533
We conclude on the basis of our results and those of othersl,9,10 that lithium is not a major human teratogen. We believe that women with major affective disorders who wish to have children may continue lithium during pregnancy, and do not need to terminate pregnancy provided’that level II ultrasound and fetal echocardiography are done. REFERENCES 1. Zalstein E, Koren G, Einarson T, et al. A case-control study on the association between first trimester exposure to lithium and Ebstein’s anomaly. Am J Cardiol 1990; 65: 817-18. 2. Smithberg M, Dixet PK. Teratogenic effects of lithium in mice. Teratology 1982; 26: 239-46. 3. Gralla EJ, McIlhenny HM. Studies in pregnant rats, rabbits and monkeys with lithium carbonate. Toxicol Appl Pharmacol 1972; 21: 428-33. 4. Nora JJ, Nora AH, Toews WH. Lithium, Ebstein’s anomaly, and other congenital heart defects. Lancet 1974; ii: 594-95. 5. Long WA, Park WW. Maternal lithium and neonatal Ebstein’s anomaly: evaluation with cross-sectional echocardiography. Am J Perinatol 1984; 1: 182-84. 6. Fries H. Lithium in pregnancy. Lancet 1970; i: 1233. 7. Frankenberg FR, Lipinski JF. Congenital malformations. N Engl J Med
1983; 309: 311-12.
8. Koren G. Retinoid embryopathy. N Engl J Med 1986; 315: 262. 9. Kallen B. Comments on teratogen update: lithium. Teratology 1988; 38: 597-98. 10. Kallen B, Tandberg A. Lithium and pregnancy. Acta Psychiatr Scand 1983; 68: 134-39. 11. American Hospital Formulary Service. In: McEvoy GK, ed. New York: American Society of Hospital Pharmacists, 1989: 1245. 12. Marden PM, Smith DW, McDonald MJ. Congenital anomalies in the newborn infant, including minor variations. J Pediatr 1964; 64: 357-71. 13. Koren G. Teratogemc drugs and chemicals in humans. In: Koren G, ed. Maternal-fetal toxicology. New York: Marcel Dekker, 1990: 17. 14. Rosa F. Spina bifida in infants of woman treated with carbamazepine during pregnancy. N Engl J Med 1991; 324: 674-77. 15. Goodman L, Gilman A, eds. The pharmacological basis of therapeutics. 7th ed. New York: Macmillan, 1985: 429. 16. Koren G, Bologa M, Pastuszak A. The way women perceive teratogenic risk: the decision to terminate pregnancy. In: Koren G, ed. Maternal fetal toxicology. New York: Marcel Dekker, 1990: 373-81. 17. Belik J, Yoder M, Pereira GR. Fetal macrosomia: an unrecognised adverse effect of maternal lithium therapy. Pediatr Res 1983; 17: 304A. 18. Yoder MC, Belik J, Lannon RA, et al. Infants of mothers treated with lithium during pregnancy have an increased incidence of prematurity, macrosomia and perinatal mortality. Pediatr Res 1984; 18: 404A. 19. Schou M. What happened later to the lithium babies? Acta Psychiatr Scand 1976; 54: 193-97.
STROKE OCTET Pathophysiology of acute ischaemic stroke WILLIAM PULSINELLI The pathogenesis of brain damage from cerebrovascular occlusion may be separated into two sequential processes: (a) vascular and haematological events that cause the initial reduction and subsequent alteration of local cerebral blood flow; and (b) ischaemia-induced abnormalities of cellular chemistry that produce necrosis of neurons, glia, and other supportive brain cells. In this article I will summarise important mechanisms of cellular necrosis. The molecular consequences of brain ischaemia include changes in cell signalling (neurotransmitters, neuromodulators) ; in signal transduction (receptors, ion channels, second messengers, phosphorylation reactions); in metabolism (carbohydrate, protein, fatty acid, free radicals); and in gene regulation/expression. Investigators who seek to amelioriate or prevent stroke damage must distinguish reversible changes that cause only cellular dysfunction from processes that cause irreversible injury. Moreover, despite a common ischaemic insult, different mechanisms underlie necrosis of neurons and glia, and probably even necrosis of distinct neuronal types.
Histopathological types of ischaemic brain damage Histopathological damage from cerebrovascular occlusion depends on the degree and duration of impaired blood flow. In its mildest form, ischaemia kills uniquely vulnerable neurons such as the pyramidal neurons in the CA1 and CA4 zones of hippocampus, while sparing other neurons and all glial cells.’ Although such injury is usually encountered after transient global ischaemia in patients resuscitated from cardiac arrest, brief focal ischaemia may also destroy these "selectively vulnerable" neurons. By contrast, about 1 h of focal ischaemia causes cerebral infarction, which is characterised by the death of neurons, glia, and other supportive cells within the affected vascular
bed. The margins of the infarct are separated from normal brain by a rim of neuronal necrosis that spares glial cells but affects all neurons irrespective of phenotype. I will not discuss ischaemic injury to white matter, which may occur via mechanisms distinct from those in grey matter.
Spatial and temporal dynamics of severe and moderate ischaemia Severe focal ischaemia Occlusion of a cerebral blood vessel reduces but seldom abolishes the delivery of oxygen and the brain’s preferred fuel, glucose, to the affected vascular territory. Since dense vascular collaterals partly maintain blood flow in the ischaemic territory, regions nearest the collateral vessels are less severely affected than more distant areas (fig 1). This incomplete or partial ischaemia is responsible for the spatial and temporal dynamics of cerebral infarction. Spontaneous or pharmacological lysis of the occluding thrombus initiates reperfusion of the ischaemic area,recovery mechanisms in reversibly affected cells, and new mechanisms that either kill cells directly or facilitate lethal processes previously triggered by ischaemia. Ischaemia that causes persistent loss of membrane potentials (persistent anoxic depolarisation), lasting at least 5 min but less than 1 h, kills some or all of the selectively vulnerable neurons within the affected vascular bed.3 When ischaemia lasts more than 1 h, infarction begins in the central zone of lowest cerebral blood flow (fig 1) and progressively enlarges in a circumferential fashion towards its maximum ADDRESS:
Cerebrovascular
Disease
Research
Center,
Department of Neurology and Neuroscience, Cornell University Medical College, 1300 York Avenue, New York, NY 10021, USA (Dr W. Pulsinelli, MD, PhD).
534
Fig 1-Embolic occlusion of the pre-Rolandic branch of
the left
middle cerebral artery. An area of severe ischaemia N is surrounded by a rim of moderate ischaemia St proximal to collateral vessels (Modified with permission from Powers W, Raichle M. Stroke In. Pearlman A, Collins R, eds. Neurological pathophysiology. New York. Oxford University Press, 1984 )
3-4 h in
rodent6-8 h in non-human undetermined time in human beings. Attempts to attenuate the volume of an infarct via pharmacological or other means are critically dependent on the timeframe over which infarction is initiated and volume
over
primates,and
an
completed. Moderate focal ischaemia There is a rim of mild to moderately ischaemic tissue between normally perfused brain and the evolving infarct in which pathophysiological mechanisms are most dynamic, cell death occurs last, and pharmacological intervention has been most successful. In this border-zone, poorly understood mechanisms either suppress or completely block of normal transmission. Periods synaptic electrophysiological suppression or silence are interspersed with irregularly spaced episodes of abnormal membrane ion conductance manifested as depolarisation/repolarisation6 (recurrent anoxic depolarisation). The physical dimensions of this dynamic rim, sometimes known as the ischaemic penumbra,’ vary considerably but are inversely related to the steepness of the ischaemia gradient between normally perfused and severely ischaemic brain.
hippocampal neurons recover normal ATP and phosphocreatine concentrations previously depleted by transient, severe ischaemia only to succumb days later9 to events initiated by, but no longer requiring, energy depletion. 10 Cerebral blood flow below about 10-15 ml/100 g per min (normal 50-60 ml/ 100 g per min) in primates deprives brain of substrate (glucose) and the mitochondrial electron acceptor (oxygen) necessary for normal oxidative metabolism. Within minutes of the onset of ischaemia, energy demands exceed the brain’s capacity to synthesise ATP anaerobically from its meagre stores of glucose and glycogen, and high-energy phosphates and fuels for their synthesis are depleted. Lactate and unbuffered hydrogen ions accumulate in tissue in proportion to the carbohydrate stores present at the onset of ischaemia. Toxicity of hydrogen ions, especially their ability to facilitate ferrous iron-mediated free-radical mechanisms,l1 may be important in astroglial injury. The latter mechanism may partly explain why an increase of brain carbohydrates before the onset of ischaemia greatly augments and/or accelerates infarction in animals subjected to severe ischaemia." In addition to the rapid change in tissue acid-base status, failure of all energy-dependent mechanisms, including ion pumps, leads to deterioration of membrane ion gradients, opening of selective and unselective ion channels, and equilibration of most intracellular and extracellular ions (anoxic depolarisation). As a consequence of anoxic depolarisation, potassium ions leave the cell, sodium, and many neurotransmitters, including excitatory aminoacids (glutamate, aspartate), are released in potentially toxic concentrations.13 One known exception to this pattern is the
chloride,
and
calcium
ions
Ischaemia
enter,
(0;,, glucose)
Pathogenesis of severe ischaemia The flow
diagram shown in fig 2 depicts multiple, branching pathways that may be important in ischaemic brain damage. This representation helps one to understand the complexity of the process, with its many potential sites of interaction, and emphasises the fundamental importance of energy depletion in the genesis of subsequent injurious events. In severely ischaemic brain persistent shortage of high-energy phosphates is an overwhelming determinant of injury: unless cerebral blood flow and the tissue’s medium for energy exchange (ATP) are restored, necrosis is inevitable. Nevertheless, energy failure is not the immediate cause of cell death since (a) all brain cells tolerate loss of ATP for several minutes and the great majority of neurons and glia recover fully when blood flow is restored even after an hour of complete ischaemia;8 (b) one or more of the branching mechansms (fig 2) may independently kill brain cells; and (c) once initiated, such mechanisms may no longer require the triggering event. For example, CAI
Fig 2-Potential mechanisms of ischaemic brain damage. VRC=vo!tage-regu!ated calcium channels; LRC=iigand-regu!ated calcium channels; NA= noradrenaline (norepinephrine), DA=dopamine; NO synth = nitric oxide synthase.
535
Caz+
Ca2+
3Na+
Fig 3-Calcium homoeostasis in
a neuron.
Calcium influx is regulated by voltage-sensitive and ligand (glutamate) -sensitive channels named for their most potent synthetic agonists (NMDA andAMPA) PIP2= phosphatidyl inositol diphosphate; PLC = phospholipase C, DG = diacylglycerol, I P 3 = inositol triphosphate. Energy-dependent regulation of intracellular calcium [Ca2+]is via an ATP-dependent pump, translocation for Na+ ions, and uptake into endoplasmic reticulum (ER) and mitochondria. Energy-mdependent calcium homoeostasis occurs via buffering of calcium ions by calmodulin and other intracellular proteins (calbindin, parvalbumin). (Modified with permission from Swanson P, Schellenberg G, Clark A, et al Calcium buffering systems in brain In. Rodnight R, Bachelard H, Stahl W, eds. Chemisms of brain London: Churchill Livingstone, 1981.)
intracellular compartmentalisation of hydrogen ions in some astroglial cells.14 Selective intracellular acidosis of these cells may contribute to their demise and to infarction.11 Despite in-vitro evidence that cell death in anoxic, energy-depleted brain cells proceeds without calcium, other experiments with cultured neurons indicate that raised intracellular calcium accelerates many potentially injurious processes.16 Calcium activates phospholipases, which hydrolyse membrane-bound glycerophospholipids to freefatty acids, and these in turn facilitate free-radical peroxidation of other membrane lipids. Other examples of the potential catalytic role of calcium in cell injury include activation of proteases that lyse structural proteins17 and activation of nitric oxide synthasel8 to initiate free-radical mechanisms. Much research has focused on calcium dyshomoeostasis and on developing pharmacological methods to block influx of calcium or its release intracellularly. Since the extracellular calcium ion concentration is 104 105 times greater than its intracellular concentration, and since most mechanisms that maintain this gradient are either directly or indirectly energy dependent (fig 3), loss of ATP rapidly leads to a massive calcium influx and release of calcium from intracellular compartments.l9 Calcium flux through both ligand-regulated and voltage-regulated ion channels contributes to the intracellular accumulation of calcium. However, the failure of potent antagonists of either channel type to block cell death in severely ischaemic tissue indicates either that other pathological mechanisms of calcium overload are quantitatively more important in severe ischaemia or that cell death in such regions is not calcium
dependent.
Pathogenesis of moderate ischaemia During moderate ischaemia, in contrast to the catastrophic events that occur in severely ischaemic tissue, several compensatory mechanisms act in concert to maintain normal ATP concentrations and membrane ion
near
gradients, and to preserve, at least temporarily, cell viability. Reduction of cerebral blood flow
to
about 50% of normal
electroencephalographic activity, and only slightly greater ischaemia completely inhibits synaptic transmission and leads to an isoelectric encephalogram. The conserved energy, which is normally spent on restoring membrane ion gradients dissipated during synaptic activity, coupled with the continued, if reduced anaerobic synthesis suppresses
of ATP, maintains near normal tissue energy status. Thus, failure of energy-dependent mechanisms cannot explain either suppression or silence of the electroencephalogram during moderate ischaemia. The observed slight increase in membrane potassium ion conductance may hyperpolarise presynaptic and postsynaptic membranes, thereby reducing neurotransmitter release and the responsiveness of postsynaptic receptors to neurotransmitters. The mechanisms responsible for increased potassium ion conductance may involve modulation of ATP-regulated and/or calcium-regulated potassium channels.13 Despite compensatory mechanisms that sacrifice electrophysiological activity to reduce energy use and preserve cell viability, cell death occurs if moderate ischaemia lasts for several hours. Unknown processes sporadically overcome the hyperpolarised membranes to brief but recurrent episodes of membrane cause depolarisation (recurrent anoxic depolarisation), large ionic shifts, and a recurrent expenditure of energy to restore normal membrane ion gradients. The presumed but unproven cause for these recurrent depolarisations is of calcium-mediated release large quanta of neurotransmitters from their presynaptic storage sites. Dysregulation of calcium ion homoeostasis features prominently in the cell death of both moderately and severely ischaemic brain. However, the partial preservation of energy-dependent mechanisms that continue to regulate intracellular calcium ion concentrations in moderate ischaemia is strikingly different from the depletion of ATP and the total collapse of calcium homoeostasis in severe
536
ischaemia. Abnormal calcium flux through voltageregulated and ligand-regulated membrane channels contributes importantly to the increase in intracellular calcium in moderate ischaemia but less so in severe ischaemia, in which many failed mechanisms lead to calcium overload.l9 Thus, pharmacological blockade of membrane channels permeable to calcium may reduce intracellular calcium below toxic concentrations in moderate but not in severe ischaemia. In-vivo and in-vitro studies with postsynaptic (L-type) voltage-regulated and glutamate-regulated membrane channels indicate that calcium movement through the latter may be more directly involved in cell injury.20 The endogenous excitatory aminoacid neurotransmitter, glutamate, activates several postsynaptic receptor/channel complexes which are named for their most potent agonist molecule. Of these, the N-methyl-D-aspartate (NMDA) and the quisqualate (Q) receptor/channel complexes are permeable to calcium ions. Blockade of either channel type with selective agonists leads to a striking reduction in infarction volume in laboratory animals with focal brain ischaemia.21 For unexplained reasons, blockade of the Q receptor (also known as the AMPA receptor) but not of the NMDA receptor protects against necrosis of selectively vulnerable neurons after transient, severe ischaemia.21 In contrast to the potential neurotoxicity of hydrogen ions in severely ischaemic tissue, these same ions, when located in the extracellular space of moderately ischaemic brain, may be neuroprotective. Extracellular hydrogen ions, at a concentration equal to pH 6-9 or less, profoundly attenuate calcium conductance through the NMDA-regulated channel and reduce injury induced by oxygen-glucose deprivation in neuronal cultures.22 In moderate ischaemia, in which injury is partly dependent on the influx of calcium ions, extracellular pH falls rapidly to 6-9 or less and hydrogen ions may serve to protect the brain.
Therapeutic implications Therapies effective in experimental stroke Use of certain drugs in experimental ischaemia can clarify greatly the pathogenesis of cerebral infarction. Curiously, diverse classes of drugs reduce infarction volume in well-controlled animal models of focal brain ischaemia. Antagonists of the L-type and presynaptic (N-type) voltageregulated calcium channels; aminoacid neurotransmitter receptor antagonists of the NMDA and Q type that regulate independent calcium channels; drugs that reduce actions of free radicals or that bind to imidazole receptors; and anticonvulsants such as barbiturates and phenytoin all lessen brain injury when given either before or shortly after the onset of experimental ischaemia. The diverse nature of these drugs, plus the observation that they are effective principally in areas of moderate ischaemia, suggests that several pathogenetic mechanisms that are active in these areas are amenable to drug intervention. In addition to protecting against cellular mechanisms of injury, the L-type calcium-channel blockers (eg, nimodipine) which relax contraction of vascular smooth muscle; free-radical agents (eg, superoxide dismutase) which may prolong the half-life of endothelium-derived relaxing factor (nitric oxide); and some of the NMDA receptor/channel antagonists (eg, MK-801) have all been shown to increase cerebral blood flow in the ischaemic
territory.
Treatment strategies
strategies evolve from this discussion of pathogenesis. Firstly, therapy with neuroprotective agents must begin shortly after the onset of cerebral ischaemia. Treatment begun at or after the time of maturation of cerebral infarction in human beings, which probably differs little from the 6-8 h interval observed in primates, will be ineffective. Although infarction evolves over hours,4 treatment should continue for days to protect against possible recurrent ischaemia and against slowly evolving injury that occurs in some neurons.9 Second, clinical trials of neuroprotective drugs should also include therapy simultaneously to improve cerebral blood flow. Even a small increase in blood flow may optimise the volume of moderately ischaemic brain that is responsive to neuroprotective agents. Finally, since the bulk of the infarct is largely composed of tissue unresponsive to any neuroprotective drugs, research is needed to clarify the mechanisms of cell death in severely ischaemic brain and to identify methods to prolong the survival of such tissue until Three
treatment
4
blood flow
can
be restored. REFERENCES
1. Brierley J. Cerebral hypoxia. In: Blackwood W, Corsellis J, eds. Greenfield’s neuropathology. London: Edward Arnold, 1976: 43-85. 2. Fieschi C, Argentino C, Lenzi G, et al. Clinical and instrumental evaluation of patients with ischaemic stroke within the first six hours. J Neurol Sci 1989; 91: 311-22. 3. Levy D, Brierley J, Plum F. Ischaemic brain damage in the gerbil in the absence of "no-reflow". J Neurol Neurosurg Psychiatry 1975; 38: 1197-205. 4.
Kaplan B, Brint S, Tanabe J, Jacewicz M, Wang X. Pulsinelli W. Temporal thresholds for neocortical infarction in rats subjected to
reversible focal cerebral ischemia. Stroke 1991; 22: 1032-39. Jones T, Morawetz R, Crowell R, et al. Thresholds of focal cerebral ischemia in awake monkeys. J Neurosurg 1981; 54: 773-82. 6. Nedergaard M, Astrup J. Infarct rim: effect of hyperglycemia on direct current potential and [14C]2-deoxyglucose phosphorylation. J Cereb Blood Flow Metab 1986; 6: 607-15. 7. Astrup J, Siesjo B, Symon L. Thresholds in cerebral ischemia: the ischemic penumbra. Stroke 1981; 12: 723-25. 8. Hossmann K, Kleihues P. Reversibility of ischemic brain damage. Arch 5.
Neurol 1973; 29: 375-84. 9. Pulsinelli W, Brierley J, Plum F. Temporal profile of neuronal damage in a model of transient forebrain ischemia. Ann Neurol 1982; 11: 491-98. 10. Pulsinelli W, Duffy T. Regional energy balance in rat brain after transient forebrain ischemia. J Neurochem 1983; 40: 1500-03. 11. Siesjo B, Agardh C-D, Bengtsson F. Free radicals and brain damage. Cerebrovasc Brain Metab Rev 1989; 1: 165-211. 12. Ginsberg M. Metabolic responses to cerebral ischemia. Cerebrovasc Brain Metab Rev 1990; 2: 58-93. 13. Obrenovitch T, Sarna G, Symon L. Ionic homeostasis and neurotransmitter changes in ischemia. In: Krieglstein J, Oberpichler H, eds. Pharmacology of cerebral ischemia. Stuttgart: WVS, 1990: 97-112. 14. Kraig R, Chesler M. Astrocytic acidosis in hyperglycemia and complete ischemia. J Cereb Blood Flow Metab 1990; 10: 104-14. 15. Plum F. What causes infarction in the ischemic brain? The Robert Wartenberg Lecture. Neurology 1983; 33: 222-33. 16. Choi D. Ionic dependence of glutamate neurotoxicity. J Neurosci 1987; 7: 369-79. 17. Seubert P, Lee K, Lynch G. Ischemia triggers NMDA receptor-linked cytoskeletal proteolysis in hippocampus. Brain Res 1989; 492: 366-70. 18. Garthwaite J. Glutamate, nitric oxide and cell signaling in the nervous system. Trends Neurosci 1991; 14: 60-67. 19. Siesjo B, Bengtsson F. Calcium fluxes, calcium antagonists, and calcium-related pathology in brain ischemia, hypoglycemia, and spreading depression: a unifying hypothesis. J Cereb Blood Flow Metab 1989; 9: 127-40. 20. Choi D. Methods for antagonizing glutamate neurotoxicity. Cerebrovasc Brain Metab Rev 1990; 2: 105-47. 21. Pulsinelli W, Sarokin A, Buchan A. Antagonism of the NMDA and non-NMDA receptors in global versus focal brain ischemia. Prog Brain Res (in press). 22. Giffard R, Monyer H, Christine C, Choi D. Acidosis reduces NMDA receptor activation, glutamate neurotoxicity, and oxygen-glucose depnvation neuronal injury in cortical cultures. Brain Res 1990; 506: 339-42.
We conclude on the basis of our results and those of othersl,9,10 that lithium is not a major human teratogen. We believe that women with major affective disorders who wish to have children may continue lithium during pregnancy, and do not need to terminate pregnancy provided’that level II ultrasound and fetal echocardiography are done. REFERENCES 1. Zalstein E, Koren G, Einarson T, et al. A case-control study on the association between first trimester exposure to lithium and Ebstein’s anomaly. Am J Cardiol 1990; 65: 817-18. 2. Smithberg M, Dixet PK. Teratogenic effects of lithium in mice. Teratology 1982; 26: 239-46. 3. Gralla EJ, McIlhenny HM. Studies in pregnant rats, rabbits and monkeys with lithium carbonate. Toxicol Appl Pharmacol 1972; 21: 428-33. 4. Nora JJ, Nora AH, Toews WH. Lithium, Ebstein’s anomaly, and other congenital heart defects. Lancet 1974; ii: 594-95. 5. Long WA, Park WW. Maternal lithium and neonatal Ebstein’s anomaly: evaluation with cross-sectional echocardiography. Am J Perinatol 1984; 1: 182-84. 6. Fries H. Lithium in pregnancy. Lancet 1970; i: 1233. 7. Frankenberg FR, Lipinski JF. Congenital malformations. N Engl J Med
1983; 309: 311-12.
8. Koren G. Retinoid embryopathy. N Engl J Med 1986; 315: 262. 9. Kallen B. Comments on teratogen update: lithium. Teratology 1988; 38: 597-98. 10. Kallen B, Tandberg A. Lithium and pregnancy. Acta Psychiatr Scand 1983; 68: 134-39. 11. American Hospital Formulary Service. In: McEvoy GK, ed. New York: American Society of Hospital Pharmacists, 1989: 1245. 12. Marden PM, Smith DW, McDonald MJ. Congenital anomalies in the newborn infant, including minor variations. J Pediatr 1964; 64: 357-71. 13. Koren G. Teratogemc drugs and chemicals in humans. In: Koren G, ed. Maternal-fetal toxicology. New York: Marcel Dekker, 1990: 17. 14. Rosa F. Spina bifida in infants of woman treated with carbamazepine during pregnancy. N Engl J Med 1991; 324: 674-77. 15. Goodman L, Gilman A, eds. The pharmacological basis of therapeutics. 7th ed. New York: Macmillan, 1985: 429. 16. Koren G, Bologa M, Pastuszak A. The way women perceive teratogenic risk: the decision to terminate pregnancy. In: Koren G, ed. Maternal fetal toxicology. New York: Marcel Dekker, 1990: 373-81. 17. Belik J, Yoder M, Pereira GR. Fetal macrosomia: an unrecognised adverse effect of maternal lithium therapy. Pediatr Res 1983; 17: 304A. 18. Yoder MC, Belik J, Lannon RA, et al. Infants of mothers treated with lithium during pregnancy have an increased incidence of prematurity, macrosomia and perinatal mortality. Pediatr Res 1984; 18: 404A. 19. Schou M. What happened later to the lithium babies? Acta Psychiatr Scand 1976; 54: 193-97.
STROKE OCTET Pathophysiology of acute ischaemic stroke WILLIAM PULSINELLI The pathogenesis of brain damage from cerebrovascular occlusion may be separated into two sequential processes: (a) vascular and haematological events that cause the initial reduction and subsequent alteration of local cerebral blood flow; and (b) ischaemia-induced abnormalities of cellular chemistry that produce necrosis of neurons, glia, and other supportive brain cells. In this article I will summarise important mechanisms of cellular necrosis. The molecular consequences of brain ischaemia include changes in cell signalling (neurotransmitters, neuromodulators) ; in signal transduction (receptors, ion channels, second messengers, phosphorylation reactions); in metabolism (carbohydrate, protein, fatty acid, free radicals); and in gene regulation/expression. Investigators who seek to amelioriate or prevent stroke damage must distinguish reversible changes that cause only cellular dysfunction from processes that cause irreversible injury. Moreover, despite a common ischaemic insult, different mechanisms underlie necrosis of neurons and glia, and probably even necrosis of distinct neuronal types.
Histopathological types of ischaemic brain damage Histopathological damage from cerebrovascular occlusion depends on the degree and duration of impaired blood flow. In its mildest form, ischaemia kills uniquely vulnerable neurons such as the pyramidal neurons in the CA1 and CA4 zones of hippocampus, while sparing other neurons and all glial cells.’ Although such injury is usually encountered after transient global ischaemia in patients resuscitated from cardiac arrest, brief focal ischaemia may also destroy these "selectively vulnerable" neurons. By contrast, about 1 h of focal ischaemia causes cerebral infarction, which is characterised by the death of neurons, glia, and other supportive cells within the affected vascular
bed. The margins of the infarct are separated from normal brain by a rim of neuronal necrosis that spares glial cells but affects all neurons irrespective of phenotype. I will not discuss ischaemic injury to white matter, which may occur via mechanisms distinct from those in grey matter.
Spatial and temporal dynamics of severe and moderate ischaemia Severe focal ischaemia Occlusion of a cerebral blood vessel reduces but seldom abolishes the delivery of oxygen and the brain’s preferred fuel, glucose, to the affected vascular territory. Since dense vascular collaterals partly maintain blood flow in the ischaemic territory, regions nearest the collateral vessels are less severely affected than more distant areas (fig 1). This incomplete or partial ischaemia is responsible for the spatial and temporal dynamics of cerebral infarction. Spontaneous or pharmacological lysis of the occluding thrombus initiates reperfusion of the ischaemic area,recovery mechanisms in reversibly affected cells, and new mechanisms that either kill cells directly or facilitate lethal processes previously triggered by ischaemia. Ischaemia that causes persistent loss of membrane potentials (persistent anoxic depolarisation), lasting at least 5 min but less than 1 h, kills some or all of the selectively vulnerable neurons within the affected vascular bed.3 When ischaemia lasts more than 1 h, infarction begins in the central zone of lowest cerebral blood flow (fig 1) and progressively enlarges in a circumferential fashion towards its maximum ADDRESS:
Cerebrovascular
Disease
Research
Center,
Department of Neurology and Neuroscience, Cornell University Medical College, 1300 York Avenue, New York, NY 10021, USA (Dr W. Pulsinelli, MD, PhD).
534
Fig 1-Embolic occlusion of the pre-Rolandic branch of
the left
middle cerebral artery. An area of severe ischaemia N is surrounded by a rim of moderate ischaemia St proximal to collateral vessels (Modified with permission from Powers W, Raichle M. Stroke In. Pearlman A, Collins R, eds. Neurological pathophysiology. New York. Oxford University Press, 1984 )
3-4 h in
rodent6-8 h in non-human undetermined time in human beings. Attempts to attenuate the volume of an infarct via pharmacological or other means are critically dependent on the timeframe over which infarction is initiated and volume
over
primates,and
an
completed. Moderate focal ischaemia There is a rim of mild to moderately ischaemic tissue between normally perfused brain and the evolving infarct in which pathophysiological mechanisms are most dynamic, cell death occurs last, and pharmacological intervention has been most successful. In this border-zone, poorly understood mechanisms either suppress or completely block of normal transmission. Periods synaptic electrophysiological suppression or silence are interspersed with irregularly spaced episodes of abnormal membrane ion conductance manifested as depolarisation/repolarisation6 (recurrent anoxic depolarisation). The physical dimensions of this dynamic rim, sometimes known as the ischaemic penumbra,’ vary considerably but are inversely related to the steepness of the ischaemia gradient between normally perfused and severely ischaemic brain.
hippocampal neurons recover normal ATP and phosphocreatine concentrations previously depleted by transient, severe ischaemia only to succumb days later9 to events initiated by, but no longer requiring, energy depletion. 10 Cerebral blood flow below about 10-15 ml/100 g per min (normal 50-60 ml/ 100 g per min) in primates deprives brain of substrate (glucose) and the mitochondrial electron acceptor (oxygen) necessary for normal oxidative metabolism. Within minutes of the onset of ischaemia, energy demands exceed the brain’s capacity to synthesise ATP anaerobically from its meagre stores of glucose and glycogen, and high-energy phosphates and fuels for their synthesis are depleted. Lactate and unbuffered hydrogen ions accumulate in tissue in proportion to the carbohydrate stores present at the onset of ischaemia. Toxicity of hydrogen ions, especially their ability to facilitate ferrous iron-mediated free-radical mechanisms,l1 may be important in astroglial injury. The latter mechanism may partly explain why an increase of brain carbohydrates before the onset of ischaemia greatly augments and/or accelerates infarction in animals subjected to severe ischaemia." In addition to the rapid change in tissue acid-base status, failure of all energy-dependent mechanisms, including ion pumps, leads to deterioration of membrane ion gradients, opening of selective and unselective ion channels, and equilibration of most intracellular and extracellular ions (anoxic depolarisation). As a consequence of anoxic depolarisation, potassium ions leave the cell, sodium, and many neurotransmitters, including excitatory aminoacids (glutamate, aspartate), are released in potentially toxic concentrations.13 One known exception to this pattern is the
chloride,
and
calcium
ions
Ischaemia
enter,
(0;,, glucose)
Pathogenesis of severe ischaemia The flow
diagram shown in fig 2 depicts multiple, branching pathways that may be important in ischaemic brain damage. This representation helps one to understand the complexity of the process, with its many potential sites of interaction, and emphasises the fundamental importance of energy depletion in the genesis of subsequent injurious events. In severely ischaemic brain persistent shortage of high-energy phosphates is an overwhelming determinant of injury: unless cerebral blood flow and the tissue’s medium for energy exchange (ATP) are restored, necrosis is inevitable. Nevertheless, energy failure is not the immediate cause of cell death since (a) all brain cells tolerate loss of ATP for several minutes and the great majority of neurons and glia recover fully when blood flow is restored even after an hour of complete ischaemia;8 (b) one or more of the branching mechansms (fig 2) may independently kill brain cells; and (c) once initiated, such mechanisms may no longer require the triggering event. For example, CAI
Fig 2-Potential mechanisms of ischaemic brain damage. VRC=vo!tage-regu!ated calcium channels; LRC=iigand-regu!ated calcium channels; NA= noradrenaline (norepinephrine), DA=dopamine; NO synth = nitric oxide synthase.
535
Caz+
Ca2+
3Na+
Fig 3-Calcium homoeostasis in
a neuron.
Calcium influx is regulated by voltage-sensitive and ligand (glutamate) -sensitive channels named for their most potent synthetic agonists (NMDA andAMPA) PIP2= phosphatidyl inositol diphosphate; PLC = phospholipase C, DG = diacylglycerol, I P 3 = inositol triphosphate. Energy-dependent regulation of intracellular calcium [Ca2+]is via an ATP-dependent pump, translocation for Na+ ions, and uptake into endoplasmic reticulum (ER) and mitochondria. Energy-mdependent calcium homoeostasis occurs via buffering of calcium ions by calmodulin and other intracellular proteins (calbindin, parvalbumin). (Modified with permission from Swanson P, Schellenberg G, Clark A, et al Calcium buffering systems in brain In. Rodnight R, Bachelard H, Stahl W, eds. Chemisms of brain London: Churchill Livingstone, 1981.)
intracellular compartmentalisation of hydrogen ions in some astroglial cells.14 Selective intracellular acidosis of these cells may contribute to their demise and to infarction.11 Despite in-vitro evidence that cell death in anoxic, energy-depleted brain cells proceeds without calcium, other experiments with cultured neurons indicate that raised intracellular calcium accelerates many potentially injurious processes.16 Calcium activates phospholipases, which hydrolyse membrane-bound glycerophospholipids to freefatty acids, and these in turn facilitate free-radical peroxidation of other membrane lipids. Other examples of the potential catalytic role of calcium in cell injury include activation of proteases that lyse structural proteins17 and activation of nitric oxide synthasel8 to initiate free-radical mechanisms. Much research has focused on calcium dyshomoeostasis and on developing pharmacological methods to block influx of calcium or its release intracellularly. Since the extracellular calcium ion concentration is 104 105 times greater than its intracellular concentration, and since most mechanisms that maintain this gradient are either directly or indirectly energy dependent (fig 3), loss of ATP rapidly leads to a massive calcium influx and release of calcium from intracellular compartments.l9 Calcium flux through both ligand-regulated and voltage-regulated ion channels contributes to the intracellular accumulation of calcium. However, the failure of potent antagonists of either channel type to block cell death in severely ischaemic tissue indicates either that other pathological mechanisms of calcium overload are quantitatively more important in severe ischaemia or that cell death in such regions is not calcium
dependent.
Pathogenesis of moderate ischaemia During moderate ischaemia, in contrast to the catastrophic events that occur in severely ischaemic tissue, several compensatory mechanisms act in concert to maintain normal ATP concentrations and membrane ion
near
gradients, and to preserve, at least temporarily, cell viability. Reduction of cerebral blood flow
to
about 50% of normal
electroencephalographic activity, and only slightly greater ischaemia completely inhibits synaptic transmission and leads to an isoelectric encephalogram. The conserved energy, which is normally spent on restoring membrane ion gradients dissipated during synaptic activity, coupled with the continued, if reduced anaerobic synthesis suppresses
of ATP, maintains near normal tissue energy status. Thus, failure of energy-dependent mechanisms cannot explain either suppression or silence of the electroencephalogram during moderate ischaemia. The observed slight increase in membrane potassium ion conductance may hyperpolarise presynaptic and postsynaptic membranes, thereby reducing neurotransmitter release and the responsiveness of postsynaptic receptors to neurotransmitters. The mechanisms responsible for increased potassium ion conductance may involve modulation of ATP-regulated and/or calcium-regulated potassium channels.13 Despite compensatory mechanisms that sacrifice electrophysiological activity to reduce energy use and preserve cell viability, cell death occurs if moderate ischaemia lasts for several hours. Unknown processes sporadically overcome the hyperpolarised membranes to brief but recurrent episodes of membrane cause depolarisation (recurrent anoxic depolarisation), large ionic shifts, and a recurrent expenditure of energy to restore normal membrane ion gradients. The presumed but unproven cause for these recurrent depolarisations is of calcium-mediated release large quanta of neurotransmitters from their presynaptic storage sites. Dysregulation of calcium ion homoeostasis features prominently in the cell death of both moderately and severely ischaemic brain. However, the partial preservation of energy-dependent mechanisms that continue to regulate intracellular calcium ion concentrations in moderate ischaemia is strikingly different from the depletion of ATP and the total collapse of calcium homoeostasis in severe
536
ischaemia. Abnormal calcium flux through voltageregulated and ligand-regulated membrane channels contributes importantly to the increase in intracellular calcium in moderate ischaemia but less so in severe ischaemia, in which many failed mechanisms lead to calcium overload.l9 Thus, pharmacological blockade of membrane channels permeable to calcium may reduce intracellular calcium below toxic concentrations in moderate but not in severe ischaemia. In-vivo and in-vitro studies with postsynaptic (L-type) voltage-regulated and glutamate-regulated membrane channels indicate that calcium movement through the latter may be more directly involved in cell injury.20 The endogenous excitatory aminoacid neurotransmitter, glutamate, activates several postsynaptic receptor/channel complexes which are named for their most potent agonist molecule. Of these, the N-methyl-D-aspartate (NMDA) and the quisqualate (Q) receptor/channel complexes are permeable to calcium ions. Blockade of either channel type with selective agonists leads to a striking reduction in infarction volume in laboratory animals with focal brain ischaemia.21 For unexplained reasons, blockade of the Q receptor (also known as the AMPA receptor) but not of the NMDA receptor protects against necrosis of selectively vulnerable neurons after transient, severe ischaemia.21 In contrast to the potential neurotoxicity of hydrogen ions in severely ischaemic tissue, these same ions, when located in the extracellular space of moderately ischaemic brain, may be neuroprotective. Extracellular hydrogen ions, at a concentration equal to pH 6-9 or less, profoundly attenuate calcium conductance through the NMDA-regulated channel and reduce injury induced by oxygen-glucose deprivation in neuronal cultures.22 In moderate ischaemia, in which injury is partly dependent on the influx of calcium ions, extracellular pH falls rapidly to 6-9 or less and hydrogen ions may serve to protect the brain.
Therapeutic implications Therapies effective in experimental stroke Use of certain drugs in experimental ischaemia can clarify greatly the pathogenesis of cerebral infarction. Curiously, diverse classes of drugs reduce infarction volume in well-controlled animal models of focal brain ischaemia. Antagonists of the L-type and presynaptic (N-type) voltageregulated calcium channels; aminoacid neurotransmitter receptor antagonists of the NMDA and Q type that regulate independent calcium channels; drugs that reduce actions of free radicals or that bind to imidazole receptors; and anticonvulsants such as barbiturates and phenytoin all lessen brain injury when given either before or shortly after the onset of experimental ischaemia. The diverse nature of these drugs, plus the observation that they are effective principally in areas of moderate ischaemia, suggests that several pathogenetic mechanisms that are active in these areas are amenable to drug intervention. In addition to protecting against cellular mechanisms of injury, the L-type calcium-channel blockers (eg, nimodipine) which relax contraction of vascular smooth muscle; free-radical agents (eg, superoxide dismutase) which may prolong the half-life of endothelium-derived relaxing factor (nitric oxide); and some of the NMDA receptor/channel antagonists (eg, MK-801) have all been shown to increase cerebral blood flow in the ischaemic
territory.
Treatment strategies
strategies evolve from this discussion of pathogenesis. Firstly, therapy with neuroprotective agents must begin shortly after the onset of cerebral ischaemia. Treatment begun at or after the time of maturation of cerebral infarction in human beings, which probably differs little from the 6-8 h interval observed in primates, will be ineffective. Although infarction evolves over hours,4 treatment should continue for days to protect against possible recurrent ischaemia and against slowly evolving injury that occurs in some neurons.9 Second, clinical trials of neuroprotective drugs should also include therapy simultaneously to improve cerebral blood flow. Even a small increase in blood flow may optimise the volume of moderately ischaemic brain that is responsive to neuroprotective agents. Finally, since the bulk of the infarct is largely composed of tissue unresponsive to any neuroprotective drugs, research is needed to clarify the mechanisms of cell death in severely ischaemic brain and to identify methods to prolong the survival of such tissue until Three
treatment
4
blood flow
can
be restored. REFERENCES
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