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We conclude on the basis of our results and those ofothersl,9,10 that lithium is not a major human teratogen. Webelieve that women with major affective disorders who wishto have children may continue lithium during pregnancy,and do not need to terminate pregnancy provided’that levelII ultrasound and fetal echocardiography are done.

REFERENCES

1. Zalstein E, Koren G, Einarson T, et al. A case-control study on theassociation 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 othercongenital 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 carbamazepineduring 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 teratogenicrisk: the decision to terminate pregnancy. In: Koren G, ed. Maternalfetal toxicology. New York: Marcel Dekker, 1990: 373-81.

17. Belik J, Yoder M, Pereira GR. Fetal macrosomia: an unrecognisedadverse effect of maternal lithium therapy. Pediatr Res 1983; 17: 304A.

18. Yoder MC, Belik J, Lannon RA, et al. Infants of mothers treated withlithium 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 PsychiatrScand 1976; 54: 193-97.

STROKE OCTET

Pathophysiology of acute ischaemic stroke

WILLIAM PULSINELLI

The pathogenesis of brain damage from cerebrovascularocclusion may be separated into two sequential processes:(a) vascular and haematological events that cause the initialreduction and subsequent alteration of local cerebral bloodflow; and (b) ischaemia-induced abnormalities of cellularchemistry that produce necrosis of neurons, glia, and othersupportive 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, neuro-

modulators) ; in signal transduction (receptors, ion channels,second messengers, phosphorylation reactions); inmetabolism (carbohydrate, protein, fatty acid, free radicals);and in gene regulation/expression. Investigators who seek toamelioriate or prevent stroke damage must distinguishreversible changes that cause only cellular dysfunction fromprocesses that cause irreversible injury. Moreover, despite acommon ischaemic insult, different mechanisms underlienecrosis of neurons and glia, and probably even necrosis ofdistinct neuronal types.

Histopathological types of ischaemic braindamage

Histopathological damage from cerebrovascularocclusion depends on the degree and duration of impairedblood flow. In its mildest form, ischaemia kills uniquelyvulnerable neurons such as the pyramidal neurons in theCA1 and CA4 zones of hippocampus, while sparing otherneurons and all glial cells.’ Although such injury is usuallyencountered after transient global ischaemia in patientsresuscitated from cardiac arrest, brief focal ischaemia mayalso destroy these "selectively vulnerable" neurons. Bycontrast, 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 normalbrain by a rim of neuronal necrosis that spares glial cells butaffects all neurons irrespective of phenotype. I will notdiscuss ischaemic injury to white matter, which may occurvia mechanisms distinct from those in grey matter.

Spatial and temporal dynamics of severe andmoderate ischaemia

Severe focal ischaemia

Occlusion of a cerebral blood vessel reduces but seldomabolishes the delivery of oxygen and the brain’s preferredfuel, glucose, to the affected vascular territory. Since densevascular collaterals partly maintain blood flow in theischaemic territory, regions nearest the collateral vessels areless severely affected than more distant areas (fig 1). Thisincomplete or partial ischaemia is responsible for the spatialand temporal dynamics of cerebral infarction. Spontaneousor pharmacological lysis of the occluding thrombus initiatesreperfusion of the ischaemic area, recovery mechanisms inreversibly affected cells, and new mechanisms that either killcells directly or facilitate lethal processes previouslytriggered by ischaemia.

Ischaemia that causes persistent loss of membrane

potentials (persistent anoxic depolarisation), lasting at least 5min but less than 1 h, kills some or all of the selectivelyvulnerable neurons within the affected vascular bed.3 Whenischaemia lasts more than 1 h, infarction begins in the centralzone of lowest cerebral blood flow (fig 1) and progressivelyenlarges in a circumferential fashion towards its maximum

ADDRESS: Cerebrovascular Disease Research Center,Department of Neurology and Neuroscience, Cornell UniversityMedical College, 1300 York Avenue, New York, NY 10021, USA(Dr W. Pulsinelli, MD, PhD).

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Fig 1-Embolic occlusion of the pre-Rolandic branch of the leftmiddle cerebral artery.

An area of severe ischaemia N is surrounded by a rim of moderateischaemia St proximal to collateral vessels (Modified with permission fromPowers W, Raichle M. Stroke In. Pearlman A, Collins R, eds. Neurologicalpathophysiology. New York. Oxford University Press, 1984 )

volume over 3-4 h in rodent 6-8 h in non-human

primates, and an undetermined time in human beings.Attempts to attenuate the volume of an infarct via

pharmacological or other means are critically dependent onthe timeframe over which infarction is initiated and

completed.

Moderate focal ischaemia

There is a rim of mild to moderately ischaemic tissuebetween normally perfused brain and the evolving infarct inwhich pathophysiological mechanisms are most dynamic,cell death occurs last, and pharmacological intervention hasbeen most successful. In this border-zone, poorlyunderstood mechanisms either suppress or completely blocknormal synaptic transmission. Periods of

electrophysiological suppression or silence are interspersedwith irregularly spaced episodes of abnormal membrane ionconductance manifested as depolarisation/repolarisation 6

(recurrent anoxic depolarisation). The physical dimensions ofthis dynamic rim, sometimes known as the ischaemic

penumbra,’ vary considerably but are inversely related to thesteepness of the ischaemia gradient between normallyperfused and severely ischaemic brain.

Pathogenesis of severe ischaemiaThe flow diagram shown in fig 2 depicts multiple,

branching pathways that may be important in ischaemicbrain damage. This representation helps one to understandthe complexity of the process, with its many potential sites ofinteraction, and emphasises the fundamental importance ofenergy depletion in the genesis of subsequent injuriousevents. In severely ischaemic brain persistent shortage ofhigh-energy phosphates is an overwhelming determinant ofinjury: unless cerebral blood flow and the tissue’s mediumfor energy exchange (ATP) are restored, necrosis isinevitable. Nevertheless, energy failure is not the immediatecause of cell death since (a) all brain cells tolerate loss of ATPfor several minutes and the great majority of neurons andglia recover fully when blood flow is restored even after anhour of complete ischaemia;8 (b) one or more of thebranching mechansms (fig 2) may independently kill braincells; and (c) once initiated, such mechanisms may no longerrequire the triggering event. For example, CAI

hippocampal neurons recover normal ATP and

phosphocreatine concentrations previously depleted bytransient, severe ischaemia only to succumb days later9 toevents initiated by, but no longer requiring, energydepletion. 10

Cerebral blood flow below about 10-15 ml/100 g per min(normal 50-60 ml/ 100 g per min) in primates deprives brainof substrate (glucose) and the mitochondrial electron

acceptor (oxygen) necessary for normal oxidativemetabolism. Within minutes of the onset of ischaemia,energy demands exceed the brain’s capacity to synthesiseATP anaerobically from its meagre stores of glucose andglycogen, and high-energy phosphates and fuels for theirsynthesis are depleted. Lactate and unbuffered hydrogenions accumulate in tissue in proportion to the carbohydratestores present at the onset of ischaemia. Toxicity of

hydrogen ions, especially their ability to facilitate ferrousiron-mediated free-radical mechanisms,l1 may be importantin astroglial injury. The latter mechanism may partlyexplain why an increase of brain carbohydrates before theonset of ischaemia greatly augments and/or acceleratesinfarction in animals subjected to severe ischaemia."

In addition to the rapid change in tissue acid-base status,failure of all energy-dependent mechanisms, including ionpumps, leads to deterioration of membrane ion gradients,opening of selective and unselective ion channels, andequilibration of most intracellular and extracellular ions(anoxic depolarisation). As a consequence of anoxic

depolarisation, potassium ions leave the cell, sodium,chloride, and calcium ions enter, and many

neurotransmitters, including excitatory aminoacids

(glutamate, aspartate), are released in potentially toxicconcentrations.13 One known exception to this pattern is the

Ischaemia (0;,, glucose)

Fig 2-Potential mechanisms of ischaemic brain damage.

VRC=vo!tage-regu!ated calcium channels; LRC=iigand-regu!atedcalcium 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 (NMDAandAMPA) PIP2= phosphatidyl inositol diphosphate; PLC = phospholipase C, DG = diacylglycerol, I P 3 = inositol triphosphate. Energy-dependentregulation 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 someastroglial cells.14 Selective intracellular acidosis of these cellsmay contribute to their demise and to infarction.11

Despite in-vitro evidence that cell death in anoxic,energy-depleted brain cells proceeds without calcium, otherexperiments with cultured neurons indicate that raisedintracellular calcium accelerates many potentially injuriousprocesses.16 Calcium activates phospholipases, which

hydrolyse membrane-bound glycerophospholipids to free-fatty acids, and these in turn facilitate free-radical

peroxidation of other membrane lipids. Other examples ofthe potential catalytic role of calcium in cell injury includeactivation of proteases that lyse structural proteins17 andactivation of nitric oxide synthasel8 to initiate free-radicalmechanisms.Much research has focused on calcium dyshomoeostasis

and on developing pharmacological methods to block influxof calcium or its release intracellularly. Since theextracellular calcium ion concentration is 104 105 times

greater than its intracellular concentration, and since mostmechanisms that maintain this gradient are either directly orindirectly energy dependent (fig 3), loss of ATP rapidlyleads to a massive calcium influx and release of calcium fromintracellular compartments.l9 Calcium flux through bothligand-regulated and voltage-regulated ion channelscontributes to the intracellular accumulation of calcium.

However, the failure of potent antagonists of either channeltype to block cell death in severely ischaemic tissue indicateseither that other pathological mechanisms of calciumoverload are quantitatively more important in severe

ischaemia or that cell death in such regions is not calciumdependent.

Pathogenesis of moderate ischaemiaDuring moderate ischaemia, in contrast to the

catastrophic events that occur in severely ischaemic tissue,several compensatory mechanisms act in concert to maintainnear normal ATP concentrations and membrane ion

gradients, and to preserve, at least temporarily, cell viability.Reduction of cerebral blood flow to about 50% of normal

suppresses electroencephalographic activity, and onlyslightly greater ischaemia completely inhibits synaptictransmission and leads to an isoelectric encephalogram. Theconserved energy, which is normally spent on restoringmembrane ion gradients dissipated during synaptic activity,coupled with the continued, if reduced anaerobic synthesisof ATP, maintains near normal tissue energy status. Thus,failure of energy-dependent mechanisms cannot explaineither suppression or silence of the electroencephalogramduring moderate ischaemia. The observed slight increase inmembrane potassium ion conductance may hyperpolarisepresynaptic and postsynaptic membranes, thereby reducingneurotransmitter release and the responsiveness of

postsynaptic receptors to neurotransmitters. The

mechanisms responsible for increased potassium ionconductance may involve modulation of ATP-regulatedand/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 moderateischaemia lasts for several hours. Unknown processessporadically overcome the hyperpolarised membranes tocause brief but recurrent episodes of membrane

depolarisation (recurrent anoxic depolarisation), large ionicshifts, and a recurrent expenditure of energy to restorenormal membrane ion gradients. The presumed but

unproven cause for these recurrent depolarisations iscalcium-mediated release of large quanta ofneurotransmitters from their presynaptic storage sites.

Dysregulation of calcium ion homoeostasis features

prominently in the cell death of both moderately andseverely ischaemic brain. However, the partial preservationof energy-dependent mechanisms that continue to regulateintracellular calcium ion concentrations in moderateischaemia is strikingly different from the depletion of ATPand the total collapse of calcium homoeostasis in severe

536

ischaemia. Abnormal calcium flux through voltage-regulated and ligand-regulated membrane channelscontributes importantly to the increase in intracellularcalcium in moderate ischaemia but less so in severe

ischaemia, in which many failed mechanisms lead to calciumoverload.l9 Thus, pharmacological blockade of membranechannels permeable to calcium may reduce intracellularcalcium below toxic concentrations in moderate but not insevere ischaemia.

In-vivo and in-vitro studies with postsynaptic (L-type)voltage-regulated and glutamate-regulated membranechannels indicate that calcium movement through the lattermay be more directly involved in cell injury.20 Theendogenous excitatory aminoacid neurotransmitter,glutamate, activates several postsynaptic receptor/channelcomplexes which are named for their most potent agonistmolecule. Of these, the N-methyl-D-aspartate (NMDA)and the quisqualate (Q) receptor/channel complexes arepermeable to calcium ions. Blockade of either channel typewith selective agonists leads to a striking reduction ininfarction volume in laboratory animals with focal brainischaemia.21 For unexplained reasons, blockade of the

Q receptor (also known as the AMPA receptor) but notof the NMDA receptor protects against necrosis of

selectively vulnerable neurons after transient, severe

ischaemia.21In contrast to the potential neurotoxicity of hydrogen ions

in severely ischaemic tissue, these same ions, when located inthe extracellular space of moderately ischaemic brain, maybe neuroprotective. Extracellular hydrogen ions, at a

concentration equal to pH 6-9 or less, profoundly attenuatecalcium conductance through the NMDA-regulatedchannel and reduce injury induced by oxygen-glucosedeprivation in neuronal cultures.22 In moderate ischaemia,in which injury is partly dependent on the influx of calciumions, extracellular pH falls rapidly to 6-9 or less and

hydrogen ions may serve to protect the brain.

Therapeutic implicationsTherapies effective in experimental stroke

Use of certain drugs in experimental ischaemia can clarifygreatly the pathogenesis of cerebral infarction. Curiously,diverse classes of drugs reduce infarction volume inwell-controlled animal models of focal brain ischaemia.

Antagonists of the L-type and presynaptic (N-type) voltage-regulated calcium channels; aminoacid neurotransmitterreceptor antagonists of the NMDA and Q type that regulateindependent calcium channels; drugs that reduce actions offree radicals or that bind to imidazole receptors; andanticonvulsants such as barbiturates and phenytoin alllessen brain injury when given either before or shortlyafter the onset of experimental ischaemia. The diversenature of these drugs, plus the observation that theyare effective principally in areas of moderate ischaemia,suggests that several pathogenetic mechanisms that areactive in these areas are amenable to drug intervention.In addition to protecting against cellular mechanismsof injury, the L-type calcium-channel blockers (eg,nimodipine) which relax contraction of vascular smoothmuscle; free-radical agents (eg, superoxide dismutase)which may prolong the half-life of endothelium-derivedrelaxing factor (nitric oxide); and some of the NMDAreceptor/channel antagonists (eg, MK-801) have all beenshown to increase cerebral blood flow in the ischaemic

territory.

Treatment strategiesThree treatment strategies evolve from this discussion of

pathogenesis. Firstly, therapy with neuroprotective agentsmust begin shortly after the onset of cerebral ischaemia.Treatment begun at or after the time of maturation ofcerebral infarction in human beings, which probably differslittle from the 6-8 h interval observed in primates, will beineffective. Although infarction evolves over hours,4 4

treatment should continue for days to protect againstpossible recurrent ischaemia and against slowly evolvinginjury that occurs in some neurons.9 Second, clinical trials ofneuroprotective drugs should also include therapysimultaneously to improve cerebral blood flow. Even a smallincrease in blood flow may optimise the volume of

moderately ischaemic brain that is responsive to

neuroprotective agents. Finally, since the bulk of the infarctis largely composed of tissue unresponsive to any

neuroprotective drugs, research is needed to clarify themechanisms of cell death in severely ischaemic brain and toidentify methods to prolong the survival of such tissue untilblood 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 instrumentalevaluation 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 theabsence 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 toreversible focal cerebral ischemia. Stroke 1991; 22: 1032-39.

5. Jones T, Morawetz R, Crowell R, et al. Thresholds of focal cerebralischemia in awake monkeys. J Neurosurg 1981; 54: 773-82.

6. Nedergaard M, Astrup J. Infarct rim: effect of hyperglycemia on directcurrent potential and [14C]2-deoxyglucose phosphorylation. J CerebBlood Flow Metab 1986; 6: 607-15.

7. Astrup J, Siesjo B, Symon L. Thresholds in cerebral ischemia: theischemic penumbra. Stroke 1981; 12: 723-25.

8. Hossmann K, Kleihues P. Reversibility of ischemic brain damage. ArchNeurol 1973; 29: 375-84.

9. Pulsinelli W, Brierley J, Plum F. Temporal profile of neuronal damage ina model of transient forebrain ischemia. Ann Neurol 1982; 11: 491-98.

10. Pulsinelli W, Duffy T. Regional energy balance in rat brain after transientforebrain 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 BrainMetab Rev 1990; 2: 58-93.

13. Obrenovitch T, Sarna G, Symon L. Ionic homeostasis andneurotransmitter changes in ischemia. In: Krieglstein J, OberpichlerH, eds. Pharmacology of cerebral ischemia. Stuttgart: WVS, 1990:97-112.

14. Kraig R, Chesler M. Astrocytic acidosis in hyperglycemia and completeischemia. 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, andspreading depression: a unifying hypothesis. J Cereb Blood Flow Metab1989; 9: 127-40.

20. Choi D. Methods for antagonizing glutamate neurotoxicity. CerebrovascBrain Metab Rev 1990; 2: 105-47.

21. Pulsinelli W, Sarokin A, Buchan A. Antagonism of the NMDA andnon-NMDA receptors in global versus focal brain ischemia. Prog BrainRes (in press).

22. Giffard R, Monyer H, Christine C, Choi D. Acidosis reduces NMDAreceptor activation, glutamate neurotoxicity, and oxygen-glucosedepnvation neuronal injury in cortical cultures. Brain Res 1990; 506:339-42.