tesla Steady Magnetic Field. Bioelectromagnetics, 1999; 20(2): 123-128. 12. Parton G.R. and Simons K. The multiple faces of caveolae. Nature Reviews, 2007; 8: 185-194. 13. Ayrapetyan SN. On The Physiological Significant of Pump Induced Cell Volume Changes. Adv Physiolo Sci 1980; 23: 67-82. 14. Iwasa K, Tasaki I, Gibbons RC. Mechanical changes in crab

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nerve fibers during action potentials. Jpn J Physiol 1980; 30: 897905. 15. Cooke KR. Ouabain and regulation of cellular volume in freshly prepared slices of rabbit renal cortex. J Physiol 1978; 279: 361-374. 16. Ayrapetyan SN, Suleymanyan MA. On The Pump-Induced Cell Volume Changes. Comp Biochem Physiol 1979; 64A: 571-575. 17. Haussinger D. Regulation of Cell Function by Hydration. Biochem J, 1996; 313: 697-710.

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Flow Dynamics Men Exposed To Toluene Cemil Sert and et al

THE INVESTIGATION BY DOPPLER ULTRASONOGRAPY OF BLOOD FLOW DYNAMICS OF TESTES AND LIVER IN MEN EXPOSED CHRONICALLY TO TOLUENE Cemil Sert1, Ocal Sirmatel2, Erkan Yildiz3, Ferat Oruc2 1. Department of Biophysics, Faculty of Medicine, Harran University Sanliurfa / TURKEY. 2. Department of Radiodiagnostic, Faculty of Medicine, Harran University, Sanliurfa / TURKEY . 3. Department of Anatomy, Faculty of Medicine, Harran University, Sanliurfa / TURKEY.

Abstract Organic solvents used in various industrial processes may cause toxic effects in various biological tissues of human. Many previous studies have demonstrated, histopathologically, that toluene produced toxic effects in liver and testes. The aim of this study was to investigate the arterial and venous blood flow rate in the liver and testes of male workers who experienced occupational exposure to toluene. The experimental group comprised 30 male painters who had experienced occupational exposure to toluene inhalation for a minimum of 10 years; a control group comprised 30 healthy, age-matched male volunteers. The blood flow rate of the testes and liver of both groups were determined by Doppler ultrasonography. We determined that some arterial and venous blood flow rates of liver and testes changed in painters exposed occupationally to toluene (p<0.05). The results indicated significant changes in arterial and venous blood flow rate of right testes, left testes arterial end diastolic flow, left testes arterial flow and main hepatic venous blood flow rate of the right lobe of the liver. Other parameters showed no statistically significant difference. We conclude that long-term occupational exposure to toluene can affect arterial and venous blood flow. The results indicate testes and liver tissue damage in these subjects. (J Int Dent Med Res 2010; 0: (0), pp. 100-103 ) Key words: Toluene, Liver, Testes, Blood flow rate, ultrasonography. Received date: 12 March 2010 Introduction Toluene (C 7 H 8 ; methylbenzene, phenylmethane, and toluol) is a clear, colorless, volatile aromatic hydrocarbon and is an organic solvent. Approximately 92% of toluene produced is used in gasoline; the remaining 8%, purified as commercial toluene, is used in the production of industrial chemicals 1. Organic solvents are used in various *Corresponding author: Dr Cemil Sert Harran University Faculty of Medicine Department of Biophysics Yenisehir Kampusu 63100, Sanliurfa / TURKEY. E-mail: csert@harran.edu.tr

Volume 3 ∙ Number ∙ 2 ∙ 2010

Accept date: 07 June 2010 industrial processes, such as paint manufacturing, spray painting, shoe making, degreasing, metal processing, and auto manufacturing 2. Toluene vapor is rapidly absorbed from the respiratory tract; oral and dermal absorption occur more slowly. The half-life elimination of toluene in human blood is approximately 3-4 hours 3. Toluene is a significant social and public health problem in many countries. Because of its low cost and easy availability, toluene is a common source of substance abuse in adolescents and younger children. Only limited research has been conducted on the effects of toluene on human fertility. The effect of toluene in men is even more difficult to evaluate. In an occupational study, Merck et al. reported sexual disturbances and an increase in plasma levels of follicle –stimulating hormone (FSH) in men exposed to toluene 4. Toluene may influence the endocrine system of the developing Page 100


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fetus, and this effect could also influence the reproductive capacity in adulthood 3. It was reported that, toluene may adversely effect male reproductive functions. Testicular atrophy and reduced spermatogenesis were observed in one case involving chronic toluene abuse 3. Women whose husbands inhaled toluene-based solvents at work have an increased incidence of spontaneous abortion 5, while Rendon et al. showed that workers in rubber factories have elevated numbers of abnormal sperm 6. Contrary to these findings, Ono et al. observed that exposure to 2000 ppm toluene for days (6h/day) resulted in decreased sperm counts and epididymal weight in male rats, although it did not affect fertility 7. Murata et al. observed that toluene has reproductive toxic potential 8. Exposure to hepatotoxic solvents can occur in 1) occupational setting, through either daily inhalation or skin absorption of solvents; 2) residential setting, during either accidental or intentional ingestion in food, or as a toxic contaminant of food, or exposure to toxic agents such as in the form of glue sniffing; 3) environmental setting, commonly residential, usually through groundwater contamination, which includes ingestion of the water, skin contact through bathing in the water, and absorption, and volatilization of the solvents through heated bathing water 2. Several factors have been shown to affect the handling of solvents by the liver and testes final toxicity effects. The most important determining factors are 1) species difference; 2) liver blood flow; 3) protein binding; 4) points of binding inside the liver and testes intracellularly. Various solvents were used for many years until they were found to cause liver tumors. Scientist and physicians have to keep an open mind and constantly evaluate exposures in either of the above settings to assess liver effects 2. This study was designed to investigate effects on the testes and liver blood flow rate of subjects with chronic occupational toluene inhalation. In many studies, it was reported that toluene had a pathological effect on the liver and testes.

Flow Dynamics Men Exposed To Toluene Cemil Sert and et al

and; a control group of thirty male volunteers. None of the subjects reported acute or chronic illnesses. The study was approved by the Human Subjects Research Committee of Harran University. Measurements of arterial and venous blood flow rate of liver (V p , V end , V m , RI) and right and left testes of all subjects were measured by Doppler ultrasonography. (Esaote, Technos MPX 796FDII, CHINA). The results were analyzed using paired samples t-tests in SPSS (version 11.5 for Windows). The experimental group and control group were compared with one another. P-values below 0.5 were considered to be statistically significant.

Materials and Methods The present study included thirty volunteer males, all of whom worked as painters, Volume 3 ∙ Number ∙ 2 ∙ 2010

Table 1. Arterial and Venous blood flow rate (m/s) of testes. Page 101


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Flow Dynamics Men Exposed To Toluene Cemil Sert and et al

Results

function and morphology. In published studies, it was claimed that toluene did not induce adverse No significant difference was observed effects on fertility of rats 9 or human males between the groups in terms of the dimensions of (American Petroleum Institute). Roberts et al. liver, right and left testes (p>0.05; table 1). reported that the effects on the 2-regeneration Arterial and venous blood flow rate were reproductive toxicity of toluene did not adversely measured in both groups. Right testes venous affect fertility and reproductive 1. flow rate, left testes arterial end diastolic flow rate, Toluene is metabolized by the liver; and left testes arterial flow rate differed however, the liver does not appear to be primary significantly between the experimental and target for toluene toxicity. In study of Guzelian et control groups (p<0.05, p<0.01, p<0.01; table 2). al. seven of the patients had liver biopsies, which Other parameters were not significantly showed some centrally lobular and periportal fat different between the experimental and control accumulation, and Kupffer cell hyperplasia 10. A groups (table 3). study by Swensson et al. has looked at 47 rotogravure workers occupationally exposed to toluene and showed elevation of liver enzymes and chemical hepatitis 11. Experimental animals exposed to toluene at concentrations of 500 to 800 ppm for 7 days showed increased liver weights, but no significant morphological changes by microscopy. Electron microscopical studies revealed ultrastructural changes which were compatible with changes in cytochrome p-450 concentrations. Other studies have shown no effect on liver size or liver function 12,13. It is highly likely that, in predisposed individuals, toluene can cause liver damage, especially in those patients who have fatty liver changes from other causes. Tomei et al. looked at liver damage among shoe repairers who use toluene, among other solvents 14. Hepatotoxicity has been reported throughout the literature in individuals exposed to xylene and toluene 15. Dalgaard et al. observed no effect on sperm parameters in rats which were exposed Table 2. Arterial and Venous blood flow rate pre-and postnatally to 1200 ppm toluene; They (m/s) of liver. found no significant differences in mating, fertility, or pregnancy indices 3. Any differences in mating, fertility or pregnancy indices were found after in utero exposure to 1200 ppm toluene. This finding is in accordance with a study by Theil and Chahoud 16. However, Ono et al. reported that two studies of rats exposed to high levels (2000 or 6000 ppm) of toluene resulted in direct toxic 17 Table 3. Dimensions of liver as milimeter, right effects on the epididymis . The findings of Ono et al. indicate that and left testes as milimeter square. toluene inhalation may adversely affect male reproductive functions, although no exposureDiscussion related effects were noted on the weights or features of the male Several studies have reported that histopathological reproductive organs. They reported that toluene exposure produces adverse effects on the testes, sperm morphology, and both liver epididymal sperm counts were significantly Volume 3 ∙ Number ∙ 2 ∙ 2010

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reduced in rats exposed to 6000 ppm toluene 17. At the same time, the sperm motion parameters were also suppressed on the whole in 600 ppm groups, although the differences were not significant. These findings reveal that toluene inhalation at 6000 ppm, 2h/day for 5 weeks, suppresses the number of sperm, the sperm quality and sperm activity. The results of their study emphasize that toluene exposure did not induce morphologic changes in testes or alter spermatogenesis within the testes. Their findings indicated that toluene does not directly affect spermatogenic cells within the testes, but may act on spermatozoa within the epididymis. These observations are consistent with our previous finding that inhalation of toluene (2000 ppm 6 h/ day) for 90 days decreases sperm counts and sperm motility in rats, but dose not affect the spermatogenesis in testes or in vivo fertility 6. Conclusions This study did not detect testicular atrophy, but found that males occupationally exposed to toluene experienced blood flow problems, especially within the testes. This study also found that Doppler ultrasonography may be used as a routine procedure to measure blood flow in workers occupationally exposed to toluene. Declaration of Interest

Flow Dynamics Men Exposed To Toluene Cemil Sert and et al

chromosome aberrations and in abnormal sperm morphology in rubber factory workers. Mutat Res 1994;323, 151-157. 7. Ono A, Sekita K,Ogawa Y, Hirose A, Saito, M, Naito K, Kaneko T, Furuya T, Kawashima K, Yasuhara K, Matsurnoto K, Tanaka S, Inoue T, Kurokawa Y. Reproductive and developmental toxicity studies of toluene. Effects of inhalation exposure on fertility in rats. J Environ. Pathol.Toxicol. Oncol.1996;15: 9-20. 8. Murata M, Tsujikawa M, Kawanishi S. Oxidative DNA Damage by Minor Metabolites of Toluene May Lead to Carcinogenesis and Reproductive Dysfunction. Biochemical and Biophysical Research Communications. 1999; 261: 478473. 9. Tap O, Solmaz S, Polat S, Mete UO, Ozbilgin MK, Kaya M . The effect of toluene on the rat ovary: an ultrastructural study. J Submicrosc Cytol Pathol 1996;28:553-558. 10. Guzelian P, Mills S, Fallon JJ. Liver structure and function in print workers exposed to toluene. J Occup Med 1998;30: 791796. 11. Svensson BG, Nise G, Erfurth EM, Olsson J. Neuroendocrine effects in printing workers exposed to toluene. Br J Ind Med 1992;49: 402-408. 12. Kjellstrand P, Bjerkemo M, Adler-Maihofer, Holmquist B. Effects of solvent exposure on testesterone levels and butryrylcholinesterase activity in mice. Acta Pharmacol Toxicol 1985;57:242-249. 13. NTP-National Toxicology Program-Technical Report series, Toxicology and Carcinogenesis Studies of Toluene. (CAS No.108-88-3) in F344/N rats and 86C3F mice (inhalation studies), research Triangle Park, NC, U.S. Environmental Protection Agency, U.S. Dept of Health and Human Services. 1990; No.371. PB90-256371. 14. Tomei F,Giuntoli P, Biagi M, Baccolo T, Tomao E, Rosati, M. Liver damage amongs shoe repairers. Amer. Journ. Of Ind. Med 1999;36: 541-547. 15. Chen JD, Wang JD, Jang JP, Chen YY. Exposure to mixtures of solvents among paint workers and biochemical alterations in liver function. Br J Ind Med 1991;48: 696-701. 16. Theil R, Chahoud I. Postnatal development and behaviour of Wistar rats after toluene exposure. Arch Toxicol 1997;71: 258-265. 17. Ono A, Sekita K, Hirose A, Ogawa Y, Saito M, Naito K, Yasuhara K, Kaneko T, Furuya T, Onoue T, Kurokawa Y (). Toluene inhalation induced epididymal sperm dysfunction in rats. Toxicology. 1999;139: 193-205.

The authors report no conflict of interest and the article is not funded or supported by any research grant. References 1. Roberts LG,Bevans AC,Schreiner CA. Developmental and reproductive toxicity evaluation of toluene vapor in the rat I. Reproductive toxicity. Reproductive Toxicology 2003; 17: 649658. 2. Brautbar N,Williams J. Industrial solvents and liver toxicity: Risk assesment, risk factors and mechanisms. International journal of hygiene and Environmental Health 2002; 205: 479491. 3. Dalgaard M, Hossaini A, Hougaard KS, Hass U, Ladefoged O. () Developmental toxicity of toluene in male rats: effects on semen quqlity, testis morphology, and apoptotic neurodegeneration. Arch Toxicology 2001;75: 103-109. 4. Mǿrck HI, Winkel P, Gyntelberg F (). Health effects of toluene exposure. Dan Med Bull 1988;35: 196-200. 5. Taskinen H, Anttila A,Lindbohm ML, Sallmen M, Hermminki K (). Spontaneous bortions and congenital malformations among the wives of men occupationally exposed to organic solvents. Scan.J. Work Environ.Health 1989;15: 345-352. 6. Rendon A, Rojas A, Fernandez SI, Pineda I. Increase in

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