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.

No abstract

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 ...

Many central nervous system (CNS) diseases display sexual dimorphism. Exposure to circulating sex steroids is felt to be a chief contributor to this phenomenon; however, CNS diseases of childhood and the elderly also demonstrate gender predominance and/or a sexually dimorphic response to therapies. Here we show that XY and XX neurons cultured separately are differentially susceptible to various cytotoxic agents and treatments. XY neurons were more sensitive to nitrosative stress and excitotoxicity versus XX neurons. In contrast, XX neurons were more sensitive to etoposide-and staurosporine-induced apoptosis versus XY neurons. The responses to specific therapies were also sexually dimorphic. Moreover, gender proclivity in programmed cell death pathway was observed. After cytotoxic challenge, programmed cell death proceeded predominately via an apoptosis-inducing factor-dependent pathway in XY neurons versus a cytochrome c-dependent pathway in XX neurons. This gender-dependent susceptibility is related to the incapacity of XY neurons to maintain intracellular levels of reduced glutathione. In vivo studies further demonstrated an incapacity for male, but not female, 17-day-old rats to maintain reduced glutathione levels within cerebral cortex acutely after an 8-min asphyxial cardiac arrest. This gender difference in sensitivity to cytotoxic agents may be generalized to nonneuronal cells, as splenocytes from male and female 16 -18-day-old rats show similar gender-dependent responses to nitrosative stress and staurosporine-induced apoptosis. These data support gender stratification in the evaluation of mechanisms and treatment of CNS disease, particularly those where glutathione may play a role in detoxification, such as Parkinson's disease, traumatic brain injury, and conditions producing cerebral ischemia, and may apply to non-CNS diseases as well.