To elucidate further the involvement of nitric oxide (NO) and/or participation of free radicals in cerebral ischemic damage, the investigations explored the effect of nitric oxide synthase (NOS) inhibition on the formation of cerebral ischemic edema and the capacity of rat brain endothelial cells (RBEC) to form free radical species (ROS). The involvement of NO in the development of ischemic cytotoxic edema was investigated by inhibiting nitric oxide synthase activity with Na-nitro-L-arginine (NLA). Bilateral carotid artery occlusion (15 min) alone or with release (15 and 60 minutes) served as a model for edema induction. NLA, Na-nitro-D-arginine methyl ester (D-NAME) or Ringer's solution were administered 4 hours prior to ischemia or sham operation. Treatment with a stable nitroxide radical, 4-hydroxy-2,2,6,6- tetramethylpiperidine-L-oxyl (TPL), was used to assess free radical involvement in edema. Accumulation of tissue water was evaluated by measuring specific gravity (SG) of brain cortex. There was a greater reduction of cortical SG in early reperfusion (15 minutes) and a lesser decrease in SG (60 minutes later) in NLA-than in D-NAME- or Ringer's- treated gerbils (confirmed by histological examination of the brain tissue). TPL treatment (pre- and postischemic) ameliorated the formation of edema to the same degree as NLA. The findings indicate a biphasic NLA modulation of cytotoxic edema most likely mediated through the respective absence or presence of NO-derived free radicals. RBEC were exposed to an oxygen-depleted atmosphere (containing 95% N2 and 5% CO2) for 4 hours at 37oC and air (10 minutes) at room temperature to simulate """"""""ischemia/reperfusion."""""""" Nitroblue tetrazolium (NBT) reduction [formation of nitroblue formazan (NBF)] served as a marker for the production of ROS. The release of lactate dehydrogenase (LDH) and [3H]arachidonic acid (AA) was used to assess cellular integrity. RBEC exposed to hypoxia/reoxygenation produced up to 59% greater NBF formation than controls without affecting LDH or AA release. The production of ROS was calcium-dependent and not affected by AA or its metabolites. The findings indicate that the RBEC can produce superoxide dismutase (SOD)-inhibitable ROS which are augmented by hyppoxia/ reoxygenation. It is suggested that in vivo cerebromicrovascular endothelium may contribute to the formation of ROS and play a role in ischemic brain injury.
