w w w . j c o l . o r g . b r
Journal
of
Coloproctology
Original
Article
Using
amniotic
membrane
for
anal
sphincter
repair
in
animal
model
Ahmad
Izadpanah
a,b,
Mohammad
Rezazadehkermani
b,
Mohammadali
Ghaderi
c,
Salar
Rahimikazerooni
a,
Alireza
Safarpour
a,
Kaynoosh
Homayouni
d,
Maral
Mokhtari
e,
Alimohammad
Bananzadeh
a,b,∗aColorectalResearchCenter,ShirazUniversityofMedicalSciences,Shiraz,Iran
bDepartmentofGeneralSurgery,SchoolofMedicine,ShirazUniversityofMedicalSciences,Shiraz,Iran
cEmergencyManagementCenter,ShirazUniversityofMedicalSciences,Shiraz,Iran
dDepartmentofPhysicalMedicineandRehabilitation,SchoolofMedicine,ShirazUniversityofMedicalSciences,Shiraz,Iran
eDepartmentofPathology,SchoolofMedicine,ShirazUniversityofMedicalSciences,Shiraz,Iran
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Articlehistory:
Received9November2015 Accepted10December2015 Availableonline1February2016
Keywords:
Analsphincter Sphincteroplasty Amnioticmembrane Electromyography Fecalincontinence
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Purpose:Sphincterrepairistheprimarymanagementforfecalincontinenceespeciallyin traumaticcauses.Regardlessofprogressioninthemethodandmaterialofsphincterrepair, theresultsarestilldisappointing.Thisstudyevaluatestheefficacyofusingamniotic mem-braneduringsphincteroplastyregardingitseffectsinhealingofvarioustissues.
Methods:Rabbitsundergonesphincterotomyandafterthreeweeksendtoend sphinctero-plastywasdone.Animalsdividedtothreegroups:classicsphincteroplasty,sphincteroplasty withfreshamnioticmembraneandsphincteroplastywithdecellularizedamniotic mem-brane.Threeweeksaftersphincteroplastyanimalsweresacrificedandsphinctercomplex wassentforhistopathologicevaluation.Sphinctermusclediameterandcompositionof sphincterwasevaluated.Beforesphincterotomy,beforeandaftersphincteroplasty elec-tromyographyofsphincteratthesiteofrepairwererecorded.
Results:Nostatisticalsignificantdifferencewasseenbetweengroupseveninhistopathology orelectromyography.
Conclusion:Althoughamnioticshowedpromisingeffectsinthehealingofdifferenttissuein animalandhumanstudiesitwasnoteffectiveinhealingofinjuredsphincter.
©2016SociedadeBrasileiradeColoproctologia.PublishedbyElsevierEditoraLtda.All rightsreserved.
∗ Correspondingauthor.
E-mail:[email protected](A.Bananzadeh). http://dx.doi.org/10.1016/j.jcol.2015.12.004
Uso
de
membrana
amniótica
para
reparo
de
esfíncter
anal
em
modelo
animal
Palavras-chave:
Esfíncteranal Esfincteroplastia Membranaamniótica Eletromiografia Incontinênciafecal
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e
s
u
m
o
Objetivo: Reparodoesfíncteréotratamentoprimárioparacasosdeincontinênciafecal, especialmenteemcausastraumáticas.Independentementedaprogressãonométodoedo materialdereparodoesfíncter,osresultadossãoaindadesapontadores.Esseestudoavalia aeficáciadousodamembranaamnióticaduranteaesfincteroplastia,comrelac¸ãoaosseus efeitosnacuradediversostecidos.
Métodos: Coelhos foramsubmetidos a um procedimento de esfincterotomia e, depois detranscorridastrêssemanas,foirealizadaumaesfincteroplastiatérmino-terminal.Os animaisforamdivididosemtrêsgrupos:esfincteroplastiaclássica,esfincteroplastiacom membranaamnióticafresca,eesfincteroplastiacommembranaamnióticadescelularizada. Trêssemanasapósarealizac¸ãodaesfincteroplastia,osanimaisforamsacrificadoseo com-plexoesfinctéricofoiencaminhadoparaavaliac¸ãohistopatológica.Odiâmetrodomúsculo esfinctéricoeacomposic¸ãodoesfíncterforamavaliados.Antesdaesfincterotomia,eantes edepoisdaesfincteroplastia,foiregistradaaeletromiografiadoesfíncternolocaldoreparo.
Resultados: Nãofoi observada diferenc¸aestatisticamentesignificativa entreos grupos, mesmonahistopatologia,ounaeletromiografia.
Conclusão: Emboraamembranaamnióticatenhademonstradoefeitospromissoresem ter-mosdacicatrizac¸ãodosdiferentestecidosemestudoscomanimaiseemhumanos,nãofoi observadaeficácianacuradoesfíncterlesionado.
©2016SociedadeBrasileiradeColoproctologia.PublicadoporElsevierEditoraLtda. Todososdireitosreservados.
Introduction
Overall prevalence of fecal incontinence in the general populationis2%andsurgicalrepairisthemainstayof treat-mentespeciallyinthetraumaticcausesofsphincterdefect. Althoughthetechniquesofsphincterrepairwasevolved dur-inglast decades but theresult isstilldisappointing.1 With theemergingofanalultrasoundevaluationofsphincter struc-tureanddetectingthesphincterdefectbecamepossible.The sphincterdefectcouldbedetectedinmostofthecasesafter the repairand the scoreofcontinence would declinewith time.Itisstillunknownthatthisdefectisduetothetechnique ofrepairorinfectionornaturalprocessofhealing.2
Comparing overlapping or end to end repair of sphinc-ter defectdid notmakesignificant difference inthe result ofsphincterrepair.3ACochranehasshownnoimprovement intheoutcomeofsimplesphincterrepairoradding divert-ing ostoma to the repair procedure.4 Studies also showed theefficacyofsphincterrepairwithProleneorPDSsutures is higher than traditional repair with chromic or Vicryl suture.5
Amniotic membrane had shown promising results in healingpathwaysincluding:(1)proliferation,migrationand chemotaxisofdifferentcelltypes,(2)productionand remodel-ingofextracellularmatrix,(3)releaseofcytokinesandgrowth factors.Ithasbeenusedinmanyregenerativeprocessessince 1910whenfirstusedinskingraft.6 Amnioticmembrane is widelyusedinophthalmologyforcornea,conjunctiva,ororbit pathologies.7Therearemultiplemolecularbasisfor explain-ingthepossiblemechanismofamnioticmembranehealing pathways.AmnioticmembranecontainhighamountofIL-8
whichisastrongstimulatorforangiogenesis.Italsocontain plateletderivedgrowthfactorandbasicfibroblastgrowth fac-torinrelativelyhighconcentration.8
Regardingmentionedevidencesusingamnioticmembrane in sphincter repair, seemed to be reasonable to improve outcome.Inthisstudy weinvestigatetheefficacy of amni-oticmembraneapplicationduringsphincterrepairinanimal modelofsphincterdamage.
Methods
and
materials
Amnioticmembranepreparation
Inthisstudyweusedtwoformsoffreshanddecellularized amnioticmembrane.Freshamnionwasgatheredfrom elec-tivetermcesareansectionsinordertousenormalamniotic membranewithleastriskofinfection.Thisamnionwaskept in 4◦Cand used atleast 8hours afterharvesting. Decellu-larizedamnioticmembranewasprovidedbyGhotbodinBurn hospitalwiththestandardprocedure.
Animals
Fig.1–Siteofsphincteroplasty.(A)Simplesphincteroplastygroup;(B)amnioticmembraneiswrappedaroundthesiteof sphincteroplastyandfixedwithsphincteroplastysuture.
Forcontrolgroupsimpleendtoendsphincterrepairwasdone andinothertwogroupsrepairwithfreshordecellularized amnioticmembranewasdone.
Electromyographicstudies
BeforeanysurgicalinterventionanEMGtracewastakenat theposteriormidlineofsphincterasbaseline.Threeweeks aftersphincterotomyandthreeweeksaftersphincteroplasty alsoanEMGtracewastakenfromeachanimal.TheEMGwas takenwithconcentric26gaugeneedlesinlithotomyposition afterattachmentofgroundconnectiontotherabbitback. Fre-quencyintervalwasrecordedandtheleastfrequencyinterval wasassociatedwithmorecompetentsphinctermuscle.
Surgicalprocedure
Animals were anesthetized with 80mg/kg Ketamine and 12mg/kg Xylazine before any intervention then they were fixedinlithotomypositionand standardhairclipping,prep anddrapeweredone.Thensphincterotomywasdonein pos-teriormidlineusing No.11blade.All fibersofexternaland internalsphincterwerecutandseparated.Threeweeksafter thesphincterotomysphincterrepairwasdoneandanimals categorizedbasedon thewayofsphincterrepairasfollow. Afterinductionofanesthesiathe rabbit wasfixedin litho-tomypositionandatransverseincisionwasgivenposterior totheanus.Thenfreeendsofsphinctermusclewerefound. Inthecontrolgroupsimpleendtoendsphincterrepairwas donewith4-0Prolenesuture(Fig.1A)andtheskinoverthe siteofrepairclosedwithchromic4-0.Foramnioticmembrane groupsa4×2cmshitofamnionwaspreparedandrepairwas doneovertheshitandattheendofrepairsiteofrepairwas wrappedwiththeshitandfixedtotherepairsitewith sphinc-teroplastysutureties(Fig.1B)andskinclosedwithchromic suture.
Three weeks after sphincteroplasty, after recording the finalEMGtraceandinductionofanesthesiaanusand sphinc-tercomplexenblocresectedandsiteofrepairmarkedwith a5-0nylonsuturethensent forpathologicexaminationin formalin solution. After that animals were sacrificed with intracardiacinjectionofKClsolution.
Histopathologyevaluation
Afterfixingthesamplesintheformalin,5mthicksectionsof thesamplewastakenandslideswerereviewedbyablinded pathologist.Atfirstsiteofsphincterrepairevaluatedandsite ofrepairreportedasmuscledominant,fibrosisdominantor mixed regardingpreviousliterature.9 Thenthe diameterof sphincterwasmeasuredatthesiteofrepairinposterior quad-rant.Thediameterwasalsomeasuredinanteriorandlateral quadrants.
Statisticalanalysis
ANOVA was used to compare sphincter muscle diameter betweengroupsandmultivariateanalysiswasusedto evalu-atetheEMGresultsbetweenandwithinthedifferentgroups. AllanalysesweredonebySPSSsoftware.
Results
All ofthe animalstoleratethecourseofstudywell andno mortalitywasobservedduringthestudyperiod.
Table1brieflydemonstratestheresultofelectromyography study ofsphincter reportedas frequencyinterval in differ-entstagesofthestudy.Aone-wayrepeatedmeasuresANOVA was conductedtocompare theresultsofEMGofsphincter muscles at time 1 (prior to the intervention), time 2 (fol-lowingtheintervention)andtime3(three-monthfollow-up). Therewasasignificanteffectfortime,Wilks’Lambda=0.316,
F=11.90,p=0.002.Mauchly’stestofsphericitywasnotviolated (p=0.427).
Table2demonstratesthe mean musclediameterin dif-ferent groups.AKruskal–WallisTest revealedastatistically significant difference in lateral muscle diameter in three groups(p=0.01).Mann–Whitneytestsrevealedthat the sig-nificancedifferencewasbetweenthelateralmusclediameter andtwootheranteriorandposteriormusclesdiameters.
Table1–Summeryofelectromyographyresults.a
Group Pre-sphincterotomy(time1) Pre-sphincteroplasty(time2) Post-sphincteroplasty(time3)
Decellularizedamnioticmembrane 21±5.48 35±6.12 29±5.48
Freshamnioticmembrane 12±4.47 23±4.47 16±5.47
Control 28±4.47 24±5.48 22±4.47
a Mean±standarddeviationoffrequencyintervalrecodedatthesiteofsphincteroplasty.
Table2–Musclediametersinsphincterquadrants.a
Anteriormusclediameter Lateralmusclediameterb Posteriormusclediameter
Decellularizedamnioticmembrane 2.3±0.57 1.35±0.28 2.2±0.27 Freshamnioticmembrane 2.0±0.0 2.4±0.65 2.5±0.5
Control 2.16±0.45 2.4±0.67 3.0±0.79
a Mean±standarddeviationofmusclediameter.
b ThedifferencebetweenGroupswasstatisticallysignificant(pvalue=0.01).
Discussion
This study evaluates the efficacy of using amniotic mem-braneinsphincteroplastyofrabbit’sinjuredanal sphincter. Althoughthe result ofelectromyography showed improve-mentinthesphinctermusclefunction;especiallyinthefresh amnioticmembranegroup,butthisdifferencewasnot sta-tisticallysignificant.Histopathologyevaluationofsphincter muscledidnotshowasignificantdifferencebetweengroups. Howeveritseemsthathighleveloffrequencyinterval varia-tioncausedthisnon-significantstatisticallydifference.
Amnioticmembranewaspreviouslyusedforrepairof vari-oustissuedefectssuchascornea,skinandduodenum.10Some studiesalsoshowedseverfibrosisatthesiteofrepairinhuman subjects11andthismayleadstoimprovetheoutcomeof rec-tovaginalfistularepairwithusingit asaprosthesisduring therepair.12Enhance fibrosisalsomightbeassociatedwith bettersphincteroutcomeasitcausethefreeendsof sphinc-terattachedstronger butthis wasnotprovenbyourstudy asthecomposition ofsphincterwasnotdifferent between groups.
Amnioticmembrane contains various levelsofdifferent cytokineand also stem cells;that may havepotential role inthehealingofinjuredsphincter.Sincecellsandsensitive structuresmightbedamagewithtimeweusedfreshamniotic membraneandcomparetheresultswithprocessedamniotic membranewhichalsodidnotrevealedasignificantdifference. In this study we used histopathology evaluation as a markerofanatomicrepairandelectromyographyasamarker of physiologic repair to evaluate the outcome of sphinc-teroplastybut the goldstandard ofevaluationofsphincter functionismanometry. Applicationofmanometryinsmall animalsisnoteasilypossibleduetosmallsizeofanusand alsolackofvoluntarycontractionandthisisoneoftheweek pointofthestudy.
Althoughapplicationofamnioticmembraneinrepairof differenttissuedefectespeciallyinthefieldofcoloproctology butapplicationofamnioticmembraneinrabbit sphinctero-plastywasnotassociatedwithpromisingresult.Forfurther studiesitissuggestedtoapplylargeranimalmodeland dif-ferentwayofevaluatingsphincterfunction.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgments
Thestudy wasfunded byVice Chancellorforresearch and technologyShirazUniversityofMedicalSciences(GrantNo. 92-01-01-6232).
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