Effects
NEURALREGENERATIONRESEARCH
;Volume3,Issue12,December2008www.
;zsyJconl
;Effectsofglutamateandnimodipineonsurvivalrateof
;embryonicratneuronalstemcellsculturedinvitro?
;XiaohongLtt,HailongFu,QiangSun,LiCui,DihuiMa,WeihongLin
;DepartmentofNeurology,FstHospital,JinlinUniversity,Changchun1300021,JilinPrownce,China
;Abstract
;BACKGRoUND:AtleastthreetypesofcalciumionchannelfTN,andL)havebeenrecognizedinnerve
;ccIls.butonlytheLtypeofchannelissensitivetodrugs.Theoretically.nimodipinecanleadtoL—typechannel
;inactivationandpreventcalciumi0ninflow.therebyexhibitingprotectiveeffectsonnervecells.
;oBJECTIVE:Toobservetheprotectiveeffectsofnimodipineonglutamate.inducedinjurytoembryonicrat
;neura】stemcells.andtomakeacomparisonwithMK一
801.anonselectiveglutamatereceptorantagonist.
;DESIGN.TIMEANDSETTING:Thepresentinvitroexperimentpertainingtoneura1stemcellswas
;performedattheDepartmentofNeurologyFirstHospital,JilinUniversitybetweenJanuary2005and
;December2006.
;MATERIALS:GlutamatewassourcedfromtheShanghaiBiologicalResearchInstituteoftheChinese
;AcademyofSciences.NimodipinewasprovidedbyBayerCompany,Germany.Braintissuewastakenfrom
;Wistarratsonday15ofgestationforisolationandcultureofneuralstemcells. ;METH0DS:Passage2neura1stemcellspheresweretakenf0rpreparationofsinglecel1suspensionThe
;preparedsinglecellsuspensionwasdividedinto4groups:(1)Normalcontrol,normallycultured.(2)
;Glutamateculturedwith5O,1?,200,5OHD,and】
0o()umol/Lglutamate(3)Nimodipinc,receiveda30一
;minutenimodipine[1x(10(一
10.)g,L]culturefollowedbyaglutamate(200umol/L)treatmentstep.(4) ;MK-801.givenas30一minuteMK一
8O1f100umol/L)culture.followedbyaglutamate(200utool/L)
;treatmentstep.
;MAIN0UTC0MEMEASURES:Determinationofglutamate—inducedcel1deathbymethylthiazolyl
;tetrazo”um(MTT)assay;calculationofneuralstemccIlsurvivairatefollowingadditionofnimodipine.
;RESULTS:Thesurvivalrateofneura1stemcellswasapproximately25.26%following24
hour50utool/L
;glutamatecultureandgraduallydecreasedastheglutamatedoseincreasedfP<0.()5O.0
1).Only9.27%of
;neuralstemcellssurvivedwhentheglutamatedosewas1000mol/L.Thesurvivalrateofneu
ralstemcells
;wassignificantlyhigher,inadose—dependentmanner,inthenimodipine[1x(1010)g/L】
groupthaninthe
;glutamategroup.Inaddition.theMKI801groupexhibitedahighersurvivalrateafter24hou
rtreatmentthan
;theglutamategroupr尸<0.0l1
;C0NCLUS10N:Glutamate(50(l000”mol/L)jnducesinjurytoneura】
stemcellsdose—dependently.
;Nimodipineexhibitsprotectiveerfectsoninjurytoneuralstemcellsandpresentstheeffects
ina
;dose(dependentmannerat1x(10一一
10.1g/L.Nimodipinedisplaysneuroprotectiveeffectsequivalentto ;MK一801_
;KeyWords:neuralstemcells;culture;glutamate;injury;nimodipine;protectiveeffects
;XiaohongLn?,MD,
;Professo5Departmentof
;Neurology,FirstHospital,
;JinlinUniversity,Changchun
;1300021,JilinProvince,
;China
;L?XD,FuHLlSunQ,CuIL,
;MaDHLinWHEffectsof
;glutamateandnimodipJneon
;sun,IvaIrateofembrvonicral
;neurona1stemcellscultured
;lnvitroNeuraIRegenRes
;2008;3(12):1286-9
;wwWCnerCn
;WWWSlZSYlcom
;lNTRODUCTl0N
;Duringinvestigations.neuralstemcellshavebeenisolated ;andpurifiedfromembryonicandadultbraintissue.NeuraI ;stemcellshavebeenshowntobeanewstrategyfortreatment ;ofnervoussystemdiseasesduetotheirdivisionand
;proliferationcapabmty.andmulti—directionaldifferentiation
;andself-renewalpotential1-21.
;Glutamate.asaprimary
;excitatoryneurotransmitter,playsanimportantrolein ;dopamine—mediatedsynapticexcitabilityandsynaptic ;plasticityinneuralstemcellculture.Inaddition.the ;substantialaccumulationofglutamateisakeyfactorin ;neurona1iniury.Thereisstrongevidencedemonstratingthat ;glutamatecaninducedamagetocerebellargranulosacells, ;andtostriata1.cortica1.andhippocampaIneurons.’”.
;Glutamatemediatesneurotoxicityprimarilythroughcalcium ;overloadl,』_oxygenfleeradicals,decreasedglutathione,and ;mitochondria?niury.Calciumoverloadparticipatesinthe ;mechanismofoxygenradical—induceddamage.Atleast
;threetypesofcalciumionchannels(T.N,andL)havebeen ;recognizedinnervecells.butonlytheLtypeofchanne】is
;sensitivetodrugs.Theoretically.nimodipinenotonlyleadsto ;L—tvDechanne1inactivationandpreventscalciumioninflow, ;therebyexhibitingprotectiveeffectsonnervecells,butalso ;improvescerebralbloodflowbyinhibitionofcerebrOvascular ;Received:2008—08—01;Accepted:200810—21(15200808010001/H)
;COrresp0ndingauthor:XiaohongLn,MD.,Professor,DepartmentofNeurology,FirstHo
spital,JinlinUniversity
;Changchun1300021,JilinProvince,China
;E-mail:lvxiaohongstudent@sina.com
;1286
;LaXH,eta1./NeuralRegenerationResearch,2008,3(12):1286—9
;contraction.Previousinvivoandinvitrostudiesmainly ;investigatedtheprotectiveeffectsofnimodipineoninjuryto ;vascularendothelia1cells.cerebra1corticalneurons.and ;hippocampalfrontalcellsLittleisknownaboutthe ;protectiveeffectsofnimodipineonglutamate—inducedinjury
;toneuralstemcells.Thepresentstudyexaminedthe ;protectiveeffectsofnimodipineonglutamate—inducedneura1
;stemcellinjury.
;MATERlALSANDMEn_10DS
;Design
;/nvitroexperimentpertainingtoneuralstemcells. ;Timeandsetting
;PerformedattheDepartmentofNeurology,FirstHospital, ;JilinUniversitybetweenJanuary2005andDecember2006. ;Materials
;CleanWistarratsonday15ofgestationwereprovidedbythe ;LaboratoryAnimalCenter.BasicMedicalCollege.Jilin
;University【permissionNo.SCXK一(J)2003—0001].The
;followingprotocolcompliedwiththeRegulationsforthe ;AdministrationofAffairsConcerningExperimentalAnimals, ;formulatedbytheMinistryofScienceandTechnologyofthe ;People’sRepublicofChinaiU1.
;Detailsofmaterialsareshownasfollows.
;MaterialSource
;Dulbecco’smodifiedeagle’smediumGibco,USA
;(DMEM)/Fl2culturemedium.basic
;fibroblastgrowthfactors(bFGF),
;epidermalgrowthfactor(EGF)
;Poly—L—lysine(PLL),streptomycinSigma,uSA
;sulfate.methylthiazolyltetrazolium
;fMTT).sodiumdodecylsulfate(SDS)
;DNaseIenzyme,PLL(Poly—L一sine),PekingDingguo
;3-3’-diaminobenzidine(DAB1Biotechnology
;developingreagent(Amersco),SPkitCo.,Ltd.,China ;GlutamateShanghaiBiological
;ResearchInstituteof
;theChinese
;Academyof
;Sciences,China
;NimodipineBayer,Germany
;B10一RADModel550enzymereaderBio—Rad.USA
;Methods
;Invitroisolationandcultureofneuralstemcells ;Followinganesthesiabyintraperitonealinjectionof0.5g/kg ;chloralhydrate,ratembryosweretakenoutunderaseptic ;conditionsandplacedintosterilephosphatebufferedsaline ;(PBS).Preparedbraintissuewasdigestedfor30minuteswith ;0.1%trypsinr1:250)and40mg/LDNaseIenzymeinawater ;bathat37?.Followingcentrifugation.thecellsuspension ;wasaddedtoastemcellculturemediumcontaining ;DMEM/F12.bFGF.andEGFandfilteredwitha200一mesh
;cel1sieve.Theresultingsinglecellsuspensionwasusedfor ;cel1countingwithtrypanblue.Singlecellswereinoculated ;ontoaT25culturcflaskat3x10/mL.Halftheculturemedium ;wasrenewedevery3—4days.Oneweeklater.thenumbers
;andsizesofsuspendedcellsphereswereobserved.andthen ;cellsphereswerecollectedandmadeintosinglecel1 ;suspensionsforfurtherculture.Passagewasperformedif ;permissionwasgiven…】.
;Cellcloning,continuouspassage,andinductionof ;differenfjation
;Asingleneura1stemcellspherewithadiameterof ;approximately20011mwastakenfromthecultureflask ;usingaPasteurtubeandmadeintoasinglecellsuspension. ;Subsequently,thesinglecellsuspensionwasinoculatedinto ;stemcellculturemediumtopreventcellaggregation.Seven ;dayslater,asinglestemcel1spherewastakenoutfromthe ;cultureflaskandwasgentlysuspendedinthedifferentia1 ;mediumcontainingDMEM/F12andfetalbovineserum.Next.
pretreatedslides ;thedispersedcellswereinoculatedontoPLL—
;ina24一wellcultureplateforanadditional7daysofculture. ;Duringthisprocess,halftheculturemediumwasrenewed ;every3—4days.
;Groupmanagementandmedication
;Passage2stemeel1spheresweredispersedandmadeintoa ;singlecellsuspension.Afterbeingresuspendedingrowth ;medium.cellswereinoculatedontoa0.01%PLL—coated
;96一wellplasticcultureplateatadensicyof20000cellsper ;wel1.Fourgroupswereformedasfollows.
;fl1Normalcontrolgroup,95uLoftheabove—mentioned
;singleeellsuspensionand5uLstemcellculturemedium ;e..Hankssolution,pHvalue7.27.41wereaddedto13 ;wells.
;(2)Glutamategroup,95uLoftheabove—mentionedsingle
;cellsuspensionand5Lglutamate(50,100,200,500,and ;1000umol/Lwereaddedto13wells.
;(3)Nimodipinegroup,95uLoftheabove—mentionedsingle
;cellsuspensionand5Lnimodipinesolutionf1x(10(一
;10.)g/L]wereaddedto11wells;30minutes1ater,5L ;glutamate(200mo1/L1wasaddedtoeachwel1.
;(4)MK一801group,95uLoftheabove—mentionedsinglecell
;suspensionand5uLglutamate(200umol/L1wereadded ;to11wells[-H:30minutes1ater,5Lglutamate(200umol/L) ;wasaddedtoeachwel1.
;Followingtreatment,eachgroupreceivedanadditional24 ;hoursofculture.
;DeterminationofcellularsurvivalratebyMTTassay ;Mitochondria1succinicaciddehvdrogenasereduced ;exogenousMTTtoslightlysolubleformazan.whichdeposited ;inthecells,givingthemapurplecolor.Methylsulfoxide ;coulddissolvetheintracellularpurpleformazan.Thedegree ;ofcolorwaspositivelycorrelatedtoenzymeactivity.So ;absorbancemeasuredat550nmcouldreflectsuccinicacid ;dehydrogenaseactivity.Fourhoursbeforeculturetermination, ;10L0.5%MTTPBSwasaddedtOthecultureanditwas
;furtherincubatedin0.05%CO,Followingculture.10% ;sodiumdodecylsulfate(SDS)and0.01mol/LHClwere ;added.resultingindarkblueformazan.whichindirectly ;reflectsthenumberoflivingcells.Absorbanceofdarkblue ;formazanwasmeasuredat550nmonanenzymereader. ;Mainoutcomemeasures
;Survivalrateofneura1stemcells.
;StatisticaIanalysis
;Al1datawerestatisticallyprocessedusingSPSSl0.0software ;1287
;LUXH,ela1./NeuralRegenerationResearch,2008,3(12):1286—9
;(SPSS,USA)andwereexpressedasMean?SD.t-testswere ;usedforcomparisonbetweengroups.
;RESUU-s
;Protectiveeffectsofnimodipineonglutamate-induced ;neuralstemcellinjury(Table1)
;Table1iSurvivalrateneuralstemcellsafterglu~te|
.-一|0|氆熬 ;administration.|一
;GrouptGlutamatedusenCellularsurvivalrate ;Normalconlrol13|一27.86~5,~30l|
;Glutamate50Itmol/L013囊25?sl65||
;—100mol/L1322西
;200umo1/L13l1858~6qb5.
;,
;~
;500mol/L1313.39+--5..’~9b.
;1000moIlL9.27~3.:;1bl.
;MK-8011130.55~1.~5
;NimodipineIx强L1?鹞6
;lxl0’/g/L11A似
;.嚣耋嚣--lxlWeJLl124.12~3.35~
;lxl0-3扎1?萋垃懵
;lxl0”z!129,27~2.71e
;《0.05,J(0.01坩.normaIcontrolgroup;cp<0,01.,vs.glutamate ;gro
;Following24hoursofglutamate(inparticular200umol/L1 ;treatment.thesurvivalrateofneuralstemcellswas ;significantlydecreasedcomparedwiththenormalcontro1 ;groupfJp<0.O1,.Thesurvivalrateofneuralstemcellswas ;approximately25.26%when50umol/Lglutamatewas ;administeredandgraduallydecreasedastheglutamatedose ;increasedfP<0.050_(】1).Only9_27%ofneuralstemcells ;werefoundtosurvivewhentheglutamatedosewas ;1000?mol/L.Theneuralstemcel1survivalratewas
;significantlyhigher,inadose—dependentmanner,inthe
;nimodipine【1x(1O’-10.)g,L]groupthanintheglutamate
;group(P<0.01).Inaddition,theMK一801groupexhibiteda
;highersurvivalrateofneura1stemcellsthantheglutamate ;groupafter24hourtreatmentf尸<0.01,.
;DISCUSS10N
;Maintenanceofglutamatephysiologicalconcentrationsin ;thecentralnervoussystem
;Thecentralnervoussystemmainlyreliesontheglutamate ;transporterinneuralcellsandneuroglialcellstomaintainthe ;physiologicalconcentrationofglutamate.Changesin ;transporterstructureandfunctioncancauseglutamatetopile ;uporglutamateconcentrationtorisedirectlyandactivate ;glutamatereceptors.whileconcomitantlyinhibitingthe ;glutamate/cystinetransporter.Thesetwomechanisms ;co—participatejnneura1eelljnjury.Glutamatemediates ;neurotoxicityprimarilythroughcalciumoverload,oxygenflee ;radicals,decreasedglutathione,mitochondrialinjury ;sphingomyelinase.ceramideandcytochromeC..Calcium ;overloadisinvolvedinthemechanismofoxygen
;l288
;radical—induceddamage.Thepresentresultsconfirmedthat ;glutamate(50—1000umol/L,inducedneuralstemcell
;injuryinadose—dependentmanner.Thrdtistosay.thesurvival ;rateofneuralstemcellsisgraduallYdecreasedasthe ;glutamatedoseincreases.
;ProtectiveeffectsofnimodipineonneuraIstemeell ;injury
;Thepresentstudyused200mol/Lglutamateasan
;experimentalcondition.Thirtyminutespriortoadditionof ;200umol/Lglutamate,nimodipinewasappliedatseven ;diflferentconcentrati0ns.Thesurvivalrateofneuralstemcells ;wassignificantlyhigher,inadose—dependentmanner,inthe
;nimodipinef】x(10-J10)g,L】groupthanintheglutamate
;groupfP<0.O1).Theseresultsindicatethatnimodipine ;exhibitsprotectiveeffectsonglutamate—inducedinjuryto
;neuralstemcells,.e.,nimodipinecanalleviatesuchaninjury. ;Intracellularcalciumionimbalancehasbeenconsidereda ;“commonpathway’’tocellularinjurycausedbyvarious
;factors.Manysubstancescanalleviateglutamate—induced
;neuronalinjury.whichisassociatedwithinhibitionofcalcium ;ioninflowandstabilizationofintracellularcalciumion ;concentrationthroughdifferentpathways.Nimodipine.as ;acalciumionantagonist,exhibitsprotectiveeffectsonneural
;stemeellinjurybystabilizingcellularcalciumion ;concentration.
;Takentogether,glutamatef50(1000mol/L)caninduce
;injurytoneuralstemcellsinadose—dependentmanner,.e.,
;thesurvivalrateofneuralstemcellsisgraduallydecreasedas ;theglutamatedoseincreases.Nimodipinef1x(10(一10.)g/L]
;exhibitsdose—dependentprotectiveeffectsonneura1stemcell ;injury.
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