Effects Of Lithium On LPS Induced Rat Encephalitis And The Seizure Susceptibility Of Post-Encephalitis As Well As The Role Of GSK-3β In These Processes

Purpose: To build SD adult rat encephalitis model by intracerebroventricular injection of lipopolysaccharide(LPS)and test the inflammatory factors and GSK-3β in the tissues of hippocampus;To observe the effects of different doses of lithium salt on hippocampal pathophysiology,expression of GSK-3β and seizure sensitivity;To probe the role of GSK-3β in the process of LPS induced encephalitis and the seizure development of post encephalitis by applying GSK-3β agonist WT and its indirect inhibitor VPA to further intervene this kind of rat model.Aiming to investigate the impact of lithium salt on encephalitis and seizure of post encephalitis,as well as its possible mechanism,and hopefully provide a new strategy for the prevention of epilepsy development after intracranial infectionMethod:(1)Preparation of LPS induced intracranial infection rat model: 20 adult male SD rats were randomly divided into control group and LPS group(each group with 10 rats).Ten rats in the LPS group were injected with LPS 50μg/5ul through the lateral ventricles;the other ten rats in the control group were injected with the same volume of normal saline(NS).After 24 hours of intraventricular administration,5 rats in each group were randomly selected and were anaesthetized with 10 percent chloral hydrate followed by decapitating and separating bilateral hippocampal tissue.The proteins were extracted,and the ELISA and Western bolt were used to measure the concentration of IL-1β and TNF-α,and the expression ofp-gsk-3andgsk-3β,respectively.theremaining5 ratsineachgroupwereperfusedandtheirbrainsweretakenouttomakeparaffinsectionsaimingtotestthepathologicalchangesinthebrain.(2)effectsofdifferentdosesoflithiumsaltonlpsinducedencephalitisandseizure:48adultmalesdratswererandomlydividedintosixgroups:controlgroup,lpsgroup,lps-10 mgligroup,lps-20 mgligroup,lps-40mgligroupandlps-80mgligroup(eachgroupwith8rats).allratsexceptinthecontrolgroupwereinjectedwithlps50μg/5μlthroughthelateralventricles,whereas,ratsinthecontrolwereinjectedwiththesamevolumeofnormalsaline(ns)instead.threehourslater,theratsinabove-amentionedgroupswereinjectedonceadaywithns5ml/kg,ns,licl10mg/kg,20mg/kg,40mg/kg,80mg/kgrespectivelyfor3 days.then,theratswereinjectedintraperitoneallywithpilocarpine(pilo)toobservetheirepilepticbehaviors.(3)effectsofdifferentdosesoflithiumsaltonthehippocampalpathophysiologyandseizure,aswellasitspossiblemechanism:126adultmalesdratswererandomlydividedintolps-10 mgligroup,lps-20 mgligroup,lps-40 mgligroup,lps-80 mgligroup,lps-vpaligroup,lps-wtligroupandlpsgroup(eachgroupwith18rats).allratswereinjectedwithlps50μg/5μlthroughthelateralventricles.threehourslater,theratsinabove-amentionedgroupswereinjectedonceadaywithlicl10mg/kg,20mg/kg,40mg/kg,80mg/kg,vpa30mg/kg,wt0.6μg/kgandns5ml/kgrespectivelyfor3 days.then,8ratsineachgroupwererandomlyselectedandinjectedintraperitoneallywithpilo,followedbyintracranialelectrodeplacementandobservationofbasaleegspectrum,powerspectraldensity,changesinthehighfrequencyoscillatoryactivity,seizurelatencyanditsseverity.another5 ratswererandomlyselectedineachgroupandwereanaesthetizedtodecapitateandseparatebilateralhippocampaltissue.theproteinswereextracted,andtheelisaandwesternboltwereusedtomeasuretheconcentrationofil-1βandtnf-α,andtheexpressionofp-gsk-3andgsk-3β,respectively.theremainingratswereusedtotestthehippocampalneuronsandmicrogliaexpressionbyimmunohistochemistry.Results:(1)comparedwiththecontrolgroup:theratsinlpstreatedgroupshowedaincreasedbodytemperature,areductionindailyactivityandfoodintake,andaremarkableelevationofil-1βandtnf-αinthetissueofhippocampus(p<0.05).inaddition,theratsinlpstreatedgroupbecamemoresensitivetopilointhattheyhadmuchshorterlatencytose(p<0.05).meanwhile,theratsinlpstreatedgrouphadasignificantincreaseingsk-3β,butaremarkabledecreaseinp-gsk-3βinthetissueofhippocampus(p<0.05forboth)whencomparedwiththoseincontrolgroup.also,theratsinlpstreatedgrouphadalargenumberofproliferatedandactivatedmicrogliacells,andshowedloose,disorganizedandlighterlystainedneurons,whichareblurandunintact,intheareaofhippocampalca1.(2)afterinjectedintraperitoneallywithsmalldoseoflithium(10mgliand20mgli),theratsshowedaremarkabledecreaseinthepowerspectraldensityofrippleoscillationsinthefocalfieldpotentialofhippocampus,andalengthenedlatencytose(p<0.05forboth);afterinjectedintraperitoneallywith40mgli,theratshadalengthenedlatencytosebutwithnostatisticalsignificance(p>0.05);whereas,afterinjectedintraperitoneallywith80 mgli,theratsdemonstratedasignificantriseinthepowerspectraldensityandtheamplitudeofrippleoscillationsinthehippocampus,andashorterlatencytose(p<0.05).meanwhile,theratiosofp-gsk-3βandgsk-3β,andthecontentofinflammatorycytokinesil-1βandtnf-αinthetissueofhippocampusintheratsoflps-10 mgligroup,lps-20mgligroupandlps-vpagroupbecamehigherandlower,respectively(p<0.05forboth),buttheanti-inflammatorycytokinesofil-10increased(p<0.05),andinthesegroups,theratshadonlysmallnumberofproliferatedandactivatedmicrogliacells(p<0.05),andkeptrelativelydenseandorganizedarrangementofneurons,whichweredarklysatinedandintact,intheareaofhippocampalca1,whencomparedwithpurelylpstreatedrats.onthecontrary,theratsinlps-80mgligroupandlps-wtgrouphadtotallyoppositeresultswhencomparedwithratsinabove-mentionedgroups.Conclusions:lpscanresultinencephalitisinsdrats.thiskindofinfectedrathasdecreasednumberofneurons,andincreasednumberofactivatedmicrogliacellsinthehippocampalca1area;intracranialinfectionmaypromoteseizures,andlowdosesoflithiummayinhibittheinflammatoryactivityandmicroglialproliferation,andreducebraininjuryafterencephalitis.instead,largedosesoflithiumfunctionintheoppositeway.lowdoseoflithiumcanlengthenthelatencyofepileptiformdischarges,reducetheamplitudeofrippleoscillations,decreasetheexcitabilityofhippocampalneurons,increasethethresholdofseizure,andthusresultindecreasedsensitivitytoseizures,showinglowdoseoflithiummayhaveamodifyingeffectonthedevelopmentofpostencephalitisepilepsy.lowdosesoflithiuminhibitsboththeexpressionofgsk-3βandthereleaseofinflammatoryfactors,andreducesbraininjury,suggestinggsk-3βmightinvolveinthemechanismoflithiummodifyingpost-encephalitisepilepsy.