Thomas M. Louis

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.

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/μg protein for time controls to 77 ± 36 pixels/μg protein (75% increase) for ischemia in parietal cortex (n = 9 to 10) and 22 ± 10 for control to 40 ± 16 pixels/μg 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.

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-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%. Pretreatment 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.

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 Abdominal Vagotomy on the Estrous Cycle of the Rat and the Induction of Pseudopregnancy

Abdominal vagotomy of estrus or proestrus rats resulted in disruptions of the estrous cycle which was characterized by prolonged periods of diestrus (10-12 days in length). In contrast, vagotomy on metestrus or diestrus did not disrupt the estrous cycle. The induction of pseudopregnancy, in response to cervical stimulation on the morning of estrus, was also interrupted by abdominal vagotomy. The nocturnal and diurnal prolactin surges and elevations in serum progesterone, characteristic of pseudopregnancy, were prevented by vagotomy. Vagotomy, also, largely prevented the formation of deciduoma in response to traumatization of the uterus in cervically stimulated rats.

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.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.

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.

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.

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.

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.