March 29, 2011Lech Kiedrowski, Ph.D.UICDepartment of [email protected] agentsObjectives1. Learn about the mechanisms ofneurodegeneration caused by brain ischemia(stroke, heart attack).2. Learn about neuroprotective agents beingtested to counteract ischemic brain damage.Outline1. High susceptibility of the brain to ischemia2. Mechanisms of ischemic neuronal deathand the role of Ca2+ and Zn2+ in triggeringthese mechanisms3. Agents developed to protect the brain fromischemic damageAmerican Heart Association 2004Ischemic brain damage may occur after:• Heart attack (global ischemia)• Stroke (focal ischemia)– ischemic (occlusion of a blood vessel) 87%– hemorrhagic (bleeding in the brain) 13%Heart attack and brain damage• Brain damage can start to occur just 4-6minutes after the heart stops pumpingblood• Survival rate is only 2% if heart isarrested for more than 12 minStroke and brain damage• About 795,000 cases each year• Every 40 seconds someone in the USA has astroke and every 3 min someone dies of it• Stroke is the third leading cause of death, afterheart disease and cancer• Stroke is the leading cause of long-term disability(60% of survivors become handicapped)• The estimated direct and indirect cost of strokefor 2009 was $68.9 billion.• Lack of effective neuroprotective therapyAmerican Heart Association 2009Stroke and brain damage The only FDA-approved therapy for stroke isintravenous injection of t-PA (Tissue PlasminogenActivator, a clot-dissolving agent) However, t-PA must be applied during the first 3hours of stroke and during this time it has to bedetermined that the stroke is not hemorrhagic Often stroke victims arrive at the hospital or arediagnosed too late to apply t-PAHigh energy requirements of the brain The human brain constitutes only 2%of the body’s weight, yet it utilizesapproximately 25% of total glucoseand almost 20% of oxygenHow humans are affected whenthis supply is interrupted?Glucose and oxygen are supplied tothe brain with bloodArch. Neurol. Psych. 50 (1943) 510-528126 volunteers !Arch. Neurol. Psych. 50 (1943) 510-528The Kabat-Rossen-Anderson apparatus7 seconds of brain ischemia will make you unconscious, but will not damage your brainArch. Neurol. Psych. 50 (1943) 510-528Hansen, Acta Physiol. Scand. (1978) 102: 324-329.EEG is flat within 10 sec ofglobal brain ischemiaIschemic depolarization (highelevation in external K+) takesplace about 2 min after theonset of ischemia.Sagital section through rat brainHippocampusSelective vulnerability of CA1 neurons to ischemiaYokota et al. Stroke (1995) 26: 1901-1907.Sham operated3 days after 10-min ischemia7 days after 10-min ischemiaCA1 neurons dieCA3 and DG neuronssurviveCA = Cornu Ammonis (Ammon’s horn)DG = Dentate GyrusDGCA1CA32 min of ischemia3 min of ischemiaKato et al. Brain Res. (1991) 553: 238-242.Hippocampal CA1 regionin gerbil brain 7 days after ischemiaIschemia has to last over 2 min to kill CA1 neuronsWhat kills the CA1 neurons?The Pulsinelli et al. experimentDenervation protectedthe CA1 neurons fromischemic deathPulsinelli (1985) Prog Brain Res 63: 29-37Which hippocampuswas denervated, theleft or the right one????Death10 20 30 10 20 10 20 30 60 120 Baseline Ischemia ReperfusionExtracellular glutamate during ischemia and reperfusionGlutamate (µM) sampled from various brain regions of the rat subjectedto 20-min ischemia.Globus et al. (1988) J Neurochem 51:1455-1464Glutamate is neurotoxicOlney, J.W., Brain lesions, obesity, andother disturbances in mice treated withmonosodium glutamate. Science, 1969.164: p. 719-721.A single subcutaneous injection of glutamate (4 mg/g) produces brainlesions and kills 2 – 9 day-old mice within 1 to 48 hoursGlutamateReceptorDeath?In cultured spinal neurons, glutamate deregulates Ca2+homeostasis in a Ca-dependent mannerTymianski et al. J. Neurosci. 13 (1993) 2085-2104Some of these receptors areCa-permeable channelsinoutGlutamateNMDAAMPA(-Glu-R2)AMPA(+Glu-R2)KainatemGluRsgroup 1mGluRsgroup 2 and 3IP3 cAMP Na+ Ca2+Na+Na+Na+ Ca2+K+K+K+K+NBQX NBQXNBQXMK-801Tymianski et al. J. Neurosci. 13 (1993) 2085-2104Ca2+ deregulationDead NeuronsFraction deregulated/deadAPV – NMDA receptor inhibitorCNQX – AMPA/kainate receptor inhibitorNIM – voltage-gated Ca channel inhibitorBlocking NMDA receptors prevents glutamate-induced deregulation of Ca2+homeostasis and neuronal deathConclusion: Inhibiting NMDA receptors is sufficient toprotect the neurons against glutamate-induced deathFailure of clinical stroke trials with glutamate receptor antagonistDrugs Mode of action ResultSelfotel competitive NMDA antagonist trial discontinued Aptiganel noncompetitive NMDA antagonist adverse effectsMK-801 noncompetitive NMDA antagonist adverse effectsDextrorfan noncompetitive NMDA antagonist adverse effects GV150526 glycine site antagonist of NMDA rec. no efficacyEliprodil polyamine site antagonist of NMDA rec. no efficacyNBQX competitive AMPA receptor antagonist trial discontinuedadverse effectsrenal toxicityCerebrovasc. Dis. 11, suppl 1 (2001) 60-70!Zinc-specific fluorescence in rathippocampus before ischemiaCA1 region 3 daysafter 10-min ischemiaZinc-specificfluorescenceFuchsin staining (pink)of degenerating neuronsKoh et al. Science 272 (1996) 1013-1016CaEDTA but not ZnEDTA protectsCA1 neurons against ischemic deathThe role of zinc in ischemic neuronal deathThe data indicate that preventing zinc translocation,using CaEDTA,prevents the ischemic death of CA1 neuronsWhat changes occur in the brain during ischemia?What role does zinc play in these changes?Poly(ADP-ribose) polymerase-1 (PARP-1) gets activated inischemic brain and may lead to neuronal deathKauppinen and Swanson, Neuroscience 145 (2007) 1267-1272Zhang et al. Science 263 (1994) 686 - 689NO – nitric oxidePARS – Poly(ADP-ribose) synthetaseNAm – nicotinamideNMN – nicotinamide mononucleotidePRPP – phosphoribosyl pyrophosphatePPi – inorganic phosphatePARP-1 (called also PARS)-mediated NAD- and ATP-depletion leads to cell deathDoes PARP play a role in ischemic neuronal death?Endres et al. J Cereb Blood Flow Metab 17 (1997) 1143-1151PBS = phosphate buffered saline (control)3-AB = 3-aminobenzamide (PARP inhibitor)PARP-1 knockout PARP-1 inhibitionPARP knockout or PARP inhibition reduce the size of ischemic braininfarct caused by the middle cerebral artery occlusion (MCAO)PARP plays a role in ischemic
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