Murine Colitis Modeling using Dextran Sulfate Sodium (DSS).

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by  Whittem, Caitlyn G.

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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.
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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.
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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).
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