Rev. Bras. Anestesiol. vol.66 número5

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REVISTA

BRASILEIRA

DE

ANESTESIOLOGIA

PublicaçãoOficialdaSociedadeBrasileiradeAnestesiologia

www.sba.com.br

SCIENTIFIC

ARTICLE

Comparative

immunohistochemical

assessment

of

the

effect

of

repetitive

anesthesia

with

isoflurane

and

sevoflurane

on

rat

liver

夽

Flavia

Ruxanda

a

_,

_Adrian

_Florin

_Gal

b,∗

_,

_Cristian

_Rat

_¸iu

c

_,

_Viorel

_Micl˘

_aus

_¸

a

_,

_Vasile

_Rus

a

_,

Liviu

Ioan

Oana

d

a_Department_of_Cell_Biology,_Histology_and_Embriology,_University_of_Agricultural_Sciences_and_Veterinary_Medicine,_Cluj-Napoca, Romania

b_Department_of_Pathological_Anatomy,_University_of_Agricultural_Sciences_and_Veterinary_Medicine,_Cluj-Napoca,_Romania c_Department_of_{Implantology,}_Faculty_of_Medicine_and_Pharmacy,_University_of_Oradea,_Oradea,_Romania

d_Department_of_{Anesthesiology}_and_Surgical_{Propedeutics,}_University_of_Agricultural_Sciences_and_Veterinary_Medicine, Cluj-Napoca,Romania

Received14January2015;accepted11February2015 Availableonline26September2015

KEYWORDS

Anesthesia; Caspase-3; Isoflurane; Liver; Sevoflurane

Abstract

Backgroundandobjectives: Inhalationanesthetics areusedin human,aswell asveterinary medicalpractice.Inthepresentstudyweinvestigatedtheeffectofisofluraneandsevoflurane onrathepatocytes.

Methods:A total of40Wistarfemaleratswere usedinthisstudy.Animalswere dividedin groupsof5rats.GroupsIM,SMservedascontrolgroups.GroupsI1,I2,I3wereusedtostudy isofluraneandS1,S2,S3forsevofluranestudy.Theywereanesthetized3times,for2hlong,at 2daysintervalwithaconcentrationof:1.5%isoflurane(I1,I2,I3)and2%sevoflurane(S1,S2, S3).Theoxygensupplythroughouttheanesthesiawas1LO2/min.GroupsIM,IS,I1,S1were

sacrificedimmediatelyafterthelastanesthesia.GroupsI2,S2weresacrificed6hafterthelast anesthesia,andgroupsI3,S3,24hpost-anesthesia.Liversampleswereharvestedtohighlight caspase-3inapoptotichepatocytes.

Results:Followingisofluraneadministration,therewerelessthan1%cellsinapoptosis high-lighted in ratlivers from groups IM, I1 and I2. At24h post-anesthesia(group I3), a small number of apoptotic hepatocyteswas highlighted(around 3.23% cellsin apoptosis),with a strictlyperiacinardisposition,randomlydistributedinasmallnumberofhepaticlobules.After sevofluraneadministration,lessthan1%apoptotichepatocyteswereidentifiedatallcontrol momentsthroughoutthestudy.

夽 _The_study_was _carried_out_at _the_Department_of _{Anesthesiology}_and _Surgical _{Propedeutics,}_University_of _Agricultural_Sciences _and

VeterinaryMedicine,Cluj-Napoca,Romania.

∗_{Corresponding}_author.

E-mail:adrianfloringal@yahoo.com(A.F.Gal).

http://dx.doi.org/10.1016/j.bjane.2015.02.003

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Conclusions:Theresultssuggestthattheanestheticsdonotpresentaconsiderable hepatotox-icity.Thecomparativeassessmentofthetwoanestheticsshowsthatsevofluraneissuperiorto isoflurane.

http://creativecommons.org/licenses/by-nc-nd/4.0/).

PALAVRAS-CHAVE

Anestesia; Caspase-3; Isoflurano; Fígado; Sevoflurano

Avaliac¸ãoimuno-histoquímicacomparativadoefeitodaanestesiarepetitiva

comisofluranoesevofluranosobreofígadoderato

Resumo

Justificativaeobjetivos: Anestésicosinalatóriossãousadosemhumanosetambémnaprática médicaveterinária.Nopresenteestudoinvestigamosoefeitodeisofluranoesevofluranoem hepatócitosderato.

Métodos: Nototal,40ratosWistarfêmeasforamusadosnesteestudo.Osanimaisforam dividi-dosemgruposdecincoratoscada.OsgruposIMeSMserviramcomogruposcontrole.Osgrupos I1,I2eI3foramusadosparaoestudodeisofluranoeosgruposS1,S2eS3paraoestudode sevoflurano.Osratosforamanestesiadostrêsvezes,duranteoperíodode2horasemintervalos dedoisdias,comumaconcentrac¸ãode1,5%deisoflurano(I1,I2,I3)e2%desevoflurano(S1,S2, S3).Ofornecimentodeoxigênioduranteaanestesiafoide1LO2/min.OsgruposIM,IS,I1eS1

foramsacrificadosimediatamenteapósaúltimaanestesia.OsgruposI2eS2foramsacrificados 6horasapósaúltimaanestesiaeosgruposI3eS3foramsacrificados24horasapósaanestesia. Amostrasdosfígadosforamcolhidaspararessaltaracaspase-3emhepatócitosapoptóticos.

Resultados: Apósaadministraçãodeisoflurano,haviamenosde1%dascélulasemapoptose emdestaquenosfígadosdosratosdosgruposIM,I1eI2.Às24horasapósaanestesia(grupo I3),umpequenonúmerodehepatócitosapoptóticosfoidestacado(cercade3,23%decélulas emapoptose),comumadisposiçãoestritamenteperiacinar,distribuídosaleatoriamenteemum pequenonúmerodelóbuloshepáticos.Apósaadministraçãodosevoflurano,menosde1%de hepatócitosapoptóticosfoiidentificadoemtodososmomentosdecontroleaolongodoestudo.

Conclusões:Osresultadossugeremqueosanestésicosnãoapresentamumahepatotoxicidade considerável.Aavaliac¸ãocomparativadosdoisanestésicosmostraquesevofluranoésuperior aoisoflurano.

http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Inhalational anesthetics are widely used in both human and veterinary medicine. They can be utilized in anes-theticmanagementinalargenumberofspecies,including reptiles, birds and wild animals.1---5 _The _primary _site _for metabolism of inhalational anesthetics is represented by theliver.6,7 _Under _these_{circumstances,} _it _is_possible _that somephysiologicalormorphologicalchangesappearinthe liver,duetoeitherthedirectactionoftheseanestheticsor someintermediarymetabolitsresultedduringtheir degra-dation.These aspects havemade the objectof numerous studieswhich broughtmuchusefulinformation, but there arestillcontroversiessurroundingtheinhalational anesthet-ics’actionontheliver.Thus,therearestudiesthatreport aminorliverenzymeselevation,whileothersreport fulmi-nantnecrotichepatitis,resultingeveninpatient’sdeath.8---10 Thegreatmajorityoftheinvestigationshavestudiedthe inhalationalanestheticeffectsonliverfunction.11_Macro_and especiallymicroscopicaspectsweretheobjectofstudyof

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fatalsubacuteliverfailure,withmassivenecrosisof hepa-tocytes,atrepeatedsevofluraneanesthesia.

Consideringthesmallnumberofmorphological investiga-tionsandespeciallytheconflictingavailableinformationin thespecialty literature,we considered opportuneto con-duct histological and immunohistochemical investigations regardingtheisofluraneandsevofluraneeffectonratliver.

Methods

The experimentalprotocol wasapprovedby theScientific Research Ethics Committee of UASVM (University of Agri-culturalSciencesandVeterinaryMedicine)Cluj-Napocaand wasconducted in accordance with theMinistry of Health force and with the pertained European legislation. The experimentalstudywasconductedintheBiobaseofUASVM Cluj-Napoca andinthe DepartmentofAnesthesiology and SurgicalPropedeutics,UASVMCluj-Napoca.

Atotalof406week-oldWistarfemaleratswereusedin theexperimentalstudy.Theratswererandomlydividedin 8groups,5ratseach.Twoofthemservedascontrolgroups: IM(controlisoflurane)andSM(controlsevoflurane),ratsin thisgroupbeingexposedtooxygenalone(1L/min)3times, for2hlong,at2daysinterval.Threegroups(I1,I2andI3) weresubjectedtoisofluraneanesthesiaataconcentration of1.5%andtheotherthree(S1, S2andS3)tosevoflurane at a2%concentration.At theutilizedconcentrations, the minimumalveolar concentration(MAC)wasapproximately 1inthecase ofisoflurane,18 _as_well_as_sevoflurane.19 _The concentrationofanestheticwasadjustedbyusinga vapor-izer.

Weusedaninductionboxforanesthesias.Theratswere introducedintheinductionbox15minbeforeinitiatingthe anesthesia,inordertoaccommodate.Experimentalgroups wereanesthetised3times,for2hlong,at2daysinterval. Theoxygensupplythroughouttheanesthesiawas1LO2/min

for both isofluraneand sevoflurane.We followedthe pro-tocolrecommendedbyHondaetal.,15 _mentioning_that_the anesthesiaswererepeated3timesinordertoseetheeffects ofrepeatedanesthesiawithisofluraneandsevoflurane.

Aftercompletingtheanesthesias,theratsweresacrificed through cervical dislocation, at different post-anesthetic times:groupsIM,SM,I1andS2immediatelyafterthe anes-thesia,groupsI2,S2---6hpost-anesthesiaandI3,S3groups ---24hpost-anesthesia.

Immediately after the euthanasia, liver samples were harvested,introducedin10%bufferedformalinforfixation. After thefixation period,the samples wereembedded in paraffinandthensectionedata5␮mthicknessandmounted

onpoly-l-lysinecoatedmicroscopicslides(forthe

immuno-histochemicalreaction).

In ordertodetect theapoptotic hepatocytes,we used theimmunohistochemicalreactiontohighlightthe caspase-3expression,asmarkerofprogrammedcelldeath.Withthis purposeinview,LeicaBond-maxTM _{Immunohostochemistry}

system(LeicaBiosystems Melbourne,BondMax model,M2 12154 series) wasused. The immunomarcation was made withRabbit Anti-Caspase-3 PolyclonalAntibodies (dilution 1/75,rabbitanti-caspase3polyclonal,Linaris,Biologische Produkte,Dossenheim,Germany---cat.n◦_RB-1197-R7)._The steps taken by the system in order to stain the sections

werethe following: (a) deparaffinizationand rinsing with anEDTA solutionat8.8 pH (EpitopeRetrievalSolution2), 98◦_C_for₂₀_min;_(b)_after_rinsing,_the_DC₉₈₀₀_kit_was_used for10min(LeicaMicrosystemGmbH)inordertoblockthe endogenousperoxidase;(c)rinsing,afterwhichthe‘‘rabbit anti-caspase-3’’primary antibody wasadded and incuba-tionfor 30min; (d) polymerase addingand incubation for 10min, obtainingthe color reactionwiththe help ofDAB (diaminobenzidine).

Evaluationofthemarkerwasmadethrough quantifica-tionofimmunomarkedcells.Cellswithadifferentintensity browncytoplasmwereconsideredpositive.Immunomarked cellsfrom5microscopicfields,at400×magnification,were counted.The followinggrading methodwasusedfor each case: (I)less than 1% cells in apoptosis, (II) 1---2% cells in apoptosis, and (III) more than 2% cells in apoptosis. The microscopicimageswereobtainedusingtheOlympusBX51 lightmicroscopeconnectedtoanOlympusDP-25digital pho-tocamera,imagesbeingstoredonadigitalmemorycardand shownonthemonitor.

Results

Immunohistochemicalreaction forcaspase-3 highlighted a smallnumberof caspase-3positivecells inratlivers from groupsIMandSM(i.e.,lessthan1%cellsinapoptosis), no morethatthenormalhepatocyteexchangerate.Apartfrom thefacttheywerescarce,thecaspase-3positivecellswere isolatedandscattered throughoutthe hepaticlobules.On mostofthesectionarea,thehepaticlobuleshadanormal structureanddidnotcontainapoptoticcells(Fig.1).

LiversfromratsinI1groupdidnotpresentmore caspase-3positive cells than the control group (i.e.,less than 1% cellsinapoptosis),sothatinthegreatmajorityofthe lob-ulestheywerenotpresent,notevenisolated.Thesituation maintainsinI2group,regardingboththegeneralaspectof theorganandthesmallnumberofapoptoticcells, compara-bletothecontrolgroup(i.e.,lessthan1%cellsinapoptosis). FirstnoticeablechangesappearonlyingroupI3andconsist ofthepresenceofcaspase-3positivecellsinsomelobules

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Figure2 Experimental groupI3 ---centrilobular distributed apoptotic cells (suggested by a darker cytoplasm) --- black arrows; immunohistochemical reaction anti-caspase-3 poly-clonalantibodies,Mayerhematoxylincounterstain.

(around3.23%cellsinapoptosis,whichrepresentmorethan 2% cells in apoptosis according to the suggested grading method).Someofthecellsintheclosevicinityofthe cen-trilobularveinappearaffected(Fig.2).Wementionthatthe phenomenonisnotpresentinallofthehepaticlobules,but onlyinsomeofthem.Therewerenoinflammatorychanges orcirculatorydisordersobservedafterexposureoftherats toisoflurane.

IntheliversharvestedfromratsingroupS1,thesituation iscomparabletotheoneinthecontrolgroup,thenumberof caspase-3positivecellsbeingverysmall.Amajorincrease ofthecaspase-3positivecellsisnoticedinneitherS2,nor S3groups.Nootherassociatedlesionswereobserved(e.g., dystrophic,inflammatorylesionsorcirculatorydisorders)in theliverafterexposureofthesubjectstosevoflurane.

Discussion

Liversfromrats inthe controlgroups presented anormal aspect regarding the lobulation and the lobular pattern. Caspase-3positivecells(i.e.,inapoptosis)wereidentified, buttheirnumberwassmall,actuallyrepresentingthe nor-mal hepatocyte exchange rate (aspect suggested by the isolateddisposition,scattered throughoutthehepatic lob-ules,withnumeroushepaticlobuleswithoutimmunomarked cells).

LiversfromratsingroupI1werecomparabletotheones from rats in the control group, from all points of view. Caspase-3positivecellswerepresentinsmallnumbersand onlyinsomelobules,sothatwecannotstatethatafterthe anesthetic administration there were immediate adverse reactions,notevendiscreteones. IngroupI2,nochanges wereobservedregardingtheapoptoses’distributioninliver in comparison to the two groups presented anteriorly. In groupI3aslightincreaseofthenumberofcaspase-3 posi-tivecellswasobserved,incomparisontothecontrol,I1and I2groups.ComparativetothethingsobservedingroupIM,I1 andI2,inthecaseofsubjectsfromgroupI3alargernumber ofimmunomarkedcells(inapoptosis)wasidentified,focally

disposed,prevailinglycentroacinar. Apossibleexplanation oftheaffectationofthecellsintheimmediatevicinityof thecentrilobularveincouldbethefactthattheanesthetic reachestheliverthroughthecirculatorysystem,butinthis situation,hepatocytesattheperipheryofthelobulesshould bethefirstcellstogivein,becausethatistheplacethat thesinusoidsenterthehepaticlobules.Buthepatocytesat theperipheryofthelobulesarefavoredintermsofnutrient andoxygensupplies,beingthefirsttobenefitduetotheir disposal,whichseemstoconferresistancetotheanesthetic action.Cellsinthevicinityofthecentrilobularveinarethe lasttosupply.Apparently,thismakesthemsomewhat vul-nerable,someofthementeringapoptosisunderanesthetic action.Otherreportssuggestthattheperiacinarzoneis par-ticularlyvulnerabletoapoptosis/necrosis(knownaszone3 or periacinar apoptosis/necrosis),partlybecausetheyare farthest from incoming arterial and portal venous blood, bearingoxygenandessentialnutrients.20,21

However,thenumberof apoptoticcells issosmall;we canstate that isofluraneisan anestheticwithagood tol-erance. Theadverse reactions appeared in theliver after itsadministrationin therapeutic dose is minimal,without other cellular lesions(e.g., dystrophies, necroses, vascu-lar disorders) that induce changes in the pattern of the organ.

Sevofluraneadministrationdidnotresultinthe increas-ingofcaspase-3positivehepatocytenumberinexperimental groups immediatelyaftertheanesthesiaortheother con-troltimes(6h and24hpost-anesthesia).At noneofthese moments,thecaspase-3positivecellswereatsuchalevel whichwouldsuggestthattheanestheticwouldtrigger hepa-tocyteapoptosis.Apoptoseswerepresentinasmallnumber ofcells,somehowcomparableinallgroups,whichmakesus statethattheirpresenceisabsolutelynormal,beinglinked to thenormal hepatocyte exchangerate. In other terms, sevoflurane does not seem to have adverse reactions on ratliver,detectablethroughhistologicalexamination.This demonstratesaverygoodtolerabilityofsevofluraneinthe liver.

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knowingthatnecrosisappearswhenthehypoxiaissevere, whileapoptosistakesplacewhenthehypoxiaismild.24

The exact path of apoptosis induction by inhalational anesthetics is not entirely known, but there are studies which propose different hypotheses. Thus, Zhang et al.25 have shown that 2% isoflurane treatment for 6h long can cause apoptosis. Their study was conducted on cell cul-tures,primaryneuronsandmice.Theystatedthatisoflurane can increasethe pro-apoptoticBax factor level, decrease the anti-apoptotic Bcl-2 factor level, increase the accu-mulationofreactiveoxygenspecies,enablethereleaseof cytochromecfrommitochondriaincytosolandinducethe activation of caspase-9 andcaspase-3, thus inducing apo-ptosisthroughBcl-2proteinfamilyandmitochondrialpath. Wei et al.26 _propose _another _hypothesis: _activation _of inositol1,4,5-triphosphate(IP3)receptorsonthe

endoplas-mic reticulum membranes by isoflurane. This fact causes the excessive release of calcium, triggering apoptosis. The authors specify that isoflurane induced apoptosis, dependent on the anestheticconcentration and duration, increasing the cytosolic and then the mitochondrial cal-cium. Turrilazzi et al.27 _report _a _case _of _fatal _fulminant hepaticnecrosisaftersevofluraneanesthesia,incriminating thecytoplasmiccalciumincreasebysevofluraneascause.

In conclusion, our results show that sevoflurane does nottriggerhepatocyteapoptosis,whereasisoflurane deter-mines a moderate inconstant periacinar apoptosis, 24h post-anesthesia. Our results suggest that the tested anesthetics do not present a significant hepatotoxicity, sevofluraneprovingtohaveasuperiortolerabilityin com-parisontoisoflurane.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgement

ThispaperwaspublishedundertheframeofEuropeanSocial Fund, Human Resources Development Operational Pro-gramme2007---2013,projectno.POSDRU/159/1.5/S/136893

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