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CerebralIschemicInjury
and
Neuroprotection缺血性神經(jīng)元損傷及其修復(fù)ReferencesMolecularNeuropharmacology.EricJ.Nestler,etal.McGraw-HillCompanies.《醫(yī)學(xué)神經(jīng)生物學(xué)》第25章。孫鳳艷主編,上??茖W(xué)技術(shù)出版社。Contents
BriefintroductionaboutischemiaExperimentalmodelsusedinresearchMechanismsunderlyingischemicneuronalinjuryNeuroprotection
BriefIntroductionAdecreaseinthebloodsupplytoabodilyorgan,tissue,orpartcausedbyconstrictionorobstructionofthebloodvessels.ISCHEMIAis:
(NewLatinischaemia,fromGreekiskhaimos,astoppingoftheblood:iskhein,tokeepback;seesegh-inIndo-Europeanroots+haima,blood)/ischemiaMayalsobespelledas:ischaemiaorisch?mia
Whatisischemia?Related
TermsIschemia
缺血:shortageofthebloodsupply(oxygen,glucose,etc)Hypoxia
缺氧:shortageofoxygenAnoxia
缺氧:anextremeformofhypoxia,absenceofoxygenIschemiccascade
缺血級(jí)聯(lián)反應(yīng):aseriesofbiochemicalreactionsthattakeplaceaftersecondstominutesofischemia.Brainischemia
腦缺血:
insufficientbloodflowtothebrain(ischemicstroke)StroketypesandincidenceTheBloodSupplyoftheBrain
TheBloodSupplyoftheBrainBloodissuppliedtotheentirebrainby2pairsofarteries:internalcarotidarteries
頸內(nèi)動(dòng)脈vertebralarteries
椎動(dòng)脈Fewseconds:littleornodamage10seconds:nooxygensupply30seconds:changesinbrainmetabolism1minute:noneuronalfunctionalactivities6-8minutes>neuronaldeath,InfarctionExperimentalischemiaAnimalModelsFocalbrainischemia(transient/permanent)
Middlecerebralarteryocclusion(MCAO)
大腦中動(dòng)脈阻塞
Photothrombosis光化學(xué)誘導(dǎo)
Spontaneousbraininfarction(inspontaneouslyhypertensiverats)
Four-vesselocclusion四血管結(jié)扎
CommoncarotidarteryocclusioningerbilsTwo-vesselocclusionplushypotention
兩血管結(jié)扎并低血壓
Cardiacarrestcerebralischemia(CACI)
心臟驟停
Hypoxicischemia低氧缺血GlobalbrainischemiaLonga,E.Z.,P.R.Weinstein,S.Carlson,R.Cummins(1989)."Reversiblemiddlecerebralarteryocclusionwithoutcraniectomyinrats".Stroke20(1):84–91.PMID2643202IntraluminalsutureMCAO線栓法致大腦中動(dòng)脈缺血MCAOinducesbrainedema(bottomleft)andinfarction(right).TopleftshowsMCAO-affectedregioninhumanbrain.MCAO-inducedstrokemodelinrhesusmonkeyrosebengal玫瑰紅B四碘四氯熒光素二鈉PhotothrombosisstrokemodelWatsonBD,DietrichWD,BustoR,WachtelMS,GinsbergMD.(1985).Inductionofreproduciblebraininfarctionbyphotochemicallyinitiatedthrombosis.AnnNeurol.17(5):497-504Photosensitivedye:rosebengalSpecificlighting:鹵素?zé)艋螂療鬚hotochemicalreactionproducts:
reactiveoxygenspeciesDamage:VascularendothelialcellinjuryThrombosisformation光化學(xué)誘導(dǎo)Left:Cortical“spot”lesion,carbonblackviewat4hRight:Occludedarterioleinpia,perfusedat2min;SEMx4160Left:562nmdyelaserbeam
positionedondMCARight:Rosebengaldye-photosensitized
dMCAthrombusImagesfrom/x301.xml光化學(xué)誘導(dǎo)模型可用于研究溶栓藥物的作用Imagesfrom/lawrence.lab/files/page_4_01.html全腦缺血模型中,海馬神經(jīng)元最易受影響HypoxiachamberImagesfromJournalofCerebralBloodFlow&Metabolism(2004)24,259–270ligationoftheright(orleft)commoncarotidarteryof7-day-oldpups,followedby90minuteofhypoxia(8%02and92%N2)at37°C.
Hypoxia-ischemiamodel
CellcultureOrganotypicbrainslicecultureIn
VitrocultureOxygen-glucosedeprivation(OGD)
hypoxiachemicalhypoxia
NeuronalcultureOrganotypicslice——maintainneuronalorganizationforextendedperiodsoftime
更接近在體狀態(tài)數(shù)周至數(shù)月(A)Brightfield,(B)and(C)PI-fluorescenceimagesofratorganotypichippocampalslicecultures(labelledareCA1,CA3subfieldsandDG:dentategyrus).(B)Neuronalcelldeath24hafter‘test’ischemia(40minofOGD).(C)Reducedcelldeathinapreconditionedslice(15minofOGD48hpriortotestischemia).Barindicates0.2mm.Neuroscienceletters,384(2005):87-92MechanismsunderlyingischemicneuronalinjuryReductionsincerebralbloodflowleadtoincreasingneuronaldysfunctionIschemicinjuryandcelldeathcontinuesinstagesforminutes,hours,andevendaysofvesselocclusion.minutesdayshoursweeksinjuryprotectionexcitotoxicityInflammation&apoptosisAntiexcitatoxicityGABAAdenosineKATPopenAntiinflammationAntiapoptosisIL-10,IL-RaBcl-2EPO,HIFVEGF…Repair®eneration孫鳳艷,醫(yī)學(xué)神經(jīng)生物學(xué),2009,pp346SeverelydamagedtissueLowperfusiontissueAfterhourstodays,ischemiccoreterritoryexpands.Ischemiccoreappearswithinminutestohours.Salvageabletissueisthetargetfortherapy.Whathappenedinischemicneurons?
Glutamateexcitotoxicity谷氨酸興奮毒INearlystagesofischemicneuronalinjuryNeuronaldeathLucasD.R.OlneyJ.SchwarczR....KainicacidQuinolinicacidNMDAGlutamate(exogenous)Agonistsofglutamate-mediatedionotropicreceptorsPre-1984Post-19841984LUCASDR,NEWHOUSEJP.(1957).ThetoxiceffectofsodiumL-glutamateontheinnerlayersoftheretina.AMAArchOphthalmol.58(2):193-201OlneyJW.(1969).Brainlesions,obesity,andotherdisturbancesinmicetreatedwithmonosodiumglutamate.Science.164(880):719-21Glutamateneurotoxicity1984
Extracellular
levelsofglutamateincreaseduringischemia(Benvenisteetal.).
Glutamate-receptorantagonistsprotectsculturedneuronsagainstbothanoxiaandthetoxicityofexogenousglutamateandaspartate(Rothman).
N-methyl-D-aspartate(NMDA)-receptorantagonistsreducedischaemia-inducedneuronaldamage
invivo
(Simonetal.).
Post-19841984Pre-1984腦缺血后細(xì)胞間隙谷氨酸含量顯著升高corepenumbraThesearethedataattheoriginofthedogma:Highextracellularglutamateunderliesendogenousexcitotoxicity.Ishighextracellularglutamatetheonlyabnormalityofglutamate-mediatedsynaptictransmission,thatmayunderlieexcitotoxicityassociatedwithneurologicaldisorders?BUT!!!GLUCytosol10mMVesicles100mMExtracellular1MTGLUGLUSynapticspace1mM?Glutamateconcentrationgradients谷氨酸轉(zhuǎn)運(yùn)體的作用?excitotoxicityEndogenousPresynapticPostsynapticGliaGlu-RGluvGlucGlucs.c.e.s.e.s.Glia1984Pre-1984Post-1984in:2-3Na+,1H+withGluout:1K+內(nèi)向電流谷氨酸的重?cái)z取是生電過(guò)程(electrogenic)引自BrainResBrainResRev.2004Jul;45(3):250-65.ModelreportedbyAmara’sgroupinUSA
Deficientglutamateuptake
OutInATPNa+K+
K+Na+
K+CGlu2Na+DeficientglutamateuptakeDeficientenergysupply(ischaemia,mitochondrialdamage)LossoftheNa/K-transmembranegradient(drivingforceofthecarrier)Glutransporter谷氨酸受體分型引自PurvesD,AugustineGJ,FitzpatrickD,etal.,Neuroscience.2ndedition配體門控陽(yáng)離子通道G
蛋白偶聯(lián)受體離子型受體ionotropicreceptor,iGluR代謝型受體metabotropicreceptor,mGluRNMDAR非NMDAR(AMPAR,KAR)mGluR1-8AMPA-RPostsynapticabnormalitiesleadingtoexcessiveexcitationIonotropicreceptors(GluR)Metabotropicreceptors(mGluR)Ca2+NMDA-RNa+Na+Kainate-RG
Increasedaffinityoftheglutamatebindingsite;
Increaseddensityofglutamatereceptor;
Deficientcationselectivityoftheionophore;
Abnormal(positive)modulationofGluRfunction.NMDAreceptor:locationandsubunitcompositiondifferentiallyregulateneuronalsurvivalordeathSynapticNMDAR:GluN2A>>GluN2BNeuronalsurvivalExtrasynapticNMDAR:GluN2B>>GluN2ANeuronaldeathGluN2BGluN2ADPAK:death-associatedproteinkinaseExcitotoxicityrecruitsdeath-associatedproteinkinase1(DAPK1)tothecytoplasmictailoftheGluN2Bsubuni興奮性突觸的突觸后致密帶(postsynapticdensity,PSD)NMDARPSD95nNOSNO·Na+內(nèi)流Na+內(nèi)流Ca2+內(nèi)流內(nèi)鈣釋放Ca2+內(nèi)流NMDA受體AMPA受體KA受體mGlu受體谷氨酸水腫內(nèi)鈣超載intracellularcalciumoverload胞內(nèi)鈣超載INearlystagesofischemicneuronalinjuryextracellularcalcium:[Ca2+]o=1~10mMintracellularcalcium:
cytosolic:
0.1%ofintracellularcalcium[Ca2+]i=100nM=0.1M
calciumstores:99.9%
calciumbindingprotein:calmodulin,calbindin,parvalbumin,calretinin
endoplasmicreticulum
mitochondriaCalciumhomeostasisinneuronsoutfluxinfluxreleasefromCa2+pool[Ca2+]iERmitochondria3Na+2Ca2+Na+-Ca2+exchangerCa2+ATP-dependentCa2+pumpsIP3receptoruniporterCa2+pumpsNMDARmGluRsvoltage-dependentCa2+channels(VDCC)IP3GqPLCAMPARNicholsonC,BruggencateGT,SteinbergR,St?ckleH.(1977).Calciummodulationinbrainextracellularmicroenvironmentdemonstratedwithion-selectivemicropipette.ProcNatlAcadSciUSA.74(3):1287-90.
anoxiatriggersrapidtranslocationofcalciumfromextratointracellularspacesinneuraltissue.Thisworkpromptedspeculationaboutwhycertainneuronsareselectivelymoresensitivetoischemia,namelybecauseofahigherdensityofcalciumchannelsintheirplasmamembranes.1977;Nicholsonetal:directlydetectchangesofextracellularca2+concentrationGlutamate-inducedCa2+transientsincorticalneuronsobservedbyconfocallasermicroscopy.Thetimecourseofthenormalizedfluorescenceintensity.Zerotimeindicatesbeginningofpostexposurephase.Eachcurverepresentsapointrecordingfromadifferentcellinthedish.Fluo-3flunorescenceintensitywereobtainedbeforeandduringstimulationwith1mMglutamateatintervalsof4sec.IntracellularcalciumoverloadingnormalaspartatewashAsp+tauERmitochondria3Na+2Ca2+Na+-Ca2+exchangerCa2+ATP-dependentCa2+pumpsIP3receptoruniporterCa2+pumpsNMDARmGluRsvoltage-dependentCa2+channels(VDCC)IP3GqPLCAMPARIschemia!!Normally,calciumparticipatesinmanyimportantcellularactivities:ModulationofenzymeactivityCellgrowthanddifferentiationMembraneexcitabilityNeurotransmitterreleaseSynapticplasticity細(xì)胞內(nèi)游離鈣離子激活一系列鈣離子依賴性酶反應(yīng)Ischemiainducescalcium-dependenttoxicityNOS:NOROSPLA2:membraneintegrityEndonuclease/caspases:apoptosis,DNAdegradationcalpain:MAP2…Oxidative/nitrosativestress氧化/硝化應(yīng)激Whatisoxidativestress?Oxidativestressistheimbalancebetweencellularproductionofreactiveoxygenspecies(ROS)andtheabilityofcellstodefendagainstthem.氧化應(yīng)激:oxidativestress體內(nèi)氧化與抗氧化作用失衡,氧化作用增加,產(chǎn)生大量氧化中間產(chǎn)物。硝化應(yīng)激:nitrosativestress由NO或NO衍生的活性氮族與活性氧族共同聯(lián)合發(fā)生的反應(yīng),可使蛋白質(zhì)的酪氨酸硝化成硝基酪氨酸,或者使半胱氨酸巰基發(fā)生S-亞硝基化。Reactiveoxygenspecies(ROS)
活性氧自由基
Freeradicals:自由基
AnychemicalspecieswithoneormoreunpairedelectronsReactivenitrogenspecies(RNS)
活性氮自由基
themostcommoncellularfreeradicals:-superoxideradicalO2-(超氧陰離子)-hydroxylradical?OH(羥自由基)-nitricoxideradicalNO?Others:(notfreeradicalsbutcanleadtothegenerationoffreeradicals)-Hydrogenperoxide
H2O2-PeroxynitriteONOO-
過(guò)氧亞硝酸陰離子ROSarephysiologicallygeneratedandmaintainedatrelativelylowlevels.Nitricoxideisafreeradical!Itcontainsanunpairedelectron
.N=ORoleinmacrophagekillingofpathogensNOalsoactsasasecondmessengerthatcausesrelaxationofsmoothmuscle
N=O
N=O
TheNobelPrizeinPhysiologyorMedicine1998RobertF.Furchgott
SUNYHealthScienceCenter
Brooklyn,NY,USA
b.1916LouisJ.Ignarro
UniversityofCaliforniaSchoolofMedicine
LosAngeles,CA,USAb.1941FeridMurad
UniversityofTexasMedicalSchoolatHouston
Houston,TX,USAb.1936Http:///research/medicine/biochemistry/bioc800/sig02-06.htmTypesofNOSNOSI(nNOS)CentralandperipheralneuronalcellsCa2+dependent,usedforneuronalcommunicationNOSII(iNOS)Mostnucleatedcells,particularlymacrophagesIndependentofintracellularCa2+
InducibleinpresenceofinflammatorycytokinesNOSIII(eNOS)VascularendothelialcellsCa2+dependentVascularregulation
H+
NO
+O2
ONOO-
ONOOH
NO2?+OH?
協(xié)同花生四烯酸損Pr、核酸、脂質(zhì)膜損Syn.前Glu轉(zhuǎn)運(yùn)體
Glureuptake
Glu濃度NMDARPSD95nNOSNO·SH→SNOSH→SNOFromCalabresietal.,2003Celldeath細(xì)胞死亡NecrosisApoptosisNecroptosisautophageApoptosisandnecrosisMethodsofdetectingapoptosisMorphologyElectronmicroscopyLightmicroscopyChromatincondensationStainingnucleiwithfluorescentdyessuchasDAPI,Hoechst,acridineorangeDNAfragmentationTUNEL(TdT-mediateddUTP-biotinnickendlabeling)InternucleosomalDNAladderFACSanalysisofDNAcontent(<2NDNA)Viabilitydyeexclusion,trypanblue,propidiumiodideAnnexinVbindingtophosphatidylserinewhichisflippedtooutsideofplasmamembrane earlyinapoptosisMTT-assaysabilityofactivemitochondriatocleavecolorimetricsubstrateCaspaseActivation--caspasecleavageeventsCytochromecreleasefrommitochondria
UVApoptosisinResponsetoUV-irradiationAnnexin-VstainingHoechststainingDNAFragmentationApoptosissignals
FasligandFasCaspase8BidCytochromecCaspase3Caspase7FADDIntrinsicpathwayExtrinsicpathwayAPOPTOSISBclBax細(xì)胞內(nèi)源性通路細(xì)胞外通路TakahashiA,MasudaA,SunM,CentonzeVE,HermanB.(2004).Oxidativestress-inducedapoptosisisassociatedwithalterationsinmitochondrialcaspaseactivityandBcl-2-dependentalterationsinmitochondrialpH(pHm).BrainResBull.62(6):497-504.LinksnecrosiscanoccurinahighlyregulatedandgeneticallycontrolledmannerAutophagy:自噬Energydependentprocess“自食”
LC3dependentOrLC3independentIschaemia(lowOxygenandglucosesupply)LowATPmembranedepolarisation[Ca2+]i
IncreasedLactateNeurotransmittersreleasedGlutamate
NA,DA
[FreeFe]Freeradicals
LipolysisNOsynth.ProteolysisArachidonicacidFreeradicalsNeuronalandglialinjuryAuto-oxidation
CelldeathWBCadhesiveness
Neuroprotection谷氨酸谷氨酸受體Na+內(nèi)流Ca2+內(nèi)流水腫鈣超載谷氨酸受體拮抗劑鈣通道阻斷劑鈉通道阻斷劑去水腫TreatmentofstrokeCategoryNameClinicalefficacyAnticoagulation/thrombolysisAspirin/heparin/tPA/urokinaseModestGlutamatereceptorblockadeAptiganel/dextrophan/EliprodilNoneVoltage-gatedCa2+channelblockersNimodipine/lifarizine/flunarizineNoneNa+channelblockersLubeluzole/riluzolUnclearornoneVoltage-dependentK+-channelagonistBMS-204352BeingtestedEnhancementofinhibitoryneurotransmissionClormethiazoleBeingtestedFreeradicalscavengers/antioxidantsPergorgotein/tirilazad/ebselenNoneorminimalNeuralrepairCiticholine/troferminUnclearorminimalEverythingworksinanimalsbutnothingworksinpeople.O’collinsetal,2006
Neurogenesisaftercerebralischemia!
SVZ:subventricularzoneSGZ:subgranularzoneNeurogenesisinthestriatuma,NeuralstemcellsorprogenitorcellsresideintheSVZ.b,Focalischemicinsultsgiverisetoincreasedproliferationofprogenitors.c,Neuroblastsformedafterandtosomeextentalsobeforethestrokethenmigratetothedamagedpartofthestriatum.d,Theyexpressmarkersspecificforstriatalprojectionneurons.CurrentopinioninNeurobiology,2003,13:127-132SelfrepairmechanismNaturemedicine,2002,8(9):963-970Dcx(green)andBrdu(red)immucoreactivityintheipsilateralstriatuminfusedwithsaline(upperlayer)andAra-c(lowlayer).Naturemedicine,2002,8(9):963-970Strokegeneratedcellsexpressmarkersofdevelopingstriatalneurons2weeksafterstrokeandmarkersofmaturestriatalneurons5weeksafterstroke..DARPP32BrdUDARPP32-BrdU如何延長(zhǎng)新生神經(jīng)元的存活?如何促進(jìn)新生神經(jīng)元分化為成熟的神經(jīng)元?如何建立有功能的成熟神經(jīng)元?如何促進(jìn)新生神經(jīng)元的整合?
BerislavV.ZlokovicNeurovascularpathwaystoneurodegenerationinAlzheimer’sdiseaseandotherdisordersCerebralmicrocirculationNVUstructuralandfunctionalunitneuronastrocyteBasementmembraneExtracellularmatrixmicrogliaPericyte/VSMCMicrovascularendothelialcellsBrainbloodbarrier(BBB)BBBisanessentialpartoftheNVUAdhesionreceptorsoftheBBBInter-endothelialcohesion:ComplexesofZO-1,claudin-5,andoccludin.Junctionaladhesionmolecules(JAMs)E-cadherinEndothelium-matrixorastrocyte-matrixadhesion:Integrinreceptorsanddystroglycan
Theimpactofocclusionofthemiddlecerebralarteryontheneurovascularunit(Sprague-Dawleyrat).Separationofastrocyteend-feetfromthe
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