医学神经生物学：缺血性神经元损伤及其修复

CerebralIschemicInjury

and

Neuroprotection缺血性神经元损伤及其修复ReferencesMolecularNeuropharmacology.EricJ.Nestler,etal.McGraw-HillCompanies.《医学神经生物学》第25章。孙凤艳主编，上海科学技术出版社。Contents

BriefintroductionaboutischemiaExperimentalmodelsusedinresearchMechanismsunderlyingischemicneuronalinjuryNeuroprotection

BriefIntroductionAdecreaseinthebloodsupplytoabodilyorgan,tissue,orpartcausedbyconstrictionorobstructionofthebloodvessels.ISCHEMIAis:

(NewLatinischaemia,fromGreekiskhaimos,astoppingoftheblood:iskhein,tokeepback;seesegh-inIndo-Europeanroots+haima,blood)/ischemiaMayalsobespelledas:ischaemiaorischæmia

Whatisischemia?Related

TermsIschemia

缺血:shortageofthebloodsupply(oxygen,glucose,etc)Hypoxia

缺氧:shortageofoxygenAnoxia

缺氧:anextremeformofhypoxia,absenceofoxygenIschemiccascade

缺血级联反应:aseriesofbiochemicalreactionsthattakeplaceaftersecondstominutesofischemia.Brainischemia

脑缺血:

insufficientbloodflowtothebrain(ischemicstroke)StroketypesandincidenceTheBloodSupplyoftheBrain

TheBloodSupplyoftheBrainBloodissuppliedtotheentirebrainby2pairsofarteries:internalcarotidarteries

颈内动脉vertebralarteries

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

Middlecerebralarteryocclusion(MCAO)

大脑中动脉阻塞

Photothrombosis光化学诱导

Spontaneousbraininfarction(inspontaneouslyhypertensiverats)

Four-vesselocclusion四血管结扎

CommoncarotidarteryocclusioningerbilsTwo-vesselocclusionplushypotention

两血管结扎并低血压

Cardiacarrestcerebralischemia(CACI)

心脏骤停

Hypoxicischemia低氧缺血GlobalbrainischemiaLonga,E.Z.,P.R.Weinstein,S.Carlson,R.Cummins(1989)."Reversiblemiddlecerebralarteryocclusionwithoutcraniectomyinrats".Stroke20(1):84–91.PMID2643202IntraluminalsutureMCAO线栓法致大脑中动脉缺血MCAOinducesbrainedema(bottomleft)andinfarction(right).TopleftshowsMCAO-affectedregioninhumanbrain.MCAO-inducedstrokemodelinrhesusmonkeyrosebengal玫瑰红B四碘四氯荧光素二钠PhotothrombosisstrokemodelWatsonBD,DietrichWD,BustoR,WachtelMS,GinsbergMD.(1985).Inductionofreproduciblebraininfarctionbyphotochemicallyinitiatedthrombosis.AnnNeurol.17(5):497-504Photosensitivedye:rosebengalSpecificlighting:卤素灯或氙灯Photochemicalreactionproducts:

reactiveoxygenspeciesDamage:VascularendothelialcellinjuryThrombosisformation光化学诱导Left:Cortical“spot”lesion,carbonblackviewat4hRight:Occludedarterioleinpia,perfusedat2min;SEMx4160Left:562nmdyelaserbeam

positionedondMCARight:Rosebengaldye-photosensitized

dMCAthrombusImagesfrom/x301.xml光化学诱导模型可用于研究溶栓药物的作用Imagesfrom/lawrence.lab/files/page_4_01.html全脑缺血模型中，海马神经元最易受影响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

更接近在体状态数周至数月(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孙凤艳，医学神经生物学，2009，pp346SeverelydamagedtissueLowperfusiontissueAfterhourstodays,ischemiccoreterritoryexpands.Ischemiccoreappearswithinminutestohours.Salvageabletissueisthetargetfortherapy.Whathappenedinischemicneurons?

Glutamateexcitotoxicity谷氨酸兴奋毒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脑缺血后细胞间隙谷氨酸含量显著升高corepenumbraThesearethedataattheoriginofthedogma:Highextracellularglutamateunderliesendogenousexcitotoxicity.Ishighextracellularglutamatetheonlyabnormalityofglutamate-mediatedsynaptictransmission,thatmayunderlieexcitotoxicityassociatedwithneurologicaldisorders?BUT!!!GLUCytosol10mMVesicles100mMExtracellular1MTGLUGLUSynapticspace1mM?Glutamateconcentrationgradients谷氨酸转运体的作用？excitotoxicityEndogenousPresynapticPostsynapticGliaGlu-RGluvGlucGlucs.c.e.s.e.s.Glia1984Pre-1984Post-1984in:2-3Na+,1H+withGluout:1K+内向电流谷氨酸的重摄取是生电过程（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谷氨酸受体分型引自PurvesD,AugustineGJ,FitzpatrickD,etal.,Neuroscience.2ndedition配体门控阳离子通道G

蛋白偶联受体离子型受体ionotropicreceptor,iGluR代谢型受体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兴奋性突触的突触后致密带（postsynapticdensity,PSD)NMDARPSD95nNOSNO·Na+内流Na+内流Ca2+内流内钙释放Ca2+内流NMDA受体AMPA受体KA受体mGlu受体谷氨酸水肿内钙超载intracellularcalciumoverload胞内钙超载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,StöckleH.(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细胞内游离钙离子激活一系列钙离子依赖性酶反应Ischemiainducescalcium-dependenttoxicityNOS:NOROSPLA2:membraneintegrityEndonuclease/caspases:apoptosis,DNAdegradationcalpain:MAP2…Oxidative/nitrosativestress氧化/硝化应激Whatisoxidativestress?Oxidativestressistheimbalancebetweencellularproductionofreactiveoxygenspecies(ROS)andtheabilityofcellstodefendagainstthem.氧化应激：oxidativestress体内氧化与抗氧化作用失衡，氧化作用增加，产生大量氧化中间产物。硝化应激：nitrosativestress由NO或NO衍生的活性氮族与活性氧族共同联合发生的反应，可使蛋白质的酪氨酸硝化成硝基酪氨酸，或者使半胱氨酸巯基发生S-亚硝基化。Reactiveoxygenspecies(ROS)

活性氧自由基

Freeradicals:自由基

AnychemicalspecieswithoneormoreunpairedelectronsReactivenitrogenspecies(RNS)

活性氮自由基

themostcommoncellularfreeradicals:-superoxideradicalO2-（超氧阴离子）-hydroxylradical•OH（羟自由基）-nitricoxideradicalNO•Others:(notfreeradicalsbutcanleadtothegenerationoffreeradicals)-Hydrogenperoxide

H2O2-PeroxynitriteONOO-

过氧亚硝酸阴离子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•

协同花生四烯酸损Pr、核酸、脂质膜损Syn.前Glu转运体

Glureuptake

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

UVApoptosisinResponsetoUV-irradiationAnnexin-VstainingHoechststainingDNAFragmentationApoptosissignals

FasligandFasCaspase8BidCytochromecCaspase3Caspase7FADDIntrinsicpathwayExtrinsicpathwayAPOPTOSISBclBax细胞内源性通路细胞外通路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谷氨酸谷氨酸受体Na+内流Ca2+内流水肿钙超载谷氨酸受体拮抗剂钙通道阻断剂钠通道阻断剂去水肿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如何延长新生神经元的存活？如何促进新生神经元分化为成熟的神经元？如何建立有功能的成熟神经元？如何促进新生神经元的整合？

BerislavV.ZlokovicNeurovascularpathwaystoneurodegenerationinAlzheimer’sdiseaseandotherdisordersCerebralmicrocirculationNVUstructuralandfunctionalunitneuronastrocyteBasementmembraneExtracellularmatrixmicrogliaPericyte/VSMCMicrovascularendothelialcellsBrainbloodbarrier(BBB)BBBisanessentialpartoftheNVUAdhesionreceptorsoftheBBBInter-endothelialcohesion:ComplexesofZO-1,claudin-5,andoccludin.Junctionaladhesionmolecules(JAMs)E-cadherinEndothelium-matrixorastrocyte-matrixadhesion:Integrinreceptorsanddystroglycan

Theimpactofocclusionofthemiddlecerebralarteryontheneurovascularunit(Sprague-Dawleyrat).Separationofastrocyteend-feetfromthe