Miguel A. Perez-Pinzon

RESVERATROL PRETREATMENT PROTECTS RAT BRAIN FROM CEREBRAL ISCHEMIC DAMAGE VIA A SIRTUIN 1-UNCOUPLING PROTEIN 2 PATHWAY

Resveratrol is a natural polyphenol found in grapes and wine and has been associated with protective effects against cardiovascular diseases. In vitro, both resveratrol preconditioning (RPC) and ischemic preconditioning (IPC) require activation of sirtuin 1 (SIRT1), a nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase, to induce neuroprotection against cerebral ischemia. In the present study, we tested two hypotheses: (a) that neuroprotection against cerebral ischemia can be induced by RPC in vivo; and (b) that RPC neuroprotection involves alterations in mitochondrial function via the SIRT1 target mitochondrial uncoupling protein 2 (UCP2). IPC was induced by 2 min of global ischemia (temporary bilateral carotid artery occlusion with hypotension), and RPC, by i.p. injection of resveratrol at 10, 50 and 100 mg/kg dosages. Forty-eight hours later, we compared the neuroprotective efficacy of RPC and IPC in vulnerable cornu ammonis 1 hippocampal pyramidal neurons using a rat model of asphyxial cardiac arrest (ACA). SIRT1 activity was measured using a SIRT1-specific fluorescent enzyme activity assay. In hippocampal mitochondria isolated 48 h after IPC or RPC, we measured UCP2 levels, membrane potential, respiration, and the mitochondrial ATP synthesis efficiency (ADP/O ratio). Both IPC and RPC induced tolerance against brain injury induced by cardiac arrest in this in vivo model. IPC increased SIRT1 activity at 48 h, while RPC increased SIRT1 activity at 1 h but not 48 h after treatment in hippocampus. Resveratrol significantly decreased UCP2 levels by 35% compared to sham-treated rats. The SIRT1-specific inhibitor sirtinol abolished the neuroprotection afforded by RPC and the decrease in UCP2 levels. Finally, RPC significantly increased the ADP/O ratio in hippocampal mitochondria reflecting enhanced ATP synthesis efficiency. In conclusion, in vivo resveratrol pretreatment confers neuroprotection similar to IPC via the SIRT1-UCP2 pathway.

Rapid preconditioning protects rats against ischemic neuronal damage after 3 but not 7 days of reperfusion following global cerebral ischemia

Earlier studies indicated that sublethal ischemic insults separated by many hours may “precondition” and, thereby, protect tissues from subsequent insults. In Wistar rats, we examined the hypothesis that ischemic preconditioning (IPC) can improve histopathological outcome even if the “conditioning” and “test” ischemic insults are separated by only 30 min. Normothermic (36.5–37°C) global cerebral ischemia was produced by bilateral carotid artery ligation after lowering mean systemic blood pressure. The conditioning ischemic insult lasted 2 min and was associated with a time sufficient to provoke “anoxic depolarization” (AD) (i.e., the abrupt maximal increase in extracellular potassium ion activity). After 30 min of reperfusion, 10-min test ischemia was produced, and histopathology was assessed 3 and 7 days later. After 3 days of reperfusion, neuroprotection was most robust in the left lateral, middle and medial subsections of the hippocampal CA1 subfield and in the cortex, where protection was 91, 76, 70 and 86%, respectively. IPC also protected the right lateral, middle and medial subsections of the hippocampal CA1 region. These data demonstrate that neuroprotection against acute neuronal injury can be achieved by conditioning insults followed by only short (30 min) periods of reperfusion. However, neuroprotection almost disappeared when reperfusion was continued for 7 days. When test ischemia was decreased to 7 min, a clear trend of neuroprotection by IPC was observed. These data suggest that subsequent rescue of neuronal populations could be achieved with better understanding of the neuroprotective mechanisms involved in this rapid IPC model.

RESVERATROL MIMICS ISCHEMIC PRECONDITIONING IN THE BRAIN

Recent studies in a variety of species including mammals showed that resveratrol (trans-3, 5, 4″-trihydroxystibene) treatment and caloric restriction increased silent information regulator 2/sirtuin 1 activity, which mediated increase in life span/cell survival. Resveratrol is a naturally occurring phytoalexin and a well-documented cardioprotective agent. Similarly, ischemic preconditioning (IPC) has been shown to be both cardio- and cerebroprotective against subsequent ischemic insults. A major emphasis in this field is to understand the molecular mechanisms that mediate this phenomenon. The goal of this study was to define whether resveratrol can emulate IPC neuroprotection against cerebral ischemia. Employing an in vitro model of cerebral ischemia, the organotypic hippocampal slice culture, we report that resveratrol pretreatment mimics IPC via the SIRT1 pathway. Blockade of SIRT1 activation by sirtinol after IPC or resveratrol pretreatment abolished their neuroprotection. A better understanding of the mechanisms by which resveratrol induces ischemic tolerance in a prophylactic manner may provide a novel therapy against stroke or neurosurgical procedures.

Protein Kinase C δ Mediates Cerebral Reperfusion Injury In Vivo

Protein kinase C (PKC) has been implicated in mediating ischemic and reperfusion damage in multiple organs. However, conflicting reports exist on the role of individual PKC isozymes in cerebral ischemic injury. Using a peptide inhibitor selective for δPKC, δV1-1, we found that δPKC inhibition reduced cellular injury in a rat hippocampal slice model of cerebral ischemia [oxygen-glucose deprivation (OGD)] when present both during OGD and for the first 3 hr of reperfusion. We next demonstrated peptide delivery to the brain parenchyma after in vivo delivery by detecting biotin-conjugatedδV1-1 and by measuring inhibition of intracellular δPKC translocation, an indicator of δPKC activity. Delivery of δV1-1 decreased infarct size in an in vivo rat stroke model of transient middle cerebral artery occlusion. Importantly, δV1-1 had no effect when delivered immediately before ischemia. However, delivery at the onset, at 1 hr, or at 6 hr of reperfusion reduced injury by 68, 47, and 58%, respectively. Previous work has implicated δPKC in mediating apoptotic processes. We therefore determined whether δPKC inhibition altered apoptotic cell death or cell survival pathways in our models. We found that δV1-1 reduced numbers of terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling-positive cells, indicating decreased apoptosis, increased levels of phospho-Akt, a kinase involved in cell survival pathways, and inhibited BAD (Bcl-2-associated death protein) protein translocation from the cell cytosol to the membrane, indicating inhibition of proapoptotic signaling. These data support a deleterious role for δPKC during reperfusion and suggest that δV1-1 delivery, even hours after commencement of reperfusion, may provide a therapeutic advantage after cerebral ischemia.

Cytochrome c Association with the Inner Mitochondrial Membrane Is Impaired in the CNS of G93A-SOD1 Mice

A “gain-of-function” toxic property of mutant Cu-Zn superoxide dismutase 1 (SOD1) is involved in the pathogenesis of some familial cases of amyotrophic lateral sclerosis (ALS). Expression of a mutant form of the human SOD1 gene in mice causes a degeneration of motor neurons, leading to progressive muscle weakness and hindlimb paralysis. Transgenic mice overexpressing a mutant human SOD1 gene (G93A-SOD1) were used to examine the mitochondrial involvement in familial ALS. We observed a decrease in mitochondrial respiration in brain and spinal cord of the G93A-SOD1 mice. This decrease was significant only at the last step of the respiratory chain (complex IV), and it was not observed in transgenic wild-type SOD1 and nontransgenic mice. Interestingly, this decrease was evident even at a very early age in mice, long before any clinical symptoms arose. The effect seemed to be CNS specific, because no decrease was observed in liver mitochondria. Differences in complex IV respiration between brain mitochondria of G93A-SOD1 and control mice were abolished when reduced cytochrome c was used as an electron donor, pinpointing the defect to cytochrome c. Submitochondrial studies showed that cytochrome c in the brain of G93A-SOD1 mice had a reduced association with the inner mitochondrial membrane (IMM). Brain mitochondrial lipids, including cardiolipin, had increased peroxidation in G93A-SOD1 mice. These results suggest a mechanism by which mutant SOD1 can disrupt the association of cytochrome c with the IMM, thereby priming an apoptotic program.

Protein Aggregation after Focal Brain Ischemia and Reperfusion

Two hours of transient focal brain ischemia causes acute neuronal death in the striatal core region and a somewhat more delayed type of neuronal death in neocortex. The objective of the current study was to investigate protein aggregation and neuronal death after focal brain ischemia in rats. Brain ischemia was induced by 2 hours of middle cerebral artery occlusion. Protein aggregation was analyzed by electron microscopy, laser-scanning confocal microscopy, and Western blotting. Two hours of focal brain ischemia induced protein aggregation in ischemic neocortical neurons at 1 hour of reperfusion, and protein aggregation persisted until neuronal death at 24 hours of reperfusion. Protein aggregates were found in the neuronal soma, dendrites, and axons, and they were associated with intracellular membranous structures during the postischemic phase. High-resolution confocal microscopy showed that clumped protein aggregates surrounding nuclei and along dendrites were formed after brain ischemia. On Western blots, ubiquitinated proteins (ubi-proteins) were dramatically increased in neocortical tissues in the postischemic phase. The ubi-proteins were Triton-insoluble, indicating that they might be irreversibly aggregated. The formation of ubi-protein aggregates after ischemia correlated well with the observed decrease in free ubiquitin and neuronal death. The authors concluded that proteins are severely damaged and aggregated in neurons after focal ischemia. The authors propose that protein damage or aggregation may contribute to ischemic neuronal death.

Focal Ischemic Preconditioning Induces Rapid Tolerance to Middle Cerebral Artery Occlusion in Mice

In a process called ischemic preconditioning, a brief, sublethal ischemic insult protects tissue from subsequent, more severe injury. There have been no reports of rapidly induced ischemic preconditioning. The authors sought to develop a model of cerebral ischemic preconditioning in the mouse that can be applied to transgenic and knockout animals. They found that brief middle cerebral artery (MCA) occlusion only minutes before a severe ischemic insult can induce protection from that insult. Here the investigators describe a mouse model of preconditioning using intraluminal MCA occlusion as both the conditioning and the test stimulus. One or three 5-minute episodes of ischemia given 30 minutes before MCA occlusion for 1 or 24 hours (permanent occlusion) confer significant protection as assessed by infarct volume measurements 24 hours later.

Metabolic Downregulation A Key to Successful Neuroprotection

Background and Purpose— The search for effective neuroprotectants remains frustrating, particularly with regard to specific pharmaceuticals. However, laboratory studies have consistently shown remarkable neuroprotection with 2 nonpharmacological strategies—therapeutic hypothermia and ischemic preconditioning. Recent studies have shown that the mechanism of protection underlying both of these treatments is correlated to downregulation of cellular and tissue metabolism. Thus, understanding the mechanisms underlying such robust protective effects could lead to appropriate translation at the clinical level. In fact, hypothermia is already being used at many centers to improve neurological outcome from cardiac arrest. Methods— A systematic review of both topics is presented in terms of underlying pathophysiological mechanisms and application at the clinical level. Results— Although the mechanisms of protection for both therapeutic strategies are multifold, both share features of downregulating metabolism. Both therapeutic strategies are robust neuroprotectants, but translating them to the clinical arena is challenging, though not impossible, and clinical studies have shown or suggest benefits of both treatments. Conclusions— The strategy of metabolic downregulation should be further explored to identify effective neuroprotectants that can be easily applied clinically.

Cytochrome C Is Released From Mitochondria Into the Cytosol After Cerebral Anoxia or Ischemia

Mitochondrial dysfunction may underlie both acute and delayed neuronal cell death resulting from cerebral ischemia. Specifically, postischemic release of mitochondrial constituents such as the pro-apoptotic respiratory chain component cytochrome c could contribute acutely to further mitochondrial dysfunction and to promote delayed neuronal death. Experiments reported here tested the hypothesis that ischemia or severe hypoxia results in release of cytochrome c from mitochondria. Cytochrome c was measured spectrophotometrically from either the cytosolic fraction of cortical brain homogenates after global ischemia plus reperfusion, or from brain slices subjected to severe hypoxia plus reoxygenation. Cytochrome c content in cytosol derived from cerebral cortex was increased after ischemia and reperfusion. In intact hippocampal slices, there was a loss of reducible cytochrome c after hypoxia/reoxygenation, which is consistent with a decrease of this redox carrier in the mitochondrial pool. These results suggest that cytochrome c is lost to the cytosol after cerebral ischemia in a manner that may contribute to postischemic mitochondrial dysfunction and to delayed neuronal death.

Ischemic Preconditioning Targets the Respiration of Synaptic Mitochondria via Protein Kinase Cε

In the brain, ischemic preconditioning (IPC) diminishes mitochondrial dysfunction after ischemia and confers neuroprotection. Activation of ε protein kinase C (εPKC) has been proposed to be a key neuroprotective pathway during IPC. We tested the hypothesis that IPC increases the levels of εPKC in synaptosomes from rat hippocampus, resulting in improved synaptic mitochondrial respiration. Preconditioning significantly increased the level of hippocampal synaptosomal εPKC to 152% of sham-operated animals at 2 d of reperfusion, the time of peak neuroprotection. We tested the effect of εPKC activation on hippocampal synaptic mitochondrial respiration 2 d after preconditioning. Treatment with the specific εPKC activating peptide, tat-ψεRACK (tat-ψε-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 εPKC 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 H2O2 production. In brief, ischemic preconditioning promoted significant increases in the level of synaptosomal εPKC. Activation of εPKC increased synaptosomal mitochondrial respiration and phosphorylation of mitochondrial respiratory chain proteins. We propose that, at 48 h of reperfusion after ischemic preconditioning, εPKC is poised at synaptic mitochondria to respond to ischemia either by direct phosphorylation or activation of the εPKC signaling pathway.