Serbian Journal of Experimental and Clinical Research Vol11 No3

LITERATURE:1. Cloninger CR, Svrakic D, Przybeck TR. A PsychobiologicalModel of Temperament and Character. Archivesof General Psychiatry 1993; 50: 975-9902. Zuckerman M. The sensation seeking motive. In: MaherBA. Progress in Experimental Personality Research.Vol. 7. New York: Academic Press, 1974.3. Masse LC, Tremblay RE. Behavior of boys in kindergartenand the onset of substance use during adolescence.Arch. Gen. Psychiatry 1997; 54 (1): 62–68.4. Malatesta V, Sutker PB, Treiber FA. Sensation seekingand chronic public drunkenness. J. Consult. Clin. Psychol1981; 49: 292–294.5. Cloninger CR, Bohman M, Sigvardsson S. Inheritanceof alcohol abuse. Cross-fostering analysis of adoptedmen. Arch Gen Psychiatry 1981; Aug 38 (8): 861-8.6. LeBon O, Basiaux P, Streel E, et al. Personality profile anddrug of choice; a multivariate analysis using Cloninger’sTCI on heroin addicts, alcoholics, and a random populationgroup. Drug Alcohol Depend 2004; 73 (2): 175–182.7. Cloninger CR, Przybeck TR, Svrakic D, Wetzel R. TheTemperament and Character Inventory (TCI): A guideto its development and use, Washington UniversitySchool of Medicine. St. Louis: Department of PsychiatryMO, 1994.8. Dukanac V. Cross Cultural Validation of TCI – Belgradedata. Master Thesis; unpublished data, 1995.9. Svrakic D, Whitehead CA, Przybeck TR, Cloninger CR.Differential Diagnosis of Personality Disorders by theSeven-Factor Temperament and Character Inventory.Archives of General Psychiatry 1993; 50: 991-999.10. SAS 9.1. SAS Institute Inc. USA: NC, Cary, 2002-2003.11. Cloninger CR, Sigvardsson S, Przybeck D, SvrakicD. Personality Antecedents Of Alcoholism in a NationalArea Probability Sample. European Archivesof Psychiatry and Clinical Neurosciense 1995; 245(4-5): 239-244.98

ORIGINAL ARTICLE ORIGINALNI NAUČNI RAD ORIGINAL ARTICLE ORIGINALNI NAUČNI RADINFLUENCE OF MODULATORS OF RELAXANT EFFECTOF PENTOXYPHYLLINE IN ISOLATED RAT UTERUSJelena Kordic-Bojinovic 1 , Dragana Jokanovic 1 , Dragana Stankovic 1 , Slobodan Jankovic 2 and Slobodan R. Milovanovic 11Department of Pharmacology, Toxicology and Clinical Pharmacology,Faculty of Medicine, University of East Sarajevo, Foca, Republic of Srpska, Bosnia and Herzegovina2Department of Pharmacology and Clinical Pharmacology, Faculty of Medicine, Kragujevac, SerbijaUTICAJ MODULATORA RELAKSANTNOGEFEKTA PENTOKSIFILINA NA IZOLOVANOM UTERUSU PACOVAKordić-Bojinović 1 , Dragana Jokanović 1 , Dragana Stanković 1 , Slobodan Jankovićc 2 i Slobodan R. Milovanović 11Odsek za farmaciju, toksikologiju i kliničku farmakologijuMedicinski fakultet, Univerzitet u Istočnom Sarajevu, Foča, Republika Srpska, Bosna i Hercegovina2Odsek za farmaciju, Medicinski fakultet Univerziteta u Kragujevcu, SrbijaReceived / Primljen: 9. 6. 2010 . Accepted / Prihvaćen: 10. 7. 2010.ABSTRACTBackground. Pentoxyphylline is a methylxanthine derivativeused in the treatment of peripheral vascular diseases.One effect of pentoxyphylline action is the vasodilatation ofblood vessels. In this study, the effect of increasing concentrationsof pentoxyphylline on contractility of isolated rat uteriwas examined.Methods. Uteri were isolated from virgin Wistar rats(180–220 g) and suspended in an isolated organ bath chambercontaining De Jalon’s solution and aerated with 95% O2and 5% CO2. The temperature was maintained at 37ºC. Isometriccontractions were recorded using an isometric forcetransducer (Ugo Basile). The preload of the preparation wasabout 1 g. Uteri were allowed to contract spontaneously orin the presence of Ca2+ (6 mM) and were treated with pentoxyphylline.Results. Pentoxyphylline caused concentration-dependentinhibition of spontaneous rhythmic uterine activity anduterine activity induced by calcium. We showed that the inhibitoryeffect of pentoxyphylline depends on the type of musclecontraction activation, and that it is significantly strongerin spontaneous contractions induced by calcium Ca2+. Asopposed to methylene blue, L-arginine and glibenclamidedid not antagonise the relaxing effect of pentoxyphylline onthe isolated rat uterus.Conclusion. Our results suggest that the signaling pathwayby which pentoxyphylline causes relaxation of uterinemuscle cells does not involve NO because the presence of L-arginine did not affect the action of the drug; however, it maydepend on an NO-independent cGMP signaling pathwaybecause the presence of methylene blue significantly antagonisedthe effect of pentoxyphylline. These results indicate thatpentoxyphylline could be a potential tocolytic drug.Key words: pentoxyphylline, rat uterus, L-arginine, glibenclamide,methylene blueRunning title: Pentoxyphylline Inhibits Contractility ofRat UterusSAŽETAKCilj. Pentoksifilin, koji se koristi za lečenje perifernihvaskularnih obolenja, je derivat metilksantina. Jedan odnačina delovanja pentoksifilina je prouzrokovanje vazodilatacijekrvnih sudova. U ovom radu ispitivali smo efektrastućih koncentracija pentoksifilina na kontraktilnostizolovanog uterusa pacova.Metode. Uterusi, koji su izolovani od neparenih ženkipacova Wistar soja (180-220 g),držani su u kupatilu zaizolovane organe na temperaturi od 37ºC, u De Jalon-ovomrastvoru kroz koji je propuštana mešavina od 95% kiseonikai 5% ugljendioksida. Izometrijske kontrakcije su registrovanekorišćenjem izometrijskog transducera Ugo Basile, priopterećenju preparata od 1 g. Efekt pentoksifilina je ispitivanna kontrakcije za vreme spontane ritmičke aktivnosti iu prisustvu kalcijuma (6 mM),Rezultati. Pentoksifilin je prouzrokovao koncentracijskizavisnuinhibciju spontane rithmičke aktivnosti, kao i fazneaktivnosti prouzrokovane kalcijumom. Inhibitorni efekt pentoksifilinazavisio je od tipa aktivacije glatkog mišića uterusa.On je ispoljio značajno jači relaksantni efekt na kontrakcijeprouzrokovane kalcijumom. Nasuprot metilenskom plavilu,L-arginin i glibenklamid ne antagonizuju relaksantni efektpentoksifilina na izolovanom uterusu pacova.Zaključak. Dobijenii rezultati sugerišu da signaliniputevi sa kojima pentoksifilin prouzrokuje relaksaciju glatkihmišičnih ćelija uterusa, za razliku izolovanih krvnihsudova, verovatno ne uključuje u većoj meri prisustvo NO(jer prisustvo L-arginine nije menjalo efekt ovog leka), alizavisi od cGMP signalinih puteva nezavisnih od NO (zbogtoga što prisustvo metilenskog plavila značajno antagonizujenjegov efekt). Ovi rezultatia ukazuju da bi pentoksifilinmogao da bude potencijalni tokolitički lek.Ključne reči: pentoksifilin, uterus pacova, L-arginin glibenclamidi metilensko plaviloKratki naslov: pentoksifilin inhibiše kontrakcije uterusapacovaUDK 615.256 / Ser J Exp Clin Res 2010; 11 (3): 99-104Correspondence to: Slobodan R. Milovanovic, M.D., Ph.D. / Department of Pharmacology, Toxicology, and Clinical Pharmacology, School of Medicine,University of East Sarajevo, Foca, Republic of Srpska, Bosnia and Herzegovina / E-mail: milarados@ptt.rs / Phone: + 387 651 560-50499

INTRODUCTIONTocolytics, such as β2-adrenergic agonists, are frequentlyused to prevent miscarriage and premature birth,however these compounds are associated with insufficientefficacy and excessive side-effects. 1 Accordingly, there is aneed to identify novel drugs with tocolytic characteristics,such as calcium agonists, potassium channel openers andother vasodilators. 2 It has been shown that even otomolarconcentrations of nicardipine inhibit spontaneous rhythmicalactivity of the isolated uterus. 3 Nitric oxide (NO) isinvolved in numerous physiological processes and pathologicalconditions, and it mediates the relaxing effect ofprotamine sulphate and other smooth muscle vasodilators.4 In cells, NO is created under the influence of NOsynthesis.5 High doses of L-arginine increase blood flow inthe heart, mesenterium, lungs and liver without affectingtotal peripheral resistance and blood pressure. 6 L-arginine,however, can cause significant hypotension in normotensiverats pretreated with physostigmin. 7 Previous reportsshowed increased NO synthesis during normal pregnancyin animals. In humans, lack of NO causes vasoconstrictionand pre-eclampsia. NO is characterised by extreme reactivityto intracellular enzymes and has been shown to affectactivity of guanylate cyclase (GC). Reaction betweenNO and GC can be inhibited with methylene blue. 8Pentoxyphylline is a methyl xanthine derivative used totreat peripheral vascular diseases. Potential indications forthis drug, as well as its mechanism of action at the molecularlevel, are being intensively studied. 9In previous studies, we showed that the endotheliumplays a significant role in the relaxing mechanism of pentoxyphyllinein isolated rat mesenteric arteries. In thepresent study, we examined the effects of increasing pentoxyphyllineconcentrations on spontaneous rhythmicalactivity of isolated uteri and on calcium chloride-causedactivity in the uterus. To elucidate the mechanism of actionof pentoxyphylline in uterine smooth muscle, we studiedthe drug’s effects in the presence of L-arginine (an NOprecursor), glibenclamide (a potassium channel antagonist)and methylene blue (a GC inhibitor).MATERIALS AND METHODSAll protocols for handling rats were approved by thelocal Ethical Committee for Animal Experiments, whichstrictly follows international regulations. Isolated uteri ofvirgin Wistar rats (200–250 g) in oestrus, as determinedby daily examination of vaginal lavage, were used in thisstudy. Each uterus was suspended in an isolated organbath chamber (Ugo Basile) containing De Jalon’s solution(NaCl 9.0 g/l, KCl 0.42 g/l, NaHCO 30.5 g/l, CaCl 20.06 g/l,glucose 0.5 g/l) and aerated with 95% O 2and 5% CO 2. Thetemperature was maintained at 37°C. Isometric contractionswere recorded using an isometric force transducer(Ugo Basile). The uteri, which were either spontaneouslyactive or induced with 6 mM Ca 2+ , were allowed to equilibrateat 1 g of tension before experimental drugs wereadded. After establishing stable spontaneous contractions(approx. 20 min), uteri were treated with increasingconcentrations of pentoxyphylline (4.2 mM, 12.8 mM,29.9 mM, 64.1 mM, 106.9 mM and 192.3 mM) until totalcessation of contractions. To explore the mechanism ofaction of pentoxyphylline on the uterus smooth muscle,we studied its effects in the presence of L-arginine (0.3μmol), glibenclamide (2 × 10 –6 mol/l) and methylene blue(0.9 × 10 –6 mol/l), which were added to De Jalon’s solution10 min before pentoxyphylline.The effects of these treatments on uterine contractionswere calculated as the percentage of control contractions in untreateduteri. All data are expressed as the mean ± SEM. Differencesbetween groups were tested by two-way ANOVA withtreatment and dose as factors. Differences between groupswere considered statistically significant when p < 0.05.Pentoxyphylline, methylene blue, L-arginine and glibenclamidewere purchased from Sigma-Aldrich (St. Louis,MO, USA). Salts for De Jalon’s solution were obtainedfrom ZORKA Pharma (Sabac, Serbia) and Merck (Darmstadt,Germany). All drugs were dissolved in distilled waterexcept for glibenclamide, which was dissolved in polyethyleneglycol.RE SULTSEffect of pentoxyphylline on spontaneous rhythmical activityand Ca 2+ -induced contractions of isolated rat uterusIncreasing pentoxyphylline concentrations (4.2 mM,12.8 mM, 29.9 mM, 64.1 mM, 106.9 mM and 192.3 mM) resultedin concentration-dependent inhibition of spontaneousrhythmical activity and calcium-induced contractionsof the isolated rat uterus. The degree of pentoxyphyllineinhibitory effect depended on activation type. Pentoxyphyllineexhibited a stronger relaxing effect on calciuminduceduterus contractility. For instance, pentoxyphyllineconcentration of 106.9 mM, which completely inhibitedcalcium-induced contractions (98.08%), only partially inhibitedspontaneous rhythmical activity of the isolated ratuterus (52.94%). Total inhibition of calcium-induced contractionswas achieved with lower concentrations of pentoxyphylline(64.1 mM). In the case of calcium-inducedcontractions, pentoxyphylline caused time-dependentcontraction inhibition; increasing pentoxyphylline concentrationsresulted in increased duration of the calciuminducedcontractility (Figure 1A, B and C).Effect of pentoxyphylline on spontaneous rhythmical activityof the isolated rat uterus in the presence of glibenclamideand L-arginineIn this series of experiments, we showed that the presenceof glibenclamide (2 × 10 –6 mol/l) stimulates the relaxanteffect of increasing concentrations of pentoxyphyllineon the spontaneous rhythmical activity of the isolatedrat uterus. In control experiments without glibenclamide,100

In these experiments we studied the effects of increasingconcentrations of pentoxyphylline on spontaneous rhythmicalactivity of the isolated rat uterus in the presence ofmethylene blue (0.9 × 10 –6 mol/l). Methylene blue antagonisedthe relaxing effect of pentoxyphylline on spontaneousrhythmical activity of the isolated rat uterus. For example,in the presence of methylene blue, even the highestconcentration of pentoxyphylline, did not cause completeinhibition of uterine contractions (Figure 3A and B). Interestingly,the addition of pentoxyphylline inhibited uterinecontractions, however this inhibition quickly disappeared,and contractions returned to control levels (Figure 3B.).DISCUSSIONIn this study, we examined the effects of increasing concentrationof pentoxyphylline on the spontaneous rhythmicalactivity and Ca 2+ -induced contractions of the isolatedrat uterus. Pentoxyphylline induced concentration-dependentinhibition of spontaneous rhythmic uterine activityand uterine activity caused by calcium. We showed that thedegree of inhibition effect by pentoxyphylline depends onthe type of muscle contraction activation, and that it is sig-Figure 1.(A) Concentration–response curves for pentoxyphylline relaxation onthe spontaneous rhythmic contractions (closed square ■ ) and on the contractionsprovoked by Ca 2+ (closed diamond♦) of the isolated rat uterus.The amplitude of contractions just before addition of pentoxyphylline wastaken as 100%. The data points represent mean values and the verticallines indicate the S.E.M. (n = 8–12).(B) A representative original trace of spontaneous uterine contractionsinduced by pentoxyphylline at various concentrations (4.2 mM, 12.8 mM,29.9 mM, 64.1 mM, 106.9 mM and 192.3 mM).(C) A representative original trace of Ca 2+ -induced contractions of the ratuterus treated with pentoxyphylline (4.2 mM, 12.8 mM, 29.9 mM, 64.1mM and 106.9 mM).pentoxyphylline caused significant inhibition of spontaneousrhythmical activity at a concentration of 192.3 mM.On the other hand, in most experiments a pentoxyphyllineconcentration of 66.3 mM was sufficient for total inhibitionof spontaneous rhythmical contractions in the presenceof glibenclamide (Figure 2A and B).We also studied the effects of increasing concentrationsof pentoxyphylline on spontaneous rhythmical activity ofisolated the rat uterus and calcium-induced contractionsin the presence of L-arginine (0.3 μmol). In the presenceof L-arginine, pentoxyphylline did not change its inhibitoryeffect on spontaneous rhythmical activity or calciuminducedactivity.Influence of pentoxyphylline on spontaneous rhythmicalactivity of the isolated rat uterus in the presence ofmethylene blueFigure 2.(A) Effect of pentoxyphylline (4,2 mM, 12,8 mM, 29,9 mM, 64,1 mM,106,9 mM and 192,3 mM) on the spontaneous rhythmic contractions ofrat uteri in the presence (closed diamond♦) and absence of glibenclamide(2 × 10 –6 mol/l) ( ■ closed square). Contractile activity was expressedas the relative ratio between mean height peak of untreated control andtreated uteri. Data are expressed as the mean ± s.e.mean (n = 8 –12 ). Pretreatmentwith glibenclamide significantly increased the relaxing effect ofpentoxyphylline (p < 0.0001).(B) A representative original trace showing the effect of (2.1 mM, 6.4 mM,14.9 mM, 32 mM and 66,3 mM) on the spontaneous rhythmic contractionsof the rat uterus, in the presence of glibenclamide.101

levels necessary for birth contractions. 14 ATP-dependentpotassium channels (K ATP) also play an important role inuterine smooth muscle physiology; these channels formthe connection between the metabolic state of the cell andcell excitation (i.e., contractility). 15Changes in the expression and activity of potassiumchannels are very important for uterus contractility control.2 For example, H 2O 2induces concentration-dependentrelaxation of the isolated rat uterus via voltage-dependentpotassium channels. 16 Based on these findings, studyingpotassium channel modulators and their effect on NO inuterine tissue is important to better understand uterinephysiology and pathophysiology and to help identify newtherapeutic concepts in the treatment of uterus contractilitydisturbances. 2,3 Together with detailed information aboutpotassium channels, there is a pronounced pharmaceuticalinterest in the synthesis and development of selectivepotassium channel modulators, as well as re-evaluation ofthe spectrum of choice of existing drugs and substances influencingpermeability of smooth muscle cells membranesfor potassium ions (especially vasodilators with so-calleddirect effects, including monoxidil and diazoxide).It has been shown that glibenclamide leads to inhibitionof contractions of arterial muscle elements, probablyby inter-reacting with voltage-dependent calcium channelsand by blocking. 17 Our results could be interpreted inthe same way. It is possible that glibenclamide stimulatesthe relaxing effect of pentoxyphylline by blocking voltagedependentcalcium channels, thereby decreasing calciumentry into the cell, causing musMethylene blue inhibits production of cGMP by preventinginteraction of NO with guanylate cyclase. In ourexperiments, methylene blue decreased the relaxing effectof pentoxyphylline. Not even the highest applied concentrationof pentoxyphylline achieved complete discontinuationof contractions. It is interesting that inhibition causedby pentoxyphylline in the presence of methylene blue hasa short duration. That is, while pentoxyphylline inhibiteduterus contractions, the duration of this inhibition was briefand contractions returned to the values of the control level.Data showing that methylene blue antagonises therelaxing effect of sodium nitroprusside are consistentwith our results. 5 Methylene blue blocks the activation ofguanylate cyclases, thereby preventing muscle relaxation.cGMP activates cGMP-dependent protein kinase, whichblocks the entrance of calcium ions, activates potassiumchannels and decreases levels of IP 3, leading to vasodilatation.However, it has also been shown that methyleneblue fails to antagonise the relaxing effect of sodium azideand of nitroglycerol. 18Out results suggest that NO is not significantly involvedin the realisation of pentoxyphylline effects. There are dataabout the existence of signaling pathways in the cell, includingNO-independent cGMP creation, which does not leadto relaxation. It is possible that methylene blue influencesthese signaling pathways, leading to weaker relaxing effectsof pentoxyphylline in interaction with methylene blue.According to previous reports, pentoxyphylline actsas a phosphodiesterase blocker. 19 By blocking phosphodiesterase,pentoxyphylline directly increases cAMP levels inmuscle cells, leading to muscle relaxation. Since methyleneblue, a known inhibitor of guanylate cyclase, decreased theinhibitory effect of pentoxyphylline, it is possible that evencGMP is included in muscle relaxation via NO-independentsignaling pathways.CONCLUSIONSPentoxyphylline caused concentration-dependent inhibitionof spontaneous rhythmical activity and calciuminducedcontractions in isolated rat uteri. The magnitudeof the relaxing effect of pentoxyphylline depended on thetype of uterus smooth muscle activation. As opposed tomethylene blue, L-arginine and glibenclamide did not antagonisethe relaxing effect of pentoxyphylline on spontaneousrhythmical activity of the isolated rat uteri. Theseobservations suggest a possible mechanism of action ofpentoxyphylline in uterine smooth muscle cells. NO isprobably not significantly involved in the relaxing effect ofpentoxyphylline, because pentoxyphylline, in the presenceof L-arginine, did not significantly affect the degree of contractioninhibition. Because methylene blue antagonisedthe relaxing effect of pentoxyphylline, we conclude that asignalling pathway that involves cGMP and is independentof NO is possibly involved in the mechanism of its effectson uterine smooth muscle. Our results also suggest thatpentoxyphylline could be a potential tocolytic drug.REFERENCES1. Kalezić I, Rodić V, Kitanović S, Milovanović G,Zgradić I and Milovanović S. The Effects of Ritodrine,on a receptors in smooth uterine muscle andheart atria of rats. Arch Toxicol Kinet Xenobiot Metab1993; 1:112-118.2. Novaković R, Milovanović SR, Heinle H, Protić D, andGojković-Bukarica Lj. The effect of potassium channelopener pinacidil on non-pregnant rat uterus. Basic &Clinical Pharmacology & Toxicology 2007;1742-84.3. Milovanovic S R, Ognjanović J, Varagic VM, BoskovićB. Effect of nicardipine of the isolated rat uterus andother smooth muscles of the rat. Archives Internationalesde Pharmacodynamic et de Therapie 1988;295: 1348-58.4. Oreščanin-Dušić Z, Milovanović S, Spasić M,Radojičić R, Blagojević D. Effect of protamine sulfateon the isolated mesenteric arteries of normotensiveand spontaneously hypertensive rats. Arch Biol Sci2008;60:163-168.5. Orescanin Z, Milovanović SR, Spasic DS, Jones DR,Spasić MB. Different responses of mesenteric arteryfrom normotensive and spontaneousely hypertensive103

ats to nitric oxide and its redox congeners. PolishPharmacological Reports 2007; 59: 322-329.6. Van Geldern EM, Heilingers JPC, Saxena PR. Hemodynamicchanges and acetylholine-induced hypotensiveresponses after N-nitro-L-arginine-methyl-ester in ratsand cats. Br J Pharmacol 1991;103:1899-1904.7. Prostran M, Varagic VM, Todorovic Z, Jezdimirovic M.The effects of physostigmine, L-arginine and N-nitro-L-arginine-methyl-ester (L-NAME) on the mean arterialblood pressure of the rat. J Basic Clin Physiol Pharmacol1994; 5:151-166.8. Choi JW, Im MW, Pai SH. Nitric oxide productionincreases during normal pregnancy and decreases inpreeclampsia. Ann Clin Lab Sci 2002; 3:257-63.9. Matson, PL, Yovich, JM, Edirisinghe, WR et al. An argumentfor the past and continued use of pentoxyphyllinein assisted reproductive technology. Hum Reprod1995 (Suppl): 10; 67–71.10. Varagić, VM, Milovanović SR, Srkalović, G. The effectof calcium-channel-blocking agents on the varioustypes of smooth muscle activation of the isolated ratuterus. Arch Int Pharmacodyn 1984; 270; 79-87.11. Oreščanin Z, Milovanović S. Effect of L-arginine on therelaxation caused by sodium nitroprusside on isolatedrat renal artery. Acta Physiologica Hungarica 2006;93:271–283.12. Gojković-Bukarica L, Kazić T. Differential effects ofpinacidil and levcromakalim on the contractions elicitedelectrically or by noradrenaline in the portal vein ofthe rabbit. Fundam Clin Pharmacol 1999;3:527-34.13. Schubert U, Krien U, Wulfusen I. Nitric oxide donorsodium nitropruside dilates rat small arteries by activationof inward rectifier potassium channels. Hypertension2004; 43:891-96.14. Khan R N, Matharoo-Ball В, Arulkumaran S, AshfordМ LJ. Potassum channels in the human myometrium.Experimental Physiology 2001; 862: 255-64.15. Morrison ЈЈ, Ashford МLЈ, Khan RN, Smith S K. Theeffects of potassium channel openers on isolated pregnanthuman myometrium before and after the onsetof labor: potential for tocolysis. Ат Ј Obstet Gynecol1993;169:1277-85.16. Appiah I, Milovanović S, Radojicic R, Nikolic-KokićA, Orescanin-Dusić Z, Slavić M, Trbojević S, Skrbić R,Spasić MB and Blagojević D. Hydrogen peroxide affectscontractile activity and anti-oxidant enzymes in rat uterus.British Journal of Pharmacology 2009;158:1932-41.17. Crosbie AE, Vuylsteke A, Ritchie AJ, Latimer RD, CallinghamBA. (): Inhibitory effects of glibenclamide onthe contraction of human arterial conduits used incoronary artery bypass surgery. J Pharm Pharmacol2000;52:333-40.18. Milovanović SR, Varagić VM, Kovacević V. The effectof nitro compounds on the metabolism of cGMP andthe activity of the isolated rat uterus. Iugoslav PhysiolPharmacol. Acta 1985(Suppl. 3); 21:12-16.19. Calogero, AE, Fishel, S, Hall, J. Correlation between intracellularcAMP content, kinematic parameters andhyperactivation of human spermatozoa after incubationwith pentoxyphylline. Hum Reprod 1998;13: 911-15....104

Excluded from the study were all patients diagnosedwith the following:- the presence of active infection of the cervical canaland vagina- the presence of an active urinary tract infection- the presence of ovarian cysts- ovarian endometriosis- uterine myomas located so that they could be a cause ofobstruction of tube mobilityThe distribution of pregnancies and their outcomes inthe period prior to our operation is shown in Table 1.Table 1All examined women were divided into three groupsdepending on which type of surgery was done:- unilateral removal of tubes (salpingectomy) at 5–7 mmfrom the uterine horns- electrocoagulation of one fallopian tube at 5–7 mm fromthe uterine horns with the evacuation of the sactosalpinxcontents- reconstruction of an abdominal mouth of one of the tubeswith adhesiolysisAfter the patients were informed in detail about thetype, benefits and risks of operative treatment, their writtenconsent was obtained for the appropriate operationalprocedure. According to the protocol of preoperationalpreparation, all patients were ambulatorily prepared.The success or failure of surgical procedures for all patientswas determined through an analysis of completedpregnancies. The results of biochemical, clinical and ultrasounddata of verified pregnancies was compared. Mostimportant, the number of realised pregnancies were analysed.The following were parameters: age, duration of infertility,number and type of previous operations and numberand type of previous assisted procedures. The condition ofpatients’ infants were analysed according to Apgar score.Assisted procedures were attempted 60 days after operativetreatment in the period of endometrial renewal.The health condition of all patients, the way in whichpregnancy was achieved, and the type and number of assistedprocedures were followed for at least one year afterthe laparoscopic operations.Patients who achieved and realised pregnancy werechecked until their children’s births and again seven daysafter the births.StatisticsIn the statistical analysis, the following methods wereused: descriptive statistics, the relative numbers and theabsolute numbers.Out of 30 patients, 20 of them became pregnant. Elevenpatients had ectopic pregnancies, eight had spontaneousabortions, one patient had an ectopic pregnancy anda spontaneous abortion, and 10 patients did not becomepregnant.The distribution of patients with a unilateral hydrosalpinxaccording to the type of previous operations is shownin Table 2.Table 2Of the 14 patients who had operations, 8 had salpingectomies,5 had salpingoneostoma formations and one patienthad both operations. In total, nine salpingectomieswere done because of ectopic pregnancies, and three ectopicpregnancies were treated with ampullary methotrexate.Sixteen patients did not have operations.In addition to the type of previous surgery, the distributionof women with unilateral sactosalpinx by the typeof previous assisted procedure has been analysed, and theresults are shown in Table 3.Table 3.RE SULTSData from 30 patients who had a unilateral hydrosalpinxwere analysed.The average age of these women was 34.8 ± 4.4 years,while the average duration of infertility was 8.0 ± 3.7 years.Out of the 30 patients, one or both assisted procedureshad been completed in 22. IVF was applied in 19 women,107

IUI in 2 women, and both IVF and IUI in 1 woman. In eightwomen, we applied no assisted procedures.All women were analysed in relation to the type of ouroperations we applied based on indications and the patient’sown wishes, as well as according to treatment outcome.The distribution of these patients in relation to thetype of operation and the outcome of treatment is shownin Table 4.Table 4.From a total of 30 patients, 5 had failed fertilisation, 3had spontaneous abortions and 4 had ectopic pregnancies,while 18 patients had realised pregnancies. In relation tothe type of operation, it is evident that the greatest successwas with women who had salpingectomies (15 of 17 women)or proximal tube electrocoagulation (2 of 3 women).DISCUSSIONTube pathology affects the expression of the endometrialintegrity during implantation windows, and in somecases, the expression may be reestablished after removingthe pathological tubes (3,12,13). Tube pathology increasesthe number of macrophages in the endometrium comparedwith the number of macrophages in the endometriaof fertile women. Despite the large number of studies concerningtube pathology with a hydrosalpinx and embryoimplantation, the pathophysiology remains unclear (3).A hydrosalpinx is formed after the destruction of fimbriaand, consequently, by the accumulation of differenttube secretions. These secretions can reach the uterus.The resulting dysfunction of endometrial receptivity caninterrupt the process of embryo implantation. If these hypothesesare correct, bilateral salpingectomy directly affectsthe cause of endometrial alteration and reestablishesits receptivity (3,12,13). The consequence is the possibilityof embryo implantation, which becomes possible after thisprocedure. The result is spontaneous pregnancy soon afterunilateral salpingectomy in hydrosalpinx (3,12,14,15).In our research, we chose 30 patients who had sactosalpingeswith presently severe pathology of their tubes. Thepatient selections were made after reviewing the internalgenital organs with adequate complementary diagnosticprocedures: transvaginal sonography; diagnostic laparoscopy;and hysterosalpingography, which is also a platform forthe implementation of operational procedures (salpingectomy,proximal tube electrocoagulation with evacuation of theliquid of a hydrosalpinx and salpingoneostoma formation).Access to surgical treatment was indicated and conditioned,in our opinion, by irreversible pathological changesin the fallopian tubes (sactosalpinx is visible with ultrasoundwith a diameter of two or more centimetres; it appearsas a fallopian tube with severe inflammatory pathologyand very thick wall with completely reduced fimbrialdevice and obliterant mouth with or without the presenceof adhesions) and the patient’s own consent to the recommendedoperating procedure.Other observed parameters were patient age, durationof infertility, number and type of previous operations andassisted procedures, new types of operations, number andtype of new assisted process and final outcome.In many other studies, which had a self-control group,before surgical treatment, the selection for salpingectomywas only based on the number of failures in the applicationof IVF. These data are taken into account although therewas no control group.Our research was a clinically controlled experiment involvingpatients whose homogeneity was conditioned bypathological tube substratum, and who are themselves aself-control group.In this way, optimal conditions were created for observingthe desired variables by the type of applied surgicalmethods.Due to the high success of the IVF method, which, inall reproductive periods of life, surpasses the performanceof spontaneous conception, the indications for ART methodsare broad. Among the most important, of course, isemergency IVF, IVF in late reproductive age, and IVF asa substitute for reconstructive surgical techniques of tubeinfertility treatment in the second half of the reproductiveperiod of life (16).From a total of 30 patients with unilateral hydrosalpinx,5 had failed fertilisations, 3 had spontaneous abortions, 4had ectopic pregnancies and 18 patients had realised pregnancies.In relation to the type of operation, the most successwas seen in women who had salpingectomies (15 of17 women) (Table 4). This result favours the hypothesis ofdysfunction of endometrial receptivity and its alterationafter the removal of the hydrosalpinx (3,12,14,15).The largest number of patients had a salpingectomy %),while the tubes were electrocoagulated in the rarest typesof operations %). Salpingectomy and proximal electrocoagulationof tubes are procedures that have the same goal,but we performed one or the other according to indications.This type of operation was two times more frequentthan formation and is applied only when the patient desiresthe operational technique. (Table 4).In all 30 patients, IVF was applied. To patients who requiredconservation tubes (salpingoneostomas), IVF wasalso applied by their own choice. The other 25 patients(83.3%) remained pregnant, while 5 patients (16.7%) didnot become pregnant. Out of 25 women who became pregnant,18 women had realised pregnancies (72%), 3 womenhad complications in the form of spontaneous abortion(12%) and 4 women had ectopic pregnancies (16%).108

CONCLUSIONPatients who underwent unilateral laparoscopic salpingectomiesor proximal tube electrocoagulations becauseof unilateral hydrosalpinges as pretreatment forIVF had a greater ability to generate and realise pregnanciescompared with patients who used IVF with the hydrosalpingesstill present.After applied laparoscopic procedures before IVF, thenumber of complications, spontaneous abortions and ectopicpregnancies was significantly reduced.LITERATURE1. Winston L. Microsurgery of the fallopian tube; Fromfantasy to reality. Fertil Steril 1980; 34: 521-302. Ljubić A, Šulović V. Poremećaji reprodukcije i belakuga. Kako zaustaviti belu kugu u Srbiji 2006; 139-1723. Strandel A ,Waldenstrom U, Nilsson L , et al. Hydrosalpinxreduces in-vitro fertilization/embryo transferrates. Hum Reprod 1994; 9: 861-34. Andersen AN , Yue Z , Meng FJ , et al. Low implantationrate after in vitro fertilisation in patients with hydrosalpinxdiagnosed by ultrasonography. Hum Reprod1994; 9: 1935-85. Murray DL, Sagoskin AW, Vidra EA, et al. The adverseeffect of hidrosalpinges on in vitro fertilization pregnancyrates and the benefit of surgical corection. FertilSteril 1998; 69:41-56. Strandell A. The influence of hydrosalpinx on in-vitrofertilisation and embryo transfer: A review. Hum ReprodUpdate 2000; 6: 387-957. Puttemans P, Campo R, Gordts R, Brosens I. Hydrosalpinxand ART. Hum Reprod 2000;15:1427-308. Camus E, Poncelet C, Goffinet F, Wainer B, Merlet F,Nisand I, et al.Peregnancy rates after in-vitro fertilizationin cases of of tubal infertility with and without hydrosalpinx:A meta-analysis of published comparativestudies. Hum reprod 1999;14:1243-99. Puttemans PJ, Brosens IA,. Salpingectomy improves invitro fertilisation outcome in patients with a hydrosalpinx:blind victimisation of the fallopian tube? HumanReprod.1996;11:2079-8110. Bontis JN. Laparoscopic management of hydrosalpinx.Ann N Y Acad Sci 2006;1092:199-21011. Camus E, Poncelet C, Goffinet F, Wainer B, Merlet F,Nisand I, et al . Pregnancy rates after in-vitro fertilizationin cases of tubal infertility with and without hydrosalpinx:A meta-analysis of published comparativestudies. Hum Reprod 1999;14:1243-912. Strandell A , Sjogren A , Bentin-Ley U , et al. Hydrosalpinxfluid does not adversely affect the normal divelopmentof human embryos and implantation invitro. Hum Reprod, 1998; Vol.13, No.10, 2921-2925 13.Zeyneloglu HB, Arici A, Olive DL, Adverse effect of hydrosalpinxon pregnacy rete after in vitro fertilisationembryotransfer. Ferti Steril 1998; 70: 492-914. Strandell A , Thorburn J , Wallin A. The presence ofcytokines and growth factors in hydrosalpingeal fluid. JAssist Reprod Genet 2004; 21: 241-715. Johnson NP, Mak W, Sowter MC. Laparoscopic salpingectomyfor women with hydrosalpinges enhances the successof IVF: A Cochrane review. Hum Reprod 2002;17:543-8.16. Vlaisavljević V. Doprinos asistirane reprodukcije demografskimpromenama. Kako zaustaviti belu kugu uSrbiji 2006; 121-137109

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REVIEW ARTICLE REVIJALNI ČLANAK REVIEW ARTICLE REVIJALNI ČLANAKTHE REVISED TNM STAGING SYSTEMFOR LUNG CANCER NON - SMALL CELL LUNG CANCERSlobodan Milisavljevic 1 , Branislav Jeremic 21Clinical Center Kragujevac2Institute of Pulmonary Diseases, Sremska Kamenica, SerbiaNOVI SISTEM STAŽIRANJA KARCINOMA PLUĆA MEĐUNARODNEASOCIJACIJE ZA ISTRAŽIVANJE KARCINOMA PLUĆANEMIKROCELULARNI KARCINOMI PLUĆA (NSCLC)Slobodan Milisavljević 1 , Branislav Jeremić 21Klinički centar Kragujevac2Institut za plućne bolesti Sremska Kamenica, SrbijaReceived / Primljen: 11. 2. 2010. Accepted / Prihvaćen: 5. 6. 2010.ABSTRACTMalignant tumours represent one of the most seriousdiseases of our time, both in developed countries and indeveloping countries. Despite significant advances in prevention,early diagnosis and therapy, mortality from malignanttumours is still very high. The mortality rate associatedwith lung cancer is the highest among all cancers inmen. New diagnostic methods (e.g., positron emission tomography)and innovations in surgical approaches (videoassistedthoracic surgery), radiotherapy (three-dimensionalradiotherapy) and chemotherapy (third-generation drugsand targeted therapy) have contributed to improved resultsin the treatment of lung cancer. A new revision of the internationalsystem of staging for lung cancer, which aims tofurther optimise our approach to this disease, was recentlyreleased by the International Association for the Study ofLung Cancer (IASLC).Key words: NSCLC, TNM classification, revisionSAŽETAKMaligni tumori predstavljaju jednu od najtežih bolestidanašnjice, kako u razvijenim zemljama, tako i u zemljamau razvoju. I pored značajnih napredaka u oblasti prevencije,rane dijagnostike i terapije, smrtnost od malignih tumora je idalje visoka. Karcinom pluća je vodeći među karcinomimapo smrtnosti kod muškaraca. Nove dijagnostičke metode (npr.Pozitronska Emisiona Tomografija), noviteti u hirurškompristupu (Video Assisted Thoracoscopic Surgery -VATS),radioterapiji (trodimenzionalna radioterapija) i hemioterapiji(treća generacija lekova i target - terapija) su doprinelipoboljšanim rezultatima u lečenju karcinoma pluća. Nova revizijainternacionalnog sistema stažiranja karcinoma pluća jejedna od skorašnjih inicijativa Internacionalne Asocijacije zaIstraživanje Karcinoma Pluća (International Association forthe Study of Lung Cancer) IASLC) sa ciljem dalje optimizacijenaših pristupa ovoj bolesti.Ključne reči: NSCLC, TNM klasifikacija, revizijaUDK 616.24-006.6 / Ser J Exp Clin Res 2010; 11 (3): 111-113Correspondence to: Dr. Slobodan Milisavljević, Thoracic Surgeon / Clinical Center Kragujevac, Zmaj Jovina Street, KragujevacTel.: 034/ 505315, cell: 064 143 76 45 / E-mail: s.milisavljevic65@gmail.com111

The TNM system, based on classification of primarytumours (T), lymph node metastases (N) and distantmetastases (M), has been in use by oncologists since1953, when the Committee on Nomenclature and Statisticsof the International Union Against Cancer (UICC)adopted the TNM system as the basis for the anatomicaldiagnosis of cancer (1). Since 1973, lung cancer stagingup to and including the fifth revision of the staging systemmade in 1997 has been based on databases takenfrom surgical series (2). At that time, an internationaldatabase was created. The IASLC led a coordinated effortlasting more than a decade to find financial supportand identify institutions and individuals who wouldcontribute to the realisation of a common internationaldatabase of patients. Upon completion, this database included100,869 cases, of which 81,495 cases included adequatetime tracking. A total of 68,463 cases were in thenon-small cell lung cancer (NSCLC) group, and 13,032cases were in the small cell lung cancer (SCLC) group(3-8). Of these, surgical treatment was used in only 41%of cases (with radiotherapy for only 11% and chemotherapyfor only 23%), whereas the remaining cases weretreated by a combined therapeutic approach.The extremely large number of cases has enabledmore analysis, and thus, several sub-committees wereformed (focusing on T, N and M indicators, prognosticfactors, SCLC, surgical site, validation and methodology).The work of these sub-committees included theanalysis of various end results, which were publishedas a series of articles in the Journal of Thoracic Oncology(3-8), the official journal of the IASLC. The ultimategoal was to revise the TNM classification of lungcancer in January 2009 under the network of the seventhreview.Based on data from 18,198 cases with sufficient datato determine the impact of T stage on NSCLC (3-6), thefollowing changes were made to the staging classificationin 1997 (3): T1 tumours are subclassified as T1a orT1b, with a T1a tumour being up to 2 cm in maximaldiameter and a T1b tumour being of maximal diameterbetween 2 and 3 cm. T2 tumours are subclassified intoT2a and T2b, with T2 tumours being > 3 cm but ≤ 5 cmin maximal diameter and T2b tumours being > 5 cm but≤ 7 cm in maximal diameter. Also, tumours bigger than7 cm in maximal diameter are reclassified as T3 insteadof T2, and the T3 category also includes tumours withseparate tumour nodule(s) in the same lobe. T4 tumoursnow include those with separate tumour nodule(s) in adifferent ipsilateral lobe.Data from 38,265 cases without clinical evidence ofdistant metastases (cM0), for which information about theclinical status of lymph nodes (cN) was available, and datafrom 28,371 surgically treated cases with information onthe pathological N stage (pN) were analysed. It was concludedthat the existing classification of N stage (N0 - N3)should not be changed.Regarding metastatic lesions (M+), they are now dividedinto M1a and M1b, of which the first is defined asa separate tumour nodule (i) in the contralateral lobe, atumour with pleural nodes, or malignant pleural or pericardialeffusion.Several changes were made to the process of stagingbased on certain combinations of the descriptors T, N andM. Namely, T2bN0M0 cases were moved from stage IB tostage IIA, and T2aN1M0 cases were moved from stage IIBto stage IIA. Cases categorised as T4N0 - 1M0 were movedfrom stage IIIB to stage IIIA (Table 1).Table 1: New (7th) revision of the IASLC staging system.Grouping of stages:Occult Tx N0 M0carcinomaStage 0 Tis N0 M0Stage IA T1a,b N0 M0Stage IB T2a N0 M0Stage IIA T1a,b N1 M0T2a N1 M0T2b N0 M0Stage IIB T2b N1 M0T3 N0 M0Stage IIIA T1, T2 N2 M0T3 N1, N2 M0T4 N0, N1 M0Stage IIIB T4 N2 M0Any T N3 M0Stage IV Any T Any N M1a,bThese changes in staging for NSCLC made by the IASLCand the UICC are the latest contributions to a better understandingof this disease. The staging system, as with other systemsfor other malignancies, will continue to contribute to anadequate comparison of results between individual institutionsand to adequate interpretation of the results of clinical studiesin this area by using a “common” language during diagnosis andtherapy. This system will also be helpful in making decisionsabout the optimal treatment of patients and in predicting prognosisafter treatment with individual or combined therapeuticmodalities. Its immediate application is imperative for thoraciconcologists across the world who wish to further optimise andcoordinate efforts in treating patients with NSCLC.REFERENCES1. UICC International Union Against cancer. TNM classificationof malignant tumours, 6th ed; New York:Willey-Liss, 2002.2. .Mountaiun CF. Revisions in the International Systemfor Staging Lung Cancer. Chest 111: 1710-1717, 1997.112

3. Rami-Porta R, Ball D, Crowley et al. The IASLC lungcancer staging project: proposals for the revision of theT descriptors in the forthcoming (seventh) edition ofthe TNM classification of lung cancer. J Thorac Oncol2: 593-602, 2007.4. Rusch VW, Crowley J, Giroux DJ et al. The IASLC lungcancer staging project: proposals for the revision of theN descriptors in the forthcoming (seventh) edition ofthe TNM classificatrion of lung cancer. J Thorac Oncol2: 603-612, 2007.5. Groome PA, Bolejack V, Crowley JJ et al. The IASLClung cancer staging project: Validation of the proposalsfor revision of the T, N, and M descriptors and consequentstage groupings in the forthcoming (seventh) editionof the TNM classification of malignant tumours. JThorac Oncol 2: 694-705.6. Goldstraw P, Crowley J, Chansky et al. The IASLC lungcancer staging project: proposals for the revision of theTNM stage groupings in the forthcoming (seventh) editionof the TNM classification of malignant tumours. JThorac Oncol 2: 706-714, 2007.7. Shepherd FA, Crowley J, van Houtte P et al. The InternationalAssociation for the Study of Lung Cancerstaging project: proposals regarding the clinical stagingof small cell lung cancer in the forthcoming (seventh)edition of the tumor, node, metastasis classification. JThorac Oncol 2: 1067-1077, 2007.8. Velliers E, Shepherd FA, Crowley J et al. The IASLC lungcancer staging project. Proposals regarding the relevance ofTNM in the pathologic staging of small cell lung cancer inthe forthcoming (seventh) edition of the TNM classificationfor lung cancer. J Thorac Oncol 2: 1049-1059, 2007.113

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ABSTACT OF THE LITERATURE PREGLED LITERATURE ABSTACT OF THE LITERATURECYTOTOXICITY OF GLASS IONOMER CEMENTSON HUMAN PULP CELLSTatjana Kanjevac 1 * and Ana Volarevic 1 *1Faculty of Medicine University of Kragujevac*T. Kanjevac and A. Volarevic contributed equally (50% each) to this work and both should be considered as first authors.CITOTOKSIČNOST GLAS JONOMER CEMENTA ISPITIVANANA ĆELIJAMA ZUBNE PULPE LJUDITatjana Kanjevac 1 * i Ana Volarević 1 *1Medicinski fakultet, Univerzitet u Kragujevcu*T. Kanjevac i A. Volarević su podjednako (sa 50%) učestvovale u pisanju ovog rada, te se smatraju prvim autorima.Received / Primljen: 17. 7. 2010. Accepted / Prihvaćen: 15. 8. 2010.ABSTRACTThe aim of restorative dentistry is the successful restorationand maintenance of the health of the tooth by an adequate reconstructivetreatment to re-establish the function of damagedpulp. The potential cytotoxicity of specific materials used in restorativedentistry has been widely studied, and the aim of thisreview article is to summarise and discuss the cytotoxicity ofglass ionomer cements when they are in direct or indirect contactwith the pulp tissue. Resin modified and metal reinforcedglass ionomer cements, in comparison to conventional glassionomer cements, showed higher cytotoxic effects on pulp cellsin vitro. In vivo, the dentin barrier between toxic glass ionomercements and the pulp cells may prevent pulp cell damage. Potentiallytoxic resin modified and metal reinforced glass ionomercements should not be applied directly to the pulp tissue.Keywords: cytotoxicity, pulp cells, glass ionomer cementsSAŽETAKČuvanje funkcije zubne pulpe je važno za zdravljezuba. Cilj ovog preglednog rada je predstavljanje do danaspoznatih rezultata in vitro i in vivo studija koje su ispitivalecitotoksičnost glas jonomer cementa, materijala koji se koristeu stomatologiji i koji su u direktnom ili indirektnomkontaktu sa zubnom pulpom., ,,Smolom modifikovani“ i,,,metalom ojačani“ glas jonomer cementi, su in vitro pokazaliznatno veću citotoksičnost, u poređenju sa konvencionalnimglas jonomer cementima. In vivo, dentin predstavlja barijerukoja štiti ćelije zubne pulpe od toksičnog dejstva glas jonomercementa i zato potencijalno toksične ,,smolom modifikovane“i ,,metalom ojačane“ glas jonomer cemente, nikada ne trebadirektno aplikovati na zubnu pulpu.Ključne reči: citotoksičnost, ćelije zubne pulpe, glasjonomer cementiINTRODUCTIONThe aim of restorative dentistry is the successful restorationand maintenance of the health of the tooth by an adequatereconstructive treatment to re-establish the functionof damaged pulp (1).The main role of pulp is dentin formation and nutritionas well as the innervation of teeth. The primary function ofpulp is dentin formation, which begins when the mesenchymalcells differentiate into odontoblasts and ends when thetooth is completely formed. The channels created by the odontoblastsfunction in dental nutrition; the continuous transportof nutrients and fluids maintains the vitality of the pulp.Throughout an individual’s lifetime, pulp continuously producesdentin in physiological condition as well as in responseto physical and chemical injuries. Pulp also serves as a defencebarrier. Increased dilatation and permeability of blood vesselsand intensive migration of inflammatory cells are usually resultsof pulp response to different noxious stimuli (2).In restorative dentistry, the protection of the dentinpulpcomplex consists of the application of one or morelayers of specific materials (e.g., varnishes, calcium hydroxide-basedproducts, glass ionomer cements (GICs) and adhesivesystems) between the restorative material and dentaltissue to avoid additional damage of pulp tissue causedby operative procedures, toxicity of restorative materialsand bacterial penetration due to microleakage (1, 3).The potential cytotoxicity of the specific materials usedin restorative dentistry has been widely studied, and theaim of this review article is to summarise and discuss thecytotoxicity of GICs when they are in direct or indirectcontact with the pulp tissue.UDK 616.24-006.6 / Ser J Exp Clin Res 2010; 11 (3): 115-117Correspondence: Ana Volarevic, volarevic_ana@yahoo.com, mob.tel: 064/38-58-629115

THE CHARACTERISTIC AND CLASSIFICATIONOF GLASS IONOMER CEMENTSGlass ionomer cements (GICs), invented and originallydescribed by Wilson and Kent (4), consist of a basic glasspowder (calcium or strontium aluminofluorosilicate) anda water-soluble acidic polymer, such as polyacrylic acid(5). GICs are classified into three categories: conventional,metal-reinforced and resin modified [6-7]. Metal-reinforcedGICs are strengthened by the inclusion of finely dividedmetal powders, typically the silver-tin alloy of dentalamalgams (8).Although conventional GICs, because of their similarityto dentin in terms of biocompatibility, elasticity andability to release fluoride, have advantages in comparisonwith other materials used in restorative dentistry, theyhave several limitations, such as susceptibility to dehydrationand poor physical properties (i.e., high solubility andslow setting rate) (1, 9-10).The incorporation of polymerisable water-compatiblemonomers such as 2-hydoxyethyl methacrylate (HEMA)led to the introduction of hybrid versions of conventionalGICs named resin-modified GICs (RMGICs) (1, 8). Incomparison with conventional GICs, RMGICs show enhancedflexural strength, diametral tensile strength, elasticmodulus and wear resistance, but they are not as biocompatibleas conventional GICs (11).THE CYTOTOXIC EFFECTS OF GLASSIONOMER CEMENTSThe main disadvantage of metal-reinforced GICs andRMGICs is their higher cytotoxicity in comparison withconventional GICs (1, 12).The cytotoxicity of RMGICs could be due to their constituentHEMA which, because of their hydrophilicity andlow molecular weight, can easily diffuse through the dentinaltubules; reach dental pulp cells; damage pulp cellsby suppressing cell growth and proliferation; and induceapoptosis of dental pulp cells (12).It has been shown that methacrylate monomerspresent in resin based materials such as Vitrebond(3M/ESPE Dental Products, St. Paul, MN, USA) and Vitremer(3M/ESPE Dental Products, St. Paul, MN, USA)are incorporated in the lipid bilayers of cell membranesthat can cause dysfunction of cellular membranes andconsequently induce cell death (13). This was documentedby Costa et al. in a study in which the cytotoxicityof several GICs was tested on an odontoblast cellline (MDPC-23) by a 3-(4, 5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay (13). Itwas shown that two GICs, Fuji IX GP (GC, Tokyo, Japan)and Ketac Molar (3M/ESPE Dental Products, St.Paul, MN, USA), were the least cytotoxic among thetested materials while two other RMGICs, Vitrebondand Vitremer, caused intense cytopathic effects on thecultured cells by significantly decreasing cell metabolismand by causing remarkable cell death (13). Thehighly cytotoxic effects of Vitremer on cultured humanosteoblastic cells was confirmed by Oliva et al. (14),who showed in vitro that HEMA is mainly responsiblefor the cytotoxicity of Vitremer.To compare the cytotoxic effects of different RMGICsand metal reinforced GICs, Stanislawski et al. carried outan in vitro pulp cell viability assay in which metal reinforcedGICs were shown to be highly toxic materials. Vitremerwas the most toxic material among the RMGICs while theleast toxic were Compoglass (Ivoclar Vivadent Ltda., SãoPaulo, SP, Brasil) and Photac Fil (3M/ESPE Dental Products,St. Paul, MN, USA) (11).It was documented that HEMA, along with other unpolymerisedmonomers like triethylene glycol dimethacrylate(TEGDMA), is responsible for the cytotoxic effects ofRMGICs and metal reinforced GICs.However, the presence of some ions in significantamounts in metal reinforced GICs and also in RMGICsalso could be responsible for their cytotoxicities. The ionsCu2+ and Ag+, which were present in toxic concentrations,could be the main elements responsible for the toxicityof the metal-reinforced GICs along with HEMA andTEGDMA (11). Further, the possible cytotoxic effects ofF-, Al3+, Zn2+ and Sr2+ ions, which are also present insignificant amounts in GICs, were tested. It was concludedthat among the tested ions, only the zinc ion was found tobe associated with a high enough concentration to inducecytotoxicity of metal reinforced GICs and RMGICs (11).Conversely, Soheili Madj et al. demonstrated that thecytotoxic effects of GICs might be caused by the metalcomponents, or the small amounts of aluminium and/oriron ions present in their composition, which may causecytotoxic effects on cultured cells by oxidative stress (15).The potential toxic effects of the organic components ofGICs also have been tested. Leyhausen et al. suggested thatthe cytotoxicity of Vitrebond may be caused by chlorinebenzene, iodine benzene, and bromide benzene, which aredecomposition products of the initiator diphenyliodoniumchloride (DPICl) (16).Complementing the results presented above, theRMGICs and metal reinforced GICs showed higher cytotoxiceffects on cultured fibroblasts and osteoblasts in vitroin comparison with conventional GICs. It has been shownthat RMGICs are able to cause intense cytophatic effectson the cultured cells by significantly decreasing cell metabolismas well as by causing remarkable cell death (1).However, it is most important to point out that the toxiceffects of GICs were tested mainly in vitro. Although invitro tests are simple to perform, cost-effective and suitableas an alternative to in vivo experiments, the results of invitro studies cannot be automatically extrapolated to clinicalsituations (17-19).It has been shown that the sensitivity of human pulpcells to cytotoxicity depended on the differences in thecontent, specifically the component monomers or additivesof the tested RMGICs. Additionally, the sensitiv-116

ity of human pulp cells to cytotoxicity depended on theconcentration of the elutes tested. The cytotoxicities ofthe tested RMGICs decreased as the dilution concentrationof the elutes increased because an increase of dilutionconcentration of the elutes is followed by a decreasein the content of the monomers or the additives in thedilution (17).In line with this observation are the results of in vivostudies performed in human teeth, which have demonstratedthat the RMGIC Vitrebond, the GIC which showedtoxic effects in vitro, caused no inflammatory pulp responsewhen it was applied in vivo as a liner in very deep class Vcavities, (18-19). It seems that pulp cell damage documentedin in vitro studies is prevented in vivo by the presenceof a dentin barrier between the RMGIC Vitrebond and thepulp cells.CONCLUSIONBased on this literature review, it may be concludedthat RMGICs and metal reinforced GICs, in comparisonwith conventional GICs, showed higher cytotoxic effectson pulp cells in vitro. Monomers (HEMA and TEGDMA)present in resin composites of RMGICs, as well as Cu2+,Ag+ and Zn2+ ions, present in significant amounts in metalreinforced GICs and are the components mainly responsiblefor the toxic effects of these GICs.In vivo experiments showed that the presence of a dentinbarrier between toxic GICs and the pulp cells may preventpulp cell damage. Potentially toxic RMGICs and metalreinforced GICs, because of their cytotoxicity, should notbe applied directly to the pulp tissue.REFERENCES1. Modena K, Casas Apayco L, Atta M, Costa C, HeblingJ, Sipert C, Navarro M, Santos C. Cytotoxicity and biocompatibilityof direct and indirect pulp capping materials.J Appl Oral Sci. 2009; 17(6):544-54.2. Pashley DH. Dynamics of the pulpo-dentin comples.Crit Rev Oral Biol Med. 1996; 7(2):104-33.3. Briso ALF, Rahal V, Mestrener SR, Dezan E Jr. Biologicalresponse of pulps submitted to different cappingmaterials. Braz Oral Res. 2006; 20(3):219-25.4. Wilson AD, Kent BE. The glass ionomer cement, a newtransluscent dental filling material. J Appl Chem Biotechnol.1971; 21:313.5. Hill RG, Wilson AD. Some structural aspects ofglasses used in ionomer cements, Glass Technol.1988; 29:150-167.6. Matsuya S, Maeda T, Ohta M. IR and NMR analyses ofhardening and maturation of glass-ionomer cement. JDent Res 1996; 75:1920-1927.7. Burgess J, Norling M, Summit J. Resin ionomer restorativematerials: the new generation. J. Esthet. Dent.1994; 6:207-215.8. Wasson EA. Reinforced glass-ionomer cements - a reviewof properties and clinical use, Clin. Mater. 1993;12:181-190.9. Mount GJ. Glass-ionomer cements: past, present andfuture. Oper Dent. 1994; 19:82-90.10. Chan C, Lan W, Chang M, Chen Y, Lan W, Chang H.Effects of TGF betas on the growth, collagen synthesisand collagen lattice contraction of human dental pulpfibroblasts in vitro. Arch Oral Biol. 2005; 50(5):469-79.11. Stanislawski L, Daniau X, Lauti A, Goldberg M. Factorsresponsible for pulp cell cytotoxicity induced by resinmodifiedglass-ionomer cements. J Biomed Mater Res.1999; 48:277-88.12. Aranha AMF, Giro EMA, Souza PPC, Hebling J, CostaCAS. Effect of curing regime on the cytotoxicity of resin-modifiedglass-ionomer lining cements applied to anodontoblast-cell line. Dent Mater. 2006; 22:864-69.13. Costa C, Hebling J, Godoy-Garcia F, Hanks C. In vitrocytotoxicity of five glass ionomer cements. Biomaterials2003; 24:3853-58.14. Oliva A, Ragione D, Salerno A, Riccio V, Tartaro G,Gozzolino A. Biocompatibility studies on glass ionomercements by primary cultures o human osteoblasts.Biomaterials 1996; 17:1351–6.15. Soheili Majd E, Goldberg M, Stanislawski L. In vitro effectsof ascorbate and Trolox on the biocompatibility ofdental restorative materials. Biomaterials 2003; 24:3–9.16. Leyhausen G, Abtahi M, Karbakhsch M, Sapotnick A,Geurtsen W. The biocompatibility of various resinmodifiedand one conventional glass-ionomer cement.Biomaterials 1998; 19:559–64.17. Kong N, Jiang T, Zhou Z, Fu J. Cytotoxicity of polymerizedresin cements on human dental pulp cells in vitro.Dental materials 2009; 25: 1371-75.18. Costa C, Hebling J, Hanks CT. Current status of pulpcapping with dentin adhesive systems: a review. DentMater 2000; 16:188–97.19. Costa CA, Giro EM, Nascimento AB, Teixeira HM,Hebling J. Shortterm evaluation of the pulpo-dentincomplex response to a resin-modified glass-ionomercement and a bonding agent applied in deep cavities.Dent Mater. 2003; 19(8):739-46.20. Costa CAS, Teixeira HM, Lopes do Nascimento AB, HeblingJ. Biocompatibility of resin-based dental materialsapplied as liners in deep cavities prepared in human teeth.J Biomed Mater Res B Appl Biomater. 2007; 81(1):175-84.117

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CASE REPORT PRIKAZ SLUČAJA CASE REPORT PRIKAZ SLUČAJA CASE REPORTBILATERAL RENAL ANGIOMYOLIPOMA IN A PATIENTWITH TUBEROUS SCLEROSIS – CASE REPORTNatasa Rakonjac, Ivana Blazic, Dragutin Lomic, Slobodan MarkovskiClinical centre Zemun, BelgradeBILATERALNI RENALNI ANGIMIOLIPOMKOD PACIJENTA SA TUBEROZNOM SKLEROZOM-PRIKAZ SLUČAJANataša Rakonjac, Ivana Blažić, Dragutin Lomić, Slobodan MarkovskiKlinički centar Zemun, BeogradReceived / Primljen: 31. 8. 2009. Accepted / Prihvaćen: 21. 10. 2009.ABSTRACTTuberous sclerosis (Bourneville disease) is a dominantautosomal congenital disease. It is caused by alterationsin the ninth or sixteenth chromosome and normally ischaracterised by a classical triad of mental retardation,epilepsy and sebaceous adenoma. The radiological diagnosisis made by observing some combination of findings:subependymal and periventricular calcifications,cortical tubers, cardiac tumours, renal cysts, and angiomyolipomas.Renal angiomyolipoma (AML) is a benignrenal neoplasm, which is composed of fat as wellas vascular and smooth muscle elements. AMLs occur inassociation with tuberous sclerosis in 20% of cases, andthey are larger and bilateral, and they affect a youngerage group. We report on the case of a 52-year-old womanwith abdominal pain, vomiting and anaemia. The initialultrasound of the abdomen showed bilateral, large, heterogenic,echogenic masses that occupied the renal fosses,and the kidneys were not observable. The patient washospitalised for observation and additional radiologicaldiagnostic. The diagnosis of tuberous sclerosis with bilateralrenal AML was made based on the presence oftypical skin lesions and findings on abdominal and headCT as well as intravenous urography. Currently, despitelarge bilateral renal AML, her laboratory results showedmild anaemia without renal failure, so the patient hasnot required surgery.Key words:angiomyolipoma, tuberous sclerosis, kidneysSAŽETAKTuberozna skleroza (Bourneville bolest) je autozomalnodominantno nasledno oboljenje. Nastaje kao rezultat alteracijana devetom i šesnaestom hromozomu i najčešće se karakterišetrijadom koja obuhvata mentalnu retardaciju, epilepsijui prisustvo sebaceoznih adenoma. Dijagnoza se postavljakombinacijom radioloških nalaza koji uključuju prisustvoparaventrikularnih i subependimalnih kalcifikacija, kao i kortikalnihtubera u mozgu, kardioloških tumora, renalnih cistai angimiolipoma. Ranalni angimiolipom (AML) je benigni tumorbubrega, koji se sastoji od masti, vaskularnih elemenata iglatkih mišićnih vlakana. Kod bolesnika sa tuberoznom sklerozomrenalni AML se javlja u 20% slučajeva i u tom slučajuje obično bilateralni, veći i zahvata mladju populaciju, negokad se javlja samostalno. Predstavljamo slučaj pacijentkinjestare 52 godine kod koje se javlja abdominalni bol, mučnina ianemija. Inicijalno, ultazvuk je pokazao ogromnu, bilateralnu,hiperehogenu masu koja se nalazila u projekciji bubrega, kojinisu mogli biti diferencirani. Pacijentkinja je hospitalizovanau cilju observacije i dodatne radiološke dijagnostike. U skladusa nalazima kompjuterizovne tomografije abdomena i glave,intravenske urografije, kao i zbog postojanja tipičnih promenana koži, dijagnoza tuberozne skleroze sa renalnim angimiolipomomje postavljena. Medjutim, uprkos ogromnim bilateralnimangimiolipomima bubrega, laboratorijski nalazi su ukazivalina blagu anemiju, bez poremećaja bubrežne funkcije,tepacijentkinji nije bila neophodna hirurška intervencija.Ključne reči:angimiolipom, bubrezi,tuberozna sklerozaUDK 616.61-006.03 ; 616-056.7 / Ser J Exp Clin Res 2010; 11 (3): 119-125Correspondence: Natasa Rakonjac, dr / Clinical centre Zemun, Belgrade / Tel. 011 3772629 / Mob. 064 1950350 / E mail: natasarakonjac@yahoo.com119

INTRODUCTIONTuberous sclerosis complex (TSC) is a rare, inheritedautosomal dominant multisystem disorder characterisedby the potential for the presence of hamartomas(1). Molecular genetic studies have identified two loci forTSC; TSC1 is located on the long arm of chromosome 9(9q34); TSC2 is located on the short arm of chromosome16 (16p13.3). Both TSC1 and TSC2 have tumour suppressoractivity that, when not activated, leads to uncontrolledcell cycle progression and the proliferation of hamartomasthroughout the body (2).A hamartoma is a benign tumour comprised of an overgrowthof mature cells and tissues that normally occur in theaffected tissue, but typically, one element is predominant.Tuberous sclerosis symptoms include adenoma sebaceumof the skin, angiomyolipomas of the kidney, corticaland subependymal tubers of the brain, rhabdomyomasof the heart and pulmonary lymphangioleiomyomatosis(LAM). Individual hamartomas are rare in the non-TSCpopulation, so the presence of hamartomas in two differentorgan systems is considered by some clinicians to besufficient for the diagnosis (3).Dermatological Manifestations. The most commondermatological manifestations are hypomelanotic maculesor “ash leaf spots” (4). Typically, these lesions aredifficult to visualise without the aid of an ultraviolet light(Wood’s lamp). Hypomelanotic macules usually becomemore apparent with age. Facial angiofibromas (adenomasebaceum) are composed of vascular and connective tissueelements and typically appear on the face as small pink tored dome-shaped papules in a “butterfly distribution.” Thelesions enlarge gradually and increase in number with age.Periungual and ungual fibromas (Koenen tumours) aresmooth, firm, nodular or fleshy lesions that are adjacent toor underneath the nails (5). Toenails are more commonlyinvolved than fingernails (4, 6). Café au lait spots are seenin up to 30% of patients with TSC (7).A thorough dermatological examination is essential becausemany TSC’s major features are cutaneous. If hypopigmentedmacules are not obvious under ambient light, aWood’s lamp illumination of the skin should be done forevery child with infantile spasms, cardiac rhabdomyoma,or renal angiomyolipoma.Neurological Manifestations. The neurologicalmanifestations are heterogeneous. The spectrum ofmanifestation ranges from patients with normal intellectand no seizures to those with severe mental retardationand incapacitating seizures. Neurological complicationsare the most common cause of mortality and morbidityas well as the most likely to affect the quality of life.The most common seizure types are infantile spasms,partial motor seizures, and generalised tonic-clonic seizures(8, 9). Autism, attention deficit, hyperactivity, andsleep problems are the most frequent behavioural disorders(8, 10, 11). The intracranial abnormalities includetubers, subependymal nodules, and subependymal giantcell astrocytomas (12). No correlation has been foundbetween the number of subependymal lesions and theclinical severity of TSC (13).Renal Manifestations. Renal complications are thesecond most common cause of mortality (14). The mostcommon renal lesion is an angiomyolipoma, which occursin approximately in 80% of cases. Angiomyolipomasare benign tumours comprised of blood vessels withthickened walls, immature smooth muscle cells, andadipose tissue (14, 15). Angiomyolipomas in tuberoussclerosis are larger than the isolated variety, often occuras multiples, are often bilateral, and affect an earlierage group (16, 17). Smaller angiomyolipomas usually donot cause symptoms, but lesions larger than 4 cm in diameterare associated with an increased risk of serioushaemorrhage (5, 15). The second most common renalmanifestation is a renal cyst, and renal cysts combinedangiomyolipomas are characteristic of TSC (5). Renalcarcinomas are rare and tend to grow more slowly inpatients with TSC than in those found in the generalpopulation (15).Cardiac Manifestations. Cardiac rhabdomyomasusually occur in multiples and are asymptomatic (5, 9).However, these lesions can result in an outflow obstruction,valvular dysfunction, arrhythmias (especially Wolff-Parkinson-White syndrome), and cerebral thromboembolism(13, 18).Ophthalmic Manifestations. Retinal hamartomas arebilateral and asymptomatic, but some patients have visualimpairment as a result of a large macular lesion (13). Angiofibromasmay develop on the eyelids (19).Oral Manifestations. Gingival fibroma occurs in 50%of adults with TSC (19).Vascular Manifestations. Patients with TSC are at increasedrisk for arterial aneurysms, which affect the aortaand peripheral arteries (e.g., carotid, renal, intracranial)with potentially appreciable morbid or mortal consequences(18, 19). Histologically, the arterial walls demonstrate aloss of elastin fibres similar to that seen in patients withMarfan Syndrome (18).Pulmonary Manifestations. The classic pulmonarylesion is lymphangioleiomyomatosis (LAN), a progressivelung disease seen mainly in adult females (20).Osseous Manifestations. Osseous lesions on radiographsinclude bone cysts found mainly in the phalangesof the hands and feet, sclerotic lesions, and periosteal newbone formation (21).Gastrointestinal Manifestations. Hamartomatous polypsin the rectum are common and are usually asymptomatic (4).RADIOLOGY FINDINGSAbnormal radiological findings are important in diagnosingthis disease and include lesions found in the CNS,heart, lungs, kidneys, skeleton, and, occasionally, liver,spleen and pancreas.120

CT findingsOverall, CT reveals intracranial abnormalities in 85%of patients with tuberous sclerosis. CT readily depicts calcifiedcortical tubers and calcified subependymal nodules.The frequency of their calcification increases with patientage. Subependymal nodules are found mostly along thelateral ventricles. These nodules may enhance after the intravenousadministration of contrast material, but contrastenhancement is more difficult to recognise on CT scansthan on other images, particularly in calcified lesions. In10%-15% of patients, subependymal nodules may transforminto giant cell astrocytomas. These tumours are benignand usually occur at or near the foramen of Monro.These lesions typically appear inhomogeneous and usuallyhave an inhomogeneous enhancement pattern after the intravenousadministration of contrast material. Frequently,they are calcified, they usually enlarge over time, and theycommonly cause obstructive hydrocephalus.Renal angiomyolipomas often have low attenuationvalues if they contain sufficient fat, but they are indistinguishablefrom other renal tumours if they containlittle or no lipids. Varying amounts of non-lipid tissueand haemorrhage can be visualised on CT scans of angiomyolipomas.Generally, calcification is not seen inangiomyolipomas. Cystic lesions commonly occur forthis disease, and they are well characterised with CT.Multiple cysts can distort the renal collecting system;with this finding alone, tuberous sclerosis is indistinguishablefrom polycystic kidney disease.ULTRASOUND findingsOn sonograms, the lesions are highly echogenic becauseof their high fat content. A finding of multiple angiomyolipomaswith a high fat content is highly suggestive oftuberous sclerosis. Angiomyolipomas can bleed and causerenal parenchymal haemorrhage as well as subcapsular orretroperitoneal haemorrhage. Cysts almost always occur inmultiples; typically, they are bilateral, and on sonograms,cysts are anechoic.Figure 1. An abdominal ultrasound shows a hyperechogenic mass underthe liver in the right abdomen that compresses, which implies intestine.A 52-year-old woman at a medical check-up presentedwith a dull, temporary pain in both flanks that radiated tothe bladder, but she had no renal failure. History was significantfor one year earlier.The initial abdominal ultrasound examination showeda large, hyperechogenic mass that occupied the right abdomenand was compressible, which implied intestine. Asimilar structure was seen in the left abdomen, posteriorlyand paravertebrally. The kidneys were not seen either byfrontal or by posterior positioning of the sonde. The patientwas prescribed additional radiological and laboratorydiagnostic and surgical examinations (figure 1).Abdominal palpation revealed bilateral tender flankmasses extending up to both iliac fosses that were insensibleon palpation and lacked peritoneal irritation. Theyeach had a smooth surface of solid consistency, low mobilityand clear limits. The patient reported frequent urinationwithout haematuria and temporary pain in both flanks.Dermatological consultation revealed erythema on theforehead, a paranasal rash with multiple sebaceous adenomasaround the nose and nasolabial folds (figure 2),some hypomelanotic macules, and fibromatous tumours(Koen) under the toenails with disfiguration of the nails.Laboratory analysis revealed mild anaemia (RBC 2, 12; Fe6, 5; Hgb 96) with normal levels of creatinine and urea (Cre89; Urea 5, 8) that suggested the renal profile was normal.IMAGING FINDINGS:Abdominal contrast-enhanced CT revealed a large tumourin the retroperitoneum with primarily fat-equivalentattenuation (image density was -50 HU) that completelyreplaced both of the kidneys. Intraperitoneal structuresCASE REPORTFigure 2. Erythema on the forehead, paranasal rash, and multiple facialangiofibromas around the nose and nasolabial folds are dermatologicalmanifestations.121

Figure 3. As seen on the abdominal CT, there is a large tumour in the retroperitoneumwith predominantly fat-equivalent attenuation that replacedboth the kidneys entirely and displaced intraperitoneal structures anteriorly.were displaced anteriorly, and the mass extended as far asthe pelvic region (figure 3).The head CT revealed cortical and subcortical reductivealteration: multiple calcified subependymal and paraventriculartubers, subcortical calcified nodules in the lefthemisphere of the cerebellum and benign white matter lesions(figure 4).The chest CT revealed bilateral, symmetrical, and diffusedistribution of small air cysts with thin regular wallsand normal lung parenchyma between the cysts.Intravenous urography showed a compressed and extendedpyelic system of both kidneys. Kidney structure was notdiscernable. The contrast was excreted, and the ureters werevisualised bilaterally. The bladder was normal (figure 5).Echocardiography and chest x-ray revealed no abnormalities.Based on these examinations, the diagnosis was Bourneville-Pringlesyndrome, tuberous sclerosis. Despite thebilateral large retroperitoneal angiomyolipoma of the kidneys,the patient’s condition was satisfactory, and the renalprofile was normal. For that reason, there will further observationwithout surgery.Figure 3. As seen on the abdominal CT, there is a large tumour in the retroperitoneumwith predominantly fat-equivalent attenuation that replacedboth the kidneys entirely and displaced intraperitoneal structures anteriorly.Figure 3. As seen on the abdominal CT, there is a large tumour in the retroperitoneumwith predominantly fat-equivalent attenuation that replacedboth the kidneys entirely and displaced intraperitoneal structures anteriorly.122

DISCUSSIONAngiomyolipomas are uncommon, benign, renal neoplasmscomprised of blood vessels with thickened walls,immature smooth muscle cells and mature adipose tissue.They occur as isolated, sporadic entities in 80% of cases.The remaining 20% develop in association with tuberoussclerosis (16, 17). This tumour is one of the major diagnosticcriteria of tuberous sclerosis (16). The occurrence ofangiomyolipomas in the tuberous sclerosis complex can beup to 80%. Although the histological appearance of angiomyolipomasin these two entities is identical, renal angiomyolipomaswith tuberous sclerosis are distinctly differentfrom those without tuberous sclerosis. Angiomyolipomasin association with tuberous sclerosis manifest at a youngerage. They are likely to be larger and bilateral and areprone to grow and need surgical treatment (17). In the seriesby Steiner et al., the average size of AML in patientswith TS is 9.6±4.8 cm and 4.1±3.4 cm in those without TS(17) (Table 1).Angiomyolipoma – benign mesenchymal tumourISOLATED (80%) ASSOCIATED WITH TS(20%)- usually solitary- unilateral (80% on theright side)- not associated with TS- mean age of incidence:40-49 years- much more common infemales- occurs in 80% of patientswith TS- commonly large- usually bilateral- usually multiple- may be only evidence ofTS- mean age of incidence:12-19 years- equal incidence in malesand femalesTable 1. Types of angiomyolipomas. Differences between the isolatedform and the form associated with TS.The hormonal influences of the steroid receptors inthe muscle cells may explain the differences in tumour behaviourseen during different periods of life, with greatertumour growth in postpubertal period and during pregnancy(22). L’Hostis et al. observed the presence of bothprogesterone and oestrogen receptors in angiomyolipomas,and they found that progesterone and oestrogenimmunoreactive angiomyolipomas were predominantlyfound in women and in patients with tuberous sclerosis(23). These findings may further explain the more aggressivenature of the disease process in patients with tuberoussclerosis, the hormonal potentiation of tumour growthand haemorrhage in conditions such as pregnancy, and theoverwhelming female predominance in the sporadic formof angiomyolipoma without tuberous sclerosis (23, 24-26).Angiomyolipomas remain silent and are commonly incidentalfindings, but they may manifest with symptoms ofabdominal and flank pain, gross haematuria, nausea, vomiting,fever, abdominal distension or simply as a mass (16,17, 27, 28). Common findings include a palpable mass, abdominaltenderness, haematuria, anaemia, shock, hypertension,urinary tract infections, and renal failure. Thesesigns and symptoms are usually a result of the effects ofthe mass and haemorrhage (16). The propensity to haemorrhageis related to multiple factors: focal deficiencies ofelastic tissue in abnormally rigid and thick blood vessels,hypervascularity, and venous invasion (29-31). The defectsin the vessel’s elastic tissue also predispose these lesionsto develop aneurysms. Our patient presented late with abdominalpain and mild anaemia but no renal failure.With advances in cross-sectional imaging, the diagnosisof renal angiomyolipoma can usually be made confidentlywithout surgery. The demonstration of fat on renal ultrasoundand CT can accurately diagnose angiomyolipoma in95% of cases (16). Angiomyolipomas are well-defined hyperechoicmasses based on ultrasonography (US) regardlessof the relative fat component (32). Angiomyolipomafat is easily identified on CT, which helps to make the radiologicaldiagnosis. Demonstration of intratumoural fatattenuation is almost pathognomonic for this lesion (16,17, 27, 28). Single or multiple well-circumscribed renalcortical tumours containing tissue with fat attenuation ofless than -20 HU are characteristic findings of angiomyolipomaon non-enhanced CT (16). In the present case, adiagnosis of AML was made based on typical ultrasoundand CT findings.Angiomyolipomas are at risk for spontaneous haemorrhage.Lesions greater than 4 cm are at greater risk of seriousspontaneous haemorrhage and need to be evaluated.Tumour diameter >4 cm has also been used as a criterionfor prophylactic treatment because many studies show ahigher frequency of haemorrhagic complications withlarger tumours (17, 33). However, according to Antonopouloset al. (34), these tumours do not have to be large(>4-cm diameter) before serious life-threatening haemorrhagecan occur, as previous studies have suggested, andat least one study has shown that smaller tumours 10 cm, the preferred route of treatment is partialnephrectomy or selective arterial embolisation (16, 17).Massive retroperitoneal haemorrhage due to AML, alsoknown as Wunderlich’s Syndrome, has been found in up to10% of patients (36). Although many angiomyolipomas donot show growth over time, those that occur with TSC aremore likely to show progressive evolution. Conservativeobservation and follow-up examinations of asymptomaticpatients with tuberous sclerosis and angiomyolipoma arerecommended with bi-annual or annual imaging: US, CT,or MR imaging.Selective arterial embolisation has been effective in thetreatment of acute haemorrhage, with or without later surgery,or as initial treatment of angiomyolipoma (37-39). Althougharterial embolisation is minimally invasive, it doesnot preserve renal function. It only has a temporary effect,it requires close clinical observation because of associated123

complications, and, as a rule, it is ineffective when usedalone. Regarding surgical treatment, a tumourectomy, partialnephrectomy, or total nephrectomy may be done. Thesurgical treatment that preserves the largest amount of renaltissue is tumour enucleation, which has been performedwith excellent results, even for large angiomyolipomas(>20 cm). Tumour enucleation is practically applicable forpatients with tuberous sclerosis who present with multipleand bilateral lesions (39-41). Total nephrectomy should beused very rarely; it is only justified in cases of uncontrollablebleeding, when there is risk to the patient’s life, in centraltumours, in the presence of extensive necrosis, whenthere is inflammation of the renal tissue, or when there is adiagnosis of renal carcinoma in the same kidney. Recently,cryotherapy has been suggested as a therapeutic optionand may be associated with laparoscopy (42). The primarytreatment objective of angiomyolipoma is the preservationof renal function, principally in those cases in which it isassociated with tuberous sclerosis and the lesions are generallylarger, multiple, bilateral, and recurring. Amongstthe therapeutic options, tumour enucleation increases thepotential for preservation of the renal tissue, followed byselective arterial embolisation and cryotherapy. Embolisationprimarily controls the bleeding in the acute phase butmay lead to greater loss of the renal function.In conclusion, the basis of management for angiomyolipomais to preserve of renal tissue and function,which can be effectively achieved with nephron-sparingsurgical procedures such as tumour enucleation. Howeverin some circumstances, it is necessary to do selectiveangioembolisation, partial nephrectomy, or eventotal nephrectomy. Especially in patients with tuberoussclerosis with large bilateral and multiple tumours, theaim of treatment is to preserve the greatest amount ofefficient renal function.REFERENCES1. Barron RP, Kainulainen VT, Forrest CR et al. Tuberous sclerosis:Clinopathologic features and review of the literature.Journal of cranio-maxillofacial surgery. 2002; 300:361-3662. Jozwiak J. Hamartin and tuberin: Working together fortumor progression. International Journal of Cancer.2006; 118:1-53. Callaghan FJ, Osborne JP. Advances in the understandingof tuberous sclerosis. Archives of Disease in Childhood.2000; 83:140-1424. Tsao H. Tuberous sclerosis. In: Bologna JL, Jorizzio JL,Rapini RP editor. Dermatology. Philadelphia: Mosby;2003: 858-8675. Roach ES, Sparagana SP. Diagnosis of tuberous sclerosiscomplex. Journal of Child Neurology. 2004; 19:643-6496. Osborne JP. Tuberous sclerosis. In: Harper J, OranjeA, Prose N, editor. Textbook of Dermatology. OxfordBlackwells Publishing: 2006;1491-15027. 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Musculosceletal and visceral findings).Journal of the American Academy of Dermatology.2001; 45:450-45222. Ewalt DH, Shepheild E, Sparagana SP et al. Renal lesiongrowth in children with tuberous sclerosis complex.Journal of Urology. 1998; 160:141-14523. L Hostis H, Deminiere C, Ferriere JM et al. Renal angiomyolipoma:a clinicopathologic, immunohistochemicaland follow up study of 46 cases. American Journal ofSurgical Pathology. 1999; 23:1011-102024. Cibas ES, Goss GA, Kulke MH et al. Malignant epiteliolidangiomyolipoma (sarcoma ex angimyolipoma) of thekidney, a case report and review of the literature. AmericanJournal of Surgical Pathology. 2001; 25:121-126124

25. Desai S, Hejmadi R, Krishnamurty S et al. Renal angiomyolipoma:a clinicopathologic, immunohistohemicaland follow-up study of 46 cases. American Journal ofSurgical Pathology. 2001; 25:972-97326. Eble JN. Angiomyolipoma of kidney. Seminars in DiagnosticsPatholology 1998; 15:21-4027. Maziak DE, Kesten S, Rappaport DC et al. Extra thoracicangiomyolipomas in lymphangioleiomyomathosis.European Respiratory Journal 1996; 9:402-40528. Lloyd GL, Robin EA, Anna ES. Angiomyolipomas intuberous sclerosis. Radiographics 2003; 23:241-24629. Gentry LR, Gould HR, Alter AJ et al. Hemorrhagic angimyolipoma:demonstration by computed tomography.Journal of Computer Assisted Tomography 1981;5:861-86530. Mouded IM, Tolia BM, Bernie JE et al. Symptomaticrenal angiomyolipoma: report of 8 cases, two withspontaneous rupture. The Journal of Urology 1978;119: 684-68831. Price EB, Mostofi FK. Symptomatic angiomyolipoma inthe kidney. Cancer 1965; 18: 761-77432. Helenon O, Merran S, Paraf F et al. Unusual fat containingtumors of the kidney: diagnostic dilemma. Radiographics1997; 17:129-14433. Oesterling JE, Fishman EK, Goldman SM et al. Themanagement of renal angimyolipoma. The Journal ofUrology 1986; 135:1121-112434. Antonopoulos P, Drossos C, Triantopoulou C et al.Complications of renal angiomyolipomas CT evaluation.Abdominal Imaging 1996; 21:357-36035. Yamamoto S, Nakamura K, Kawanami S et al. Renal angimyolipoma:evolutional changes of its internal structureon CT. Abdominal Imaging 2000; 25:651-65436. Noor N, Aatif HS, Salman EK. Tuberous sclerosis withbilateral renal angiomyolipoma and Wunderlich’s syndrome.Journal of College of Physicians and SurgeonsPakistan 2007; 17(11): 706-70737. Tongaonkar HB, Sampat MB, Dalal AV et al. Bilateralrenal angiomyolipoma. Journal of Surgical Oncology1994; 57:65-7038. Bosniak MA. Angiomyolipoma (hamartoma) of thekidney: a preoperative diagnosis is possible in virtuallyevery case. TheUrological Radiology 1981; 3:135-14239. Oesterling JE, Fishman EK, Goldman SM et al. Themanagement of renal angiomyolipoma. The Journal ofUrology 1986; 135:1121-112440. Cen CH, Yu TJ, Hsu K. Unusual presentation of angiomyolipoma.Changgeng Yie Xue Za Zhi 1991; 14:269-27241. Meiri H, Soejima K, Tokuda Y et al. The managementselection of renal angiomyolipoma. Nippon HinyokikaGakkai Zasshi 1996; 87:1197-120042. Delworth MG, Pisters LL, Fornage BD et al. Cryoteraphyfor renal cell carcinoma and angiomyolipoma. TheJournal of Urology 1996; 155:252-255125

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INSTRUCTION TO AUTHORSFOR MANUSCRIPT PREPARATIONSerbian Journal of Experimental and Clinical Researchis a peer-reviewed, general biomedical journal. It publishesoriginal basic and clinical research, clinical practice articles,critical reviews, case reports, evaluations of scientificmethods, works dealing with ethical and social aspects ofbiomedicine as well as letters to the editor, reports of associationactivities, book reviews, news in biomedicine, andany other article and information concerned with practiceand research in biomedicine, written in the English.Original manuscripts will be accepted with the understandingthat they are solely contributed to the Journal.The papers will be not accepted if they contain the materialthat has already been published or has been submittedor accepted for publication elsewhere, except of preliminaryreports, such as an abstract, poster or press reportpresented at a professional or scientific meetings and notexceeding 400 words. Any previous publication in suchform must be disclosed in a footnote. In rare exceptionsa secondary publication will acceptable, but authors arerequired to contact Editor-in-chief before submission ofsuch manuscript. the Journal is devoted to the Guidelineson Good Publication Practice as established by Committeeon Publication Ethics-COPE (posted at www.publicationethics.org.uk).Manuscripts are prepared in accordance with „UniformRequirements for Manuscripts submitted to BiomedicalJournals“ developed by the International Committee ofMedical Journal Editors. Consult a current version of theinstructions, which has been published in several journals(for example: Ann Intern Med 1997;126:36-47) and posted atwww.icmje.org, and a recent issue of the Journal in preparingyour manuscript. For articles of randomized controlledtrials authors should refer to the „Consort statement“ (www.consort-statement.org). Manuscripts must be accompaniedby a cover letter, signed by all authors, with a statement thatthe manuscript has been read and approved by them, and notpublished, submitted or accepted elsewhere. Manuscripts,which are accepted for publication in the Journal, become theproperty of the Journal, and may not be published anywhereelse without written permission from the publisher.Serbian Journal of Experimental and Clinical Researchis owned and published by Medical Faculty University ofKragujevac. However, Editors have full academic freedomand authority for determining the content of the journal, accordingto their scientific, professional and ethical judgment.Editorial policy and decision making follow procedureswhich are endeavoring to ensure scientific credibility of publishedcontent, confidentiality and integrity of auth ors, reviewers,and review process, protection of patients’ rights toprivacy and disclosing of conflict of interests. For difficultieswhich might appear in the Journal content such as errors inpublished articles or scientific concerns about research findings,appropriate handling is provided. The requirements forthe content, which appears on the Journal internet site orSupplements, are, in general, the same as for the master version.Advertising which appears in the Journal or its internetsite is not allowed to influence editorial decisions.Ad dress ma nu scripts to:Serbian Journal of Experimental andClinical ResearchThe Me di cal Fa culty Kra gu je vacP.O. Box 124, Sve to za ra Mar ko vi ca 6934000 Kra gu je vac, Ser biaTel. +381 (0)34 30 68 00;Tfx. +381 (0)34 30 68 00 ext. 112E-mail: sjecr@medf.kg.ac.rsManuscript can also be submitted to web address ofjournal: www.medf.kg.ac.rs/journalMA NU SCRIPTOriginal and two anonymous copies of a ma nu script,typed do u ble-spa ced thro ug ho ut (in clu ding re fe ren ces, tables,fi gu re le gends and fo ot no tes) on A4 (21 cm x 29,7 cm)pa per with wi de mar gins, should be submitted for considerationfor publication in Serbian Journal of Experimentaland Clinical Research. Use Ti mes New Ro man font, 12pt. Ma nu script sho uld be sent al so on an IBM com pa ti ble127

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Ar tic le: (all aut hors are li sted if the re are six or fe wer;ot her wi se only the first three are li sted fol lo wed by “et al.”)12. Tal ley NJ, Zin sme i ster AR, Schleck CD, Mel ton LJ.Dyspep sia and dyspep tic sub gro ups: a po pu la tion-ba sedstudy. Ga stro en te ro logy 1992; 102: 1259-68.Bo ok: 17. Sher lock S. Di se a ses of the li ver and bi li arysystem. 8th ed. Ox ford: Blac kwell Sc Publ, 1989.Chap ter or ar tic le in a bo ok: 24. Tri er JJ. Ce li ac sprue.In: Sle i sen ger MH, For dtran JS, eds. Ga stro-in te sti nal di sease. 4th ed. Phi la delp hia: WB Sa un ders Co, 1989: 1134-52.The aut hors are re spon si ble for the exac tness of referen ce da ta.For other types of references, style and interpunction,the authors should refer to a recent issue of Serbian Journalof Experimental and Clinical Research or contact theeditorial staff.Non-English citation should be preferably translated toEnglish language adding at the end in the brackets nativelangauage source, e.g. (in Serbian). Citation in old languagerecognised in medicine (eg. Latin, Greek) should beleft in their own. For internet soruces add at the end insmall bracckets ULR address and date of access, eg. (Accessedin Sep 2007 at www.medf.kg.ac.yu). If available, insteadof ULR cite DOI code e.g. (doi: 10.1111/j.1442-2042.2007.01834.x)TA BLESTa bles should be typed on se pa ra te she ets with ta blenum bers (Ara bic) and ti tle abo ve the ta ble and ex pla na toryno tes, if any, be low the ta ble.FI GU RES AND FI GU RE LE GENDSAll il lu stra ti ons (pho to graphs, graphs, di a grams) willbe con si de red as fi gu res, and num be red con se cu ti vely inAra bic nu me rals. The num ber of fi gu res in clu ded shouldbe the le ast re qu i red to con vey the mes sa ge of the pa per,and no fi gu re should du pli ca te the da ta pre sented in theta bles or text. Fi gu res should not ha ve ti tles. Let ters, numerals and symbols must be cle ar, in pro por tion to eachot her, and lar ge eno ugh to be readable when re du ced forpu bli ca tion. Fi gu res should be sub mit ted as ne ar to the irprin ted si ze as pos si ble. Fi gu res are re pro du ced in one ofthe fol lo wing width si zes: 8 cm, 12 cm or 17 cm, and witha ma xi mal length of 20 cm. Le gends for fi gu res sho uld begi ven on se pa ra te pa ges.If mag ni fi ca tion is sig ni fi cant (pho to mic ro graphs)it should be in di ca ted by a ca li bra tion bar on the print,not by a mag ni fi ca tion fac tor in the fi gu re le gend. Thelength of the bar should be in di ca ted on the fi gu re or inthe fi gu re le gend.Two com ple te sets of high qu a lity un mo un ted glossyprints should be sub mit ted in two se pa ra te en ve lo pes, andshi el ded by an ap pro pri a te card bo ard. The backs of singleor gro u ped il lu stra ti ons (pla tes) should be ar the firstaut hors last na me, fi gu re num ber, and an ar row in di ca tingthe top. This in for ma tion should be pen ci led in lightly orpla ced on a typed self-ad he si ve la bel in or der to pre ventmar king the front sur fa ce of the il lu stra tion.Pho to graphs of iden ti fi a ble pa ti ents must be ac com paniedby writ ten per mis sion from the pa ti ent.For fi gu res pu blis hed pre vi o usly the ori gi nal so ur ceshould be ac know led ged, and writ ten per mis sion from thecopyright hol der to re pro du ce it sub mit ted.Co lor prints are ava i la ble by re qu est at the aut horsex pen se.LET TERS TO THE EDI TORBoth let ters con cer ning and tho se not con cer ningthe ar tic les that ha ve been pu blis hed in Serbian Journalof Experimental and Clinical Research will be con si deredfor pu bli ca tion. They may con tain one ta ble or fi gureand up to fi ve re fe ren ces.PROOFSAll ma nu scripts will be ca re fully re vi sed by the publisher desk edi tor. Only in ca se of ex ten si ve cor rec ti onswill the ma nu script be re tur ned to the aut hors for fi nalap pro val. In or der to speed up pu bli ca tion no pro of willbe sent to the aut hors, but will be read by the edi tor andthe desk edi tor.129

CIP – Каталогизација у публикацијиНародна библиотека Србије, Београд61SERBIAN Journal of Experimental and Clinical Researcheditor - in - chief SlobodanJanković. Vol. 9, no. 1 (2008) -Kragujevac (Svetozara Markovića 69):Medical faculty, 2008 - (Kragujevac: Medical faculty). - 29 cmJe nastavak: Medicus (Kragujevac) = ISSN 1450 – 7994ISSN 1820 – 8665 = Serbian Journal ofExperimental and Clinical ResearchCOBISS.SR-ID 149695244130