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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.

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., (© 2017 American Heart Association, Inc.)

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., (© 2015 American Heart Association, Inc.)

Background and Purpose: Nuclear erythroid 2 related factor 2 (Nrf2) is an astrocyte-enriched transcription factor that has previously been shown to upregulate cellular antioxidant systems in response to ischemia. Although resveratrol preconditioning (RPC) has emerged as a potential neuroprotective therapy, the involvement of Nrf2 in RPC-induced neuroprotection and mitochondrial reactive oxygen species production after cerebral ischemia remains unclear. The goal of our study was to study the contribution of Nrf2 to RPC and its effects on mitochondrial function., Methods: We used rodent astrocyte cultures and an in vivo stroke model with RPC. An Nrf2 DNA binding ELISA and protein analysis via Western blotting of downstream Nrf2 targets were performed to determine RPC-induced activation of Nrf2 in rat and mouse astrocytes. After RPC, mitochondrial function was determined by measuring reactive oxygen species production and mitochondrial respiration in both wild-type and Nrf2-/- mice. Infarct volume was measured to determine neuroprotection, whereas protein levels were measured by immunoblotting., Results: We report that Nrf2 is activated by RPC in rodent astrocyte cultures, and that loss of Nrf2 reduced RPC-mediated neuroprotection in a mouse model of focal cerebral ischemia. In addition, we observed that wild-type and Nrf2-/- cortical mitochondria exhibited increased uncoupling and reactive oxygen species production after RPC treatments. Finally, Nrf2-/- astrocytes exhibited decreased mitochondrial antioxidant expression and were unable to upregulate cellular antioxidants after RPC treatment., Conclusions: Nrf2 contributes to RPC-induced neuroprotection through maintaining mitochondrial coupling and antioxidant protein expression., (© 2015 American Heart Association, Inc.)

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.

Cardiac arrest results in significant mortality after initial resuscitation due in most cases to ischemia-reperfusion induced brain injury and to a lesser degree myocardial dysfunction. Nitrite has previously been shown to protect against reperfusion injury in animal models of focal cerebral and heart ischemia. Nitrite therapy after murine cardiac arrest improved 22 h survival through improvements in myocardial contractility. These improvements accompanied transient mitochondrial inhibition which reduced oxidative injury to the heart. Based on preliminary evidence that nitrite may also protect against ischemic brain injury, we sought to test this hypothesis in a rat model of asphyxia cardiac arrest with prolonged survival (7d). Cardiac arrest resulted in hippocampal CA1 delayed neuronal death well characterized in this and other cardiac arrest models. Nitrite therapy did not alter post-arrest hemodynamics but did result in significant (75%) increases in CA1 neuron survival. This was associated with increases in hippocampal nitrite and S-nitrosothiol levels but not cGMP shortly after therapy. Mitochondrial function 1h after resuscitation trended towards improvement with nitrite therapy. Based on promising preclinical data, the first ever phase I trial of nitrite infusions in human cardiac arrest survivors has been undertaken. We present preliminary data showing low dose nitrite infusion did not result in hypotension or cause methemoglobinemia. Nitrite thus appears safe and effective for clinical translation as a promising therapy against cardiac arrest mediated heart and brain injury., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Mammals that hibernate experience extreme metabolic states and body temperatures as they transition between euthermia, a state resembling typical warm blooded mammals, and prolonged torpor, a state of suspended animation where the brain receives as low as 10% of normal cerebral blood flow. Transitions into and out of torpor are more physiologically challenging than the extreme metabolic suppression and cold body temperatures of torpor per se. Mammals that hibernate show unprecedented capacities to tolerate cerebral ischemia, a decrease in blood flow to the brain caused by stroke, cardiac arrest or brain trauma. While cerebral ischemia often leads to death or disability in humans and most other mammals, hibernating mammals suffer no ill effects when blood flow to the brain is dramatically decreased during torpor or experimentally induced during euthermia. These animals, as adults, also display rapid and pronounced synaptic flexibility where synapses retract during torpor and rapidly re-emerge upon arousal. A variety of coordinated adaptations contribute to tolerance of cerebral ischemia in these animals. In this review we discuss adaptations in heterothermic mammals that may suggest novel therapeutic targets and strategies to protect the human brain against cerebral ischemic damage and neurodegenerative disease., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Estradiol-17beta is released from the ovaries in a cyclic manner during the normal estrous cycle in rats. During the transition from the diestrous to proestrous stage, the 17beta-estradiol increases in blood circulation. We hypothesized that a higher serum level of endogenous 17beta-estradiol would protect hippocampal pyramidal neurons against global cerebral ischemia via activation of the cyclic-AMP response element binding protein (CREB)-mediated signaling cascade. Furthermore, we asked if a single 17beta-estradiol bolus provides protection against ischemia in the absence of endogenous estradiol. To test these hypotheses, rats were subjected to global cerebral ischemia at different stages of the estrous cycle. Ischemia was produced by bilateral carotid occlusion and systemic hypotension. Brains were examined for histopathology at 7 days of reperfusion. Higher serum levels of 17beta-estradiol (at proestrus and estrus stages) correlated with increased immunoreactivity of pCREB in hippocampus and ischemic tolerance. At diestrus, when circulating gonadal hormone concentrations were lowest, the pCREB protein content of hippocampus was reduced and showed the least number of normal neurons after ischemia compared to other stages of the estrous cycle. A similar phosphorylation pattern was also observed for mitogen-activated protein kinase (MAPK) and calcium-calmodulin-dependent protein kinase (CaMKII) in hippocampus. The cyclic variation in ovarian hormones did not reflect phosphorylation of protein kinase B (Akt). To test the efficacy of a single bolus of 17beta-estradiol before ischemia, ovariectomized rats were treated with 17beta-estradiol (5/10/50 microg/kg) or vehicle (oil) and 48/72/96 h later rats were exposed to cerebral ischemia. A single 17beta-estradiol bolus treatment in ovariectomized rats significantly increased CREB mRNA activation and protected CA1 pyramidal neurons against ischemia. These results suggest that an exogenous bolus of 17beta-estradiol to ovariectomized rats protects hippocampus against ischemia via activation of the CREB pathway in a manner similar to the endogenous estrous cycle.

Cardiovascular pathologies in the French are not prevalent despite high dietary saturated fat consumption. This is commonly referred to as the "French Paradox" attributing its anti-lipidemic effects to moderate consumption of red wine. Resveratrol, a phytoalexin found in red wine, is currently the focus of intense research both in the cardiovascular system and the brain. Current research suggests resveratrol may enhance prognosis of neurological disorders such as, Parkinson's, Huntington's, Alzheimer's diseases and stroke. The beneficial effects of resveratrol include: antioxidation, free radical scavenger, and modulation of neuronal energy homeostasis and glutamatergic receptors/ion channels. Resveratrol directly increases sirtuin 1 (SIRT1) activity, a NAD(+) (oxidized form of nicotinamide adenine dinucleotide)-dependent histone deacetylase related to increased lifespan in various species similar to calorie restriction. We recently demonstrated that brief resveratrol pretreatment conferred neuroprotection against cerebral ischemia via SIRT1 activation. This neuroprotective effect produced by resveratrol was similar to ischemic preconditioning-induced neuroprotection, which protects against lethal ischemic insults in the brain and other organ systems. Inhibition of SIRT1 abolished ischemic preconditioning-induced neuroprotection in CA1 region of the hippocampus. Since resveratrol and ischemic preconditioning-induced neuroprotection require activation of SIRT1, this common signaling pathway may provide targeted therapeutic treatment modalities as it relates to stroke and other brain pathologies. In this review, we will examine common signaling pathways, cellular targets of resveratrol, and ischemic preconditioning-induced neuroprotection as it relates to the brain.

Cytosolic Ca2+ overload has been proposed as a main cause of neuronal injury during cerebral ischemia. SNX-111, a synthetic product of the naturally occurring omega-conotoxin MVIIA, is a novel, presynaptic N-type Ca2+ channel antagonist and has been reported to be neuroprotective against cerebral ischemia. We studied the neuroprotective effects of SNX-111 in a rabbit model of focal cerebral ischemia. New Zealand white male rabbits (2.5-3.5 kg) were given 1 mg/kg/h i.v. SNX-111 (n=8) or normal saline (n=8) 10 min after onset of a 2-h period of transient focal cerebral ischemia induced by occlusion of the left middle cerebral, anterior cerebral and internal carotid arteries followed by 4 h reperfusion. SNX-111 significantly attenuated overall cortical ischemic neuronal damage by 44% (saline, 38.7+/-3.0%; SNX-111, 21.5+/-6.0%, P<0.05) and regions of hyperintensity on T2-weighted MRI by 30% (saline, 70.6+/-4.0%; SNX-111, 49.3+/-11.0%, P<0.05). No significant difference in (regional cerebral blood flow) rCBF or MAP (mean arterial blood pressure) was found between SNX-111- and saline-treated rabbits suggesting that neuroprotection is due to a cellular effect. We conclude that SNX-111 reduces ischemic injury in this model. Its use as a clinical neuroprotective agent for cerebrovascular surgery or stroke should be investigated further.