Epidemiologic studies have shown that foods rich in polyphenols, such as flavanols, can lower the risk of ischemic heart disease; however, the mechanism of protection has not been clearly established. In this study, we investigated whether epicatechin (EC), a flavanol in cocoa and tea, is protective against brain ischemic damage in mice. Wild-type mice pretreated orally with 5, 15, or 30 mg/kg EC before middle cerebral artery occlusion (MCAO) had significantly smaller brain infarcts and decreased neurologic deficit scores (NDS) than did the vehicle-treated group. Mice that were posttreated with 30 mg/kg of EC at 3.5 hours after MCAO also had significantly smaller brain infarcts and decreased NDS. Similarly, WT mice pretreated with 30 mg/kg of EC and subjected to N-methyl-D-aspartate (NMDA)-induced excitotoxicity had significantly smaller lesion volumes. Cell viability assays with neuronal cultures further confirmed that EC could protect neurons against oxidative insults. Interestingly, the EC-associated neuroprotection was mostly abolished in mice lacking the enzyme heme oxygenase 1 (HO1) or the transcriptional factor Nrf2, and in neurons derived from these knockout mice. These results suggest that EC exerts part of its beneficial effect through activation of Nrf2 and an increase in the neuroprotective HO1 enzyme.

The transcriptional factor Nrf2 has a unique role in various physiological stress conditions, but its contribution to ischemia/reperfusion injury has not been fully explored. Therefore, wildtype (WT) and Nrf2 knockout (Nrf2 -/-) mice were subjected to 90-min occlusion of the middle cerebral artery (MCA) followed by 24-h reperfusion to elucidate Nrf2 contribution in protecting against ischemia/ reperfusion injury. Infarct volume, represented as percent of hemispheric volume, was significantly (P<0.05) larger in Nrf2 -/-mice than in WT mice (30.8 ± 6.1% vs 17.0 ± 5.1). Furthermore, neurological deficit was significantly greater in the Nrf2 -/-mice. To examine whether neuronal protection was mediated by Nrf2, neurons were treated with various compounds to induce excitotoxic or oxidative stress. Translocation of Nrf2 into the nucleus was increased by the free-radical donor tert-butylhydroperoxide, but not by glutamate or NMDA. In addition, a common Nrf2 inducer, tertbutylhydroquinone, significantly attenuated neuronal cell death induced by tert-butylhydroperoxide (83.6 ± 1.6 vs 62.0 ± 7.7%) but not as substantially as when excitotoxicity was induced by NMDA (91.9 ± 1.6 vs 79.3 ± 3.3%) or glutamate (87.8 ± 1.5 vs 80.2 ± 2.6%). The results suggest that Nrf2 reduces ischemic brain injury by protecting against oxidative stress. Keywords free radicals; MCA occlusion/reperfusion; NF-E2-related factor 2; stroke Oxidative stress from reactive oxygen species (ROS) enhances inflammatory responses during tissue injury, possibly through activation of redox-sensitive chemokines and transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2 has been reported to be a key Address all correspondence to Sylvain Doré, PhD, Departments of Anesthesiology/Critical Care Medicine and Neuroscience, Johns Hopkins University, 720 N Rutland Ave. Ross 365, Baltimore, MD 21205 (Tel: 410.614.4859; Fax: 410.955.7271; sdore@jhmi.edu; www.hopkinsmedicine.org/dorelab) * These authors contributed equally. Section Editor: Professor Constantino Sotelo Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. (Owuor and Kong, 2002). It induces expression and upregulation of cytoprotective and antioxidant genes that attenuate tissue injury (Zhang et al., 2005). Under basal conditions, cytoplasmic Nrf2 is bound to the Kelch-like ECH-associated protein 1 (Keap 1), but when cells are exposed to oxidative or xenobiotic stress, Nrf2 dissociates and traverses to the nucleus (Wakabayashi et al., 2003;Li et al., 2004a). NIH Public AccessNrf2 is considered to be a multi-organ protector (for review see Lee...

Ginkgo biloba extract, EGb 761, a popular and standardized natural extract, contains 24% ginkgoflavonol glycosides and 6% terpene lactones. EGb 761 is used worldwide to treat many ailments, and while a number of studies have shown its neuroprotective properties, the mechanisms of action have not been elucidated fully. We hypothesize that EGb 761 and some of its bioactive components [Bilobalide (BB), Ginkgolide A (GA), Ginkgolide B (GB), and Terpene Free Material (TFM)] could provide neuroprotection ischemic conditions through heme oxygenase 1 (HO1). Mice were subjected permanent distal middle cerebral artery occlusion (pMCAO) and survived for 7 days. HO1 -/-mice showed significantly higher (p<0.05) infarct volume and Neurologic Deficit Scores (NDS) as compared to their wildtype (WT) counterparts. In another cohort, mice subjected to pMCAO and treated at 4 h of pMCAO with 100mg/kg EGb 761 6mg/kg BB, GA, GB, or 10mg/ kg TFM showed significantly lower (p<0.05) infarct volumes (BB; 29.0±3.9%, GA; 31.3±4.0%, GB; 32.0±3.8%, TFM; 32.5±3.5%, and 761; 27.4±4.5%) than those in the vehicle-treated mice (46.0±3.7%). Similarly, were lower in BB: 7.1±1.8, GA; 7.4±2.1, GB; 7.9±1.8, TFM; 7.7±1.7, and EGb 6.8±2.0 groups as compared with the vehicle-treated group (13.8±1.5). Interestingly, the protective effect of EGb 761 was essentially lost when HO1 knockout mice were treated with EGb 761. In another cohort, HO1, VEGF and eNOS protein levels in the cortices appeared to be higher in EGb 761 and BB but not in GA, GB and TFM treated groups. Together, these results suggest that HO1 plays, at least in part, an important role in the neuroprotective mechanism of EGb 761 and in delayed ischemia. Targeting this pathway could lead to neuroprotective agents against ischemic stroke. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptNeuroscience. Author manuscript; available in PMC 2012 April 28. (Chan et al., 2007). At present, EGb 761 extract is one of the most renowned and commonly used natural compounds, and it has been used in many preclinical and clinical studies to evaluate its efficacy (Snitz et al., 2009). It contains: 24% ginkgo-flavanol glycosides; 6% terpene lactones such as ginkgolides A, B, C, J, and bilobalide; 5-10% organic acids; and >0.5% proanthocyanidins, defined as flavonoidbased polymers (van Beek, 2002).Numerous studies have shown neuroprotective properties of EGb 761 and its different constituents, but the mechanism underlying its neuroprotection has not been studied fully (Bastianetto et al., 2000, Lee et al., 2002, Chandrasekaran et ...

Ischemic preconditioning (IPC) protects brain against ischemic injury by activating specific mechanisms. Our goal was to determine if the inducible heme oxygenase 1 (HO1) is required for such protection. IPC before transient or permanent ischemia reduced cortical infarct volumes by 57.4% and 33.9%, respectively at 48 h in wildtype adult mice. Interestingly, IPC failed to protect the HO1 gene deleted mice against permanent ischemic brain injury. IPC also resulted in a significant increase in HO1 protein levels in the brain and correlated with reduced neurological deficits after permanent and transient brain ischemia. Our study demonstrates that neuroprotective effects of IPC are at least partially mediated via HO1. Elucidating the physiological/cellular role by which HO1 is protective against brain ischemia may aid the development of selective drugs to treat stroke and its associated neurological disorders.

Ginkgo biloba extract is an alternative medicine available as a standardized formulation, EGb 761®, which consists of ginkgolides, bilobalide, and flavonoids. The individual constituents have varying therapeutic mechanisms that contribute to the pharmacological activity of the extract as a whole. Recent studies show anxiolytic properties of ginkgolide A, migraine with aura treatment by ginkgolide B, a reduction in ischemia-induced glutamate excitotoxicity by bilobalide, and an alternative antihypertensive property of quercetin, among others. These findings have been observed in EGb 761 as well and have led to clinical investigation into its use as a therapeutic for conditions such as cognition, dementia, cardiovascular, and cerebrovascular diseases. This review explores the therapeutic mechanisms of the individual EGb 761 constituents to explain the pharmacology as a whole and its clinical application to cardiovascular and neurological disorders, in particular ischemic stroke.

Microglial cells are activated in response to different types of injuries or stress in the CNS. Such activation is necessary to get rid of the injurious agents and restore tissue homeostasis. However, excessive activation of microglial cells is harmful and contributes to secondary injury. Pertinently, microglial cell activity was targeted in many preclinical and clinical studies but such strategy failed in clinical trials. The main reason behind the failed attempts is the complexity of the injury mechanisms which needs either a combination therapy or targeting a process that is involved in multiple pathways. Cofilin is a cytoskeleton-associated protein involved in actin dynamics. In our previous study, we demonstrated the role of cofilin in mediating neuronal apoptosis during OGD conditions. Previous studies on microglia have shown the involvement of cofilin in ROS formation and phagocytosis. However, additional studies are needed to delineate the role of cofilin in microglial cell activation. Therefore, in the current study, we investigated the role of cofilin in LPS-induced microglial cell activation using cofilin siRNA knockdown paradigms. The viability of differentiated PC12 cells was used as a measure of the neurotoxic potential of conditioned medium derived from cofilin siRNA-transfected and LPS-activated microglial cells. Cofilin knockdown significantly inhibited LPS-induced microglial cell activation through NF-κB and JAK-STAT pathways. The release of proinflammatory mediators (NO, TNF-α, iNOS, and COX2) as well as microglial proliferation and migration rates were significantly reduced by cofilin knockdown. Furthermore, differentiated PC12 cells were protected from the neurotoxicity induced by conditioned medium derived from cofilin-transfected and LPS-activated microglial cells. In conclusion, we demonstrated that cofilin is involved in the cascade of microglial cell activation and further validates our previous study on cofilin's role in mediating neuronal apoptosis. Together, our results suggest that cofilin could present a common target in neurons and microglial cells and might prove to be a promising therapy for different brain injury mechanisms including stroke.

Tyrosine hydroxylase, a hypoxia-regulated gene, may be involved in tissue adaptation to hypoxia. Intermittent hypoxia, a characteristic feature of sleep apnea, leads to significant memory deficits, as well as to cortex and hippocampal apoptosis that are absent after sustained hypoxia. To examine the hypothesis that sustained and intermittent hypoxia induce different catecholaminergic responses, changes in tyrosine hydroxylase mRNA, protein expression, and activity were compared in various brain regions of male rats exposed for 6 h, 1 day, 3 days, and 7 days to sustained hypoxia (10% O(2)), intermittent hypoxia (alternating room air and 10% O(2)), or normoxia. Tyrosine hydroxylase activity, measured at 7 days, increased in the cortex as follows: sustained > intermittent > normoxia. Furthermore, activity decreased in the brain stem and was unchanged in other brain regions of sustained hypoxia-exposed rats, as well as in all regions from animals exposed to intermittent hypoxia, suggesting stimulus-specific and heterotopic catecholamine regulation. In the cortex, tyrosine hydroxylase mRNA expression was increased, whereas protein expression remained unchanged. In addition, significant differences in the time course of cortical Ser(40) tyrosine hydroxylase phosphorylation were present in the cortex, suggesting that intermittent and sustained hypoxia-induced enzymatic activity differences are related to different phosphorylation patterns. We conclude that long-term hypoxia induces site-specific changes in tyrosine hydroxylase activity and that intermittent hypoxia elicits reduced tyrosine hydroxylase recruitment and phosphorylation compared with sustained hypoxia. Such changes may not only account for differences in enzyme activity but also suggest that, with differential regional brain susceptibility to hypoxia, recruitment of different mechanisms in response to hypoxia will elicit region-specific modulation of catecholamine response.

CIH during sleep is associated with decreased libido in mice. The decreased expression of endothelial NOS protein in erectile tissue and the favorable response to tadalafil suggest that altered nitric oxide mechanisms underlie CIH-mediated ED. No changes in testosterone emerge after intermittent hypoxia.