醫(yī)學神經(jīng)生物學：缺血性神經(jīng)元損傷及其修復

CerebralIschemicInjury

and

Neuroprotection缺血性神經(jīng)元損傷及其修復ReferencesMolecularNeuropharmacology.EricJ.Nestler,etal.McGraw-HillCompanies.《醫(yī)學神經(jīng)生物學》第25章。孫鳳艷主編，上?？茖W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

缺血級聯(lián)反應:aseriesofbiochemicalreactionsthattakeplaceaftersecondstominutesofischemia.Brainischemia

腦缺血:

insufficientbloodflowtothebrain(ischemicstroke)StroketypesandincidenceTheBloodSupplyoftheBrain

TheBloodSupplyoftheBrainBloodissuppliedtotheentirebrainby2pairsofarteries:internalcarotidarteries

頸內(nèi)動脈vertebralarteries

椎動脈Fewseconds:littleornodamage10seconds:nooxygensupply30seconds:changesinbrainmetabolism1minute:noneuronalfunctionalactivities6-8minutes>neuronaldeath,InfarctionExperimentalischemiaAnimalModelsFocalbrainischemia(transient/permanent)

Middlecerebralarteryocclusion(MCAO)

大腦中動脈阻塞

Photothrombosis光化學誘導

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:鹵素燈或氙燈Photochemicalreactionproducts:

reactiveoxygenspeciesDamage:VascularendothelialcellinjuryThrombosisformation光化學誘導Left:Cortical“spot”lesion,carbonblackviewat4hRight:Occludedarterioleinpia,perfusedat2min;SEMx4160Left:562nmdyelaserbeam

positionedondMCARight:Rosebengaldye-photosensitized

dMCAthrombusImagesfrom/x301.xml光化學誘導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)學神經(jīng)生物學，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)向電流谷氨酸的重攝取是生電過程（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細胞內(nèi)游離鈣離子激活一系列鈣離子依賴性酶反應Ischemiainducescalcium-dependenttoxicityNOS:NOROSPLA2:membraneintegrityEndonuclease/caspases:apoptosis,DNAdegradationcalpain:MAP2…Oxidative/nitrosativestress氧化/硝化應激Whatisoxidativestress?Oxidativestressistheimbalancebetweencellularproductionofreactiveoxygenspecies(ROS)andtheabilityofcellstodefendagainstthem.氧化應激：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，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)運體

Glureuptake

Glu濃度NMDARPSD95nNOSNO·SH→SNOSH→SNOFromCalabresietal.,2003Celldeath細胞死亡NecrosisApoptosisNecroptosisautophageApoptosisandnecrosisMethodsofdetectingapoptosisMorphologyElectronmicroscopyLightmicroscopyChromatincondensationStainingnucleiwithfluorescentdyessuchasDAPI,Hoechst,acridineorangeDNAfragmentationTUNEL(TdT-mediateddUTP-biotinnickendlabeling)InternucleosomalDNAladderFACSanalysisofDNAcontent(<2NDNA)Viabilitydyeexclusion,trypanblue,propidiumiodideAnnexinVbindingtophosphatidylserinewhichisflippedtooutsideofplasmamembrane earlyinapoptosisMTT-assaysabilityofactivemitochondriatocleavecolorimetricsubstrateCaspaseActivation--caspasecleavageeventsCytochromecreleasefrommitochondria

UVApoptosisinResponsetoUV-irradiationAnnexin-VstainingHoechststainingDNAFragmentationApoptosissignals

FasligandFasCaspase8BidCytochromecCaspase3Caspase7FADDIntrinsicpathwayExtrinsicpathwayAPOPTOSISBclBax細胞內(nèi)源性通路細胞外通路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如何延長新生神經(jīng)元的存活？如何促進新生神經(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