Despite four decades of research concerning cardiac arrest/cardiopulmonary resuscitation (CPR), clinical outcome remains poor. Much of this research has taken the form of developing new methodologic or pharmacologic approaches to CPR. However, now, novel basic science and molecular genetic approaches to CPR are needed to improve our understanding of critical mechanisms of injury during cardiac arrest/CPR to develop new therapeutic interventions. Poly (ADP-ribose) polymerase (PARP) is an abundant nuclear enzyme which helps maintain genomic integrity in neurons and numerous other cell types. Inhibition of PARP with pharmacologic agents or PARP deficiency via PARP knockout animals reduces injury after focal cerebral ischemia, but there is little information concerning PARP and cardiac arrest/CPR. Our preliminary studies show that cell injury after cardiac arrest/CPR in PARP null mutants (PARP-/-) is markedly reduced relative to wild type mice.
Aim 1 will investigate this apparent neuroprotection observed in PARP deficient mice, characterizing extent and longevity of protection in intact animals over days of the evolving injury. We will use a combination of functional behavioral evaluation over time and terminal histopathology.
Aim 2 will establish the importance of nitric oxide generation to PARP activation in vivo and subsequent injury during cardiac arrest/CPR. The relative consequences of inhibiting PARP vs neuronal or inducible nitric oxide synthase will be examined in the cardiac arrest/CPR mouse model. Lastly, using a strategy of transfecting virus carrying PARP mutant at two distinct catalytic and cleavage sites, we will begin to examine the molecular mechanism of PARP's importance in cardiac arrest/CPR. The role of the C-terminal NAD binding domain in PARP-mediated neuroprotection will be determined by comparison of cardiac arrest/CPR outcome in PARP-/- with or without transfection of a mutant form of PARP with inactive catalytic domain. Also, we will examine cysteine aspartase (CASPASE)-PARP interactions by comparison of cardiac arrest/CPR outcome in PARP-/- with and without transfection of a mutant form of PARP lacking in CASPASE 3 cleavage site (D214G mutant). These experiments will further our understanding of PARP-mediated mechanisms of in vivo ischemic brain injury and may provide a rational basis for development of effective, new therapeutic strategies to preserve neurological function atter cardiac arrest/CPR.
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