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Effects_11

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Effects_11Effect

    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.nimodipinecanleadtoLtypechannel

    ;inactivationandpreventcalciumi0ninflow.therebyexhibitingprotectiveeffectsonnervecells.

    ;oBJECTIVE:Toobservetheprotectiveeffectsofnimodipineonglutamate.inducedinjurytoembryonicrat

    ;neurastemcells.andtomakeacomparisonwithMK

    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

    ;minutenimodipine[1x(10(一

    10.)g,L]culturefollowedbyaglutamate(200umol/L)treatmentstep.(4) ;MK-801.givenas30minuteMK

    8O1f100umol/L)culture.followedbyaglutamate(200utool/L)

;treatmentstep.

    ;MAIN0UTC0MEMEASURES:Determinationofglutamateinducedcel1deathbymethylthiazolyl

    ;tetrazo”um(MTT)assay;calculationofneuralstemccIlsurvivairatefollowingadditionofnimodipine.

    ;RESULTS:Thesurvivalrateofneura1stemcellswasapproximately25.26%following24

    hour50utool/L

    ;glutamatecultureandgraduallydecreasedastheglutamatedoseincreasedfP<0.()5O.0

    1).Only9.27%of

    ;neuralstemcellssurvivedwhentheglutamatedosewas1000mol/L.Thesurvivalrateofneu

    ralstemcells

    ;wassignificantlyhigher,inadosedependentmanner,inthenimodipine[1x(1010)g/L

    groupthaninthe

    ;glutamategroup.Inaddition.theMKI801groupexhibitedahighersurvivalrateafter24hou

    rtreatmentthan

    ;theglutamategroupr<0.0l1

    ;C0NCLUS10N:Glutamate(50(l000”mol/L)jnducesinjurytoneura

    stemcellsdosedependently.

    ;Nimodipineexhibitsprotectiveerfectsoninjurytoneuralstemcellsandpresentstheeffects

    ina

    ;dose(dependentmannerat1x(10一一

    10.1g/L.Nimodipinedisplaysneuroprotectiveeffectsequivalentto ;MK801_

    ;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.andmultidirectionaldifferentiation

    ;andself-renewalpotential1-21.

    ;Glutamate.asaprimary

    ;excitatoryneurotransmitter,playsanimportantrolein ;dopaminemediatedsynapticexcitabilityandsynaptic ;plasticityinneuralstemcellculture.Inaddition.the ;substantialaccumulationofglutamateisakeyfactorin ;neurona1iniury.Thereisstrongevidencedemonstratingthat ;glutamatecaninducedamagetocerebellargranulosacells, ;andtostriata1.cortica1.andhippocampaIneurons.’”.

    ;Glutamatemediatesneurotoxicityprimarilythroughcalcium ;overloadl,_oxygenfleeradicals,decreasedglutathione,and ;mitochondria?niury.Calciumoverloadparticipatesinthe ;mechanismofoxygenradicalinduceddamage.Atleast

    ;threetypesofcalciumionchannels(T.N,andL)havebeen ;recognizedinnervecells.butonlytheLtypeofchanneis

    ;sensitivetodrugs.Theoretically.nimodipinenotonlyleadsto ;LtvDechanne1inactivationandpreventscalciumioninflow, ;therebyexhibitingprotectiveeffectsonnervecells,butalso ;improvescerebralbloodflowbyinhibitionofcerebrOvascular ;Received:20080801;Accepted:20081021(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):12869

    ;contraction.Previousinvivoandinvitrostudiesmainly ;investigatedtheprotectiveeffectsofnimodipineoninjuryto ;vascularendothelia1cells.cerebra1corticalneurons.and ;hippocampalfrontalcellsLittleisknownaboutthe ;protectiveeffectsofnimodipineonglutamateinducedinjury

    ;toneuralstemcells.Thepresentstudyexaminedthe ;protectiveeffectsofnimodipineonglutamateinducedneura1

    ;stemcellinjury.

    ;MATERlALSANDMEn_10DS

    ;Design

    ;/nvitroexperimentpertainingtoneuralstemcells. ;Timeandsetting

    ;PerformedattheDepartmentofNeurology,FirstHospital, ;JilinUniversitybetweenJanuary2005andDecember2006. ;Materials

    ;CleanWistarratsonday15ofgestationwereprovidedbythe ;LaboratoryAnimalCenter.BasicMedicalCollege.Jilin

;UniversitypermissionNo.SCXK(J)20030001].The

    ;followingprotocolcompliedwiththeRegulationsforthe ;AdministrationofAffairsConcerningExperimentalAnimals, ;formulatedbytheMinistryofScienceandTechnologyofthe ;People’sRepublicofChinaiU1.

    ;Detailsofmaterialsareshownasfollows.

    ;MaterialSource

    ;Dulbecco’smodifiedeagle’smediumGibco,USA

    ;(DMEM)/Fl2culturemedium.basic

    ;fibroblastgrowthfactors(bFGF),

    ;epidermalgrowthfactor(EGF)

    ;PolyLlysine(PLL),streptomycinSigma,uSA

    ;sulfate.methylthiazolyltetrazolium

    ;fMTT).sodiumdodecylsulfate(SDS)

    ;DNaseIenzyme,PLL(PolyLsine),PekingDingguo

    ;3-3’-diaminobenzidine(DAB1Biotechnology

    ;developingreagent(Amersco),SPkitCo.,Ltd.,China ;GlutamateShanghaiBiological

    ;ResearchInstituteof

    ;theChinese

    ;Academyof

    ;Sciences,China

    ;NimodipineBayer,Germany

    ;B10RADModel550enzymereaderBioRad.USA

    ;Methods

    ;Invitroisolationandcultureofneuralstemcells ;Followinganesthesiabyintraperitonealinjectionof0.5g/kg ;chloralhydrate,ratembryosweretakenoutunderaseptic ;conditionsandplacedintosterilephosphatebufferedsaline ;(PBS).Preparedbraintissuewasdigestedfor30minuteswith ;0.1%trypsinr1:250)and40mg/LDNaseIenzymeinawater ;bathat37?.Followingcentrifugation.thecellsuspension ;wasaddedtoastemcellculturemediumcontaining ;DMEM/F12.bFGF.andEGFandfilteredwitha200mesh

    ;cel1sieve.Theresultingsinglecellsuspensionwasusedfor ;cel1countingwithtrypanblue.Singlecellswereinoculated ;ontoaT25culturcflaskat3x10/mL.Halftheculturemedium ;wasrenewedevery34days.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

    ;ina24wellcultureplateforanadditional7daysofculture. ;Duringthisprocess,halftheculturemediumwasrenewed ;every34days.

    ;Groupmanagementandmedication

    ;Passage2stemeel1spheresweredispersedandmadeintoa ;singlecellsuspension.Afterbeingresuspendedingrowth ;medium.cellswereinoculatedontoa0.01%PLLcoated

    ;96wellplasticcultureplateatadensicyof20000cellsper ;wel1.Fourgroupswereformedasfollows.

    ;fl1Normalcontrolgroup,95uLoftheabovementioned

    ;singleeellsuspensionand5uLstemcellculturemedium ;e..Hankssolution,pHvalue7.27.41wereaddedto13 ;wells.

    ;(2)Glutamategroup,95uLoftheabovementionedsingle

    ;cellsuspensionand5Lglutamate(50,100,200,500,and ;1000umol/Lwereaddedto13wells.

    ;(3)Nimodipinegroup,95uLoftheabovementionedsingle

    ;cellsuspensionand5Lnimodipinesolutionf1x(10(一

    ;10.)g/L]wereaddedto11wells;30minutes1ater,5L ;glutamate(200mo1/L1wasaddedtoeachwel1.

    ;(4)MK801group,95uLoftheabovementionedsinglecell

    ;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):12869

    ;(SPSS,USA)andwereexpressedasMean?SD.t-testswere ;usedforcomparisonbetweengroups.

    ;RESUU-s

    ;Protectiveeffectsofnimodipineonglutamate-induced ;neuralstemcellinjury(Table1)

    ;Table1iSurvivalrateneuralstemcellsafterglu~te|

    .-|0|氆熬 ;administration.|

    ;GrouptGlutamatedusenCellularsurvivalrate ;Normalconlrol13|27.86~5,~30l|

    ;Glutamate50Itmol/L01325?sl65||

    ;100mol/L1322西

    ;200umo1/L13l1858~6qb5.

    ;,

    ;~

    ;500mol/L1313.39+--5..’~9b.

    ;1000moIlL9.27~3.:;1bl.

    ;MK-8011130.55~1.~5

    ;NimodipineIxL1?6

    ;lxl0’/g/L11A

    ;.嚣耋嚣--lxlWeJLl124.12~3.35~

    ;lxl0-31?萋垃懵

    ;lxl0”z!129,27~2.71e

    ;0.05,J(0.01.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,inadosedependentmanner,inthe

    ;nimodipine1x(1O’-10.)g,L]groupthanintheglutamate

    ;group(P<0.01).Inaddition,theMK801groupexhibiteda

    ;highersurvivalrateofneura1stemcellsthantheglutamate ;groupafter24hourtreatmentf<0.01,.

    ;DISCUSS10N

    ;Maintenanceofglutamatephysiologicalconcentrationsin ;thecentralnervoussystem

    ;Thecentralnervoussystemmainlyreliesontheglutamate ;transporterinneuralcellsandneuroglialcellstomaintainthe ;physiologicalconcentrationofglutamate.Changesin ;transporterstructureandfunctioncancauseglutamatetopile ;uporglutamateconcentrationtorisedirectlyandactivate ;glutamatereceptors.whileconcomitantlyinhibitingthe ;glutamate/cystinetransporter.Thesetwomechanisms ;coparticipatejnneura1eelljnjury.Glutamatemediates ;neurotoxicityprimarilythroughcalciumoverload,oxygenflee ;radicals,decreasedglutathione,mitochondrialinjury ;sphingomyelinase.ceramideandcytochromeC..Calcium ;overloadisinvolvedinthemechanismofoxygen

    ;l288

    ;radicalinduceddamage.Thepresentresultsconfirmedthat ;glutamate(501000umol/L,inducedneuralstemcell

    ;injuryinadosedependentmanner.Thrdtistosay.thesurvival ;rateofneuralstemcellsisgraduallYdecreasedasthe ;glutamatedoseincreases.

    ;ProtectiveeffectsofnimodipineonneuraIstemeell ;injury

    ;Thepresentstudyused200mol/Lglutamateasan

    ;experimentalcondition.Thirtyminutespriortoadditionof ;200umol/Lglutamate,nimodipinewasappliedatseven ;diflferentconcentrati0ns.Thesurvivalrateofneuralstemcells ;wassignificantlyhigher,inadosedependentmanner,inthe

    ;nimodipinefx(10-J10)g,Lgroupthanintheglutamate

    ;groupfP<0.O1).Theseresultsindicatethatnimodipine ;exhibitsprotectiveeffectsonglutamateinducedinjuryto

    ;neuralstemcells,.e.,nimodipinecanalleviatesuchaninjury. ;Intracellularcalciumionimbalancehasbeenconsidereda ;“commonpathway’’tocellularinjurycausedbyvarious

    ;factors.Manysubstancescanalleviateglutamateinduced

    ;neuronalinjury.whichisassociatedwithinhibitionofcalcium ;ioninflowandstabilizationofintracellularcalciumion ;concentrationthroughdifferentpathways.Nimodipine.as ;acalciumionantagonist,exhibitsprotectiveeffectsonneural

    ;stemeellinjurybystabilizingcellularcalciumion ;concentration.

    ;Takentogether,glutamatef50(1000mol/L)caninduce

    ;injurytoneuralstemcellsinadosedependentmanner,.e.,

    ;thesurvivalrateofneuralstemcellsisgraduallydecreasedas ;theglutamatedoseincreases.Nimodipinef1x(10(一10.)g/L]

    ;exhibitsdosedependentprotectiveeffectsonneura1stemcell ;injury.

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