Cerebral ischemia/reperfusion increases endothelial nitric oxide synthase levels by an indomethacin-sensitive mechanism.

In anesthetized piglets, endothelial and neuronal nitric oxide synthase (eNOS and nNOS, respectively) levels were investigated after global cerebral ischemia. Increased intracranial pressure was used to produce 5 or 10 minutes of global ischemia, which was verified visually by observing pial arteriolar blood flow and by a microsphere technique. At 4 to 6 hours of reperfusion, parietal cortex, hippocampus, and cerebellum were collected for immunohistochemical or immunoblot analysis. Immunohistochemical examination localized eNOS only to blood vessels and nNOS only to nonvascular cells, which were primarily neurons in all regions examined. Analysis of immunoblot data revealed significant increases in eNOS levels from 47 +/- 22 pixels/micrograms protein for time controls to 77 +/- 36 pixels/micrograms protein (75% increase) for ischemia in parietal cortex (n = 9 to 10) and 22 +/- 10 for control to 40 +/- 16 pixels/micrograms protein (40% increase) for ischemia in hippocampus (n = 7 to 8). Levels of eNOS in cerebellum also tended to be higher but were variable and not significant (n = 5 to 6). In contrast, changes in nNOS levels were not detected at 4 or 6 hours. The increase in eNOS levels detected on immunoblots also was apparent on tissue sections as an increase in intensity of staining. Cyclooxygenase-dependent mechanisms were investigated with respect to the ischemia-induced increase in eNOS levels. Pretreatment with the cyclooxygenase inhibitor indomethacin (5 mg/kg intravenously) abolished the ischemia-induced eNOS increase in parietal cortex and hippocampus (n = 7). Thus, we conclude that the eNOS response is rapid, specific to vessels, and involves an indomethacin-sensitive mechanism.

Kainic acid rapidly induces cyclooxygenase (COX)-2 in piglet cerebral cortex.

Ischemia/reperfusion (I/R) results in a robust induction of cyclooxygenase (COX)-2 in the newborn brain via unknown mechanisms, but glutamate release and activation of KA receptors may be involved. We examined effects of local KA (3-300 micromol/l for 10 min) treatment on cortical COX-2 expression in anesthetized piglets using a closed cranial window. Treated and corresponding control tissue samples were collected 0.5-10 h after treatment. COX-2 mRNA and protein levels were assessed using RNase protection assay and immunohistochemistry, respectively. KA elicited reproducible dose-dependent increases in cortical COX-2 mRNA unaffected by indomethacin or N(G)-nitro-L-arginine methyl ester pretreatment. COX-2 mRNA levels were elevated at 30 min, peaked at 2 h, but remained enhanced for up to 10 h after KA. Neuronal COX-2 immunoreactivity was also enhanced compared with the control side in all cortical layers 8h after KA. In summary, activation of KA receptors may be involved in the neuronal induction of COX-2 after I/R in the newborn.

Indomethacin attenuates early increases in inducible heat shock protein 70 after cerebral ischemia/reperfusion in piglets.

Indomethacin-sensitive mechanisms involved in inducible heat shock protein 70 (iHSP 70) synthesis were investigated at 6 h after global cerebral ischemia in parietal cortex and hippocampus. In anesthetized piglets, increased intracranial pressure was used to produce 5 or 10 min of cerebral ischemia. Brain regions were sampled for immunoblot analysis, immunohistochemistry and morphology. Immunoblots revealed differential expression of iHSP 70 in untreated brains. Cerebellum contained substantial amounts of iHSP 70 while lower levels were present in parietal cortex and hippocampus. Detectable increases in iHSP 70 were observed at 2 h after ischemia in parietal cortex and hippocampus. Using immunoblot data, calculation of percent change from control at 6 h after ischemia revealed significant (p < 0.05) increases in iHSP 70 of 111 +/- 39% (x +/- sem) (n = 6) in parietal cortex and 195 +/- 69% (n = 8) in hippocampus. Increased iHSP 70 immunoreactivity occurred primarily in the granular/subgranular area of the dentate gyrus 6 h after ischemia. Histological staining revealed little cellular injury at 6 h after ischemia in the granular/subgranular region injury whereas the CA3 region, which lacked iHSP 70 staining, displayed modest cellular injury. Cellular injury was also observed in cortical layers II/III and VI. At 6 h after ischemia, indomethacin pretreatment (5 mg/kg, i.v.) attenuated the iHSP 70 increases in parietal cortex and hippocampus (7 +/- 30% and 89 +/- 30%, respectively n = 5; p < 0.05 compared to ischemia). Also, the increase in iHSP 70 immunoreactivity and appearance of cellular injury were not detected with indomethacin pretreatment. Thus, prior administration of indomethacin is associated with attenuation of ischemia-induced increases in iHSP 70 and cellular injury.

Effects of anoxic stress on prostaglandin H synthase isoforms in piglet brain.

We examined effects of ischemia and asphyxia on levels of prostaglandin H synthase-1 (PGHS-1) and prostaglandin H synthase-2 (PGHS-2) in piglet brain. Ischemia was induced by increasing intracranial pressure and asphyxia was induced by turning off the respirator. Duration of anoxic stress was 10 min. In some animals, indomethacin (5 mg/kg, i.v.) or 7-nitroindazole (7-NI) was administered prior to ischemia to block PGHS or brain nitric oxide synthase (bNOS), respectively. Tissues from cerebral cortex and hippocampus were removed and fixed and/or frozen after 1, 2, 4 and 8 h of recovery from anoxic stress. In addition, tissues were obtained from untreated animals or from time control animals. Levels of mRNA and proteins were determined using RNase protection assay and immunohistochemical approaches, respectively. In the tissues studied, only a few neurons were immunopositive for PGHS-1, and neither ischemia or asphyxia affected PGHS-1 immunostaining at 8 h after recovery. Likewise, PGHS-1 mRNA did not increase following anoxic stress. In contrast, substantial PGHS-2 immunoreactivity was present in neurons and glial cells in the cerebral cortex and hippocampus and there was no difference between time control and non treated animals. PGHS-2 mRNA increased by 2-4 h after ischemia, and heightened immunoreactivity for PGHS-2 was present at 8 h after ischemia in cerebral cortex and hippocampus. However, asphyxia did not increase PGHS-2 mRNA or immunostaining. Indomethacin pretreatment inhibited increases in mRNA and protein for PGHS-2 after ischemia, while 7-NI had little effect on increases in PGHS-2 immunoreactivity. We conclude that: (1) PGHS-2 is the predominant isoform present in piglet cerebral cortex and hippocampus; (2) Ischemia but not asphyxia increases levels of PGHS-2; (3) Ischemia does not increase levels of PGHS-1; and (4) Indomethacin but not 7-NI attenuates ischemia-induced increases in PGHS-2.

Indomethacin attenuates early increases in inducible heat shock protein 70 after cerebral ischemia/reperfusion in piglets

Indomethacin-sensitive mechanisms involved in inducible heat shock protein 70 (iHSP 70) synthesis were investigated at 6 h after global cerebral ischemia in parietal cortex and hippocampus. In anesthetized piglets, increased intracranial pressure was used to produce 5 or 10 min of cerebral ischemia. Brain regions were sampled for immunoblot analysis, immunohistochemistry and morphology. Immunoblots revealed differential expression of iHSP 70 in untreated brains. Cerebellum contained substantial amounts of iHSP 70 while lower levels were present in parietal cortex and hippocampus. Detectable increases in iHSP 70 were observed at 2 h after ischemia in parietal cortex and hippocampus. Using immunoblot data, calculation of percent change from control at 6 h after ischemia revealed significant (p<0.05) increases in iHSP 70 of 111±39% (&xmacr;±sem) (n=6) in parietal cortex and 195±69% (n=8) in hippocampus. Increased iHSP 70 immunoreactivity occurred primarily in the granular/subgranular area of the dentate gyrus 6 h after ischemia. Histological staining revealed little cellular injury at 6 h after ischemia in the granular/subgranular region injury whereas the CA3 region, which lacked iHSP 70 staining, displayed modest cellular injury. Cellular injury was also observed in cortical layers II/III and VI. At 6 h after ischemia, indomethacin pretreatment (5 mg/kg, i.v.) attenuated the iHSP 70 increases in parietal cortex and hippocampus (7±30% and 89±30%, respectively n=5; p<0.05 compared to ischemia). Also, the increase in iHSP 70 immunoreactivity and appearance of cellular injury were not detected with indomethacin pretreatment. Thus, prior administration of indomethacin is associated with attenuation of ischemia-induced increases in iHSP 70 and cellular injury.

Effects of ischemia on prostaglandin H synthase-2 expression in piglet choroid plexus.

We examined effects of ischemia on expression of prostaglandin H synthase-1 (PGHS-1) and prostaglandin H synthase-2 (PGHS-2) in piglet choroid plexus. Ten minutes of ischemia was induced by increasing intracranial pressure. Whole choroid plexus was removed and fixed and/or frozen after 1, 2, 4, and 8 h of recovery from anoxic stress. In addition, tissues were obtained from untreated animals or from time control animals. Tissues were analyzed for mRNA, using RNase protection assays, and for proteins, using immunohistochemical approaches. Limited, but detectable PGHS-2 immunoreactivity was present in choroid plexus under normal conditions, and there was no difference between time-control and non-treated animals. Further, PGHS-2 mRNA increased by 2-4 h after ischemia, and enhanced immunoreactivity for PGHS-2 was present at 8 h after ischemia. Enhanced immunoreactivity for PGHS-2 was present in vascular endothelial cells as well as cuboidal epithelial cells and macrophages. In contrast, PGHS-1 mRNA did not increase following ischemia. We conclude that PGHS-2 is present in piglet choroid plexus under normal conditions and that ischemia increases levels of PGHS-2 in choroid plexus.

Ischemia-reperfusion rapidly increases COX-2 expression in piglet cerebral arteries.

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.

Regional distribution of prostaglandin H synthase-2 and neuronal nitric oxide synthase in piglet brain.

Immunohistochemical techniques were used to examine the distribution of prostaglandin H synthase (PGHS)-2 and neuronal nitric oxide synthase (nNOS) in piglet brain. Samples from parietal cortex, hippocampus, and cerebellum were immersion fixed in 10% formalin, sectioned at 50 microm, and immunostained using specific antibodies against PGHS-2 and nNOS. Immunoreactivity for PGHS-2 was extensive throughout the areas examined. For example, PGHS-2 immunoreactive cells were present in all layers of the cortex, but were particularly dense among neurons in layers II/II, V, and VI. In addition, glial cells associated with microvessels in white matter showed PGHS-2 immunoreactivity. In contrast, nNOS immunoreactive neurons were limited in number and widely dispersed across all layers of the cortex and thus did not form a definable pattern. In the hippocampus, heavy PGHS-2 immunoreactivity was present in neurons and glial cells in the subgranular region, stratum radiatum, adjacent to the hippocampal sulcus, and in CA1 and CA3 pyramidal cells. Immunostaining for nNOS displayed a different pattern from PGHS-2 in the hippocampus, and was mainly localized to the granule cell layer of the dentate gyrus and the mossy fiber layer. In the cerebellum, PGHS-2 immunoreactivity was heavily represented in the Bergmann glia and to a lesser extent in cells of the granular layer, whereas nNOS was detected only in Basket cells. There are four conclusions from this study. First, PGHS-2 immunoreactivity is widely represented in the cerebral cortex, hippocampus, and cerebellum of neonatal pigs. Second, glia cells as well as neurons can show immunoreactivity for PGHS-2. And third, the distribution of nNOS is different from PGHS-2 immunoreactivity in the cerebral cortex, hippocampus, and cerebellum.

Ischemia increases prostaglandin H synthase-2 levels in retina and visual cortex in piglets.

BACKGROUND: Ischemia increases levels of prostaglandin H synthase-2 (PGHS-2) in neonatal brain and cerebral vasculature, but effects on the developing visual system are unknown. We examined the effects of ischemia on PGHS-2 mRNA and protein levels in the retina and visual cortex in anesthetized piglets. METHODS: Ten minutes of complete retinal and brain ischemia was induced by increasing intracranial pressure. After 2-12 h of reperfusion, samples of retina and visual cortex were collected for determinations of levels of PGHS-2 mRNA (RNase protection assay) or protein (immunohistochemistry and western blotting). Tissues also were obtained from control animals. RESULTS: Levels of PGHS-2 mRNA were undetectable in control animals but showed a dramatic increase at 2-4 h in the cortex and retina in animals exposed to ischemia. Detectable but limited PGHS-2 immunoreactivity (IR) was present in the retina and visual cortex from control animals. In piglets not subjected to ischemia, PGHS-2 IR was localized mainly to the outer limiting membrane and to the Muller cells. Ischemia induced a marked increase in PGHS-2 IR in the neural retina, with the greatest increase in the photoreceptor layer. PGHS-2 levels in whole retina also increased at 8 h after ischemia. In the intact visual cortex PGHS-2 IR was evident in layers II and V. Ischemia increased the intensity of IR in layers II/III as well as layer V. CONCLUSIONS: 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.

Endothelial nitric oxide synthase gene transfer enhances dilation of newborn piglet pulmonary arteries.

We determined the expression and functional correlate of in vitro transfection with a recombinant adenoviral vector encoding the gene for bovine endothelial nitric oxide synthase (AdCMVeNOS) or Escherichia coli beta-galactosidase (AdCMVLacZ) in pulmonary endothelial cells (EC), vascular smooth muscle cells (VSMC), and pulmonary arteries (PA) from newborn piglets. AdCMVeNOS and AdCMVeLacZ vectors, grown in 293-cell monolayers, were purified by double-cesium gradient ultracentrifugation. Cell cultures and PA were incubated with increasing vector titers for 30 or 60 min, followed by incubation in fresh medium for 18 h at 37 degrees C. LacZ expression was assessed by histochemical staining; eNOS expression was evaluated by Western blot analysis. Functional eNOS expression was determined by measurement of cGMP and quantification of the relaxation response to bradykinin (BK). In PA, LacZ transgene expression was preferentially localized to the adventitia and endothelium. Increased eNOS protein expression was observed in EC and VSMC transfected with AdCMVeNOS. Functional studies revealed increased cGMP abundance in cultured cells and enhanced relaxation to BK in AdCMVeNOS-transfected PA. These studies demonstrate that gene transfer with AdCMVeNOS results in functional expression and altered vasoactive responses in the neonatal pulmonary vasculature. Gene transfer with replication-deficient adenovirus vectors is a useful tool for the study of targeted genes in vascular biology.