TraumaticbraininjuryTBIisacommoncauseof morbidityandmortalityacrossboththecivilianand militarypopulationswithareportedworldwideannual incidenceofsometenmillioncases1Indeedwithinthe USaloneTBIa ID: 198112
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Introduction Traumaticbraininjury(TBI)isacommoncauseof morbidityandmortalityacrossboththecivilianand militarypopulations,withareportedworldwideannual incidenceofsometenmillioncases[1].Indeed,withinthe USalone,TBIaccountsforsome1.7millionemergency departmentvisits-anumberthatlikelyunderestimatesits trueincidence[2]-andiscreditedwithsome30%ofall injury-relateddeaths[3].Inessence,TBIiselicited followingtheunexpectedapplicationofanexternal forcetothehead.Patientswhosurvivesuchinjury oftenpresentwithpersistentlong-termdisabilities thatrequirerehabilitation-acostly52billiondollars annualexpenseintheUSalone[4-6].Theseverityof ensuingdisabilitiesvariesandoftenmaybeassociated withtheseverityoftheinjuryitself[7].MildTBI (mTBI)accountsforsome80%to90%ofcases,and arisingcommondisabilitiesincludesensory-motor problems,learningandmemorydeficits,anxiety,and depression[8,9].Ofsignificantadditionalconcern, mTBImaypredisposelong-termsurvivorstoage-related neurodegenerativedisordersbyprovidingariskfactorfor thedevelopmentofAlzheimer sdisease,Parkinson s disease,andpost-traumaticdementia[10-14],withthe olderpeoplebeingmostvulnerable[15,16].Despite significantongoingresearchandadvancementsinour understandingofthemolecularandcellularchangesthat occurafterTBI,noeffectivepharmacologicaltreatmentis currentlyavailable[17,18]. mTBI-associatedbraindamagecanbesubdividedinto twophases:aninitialprimaryphasethatisimmediateand resultsfromthemechanicalforce(s)appliedtotheskull andbrainatthetimeofimpact,potentiallyinducing shearingandcompressionofneuronalandvasculartissue thatresultsinbraincontusion,axonalinjury,bloodvessel rupture,andhemorrhage.Thisisfollowedbyanextended secondphasethatinvolvescascadesofbiological processesinitiatedatthetimeofinjurythatmaypersist oversubsequentdays,weeks,andpossiblymonths,conse- quenttoischemia,neuroinflammation,glutamatetoxicity, alteredblood-brainbarrierpermeability,oxidativestress, astrocytereactivity,cellulardysfunction,andapoptosis [19-22].Assecondarybraininjurymaybereversible,in ordertodevelopaneffectivetreatment,itisimperativeto understandthebiologicalcascadesthatdrivethedelayed secondaryphasethatoccursfollowingTBI[23-25]. Itiswidelyrecognizedthatinflammatorycytokines, chemokines,andgrowthfactorsplaysignificantrolesin thepathophysiologyofTBI.Albeitthatinitiationofan inflammatoryresponsecanbeessentialtopromote neuroreparativemechanismsi nresponsetoaphysiological insult[26-28],ifthisisexcess iveorunregulated,itcanaug- mentneuronaldysfunctionanddegenerationbyinducinga self-propagatingpathological cascadeofneuroinflammation [29-31].ShortlyfollowingTBI,substantialsynthesisandre- leaseofproinflammatorycyt okinesoccurfromastrocytes andmicroglia,particularlytumornecrosisfactor- (TNF- ) withmRNAandproteinlevelsbecomingacutelyelevated withinaslittleas17minafterinjuryseeninpost-mortem brainsfrompatientswhodiedshortlyafterTBI[32].A parallelrapidsequencehasbeendescribedinrodentTBI animalmodelsinwhichaTNF- riseprecedesthe appearanceofensuingcytokines[33-35].Depending onitssignalingpathway,TNF- canexacerbatetrauma andoxidativestresswithinthebrainandcontributeto glutamatereleaseandblood-brainbarrierdysfunctionthat canleadtofurtherinfluxofinflammatoryfactorsfrom bloodtobrain[36]. InhibitingthegenerationofTNF- maythusreinforce itsroleinmTBIanddefineitsvalueasapotentialtreatment target,asitisconsideredamasterregulatoroftheinflam- matoryresponse.SuddenandsubstantialrisesinTNF- caninduceadiversearrayofcelldeathprocesses,including NF-kBactivation,apoptosis,andnecrosis[37].Inaddition, anincreaseinTNF- levelstriggerglutamatereleasefrom astrocytes,whichcanleadtogl utamateexcitotoxicity[38]. AlthoughtheelevationofTNF- levelsintheearlyhours postTBIcanbeharmful[39-41],cytokinebalancehasbeen reportedasessentialforlong-termrecoveryfrominjury [40-42].Inthiscurrentstudy,ratherthanutilizingaTNF- antibodyapproachtocaptu reandclearitbeforeitcan potentiallyreachitstarget,asiseffectivelyachievedinthe treatmentofrheumatoidarthritis,theexperimentaldrug 3,6 -dithiothalidomidewasemployedtoreduceTNF- synthesis[43]andtherebymaintainbutdramaticallylower itsphysiologicalreleasepattern.Inourpreviousstudies,we effectivelyused3,6 -dithiothalidomidetoameliorate cognitivedeficitfollowingmTBI[44].However,our previousworkdidnotdefinethetherapeuticwindow for3,6 -dithiothalidomide,theextendedtimecourse ofTNF- overproduction,andthehistochemicalchanges inneuronsandgliacorrelatedwithinjury.Weextendour previousfindinginthepresentstudy,correlatingthe potentialroleofmTBI-inducedTNF- releasewith neuronalloss,apoptosis,andastrocyteelevation,and definingawindowofopportunityforpotentialtreatment. Materialsandmethods Animals MaleICRmice(30to40gofweightand6to8weeks ofage)werebredandraisedwithinthevivariumof TelAvivUniversity,Israel,originallyderivedfrom breedingpairspurchasedfromHSDJerusalem,Israel. Theywerehousedfourtosixpercage,maintainedat aconstant22±1°C,had adlibitum accesstofood andwater,andkeptona12:12hlight/darkcycle. Lightingduringthelightphaseremainedconstant, andallexperimentalmanipulationswereundertaken Baratz etal.JournalofNeuroinflammation (2015) 12:45 Page2of14 duringthislightphaseofthecycle.Aminimumnumber ofanimalswereincludedintostudies,andalleffortswere madetominimizepotentialsuffering.Eachanimalwas usedforonlyasingleexperiment,andallexperimental proceduresandhousingconditionswereapprovedby theInstitutionalAnimalCareandUseCommitteeof TelAvivUniversity(M-10-006). Mildtraumaticbraininjury MiceweresubjectedtomTBIusingaweightdropdevice thathaspreviouslybeendescribed[44-46].Micewere anesthetizedwithisoflurane(Merck&Co.,Inc., WhitehouseStation,NJ,USA)andthenplacedunder thedevice.Theweightdropapparatuscomprisedofa cylindrical-shaped50-gpieceofmetalwitharounded sphericaltip,whichwasdroppedthroughavertical metalguidetube(diameter13mm×length80cm). Anesthetizedmicewerecare fullypositionedwiththeir headsupportedandimmobilizedbyaspongesothat therighttemporalsideofthehead,betweenthecornerof theeyeandtheear,wasdirectlybelowtheguidetube opening.Thespongeallowedanterior/posteriormotionof theheadwithoutrotationalmovementatthemomentof impactfollowingweightdrop[44-46].Shammicewere submittedtothesameprocedureasdescribedformTBI, butwithoutreleaseoftheweight. Drugadministration Synthesisof3,6 -dithiothalidomide(Merck&Co.,Inc., WhitehouseStation,NJ,USA)wasachievedbyapub- lishedsyntheticroute[43],andchemicalcharacterization confirmedthestructureofthefinalproductwitha chemicalpurityof99.8%.T heagentwaspreparedas asuspensionin1%carboxymethylcellulose(formulated inisotonicsaline;Merck&Co.,Inc.,WhitehouseStation, NJ,USA)immediatelypriortodailyuseineachstudyto provideafinaldoseof28mg/kg(0.1ml/10g)body weight.Either3,6 -dithiothalidomideorsimilarlyprepared vehiclewasadministeredbytheintraperitoneal(i.p.)route from1to18hpostinjuryorshamprocedure,depending onthemeasuresevaluated(whetherforELISA,immuno- histochemistry,orbehavioralstudies). TNF- analysisbyELISA ToverifytheoccurrenceofTNF- elevationinourmTBI modelanddefineitstimedependence,micewere subjectedtomTBIandbrainswereremovedatspecific timesthereafter(1to18h; n =4to5pertime).Theright cortexwasimmediatelyfrozeninliquidnitrogenand homogenizedwithappropriateproteaseinhibitors (HaltProteaseInhibitorCocktail;Sigma-Aldrich,St. Louis,MO,USA).Thesampleswerethenquantified forTNF- levelsbyELISAassay(BioLegend,SanDiego, CA,USA). Physiologicalparametersofwell-being Rectaltemperaturewasrecordedwithamouse thermometer.Baselinevalues(°C)wereevaluated30min before3,6 -dithiothalidomideadministrationandat1and 4hfollowingmTBIorshamprocedure. Anxiety-likebehaviorandmotoractivitywereevaluated byelevatedplusmaze.Themazewaselevated60cm abovethefloorlevelandcomprisedof4arms(30×5× 15cm)alongwhichmicecouldwalkthatformeda + shape[47].Twoconjoinedarmswereopen(withoutwalls) andtheothertwowereclosed(withwallsbutnoceiling). Onevaluationdays,micewereplacedatthecenterofthe plus-maze,facingoneoftheopenarmsandtheirtime spentwithintheopenarmswasrecordedovera5-min period.Themazewascleansedwith70%ethanol(ETOH; v / v )betweenanimals. Cognitivebehavioraltests Twobehavioralparadigmswereevaluated:Y-mazeand novelobjectrecognition(NOR). Y-mazetest SpatialmemorywasevaluatedbyY-maze,asinitially describedbyDelluandcolleagues[48],andisataskthat takesadvantageofapreferenceofrodentstoexplore novelratherthanfamiliarplaces.TheY-mazewas erectedfromblackPlexiglasandcomprisedofthree alikearms(30×8×15cmlength,setatanangleof120° fromoneanother).Evaluationcomprisedoftwotrials separatedbya2-mininterval(duringwhichthemouse wasreturnedtoitshomecage).Theinitial familiarization trialwasof5-mindurationwithonlytwoarmsopen (onetermedthe start armandtheotherthe old arm),withthethird( novel )armblockedbyadoor. Thesecondtrialwasof2-minduration,andallthree armswereopen.Thetimespentineachofthearms wasrecorded,anddiscriminationofspatialnovelty wasdeterminedasapreferenceindex[49]calculated as(timeinthenovel timeintheoldarm)/(timein thenovel+timeintheoldarm).Theapparatuswas cleansedbetweentrialswith70%ETOH( v / v ). NORtest Anobjectrecognitiontesttoevaluateshort-termrecogni- tionmemory[50]wasundertakenwithinanopenfieldthat comprisedablackPlexiglasarena(59×59cmsize) surroundedby20-cmblackwalls.Thetasktakesadvantage ofapredispositionforrodentstoexplorenewobjectsand includedthreetrialsof5-mindurationseparatedbya24-h interval.Ontheinitialdayofevaluation,micewereindi- viduallyplacedwithintheemptyarenaforhabituation. Thefollowingday,micewereplacedintothesamearena thathadtwoidenticalobjects,AandB,positioned40cm fromoneanotherand10cmfromthewalls.Onthethird Baratz etal.JournalofNeuroinflammation (2015) 12:45 Page3of14 day,micewereagainplacedintothearena;however, objectAremainedthesameastheprecedingdayand newobjectCreplacedpriorobjectB.Thearenaand objectswerethoroughlycleansed(70%ETOH v / v ) betweeneachtrial.Objectexploration(definedas rearingontheobjectorsniffingitatadistanceof lessthan2cmand/ornosetouchingit)wasrecorded anddiscriminationofrecognitionnoveltywasdeter- minedasapreferenceindex[49]:(timeexploringthe newobject timeexploringtheoldobject)/(totaltime exploringanobject).Micethatexploredobjectsfor lessthan10%ofthetotalavailabletimewereexcluded fromanalyses. Immunohistochemistry/immunofluorescencebrainslice studies AcohortofmTBIandshammicewereanesthetizedat 72hfollowingtheprocedurebyexcessketamine+xyla- zineadministrationandwereimmediatelyperfusedtrans- cardiallywithPBSfollowedby4%paraformaldehyde ((PFA)in0.1Mphosphatebuffer,pH7.4).Theirbrains wereremoved,fixedovernight(4%PFAin0.1Mphos- phatebuffer,pH7.4),andthenplacedin30%sucrosefor 48h.Coronalsections(30 m)werecutonacryostat, placedincryoprotectant,andstoredat 20°Cuntiluse. Thereafter,5sectionsofcortexand5ofhippocampus wereblockedbyincubationwith0.1%TritonX-100in phosphate-bufferedsaline(PBST)and10%normalhorse serumfor1hat25°C.Theprimaryantibodies,mouse anti-neuronalnuclei(NeuN;1:50,Millipore,Danvers, MA,USA,Cat#MAB3377),mouseanti-glialfibrillary acidicprotein(GFAP;1:10,000,Millipore,Cat#MAB3402), andrabbitanti-BH3-interactingdomaindeathagonist (BID;1:50,CellSignaling,Danvers,MA,USA,Cat#9942), werethendissolvedinPBSTand2%normalhorseserum andincubatedwiththesectionsfor48hat4°C.Following rinsinginPBST,sectionswereincubatedfor1hat25°C withDyLight 594-conjugatedAffinityPureDonkey Anti-rabbitIgGandDyLight 488-conjugatedAffinityPure DonkeyAnti-mouseIgG(1:300;JacksonLaboratories, BarHarbor,ME,USA).AfterrinsesinPBST,sections weremountedondrygelatin-coatedslidesandevalu- atedforfluorescencewithaZeissLSM510confocal microscopewith×20and×63lens(CarlZeiss,Jena, Germany).Foreachbrain,threetofivesectionswere takenandtheaveragenumbersofcellswithinthe hippocampusandthetemporalcortexwerecalculated withindefinedfieldsofeither140 2 or440 2 M.Evaluation ofimmunohistochemicalslidesforimmunofluorescence wasundertakeninablindedmanner,andtheomissionof primaryantibodieswasroutinelyundertakeninthe generationofnegativecontrolsections.Analyseswere performedbyImarisprogramforcolorquantification (BitplaneAG,Zurich,Switzerland). Dataanalyses Resultsthroughoutarepresentedasmean±SEMvalues andwereanalyzedbySPSS18software(GeniusSystems, PetahTikva,Israel).One-wayANOVAswereperformed tocomparebetweenallgroups, followedbyleastsignificant difference(LSD) posthoc tests.ANOVA-repeatedmeasures wereperformedtocomparerectaltemperatures. Results Evaluationofwell-being Basicwell-being, aconceptthatunderliesthecombined healthandwellnessofananimal[51],wasevaluated acrossallmicegroupsandcombinedsubjectivemeasures, suchasthegroomingandappearance,rightingskills, ambulation,andblinkingreflex,withobjectiveonesthat includedtheparametersofweight,bodytemperature, anxiety-likebehavior,andmotorskills. Subjectivelyandinaccordwithpriorstudies[45],mice subjectedtothistypeofmTBIwereindistinguishable fromthosesubjectedtotheshamprocedurewhen evaluatedat1or24hlater,irrespectiveof3,6 - dithiothalidomideorvehicleadministration.Rectal temperaturemeasurementswereusedtomonitor potentialcoretemperaturechangesinducedbyeither braininjuryor3,6 -dithiothalidomideadministration, andnosignificantdifference(NS)wasfoundeither betweenanimalgroups[ F (2,12)=0.084,NS]oracross measurementtimes(30minbeforeinjuryand1and4h post-mTBI/injection)[ F (2,12)=3.630,NS](datanotshown). Theelevatedplusmazewasusedtoexamineanxiety-like behaviorandmotoractivity.Nodifferenceswerefound betweenanygroupsinanxiety-likebehaviorat72hand 7dayspost-injury[ F (5,56)=0.791,NS][ F (5,47)=0.765, NS],respectively(datanotshown).Likewise,nodifferences wereevidentbetweenanygroupsinrelationtomotorskills evaluatedat72hand7dayspost-injury[ F (5,56)=1.13NS] [ F (5,47)=0.798,NS],respectively(datanotshown). Togethertheseresultindicatethatmicewerehealthyand thatneithermTBInor3,6 -dithiothalidomideimpacted theirwell-being. Time-dependentchangesinTNF- levelsinbraintissue AsillustratedinFigure1,micechallengedwithmTBI demonstratedatime-dependentriseinbrainprotein levelsofTNF- thatwereincreasedby2.5-fold, peakedat12hpostinjury,andreturnedtobaseline by18h[ F (3,13)=30.529, p 0.0001].LSD posthoc analysesconfirmedthatthe12-hmTBIgroupwassig- nificantlydifferentfromallothergroups( p 0.0001).Levels wereelevatedto132.9pg/mlat12hversusabaselinevalue of53.4pg/ml.InanimalssubjectedtomTBIandadminis- tered3,6 -dithiothalidomide1hpostinjury,theelevated TNF- 12hpostinjuryresponsewasameliorated. Specifically,micetreatedwith3,6 -dithiothalidomidepost Baratz etal.JournalofNeuroinflammation (2015) 12:45 Page4of14 injuryhadsimilarbrainTNF- levelsasthesham group,67.0and53.4pg/ml,respectively, F (4,17)=14.579, p 0.0001,Figure1B.LSD posthoc analysesconfirmed thatthemTBI12-hgroupwassignificantlydifferentfrom allothergroups( p 0.0001). mTBI-andtreatment-inducedchangesincognitive function WhenevaluatedbyY-mazeat7dayspostprocedure, vehicle-treatedmTBI-challengedmicedemonstrateda significantimpairmentinspatialmemory,ascompared toshamcontrolanimals.ThismTBI-induceddeficitwas amelioratedbyasingledoseof3,6 -dithiothalidomide administeredeither1or12hpostinjury.However,when 3,6 -dithiothalidomideadministrationwaswithhelduntil 18h,micedisplayedimpairmentand,togetherwiththe mTBIvehiclegroup,theirpreferenceindexwassignifi- cantlyreducedcompar edtoshamcontrols[ F (4,57)=6.462, p 0.01](Figure2A).LSD posthoc analysesconfirmed thatthemTBI+vehicleandthemTBI+18h3,6 - dithiothalidomidegroupsweresignificantlydifferent fromallothergroups( p 0.05). AsillustratedinFigure2B,thespatialdeficitevident invehicle-treatedmTBImiceintheY-mazewasalsoseen withtheNORparadigm.Heretoo,theadministrationofa singledoseof3,6 -dithiothalidomidetomTBImice1or 12hfollowinginjuryfullymitigatedthedeficit,butdelaying administrationto18hpostinjurydidnot.Specifically,the mTBIvehicleandmTBI+18h3,6 -dithiothalidomide groupsdisplayedasignificantlyreducedindexpreference versusshamcontrols[ F (4,57)=8.975, p 0.001].LSD post hoc analysesestablishedthatthemTBI+vehicleandthe Figure1 mTBIinducesatime-dependentriseinbrainTNF- levels. Right(ipsilateraltomTBI)cerebralcortexproteinextractswereprepared fromshamormTBImiceattheindicatedtimepointspostinjury. (A) Time-dependentbrainlevelsofTNF- atbaseline(sham)andpostinjury.At 12hpostmTBI,TNF- levelspeaked(132.8vs.53.4(sham)pg/ml, p 0.0001).By18hpostinjury,TNF- levelsreturnedtobaseline(50.5pg/ml). (B) Treatmentwith3,6 -dithiothalidomide(3,6-DT)at1haftermTBIpreventedtheTNF- elevationevidentat12hpostmTBI(3,6 -DT+mTBI 67.1pg/mlvs.mTBI132.8pg/ml, p 0.0001).Inboth (A) and (B) ,****wassignificantlydifferentfromallothergroups( p 0.0001). Baratz etal.JournalofNeuroinflammation (2015) 12:45 Page5of14 mTBI+18h3,6 -dithiothalidomidegroupsweresignifi- cantlydifferentfromallothergroups( p .05). TogethertheseresultsextendtheworkofBaratzand colleagues[44]anddefineatherapeuticwindowofupto 12hpostmTBItomitigatecognitivedeficitsbylowering TNF- generation,aswellasdocumentingthetimecourse ofTNF- elevation. Immunofluorescence ToevaluatetheimpactofmTBIatthecellularlevel, particularlyinrelationtothedescribedameliorationof cognitivedeficitsimpartedbyloweringTNF- generation, immunohistochemicalanalys eswereundertakenat72h postinjury.Thesefocusedont wokeybrainareasipsilateral toinjury:thecerebralcortex,astheareaclosesttoimpact, andthedentategyrus,aregionofthehippocampal formationconsideredtocontributetotheformation ofnewepisodicmemory[52,53],thespontaneousex- plorationofnovelenvironments,andothermnemonic functions[53,54]. IllustratedinFigures3Aand4Aarebrainregions (cerebralcortexanddentategyrus,respectively)displaying immunofluorescenceassociatedwith(i)NeuN,aneuronal nuclearproteinthatiswidelyusedasamarkerofadult neurons,andwith(ii)BID,aproapoptoticBcl-2protein. QuantificationofNeuNstainingrevealedaneuronalloss inboththecortex[ F (3,13)=7.198, p .005,Figure3B] anddentategyrus[ F (3,15)=5.641, p 0.05,Figure4B]. Posthoc analysesrevealedthatthemTBIalonegroupwas differentfromallothergroups( p 0.05)inbothbrain regionsandwasreducedby42.5%and22.3%versus shamvaluesincortexanddentategyrus,respectively. Correlatedwiththiswasanelevationinapoptoticcell number,asrevealedfromBIDstaininginthecortex [ F (3,13)=23.067, p 0.0001,Figure3C]andindentate gyrus[ F (3,13)=6.301, p 0.05,Figure4C].Likewise, post hoc analysesdemonstratedthatthemTBIgroupwas differentfromallothergroups( p 0.0001, p .05, respectively;and2.76-and1.91-foldcomparedtotheir respectiveshamvalues).Inadditionandillustratedin Figures5Aand6A,mTBI-challengedmicehadanelevation inastrocytenumber(3.37-and1.39-fold,respect- ively),asrevealedbyGFAPstaining,withinthecortex [ F (3,13)=37.641, p 0.0001,Figure5B]anddentate Figure2 mTBIinducesimpairmentsinperformanceinbothaY-mazeandnovelobjectrecognition(NOR)preferenceindexparadigmsthat areamelioratedby3,6 -dithiothalidomidewhenadministeredupto12butnot18hpostinjury.(A) Performanceofmicewasquantitatively assessedinaY-mazeand (B) inaNORparadigmat7daysfollowingmTBIasapreferenceindexthatwascalculatedas(timeassociatedwiththe novel timewiththeoldarmorobject)/(timewiththenovel+timewiththeoldarmorobject).Valuesaremean±SEMvalues;aone-wayANOVA indicatesthatmTBIanimalshadadeficitinspatial(Y-maze)andvisual(NOR)memoryperformancecomparedwithalltheothergroups(* p .05)with theexceptionofanimalsdosedwith3,6 -dithiothalidomideat18hpostinjury.Nodifferences werefoundbetweenanyoftheothergroups(control (sham)1and12h3,6 -dithiothalidomidedosing),suggestingcompleteameliorationby3,6 -dithiothalidomidewhenadministeredwithin12hofinjury. Baratz etal.JournalofNeuroinflammation (2015) 12:45 Page6of14 gyrus[ F (3,13)=13.284, p 0.001,Figure6B].Theadminis- trationof3,6 -dithiothalidomide1hpostinjuryamelio- ratedallmTBI-inducedchangesinneuron,BID,and astrocytenumberas,notably,nodifferenceswerefound betweenthemTBI+3,6 -dithiothalidomideandthesham groups.Finally,nochangeswereevidentbetweenany groups(sham,mTBI,andmTBI+drug)inthetotalcell numbers,asrevealedfromDAPIstaining,withinthe cortexanddentategyrus[ F (3,15)=1.009,NS,Figure5C; F (3,15)=2.251,NS,Figure6C]. Inconclusion,theearlyadministration(1hpostinjury) ofasingledoseoftheTNF- synthesisinhibitor3,6 - dithiothalidomideinhibitedcellularchangesinduced bymTBIintwokeybrainregionsevaluated,cerebral cortexanddentategyrus. Discussion TNF- hasbeenimplicatedinthepathogenesisofawide numberofneurologicaldisordersthatdevelopbothacutely, asinTBIandstroke,andchronically,asinAlzheimer s diseaseandParkinson sdisease[29-36,40-42,55,56].The currentstudyconfirmstherap idgenerationandreleaseof TNF- inamouseclosedhead50gweightdrop mTBImodel,emulatingaconcussiveheadinjuryin humans,whichledtoneuronallossandspecificcog- nitivedeficits.TheinhibitionofTNF- synthesis blockedthemTBI-inducedriseinbrainTNF- and protectedagainstneuronallossandcognitivedeficitswith atherapeuticwindowof12h.Theseresultsunderlinea roleforTNF- asakeyregulatorofcascadesleadingto neuronallossandcognitiveimpairmentinmTBIand highlightsTNF- asanamenabledrugtargetforfuture mTBItreatment. Inlightof(i)thehighincidenceofmTBI(approximately 600per100,000people);(ii)theincreasedriskofdementia resultingfrommTBI,particularlyintheolderpeople[15]; (iii)theupregulationofpathwaysleadingtochronic neurodegenerativedisordersinducedbymTBI[12,20,57,58]; (iv)thelong-termcare,suffering,andeconomicdebt associatedwithmTBIpatients[59];and(v)thelack ofanyavailabletherapeutic[60],itisimportantto understandthemechanismsthatunderlieheadinjury. Figure3 NeuronallossandapoptosisisinducedbymTBIincerebralcortexipsilateraltoinjuryandmitigatedby3,6 -dithiothalidomide. At72hpostinjury,cerebralcortexipsilateraltomTBIwasassessedforcellularchanges. (A) and (B) Adeclineinneuronalnumberindicativeof neuronalloss(NeuN-green)wasevidentpostmTBI( p 0.01).Treatmentwith3,6 -dithiothalidomideat1hpost-injurypreventedsuchachange. (A) and (C) AnelevationinBID(amarkerforapoptosis-red)wasevidentwithinmTBIbrains( p 0.001).Nochangesinapoptoticcelldeathwere foundinanimalsthatweretreatedwith3,6 -dithiothalidomide(ascomparedtoshamanimals).Within (A) (representativesectionswithinthecerebral cortex),thebarisequalto20 minlength. Baratz etal.JournalofNeuroinflammation (2015) 12:45 Page7of14 TNF- isawell-characterizedproteinthatregulates numerouscellularprocesses,includinginflammationand celldeathaswellascellulardifferentiationandsurvival,by bindingtoandactivationoftwocognatereceptors:TNF- receptor1(TNFR1)(p55)andTNFR2(p75)[29-31,61]. TNFR1isexpressedubiquitously,includingneurons, astrocytes,andmicrogliathroughoutthebrain.Withits intracellulardeathdomain,itcontributestoneuronal dysfunctionanddeathandprimarilyisactivatedbysoluble TNF- [62].TNFR2,ontheotherhand,isprincipally expressedonhematopoieticcellsbutalsoispresenton othercelltypes,includingneurons,hasbeenassociated withcellsurvival[61,63-65]andchieflyrespondsto membrane-boundTNF- [66,67].Theengagementof homotrimericTNF- (eithersolubleormembranebound) toeitherreceptorcanactivatethreemajorsignaling pathways:anapoptoticcascadeinitiatedviatheTNF- receptor-associateddeathdomain,anuclearfactorkappa B(NF B)signalingapro-survivalpathwayimplemented viaNF B-mediatedgenetranscriptionalactions,anda c-JunN-terminalkinase(JNK)cascadeinvolvedin cellulardifferentiationandproliferationthatisgenerally proapoptotic[38,68].Inlargepart,thecontrastingpro- survivalversusdeath-inducedactionsofTNF- plausibly relyonwhichTNF- receptorsubtypeisactivated,the targetcelltypesinvolvedandtheirexpressionratioof TNFR1/2andassociatedcouplingproteins,andthe temporalconcentrationsofav ailablesolubleandmembrane- boundTNF- [64].However,crosstalkbetweenthe differentsignalingpathwaysandthedegreeanddurationof neuroinflammationcombineindeterminingtheeventual physiologicalconsequencesofTNF- receptoractivation [69].ConsequenttothediverseactionsofTNF- andthe influenceofthebrainmicroenvironmentinwhichthey occur,itisnotalwaysclearunderwhichconditionsTNF- promotesbeneficialversusdeleteriousneuronaleffects. Thisexplainsthesometimescontradictoryliteratureinthe TNF- neurosciencefield[ 29-31,36,38,55,69]andits involvementincascadespromotingneuronaldysfunction andlossinbothacuteandlon g-termneurodegenerative disorders.Inthepresentstudy,nodifferenceswereevident acrosstheshamandmTBIgroupsinrelationtothebroad measureof wellbeing orintheevaluationofbody temperature,anxiety-relatedbehavior,andmotoractivity, Figure4 NeuronallossandapoptosisisinducedbymTBIinthedentategyrusipsilateraltoinjuryandmitigatedby3,6 -dithiothalidomide. At72hpostinjury,thedentategyrusofthehippocampusipsilateraltomTBIwasevaluatedforcellularchanges. (A) and (B) Neuronal loss(NeuN-green)wasfoundpostmTBI( p 0.05).Treatmentwiththe3,6 -dithiothalidomideat1hpost-injurypreventedthisloss. (A) and (C) AnincreaseinBID(amarkerforapoptosisinred)wasevidentinthemTBIbrains( p 0.01).Nochangeinapoptoticcelldeath wasapparentinanimalstreatedwith3,6 -dithiothalidomide(ascomparedtoshamanimals).Within (A) (representativesectionswithinthedentate gyrus),thebarisequalto100 minlength. Baratz etal.JournalofNeuroinflammation (2015) 12:45 Page8of14 whichisinaccordwithpreviousresultsinrodents[51]and humans[70].Althoughindis tinguishableacrossawide numberofmeasures,importantly,deficitsincognitive performancewereapparentinmTBImiceinaccordance withpaststudiesinmice[24,44-46]andhumans[71].In evaluatingpotentialmechanismsresponsibleforthese cognitivechanges,amTBI-triggeredinflammatorycascade mediatedbythegenerationofproinflammatorycytokines appearslikely[72].Inthisregard,theproinflammatory cytokineTNF- isconsideredessentialforbothinitiating andregulatinganinflammatoryresponsetotrauma,and earlytransientelevationsinbrainmRNAexpressionof TNF- aswellasrisesinIL-1 andIL-6havebeen describedinrodentclosedheadTBI,andassociatedadverse events[33,35,73].Inthecurrentstudy,atime-dependent elevationinbrainTNF- proteinlevelswasapparentin mTBI-challengedmicethatpeakedat12handdeclinedto baselineby18h.Inlinewiththis,elevatedbrainprotein levelsofTNF- ,IL-1 ,andIL-6havebeenreportedin rodentmTBImodelsaswellaswithinhumanCSFwithin hoursofinjury[74-78],astheyhaveinotherneurological disorders[79-81].Inhibitingsuchanelevationinbrain TNF- inthismodelallowedtheevaluationoftheroleof thistransientTNF- riseinneuronalcellloss,neuroinflam- mation,andcognitivedeficitsknowntoaccompanymTBI. TodefinetherelationshipbetweenthemTBI-induced elevationinTNF- andcognitiveimpairmentevident 7dayslater,3,6 -dithiothalidomidewasadministered1, 12,and18hfollowingmTBI,extendingourinitial concentration-dependentstudiesofthecompoundin thissamemTBImodel[44].Notably,mTBI-induced impairmentsinboththeY-mazeandNORparadigms wereblockedbyasingledrugdoseeitherat1or12h postinjury,thepeakofTNF- generationinbrain,but werenotmitigatedwhenadministrationwasdelayedto 18h,therebydefiningatreatmentwindowofopportunity. ToevaluatethebasisofthemTBI-inducedcognitive impairment,brainregionsipsilateraltothesideofinjury wereevaluatedat72h,asthistimecoincideswiththe substantialoccurrenceofmarkersofneuronalapoptosis [24,82].Assessmentofthecerebralcortex,theareaclosest tothesiteofimpact,anddentategyrusofthehippocampus wasperformed,asdysfunctionintheformerandlatter mightexplainthedeclineinperformanceinvisualmemory evaluatedbyNOR[83]andinspatiallearningasassessed bytheY-maze[48],respectively.Neuronalloss(NeuN),an Figure5 mTBIinducesanelevationinastrocytenumberinipsilateralcerebralcortexthatisinhibitedby3,6 -dithiothalidomide. At 72hpostinjury,cerebralcortexipsilateraltomTBIwasassessedforcellularchanges. (A) and (B) Astrocytenumber(GFAP-red)wasincreased postmTBI( p 0.001).Treatmentwith3,6 -dithiothalidomideat1hpost-injurypreventedthis. (A) and (C) Nodifferenceintotalnumberofcells wasevidentbetweengroups,asrevealedfromDAPI(blue)staining.Within (A) (representativesectionswithinthecerebralcortex),thebarisequal to100 minlength. Baratz etal.JournalofNeuroinflammation (2015) 12:45 Page9of14 increaseneuronalapoptosi s(BID),andanelevationin astrocytenumber(GFAP)wereevidentinbothbrain regions,whichisinaccordwithpriorstudiesinthis mTBImodeldescribingelevationsinapoptoticproteins (p53,c-Jun,andBcl-2)aswellasTUNEL-positiveand silverstain-impregnateddegeneratingneurons[82,84],as wellasotheranimalmodelsofbraininjury[20,60,85,86]. Importantly,earlypostinjurytreatmentwith3,6 - dithiothalidomidefullypreventedthesechanges.Inline withthis,thissameagenthasrecentlybeenreportedto ameliorateneuroinflammationandalleviatecognitive deficitsarisingfromintracerebraladministrationof LPSoramyloid- peptide[79,87,88].3,6 -Dithiothalidomide isalsoreportedtoattenuateinflammatorymarkers, Alzheimer sdiseasepathology,andbehavioraldeficits evidentinagedAlzheimertr ansgenicmice[79,80],as wellasmitigateneuroinflammationandapoptosiswithin thepenumbraoffocalischemicstrokeinmice[81]. Additionally,3,6 -dithiothalidomidehasrecentlybeen describedtolowerTNF- andcerebralaneurysmforma- tionandprogressiontoruptureinmice[89,90]. Takentogether,thesestudiessupportanimportant roleforTNF- inneuroinflammationandthemodulation ofneuronalfunctionandviabilityacrossabroadrangeof neurologicaldisorders.Consequenttotheavailabilityof bothbiologicalandsmallmolecularweightTNF- inhibitorsinpreclinicalandclinicalresearch,thereis growingevidencethatwhereasphysiologicalTNF- levelsarecriticalinnormalbrainphysiology[37,38,55], excessTNF- playsakeyroleinbraindysfunction [29-31,69].Inrelationtotheformer,amongahostof functionsinbrain,TNF- servesasagliotransmitterthat, whensecretedfromglialcellssurroundingsynapses,can regulatesynapticcommunicationbetweenneuronsaswell asneuronalnetworks[36-38].Withrespecttothelatter, TNF- reductionsachievedwiththeclinicalTNF- binding proteinetanercept,whenadmi nisteredi.p.followingfluid percussioninjury-induced TBI,attenuatedTBI-induced contusion,ischemia,andre sultingmotorandcognitive deficits[91].Asinourstudies,thisbrainTNF- lowering approachalsomitigatedTBI-i nducedelevationsin[91]. Albeitthattheseanimalstudiesutilizedafarhigher etanerceptdosethanachievableinhumans[91],inan open-labelanalysisof12TBIpatientsgivenperispinal etanerceptuptomorethan1 0yearsfollowinginjury, motorimpairmentandspasticitywerereportedsignificantly reduced[55],supportingbot hclinicalandtranslational relevance.Additionally,inratstudiesusingaTBIweight Figure6 mTBIinducesanelevationinastrocytenumberinipsilateraldentategyrusthatisinhibitedby3,6 -dithiothalidomide. At72hpost injury,dentategyrusipsilateralinjurywasassessedforcellularchanges. (A) and (B) Astrocytenumber(GFAP-red)waselevatedpost mTBI( p 0.001).Treatmentwith3,6 -dithiothalidomideat1hpos t-injuryinhi bitedthis. (A) and (C) Nodifferenceintotalnumberofcellswasapparent betweengroups,asevaluatedbyDAPI(blue)staining.Within (A) (representativesectionswithinthedentategyrus),thebarisequalto100 minlength. Baratz etal.JournalofNeuroinflammation (2015) 12:45 Page10of14 dropparadigmwithsomeparallelstoourstudiesinmice, immediatei.v.administrationofaTNF- bindingprotein orthecompetitivenonselectivephosphodiesteraseinhibitor, pentoxifylline,thatlowersTNF- atatranscriptionallevel, hasbeenreportedtomitigatemTBI-inducedbrainedema at24handneurologicaldysfunctionevaluatedupto 14days[75].Finally,inother animalmodelsthatinclude ischemicandspinalcordinjury,thalidomidehasbeen reportedtoeffectivelyreduceinflammationandimprove theinjuryoutcomewhenadministeredeitherbefore[92], immediatelyafter[93],orwithinanhourofinjury[94]in dosesthatvariedbetween20and300mg/kg. Thesingledoseof3,6 -dithiothalidomideusedinthe currentstudy(28mg/kginmouse)comparesfavorably withformerstudiesofthalidomide(20to300mg/kg) andequatestoadoseof150mgina65kghuman (2.3mg/kg),followingnormalizationofbodysurface areainaccordwithFDAguidelines[95].Priorcellular [43,79]andanimalstudies[80,81]indicatethat3,6 - dithiothalidomide(albeitadministeredsystemicallyby thei.p.route,asinthecurrentstudy)ismorepotentin reducingTNF- elevationsthanthalidomideandthatit entersthebrainbutdoesnotappeartobesoporific [44,79,80].Inlightofrecentstudiessuggestingthat thalidomideanaloguescanexpressmorepotentanti- inflammatoryactionwithlessneurotoxicitythanthe parentcompound[96,97],thedevelopmentofnewand well-toleratedsmallmolecularweightTNF- inhibitors thatcanbeadministeredorallymaybeofgreatclinical potential.Paststudiesevaluatinggeneticallyengineered micethateitherlackTNF- oritsreceptorshave suggesteda JekyllandHyde scenarioinwhichelevated TNF- isdetrimentalduringtheacutephaseafteraTBI incident,butapartoftheregenerativeprocessesduring thelaterchronicpost-injuryphase[42,98,99].More recentstudiesinwhichthetwoindividualreceptors, TNFR1(p55)andTNFR2(p75),havebeenseparately deletedsuggestthateachmayhaveadistincttime- dependentfunctioninTBI[40,41].TNFR1knockout micepossessedasmallercontusionvolumeandaclearly improvedneurobehavioralperformanceforupto4weeks followingacontrolledcorticalimpactTBI,ascompared withwild-typemice,whereasTNFR2knockoutmice demonstratedsignificantworseningpostinjury[42]. ThisimplicatesTNFR1involvementintheimmediate deleteriousactionsassociatedwithacuteTNF- release followinganinjuryandaninvolvementofTNFR2in latertissuerepair. Insummary,ourstudiessuggestthattheadministration ofaTNF- synthesisinhibitor,3,6 -dithiothalidomide, withintheinitial12-hwindowofamTBievent,maybe therapeuticallyvaluableatatimewhenelevatedTNF- interactswithTNFR1todrivethedevelopmentofneuro- inflammation,neuronaldysfunction,andapoptosis.But suchatherapeuticstrategyshouldbestbeacuteto allowlaterpotentiallybeneficialactionsofTNF- medi- atedviaTNFR2.Ourresults,togetherwithotherstudies [33,36,39-41,44,56,74-78,97],underscorethepotentialof TNF- asapotentialtherapeutictargetinTBIandother neurologicaldisorders. Conclusion ThisstudyimplicatesTNF- inthedelayedneuronalcell deathandgliosisthatoccurswithinthebrainfollowing mTBI,whichleadstocognitivedeficits.Itadditionally indicatesthatpharmacologicallylimitingtheelevationof TNF- within12hofthemTBIeventmarkedlyreduces suchsecondarydamageandleadstoimprovedcognitive outcomemeasures.Suchawindowprovidesanoppor- tunityfortranslationalstudiesinmTBIthatismore difficulttodefineforotherneurologicaldisorders[100]. BuildingonthegrowingliteratureontheroleofTNF- intheinitiationandperpetuationoftheneuroinflamma- tionthatcandrivetheprogressionofacuteandchronic neurologicaldisorders[29-31,36,55,56,68,69,101,102],the presentstudyunderscoresthevalueoftargetingofTNF- asatreatmentstrategyforTBIandthedevelopment ofnewandwell-toleratedoralsmallmolecularweight TNF- inhibitorsandrelatedapproachesasclinical treatmentoptions. Competinginterests ThesmallmoleculeTNF- synthesisinhibitor3,6 -dithiothalidomidewas originallysynthesizedwithinthelaboratoryofNHGandcolleagueswithin theIntramuralResearchProgramofNIA,NIH.WhereasNHGisaco-inventor oftheagent,neitherhenoranyoftheauthorsholdanyrightstotheagent. Allauthorsdeclarethattheyarewithoutanyfinancialandnon-financial competinginterests(political,personal,religious,ideological,academic, intellectual,commercial,oranyother)inrelationtothisarticle. Authors contributions RB,DT,VR,andWLundertook hands-on experimentalstudiesgenerating datausedwithinthemanuscript,preliminarydataonwhichstudieswere thenoptimized,andsyntheticchemistrytoprovidecompoundsusedwithin thestudies.RD,DT,JYW,VR,WL,BJH,NHG,andCGPanalyzed immunohistochemical,ELISA,behavioral,orchemicalcharacterizationdata essentialtothestudieswithinthemanuscript.CGP,BJH,andNHGconceived andplannedthestudies.RB,DT,JYW,BJH,CGP,andNHGpreparedthe manuscript.Allauthorshavereadthemanuscriptinfullandarein agreementwiththedataanditsinterpretation. Acknowledgements ThisstudywassupportedinpartbytheIntramuralResearchProgram, NationalInstituteonAging,NIH,Baltimore,USA;byTel-AvivUniversity, Tel-Aviv,Israel;andbyagrantfromtheMinistryofScienceandTechnology, Taiwan(MOST103-2321-B-038-002toJYWang).Noneoftheauthorshave anycompetinginterestsinthemanuscript. Authordetails 1 DepartmentofAnatomyandAnthropology,SacklerSchoolofMedicine, Tel-AvivUniversity,Tel-Aviv,Israel. 2 DrugDesignandDevelopmentSection, TranslationalGerontologyBranch,IntramuralResearchProgram,National InstituteonAging,NationalInstitutesofHealth,BRCRoom05C220,251 BayviewBlvd.,Baltimore,MD21224,USA. 3 GraduateInstituteofMedical Sciences,CollegeofMedicine,TaipeiMedicalUniversity,Taipei,Taiwan. 4 DepartmentofNeurosurgery,CaseWesternReserveUniversitySchoolof Medicine,Cleveland,OH,USA. 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Submit your next manuscript to BioMed Central and take full advantage of: Convenient online submission Thorough peer review No space constraints or color gure charges Immediate publication on acceptance Inclusion in PubMed, CAS, Scopus and Google Scholar Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Baratz etal.JournalofNeuroinflammation (2015) 12:45 Page14of14 RESEARCHOpenAccess Transientlyloweringtumornecrosisfactor- synthesisamelioratesneuronalcelllossand cognitiveimpairmentsinducedbyminimal traumaticbraininjuryinmice RenanaBaratz 1 ,DavidTweedie 2 ,Jia-YiWang 3 ,VarditRubovitch ,WeimingLuo 2 ,BarryJHoffer 4 , NigelHGreig 2* andChaimGPick 1 Abstract Background: Thetreatmentoftraumaticbraininjury(TBI)representsanunmetmedicalneed,asnoeffective pharmacologicaltreatmentcurrentlyexists.Thedevelopmen tofsuchatreatmentrequiresafundamentalunderstanding ofthepathophysiologicalmechanismsthatunderpinthesequelaeresultingfromTBI,particularlytheensuingneuronal celldeathandcognitiveimpairments.Tumornecrosisfactor-alpha(TNF- )isacytokinethatisamasterregulatorof systemicandneuroinflammatoryprocesses.TNF- levelsarereportedtobecomerapidlyelevatedpostTBI and,potentially,canleadtosecondaryneuronaldamage. Methods: ToelucidatetheroleofTNF- time-dependentTNF- levelsand(ii)markersofapoptosisandgliosiswithinthebrainandrelatedtheseto behavioralmeasuresof wellbeing andcognitioninamouseclosedhead50gweightdropmildTBI(mTBI)modelin thepresenceandabsenceofpost-treatmentwithanexperimentalTNF- synthesisinhibitor,3,6 -dithiothalidomide. Results: mTBIelevatedbrainTNF- levels,whichpeakedat12hpostinjuryandreturnedtobaselineby18h.Thiswas accompaniedbyaneuronallossandanincreaseinastrocytenumber(evaluatedbyneuronalnuclei(NeuN)andglial fibrillaryacidicprotein(GFAP)immunostaining),aswellasanelevationintheapoptoticdeathmarkerBH3-interacting domaindeathagonist(BID)at72h.Selectiveimpairmentsinm andpassiveavoidanceparadigms-withoutchangesinwellb eing,wereevidentat7daysafterinjury.Asinglesystemic treatmentwiththeTNF- synthesisinhibitor3,6 -dithiothalidomide1hpostinjurypreventedthemTBI-inducedTNF- elevationandfullyamelioratedtheneuronalloss(NeuN),e levationsinastrocytenumber(GFAP)andBID,andcognitive impairments.Cognitiveimpairmentsevidentat7daysafterinjurywerepreventedbytreatmentaslateas12h postmTBIbutwerenotreversedwhentreatmentwasdelayeduntil18h. Conclusions: TheseresultsimplicatethatTNF- inmTBIinducedsecondarybraindamageandindicatethat pharmacologicallylimitingthegenerationofTNF- postmTBImaymitigatesuchdamage,definingatime-dependent windowofupto12htoachievethisreversal. *Correspondence: greign@grc.nia.nih.gov Equalcontributors 2 DrugDesignandDevelopmentSection,TranslationalGerontologyBranch, IntramuralResearchProgram,NationalInstituteonAging,NationalInstitutes ofHealth,BRCRoom05C220,251BayviewBlvd.,Baltimore,MD21224,USA Fulllistofauthorinformationisavailableattheendofthearticle JOURNAL OF NEUROINFLAMMATION ©2015Baratzetal.;licenseeBioMedCentral.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/4.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycredited.TheCreativeCommonsPublicDomain Dedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle, unlessotherwisestated. Baratz etal.JournalofNeuroinflammation (2015) 12:45 DOI10.1186/s12974-015-0237-4