Ischemic stroke is caused by acute neuronal degeneration provoked by interruption of cerebral blood flow. Although the mechanisms contributing to ischemic neuronal degeneration are myriad, mitochondrial dysfunction is now recognized as a pivotal event that can lead to either necrotic or apoptotic neuronal death. Lack of suitable 'upstream' targets to prevent loss of mitochondrial homeostasis has, so far, restricted the development of mechanistically based interventions to promote neuronal survival. Here, we show that the uncoupling agent 2,4 dinitrophenol (DNP) reduces infarct volume approximately 40% in a model of focal ischemia-reperfusion injury in the rat brain. The mechanism of protection involves an early decrease in mitochondrial reactive oxygen species formation and calcium uptake leading to improved mitochondrial function and a reduction in the release of cytochrome c into the cytoplasm. The observed effects of DNP were not associated with enhanced cerebral perfusion. These findings indicate that compounds with uncoupling properties may confer neuroprotection through a mechanism involving stabilization of mitochondrial function. Stroke occurs as a consequence of irreversible brain injury resulting from cerebral ischemia. Reduction or interruption of regional blood flow in the brain produces ischemic injury when delivery of metabolic substrates (glucose and oxygen) fails to meet the energy demand of the affected tissue. The actual blood flow rate at which local tissue ischemia is produced in the brain is variable, because cerebral metabolic demand varies from region to region and under different physiological conditions (Siesjö et al. 1995). While considerable progress has been made in the understanding of mechanisms contributing to neuronal injury following cerebral ischemia, the only approved therapy for the treatment of acute stroke remains the use of the thrombolytic agent, tissue plasminogen activator (tPA) (Fisher and Ratan 2003). Timely administration of tPA will lyse intra-arterial thrombus, reperfuse ischemic brain, and hasten clinical recovery. However, the implementation of neuroprotective strategies targeted at intracellular pathways activated in the aftermath of blood-flow impediment remains elusive. To the present date, there is no mechanistically based treatment of ischemic neuronal injury that has been successfully validated in clinical trials, even after demonstration of efficacy in laboratory studies (DeGraba and Pettigrew 2000).While numerous 'cell death' pathways are activated following an episode of transient brain ischemia, the ability of neurons to maintain mitochondrial homeostasis seems to be a critical factor in the 'life/death' decision (Fiskum 2000;Zipfel et al. 2000). Two of the earliest events that can tip the balance in favor of neurodegeneration following transient ischemia are the accumulation of calcium (Ca 2+ ) and (Gunter et al. 1994;White and Reynolds 1996) and production of ROS are attenuated following a decrease in DY m (Korshunov et al. 1997;Liu 1997;...

Calpain, a neutral protease activated by calcium, may promote microtubular proteolysis in ischemic brain. We tested this hypothesis in an animal model of focal cerebral ischemia without reperfusion. The earliest sign of tissue injury was observed after no more than 15 min of ischemia, with coiling of apical dendrites immunolabeled to show microtubule-associated protein 2 (MAP2). After 6 h of ischemia, MAP2 immunoreactivity was markedly diminished in the infarct zone. Quantitative Western analysis demonstrated that MAP2 was almost unmeasurable after 24 h of ischemia. An increase in calpain activity, shown by an antibody recognizing calpain-cleaved spectrin fragments, paralleled the loss of MAP2 immunostaining. Double-labeled immunofluorescent studies showed that intraneuronal calpain activity preceded evidence of MAP2 proteolysis. Perikaryal immunolabeling of τ protein became increasingly prominent between 1 and 6 h in neurons located within the transition zone between ischemic and unaffected tissue. Western blot experiments confirmed that dephosphorylation of τ protein occurred during 24 h of ischemia, but was not associated with significant loss of τ antigen. We conclude that focal cerebral ischemia is associated with early microtubular proteolysis caused by calpain.

Increasing evidence indicates that glucocorticoids (GCs), produced in response to physical/emotional stressors, can exacerbate brain damage resulting from cerebral ischemia and severe seizure activity. However, much of the supporting evidence has come from studies employing nonphysiological paradigms in which adrenalectomized rats were compared with those exposed to constant GC concentrations in the upper physiological range. Cerebral ischemia and seizures can induce considerable GC secretion. We now present data from experiments using metyrapone (an 11-beta-hydroxylase inhibitor of GC production), which demonstrate that the GC stress-response worsens subsequent brain damage induced by ischemia and seizures in rats. Three different paradigms of brain injury were employed: middle cerebral artery occlusion (MCAO) model of focal cerebral ischemia; four-vessel occlusion (4VO) model of transient global forebrain ischemia; and kainic acid (KA)-induced (seizure-mediated) excitotoxic damage to hippocampal CA3 and CA1 neurons. Metyrapone (200 mg/kg body wt) was administered systemically in a single i.p. bolus 30 min prior to each insult. In the MCAO model, metyrapone treatment significantly reduced infarct volume and also preserved cells within the infarct. In the 4VO model, neuronal loss in region CA1 of the hippocampus was significantly reduced in rats administered metyrapone. Seizure-induced damage to hippocampal pyramidal neurons (assessed by cell counts and immunochemical analyses of cytoskeletal alterations) was significantly reduced in rats administered metyrapone. Measurement of plasma levels of corticosterone (the species-typical GC of rats) after each insult showed that metyrapone significantly suppressed the injury-induced rise in levels of circulating corticosterone. These findings indicate that endogenous corticosterone contributes to the basal level of brain injury resulting from cerebral ischemia and excitotoxic seizure activity and suggest that drugs that suppress glucocorticoid production may be effective in reducing brain damage in stroke and epilepsy patients.

The β‐amyloid precursor protein (βAPP) is the source of the amyloid β‐peptide that accumulates in the brain in Alzheimer's disease. A major processing pathway for βAPP involves an enzymatic cleavage within the amyloid β‐peptide sequence that liberates secreted forms of βAPP (APPSs) into the extracellular milieu. We now report that postischemic administration of these APPSs intracerebroventricularly protects neurons in the CA1 region of rat hippocampus against ischemic injury. Treatment with APPS695 or APPS751 resulted in increased neuronal survival, and the surviving cells were functional as demonstrated by their ability to synthesize protein. These data provide direct evidence for a neuroprotective action of APPSs in vivo.

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