Delayed neuronal death after transient cerebral ischemia may be mediated, in part, by the induction of apoptosis-regulatory gene products. Caspase-3 is a newly characterized mammalian cysteine protease that promotes cell death during brain development, in neuronal cultures, and in other cell types under many different conditions. To determine whether caspase-3 serves to regulate neuronal death after cerebral ischemia, we have (1) cloned a cDNA encoding the rat brain caspase-3; (2) examined caspase-3 mRNA and protein expression in the brain using in situ hybridization, Northern and Western blot analyses, and double-labeled immunohistochemistry; (3) determined caspase-3-like activity in brain cell extracts; and (4) studied the effect of caspase-3 inhibition on cell survival and DNA fragmentation in the hippocampus in a rat model of transient global ischemia. At 8-72 hr after ischemia, caspase-3 mRNA and protein were induced in the hippocampus and caudate-putamen (CPu), accompanied by increased caspase-3-like protease activity. In the hippocampus, caspase-3 mRNA and protein were predominantly increased in degenerating CA1 pyramidal neurons. Proteolytic activation of the caspase-3 precursor was detected in hippocampus and CPu but not in cortex at 4-72 hr after ischemia. Double-label experiments detected DNA fragmentation in the majority of CA1 neurons and selective CPu neurons that overexpressed caspase-3. Furthermore, ventricular infusion of Z-DEVD-FMK, a caspase-3 inhibitor, decreased caspase-3 activity in the hippocampus and significantly reduced cell death and DNA fragmentation in the CA1 sector up to 7 d after ischemia. These data strongly suggest that caspase-3 activity contributes to delayed neuronal death after transient ischemia.

The inducible isoform of the enzyme cyclooxygenase-2 (COX2) is an immediate early gene induced by synaptic activity in the brain. COX2 activity is an important mediator of inflammation, but it is not known whether COX2 activity is pathogenic in brain. To study the role of COX2 activity in ischemic injury in brain, expression of COX2 mRNA and protein and the effect of treatment with a COX2 inhibitor on neuronal survival in a rat model of global ischemia were determined. Expression of both COX2 mRNA and protein was increased after ischemia in CA1 hippocampal neurons before their death. There was increased survival of CA1 neurons in rats treated with the COX2-selective inhibitor SC58125 {1-[(4-methylsulfonyl) phenyl]-3-trifluoro-methyl-5-[(4-fluoro)phenyl] pyrazole} before or after global ischemia compared with vehicle controls. Furthermore, hippocampal prostaglandin E 2 concentrations 24 h after global ischemia were decreased in drug-treated animals compared with vehicle-treated controls. These results suggest that COX2 activity contributes to CA1 neuronal death after global ischemia.Cyclooxygenase (prostaglandin G͞H synthase) is the first committed step in the production of prostaglandins and thromboxanes. Two forms of the cyclooxygenase enzyme have been cloned. Cyclooxygenase-1 (COX1) is constitutively expressed in many tissues including platelets, gastrointestinal mucosa, and kidney (1-3). The inducible form, cyclooxygenase-2 (COX2), is primarily expressed in leukocytes and brain (4). Its expression is induced by cytokines and inhibited by glucocorticoids, and it is an important mediator of cell injury in inflammation (5-7). Transcription of COX2 mRNA does not require new protein synthesis; therefore, it is an immediate early gene (6). The rat brain COX2 is identical to the nonnervous system COX2 (8). Its mRNA expression is rapidly induced by synaptic activity but blocked by the N-methyl-Daspartate receptor antagonist MK801 (8). This finding suggests that COX2 transcription is induced by increased intraneuronal Ca 2ϩ . COX2 is found in dendrites of neurons that receive excitatory input (9). Thus, COX2 may produce rapid neuronal responses to synaptic activity.Neuronal excitation and increased intracellular calcium, two stimuli that induce expression of COX2, are also important in the pathophysiology of neuronal death in ischemia and a variety of neurodegenerative diseases (10, 11). In nonneural cells, COX2 activity mediates inflammatory injury (12); however, COX2 overexpression may prevent apoptosis in intestinal epithelium (13). What role COX2 expression and activity have in mediating injury after global ischemia in brain is unknown. To address this question, the expression of COX2 mRNA and protein was studied in rat brain after global ischemia, and the effect of treatment with a selective inhibitor of COX2 {SC58125; 1-[(4-methylsulfonyl)phenyl]-3-trifluoro-methyl-5-[(4-fluoro)phenyl] pyrazole} on hippocampal neuronal survival and prostaglandin E 2 (PGE 2 ) concentrations was determined. METHODSAnimal M...

Summary: Stress proteins are induced after a variety of neuronal injuries. The inducible 72-kDa heat shock pro tein (hsp70) is a stress protein that protects neurons from glutamate toxicity in vitro. Hsp70 has also been proposed to underlie the phenomenon of ischemic tolerance whereby brief sublethal intervals of global ischemia pro tect the hippocampus from subsequent lethal prolonged ischemia. To determine if the phenomenon of tolerance occurs in cortex after focal ischemia, the rat middle ce rebral artery (MCA) was occluded by the suture method. Three lO-min intervals of transient ischemia (3 x lO-isc) separated by 45-min periods of reperfusion made up the most effective paradigm of preconditioning ischemia studied, and substantially reduced the volume of infarc tion 72 h after subsequent IOO-min MCA occlusion. This approach induced protection if the interval between the 3 x lO-isc and the IOO-min ischemia was 2, 3, or 5 days but not 1 or 7 days. Three lO-min intervals of transient ischemia alone produced minimal histological changes in the cortex at 72 h. Moreover, there were no significant changes in regional cerebral blood flow in the tolerant regions at 72 h after 3 x lO-isc before or during MCA occlusion. To explore the role of stress proteins in the induction of tolerance, expression of hsp70 and the gluIschemic tolerance is the phenomenon whereby a preconditioning ischemia of brief duration, inade quate to infarct the brain, protects it from subse quent severe ischemia. Global ischemia was the first model used to describe ischemic tolerance, which protected hippocampal neurons from subse quent ischemic necrosis (Kato et aI. , 199 1; Kita-

The proto-oncogenes bcl-2 and bcl-x-long have been shown to suppress apoptotic cell death in a variety of in vitro systems and cell lines, including neurons. An alternatively spliced from of bcl-x, bcl-x-short, is a promoter of apoptotic death. Whether these genes are induced after ischemia or play any role in determining the fate of ischemic neurons is unknown. To begin to address this issue, we studied the expression of bcl-2, and bcl-x mRNA and protein after global ischemia in the rat. Ischemia was induced in isoflurane-anesthetized rats by the four-vessel occlusion method. mRNA expression was studied by Northern blot analysis at 24 h after ischemia and by in situ hybridization at 2, 4, 8, 24, and 72 h after 15 min of global ischemia. Protein expression was studied using both immunocytochemistry at 4, 8, 16, 24, and 72 h after ischemia and Western blot analysis from tissue harvested at 16, 24, and 72 h after ischemia. Western blots showed that bcl-x-long is the predominant form of bcl-x protein expressed in both normal and ischemic brain. Both bcl-2 and bcl-x-long mRNA were expressed in CA1, CA3, and the molecular layer of the dentate after ischemia. However, bcl-2 and bcl-x protein were expressed only in CA3 and dentate. Thus, while bcl-2 and bcl-x-long mRNA were expressed in both surviving and dying neurons, their proteins were expressed in neurons destined to survive. These results support potential roles for these two apoptosis suppressor proteins in promoting survival after cerebral ischemia.