Study On Inhibition Of Neuronal Apoptosis Induced By Hypoxia/ischemia Through RAAV Vector Delivery Of NOS Antisense RNA(rAAV-AsnNOS And RAAV-AsiNOS) In Vivo And In Vitro

Oxygen deprivation (hypoxia or ischemia) in the brain can result in profound cellular and tissue damages. Nitric oxide (NO) is synthesized by a family of NO synthases (NOS) composed of three isoforms, including the endothelial (eNOS), the neuronal (nNOS) and the inducible isoform (iNOS). NO may function as an important neuronal messenger molecule in cerebral ischemia. nNOS contributes to early-phase damage while iNOS to late-phase. Numerous transmitter and second messenger pathways are inappropriately activated after the initial ischemic event. P38 MAPK is one of the major pathways leading toward neuronal injury by initiating the apoptotic cell death which results in the activation of Caspase-3. And all the pathways leading to apoptosis hinge on Caspase-3.Presently, occlusion of middle cerebral artery (MCAO) using the intraluminal filament technique is the most popular in vivo model of cerebral ischemia. The pheochromocytoma cell line PC12 is an oxygen-sensitive cell type that has been extensively used as an in vitro model for neural cells to study the effects of hypoxia. As the only safe vector, rAAV, with its specific characteristics, has become a novel genetic tool to treat nervous system diseases.Objective: to investigate the expression of NOS and NO in the cerebral ischemic tissue, their neurotoxicity and the potential pathway resulting in neuronal apoptosis. To explore the mechanisms of resistance to ischemic /hypoxic injury of neurons and inhibition for neuronal apoptosis after rAAV-AsNOS transfection. The methods ,results and Conclusions are as follows:1.TO establish the Focal cerebral ischemia model and investigate the expression of NOS and NO in the cerebral ischemic tissue. 1.1 Methods: Focal cerebral ischemia model was established via MCAO with the intraluminal filament method. NO and NOS were evaluated respectively with nitrate...