1April 4, 2007L. Kiedrowski, Ph.D.UICDepartment of [email protected] agentsOutline1. Extreme vulnerability of brain to ischemia2. Early stages of brain ischemia3. Delayed neuronal death after ischemia4. Neuroprotective agents and strategiesAmerican Heart Association 2004Ischemic brain damage may occur after:• Heart attack (global ischemia)• Stroke (focal ischemia)– ischemic (occlusion of a blood vessel) 88%– hemorrhagic (bleeding in the brain) 12%American Heart Association 2004Heart attack and brain damage• Brain damage can start to occur just 4-6 minafter the heart stops pumping blood• Survival rate is only 2% if heart is arrestedfor more than 12 min2Stroke and brain damage• 600,000 new cases each year• Every 45 seconds someone in the USA has a stroke andevery 3 min someone dies of it• Stroke is the third leading cause of death, behind heartdisease and cancer• Stroke is the leading cause of long-term disability (60% ofsurvivors become handicapped)• In 2004, the overall cost of stroke-induced brain injury wasover $53.6 billion• Lack of effective neuroptotective agents• The only currently available therapy: intravenous injectionof t-PA (Tissue Plasminogen Activator, a clot-dissolvingagent)High energy requirements of the brain• The human brain constitutes only 2% ofthe body weight, yet it utilizesapproximately 25% of total glucose andalmost 20% of oxygen.Arch. Neurol. Psych. 50 (1943) 510-528“This is the first controlled investigation on the effects ofacute arrest of the circulation to the human brain.”Arch. Neurol. Psych. 50 (1943) 510-528Humans become unconscious within7 seconds of brain ischemia3Where does the consciousness reside?ILN – intralaminarnuclei of thalamusMRF – mesencephalicreticular formationConsciousness requiresMRF activityPermanent post-strokecoma results frombilateral lesions withinMRFfrom Hansen, (1978) Acta Physiol. Scand. EEG is flat within 10 sec ofglobal brain ischemiaIschemic depolarization (highelevation in external K+) takesplace about 2 min after theonset of ischemia.Sagital section of rat brainHippocampusSelective vulnerability of hippocampal CA1 neurons to ischemiaFrom Yokota 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 GyrusDGCA1CA34Ischemia2 min3 minFrom Kato et al. Brain Res. (1991) 238-242Hippocampal CA1 regionin gerbil brain 7 days after ischemiaIschemia has to last over 2 min to kill CA1 neuronsDenervation protected CA1neurons from ischemic death.This indicates that CA1neurons are damagedindirectly.from Pulsinelli (1985) Prog Brain Res 63:29-37.???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.From Globus et al. (1988) J Neurochem 51:1455-1464.5Glutamate 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) producesbrain lesions and kills 2 – 9 day-old mice within 1 to 48 hours.GlutamateReceptorDeath?In cultured spinal neurons, application of glutamatederegulates Ca2+ homeostasis. This deregulation dependson extracellular Ca2+ concentrationFrom Tymianski et al. J. Neurosci. 13 (1993) 2085-2104The data imply that Ca2+ homeostasis isderegulated by glutamate-induced Ca2+ influxTwo classes of glutamate receptors:ionotropic and metabotropicGlutamateNMDAAMPA(-Glu-R2)AMPA(+Glu-R2)KainatemGluRsgroup 1mGluRsgroup 2 and 3inoutIonotropic receptorsMetabotropic receptors6Some ionotropic glutamate receptorsmediate Ca2+ influxinoutGlutamateNMDAAMPA(-Glu-R2)AMPA(+Glu-R2)KainatemGluRsgroup 1mGluRsgroup 2 and 3IP3 cAMP Na+ Ca2+Na+Na+Na+ Ca2+K+K+K+K+MK-801 and NBQX inhibit NMDA andAMPA/kainate receptors, respectivelyinoutGlutamateNMDAAMPA(-Glu-R2)AMPA(+Glu-R2)KainatemGluRsgroup 1mGluRsgroup 2 and 3IP3 cAMP NBQXCNQXNBQXCNQXNBQXCNQXMK-801APVFrom Tymianski 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 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 Racemide noncompetitive NMDA antagonist phase III planedGV150526 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) 60-707!What methodological problems?Answer:Many in vitro studies underestimated the impact of earlyischemic events on the mechanism of toxic Ca2+ influx.What early events ?1. Oxygen depletion2. Drop in pH3. Plasma membrane depolarization by K+ efflux4. Cytosolic [Na+] elevationThese events affect the mechanismby which glutamate kills neurons!Early Stages of Global Brain IschemiaOxygen DepletionFrom Halsey et al. Microvasc. Res. (1977)8In the presence of oxygen and glucose,Ca2+ accumulates in the mitochondriaGlucosePyruvateKrebscycle4NADHGlycolysise-2H2OO2H+H+H+ΔΨ-180 mVCa2+Ca2+PiCa-phosphateGlutamate+ATPCa-pumpATPendoplasmicreticulumplasmalemmamitochondrionPyruvateKrebscycle4NADHGlycolysise-ΔΨ-180 mVCa2+Ca2+PiCa-phosphateGlutamate+Ca-pumpLactateCa2+In the absence of oxygen and glucose,Ca2+ accumulates in the cytosolcalpain+Mg2+ blocks NMDA channelinoutNMDA rec.Na+ Ca2+GluMgNa+ influx via AMPA channel depolarizes the plasmamembrane and removes the Mg2+ blockAMPA rec.Na+ Ca2+Na+DepolarizationGluGluinoutNMDA rec.9Many in vitro studies were performed in the absence of Mg2+.GlucosePyruvateKrebscycle4NADHGlycolysise-2H2OO2H+H+H+ΔΨ-180 mVCa2+Ca2+PiCa-phosphateGlutamate+NMDA channelnot blocked byMg2+Under Mg-free
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