1985 — 1990 |
Fiskum, Gary |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Transport-Regulated Calcium Metabolism in Tumor Cells @ George Washington University
The long-term objective is to identify the alterations of Ca2+ homeostasis in hepatoma cells and characterize the biochemical basis for these alterations. The approach to this problem is based on the hypothesis that the regulation of intracellular Ca2+ and (or) the metabolic sensitivity to Ca2+ in tumor cells is modified so that cell survival and proliferation are promoted even under adverse environmental conditions. The specific aims of this study are to: 1. Accurately compare the cytosolic free Ca2+ concentration of rat hepatoma cells and rat hepatocytes under normal and metabolically-stressful conditions; 2. Compare the influence of extramitochondrial or cytosolic free Ca2+ on the activity of Ca2+- sensitive dehydrogenases and the TCA cycle in mitochondria from normal liver and hepatoma cells; 3. Test the possibility that abnormally high levels of membrane cholesterol are responsible for the abnormal Ca2+ buffering properties of hepatoma mitochondria; 4. Further characterize the difference in the response of rat liver and hepatoma microsomes to release of Ca2+ induced by inositol trisphosphate; 5. Compare the effects of transient elevation of intracellular Ca2+ and depletion of ATP on the respiratory and Ca2+-buffering characteristics of mitochondria and the Ca2+ transport activities of other Ca2+-sequestering organelles. The methods of approach will include the use of digitonin cell permeabilization, fluorescent Ca2+ indicators, O2 and Ca2+ electrode measurements, dual beam and dual wavelength spectrophotometry, steady-state measurements of 14CO2 ratios using metabolites labeled at different carbons, and TLC-flame ionization determinations of membrane phospholipids. The significance of this work is that: a. It will help explain how tumor cells are adapted for survival in harsh environments; b. It will describe the relationships between Ca2+ homeostasis and energy metabolism in tumor cells; c. It relates to recent evidence that Ca2+ homeostasis is linked to the activity of oncogene products via turnover of phosphatidylinositol; d. It may ultimately lead to improved modes of cancer chemotherapy based on manipulations of cellular Ca2+ metabolism.
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0.936 |
1995 — 1998 |
Fiskum, Gary |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Mechanisms--Ischemia/Reperfusion Brain Injury @ University of Maryland Baltimore
DESCRIPTION: (Adapted from investigator's abstract) Free radical- mediated damage to lipids and proteins, altered mitochondrial electron transport chain activities and abnormal patterns of tissue metabolism are strongly implicated in the pathophysiology of neurological morbidity caused by global cerebral ischemia and reperfusion. The overall objective of this project is to characterize the relationships between these factors and to relate them to the neurological impairment observed with animals subjected to a clinically relevant scenario consisting of cardiac arrest followed by 30 minutes to 24 hours of restoration of spontaneous circulation. The specific aims of these studies are to test the following hypotheses: (1) A short period of global ischemia causes immediate and reversible alterations in mitochondrial respiratory and Ca2+ uptake activities that are distinguishable from delayed, reperfusion-induced alterations. (2) Free radical damage due to cerebral ischemia/reperfusion adversely affects proteins as well as lipids present within mitochondria and other cellular compartments and membranes. (3) Mitochondrial membrane damage and enzyme inactivation result in immediate and delayed alterations in cerebral energy metabolism following cardiac arrest and ROSC. (4) Clinically-feasible manipulations of inspired and brain 02 concentrations alter the degree of molecular, subcellular, and metabolic injury, corresponding to differences in the extent of neurological injury observed following these different resuscitation protocols. Methods of approach to these aims will include measurements of 02 consumption, peroxide formation and Ca2+ uptake by isolated brain mitochondria and digitonin-permeabilized synaptoneurosomes and PC12 pheochromocytoma cells; determinations of lipid and protein oxidation, measurements of phospholipid and free fatty acid moieties and determinations of metabolic activities associated with glycolysis and the TCA cycle. Additional support for involvement of specific subcellular and metabolic alterations in the pathogenesis of ischemia/reperfusion brain injury will come from comparison of results obtained with different animal treatment protocols as well as with neurochemical measurements and histological testes. In vitro modeling of cellular and subcellular injury will also assist in the elucidation of cause and effect relationships. The significance of these studies is that they will provide unique molecular insight into the roles that altered bioenergetics and free-radical-metabolism play in brain injury associated with cardiac arrest and resuscitation as well as other forms of cerebral ischemia including stroke and trauma.
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1 |
1999 — 2003 |
Fiskum, Gary |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Mechanisms Ischemia Reperfusion Brain Injury @ University of Maryland Baltimore
The overall goal of this ongoing project is to gain a more complete understanding of the roles that mitochondrial dysfunction and oxidative molecular alterations play in ischemia/reperfusion brain injury. The specific aims are designed to amplify the most important conclusions made during the last few years and to test the following hypotheses using a combination of a clinically relevant animal model of transient global ischemia, neuronal and astrocytic cell culture models of hypoxic and excitotoxic delayed cell death, and subcellular models of mitochondrial stress. 1. Brain mitochondria release the apoptosis factor cytochrome c in response to elevated Ca2+, oxidative stress, and the presence of specific cell death proteins by membrane permeability transition independent and dependent mechanisms that can be controlled by unique neuroprotective agents. 2. Oxidative alterations to mitochondrial proteins and lipids is a common pathway by which elevated intracellular Ca2+, reactive oxygen species and metabolic derangements cause mitochondrial functional alterations during both acute ischemia and during reperfusion. 3. Early reperfusion-dependent loss of pyruvate dehydrogenase is a sensitive marker of selective neuronal vulnerability to oxidative stress and delayed cell death. 4. Delayed, post-ischemic hyperbaric oxygen therapy reduces oxidative injury and death in selectively vulnerable neurons through increasing expression of antioxidant defense mechanisms present in mitochondria and in other cellular compartments. The significance of these studies is that they will define the molecular mechanisms by which mitochondria are injured during cerebral ischemia and reperfusion, they will explicate the modes by which mitochondrial dysfunction promotes neural cell death, they will help identify novel targets for neuroprotection, and they will further test the neuroprotective potential of both pre- and postischemic hyperbaric oxygen therapy.
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1 |
2002 — 2003 |
Fiskum, Gary |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Novel Mechanisms of Mitochondrial Free Radial Generation @ University of Maryland Baltimore
DESCRIPTION (provided by applicant) Although evidence suggests that mitochondrial dysfunction stimulates the production of reactive oxygen species (ROS) that trigger dopaminergic cell death in Parkinson's disease (PD) the molecular mechanisms responsible for mitochondrial ROS production are unknown. We have recently discovered that the multi-subunit enzyme alpha-ketoglutarate dehydrogenase (alpha-KGDC) is a substantial source of ROS production in brain mitochondria. The activity and immunoreactivity of this protein has been shown to be altered in neurons and in the brains of animals treated with MPP' and in the substantia nigra of patients with PD. We hypothesize that dysregulation in the intramolecular electron transfer within the subunits of alpha-KGDC is a primary mediator of oxidative stress associated with PD and to ROS-mediated neuronal cell death. The specific aims of this exploratory project are: 1. Quantify the contribution of alpha-KGDC to ROS production in isolated brain mitochondria in the absence and presence of PD-associated neurotoxins. We will compare the ROS production by alpha-KGDC, other mitochondrial dehydrogenases and electron transport chain Complex I. We will also determine if the ROS generated by alpha-KGDC and Complex I interact to decrease normal enzyme activity while increasing production of ROS. 2. Explore possible chemical mechanisms of ROS production by different enzyme subunits of the alpha-KGDC. 3. Develop a cell culture model for assessing the contribution of alpha-KGDC to oxidative stress and the interactions of alpha-KGDC and Complex I in the absence and presence of neurotoxins. We will measure the effects of MPP' in the absence and presence of high extracellular alpha-ketoglutarate and alpha-KGDC inhibitors on markers of protein and DNA oxidation. The effects of different culture conditions on alpha-KGDC and Complex I enzyme activities and on H2O2 production will be measured using mitochondria isolated from these cells. This project will lay the foundation for the molecular etiology of cell death in PD which could be activated by genetic and (or) environmental determinants. Verification of the role of alpha-KGDC in mitochondrial ROS generation and in oxidative cell death could lead to the development of genetic animal models of susceptibility to PD disease leading to the development of targeted neuroprotective interventions that will minimize the incidence or slow the progression of Parkinson's disease.
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1 |
2003 — 2004 |
Fiskum, Gary |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Novel Delivery of Bcl-2 For Neuroprotection @ University of Maryland Baltimore
DESCRIPTION (provided by applicant): The overall goal of this exploratory project is to develop a novel strategy for in vivo neuroprotection based on the new concept of protein transduction. This model will be used to deliver full-length, anti-death Bcl-2 proteins into the brain to test the hypothesis that postischemic neuroprotection by Bcl-2 can be achieved by a realistic method for delivering exogenous protein into brain cells. A second objective is to elucidate the contribution of the anti-oxidant vs. anti-Bax mechanisms in different neural cell death paradigms and to investigate the specific role of phosphorylation in regulating these mechanisms and in neuroprotection. The specific aims of the project are to; 1) Test the hypothesis that the anti-death activity of Bcl-2 is inhibited by post-translational mechanisms activated in response to chemical hypoxia and glucose deprivation in vitro. 2) Determine the role of phosphoQrlation in regulating the mechanisms by which Bcl-2 protects against mitochondrial dysfunction caused by Ce plus oxidative stress compared to the interaction of proapoptotic proteins, i.e., Bax plus BH3 death domain only protein. 3) Test the hypothesis that delivery of full-length Bcl-2, as a TAT-Bcl-2 fusion protein, into the brain is protective in a rat transient focal cerebral ischemia model. The methods of approach to these aims will utilize cloning of both the normal Bcl-2 gene and the gene with mutations in specific phosphorylation sites, each ligated to the TAT protein transduction domain. TATBcl-2 fusion proteins will be isolated and tested for their ability to inhibit hypoxic neural cell death in vitro and to reduce cerebral infarct volume in a rat reversible focal ischemia model. The effects of exogenous Bcl-2 constructs on mitochondrial dysfunction will also be assessed using measures of mitochondrial cvtochrome c release, membrane potential, redox potential, and reactive 07 species production. The influence of phosphorylation state on the ability of endogenous and exogenous Bcl-2 to protect against mitochondrial dysfunction and cell death will also be investigated. The significance of these studies is 1. They could establish the foundation for a novel neuroprotective treatment strategy targeting both necrotic and apoptotic cell death and, 2. They will provide completely new insight into the molecular mechanisms by which the antideath activities of proteins like Bcl-2 are regulated under pathological conditions.
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1 |
2004 — 2013 |
Fiskum, Gary M |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Mitochondrial Mechanisms of Hypoxic Ischemic Neonatal Brain Injury @ University of Maryland Baltimore
The pathophysiology of hypoxia-induced injury to the immature brain is not well understood but is linked to a vicious cycle between oxidative stress and mitochondrial dysfunction. In an effort to promote aerobic energy metabolism, neonates often receive hyperoxic ventilation following global cerebral hypoxia or ischemia. We have found that hyperoxic reoxygenation promotes oxidative stress, damages metabolic enzymes, and worsens energy metabolism and neurologic outcome. These studies have uncovered mechanisms of metabolic failure, including impaired pyruvate dehydrogenase complex (PDHC) activity and loss of mitochondrial NAD(H). Preliminary evidence also indicates that combination therapeutic approaches are available that both ameliorate the metabolic abnormalities and improve outcome. These approaches include genomic post-conditioning by administration of agents, e.g., sulforaphane (SFP), which stimulate the Nr^ mediated transcriptional activation of Phase 2 response, antioxidant and anti-inflammatory genes. We therefore hypothesize that optimal neuroprotection following neonatal cerebral hypoxic ischemia (HII) can be achieved by eariy minimization of oxidative stress and optimization of mitochondrial energy metabolism through the combined interventions of avoiding unnecessary hyperoxia and post-administration of SFP. There are two main aims for this grant period: 1. Determine If both early and delayed oxidative stress, neuronal death, and neurologic Impairment after HII are alleviated by the individual and combined approaches of normoxic re-oxygenatlon and sulforaphane administration. 2. Identify the mechanisms by which mitochondrial metabolic dysfunction contributes to neurodegeneration after neonatal HII, and determine how they are Influenced by gender, levels of ambient [Ozj and activation of the Nrt2-regulated pathway of gene expression. Methods of approach to these aims include the use of male and female rats and Nrf2 +/+ and -/- mice, behavioral outcomes, stereologic histopathology, immunohistochemistry, measurements of mitochondrial proteins and bioenergetic activities, and exposure of cultured Nrf2 +/+ and - /-neurons to transient O2 and glucose deprivation (OGD), and unique quantitative measures of neuronal respiration and glycolysis. The use of both animal and cellular models of H/l in collaboration with Projects 2 and 3 will allow us to elucidate mitochondrial mechanisms of ischemic neuronal dysfunction and death and the molecular pathways through which normoxic re-oxygenation and genomic post-conditioning by SFP reduce neurodegeneration in the immature brain.
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1 |
2004 — 2006 |
Fiskum, Gary |
U01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Neuroprotection After Cardiac Arrest @ University of Maryland Baltimore
DESCRIPTION (provided by applicant):Comparisons of pre-lethal neurochemical alterations to neurologic outcome and neuropathology following cardiac arrest (CA) and resuscitation in animals using hyperoxic and normoxic ventilation strongly implicate early, oxidative modification to mitochrondial proteins and associated metabolic dysfunction in the etiology of delayed neural cell death and permanent brain injury. To expedite progress in improving neurologic outcome following human CA, we propose a change in standard resuscitative protocols that dramatically lowers the concentration of inspired 02 (Fi02) to a level that sufficiently oxygenates the brain and other tissues but that minimizes oxidative stress, cell death, and neurologic impairment. Our specific aims test the hypothesis that neuronal cell death and both short- and long-term neurologic impairment following CA and resuscitation are minimized by maintaining normal post-ischemic PaO2, as guided by oximetry-based measurements of hemoglobin 02 saturation (SpO2) that are practical for use in the field. Aim 1. Compare neurologic outcome following canine cardiac arrest and resuscitation using:* Hyperoxic Resuscitation - 100% FiO2 for 1 hr then adjust FiO2 to maintain normal PaO2 * Normoxic Resuscitation - 21% FiO2 for 1 hr then adjust FiO2 to maintain normal PaO2 * Oximetry-Based Resuscitation - Adjust FiO2 to maintain SpO2 at 94% to 96% *Aim 2. Establish relationships between resuscitative FiO2 and neuronal cell death using the canine model and three resuscitation protocols. Aim 3. Extend the results obtained with the short-term canine CA model using a transient global cerebral ischemia model with long-term outcome measures performed on mature and aged rats. We will compare both neurologic outcome and neuronal cell death using different protocols in a clinically very relevant canine model of cardiac arrest. In addition, a rat model of transient global cerebral ischemia is utilized to measure long-term outcome and to test different protocols on aged animals, representing the typical CA patient age-group. Within the first two - three years of this project, our results could be used to design clinical trials to improve neurologic outcome following cardiac arrest and resuscitation. With over 250,000 cardiac arrest victims resuscitated each year in the U.S. alone, and over half of these suffering from neurological morbidity and mortality, the potential impact of the proposed translational research project is highly significant.
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1 |
2005 — 2007 |
Fiskum, Gary |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Mechanisms of Ischemia Reperfusion Brain Injur @ University of Maryland Baltimore
DESCRIPTION (provided by applicant): Comparisons of pre-lethal neurochemical alterations to neurologic outcome and neuropathology following cardiac arrest (CA) and resuscitation using hyperoxic and normoxic ventilation strongly implicate oxidative modification to mitochondrial proteins and associated bioenergetic dysfunction in the etiology of delayed, selective neural cell death. These findings also question the indiscriminate use of 100% ventilatory O2 (FiO2) with patients following CA and suggest that modification of existing resuscitation guidelines may significantly improve neurologic outcome. Our primary goal is to reduce neurologic morbidity and mortality following CA through by minimizing oxidative stress and maximizing cerebral energy metabolism. Our specific aims are to test the following hypotheses focusing on mitochondrial mechanisms of oxidative brain injury in young and aged animals, and on optimizing neurologic outcome using oximetry-based adjustments to FiO2 that are practical for use in out-of-hospital CA. 1. Oxidative brain injury and neurologic impairment following cerebral ischemia are minimized by maintaining postischemic hemoglobin O2 saturation at 94 - 98%. 2. Post-resuscitative cerebral hyper-oxygenation worsens neurologic and histopathologic outcome as a consequence of impaired cerebral energy metabolism, delayed neuronal Ca2+ dysregulation, and exacerbated expression and subcellular redistribution of pro-apoptotic proteins. 3. Neuronal survival following in vitro hypoxia and re-oxygenation is optimized using moderate post-hypoxic oxygenation, due to reduced oxidative stress-mediated mitochondrial dysfunction. 4. Aged animals are sensitive to exacerbation of oxidative stress, cell death, and neurologic impairment by post-resuscitative hyper-oxygenation. Methods of approach include the use of mature and aged animals in models of global cerebral ischemia, models of cell death using primary neuronal cultures, measurements of mitochondrial Ca2+ transport, membrane potential, and production of reactive O2 species with brain mitochondria and neurons, immunohistochemical and immunoblot analysis of changes in nitrotyrosine and the levels and intracellular distribution of metabolic and apoptotic proteins, 13C NMR spectroscopic analysis of altered metabolism in the post-ischemic brain and in primary cultures of neurons exposed to stress, and short- and long-term tests of neurologic impairment.
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1 |
2007 — 2008 |
Fiskum, Gary |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Mechanisms of Ischemia Reperfusion Brain Injury @ University of Maryland Baltimore
Comparisons of pre-lethal neurochemical alterations to neurologic outcome and neuropathology following cardiac arrest (CA) and resuscitation using hyperoxic and normoxic ventilation strongly implicate oxidative modification to mitochondrial proteins and associated bioenergetic dysfunction in the etiology of delayed, selective neural cell death. These findings also question the indiscriminate use of 100% ventilatory O2 (FiO2) with patients following CA and suggest that modification of existing resuscitation guidelines may significantly improve neurologic outcome. Our primary goal is to reduce neurologic morbidity and mortality following CA through by minimizing oxidative stress and maximizing cerebral energy metabolism. Our specific aims are to test the following hypotheses focusing on mitochondrial mechanisms of oxidative brain injury in young and aged animals, and on optimizing neurologic outcome using oximetry-based adjustments to FiO2 that are practical for use in out-of-hospital CA. 1. Oxidative brain injury and neurologic impairment following cerebral ischemia are minimized by maintaining postischemic hemoglobin O2 saturation at 94 - 98%. 2. Post-resuscitative cerebral hyper-oxygenation worsens neurologic and histopathologic outcome as a consequence of impaired cerebral energy metabolism, delayed neuronal Ca2* dysregulation, and exacerbated expression and subcellular redistribution of pro-apoptotic proteins. 3. Neuronal survival following in vitro hypoxia and re-oxygenation is optimized using moderate post-hypoxic oxygenation, due to reduced oxidative stress-mediated mitochondrial dysfunction. 4. Aged animals are sensitive to exacerbation of oxidative stress, cell death, and neurologic impairment by post- resuscitative hyper-oxygenation. Methods of approach include the use of mature and aged animals in models of global cerebral ischemia, models of cell death using primary neuronal cultures, measurements of mitochondrial Ca2+ transport, membrane potential, and production of reactive 02 species with brain mitochondria and neurons, immunohistochemical and immunoblot analysis of changes in nitrotyrosine and the levels and intracellular distribution of metabolic and apoptotic proteins, 13C NMR spectroscopic analysis of altered metabolism in the post-ischemic brain and in primary cultures of neurons exposed to stress, and short- and long-term tests of neurologic impairment.
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1 |
2015 — 2019 |
Fiskum, Gary M Rosenthal, Robert Edward (co-PI) [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Optimal Oxygenation and Gene Expression During Critical Care After Cardiac Arrest @ University of Maryland Baltimore
? DESCRIPTION (provided by applicant): Less than 50% of cardiac arrest (CA) survivors exhibit good neurologic outcome, emphasizing the need for new neuroprotective strategies in addition to meticulous management of temperature. Our research performed with a canine model of CA and resuscitation (ROSC) demonstrated neuroprotection with oximetry-guided normoxic resuscitation compared to the previously standard practice of hyperoxic resuscitation. These results contributed to a major change in AHA/ACLS guidelines for CA/ROSC; i.e., minimize ventilatory O2, maintaining hemoglobin oxygen saturation >94%. While these procedures can be safely used in-hospital for CA/ROSC, the risk of hypoxia associated with rapidly lowering inspired O2 makes this paradigm dangerous in pre-hospital resuscitation. In light of these limitations, our primary aim is to determine the level of O2 inspired during the firt 2 hr of critical care in a hospital setting that optimizes neurologic outcome following pre-hospita resuscitation. We hypothesize that in contrast to the benefit of normoxia during early resuscitation, maintenance of moderate hyperoxemia at the period following the initial reperfusion-induced free radical surge, and prior to the onset of inflammation, will improve clinical outcome. Our related, albeit independent secondary aim is to test the hypothesis that inflammation, oxidative stress, and brain mitochondrial dysfunction contribute substantially to post-ischemic brain injury. Comparisons will be made of neurologic, histologic and biochemical outcomes following normoxic, mildly hyperoxic, and severely hyperoxic ventilation and in the absence or presence of sulforaphane-induced expression of cytoprotective genes whose products protect against these injury mechanisms. Methods of approach include use of our highly clinically relevant canine model of CA/ROSC for short-term outcomes, and a rat CA and resuscitation model for long-term outcomes. Additional comparisons between males and females will enhance potential for clinical translation and detect any sexually dimorphic mechanisms of brain injury and responses to different O2 levels or sulforaphane treatment. Translational outcome measures include advanced histopathology and neurobehavioral tests. Mechanistic outcomes include measurements of mitochondrial bioenergetics, cerebral metabolism of 13C-labeled glucose, proton NMR of energy metabolite levels, inflammatory microglial activation, and markers of oxidative stress. Relevance: Results from our studies will provide fresh new insight into the levels of inspired O2 used in a hospital setting that result in best neurologic outcome after out-of-hospital CA/ROSC. These experiments will also determine if treatment with sulforaphane after resuscitation further improves neurologic function, based on stimulated expression of cytoprotective gene products that inhibit oxidative stress, inflammation, and mitochondrial dysfunction. Either approach toward neuroprotection could be safely translated to clinical trials, eventually improving the quality of life experienced by the hundreds of thousands who survive CA each year.
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0.929 |