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Developmental Psychobiology Masoud Asiaei 1. Jalal Solati 1 Ali-Akbar Salari 2 1 Faculty of Science, Karaj Branch Islamic Azad University P.O. Box 31485-313, Karaj, Iran E-mail: solati@kiau.ac.ir 2. Prenatal Exposure to LPS Leads to Long-Lasting Physiological Consequences in Male
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DevelopmentalPsychobiologyMasoudAsiaei1JalalSolati1Ali-AkbarSalari21FacultyofScience,KarajBranchIslamicAzadUniversityP.O.Box31485-313,Karaj,IranE-mail:solati@kiau.ac.ir2PrenatalExposuretoLPSLeadstoLong-LastingPhysiologicalConsequencesinMaleOffspringDepartmentofBiologyDepartmentofBiologyIslamicAzadUniversityNorthTehranBranch,Tehran,IranABSTRACT:Growingevidencesuggeststhatearlylifeeventsarecriticaldeterminantsfordisorderslaterinlife.Accordingtoacomprehensivenumberofepidemiological/animalstudies,exposuretolipopolysaccharide,causesalterationinpro-inflammatorycytokinelevels,hypothalamic–pituitary–adrenalfunctioningandthehormonalsystemwhichmaycontributetobehavioralandneurologicalinjuries.Inthisstudyweinvestigatedtheeffectsoflipopolysaccharideadminis-trationonphysiologicalparametersinpregnantdamsandtheirmaleoffspringaged9weeks.IngestationalDay10,pregnantmicewereinjectedintraprito-neallywithSalmonellaentericalipopolysaccharidetomodelprenatalexposuretoinfection.Thefollowingresultswereobtainedforoffspringfromdamsstressedduringpregnancy:(a)reducedanxiety-relatedbehaviorintheelevatedplusmaze;(b)reducedfoodandwaterintake;(c)reducedbodyweightfrombirthuptopostnatalDay40.Theobserveddataprovideexperimentalevidenceshowingthatprenatalstresscanhavecomplexandlong-lastingphysiological/behavioralconsequencesinoffspring.ß2011WileyPeriodicals,Inc.DevPsychobiol53:828–838,2011.Keywords:lipopolysaccharide;prenatalstress;pro-inflammatorycytokine;corti-costerone;bodyweight;foodintake;waterintake;C57BL/6miceINTRODUCTIONLipopolysaccharide(LPS),anendotoxinproducedfromthecellwallsofgram-negativebacteria,actsasanon-specificimmunostimulanttoinduceasevereinflamma-toryresponsebyinitiatingmultipleintracellularsignalingevents,includingtheactivationofnuclearfactorkb(NF-kb),whichultimatelyleadstothesyn-thesisandreleaseofcytokines,suchaspro-inflamma-torycytokinetumornecrosisfactoralpha(TNF-a),interleukin1b(IL-1b)andinterleukin6(IL-6)frommacrophages(Anisman,Hayley,Turrin,&Merali,2002;Anisman,Kokkinidis,&Merali,2002;Anisman,Received8December2010;Accepted25April2011Correspondenceto:J.SolatiPublishedonline31May2011inWileyOnlineLibrary(wileyonlinelibrary.com).DOI10.1002/dev.20568ß2011WileyPeriodicals,Inc.Zaharia,&Meaney,1998;Bachmanov,Reed,Beau-champ,&Tordoff,2002).LPSandsomecytokinescanactivatethehypothalamic–pituitary–adrenal(HPA)axiswhichleadstoincreaseinplasmaconcentrationsofadrenocorticotropichormone(ACTH),glucocorticoidsandalsocorticotrophinreleasingfactor(CRF)(Banetal.,1993;Barkeretal.,1993;Becskeietal.,2008;Bell&Hallenbeck,2002),causingchangesinthebrainneurochemistry(Betancur,Lledo,Borrell,&Guaza,1994;Chen,Zhou,Beltran,Malellari,&Chang,2005).Thisimmuneandneuroendocrineactivationcouldinfluencetheeffectivestateincludinganxiety-relatedbehavior,mood,andcognitionthathaveclinicalimpor-tance(Degroot,Kashluba,&Treit,2001;Delrue,Dele-planque,Rougepont,Vitiello,&Neveu,1994).Animalandhumanstudiesshowedthatthereisanincreaseofabove-mentionedcytokinesbothinsystemicandmRNAlevels,especiallyinthebrainofrodentsfollow-ingperipheralexposuretoLPS(Dunn,1988,1989;Dunn&Berridge,1990).DevelopmentalPsychobiologyPrenatalLPSExposureandMiceBehaviors829Ontheotherhand,agrowingbodyofscientificliter-atureofanimalandepidemiologicalstudiessuggeststhatbesidesgeneticfactors,environmentalfactors,likematernalstresscanhavelong-lastingeffectsonphysicaldevelopment,neurochemistry,behaviorandimmunocompetenceoftheoffspring,hencethisphenomenonhasbeendenotedas‘‘fetalprogramming’’(Barkeretal.,1993;File,1996,2001).Accordingtoseveralscientificliteratures,thesematernalstressesareveryimportantelementsintheprovocationorexacer-bationofawiderangeofphysiological/behavioralandpsychological/mentaldisturbances(Fride&Weinstock,1988;Golan,Lev,Hallak,Sorokin,&Huleihel,2005;Harbuz&Lightman,1992;Hava,Vered,Yael,Morde-chai,&Mahoud,2006).Maternalstresscanleadtoelevatedlevelsofmaternalstresshormones,notably,itiswellestablishedthatHPAhormonesplayacriticalroleinthestressresponse(Johnson,Kamilaris,Chrou-sos,&Gold,1992).Inrodentsandnon-humanprimatespecies,itwasfoundthatprenatalstress,includingexposuretoendotoxin,canalterHPAaxisandbrainneurotransmittersystemsintheoffspring(Kapoor,Dunn,Kostaki,Andrews,&Matthews,2006;Karrow,2006),andtheseeffectsaremediatedbycytokineinductionwithinthematernalcirculationandplacenta(Kirsten,Taricano,Flo´rio,Palermo-Neto,&Bernardi,2010;Klein&Rager,1995;Kofman,2002).Thesephysiologicalandneurochemical/hormonalchangescaninfluencesomeotherbehaviorslikeeatinganddrink-ing.Foodandwaterintakescorrelatedpositively,andthismaybeduetotheirmutualdependenceonbodysize,butanadditionalmechanismdirectlylinkingfoodandwaterintakesmayalsobeinvolved(Bachmanovetal.,2002;Kraly,1984).Itshouldbenotedthatthenatureandthepersistenceofprenatalstresseffectsprobablydependonthetimeofapplicationofmaternalstressrelativetothefetalstageofdevelopment(Merlot,Couret,&Otten,2008).Thereisemergingevidencesuggestingthatinflamma-toryeventsassociatedwithimmunologicaleventsinearly/middlefetallife(e.g.,GD8-10inratsandmice)arelikelytohavemorestrongerneurodevelopmentalimpactsthanlate-pregnancyinflammations.Thesematernalimmuneactivationsduringearly/middlepreg-nancyimpedewithcellproliferation,differentiation,migration,targetselection,andsynapsematuration,finallyleadingtoseveralbrainandbehavioralabnor-malitiesinadulthood(Kirstenetal.,2010).Thepresentstudyproceededtoinvestigatesomeoftheneuroendo-crineandbehavioralconsequencesofprenatalexposuretoLPSonbothpregnantC57BL/6miceingestationalday(GD)10andtheirmaleoffspringaged9weeks.Therefore,weassessedtheeffectofprenatallyadminis-teredLPSontheconcentrationofcorticosteroneandpro-inflammatorycytokines,anxiety-relatedbehaviorintheelevatedplusmaze(EPM),foodandwaterintakeofbothpregnantdamsandtheirmaleoffspringatadulthoodandalsothebodyweightofoffspringfrombirthtopostnatalday(PND)40.METHODSAnimalsMaleandfemaleC57BL/6mice(PasteurInstituteofIran)aged6–8weeksuponarrivalandwerelefttoacclimatizetothelaboratoryfor10dayspriortotesting.Miceweremain-tainedingroupsof5instandardpolypropylenecages,toavoidbehavioralchangesthatmayresultfromsinglehousingwhichcanusuallybeanincreaseinaggressiveandfear-likebehavior.Theanimalswereallowedfreeaccesstofoodandwateratalltimesandweremaintainedona12hrlight/darkschedule(lightson07:00hr)inacontrolledtemperature(23Æ18C).Formatingpurposes,threefemaleswerehousedovernightwithtwomalesstartingat19:00hr.Eachfemalemousewasvisuallyinspectedforthepresenceofavaginalplugthenextmorningat07:00hr.Thepresenceofplugwasdesignedasday0ofgestation.Thepregnantmice(N¼80)weredividedrandomlyintofourgroups.Fortyofthesemice(N¼10ineachgroup)werescarifiedformeasuringcyto-kinesandcorticosteroneandotherswerelefttolabor(N¼10ineachgroup).Followingdelivery,littersremainedintacttoavoidconfoundingchangesinmaternalbehavior.Thelittersremainedwiththeirmothersuntilweaning(Day21)andwereseparatedaccordingtosexonDay36.Themaleoffspringmaintainedingroupsof3–4intheabove-mentionedconditions.Themaleoffspringweredistributedintocontrolandexperimentalgroups(N¼10/group;twopupswereselectedfromeachmotherforthenextexperiments).ThestudywasapprovedbytheEthicsCommitteeofKarajIslamicAzadUniversityandexperimentalprotocolisincompliancewiththeNationalInstitutesofHealthGuideforCareandUseofLaboratoryAnimals(PublicationNo.85-23,revised1985).PrenatalAdministrationofLPSLipopolysaccharide(Salmonellaentericaserotypeentritidis,L6011,SigmaAldrich,SaintLouis,MO)wasdissolvedinster-ilepyrogen-freesalinebeforeuse.ThedamswererandomlyassignedtoasalinecontrolgroupandLPSgroups.ThedamsintheLPSgroupswereadministeredasingleintraperitoneal(i.p.)injectionof50,100,or150mg/kgLPSonday10ofpregnancy.Thedamsinthecontrolgroupwereadministeredasinglei.p.injectionofsalineonday10ofpregnancy.ItisimportanttonotethatalthoughmaternalexposuretoLPScausessomedisturbances,itcanvarydependingondosesofLPS,potencyofLPS,age,sex,andinteractionswithenviron-mentalandgeneticriskfactors(Bell&Hallenbeck,2002;Urakubo,Jarskog,Lieberman,&Gilmore,2001).ThedosageofLPSwechosecouldinducesystemicinflammation,resulting830Asiaei,SolatiandSalariDevelopmentalPsychobiologyplatform(5cmÂ5cm)eachwithanopenroof.Themazewasplacedinthecenterofaquietanddimlylitroom.Themicebehaviorwasdirectlyobservedusingamirror,sus-pendedatanangleabovethemaze.Behavioraldatawerecollectedbya‘‘blind’’observerwhoquietlysat1mbehindoneoftheclosedarmsofthemaze,usingachronometer.TheanxietytestoftheoffspringwascarriedoutatpostnatalDay61.Itwasrepeatedthreetimes,threeseparatecohortsofmaleoffspringwereusedfortestsandeachmousewasonlyusedonEPMonce.WerepeatedthistestthreetimesduetoourdifferentresultsonEPMfromotherinvestigationsandtheresultsrepresentanaverageofthethreetestsessions.Fortest-ingpurpose,maleoffspringwereplacedindividuallyinthecenterportionoftheplus-maze,facingoneoftheopenarms.Theobservermeasured:(1)thetimespentintheopenarms,(2)thetimespentintheclosedarms,(3)thenumberofentriesintotheopenarms,and(4)thenumberofentriesintotheclosedarmsduringthe5-mintestperiod.Anentrywasdefinedasallfourpawsinthearm.Theelevatedplus-mazewasthoroughlycleanedwithdistilledwaterfollowingthetestingofeachanimaltoavoidpossiblebiasingeffectsduetoodorcluesleftbypreviousmice.Forthepurposeofanalysis,open-armactivitywasquantifiedastheamountoftimethatthemicespentintheopenarms(OAT)relativetothetotalamountoftimespentinanyarm(open/totalÂ100),andthenumberofentriesintotheopenarms(OAE)wasquantifiedrelativetothetotalnumberofentriesintoanyarm(open/totalÂ100).Thetotalnumberofopenarmsentered,aswellasthetotalnumberofclosedarmsenteredwereusedasindexesofgenerallocomotoractivity(LMA)(Solati,Zarrindast,&Salari,2010;Zarrindast,Solati,Oryan,&Parivar,2008).Alltestingwasconductedbetween13:00and16:00hr.BodyWeightThebodyweightsofmaleoffspring(Æ0.1g)wereregularlymonitoredat13:00hrevery10daysduringtheexperimentfrombirth(PND0)toPND40.FoodandWaterIntakeInordertofindoutwhetheri.p.exposuretoLPSduringpreg-nancyaffectsfood/waterintakeindamsorintheirmaleoff-springunderourexperimentalconditions,afeeding/drinkingstudywasconducted.Micehadfreeaccesstofoodandwaterbeforetheexperimentsbegan.Eachcagecontained3or4micewhichweregivenwiththesameamountoffoodandwater.Theirfoodintakewasmeasuredthefollowingdaybysubtractingtheuneatenfoodmanually.Theamountofwateringestedinourexperimentwasmeasuredwith0.2mlgradu-atedglassburettesadaptedwithametaldrinkingspout.ImmediatelyafterinjectionofLPS/salineinpregnantdams,eachmousewasreturnedtoitscageandwemeasuredthecumulativewaterintakemanuallythefollowingday.Thesemeasurementsweredonein3daysafterinjectionoftheLPS/salineinrespecttopregnantdams(N¼10ineachgroup)andin5daysinPND56-60inmaleoffspring(N¼10ineachgroup)anditwascalculatedasfood(ingramsÆ0.1g)/water(inmlÆ0.2ml)intakepermiceperday.Measurementsinalowpercentageoffetalanomalies,butnotabortionandpossibleintra-uterinefetaldeath(IUFD)(Bell&Hallenbeck,2002).Therationaleforchoosinggestationalday10wasthatthisperiodcorrespondstothefirst-to-secondtrimestersofhumanpregnancy,withrespecttodevelopmentalbiologyandpercentageofgestationfrommicetohuman(Kaufman,1992).Otherscientificliteraturesstatethatthistimephaseistheperiodofearlyfetalbraindevelopment(Wei,Li,&Zhou,2007),cerebralorganogenesisinmice,especiallyneural-plateformation(Kirstenetal.,2010)andalsoembryonicstemcellformation(gestationalperiod0.38–0.53inrodents)whichisoneofthemainperiodsofvulnerabilityoftheimmunesystemtoenvironmentalinsults(Merlotetal.,2008).Inaddition,otherinvestigatorssuggestthatmaternalinfectionfromearlytomidpregnancyismorelikelytoberelatedtolong-lastingdevelop-mentalbrainandbehavioralabnormalitiesintheoffspring(Mednick,Machon,Huttunen,&Bonett,1988;Rodier&Hyman,1998).Eachdamwasadministered50mlsalineorLPSsolution.FollowinginjectionwithLPSorsaline,thedamscontinuedtobehousedintheabove-mentionedconditions.Asweknow,LPScandisrupttheblood–brainbarrier(BBB)ifadminis-teredtopicallytothecerebralmicrocirculationorintracister-nally,anditispossiblethathighdosesofLPScrosstheplacentaintothefetalcirculation(Urakuboetal.,2001).LowdosesofLPSwereselectedinordertopreventpossibledirectexposureoffetustoLPS.Withthisdoseandrouteofapplicationweobservednoabortioninourendotoxingroups.Allinjectionsweregivenbetween13:00and14:00hr.MeasurementofSerumCorticosteroneandCytokineConcentrationMaternalserumfromtrunkbloodofpregnantdamswaspre-pared1.5hrafterinjectionofLPS/saline(N¼10ineachgroup)bycentrifugationat5,000gfor12min,aliquotedandthenstoredatÀ808Cuntilthecytokine/corticosteroneassayswereperformed.Concentrationsofcorticosterone(KT-510,KamiyaBiomedicalCompany,Seattle,WA),IL-1b(IB49700,Immuno-BiologicalLaboratories,Minneapolis,MN),IL-6(BioSourceInternational,CA),andTNF-a(CT302,Ucytech,Utrecht,TheNetherlands)weredeterminedbyusingcommer-cialELISAkitsaccordingtothemanufacturer’sprotocol.Trunkbloodwascollectedforpreparationofbloodserumofmaleoffspringintheir9thweek(PND62)andmeasurementofserumcorticosteroneandcytokineconcentrationwascarriedoutagaininthesameway.Allsamplesandstandardswereassayedinduplicate.Thepregnantmiceandtheirmaleoffspringwereselectedrandomly.AnxietyTestOneofthemostpopulartestsofanxiety-likebehaviorinmiceandratsistheEPM,inwhichthereducednumberofentriesortimespentintheopenarmsoftheEPMsuggeststheoperationofanxiety-likeprocesses.Thiswooden,plus-shapedapparatuswaselevatedtotheheightof50cmabovethefloorandconsistsoftwoopenarms(30cmÂ5cm),twoenclosedarms(30cmÂ5cmÂ15cm),andcentralDevelopmentalPsychobiologywereperformedbythesameexperimenterandweusedsepar-atecohortsofpregnantdamsforfood/waterintake,butthesamegroupsofmaleoffspringwereusedforfood/waterintakeandanxietytestonEPM.StatisticalAnalysisDatawereanalyzedusingSPSS.Sincedatadisplayednormaldistributionandhomogeneityofvariance,one-wayANOVAwasusedforcomparisonbetweentheeffectsofdifferentdosesofLPSwithvehicle.Differenceswithp<0.05betweenexperimentalgroupsateachpointwereconsideredstatisticallysignificant.RESULTSEffectsofLPSonSerumConcentrationsofTNF-a,IL-1b,andIL-6inPregnantMiceandTheirMaleOffspringTheeffectsofLPStreatmentonTNF-a,IL-1b,andIL-6inmaternalserumwereanalyzed.InresponsetoLPSPrenatalLPSExposureandMiceBehaviors831challenge,TNF-a(F3,36¼161.825,p<0.001),IL-1b(F3,36¼39.468,p<0.000)andIL-6(F3,36¼9.968,p<0.001)inmaternalserumwereincreased1.5hrafterLPSadministration(exceptforIL-6in50mg/kgLPS;Fig.1).Table1showsthattherewerenosignifi-cantdifferencesbetweenprenatallyLPSandsaline-treatedoffspringinserumconcentrationsofTNF-a(F3,36¼1.971,p<0.145),IL-1b(F3,36¼1.403,p<0.266)andIL-6(F3,36¼1.496,p<0.241)intheir9thweek(PND62).EffectsofLPSonSerumConcentrationsofCorticosteroneinPregnantMiceandTheirMaleOffspringTheeffectsofLPStreatmentonserumcorticosteronelevelofpregnantdamsandtheirmaleoffspringareshowninFigure1andTable1.OnewayANOVAconfirmedthatinpregnantdams,highdosesofLPS(100or150mg/kg)increasedserumcorticosteronelevelsrelativetosaline-treatedanimals(F3,36¼8.937,p<0.002)(Fig.1).ButprenatalLPSexposuredoesnothaveanysignificanteffectonserumconcentrationsofcorticosteroneinmaleoffspringintheir9thweek(PND62)(F3,36¼3.442,p<0.052)(Tab.1).EffectsofLPSonAnxiety-RelatedBehaviorsinMaleOffspringFigure2showsdataontheeffectsofprenatalexposuretoLPSonanxiety-relatedbehaviorofmaleoffspringintheEPM.One-wayANOVArevealedthatprenatalexposuretoLPShasincreasedthepercentageofopenFIGURE1Effectofintraperitonealinjectionofsaline(50ml/mouse)orLPS(50,100,or150mg/kg)onserumlevelofTNF-a,IL-1b,IL-6(a)andcorticosterone(b)inpregnantdams.EachbarismeanÆSEM.N¼10.Ãp<0.05,ÃÃtreatedgroup.armtime(F3,36¼49.396,p<0.000)andopenarmentries(F3,36¼8.843,p<0.000).Nodifferenceswerefoundinthetotalnumberofarmentriesintheplus-maze(open-armsþclosed-arms)(F3,36¼1.604,p<0.215).Consideringalloftheseeffects,thepres-entlyobservedbehavioraldatasuggestlowlevelsofanxietyinprenatallyLPS-treatedoffspringwhichwasnotreportedlikethisbefore.EffectsofPrenatalExposuretoLPSonBodyWeightinMaleOffspringDuringpostnataldevelopment,allthepupsgainedbodyweightcontinuously,butindifferentwaysamongourexperimentalgroups.Thebirthweight(PND0)ofprenatallyLPS-treatedoffspring(LPS50:1.31Æ0.33g;LPS100:1.20Æ0.33g;LPS150:1.20Æ0.32g)waslowerthanprenatallysaline-treatedoffspring(1.62Æ0.31g)(F3,36¼36.282,p<0.000).Furthermore,thetotalbodyweightofprenatallyLPS-treatedoffspringwaslowerthantheprenatallysaline-treatedoffspringduring40daysofage[PND10(F3,36¼26.399,p<0.01,andÃÃÃp<0.001,whencomparedtothesaline832Asiaei,SolatiandSalariDevelopmentalPsychobiologyTable1.EffectofPrenatalExposuretoLPSonSerumLevelofPro-InflammatoryCytokinesandCorticosteroneinMaleOffspringinPND62(MeanÆSEM,N¼10inEachGroup)LPS50mg/kgLPS100mg/kgLPS150mg/kgControl(Saline)TNF-a(pg/ml)IL-1b(pg/ml)IL-6(pg/ml)26.00Æ1.41484.25Æ3.705285.5Æ3.66288.50Æ3.379118.75Æ2.750297.00Æ5.552114.25Æ4.211123.75Æ4.871317.00Æ6.819130.50Æ4.113130.50Æ3.617320.50Æ5.781p<0.000),PND20(F3,36¼25.849,p<0.000),PND30(F3,36¼8.174,p<0.001),PND40(F3,36¼5.366,p<0.006)](Fig.3).Itisimportanttonotethatweobservednomiscarriageorfetallossamongourgroupsduringexperiment.EffectsofLPSonFoodandWaterIntakeinPregnantMiceandTheirMaleOffspringAscanbeclearlyseeninFigure4,i.p.administrationofLPSreducedfoodintakeinthefirstdayafterinjec-tioninpregnantdams(F3,36¼13.465,p<0.000).Duringthe2nd(F3,36¼2.375,p<0.095)and3rd(F3,36¼1.685,p<0.197)dayafterchallenge,therewerenosignificantdifferencesbetweenLPSandsaline-treateddamsinfoodintake.Inotherwords,feedingthenreturnedtonormaloverthenext48–72hrastheLPSeffectdissipated.ItisalsoimportanttonotethatthereweresignificantdecreasesinfoodintakebetweenprenatallyLPSorsaline-treatedmaleoffspringatpostnatalDay56–60[PND56(F3,36¼37.029,p<0.000),PND57(F3,36¼20.638,p<0.000),PND58(F3,36¼30.582,p<0.000),PND59(F3,36¼45.155,p<0.000),PND60(F3,36¼36.505,p<0.000)](Fig.4).TheintraperitonealinjectionofsmallamountsofendotoxintakenfromSalmonellaentericasignificantlydecreasedandalmostcompletelyinterruptedthewaterintakeofpregnantdams.OnedayafterLPSexposuretherewasasignificantdecreaseinwaterintakeamongalldams(F3,36¼47.734,p<0.000).Inotherwords,intheexperimentpresentedinFigure5,50mg/kgofendotoxinwassufficienttoalterwaterintake.Therewerenosignificantdifferencesinthewaterintakeofdamswhichreceivedthesmallestdoseofendotoxin,comparedtothecontrolgroup,buttheinhibitionper-sistedlongerasthedosewasincreased(F3,36¼20.714,p<0.000).With100or150mg/kgofendo-toxin,thedailywaterintakeinpregnantdamsdidnotreturntonormaluntiltheendofthe3rddayafterinjec-tion(F3,36¼4.975,p<0.008),(seeFig.5).ThewaterFIGURE2EffectsofprenatalexposuretoLPSonanxiety-relatedbehaviorinoffspringintheelevatedplus-maze.Maleoffspringprenatallyexposedtosaline(50ml/mouse)orLPSEffectofprenatalexposuretosaline(50ml/FIGURE3(50,100,or150mg/kg)andanxietytestoftheseoffspringcarriedoutatpostnatalDay61.EachbarismeanÆSEM.Openarmtime(OAT),openarmentries(OAE)orlocomotorÃÃÃÃÃÃmouse)orLPS(50,100,or150mg/kg)onbodyweightofmaleoffspringfrombirthdaytoPND40.TheendotoxinoffspringhadalowerbodyweightfrombirthdayuptoPND40.EachbarismeanÆSEM.N¼10.ÃÃp<0.01andÃÃÃactivity(LMA).N¼10.p<0.05,p<0.01,andp<0.001whencomparedtothesalinetreatedgroup.p<0.001whencomparedtothesalinetreatedgroup.DevelopmentalPsychobiologyPrenatalLPSExposureandMiceBehaviors833FIGURE4Effectofintraperitonealinjectionorprenatalexposuretosaline(50ml/mouse)orLPS(50,100,or150mg/kg)onfoodintakeinpregnantdamsandtheirmaleoffspring.EndotoxindamsatelessthansalineanimalsjustinfirstdayafterLPSinjection,whileendo-toxinoffspringatelessthansalineoffspringduringexperiment.EachbarismeanÆSEM.N¼10.ÃÃÃp<0.001whencomparedtothesalinetreatedgroup.p<0.000),PND60(F3,36¼6.552,p<0.002)](Fig.5).Differentstudiesshowedthattheinhibitionofwaterintakewasessentiallythesame,despitethestrainofGram-negativespeciesfromwhichtheendotoxinwasprepared.intakeinprenatallyLSP-treatedmaleoffspringwaslowercomparedtoprenatally-salinetreatedoffspringinPND56–60[PND56(F3,36¼78.004,p<0.000),PND57(F3,36¼109.789,p<0.000),PND58(F3,36¼137.651,p<0.000),PND59(F3,36¼161.790,FIGURE5Effectofintraperitonealinjectionorprenatalexposuretosaline(50ml/mouse)orLPS(50,100,or150mg/kg)onwaterintakeinpregnantdamsandtheirmaleoffs
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