Background and Purpose-Production of NO by endothelial NO synthase (eNOS) plays a protective role in cerebral ischemia. We studied the effects of transient focal ischemia on eNOS expression. Methods-Wistar rats (nϭ72) underwent reversible filament occlusion of the right middle cerebral artery for 75 minutes.After 6, 24, 72, or 168 hours of reperfusion, brains were removed and coronal sections cut for eNOS immunohistochemistry, eNOS-alkaline phosphatase costaining, and hematoxylin-eosin staining. Samples for eNOS immunoblots were taken from corresponding striatum and overlying parietal cortex bilaterally. Results-eNOS protein occurred in virtually all blood vessels and was consistently increased in microvessels in the ischemic striatum after 24 to 168 hours of reperfusion but not at 6 hours. eNOS upregulation in the parietal cortex was only present in animals with evidence of cortical infarcts documented on adjacent HE-stained sections. Costaining of endogenous alkaline phosphatase and eNOS demonstrated eNOS expression in all segments of cerebral microvessels. Quantitative analysis of eNOS immunostaining and immunoblots showed no attenuated increase in animals that were treated with indomethacin (5 mg/kg IP), NS398 (20 mg/kg IP), or L-arginine-methyl ester (10 mg/kg IP). In contrast to eNOS, levels of brain NOS did not increase after ischemia. Conclusion-eNOS protein is upregulated in pre-and postcapillary microvessels and upregulation appears slower after transient compared with permanent ischemia. Cyclooxygenase and NOS products do not play a major role in postischemic eNOS induction.

In the newborn, cyclooxygenase (COX)-derived products play an important role in the cerebrovascular dysfunction after ischemia-reperfusion (I/R). We examined effects of I/R on expression of COX-1 and COX-2 isoforms in large cerebral arteries of anesthetized piglets. The circle of Willis, the basilar, and the middle cerebral arteries were collected from piglets at 0.5–12 h after global ischemia (2.5–10 min, n = 50), hypoxia ( n = 3), or hypercapnia ( n = 2) and from time-control ( n = 19) or untreated animals ( n = 7). Tissues were analyzed for COX-1 and COX-2 mRNA and protein using RNase protection assay and immunoblot analysis, respectively. Ischemia increased COX-2 mRNA by 30 min, and maximal levels were reached at 2 h. Hypoxia or hypercapnia had minimal effects on COX-2 mRNA. COX-2 protein levels were also consistently elevated by 8 h after I/R. Increases in COX-2 mRNA or protein were not influenced by pretreatment with either indomethacin (5 mg/kg iv, n = 5) or nitro-l-arginine methyl ester (15 mg/kg iv, n = 7). COX-1 mRNA levels were low in time controls, and ischemic stress had no significant effect on COX-1 expression. Thus ischemic stress leads to relatively rapid, selective induction of COX-2 in cerebral arteries.

Detectable amounts of PGHS-2 protein are present in the piglet retina and visual cortex under normal conditions, but levels are markedly increased 8-12 h after ischemic stress. Enhanced PGHS-2 levels after ischemic stress may contribute to delayed pathological changes of the visual system in the neonate.

We have previously shown that cycloheximide (CHX) preserved neuronal function after global cerebral ischemia in piglets, in a manner similar to indomethacin. To elucidate the mechanism of this protection, we tested the hypothesis that CHX would inhibit cyclooxygenase (COX) activity in the piglet cerebral cortex and vasculature. Pial arteriolar responses to hypercapnia, arterial hypotension, and sodium nitroprusside (SNP) were determined before and 20 min after treatment with CHX (0.3–1 mg/kg iv) using a closed cranial window and intravital microscopy. We also determined baseline and arachidonic acid (AA)-stimulated cortical PGF2αand 6-keto-PGF1α production before and 20–60 min after CHX (1 mg/kg iv) treatment, using ELISA kits. CHX did not affect baseline diameters (∼100 μm) but significantly decreased arteriolar dilation to COX-dependent stimuli, such as hypercapnia and hypotension, but not to COX-independent SNP. In the 1 mg/kg CHX-treated group, increases in vascular diameters were reduced from 22 ± 2 to 10 ± 2%, from 49 ± 5 to 31 ± 3% (means ± SE, 5 and 10% CO2, respectively, n = 8), from 12 ± 3 to 3 ± 1%, and from 26 ± 5 to 6 ± 2% (∼25 and 40% decreases in blood pressure, respectively, n = 6). CHX also inhibited conversion of exogenous AA to both PGF2αand 6-keto-PGF1α; for example, 20 min after CHX treatment 10 μg/ml AA-stimulated PGF2α concentrations in the artificial cerebrospinal fluid decreased from 14.28 ± 3.04 to 5.90 ± 1.26 ng/ml ( n = 9). Thus CHX rapidly decreases COX activity in the piglet cerebral cortex. This result may explain in part the preservation of neuronal function of CHX in cerebral ischemia.