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Volume46,Issue10
October2005
Pages16031612

GenderDifferencesinFebrileSeizureinducedProliferationandSurvivalin
theRatDentateGyrus
EviM.P.Lemmens,TimLubbers,OlafE.M.G.Schijns,EmileA.M.Beuls,GovertHoogland

Firstpublished:
28September2005

Fullpublicationhistory

DOI:
10.1111/j.15281167.2005.00252.x
Citedby:
32articles

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AddresscorrespondenceandreprintrequeststoDr.E.M.P.LemmensatDepartmentofPsychiatryand
Neuropsychology,DivisionofCellularNeuroscience,MaastrichtUniversity,POBox616,6200MDMaastricht,
TheNetherlands.Email:evi.lemmens@np.unimaas.nl

Abstract
Summary:Purpose:Febrileseizuresarefeverassociatedearlylifeseizuresthatarethoughtplayarolein
thedevelopmentofepilepsy.Seizureinducedproliferationofdentategranulecellshasbeendemonstrated
inseveraladultanimalmodelsandisthoughttobeanintegralpartofepileptogenesis.Theaimofthe
presentstudywastoinvestigateproliferationandsurvivalofdentategyrus(DG)cellsbornafterearlylife
hyperthermia(HT)inducedseizuresinmaleandfemalerats.
Methods:Atpostnatalday(PN)10,maleandfemaleratswereexposedtoheatedairtoinduceseizures.
Littermateswereusedasnormothermiacontrols.Convulsivebehaviorwasobservedbytworesearchers.
FromPN11toPN16,ratswereinjectedwithbromodeoxyuridine(BrdU)tolabeldividingcells.Thenumber
ofBrdUimmunoreactivecellsintheDGwascountedatPN17andPN66.
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Results:AtPN17,maleaswellasfemaleHTratshadthesameamountofBrdUpositivecellscompared
withcontrols.AtPN66,significantlymoreBrdUpositivecellswereleftinHTfemales(53%)thanincontrols
(44%,percentageofBrdUpositivecellsatPN17),whereasnodifferencewasfoundbetweenHTmales
andmalecontrols.ThenetresultofproliferationandsurvivalatPN66wasthatfemaleHTratshadthe
samenumberofBrdUimmunoreactivecellsascontrols,whereasmaleHTratshad25%moreBrdU
immunoreactivecellsthandidcontrols(p<0.05).
Conclusions:Earlylifeseizurescauseasexuallydimorphiccytogenicresponsethatresultsinanincreased
populationofnewbornDGcellsinyoungadultmales,whileleavingthatofyoungadultfemalesunaltered.
Febrileseizures(FSs)arethemostcommonseizuretypeinyoungchildren.Theyoccurin34%ofchildren
betweentheageof3monthsand5yearsandareassociatedwithafebrileillness.Anumberofgenetic
defectshavebeenassociatedwithahigherincidenceofFSs,thesocalledchannelopathies,mutationsin
genescodingforionchannels,representingthelargestgroup(13).Environmentalfactorshavebeenfound
toincreasetheriskofdevelopingFSs.Forinstance,inapopulationbasedstudy,Liebermanetal.(4)found
almostafourfoldincreaseintheriskofFSs,whenmaternalfeverof>100.4F(or38C)wasmeasuredduring
labor.
AlthoughanumberoffactorscontributingtoFSshavebeenidentified,stillmuchdebateexistsonthe
consequences.SomestudiessuggestthatFSsarebenign,causingnodamagetothechild(57).However,
thisideaisbasedonstudiesthatarelimitedbytheuseofnoninvasivetechniques,thefollowuptime,and
oftendidnotclassifythetypesofepilepsy.Inaprospectivestudy,Tarkkaetal.(8)failedtoshowstructuralor
behavioraldifferencesinchildrenthatexperiencedFSswhencomparingthemwithagematched,seizurefree
controls.Incontrast,Cendesetal.(9)founda10foldincreasedincidenceofFSs(40%)amongpatientswith
mesialtemporalsclerosisassociatedtemporallobeepilepsy(TLE).Similarly,Camfieldetal.(10)foundan
8.8foldincreasedriskfordevelopingintractableepilepsyafterprolongedFSs.ThesuggestionthatFSsare
causallyrelatedtothedevelopmentofTLEisinlinewiththelongstandingnotionthatseizuresbeget
seizures.AnumberofanimalmodelsforFSshavebeendevelopedtostudywhetherFSsinduce
epileptogenesis.MostanimalmodelsforFSsaimtomimicfeverbyincreasingthebodytemperature,which
canbeachievedbyexposingtheanimalstoaheatsourcesuchasamicrowave(11,12),infraredirradiation(
13),orwarmwater(14).Inthepresentstudy,weusedtheimmatureratmodeldevelopedbyBarametal.(15
).Themainadvantageofthismodelisthatitusesratsduringabraindevelopmentagecomparabletothatof
ahumanchildwhenitismostsusceptibletoseizures.Furthermore,themortalityandmorbidityislow,which
makesthismodelhighlysuitableforlongtermfollowupstudies.
NoneoftheanimalstudiesthatusedthemodelofBarametal.(15)couldconcludethathyperthermia(HT)
inducedseizurescauseTLE.TheywereunabletofindhistologicevidenceforthepresenceofTLE.For
instance,hippocampalcelldeath,ahistopathologichallmarkofTLE,wasnotseenintheseanimals.Still,
acutecelldamage,evidentbyargyrophilicneurons,waspresent,butnochangewasseeninthetotalnumber
ofneurons(16).Chenetal.(17)showedthatHTinducedseizuresrenderthehippocampalnetwork
hyperexcitablebyraisingthedepolarizationcurrentofneurons,therebyloweringtheseizurethreshold.
Previously,daytimebehavioralandelectrophysiologicmonitoringfailedtodemonstratespontaneousseizures
inadultratsafterearlylifeHTinducedseizures.However,theseanimalshadalowerthresholdforseizures
inducedbyadministrationofasubconvulsivedoseofkainicacid(18).Recently,Barametal.(19)showedthat
HTinducedseizuresareabletoelicitspontaneousnocturnalseizures.Anotherimportantfindingisincreased
mossyfibersproutingafterHTinducedseizures,whichmightalsocontributetoincreasedexcitabilityof
hippocampalneurons(20).
Inthepastfewyears,alteredneurogenesishasbeenproposedasamechanismbywhichseizuresmodulate
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thehippocampalnetwork.Severalstudiesshowthatpilocarpine(2123)orkainateinducedseizures(24,25
),perforantpathwaystimulation(25),oramygdalakindling(26,27)causesanincreasedproliferationof
neuronsinadultrats.Mostofthenewborncellsdifferentiateintoneuronsinthedentategranulecelllayerand
formconnectionswiththeCA3areaofthehippocampusandotherdentategranulecells(21).
Genderdifferencesincellproliferation,differentiation,andsurvival(28,29)havebeenattributedtotheroleof
hormonesinneurogenesis(30).Evidencealsoexistsofgenderdifferencesinseizuresusceptibility.Malesare
foundtobemoresusceptibletotemporallobelikeseizuresbecauseofhighlevelsoftestosterone(31).After
administrationoftheconvulsant N methylDaspartate,maleratsshowmoresevereseizureactivitythan
femalerats(32).Hence,theaimofthepresentstudywastoinvestigateproliferationofDGcellsafterearlylife
HTinducedseizuresinmaleandfemalerats.

MATERIALSANDMETHODS
Animals
SpragueDawleyrats(Harlan,TheNetherlands)werehousedunderstandardconditions(212Cambient
temperature,a12hlight/darkschedule,backgroundnoiseprovidedbyaradio,andfoodandwaterad
libitum).Intotal,21maleand24femaleratsweredividedintothefollowingfourgroups:(a)shortterm
normothermia(STNT),killedatpostnatalday(PN)17(n=5males,n=5females)(b)shortterm
hyperthermia(STHTn=6males,n=6females)(c)longtermnormothermia(LTNT),killedatPN66(n=4
males,n=6females)and(d)longtermhyperthermia(LTHTn=6males,n=7females).Ratswere
weigheddailyfromPN1to17,andonceatPN66.AllexperimentswereapprovedbytheAnimalExperiments
Committee(DEC)oftheUniversityofMaastricht,TheNetherlands.

Hyperthermiaparadigm
HTwasinducedasdescribedpreviouslybyBarametal.(15),withminormodifications.Inbrief,PN10rat
pupswereinjectedsubcutaneouslywith0.2ml0.9%salinetopreventdehydrationandplacedinaPerspex
cylinderwithadiameterof10cm(onerat/cylinder).Anadjustablestreamofheatedair(5052C)wasblown
intothecylinderbyacommercialhairdryer,placedat50cmabovetherats,toraisethebodytemperatureof
theratpupsfromabasalvalueof35Cto4142.5C.Coretemperaturesweremeasuredbeforeandevery
2.5minduringtheHTtreatment,witharectalprobe(KtypethermocouplebeadprobeconnectedtoaST
9612digitalthermometerVellemanComponents,Gavere,Belgium).Whenthecoretemperatureoftherats
reached39.5C,usuallyafter510min,thetemperatureandvolumeoftheairwereadjustedsothatacore
temperatureof4142.5Cwasmaintainedfor30min.Ifthecoretemperatureexceeded42.5C,theratwas
removedfromthecylinderuntilthetemperaturedroppedto4042.5C.Theoccurrenceofseizureswas
monitoredbehaviorallybytwoobservers.Thebehavioralseizureswerestereotypedandpreviouslyshownto
correlatewithEEGdischargesinthehippocampus(15,18).Behaviorally,theseizuresconsistedofarrestof
heatinducedhyperkinesis,followedbybodyflexion,andoccasionallyfollowedbycloniccontractionsofthe
limbs.Themomenttheratsshowedbodyflexionwastakenasthestartofaseizure,whereastheendofthe
seizurewasmarkedeitherbytheendofthe30minhyperthermiatreatmentorbyregainingnormalbehavior.
Thesetimevalueswereusedtoestimatetheaverageseizureduration.Immediatelyafterthe30min
treatment,theratswereplacedinwater(roomtemperature,RT)withtheirheadsabovethesurfacetoregain
theirnormalbodytemperature,afterwhichtheywerereturnedtotheirmother.
Normothermia(NT)controlsfromthesamelitterastheHTratswereexposedtothesameconditions,except
thatthestreamofairwasusedtomaintainthecoretemperatureoftherats.AtPN22,allpupswereweaned
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andrandomlyhousedtwotothreepercage.Aschematicoverviewoftheexperimentaldesignispresentedin
Fig.1.

Figure1.
OpenFigure
Timescheduleoftheexperimentaldesign.Atpostnatalday(PN)10,ratsreceivedahyperthermia
(HT)ornormothermia(NT)treatment.FromPN11to16,ratswereinjectedtwicedaily,
intraperitoneally(i.p.),with25mgbromodeoxyuridine(BrdU)/kgbodyweight.Immunohistochemical
(IHC)stainingforBrdUwascarriedoutatPN17(shortterm,ST)orPN66(longterm,LT).

BrdUimmunohistochemistry
FromPN1116,allratsreceivedtwicedaily(minimum6hapart)intraperitonealinjectionsofthethymidine
analog5bromo2deoxyuridine[(BrdU25mg/kgSigma,St.Louis,MO,U.S.A.),2mg/mlin0.9%saline(pH
7.6)].AtPN17orPN66,theratsreceivedanoverdoseofpentobarbital(Nembutal),followedbyperfusion
fixationwith4%paraformaldehydein0.1 M phosphatebuffer(PB,pH7.6).Afterthebrainswereremoved,
theywerepostfixedin4%paraformaldehyde/0.1 M PBfor48h(4C),andcryoprotectedin20%sucrose/0.1
M PBfor24h(4C).Coronalserialsectionsof10mwerecutonacryostat,andsixsectionsperratwere
mountedonSuperfrostslides.Thefirstandlastsectionsweremaximally100mapartfromeachother.
Forbromodeoxyuridine(BrdU)detection,thesectionswerewashedinTBS(0.1 M Trisbase,0.15 M NaCl,
pH7.4),treatedwithTBScontaining0.6%H 2 O 2 for30min,andwashedagaininTBS(210minatRT,1
10minat65C).ForDNAdenaturation,thesectionswereincubatedin50%formamide/2SSCbuffer(0.3
M NaCl,0.03 M sodiumcitrate,pH7.0)for2hat65C,rinsedin2SSCbuffer(RT),incubatedin2NHCl
for30minat37C,andincubatedin0.1 M boratebuffer(pH8.5)for10min(RT).Afterthispretreatment,the
sectionswerewashed6timesinTBS,incubatedinTBSTS(TBScontaining0.25%TritonX100and3%
normaldonkeyserum)for60min(RT),andthenincubatedovernightinprimaryantiBrdUantibody
(monoclonalmouseRoche,Almere,TheNetherlands1:800inTBSTS).
AfterrinsingthesectionsinTBS,theywereincubatedfor1hinsecondarybiotinylateddonkeyantimouse
antibody(JacksonImmunoresearchLaboratories,WestGrove,PA,U.S.A.1:400inTBSTS).Thestaining
wasvisualizedwithaVectastainABC/Elitestandardkit(VectorLaboratories,Burlingame,CA,U.S.A.),based
ontheavidinbiotinperoxidasereaction,withdiaminobenzidineaschromogen,andNiCl 2 asasignal
enhancer.Finally,thesectionswerecounterstainedwith0.2%cresylvioletfor1h,dehydrated,and
coverslippedwithDePeX.SectionsoftheNTandHTgroupsfrombothgenderswereprocessed
simultaneouslytominimizeinterassayvariability.

Quantitativeanalysis
Perrat,sixcoronalsectionscutbetweenbregma2.12and2.30mmwereusedasseparatevaluesfor
quantitativeanalysisoftheBrdUstaining.ThisanalysiswascarriedoutbyusinganOlympusBX51
microscopecoupledtoacomputersupportedbytheStereoInvestigatorprogram(MicroBrightFieldInc.,
Williston,VT,U.S.A.).Ineachsection,theleftorrightDG(randomlychosen)wasdelineatedasdepictedin
Fig.2A,thenumberofBrdUpositivecellsinthedelineatedareawascounted,andthesurfaceofthatarea
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wascalculatedbytheStereoInvestigatorprogram.Acellwasincludedifitwasuniformlyorfragmentally
stained(seeFig.2B)andhadthemorphologicappearanceofanovalorroundnucleus.Thereasonfor
countingfragmentallystainedcellsisthattheBrdUlabeldiluteswitheachcelldivision(33).Thenumberof
BrdUpositivecellsper100,000m 2 wascalculated,andthecellcountsoftheHTgroupswerealso
expressedaspercentageofNT(meanof100%).Cellcountswereperformedata500magnificationbytwo
observersblindedtothetreatmentstatusofthesectionsandshowednosignificantinterobservervariation.

Figure2.
OpenFigure
Representativephotomicrographsofacoronalsectionofthehippocampusofa66dayoldmale
controlrat.Afterimmunohistochemicaldetectionofbromodeoxyuridine(BrdU),thesectionwas
counterstainedwithcresylviolet.A: Dottedline ,Thedelineationofthedentategyrus(DG).B:
DetailedpictureofthegranulecelllayeroftheDG.Cellsthatwereuniformlyorfragmentallystained
(soccerballpattern)werecounted( arrows ).Scalebars,100m.

Figure2.
OpenFigure
Representativephotomicrographsofacoronalsectionofthehippocampusofa66dayoldmale
controlrat.Afterimmunohistochemicaldetectionofbromodeoxyuridine(BrdU),thesectionwas
counterstainedwithcresylviolet.A: Dottedline ,Thedelineationofthedentategyrus(DG).B:
DetailedpictureofthegranulecelllayeroftheDG.Cellsthatwereuniformlyorfragmentallystained
(soccerballpattern)werecounted( arrows ).Scalebars,100m.
ToavoidbiasofpotentialchangesduetotheHTtreatmentorgender,thesizeoftheDG(meansurfacearea
ofsixsections)aswellasthesizeofthecells(meanof100BrdUlabeledcellsperrat)andcelldensityofthe
DGlayer(DGL,totalofBrdUlabeledandcresylvioletstainedcells)weremeasuredbyusingtheStereo
Investigatorprogram(MicroBrightField).

Statisticalanalysis
Bodyweightmeasurementsandgrowthcurveswereanalyzedbyusingrepeatedmeasuresanalysisof
variance(ANOVA)andBonferroni'smultiplecomparisonposthoctest.Toanalyzedifferencesinbodyweight
betweentreatmentgroupsbutwithingendergroupsateachindividualday,aStudent's t testwasused.
Treatment,gender,andtimeeffectonthenumberofBrdUlabeledcells,cellsize,surfaceareaoftheDG,and
cellulardensitywereanalyzedbyusingANOVAwithBonferroni'sposthoctest.Dataarepresentedasmean
SEM.Significancelevelsweresetatp<0.05.

RESULTS
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Inthepresentstudy,theeffectofHTinducedseizuresonproliferationandsurvivalofDGcellswas
determinedinimmaturemaleandfemalerats.
AttheendoftheHTphase,allHTratsdisplayedseverebehavioralseizures,characterizedbyarrestofthe
heatinducedhyperkinesis,followedbybodyflexionandoccasionallybyclonicmovementsofthelimbs.The
averageseizuredurationinmales(7.860.81min)wasnotsignificantlydifferentfromthatinfemales(10
1.54min).NoneoftheNTratshadbehavioralseizuresorshowedabnormalbehaviorduringtheirstayinthe
cylinder.
BodyweightmeasurementsshowedthattheHTtreatmenthadnooveralleffectonthegrowthcurve(from
PN12onward,theslopeofNTratsisnotsignificantlydifferentfromthatofHTrats)butdidreduceweightgain
inthefirst12daysafterHT(Fig.3).ANOVAshowedaninteractionbetweenDayandTreatmentwhenbody
weightsfromPN10to16wereanalyzed(F 6,246 =5.42,p<0.001),butfromPN12to16,thisinteractionwas
notpresent.AlthoughpretreatmentbodyweightsofHTandNTratswerethesame,thepostHTdipinweight
gainresultedinatransientlyreducedbodyweightofHTratscomparedwiththeirNTcounterpart(F 1,41 =
13.32,p<0.01).FromPN10to16,nooveralleffectofGenderwasseen,andnointeractionbetweenGender
andTreatmentonbodyweight.

Figure3.
OpenFigure
Growthcurvesfrompostnatal(PN)days1016.Meanbodyweightof(A)malenormothermia(NT,n
=9)andhyperthermiarats(HT,n=12),andof(B)femaleNT(n=11)andHT(n=13)rats.
Althoughpretreatment(PN10)bodyweightsofNTandHTratsdidnotdiffer,posttreatment(PN11
16)bodyweightsofHTratsweresignificantlyreducedcomparedwiththeirNTcounterparts.From
PN12to16,weightgainofNTratswasthesameasthatofHTrats(slopesoftheNTandHTcurves
arenotsignificantlydifferent).DataareexpressedasmeanSEM.*p<0.05(differencebetween
NTandHT,Student's t test).

Figure3.
OpenFigure
Growthcurvesfrompostnatal(PN)days1016.Meanbodyweightof(A)malenormothermia(NT,n
=9)andhyperthermiarats(HT,n=12),andof(B)femaleNT(n=11)andHT(n=13)rats.
Althoughpretreatment(PN10)bodyweightsofNTandHTratsdidnotdiffer,posttreatment(PN11
16)bodyweightsofHTratsweresignificantlyreducedcomparedwiththeirNTcounterparts.From
PN12to16,weightgainofNTratswasthesameasthatofHTrats(slopesoftheNTandHTcurves
arenotsignificantlydifferent).DataareexpressedasmeanSEM.*p<0.05(differencebetween
NTandHT,Student's t test).
AtPN66,theTreatmenteffectwasnolongerpresent,butthenaGendereffectappeared(F 1,14 =499.24,p<
0.001),inwhichmaleratsweighedsignificantlymorethanfemaleratswithineachtreatmentgroup(posthoc
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NT:male2794gvs.female1774.43gp<0.001HT:male2822.71gvs.female1764.42gp<
0.001).
TodeterminetheeffectofHTinducedseizuresonproliferationandonsurvivalofnewborncells,welabeled
postseizuredividingcellswithBrdUandquantifiedthenumberofBrdUimmunoreactivecellseithershortly
afterlabeling(i.e.,atPN17)or7weeksafterthelastBrdUinjection(i.e.,atPN66).Figure4showstypical
examplesoftheBrdUstainingpatternsfoundintheDGofthedifferenttreatmentgroups.Theyillustratea
cleardecreaseinBrdUlabelingwithincreasingage(STvs.LT).Onlypicturesofthemalegroupsare
presented,becausemaleandfemaleBrdUstainingpatternslookedalike.

Figure4.
OpenFigure
Photomicrographsofbromodeoxyuridine(BrdU)immunolabelinginthedentategyrus(DG)ofthe
malerats.Thesectionswerecounterstainedwithcresylviolettovisualizethehippocampalstructure.
FiftydaysafterthelastBrdUinjection(LT),anunambiguousdecreaseinBrdUlabelingisseeninthe
DGcomparedwithlabeling1dayafterthelastinjection(ST).BrdUstainingpatternsweresimilarin
thefemalegroups(picturesnotshown).NT,normothermiaHT,hyperthermiaST,shortterm,PN17
LT,longterm,PN66.Scalebar,250m.
BrdUquantificationrevealedthatHTinducesagenderspecificcytogenicresponseintheDG.Tobeableto
compareBrdUcountsbetweenthepresentstudyandothers,rawdataofthenumberofBrdUlabeled
cells/100,000m 2 areasfollows:atPN17,maleNT=116.133.22maleHT=126.734.6femaleNT=
79.212.35andfemaleHT=71.462.35atPN66,maleNT=23.091.79maleHT=29.211.66
femaleNT=34.732.15andfemaleHT=37.781.67.AtPN17,femaleNTandHTratshadsignificantly
fewerBrdUlabeledcellsper100,000m 2 thantheirmalecounterparts(ANOVA,p<0.001foreachtreatment
group).AtPN66,nogendereffectwasnoted.NoticethedecreaseinBrdUlabelingatPN66comparedwith
PN17.ANOVAshowedasignificanttimeeffectwithineachgender(ANOVA,p<0.001foreachgender).The
dataoftheNTgroupsalsoarepresentedinFig.5.

Figure5.
OpenFigure
Absolutenumberofbromodeoxyuridine(BrdU)positivecellsinthenormothermiacontrols.AtPN17,
femaleratshadsignificantlyfewerBrdUpositivecellsthandidmalerats,whereasatPN66,this
genderdifferencewasnolongerpresent.WhencomparingthenumberofBrdUlabeledcellsat
PN66withthatatPN17,asignificantdecreaseisobservedinthemaleandfemalegroup(PN17,
male:n=5andfemale:n=5PN66,male:n=4andfemale:n=7).Dataarepresentedasmean
SEM*Malevs.femaleatPN17,p<0.001. MalePN66vs.PN17,p<0.001. FemalePN66vs.
PN17,p<0.001.
TocomparetheHTeffectbetweengenders,thenumberofBrdUlabeledcells/100,000m 2 oftheHTgroups
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ispresentedaspercentageoftheircontrolgroupinwhichthecontrolgroupshaveameanof100%(Fig.6).At
PN17,maleandfemaleHTratshadthesamepercentageofnewborncellsasdidtheircontrols.

Figure6.
OpenFigure
Bromodeoxyuridine(BrdU)quantificationinthedentategyrusatPN17.Afterhyperthermia(HT)
treatment,maleandfemaleratsshowedthesamepercentageofBrdUpositivecellsastheir
normothermia(NT)controlrats(male:NT,n=5/HT,n=6female:NT,n=5/HT,n=6).Dataare
presentedasmeanSEM.
Tocomparegenderdifferencesinsurvival,weexpressedthenumberofBrdUpositivecellsatPN66asa
percentageofthenumberofBrdUpositivecellscountedatPN17(Fig.7).FromallthenewborncellsatPN17
thatwereBrdUlabeledduringthefirst6daysaftertreatment,7weekslater,23%wereleftinHTmalesand
20%inNTmales.Infemales,however,53%survivedinHTratscomparedwith44%inNTrats(ANOVAwith
posthoc,p<0.05).

Figure7.
OpenFigure
SurvivalofnewborncellsatPN66.Malehyperthermia(HT)ratsshowednodifferenceinsurviving
cellscomparedwithmalenormothermia(NT)controls,whereasfemaleHTratshadsignificantly
moresurvivingcellsthandidfemaleNTrats(male:NT,n=4/HT,n=6female:NT,n=6/HT,n=7).
DataarepresentedasmeanSEM. MaleNTvs.femaleNT,p<0.001. MaleHTvs.femaleHT,p
<0.001.*FemaleNTvs.femaleHT,p<0.05).
BrdUquantificationatPN66(Fig.8)showsthenetresultofproliferationandsurvival.About25%moreBrdU
positivecellswerefoundintheDGofmaleHTratsthanintheDGofmaleNTrats(ANOVAwithposthoc,p<
0.05).Atthisage,thenumberofBrdUpositivecellsdidnotdifferbetweenfemaleHTandfemaleNTrats.

Figure8.
OpenFigure
Bromodeoxyuridine(BrdU)quantificationinthedentategyrusatPN66.Thenetresultofproliferation
andsurvivalshowedthatmalehyperthermia(HT)ratshad25%moreBrdUpositivecellsthandid
malenormothermia(NT)rats,whereastheamountofBrdUpositivecellsdidnotdifferbetween
femaleHTandNTrats(male:NT,n=4/HT,n=6female:NT,n=6/HT,n=7).Dataarepresented
asmeanSEM.*p<0.05.
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ToavoidbiasfrompotentialchangesinDGsize,cellsizeandcelldensityoftheDGL,associatedwithHT
treatmentorgender,thesethreeparameterswerecomparedbetweenthegroups.Theresultswereas
follows:atPN17,neitherHTtreatmentnorgenderhadaneffectonthesurfaceareaoftheDG,cellsize,or
celldensityatPN66,HTtreatmentresultedinasignificantlysmallerDGsurfaceareainfemaleHTrats
comparedwithfemaleNTrats(ANOVAwithposthoc,p<0.05),whereasinmales,HTtreatmenthadno
effect.CellsizeandcelldensitywerenotaffectedbytreatmentorgenderatPN66.Withrespecttogender
differencesingrowth,PN66maleratshadalargerDGthandidfemales,irrespectiveoftreatment.When
comparingthevaluesfromPN66withPN17,celldensitydidnotdifferbetweenanyofthegroups,whereasthe
surfaceareaoftheDGwaslargeratPN66.Thiswasirrespectiveofgenderandtreatmentandwas
accompaniedbyanincreasedcellsizeatPN66.

DISCUSSION
Oneofthemainfeaturesofepilepsyisadisturbedbalancebetweenexcitationandinhibition,resultingin
hyperexcitability.Anumberofhippocampalabnormalities,suchasmossyfibersprouting,astrogliosis,and
neuronalloss,mayaccountforthishyperexcitability.Inadditiontothesehistopathologicfindings,itwas
recentlyshownthatseizurescanalterthenumberofnewbornDGcells(2127).Thisobservationhasledto
thehypothesisthatseizureinducedneurogenesismayhaveapermanenteffectontheconstituentsofthe
hippocampalnetwork,therebycontributingtotheprocessofepileptogenesis.Herewestudiedseizure
inducedcytogenesisintheDGofdevelopingmaleandfemaleratsbecause,undernormalphysiologic
conditions,neurogenesisisdependentonfactorssuchasageandsexhormones(31,32).
Themajorfindingsfromthepresentstudyarethefollowing:(a)earlylifeseizuresdonotaltercytogenesisin
theDGofmaleorfemalerats,(b)thesurvivalofDGcellsbornafterearlylifeseizuresis,comparedwiththat
oflittermatecontrols,increasedinfemaleratsandunchangedinmalerats,and(c)thenetresultof
proliferationandsurvivalisthat8weeksaftertreatment,HTmaleshad25%moreBrdUpositivecellsthandid
controls,whereasinfemales,nodifferenceappearedbetweenHTandcontrolrats.

Genderdifferencesinproliferationandsurvivalofcellsinnormothermiacontrols
Sexhormonesareknowntoplayaroleinproliferationandsurvival.ThustostudywhetherHTinduced
seizuresaffecttheseprocesses,wefirstinvestigatedgenderdifferencesinnormothermiacontrols(Fig.5).
TheanalysisshowedthatatPN17,femaleratshadsignificantlyfewernewborncellsthandidmalerats,
whereasthisdifferencewasnolongerpresentatPN66.
ThelowerproliferationinfemalesfoundinthepresentstudyisinagreementwiththeresultsofPerfilievaetal.
(29),whoinjectedyoungnaveSpragueDawleyratswithBrdUfor7consecutivedays.Theyalsofoundthat
1dayafterthelastinjection,femaleratshadsignificantlyfewerBrdUlabeledDGcellsthandidtheirmale
counterparts.ApparentlyagenderdifferenceinbasalproliferativerateofDGcellsexistsinimmaturerats.In
linewiththepresentstudy,theyalsoevaluatedthenumberofBrdUlabeledcells30daysafterthelastBrdU
injectionandfoundnodifferencebetweenmalesandfemales.BecausePN17femalesshowedsignificantly
fewernewborncellsthandidmales,andPN66femaleshadthesameamountofBrdUpositivecellsasPN66
males(Fig.5),thesurvival(PN66asapercentageofPN17Fig.7)wassignificantlyhigherinfemales(44%)
thaninmales(20%).ThisresultissimilartothatfoundbyPerfilievaetal.(29).
Toourknowledge,genderdifferencesinproliferationandsurvivalhavenotbeenstudiedinratsofcomparable
ageandattimeintervalssimilartothoseusedinthepresentstudy.Althoughsomeinformationexistsabout
theinfluenceofgenderonproliferationinadults,thenumberofstudiesislimited.Afewstudies,suchasthat
byTanapatetal.(28),foundatransientincreaseinthenumberofnewborncellsinadultfemalerats.This
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wasattributedtoincreasedlevelsofestrogenbecauseestrogentreatedovariectomizedratsshowedan
increasedproliferation.Becausetheratsinthepresentstudyweretreatedduringthefirst2weeksoftheir
livesandthereforehadnotreachedsexualmaturitywhenfirstanalyzedatPN17,itisunlikelythatahigher
estrogenlevelinthefemaleswasthecauseofthegenderdifferenceinproliferationfoundhere.Conversely,
sexdifferencesinestrogenreceptordensity,orperhapsbindingcapacity,mightexplainourdata.However,
O'Keefeetal.(34)foundnosexdifferencesintheconcentrationofestrogenreceptorsorinestradiolreceptor
bindinginyoungSpragueDawleyrats.Analternativeexplanationforthegenderdifferenceinproliferation
comesfromastudybyPangetal.(35).TheyfoundthatserumconcentrationsoftestosteronefromPN1to10
were3timeshigherinmaleratsthaninfemales,whereasserumlevelsofestrogensdidnotdifferbetween
thegenders.Becauseneonataladministrationoftestosteronehasbeenfoundalsotoincreasepostnatalcell
proliferation(36),thismightexplainthehigherproliferationinmalescomparedwithfemalesinourstudy.
Conversely,thetimepointatwhichsurvivalwasanalyzeddoescoincidewiththeageatwhichtheratshad
attainedsexualmaturity(PN66).Althoughthemolecularmechanismsofsurvivalofnewborncellsarenotwell
understood,genderdifferencessuggestthatsexhormonesareinvolvedinthisprocessaswell.Forexample,
estrogenhasbeenfoundtoexertaneuroprotectiveeffect(37,38).Thereforetheincreasedsurvivalin
femalesmayoccurasaresultofanincreaseinestrogenlevelsandconsequentlyareducedcelldeath.

Absenceofgenderdifferencesinseizureinducedproliferation
Becauseseizurescanalterproliferationandbecause,asmentionedearlier,agenderdifferenceisfoundin
basalproliferativerate,westudiedtheeffectofHTinducedseizuresonproliferationinbothmaleandfemale
rats.WefoundnoeffectofHTinducedseizuresonproliferationineitherofthetwogenders(Fig.6).Itis
knownthatpostnatalneurogenesiscanbemodifiedbymanyfactors.Here,wediscussseveralfactorsthat
mayhaveparticularimportanceforthepresentstudy.
First,factorsrelatedtothetreatment,suchasseizureduration,weightloss,andstress,mayaffecttheBrdU
cellcounts.TheobservationthatHTinducedseizuresdidnotalterthebirthrateofDGcells1weekafterthe
seizures(PN17)isinagreementwiththeresultsofapreviousstudybyBenderatal.(20).Theauthors
suggestedthatthedurationoftheHTinducedseizuresisinsufficienttoprovokeDGcellproliferationinthe
immaturehippocampus.Thisideawasbasedontheobservationthatlonglasting(>120min)KAinduced
seizuresincreasedthenumberofnewborncellswhencomparedwithlittermatecontrols,andHTinduced
seizureswereunabletochangethenumberofproliferatingcells.However,adifferenceinnewborncellswas
presentinthecontrolgroups(i.e.,thecontrolgroupthatwasusedtocomparewiththeKAtreatedanimals
hadfewerBrdUlabeledcellsthanthecontrolgroupthatwasusedforcomparisonwiththeHTtreated
animals).TheHTandKAtreatedanimals,incontrast,showedthesameamountofBrdUlabeledcells.
Weightlossalsomayhaveaffectedourresults.Forinstance,fooddeprivationresultinginreducedweightgain
isassociatedwithdecreasedproliferation(39).InlinewiththeresultsofSternetal.(40),wefoundthatHT
treatmentcausedatransientlyreducedbodyweight.Thiswasprobablyduetoareducedfoodintakeduring
thefirst24haftertreatment,becausefromPN12on,HTandcontrolratsshowedasimilarweightgain,and
onPN66,adifferenceinbodyweightwasnolongerseenbetweenHTandcontrolanimals.Moreimportant,
thiseffectwassimilarinmaleandfemalerats,andatthisearlyage,nodifferenceinbodyweightwasfound
betweenmaleandfemalecontrols,whichisinagreementwithfindingsofPerfilievaetal.(29).Wethushave
noindicationsthatHTinducedweightlossaffectedourresults.Finally,stressisanotherwellknownfactor
abletoinfluencetheproliferativerate.Stressisknowntodecreaseproliferationintheadultrat(41,42).The
stressinthepresentstudymightderivefromthetreatmentitself,forinstance,frommaternalseparation.
Althoughwecannotexcludethatisolationfromthemotherplayedaroleintheneurogenesisprocessinthese
pups,forseveralreasons,itisunlikelythatthestressinducedbyisolationaffectedourfindings.First,the
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separationduringtheNTorHTtreatmentonlylasted30min,whereasinmoststudiespupsandmothersare
separatedfor3to24h.Inthesestudies,itseemsthatthelackoftactilestimulationbythemotherandmilk
deprivationalterphysiologicresponses[forreview,see(43)].Duringtheisolation,wefrequentlymeasured
bodytemperature,whichinvolvedtactilestimulation,andreduceddehydrationbyinjectingsalinebefore
separation.Moreover,Severinoetal.(44)studiedgenderdifferencesinstressresponseatdifferentstagesin
theanimal'slifespanafterneonatalhandling.TheyhandledtheanimalsfromPN1to10for1min/day.At
PN11,theyfoundnodifferenceinthestressresponsebetweenmalesandfemales.Additionally,andmost
important,maleandfemalecontrolsandHTtreatedanimalswereexposedtothesamelevelofstressdueto
isolation.Inadditiontomaternalseparation,seizuresalsomayhavecausedsomestressinthismodel.Liuet
al.(45)showedthatsuppressionofhippocampalneurogenesisinimmatureratsisassociatedwiththe
numberofseizureepisodesandglucocorticosteroidlevels.WeinducedHTseizuresonlyonceandfoundno
changeinproliferation.ThisisinagreementwiththeresultsofLiuetal.,whoalsofoundnochangeinthe
numberofnewborncellsafteroneseizureepisode.Tosummarize,treatmenteffectssuchasseizureduration,
weightloss,andseizureinducedstressdonotseemtohaveaffectedproliferationinthismodel.
Asecondimportantfactorthatmayhaveaffectedourresultsisthemethodapplied.Inthepresentstudy,
proliferatingcellswerelabeledwithBrdUfor6consecutivedaysaftertheNTorHTtreatment.Thusevaluation
ofthenumberofBrdUpositivecells1dayafterthelastinjection(1weekafterthetreatment)cannotexclude
thatsomenewborncellshadalreadydied.ItissuggestedbyHayesandNowakowski(33)thatamore
accurateestimationofpureproliferationcanbegivenwheninjectingasingledoseofBrdUimmediatelyafter
treatment,followedbyevaluationofthenumberofBrdUpositivecellswithin24h.However,Benderetal.(20)
followedthissuggestionanddidnotfinddifferencesinproliferationafterHTinducedseizureseither.The
absenceofanalteredproliferationrateafterHTinducedseizuresinthepresentstudymightderivefrom
increasedcelldeathaccompanyingincreasedproliferation.However,likeBenderetal.,othershaveusedthe
HTmodeltostudycelldeathafterseizuresandfailedtodetectincreasedcelldeath(16,20,46).Wetherefore
concludethatindeednochangeinproliferationoccurredinourstudy.Toourknowledge,itisnotyetknown
whethergenderdifferencesincelldeathexistintheHTmodelforearlylifeseizures.Furthermore,toavoid
biasfrompotentialdifferencesinDGsize,cellsize,andcelldensity,weanalyzedwhethergenderortreatment
affectedtheseparameters.WefoundnooverallgenderortreatmenteffectontheDGsize,cellsize,orcell
densityatPN17.

GenderdifferencesinsurvivalofcellsbornafterHTinducedseizures
ThisisthefirstreportthathasquantifiedthesurvivalofDGcellsbornafterHTinducedseizuresinimmature
maleandfemalerats.AtPN66,malecontrolshad20%andHTmaleshad23%BrdUlabeledcellscompared
withthoseatPN17.HTinducedseizuresalsoincreasedthesurvivalofnewborncellsinfemales,becauseat
PN66,femalecontrolshad44%,whereasHTfemaleshad53%BrdUlabeledcellscomparedwiththoseat
PN17(Fig.7).ThusHTinducedseizureshardlyaffectedthesurvivalofnewbornDGcellsinmalesand
significantlyincreasedthatinfemalerats.AtPN66,cellsizeandcelldensitywerenotaffectedbygenderor
treatment.TheDGsizewasnotaffectedbytreatmentinmaleshowever,femaleHTratshada14%smaller
DGthantheirNTcounterparts.Thisresultissomewhatdifficulttoexplainbutisprobablynotduetothe
treatmentbecausesuchadifferencewasnotfoundatPN17orinmales.Mostimportant,cellulardensityand
cellsizewerethesameinbothgroups,andbyvisualinspectionofcresylvioletstainedsections,wedidnot
findanyevidenceofdamageintheDGofthefemaleHTrats.Themostlikelyexplanationthereforeis
utilizationofsectionsfromdifferentlevels,withsectionsfromNTfemalestakensomewhatclosertobregma
2.30thanthoseoffemaleHTrats.ThemainlimitationofthisfindingmightbethatthesamplesizeintheHT
groupisnotasaccurateasintheNTgroup.
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Ourresultsshowedsubstantialgenderdifferencesinsurvival.Asdescribedearlier,survivalofnewborncellsin
femalecontrolswassignificantlyhigherthanthatinmalecontrols.Inadditiontothegenderrelateddifference
insurvival,HTinducedseizurescausedanevenhigherincreaseinthesurvivalrateinfemales,althoughthey
didnotaffectthesurvivalrateinmales.Thismightbeexplainedbythefactthatseizuresareknowntochange
thelevelofreproductivehormones[forreview(47,48)],suchasanincreaseinestrogenlevels.Recently,
Barametal.(19)showedthatadultrats(PN70)developnocturnalspontaneousseizuresafterearlylifeHT
inducedseizures.Sothesespontaneousseizures,ifpresentinourrats,mayberesponsibleforthesexually
dimorphicsurvivalrate.AtPN17,wedidnotfindadifferenteffectoftheHTtreatmentbetweenthetwo
genders.ThissuggeststhattheHTinducedseizuresatPN10arenotabletoaffectthelevelsofsex
hormonesascanspontaneousseizures.

ConsequencesofalteredproliferationandsurvivalafterHTinducedseizures
Thenetresultofproliferationandsurvivalwasthat8weeksafterHTinducedseizures,femaleHTratshad
similaramountsofBrdUlabeledcellscomparedwithcontrols,whereasmaleHTratshad25%moreBrdU
labeledDGcellsthandidcontrols(Fig.8).Porterandcolleagues(49)recentlyassessedthesurvivalofcells
bornafterpilocarpineinducedstatusepilepticusinPN20rats,usingBrdUinjectionstolabelcellsdividing4,6,
and8daysafterseizure.ThreeweeksafterthelastBrdUinjection,theyalsofoundsignificantlymoreBrdU
labeledDGcellsinstatusepilepticusratsthanincontrols.Furthercharacterizationofthesesurvivingcells
revealedthat90%coexpressedtheneuronalmarkerNeuNandthuswereneurons.Theyalsofoundthatthe
percentageofBrdUlabeledcellsthatcoexpressedNeuNdidnotdifferbetweencontrolsandratsthathad
experiencedseizures.Sotheyconcludedthatstatusepilepticusdidnotaltertheneuronalfateofthesurviving
cells.Theobservationthatseizureinducedcytogenesisinimmatureratsmostlyresultsinthebirthofneurons
confirmspreviousstudies(23,50).ArecentstudybyDayeretal.(51)suggeststhatthesesurvivingneurons
areverystableandmaypermanentlyreplaceneuronsthatareintegratedintocircuits.Previousstudiesusing
theHTmodelinmaleratsfailedtodetectsignificanthippocampalneuronaldropoutat4weeksafterseizures(
16,20,46),resultinginthesametotalnumberofneuronsincontrolsandseizurerats.Ourowndatafailedto
demonstrateatreatmenteffectoncelldensity.ThustheincreasednumberofBrdUlabeledcellsweobserved
8weeksafterHTinducedseizuresinmaleratssuggeststhatcelldeathdidoccur,butdeadcellshavebeen
replacedbynewborncells.
Recently,areviewarticlebyShapiroandRibak(52)describedachangeinthemigrationpatternofnewborn
DGcellsafterseizures.Thesocalledbasaldendrites,whichnormallyretractearlyinDGcelldevelopment,fail
todothisafterseizures,resultinginanectopiclocationofthesecellsinthehilus.Thisideawasbasedonthe
resultsofseveralstudies,inwhichitwasshownthatseizurescausedanincreasednumberofnewborn
granulecellsthatresidedinthehilus.Theseectopiccells,althoughpresentinsmallnumbers,aresuggested
toplayaroleinhippocampalhyperexcitabilityseeninepilepticrodents.Thefunctionalconsequencesofthe
increaseincellsinthemaleHTratsseeninthepresentstudyremaintobeelucidatedbutmayberesolvedin
partbydeterminingtheidentityandmigrationofthesenewborncells.
Tosummarize,itwasshownthatHTinducedseizuresdonotchangeproliferationofDGcellsinmaleor
femalerats.Conversely,survivalofthesenewborncellswashigherinfemalethaninmalerats.Thenetresult
isanincreasedpopulationofnewbornDGcellsinyoungadultmales,whileleavingthatofyoungadult
femalesunaltered,whichmightcontributetogenderrelateddifferencesinseizuresusceptibility.

Acknowledgments
Acknowledgment:ThisresearchwassupportedbytheDutchBrainFoundation(grantH00.03toG.H.)and
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byaMarieCurieFellowshipoftheEuropeanCommunityprogramQualityofLifeandManagementofLiving
ResourcesundercontractnumberQLK6CT200060042andreferencenumberQLK6GH006004242.

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