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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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DevelopmentalPsychobiology MasoudAsiaei1 JalalSolati1 Ali-AkbarSalari2 1 FacultyofScience,KarajBranch IslamicAzadUniversity P.O.Box31485-313,Karaj,Iran E-mail:solati@kiau.ac.ir 2 PrenatalExposuretoLPSLeads toLong-LastingPhysiological ConsequencesinMale Offspring DepartmentofBiology DepartmentofBiology IslamicAzadUniversity NorthTehranBranch,Tehran,Iran ABSTRACT:Growingevidencesuggeststhatearlylifeeventsarecritical determinantsfordisorderslaterinlife.Accordingtoacomprehensivenumberof epidemiological/animalstudies,exposuretolipopolysaccharide,causesalteration inpro-inflammatorycytokinelevels,hypothalamic–pituitary–adrenalfunctioning andthehormonalsystemwhichmaycontributetobehavioralandneurological injuries.Inthisstudyweinvestigatedtheeffectsoflipopolysaccharideadminis- trationonphysiologicalparametersinpregnantdamsandtheirmaleoffspring aged9weeks.IngestationalDay10,pregnantmicewereinjectedintraprito- neallywithSalmonellaentericalipopolysaccharidetomodelprenatalexposure toinfection.Thefollowingresultswereobtainedforoffspringfromdams stressedduringpregnancy:(a)reducedanxiety-relatedbehaviorintheelevated plusmaze;(b)reducedfoodandwaterintake;(c)reducedbodyweightfrom birthuptopostnatalDay40.Theobserveddataprovideexperimentalevidence showingthatprenatalstresscanhavecomplexandlong-lastingphysiological/ behavioralconsequencesinoffspring.ß2011WileyPeriodicals,Inc.Dev Psychobiol53:828–838,2011. Keywords:lipopolysaccharide;prenatalstress;pro-inflammatorycytokine;corti- costerone;bodyweight;foodintake;waterintake;C57BL/6mice INTRODUCTION Lipopolysaccharide(LPS),anendotoxinproducedfrom thecellwallsofgram-negativebacteria,actsasanon- specificimmunostimulanttoinduceasevereinflamma- toryresponsebyinitiatingmultipleintracellular signalingevents,includingtheactivationofnuclear factorkb(NF-kb),whichultimatelyleadstothesyn- thesisandreleaseofcytokines,suchaspro-inflamma- torycytokinetumornecrosisfactoralpha(TNF-a), interleukin1b(IL-1b)andinterleukin6(IL-6)from macrophages(Anisman,Hayley,Turrin,&Merali, 2002;Anisman,Kokkinidis,&Merali,2002;Anisman, Received8December2010;Accepted25April2011 Correspondenceto:J.Solati Publishedonline31May2011inWileyOnlineLibrary (wileyonlinelibrary.com).DOI10.1002/dev.20568 ß2011WileyPeriodicals,Inc. Zaharia,&Meaney,1998;Bachmanov,Reed,Beau- champ,&Tordoff,2002).LPSandsomecytokinescan activatethehypothalamic–pituitary–adrenal(HPA)axis whichleadstoincreaseinplasmaconcentrationsof adrenocorticotropichormone(ACTH),glucocorticoids andalsocorticotrophinreleasingfactor(CRF)(Ban etal.,1993;Barkeretal.,1993;Becskeietal.,2008; Bell&Hallenbeck,2002),causingchangesinthebrain neurochemistry(Betancur,Lledo,Borrell,&Guaza, 1994;Chen,Zhou,Beltran,Malellari,&Chang,2005). Thisimmuneandneuroendocrineactivationcould influencetheeffectivestateincludinganxiety-related behavior,mood,andcognitionthathaveclinicalimpor- tance(Degroot,Kashluba,&Treit,2001;Delrue,Dele- planque,Rougepont,Vitiello,&Neveu,1994).Animal andhumanstudiesshowedthatthereisanincrease ofabove-mentionedcytokinesbothinsystemicand mRNAlevels,especiallyinthebrainofrodentsfollow- ingperipheralexposuretoLPS(Dunn,1988,1989; Dunn&Berridge,1990).
DevelopmentalPsychobiology PrenatalLPSExposureandMiceBehaviors 829 Ontheotherhand,agrowingbodyofscientificliter- atureofanimalandepidemiologicalstudiessuggests thatbesidesgeneticfactors,environmentalfactors,like maternalstresscanhavelong-lastingeffectson physicaldevelopment,neurochemistry,behaviorand immunocompetenceoftheoffspring,hencethis phenomenonhasbeendenotedas‘‘fetalprogramming’’ (Barkeretal.,1993;File,1996,2001).Accordingto severalscientificliteratures,thesematernalstressesare veryimportantelementsintheprovocationorexacer- bationofawiderangeofphysiological/behavioraland psychological/mentaldisturbances(Fride&Weinstock, 1988;Golan,Lev,Hallak,Sorokin,&Huleihel,2005; Harbuz&Lightman,1992;Hava,Vered,Yael,Morde- chai,&Mahoud,2006).Maternalstresscanleadto elevatedlevelsofmaternalstresshormones,notably,it iswellestablishedthatHPAhormonesplayacritical roleinthestressresponse(Johnson,Kamilaris,Chrou- sos,&Gold,1992).Inrodentsandnon-humanprimate species,itwasfoundthatprenatalstress,including exposuretoendotoxin,canalterHPAaxisandbrain neurotransmittersystemsintheoffspring(Kapoor, Dunn,Kostaki,Andrews,&Matthews,2006;Karrow, 2006),andtheseeffectsaremediatedbycytokine inductionwithinthematernalcirculationandplacenta (Kirsten,Taricano,Flo´rio,Palermo-Neto,&Bernardi, 2010;Klein&Rager,1995;Kofman,2002).These physiologicalandneurochemical/hormonalchangescan influencesomeotherbehaviorslikeeatinganddrink- ing.Foodandwaterintakescorrelatedpositively,and thismaybeduetotheirmutualdependenceonbody size,butanadditionalmechanismdirectlylinkingfood andwaterintakesmayalsobeinvolved(Bachmanov etal.,2002;Kraly,1984). Itshouldbenotedthatthenatureandthepersistence ofprenatalstresseffectsprobablydependonthetime ofapplicationofmaternalstressrelativetothefetal stageofdevelopment(Merlot,Couret,&Otten,2008). Thereisemergingevidencesuggestingthatinflamma- toryeventsassociatedwithimmunologicaleventsin early/middlefetallife(e.g.,GD8-10inratsandmice) arelikelytohavemorestrongerneurodevelopmental impactsthanlate-pregnancyinflammations.These maternalimmuneactivationsduringearly/middlepreg- nancyimpedewithcellproliferation,differentiation, migration,targetselection,andsynapsematuration, finallyleadingtoseveralbrainandbehavioralabnor- malitiesinadulthood(Kirstenetal.,2010).Thepresent studyproceededtoinvestigatesomeoftheneuroendo- crineandbehavioralconsequencesofprenatalexposure toLPSonbothpregnantC57BL/6miceingestational day(GD)10andtheirmaleoffspringaged9weeks. Therefore,weassessedtheeffectofprenatallyadminis- teredLPSontheconcentrationofcorticosteroneand pro-inflammatorycytokines,anxiety-relatedbehaviorin theelevatedplusmaze(EPM),foodandwaterintake ofbothpregnantdamsandtheirmaleoffspringat adulthoodandalsothebodyweightofoffspringfrom birthtopostnatalday(PND)40. METHODS Animals MaleandfemaleC57BL/6mice(PasteurInstituteofIran) aged6–8weeksuponarrivalandwerelefttoacclimatizeto thelaboratoryfor10dayspriortotesting.Miceweremain- tainedingroupsof5instandardpolypropylenecages,to avoidbehavioralchangesthatmayresultfromsinglehousing whichcanusuallybeanincreaseinaggressiveandfear-like behavior.Theanimalswereallowedfreeaccesstofoodand wateratalltimesandweremaintainedona12hrlight/dark schedule(lightson07:00hr)inacontrolledtemperature (23Æ18C).Formatingpurposes,threefemaleswerehoused overnightwithtwomalesstartingat19:00hr.Eachfemale mousewasvisuallyinspectedforthepresenceofavaginal plugthenextmorningat07:00hr.Thepresenceofplugwas designedasday0ofgestation.Thepregnantmice(N¼80) weredividedrandomlyintofourgroups.Fortyofthesemice (N¼10ineachgroup)werescarifiedformeasuringcyto- kinesandcorticosteroneandotherswerelefttolabor (N¼10ineachgroup).Followingdelivery,littersremained intacttoavoidconfoundingchangesinmaternalbehavior. Thelittersremainedwiththeirmothersuntilweaning(Day 21)andwereseparatedaccordingtosexonDay36.Themale offspringmaintainedingroupsof3–4intheabove-mentioned conditions.Themaleoffspringweredistributedintocontrol andexperimentalgroups(N¼10/group;twopupswere selectedfromeachmotherforthenextexperiments).The studywasapprovedbytheEthicsCommitteeofKarajIslamic AzadUniversityandexperimentalprotocolisincompliance withtheNationalInstitutesofHealthGuideforCareand UseofLaboratoryAnimals(PublicationNo.85-23,revised 1985). PrenatalAdministrationofLPS Lipopolysaccharide(Salmonellaentericaserotypeentritidis, L6011,SigmaAldrich,SaintLouis,MO)wasdissolvedinster- ilepyrogen-freesalinebeforeuse.Thedamswererandomly assignedtoasalinecontrolgroupandLPSgroups.Thedams intheLPSgroupswereadministeredasingleintraperitoneal (i.p.)injectionof50,100,or150mg/kgLPSonday10of pregnancy.Thedamsinthecontrolgroupwereadministereda singlei.p.injectionofsalineonday10ofpregnancy.Itis importanttonotethatalthoughmaternalexposuretoLPS causessomedisturbances,itcanvarydependingondosesof LPS,potencyofLPS,age,sex,andinteractionswithenviron- mentalandgeneticriskfactors(Bell&Hallenbeck,2002; Urakubo,Jarskog,Lieberman,&Gilmore,2001).Thedosage ofLPSwechosecouldinducesystemicinflammation,resulting
830 Asiaei,SolatiandSalari DevelopmentalPsychobiology platform(5cmÂ5cm)eachwithanopenroof.Themaze wasplacedinthecenterofaquietanddimlylitroom.The micebehaviorwasdirectlyobservedusingamirror,sus- pendedatanangleabovethemaze.Behavioraldatawere collectedbya‘‘blind’’observerwhoquietlysat1mbehind oneoftheclosedarmsofthemaze,usingachronometer.The anxietytestoftheoffspringwascarriedoutatpostnatalDay 61.Itwasrepeatedthreetimes,threeseparatecohortsofmale offspringwereusedfortestsandeachmousewasonlyused onEPMonce.Werepeatedthistestthreetimesduetoour differentresultsonEPMfromotherinvestigationsandthe resultsrepresentanaverageofthethreetestsessions.Fortest- ingpurpose,maleoffspringwereplacedindividuallyinthe centerportionoftheplus-maze,facingoneoftheopenarms. Theobservermeasured:(1)thetimespentintheopenarms, (2)thetimespentintheclosedarms,(3)thenumberof entriesintotheopenarms,and(4)thenumberofentriesinto theclosedarmsduringthe5-mintestperiod.Anentrywas definedasallfourpawsinthearm.Theelevatedplus-maze wasthoroughlycleanedwithdistilledwaterfollowingthe testingofeachanimaltoavoidpossiblebiasingeffectsdueto odorcluesleftbypreviousmice.Forthepurposeofanalysis, open-armactivitywasquantifiedastheamountoftimethat themicespentintheopenarms(OAT)relativetothetotal amountoftimespentinanyarm(open/totalÂ100),andthe numberofentriesintotheopenarms(OAE)wasquantified relativetothetotalnumberofentriesintoanyarm(open/ totalÂ100).Thetotalnumberofopenarmsentered,aswell asthetotalnumberofclosedarmsenteredwereusedas indexesofgenerallocomotoractivity(LMA)(Solati,Zarrindast, &Salari,2010;Zarrindast,Solati,Oryan,&Parivar,2008). Alltestingwasconductedbetween13:00and16:00hr. BodyWeight Thebodyweightsofmaleoffspring(Æ0.1g)wereregularly monitoredat13:00hrevery10daysduringtheexperiment frombirth(PND0)toPND40. FoodandWaterIntake Inordertofindoutwhetheri.p.exposuretoLPSduringpreg- nancyaffectsfood/waterintakeindamsorintheirmaleoff- springunderourexperimentalconditions,afeeding/drinking studywasconducted.Micehadfreeaccesstofoodandwater beforetheexperimentsbegan.Eachcagecontained3or4 micewhichweregivenwiththesameamountoffoodand water.Theirfoodintakewasmeasuredthefollowingdayby subtractingtheuneatenfoodmanually.Theamountofwater ingestedinourexperimentwasmeasuredwith0.2mlgradu- atedglassburettesadaptedwithametaldrinkingspout. ImmediatelyafterinjectionofLPS/salineinpregnantdams, eachmousewasreturnedtoitscageandwemeasuredthe cumulativewaterintakemanuallythefollowingday.These measurementsweredonein3daysafterinjectionoftheLPS/ salineinrespecttopregnantdams(N¼10ineachgroup) andin5daysinPND56-60inmaleoffspring(N¼10in eachgroup)anditwascalculatedasfood(ingramsÆ0.1g)/ water(inmlÆ0.2ml)intakepermiceperday.Measurements inalowpercentageoffetalanomalies,butnotabortionand possibleintra-uterinefetaldeath(IUFD)(Bell&Hallenbeck, 2002).Therationaleforchoosinggestationalday10wasthat thisperiodcorrespondstothefirst-to-secondtrimestersof humanpregnancy,withrespecttodevelopmentalbiologyand percentageofgestationfrommicetohuman(Kaufman,1992). Otherscientificliteraturesstatethatthistimephaseisthe periodofearlyfetalbraindevelopment(Wei,Li,&Zhou, 2007),cerebralorganogenesisinmice,especiallyneural-plate formation(Kirstenetal.,2010)andalsoembryonicstemcell formation(gestationalperiod0.38–0.53inrodents)whichis oneofthemainperiodsofvulnerabilityoftheimmunesystem toenvironmentalinsults(Merlotetal.,2008).Inaddition,other investigatorssuggestthatmaternalinfectionfromearlytomid pregnancyismorelikelytoberelatedtolong-lastingdevelop- mentalbrainandbehavioralabnormalitiesintheoffspring (Mednick,Machon,Huttunen,&Bonett,1988;Rodier& Hyman,1998). Eachdamwasadministered50mlsalineorLPSsolution. FollowinginjectionwithLPSorsaline,thedamscontinuedto behousedintheabove-mentionedconditions.Asweknow, LPScandisrupttheblood–brainbarrier(BBB)ifadminis- teredtopicallytothecerebralmicrocirculationorintracister- nally,anditispossiblethathighdosesofLPScrossthe placentaintothefetalcirculation(Urakuboetal.,2001). LowdosesofLPSwereselectedinordertopreventpossible directexposureoffetustoLPS.Withthisdoseandrouteof applicationweobservednoabortioninourendotoxingroups. Allinjectionsweregivenbetween13:00and14:00hr. MeasurementofSerumCorticosterone andCytokineConcentration Maternalserumfromtrunkbloodofpregnantdamswaspre- pared1.5hrafterinjectionofLPS/saline(N¼10ineach group)bycentrifugationat5,000gfor12min,aliquotedand thenstoredatÀ808Cuntilthecytokine/corticosteroneassays wereperformed.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. Trunkbloodwascollectedforpreparationofbloodserumof maleoffspringintheir9thweek(PND62)andmeasurement ofserumcorticosteroneandcytokineconcentrationwas carriedoutagaininthesameway.Allsamplesandstandards wereassayedinduplicate.Thepregnantmiceandtheirmale offspringwereselectedrandomly. AnxietyTest Oneofthemostpopulartestsofanxiety-likebehaviorin miceandratsistheEPM,inwhichthereducednumberof entriesortimespentintheopenarmsoftheEPMsuggests theoperationofanxiety-likeprocesses.Thiswooden,plus- shapedapparatuswaselevatedtotheheightof50cmabove thefloorandconsistsoftwoopenarms(30cmÂ5cm), twoenclosedarms(30cmÂ5cmÂ15cm),andcentral
DevelopmentalPsychobiology wereperformedbythesameexperimenterandweusedsepar- atecohortsofpregnantdamsforfood/waterintake,butthe samegroupsofmaleoffspringwereusedforfood/water intakeandanxietytestonEPM. StatisticalAnalysis DatawereanalyzedusingSPSS.Sincedatadisplayednormal distributionandhomogeneityofvariance,one-wayANOVA wasusedforcomparisonbetweentheeffectsofdifferent dosesofLPSwithvehicle.Differenceswithp<0.05 betweenexperimentalgroupsateachpointwereconsidered statisticallysignificant. RESULTS EffectsofLPSonSerumConcentrationsof TNF-a,IL-1b,andIL-6inPregnantMice andTheirMaleOffspring TheeffectsofLPStreatmentonTNF-a,IL-1b,andIL-6 inmaternalserumwereanalyzed.InresponsetoLPS PrenatalLPSExposureandMiceBehaviors 831 challenge,TNF-a(F3,36¼161.825,p<0.001),IL-1b (F3,36¼39.468,p<0.000)andIL-6(F3,36¼9.968, p<0.001)inmaternalserumwereincreased1.5hr afterLPSadministration(exceptforIL-6in50mg/kg LPS;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)in their9thweek(PND62). EffectsofLPSonSerumConcentrations ofCorticosteroneinPregnantMiceand TheirMaleOffspring TheeffectsofLPStreatmentonserumcorticosterone levelofpregnantdamsandtheirmaleoffspringare showninFigure1andTable1.OnewayANOVA confirmedthatinpregnantdams,highdosesofLPS (100or150mg/kg)increasedserumcorticosterone levelsrelativetosaline-treatedanimals(F3,36¼8.937, p<0.002)(Fig.1).ButprenatalLPSexposuredoes nothaveanysignificanteffectonserumconcentrations ofcorticosteroneinmaleoffspringintheir9thweek (PND62)(F3,36¼3.442,p<0.052)(Tab.1). EffectsofLPSonAnxiety-RelatedBehaviorsin MaleOffspring Figure2showsdataontheeffectsofprenatalexposure toLPSonanxiety-relatedbehaviorofmaleoffspringin theEPM.One-wayANOVArevealedthatprenatal exposuretoLPShasincreasedthepercentageofopen FIGURE1Effectofintraperitonealinjectionofsaline (50ml/mouse)orLPS(50,100,or150mg/kg)onserumlevel ofTNF-a,IL-1b,IL-6(a)andcorticosterone(b)inpregnant dams.EachbarismeanÆSEM.N¼10.Ãp<0.05, ÃÃ treatedgroup. armtime(F3,36¼49.396,p<0.000)andopenarm entries(F3,36¼8.843,p<0.000).Nodifferences werefoundinthetotalnumberofarmentriesinthe plus-maze(open-armsþclosed-arms)(F3,36¼1.604, p<0.215).Consideringalloftheseeffects,thepres- entlyobservedbehavioraldatasuggestlowlevelsof anxietyinprenatallyLPS-treatedoffspringwhichwas notreportedlikethisbefore. EffectsofPrenatalExposuretoLPSon BodyWeightinMaleOffspring Duringpostnataldevelopment,allthepupsgained bodyweightcontinuously,butindifferentwaysamong ourexperimentalgroups.Thebirthweight(PND0)of prenatallyLPS-treatedoffspring(LPS50:1.31Æ0.33g; LPS100:1.20Æ0.33g;LPS150:1.20Æ0.32g)was lowerthanprenatallysaline-treatedoffspring(1.62Æ 0.31g)(F3,36¼36.282,p<0.000).Furthermore,the totalbodyweightofprenatallyLPS-treatedoffspring waslowerthantheprenatallysaline-treatedoffspring during40daysofage[PND10(F3,36¼26.399, p<0.01,andÃÃÃp<0.001,whencomparedtothesaline
832 Asiaei,SolatiandSalari DevelopmentalPsychobiology Table1.EffectofPrenatalExposuretoLPSonSerumLevelofPro-InflammatoryCytokinesandCorticosteroneinMale OffspringinPND62(MeanÆSEM,N¼10inEachGroup) LPS50mg/kg LPS100mg/kg LPS150mg/kg Control(Saline) TNF-a(pg/ml) IL-1b(pg/ml) IL-6(pg/ml) 26.00Æ1.414 84.25Æ3.705 285.5Æ3.662 88.50Æ3.379 118.75Æ2.750 297.00Æ5.552 114.25Æ4.211 123.75Æ4.871 317.00Æ6.819 130.50Æ4.113 130.50Æ3.617 320.50Æ5.781 p<0.000),PND20(F3,36¼25.849,p<0.000),PND 30(F3,36¼8.174,p<0.001),PND40(F3,36¼5.366, p<0.006)](Fig.3).Itisimportanttonotethatwe observednomiscarriageorfetallossamongourgroups duringexperiment. EffectsofLPSonFoodandWaterIntakein PregnantMiceandTheirMaleOffspring AscanbeclearlyseeninFigure4,i.p.administration ofLPSreducedfoodintakeinthefirstdayafterinjec- tioninpregnantdams(F3,36¼13.465,p<0.000). Duringthe2nd(F3,36¼2.375,p<0.095)and3rd (F3,36¼1.685,p<0.197)dayafterchallenge,there werenosignificantdifferencesbetweenLPSand saline-treateddamsinfoodintake.Inotherwords, feedingthenreturnedtonormaloverthenext48–72hr astheLPSeffectdissipated.Itisalsoimportanttonote thatthereweresignificantdecreasesinfoodintake betweenprenatallyLPSorsaline-treatedmaleoffspring atpostnatalDay56–60[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). Theintraperitonealinjectionofsmallamountsof endotoxintakenfromSalmonellaentericasignificantly decreasedandalmostcompletelyinterruptedthewater intakeofpregnantdams.OnedayafterLPSexposure therewasasignificantdecreaseinwaterintakeamong alldams(F3,36¼47.734,p<0.000).Inotherwords, intheexperimentpresentedinFigure5,50mg/kgof endotoxinwassufficienttoalterwaterintake.There werenosignificantdifferencesinthewaterintakeof damswhichreceivedthesmallestdoseofendotoxin, comparedtothecontrolgroup,buttheinhibitionper- sistedlongerasthedosewasincreased(F3,36¼ 20.714,p<0.000).With100or150mg/kgofendo- toxin,thedailywaterintakeinpregnantdamsdidnot returntonormaluntiltheendofthe3rddayafterinjec- tion(F3,36¼4.975,p<0.008),(seeFig.5).Thewater FIGURE2EffectsofprenatalexposuretoLPSonanxiety- relatedbehaviorinoffspringintheelevatedplus-maze.Male offspringprenatallyexposedtosaline(50ml/mouse)orLPS Effectofprenatalexposuretosaline(50ml/ FIGURE3 (50,100,or150mg/kg)andanxietytestoftheseoffspring carriedoutatpostnatalDay61.EachbarismeanÆSEM. Openarmtime(OAT),openarmentries(OAE)orlocomotor ÃÃÃ ÃÃÃ mouse)orLPS(50,100,or150mg/kg)onbodyweightof maleoffspringfrombirthdaytoPND40.Theendotoxin offspringhadalowerbodyweightfrombirthdayupto PND40.EachbarismeanÆSEM.N¼10.ÃÃp<0.01and ÃÃÃ activity(LMA).N¼10. p<0.05, p<0.01,and p<0.001whencomparedtothesalinetreatedgroup. p<0.001whencomparedtothesalinetreatedgroup.
DevelopmentalPsychobiology PrenatalLPSExposureandMiceBehaviors 833 FIGURE4Effectofintraperitonealinjectionorprenatalexposuretosaline(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).Differentstudiesshowedthattheinhibitionof waterintakewasessentiallythesame,despitethestrain ofGram-negativespeciesfromwhichtheendotoxin wasprepared. intakeinprenatallyLSP-treatedmaleoffspringwas lowercomparedtoprenatally-salinetreatedoffspring inPND56–60[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)onwaterintakeinpregnantdamsandtheirmaleoffspring. EachbarismeanÆSEM.N¼10.Ãp<0.05,ÃÃp<0.01,andÃÃÃp<0.001,whencom- paredtothesalinetreatedgroup.
834 Asiaei,SolatiandSalari DevelopmentalPsychobiology DISCUSSION Thereisaconsensusthatsevereenvironmentalor psychologicalstress,includingexposuretoLPS,results inthereleaseofglucocorticoids,pro-inflammatory cytokines,ACTHandCRFinmaternalbloodstream. Thesechangescouldoperatedirectlyorindirectlyby regulatingtheactivationofHPAaxisandchangesin placentalmetabolismonfetaldevelopment.Among thesecandidates,maternalglucocorticoidsandpro- inflammatorycytokinesarethemaincandidatesforthe mediationofprenatalstresstothefetus(Harbuz& Lightman,1992;Johnsonetal.,1992;Weinstock,Pol- tyrev,Schorer-Apelbaum,Men,&McCarty,1998). Glucocorticoidsareknownfortheircentralrolesin immunocompetenceinanxietycircumstances(Dunn, 1989;Dunn&Berridge,1990;Merlotetal.,2008; Weinstocketal.,1998)andtheycanapplymanyorgan- izationaleffectsonprenataltissuedevelopment,includ- ingHPAdevelopment,leadingtochangesintheHPA axisfunctionthatpersistthroughoutlife(Merlotetal., 2008;Muglia,Jacobson,Dikkes,&Majzoub,1995). Inthisstudy,wemeasuredthelevelofcorticosterone, majormurineglucocorticoidhormone,andpro-inflam- matorycytokinesinbothpregnantdamsandtheirmale offspring. Asdescribedintheresults,weobservedan increasedlevelofpro-inflammatorycytokines(except forIL-6in50mg/kgLPS)andcorticosteroneinpreg- nantdamswhichreceived100or150mg/kgLPS,but notintheirmaleoffspring.Theseelevationsof maternalcytokinesandglucocorticoidscanresultin growthretardationandbehavioralalterationsintheoff- spring(Anisman,Hayley,etal.,2002;Anisman,Kokki- nidis,etal.,2002).Thepossiblemechanismisthat prenatalstresssuchasexposuretohighlevelofgluco- corticoidshaslong-termconsequencesonchild,includ- ingonneurodevelopment(Talge,Neal,&Glover, 2007;VandenBergh,Mulder,Mennes,&Glover, 2005).Glucocorticoidsinhibithippocampalcellpro- liferation(Gould,Cameron,Daniels,Woolley,&McE- wen,1992;Reuletal.,1994)andexerttheiractionvia directinfluenceonontogenyoffetalcellsbyreaching thefetalorgans.Inthisrespecthumanstudiesshow thatincreasedcortisolexposureaffectstheexpression ofoverathousandgenesinfetalbraincellsandthis exposureaffectthefunctionoftheplacenta,including theexpressionandactivityof11beta-hydroxysteroid dehydrogenase2(11b-HSD2),themainbarriertothe placentalpassageofglucocorticoids(Glover,Bergman, Sarkar,&O’Connor,2009;Mairesseetal.,2007; Salariaetal.,2006;Welberg,Thrivikraman,&Plotsky, 2005).Inhumanslowerplacentallevelsof11b-HSD2 havebeenfoundtobeassociatedwithintrauterine growthrestriction(Dy,Guan,Sampath-Kumar, Richardson,&Yang,2008;Gloveretal.,2009).Other humanstudiesshowthatsyntheticglucocorticoidsthat crosstheplacentacanaffectinfantneurodevelopment (Sizonenkoetal.,2006).Ontheotherhand,animal studiesalsodemonstratelong-termeffectsofprenatal administrationofsyntheticglucocorticoids,suchas dexamethasone,onoffspringbraindevelopmentand behavior.Forexample,someanimalworksshowthat prenatalexposuretoraisedglucocorticoidscandamage thebrainandcausedareductioninhippocampalvol- umeinrodentsandmonkeys(Bergman,Sarkar,Glover, &O’Connor,2010;Mitra&Sapolsky,2008;Uno etal.,1994).Thus,maternalhormonesseemtobegood candidatesforcommunicationbetweenthedamand developingfetus(Joffe,1969;Merlotetal.,2008). Althoughdirectevidenceforthesecausallinksislack- ing,andthemechanismsbywhichinfection-induced elevationofcytokineandglucocorticoidlevelsduring fetallifecaninterferewithneurodevelopmentalevents remainpoorlyunderstood. Ithasbeensuggestedthatinfectionsandinflamma- toryprocessesmaybecausativefactorsinemotional disorders,includinganxiety(Belzung&Griebel,2001; File,2001;Hogg,1996).Toexplorethishypothesis,we measuredtheanxiety-relatedbehaviorofmaleoff- springonEPM.PrenatalLPSexposurewithoutloco- motorimpairmentintheelevatedplusmaze,increased thepercentageofopenarmtimesandopenarmentries inadultmaleoffspring,indicatingtheinductionof anxiolyticresponsebyprenatalimmunechallenge, whichdonotagreewithothers’obtainedinsimilar contexts(Dunn&Berridge,1990;Fride&Weinstock, 1988,1989).Though,atfirsttheseresultsappearcon- tradictory,thediscrepancymayberelatedtotheg-ami- nobutyricacid(GABA)system.Prenatalstresshas beenfoundtoaffecttheGABAsystemindifferent brainregions,however,differentstressparadigms havedifferenteffectsontheGABAsystem(Bowers, Cullinan,&Herman,1998;Gruen,Wenberg,Elahi,& Friedhoff,1995;Montpiedetal.,1993).GABA,which isthemaininhibitoryneurotransmitterofbrain,hasa wellknownandveryimportantroleinthemodulation ofanxiety.Inaddition,itiswellknownthatGABA- ergicagentsanddrugshavestronganxiolyticeffectsin humansandanimals.Onefeasiblemechanismisthat prenatalexposuretoendotoxinchangesthesensitivity ofGABAAreceptorandfinallymodifiesthebehavioral responsestostressinadulthood(Jaiswal&Bhatta- charya,1993;Kellogg,Taylor,Rodriguez-Zafra,& Pleger,1993).Infact,LPSmayhavenegatedthe effectsofstressonneonatemice,forexample,through suppressionofastress-inducedsurgeofcorticosterone andalteringthecompositionandfunctionofthe
DevelopmentalPsychobiology PrenatalLPSExposureandMiceBehaviors 835 GABAAreceptorcomplexbymeansofatranscriptional mechanism(Stoneetal.,2001).Stoneetal.(2001) demonstratedthatelevationinthelevelofglucocorti- coidscanaffecttheGABAergicsystemandchangethe expressionofGABAAreceptorsubunitmRNAlevelsin thebrainregionsthatareinvolvedinmodulation anxiety,likehippocampus.Thiselevationalsoaffects mRNAsforglutamicaciddecarboxylase(GAD)iso- forms,theenzymethatconvertsglutamatetoGABA. Prenatalincreaseofcorticosteronelevelincreases GAD67mRNAinhippocampus.Increasedinhibitory inputbylocalGABAneuronsmaydiscusstheeffects ofprenatalstressesonreducedanxiety-relatedbehav- iorsinadulthood.Therefore,thereisapossibilitythat prenatalactivationofHPAaxisbyLPSmayincrease theactivityofGABAergicsysteminthebrainregions likehippocampusandinthiswaydecreasetheanxiety levelofoffspring(Stoneetal.,2001).Insum,itisclear thatourresultscannotdirectlyprovethisphenomenon andsurelyneedtobereplicatedbeforefirmconclusions canbedrawn. Inotherpartsofourstudyweexaminedtheeffects ofLPSexposureoningestivebehaviorsofpregnant damsandtheirmaleoffspring.Inthispartofthestudy weobservedthereductionoffoodandwaterintake inbothpregnantdamsandtheirmaleoffspring(PND 56–60).Thecentralmechanismsunderlyinganorexia duringexposuretobacterialcomponentsarenotfully understood.Severalstudiesconfirmedthatallsystems, signals,andswitches(physiologic,metabolic,neuro- logic,anatomic,endocrine,behavioral,etc.)caninflu- encefoodandwaterintake(Bachmanovetal.,2002; Kraly,1984)andMoyer,Herrenkohl,andJacobowitz (1978)suggestedthatprenatalstressmaycauseperma- nentchangesinnoradrenalinehypothalamiccontent withapossibleanorexiceffect(Moyeretal.,1978). Ourresults,whichconcurwithotherreportsinthe literature(Maccarietal.,2003;Maccari&Morley- Fletcher,2007;Seckl,2001;Tamashiro,Terrillion, Hyun,Koenig,&Moran,2009),demonstratedthat exposuretoincreasedlevelsofcorticosteroneandpro- inflammatorycytokinesduringprenatalstage,induce adverseeffectsoningestivebehaviorsinlaterlife. Theseresultsthushighlightthelong-terminfluences ofprenatalexposuretoLPS(whichleadstomaternal malnutrition,anxietyandhormonalimbalanceduring pregnancy)oningestivebehaviorsinmice.Although itneedsreplicationinlargercohortsbeforefirmcon- clusionscanbedrawn. Previousreportsontheeffectsofprenatalstresses onbodyweightarecontradictory.Someauthors reportedthatprenatalstressleadstoreducedbody weightofpups(Armario,Restrepo,Castellanos,& Balasch,1985;Klein&Rager,1995;Patinetal.,2002; Pollard,1984;Salgado,Martinez,&Tarres,1977; Valle´e,Mayo,Maccari,LeMoal,&Simon,1996), whereasotherstudiesfailedtofindaneffect(Fride, Dan,Feldon,Halevy,&Weinstock,1986;Glo¨ckner& Karge,1991;Hughes&Beveridge,1990).Thediscrep- anciesbetweenstudiescouldbeduetodifferencesin theprenatalstressprotocolsemployed.Ourstudy indicatesthatprenatalexposuretoSalmonellaendo- toxindecreasesfetalbodyweightatterm,indicatinga fetalgrowthrestriction.Inaddition,themaleoffspring withprenatalexposuretoLPSshoweddecreasein bodyweightswhentheygrewup,inwhichtheduration oftheeffectswasdirectlyrelatedtothedoseinjected. Thepossiblemechanism(s)isthatdecreaseinbody weightoftheoffspringmighthavebeeninducedby long-termeffectsofmaternalnutritionaldeficiency, hyperactivityoftheHPAaxis(Levittetal.,2000;Phil- lipsetal.,1998;Reynolds&Phillips,2000),excessive glucocorticoidlevelinfetalenvironment(Kriegetal., 1992)and/oralterationinorganmaturation(Novy& Walsh,1983;Reinisch,Simon,Karow,&Gandelman, 1978).Althoughthemechanismsunderlyingthese phenomenaarestillunclear,furtherunderstanding couldbegainedbyinvestigatingtheconsequencesof prenatalmanipulations. CONCLUSION Insummary,thisstudydemonstratedthatexposureto endotoxinduringfetallifecanleadtolong-lasting physiologicalconsequencesmostofthemdetrimental fortheanimalduringadulthood.Thisconclusionwas basedonthefollowingdataobservedindamsexposed toLPSinGD10duringpregnancyandtheirmaleoff- springversusthecontrolgroup:(1)Increasedpro- inflammatorycytokineandcorticosteronelevelsin maternalserum,(2)Decreaseinanxiety-relatedbehav- iorinmaleoffspring,(3)Decreaseinfoodandwater intakeinpregnantdams,andinterestinglyintheirmale offspring,(4)Restrictioninthebodyweightofmale offspringuptoPND40. REFERENCES Anisman,H.,Hayley,S.,Turrin,N.,&Merali,Z.(2002). Cytokinesasastressor:Implicationsfordepressiveillness. TheInternationalJournalofNeuropsychopharmacology, 5(04),357–373. Anisman,H.,Kokkinidis,L.,&Merali,Z.(2002).Further evidenceforthedepressiveeffectsofcytokines:Anhedo- niaandneurochemicalchanges.Brain,Behavior,and Immunity,16(5),544–556.
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