This application proposes four projects and core facilities to investigate mechanisms of CNS injury. Project 1 Experimental Therapy of Spinal Cord Injury. We recently discovered that cyclosporin A (CsA) reduces responses, releases vasodilatory and inflammatory substances, and modulates neurotransmitter receptors and release. We propose to study the role of calcineurin in spinal cord injury. The experiments will compare the effects of CsA, FK506 (another calcineurin inhibitor), alone and in combination with rapamycin (an antagonist of K506 (another calcineurin inhibitor), alone and in combination with rapamycin (an antagonist of FK506 inhibition of calcineurin), on spinal cord ionic shifts, lesion volumes, motor recovery, blood flow, and serotonin-,GABA- and glutamate-induced spinal excitability and injury. Project 2. Acid-base homeostasis in brain injury. The mechanisms and consequences of pH changes in injured tissues are not well-understood. We will measure pH, PCO2, HCO3, and CO32 in ischemic brains and injured spinal cords, to determine the role of buffer capacity, lactoacidosis, and carbonic anhydrase in the pH changes. We propose that pH shifts modulate NMDA receptors and that interstitial buffering influences the rate and magnitude of spreading depression and ischemic injury in brain slices. Using on-selective microelectrodes, voltage clamp, and spectrophotometric methods, we will test the hypothesis that voltage-dependent Na:HCO3 co-transport governs H+, Na+, and HCO3- distribution across astrocytic membranes in normo- and hyperglycemic ischemia. Project 3. Ascorbate and glutathione in CNS Injury. We hypothesize that ascorbate (Asc) and glutathione (GSH) loss contributes to brain cell vulnerability to oxidative damage. Extracellular Asc will be measured in hypoxic brain slices and in ischemic cerebral cortex after middle cerebral artery occlusion. Intracellular Asc will be estimated from extracellular volumes and total tissue levels. Preliminary studies suggest that Asc and GSH are localized in neurons and astrocytes respectively, that both Asc and GSH decline with age and are lower in females. We will confirm these results, determine whether hypoxia and age compromises Asc homeostasis, assess protective effects of Asc and GSH in hypoxic brain slices, identify mechanisms of Asc and GSH loss in ischemia, and correlate tissue damage with Asc loss in male and females. Project 4. Water compartmentalization in Ischemia. The objective of this project is to understand how water shifts between extra- and intracellular compartments in ischemic brain tissues. Water shifts will be assessed with extracellular tracers, ion-elective microelectrodes, and weighing-drying method. We will manipulate extracellular ionic levels and block ionic transport and glutamate receptors. Water movements will be investigated in slices from aged animals. The role of the Na:HCO3 cotransport will be assessed. Light scattering and Na-K changes will be compared with wet-dry weight and tracer measurements.
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