Because neurogenesis persists in the adult mammalian brain and can be regulated by physiological and pathological events, we investigated its possible involvement in the brain's response to focal cerebral ischemia. Ischemia was induced by occlusion of the middle cerebral artery in the rat for 90 min, and proliferating cells were labeled with 5-bromo-2 -deoxyuridine-5 -monophosphate (BrdUrd) over 2-day periods before sacrificing animals 1, 2 or 3 weeks after ischemia. Ischemia increased the incorporation of BrdUrd into cells in two neuroproliferative regions-the subgranular zone of the dentate gyrus and the rostral subventricular zone. Both effects were bilateral, but that in the subgranular zone was more prominent on the ischemic side. Cells labeled with BrdUrd coexpressed the immature neuronal markers doublecortin and proliferating cell nuclear antigen but did not express the more mature cell markers NeuN and Hu, suggesting that they were nascent neurons. These results support a role for ischemia-induced neurogenesis in what may be adaptive processes that contribute to recovery after stroke. D iseases of the brain have singularly adverse effects on the quality and duration of life. Unlike many other tissues, the mature brain has limited regenerative capacity, and its unusual degree of cellular specialization restricts the extent to which residual healthy tissue can assume the function of damaged brain. However, cerebral neurons are derived from precursor cells that persist in the adult brain (1-5), so stimulation of endogenous neural precursors in the adult brain could have therapeutic potential (6).Neurogenesis occurs in discrete regions of the adult brain, including the rostral subventricular zone (SVZ) of the lateral ventricles (7) and the subgranular zone (SGZ) of the dentate gyrus (DG) (8). Neurons that arise in the SVZ travel via the rostral migratory stream to the olfactory bulb (9) and also enter association neocortex (10), and new neurons leaving the SGZ migrate into the adjacent DG granule cell layer. Neurogenesis in these regions is subject to physiological regulation by glucocorticoids (11), sex hormones (12), growth factors (13-16), excitatory neurotransmission (17), learning (18), and stress (19) and can be modified pharmacologically (20).Pathological events can also stimulate neurogenesis in the adult brain. Mechanical injury to the DG granule cell layer in the rat increases proliferation of granule neuron precursors in the adjacent SGZ, as shown by enhanced incorporation of [ 3 H]thymidine and 5-bromo-2Ј-deoxyuridine-5Ј-monophosphate (BrdUrd), induction of proliferating cell nuclear antigen (PCNA), and coexpression of mature neuronal markers in [ 3 H]thymidinelabeled cells (21). Seizures also trigger neurogenesis in the SGZ, with BrdUrd-labeled cells expressing the neuronal markers TOAD-64, class III␤-tubulin, and microtubule-associated protein 2 (MAP-2) (22). Finally, apoptosis induced by oxidative stress in mouse corticothalamic neurons increases BrdUrd labeling in cells that go on to express ...

Ca2+ toxicity remains the central focus of ischemic brain injury. The mechanism by which toxic Ca2+ loading of cells occurs in the ischemic brain has become less clear as multiple human trials of glutamate antagonists have failed to show effective neuroprotection in stroke. Acidosis is a common feature of ischemia and is assumed to play a critical role in brain injury; however, the mechanism(s) remain ill defined. Here, we show that acidosis activates Ca2+ -permeable acid-sensing ion channels (ASICs), inducing glutamate receptor-independent, Ca2+ -dependent, neuronal injury inhibited by ASIC blockers. Cells lacking endogenous ASICs are resistant to acid injury, while transfection of Ca2+ -permeable ASIC1a establishes sensitivity. In focal ischemia, intracerebroventricular injection of ASIC1a blockers or knockout of the ASIC1a gene protects the brain from ischemic injury and does so more potently than glutamate antagonism. Thus, acidosis injures the brain via membrane receptor-based mechanisms with resultant toxicity of [Ca2+]i, disclosing new potential therapeutic targets for stroke.

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In the United States stroke is the third leading cause of death and the leading cause of disability. Brain injury following stroke results from the complex interplay of multiple pathways including excitotoxicity, acidotoxicity, ionic imbalance, peri-infarct depolarization, oxidative and nitrative stress, inflammation and apoptosis. There are very few treatments for stroke and the development of new treatments requires a comprehensive understanding of the diverse mechanisms of ischemic brain damage that are responsible for neuronal death. Here, we discuss the underlying pathophysiology of this devastating disease and reveal the intertwined pathways that are the target of therapeutic intervention.

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.

Astrogliosis is a pathological hallmark of the epileptic brain. The identification of mechanisms that link astrogliosis to neuronal dysfunction in epilepsy may provide new avenues for therapeutic intervention. Here we show that astrocyte-expressed adenosine kinase (ADK), a key negative regulator of the brain inhibitory molecule adenosine, is a potential predictor and modulator of epileptogenesis. In a mouse model of focal epileptogenesis, in which astrogliosis is restricted to the CA3 region of the hippocampus, we demonstrate that upregulation of ADK and spontaneous focal electroencephalographic seizures were both restricted to the affected CA3. Furthermore, spontaneous seizures in CA3 were mimicked in transgenic mice by overexpression of ADK in this brain region, implying that overexpression of ADK without astrogliosis is sufficient to cause seizures. Conversely, after pharmacological induction of an otherwise epileptogenesis-precipitating acute brain injury, transgenic mice with reduced forebrain ADK were resistant to subsequent epileptogenesis. Likewise, ADK-deficient ES cell-derived brain implants suppressed astrogliosis, upregulation of ADK, and spontaneous seizures in WT mice when implanted after the epileptogenesis-precipitating brain injury. Our findings suggest that astrocyte-based ADK provides a critical link between astrogliosis and neuronal dysfunction in epilepsy.

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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 ...