There is significant data indicating the cerebral ischemia is accompanied by an increase in extracellular excitatory amino acids. Evidence has recently accumulated linking the increase in these excitatory amino acids with the production of nitric oxide in neuronal tissue. The in vitro data strongly suggests a significant role for nitric oxide int he cerebral ischemia, while the limited data that has been obtained in vivo, is equivocal. The principal aim of this project is to resolve the apparent discrepancies int he literature by performing careful studies looking at a variety of parameters. In a focal ischemia model in the cat, nitric oxide production in ischemic and postischemic tissue will be monitored by a variety of techniques including the direct measurement of nitric oxide with a microelectrode, measurement of nitric oxide synthase activity, nitric oxide synthase immunohistochemical staining (for neuronal, endothelial, and macrophage NOS), and NADPH diaphorase staining. Cerebral blood flow will be measured using both laser doppler for continuous measurements in one region, and radiolabeled microspheres for discrete measurements in a number of regions. To further investigate the mechanism by which nitric oxide may function in cerebral ischemia, measurements of microvascular permeability will also be made. Focal ischemia will be produced by temporary occlusion of the left middle cerebral artery int he cat, and measurements will be undertaken in both the acute and the chronic stage of ischemia. Utilizing compounds that inhibit the synthesis of nitric oxide, or compounds that increase tissue nitric oxide levels, we will determine if the extent of neuronal tissue damage can be altered by altering nitric oxide production. The primary hypothesis of this project is that local nitric oxide in cerebral tissue produces a balance of opposing forces, some acting to reduce damage in cerebral ischemia (ie. vasodilation, due to the action of nitric oxide ont he vascular smooth muscle), and some acting to augment cerebral ischemic damage (ie. toxic action of nitric oxide in the tissue). By appropriately modifying the activity of nitric oxide synthase so as to attenuate the tissue nitric oxide increases that occur in ischemia without adversely affecting blood flow, we will be able to reduce the extent of neuronal damage following cerebral ischemia. Using drugs which act preferentially on the neuronal and endothelial constitutive nitric oxide synthase, and on the inducible form of the enzyme, we will obtain information on the relateive importance of neuronal, endothelial, and inducible nitric oxide synthesis. Only by using a multi-prong approach, where a number of parameters are measured int he same animal model, will we be able to sort out the true role that nitric oxide plays in cerebral ischemia. With a better understanding of the mechanisms of cerebral ischemic injury, treatment protocols can more directly be designed. If compounds that act on the nitric oxide/arginine system are shown to be neuroprotective, it will provide another potential avenue for the treatment of cerebral ischemia.
