NMR OF FBP PROTECTION IN HYPOXIC RAT BRAIN SLICES

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It has been known for years that pre- and post-treatment with fructose-1,6-bisphosphate (FBP) can dramatically improve hypoxic/ ischemic tolerance in vivo in brain, muscle, and intestinal tissues, suggesting huge potential benefits in high risk childbirth, surgeries where there is major blood flow interruption or total circulatory arrest, and organ transplantation. Primary mechanisms of FBP protection affect intracellular metabolism, which is now easier to explore because of advances in high resolution nuclear magnetic resonance (NMR) spectroscopy. The Specific Aims of 14.1 Tesla ex vivo and in vitro multinuclear NMR spectroscopy studies of neonatal rat brain slices are to determine: 1) if [1-13C]fructose-1,6-bisphosphate enters oxygenated and/or hypoxic cells, and if so, its metabolic fate and influence. 2) if FBP-induces metabolic changes in the intracellular metabolism of glucose, particularly during oxygen deprivation. [U-13C]glucose will be used to distinguish glial from neuronal TCA cycle activity. [2-13C]glucose will probe the activity of the pentose phosphate pathway (PPP). [1-13C]glucose will be used to determine total glucose utilization. 3) if FBP preservation of ATP is secondary to its prevention of glutamate toxicity and/or its prevention of damage from PARS (polyadenosine 5'-diphosphoribose synthetase, also know as PARP.) During hypoxia FBP increases glucose metabolism by the PPP, a source of ribose. Hypoxia studies will be performed with nontoxic glutamate receptor blockade and nontoxic inhibition of glutamata release, and with inhibitors of PARS. 4) if hypoxia-induced changes in ATP are associated with concomitant changes in the apparent diffusion coefficient of brain slice water, ADCw, which is commonly used clinically; increases in brain slice water; histological measures of cell swelling; and immunohistological measures of cell and mitochondrial injury. The hypotheses tested are that: 1) FBP enters cells more readily during hypoxia and serves as a metabolic modulator and substrate; 2) Because of PARS, ATP maintenance by FBP during hypoxia requires increased glucose metabolism by the PPP; 3) apparent intracellular diffusion coefficients can be used to accurately estimate cell swelling and the integrity of intracellular metabolism; and 4) when FBP sustains ATP levels during hypoxia, mitochondrial viability is also sustained.