Neuroprotection In Stroke Research Articles

Metabolic Reconstruction of Glutamate-Glutamine Cycling: A Flux Balance Approach

Event Abstract Back to Event Metabolic Reconstruction of Glutamate-Glutamine Cycling: A Flux Balance Approach Nelson E. Vega-Vela1, Cynthia M. Jimenez1, George E. Barreto1* and Janneth Gonzalez1 1 Pontificia Universidad Javeriana, Colombia Outline - Astrocyte/Neuron metabolic relationships: A short overview - Metabolic reconstruction: Basic principles - Computational systems biology databases and toolkits: Short review - Metabolic modelling and flux balance analysis of glutamate-glutamine cycle between neurons and astrocytes - Metabolic network analysis and visualization Abstract Cells, tissues, organs and organisms can be understood as a large number of interconnected networks of biochemical pathways, genes, transcripts and proteins which give rise to emergent and specific functions and behaviors in a complex biological system. The complexity exhibited by these systems imposes considerable challenges to understand how they behave and work. For instance, diseases or patho-physiological states are complex conditions that must be considered in a holistic approach because they involve intricate interactions between thousands of components such as genes, transcripts and proteins. In this context, diseases are considered as perturbed states of these networks which can be used to identify components (e.g. metabolites, enzymes, etc.) and interactions that are gained or lost, as well as the biochemical pathways involved. Therefore, discrimination of components and interactions that are not relevant or informative is important to embrace the complexity for understanding biological processes. Systems biology, Network medicine and the recent Systems neuroscience has been successfully applied to make this complexity comprehensible in the different fields of biology, physiology, medicine and neuroscience. To illustrate these approaches within the framework of the workshop “Latin-American School on glial cells in the diseased brain”, we reconstruct the well-known glutamate/glutamine cycle to understand how glutamate can be a neurotoxic agent through glutamate-induced excitotoxicity in neurons and astrocytes. Figure 1 References Barreto GE, Santos-Galindo M, Garcia-Segura LM. Selective estrogen receptor modulators regulate reactive microglia after penetrating brain injury. Front Aging Neurosci, 6:132, 2014. Cabezas R, Ávila M, Gonzalez J, El-Bachá RS, Báez E, Garcia-Segura LM, Jurado Coronel JC, Capani F, Cardona-Gomez GP, Barreto GE. Astrocytic modulation of blood brain barrier: perspectives on Parkinson’s disease. Front. Cell. Neurosci. 8:211, 2014. Barreto GE, White RE, Xu L, Palm CJ, Giffard RG. Effects of heat shock protein 72 (Hsp72) on evolution of astrocyte activation following stroke in the mouse. Exp Neurol. 238(2):284-296, 2012. Barreto G , White RE, Ouyang Y, Xu L, Giffard RG. Astrocytes: targets for neuroprotection in stroke. Cent Nerv Syst Agents Med Chem, 11(2):164-73, 2011. Barreto GE , González J, Torres Y, Morales L. Astrocyte-neuronal crosstalk: Implications for neuroprotection from brain injury. Neurosc Res, 71(2):107-13, 2011. Keywords: astrocyte, Neurons, Metabolic Networks, Flux balance analysis, Neurotoxicity, Computational Biology Conference: Latin-American School on glial cells in the diseased brain (IBRO), Bogotá, Colombia, 13 Jul - 17 Jul, 2015. Presentation Type: Oral Presentation Topic: Neural excitability, synaptic transmission, glia: Cellular and molecular mechanisms Citation: Vega-Vela N, Jimenez CM, Barreto G and Gonzalez J (2015). Metabolic Reconstruction of Glutamate-Glutamine Cycling: A Flux Balance Approach. Conference Abstract: Latin-American School on glial cells in the diseased brain (IBRO). doi: 10.3389/conf.fncel.2015.35.00008 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 22 Feb 2015; Published Online: 16 May 2015. * Correspondence: Prof. George E. Barreto, Pontificia Universidad Javeriana, Bogotá, Colombia, gesbarreto@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Nelson E. Vega-Vela Cynthia M Jimenez George E. Barreto Janneth Gonzalez Google Nelson E. Vega-Vela Cynthia M Jimenez George E. Barreto Janneth Gonzalez Google Scholar Nelson E. Vega-Vela Cynthia M Jimenez George E. Barreto Janneth Gonzalez PubMed Nelson E. Vega-Vela Cynthia M Jimenez George E. Barreto Janneth Gonzalez Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Towards reperfusion-centric preclinical stroke research: outside the box of "reperfusion injury".

Stroke is a major health issue of increasing significance for any society with an aging population. Globally, stroke is the second-leading cause of death with approximately 5.9 million fatal events in 2010, equivalent to 11.1% of all deaths. Yet, despite years of preclinical research on neuroprotection and a multitude of clinical trials, tissue plasminogen activator (tPA)-mediated recanalization remains the mainstay of acute ischemic stroke therapy, whereas tPA thrombolysis rarely provides benefits in the mechanical occlusion-based stroke models. This split between the bench and bedside raised the concern over the clinical applicability of neuroprotection in acute ischemic stroke. In this perspective commentary, we call for attention to the differences between mechanical-occlusion and thromboembolic stroke models in cerebral hemodynamics (Figure ​Figure1A,1A, ​,BB), the implications of these differences in view of progressive pathobiology of ischemic stroke (Figure 1C), and the need and strategies towards reperfusion-centric preclinical stroke research.

Squalenoyl adenosine nanoparticles provide neuroprotection after stroke and spinal cord injury.

There is an urgent need to develop new therapeutic approaches for the treatment of severe neurological trauma, such as stroke and spinal cord injuries. However, many drugs with potential neuropharmacological activity, like adenosine, are inefficient upon systemic administration because of their fast metabolisation and rapid clearance from the bloodstream. Here, we show that the conjugation of adenosine to the lipid squalene and the subsequent formation of nanoassemblies allow a prolonged circulation of this nucleoside, to provide neuroprotection in mouse stroke and rat spinal cord injury models. The animals receiving systemic administration of squalenoyl adenosine nanoassemblies showed a significant improvement of their neurologic deficit score in the case of cerebral ischaemia, and an early motor recovery of the hindlimbs in the case of spinal cord injury. Moreover, in vitro and in vivo studies demonstrated that the nanoassemblies were able to extend adenosine circulation and its interaction with the neurovascular unit. This paper shows, for the first time, that a hydrophilic and rapidly metabolised molecule like adenosine may become pharmacologically efficient owing to a single conjugation with the lipid squalene.

Neuroprotection by acetyl-11-keto-β-Boswellic acid, in ischemic brain injury involves the Nrf2/HO-1 defense pathway.

Stroke is a complex disease involved oxidative stress-related pathways in its pathogenesis. The nuclear factor erythroid-2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway has been considered a potential target for neuroprotection in stroke. Acetyl-11-Keto-β-Boswellic Acid (AKBA) is an active triterpenoid compound from the extract of Boswellia serrate. The present study was to determine whether AKBA, a novel Nrf2 activator, can protect against cerebral ischemic injury. The stroke model was produced in Sprague–Dawley rats via middle cerebral artery occlusion. To model ischemia-like conditions in vitro, primary cultured cortical neurons were exposed to transient oxygen and glucose deprivation (OGD). Treatment of AKBA significantly reduced infarct volumes and apoptotic cells, and also increased neurologic scores by elevating the Nrf2 and HO-1 expression in brain tissues in middle cerebral artery occlusion (MCAO) rats at 48 hours post reperfusion. In primary cultured neurons, AKBA increased the Nrf2 and HO-1 expression, which provided protection against OGD-induced oxidative insult. Additionally, AKBA treatment increased Nrf2 binding activity to antioxidant-response elements (ARE). The protective effect of AKBA was attenuated by knockdown of Nrf2 or HO-1. In conclusion, these findings provide evidence that AKBA protects neurons against ischemic injury, and this neuroprotective effect involves the Nrf2/HO-1 pathway.

Posttreatment with 11-Keto-β-Boswellic Acid Ameliorates Cerebral Ischemia-Reperfusion Injury: Nrf2/HO-1 Pathway as a Potential Mechanism.

Oxidative stress is well known to play a pivotal role in cerebral ischemia-reperfusion injury. The nuclear factor erythroid-2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway has been considered a potential target for neuroprotection in stroke. 11-Keto-β-boswellic acid (KBA) is a triterpenoid compound from extracts of Boswellia serrata. The aim of the present study was to determine whether KBA, a novel Nrf2 activator, can protect against cerebral ischemic injury. Middle cerebral artery occlusion (MCAO) was operated on male Sprague-Dawley rats. KBA (25 mg/kg) applied 1 h after reperfusion significantly reduced infarct volumes and apoptotic cells as well as increased neurologic scores at 48 h after reperfusion. Meanwhile, posttreatment with KBA significantly decreased malondialdehyde (MDA) levels, restored the superoxide dismutase (SOD) activity, and increased the protein Nrf2 and HO-1 expression in brain tissues. In primary cultured astrocytes, KBA increased the Nrf2 and HO-1 expression, which provided protection against oxygen and glucose deprivation (OGD)-induced oxidative insult. But knockdown of Nrf2 or HO-1 attenuated the protective effect of KBA. In conclusion, these findings provide evidence that the neuroprotection of KBA against oxidative stress-induced ischemic injury involves the Nrf2/HO-1 pathway.

Drug-induced Hypothermia by 5HT1A Agonists Provide Neuroprotection in Experimental Stroke: New Perspectives for Acute Patient Treatment

Drug-induced hypothermia reduces brain damage in animal stroke models and is an undiscovered potential in human stroke treatment. We studied hypothermia induced by the serotonergic agonists S14671 (1-[2-(2-thenoylamino)ethyl]-4[1-(7- methoxynaphtyl)]piperazine) and ipsapirone in a rat stroke model and in man by literature meta-analysis. Rats had 60 minutes of middle cerebral artery occlusion (MCAO) and then 7 days of survival. Body temperatures were monitored for 22 hours. Thirty minutes after MCAO, 1 group (n = 9) received bolus of S14671 (.75 mg/kg) and continuous infusion of .06 mg/kg hour(-1) S14671 for 20 hours. Other MCAO rats (n = 7) had bolus of ipsapirone (.75 mg/kg) and continuous infusion of .25 mg/kg hour(-1) ipsapirone for 3 hours. Controls (n = 9; n = 5) received similar amounts of vehicle as bolus and continuous infusion for 20 hours/3 hours. Additional controls of the S14761 effect in MCAO were performed as previously mentioned (n = 10) but with rats kept normothermic by a heating lamp for 22 hours. Finally, a meta-analysis of ipsapirone-induced hypothermia in man was included. Infarct volumes were reduced by 50% in hypothermic rats versus controls (P < .05). S14671 rats kept normothermic did not show infarct reduction (P > .05). The body temperature after stroke was reduced 1.0-3.0°C compared with controls for 20 hours with S14671 treatment and for 6 hours with ipsapirone treatment. In humans, ipsapirone reduced temperature in average with .55 °C ranging between .1-1.4 °C. 5-hydroxytryptamine receptor 1A (5HT(1A)) agonists significantly reduce infarct volumes in MCAO rats primarily because of the hypothermic drug effect. 5HT(1A) agonists may be introduced to reduce body temperatures rapidly and prepare patients for further therapeutic hypothermia.

Neuroprotective effect of guggulipid alone and in combination with aspirin on middle cerebral artery occlusion (MCAO) model of focal cerebral ischemia in rats

This study was designed to test the pre-treatment doses of guggulipid (50 mg/kg), aspirin (100 mg/kg) per orally and co-administration of both drugs for 28 days followed by middle cerebral artery occlusion – a model of focal cerebral ischemia in rats. Middle cerebral artery was occluded for two hours, followed by reperfusion for 22 hours for the induction of focal cerebral ischemia in rats. Neurobehavioral tests like locomotor activity and grip strength tests were performed before sacrificing the animal. After neurobehavioral tests, the animals were sacrificed for the measurement of infarction areas and biochemical estimations in brain. Locomotor activity and grip strength were significantly improved in guggulipid and aspirin pre-treated rats. Guggulipid and aspirin pre-treatment reduced the infarction areas as compared with middle cerebral occluded (MCAO) rats. An elevation of nitrite, thiobarbituric acid reactive substance (TBARS), acetylcholine esterase activity (AchE) and reduction in antioxidant enzymes like superoxide dismutase (SOD), glutathione (GSH) and catalase were observed following MCAO. Pre-treatment with guggulipid and aspirin caused a reduction in TBARS and nitrite levels, AchE, but elevated GSH level, SOD and catalase activities as compared with MCAO rats. The protective effects observed in this study were due to antioxidant, anti-inflammatory and anti-hyperlipidemic properties of guggulipid. The protective effect of guggulipid in cerebral ischemia, that it may have a role in reversing the symptoms and may offer significant neuroprotection in stroke.

Vagus nerve stimulation in ischemic stroke: old wine in a new bottle.

Vagus nerve stimulation (VNS) is currently Food and Drug Administration-approved for treatment of both medically refractory partial-onset seizures and severe, recurrent refractory depression, which has failed to respond to medical interventions. Because of its ability to regulate mechanisms well-studied in neuroscience, such as norepinephrine and serotonin release, the vagus nerve may play an important role in regulating cerebral blood flow, edema, inflammation, glutamate excitotoxicity, and neurotrophic processes. There is strong evidence that these same processes are important in stroke pathophysiology. We reviewed the literature for the role of VNS in improving ischemic stroke outcomes by performing a systematic search for publications in Medline (1966–2014) with keywords “VNS AND stroke” in subject headings and key words with no language restrictions. Of the 73 publications retrieved, we identified 7 studies from 3 different research groups that met our final inclusion criteria of research studies addressing the role of VNS in ischemic stroke. Results from these studies suggest that VNS has promising efficacy in reducing stroke volume and attenuating neurological deficits in ischemic stroke models. Given the lack of success in Phase III trials for stroke neuroprotection, it is important to develop new therapies targeting different neuroprotective pathways. Further studies of the possible role of VNS, through normally physiologically active mechanisms, in ischemic stroke therapeutics should be conducted in both animal models and clinical studies. In addition, recent advent of a non-invasive, transcutaneous VNS could provide the potential for easier clinical translation.

Thioredoxin-interacting protein: a novel target for neuroprotection in experimental thromboembolic stroke in mice.

Redox imbalance in the brain significantly contributes to ischemic stroke pathogenesis, but antioxidant therapies have failed in clinical trials. Activation of endogenous defense mechanisms may provide better protection against stroke-induced oxidative injury. TXNIP (thioredoxin-interacting protein) is an endogenous inhibitor of thioredoxin (TRX), a key antioxidant system. We hypothesize that TXNIP inhibition attenuates redox imbalance and inflammation and provides protection against a clinically relevant model of embolic stroke. Male TXNIP-knockout (TKO), wild-type (WT), and WT mice treated with a pharmacological inhibitor of TXNIP, resveratrol (RES; 5 mg/kg body weight), were subjected to embolic middle cerebral artery occlusion (eMCAO). Behavior outcomes were monitored using neurological deficits score and grip strength meter at 24 h after eMCAO. Expression of oxidative, inflammatory, and apoptotic markers was analyzed by Western blot, immunohistochemistry, and slot blot at 24 h post-eMCAO. Our result showed that ischemic injury increases TXNIP in WT mice and that RES inhibits TXNIP expression and protects the brain against ischemic damage. TKO and RES-treated mice exhibited a 39.26 and 41.11 % decrease in infarct size and improved neurological score and grip strength compared to WT mice after eMCAO. Furthermore, the levels of TRX, nitrotyrosine, NOD-like receptor protein (NLRP3), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and activations of caspase-1, caspase-3, and poly-ADP-ribose polymerase (PARP) were significantly (P < 0.05) attenuated in TKO and RES-treated mice. The present study suggests that TXNIP is contributing to acute ischemic stroke through redox imbalance and inflammasome activation and inhibition of TXNIP may provide a new target for therapeutic interventions. This study also affirms the importance of the antioxidant effect of RES on the TRX/TXNIP system.

Identification of isoxsuprine hydrochloride as a neuroprotectant in ischemic stroke through cell-based high-throughput screening.

Stroke is a leading cause of death and disability and treatment options are limited. A promising approach to accelerate the development of new therapeutics is the use of high-throughput screening of chemical libraries. Using a cell-based high-throughput oxygen-glucose deprivation (OGD) model, we evaluated 1,200 small molecules for repurposed application in stroke therapy. Isoxsuprine hydrochloride was identified as a potent neuroprotective compound in primary neurons exposed to OGD. Isoxsuprine, a β2-adrenergic agonist and NR2B subtype-selective N-methyl-D-aspartate (NMDA) receptor antagonist, demonstrated no loss of efficacy when administered up to an hour after reoxygenation in an in vitro stroke model. In an animal model of transient focal ischemia, isoxsuprine significantly reduced infarct volume compared to vehicle (137±18 mm3 versus 279±25 mm3, p<0.001). Isoxsuprine, a peripheral vasodilator, was FDA approved for the treatment of cerebrovascular insufficiency and peripheral vascular disease. Our demonstration of the significant and novel neuroprotective action of isoxsuprine hydrochloride in an in vivo stroke model and its history of human use suggest that isoxsuprine may be an ideal candidate for further investigation as a potential stroke therapeutic.

Importance of Preclinical Research in the Development of Neuroprotective Strategies for Ischemic Stroke

Preclinical stroke research has had a remarkably low translational success rate, and the clinical need for novel neuroprotective therapeutics has gone largely unmet, especially in light of the severe underuse of thrombolysis in acute ischemic stroke. In this review, we aim to provide a brief overview of the commonly used stroke models, their merits and shortcomings, and how these have contributed to translational failures. We review some recent developments in preclinical stroke, providing examples of how improved study quality and the use of novel methods can facilitate translation into the clinical setting. This is a narrative review of ischemic stroke neuroprotection based on electronic database searches, references of previous publications, and personal libraries. The stroke research community has not been complacent in its response to criticism: preclinical stroke studies now demonstrate considerable rigor, standardization, and emphasis on minimization of experimenter bias. In addition, numerous innovative methods and strategies are providing novel avenues for investigating neuroprotection, as well as more extensive characterization of established models. The improvements in preclinical stroke models and methods will make stroke research a good example for preclinical medicine, in general, and will hopefully instill greater confidence in the clinical community regarding which compounds are worthy of further investigation in a clinical setting.

Pharmacological hypothermia by Trpv1 agonism provides neuroprotection following ischemic stroke (877.13)

Stroke is the leading cause of death and major cause of long‐term disability. Current therapeutic hypothermia by physical cooling shows the potential for neuroprotection in animal ischemic stroke, but causes complications and often requires sedation and muscle paralysis. The limitations restrict the translational application in clinic. Our previous studies show agonism of transient receptor potential vanilloid 1 (TRPV1) by dihydrocapsaicin (DHC) induces reliable and reversible pharmacological hypothermia (PH). We hypothesize that DHC induced hypothermia provides neuroprotection in ischemic stroke mice.Acute response of HR, BP and cerebral perfusion to the therapeutic dose of DHC were measured. C57BL/6 WT and TRPV1 KO mice were treated with therapeutic doses (1.25, 1.25 mg/kg) and high doses (30, 15 mg/kg) at two consecutive days. After one month recovery, the temperature change response of DHC (1.25 mg/kg) was applied to evaluate the potential TRPV1 desensitization. Focal cerebral I/R (one hour ischemia + 24 hours reperfusion) was induced by distal middle cerebral artery plus common carotid artery transient occlusion in conscious WT and TRPV1 KO mice. PH (8 hours) was initiated 90 minutes after start of reperfusion by DHC infusion (osmotic pump). Neurofunction (by behavioral tests) and infarct volume (by TTC staining) were measured at 24 hours.DHC (1.25 mg/kg) had no measurable effect on HR and cerebral perfusion, did not desensitize TRPV1 channels, but produced a slight transient drop in BP (&lt;6mmHg). In I/R mice, DHC infusion produced mild hypothermia, decreased infarct volume by 87%, and improved neurofunctional score. The hypothermic and neuroprotective effects were absent in TRPV1 KO mice or mice maintained normothermic with heat support.TRPV1 agonism provides an effective and well‐tolerated PH for acute cerebral I/R neuroprotection. Hypothermic and neuroprotective effects are specifically through TRPV1 agonism.Grant Funding Source: Supported by NIH R21 NS077413, R01 HL088435 (SPM) and AHA Pre Doctoral fellowship 16900006 (ZJC).

Hypoxia-Induced Gene Expression of Aquaporin-4, Cyclooxygenase-2 and Hypoxia-Inducible Factor 1α in Rat Cortical Astroglia Is Inhibited by 17β-Estradiol and Progesterone

17β-Estradiol (E₂) and progesterone (P) are neuroprotective in acute brain injury by attenuating neuropathophysiological processes and regulating local glial function. Besides controlling brain-intrinsic immune responses, astrocytes are cellular targets for sex steroids in health and disease and typically resist to hypoxic damage. In this in vitro study, we aimed at uncovering astroglia-specific reactions to sublethal hypoxic conditions and astroglia-specific effects of both sex steroid hormones on these parameters. Short-term hypoxia for 3 h increased reactive oxygen species production, but had no influence on cell viability of cerebral cortical rat astroglia. Astrocytes expressed classical estrogen receptors (ER), progesterone receptor (PR), and a set of nonclassical steroid hormone receptors. Hypoxia specifically induced ERα and PR isoform A gene expression. Oxygen deprivation increased gene expression of aquaporin-4 (AQP4), hypoxia-inducible factor 1α (Hif1α), and cyclooxygenase-2 (COX2). The application of E₂ and P selectively prevented this induction. Effects on protein levels of these genes appeared to be delayed. These data show that astrocytes change their receptivity for sex steroid hormones by switching steroid hormone receptor expression and that E₂ and P modify or antagonize proinflammatory COX2 synthesis, edema-promoting AQP4 expression, and the Hif1α increase. In vivo studies have to address whether these cell responses contribute to steroid-mediated neuroprotection in stroke.

Blood Brain Barrier and Neuroinflammation Are Critical Targets of IGF-1-Mediated Neuroprotection in Stroke for Middle-Aged Female Rats

Ischemia-induced cerebral infarction is more severe in older animals as compared to younger animals, and is associated with reduced availability of insulin-like growth factor (IGF)-1. This study determined the effect of post-stroke IGF-1 treatment, and used microRNA profiling to identify mechanisms underlying IGF-1’s neuroprotective actions. Post-stroke ICV administration of IGF-1 to middle-aged female rats reduced infarct volume by 39% when measured 24h later. MicroRNA analyses of ischemic tissue collected at the early post-stroke phase (4h) indicated that 8 out of 168 disease-related miRNA were significantly downregulated by IGF-1. KEGG pathway analysis implicated these miRNA in PI3K-Akt signaling, cell adhesion/ECM receptor pathways and T-and B-cell signaling. Specific components of these pathways were subsequently analyzed in vehicle and IGF-1 treated middle-aged females. Phospho-Akt was reduced by ischemia at 4h, but elevated by IGF-1 treatment at 24h. IGF-1 induced Akt activation was preceded by a reduction of blood brain barrier permeability at 4h post-stroke and global suppression of cytokines including IL-6, IL-10 and TNF-α. A subset of these cytokines including IL-6 was also suppressed by IGF-1 at 24h post-stroke. These data are the first to show that the temporal and mechanistic components of post-stroke IGF-1 treatment in older animals, and that cellular components of the blood brain barrier may serve as critical targets of IGF-1 in the aging brain.

Analysis of early phase and subsequent phase III stroke studies of neuroprotectants: outcomes and predictors for success

BackgroundEfficacy of neuroprotective treatments for ischemic stroke was not convincingly demonstrated in clinical phase III trials so far, whereas some preceding early phase studies found neuroprotection to be beneficial. We aimed to determine the frequency with which phase III studies are preceded by positive early phase studies, and to identify characteristics of early phase studies that are associated with correct prediction of phase III studies.MethodsWe identified phase III studies and corresponding early phase studies of neuroprotective treatments for stroke. Data on study characteristics of early phase trials were extracted and compared between studies that were classified according to their results as “false positive” and “true neutral” using logistic regression analysis.ResultsForty-six phase III studies and 59 corresponding early phase studies were identified. Only one phase III study was positive and this study was followed by a larger negative study. Twenty-two (37.3%) early phase studies were considered to be false positive and 37 (62.7%) to be true neutral. None of the early phase study characteristics were significantly associated with correct prediction of phase III studies.ConclusionsMore than one third of early phase studies on neuroprotective stroke treatments are false positive. Neither the results nor specific study design characteristics of early phase stroke studies reliably predict success in phase III trials. Further efforts are needed to improve early phase studies regarding its predictability and to identify those early studies that should be advanced to phase III trials.

Abstract T P223: Role of GSK-3ß and Transforming Growth Factor-ß-activated Kinase in Ischemic Neuroprotection

Objectives: Glycogen synthase kinase-3β (GSK-3β) is involved in several essential intracellular signaling pathways involved in both cell survival and cell death. GSK-3β activation promotes inflammation and apoptosis which are detrimental in stroke. GSK-3β inhibition enhances cognition in many animal models; however the role of GSK-3β in stroke and the mechanism by which activation is detrimental remains to be elucidated. It was recently found that GSK inhibits the function of the master energy sensor AMP-activated kinase (AMPK). We have also recently shown that TAK1, an upstream kinase that activates AMPK, is also involved in regulating ischemic injury. Inhibition of TAK leads to dramatic protection, even when treatment is delayed, and this effect is independent of AMPK signaling. We hypothesize that TAK1 regulates GSK-3β. The aim of this study was to determine whether GSK-3β inhibition confers ischemic neuroprotection via TAK1 signaling. Methods: C57BL/6 male mice were randomly assigned to treatment groups and subjected to right middle cerebral artery occlusion The GSK-3β inhibitor VIII (4 mg/kg); and/or the TAK1 inhibitor, 5Z-7-oxozeaenol (5 mg/kg) were administered i.p. either immediately or 2h after stroke. Control mice were treated vehicle. Western blotting was performed for on brains obtained 6h after stroke. Data were presented as mean ± SEM. Results: GSK-3β inhibition was protective when delivered immediately after stroke (Total hemisphere: 44.2±3.2 vs 36.2±3.1; n=8, p&lt;0.05) or if treatment was delayed for 2h (Total: 46.2±3.0 vs 32.2±4.3; n=7, p&lt;0.05). Beneficial effects of GSK-3β inhibition in NDS (p&lt;0.05) was also seen. TAK inhibitor reduced pTAK1 in parallel to GSK-3β inhibition. But, GSK-3β inhibitor treated brains has no effect on pTAK1 levels p&gt;0.05. Finally, compared to GSK-3β inhibition alone, simultaneous GSK-3β+TAK1 inhibition did not offer any additional neuroprotection in stroke (Total: 33.6±2.8 vs 30.7±3.9; p&gt;0.05). Conclusion: Our findings indicate that GSK-3β inhibition is protective even with delayed administration after stroke. In this study, we identified TAK1 as a potential upstream regulator of GSK-3β. Directly targeting TAK1 and GSK-3β inhibition may develop into a novel and powerful neuroprotective strategy.

Abstract T P213: Post-stroke Administration of Ethanol and Normobaric Oxygen Preserves Oxidative Metabolism by Modulating Pyruvate Dehydrogenase Complex in Transient and Permanent Ischemic Stroke

Background: Ischemic stroke induces metabolic disarray where the entire framework of cellular metabolism is altered. A central regulatory site, Pyruvate Dehydrogenase Complex (PDHC) sits at the cross-roads of two fundamental metabolic systems: the citric acid cycle and glycolysis. Down regulation of PDHC has been proposed to explain impaired brain catabolism after ischemic injury. In this study, we combined ethanol and normobaric oxygen (NBO) to develop a powerful non-drug treatment to modulate PDHC and its regulatory proteins, namely PDPhosphatase and PDKinase, leading to improved metabolism and reduced oxidative damage. Methods: Sprague-Dawley rats were subjected to transient (4h) or permanent (24h) middle cerebral artery occlusion followed by 24 hours reperfusion or no reperfusion. Rats received IP injections of saline, two doses of Ethanol (EtOH, 1.5 at onset of reperfusion followed by 1.0 g/kg in 2 hours) for transient stroke or EtOH+95%NBO (for 6 hours) for permanent stroke. Infract volume and the protein levels of PDHC, PDK, and PDP were all assessed. Oxidative metabolism was determined by ADP/ATP ratios and ROS levels. Results: Our therapy enhanced the oxidative metabolism profile of the cell by decreasing ADP/ATP ratios and ROS levels. In transient ischemia, administration of EtOH significantly raised the levels of PDHC and PDP in the ischemic brain issue and simultaneously decreased PDK levels. NBO combined with EtOH synergistically had better outcomes in the permanent ischemic stroke group, as compared to EtOH or NBO alone. Conclusion: EtOH and NBO treatment confirms neuroprotection in stroke by affecting brain catabolism. It modulates the damaging cascade of events by bringing the concentration of active PDHC activity back to metabolic levels.

Abstract T P204: Thioredoxin Interacting Protein: A Novel Target for Neuroprotection in Experimental Thromboembolic Stroke in Mice

Background &amp; Objective: Redox imbalance in the brain significantly contributes to ischemic stroke pathogenesis but antioxidant therapies have failed in clinical trials. Activation of endogenous antioxidant defense mechanisms may provide better protection against stroke-induced oxidative injury. TXNIP (thioredoxin-interacting protein) is an endogenous inhibitor of thioredoxin (TRX), a key antioxidant system. We tested the hypothesis that inhibition of TXNIP is neuroprotective in a clinically relevant model of embolic stroke. Methods: We determined the contribution of TXNIP inhibition to neuronal injury and functional outcome after embolic middle cerebral artery occlusion (eMCAO) using male TXNIP-knockout (TKO), wild-type (WT) and WT mice treated with a pharmacological inhibitor of TXNIP, resveratrol (RES; 5mg/kg body weight). Results: TKO and RES-treated mice exhibited 45.2% and 52.4% decrease in infarct size and improved neurological score and grip strength compared to WT mice after eMCAO. Moreover, the levels of nitrotyrosine, a marker of oxidative damage increased significantly after eMCAO, indicating oxidative brain injury. TXNIP inhibition reduced oxidative marker generation. Furthermore, TXNIP was involved in ischemic brain damage, as less TXNIP expression was found in the RES-treated mice following eMCAO. Conclusions: The present study suggests that unregulated TXNIP is contributes to acute ischemic stroke, and that inhibition of TXNIP may provide a new target for therapeutic interventions. This study also affirms the importance of the antioxidant effect of RES on the TRX/TXNIP system. Table 1 . Summary of experimental stroke data presented as mean ±SE. # p&lt;0.05 * p&lt;0.05 vs. respective control.

Abstract W P196: Post-Stroke Activation of Angiotensin Converting Enzyme 2 is Neuroprotective

Background: Recent discoveries point to the angiotensin converting enzyme 2/angiotensin-(1-7)/mas (ACE2-Ang-(1-7)-Mas) axis as a potential target for neuroprotective stroke therapies. Central pre-stroke activation of this axis by infusion of diminazene aceturate (DIZE), an activator of ACE2, improves stroke outcomes in rats. This led us to hypothesize that intraperitoneal (IP) post-stroke injections of DIZE may exert similar Mas-mediated neuroprotection and thus offer additional clinically-relevant evidence to support this approach. We also assessed the direct effects of DIZE on ACE2 activity in brain tissue. Methods: Male SD rats underwent ischemic stroke by endothelin-1-induced middle cerebral artery occlusion and were randomly divided into groups (n=~12/group) that received IP injections of dH2O or DIZE (0.75, 2.5, or 7.5 mg/kg) at 4, 24, and 48 h after stroke, as well as blinded neurological assessments at 4, 24, and 72 h after stroke. Additional groups received pre- and post-stroke intracerebroventricular (ICV) infusion of sterile 0.9% saline or Mas receptor antagonist A-779 in addition to post-stroke control or drug therapy. Immediately after the 72 h tests, animals were euthanized, cerebral infarct size assessed by TTC staining, and tissue harvested for ACE2 activity assay. Data are expressed as mean ± SEM with significance inferred at p&lt;0.05. Results: Mean infarct sizes (%) were significantly decreased by post-stroke IP injections of all doses of DIZE (0.75 mg/kg: 29.9±5.0; 2.5 mg/kg: 27.9±4.2; 7.5 mg/kg: 31.36±4.1) vs. dH2O (41.9±3.8). At 24 h post-stroke, neurologic deficits (Garcia and Bederson scales) were significantly improved in DIZE-treated rats (7.5 mg/kg: 16.7±0.2 and 0.5±0.1) vs. controls (15.5±0.4 and 1.1±0.2). ICV infusion of A-779 in DIZE-treated rats abolished the improvement in infarct size observed in saline infused DIZE-treated rats (A-779: 43.8%±7.5; saline: 25.8±6.7). Addition of DIZE (4 nmol/μg protein) to brain homogenate increased ACE2 activity by more than 3-fold. Conclusions: This suggests that targeting the ACE2/Ang-(1-7)/Mas axis by post-stroke DIZE injections exerts neuroprotection in a Mas-dependent fashion.

Abstract T MP52: Treatment Times are Reduced by Prehospital Initiation of Neuroprotective Stroke Therapy

Background: In a typical stroke, every minute the brain loses 1.9 million neurons. An increasing focus of stroke treatment is early initiation. Paramedic initiation of stroke therapy can improve the time from symptom onset to treatment in patients suffering an acute stroke. We sought to characterize the time of prehospital therapy initiation relative to symptom onset, paramedic on-scene arrival and emergency department arrival. Methods: All subjects were enrolled in the Field Administration of Stroke Therapy- Magnesium (FAST-MAG) clinical trial, a phase 3 trial of prehospital treatment with magnesium or placebo for patients with a symptom onset less than two hours. Subjects were assessed in the field by paramedics and provided consent via cellular contact with study investigator-physician prior to treatment. Results: The 1700 patients enrolled in the FAST-MAG clinical trial, had a mean age of 69 [SD 14] years, 57% were male, 23% were of Hispanic ethnicity, 78% White race, 78% had pre- existing hypertension, 22% with diabetes, and 49% with hyperlipidemia. They called 911 a median [IQR] of 16 [8-36] minutes, and were assessed by on-scene paramedics 24 [15-43] minutes after symptom onset. Paramedics initiated treatment 23 [18-28] minutes after they were on-scene and 10 [5-14] minutes prior to ED arrival. Initial imaging was obtained 28 [19-40] minutes after arrival to the ED. The treatment initiation was 43 [33-58] minutes after symptom onset. Conclusion: Pre- hospital initiation of the stroke therapy achieved a time to treatment within 60 minutes in over 75% of cases and led to the earliest recorded treatment trials in a stroke study. The strategy of prehospital initiation is an effective means in reducing time from symptom onset to treatment.