Cycloheximide rapidly inhibits cortical COX activity and COX-dependent pial arteriolar dilation in piglets.

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 PGF(2alpha) and 6-keto-PGF(1alpha) production before and 20-60 min after CHX (1 mg/kg iv) treatment, using ELISA kits. CHX did not affect baseline diameters (approximately 100 microm) 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% ( approximately 25 and 40% decreases in blood pressure, respectively, n = 6). CHX also inhibited conversion of exogenous AA to both PGF(2alpha) and 6-keto-PGF(1alpha); for example, 20 min after CHX treatment 10 microg/ml AA-stimulated PGF(2alpha) 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.

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.

Cerebrovascular reactivity remains intact after cortical depolarization in newborn piglets.

Cerebrovascular reactivity is severely affected by ischemia, and changes in vascular responses have been reported after cortical spreading depression and head trauma as well. Cortical depolarization (CD) occurs during ischemia, cortical spreading depression, and head trauma, but its effects on cerebrovascular reactivity are unclear. We tested the hypothesis that CD induced by KCl diminishes the vascular responsiveness to various vasodilatory stimuli in piglets. Responses of pial arterioles were determined by changes in vascular diameter by use of a closed cranial window and intravital microscopy. Baseline arteriolar diameters were 105 +/- 3 microm (mean +/- SEM, n = 27). CD was elicited by topical administration of 1 mol/L KCl for 3 min. Vascular responses were measured before and 1 h after CD. KCl elicited CD and constricted arterioles by 54 +/- 4% (n = 27). N-methyl-D-aspartate induced dose-dependent vasodilation that was unaffected by CD; the percent changes were 9 +/- 1 versus 8 +/- 1 (before and after CD) at 10(-5) mol/L, 19 +/- 2 versus 18 +/- 3 at 5 x 10(-5) mol/L, and 29 +/- 2 versus 26 +/- 3 at 10(-4) mol/L (n = 9). Hypercapnic vasodilation was not diminished by CD; the percent changes were 15 +/- 2 versus 16 +/- 4 at 5%, and 27 +/- 5 versus 27 +/- 6 at 10% inspired CO2 (n = 8). Aprikalim and forskolin caused dilation that was also resistant to prior CD; the percent change values were 21 +/- 4 versus 18 +/- 3 and 16 +/- 2 versus 16 +/- 4 at 10(-6) mol/L, 36 +/- 5 versus 34 +/- 5 and 34 +/- 7 versus 37 +/- 7 at 10(-5) mol/L (n = 8), respectively. Finally, calcitonin gene-related peptide-induced vasodilation was unaffected by CD; percent changes were 15 +/- 3 versus 16 +/- 2 at 10(-7) mol/L and 26 +/- 4 versus 22 +/- 3 at 10(-6) mol/L (n = 8). The intact vascular responses after CD suggest that this component is not responsible for decreased cerebrovascular reactivity after ischemia, head trauma, or cortical spreading depression.

Inhibitors of protein synthesis preserve the N-methyl-D-aspartate-induced cerebral arteriolar dilation after ischemia in piglets.

BACKGROUND AND PURPOSE--Cerebral arteriolar dilation to N-methyl-D-aspartate (NMDA) is a neuronally mediated process that is sensitive to cerebral ischemia. We tested the hypothesis that pretreatment with transcription or translation inhibitors preserves the vascular response to NMDA after global cerebral ischemia. METHODS--Pial arteriolar diameters were measured in anesthetized piglets by use of a closed cranial window and intravital microscopy. Arteriolar responses to NMDA (10(-5) and 10(-4) mol/L) were measured before and 1, 2, and 4 hours after 10 minutes of ischemia. Ischemia was induced by increasing intracranial pressure. Subgroups were pretreated with vehicle, topical actinomycin D (Act-D) 10(-5) or 10(-6) mol/L, or intravenous cycloheximide (CHX) 1.0 or 0.3 mg/kg 15 minutes before ischemia. The effects of Act-D and CHX on vascular responses to NMDA without preceding ischemia were also examined. RESULTS--In the vehicle group, arteriolar responses to NMDA were clearly attenuated 1 hour after ischemia but returned to baseline at 2 to 4 hours. Preischemic compared with 1 hour postischemic arteriolar dilation to NMDA was 10+/-2% versus 1+/-0% at 10(-5) mol/L and 40+/-4% versus 20+/-4% at 10(-4) mol/L NMDA (mean+/-SEM; both P<0.05, n=7). In contrast, pretreatment with Act-D resulted in preservation of the arteriolar responses to NMDA 1 hour after ischemia. For 10(-6) mol/L (n=5) of Act-D, dilations were 6+/-2% versus 6+/-2% at 10(-5) mol/L and 51+/-9% versus 39+/-10% at 10(-4) mol/L of NMDA. For 10(-5) mol/L (n=5) of Act-D, arterioles dilated by 7+/-2% versus 7+/-2% at 10(-5) mol/L and 38+/-4% versus 35+/-4% at 10(-4) mol/L NMDA. Similarly, CHX preserved NMDA-induced vasodilation. For 0.3 mg/kg of CHX (n=5), dilations were 8+/-2% versus 8+/-1% at 10(-5) mol/L and 39+/-4% versus 28+/-6% at 10(-4) mol/L NMDA. For 1.0 mg/kg of CHX (n=5), arterioles dilated by 10+/-2% versus 6+/-2% at 10(-5) mol/L and 37+/-7% versus 35+/-6% at 10(-4) mol/L NMDA. In experiments without ischemia, NMDA-induced vasodilation before and 85 minutes after administration of Act-D or CHX was not significantly different. CONCLUSIONS--Vascular responses of cerebral arterioles to NMDA after ischemia are preserved by pretreatment with either Act-D or CHX. Without preceding ischemia, Act-D and CHX do not potentiate neuronal-vascular responses to NMDA. Our results suggest that continued or augmented protein synthesis is involved in the transient attenuation of NMDA-induced dilation during the early reperfusion phase and that inhibitors of protein synthesis may protect neurons against ischemic stress.

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.

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.

Inhibitory effects of hypoxia and adenosine on N-methyl-D-aspartate-induced pial arteriolar dilation in piglets.

Our previous studies have indicated that oxygen radicals, produced during reoxygenation following short-term arterial hypoxia, lead to sustained suppression of cerebral arteriolar responses to N-methyl-D-aspartate (NMDA). However, whether arteriolar dilator responses to NMDA are reduced during arterial hypoxia has never been examined. In this study, we determined whether hypoxia or hypoxia-related metabolites such as adenosine or nitric oxide (NO) will reduce NMDA-induced arteriolar dilation. We have also determined the location of NMDA receptor- and brain nitric oxide synthase (bNOS)-positive neurons in the cerebral cortex. In anesthetized piglets, pial arteriolar diameters were determined using intravital microscopy. Baseline arteriolar diameters were approximately 100 microns. Topical application of NMDA at concentrations of 10(-5), 5 x 10(-5) and 10(-4) M resulted in dose-dependent vasodilation (9 +/- 2, 18 +/- 2 and 29 +/- 2% above baseline, respectively, n = 21). Administration of theophylline (20 mg/kg, i.v.) had no effect on NMDA-dependent vasodilation, but it did block dilation to hypoxia (inhalation of 8.5% O2). In theophylline-treated animals, NMDA responses were completely abolished during hypoxia (28 +/- 2 vs. 2 +/- 1%, respectively to 10(-4) M, n = 7) while sodium nitroprusside (SNP, 10(-4) M) still dilated pial arterioles normally. NMDA-induced vasodilation was not modified after application and removal of adenosine (10(-4) M; n = 5) or SNP (10(-5) M; n = 4), or when SNP (10(-7) M) was coapplied with NMDA (n = 6). Conversely, coapplication of adenosine (10(-6) M) attenuated NMDA responses (31 +/- 5 vs. 20 +/- 3%, n = 7). We also found that NMDA receptor- and bNOS-containing neurons were located predominantly in layers II/III of the cortex. Proximity of these neurons to the cortical surface is consistent with diffusion of NO to pial arterioles as the mechanism of dilation to NMDA. We conclude that NMDA-induced cerebral arteriolar dilation is inhibited by hypoxia alone and by exogenous adenosine, but not by NO.

Effects of ischemia on cerebral arteriolar dilation to arterial hypoxia in piglets.

BACKGROUND AND PURPOSE: Arterial hypoxia mediates cerebral arteriolar dilation primarily via mechanisms involving activation of ATP-sensitive K+ channels (K[ATP]), which we have shown to be sensitive to ischemic stress. In this study, we determined whether ischemia/reperfusion alters cerebral arteriolar responses to arterial hypoxia in anesthetized piglets. Since adenosine plays an important role in cerebrovascular responses to hypoxia, we also determined whether adenosine-induced arteriolar dilation is affected by ischemic stress. We tested the hypothesis that reductions in cerebral arteriolar dilator responses after ischemia would be proportional to the contribution of K(ATP) to hypoxia and adenosine. METHODS: Pial arteriolar diameters were measured using a cranial window and intravital microscopy. We examined arteriolar responses to arterial hypoxia (inhalation of 8.5% and 7.5% O2), to topical adenosine (10[-5] and 10[-4] mol/L) and to arterial hypercapnia (inhalation of 5% and 10% CO2 in air) before and after 10 minutes of global ischemia. Ischemia was achieved by increasing intracranial pressure. Arterial hypercapnia was used as a positive control for the effectiveness of the ischemic insult. In addition, we evaluated cerebral arteriolar responses to 10(-5) and 10(-4) mol/L adenosine applied topically with or without glibenclamide, a selective inhibitor of K(ATP) (10[-5] and 10[-6] mol/L). Finally, we administered theophylline (20 mg/kg, i.v.) to assess the contribution of adenosine to cerebral arteriolar dilation to arterial hypoxia. RESULTS: Before ischemia, cerebral arterioles dilated by 19+/-3% to moderate and 29+/-4% to severe hypoxia (n=7; P<.05); 13+/-2% to 10(-5) and 20+/-1% to 10(-4) mol/L adenosine (n=9; P<.05); and by 17+/-2% to moderate and 28+/-3% to severe hypercapnia (n=6; P<.05). After ischemia, cerebral arteriolar responses to hypoxia and adenosine were unchanged. In contrast, cerebral arteriolar dilation to hypercapnia was impaired by ischemia (1+/-1% and 2+/-1% at 1 hour; n=6). Glibenclamide reduced cerebral arteriolar dilation to adenosine by approximately one half (n= 7). In addition, blockade of adenosine receptors by theophylline (20 mg/kg, i.v.) almost totally suppressed cerebral arteriolar dilation to arterial hypoxia (n = 6). CONCLUSIONS: Cerebrovascular responsiveness is selectively affected by anoxic stress. In addition, cerebral arteriolar dilation to hypoxia and adenosine is maintained after ischemia despite the expected impairment in K(ATP) function.

Calcium-activated K+ channels in cerebral arterioles in piglets are resistant to ischemia.

Our previous studies indicate that function of ATP-dependent K+ channels (K(ATP)) in cerebral arterioles is suppressed after ischemia. In the current study, we examined pial arteriolar responses to forskolin, dibutyryl-cAMP, NS-1619, and methionine (met)-enkephalin, activators of calcium-dependent K+ channels (K(Ca)) before and 1 hour after 10 minutes of total, global ischemia in anesthetized piglets. Arteriolar diameters were measured using a closed cranial window and intravital microscopy. All pharmacologic agents were given topically. Baseline diameters were approximately 100 microm, and diameters had returned to normal by 1 hour after ischemia. Forskolin dilated arterioles by 9 +/- 3%, 18 +/- 4%, and 31 +/- 12% at 5 x 10(-8), 5 x 10(-7), and 10(-6) mol/L, respectively (P < 0.05, n = 10). In addition, dibutyryl-cAMP dilated arterioles by 8 +/- 2% at 10(-4) mol/L and 14 +/- 2% at 3 x 10(-4) mol/L (P < 0.05, n = 6). Also, NS-1619 increased diameter of arterioles by 9 +/- 2% at 10(-7) mol/L and 17 +/- 9% at 10(-5) mol/L (P < 0.05, n = 5). Finally, met-enkephalin dilated arterioles by 9 +/- 2% at 10(-8) mol/L and 16 +/- 3% at 10(-6) mol/L (P < 0.05, n = 5). At 1 hour after ischemia, arteriolar dilator effects to forskolin, dibutyryl-cAMP and NS-1619, and met-enkephalin were intact. Thus, in contrast to K(ATP), K(Ca) in cerebral arterioles are resistant to ischemic stress.

Influence of hypoxia/ischemia on cerebrovascular responses to oxytocin in piglets.

We examined the effects of hypoxic/ischemic stress on cerebral arteriolar responses to oxytocin in anesthetized piglets. Pial arteriolar diameters were measured using a cranial window and intravital microscopy. First, we evaluated arteriolar responses to topical application of oxytocin during normoxic conditions. We then determined whether 5-10 min of arterial hypoxia, ischemia, or asphyxia alters oxytocin-induced responses. Arterial hypoxia was produced by inhalation of 7.5% O2-92.5% N2 for 10 min. Ischemia was achieved by increasing intracranial pressure for 10 min. Asphyxia was achieved by turning off the ventilator for 5 min. During normoxic conditions, oxytocin dilated pial arterioles by 9 +/- 1% at 10(-8) and by 16 +/- 1% at 10(-6) mol/l (n = 47, p < 0.05). Arteriolar responses to oxytocin did not change with repeated applications (n = 10). Following hypoxia, dilator effect of oxytocin was not changed at 10(-8) (8 +/- 2%) but it was reduced at 10(-6) mol/l (7 +/- 2%; p < 0.05, n = 8). After asphyxia or ischemia, oxytocin did not dilate arterioles at 10(-8) mol/l, whereas 10(-6) mol/l resulted in a mild vasoconstriction (-4 +/- 3 to -6 +/- 4%, n = 6 and 8). Topically applied superoxide dismutase did not preserve arteriolar responses to oxytocin after asphyxia although the arterioles did not constrict to 10(-6) mol/l oxytocin (n = 5). Dilatation of cerebral arterioles in response to oxytocin was reversed to constriction by N(omega)-nitro-L-arginine methyl ester (L-NAME) (15 mg/kg, i.v.; n = 5) and by endothelial impairment by intra-arterial infusion of phorbol ester (10[-5] mol/l; n = 5). We conclude that the absence of pial arteriolar dilation to oxytocin after ischemia and asphyxia indicates endothelial dysfunction which may be involved in the pathology of perinatal brain injury.

Kainate-induced cerebrovascular dilation is resistant to ischemia in piglets.

BACKGROUND AND PURPOSE: Cerebral arteriolar dilation to N-methyl-D-aspartate (NMDA) is drastically reduced by anoxic stress. The effects of anoxic stress on cerebrovascular dilation to activation of other types of glutamate receptors are unknown. The purpose of this study was to examine the effects of ischemia on cerebral arteriolar responses to kainate in anesthetized piglets. METHODS: Arteriolar responses to 5 x 10(-5) mol/L and 10(-4) mol/L kainate were evaluated before and 10 minutes after total, global ischemia. Ischemia was induced by increasing intracranial pressure. We measured pial arteriolar diameters (approximately 100 microns) using a cranial window and intravital microscopy. RESULTS: Before ischemia, kainate dilated arterioles by 16 +/- 2% at 5 x 10% mol/L and 30 +/- 2% at 10(-4) mol/L (mean +/- SEM; n = 6). After ischemia, the diameter of arterioles increased by 17 +/- 3% and 26 +/- 3% to 5 x 10% and 10(-4) mol/L kainate, respectively (P > .05). We also investigated the mechanisms involved in mediating arteriolar dilation to kainate. Intravenous administration of N omega-nitro-L-arginine methyl ester (L-NAME) (15 mg/kg) (n = 7) or indomethacin (10 mg/kg) (n = 6) individually reduced arteriolar dilation to kainate by approximately one half. Coadministration of L-NAME and indomethacin almost completely eliminated arteriolar dilation to kainate (n = 10). Administration of theophylline (20 mg/kg IV) did not affect dilator responses to kainate (n = 7). Blockade of NMDA receptors by MK801 had minimal effects on arteriolar dilation to kainate (n = 6). CONCLUSIONS: There are three main findings from this study: (1) kainate is a potent dilator agent in the neonatal cerebral circulation; (2) nitric oxide and prostaglandins both participate in the vasodilator response to kainate; and (3) in contrast to NMDA, cerebral arteriolar dilator responses to kainate are resistant to ischemic stress.

Interaction between ATP-sensitive K+ channels and nitric oxide on pial arterioles in piglets.

The interaction between ATP-sensitive K+ channels (KATP) and nitric oxide (NO) was studied in pial arterioles of piglets. We examined the effects of N omega-nitro-L-arginine methyl ester (L-NAME), a general inhibitor of nitric oxide synthase (NOS), and 7-nitroindazole (7-NI), a selective inhibitor of neuronal NOS, on aprikalim-induced cerebral vasodilation. Topically applied, aprikalim, a selective activator of KATP, dilated arterioles by 11 +/- 7% at 10(-8) M and 17 +/- 6% at 10(-6) M. After L-NAME treatment (15 mg/kg, i.v.), the response was reduced (4 +/- 4% and 12 +/- 7%, respectively; n = 8, p < 0.05). Administration of 7-NI (50 mg/kg, i.p.) did not change pial arteriolar responsiveness to aprikalim. However, both L-NAME and 7-NI reduced the vasodilator responses to 10(-4) M N-methyl-D-aspartate (NMDA) (by 73% and by 36%, respectively). Furthermore, 7-NI treatment abolished the glutamate-induced dilatation of pial arterioles. Administration of L-NAME reduced the NOS activity in the cerebral cortex by 88%, whereas the reduction after the 7-NI treatment was 44%. Pre-treatment and coadministration of 10(-5) M glibenclaminde, a specific inhibitor of KATP or L-NAME administration, did not change the dilatory response to sodium nitroprusside. We conclude that NO may be involved in aprikalim-induced dilation of pial arterioles.

Global ischemia impairs ATP-sensitive K+ channel function in cerebral arterioles in piglets.

BACKGROUND AND PURPOSE: Indirect evidence from studies in which calcitonin gene-related peptide was used indicates that anoxic stress suppresses functioning of cerebral vascular ATP-sensitive K+ channels. The purpose of this study was to directly examine effects of total global ischemia on cerebral arteriolar dilator responses to activators of ATP-sensitive K+ channels. METHODS: We measured pial arteriolar diameters in anesthetized piglets using a closed cranial window and intravital microscopy. Baseline diameters were approximately 100 microns. Arteriolar responses to aprikalim (10(-8) and 10(-6) mol/L), a pharmacological activator of ATP-sensitive K+ channels, and iloprost (0.1 and 1 microgram/mL), a physiological activator of these channels, were determined before and 1, 2, and 4 hours after a 10-minute period of total global ischemia. Ischemia was caused by increasing intracranial pressure. RESULTS: Before ischemia, aprikalim dilated cerebral arterioles by 7 +/- 2% at 10(-8) mol/L and by 25 +/- 4% at 10(-6) mol/L (n = 5). At 1 hour after ischemia, aprikalim did not cause significant dilation at either dose (3 +/- 2% at 10(-8) mol/L and 7 +/- 4% at 10(-6) mol/L; P < .05 compared with corresponding preischemic response). Arteriolar dilation returned toward normal values at 2 and 4 hours. Similar results were found with iloprost. Furthermore, prior treatment with indomethacin (5 mg/kg) preserved normal arteriolar dilation to aprikalim and iloprost after ischemia. In contrast, arteriolar dilator responses to prostaglandin E2 were intact after ischemia. CONCLUSIONS: Ischemia transiently eliminates cerebral arteriolar dilation to activation of ATP-sensitive K+ channels; arteriolar responses are suppressed at 1 hour and return toward normal over 2 to 4 hours. In addition, reduced responsiveness can be prevented by prior treatment with indomethacin.

Differential effects of short-term hypoxia and hypercapnia on N-methyl-D-aspartate-induced cerebral vasodilatation in piglets.

BACKGROUND AND PURPOSE: Recent studies in piglets show that either asphyxia or global cerebral ischemia, which combines effects of hypoxia and hypercapnia, transiently attenuates N-methyl-D-aspartate (NMDA)-induced pial arteriolar dilation. The purpose of this study was to determine individually the effects of hypoxic hypoxia and normoxic hypercapnia on NMDA-dependent cerebrovascular reactivity. In addition, we examined mechanisms involved in reduced cerebral vascular dilation to NMDA. METHODS: In anesthetized piglets, we examined pial arteriolar diameters using a cranial window and intravital microscopy. Arteriolar responses to topically applied NMDA were determined under control conditions and after arterial hypoxia or arterial hypercapnia. In addition, arteriolar responses to NMDA were examined in animals given indomethacin (10 mg/kg IV) or superoxide dismutase (100 U/mL, topical application) before hypoxia. RESULTS: Under control conditions, application of NMDA produced a dose-related dilation of pial arterioles (eg, 9 +/- 1% to 10(-5), 15 +/- 2% to 5 x 10(-5), and 28 +/- 5% to 10(-4) mol/L NMDA above baseline, respectively, in the hypoxic group; n = 6, P < .05). After transient exposure to 15 minutes of hypoxic hypoxia, arteriolar responses to NMDA were reduced at 30 minutes and at 60 minutes (10(-4) mol/L NMDA dilated by 12 +/- 5% and 18 +/- 5%, respectively; n = 6, P < .05). Five minutes of hypoxic hypoxia also reduced dilatation to NMDA. Indomethacin or superoxide dismutase preserved arteriolar responses to NMDA after 15 minutes of hypoxia. Pial arteriolar responses to NMDA remained unimpaired during and after hypercapnia. CONCLUSIONS: Short-term severe hypoxic hypoxia and reventilation impair the NMDA-induced dilatation of pial arterioles. Respiratory acidosis alone does not modify pial arteriolar reactivity to NMDA. The reduced responsiveness of the cerebral blood vessels to NMDA caused by hypoxia appears to be due to action of oxygen radicals.

Effects of ischemia on cerebrovascular responses to N-methyl-D-aspartate in piglets.

We examined the effects of total global ischemia on cerebral arteriolar responses to N-methyl-D-aspartate (NMDA) in anesthetized newborn pigs. Arteriolar responses to 10(-4) M NMDA were determined before and after 10 to 20 min of ischemia caused by increasing intracranial pressure. Before ischemia, NMDA dilated arterioles by 30 +/- 5% (baseline = 88 +/- 2 microns; n = 6). However, after 10 min of ischemia, arteriolar dilation was reduced to 10 +/- 3% at 1 h (P < 0.05). At 2 and 4 h, NMDA-induced dilation was not different from preischemia values. Twenty minutes of ischemia had similar effects. Coadministration of 100 U/ml of superoxide dismutase did not restore arteriolar dilation to NMDA at 1 h after ischemia. Sodium nitroprusside dilated by 14 +/- 3 and 40 +/- 5% at 10(-6) and 10(-5) M before ischemia, respectively, and arteriolar responsiveness was not changed by ischemia (n = 6). Cortical nitric oxide synthase (NOS) activity, measured by the in vitro conversion of L-[14C]arginine to L-[14C]citrulline, was unaffected by ischemia (n = 12). We conclude that decreases in cerebral arteriolar responsiveness to NMDA are not due to impairment of NOS activity, enhanced degradation or chelation of nitric oxide (NO), or reduced vascular smooth muscle responsiveness to NO.

Ischemia reduces CGRP-induced cerebral vascular dilation in piglets.

BACKGROUND AND PURPOSE: Effects of anoxic stress on cerebrovascular responses to calcitonin gene-related peptide (CGRP) have not been examined previously. We determined the effects of total global ischemia on cerebral arteriolar responses to CGRP in newborn pigs. METHODS: Piglets were anesthetized and ventilated with a respirator. Pial arteriolar diameter was determined using a closed cranial window and intravital microscopy. Baseline arteriolar diameters ranged from 80 to 100 microns. Arteriolar responses to 10(-9) and 10(-8) mmol/L CGRP applied topically were determined before and 1, 2, and 4 hours after a 10-minute period of total global ischemia. Ischemia was caused by increasing intracranial pressure. RESULTS: Before ischemia, CGRP dilated arterioles by 14 +/- 2% (n = 6) and 24 +/- 3% (n = 7) at 10(-9) and 10(-8) mmol/L, respectively. However, after ischemia, arteriolar responses to 10(-9) mmol/L CGRP were reduced at 1 hour to 4 +/- 1%, at 2 hours to 3 +/- 2%, and at 4 hours to 5 +/- 4% (P < .05 for all comparisons). Similarly, arteriolar responses to 10(-8) mmol/L CGRP were reduced to 5 +/- 2% at 1 hour, 5 +/- 2% at 2 hours, and 10 +/- 6% at 4 hours (P < .05 for all comparisons). In time control animals, arteriolar responses to CGRP did not change over time. In other animals, we examined effects of pretreatment with indomethacin (5 mg/kg IV) on ischemia-induced decreases in arteriolar responses to CGRP. Indomethacin administration did not preserve arteriolar dilation to CGRP at 1 hour after ischemia, but responses were normal at 2 hours. CONCLUSIONS: Total global ischemia leads to prolonged attenuated dilator responses of cerebral arterioles to CGRP. In addition, indomethacin treatment alters effects of ischemia on CGRP-induced dilation.

The development of preatherosclerotic coronary artery lesions in perinatal piglets.

The fine structure and ultrastructure of the anterior descending coronary artery were studied in a series of perinatal piglets at 1 week prior to birth, and at 8, 24, 72 and 168 h after birth. In the anterior descending coronary artery, at or just distal to the branch point of the left circumflex artery, early plaque-like intimal lesions were present in the majority of animals. These consisted of subendothelial edema, fragmentation and dissolution of the internal elastic lamella, and the appearance of intimal myoid cells known as modified smooth muscle cells (MSMCs). These changes were present in all piglets at and before 8 h of age. They persisted and progressed during the first week of life in about half of the piglets. Beginning at 72 h and continuing through 168 h, there was an increase in MSMCs and the appearance of fibroblasts. Both fibroblasts and MSMCs were associated with the elaboration of dense collagen fibrils. Foamy macrophages appeared within the subendothelial intima having the appearance of lipophages. While the prevalence of these changes at birth indicates that they may be part of normal development, their persistence in half the piglets and structural features suggest reaction to intimal injury beginning prenatally. The lesions may be precursive of coronary atherosclerosis later in life and may parallel the early stages of atherogenesis in humans.

The effects of acute norepinephrine-induced hypertension on the coronary arteries of newborn piglets.

Newborn piglets were subjected to 45 min of sustained norepinephrine-induced hypertension and then monitored for 4 hr at baseline conditions. They were then sacrificed and the anterior descending coronary artery was serially sectioned for study by light and electron microscopy. Other groups were sacrificed after 72 and 168 hr of baseline conditions. Changes were limited to the endothelium and subendothelial intima of the most proximal segment of the anterior descending coronary artery. As similar changes are normally present in perinatal piglets, the experimental animals were compared with sham-operated controls to determine if there was a modification of the naturally occurring congenital lesions. Although the prevalence of coronary lesions in control and experimental animals was not significantly different, the experimental groups showed unique features. At 4 hr, there was marked intimal edema and disruption of the endothelium with fragmentation and dissolution of the internal elastic lamina. There was selective invasion of the intima by platelets and monocytemacrophages. After 72 and 168 hr, there was an increase and progression in preexisting modified smooth muscle cell plaques in which there developed prominent fibroplasia and collagenization. It is proposed that acute hypertension may be responsible for these changes. Such perinatal surges in blood pressure may be involved in the initiation of atherogenesis.

Effects of nimodipine on brain blood flow following acute brain ischemia in the newborn piglet.

We examined the effect of the calcium channel blocker nimodipine on postischemic hypoperfusion in the newborn piglet brain. A severe pneumothorax (SP) was induced by injecting air into the right thorax until the mean arterial blood pressure fell to 25% of baseline and was maintained for 4 min. Blood flow was immediately reduced 70-90% from baseline in each brain region during SP. In untreated animals postischemic hypoperfusion existed at 60 min, following recovery from SP with regional brain blood flow reduced 20-30% from baseline. Nimodipine infusion after SP prevented postischemic hypoperfusion in all brain regions and increased blood flows by as much as 40% above baseline in midbrain and brainstem structures. Nimodipine infusion began after severe brain ischemia prevented postischemic hypoperfusion and enhanced brain blood flow in this model.