CYTOKINE-MEDIATED ENHANCEMENT OF NEURONAL INJURY

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DESCRIPTION (Adapted from Applicant's Abstract): The pathophysiology of delayed neuronal death as it occurs hours or even days following cerebral ischemia is poorly understood. Recent study in animal models indicate that cytokine-inducible nitric oxide synthase (iNOS) is strongly induced in astrocytes surrounding areas of extensive neuronal degeneration 1 to 3 days following cerebral ischemic insult. Previous work by Dr Hewett demonstrated that NO derived from cytokine induction of astrocyte iNOS while not toxic alone dramatically increased the magnitude of N-methyl-D-aspartate and oxygen glucose deprivation induced neuronal injury in vitro suggesting that in vivo activation of iNOS could have the dangerous consequence of enhancing excitotoxic neuronal injury. Further it was shown that the potentiation was associated with an increase in extracellular glutamate levels and was dependent on reactive oxygen species as well as NO. Thus, the goal of this project is to elucidate specific cellular and molecular events by which astrocytic NO and reactive oxygen species contribute to the cytokine-mediated enhancement of excitotoxic neuronal injury. Experiments will be performed in vitro in primary cortical cell cultures. Astrocytic iNOS will be induced by exogenous addition of pro-inflammatory cytokines to cultures. Combined oxygen glucose deprivation as well as excitatory amino acid administration will be used in in vitro models of cerebral ischemia. Studies will be designed to answer the following questions: 1. How do NO and reactive oxygen species interact to augment excitotoxicity and what is the cellular source and enzymatic source of reactive oxygen species? 2. How does astrocytic iNOS induction lead to enhancement of extracellular glutamate? Specifically, the ability of cytokine stimulation to alter glutamate e-flux and/or re-uptake will be assessed. The long-term objectives of this study is to better understand the pathways and mechanisms by which inflammatory cytokines contribute to the enhancement of excitotoxic neuronal injury. Improved definition of these events could lead to the development of new therapeutic strategies designed to attenuate the progression of neuronal destruction following stroke.