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Background-This review article deals with the role of calcium in ischemic cell death. A calcium-related mechanism was proposed more than two decades ago to explain cell necrosis incurred in cardiac ischemia and muscular dystrophy. In fact, an excitotoxic hypothesis was advanced to explain the acetylcholine-related death of muscle end plates. A similar hypothesis was proposed to explain selective neuronal damage in the brain in ischemia, hypoglycemic coma, and status epilepticus. Summary of Review-The original concepts encompass the hypothesis that cell damage in ischemia-reperfusion is due to enhanced activity of phospholipases and proteases, leading to release of free fatty acids and their breakdown products and to degradation of cytoskeletal proteins. It is equally clear that a coupling exists between influx of calcium into cells and their production of reactive oxygen species, such as ⅐O 2 Ϫ , H 2 O 2 , and ⅐OH. Recent results have underscored the role of calcium in ischemic cell death. A coupling has been demonstrated among glutamate release, calcium influx, and enhanced production of reactive metabolites such as ⅐O 2 Ϫ , ⅐OH, and nitric oxide. It has become equally clear that the combination of ⅐O 2 Ϫ and nitric oxide can yield peroxynitrate, a metabolite with potentially devastating effects. The mitochondria have again come into the focus of interest. This is because certain conditions, notably mitochondrial calcium accumulation and oxidative stress, can trigger the assembly (opening) of a high-conductance pore in the inner mitochondrial membrane. The mitochondrial permeability transition (MPT) pore leads to a collapse of the electrochemical potential for H ϩ , thereby arresting ATP production and triggering production of reactive oxygen species. The occurrence of an MPT in vivo is suggested by the dramatic anti-ischemic effect of cyclosporin A, a virtually specific blocker of the MPT in vitro in transient forebrain ischemia. However, cyclosporin A has limited effect on the cell damage incurred as a result of 2 hours of focal cerebral ischemia, suggesting that factors other than MPT play a role. It is discussed whether this could reflect the operation of phospholipase A 2 activity and degradation of the lipid skeleton of the inner mitochondrial membrane. Conclusions-Calcium is one of the triggers involved in ischemic cell death, whatever the mechanism. (Stroke.1998;29:705-718.)Key Words: calcium Ⅲ cerebral ischemia Ⅲ free radicals Ⅲ mitochondria T he calcium hypothesis of ischemic cell death was originally launched to explain the relationship between excessive calcium influx and the cell damage that is incurred in myocardial ischemia 1 as well as in muscle dystrophy. 2 In fact, studies conducted at that time led to the formulation of an excitotoxic hypothesis, predicting that excessive release of acetylcholine at the motor end plate was what caused damage to skeletal muscle. 3,4 The work performed at that time on ischemic muscle damage was almost visionary since it forestalled the pivotal role of release o...

This article examines the pathophysiology of lesions caused by focal cerebral ischemia. Ischemia due to middle cerebral artery occlusion encompasses a densely ischemic focus and a less densely ischemic penumbral zone. Cells in the focus are usually doomed unless reperfusion is quickly instituted. In contrast, although the penumbra contains cells "at risk," these may remain viable for at least 4 to 8 hours. Cells in the penumbra may be salvaged by reperfusion or by drugs that prevent an extension of the infarction into the penumbral zone. Factors responsible for such an extension probably include acidosis, edema, K+/Ca++ transients, and inhibition of protein synthesis. Central to any discussion of the pathophysiology of ischemic lesions is energy depletion. This is because failure to maintain cellular adenosine triphosphate (ATP) levels leads to degradation of macromolecules of key importance to membrane and cytoskeletal integrity, to loss of ion homeostasis, involving cellular accumulation of Ca++, Na+, and Cl-, with osmotically obligated water, and to production of metabolic acids with a resulting decrease in intra- and extracellular pH. In all probability, loss of cellular calcium homeostasis plays an important role in the pathogenesis of ischemic cell damage. The resulting rise in the free cytosolic intracellular calcium concentration (Ca++) depends on both the loss of calcium pump function (due to ATP depletion), and the rise in membrane permeability to calcium. In ischemia, calcium influx occurs via multiple pathways. Some of the most important routes depend on activation of receptors by glutamate and associated excitatory amino acids released from depolarized presynaptic endings. However, ischemia also interfers with the intracellular sequestration and binding of calcium, thereby contributing to the rise in intracellular Ca++. A second key event in the ischemic tissue is activation of anaerobic glucolysis. The main reason for this activation is inhibition of mitochondrial metabolism by lack of oxygen; however, other factors probably contribute. For example, there is a complex interplay between loss of cellular calcium homeostasis and acidosis. On the one hand, a rise in intracellular Ca++ is apt to cause mitochondrial accumulation of calcium. This must interfere with ATP production and enhance anaerobic glucolysis. On the other hand, acidosis must interfere with calcium binding, thereby contributing to the rise in intracellular Ca++.

The density and distribution of brain damage after 2-10 min of cerebral ischemia was studied in the rat. Ischemia was produced by a combination of carotid clamping and hypotension, followed by 1 week recovery. The brains were perfusion-fixed with formaldehyde, embedded in paraffin, subserially sectioned, and stained with acid fuchsin/cresyl violet. The number of necrotic neurons in the cerebral cortex, hippocampus, and caudate nucleus was assessed by direct visual counting. Somewhat unexpectedly, mild brain damage was observed in some animals already after 2 min, and more consistently after 4 min of ischemia. This damage affected CA4 and CA1 pyramids in the hippocampus, and neurons in the subiculum. Necrosis of neocortical cells began to appear after 4 min and CA3 hippocampal damage after 6 min of ischemia, while neurons in the caudoputamen were affected first after 8-10 min. Selective neuronal necrosis of the cerebral cortex worsened into infarction after higher doses of insult. Damage was worst over the superolateral convexity of the hemisphere, in the middle laminae of the cerebral cortex. The caudate nucleus showed geographically demarcated zones of selective neuronal necrosis, damage to neurons in the dorsolateral portion showing an all-or-none pattern. Other structures involved included the amygdaloid, the thalamic reticular nucleus, the septal nuclei, the pars reticularis of the substantia nigra, and the cerebellar vermis.

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A model is described in which transient ischemia is induced in rats anaesthetized with N2O:O2 (70:30) by bilateral carotid artery clamping combined with a lowering of mean arterial blood pressure to 50 mm Hg, the latter being achieved by bleeding, or by bleeding supplemented with administration of trimetaphan or phentolamine. By the use of intubation, muscle paralysis with suxamethonium chloride, and insertion of tail arterial and venous catheters, it was possible to induce reversible ischemia for long-term recovery studies. Autoradiographic measurements of local CBF showed that the procedure reduced CBF in neocortical areas, hippocampus, and caudoputamen to near-zero values, flow rates in a number of subcortical areas being variable. Administration of trimethaphane or phentolamine did not affect ischemic and postischemic flow rates, nor did they alter recovery of EEG and sensory-evoked responses, but trimetaphan blunted the changes in plasma concentrations of adrenaline and noradrenaline. Recovery experiments showed that 10 min of ischemia gave rise to clear signs of permanent brain damage, with a small number of animals developing postischemic seizures that led to the death of the animals in status epilepticus. After 15 min of ischemia, such alterations were more pronounced, and the majority of animals died. It is concluded that the short revival times noted are explained by the fact that the model induces near-complete ischemia, and that recovery following forebrain ischemia is critically dependent on residual flow rates during the period of ischemia.

Summary:The involvement of caspase-3 in cell death after hypoxia-ischemia (HI) was studied during brain maturation.Unilateral HI was produced in rats at postnatal day 7 (P7), 15 (PIS), 26 (P26), and 60 (P60) by a combination of left carotid artery ligation and systemic hypoxia (8% 02)' Activation of caspase-3 and cell death was examined in situ by high resolution confocal microscopy with anti-active caspase-3 an tibody and propidium iodide and by biochemical analysis. The active caspase-3 positive neurons were composed of more than 90% HI damaged striatal and neocortical neurons in P7 pups, but that number was reduced to approximately 65% in striatum and 34% in the neocortex of PIS pups, and approximately 26% in striatum and 2% in neocortex of P26 rats. In P60 rats, less than 4% of the damaged neurons in striatum and less than 1 % in neocortex were positive for active caspase-3. Western blot