Increased expression of heat shock protein 70 (HSP70) in the brain has been extensively documented in association with a variety of insults, including ischemia, and is suggested to play a role in cell survival and recovery after ischemic injury. To more directly assess the protective role of HSP70 during ischemic brain damage, we used transgenic mice overexpressing the rat HSP70 (HSP70tg mice). In contrast to wild‐type (wt) littermates, high levels of HSP70 messenger RNA and protein were detected in brains of HSP70tg mice under normal conditions, and immunohistochemical analysis revealed primarily neuronal expression of HSP70. Heterozygous HSP70tg mice and their wt littermates were subjected to permanent focal cerebral ischemia by intraluminal blockade of the middle cerebral artery. Cerebral infarction after 6 hours of ischemia, as evaluated by Nissl staining, was significantly less in HSP70tg mice compared with wt mice. This reduction in infarction volume in HSP70tg mice was not attributable to an altered cardiovascular anatomy or to initial differences in body temperature or hemodynamic parameters. The HSP70tg mice were still protected against cerebral infarction 24 hours after permanent focal ischemia. The data suggest that HSP70 can markedly protect the brain against ischemic damage and that approaches aimed at inducing HSP70 may lead to new therapeutic interventions in cerebrovascular injuries. Ann Neurol 2000;47:782–791

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Accumulation of intracellular Ca2~is known to be critically important for the expression of NMDA receptor-mediated glutamate neurotoxicity. We have observed, however, that glutamate can also increase the neuronal intracellular Mg2~concentration on activation of NMDA receptors. Here, we used conditions that elevate intracellular Mg2~content independently of Ca2~to investigate the potential role of Mg2~in excitotoxicity in rat cortical neurons in vitro. In Ca2 -free solutions in which the Naw as replaced by N-methyl-D-glucamine or Tris (but not choline), which also contained 9mM Mg2~, exposure to 100~Mglutamate or 200 ,uM NMDA for 20 mm produced delayed neuronal cell death. Neurotoxicity was correlated to the extracellular Mg2~concentration and could be blocked by addition of NMDA receptor antagonists during, but not immediately following, agonist exposure. Finally, we observed that rat cortical neurons grown under different serum conditions develop an altered sensitivity to Mg2~-dependentNMDA receptor-mediated toxicity. Thus, the increase in intracellular Mg2~concentration following NMDA receptor stimulation may be an underestimated component critical for the expression of certain forms of excitotoxic injury. Key Words: N-Methyl-Daspartate-Excitotoxicity-Cations-Cell culture-Neurodegeneration. J. Neurochem. 68, 1836Neurochem. 68, -1845Neurochem. 68, (1997.Overstimulation of glutamate receptors can induce excitotoxic cell death in cortical neurons in vitro (Choi, 1988). Two types of glutamate receptor-mediated neurotoxicity have been characterized. First, the acute toxic effects of a brief, intense glutamate exposure are thought to be triggered by the excessive entry of Ca2t hrough the Ca2-permeable NMDA receptor-gated channel (Choi, 1992). A second form of glutamate toxicity, also characterized by neuronal degeneration, is induced by the prolonged stimulation of ct-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) / kainate receptors, which may or may not be dependent on the extracellular presence of Ca2~ (Choi, 1992). It is interesting that it has been shown that elevations in the intracellular free Ca2~concentration (Ca~I~) during glutamate exposure in hippocampal neurons can be blocked without preventing subsequent cell death (Michaels and Rothman, 1990), and, conversely, various stimuli, e.g., treatment with sodium cyanide or a high extracellular K + concentration, increase [Ca 2± without being neurotoxic (Rothman et al., 1987;Dubinsky and Rothman, 1991). More recently, however, Hartley et al. (1993) showed a direct correlation between the accumulation of 45Ca2~and subsequent neuronal degeneration induced by brief, intense glutamate exposure. Thus, Ca2~is an important component of excitotoxicity, although the proportional change in [Ca2~I~may not correlate with the toxic potential of a particular agonist (Michaels and Rothman, 1990;Rajdev and Reynolds, 1994).Mg 2±is an abundant intracellular cation critical for the regulation of many physiological processes, such as cell metabolism, ion ch...

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