REVISTA
BRASILEIRA
DE
ANESTESIOLOGIA
OfficialPublicationoftheBrazilianSocietyofAnesthesiologywww.sba.com.br
SCIENTIFIC
ARTICLE
Pretreatment
with
remifentanil
protects
against
the
reduced-intestinal
contractility
related
to
the
ischemia
and
reperfusion
injury
in
rat
Hale
Sayan-Ozacmak
a,
Veysel
Haktan
Ozacmak
a,∗,
Inci
Turan
a,
Figen
Barut
b,
Volkan
Hanci
caDepartmentofPhysiology,BulentEcevitUniversityMedicalSchool,Kozlu,Zonguldak,Turkey bBulentEcevitUniversityMedicalSchoolDepartmentofPathology,67600Kozlu,Zonguldak,Turkey
cDepartmentofAnesthesiologyandReanimation,DokuzEylulUniversitySchoolofMedicine,Inciralti,Izmir,Turkey
Received18July2013;accepted2September2013 Availableonline6November2014
KEYWORDS
Intestinal contractility; Intestinal
ischemia---reperfusion injury;
Rat;
Remifentanil
Abstract
Backgroundandobjectives: Serious functional and structural alterations of gastrointestinal tractareobservedinfailureofbloodsupply,leadingtogastrointestinaldismotility.Activation ofopioidreceptorsprovidescardioprotectiveeffectagainstischemia---reperfusion(I/R)injury. TheaimofthepresentstudywastodeterminewhetherornotremifentanilcouldreduceI/R injuryofsmallintestine.
Methods:MaleWistarAlbinoratsweresubjectedtomesentericischemia(30min)followedby reperfusion(3h).Fourgroupsweredesigned:shamcontrol;remifentanilalone;I/Rcontrol;and remifentanil+I/R.Animalsinremifentanil+I/Rgroupweresubjectedtoinfusionofremifentanil (2ugkg−1min−1)for 60min,half ofwhich started before inducing ischemia. Collectingthe
ileumtissues,evaluationofdamagewasbasedoncontractileresponsestocarbachol,levelsof lipidperoxidationandneutrophilinfiltration,andobservationofhistopathologicalfeaturesin intestinaltissue.
Results:Following reperfusion, a significant decrease in carbachol-induced contractile response,aremarkableincreaseinbothlipidperoxidationandneutrophilinfiltration,anda sig-nificantinjuryinmucosawereobserved.Anaveragecontractileresponseofremifentanil+I/R groupwassignificantlydifferentfromthatoftheI/Rgroup.Lipidperoxidationandneutrophil infiltrationwerealsosignificantlysuppressedbythetreatment.ThetissuesamplesoftheI/R groupweregrade4inhistopathologicalevaluation.Inremifentanil+I/Rgroup,ontheother hand,themucosaldamagewasmoderate,stagingasgrade1.
Conclusions: ThepretreatmentwithremifentanilcanattenuatetheintestinalI/Rinjuryata remarkabledegreepossiblybyloweringlipidperoxidationandleukocyteinfiltration.
© 2013SociedadeBrasileirade Anestesiologia.Publishedby ElsevierEditoraLtda.Allrights reserved.
∗Correspondingauthor.
E-mail:vhaktan@yahoo.com(V.H.Ozacmak).
PALAVRAS-CHAVE
Contratilidade intestinal;
Lesãointestinalde isquemia/reperfusão; Rato;
Remifentanil
Pré-tratamentocomremifentanilprotegecontraareduc¸ãodacontratilidade intestinalrelacionadaàlesãodeisquemiaereperfusãoemratos
Resumo
Justificativaeobjetivos: Alterac¸õesfuncionaiseestruturaissériasdotratogastrointestinalsão observadasnainsuficiênciadeirrigac¸ãosanguínea,levandoaalterac¸õesdamotilidade gastroin-testinal.Aativac¸ãodosreceptoresopioidesproporcionaumefeitocardioprotetorcontraalesão deisquemia/reperfusão (I/R).Oobjetivodopresenteestudofoideterminarseremifentanil
podeounãoreduziralesãodeI/Rdointestinodelgado.
Métodos: Ratosmachosalbinos,dalinhagemWistar,foramsubmetidosàisquemiamesentérica (30minutos)seguidadereperfusão(3horas).Quatrogruposforamdesignados:shamcontrole; remifentanilisolado;controleI/R;remifentanil+I/R. Osanimais dogrupo remifentanil+I/R
foramsubmetidos àinfusãode remifentanil(2gkg−1min−1)por 60min,metade dos quais
iniciouantesdainduc¸ãodaisquemia.Coletandoostecidosdoíleo,aavaliac¸ãodosdanosfoi baseadanasrespostascontráteisaocarbacol,nosníveisdeperoxidac¸ãolipídicaeinfiltrac¸ão deneutrófilosenaobservac¸ãodascaracterísticashistopatológicasnotecidointestinal.
Resultados: Apósareperfusão,umadiminuic¸ãosignificativadarespostacontrátilinduzidapor carbacol,umnotávelaumentotantodaperoxidac¸ãolipídicaquantodainfiltrac¸ãodeneutrófilos eumalesãosignificativadamucosaforamobservados.Amédiadarespostacontrátilnogrupo remifentanil+I/RfoisignificativamentediferentedaqueladogrupoI/R.Aperoxidac¸ãolipídica
eainfiltrac¸ãodeneutrófilostambémforamsignificativamentesuprimidaspelotratamento.As amostrasdetecidodogrupoI/Rapresentaramgrau4naavaliac¸ãohistopatológica.Nogrupo
remifentanil+I/R,poroutrolado,alesãodamucosafoimoderada,apresentandoestadiamento
degrau1.
Conclusões:Opré-tratamentocomremifentanilpodeatenuaralesãointestinaldeI/Remum
graunotável,possivelmentepelareduc¸ãodaperoxidac¸ãolipídicaedainfiltrac¸ãoleucocitária. ©2013SociedadeBrasileiradeAnestesiologia.PublicadoporElsevierEditoraLtda.Todosos direitosreservados.
Introduction
Activated opioid receptors (ORs) protects against ischemia/reperfusion (I/R) injury inheart and neurons.1---3 Opioid peptides and ORs are present in gut.4,5 An ultra-short-actingphenylpiperidineopioidanalgesic,remifentanil is cardioprotective against the I/R injury via mediating ORs,1,6 activating protein kinase C (PKC), and driving mitochondrial ATP-sensitive potassium (KATP) channels7 whichmimicischemicpreconditioning(PC).1,8
Sepsis, hemorrhage, intestinal transplantation, severe burns, and mesenteric thrombosis cause mesenteric ischemia,9,10 making a dramatic impact on the intes-tine.IntestinalI/Rhave a majorrolein the development of primary graft dysfunction.11,12 The intestinal tissue is very sensitive to the I/R. In fact, even though the gastrointestinal tract represents about 5% of the body weight, its oxygen consumption constitutes roughly 20% of the total oxygen usage.13 Interruption of the oxygen supply decreases the intracellular ATP content, dis-turbing the cellular homeostasis through the increased reactive oxygenspecies (ROS). Reperfusioninduces struc-tural and functional damage of mucosa and interstitial edema (reperfusion injury),14 which is primarily due to the generation of ROS from various sources including the electron transport chains of mitochondria, xanthine oxidase metabolism, prostaglandins, endothelial cells, and activated neutrophils.14---17 I/R damages the mucosal barrier function, causing the mucosal and vascular per-meabilities and bacterial translocation, ending up with
systemicinflammationandmultiple-organfailure.18 Italso changes thecontractile responseof the intestinalsmooth muscle,19---22 which is associated with the local inflamma-tory response augmented by the infiltrating neutrophils10 andROS.23
Somepharmacologicalagentsincludinganesthetics pro-ducePC.24---28Specifically,ORsareinvolvedinthePCofrat heart.7,8Moreover,pharmacologicpostconditioninginduced byremifentanilalsoprotectsagainsttheI/R-inducedcardiac injury.3Ontheotherhand,whetherornotthepossible ben-eficialeffectoccursintheintestinalI/Rinjuryisextremely limited. Only one study by Cho et al.29 reports that remifentanilprotectstheintestineagainstI/Rinjury.They clearlydemonstratethatremifentanilsignificantlyreduces oxidative stress,inflammatoryresponse,andtissueinjury. However,theydidnotstudydirectlytheeffecton physiologi-calfunctionsofthetissue(e.g.contractility).Therefore,our aimwastodeterminewhetherremifentanilhasbeneficial effectontheI/R-disturbedintestinalcontractility.We eval-uated thecontractile response, histopathological changes andmeasuredthetissuelevelsofmalondialdehyde(MDA), lipidperoxidationmarker,andmyeloperoxidase,neutrophil infiltrationmarker,(MPO).
Materials
and
methods
Animals
BulentEcevitUniversityAnimalCareUnitandhousedunder standard conditions. They had free access tocommercial chowandwater.Onthedaybeforethesurgicalprocedures, theanimals werefastedovernight butallowed adlibitum access to water. They were maintained in their cages at a constant room temperature usinga 12h:12h light/dark cycle.Alloftheproceduresdescribedherewereapproved bythelocalanimalethicscommitteeoftheinstitution.
Operativeprocedures
Rats were anesthetizedwith sodium thiopental intraperi-toneally (60mgkg−1) followed by conducting laparotomy
throughamidlineincisionintotheperitonealcavity.After the small bowel was exteriorized gently to the left onto moistgauze,animalsweresubjectedto30minofischemia by ligation ofthe superior mesentericartery (SMA),using 3/0 silk thread.Intestinal ischemiawas confirmed by the lack of pulsein the mesenteric arteryand the palecolor ofthe jejunumandileum.Afterward,theintestineswere returnedto the abdomen,which wasclosed with2 small clamps.Attheendof30minofischemia,theabdomenwas reopenedandthethreadwasgentlyremovedtoallow reper-fusionofthebloodflow,whichwasconfirmedbyobserving thepulsation of theartery anditsbranches onthe intes-tine.Bodytemperaturewasmaintainedat37.8◦Cbyheating
lampduringtheprocedureofI/R.
Experimentalgroups
Animalsweredividedinto4 groupseach of8 animals:(a) Shamcontrolgroup,animalsunderwentlaparotomywithout performingtheocclusionofSMA;(b)Shamplusremifentanil (remifentanilalone)group,ratsweretreatedwith remifen-tanilandashamoperation withoutSMAocclusion;(c)I/R control group, the SMA wasoccluded for 30min followed by3h ofreperfusion;(d)I/Rplusremifentanilgroup,rats subjectedtoI/Rweretreatedwithremifentanil.
Remifentanil(GlaxoGroupLimited,LakeForest,IL,USA) pretreatment wascarriedoutbyinfusion(2gkg−1min−1)
via tail vein for 30min prior tothe occlusion of SMA and foradditional30min duringthewholeperiodofischemia. Catheterizationofthetailveinwascarriedoutwitha 24-gPolyneocatheter(Polymed,PolyMedicureLtd.,Haryana, India)foradministrationoftheinfusion.Animalsinthesham controlandtheI/Rgroupswereinsteadgivensterileserum physiological solution in the same volume. Following the reperfusionperiod,theanimalswerekilledby administra-tion of anesthetic at lethal doses; thereafter, samples of ileumwerecollectedandprocessedforeachexperimental protocol.
lleallongitudinalmusclecontractility
Theileallongitudinalmusclecontractileactivitywas eval-uatedin isolatedilealsegmentsafter3h ofreperfusionin anorganbath.Stripsoflongitudinalmuscleat5-mmlength wereremoved1cmawayfromtheileocecaljunction.After removingthefirststripforhistologicalevaluation,second, third,andfourthsamplesat thesamesizewerecollected
forcontractilityandbiochemicalanalyses.Stripswere lon-gitudinallysuspendedunder 2-g loadin 20mLorgan bath filledwith Krebs solution (in mmolL−1: NaCl,118.5; KCl,
4.8;KH2PO4,1.2;MgSO4·7H2O,1.2;CaCl21.9;NaHCO3,25;
glucose,10.1).The solutionwasgassedwitha mixtureof 5%CO2 and 95% O2 andmaintained at 37◦C. After60min
equilibration with 2g load, carbachol was added to the organbathatfinalvariousconcentrationsrangingfrom10−7
to10−2molL−1.Activeforcedevelopmentwasrecordedat
each concentration to determine the dose-response rela-tionship.Isometricforce wasmonitoredby external force displacement transducer (FDA-10A, Commat Co, Ankara, Turkey) using MP 30 software (MP30 Biopac Systems Inc, SantaBarbara,CA,USA).
Histopathologicscoringofthe
ischemia---reperfusioninjurytosmallintestine
The segments of ileum were fixed in 10% formalin and embedded in paraffin. Intestinal paraffin sections were stained with hematoxylin and eosin for morphological analysis. Histopathological evaluation of the reperfused-intestinaltissuewasbasedonastagingmethoddescribedby Hierholzeretal.30andtheevaluationwasgradedfrom0to 4.Ingrade0,nospecificpathologicalchangesareobserved: normalstructureofgutwall,includingvilli,crypts,lamina propria,andmuscularis externa.In grade1,mildmucosal damageisassessed:denudation ofvilli epithelium, other-wisenormalstructure.Ingrade2,moderatedamageoccurs: lossofvilluslengthandepithelialsloughingwithevidenceof congestion,hemorrhage,andinflammationinthemucosa, butnochangeinsubmucosaormuscularisexterna.Ingrade 3, extensive damage is observed: loss of a large number ofvilli including denudation,sloughing, andthe presence ofgranulomatous tissuewiththedamagelocalizedto sub-mucosaandmuscularis.Ingrade4,thereisseveredamage andnecrosis: inflammation andnecrosisin areas through-out the thickness of the intestinal wall. The investigator whoperformed these measurements was unaware of the experimentaldesign.
DeterminationoftissueMDAactivity
Intestinallipidperoxidelevelsweremeasuredbyamethod describedbyCasinietal.31 Briefly,byusingamotor-driven pestle,tissue samples were homogenized in ice-cold TCA byadding10mL of10% TCApergramof tissue.Following centrifugation,750Lof supernatant wasadded toequal volumeof0.67%thiobarbituricacidandheatedto100◦Cfor
15min.Theabsorbanceofthesampleswasthenmeasured spectrophotometricallyat535nm.
DeterminationoftissueMPOactivity
The activity of MPO within the supernatant of the homogenatewasdeterminedfromtheH2O2-dependent
oxi-dationof o-dianisidine.32 Aliquotsof supernatant (0.1mL) were added to 2.9mL of reaction mixture containing 0.167mgmL−1ofo-dianisidine,and20mMofH
2O2solution,
addingthealiquottothemixture,thechangeinabsorbance at460nmwasmeasuredfor5min.OneunitofMPOactivity wasdefinedasthatdegrading 1molof peroxidepermin at25◦C.Theactivitywasthennormalizedasunitpermgof
tissue(Umg−1).
Dataanalysis
Valuesfor theexperimentsdealingwithcontractilitywere normalizedforpergoftissue.Eachdatapoint represents mean±S.E.M.Forstatisticalevaluation,SPSS11.0 statisti-calsoftwarepackageprogramwasused(SPSSInc.,Chicago, IL,USA).Non-parametrictestswereperformedsinceeach group consisted of eight replicate samples. Thus, using Kruskal---Wallisvariance analysis (ANOVA),all groups were comparedintermsofexistenceofheterogeneity.Once sta-tisticallymeaningfulheterogeneitywasdetectedamongall groups, individualgroups were comparedwitheach other byemployingTukey’stest. pValuesoflessthan0.05were consideredsignificant.
Results
lleallongitudinalmusclecontractility
Carbachol caused a dose-dependent contractile effect on theterminalileumsegmentsfromallgroups,providing sig-moid curves with their maximal responses (Emax values)
(Fig.1).TheEmaxvalueforcarbacholwassignificantlylower
intheI/Rcontrolthanintheshamgroup(12.83±0.76vs. 22.19±2.10g/gtissue, respectively). In other words, the contractionin responsetocarbacholwassignificantlyand dose-dependentlyreducedbytheinductionofI/R,as indi-cated by a downward shift of the curve. The statistical differencebetweenthesegroupswassignificantatalldoses
30,00
25,00
20,00
15,00
10,00
5,00
0,00 10–7
10–5 10–5 10–4 10–3 10–2
[Carbachol], mM
Contraction, g/g tissue
Sham control
Sham+Rem
IR control
IR+Rem
Figure 1 Concentration---response curves of carbachol in
longitudinal ileum muscle isolated from the sham control, sham+Rem, IRcontrol, and IR+Rem. Eachdata point isthe mean±S.E.M.ofeightexperiments.*p<0.05indicates statisti-callymeaningfuldifferencefromtheIRcontrolgroup(IRcontrol vsallotherthreegroupsateachconcentrationofcarbachol). (Rem;remifentanil,IR;ischemia---reperfusion).
of carbachol (p<0.01). Reduced contractility was signifi-cantlyameliorated bythetreatment ofremifentanil.This effectwasstatisticallysignificantateachdoseofcarbachol, compared to the I/R group. As seen in Fig. 1, contrac-tion responses of sham group, remifentanil alone group, and remifentanil+I/R group, however, provided a similar curvesatallcarbacholdoses,beingstatisticallyindifferent fromeachother(p>0.05).ComparingEmax values,theI/R
group (12.83±0.76g/g tissue)was only about 58% of the shamgroup(22.19±2.10g/gtissue).AverageEmaxvalueof
the sham group wasnotstatistically different fromthose ofthebothremifentanilalone(21.55±2.85g/gtissue)and remifentanil+I/R(24.60±2.86g/gtissue)groups.
Histopathologicalfindings
Analyzing sixsectionsper group,themostdramatic alter-ations were observed in the I/Rgroup (Fig.2C; Table 1). Inflammation,mucosaldenudation,andedemawereclearly detected on some partsof the mucosa and submucosa in addition tonecrotic areas throughout the intestinal wall. Sloughingonsomeofthesurfaceepitheliumwasalso obvi-ous. The I/Rgroup was, therefore, evaluatedasgrade 4. In the sham group, as demonstrated in Fig. 2A, normal structureofgutwallwithvilli,crypts,laminapropria,and muscularislayerwasclearlyobserved.Thegroupwasgivena grade0.Remifentanilalonegrouphasprovidedverysimilar appearancestothoseobservedintheshamgroup;namely, nospecificpathologicalchangeswereobserved;thus,itwas graded as0 (Fig.2B). Remifentanil+I/Rgrouphas moder-atemucosaldamagewithonlyminimalfocaldenudationsof thevilliepithelium;therefore,thegroupreceivedagrade 1(Fig.2D).
MDAlevels
The MDA content of the sham group animals averaged 73.29±7.46nmolg−1 tissue, while animals subjected only
totheI/Rwerefoundtohave156.82±15.74nmolg−1tissue
(Fig.3), a significant increasein MDA content(p<0.001). Pretreatment with remifentanil (remifentanil+I/R group) significantlyreducedtheI/R-inducedintestinalMDAcontent to the sham group levels. With an average value of 107.80±8.93nmolg−1 tissue, this group was statistically
indistinguishablefromtheshamgroup(p=0.143),but statis-ticallydifferentfromtheI/Rgroup(p=0.02).Ontheother hand, the average MDAcontent in theremifentanil alone
Table 1 Semi-quantitative histological grading of
cross-sectionsoftheratileum(6sections/group).
Mean±S.E.M.
Shamcontrol 0
Sham+Remifentanil 0
I/Rcontrol 3.83±0.16
I/R+Remifentanil 1.17±0.16a
Analyzingwithone-wayANOVA,statisticalsignificance among twogroups.
Figure2 Lightmicrographsoftheratintestinaltissue:(A)Sham-operatedcontrolgroup;normalarchitectureofintestinalwall withnoanypathologicalalteration(grade0);(B)Sham+Rem;normalhistomorphologicappearanceofintestinaltissue(grade0); (C)I/Rcontrol;themostextensivemorphologicalalterationsdetected,suchasulceredintestinallumen,significantlydecreased muscularlayer,lossofepitheliumandglands,andhavingsuperficialexudaandmucoidmaterial(grade4);(D)IR+Rem;the pretreat-mentimproveshistopathologicalalterationsobservedaftertheI/R;inflammatedintestinaltissuewithmoderatemucosalthinning, patchylossofepithelium,andmixtypeinflammatorycellreactionwhichisdominatedbypolimorphnuclearleukocytes(grade1). (H&Estain×200,Scalebar=50m).(Rem;remifentanil,I/R;ischemia---reperfusion).
group (84.03±14.90nmolg−1 tissue) was not statistically
differentfromthatintheshamgroup(p=0.946).
MPOactivity
TheaverageMPOenzymeactivitiesofthetissuesfrom ani-malssubjectedtoshamoperation,remifentanilalone,I/R, andremifentanil+I/Rwere(in Umg−1 tissue) 0.63
±0.17,
Sham 200
180
160
140
120
100
80
60
40
[MDA], nmol/g tissue
20
0
Sham+Rem IR IR+Rem
Figure3 Averagemalondialdehyde(MDA)content ofileum
samples from the sham control, sham+Rem control, IR con-trol,andIR+Rem.Dataareexpressedasmean±S.E.M.(n=8). Compared to the IR control group, statistically significant difference is denoted as *(p<0.05). (Rem; remifentanil, IR; ischemia---reperfusion).
0.54±0.08, 1.60±0.28, and 0.83±0.14, respectively (Fig.4).
TheI/Rcausedapproximatelya2.5-foldincreaseinthe MPOactivity of thetissue comparedtothe basallevel of the activity (p=0.004). Pretreatment with remifentanil completely abolished the increased enzymatic activity resultingfromtheI/Rinsult; theMPO activity ofsamples fromanimalspretreatedwithremifentanilwassignificantly differentfromthat intheI/Rgroup (p=0.049).No
statis-2.00
1.80
1.60
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00
Sham
[MPO] activity
, U/mg tissue
Sham+Rem IR IR+Rem
Figure 4 Average myeloperoxidase (MPO) activity of the
ticaldifferencewasobservedintheMPOactivitybetween thesham-groupandtheremifentanilalonegroup.
Discussion
Thepresentstudydemonstratesthatremifentanilmayhave strongprotectivepotentialagainsttheintestinalI/Rinjury. Evidences indicate that remifentanil attenuates the I/R-induced:(1)intestinalpathologicalchanges;(2)decreasein contractileresponse; (3)increased neutrophil infiltration; and(4)elevatedlipidperoxidation.
There aremany publishedstudies that showbeneficial effectsofpharmacologicalPCwithremifentanilon reper-fusioninjuryof varioustissues.However,thoseconcerned withpharmacologicalPCoftheintestinewithremifentanil are very limited and lacking functional aspects, such as motility and secretion. In fact, only one study has been dealing with this question. Cho et al.29 have shown that remifentanil attenuates intestinal I/R injury in mice by reducinglipidperoxidationandsystemicinflammation.Our dataconfirmedtheoutcomesofthisstudyinregardtothe inhibitionofoxidativestressandinflammationand demon-strated the direct involvement of leukocytes in tissues. However,the studydid notdirectly investigatethe effect of remifentanil on physiological functions of the tissue, suchas motility and/or secretion.Therefore, the current studytakesafurtherstep,showingthatthepretreatment with remifentanil ameliorates the reduced contractility duetoI/R----a novel finding potentially linkedtothe anti-inflammatoryandantioxidantactivityofremifentanil.
IntestinalI/Rmay causemultiple organfailure,severe morbidity, and even death.1,16 Both acute and chronic ischemia make a substantial impact on gut functionally as well as structurally, leading to dismotility, abnormal absorption, and altered barrier function against bacte-rial translocation.10,14,33 Acute intestinal I/R causes such long-term structural alterations as the development of hypertrophy,cellulardysfunction,neuronaldeath,necrosis withinthe myenteric plexus,19,22,33 and the infiltration of inflammatory cells (i.e., neutrophils).30,34 Many published studies demonstrate deleterious effects of I/Ron intesti-nal motility, such as delayed gastrointestinal transit,35---37 altered migrating motor complex,38 and impaired motor response toany stimulation.20,39,40 Inflammatory response withinthemuscularcellsanddecreaseincontractilityofthe circularsmoothmusclearetriggeredbyI/R.30,34Integrityof theentericneuronalnetwork isnecessary for normal gas-trointestinalmotility. Therefore, the I/R may disturb the motor function of the gut by altering the properties of entericneuronsortheneuro-effectortransmission.22These changes are probably the outcome of the inflammatory responseinducedbytheI/Rinjury.Theintestinaldamagewe observedmaybeassociatedwiththeinflammation,whichis markedspecificallywithanincreasedamountofneutrophils inthetissue.Inhibitingtheactivationandtheendothelial adherenceofneutrophilsreducetheI/R-induceddamageto themucosa.41 Inresponsetotheinflammationofthe mus-cle,neutrophilsareactivated,contributingpossiblytothe muscledamage.Therefore,themotilitydisturbance occur-ringaftertheI/Rwasmorelikelyresultedfromthesevere muscledamageandassociatedinflammatoryreactionthat
weobserved.Thetreatmentwithremifentanilreducednot onlythelipidperoxidationinthetissuebutalsothe leuko-cyteinfiltration, tissuedamage,and disturbedcontractile response.
Returningoftheoxygenatedbloodtotheischemic intes-tinefurtherexacerbatesthetissuedamagemainlybecause of the increased generation of ROS, causing oxidative stress.17,42 Primary sources of ROS during the reperfusion are xanthine oxidase and activated neutrophils releasing thepro-inflammatory mediatorsandcytotoxicsubstances, suchasMPO.17 Intestinalreperfusioninjury isregardedas aninflammatorydiseasecharacterizedwiththerecruitment ofleukocytes.9ROSliberatedinreperfusionfurther stimu-lateinflammatoryresponse,enhancingtherecruitmentand activation ofneutrophils.The tissueMPOactivity, propor-tional tothe amount of accumulated-neutrophils, mirrors the mucosal injury and the rate of neutrophil infiltration in postischemic tissue.17,43 Hence, following the I/R, the increasedMPOactivityinthetissueindicatedthe enhanced-inflammatory responsein the current study.The elevated histologic scoring of the tissue lesions andthe decreased contractile response coincided with the significant eleva-tion of both MPO activity and lipid peroxidation of the tissue in I/R group. One of the deleterious consequences of oxidative damage is lipid peroxidation, which eventu-ally causes structural and functional disturbances to the membranes.SignificantreductioninbothMPOactivityand MDAcontentwereobservedintheremifentanil+I/Rgroup which indicates a significant limitation to the inflamma-tion. Moreover, contractile response to carbachol in the samegroup appeared tobemaintainedveryclosetothat in theshamcontrol group.These findingsweresupported bythehistopathologicalobservationthatremifentanil suc-cessfully rescued the integrity of the tissue from I/R injury.
Theopioid-inducedPCprotectsagainst thecardiacI/R injury.Specifically,PCbyremifentanil,anultra-short-acting phenylpiperidine opioid analgesic, confirms the cardio-protection.3 Activation of - and ␦-ORs was involved in PKCactivation,openingofmitochondrialKATPchannel,and increasinganti-apoptoticproteinexpression,important tar-gets of cardio-protection in ischemic and pharmacologic PC.7,8Thetoll-likereceptorsignalingininflammatory path-way is alsoassociated withthe opioid-inducedprotection inbrainandcellcultures.Followingactivationof the toll-likereceptor2,opioidagonistshaveaninhibitoryeffecton proinflammatorycytokineproductioninhumanmonocytes. The inhibition is mediated exclusively by -ORs.2 In the current study,we recognizedthatthe integrityof intesti-nalmucosaandcontractileresponseofthetissueremained nearlynormalinremifentanil+I/Rgroup.Ourdata demon-stratedthatremifentanildecreasedbothlipidperoxidation andneutrophil accumulationinischemictissuewhichmay beaccountedforunderlyingmechanismofabovementioned findings.However,it’dhave been betterandconfirmative toevaluate theeffects of an antagonist/agonistof -ORs on the present model besides examining a dose-response patternofremifentanil.
ORs.44---46Ontheotherhand,somesuggestthemediationof OR-independentmechanism.2However,Zhangetal.5 show thattheendogenousopioidpeptidesmightplayamajorrole in ischemic PCof the small intestine. In model of croton oil-induced inflammation of mice intestine, expression of the-OR isreportedtobe up-regulatedinthe myenteric plexus of jejunum. Similarly, patients with IBD have an increasedexpressionof-ORmRNAintheirinflamedsmall bowelandcoloninrespecttothehealthyintestinaltissue.47 Inthepresentstudy,wedonotknowwhethertheprotective effect of remifentanilis mediated throughOR-dependent mechanismsornot.However,itistemptingtospeculatethat thebeneficialeffectis due tothe OR-mediatedprocesses sincethetissuehasanabundantamountofORsandsincethe inflammatory insultsinduce theup-regulation of theORs. If this is the case, then the pretreatment with remifen-tanil possibly mimics the effect of ischemic PC. Namely, acting via ORs, remifentanilmay eventually activate PKC throughtheturnoverofphosphatidylinositolinducedbythe activation of the membrane-bound phospholipase C or D. PKC then phosphorylates and stabilizes the open state of both the mitochondrial and the sarcolemmal KATP chan-nelswhichultimatelyelicitscytoprotectionasobservedin remifentanil-induced cardioprotection.7 The pretreatment withremifentanilmayalsohaveanOR-mediatedinhibitory effectonthe generation ofproinflammatory cytokinesby monocytesinresponsetothestimulationoftoll-like recep-tor2.2Inthesecases,theadministrationofanORantagonist (i.e. Naloxone) would have been detrimental tothe pre-ventiveeffectsobservedinthepresentstudy.Ontheother hand, if the mechanisms are non-receptor-mediated, the remifentanil-inducedintestinalprotectionmaybe multifac-torial.Inthiscase,asreportedonremifentanilPCofhepatic I/R,induciblenitricoxidesynthase(iNOS)maymediatethe preventive effects in intestinal tissue.For instance, both lipid peroxidation and inflammation could be attenuated by an elevated generationof the endogenousnitric oxide (NO)which wouldlimit theamount of ROS andattenuate theinflammatoryresponse.Itiswelldocumentedthatthe endogenousNOmayexhaustROSandneutrophil-mediated tissueinjury;hence,reducinginflammatoryresponse signif-icantly.Itisalsoshownthatthecellulardamageinducedby theI/RisinvolvedwithasignificantdecreaseofNO. There-fore,remifentanilmaybeassociatedwiththeinductionof iNOSexpressionandconsequentgenerationofthe endoge-nousNOin intestinaltissuechallenging tothereperfusion injury.Ifso,theprotectiveeffectswouldhavebeenblocked byaNOSinhibitorbutnotwithNaloxone.2
Inconclusion,wehavedemonstratedthatthetreatment ofratswithremifentanilpriortotheI/Rprovidedprotection against I/R-induced mucosal damage and contractile dis-turbance.Possiblemechanismresponsibleforthesesalutary activities of remifentanil may probably be multifactorial. However, as the present study has given evidences, the attenuationofneutrophil infiltration andthereductionof oxidativestressappeartobeofprimaryimportancetoget thebeneficialeffects.Furtherstudies,whichwillfocuson the mechanisms of protective effects of remifentanil in depth,arerequiredforsure.Inconclusion,wehaveshown that the administration of remifentanil during ischemia couldefficiently be protective againstthe most histologi-cal,cytological,biochemical, and physiologicalaspectsof
intestinalinjury,providing therapeutic potentialfor clini-calapplicationin intestinalischemiaandischemia-related disorders.Moreover,thecurrent study implicatesthatthe application of remifentanil as analgesic agent may be preferredforthosesufferingfromintestinalhypoperfusion andundergoinganesthesia.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgement
Wewouldliketothanktothosewhoworkattheanimalcare unitfortheirinvaluableassistance.
References
1.PeartJN,GrossER,GrossGJ.Opioid-inducedpreconditioning: Recentadvances and future perspectives.Vascul Pharmacol. 2005;42:211---8.
2.Yang LQ, Tao KM, Liu YT, et al. Remifentanil precondition-ing reduces hepatic ischemia---reperfusion injury in rats via inducible nitric oxide synthase expression. Anesthesiology. 2011;114:1036---47.
3.Chun KJ, Park YH, Kim JS, et al. Comparison of 5 different remifentanil strategies against myocardial ischemia---reperfusion injury. J Cardiothorac Vasc Anesth. 2011;25:926---30.
4.Cosola C, Albrizio M, Guaricci AC, et al. Opioid ago-nist/antagonisteffectofnaloxoneinmodulatingrabbitjejunum contractilityinvitro.JPhysiolPharmacol.2006;57:439---49. 5.Zhang Y, Wu YX, Hao YB, et al. Role of endogenous opioid
peptidesinprotectionofischemicpreconditioninginratsmall intestine.LifeSci.2001;68:1013---9.
6.Yu CK, Li YH, WongGT, et al. Remifentanil preconditioning confers delayed cardioprotection in the rat. Br J Anaesth. 2007;99:632---8.
7.Zhang Y, Chen ZW, Girwin Metal. Remifentanil mimics car-dioprotective effect of ischemic preconditioning viaprotein kinaseCactivationinopenchestofrats.ActaPharmacolSin. 2005;26:546---50.
8.Kim HS, Kim SY, Kwak YL, et al. Hyperglycemia attenu-ates myocardial preconditioningof remifentanil.JSurg Res. 2011;174:231---7.
9.Vollmar B, Menger MD. Intestinal ischemia/reperfusion: microcirculatorypathologyandfunctionalconsequences. Lan-genbecksArchSurg.2011;396:13---29.
10.Mallick IH,Yang W, WinsletMC, et al. Ischemia---reperfusion injuryoftheintestineandprotectivestrategiesagainstinjury. DigDisSci.2004;49:1359---77.
11.SutoY,OshimaK,ArakawaK,etal.Theeffectofnicorandilon smallintestinalischemia---reperfusioninjuryinacaninemodel. DigDisSci.2011;56:2276---82.
12.Cámara-LemarroyCR,Guzmán-delaGarzaFJ,Alarcón-Galván G, et al. The effects of NMDA receptor antagonists over intestinalischemia/reperfusioninjuryinrats.EurJPharmacol. 2009;621:78---85.
13.OrsenigoMN,PortaC,SironiC,etal.Effectsofcreatineina ratintestinalmodelofischemia/reperfusioninjury.EurJNutr. 2011;51:375---84.
15.ArumugamTV,ArnoldN,ProctorLM,etal.Comparative protec-tionagainstratintestinalreperfusioninjurybyanewinhibitor of sPLA2COX-1and COX-2selective inhibitors, and anLTC4 receptorantagonist.BrJPharmacol.2003;140:71---80. 16.Hsieh YH, McCartney K, Moore TA, et al. Intestinal
ischemia---reperfusioninjuryleadstoinflammatorychangesin thebrain.Shock.2011;36:424---30.
17.Margaritis EV, Yanni AE, Agrogiannis G, et al. Effects of oral administration of (l)-arginine (l)-NAME and allopurinol on intestinal ischemia/reperfusion injury in rats. Life Sci. 2011;88:1070---6.
18.AbdeenSM,MathewTC,DashtiHM,etal.Protectiveeffectsof greenteaonintestinalischemia---reperfusioninjury.Nutrition. 2011;27:598---603.
19.Rivera LR, Thacker M, Pontell L, et al. Deleterious effects ofintestinalischemia/reperfusioninjuryinthemouseenteric nervoussystemareassociatedwithproteinnitrosylation.Cell TissueRes.2011;344:111---23.
20.Ballabeni V, Barocelli E, Bertoni S, et al. Alterations of intestinal motor responsiveness in a model of mild mesenteric ischemia/reperfusion in rats. Life Sci. 2002;71: 2025---35.
21.Ługowska-Umer H, Umer A, Sein-Anand J, et al. Endothe-lin receptor blockers protect against ischemia/reperfusion impairmentofgastrointestinalmotilityinrats.PharmacolRes. 2008;57:413---8.
22.ShimojimaN,NakakiT,MorikawaY,etal.Interstitialcellsof cajalindysmotilityinintestinalischemiaandreperfusioninjury inrats.JSurgRes.2006;135:255---61.
23.Ozacmak VH, Sayan H, Arslan SO, et al. Protective effect of melatonin on contractile activity and oxidative injury induced by ischemia and reperfusion of rat ileum. Life Sci. 2005;76:1575---88.
24.Yang Q, Dong H, Deng J, et al. Sevoflurane preconditioning induces neuroprotection through reactive oxygen species-mediatedup-regulationofantioxidantenzymesinrats.Anesth Analg.2011;112:931---7.
25.Obal D, Dettwiler S, Favoccia C, et al. The influence of mitochondrialKATP-channelsinthecardioprotectionof precon-ditioningandpostconditioningbysevofluraneintheratinvivo. AnesthAnalg.2005;101:1252---60.
26.AssadAR,DelouJM,FonsecaLM,etal.TheroleofKATP chan-nels onpropofolpreconditioninginacellular modelofrenal ischemia---reperfusion.AnesthAnalg.2009;109:1486---92. 27.Xiao YY, Chang YT, Ran K, et al. Delayed preconditioning
by sevoflurane elicits changes in the mitochondrial pro-teome in ischemia---reperfused rat hearts. Anesth Analg. 2011;113:224---32.
28.Huang Z, Zhong X, Irwin MG, et al. Synergy of isoflurane preconditioningandpropofolpostconditioningreduces myocar-dialreperfusioninjuryinpatients.ClinSci(Lond).2011;121: 57---69.
29.ChoSSC,RudloffI,BergerPJ,etal.Remifentanilameliorates intestinal ischemia---reperfusion injury. BMC Gastroenterol. 2013;13:69---77.
30.Hierholzer C, Kalff JC, Audolfsson G, et al. Molecu-lar and functional contractile sequelae of rat intesti-nal ischemia/reperfusion injury. Transplantation. 1999;68: 1244---54.
31.CasiniAF,FerraliM,PompellaA,etal.Lipidperoxidationand cellular damage inextrahepatic tissuesof bromobenzene in toxicatedmice.AmJPathol.1986;123:520---31.
32.Bradley PP, Priebat DA, Christensen RD, et al. Measure-ment of cutaneous inflammation: estimation of neutrophil contentwithanenzymemarker.JInvestDermatol.1982;78: 206---9.
33.GiaroniC,ZanettiE,GiulianiD,etal.Proteinkinasecmodulates NMDAreceptorsinthemyentericplexusoftheguineapigileum duringinvitro ischemiaand reperfusion.Neurogastroenterol Motil.2011;23:e91---103.
34.DerT,BercikP,DonnellyG,etal.Interstitialcellsofcajaland inflammation-induced motor dysfunction in themouse small intestine.Gastroenterology.2000;119:1590---9.
35.AlicanI,YegenC,OlcayA,etal.Ischemia---reperfusion-induced delay in intestinal transit Role of endothelins. Digestion. 1998;59:343---8.
36.UdassinR,EimerlD,SchiffmanJ,etal.Postischemicintestinal motilityinratisinverselycorrelatedtolengthofischemia:an invivoanimalmodel.DigDisSci.1995;40:1035---8.
37.Hassoun HT, Weisbrodt NW, Mercer DW, et al. Inducible nitric oxide synthase mediates gut ischemia/reperfusion-induced ileusonly aftersevereinsults. JSurg Res.2001;97: 150---4.
38.HebraA,HongJ,McGowanKL,etal.Bacterialtranslocationin mesentericischemia---reperfusioninjury:isdysfunctional motil-itythelink?JPediatrSurg.1994;29:285---7.
39.Sayan H, Ozacmak VH, Altaner S, et al. Protective effects of l-arginine on rat terminal ileum subjected to ischemia/reperfusion. J Pediatr Gastroenterol Nutr. 2008;46:29---35.
40.BielefeldtK,ConklinJL.Intestinalmotilityduringhypoxiaand reoxygenationinvitro.DigDisSci.1997;42:878---84.
41.SchoenbergMH,PochB,YounesM,etal.Involvementof neu-trophilsinpostischaemicdamagetothesmallintestine.Gut. 1991;32:905---12.
42.Sayan H, Ozacmak VH, Sen F, et al. Pharmacological pre-conditioningwitherythropoietinreducesischemia---reperfusion injury in the small intestine of rats. Life Sci. 2009;84: 364---71.
43.TianXF,YaoJH,ZhangXS,etal.Protectiveeffectofcarnosol onlunginjuryinducedbyintestinalischemia/reperfusion.Surg Today.2010;40:858---65.
44.WongGT,LiR,JiangLL,etal.Remifentanilpost-conditioning attenuatescardiac ischemia---reperfusion injury viakappa or delta opioid receptor activation. Acta Anaesthesiol Scand. 2010;54:510---8.
45.ZattaAJ,KinH,YoshishigeD,etal.Evidencethat cardiopro-tectionbypostconditioninginvolvespreservationofmyocardial opioidcontentandselectiveopioidreceptoractivation.AmJ PhysiolHeartCircPhysiol.2008;294:H1444---51.
46.Jang Y, Xi J, Wang H, et al. Postconditioning prevents reperfusioninjurybyactivatingdelta-opioidreceptors. Anes-thesiology.2008;108:243---50.
