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.

Marijuana and related drugs (cannabinoids) have been proposed as treatments for a widening spectrum of medical disorders. R(ϩ)- [2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo [1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate (R(ϩ)-WIN 55212-2), a synthetic cannabinoid agonist, decreased hippocampal neuronal loss after transient global cerebral ischemia and reduced infarct volume after permanent focal cerebral ischemia induced by middle cerebral artery occlusion in rats. The less active enantiomer S(Ϫ)-WIN 55212-3 was ineffective, and the protective effect of R(ϩ)-WIN 55212-2 was blocked by the specific central cannabinoid (CB 1 ) cannabinoid receptor antago-55212-2 also protected cultured cerebral cortical neurons from in vitro hypoxia and glucose deprivation, but in contrast to the receptor-mediated neuroprotection observed in vivo, this in vitro effect was not stereoselective and was insensitive to CB 1 and CB 2 receptor antagonists. Cannabinoids may have therapeutic potential in disorders resulting from cerebral ischemia, including stroke, and may protect neurons from injury through a variety of mechanisms. Key words: cannabinoid; ischemia; stroke; glutamate; excitotoxicity; infarct; neuronal cultureCannabis, the marijuana plant, has been used since antiquity for its medicinal and psychoactive properties (Snyder, 1971). Both its principal active ingredient, ⌬ 9 -tetrahydrocannabinol (THC), and synthetic analogs thereof (cannabinoids) have been proposed as therapy for a variety of medical conditions, including glaucoma, cancer chemotherapy-induced nausea and vomiting, acquired immunodeficiency syndrome, inflammatory disorders, and epilepsy (Jack, 1997). This has contributed to efforts to legalize marijuana use for therapeutic purposes (Annas, 1997;Kassirer, 1997). However, concern exists about the safety of cannabinoids, including their possible role in infertility (Schmid et al., 1997) and the extent to which they share effects with narcotics (Rodríguez de Fonseca et al., 1997;Tanda et al., 1997). This controversy persists despite major advances regarding the basic molecular and cellular mechanisms of cannabinoid action (for review, see Felder and Glass, 1998), including the discovery of endogenous cannabinoids (Devane et al., 1992;Stella et al., 1997;Randall and Kendall, 1998), mechanisms for their synthesis and termination of action (Di Marzo et al., 1994;Beltramo et al., 1997), cannabinoid receptors (Matsuda et al., 1990;Kuster et al., 1993;Howlett, 1995), receptor-effector coupling pathways (Mackie and Hille, 1992;Derkinderen et al., 1996), and synthetic cannabinoid agonist and antagonist drugs (Compton et al., 1992;Rinaldi-C armona et al., 1994).Central cannabinoid (CB 1 ) receptors are coupled to several signal transduction pathways, including G-proteins that inhibit N-type voltage-gated calcium channels involved in the release of neurotransmitters (Mackie and Hille, 1992). These channels participate in release of the excitatory transmitter L-glutamate, which has been implicated in ...

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...

The brain's response to ischemia, which helps determine clinical outcome after stroke, is regulated partly by competing genetic programs that respectively promote cell survival and delayed cell death. Many genes involved in this response have been identified individually or systematically, providing insights into the molecular basis of ischemic injury and potential targets for therapy. The development of microarray systems for gene expression profiling permits screening of large numbers of genes for possible involvement in biological or pathological processes. Therefore, we used an oligodeoxynucleotide-based microarray consisting of 374 human genes, most implicated previously in apoptosis or related events, to detect alterations in gene expression in the hippocampus of rats subjected to 15 minutes of global cerebral ischemia followed by up to 72 hours of reperfusion. We found 1.7-fold or greater increases in the expression of 57 genes and 1.7-fold or greater decreases in the expression of 34 genes at 4, 24, or 72 hours after ischemia. The number of induced genes increased from 4 to 72 hours, whereas the number of repressed genes decreased. The induced genes included genes involved in protein synthesis, genes mutated in hereditary human diseases, proapoptotic genes, antiapoptotic genes, injury-response genes, receptors, ion channels, and enzymes. We detected transcriptional induction of several genes implicated previously in cerebral ischemia, including ALG2, APP, CASP3, CLU, ERCC3, GADD34, GADD153, IGFBP2, TIAR, VEGF, and VIM, as well as other genes not so implicated. We also found coinduction of several groups of related genes that might represent functional modules within the ischemic neuronal transcriptome, including VEGF and its receptor, NRP1; the IGF1 receptor and the IGF1-binding protein IGFBP2; Rb, the Rb-binding protein E2F1, and the E2F-related transcription factor, TFDP1; the CACNB3 and CACNB4 beta-subunits of the voltage-gated calcium channel; and caspase-3 and its substrates, ACINUS, FEM1, and GSN. To test the hypothesis that genes identified through this approach might have roles in the pathophysiology of cerebral ischemia, we measured expression of the products of two induced genes not heretofore implicated in cerebral ischemia-GRB2, an adapter protein involved in growth-factor signaling pathways, and SMN1, which participates in RNA processing and is deleted in most cases of spinal muscular atrophy. Western analysis showed enhanced expression of both proteins in hippocampus at 24 to 72 hours after ischemia, and SMN1 was localized by immunohistochemistry to hippocampal neurons. These results suggest that microarray analysis of gene expression may be useful for elucidating novel molecular mediators of cell death and survival in the ischemic brain.