Cerebral Ischemia overview

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Overview of the physiology of experimental cerebral ischemia research (1980-2005)

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Cerebral Ischemia overview

  1. 1. Cerebral Ischemia Cellular and Molecular Mechanisms of the Injured Brain Joseph Grenier, MD, PhD Benedictine University The Neurophysiology Foundation
  2. 2. Definition <ul><li>A decrease in blood flow to brain tissue such that vital metabolic functions fail and cell death begins. </li></ul>
  3. 3. Etiology of Stroke <ul><li>Atherosclerotic on thrombotic occlusion </li></ul><ul><li>Embolic occlusion secondary to hypercoagulable states </li></ul><ul><li>Intracerebral hemorrhage secondary to hypertension </li></ul><ul><li>Vasculitis secondary to lupus </li></ul><ul><li>Collagen vascular diseases </li></ul><ul><li>Amyloidosis </li></ul>
  4. 4. Animal Models Global Ischemia Models <ul><li>Unilateral carotid occlusion of the gerbil </li></ul><ul><li>Uni and bilateral occlusion in the rat </li></ul><ul><li>Unilateral carotid occlusion with induced hypertension </li></ul><ul><li>Seven Artery Occlusion Model in the rat </li></ul><ul><li>Focal ischemia models </li></ul><ul><ul><li>Middle cerebral artery occlusion in the rat or primate </li></ul></ul><ul><ul><li>Pterygo palatine artery occlusion in the rat </li></ul></ul>
  5. 5. Brain Tissue In Vitro Models <ul><li>Dish with cultured neurons perfused with deprived oxygen </li></ul><ul><li>Thin brain slice model perfused with nitrogen gas and CO 2 </li></ul><ul><li>Thick brain slice model perfused with nitrogen gas and CO 2 </li></ul><ul><li>En bloc (section) of brain deprived of oxygen or perfused with hypoxic solutions </li></ul>
  6. 6. Advantages/Disadvantages of Animal Models <ul><li>Can determine what the nature of the ischemic response is like in humans </li></ul><ul><li>SHR spontaneously hypertensive rats may be fed high fat diets to simulate hypercholesterolemia </li></ul><ul><li>Acute photochemical thrombosis with the rose bengal dye laser can simulate embolic disease </li></ul><ul><li>Cannot accurately simulate chronic antherosclerosis (aka atherosclerosis) </li></ul><ul><li>Anatomy of cerebrovasculature is different between animals and humans </li></ul>
  7. 7. Excitotoxicity Model <ul><li>Massive influx of calcium associated with ischemic injury is thought to poison the viability of cell mitochondria </li></ul><ul><li>NMDA AMPA receptors are activated during profuse neurotransmitter release leading to further calcium injury and therefore cell cytotoxicity </li></ul>
  8. 8. Neurotrophin Biochemistry <ul><li>Nerve growth factor, insulin-like growth factors 1 and 2, basic fibroblast growth factor, brain-derived neurotrophin factor, and neurotrophin 3 have proven useful in therapy for ischemia in vitro and in vivo </li></ul>
  9. 9. Free Radical Biochemistry <ul><li>Free radical injury has been found to occur during the reperfusion phase following ischemia </li></ul><ul><li>Mediators include: hydroxyl, superoxide, hydrogen peroxide, and other peroxy radicals </li></ul><ul><li>Antioxidants such as Vitamin E, 21 amino steroid and spin trap compounds can reduce reperfusion injury </li></ul>
  10. 10. Trace Elements Contributing to Excitotoxicity <ul><li>Zinc </li></ul><ul><li>Calcium </li></ul><ul><li>Cadmium </li></ul><ul><li>Iron </li></ul><ul><li>Other divalent cations </li></ul>
  11. 11. Apoptosis and Necrosis <ul><li>Apoptosis is genetically programmed cell death </li></ul><ul><li>Apoptosis is exhibited by compaction of the cell body, nuclear fragmentation, and the formation of cell surface blebs </li></ul><ul><li>Necrosis involves acute cell swelling, and vacuolation of the cell body and neurites with lysis of the cell and spillage into the extracellular space </li></ul>
  12. 12. Neuron Selectivity <ul><li>Hippocampal CA 1 and CA 3 are most vulnerable to transient ischemic insults </li></ul><ul><li>Ventral horn motor neurons are also susceptible </li></ul><ul><li>Cerebellar Purkinje cells are highly susceptible as well </li></ul><ul><li>Neocortical neurons are also susceptible </li></ul>
  13. 13. Heat-Shock Proteins <ul><li>Heat-shock protein 70 and 72 are induced in neurons within the penumbra </li></ul><ul><li>It is not clear if heat-shock protein 70 and 72 are neuroprotective </li></ul><ul><li>Cyclic nucleotides potentiate excitotoxic injury </li></ul>
  14. 14. Brain Ischemia and Hypothermia <ul><li>A 1 to 2 degree reduction in brain temperature has found to be neuroprotective </li></ul><ul><li>Reducing temperature from 36 degrees centigrade to 34 degrees centigrade further protects CA1 hippocampal and dorsal-lateral striatal neurons </li></ul>
  15. 15. Hypothermia - cont’d <ul><li>Kuluz et al reported improved outcome after 30 minutes of global ischemia </li></ul><ul><li>Okada et al demonstrated an extension of protection from oxygen-glucose deprivation with extended treatment </li></ul><ul><li>Busto et al determined the best temperature is approximately 35 centigrade </li></ul>
  16. 16. Hypothermia - cont’d <ul><li>Intraischemic hypothermia at 30 degrees centigrade provides chronic histopathological protection for up to 2 months following transient global ischemia </li></ul><ul><li>Chen et al demonstrated that whole body hypothermia at 30 degrees centigrade induced before ischemia and continued for 2 hours following MCA occlusion afforded significant protection </li></ul>
  17. 17. Hyperthermia <ul><li>In gerbils, intraischemic hyperthermia at 30 degrees centigrade during a 5 minute bilateral carotid artery occlusion expanded the area of damage from CA1 to CA1-CA4 in the hippocampus </li></ul><ul><li>Is detrimental in both focal and global models </li></ul><ul><li>Whole body hyperthermia at 40.6 degrees centigrade during global ischemia caused a severe tissue acidosis during recirculation and an incomplete normalization of ATP </li></ul>
  18. 18. Spinal Cord Ischemia <ul><li>Berguer et al used cold saline in the subarachnoid space to induce hypothermia begun 50 minutes before aortic cross-clamping and improved functional recovery measured 24 hours subsequently </li></ul><ul><li>Deep spinal cord hypothermia produced by epidural cooling in general protects against ischemia </li></ul>
  19. 19. Mechanisms of Hypothermic Protection <ul><li>Brain cooling slows oxygen consumption and CO 2 production by 7% per degree centigrade </li></ul><ul><li>Hypothermia also reduces the rate of metabolism </li></ul><ul><li>Welsh et al showed that decreasing head temperature to 35 degrees centigrade delayed but did not stop the depletion of ATP within the hippocampus during a 5 minute ischemic episode </li></ul><ul><li>Busto et al showed that tissue levels of glucose, glycogen, phosphocreatine, and ATP were depleted to similar levels after 20 minutes of hypothermia at 30 degrees centigrade compared to 36 degrees centigrade </li></ul>
  20. 20. Hemodynamics of Hypothermia <ul><li>Rosomoff demonstrated in 1954 that whole body hypothermia at 25 degrees C lowered cerebral blood flow in dogs </li></ul><ul><li>Sakamoto et al showed diminished blood flow at 27.7 degrees C of systemic hypothermia; however, Kuluz et al showed increased cortical flow with local brain cooling as measured by laser doppler where flow doubled </li></ul>
  21. 21. Acidosis Related Damage <ul><li>Both ischemia and hypoxia lead to both acidosis and energy failure </li></ul><ul><li>Excess lactate is formed during complete ischemia </li></ul><ul><li>Tissue lactate rises 15 micromoles/gram </li></ul><ul><li>During hyperglycemia, lactate rises to 22 micromoles/gram </li></ul><ul><li>In severe hypoglycemia, lactate is formed from glycogen stores which raises it to 7 micromoles/gram </li></ul>
  22. 22. Acidosis Damage - cont’d <ul><li>There is a linear decrease in pH with increasing lactate </li></ul><ul><li>Increased hydrogen ions correlates with an increase in calcium </li></ul><ul><li>Acidosis exacerbates intracellular calcium during ischemia </li></ul><ul><li>Correlation: acidosis/free radical formation </li></ul><ul><li>Hypercapnia worsens neuronal damage in hypoglycemic coma </li></ul>
  23. 23. Endothelins <ul><li>Release of endothelins is stimulated by factors related to atherosclerosis, thrombin, platelet aggregation, macrophage migration, transforming growth factor, and IL-1 </li></ul><ul><li>Endothelin release is also stimulated by hypoxia and vasoactive peptides </li></ul><ul><li>Endothelial cells in large vessels express E-selectin as an inflammatory marker </li></ul>
  24. 24. Cytokines <ul><li>Cytokines are polypeptides that function as modulators of hemostasis and inflammation </li></ul><ul><li>Cytokines may be thought of as universal mediators of intercellular communicators </li></ul><ul><li>They include interleukins, colony stimulating factors, growth factors, and tumor necrosis factors </li></ul><ul><li>Cultured endothelial cells bathed in IL-1 and TNF-alpha diminish the expression of thrombomodulin and IL-6 </li></ul>
  25. 25. Interleukins <ul><li>IL-6 belongs to the hematopoietin receptor gene family </li></ul><ul><li>Okada et al showed that P-selectin became up-regulated during ischemia for 24 hours whereas ICAM-1 was up-regulated between 1-4 hours of reperfusion, mostly in microvessels </li></ul><ul><li>Central injection of IL-1 inhibits ischemic and excitotoxic damage </li></ul>
  26. 26. Blood-Brain Barrier in Ischemia <ul><li>The blood-brain barrier is a lining of adjacent endothelial cells connected through tight junctions </li></ul><ul><li>The barrier can be made even tighter by certain steroid hormones </li></ul><ul><li>The brain endothelial cell can manufacture nitric oxide, endothelin, and prostaglandins which regulate vascular caliber </li></ul>
  27. 27. Blood-Brain Barriers in Ischemia cont’d <ul><li>The blood-brain barrier opens only after 3-6 hours of continual ischemia </li></ul><ul><li>After 3 weeks of continual ischemia, the blood-brain barrier returns to normal </li></ul><ul><li>Vesicles containing histamine, calcium, serotonin, and bradykinin can open by increasing intracellular calcium </li></ul>
  28. 28. BBB in Ischemia - cont’d <ul><li>Chan et al observed BBB breakdown following injection of free radical generating reagents in the brain </li></ul><ul><li>Olesen reported injury with free radical agents perfused over the surface of the brain </li></ul><ul><li>Arachidonic acid opened the BBB reversibly and it was blocked by SOD or catalase </li></ul><ul><li>Brain edema formation is more due to sodium ion influx than influx of albumin from plasma after the BBB breaks down </li></ul>
  29. 29. BBB in Ischemia <ul><li>After the BBB is disrupted permitting influx of albumin, electrolytes such as sodium enter the brain intra and extracellular spaces resulting in vasogenic edema </li></ul><ul><li>Vasogenic edema secondary to ischemia leads to an increase in brain cations, the primary cause of edema formation </li></ul><ul><li>Sodium and chloride entry is thought to be the major determinants of both vasogenic and cytotoxic edema formation </li></ul>
  30. 30. BBB in Ischemia cont’d <ul><li>There is an increase in sodium transport in the BBB before the BBB is disrupted </li></ul><ul><li>The increase in permeability is specific to sodium; this is not seen for potassium or chloride </li></ul><ul><li>Nitric oxide formation by endothelium is increased during ischemia since endothelial cells contain nitric oxide synthase and this enzyme is increased in capillary beds </li></ul>
  31. 31. Nitric Oxide and Ischemia <ul><li>Nitric Oxide reduces the adherence of platelets and neutrophils to endothelium </li></ul><ul><li>Nitric Oxide knock-out mice are resistant to ischemia; the nitric oxide synthase inhibitor nitroarginine persistently decreases blood flow and increases ischemic damage </li></ul><ul><li>Nitric Oxide is produced by both neurons and endothelium in the brain </li></ul>
  32. 32. Nitric Oxide - cont’d <ul><li>The high activity of nitric oxide synthase in the CNS suggests that nitric oxide has important functions besides regulation of blood flow </li></ul><ul><li>Nitric Oxide also modulates synaptic plasticity in the brain. </li></ul><ul><li>Its synthesis is regulated by calmodulin and is induced by the influx of calcium through the NMDA receptor. Nitric oxide can affect intracellular pools of calcium in adjacent cells by changing the release of intracellular stores via a cGMP kinase mechanism. </li></ul>
  33. 33. Nitric Oxide Interactions <ul><li>Shibuki and Okado demonstrated that brief simultaneous electrical stimulation of both ascending and parallel fibers in cerebellar slices led to a rapid production of 70-100 nM NO. </li></ul><ul><li>Many groups have shown that NO synthesis is important for the development of long term potentiation in hippocampal slices. </li></ul><ul><li>NO is widely thought to be a highly toxic hydrophobic gas, but it is not highly toxic or reactive at the concentrations produced in vivo. </li></ul><ul><li>NO itself is far less reactive with most biological molecules than has been widely assumed. </li></ul>
  34. 34. NO and Superoxide Dismutase <ul><li>NO and superoxide dismutase interact in vivo during ischemia. </li></ul><ul><li>The brain can produce large amounts of NO, higher than the intracellular concentrations of superoxide dismutase. When this occurs NO will not only compete effectively for superoxide to produce substantial fluxes of NO, but the product peroxinitrite will in turn react with superoxide dismutase to nitrate tyrosines in brain proteins. </li></ul>
  35. 35. NO and SOD <ul><li>Dawson et al has found that SOD reduces the toxicity of endogenous nitric oxide production after NMDA stimulation in their neuronal cell culture system. </li></ul><ul><li>In the middle cerebral artery (MCA) occlusion model in rats and gerbils, IV PEG-SOD reduces ischemic injury. Liposome trapped SOD is also highly protective for cold edema and ischemia. Transgenic mice overexpression, superoxide dismutase are also protected against ischemia. </li></ul><ul><li>PED-SOD is also protective in severe head trauma in humans. </li></ul>
  36. 36. NO Formation in Neurons <ul><li>In vitro cerebellar slices and in vivo thalamic nucleus studies have shown arginine, a NO precursor, to be released following electrical or afferent path stimulation. </li></ul><ul><li>The fact that some authors do not see a protective effect of NOS inhibitors against NMDA toxicity in neuronal cultures is thought to be due to the routes linking NMDA receptor activation to cell death, in addition to that involving NO, and the relative involvement of routes varies in different culture types. </li></ul>
  37. 37. Protein Kinase C and Ischemia <ul><li>Protein kinase C is an important factor in cell survival. </li></ul><ul><li>During ischemia and immediately following ischemia, conditions favor PKC activation. During a 15 min ischemia episode, the calcium dependent isoforms of PKC and in particular PKC¥ from the cytosol to the neocortex and striatum increase. </li></ul><ul><li>Modulators of PKC activity affect neuronal injury. Staurosporin, a protein kinase inhibitor administered before but not after ischemia reduces neuronal damage </li></ul>
  38. 38. Protein Kinase C cont’d <ul><li>Gangliosides are very potent PKC inhibitors and have been shown to have a beneficial effect on ischemia </li></ul><ul><li>During ischemia, MK II is redistributed to the cytosol in synaptic fractions </li></ul>
  39. 39. Effect of Ischemia on CaMKII <ul><li>CMKII activity is generally depressed by ischemia and there is now overt autophosphorylation. </li></ul><ul><li>During ischemia, CaMII is redistributed from the cytosol to the synaptosomal fraction, preferentially to the postsynaptic densities. </li></ul><ul><li>The ischemia induced downregulation of the protein kinase activities and the depression of mRNA may be a cellular feedback mechanism activated to prevent a possible detrimental influence of the kinases. These protective responses, however, may become detrimental late during reperfusion when trophic signaling is needed for neuron repair and recovery. </li></ul>
  40. 40. MAP Kinase and Ischemia <ul><li>Following temporary ischemia, cerebral tyrosine phosphorylation of membrane proteins increases during reperfusion. In contrast, cytosolic proteins such as MAP kinase phosphorylation is transiently enhanced. </li></ul><ul><li>In the vulnerable striatum phosphorylation decreases while in the CA1 zone phosphorylation declines to control levels </li></ul>
  41. 41. MAP Kinase and Ischemia <ul><li>MAP kinase phosphorylation after ischemia is depressed by glutamate antagonists as is seen in hippocampal neurons exposed to glutamate. </li></ul><ul><li>This shows glutamate activates a pathway which includes protective measures against glutamate toxicity. </li></ul><ul><li>In contrast to protein kinases, the phosphatases are less affected by ischemia. </li></ul>
  42. 42. Neurotrophins and Cerebral Ischemia <ul><li>Nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophins 1-4 (trk protein kinases 1-4), p75, ciliary neurotrophic factor (CNTF) </li></ul><ul><li>Beta fibroblast growth factors (FGF and alpha-FGF). </li></ul><ul><li>All the neurotrophins are upregulated following cerebral ischemia/hypoxia. </li></ul>
  43. 43. Experimental Therapies for Ischemia <ul><li>NMDA/AMPA antagonists </li></ul><ul><li>Calcium channel blockers </li></ul><ul><li>Neurotrophin intracisternal or stereotactic parenchymal injections </li></ul><ul><li>Neural Stem Cell + Glial Cell Transplantation </li></ul><ul><li>Bone Marrow Cell Transplantation </li></ul>
  44. 44. Experimental Therapies <ul><li>Newer generation anticonvulsants e.g. Keppra, Tegretol, Neurontin, Gabatril, etc. </li></ul><ul><li>Stem cells + priming neurotrophins </li></ul><ul><li>Whole body hypothermia </li></ul><ul><li>Brain hypothermia </li></ul><ul><li>Gene Therapy </li></ul><ul><li>Suicide Gene Therapy/Antisense Oligos </li></ul>

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