In the brain, ischemic preconditioning (IPC) diminishes mitochondrial dysfunction after ischemia and confers neuroprotection. Activation of protein kinase C (PKC) has been proposed to be a key neuroprotective pathway during IPC. We tested the hypothesis that IPC increases the levels of PKC in synaptosomes from rat hippocampus, resulting in improved synaptic mitochondrial respiration. Preconditioning significantly increased the level of hippocampal synaptosomal PKC to 152% of sham-operated animals at 2 d of reperfusion, the time of peak neuroprotection. We tested the effect of PKC activation on hippocampal synaptic mitochondrial respiration 2 d after preconditioning. Treatment with the specific PKC activating peptide, tat-RACK (tat--receptor for activated C kinase), increased the rate of oxygen consumption in the presence of substrates for complexes I, II, and IV to 157, 153, and 131% of control (tat peptide alone). In parallel, we found that PKC activation in synaptosomes from preconditioned animals resulted in altered levels of phosphorylated mitochondrial respiratory chain proteins: increased serine and tyrosine phosphorylation of 18 kDa subunit of complex I, decreased serine phosphorylation of FeS protein in complex III, increased threonine phosphorylation of COX IV (cytochrome oxidase IV), increased mitochondrial membrane potential, and decreased H 2 O 2 production. In brief, ischemic preconditioning promoted significant increases in the level of synaptosomal PKC. Activation of PKC increased synaptosomal mitochondrial respiration and phosphorylation of mitochondrial respiratory chain proteins. We propose that, at 48 h of reperfusion after ischemic preconditioning, PKC is poised at synaptic mitochondria to respond to ischemia either by direct phosphorylation or activation of the PKC signaling pathway.

Ischemic tolerance in brain develops when sublethal ischemic insults occur before "lethal" cerebral ischemia. Two windows for the induction of tolerance by ischemic preconditioning (IPC) have been proposed: one that occurs within 1 hour after IPC, and another that occurs 1 or 2 days after IPC. The authors tested the hypotheses that IPC would reduce or prevent ischemia-induced mitochondrial dysfunction. IPC and ischemia were produced by bilateral carotid occlusions and systemic hypotension (50 mm Hg) for 2 and 10 minutes, respectively. Nonsynaptosomal mitochondria were harvested 24 hours after the 10-minute "test" ischemic insult. No significant changes were observed in the oxygen consumption rates and activities for hippocampal mitochondrial complexes I to IV between the IPC and sham groups. Twenty-four hours of reperfusion after 10 minutes of global ischemia (without IPC) promoted significant decreases in the oxygen consumption rates in presence of substrates for complexes I and II compared with the IPC and sham groups. These data suggest that IPC protects the integrity of mitochondrial oxidative phosphorylation after cerebral ischemia.

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Excitotoxicity is recognized to play a major role in cerebral ischemia-induced cell death. The main goal of the present study was to define whether our model of ischemic preconditioning (IPC) promotes a shift from excitatory to inhibitory neurotransmission during the test ischemia to diminish metabolic demand during the reperfusion phase. We also determined whether gamma-aminobutyric acid (GABA) played a role in IPC-induced neuroprotection. Ten minutes of cerebral ischemia was produced by tightening the carotid ligatures bilaterally following hypotension. Samples of microdialysis perfusate, representing extracellular fluid, were analyzed for amino acid content by HPLC. IPC promoted a robust release of GABA after lethal ischemia compared with control rats. We also observed that the activity of glutamate decarboxylase (the predominant pathway of GABA synthesis in the brain) was higher in the IPC group compared with control and ischemic groups. Because GABAA receptor up-regulation has been shown to occur following IPC, and GABAA receptor activation has been implicated in neuroprotection against ischemic insults, we tested the hypothesis that GABAA or GABAB receptor activation was neuroprotective during ischemia or early reperfusion by using an in vitro model (organotypic hippocampal slice culture). Administration of the GABAB agonist baclofen during test ischemia and for 1 hr of reperfusion provided significant neuroprotection. We concluded that increased GABA release in preconditioned animals after ischemia might be one of the factors responsible for IPC neuroprotection. Specific activation of GABAB receptor contributes significantly to neuroprotection against ischemia in organotypic hippocampal slices.

Background and Purpose-A major limitation of intracerebral hemorrhage (ICH) research is the lack of reproducible animal models. The present study was conducted to validate in the mouse the double-injection method of ICH initially developed in the rat. We investigated the effect of intrastriatal injection of blood or cerebrospinal fluid (CSF) on cerebral blood flow (CBF), neurological score, hematoma volume, and brain swelling. Methods-Male C57BL/6 mice were anesthetized with halothane/nitrous oxide delivered by face mask. Rectal and cranial temperatures were regulated at 37°C to 37.5°C. Mice were placed in a stereotactic frame, and a 30-gauge stainless steel cannula was introduced through a burr hole into the left striatum. Each mouse received a 5-L injection of either whole blood or CSF (over 3 minutes), followed 7 minutes later by 10 L injected over 5 minutes. The injection cannula was slowly withdrawn 10 minutes after the second injection. Control mice had only cannula insertion. CBF was studied by laser Doppler perfusion imaging. Neurological status was evaluated on days 1 and 2. After 2 days, hematoma volume and brain swelling were calculated. Results-Physiological values were stable. Mice with ICH but not those with CSF or cannula alone had a marked, persistent neurological deficit and a highly reproducible hematoma, whose meanϮSEM volume was 2.0Ϯ0.2 mm

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During the pre-hibernation season, arctic ground squirrels (AGS) can tolerate 8 min of asphyxial cardiac arrest (CA) without detectable brain pathology. Better understanding of the mechanisms regulating innate ischemia tolerance in AGS has the potential to facilitate the development of novel prophylactic agents to induce ischemic tolerance in patients at risk of stroke or CA. We hypothesized that neuroprotection in AGS involves robust maintenance of ion homeostasis similar to anoxiatolerant turtles. Ion homeostasis was assessed by monitoring ischemic depolarization (ID) in cerebral cortex during CA in vivo and during oxygen glucose deprivation in vitro in acutely prepared hippocampal slices. In both models, the onset of ID was significantly delayed in AGS compared with rats. The epsilon protein kinase C (ePKC) is a key mediator of neuroprotection and inhibits both Na + /K + -ATPase and voltage-gated sodium channels, primary mediators of the collapse of ion homeostasis during ischemia. The selective peptide inhibitor of ePKC (eV1-2) shortened the time to ID in brain slices from AGS but not in rats despite evidence that eV1-2 decreased activation of ePKC in brain slices from both rats and AGS. These results support the hypothesis that ePKC activation delays the collapse of ion homeostasis during ischemia in AGS.

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