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Pathology and Autopsy of a mouse

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Alwaysfill-inaformforeachmouseyoufind/submitandmakeasmanynotesaspossIBLeabouthisbehaviour,healthstatusorgrossfeatures.Alwaysweighthemouse.

ifmouseisfounddead:

Almostinvariably,amousefounddeadinthecageisunsuitableformicroscopicandevengrossanalysisbecause:
- Timefromdeathisunpredictable
- Thetemperatureoftheroomfavorrapidautolysisofthetissue,andthendehydration
- Othermicechowthecadaver
Asickmouseshouldbeeuthanizedassoonaspossible,savingthetissueforanalysis.Ifthemouseisdeadisbecausehasnotbeencarefullyinspectedbefore.
Ifyoureallyliketotry,putoniceandfixinformalin(seebelowformalinfixation)immediately

ifmouseisanewbornor<1wkofage:

euthanizebyCO₂inhalation.AskyourInstituteofComparativeMedicine(ICM)forproceduresandfacilities.NewbornmicearelesssensibletoCO₂narcosis.
Carefullyverifythatyourmouseisdead,astaughtbyyourICMduringthecompulsoryintroductorycourseforresearchershandlinganimals.
gentlyopenuptheaBDomenlongitudinallyandthethoraxalongthemid-sternumuptothelower2/3.Makeaverysuperficialincisionintheskullwithablade.Makesurethattheformalingetsintothethoraxandtheabdomen,fixinformalinfor24hat+4°C.(seebelowformalinfixation)

Ifmouseisaliveormoribundandaboveoneweekofage:

euthanizebyCO₂inhalation.AskyourInstituteofComparativeMedicine(ICM)forproceduresandfacilities.
Carefullyverifythatyourmouseisdead,astaughtbyyourICMduringthecompulsoryintroductorycourseforresearchershandlinganimals.
getblood(easiestfromthecuttipofthetailimmediatelyafterdeath:seealsoFlowprotocol)andmake2ormoresmears(seebelowstorageofsmearsandslides).Bloodcanbetakenfromheart(thisproceduremustbedoneonmicetobesacrifiedimmediatelyafter),retroorbitalveinplexi(withacapillarytube)orinferiorvenacava.Use22Gneedles.Weusetoexanguinateeuthanizedmice(seeabove)bycardiacpunctureinorderto
- obtainsserumforimmunoglobulinanalysis
- betterdissectthemouseandreduceartifactualstainingofRBCbyimmunoperoxidase.
- NOTE:intracardiacpunturemayspillbloodintheperitoneum:ifyouneedtoanalyzeperitonealcells,drawbloodwithalateralapproachinthethorax.

collectperitonealcellsifindicated.

fixtheeuthanizedmouseonaflatsurface(thickstyrofoamboxcovers)with22Gand24Gneedlesinsertedobliquely,spraywith70%ethanolinordertoavoidfurallovertheplaces,cuttheskinfromlegtolegandfromperineumtochininsixeasysteps(bluelinesbelow).Pullapartthefurandexposeperitoneumandribs.

clicktoenlarge

Inordergetconsistentresults,youhavetofollowaroutine,soyoudon"tmisstosampleeveryorganyouneed.Youwillprepareinadvancethreetubes:a5mlforlymphoidtissue,a15mlforsolidorgansanda5mlforthebonemarrow,allroundbottomandallconatiningfixative.CapthetubesandLABELCAREFULLY.

Withethemousefixedandopened,startyourroutine(typicalfortheaveragemouse):

Getinguinal,axillaryandsuBMAndibularlymphnodes.
Opentheperitoneum,findthespleenunderthestomach,gentlydetachitfromtheomentum.PlaceincoldPBSormedium.
Findtheappendixandexcisethetip.
Pullapartthelargeandsmallintestineexposingthemesentery.
GrabthesigmaandvisualizethemesentericLNrunnungtowardtherootofthemesentery.Exciseit,togetherwithasmallpieceofpancreas.
CarefullyinspectthewholegutandexciseasmanyPeters"sPatchesaspossible,withsomeintestineattached.
Grabtheliveracrossthefalciformligamentunderthediaphragm,togetherwiththeabdominalaortaandremovetheliverinasingleblockatonce.Weighttheliver.
TrimapieceofliverasbigasthespleenandputincoldPBS.
Removeonekidney.
Openthethoraxalongthemidlineuptothesternum,beinggentleontop,pryopen,visualizethethymus.Grabitrightinthemiddle(isthmus),gentlypullthethymus,detachingitfromthemediastinum.PutincoldPBS.
Removeheart,lungandmediastinumen-block.
Removetheabdominalorgansandcutoutthespine.
Weighthespleen,cutinhalf,maketouch-prepson3ormorelabelledslides,put1/2informalin,and1/2inalabelledcryotube(tobefrozen).Ifthespleenisenlarged,make5ormoretouch-prepslides.
Getthethymus,maketouchprepson3ormoreslides,put1/2informalin,and1/2inacryotube(tobefrozen).
Freezethetubeswithspleenandthymusinliquidnitrogendirectly.Storeat-80°C
Fixspleen,thymusandliver(inthesametube)informalin(seebelowformalinfixation).
Fixthewholemouseinformalin(seebelowformalinfixation)

ProcedurestosamplelymphoidorgansaredetailedinthemainmenuofMousePathology,inthepopUpmenuunder"RemovalofLymphoidOrgans".

Formalinfixation

10%bufferedformalinisthefixativeofchoice.Tissuescanstayinformalinforeverwithgoodmorphologicpreservation.However,prolongedfixationaffectsimmunodetection.Thefollowingprotocolisintendedforoptimalimmunoreactivitypreservation.
Fixsmallpiecesoftissuesforminimum6h(atRT),maximum48h(at+4°C).OPTIMAL8-10h.
Thenwashonce(2-24h)withPBSandtransferto70%ethanolforlessthanaweekstorage.Biggerspecimens(e.g.wholemouse)need24to48hfixation,onedayinPBSandthentwochanges(6-12hrsapart)of70%ethanolforshortandlongtermstorage.
StoreatRTonceinethanol.
Forlongtermstorage(>1week)ofsmallpieces,soakthewashedspecimenin30%sucrosein0.1MPBS,freezeat-80°Concethepiecesreachthebottomofthetube.A1mlcryovialisenough.
Thisfreezingprotocolcanbeusedforawholemouse,providedthatthemouseisopenedupbeforefixation(includingtheskull),fixedfor24hrinformalin,washedovernightwithonePBSchange,soakedinalargevolumeofsucroseandfrozenina50mltubewithsucrose.

Storageofsmearsandslides

SmearsandtouchprepscanbestoredatRTinadryplaceforalimitedamountoftime.Storageinarefrigeratororfreezerwithoutproperhandling(seebelow)isdeleterious.
Within48hfromthepreparationtime,wraptheslidesinSaranwrap,incouples,afterhavinginsertedathreadbetweenthefacingslidesurfaces.
Sealthepacketbyfolding3to4timestheplasticfoilaroundtheslidesandbendingtheedgesunderthepacket.Labelandstoreat-20°.

Tissueembeddinginparaffin

Collectthefollowingtissuesinatubecontainingbufferedformalin,orthefixativeofyourchoice:spleen,thymus,lymphnodes(mesenteric,inguinal,axillary,submandibular,paraspinal),Peyer"spatcheswithsomeintestineattachedonbothsides(asmanyasyoucan),appendix,asmallpieceofliver.
VerysmallpiecescanbetaggedbeforefixationwithIndiaInk,thedeepblackproteinaceusink,usedinSurgicalPathologyorwithMerthiolate.Donotuseothertypeofink(e.g.stamppadetc.)
Inasecondtubecollectasingleblockofheart,lung,tracheaandmediastinum.Youwillgrosstheseorgansafterfixation.Putalsoakidneywithadrenalattachedandanyorganthatmaybeofyourhematopathologicinterest.
Collectthespineinathirdtube.Youwillprocessthisseparatelybecauseoftheneedofdecalcification.
Afterfixation(ideally8hours),collectallthelymphoidorgans(youmayliketoincludemediastinalnodesalso:seebelow)inacassette,wrappedinKimwipepaper(squaresof1/8ofaregularKimwipe)soyoudon"tlosesmallpieces,andplaceinanembeddingprocessor.
Detacheachlunglobefromtheblock,detachthetracheatogetherwithmediastinalnodes,thyroidetc.,slicetheheartandkidneylengthwiseonafrontalplane.Embedtogetherinacassette.
Seeexampleoftheresultoftheprocedureherebelow

clickonthethumbnailtoenlarge Multitissue block