Hung Wen Lin

Pathways for ischemic cytoprotection: Role of sirtuins in caloric restriction, resveratrol, and ischemic preconditioning

Caloric restriction (CR), resveratrol, and ischemic preconditioning (IPC) have been shown to promote protection against ischemic injury in the heart and brain, as well as in other tissues. The activity of sirtuins, which are enzymes that modulate diverse biologic processes, seems to be vital in the ability of these therapeutic modalities to prevent against cellular dysfunction and death. The protective mechanisms of the yeast Sir2 and the mammalian homolog sirtuin 1 have been extensively studied, but the involvement of other sirtuins in ischemic protection is not yet clear. We examine the roles of mammalian sirtuins in modulating protective pathways against oxidative stress, energy depletion, excitotoxicity, inflammation, DNA damage, and apoptosis. Although many of these sirtuins have not been directly implicated in ischemic protection, they may have unique roles in enhancing function and preventing against stress-mediated cellular damage and death. This review will include in-depth analyses of the roles of CR, resveratrol, and IPC in activating sirtuins and in mediating protection against ischemic damage in the heart and brain.

Transient Ischemic Attack Before Nonlacunar Ischemic Stroke in the Elderly

BACKGROUND Several studies suggest transient ischemic attack (TIA) may be neuroprotective against ischemic stroke analogous to preinfarction anginas protection against acute myocardial infarction. However, this protective ischemic preconditioning-like effect may not be present in all ages, especially among the elderly. The purpose of this study was to determine the neuroprotective effect of TIAs (clinical equivalent of cerebral ischemic preconditioning) to neurologic damage after cerebral ischemic injury in patients over 65 years of age. METHODS We reviewed the medical charts of patients with ischemic stroke for presence of TIAs within 72 hours before stroke onset. Stroke severity was evaluated by the National Institutes of Health Stroke Scale and disability by a modified Rankin scale. RESULTS We evaluated 203 patients (>or=65 years) with diagnosis of acute ischemic stroke and categorized them according to the presence (n = 42, 21%) or absence (n = 161, 79%) of TIAs within 72 hours of stroke onset. Patients were monitored until discharged from the hospital (length of hospital stay 14.5 +/- 4.8 days). No significant differences in the National Institutes of Health Stroke Scale and modified Rankin scale scores were observed between those patients with TIAs and those without TIAs present before stroke onset at admission or discharge. CONCLUSION These results suggest that the neuroprotective mechanism of cerebral ischemic preconditioning may not be present or functional in the elderly.

Post-ischemic activation of protein kinase C epsilon protects the hippocampus from cerebral ischemic injury via alterations in cerebral blood flow

Protein kinase C (PKC) is a family of serine/threonine-isozymes that are involved in many signaling events in normal and disease states. Previous studies from our lab have demonstrated that ɛPKC plays a pivotal role in neuroprotection induced by ischemic preconditioning. However, the role of ɛPKC during and after brain ischemia is not clearly defined. Therefore, in the present study, we tested the hypothesis that activation of ɛPKC during an ischemic event is neuroprotective. Furthermore, other studies have demonstrated that ɛPKC mediates cerebral ischemic tolerance in the rat brain by decreasing vascular tone. Thus, we also tested the effects of ɛPKC activation during ischemia on cerebral blood flow (CBF). We found that ψɛ-Receptors for Activated C Kinase (RACK), a ɛPKC-selective peptide activator, injected intravenously 30min before induction of global cerebral ischemia conferred neuroprotection in the CA1 region of the rat hippocampus. Moreover, measurements of CBF before, during, and after cerebral ischemia revealed a significant reduction in the reperfusion phase of rats pretreated with ψɛRACK as compared to Tat peptide (vehicle). Our results suggest that ɛPKC can protect the rat brain against ischemic damage by regulating CBF. Thus, ɛPKC may be one of the treatment modalities against ischemic injury.

Signal Transducers and Activators of Transcription: STATs-Mediated Mitochondrial Neuroprotection

Cerebral ischemia is defined as little or no blood flow in cerebral circulation, characterized by low tissue oxygen and glucose levels, which promotes neuronal mitochondria dysfunction leading to cell death. A strategy to counteract cerebral ischemia-induced neuronal cell death is ischemic preconditioning (IPC). IPC results in neuroprotection, which is conferred by a mild ischemic challenge prior to a normally lethal ischemic insult. Although many IPC-induced mechanisms have been described, many cellular and subcellular mechanisms remain undefined. Some reports have suggested key signal transduction pathways of IPC, such as activation of protein kinase C epsilon, mitogen-activated protein kinase, and hypoxia-inducible factors, that are likely involved in IPC-induced mitochondria mediated-neuroprotection. Moreover, recent findings suggest that signal transducers and activators of transcription (STATs), a family of transcription factors involved in many cellular activities, may be intimately involved in IPC-induced ischemic tolerance. In this review, we explore current signal transduction pathways involved in IPC-induced mitochondria mediated-neuroprotection, STAT activation in the mitochondria as it relates to IPC, and functional significance of STATs in cerebral ischemia.

Effect of cardiac arrest on cognitive impairment and hippocampal plasticity in middle-aged rats.

Cardiopulmonary arrest is a leading cause of death and disability in the United States that usually occurs in the aged population. Cardiac arrest (CA) induces global ischemia, disrupting global cerebral circulation that results in ischemic brain injury and leads to cognitive impairments in survivors. Ischemia-induced neuronal damage in the hippocampus following CA can result in the impairment of cognitive function including spatial memory. In the present study, we used a model of asphyxial CA (ACA) in nine month old male Fischer 344 rats to investigate cognitive and synaptic deficits following mild global cerebral ischemia. These experiments were performed with the goals of 1) establishing a model of CA in nine month old middle-aged rats; and 2) to test the hypothesis that learning and memory deficits develop following mild global cerebral ischemia in middle-aged rats. To test this hypothesis, spatial memory assays (Barnes circular platform maze and contextual fear conditioning) and field recordings (long-term potentiation and paired-pulse facilitation) were performed. We show that following ACA in nine month old middle-aged rats, there is significant impairment in spatial memory formation, paired-pulse facilitation n dysfunction, and a reduction in the number of non-compromised hippocampal Cornu Ammonis 1 and subiculum neurons. In conclusion, nine month old animals undergoing cardiac arrest have impaired survival, deficits in spatial memory formation, and synaptic dysfunction.

Activation of Protein Kinase C Delta following Cerebral Ischemia Leads to Release of Cytochrome C from the Mitochondria via Bad Pathway

Background The release of cytochrome c from the mitochondria following cerebral ischemia is a key event leading to cell death. The goal of the present study was to determine the mechanisms involved in post-ischemic activation of protein kinase c delta (δPKC) that lead to cytochrome c release. Methods/Findings We used a rat model of cardiac arrest as an in vivo model, and an in vitro analog, oxygen glucose deprivation (OGD) in rat hippocampal synaptosomes. Cardiac arrest triggered translocation of δPKC to the mitochondrial fraction at 1 h reperfusion. In synaptosomes, the peptide inhibitor of δPKC blocked OGD-induced translocation to the mitochondria. We tested two potential pathways by which δPKC activation could lead to cytochrome c release: phosphorylation of phospholipid scramblase-3 (PLSCR3) and/or protein phosphatase 2A (PP2A). Cardiac arrest increased levels of phosphorlyated PLSCR3; however, inhibition of δPKC translocation failed to affect the OGD-induced increase in PLSCR3 in synaptosomal mitochondria suggesting the post-ischemic phosphorylation of PLSCR3 is not mediated by δPKC. Inhibition of either δPKC or PP2A decreased cytochrome c release from synaptosomal mitochondria. Cardiac arrest results in the dephosphorylation of Bad and Bax, both downstream targets of PP2A promoting apoptosis. Inhibition of δPKC or PP2A prevented OGD-induced Bad, but not Bax, dephosphorylation. To complement these studies, we used proteomics to identify novel mitochondrial substrates of δPKC. Conclusions We conclude that δPKC initiates cytochrome c release via phosphorylation of PP2A and subsequent dephosphorylation of Bad and identified δPKC, PP2A and additional mitochondrial proteins as potential therapeutic targets for ischemic neuroprotection.

DERANGEMENTS OF POST-ISCHEMIC CEREBRAL BLOOD FLOW BY PROTEIN KINASE C DELTA

Cerebral ischemia causes blood flow derangements characterized by hyperemia (increased cerebral blood flow, CBF) and subsequent hypoperfusion (decreased CBF). We previously demonstrated that protein kinase C delta (δPKC) plays an important role in hippocampal neuronal death after ischemia. However, whether part of this protection is due to the role of δPKC on CBF following cerebral ischemia remains poorly understood. We hypothesized that δPKC exacerbates hyperemia and subsequent hypoperfusion resulting in CBF derangements following ischemia. Sprague-Dawley (SD) rats pretreated with a δPKC specific inhibitor (δV1-1, 0.5 mg/kg) exhibited attenuation of hyperemia and latent hypoperfusion characterized by vasoconstriction followed by vasodilation of microvessels after 2-vessel occlusion plus hypotension measured by 2-photon microscopy. In an asphyxial cardiac arrest model (ACA), SD rats treated with δV1-1 (pre- and post-ischemia) exhibited improved perfusion after 24 h and less hippocampal CA1 neuronal death 7 days after ACA. These results suggest possible therapeutic potential of δPKC in modulating CBF and neuronal damage after cerebral ischemia.

Protein kinase C delta modulates endothelial nitric oxide synthase after cardiac arrest.

We previously showed that inhibition of protein kinase C delta (PKCδ) improves brain perfusion 24 hours after asphyxial cardiac arrest (ACA) and confers neuroprotection in the cortex and CA1 region of the hippocampus 7 days after arrest. Therefore, in this study, we investigate the mechanism of action of PKCδ-mediated hypoperfusion after ACA in the rat by using the two-photon laser scanning microscopy (TPLSM) to observe cortical cerebral blood flow (CBF) and laser Doppler flowmetry (LDF) detecting regional CBF in the presence/absence of δV1-1 (specific PKCδ inhibitor), nitric oxide synthase (NOS) substrate (L-arginine, L-arg) and inhibitor (N ω -Nitro-L-arginine, NLA), and nitric oxide (NO) donor (sodium nitroprusside, SNP). There was an increase in regional LDF and local (TPLSM) CBF in the presence of δV1-1+L-arg, but only an increase in regional CBF under δV1-1+SNP treatments. Systemic blood nitrite levels were measured 15 minutes and 24 hours after ACA. Nitrite levels were enhanced by pretreatment with δV1-1 30 minutes before ACA possibly attributable to enhanced endothelial NOS protein levels. Our results suggest that PKCδ can modulate NO machinery in cerebral vasculature. Protein kinase C delta can depress endothelial NOS blunting CBF resulting in hypoperfusion, but can be reversed with δV1-1 improving brain perfusion, thus providing subsequent neuroprotection after ACA.

Differential effects of delta and epsilon protein kinase C in modulation of postischemic cerebral blood flow

Cerebral ischemia causes cerebral blood flow (CBF) derangements resulting in neuronal damage by enhanced protein kinase C delta (δPKC) levels leading to hippocampal and cortical neuronal death after ischemia. Contrarily, activation of ePKC mediates ischemic tolerance by decreasing vascular tone providing neuroprotection. However, whether part of this protection is due to the role of differential isozymes of PKCs on CBF following cerebral ischemia remains poorly understood. Rats pretreated with a δPKC specific inhibitor (δV1-1, 0.5 mg/kg) exhibited attenuation of hyperemia and latent hypoperfusion characterized by vasoconstriction followed by vasodilation of microvessels after two-vessel occlusion plus hypotension. In an asphyxial cardiac arrest (ACA) model, rats treated with δV1-1 (pre- and postischemia) exhibited improved perfusion after 24 h and less hippocampal CA1 and cortical neuronal death 7 days after ACA. On the contrary, ePKC-selective peptide activator, conferred neuroprotection in the CA1 region of the rat hippocampus 30 min before induction of global cerebral ischemia and decreased regional CBF during the reperfusion phase. These opposing effects of δv. ePKC suggest a possible therapeutic potential by modulating CBF preventing neuronal damage after cerebral ischemia.

The role of fatty acids in the regulation of cerebral vascular function and neuroprotection in ischemia.

Cerebral circulation is tightly regulated by vasoactive substances. There is a delicate balance among vasoconstriction and vasodilation factors. During ischemia/stroke, cerebral blood flow autoregulation may be compromised triggering hyperemia (early phase) or hypoperfusion (late phase or post-ischemia) deranging cerebral blood flow that can lead to subsequent neuronal cell death due to blood flow abnormalities. Traditional vasoactive mediators such as nitric oxide and calcitonin gene-related peptide have been well-documented to provide vasodilation and neuroprotection in the ischemic brain. An emerging field is the identification of fatty acids (polyunsaturated or saturated) that can lead to vasodilation possibly causing neuroprotection. This review investigates fatty acids such as palmitic acid methyl ester, α-linolenic acid, and docosahexaenoic acid as novel vasoactive substances that can modulate cerebral blood flow as well as offer neuroprotection after ischemia.