Glutamate (glu) toxicity accounts for one kind of neuronal injury from stroke, a major health problem that is the third leading cause of death and the most common cause of adult disability. This application proposes mechanism-oriented studies in respiring rat cerebrocortical and hippocampal slices of neuronal/glial energy failure and injury during exogenous glu toxicity and ischemia. In each experiment the time course of NMR metabolite changes are monitored in 20 adult rat brain slices (each 350 mu total wet wgt=3.2 gm). Recent P NMR studies by the P.I. found that intracellular energy failure occurs very soon after glu exposure. However, it can be ameliorated if exogenous glu is discontinue or glu receptor antagonists are give. Intracellular energy failure and Ca2+ are assessed noninvasively using interleaved P/H/F NMR spectroscopy for concurrent determinations of: ATP, PC, Pi pHi, Mg2+, Ca2+, N- acetylaspartate (NAA), and lactate. Extracellular levels of glu are determined from the use of a catheter whose tip is among the slices. Tissue injury is measured by water content determinations from wet/dry weights and light microscopy of sections stained for expression of heat shock protein (HSP72). Histological examinations of neurons and glia are also performed using Cresyl violet [Nissl] staining, and immunocytochemical stains for GFAP, and neuron-specific enolase. NO activity and cGMP levels ar measured using radioimmunoassay.
Five specific aims focus in a unified way on three molecular events in glu toxicity: 1) activation of glu receptors; 2) activation of nitric oxide synthase (NOS); 3) intracellular energy failure. The goal is to link ischemic and glutamatergic energy failure to glu receptor and NOS activation.
Aim 1) significance of energy failure during and after non- ischemic glu toxicity and ischemia. Hypotheses: (a) nonrecoverable intracellular energy failure correlates with decreased NAA, increased lactate, Ca2+, and extracellular glu, and with increased neuronal injury. (b) Hippocampal neurons are more susceptible to injury than cortical neurons.
Aim 2) effects of dizocilpine and NBQX on glutamate-induced and ischemia-induced energy failure. Hypotheses: (a) Blockade of both NMDA and AMPA receptors will reduce increases in Ca2+ and extracellular glu, reduce NMR manifestations of energy failure, and correlate with decreased neuronal injury.
Aim 3) effects of NOS inhibition by two blockers, NOLA and L-NMMA, on glutamate-induced and ischemia-induced energy failure. Hypothesis: Energy failure following NMDA and AMPA receptor activation involves both Ca2+ increases and activation of NOS, a neuronal Ca2+/calmodulin dependent enzyme.
Aim 4) search for synergism between NOS-blockade and antagonism of NMDA-type or AMPA-type receptors. Hypothesis: synergism exists.
Aim 5) effects of fructose-1,6 or AMPA- type receptors. Hypotheses: FBP protects energy levels in glia by glycolytic activation, reduces neuronal injury, and is associated with decreased extracellular glu, possibly because of increased glial uptake.
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