Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model.

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by  Engel, Odilo

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Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model
Odilo Engel,Sabine Kolodziej,Ulrich Dirnagl,Vincent Prinz
DepartmentforExperimentalNeurology,CenterforStrokeResearchBerlin,CharitéUniversitätsmedizin
Correspondenceto:[email protected]
URL:http://www.jove.com/details.php?id=2423
DOI:10.3791/2423
Citation:Engel O.,Kolodziej S.,Dirnagl U.,Prinz V.(2011).ModelingStrokeinMice-MiddleCerebralArteryOcclusionwiththeFilamentModel.JoVE.47.
http://www.jove.com/details.php?id=2423,doi:10.3791/2423
Abstract
Strokeisamongthemostfrequentcausesofdeathandadultdisability,especiallyinhighlydevelopedcountries.However,treatmentoptionsto
dateareverylimited.Tomeettheneedfornoveltherapeuticapproaches,experimentalstrokeresearchfrequentlyemploysrodentmodelsoffocal
cerebralischaemia.Mostresearchersusepermanentortransientocclusionofthemiddlecerebralartery(MCA)inmiceorrats.
Proximalocclusionofthemiddlecerebralartery(MCA)viatheintraluminalsuturetechnique(socalledfilamentorsuturemodel)isprobablythe
mostfrequentlyusedmodelinexperimentalstrokeresearch.TheintraluminalMCAOmodelofferstheadvantageofinducingreproducible
transientorpermanentischaemiaoftheMCAterritoryinarelativelynon-invasivemanner.Intraluminalapproachesinterruptthebloodflowofthe
entireterritoryofthisartery.Filamentocclusionthusarrestsflowproximaltothelenticulo-striatearteries,whichsupplythebasalganglia.Filament
occlusionoftheMCAresultsinreproduciblelesionsinthecortexandstriatumandcanbeeitherpermanentortransient.Incontrast,models
inducingdistal(tothebranchingofthelenticulo-striatearteries)MCAocclusiontypicallysparethestriatumandprimarilyinvolvetheneocortex.In
additionthesemodelsdorequirecraniectomy.Inthemodeldemonstratedinthisarticle,asiliconcoatedfilamentisintroducedintothecommon
carotidarteryandadvancedalongtheinternalcarotidarteryintotheCircleofWillis,whereitblockstheoriginofthemiddlecerebralartery.In
patients,occlusionsofthemiddlecerebralarteryareamongthemostcommoncausesofischaemicstroke.Sincevaryingischemicintervalscan
bechosenfreelyinthismodeldependingonthetimepointofreperfusion,ischaemiclesionswithvaryingdegreesofseveritycanbeproduced.
Reperfusionbyremovaloftheoccludingfilamentatleastpartiallymodelstherestorationofbloodflowafterspontaneousortherapeutic(tPA)lysis
ofathromboembolicclotinhumans.
Inthisvideowewillpresentthebasictechniqueaswellasthemajorpitfallsandconfounderswhichmaylimitthepredictivevalueofthismodel.
Protocol
Toguaranteehighqualityandreproducibility,werecommendtheuseofStandardOperatingProcedures(SOP).Inthisvideo,publishedSOPsas
developedandusedinourlaboratoryareapplied.1
1. Middle CerebralArtery Occlusion
1. Miceareanaesthetizedwithanappropriateanaestheticregimeinconsultwithveterinarystaff.(E.g.inductionwith1.5-2%Isofluraneand
maintainancewith1.0-1.5%Isofluranein2/3N2Oand1/3O2usingavaporizer).
1. Bodytemperatureofthemiceismaintainedat36.5°C±0.5°Cduringsurgerywithaheatingplate.Afeedbackcontrolledheatingpad,
whichwarmsaccordingtotherectaltemperatureofthemouse,ishighlyrecommended.
2. Disinfecttheskinandsurroundingfurwithanappropriateagent(e.g.70%ethylalcohol)anddryitafterwards.
2. Amidlineneckincisionismadeandthesofttissuesarepulledapart.
3. Theleftcommoncarotidartery(LCCA)iscarefullydissectedfreefromthesurroundingnerves(withoutharmingthevagalnerve)anda
ligatureismadeusing6.0/7.0string.5.0stringcanalsobeused.
4. Theleftexternalcarotidartery(LECA)isthenseparatedandasecondknotismade.
5. Next,theleftinternalcarotidartery(LICA)isisolatedandaknotispreparedwitha6.0filament.
6. Afterobtaininggoodviewoftheleftinternalcarotidartery(LICA)andtheleftpterygopalatineartery(LPA),botharteriesareclipped,usinga
microvascularclip.
7. AsmallholeiscutintheLCCAbeforeitbifurcatestotheLECAandtheLICA.Amonofilamentmadeof8.0nyloncoatedwithsiliconhardener
mixture(seebelow)isthenintroducedintotheLICA,untilitstopsattheclip.Attentionhastobepaidnottoentertheoccipitalartery.(Figure
1)
8. TheclippedarteriesareopenedwhilethefilamentisinsertedintotheLICAtooccludetheoriginoftheLMCAinthecircleofWillis.
9. ThethirdknotontheLICAisclosedtofixthefilamentinposition.
10. Themicereceivesaline0.5mLsubcutaneouslyasvolumereplenishment.Forpainrelief,Lidocainegelistopicallyappliedinthewound.
11. Ifreperfusionisintended,micestayfor30-90minocclusioninaheatedcage,thewoundcouldbeclosedwithasmallsutureclip.
Afterwards,asecondanaesthesiaisperformed,thethirdknotontheICAismomentaryopenedandthefilamentwithdrawn.
12. Theremainingsuturesareshortenedandtheskinisadaptedwithasurgicalsuture.
13. Allanimalsreceiveasecondvolumereplenishmentasdescribedabove.
14. Animalsareputinaheatedcagefortwohourstocontrolforbodytemperature.
1. Theanimalsmustbecheckeddailyaftersurgeryforsignsofdiscomfort.Themicecouldshowsomepostsurgicalweightloss.They
receivemashedfoodinapetri-dishplacedonthefloortoencourageeating.Thefoodisreplaceddailyforsevendays.
2. Sham Operation
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1. ForshamoperationsthefilamentisinsertedtooccludeLMCAandwithdrawnimmediatelytoallowinstantreperfusion(1.8).Thesubsequent
operationisidenticaltothatperformedontheanimalsundergoingcerebralischaemia(1.9-1.14)includingasecondanaesthesia.
3. Preparation of the Filament
1. Sterilityofthefilamentshouldbeconsidered.Theuseofsterilizedequipmentaswellasanappropriatehandlingofthefilamentafterwardsis
aprerequisiteforasterilesurgery.Disinfectionofthefilamentisdifficult,sincemanyofthecommonsterilizationmethodsmayworsenthe
qualityofthefilament.Howevermethodssuchasradiation,forexamplewithUVlightorγrays,orchemicalsterilization,forexamplewith
highlyreactivegasessuchasethyleneoxide,areapplicable.
2. 8.0nylonfilamentiscutintolengthsof11mmunderthemicroscope
3. Thefilamenttipmustbecoatedcompletelyandevenlyoveralengthof8mmwithahardenermixtureofXantoprenMMucosaandActivator
NFOptosil
4. Representative Results
Dependingonthedurationofbloodflowrestriction,differentmotorandbehaviouraldeficitsresult.Bothafter30and60minofcerebralischaemia,
animalsinmostcasesshowdecreasedresistancetolateralpushandcirclingduetodisturbanceinlocomotion.Milderlesionsmanifestasa
flexorpositioninthefrontlimbs.Theseeasilyobservablesignscanbeusedasabasicscoreforthesuccessoftheoperation.2
MorphologicallythelesioncanbeassessedusingeitherhistologyorMagneticResonanceImaging(MRI).Sixtyminutesocclusionofthemiddle
cerebralarteryproducestissuepannecrosisinanareaincludingboththestriatumandtheneocortex,whereas30minutesofischaemiacause
mainlyneuronalcelldeathlimitedtothestriatum.3(Figure2)Intermsofinfarctvolume,weexpectastandarddeviationlowerthan30%inaset
ofoperations.Mortalitydependsontheocclusiontimewitharound5%after30minofischaemiaand10-20%after60min.
AnotherminimalinvasivepossibilityistheuseofLaserDopplerflowmetry(LDF)duringtheoperation,whichallowsadirectcontrolofitssuccess.
Inanindividualanimal,reductionto10-20%ofpreocclusionvaluesclearlyindicatessuccessfulinductionoffocalcerebralischemia.4However,
LDFcannotbeusedasamethodforinterindividualcomparisons,sinceLDFcanonlymeasurequantitativechanges(inpercentages)ofblood
flowwithinasmallandlimitedtissuesamplevolume.Itdoesnotgiveinformationaboutthespatialextentofbloodflowreduction.5
Thereareseveralteststoassessbehaviouralaspectsafterstroke,includinggaitanalysis6,7,Rotarod8,Poletest9,10,adhesiveremovaltest11,12,
staircasetest13,14,ladderrungtest15,16andMorriswatermaze17.Inallthesetests,micesubjectedtoMCAoperformlesssuccessfullythan
controlanimals.
Figure1.Schemeofthevesselarchitecturesupplyingthebrain(depictedinbackground)inthemouse.Differentstrainsmayshowvariations,for
exampletheoccipitalarterysometimesleavesfromtheinternalcarotidartery.
Figure2.SchematicillustrationoftypicallesionsizesafterdifferenttimepointsofreperfusionintheproximalMCAomodel.Inthemiddle,the
typicalcourseoffunctionalactivityandcerebralbloodflowafterMCAoaredepicted.(MCAo:Middlecerebralarteryocclusion,LDF:Laser
Dopplerflowmeasurement)
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Discussion
Themodeloftransient,proximalMCAocclusion18,19presentedheremimicsoneofthemostcommontypesofischaemicstrokeinpatients.20
Basedonvariablereperfusiontimes,themodeloffersdifferentgradesofdamagerangingfromtransientischaemicattack(TIA)tolargeinfarcts
includingmajorpartsoftheischemichemisphere.Thisallowstheresearchertostudydifferentpathophysiologicalmechanismsafterstroke.20,21
Surgerycanbeperformedinashorttimeperiodandproduceshighlyreproduciblelesions.Nevertheless,thisrequiresathoroughcontrolof
confounders.22Smalldifferencesinoperationtechniquemayaccountfordifferenteffectsontheinfarct.23,24Furthermore,duetovariancesin
cerebralvascularanatomy,differentmousestrainsshowadifferentoutcome.25,26Bodytemperatureaffectsneurologicaldamage,with
hypothermialeadingtosmallerlesions27andhyperthermiatomoreseveredeficits.28Accordingly,temperaturecontrolandmaintenanceishighly
relevantinthismodel.29Inaddition,bloodpressureandbloodgasesareimportantconfoundersofoutcomeandneedtobemonitored.30,31The
useofquickandminimalinvasivemethods(non-invasivebloodpressuremeasurement,suitableandeasyaccessiblebloodcollectionsites)is
recommended.Thechoiceoftheanaestheticisalsohighlyimportant,assomemayhaveneuroprotectiveeffects,and/orbevasodilators,asfor
exampleIsoflurane.32Consequently,exposuretoanaesthesiashouldbeasshortaspossibleandstandardized.Weexcludeanimalswhichhave
undergonesurgeryforlongerthan15min.
Shavingthesurgicalsiteproducesmicroabrasionsandinflammationandreleaseshairfragments.Thismayfurtherincreaseinflammationand
fosterlocalinfection,whichmayimpactonstrokepathophysiology.Housingconditions,especiallytheuseofenrichment,mayaffectstroke
outcomeandshouldbestandardizedanddescribedinresearchreports.6,33,34Theuseofenvironmentalenrichmentanditseffecton
reproducibilityisamatterofdiscussion.35Anotherimportantconfounderisthestrokeinducedriskforinfectionespeciallyafterlongertimesof
ischaemia36,whichleadstoadditionalmorbidityandincreasedmortality.37,38Asinfectionsbecomesymptomaticataroundday3to5,thishas
importantconsequencesforlongtermfollow-upinsuchmodels.
Toproduceresultsrelevantforthedevelopmentofnoveltreatmentstrategiesforstroke,standardizationandqualitycontrolishighlyimportantin
preclinicaltranslationalstrokeresearch.39"GoodLaboratoryPractice"40,41mandates:
1. anappropriateanddetaileddescriptionofanimalsused;
2. samplesizecalculationandreportingontheexpectedeffectsize;
3. inclusionandexclusioncriteria,setupbeforethestudy;
4. randomizationoftheallocationintogroups;
5. allocationconcealmentwithrespecttotheinvestigators;
6. reportinganimalsexcludedfromanalysis;
7. blindedassessmentofoutcome;
8. reportingpotentialconflictsofinterestandstudyfunding.
Disclosures
ExperimentsonanimalswereperformedinaccordancewiththeguidelinesandregulationssetforthbyLandesamtfuerGesundheitundSoziales,
Berlin,Germany.
Acknowledgements
ThisworkwasfundedbytheEuropeanCommunity'sSeventhFrameworkProgramme(FP7/2007-2013)undergrantagreementn°201024andn°
202213(EuropeanStrokeNetwork).AdditionalfundingwasgivenbytheBundesministeriumfürBildungundForschung(CenterforStroke
ResearchBerlin),andtheDeutscheForschungsgemeinschaft(ExzellenzclusterNEUROCURE).
TheauthorswouldliketothankMareikeThielke(Dep.forExperimentalNeurology,CharitéBerlin)forthesupportduringtheoperationandElke
Ludwig(CharitéVideoServices)forproducingtheanimation.
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