1、缺血性腦損傷及腦保護Ischemic Brain Injury and Protection2013-111, What is ischemic brain injury?2, Why does ischemia cause brain injury? Experimental models Prominent Mechanisms leading to brain injury3, How does the brain repair/protect itself after stroke?4, Translational strategiesOUTLINE1, What is ischemi

2、c brain injury?2, Why does ischemia cause brain injury? Experimental models Prominent Mechanisms leading to brain injury3, How does the brain repair/protect itself after stroke?4, Translational strategiesOUTLINEA decrease in the blood supply to a bodily organ, tissue, or part caused by constriction

3、or obstruction of the blood vessels. ISCHEMIA is: (New Latin ischaemia, from Greek iskhaimos, a stopping of the blood : iskhein, to keep back; see segh- in Indo-European roots + haima, blood ) http:/ischemiaMay also be spelled as: ischaemia or ischmia Related TermsIschemia 缺血: shortage of the blood

4、supply (oxygen, glucose, etc)Hypoxia 缺氧: shortage of oxygenAnoxia 缺氧: an extreme form of hypoxia, absence of oxygenBrain ischemia 腦缺血: insufficient blood flow to the brain (eg: ischemic stroke)Ischemic cascade 缺血級聯反應: a series of biochemical reactions that take place after seconds to minutes of isch

5、emia.Stroke types and incidence中風中絕大多數是缺血性中風腦血栓形成腦栓塞Blockage of a blood vessel (in the brain or neck)Cerebral ischemiaIschemic Brain InjuryFew seconds: little or no damage10 seconds: no oxygen supply 30 seconds: changes in brain metabolism1 minute: no neuronal functional activities6-8 minutes neuron

6、al death, InfarctionNeurological dysfunction occurs within seconds to minutes of vessel occlusionIschemic injury and cell death continues in stages for minutes, hours, and even days of vessel occlusion血栓使大腦中動脈栓塞，供血區血供不足大腦中動脈缺血腦水腫Red NeuronsMacrophagesOedemaCyst formation神經細胞受損缺血1h再灌 24h，同側和對側皮層、紋狀 體

7、神經元變化（CV染色）cortexstriatumcontralateralipsilateralSeverely damaged tissueLow perfusion tissueAfter hours to days, ischemic core territory expands.Ischemic core appears within minutes to hours.腦缺血造成腦梗死缺血核心區 ischemic core缺血半影區 penumbraIschemic core: irreversibly damaged tissues distal to an occluded bl

8、ood vessels 20% of baseline blood flow levels depleted ATP stores irreversible failure of energy metabolismSalvageable tissue is the target for therapy.Ischemic penumbra: significantly depressed tissue perfusion barely sufficient to support basal ATP levels as well as normal ionic gradients in the p

9、resence of electrical silence and suppressed protein synthesis “at risk” region: functionally impaired, potentially salvageableDamage MechanismsProtective MechanismsCell Fateischemia缺血半影區的神經元病理變化：1，啟動細胞死亡機制2，激活腦內自身保護機制Live? Dead?1, What is ischemic brain injury?2, Why does ischemia cause brain injur

10、y? Experimental models Prominent Mechanisms leading to brain injury3, How does the brain repair/protect itself after stroke?4, Translational strategiesOUTLINERat, mice, and gerbil are the commonly used model animals. Monkey, cat, and rabbit are also used to establish stroke models. 外周血液經兩對動脈進入腦內：頸動脈

11、和椎動脈阻斷血流（顱內、顱外）：腦缺血Focal brain ischemia (transient/ permanent)： Middle cerebral artery occlusion (MCAO) Photothrombosis 光化學誘導 Spontaneous brain infarction (in spontaneously hypertensive rats)Global brain ischemia： Four-vessel occlusion 四血管結扎 Common carotid artery occlusion in gerbils Two-vessel occl

12、usion plus hypotention 兩血管結扎并低 血壓 Cardiac arrest cerebral ischemia (CACI) 心臟驟停 Hypoxic ischemia 低氧缺血 Intracranial hypertension and common carotid artery occlusion Longa, E. Z., P. R. Weinstein, S. Carlson, R. Cummins (1989). Reversible middle cerebral artery occlusion without craniectomy in rats. St

13、roke 20 (1): 8491. PMID 2643202Intraluminal suture MCAO大腦中動脈栓塞MCAO-induced stroke model in rhesus monkey 大鼠行為學視頻MCAO induces brain edema (bottom left) and infarction (right). Top left shows MCAO-affected region in human brain.rose bengal 玫瑰紅B四碘四氯熒光素二鈉Photothrombosis stroke modelWatson BD, Dietrich W

14、D, Busto R, Wachtel MS, Ginsberg MD. (1985). Induction of reproducible brain infarction by photochemically initiated thrombosis. Ann Neurol. 17(5):497-504Photosensitive dye: rose bengalSpecific lighting: 鹵素燈或氙燈Photochemical reaction products: reactive oxygen speciesDamage: Vascular endothelial cell

15、injury Thrombosis formationLeft: Cortical “spot” lesion, carbon black view at 4 h Right: Occluded arteriole in pia, perfused at 2 min; SEM x4160Left: 562 nm dye laser beam positioned on dMCARight: Rose bengal dye- photosensitizeddMCA thrombusImages from /x301.xml光化學誘導模型可用于研究溶栓藥物的作用Images from /lawre

16、nce.lab/files/page_4_01.html全腦缺血模型中，海馬神經元最易受影響Hypoxia chamberImages from Journal of Cerebral Blood Flow & Metabolism (2004) 24, 259270 ligation of the right (or left) common carotid artery of 7-day-old pups, followed by 90 minute of hypoxia (8% 02 and 92% N2) at 37C. Hypoxia-ischemia model Cell cult

17、ure Organotypic brain slice cultureIn Vitro cultureOxygen-glucose deprivation (OGD) hypoxiachemical hypoxia Neuronal cultureOrganotypic slice maintain neuronal organization for extended periods of time 更接近在體狀態 數周至數月(A) Bright field, (B) and (C) PI-fluorescence images of rat organotypichippocampal sl

18、ice cultures (labelled are CA1, CA3 subfields and DG:dentate gyrus). (B) Neuronal cell death 24 h after test ischemia (40 min ofOGD). (C) Reduced cell death in a preconditioned slice (15 min of OGD48 h prior to test ischemia). Bar indicates 0.2 mm.Neuroscience letters, 384(2005): 87-921, What is isc

19、hemic brain injury?2, Why does ischemia cause brain injury? Experimental models Prominent Mechanisms leading to brain injury3, How does the brain repair/protect itself after stroke?4, Translational strategiesOUTLINE缺血細胞內信號轉導細胞間通訊缺血中心區細胞死亡缺血損傷向四周擴布？AB1B2CD神經元的突觸結構CBF reductionEnergy failureAnoxic dep

20、olarization,Excitotoxicity,Oxidative stress,NecrosisPeri-infarct depolarization,Calcium overload,Mitochondrial damageInflammation,Programmed cell deathNeuronal ischemic injury cascade Glutamate excitotoxicity谷氨酸興奮毒神經元受損的早期環節Lucas D.R.Olney J.Schwarcz R.Pre-1984Post-19841984 LUCAS DR, NEWHOUSE JP. (1

21、957). The toxic effect of sodium L-glutamate on the inner layers of the retina. AMA Arch Ophthalmol. 58(2):193-201 Olney JW. (1969). Brain lesions, obesity, and other disturbances in mice treated with monosodium glutamate. Science. 164 (880): 719-21 過量谷氨酸可以破壞新生小鼠視 網膜神經元 全身應用谷氨酸可導致腦內神經 元的退化性病變 谷氨酸引起神

22、經元死亡的作用通 過興奮突觸后膜上離子通道型谷 氨酸受體1984 缺血時細胞外谷氨酸含量上升 (Benveniste et al.). 谷氨酸受體拮抗劑保護培養的神經元免受谷氨酸或者門冬氨酸興奮毒 (Rothman). N-methyl-D-aspartate (NMDA)-受體拮抗劑降低缺血誘導的神經元損傷 (Simon et al.). Post-19841984Pre-1984腦缺血后細胞間隙谷氨酸含量顯著升高corepenumbraGLUCytosol10 mMVesicles100 mMExtracellular1 MTGLUGLUSynaptic space1 mM ?Glutam

23、ateconcentration gradients谷氨酸轉運體的作用？excitotoxicityEndogenousPresynapticPostsynapticGliaGlu-RGluvGlucGlucs.c.e.s.e.s.Glia1984Pre-1984Post-1984YProcesses involved in the inactivation of synaptic transmissionYYYYYYUU = uptakeDD = diffusionYZMM = metaboliteYYYYRR = receptordesensitisation Deficient glut

24、amate uptake OutInATPNa+K+ K+Na+ K+CGlu2 Na+Deficient glutamate uptakeDeficient energy supply(ischaemia, mitochondrial damage)Loss of the Na /K -transmembrane gradient(driving force of the carrier)谷氨酸轉運體AMPA-RPostsynaptic abnormalities leading to excessive excitationIonotropic receptors (GluR)Metabo

25、tropicreceptors (mGluR)Ca2+NMDA-RNa+Na+Kainate-RG Increased affinity of the glutamate binding site; Increased density of glutamate receptor; Deficient cation selectivity of the ionophore; Abnormal (positive) modulation of GluR function.谷氨酸受體Presynaptic release increaseReuptake decreaseHigher level o

26、f extracellular glutamatePostsynaptic receptor abnormalitiesexcessive excitationNa+內流 Na+內流 Ca2+內流 內鈣釋放 Ca2+內流NMDA 受體 AMPA受體 KA 受體 mGlu受體谷氨酸水腫內鈣超載nNOSPLA2ROSCelldeath？Clinical trials failureCategoryNameClinical efficacyGlutamate receptor blockadeAptiganel/ dextrophan/ EliprodilNoneOversimplified the

27、 complexity of the cell death processesUnderestimated the diversity of expressing cell types as well as the heterogeneity of glutamate responses following receptor overstimulation in strokeFunctional diversity of NMDARs is rooted in their location and subunit diversity.Selective enhancement of synap

28、tic receptors (NR2A predominate over NR2B): promotes neuronal survivalActivating extrasynaptic receptors (NR2B predominate over NR2A): promotes cell deathCREB-dependent signaling and BDNF expression up and down is critical for cell fate detemination during NMDAR activationExample 1: Selective drug t

29、arget is important.GLIAL:GLAST/EAAT1 Retinal Muller cells 77 M Cerebellar Bergmann gliaGLT-1/EAAT2 Astrocytes throughout brain 2 MNEURONAL:EAAC1/EAAT3 Neuronal somata and dendrites 15 MEAAT4 Cerebellar Purkinje cells 3.3 MEAAT5 Retinal photoreceptors and 64 M bipolar cellsThe name of the glutamate t

30、ransporter family members. The excitatory amino acid transporter (EAAT) names are for the human transporters; other names were given when the transporters were first cloned in non human species.Example 2: Nonneuronal cell typesCerebral cortexStriatumHippocampusThalamusIn situ hybridization for EAAT1

31、 mRNA and immunohistochemistry for EAAT1 protein after H-IDouble staining with confocal laser scanning microscopic analysis in neonatal rat brainsImmunohistochemical staining in the neonatal rat brain at 48 h following H-I H-I+sense H-I+antisense0510152025cerebral cortexstriatumhippocampusthalamusEA

32、AT1-positive cells/mm2vehicleantisensesensemissense*Sham-operationH-I + senseH-I + antisenseCresyl violet staining in the neonatal rat brain at 48 h following H-I:* intracellular calcium overload胞內鈣超載神經元受損的早期環節extracellular calcium : Ca2+o=1 10mM intracellular calcium : cytosolic： 0.1% of intracellu

33、lar calcium Ca2+i=100 nM = 0.1 M calcium stores：99.9% calcium binding protein: calmodulin, calbindin, parvalbumin, calretinin endoplasmic reticulum mitochondriaCalcium homeostasis in neuronsVOCs: voltage-operated channels ROCs: receptor-operated channels SMOCs: second-messenger operated channels SOC

34、s: store-operated channels RyRs: ryanodine receptors, by calcium itself (CICR) or by voltage (DICR)SR: sarcoplasmic reticulum ER: endoplasmic reticulum S : a hypothetical calcium sensorAll currently known calcium channel proteinsCalcium influx: glutamate-dependent glutamate-independentCalcium releas

35、e from stores:Calcium efflux ERmitochondria3 Na+2 Ca2+Na+-Ca2+ exchangerCa2+ATP-dependent Ca2+ pumpsIP3 receptoruniporterCa2+ pumpsNMDA RmGluRsvoltage-dependent Ca2+ channels(VDCC)IP3GqPLCAMPA R細胞內游離鈣離子激活一系列鈣離子依賴性酶反應Normally, calcium participates in many important cellular activities: Modulation of

36、enzyme activity Cell growth and differentiation Membrane excitability Neurotransmitter release Synaptic plasticityNicholson C, Bruggencate GT, Steinberg R, Stckle H. (1977). Calcium modulation in brain extracellular microenvironment demonstrated with ion-selective micropipette. Proc Natl Acad Sci U

37、S A. 74(3):1287-90. anoxia triggers rapid translocation of calcium from extra to intracellular spaces in neural tissue. This work prompted speculation about why certain neurons are selectively more sensitive to ischemia, namely because of a higher density of calcium channels in their plasma membrane

38、s. 1977; Nicholson et al:缺血缺氧導致胞內鈣超載Glutamate-induced Ca2+ transients in cortical neurons observed by confocal laser microscopy. The time course of the normalized fluorescence intensity. Zero time indicates beginning of postexposure phase. Each curve represents a point recording from a different cel

39、l in the dish. Fluo-3 flunorescence intensity were obtained before and during stimulation with 1mM glutamate at intervals of 4 sec. Intracellular calcium overloadingnormalaspartatewashAsp+tauERmitochondria3 Na+2 Ca2+Na+-Ca2+ exchangerCa2+ATP-dependent Ca2+ pumpsIP3 receptoruniporterCa2+ pumpsNMDA Rm

40、GluRsvoltage-dependent Ca2+ channels(VDCC)IP3GqPLCAMPA RIschemia !Coupling between NMDA Receptor and Acid-Sensing Ion Channel Contributes to Ischemic Neuronal DeathASIC1a: Acid-sensing ion channels (ASICs) highly permeable to Calcium Calcium influxGlobal Ischemia Rapidly Elevated Serine Phosphorylat

41、ion of ASIC1a SubunitGlobal Ischemia Increased the Association of CaMKIIa with ASIC1aNMDAR Activation Is Required for Ischemia-Induced Enhancement of ASIC1a PhosphorylationNR2B-specific blockersNMDARAMPARActivation of NR2B-NMDARs Enhanced ASIC CurrentsNR2B Antagonist or CaMKII Inhibitor Prevented OG

42、D-Induced Enhancement of ASIC Currents in Hippocampal NeuronsNeuroprotection by ASIC1a Gene Deficiency and ASIC1a Mutants in Cultured Hippocampal Neurons or COS7 Cells NMDAR-CaMKII cascade is functionally coupled to ASICs and contributes to acidotoxicity during ischemia. Specific blockade of NMDAR/C

43、aMKII-ASIC coupling may reduce neuronal death after ischemia and other pathological conditions involving excessive glutamate release and acidosis.Thus:Oxidative/nitrosative stress 氧化/硝化應激神經元受損的分子基礎Angioplasty Keshan disease (selenium deficiency)TraumaStrokeNeurotoxinsParkinsons DiseaseAlzheimers Dis

44、easeRadiationAgingCancerInflammatory-immune injuryIschemia-reperfusionDiabetesKidneySkinHeartJointsLungBrainMulti-organVesselsGIEyeRenal graftGlomerulonephritisBurnDermatitisPsoriasisIschemic bowelEndotoxin liver InjuryAsthmaHyperoxiaVasospasmAtherosclerosisRheumatoidAthritisDegenerative retinal dam

45、ageCataractogenesisOxidative StressOxidative stress-related clinical conditionsWhat is oxidative stress ?Oxidative stress is the imbalance between cellular production of reactive oxygen species (ROS) and the ability of cells to defend against them.氧化應激： 體內氧化與抗氧化作用失衡，氧化作用增加，產生大量氧化中間產物。硝化應激： 由NO或NO衍生的

46、活性氮族與活性氧族共同聯合發生的反應，可使蛋白質的酪氨酸硝化成硝基酪氨酸，或者使半胱氨酸巰基發生S-亞硝基化。Reactive oxygen species (ROS) 活性氧自由基 Free radicals: 自由基 Any chemical species with one or more unpaired electronsReactive nitrogen species (RNS) 活性氮自由基 the most common cellular free radicals: - superoxide radical O2-（超氧陰離子） - hydroxyl radical OH（

47、羥自由基）- nitric oxide radical NOOthers: (not free radicals but can lead to the generation of free radicals)- Hydrogen peroxide H2O2- Peroxynitrite ONOO- 過氧亞硝酸陰離子ROS/RNS are physiologically generated and maintained at relatively low levels.Enzymes, antioxidantsROS的清除Different sources of ROS generation

48、leading tovarious lipid peroxides formation.腦缺血腦缺血腦缺血產生大量自由基，抗自由基系統活性下降During ischemia, ROS were generated principally by mitochondria.Recently shown: NADPH oxidase (NOX) is the primary source of superoxide induced by NMDA receptro activation.PKCnNOSXDH: 黃嘌呤脫氫酶XO：黃嘌呤氧化酶NOX：NADPH氧化酶 a membrane-bound

49、enzyme complex be found mainly in the plasma membraneNOS：NO合酶線粒體功能障礙，SOD等活性下降黃嘌呤氧化酶過度活化 次黃嘌呤 黃嘌呤 + 尿酸 + O2-花生四烯酸代謝增加 Ca2+ PLA2/ PLC AA O2- NOS NO，進一步介導自由基的生成。腦缺血后多條途徑產生自由基： The Nobel Prize in Physiology or Medicine 1998Robert F. Furchgott SUNY Health Science Center Brooklyn, NY,USA b. 1916Louis J. I

50、gnarro University of California School of Medicine Los Angeles, CA, USA b. 1941Ferid Murad University of Texas Medical School at Houston Houston, TX, USA b. 1936Http:/research/medicine/biochemistry/bioc800/sig02-06.htmNOS coupling NO NOS uncoupling O2- instead of NOTypes of NOSnNOS neuronal NOSCentr

51、al and peripheral neuronal cellsCa2+ dependent, used for neuronal communicationiNOS inducible NOSMost nucleated cells, particularly macrophagesIndependent of intracellular Ca2+ Inducible in presence of inflammatory cytokineseNOS endothelial NOSVascular endothelial cellsCa2+ dependentVascular regulat

52、ion H+ NO +O2 ONOO- ONOOH NO2 + OH 協同花生四烯酸 損Pr、核酸、脂質膜 損Syn.前Glu轉運體 Glu reuptake Glu濃度腦缺血時大量超氧化物與NO反應CategoryNameClinical efficacyFree radical scavengers/ antioxidantsPergorgotein/ tirilazad/ ebselenNone or minimalClinical trials failureTo suppress deleterious radicals without interfering with endoge

53、nous signaling will be important.From Calabresi et al., 2003Cell death 細胞死亡神經元受損的最終歸宿Apoptosis and necrosisCell shrinkage, membrane blebbing, chromatin condensation, apoptotic body formationMethods of detecting apoptosisMorphology Electron microscopy Light microscopyChromatin condensation Staining n

54、uclei with fluorescent dyes such as DAPI, Hoechst, acridine orangeDNA fragmentation TUNEL (TdT-mediated dUTP-biotin nick end labeling) Internucleosomal DNA ladder FACS analysis of DNA content (2N DNA)Viability dye exclusion, trypan blue, propidium iodide Annexin V binding to phosphatidylserine which

55、 is flipped to outside of plasma membraneearly in apoptosis MTT-assays ability of active mitochondria to cleave colorimetric substrateCaspase Activation-caspase cleavage eventsCytochrome c release from mitochondriaUVApoptosis in Response to UV-irradiationAnnexin-V stainingHoechst stainingDNA Fragmen

56、tationApoptosis signals Fas ligandFasCaspase 8BidCytochrome cCaspase 3Caspase 7FADDIntrinsicpathwayExtrinsicpathwayAPOPTOSISBclBax細胞內源性通路細胞外通路Takahashi A, Masuda A, Sun M, Centonze VE, Herman B. (2004). Oxidative stress-induced apoptosis is associated with alterations in mitochondrial caspase activi

57、ty and Bcl-2-dependent alterations in mitochondrial pH (pHm). Brain Res Bull. 62(6):497-504. LinksAutophagy: 自噬Energy dependent process“自食” LC3 dependentOrLC3 independentTreatment of strokeCategoryNameClinical efficacyAnticoagulation/ thrombolysisAspirin/ heparin/ tPA/ urokinaseModestGlutamate recep

58、tor blockadeAptiganel/ dextrophan/ EliprodilNoneVoltage-gated Ca2+ channel blockersNimodipine/ lifarizine/ flunarizineNoneNa+ channel blockersLubeluzole/ riluzolUnclear or noneVoltage-dependent K+-channel agonistBMS-204352Being testedEnhancement of inhibitory neurotransmissionClormethiazoleBeing tes

59、tedFree radical scavengers/ antioxidantsPergorgotein/ tirilazad/ ebselenNone or minimalNeural repairCiticholine/ troferminUnclear or minimalTreatment of strokeCategoryNameClinical efficacyAnticoagulation/ thrombolysisAspirin/ heparin/ tPA/ urokinaseModestGlutamate receptor blockadeAptiganel/ dextrop

60、han/ EliprodilNoneVoltage-gated Ca2+ channel blockersNimodipine/ lifarizine/ flunarizineNoneNa+ channel blockersLubeluzole/ riluzolUnclear or noneVoltage-dependent K+-channel agonistBMS-204352Being testedEnhancement of inhibitory neurotransmissionClormethiazoleBeing testedFree radical scavengers/ an