A modified microsurgical model for end-to-side selective portacaval shunt in the rat. Intraoperative microcirculatory investigations

Ac a Ci ú gica B asilei a - Vol. 28 (9) 2013 - 625 1 – ORIGINAL ARTICLE MICROSURGICAL MODEL A modi ied mic osu gical model o end- o-side selec i e po aca al shun in he a . In aope a i e mic oci cula o y in es iga ions1 Zol an Kla ikI, Eniko To hII, Fe enc KissIII, I en MikoIV , Is an Fu kaV , No be Neme hVI IMD, Pos g adua e lec u e , Depa men o Ope a i e Techniques and Su gical Resea ch, Ins i u e o Su ge y, Medical and Heal h Science Cen e , Uni e si y o Deb ecen, Hunga y. Pe o med he mic osu gical ope a ions, in ol ed wi h echnical p ocedu es, analysis and in e p e a ion o da a, manusc ip w i ing. IIMD, Pos g adua e lec u e , Depa men o Ope a i e Techniques and Su gical Resea ch, Ins i u e o Su ge y, Medical and Heal h Science Cen e , Uni e si y o Deb ecen, Hunga y. Taking pa in mic oci cula o y da a acquisi ion and da a analysis. IIIMD, PhD, Assis an lec u e , Depa men o Ope a i e Techniques and Su gical Resea ch, Ins i u e o Su ge y, Medical and Heal h Science Cen e , Uni e si y o Deb ecen, Hunga y. Taking pa in mic oci cula o y da a acquisi ion and da a analysis. IVMD, PhD, Full P o esso , Depa men o Ope a i e Techniques and Su gical Resea ch, Ins i u e o Su ge y, Medical and Heal h Science Cen e , Uni e si y o Deb ecen, Hunga y. Manusc ip w i ing, c i ical e ision. VMD, PhD, Eme i us P o esso , Depa men o Ope a i e Techniques and Su gical Resea ch, Ins i u e o Su ge y, Medical and Heal h Science Cen e , Uni e si y o Deb ecen, Hunga y. Manusc ip w i ing, c i ical e ision. VIMD, PhD, Associa e P o esso , Head o Depa men , Depa men o Ope a i e Techniques and Su gical Resea ch, Ins i u e o Su ge y, Medical and Heal h Science Cen e , Uni e si y o Deb ecen, Hunga y. Planning and conduc ing expe imen , e alua ing esul s, manusc ip w i ing. AbSTRACT PURPOSE: To in es iga e he in aope a i e mic oci cula o y changes o he a ec ed o gans (small bowel, li e and kidney) du ing he making o a modi ied selec i e po aca al (PC) shun . METHODS: On en anaes he ized Sp ague-Dawley a s he selec i e end- o-side mesoca al anas omosis was pe o med, whe e only he os al mesen e ic ein is u ilized and he po al ein wi h he splenic ein a e le in ac . Mo phome ic and mic oci cula o y in es iga ions using a LDF de ice de e mining lux uni s (BFU) we e ca ied ou . RESULTS: A e comple ing he shun s he mic oci cula o y lux alues did no eco e in he same manne on he su ace o he small in es ine, he li e o he kidney. BFU alues showed de e io a ion in he small in es ine and in he li e (p<0.001). Du ing he epe usion he BFU alues imp o ed, bu no in he same manne . The small in es ine alues le behind he kidney and li e da a. CONCLUSIONS: Technically, he ad an ages o he models include he selec i e cha ac e is ic, he mesoca al localiza ion and he ela i ely easy access o hose essels. Howe e , i s majo disad an age is he ime needed o posi ioning he essels wi hou coiling o de ini i e s e ching. In aope a i e LDF may p o ide use ul da a on he mic oci cula o y a ec ion o he o gans su e ing om hypope usion o ischemia du ing c ea ing he shun s. Key wo ds: Po aca al Shun , Su gical. Anas omosis, Su gical. Mic osu ge y. Mic oci cula ion. Ra s. Kla ik Z e al. 626 - Ac a Ci ú gica B asilei a - Vol. 28 (9) 2013 In oduc ion A i icial po o-sys emic shun s can be c ea ed by a ious echniques and localiza ions in case o unde lying po al hype ension when o he he apeu ical ways a e no e ec i e1-5, bu hese shun s may ac as suppo i e ools in o he su gical p ocedu es (li e ansplan a ion, small- o -size g a ing)6,7. So hese shun s s ill ha e impo an clinical ele ance. While c ea ing such a i icial anas omoses, he su gical sa e y is an impo an aspec s: pa ency, geome y and long- e m e ec i eness o hese shun s ha e p o ed o be signi ican 8,9. Fo analyzing he complex unc ional and mo phological changes, nume ous po aca al shun models ha e been de eloped in a s, abbi s, dogs, pigs o e en using simula ion models10-17. Lee has made he i s mic osu gical po aca al shun in he a 10. I was an end- o-side anas omosis model, which had been widely applied in a ie y o esea ch models. Besides u he e ined models3,15, side- o-side a ia ion1,3,11 and in e posi ioned H-g a me hods a e s ill used oday. In case o selec i e po aca al shun , one o he main b anches o he po al enous sys em is connec ed di ec ly in o he in e io ca al ein o d ained in o he le enal ein. The dis al spleno enal shun 4,12,14,19 is one o he example o such selec i e shun s. Based on he ana omical possibili ies u he selec i e po aca al shun s we e c ea ed, such as he mesoca al localiza ion8,11,12,15,19-21. In his s udy we desc ibe a e ined mic osu gical model o a selec i e po aca al shun model in he a , whe e only he os al mesen e ic ein is u ilized and he po al and lienal ein a e le in ac . Since po aca al shun causes hemodynamic al e a ions (p essu e-g adien di e ences, hepa ic and splanchnic low dynamic changes, selec i e edis ibu ion o ca diac ou pu )3,18,20,22-25, we also aimed o obse e he mic oci cula o y changes o he a ec ed o gans (li e , small in es ine and igh kidney) in aope a i ely. In e es ingly he e is a lack o da a in he li e a u e abou in aope a i e mic oci cula o y changes in he o gans ha a e a ec ed by hypope usion o ischemia du ing c ea ing o po aca al shun . Me hods Expe imen al animals and ope a i e echniques The expe imen s we e app o ed and egis e ed by he Uni e si y o Deb ecen Commi ee o Animal Resea ch (pe mission N .: 6/2008. UD CAR), in acco dance wi h he ele an Hunga ian Animal P o ec ion Ac (Law XVIII/1998). Ten Sp ague-Dawley a s (bodyweigh : 340.9 ± 24.52 g) we e subjec ed o he s udy. The expe imen al animals we e anes he ized using sodium- hiopen al (60 mg/kg i.p., Thiopen hal, Biocheme GmbH, Aus ia). Fo mic osu gical ope a ions a Leica Wild M650 ope a i e mic oscope was used, and ideo eco dings we e made. Du ing he ope a ion he ec al empe a u e was moni o ed (SEN-06-RTH1 S ick empe a u e p obe, Expe ime ia L d., Hunga y). The mic osu gical echnique p o ided a design o a selec i e po aca al shun , in which only he os al mesen e ic ein ( MV) was su u ed, as end- o-side anas omosis, in o he caudal ca al ein (CCV), while he o he main b anch o he po al ein (gas osplenic ein) was le in ac (Figu es 1 and 2). FIGURE 1 – Schema ic d awing o he enewed, selec i e po aca al shun model (d awing by Z. Kla ik, wi h he main con ou s om Re . 26, as a empla e). CCV: caudal ca al ein, PV: po al ein, MV: os al mesen e ic ein, SV: splenic ein, L: li e . L CCV PV SV MV A modi ied mic osu gical model o end- o-side selec i e po aca al shun in he a . In aope a i e mic oci cula o y in es iga ions Ac a Ci ú gica B asilei a - Vol. 28 (9) 2013 - 627 FIGURE 2 – A: no mal ana omical posi ions o he caudal ca al ein (CCV) and he os al mesen e ic ein ( MV). b: he comple ed end- o- side anas omosis (a ow) be ween he MV and he CCV a mesoca al localiza ion. Magni ica ion: x16 in A, x25 in B. A e median lapa o omy and an eposi ion o he in es ines, ca e ul dissec ion o he po al ein (PV) and i s ibu a ies we e p epa ed.26 The CCV was mobilized be ween he wo enal eins. A he bi u ca ion o he po al ein he p oximal pa o he MV was liga ed wi h 8/0 b aided silk. Clips we e applied on o he in es inal pa o he MV and o he CCV p oximally and dis ally. The MV was washed ou wi h physiological saline solu ion dilu ed wi h sodium-hepa in (10%) and posi ioned owa d he CCV in o de o minimize he ension. On he isola ed an e io wall o he CCV, eno omy was pe o med wi h he leng h equal o he diame e o he mesen e ic ein. The si e o he eno omy was loca ed as la e al as possible. Fi s ly, he pos e io wall o he anas omosis was con inuously su u ed, while on he an e io wall simple in e up ed su u es (6-7 s i ches) we e used wi h 10/0 polyamide mono ilamen su u e ma e ial. A e eleasing he clips, he shun pa ency was checked. The in es ines hen we e e-posi ioned and a e comple ing he in aope a i e mic oci cula o y measu emen s, he abdominal wall was closed in wo laye s. Mo phome ic analyses o he shun s The essel geome y pa ame e s we e de e mined o - line om he ideo- eco dings: ou e diame e o he CCV and he MV, as well as o he legs o he comple ed end- o-side anas omoses oge he wi h hei angle. The o line measu emen s we e ca ied ou wi h Adobe Pho oshop CS5 so wa e. The angle o he anas omosis a he heel was measu ed wi h he same so wa e. The axis o he eins was d awn and he angle was calcula ed whe e he lines we e connec ed. In aope a i e mic oci cula o y in es iga ions A non-in asi e lase Dopple issue lowme y was used (LD-01 lase Dopple issue lowme y moni o ing sys em, Expe ime ia L d., Hunga y) wi h a s anda d pencil p obe (MNP100XP, Ox o d Op onix L d., UK) placed on he su ace o he li e ’s middle lobe, on he an imesen e ic su ace o a jejunum segmen , as well as on he an e io su ace o he igh kidney. Based upon he Dopple shi e ec when he lase beam is e lec ed om mo ing ed blood cells, he de ice de e mines blood lux uni s (BFU), which we e egis e ed o 20 seconds a e s abiliza ion o he signal. Using single-channel lase Dopple lowme y, i is impo an o se he s anda ds o he e alua ion27,28-30, we ha e chosen he e alua ion o he a e age BFU alues o a s anda d ime pe iod o 20 seconds30. The lase Dopple measu emen s we e ca ied ou be o e (Base) and sho ly a e applying he clips, be o e and jus a e eleasing he clamping (Repe usion 0’) as well as in he 30 h minu es o he epe usion ( epe usion 30’). S a is ical analysis Da a a e p esen ed as mean ± s anda d de ia ion (S.D.), i is indica ed i median ± s anda d e o (S.E.) is shown. The compa ison o in aope a i e lase Dopple lowme y da a ob ained om a ious measu emen si es we e ca ied ou wi h S uden ’s - es o Mann-Whi ney ank sum es , while he changes du ing he ime- ame o expe imen wi hin measu emen si es we e analyzed by one-way ANOVA me hods (Bon e oni’s o Dunne ’s es ), depending on he da a dis ibu ion. The signi icance le el we e conside ed when p<0.05. Resul s Technical expe iences and mo phome ic da a The c ea ed mesoca al end- o-side enous anas omosis was unc ioning well; he e we e no bleeding a he si e o he anas omosis. I s pa ency was main ained and no s enosis was obse ed. The ope a ion ime was 48.75 ± 11.26 min. The longes phase was he p epa a ion and posi ioning o he essels o be, p o iding si ua ion wi hou ea ing, s e ching o up u ing he in ima. Du ing he p ocedu e (when clips we e being applied) he changing o o gans’ colo was well obse able. The small in es ine showed enous conges ion and he li e became pale . The p esen ed geome y o he shun occu ed due o he ana omical localiza ion o he gi en essels, since he e was a pe sis ed isk o he MV o ea a a ious angula ion posi ioning. In o de o minimalize such in aope a i e complica ion, he Li e MV A b CCV MV M: 16X M: 25X Kla ik Z e al. 628 - Ac a Ci ú gica B asilei a - Vol. 28 (9) 2013 anas omosis could be c ea ed ension- ee and a he angula ion o 77.13 ± 5.84°. The ou e diame e o he MV as well as he CCV dila ed jus abo e o below he shun compa ed o he no mal si ua ion (s a e be o e applying he clips) (Table 1). Dila a ion o he MV was no signi ican , and he CCV diame e was inc eased signi ican ly abo e (p<0.001 s. no mal; p=0.05 s. anas omosis si e) and below (p<0.001 s. no mal; p=0.004 s. anas omosis si e) he shun . TAbLE 1 – Diame e o he caudal ca al ein and he os al mesen e ic ein be o e and a e comple ing he end- o-side anas omosis. Vessel Diame e [mm] Be o e A e caudal ca al ein 2.41 ± 0.15 abo e he anas omosis: 3 ± 0.36 a he anas omosis: 2.44 ± 0.63 below he anas omosis: 3.51 ± 0.61 os al mesen e ic ein 1.26 ± 0.19 1.4 ± 0.33 In aope a i e mic oci cula o y in es iga ions The changes o blood lux uni (BFU) a e shown on Figu e 3. A base le el he alues o li e (32.64 ± 13.68), jejunum (35.83 ± 13.67) o kidney (36.14 ± 17.71) did no di e signi ican ly among each o he . When applying he clips acco ding o he ope a i e p o ocol, he in es inal BFU alues as well as enal ones dec eased, and as we expec ed, i was mo e exp essed on he jejunum. A e clamping he in es inal alues (17.61 ± 9.09) we e signi ican ly lowe compa ed o i s base (p<0.001), as well as e sus li e (29.13 ± 14.45, p<0.001) and kidney alues (24.75 ± 10.86, p=0.001). Renal BFU dec eased signi ican ly compa ed o i s base (p<0.001). FIGURE 3 – Al e a ions o mic oci cula o y blood lux uni (BFU) measu ed on he su ace small in es ine (jejunum-pa ), middle lobe o he li e and on he igh kidney. Median ± S.E., n=10. * p<0.05 s. Base (one way ANOVA es , Bon e oni’s/Dunne ’s), #p<0.05 s. Li e ; + p<0.05 s. Kidney ( - es /Mann-Whi ney RS es ). Jus be o e eleasing he clamps he lowe ed alues we e mo e exp essed, keeping simila ela ions be ween he in es iga ed o gans (on he jejunum: 12.03 ± 9.19, p<0.001 s. base and li e , p=0.036 s. kidney; on he li e : 23.26 ± 13.37, p<0.001 s. base, p=0.02 s. kidney; on he kidney: 16.07 ± 10.91, p<0.001 s. base). Jus a e eleasing all he clips, he BFU alues s a ed o inc ease, bu no in he same manne in he o gans. The enal BFU alues eco e ed almos comple ely (31.87 ± 12.59), while in es inal alues (21.26 ± 17.07, p<0.001 s. base, p<0.001 s. kidney) as well as hepa ic BFU alues (24.43 ± 12.06, p<0.001 s. base, p<0.001 s. kidney) we e d opped behind he enal da a. Du ing he in es iga ed epe usion pe iod, he in es inal alues we e emained lowe compa ed o he hepa ic alues, howe e , he p alues we e close o he signi icance le el (a epe usion 0’: p=0.059; a epe usion 30’: p=0.051). Discussion Expe imen al esea ches ocusing on ascula anas omosis ha e high clinical impo ance hence he in es iga ion o su gical sa e y, he mo phological ascula changes and he long e m e ec i es o hese p ocedu es a e ine i able. S udying hese aspec s is necessa y o a be e unde s anding o he cha ac e is ic o such decomp essi e po o-sys emic shun s3,10,13,31. The su gical managemen o hese shun s is echnically di icul , and because o hese ac o s, a iously localized po aca al shun s ha e been c ea ed since in he 1960s3,12,19. The pu pose o he s udy g oups was o desc ibe new su gical me hods o cons uc an anas omosis, whe e he echnical di icul ies (ana omical dis ance be ween he wo essels, sho ness o he po al ein) can be o e whelmed. Wi h he ad ance o mic osu ge y, hese no el echnique models could ha e been in es iga ed in smalle animals and b ough he possibili y o 0 5 10 15 20 25 30 35 Base A e clamping Be o e eleasing Repe usion 0' Repe usion 30' bFU Small in es ine Li e Righ kidney * # +* # + * +* + * + * + * * * A modi ied mic osu gical model o end- o-side selec i e po aca al shun in he a . In aope a i e mic oci cula o y in es iga ions Ac a Ci ú gica B asilei a - Vol. 28 (9) 2013 - 629 u he de elopmen o he su gical p o ocols. Lee e al.10 and Numa a e al.13 we e he pionee s o desc ibe po aca al shun models in small labo a o y animals, and obse e changes in he p esence o hese anas omoses. Addi ionally, labo a o y a s p o ed o be excellen models o s udy li e ci hosis11, as well as hepa ic encephalopa hy31-33. In hose expe imen s he li e a ophy and egene a ion34, mic o ascula s uc u al changes35, as well as emo e e ec s we e s udied. Howe e , mos o hese shun s we e non-selec i e ypes. Nume ous expe imen s ocused on he compa ison o he cha ac e is ics o he di e en ly localized shun s3,14,20. In hei s udy, D apanas e al.20 desc ibed p essu e and low measu emen s in he hepa ic ci cula ions and obse ed he changes ollowing side- o-side and in e posi ional mesoca al shun . By c ea ing he side- o-side po aca al shun he o al po al blood low ceased, while wi h he selec i e coun e pa only 50% o he o al blood was di e ed. Sch öde e al.14 concluded ha in case o dis al spleno-ca al shun he blood d ained om splenic ein does no signi ican ly a ec he li e compa ed o he non-selec i e po aca al shun . The p esen ed end- o-side mesoca al anas omosis be ween he caudal ca al ein and he os al mesen e ic ein is a enewed mic osu gical model o c ea ing a selec i e po aca al shun . Al hough, he ope a i e echnique is no simple, due o he ana omical dis ances, bu i gi es he possibili y o edi ec he e og ade low exis ing in case o po al hype ension back o he sys emic ci cula ion ia bypassing he li e . Du ing he design o he su gical p o ocol ou aim was o c ea e a si ua ion whe e we can minimize he ension be ween he s uc u es. The e o e, addi ional essel liga ions we e needed o u he mobilize he mesen e ic ein, so he app oxima ion o he ana omical s uc u es was unde less ension. Using he applied su u ing echnique he eins we e no s e ched and he anas omosis was secu e. A he oe o he anas omosis, he i s kno o he con inuous su u e line was a su gical kno . The ad an ages o he models include he selec i e cha ac e is ic ( he gas osplenic ein emains in ac ), he mesoca al localiza ion, and he ela i ely easy access o hose essels. Howe e , i s majo disad an age is he ime needed o posi ioning he essels wi hou coiling o de ini i e s e ching i . I was also obse ed, ha a e comple ing he shun s he mic oci cula o y lux alues did no eco e in he same manne on he su ace o he small in es ine, li e o he kidney. The mesoca al shun is also a compe en model o s udy hepa ic encephalopa hy and po al ein h ombosis. Se e al s udies used po aca al shun s ins ead o pha maceu ical induc ion o encephalopa hy o obse e he neu ological changes31,36. The desc ibed selec i e po aca al shun c ea ed be ween he os al mesen e ic ein and he caudal ena ca a wi h an end- o-side anas omosis di e s in i s su gical echnique a he han i s e ec on he issue and unc ion o he li e om o he p e iously known meso-ca al shun models. Engelb ech e al.21 s udied he e ec o meso-ca al shun s in a s whe e hey c ea ed an end- o-side anas omosis wi h he liga ion o he pylo ic ein. To in es iga e he hepa o ophic ac o s in he li e , Rozga e al.34 applied his shun , howe e hey only used con inuous su u e line. Jakab e al.15 demons a ed a echnique o exclude he possibili y o s enosis du ing he mic o ascula mesoca al shun anas omosis. By liga ing he le and igh b anches o he po al ein and ansec ing a he liga ions, hey c ea ed a “cu ” and wi h ha he diame e o he essel inc eased. Jenkins e al.23 s udied he hemodynamic di e ences be ween o al and selec i e po aca al shun s. They ound ha selec i e shun ing (mesoca al H-g a ) p ese ed li e blood low o a g ea e ex en han o al po aca al shun ing, while hey had less ma ked e ec on he wedge hepa ic p essu e. Since he p esence o any kind o po aca al shun s cause complex changes in he hemodynamic and he blood low a e o he o gans16,17,22,37,38, i was also supposed, ha he mic oci cula o y pa ame e s may show ea ly al e a ions e en sho ly a e he ope a ion24,39-41. Lase Dopple lowme y is a well-known me hod o e alua e mic oci cula o y pa e n o he issues. Wi h he necessa y c i ical e alua ion and s anda dized measu emen condi ions i is an easily applicable me hod o moni o ing o gan mic oci cula o y changes27,28,30. In aope a i e mic oci cula o y measu emen s o he a ec ed o gans wi h lase Dopple lowme y du ing p ocedu es whe e he po al ci cula ion is selec i e decomp essed is no well known in he li e a u e, al hough i can p o e o be bene icial in designing an innocuous su gical echnique since he hypope usion and/o ischemia- epe usion o he o gans can in luence he ou come o such shun s. In his model we es ed he mic oci cula o y blood lux uni s (an in eg al o e e y h ocy e eloci y and numbe ) on li e , jejunum and kidney su ace. The de e io a ion o mic oci cula o y low was p esen acco ding o he blood low cessa ion, based on he clip posi ions. A e eleasing he clamp, i was isible ha he in es inal and hepa ic alues did no eco e in he same manne compa ed o he enal alues. Mic oci cula o y changes a e widely s udied being ela ed o issue hypope usion and ischemia- epe usion. The de e io a ion o mic oci cula o y pa ame e s can be o igina ed om endo helial swelling, bleb o ma ion, Kla ik Z e al. 630 - Ac a Ci ú gica B asilei a - Vol. 28 (9) 2013 asospasm, in e s i ial edema, plugged leukocy es and pla ele s, and also wi h ma ked mic o- heological al e a ions (dec eased de o mabili y and enhanced agg ega ion o he e y h ocy es)41-43. Conce ning he ollow-up o mo phological and unc ional changes o a i icial shun s, in a p e ious wo k we demons a ed signi ican hemodynamic and no able mic o- heological ( ed blood cell de o mabili y and agg ega ion) al e a ions in case o a i icial sapheno-saphenous a e io- enous shun model in he a 44. In a ecen s udy, we ound ao o-po aca al di e ences in he ce ain hemo heological pa ame e s ( ed blood cell agg ega ion, e y h ocy e elonga ion index, osmo ic g adien ek acy ome y pa ame e s)45. By he al e a ions o hemodynamic ac o s in luencing blood low, he hemo heological p ope ies play an impo an ole in he mic oci cula o y egula ion41. The e o e, o unde s and he p ope hemodynamic p o ile o hese shun s and he mic oci cula o y pa e n, u he hemo heological examina ions a e planned. Conclusions In his model a selec i e po aca al shun was achie ed by anas omosing he os al mesen e ic ein in o he caudal ca al ein wi h a modi ied mic osu gical echnique in labo a o y a , whe e he shun s we e well easible and s able geome y was o med. The in aope a i e lase Dopple measu emen s p o ided use ul in o ma ion o he elabo a ion o a mo e secu e su gical echnique wi h he assessmen o mic oci cula o y changes on he a eas exposed o hypope usion and/o ischemia- epe usion. Re e ences 1. Blakemo e AH. The po al shun in he su gical ea men o po al hype ension. Ann Su g. 1948;128:825-41. 2. Dono an AJ. Su gical ea men o po al hype ension: a his o ical pe spec i e. Wo ld J Su g. 1984;8:626-45. 3. 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He e T, Neme h N, B a h E, To h L, Kiss F, Saj os E, Ma yas L, Szaszko J, D imba L, Pei l B, Csiki Z, Miko I, Fu ka I. Mo phological, hemodynamical and hemo heological changes o ma u e a i icial saphenous a e io- enous shun s in he a model. Mic osu ge y. 2010;30:649-56. 45. Kla ik Z, Kiss F, Miko I, Neme h N. Ao o-po o-ca al mic o- heological di e ences o ed blood cells in labo a o y a s: Fu he de o mabili y and ek acy ome ial osmoscan da a. Clin Hemo heol Mic oci c. 2013;53:217-9. Acknowledgemen s To he echnical s a o he Depa men o Ope a i e Techniques and Su gical Resea ch, and o Do ina Bodna . Co espondence: No be Neme h M.D., Ph.D. Depa men o Ope a i e Techniques and Su gical Resea ch, Ins i u e o Su ge y, Medical and Heal h Science Cen e , Uni e si y o Deb ecen H-4032 Deb ecen, Nagye dei k . 98., P.O.Box: 21., Hunga y Phone/Fax: +36-52-416-915 [email p o ec ed] Recei ed: May 27, 2013 Re iew: July 25, 2013 Accep ed: Aug 22, 2013 Con lic o in e es : none Financial sou ce: The Hunga ian Scien i ic Resea ch Fund (G an N .: OTKA K-67779), Janos Bolyai Resea ch Schola ship o he Hunga ian Academy o Sciences (N. Neme h), B idging Fund 2012 (N. Neme h), Na ional P og am o Excellence, Janos Apaczai Cse e Pos g adua e Schola ship A2-ACSJD-12-0380 (Z. Kla ik). 1Resea ch pe o med a Depa men o Ope a i e Techniques and Su gical Resea ch, Ins i u e o Su ge y, Medical and Heal h Science Cen e , Uni e si y o Deb ecen, Hunga y.