Resveratrol Preconditioning Downregulates PARP1 Protein to Alleviate PARP1-Mediated Cell Death Following Cerebral Ischemia.

Stroke remains a leading cause of mortality; however, available therapeutics are limited. The study of ischemic tolerance, in paradigms such as resveratrol preconditioning (RPC), provides promise for the development of novel prophylactic therapies. The heavily oxidative environment following stroke promotes poly-ADP-ribose polymerase 1 (PARP1)-overactivation and parthanatos, both of which are major contributors to neuronal injury. In this study, we tested the hypothesis that RPC instills ischemic tolerance through decreasing PARP1 overexpression and parthanatos following in vitro and in vivo cerebral ischemia. To test this hypothesis, we utilized rat primary neuronal cultures (PNCs) and middle cerebral artery occlusion (MCAO) in the rat as in vitro and in vivo models, respectively. RPC was administered 2 days preceding ischemic insults. RPC protected PNCs against oxygen and glucose deprivation (OGD)-induced neuronal loss, as well as increases in total PARP1 protein, implying protection against PARP1-overactivation. Twelve hours following OGD, we observed reductions in NAD+/NADH as well as an increase in AIF nuclear translocation, but RPC ameliorated NAD+/NADH loss and blocked AIF nuclear translocation. MCAO in the rat induced AIF nuclear translocation in the ischemic penumbra after 24 h, which was ameliorated with RPC. We tested the hypothesis that RPC's neuroprotection was instilled through long-term downregulation of nuclear PARP1 protein. RPC downregulated nuclear PARP1 protein for at least 6 days in PNCs, likely contributing to RPC's ischemic tolerance. This study describes a novel mechanism by which RPC instills prophylaxis against ischemia-induced PARP1 overexpression and parthanatos, through a long-term reduction of nuclear PARP1 protein.

Resveratrol Preconditioning Mitigates Ischemia-Induced Septal Cholinergic Cell Loss and Memory Impairments.

BACKGROUND: Cholinergic cells originating from the nuclei of the basal forebrain (BF) are critical for supporting various memory processes, yet BF cholinergic cell viability has not been explored in the context of focal cerebral ischemia. In the present study, we examined cell survival within several BF nuclei in rodents following transient middle cerebral artery occlusion. We tested the hypothesis that a previously established neuroprotective therapy-resveratrol preconditioning-would rescue BF cell loss, deficits in cholinergic-related memory performance, and hippocampal synaptic dysfunction after focal cerebral ischemia. METHODS: Adult (2-3-month old) male Sprague-Dawley rats or wild-type C57Bl/6J mice were injected intraperitoneally with a single dose of resveratrol or vehicle and subjected to transient middle cerebral artery occlusion using the intraluminal suture method 2 days later. Histopathological, behavioral, and electrophysiological outcomes were measured 1-week post-reperfusion. Animals with reduction in cerebral blood flow <30% of baseline were excluded. RESULTS: Cholinergic cell loss was observed in the medial septal nucleus and diagonal band of Broca following transient middle cerebral artery occlusion. This effect was prevented by resveratrol preconditioning, which also ameliorated transient middle cerebral artery occlusion-induced deficits in cognitive performance and hippocampal long-term potentiation. CONCLUSIONS: We demonstrate for the first time that focal cerebral ischemia induces cholinergic cell death within memory-relevant nuclei of the BF. The preservation of cholinergic cell viability may provide a mechanism by which resveratrol preconditioning improves memory performance and preserves functionality of memory-processing brain structures after focal cerebral ischemia.

Post cardiac arrest physical exercise mitigates cell death in the septal and thalamic nuclei and ameliorates contextual fear conditioning deficits in rats.

A major concern for cardiac arrest (CA) survivors is the manifestation of long-term cognitive impairments. Physical exercise (PE) is a well-established approach to improve cognitive functions under certain pathological conditions. We previously showed that PE post-CA mitigates cognitive deficits, but the underlying mechanisms remain unknown. To define neuroprotective mechanisms, we analyzed whether PE post-CA protects neurons involved in memory. We first performed a contextual fear conditioning (CFC) test to confirm that PE post-CA preserves memory in rats. We then conducted a cell-count analysis and determined the number of live cells in the hippocampus, and septal and thalamic nuclei, all areas involved in cognitive functions. Lastly, we performed RNA-seq to determine PE post-CA effect on gene expression. Following CA, exercised rats had preserved CFC memory than sham PE animals. Despite this outcome, PE post-CA did not protect hippocampal cells from dying. However, PE ameliorated cell death in septal and thalamic nuclei compared to sham PE animals, suggesting that these nuclei are crucial in mitigating cognitive decline post-CA. Interestingly, PE affected regulation of genes related to neuroinflammation, plasticity, and cell death. These findings reveal potential mechanisms whereby PE post-CA preserves cognitive functions by protecting septal and thalamic cells via gene regulation.

Comparing Protection of Remote Limb with Resveratrol Preconditioning following Rodent Subarachnoid Hemorrhage.

BACKGROUND: Preventing delayed cerebral ischemia (DCI) after subarachnoid hemorrhage (SAH) remains an important therapeutic target. Preconditioning stimulates multiple endogenous protective mechanisms and may be a suitable treatment for DCI following SAH. We here compare remote limb conditioning with resveratrol conditioning in a clinically relevant SAH model. METHODS: We produced a SAH in 39 male Sprague Dawley rats using a single injection model. Animals were randomized to four groups: repetitive limb conditioning with a blood pressure cuff, sham conditioning, intraperitoneal resveratrol (10 mg/kg) or intraperitoneal vehicle administered at 24, 48 and 72 h after SAH. On day 4 neurological and behavioral scores were obtained, and animals were euthanized. The cross-sectional area of the basilar artery was measured at the vertebrobasilar junction, and at the mid and distal segments. Hippocampal cells were counted in both hemispheres and normalized per mm length. We compared true limb preconditioning with sham conditioning and resveratrol with vehicle preconditioning. RESULTS: The cross-sectional area of the mid-basilar artery in the true limb preconditioning group was significantly larger by 43% (p = 0.03) when compared with the sham preconditioning group. No differences in the cross-sectional area were found in the resveratrol-treated group when compared to the vehicle-treated group. We found no differences in the neuro score, behavioral score, and in mean hippocampal neuron counts between the groups. CONCLUSION: We found beneficial vascular effects of remote limb preconditioning on SAH-induced basilar artery vasoconstriction. Our findings support further studies of limb preconditioning as a potential treatment after SAH.

Sex-Dependent Differences in Physical Exercise-Mediated Cognitive Recovery Following Middle Cerebral Artery Occlusion in Aged Rats.

Stroke remains a leading cause of death and disability in the United States. No current treatments exist to promote cognitive recovery in survivors of stroke. A previous study from our laboratory determined that an acute bout of forced treadmill exercise was able to promote cognitive recovery in 3 month old male rats after middle cerebral artery occlusion (MCAo). In this study, we tested the hypothesis that 6 days of intense acute bout of forced treadmill exercise (physical exercise - PE) promotes cognitive recovery in 11-14 month old male rats. We determined that PE was able to ameliorate cognitive deficits as determined by contextual fear conditioning. Additionally, we also tested the hypothesis that PE promotes cognitive recovery in 11-13 month old reproductive senescent female rats. In contrast to males, the same intensity of exercise that decrease cognitive deficits in males was not able to promote cognitive recovery in female rats. Additionally, we determined that exercise did not lessen infarct volume in both male and female rats. There are many factors that contribute to higher stroke mortality and morbidities in women and thus, future studies will investigate the effects of PE in aged female rats to identify sex differences.

Preconditioning with CpG-ODN1826 reduces ischemic brain injury in young male mice: a replication study.

Earlier studies established that ischemic tolerance can be induced in the brain using various strategies. An earlier study demonstrated that preconditioning with the toll-like receptor 9 ligand, CpG oligodeoxynucleotides (ODN), protects the brain against ischemic damage. To increase the potential translational value of the previous study, the goal of the present study was to replicate this earlier finding in a different animal cohort at a different site. In addition to these replication studies, following the Stroke Treatment Academic Industry Roundtable (STAIR) guidelines, we also conducted studies to evaluate the protective effect of CpG-ODN 1826 preconditioning on cerebral ischemic damage in ovariectomized (Ovx) female animals. Young male and female mice were treated with CpG-ODN 1826 or control ligand 3 days prior to the induction of transient (60 min) cerebral ischemia using a middle cerebral artery occlusion (MCAO) model. Infarct size was evaluated at ~24 h post-MCAO. We were able to replicate earlier findings that preconditioning with a low dose (20 µg/mouse) of CpG-ODN 1826 was able to lower cerebral ischemic damage in young male mice. However, we did not see any protective effect of low dose CpG-ODN 1826 preconditioning against cerebral ischemic damage in young Ovx female mice. Our study independently confirms the protective effect of CpG-ODN 1826 in inducing cerebral ischemia tolerance in male but not in Ovx female mice. Our study also demonstrates the feasibility of conducting such replication studies in rodent models of transient stroke.

Neuronal SIRT1 (Silent Information Regulator 2 Homologue 1) Regulates Glycolysis and Mediates Resveratrol-Induced Ischemic Tolerance.

BACKGROUND AND PURPOSE: Resveratrol, at least in part via SIRT1 (silent information regulator 2 homologue 1) activation, protects against cerebral ischemia when administered 2 days before injury. However, it remains unclear if SIRT1 activation must occur, and in which brain cell types, for the induction of neuroprotection. We hypothesized that neuronal SIRT1 is essential for resveratrol-induced ischemic tolerance and sought to characterize the metabolic pathways regulated by neuronal Sirt1 at the cellular level in the brain. METHODS: We assessed infarct size and functional outcome after transient 60 minute middle cerebral artery occlusion in control and inducible, neuronal-specific SIRT1 knockout mice. Nontargeted primary metabolomics analysis identified putative SIRT1-regulated pathways in brain. Glycolytic function was evaluated in acute brain slices from adult mice and primary neuronal-enriched cultures under ischemic penumbra-like conditions. RESULTS: Resveratrol-induced neuroprotection from stroke was lost in neuronal Sirt1 knockout mice. Metabolomics analysis revealed alterations in glucose metabolism on deletion of neuronal Sirt1, accompanied by transcriptional changes in glucose metabolism machinery. Furthermore, glycolytic ATP production was impaired in acute brain slices from neuronal Sirt1 knockout mice. Conversely, resveratrol increased glycolytic rate in a SIRT1-dependent manner and under ischemic penumbra-like conditions in vitro. CONCLUSIONS: Our data demonstrate that resveratrol requires neuronal SIRT1 to elicit ischemic tolerance and identify a novel role for SIRT1 in the regulation of glycolytic function in brain. Identification of robust neuroprotective mechanisms that underlie ischemia tolerance and the metabolic adaptations mediated by SIRT1 in brain are crucial for the translation of therapies in cerebral ischemia and other neurological disorders.

Protein Kinase C Epsilon Promotes Cerebral Ischemic Tolerance Via Modulation of Mitochondrial Sirt5.

Sirtuin 5 (SIRT5) is a mitochondrial-localized NAD(+)-dependent lysine desuccinylase and a major regulator of the mitochondrial succinylome. We wanted to determine whether SIRT5 is activated by protein kinase C epsilon (PKCε)-mediated increases in mitochondrial Nampt and whether SIRT5 regulates mitochondrial bioenergetics and neuroprotection against cerebral ischemia. In isolated mitochondria from rat cortical cultures, PKCε activation increased SIRT5 levels and desuccinylation activity in a Nampt-dependent manner. PKCε activation did not lead to significant modifications in SIRT3 activity, the major mitochondrial lysine deacetylase. Assessments of mitochondrial bioenergetics in the cortex of wild type (WT) and SIRT5-/- mice revealed that SIRT5 regulates oxygen consumption in the presence of complex I, complex II, and complex IV substrates. To explore the potential role of SIRT5 in PKCε-mediated protection, we compared WT and SIRT5-/- mice by employing both in vitro and in vivo ischemia paradigms. PKCε-mediated decreases in cell death following oxygen-glucose deprivation were abolished in cortical cultures harvested from SIRT5-/- mice. Furthermore, PKCε failed to prevent cortical degeneration following MCAO in SIRT5-/- mice. Collectively this demonstrates that SIRT5 is an important mitochondrial enzyme for protection against metabolic and ischemic stress following PKCε activation in the brain.

Resveratrol Preconditioning Induces a Novel Extended Window of Ischemic Tolerance in the Mouse Brain.

BACKGROUND AND PURPOSE: Prophylactic treatments that afford neuroprotection against stroke may emerge from the field of preconditioning. Resveratrol mimics ischemic preconditioning, reducing ischemic brain injury when administered 2 days before global ischemia in rats. This protection is linked to silent information regulator 2 homologue 1 (Sirt1) and enhanced mitochondrial function possibly through its repression of uncoupling protein 2. Brain-derived neurotrophic factor (BDNF) is another neuroprotective protein associated with Sirt1. In this study, we sought to identify the conditions of resveratrol preconditioning (RPC) that most robustly induce neuroprotection against focal ischemia in mice. METHODS: We tested 4 different RPC paradigms against a middle cerebral artery occlusion model of stroke. Infarct volume and neurological score were calculated 24 hours after middle cerebral artery occlusion. Sirt1-chromatin binding was evaluated by ChIP-qPCR. Percoll gradients were used to isolate synaptic fractions, and changes in protein expression were determined via Western blot analysis. BDNF concentration was measured using a BDNF-specific ELISA assay. RESULTS: Although repetitive RPC induced neuroprotection from middle cerebral artery occlusion, strikingly one application of RPC 14 days before middle cerebral artery occlusion showed the most robust protection, reducing infarct volume by 33% and improving neurological score by 28%. Fourteen days after RPC, Sirt1 protein was increased 1.5-fold and differentially bound to the uncoupling protein 2 and BDNF promoter regions. Accordingly, synaptic uncoupling protein 2 level decreased by 23% and cortical BDNF concentration increased 26%. CONCLUSIONS: RPC induces a novel extended window of ischemic tolerance in the brain that lasts for at least 14 days. Our data suggest that this tolerance may be mediated by Sirt1 through upregulation of BDNF and downregulation of uncoupling protein 2.

Fatty acid methyl esters and Solutol HS 15 confer neuroprotection after focal and global cerebral ischemia.

We previously showed that palmitic acid methyl ester (PAME) and stearic acid methyl ester (SAME) are simultaneously released from the sympathetic ganglion and PAME possesses potent vasodilatory properties which may be important in cerebral ischemia. Since PAME is a potent vasodilator simultaneously released with SAME, our hypothesis was that PAME/SAME confers neuroprotection in rat models of focal/global cerebral ischemia. We also examined the neuroprotective properties of Solutol HS15, a clinically approved excipient because it possesses similar fatty acid compositions as PAME/SAME. Asphyxial cardiac arrest (ACA, 6 min) was performed 30 min after PAME/SAME treatment (0.02 mg/kg, IV). Solutol HS15 (2 ml/kg, IP) was injected chronically for 14 days (once daily). Histopathology of hippocampal CA1 neurons was assessed 7 days after ACA. For focal ischemia experiments, PAME, SAME, or Solutol HS15 was administered following reperfusion after 2 h of middle cerebral artery occlusion (MCAO). 2,3,5-Triphenyltetrazolium staining of the brain was performed 24 h after MCAO and the infarct volume was quantified. Following ACA, the number of surviving hippocampal neurons was enhanced by PAME-treated (68%), SAME-treated (69%), and Solutol-treated HS15 (68%) rats as compared to ACA only-treated groups. Infarct volume was decreased by PAME (83%), SAME (68%), and Solutol HS15 (78%) as compared to saline (vehicle) in MCAO-treated animals. PAME, SAME, and Solutol HS15 provide robust neuroprotection in both paradigms of ischemia. This may prove therapeutically beneficial since Solutol HS15 is already administered as a solublizing agent to patients. With proper timing and dosage, administration of Solutol HS15 and PAME/SAME can be an effective therapy against cerebral ischemia.

Ventricular fibrillation-induced cardiac arrest in the rat as a model of global cerebral ischemia.

Cardiopulmonary arrest remains one of the leading causes of death and disability in Western countries. Although ventricular fibrillation (VF) models in rodents mimic the "square wave" type of insult (rapid loss of pulse and pressure) commonly observed in adult humans at the onset of cardiac arrest (CA), they are not popular because of the complicated animal procedure, poor animal survival and thermal injury. Here we present a modified, simple, reliable, ventricular fibrillation-induced rat model of CA that will be useful in studying mechanisms of CA-induced delayed neuronal death as well as the efficacy of neuroprotective drugs. CA was induced in male Sprague Dawley rats using a modified method of von Planta et al. In brief, VF was induced in anesthetized, paralyzed, mechanically ventilated rats by an alternating current delivered to the entrance of the superior vena cava into the heart. Resuscitation was initiated by administering a bolus injection of epinephrine and sodium bicarbonate followed by mechanical ventilation and manual chest compressions and countershock with a 10-J DC current. Neurologic deficit score was higher in the CA group compared to the sham group during early reperfusion periods, suggesting brain damage. Significant damage in CA1 hippocampus (21% normal neurons compared to control animals) was observed following histopathological assessment at seven days of reperfusion. We propose that this method of VF-induced CA in rat provides a tool to study the mechanism of CA-induced neuronal death without compromising heart functions.

Epsilon PKC increases brain mitochondrial SIRT1 protein levels via heat shock protein 90 following ischemic preconditioning in rats.

Ischemic preconditioning is a neuroprotective mechanism whereby a sublethal ischemic exposure is protective against a subsequent lethal ischemic attack. We previously demonstrated that SIRT1, a nuclear localized stress-activated deacetylase, is vital for ischemic preconditioning neuroprotection. However, a recent study demonstrated that SIRT1 can also localize to the mitochondria. Mitochondrial localized SIRT1 may allow for a direct protection of mitochondria following ischemic preconditioning. The objective of this study was to determine whether ischemic preconditioning increases brain mitochondrial SIRT1 protein levels and to determine the role of PKCɛ and HSP90 in targeting SIRT1 to the mitochondria. Here we report that preconditioning rats, with 2 min of global cerebral ischemia, induces a delayed increase in non-synaptic mitochondrial SIRT1 protein levels which was not observed in synaptic mitochondria. This increase in mitochondrial SIRT1 protein was found to occur only in neuronal cells and was mediated by PKCε activation. Inhibition of HSP90, a protein chaperone involved in mitochondrial protein import, prevented preconditioning induced increases in mitochondrial SIRT1 and PKCε protein. Our work provides new insights into a possible direct role of SIRT1 in modulating mitochondrial function under both normal and stress conditions, and to a possible role of mitochondrial SIRT1 in activating preconditioning induced ischemic tolerance.

Synergistic inhibitory effect of nicotine plus oral contraceptive on mitochondrial complex-IV is mediated by estrogen receptor-ß in female rats.

Chronic nicotine and oral contraceptive (NOC) exposure caused significant loss of hippocampal membrane-bound estrogen receptor-beta (ER-ß) in female rats compared with exposure to nicotine alone. Mitochondrial ER-ß regulates estrogen-mediated mitochondrial structure and function; therefore, investigating the impact of NOC on mitochondrial ER-ß and its function could help delineate the harmful synergism between nicotine and OC. In this study, we tested the hypothesis that NOC-induced loss of mitochondrial ER-ß alters the oxidative phosphorylation system protein levels and mitochondrial respiratory function. This hypothesis was tested in hippocampal mitochondria isolated from female rats exposed to saline, nicotine, OC or NOC for 16 days. NOC decreased the mitochondrial ER-ß protein levels and reduced oxygen consumption and complex IV (CIV) activity by 34% and 26% compared with saline- or nicotine-administered groups, respectively. We also observed significantly low protein levels of all mitochondrial-encoded CIV subunits after NOC as compared with the nicotine or saline groups. Similarly, the silencing of ER-ß reduced the phosphorylation of cyclic-AMP response element binding protein, and also reduced levels of CIV mitochondrial-encoded subunits after estrogen stimulation. Overall, these results suggest that mitochondrial ER-ß loss is responsible for mitochondrial malfunction after NOC.

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.

Recurrent hypoglycemia exacerbates cerebral ischemic damage in streptozotocin-induced diabetic rats.

BACKGROUND AND PURPOSE: Stroke and heart disease are the most serious complications of diabetes accounting for >65% of mortality among diabetics. Although intensive insulin therapy has significantly improved the prognosis of diabetes and its complications, it is associated with an elevated risk of recurrent hypoglycemia (RH). We tested the hypothesis that RH exacerbates cerebral ischemic damage in a rodent model of diabetes. METHOD: We determined the extent of neuronal death in CA1 hippocampus after global cerebral ischemia in control and streptozotocin-induced diabetic rats. Diabetic animals included an insulin-treated streptozotocin-diabetic (ITD) group and a group of ITD rats exposed also to 10 episodes of hypoglycemia (ITD+recurrent hypoglycemia: RH). Hypoglycemia (55 to 65 mg/dL blood glucose) was induced twice daily for 5 consecutive days. RESULTS: As expected, uncontrolled diabetes (streptozotocin-diabetes, untreated animals) resulted in a 70% increase in ischemic damage as compared with the control group. Insulin treatment was able to lower ischemic damage by 64% as compared with the diabetic group. However, ITD+RH rats had 44% more damage when compared with the ITD group. We also observed that free radical release from mitochondria is increased in ITD+RH rats. CONCLUSIONS: This is the first report on the impact of RH in exacerbating cerebral ischemic damage in diabetic animals. Our results suggest that increased free radical release from mitochondria may be responsible for observed increased ischemic damage in ITD+RH rats. RH thus may be an unexplored but important factor responsible for increased ischemic damage in diabetes.

Protein kinase C epsilon activation delays neuronal depolarization during cardiac arrest in the euthermic arctic ground squirrel.

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 anoxia-tolerant 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 (epsilonPKC) 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 epsilonPKC (epsilonV1-2) shortened the time to ID in brain slices from AGS but not in rats despite evidence that epsilonV1-2 decreased activation of epsilonPKC in brain slices from both rats and AGS. These results support the hypothesis that epsilonPKC activation delays the collapse of ion homeostasis during ischemia in AGS.

Chronic nicotine exposure inhibits 17beta-estradiol-mediated protection of the hippocampal CA1 region against cerebral ischemia in female rats.

Nicotine addiction in women increases the risk of ischemic stroke. Importantly, women who smoke and use hormone replacement therapy/oral contraceptives greatly increase their risk of coronary heart disease and ischemic stroke as compared to nonsmoking women who use occasionally oral contraceptives. Nicotine addiction disturbs the normal periodicity of the menstrual cycle and induces early onset of menopause in women; however, the mechanism of the synergistic effects of nicotine and sex hormones on cerebrovascular health is not clearly understood. In the current study based on a rat model of global cerebral ischemia, our goals are (1) to determine whether chronic nicotine exposure abrogates beneficial effects of estrogen on hippocampal neurons subjected to ischemia, and (2) to determine whether nicotine exposure antagonizes estrogen signaling by reducing the availability of estrogen receptor(s). To test the effects of chronic nicotine exposure, normally cycling or ovariectomized rats were injected with nicotine daily for 15 days. To investigate the efficacy of estrogen treatment, nicotine-exposed ovariectomized rats were injected with a bolus of 17beta-estradiol and 48h later ischemia was induced. Our results demonstrated that chronic nicotine exposure followed by ischemic insult at the proestrus stage of the estrous cycle showed that only 14% of normal neurons remained compared to the non-nicotine-treated group (p<0.05). Similarly, a bolus of 17beta-estradiol to nicotine-treated ovariectomized rats showed only 26% of normal neurons remaining as against 47% in the non-nicotine-treated group. Nicotine exposure decreased ERbeta but not ERalpha protein levels in the hippocampus, suggesting a role for ERbeta in increased post-ischemic neurodegeneration from nicotine exposure.

Ischemic preconditioning targets the respiration of synaptic mitochondria via protein kinase C epsilon.

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

Neuroprotective effect of STAZN, a novel azulenyl nitrone antioxidant, in focal cerebral ischemia in rats: dose-response and therapeutic window.

Stilbazulenyl nitrone (STAZN) is a potent antioxidant that, in a rat model of transient focal cerebral ischemia, confers significant enduring functional and morphological neuroprotection. This study investigated the influence of dose and time of administration on the neuroprotective effects of STAZN in the intraluminal suture model of middle cerebral artery occlusion (MCAo). Dose response: At 2 and 4 h after the onset of MCAo, animals received intravenously either STAZN (low dose=0.07 mg/kg, n=8; medium dose=0.7 mg/kg, n=9; high dose=3.5 mg/kg, n=9), an equivalent volume of vehicle (30% Solutol HS15 and 70% isotonic saline, 0.37 ml/kg, n=5) or saline (0.37 ml/kg, n=5). Only the medium dose improved scores (p<0.05) on a standardized neurobehavioral test at 1, 2 and 3 days after MCAo. Only the medium dose reduced the total infarction (51%, p=0.014) compared to controls. These results indicate that STAZN exhibits maximal neuroprotection at the 0.7 mg/kg dose. Therapeutic window: STAZN (0.6 mg/kg) dissolved in dimethylsulfoxide was given intra-peritoneally at 2 and 4 h (n=11), 3 and 5 h (n=10), 4 and 6 h (n=10) or 5 and 7 h (n=7) after the onset of MCAo. Additional doses were given at 24 and 48 h. Vehicle (dimethylsulfoxide, 2.0 ml/kg, n=6) was administered at 3, 5, 24 and 48 h. STAZN treatment initiated at 2 or 3 h after the onset of MCAo improved neurological scores (p<0.001) and reduced total infarction (42.2%, p<0.05) compared to controls.