Journal
of
Coloproctology
w w w . j c o l . o r g . b r
Original
article
Role
of
preoperative
retrograde
enema
on
inflammatory
and
healing
parameters
in
colonic
anastomosis:
experimental
study
in
dogs
夽
Carlos
Renato
Sales
Bezerra
a,∗,
Francisco
Sérgio
Pinheiro
Regadas
b,
Welligton
Ribeiro
Figueiredo
b,
Miguel
Augusto
Arcoverde
Nogueira
b,
Péricles
Cerqueira
de
Sousa
a,
Claudio
Manuel
Gonc¸alves
da
Silva
Leite
c,
Kaline
Fortes
e
Silva
d,
Erbert
Portela
Martins
Filho
e,
José
Alberto
Dias
Leite
baUniversidadeFederaldoCeará(UFC),Fortaleza,CE,Brazil
bSchoolofMedicine,UniversidadeFederaldoCeará(UFC),Fortaleza,CE,Brazil
cSchoolofMedicine,CentroUniversitárioChristus(UNICHRISTUS),Fortaleza,CE,Brazil
dSchoolofMedicine,DeVry,FACID,Teresina,PI,Brazil
eUniversidadeFederaldoPiauí(UFPI),Teresina,PI,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received10June2014
Accepted10July2014
Availableonline10September2014
Keywords:
Enema
Woundhealing
Inflammation Collagen
a
b
s
t
r
a
c
t
Objective:Thepurposeofthisexperimentalstudywastocomparetheinflammatoryand
woundhealingresponseofdogssubmittedtocolonicanastomosiswithandwithout
pre-operativeretrogradeenema.
Methods:Thestudyincludedtwogroupsof31femaledogs(Canisfamiliaris).G-I(control):
nopreoperativebowelpreparation;G-II(study):preoperativeretrogradeenemausinga10%
glycerinsolution.Alltheanimalsweresubmittedtolaparotomyandcolotomyat20cm
fromtheanalverge,followedbyclosurewitharunningextramucosalsingle-layersuture
(Prolene®000).Theanimalswerethenanesthetizedandeuthanizedonthe7th(n=10)or21st
(n=20)postoperativeday(POD)toremovetheanastomosedcolonsegmentforhistological
andimmunohistochemicalanalysisevaluatingtheparameters:anastomoticedema,
vaso-proliferation,abdominaladhesions,typeIandIIIcollagen,nitricoxideandmyeloperoxidase.
TheobserveddifferenceswereanalyzedwiththeMann–Whitneytestfornonparametric
dataandStudent’sttestforunpairedsamplesandparametricdata.
Results:OneanimalfromG-IandonefromG-IIdiedonPOD7andPOD10dueto
anasto-moticcomplicationsandsepsis,respectively.Thegroupsdidnotdiffersignificantlywith
regardtoinflammatoryandhealingparameters,althoughthelevelsofmaturecollagen
weresignificantlylowerintheanimalssubmittedtopreoperativebowelpreparation.
夽
StudycarriedoutatPost-graduationprogramstrictosensuinSurgery,SurgeryDepartment,FacultyofMedicine,UniversidadeFederal
doCeará(UFC),Fortaleza,CE,Brazil.
∗ Correspondingauthor.
E-mail:piripiri@uol.com.br(C.R.S.Bezerra).
http://dx.doi.org/10.1016/j.jcol.2014.08.007
Cicatrizac¸ãodeferidas Inflamac¸ão
Colágeno
retrógradapré-operatório.
Métodos: Oestudoincluiudoisgruposde31cãesfêmeas(Canisfamiliaris).G-I(controle):
sempreparointestinalpré-operatório;G-II(estudo):Enemaretrógradapré-operatóriacom
umasoluc¸ãodeglicerinaa10%.Todososanimaisforamsubmetidosàlaparotomiae
colo-tomia a20cmda bordaanal,seguido defechamentoem suturaextramucosacontínua
(Prolene®000).Osanimaisforamanestesiadose,emseguida,submetidosàeutanásiano7◦
(n=10)ou21(n=20)pós-operatório(DPO)pararemoverosegmentodecólonanastomosado
paraanálisehistológicaeimunohistoquímicaavaliandoosparâmetros:edemada
anas-tomose,vasoproliferac¸ão,aderênciasabdominais,colagéniotipoIeIII,oóxidonítricoea
mieloperoxidase.Asdiferenc¸asobservadasforamanalisadascomotestedeMann–Whitney
paraosdadosnãoparamétricosetestetdeStudentparaamostrasnãopareadasedados
paramétricos.
Resultados: UmanimaldoGIeumdoG-IImorreunodia7e10◦DPOdevidoacomplicac¸ões
deanastomoseesepse,respectivamente.Osgruposnãodiferiramsignificativamenteem
relac¸ãoaosparâmetrosinflamatóriosedecura,emboraosníveisdecolágenomaduroforam
significativamentemenoresnosanimaissubmetidosaopreparointestinalpré-operatório.
Conclusão: Demonstrou-sequeambososprocedimentossãosegurosparaseremusados,
noentanto,ogrupocomapreparac¸ãodointestinomostrouumamenorquantidadede
colágenomaduronoperíodopós-operatórioimediato,podendoserconstituídoumfator
preventivoparacomplicac¸õescirúrgicasparaestetipodeprocedimentocirúrgico,embora
nenhumaevidêncianesteestudo.
©2014SociedadeBrasileiradeColoproctologia.PublicadoporElsevierEditoraLtda.
Todososdireitosreservados.
Introduction
Historically,colorectalsurgicalprocedureshavebeen
associ-atedwithpostoperativecomplications,especiallypriortothe
adventofantibioticsinthe20thcentury.1,2 Thepracticeof
bowelpreparationwasfirstintroducedin1950andwas
fur-therpopularizedinthe1970swhencatharticsubstanceswere
combinedwithoralantibiotics(macrolids)andmetronidazole.
Nevertheless,bowelpreparationisalsoassociatedwith
com-plications,includingdehydrationandexcessiveflatulence.3In
fact,theuseofbowelpreparation(primarilyenema)toprepare
patientsforcolorectalprocedureshasbeentheobjectofmuch
discussionintheliteratureoverthepastfewyears.3
Thepurposeofthis experimentalstudywas tocompare
theinflammatoryandwoundhealingresponseofdogs
sub-mittedtocolonicanastomosiswithandwithoutpreoperative
retrogradeenemausinga10%glycerinsolution.
Methods
ThenumberoftheprotocolapprovedbytheResearchEthics
Committeewas65/2010and theresearchwasconductedin
accordancewiththeethicalstandardsrequired.
Thestudy includedtwogroupsof31female dogs(Canis
familiaris)withanaverageweightof12.01kg(range:5.75–25.0).
GroupI(control):nopreoperativebowelpreparation
admin-istered.
GroupII(study):preoperativebowelpreparation(retrograde
enema)usinga10%glycerinsolutiononthedaybeforethe
procedureandonthedayoftheprocedure.
Followinganesthesiawithketaminehydrochloride,allthe
animalsweresubmittedtoindividualdigitalrectal
Fig.1–Colonsegmentretrievedforhistologicaland
immunohistochemicalanalyses.
proposed by O’Dwyer (1989)4 (excellent=absence of
feces; good=presence of minimal fecal residue;
accept-able=presence of liquid feces; soiled=presence of solid
feces). The surgical procedure consisted of a laparotomy
using a midline transumbilical incision, identification of
the descending colon at 20cm from the anal verge and
a full thickness circumferential colotomy, followed by an
end-to-endmanualcolonicanastomosisusingasingle-layer
extramucosal running suture with polypropylene 3-0. On
the7th(n=10)andthe21st(n=20)postoperativeday(POD),
the animals were euthanized, a second laparotomy was
performedthroughthepreviousabdominalincisionandthe
anastomosis was evaluated for anastomotic complications
(fistulaanddehiscence).Adhesionsweremeasuredaccording
toKnightly’s classification: 0=no adhesions, 1=single thin
and easily separable adhesion, 2=less extensive but weak
adhesionswhichwithstandtractionpoorly,3=extensive
vis-ceraladhesionsextendingtoabdominalwall, 4=numerous
extensive and visceral adhesions involving the mesentery,
bowel, omentum and abdominal wall. Then a 6-cm colon
segment including the anastomotic site was removed for
analysis(Fig.1).
The resected segment of colon including the
anasto-motic area was prepared for histological analysis and
stainedwithhematoxylin-eosin.Edemaandvasoproliferation
were evaluated according to Sousa’s classification.5
Colla-gen wasevaluated withPicro-Sirius Red undera polarized
light microscope (Type I collagen stains red, yellow and
orange;typeIIIcollagen stainsgreen)(Fig. 2).6,7 The
histo-logical sections were submitted to immunohistochemistry
to evaluate the direct and indirect inflammatory markers
(myeloperoxidaseandinducednitricoxidesynthase,
respec-tively).
Allanimalswereevaluatedwithregardtotheparameters
weight,postoperativeclinicalrecovery,intra-abdominal
adhe-siongrade,anastomoticedema,vasoproliferation,typeIand
IIIcollagen,myeloperoxidase(MPO)andinducednitricoxide
synthase(iNOS).Theresultsofthetwogroupswerecompared
onPOD7andPOD21(Fig.3).
Fig.2–TypeIandIIIcollagenstainedwithPicro-SiriusRed.
0 20 40 60 80
Collagen I Study Collagen II Study 35.4
42.0
p=0.2712
Collagen Área (mm
2)
Fig.3–TypeIandtypeIIIcollagenlevelsinG-II(study)on
POD21.
Statisticalanalysis
DifferencesbetweenthegroupswereanalyzedwithStudent’s
t test (unpaired samples) and parametric data or with the
Mann–Whitneytest(nonparametricdata).Thelevelof
statis-ticalsignificancewassetat5%(p<0.05).
Results
In G-II, bowel preparation was considered good (n=18) or
excellent(n=12),accordingtoO’Dwyersclassification.In
G-I,23animalshadsolidstoolsand7hadliquidstoolsidentified
byrectaldigitalexam.
Thegroupsdidnotdiffersignificantlywithregardto
aver-ageweightonPOD7(G-I=11.45kg;G-II=10.86kg;p=0.493)or
POD21(G-I=13.04kg;G-II=11.86kg;p=0.306).
OneanimalfromG-Idiedon7thPODduetoanastomotic
dehiscence,andoneanimalfromG-IIdiedon10thPODdueto
infectionoftheabdominalincision,necrotizingfasciitisand
sepsis,despiteanintactanastomosis.
The groups did not differ significantly with regard to
abdominaladhesions,neitheronPOD7(p=0.734)noronPOD
21(p=0.568).However,asignificantdifferencewasobserved
betweenPOD7andPOD21inbothG-I(p<0.0001)andG-II
andsevere(n=2)inG-II.OnPOD21,vasoproliferationwasmild
(n=17),moderate(n=3)inG-I,comparedtomild(n=12),
mod-erate(n=7)andsevere(n=1)inG-II.However,withinG-I,POD
7differedsignificantlyfromPOD21(p<0.0001).
G-IandG-IIdidnotdiffersignificantlywithregardtotype
IandIIIcollagenlevelsonPOD7(p=0.891;p=0.915)orPOD
21(p=0.271;p=0.008).WhenPOD7and POD21were
com-pared,asignificantdifferenceintypeIcollagenconcentrations
wasobservedinbothG-I(p=0.009)andG-II(p=0.002).Finally,
whencomparingtypeItotypeIIIcollagen,asignificant
differ-encewasfoundinG-IonbothPOD7(p=0.0077)andPOD21
(p=0.0003).InG-II,thetwotypesofcollagenonlydifferedon
POD7(p=0.002).
ThegroupsdidnotdifferwithregardtoMPOoriNOSon
POD7(p=0.580 andp=0.089)andon POD21(p=0.775and
p=0.394)respectively.WhencomparingPOD7toPOD21,iNOS
levels differedsignificantly inG-II (p=0.003),whereas MPO
levelsdifferedinbothG-I(p=0.0004)andG-II(p<0.0001).
Discussion
Theuseofpreoperativeretrogradeenemaisstillamatterof
controversy.However,manysurgeonsperformtheprocedure
routinely,mainlyinordertoreduceexposureofthesurgical
site(anastomosis)tofecalmatter8andtherebyminimizelocal
inflammatoryreactions.
Femalemongreldogs(Canisfamilliaris)werechosenforthis
studybecauseoftheconvenientcharacteristicsofthepelvic
cavity.9Thesuturewasmadewithmonofilament
polypropyl-ene000threadbecauseofthelowindicesofinflammatory
reactionswithwhichitisassociated.10,11Thetypeofsuture
used(runningsingle-layerextramucosalsuture)isconsidered
betterthancontinuoussuturewithinterruptionswithregard
tovascularizationandhealingattheanastomoticsite.12
Pre-operativeretrogradeenemawasperformedusing10%glycerin
(ratherthanmagnesiumhydroxide,mannitolorpolyethylene
glycol/sodiumphosphate)duetoitslowcost,easeof
acquisi-tionandlowindicesofmorbidity.13,14
Inthestudygroup,bowelpreparationwasratedverygood
orexcellentinallcasesaccordingtoO’Dwyer’sclassification:
ondigitalexamination,nofeceswereobservedwhichmight
compromisetheexperiment.Likewise,duringcolotomywith
colorectalanastomosis,nofecesweredetectedinthelumen.
Inthecontrolgroup,despitetheabsenceofbowelpreparation,
the anastomotic site,adhesiongradeis usedasa
parame-tertoquantifytheinflammatoryresponseintheanastomosis
site.11,15
Theabsenceofasignificantdifferencebetweenthegroups
withregard toedemaon bothPOD7and POD21 suggests
bowelpreparationhadnoinfluenceontheintensityof
inflam-mation.Studiesbasedonotheranimalmodelshaveyielded
similarresults.5,16
OnPOD7,thetwogroupsdidnotdifferwithregardto
vaso-proliferation,whichwasmoderatetosevereinG-I.OnPOD
21,vasoproliferationwaspredominantlymildinbothgroups.
However,whencomparingPOD21toPOD7withinG-I,
vaso-proliferationwassignificantlyhigheronPOD7thanonPOD
21,indicatingthatthepresenceoffecesinthelumenmaybea
determiningfactorforvasoproliferationduetogreater
bacte-rialcontaminationintheearlypostoperativestage.Asshown
inanotherexperimentalstudy,thegreaterthevascularization,
moreviablewillbetheanastomosis.17
TypesIandIIIcollagenlevelsweresimilarinthetwogroups
on bothPOD 7and POD21, despiteexposuretofeces and
bacteriainG-I,asalsosuggestedinapreviousstudy.18
Withineachgroup,thetypeIcollagenlevelswere
signif-icantly lower onPOD 21than on POD7.Other researchers
reportedincreasedlevelsoftypeIIIcollageninthefirstfour
daysaftersurgery,followedbyadecrease,whiletypeIcollagen
levelsremainedhighuntilthe14thpostoperativeday.18
ThedifferencebetweentypeIandtypeIIIcollageninG-I
onPOD21maybeexplainedbytheexacerbationof
inflamma-toryresponseduetothepresenceofhigheramountoffecal
matterandbacteriaattheanastomoticsite,which,however,
didnotinterferewiththehealingprocess.Inanotherstudy,
theproliferationoftypeIandtypeIIIcollagenwasmore
evi-dentinanimalssubmittedtobowelpreparationduetolower
inflammatoryresponseand betterinterweavingoffibersof
bothtypes.19
In G-II, intense stainingfor iNOS was observed
signifi-cantly more often(70% ofthe animals) on POD7 than on
POD21.NosuchdifferencewasobservedinG-I,indicating
that theabsence ofbowelpreparationwasassociatedwith
highpostoperativenitricoxidelevelsuntilthe21st
postopera-tiveday.iNOSlevels(asevaluatedbyimmunohistochemical
analysis) may be high inside macrophages for up to one
monthaftertheinitialsurgery.Thesame maybeobserved
forendothelialcellsinanastomoses,evenintheabsenceof
Thegroupsdidnotdifferwithregardtomyeloperoxidase
onPOD7orPOD21,butwhencomparingPOD7toPOD21,
myeloperoxidaselevelsdifferedinbothgroupsduetohigher
inflammatoryactivityintheearlypostoperativestage.
Theincidenceofpostoperativecomplicationswassimilar
inthetwogroups,suggestingthattheuseofpreoperative
ret-rogradeenemahadnoinfluenceonthisparameter.
Conclusion
Ithasbeenshownthatbothproceduresaresafetobeused
onthepre-operativecolorectalsurgicalprocedures,however,
thegroupwithbowelpreparationshowedaloweramountof
maturecollagenintheimmediatepostoperativeperiodand
maybeconstitutedapreventivefactorforsurgical
complica-tionsforthistypeofsurgicalprocedure,althoughnoevidence
inthisstudycouldbedetermined.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
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