JournalofVisualizedExperiments www.jove.com VideoArticle Murine Colitis Modeling using Dextran Sulfate Sodium (DSS) CaitlynG. Whittem1,AmandaD. Williams2,ChristopherS. Williams2 1DepartmentofCancerBiology,VanderbiltUniversity 2DepartmentsofMedicineandCancerBiology,VanderbiltUniversity Correspondenceto:[email protected] URL:http://www.jove.com/index/Details.stp?ID=1652 DOI:10.3791/1652 Citation:Whittem C.G.,Williams A.D.,Williams C.S.(2010).MurineColitisModelingusingDextranSulfateSodium(DSS).JoVE.35.http://www.jove.com/index/Details.stp?ID=1652,doi: 10.3791/1652 Abstract Colitiscanoccurfromviralorbacterialinfections,ischemicinsult,orautoimmunedisorders;mostnotablyUlcerativeColitisandthecolonicvariant ofCrohn’sDisease-Crohn’sColitis.Acutecolitismaypresentwithabdominalpainanddistention,malabsorption,diarrhea,hematocheziaand mucusinthestool.Wearebeginningtounderstandthecomplexinteractionsbetweentheenvironment,genetics,andepithelialbarrierdysfunction inInflammatoryBowelDiseaseandanimalmodelsofcolitishavebeenessentialinadvancingourunderstandingofthisdisease.Onepopular modelinvolvessupplementingthedrinkingwaterofmicewithlow-molecularweightDextranSodiumSulfate(DSS),resultinginepithelialdamage andarobustinflammatoryresponseinthecolonlastingseveraldays1.Variationsofthisapproachcanbeusedtomodelacuteinjury,acuteinjury followedbyrepair,andrepeatedcyclesofDSSinterspersedwithrecoverymodelingchronicinflammatorydiseases2.Afterasinglefour-day treatmentof3%DSSindrinkingwater,miceshowsignsofacutecolitisincludingweightloss,bloodystools,anddiarrhea.Miceareeuthanizedat theconclusionofthetreatmentcourseandatnecropsydissectedcolonsareprocessedandcanbe'Swissrolled"3toallowmicroscopicanalysisof theentirecolonorinfusedwithformalinas"sausages"toallowmacroscopicanalysis.Tissueisthenembeddedinparaffin,sectioned,andstained forhistologicreview. Protocol Part 1: Injury-Repair Colitis Model 1. BaselineweightshouldbeobtainedforeachmousepriortobeginningDSStreatment. 2. Makea3%(w/v)DextranSulfateSodiumSaltsolutioninwaterandfilterwitha0.45μmcelluloseacetatefilter. 3. Replacedrinkingwaterinthemousecagewiththe3%DSSsolutionforfourdays.Themiceshouldnothaveaccesstoanyothersourceof water(i.e.exclusiontipsplacedonautomatedwateringsystems). 4. Ondayfour,replacetheDSSsolutionwithwaterforanadditionalthreedays,allowingsomecolonicepithelialrecovery.Themiceshouldbe weighedondayfourinordertoquantifysystemicconsequencesofcolitis.Weightlossiscommonwithsevereinjury. 5. Onday7,weighandsacrificethemice.Micecanbeeuthanizedbyinhalationaloverdoseofisoflurane,orotherinstitutionally,IACUC approvedmethods. Part 2: Necropsy and Harvesting of Colon 1. Exposetheventralsideofthemouseandsecurelegstoensureunobstructedaccesstotheabdomen.Wettheabdomencompletelywith 70%ethanol. 2. Graspthemidlineoftheabdomenwithtissueforcepsandextendupwardsthustentingtheskin.Usefine-tippedscissorstoincisethe abdomenthusexposingtheabdominalcontents.Extendtheincisiontothetipofthexyphoidprocessatthemidlineandthenextendalong theinferioraspectofthecostalmarginsbilaterally. 3. Identifythesmallintestine,cecum,andcolon.Carefullydissect/teasethecolonfromthesurroundingmesentery.Transectthecolondeepin thepelvistofreethedistalcolonandrectum.Transectthecolonatthecolonocecalmargintofreetheproximalcolon.Caremustbetaken duringthisprocessnottodamagethecolonascleaningthecolonwillbeproblematicifperforationoccurs. 4. Usinga20Gfeedingneedleand10mlsyringe,intubateandflushthecolonwithice-coldPBSuntiltheeluateiscompletelyclearofstool. 5. Atthispoint,ifmacroscopicanalysiswithadissectingmicroscopeisdesired,thecolonscanbefixedas“sausages”.Ifhistologicanalysisonly isdesiredthanproceedtoPart4“MakingSwissRollsfortheHistologicalAnalysisofAcuteColitis”. Part 3: Processing as “Sausages” for MacroscopicAnalysis of the Entire Colon 1. Itisimportanttomaintainthecorrectorientationofthecolon,therefore,keepthedistalendofthecolonclosesttotheoperator.Cuttwo piecesofnon-absorbablesuture(SP1161.5metricbraidedsilk),oneapproximately1inchinlength,theother2inches. 2. Usethe2inchpieceofsuturetotieoffthedistalendasclosetothemarginaspossiblewhilestillmaintainingagoodseal. 3. Placea20Gfeedingneedlecontaining5mlof10%bufferedformalinphosphateintotheproximalendofthecolon.Looselytietheremaining pieceofsutureimmediatelyproximaltothebulbofthefeedingneedle.Graspthecolonfirmlyatthebulbofthefeedingneedleandinfuse formalinuntilthecolonisexpanded.Tightentheknotinthesutureasyouwithdrawthefeedingneedle,thusleavingtheinfused,expanded colonasa“sausage”. 4. Filla15mlconicaltubewith10%bufferedformalinphosphateandplacethecoloninthetube.Fixfor24hours. 5. Pouroffformalinandreplacewith70%EtOHforanadditional24hrs.(Colonscanbestoredin70%EtOHindefinitelyatroomtemperature.) 6. Removethecolonfromtheconicaltubeandcutthestringsoneachendwithascalpel,beingcarefulnottodamagethecolon.Remember thatthedistalendhasthelongestpieceofstring.Cutlongitudinallyfromthedistaltoproximalendofthecolonsothatitformsalongsheet. Atthispoint,thecoloncanbeviewedunderadissectingmicroscope. Copyright©2010JournalofVisualizedExperiments Page1of3 JournalofVisualizedExperiments www.jove.com Part 4: Making Swiss Rolls for the HistologicalAnalysis ofAcute Colitis 1. Itisimportanttomaintainthecorrectorientationofthecolon,therefore,keepthedistalendofthecolonclosesttotheoperator. 2. Measurethecolonlength.Thismetricisanotherindicatoroftheseverityofinjury.Colitisincreasesedemaandshortenstheoverallcolon length. 3. Usingfine-tippedscissors,inciselongitudinallyfromdistaltoproximalendofthecolon.Usefinetippedforcepstograspeitheredgeofthe incisionandopenlaterallyworkingyourwaydistally->proximally,thusdisplayingthecolonasaflatsheet. 4. Rollingthecolonrequiresapairofforcepsandatwohandedtechnique.Graspeitheredgeofthedistalmarginwithforceps.Proceedto sequentiallyrollthecolonbyrotatingandreleasingtheforcepsproceedingtotheproximalmargin.Thusgeneratingaspiralwithathird dimensionor“SwissRoll”.Tomaintaintherolledform,grasptherollfirmlywithforcepsandtransectitwitha27G1/2needle.Securethe needlebybendingtheneedleasitexitstheroll. 5. Placetherollinanappropriatelylabeledtissuecassetteandplaceinajarof10%bufferedformalinphosphateatroomtemperaturefor24hrs toensuretissuefixation. 6. Pouroffformalinandreplacewith70%EtOHforanadditional24hrs.(Colonscanbestoredin70%EtOHindefinitelyatroomtemperature.) 7. Oncethesamplehasbeenfixed,itcanbeparaffin-embedded,sectionedandmountedonslidesforhistologicanalysis. Part 5: Representative Results TheDSSmodelforacutecolitisallowstheresearchertoobtain,fix,andanalyzeacolonthatmodelsacutecolitis.WhentheSwissrolliscutand mounted,itshouldformarepresentativesliceoftheentirecolonifrolledproperly(Figure1).ThemountedrollcanbestainedwithH&Einorderto determinetheextentofdamagetothecolon,fromthedistal(inside)endtotheproximal(outside)end(Figure2).Immunohistochemistrycanalso beperformedontheSwissrolltoidentifyandquantifyinflammatoryinfiltrates.Ifthesausagemethodhasbeenperformedcorrectly,thefixed colonwillbedilatedandtheentiremucosalsurfacecanbeeasilymanipulatedandviewedunderthedissectingmicroscope(Figure3). Figure1.Theproperly-executed"Swissroll".(A)Thecolonisrolledfromthedistaltoproximalend,transectedwithaneedleandsecuredby bendingtheendoftheneedle.Itisthenplacedinatissuecassetteforfixation.(B)H&Estained5μmsectionofaSwissrollmadefromthecolon ofamousetreatedwithDSS(d=distalcolonp=proximalcolon). Figure2.DSStreatedcolonsshowsignsofacutecolitis.InflammationandcryptdamageareapparentintheDSS-treatedcoloncomparedtoa watertreatedcontrol. Copyright©2010JournalofVisualizedExperiments Page2of3 JournalofVisualizedExperiments www.jove.com Figure3.Anexampleofa"sausage".Thesausageinfusedwithformalinandcompletelyexpanded.Aslightanglewillbepresentsecondaryto thenaturalcurvatureofthecolon.Theopenedsausageshouldlieflat. Discussion Thisprotocolcanbemodifiedtomodelacuteinjury,injury-repair,orchroniccolonicinjuryprocesses. AcuteInjuryModification: DSStreatmentadlibfor4-6daysfollowedbyimmediatesacrifice Injury-RepairModification: Injurywith4-6daysofDSStreatmentfollowedbyrecoveryperiodof3-4daysandsacrifice(asdescribedinaboveprotocol). ChronicColitisModification: Miceareplacedonthreefive-daycyclesof3%DSSwithsixteendaysofrecoverybetweeneachcycle.Micearesacrificedafterthefinal16-day restperiod. Thereareseveralissuesthatresearchersneedtobeawareofwiththismodel: 1. Variabilityinsiteofinjury:Inourhands,weseeapredominantlydistalinjurypattern,withrelativesparingoftheproximalcolonandcecum. Othershavereportedmoreproximalpredominancetotheinjurypattern. 2. Environmentalvariability.Thereissignificantenvironmentalvariability.Thisislikely,inpart,duetodifferencesinintestinalmicroflora,dietand otherenvironmentalconsiderations. 3. ThismodelisineffectiveifhighmolecularweightDSSisused(i.e.500,000Da). 4. Thereisconsiderablestrainvariabilitywiththismodel4. Biochemicalanalysisofthecoloncanbeperformedbytakingsamplesfromtheproximalordistalmargins.Dependingontheseverityofinjury thismayimpactyourabilitytogenerateahistologicinjuryscore.InvivoBrdUlabelingtomeasureproliferationcanbeachievedbyintraperitoneal injectionof16.7μg/kgBrdU2hrspriortosacrificefollowedbyα-BrdUIHCprocessing. Asanalternativetothe“sausage”,thecoloncanbefixedcompletelyflat.LinethebottomofaTupperwarecontainerwithWhatmanpaperand soakthepaperwith10%bufferedformalinphosphate.Dissectthecolonasdescribedin“Part2:NecropsyandHarvestingofColon”.Again,keep thedistalendofthecolonclosesttotheoperator.Usingfine-tippedscissors,inciselongitudinallyfromdistaltoproximalendofthecolon.With finetippedforcepsgraspeitheredgeoftheincisionandopenlaterallyworking,thusdisplayingthecolonasaflatsheet.Transferthisflatsheetto thepre-soakedWhatmanpaper.CovertheflattenedcolonwithanotherpieceofWhatmanandwetwith10%bufferedformalinphosphate.The Whatmanshouldbecompletelycoveredinformalinbutnotsomuchthatitliftsoffthecolon.SealtheTupperwareandfixfor24hours.Remove thecolonfromtheTupperwareandplaceitin10ml70%ethanolina15mlconicaltube.(Colonscanbestoredin70%EtOHindefinitelyatroom temperature.) Thereareseveralmethodsforquantifyingcolonicinjury5,6,7.Onemethod,forexample,usesamulti-parameterscaleincluding:inflammation, extentinvolvement,regeneration,cryptdamage,andpercentinvolvement8. Acknowledgements FundingprovidedbyNIH(1K08DK080221-01)andVanderbiltinstitutionaldevelopmentfunds. References 1. Okayasu,I.etal.,Anovelmethodintheinductionofreliableexperimentalacuteandchroniculcerativecolitisinmice.Gastroenterology98 (3),694-702(1990). 2. Martinez,J.A.etal.,DeletionofMtgr1sensitizesthecolonicepitheliumtodextransodiumsulfate-inducedcolitis.Gastroenterology131(2), 579-588(2006). 3. Moolenbeek,C.&Ruitenberg,E.J.,The"Swissroll":asimpletechniqueforhistologicalstudiesoftherodentintestine.LabAnim15(1),57-59 (1981). 4. Mahler,M.etal.,Differentialsusceptibilityofinbredmousestrainstodextransulfatesodium-inducedcolitis.AmJPhysiol274(3Pt1), G544-551(1998). 5. Aharoni,R.etal.,Immunomodulatorytherapeuticeffectofglatirameracetateonseveralmurinemodelsofinflammatoryboweldisease.J PharmacolExpTher318(1),68-78(2006). 6. Hahm,K.B.etal.,Lossoftransforminggrowthfactorbetasignallingintheintestinecontributestotissueinjuryininflammatoryboweldisease. Gut49(2),190-198(2001). 7. Green,B.T.etal.,AdrenergicmodulationofEscherichiacoliO157:H7adherencetothecolonicmucosa.AmJPhysiolGastrointestLiver Physiol287(6),G1238-1246(2004). 8. Dieleman,L.A.etal.,Chronicexperimentalcolitisinducedbydextransulphatesodium(DSS)ischaracterizedbyTh1andTh2cytokines.Clin ExpImmunol114(3),385-391(1998). Copyright©2010JournalofVisualizedExperiments Page3of3