April 4, 2007 L. Kiedrowski, Ph.D. UIC Department of Psychiatry lkiedr@psych.uic

1. April 4, 2007 L. Kiedrowski, Ph.D. UIC Department of Psychiatry lkiedr@psych.uic.edu Neuroprotective agents

2. Outline • Extreme vulnerability of brain to ischemia • Early stages of brain ischemia • Delayed neuronal death after ischemia • Neuroprotective agents and strategies

3. Ischemic 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 2004

4. Heart attack and brain damage • Brain damage can start to occur just 4-6 min after the heart stops pumping blood • Survival rate is only 2% if heart is arrested for more than 12 min American Heart Association 2004

5. Stroke and brain damage • 600,000 new cases each year • Every 45 seconds someone in the USA has a stroke and every 3 min someone dies of it • Stroke is the third leading cause of death, behind heart disease and cancer • Stroke is the leading cause of long-term disability (60% of survivors become handicapped) • In 2004, the overall cost of stroke-induced brain injury was over $53.6 billion • Lack of effective neuroptotective agents • The only currently available therapy: intravenous injection of t-PA (Tissue Plasminogen Activator, a clot-dissolving agent)

6. High energy requirements of the brain • The human brain constitutes only 2% of the body weight, yet it utilizes approximately 25% of total glucose and almost 20% of oxygen.

7. “This is the first controlled investigation on the effects of acute arrest of the circulation to the human brain.” Arch. Neurol. Psych. 50 (1943) 510-528

8. Humans become unconscious within 7 seconds of brain ischemia Arch. Neurol. Psych. 50 (1943) 510-528

9. Where does the consciousness reside? ILN – intralaminar nuclei of thalamus MRF – mesencephalic reticular formation Consciousness requires MRF activity Permanent post-stroke coma results from bilateral lesions within MRF

10. EEG is flat within 10 sec of global brain ischemia Ischemic depolarization (high elevation in external K+) takes place about 2 min after the onset of ischemia. from Hansen, (1978) Acta Physiol. Scand.

11. Sagital section of rat brain Hippocampus

12. Selective vulnerability of hippocampal CA1 neurons to ischemia CA1 CA = Cornu Ammonis (Ammon’s horn) DG = Dentate Gyrus Sham operated DG CA3 CA1 neurons die CA3 and DG neurons survive 3 days after 10-min ischemia 7 days after 10-min ischemia From Yokota et al. Stroke (1995) 26: 1901-1907.

13. Ischemia has to last over 2 min to kill CA1 neurons Ischemia 2 min 3 min Hippocampal CA1 region in gerbil brain 7 days after ischemia From Kato et al. Brain Res. (1991) 238-242

14. Denervation protected CA1 neurons from ischemic death.This indicates that CA1 neurons are damaged indirectly. from Pulsinelli (1985) Prog Brain Res 63:29-37.

15. ? ? ? Death

16. Extracellular glutamate during ischemia and reperfusion Baseline Ischemia Reperfusion 10 20 30 10 2010 20 30 60 120 Glutamate (µM) sampled from various brain regions of the rat subjected to 20-min ischemia. From Globus et al. (1988) J Neurochem 51:1455-1464.

17. Glutamate is neurotoxic Olney, J.W., Brain lesions, obesity, and other disturbances in mice treated with monosodium glutamate. Science, 1969. 164: p. 719-721. A single subcutaneous injection of glutamate (4 mg/g) produces brain lesions and kills 2 – 9 day-old mice within 1 to 48 hours.

18. Receptor Glutamate ? Death

19. In cultured spinal neurons, application of glutamate deregulates Ca2+ homeostasis. This deregulation depends on extracellular Ca2+ concentration The data imply that Ca2+ homeostasis is deregulated by glutamate-induced Ca2+ influx From Tymianski et al. J. Neurosci. 13 (1993) 2085-2104

20. Ionotropic receptors Metabotropic receptors Two classes of glutamate receptors: ionotropic and metabotropic Glutamate AMPA (-Glu-R2) AMPA (+Glu-R2) NMDA Kainate mGluRs group 1 mGluRs group 2 and 3 out in

21. Some ionotropic glutamate receptors mediate Ca2+ influx Glutamate AMPA (-Glu-R2) AMPA (+Glu-R2) NMDA Kainate mGluRs group 1 mGluRs group 2 and 3 Na+ Ca2+ Na+ Ca2+ Na+ Na+ out in IP3 K+ K+ K+ K+ cAMP

22. MK-801 APV NBQX CNQX NBQX CNQX NBQX CNQX MK-801 and NBQX inhibit NMDA and AMPA/kainate receptors, respectively Glutamate AMPA (-Glu-R2) AMPA (+Glu-R2) NMDA Kainate mGluRs group 1 mGluRs group 2 and 3 out in IP3 cAMP

23. Blocking NMDA receptors prevents glutamate-induced deregulation of Ca2+ homeostasis and neuronal death Ca2+ deregulation Dead Neurons Fraction deregulated/dead APV – NMDA receptor inhibitor CNQX – AMPA/kainate receptor inhibitor NIM – voltage-gated Ca channel inhibitor Conclusion: Inhibiting NMDA receptors is sufficient to protect the neurons against glutamate-induced death From Tymianski et al. J. Neurosci. 13 (1993) 2085-2104

24. Failure of clinical trials with glutamate receptor antagonist Drugs Mode of action Result Selfotel competitive NMDA antagonist trial discontinued Aptiganel noncompetitive NMDA antagonist adverse effects MK-801 noncompetitive NMDA antagonist adverse effects Dextrorfan noncompetitive NMDA antagonist adverse effects Racemide noncompetitive NMDA antagonist phase III planed GV150526 glycine site antagonist of NMDA rec. no efficacy Eliprodil polyamine site antagonist of NMDA rec. no efficacy NBQX competitive AMPA receptor antagonist trial discontinued adverse effects renal toxicity Cerebrovasc. Dis. 11, suppl 1 (2001) 60-70

25. !

26. What methodological problems? Answer: Many in vitro studies underestimated the impact of early ischemic events on the mechanism of toxic Ca2+ influx.

27. What early events ? • Oxygen depletion • Drop in pH • Plasma membrane depolarization by K+ efflux • Cytosolic [Na+] elevation These events affect the mechanism by which glutamate kills neurons!

28. Early Stages of Global Brain IschemiaOxygen Depletion From Halsey et al. Microvasc. Res. (1977)

29. Glucose Ca-pump Glycolysis ATP Pyruvate Glutamate Ca-phosphate Krebs cycle + Ca2+ Ca2+ Pi ATP -180 mV O2 4NADH e- DY 2H2O endoplasmic reticulum H+ H+ H+ In the presence of oxygen and glucose, Ca2+ accumulates in the mitochondria plasmalemma mitochondrion

30. + calpain In the absence of oxygen and glucose, Ca2+ accumulates in the cytosol Ca-pump Glycolysis Pyruvate Lactate Glutamate Ca-phosphate Krebs cycle Ca2+ + Ca2+ Ca2+ Pi -180 mV 4NADH e- DY

31. Mg2+ blocks NMDA channel Mg Glu Na+ Ca2+ out NMDA rec. in

32. Glu Glu Na+ influx via AMPA channel depolarizes the plasma membrane and removes the Mg2+ block Na+ Na+ Ca2+ out NMDA rec. AMPA rec. in Depolarization

33. Glucose Glycolysis Pyruvate Glutamate Ca-phosphate Krebs cycle + Ca2+ Ca2+ Pi -180 mV O2 4NADH e- DY 2H2O H+ H+ H+ Many in vitro studies were performed in the absence of Mg2+. NMDA channel not blocked by Mg2+ Under Mg-free conditions, the Ca2+ influx via NMDA receptors is artificially enhanced.

34. Early events of brain ischemia • Oxygen depletion • Drop in pH • Plasma membrane depolarization by K+ efflux • Cytosolic [Na+] elevation

35. Early Event of Global Brain Ischemia Low pH inhibits NMDA receptors Kainate rec. AMPA rec. NMDA rec. min Pyruvate Lactate From Traynelis and Cull-Candy (1991) Mitochondria From Siemkowicz and Hansen (1981)

36. Early events of brain ischemia • Oxygen depletion • Drop in pH • Plasma membrane depolarization by K+ efflux • Cytosolic [Na+] elevation

37. 100 50 mM [K+]o 10 3 From Hansen (1978) Early Stages of Brain IschemiaANOXIC DEPOLARIZATION Depolarization of the plasma membrane due to [K+]o increase, reduces (by about 50 mV) the electrochemical driving force for Ca2+ influx via NMDA and other Ca-permeable channels.

38. Early events of brain ischemia • Oxygen depletion • Drop in pH • Plasma membrane depolarization by K+ efflux • Cytosolic [Na+] elevation

39. 170 100 mM 100 [Na+]o 50 50 mM 5 [K+]o 1 .5 mM 10 [Ca2+]o .1 .05 3 From Hansen (1978) From Hansen and Zeuthen (1981) Early Stages of Brain Ischemia

40. Normal conditions 150 mM Na+ 5 mM K+ 3Na+ 4Na+ out NCX NCKX in -65 mV Ca2+ K+ Ca2+ 5 mM Na+ 150 mM K+ Forward mode How do the early ischemic ionic fluxes affect the plasmalemmal Na/Ca (NCX) and Na/Ca+K (NCKX) exchange operation? NCX and NCKX are electrogenic exchangers. The charge associated with Na+ transport exceeds that associated with Ca2+ transport. Therefore, NCX and NCKX generate membrane potential and are affected by the existing membrane potential. Normally, NCX and NCKX remove Ca2+ from the cytosol (forward mode to the exchange).

41. after anoxic depolarization before ischemia 170 100 mM 100 [Na+]o 50 50 mM 5 [K+]o 1 .5 mM 10 [Ca2+]o .1 .05 3 From Hansen (1978) From Hansen and Zeuthen (1981) Lets examine NCX and NCKX operation

42. Ion Out In Na+ 150 mM 5 mM Ca2+ 1300 mM 0.1 mM Em and ENCX before ischemia Em = - 65 mV ENCX = 3ENa – 2ECa ENa = 2.303 RT/F log ([Na]o/[Na]i) = 2.303 x 26.73 x log (150/5) = 61.56 x 1.477 = 90.9 mV ECa = 2.303 RT/2F log ([Ca]o/[Ca]i) = (2.303 x 26.73)/2 x log (1300/0.1) = 30.78 x 4.114 = 126.6 mV ENCX = 3ENa – 2ECa = (3 x 90.9) – (2 x 126.6) = 272.7 – 253.2 = 19.5 mV Em (- 65 mV) < ENCX (19.5 mV) When Em < ENCX, NCX operates in the forward mode

43. Ion Out In Na+ 150 mM 5 mM Ca2+ 1300 mM 0.1 mM K+ 5 mM 150 mM Em and ENCKX before ischemia Em = - 65 mV ENCKX = 4ENa – 2ECa – EK ENa = 2.303 RT/F log ([Na]o/[Na]i) = 2.303 x 26.73 x log (150/5) = 61.56 x 1.477 = 90.9 mV ECa = 2.303 RT/2F log ([Ca]o/[Ca]i) = (2.303 x 26.73)/2 x log (1300/0.1) = 30.78 x 4.114 = 126.6 mV EK = 2.303 RT/F log ([K]o/[K]i) = 2.303 x 26.73 x log (5/150) = 61.65 x -1.477 = - 90.9 mV ENCKX = 4ENa – 2ECa – EK = (4 x 90.9) – (2 x 126.6) – (– 90.9) = 201.3 mV Em (- 65 mV) < ENCKX (201.3 mV) When Em < ENCKX, NCKX operates in the forward mode

44. Ion Out In Na+ 50 mM 45 mM Ca2+ 130 mM 1 mM Em and ENCX after anoxic depolarization Em = - 15 mV ENCX = 3ENa – 2ECa ENa = 2.303 RT/F log ([Na]o/[Na]i) = 2.303 x 26.73 x log (50/45) = 61.56 x 0.0458 = 2.82 mV ECa = 2.303 RT/2F log ([Ca]o/[Ca]i) = (2.303 x 26.73)/2 x log (130/1) = 30.78 x 2.114 = 65.1 mV ENCX = 3ENa – 2ECa = (3 x 2.82) – (2 x 65.1) = 8.46 – 130.2 = –121.7 mV Em (–15 mV) > ENCX (–121.7 mV) When ENCX < Em, NCX operates in the reverse mode

45. Ion Out In Na+ 50 mM 45 mM Ca2+ 130 mM 1 mM K+ 65 mM 120 mM Em and ENCKX after anoxic depolarization Em = - 15 mV ENCKX = 4ENa – 2ECa –EK ENa = 2.303 RT/F log ([Na]o/[Na]i) = 2.303 x 26.73 x log (50/45) = 61.56 x 0.0458 = 2.82 mV ECa = 2.303 RT/2F log ([Ca]o/[Ca]i) = (2.303 x 26.73)/2 x log (130/1) = 30.78 x 2.114 = 65.1 mV EK = 2.303 RT/F log ([K]o/[K]i) = 2.303 x 26.73 x log (65/120) = 61.65 x -0.266 = -16.4 mV ENCKX = 4ENa – 2ECa – EK = (4 x 2.82) – (2 x 65.1) – (– 16.4) = – 102.5 mV Em (–15 mV) > ENCKX (–102.5 mV) When ENCKX < Em, NCKX operates in the reverse mode

46. Normal conditions Anoxic depolarization 150 mM Na+ 5 mM K+ 50 mM Na+ >65 mM K+ Ca2+ K+ 3Na+ 4Na+ Ca2+ out out NCX NCX NCKX NCKX in in K+ Ca2+ 3Na+ 4Na+ Ca2+ 45 mM Na+ 120 mM K+ 5 mM Na+ 150 mM K+ Forward mode Reverse mode NCX and NCKX reverse during anoxic depolarization

47. Ion Out In Na+ 50 mM 45 mM Ca2+ 130 mM x mM K+ 65 mM 120 mM How high do NCX and NCKX elevate [Ca2+]c? When Em > ENCKX , NCKX reverses When Em > ENCX , NCX reverses ENCKX = 4ENa – 2ECa – EK ENCX = 3ENa – 2ECa ENa = RT/F ln ([Na]o/[Na]i) EK = RT/F ln ([K]o/[K]i) ECa = RT/2F ln ([Ca]o/[Ca]i)

48. Very shortly after the onset of ischemia, in hippocampal CA1 neurons in vivo, [Ca2+]i elevates to ~50 mM 2 min From Silver and Erecinska, J. Gen. Physiol. (1990)

49. SEA0400 NCX NCKX SEA0400 is a potently inhibits NCX1 reversal AMPA/kainate rec. Ca2+ + Glutamate Krebs cycle Na+ + NMDA rec. Ca2+ 4NADH e- K+

50. Comparison of effects of SEA0400 versus MK-801 on ischemic brain damage SEA0400 cerebral cortex striatum The drugs were applied i.p. immediately after occlusion of the middle cerebral artery from Matsuda et al. J Pharmacol Exp Therap 298 (2001) 249-256