The goal of this project is to define the mechanisms of excitatory neurotransmitter mediated neuronal death during hypothermic circulatory arrest (HCA) and to develop a pharmacologic strategy to prevent it. We established a canine survival model of HCA that closely simulates clinical practice in cardiovascular surgery. Dogs subjected to 2 hours of circulatory arrest at a brain temperature of 18 degrees C suffer a consistent neurologic deficit and histopathologic pattern of selective neuronal necrosis and changes in neurotransmitter specific receptors. In the current period of the grant, we found that the NMDA antagonist, dizocilpine, prevented most of the injury when given before and over 20 hours after HCA. The AMPA antagonist NBQX was also protective even though it was administrated after rewarming from HCA at a time when electroencephalographic (EEG) changes associated with neuronal death had developed. These observations indicate the majority of glutamate mediated damage from HCA evolves in the reperfusion period and that the events mediating damage can be blocked with medications. Preliminary experiments utilizing in vivo brain microdialysis indicate that large extracellular increases in the excitatory amino acid neurotransmitters glutamate and glycine precede a 12-fold rise in the conversion of arginine to citrulline by nitric oxide synthase (NOS) during the postoperative reperfusion period. Using immunocytochemistry, we observed that the expression of neuronal NOS (nNOS) activity increases substantially after 6-18 hours of reperfusion in areas of the brain enriched in glutamate receptors such as the hippocampus, basal ganglia and cerebellum. Concomitant with this increased enzymatic activity is an accumulation of nitric oxide (NO) metabolites in the serum and urine that are effectively reduced by NOS inhibitors. Preliminary data also suggest that neuronal death following HCA occurs due to both apoptosis and necrosis during the period when nNOS is activated. NO may be directly responsible for such programmed cell death since we can reliably inhibit the apoptosis with NOS inhibitors. To explore the cascade of events that mediate brain injury following HCA we propose to: (1) determine the pattern and sequence of neurotransmitter release as well as the regulation of neurotransmitter receptor expression; (2) examine the role of NO in mediating neurotoxicity after HCA; (3) determine the relationship between excitatory neurotransmitters and NO activity; (4) examine the occurrence of both apoptosis and necrosis following HCA and the inhabitation of cell death with NOS inhibitors; (5) determine how and when manipulating NO production affects the neurologic outcome of dogs. The proposed research will expedite the clinical use of glutamate receptor antagonists, and may develop a novel therapeutic approach to neuroprotection based on the use of isoform specific NOS inhibitors. The ability to provide cerebral protection could result in better patient outcomes after circulatory arrest during cardiothoracic procedures.
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