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 5×10−5 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 (≈100 μm) using a cranial window and intravital microscopy.
Results Before ischemia, kainate dilated arterioles by 16±2% at 5×10−5 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 5×10−5 and 10−4 mol/L kainate, respectively (P>.05). We also investigated the mechanisms involved in mediating arteriolar dilation to kainate. Intravenous administration of Nω-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.
Glutamate is one of the most prevalent neurotransmitters in the central nervous system and can activate three types of ionotropic receptors: NMDA, kainate, and AMPA.1 2 Several studies have shown that stimulation of NMDA receptors can dilate arterioles in the cerebral cortex through mechanisms involving synthesis and actions of NO.3 4 5 Similarly, activation of kainate receptors dilates cortical arterioles predominantly through an NO-dependent mechanism, at least in rabbits.6 In contrast, activation of AMPA receptors in cerebral cortex apparently does not affect the diameter of cerebral arterioles.6
Excitatory amino acids appear to be potent vasodilators in both adults3 5 and infants.4 Although kainate receptors are widely distributed in the newborn brain,7 kainate-dependent cerebrovascular changes in the perinatal period have not been characterized. In newborn piglets, we have shown that NMDA-induced arteriolar dilation is sensitive to hypoxic-anoxic stress. Thus, short exposure to arterial hypoxia,8 asphyxia,9 or total, global ischemia10 is able to reduce or abolish cerebral arteriolar dilation to NMDA. The susceptibility of kainate-induced cerebral arteriolar dilation to ischemic stress is unknown. However, in vitro experiments indicate that kainate receptors are less sensitive to oxidative stress than are NMDA receptors.11
The purpose of this study was to test the hypothesis that kainate dilates cerebral arterioles in newborn pigs. We examined the effects of ischemia on cerebral arteriolar dilation to kainate. We tested the hypothesis that kainate-induced arteriolar dilation would be resistant to ischemic stress. Since the relative contribution of NO to vasodilation is inversely related to the dose of kainate in rabbits,6 we examined the effects of ischemia on arteriolar responses to two doses of this agent. In addition, we investigated the contribution of NO, adenosine, and prostanoids to kainate-induced arteriolar dilation. The roles of adenosine and prostanoids in cerebral arteriolar dilation to kainate have not been examined previously. We chose to use the newborn pig because of similarities to babies in developmental status of the brain and in cerebrovascular control mechanisms. Furthermore, hypoxic/ischemic injury is a relatively common problem in the perinatal period.
Materials and Methods
Experiments were performed on newborn pigs (1 to 7 days) of either sex weighing 1 to 2 kg. The procedures used in this study were approved by the Institutional Animal Care and Use Committee. The piglets were anesthetized with sodium thiopental (30 mg/kg IP) and then α-chloralose (75 mg/kg IV). Additional amounts of α-chloralose were given as needed to maintain a stable level of anesthesia. The piglets were intubated and artificially ventilated. A femoral artery and vein were cannulated with polyethylene tubing (PE-90). Arterial blood pressure, gases, and pH were maintained within the normal physiological range. The head of each piglet was fixed in a stereotaxic apparatus, the scalp was cut, and the connective tissue over the parietal bone was removed. A craniectomy (19 mm in diameter) was made in the parietal bone. The dura was cut and reflected over the skull. A stainless steel and glass cranial window with three ports was put into the opening, sealed with bone wax, and cemented with cyanoacrylate ester followed by one or two layers of dental acrylic. The closed window was filled with aCSF, which was warmed to 37°C and equilibrated with 6% O2, 6.5% CO2, balance N2. Arterioles were observed with a microscope (Wild M36) equipped with a television camera (Panasonic), and arteriolar diameter was measured with a video microscaler (IV-550, For-A Co).
Cerebral Ischemia/Reperfusion Injury
We produced cerebral ischemia/reperfusion injury using a hollow brass bolt implanted in the right parietal cranium 20 mm rostral to the cranial window. Immediately after placement of the cranial window, we drilled a 3-mm hole in the skull using an electric drill with a toothless bit, and the dura was exposed. A hollow bolt was inserted and secured in place with cyanoacrylate ester and dental acrylic. After implantation of the window and the bolt, aCSF was allowed to equilibrate with the periarachnoid CSF under the window for 20 minutes. To induce ischemia, aCSF was infused to maintain intracranial pressure above mean arterial pressure so that blood flow through pial vessels was stopped. Venous blood was withdrawn as necessary to maintain mean arterial blood pressure near normal values. At the end of the 10-minute period of ischemia, the infusion tube was clamped, and the intracranial pressure was allowed to return to preischemia values.
Arteriolar Effects of Kainate
At the beginning of each experiment, the cranial window was flushed with aCSF several times until a stable baseline was obtained. Then arteriolar responses were determined to two levels of topically applied kainate (5×10−5 and 10−4 mol/L). Exposure to each level of kainate was 5 minutes. These doses of kainate were similar to those used by Faraci et al.6 To allow a rapid recovery of baseline diameter, we did not use kainate in concentrations higher than 10−4 mol/L.
Effects of Ischemia
After recovery from exposure to the two doses of kainate, animals were exposed to cerebral ischemia for 10 minutes. At 1 hour after ischemia, arteriolar responses were once again examined to both levels of kainate. We only examined arteriolar responses to kainate at 1 hour after ischemia because previous studies in piglets indicate that the major changes in responsiveness occur at this time.9 10
Time control animals were exposed to both levels of kainate twice at an interval similar to that in the ischemia experiments.
Mechanisms of Dilation to Kainate
To assess the contribution of NO, prostaglandins, and adenosine, we determined arteriolar responses to kainate before and after intravenous administration of L-NAME (15 mg/kg IV; 40 minutes before second kainate application), indomethacin (10 mg/kg IV; 20 minutes before second kainate application), combined L-NAME and indomethacin treatment, or theophylline (20 mg/kg IV; 15 minutes before second kainate application). We have shown previously that these agents are effective in the cerebral circulation.8 9 10 12
Specificity of Kainate
We assessed whether kainate is able to activate NMDA receptors by coapplying kainate with topical MK801 (10−5 mol/L). We have shown previously that MK801 abolishes arteriolar dilation to NMDA or glutamate under normal conditions in piglets.4
All values are expressed as mean±SEM. When appropriate, data were analyzed with the paired t test, repeated measures ANOVA, or one-way ANOVA. When the F value was significant, pairwise comparisons were made with the Student-Newman-Keuls test. A value of P<.05 was considered statistically significant.
Arterial blood pressures were within normal limits (55 to 60 mm Hg) for piglets in each group and did not change during application of kainate.
Topical kainate dilated pial arterioles in a dose-dependent fashion (Table⇓; Figs 1 through 4⇓⇓⇓⇓.). Arteriolar dilation to kainate was sustained and lasted as long as 15 to 20 minutes after the higher dose was flushed from the cortical surface with aCSF. After recovery from the first application, repeated application of kainate resulted in similar arteriolar dilation (Table⇓ [group 1]).
Ten minutes of global ischemia had no significant effect on arterial blood pH and blood gases (before ischemia: pH, 7.45±0.02; Pco2, 33±2 mm Hg; and Po2, 92±5 mm Hg; after ischemia: pH, 7.42±0.03; Pco2, 34±3; and Po2, 89±3 mm Hg). Sixty minutes after recovery from ischemia, mean arterial blood pressure (56±3 versus 54±4 mm Hg) and arteriolar diameter (Table⇑ [group 2]) were not significantly changed compared with the initial baseline value. However, on the basis of absolute (Table⇑) or percent changes from baseline (Fig 1⇑), ischemia had no significant effect on arteriolar dilator responses to kainate.
Intravenous administration of L-NAME resulted in an immediate but transient elevation of blood pressure. In this group baseline arterial blood pressure was 56±4 mm Hg. Forty minutes after L-NAME administration, arterial blood pressure stabilized at 62±6 mm Hg. Arteriolar dilator responses to kainate were reduced by approximately 50% by L-NAME pretreatment (Table⇑ [group 3] and Fig 2⇑).
Indomethacin treatment transiently increased arterial blood pressure, and 20 minutes after drug application it was significantly higher than baseline (60±7 versus 74±5 mm Hg; P<.05). Indomethacin pretreatment also reduced arteriolar dilation to kainate by 50% (Table⇑ [group 4] and Fig 3⇑).
Combined pretreatment with both L-NAME and indomethacin did not potentiate the hypertensive effect of indomethacin and L-NAME treatment (53±4 mm Hg before and 71±3 mm Hg after). The combined drug application strongly inhibited arteriolar dilation to kainate (Table⇑ [group 5] and Fig 4⇑). Although there was a trend for combined treatment to reduce arteriolar dilation to 10−4 mol/L kainate more than treatment with L-NAME or indomethacin alone, an ANOVA on the absolute and percent change data indicated that there was no significant difference.
In contrast to L-NAME and indomethacin, arteriolar dilator effects of kainate were unaffected by intravenous administration of theophylline (Table⇑ [group 6]). Arteriolar diameter increased first by 15±1% during application of 5×10−5 mol/L kainate and 31±3% during 10−4 mol/L kainate (n=7). Fifteen minutes after theophylline administration, arteriolar diameter increased by 19±3% during application of 5×10−5 mol/L and 36±4% during 10−4 mol/L kainate.
In group 7, during baseline conditions arteriolar diameter increased by 16±3% during application of 5×10−5 mol/L kainate and 26±3% during application of 10−4 mol/L kainate (n=6). Application of MK801 did not change baseline diameter (Table⇑). During coadministration of MK801, arteriolar diameter increased by 13±3% during application of 5×10−5 mol/L kainate and 24±3% during application of 10−4 mol/L kainate.
In groups 3 through 6, blood gas values were obtained before and after systemic drug administration. Intravenous L-NAME, indomethacin, or theophylline treatment did not change blood pH, Pco2, and Po2 values. (7.46±0.03, 34±3 mm Hg, and 94±4 mm Hg before intravenous drug administration and 7.42±0.04, 35±4 mm Hg, and 89±6 mm Hg after intravenous drug administration; n=30).
There are several major, new findings from this study. First, kainate is a potent dilator agent in the cerebral circulation of the newborn pig. Second, arteriolar dilation to kainate is due to mechanisms involving NO and prostaglandins but not due to actions of adenosine or through activation of NMDA receptors. Third, cerebral arteriolar dilator responses to kainate are not reduced substantially by ischemia. Thus, our results indicate that cerebral arteriolar dilator responses to kainate are multifactorial and also largely resistant to ischemic stress.
The basis for the present study was our previous demonstration that NMDA-induced arteriolar dilation was very sensitive to hypoxic/ischemic stress. Thus, we have shown that arteriolar dilator responses to NMDA are greatly reduced at 1 hour after 10 minutes of asphyxia or total, global ischemia.9 10 Therefore, the magnitude of this decrease in arteriolar responsiveness after asphyxia or ischemia is up to 70%. In addition, we have recently demonstrated that exposure to even short periods of moderate arterial hypoxia has similar effects.8 The mechanism involved in sustained suppression of NMDA-induced arteriolar dilation appears to be due to actions of oxygen radicals on the NMDA receptor rather than on bioavailability of NO or on synthetic capacity of NOS.9 13 Thus, effects of exogenous NO are not attenuated by ischemia, and levels of neuronal NOS and total NOS activity are not decreased at this time by ischemia.10
The present experiments suggest that the cerebral arteriolar dilator responses to kainate are more resistant to anoxic stress than to NMDA. Reasons for this difference in susceptibility to ischemia/reperfusion are unclear at this time. In contrast to the NMDA receptor, little information is available about the modulatory factors and the kainate receptor. It is known that NMDA receptors are associated with several binding sites, including a redox site, that are involved in suppression of NMDA receptor function.14 15 16 In one study it was found that NMDA receptors in cultured chick retinal cells were more affected by oxidative stress than were kainate receptors.11 The maintenance of normal arteriolar dilation to kainate after ischemia lends additional, independent support to our earlier observation that NOS activity and availability of vasoactive NO are not responsible for decreased arteriolar dilator responses to NMDA at 1 hour after ischemia in piglets.10
In a previous study Faraci et al6 showed that inhibition of NOS resulted in a reduction in arteriolar dilation during kainate administration in rabbits to a degree similar to that we observed in piglets. Thus, our results support their contention that NO mediates approximately one half of the cerebral vasodilation to kainate. Our results extend these findings to indicate that indomethacin is equally as potent as L-NAME in suppressing arteriolar dilation to kainate. Thus, indomethacin administration alone reduced arteriolar dilation by approximately 50%, and coadminstration of L-NAME and indomethacin almost completely abolished arteriolar dilator responses to kainate. The codependence of cerebral arteriolar dilation to kainate on NO and prostanoid mechanisms is similar to that of cerebral cortical vasodilation to electric stimulation of the rostral ventrolateral medulla.17 In that study increases in cortical blood flow due to medullary stimulation were blocked more by combined pretreatment with l-nitro-NG-arginine and indomethacin than by only one of these drugs. Thus, l-nitro-NG-arginine– and indomethacin-dependent cerebrovascular dilator response to medullary stimulation appeared to be additive. However, Armstead18 recently has presented evidence that cerebral arteriolar dilation to prostacyclin and prostaglandin E2 are completely blocked by L-NAME administration in piglets. Based on restoration of prostaglandin-induced dilation by addition of low amounts of exogenous NO by sodium nitroprusside, Armstead18 proposed that NO was exerting a “permissive” influence on vascular responses. Thus, only a small amount of NO is needed to allow prostaglandin-induced dilation. Unfortunately, our experimental design does not allow us to differentiate between independence and synergy between L-NAME– and indomethacin-dependent mechanisms in promotion of arteriolar dilation during kainate application.
In contrast to our results with L-NAME and indomethacin, administration of theophylline did not alter arteriolar dilation to kainate. We have shown previously that the dose of theophylline used was sufficient to block arteriolar dilator responses to topical adenosine and to arterial hypoxia.19 Thus, activation of adenosine receptors apparently plays little or no role in arteriolar dilator responses to kainate application. This situation could occur if blood flow increases induced by rapid actions of NO and prostaglandins were able to satisfy augmented metabolic demands due to kainate application.
Under normal conditions, topical application of glutamate activates cortical NMDA receptors and causes dilation of pial arterioles.4 Thus, under these conditions the contribution of kainate receptor activation to vascular changes is small. The reason for preference of glutamate for NMDA rather than kainate receptors is unclear. It may reflect access of glutamate to various receptors, relative Km for receptors, or the presence of factors that normally inhibit glutamate activation of kainate receptors. However, when NMDA but not kainate receptor function is impaired after hypoxic/ischemic stress, local changes in neuronal glial status or composition of extracellular fluid may promote kainate receptor activation by topical glutamate. Thus, after ischemia and perhaps other stressful conditions, activation of kainate receptors by glutamate may play an important role in linking changes in blood flow to alterations in metabolic rate.
Although our methods do not allow us to document the types of brain cells affected by kainate administration, it appears that stimulation of both neurons and glial cells could be involved in mediating arteriolar dilation. Kainate receptors have been shown to be present in high numbers on both neurons and glial cells in cortex but not to be present on cerebral resistance vessels.1 2 6 20 In contrast to kainate receptors, NMDA receptors appear to be located only on neurons in cortex.3 Our experiments with MK801 indicate that even high doses of kainate do not activate NMDA receptors.
Babies frequently are exposed to hypoxic/anoxic stress during the perinatal period, and activation of various glutamate receptors may contribute to or potentiate development of neurological sequelae. Considerable evidence suggests that release of glutamate and activation of NMDA receptors can lead to neurological damage. Several reports indicate that kainate receptors may also play an important role in cerebral pathogenesis after anoxic stress/reperfusion.21 22 Hypoxia selectively affects kainate binding in various brain regions.23 Furthermore, direct blockade of kainate receptors affords neuroprotection in various ischemia models.21 22 24 While our previous results indicate that NMDA receptor mechanisms are rapidly inhibited by hypoxic/ischemic stress,8 9 10 our new data indicate that kainate receptor activation is resistant to anoxic insult. We speculate that kainate receptor rather than NMDA receptor mechanisms could be the initial contributor to neurological damage during and after hypoxic/ischemic stress in neonates.
In summary, kainate is a potent dilator stimulus in the neonatal cerebral circulation, and mechanisms of arteriolar dilation appear to involve release and actions of NO and prostaglandins. In addition, cerebral arteriolar dilator responses to kainate are resistant to ischemic stress.
Selected Abbreviations and Acronyms
|aCSF||=||artificial cerebrospinal fluid|
|L-NAME||=||Nω-nitro-l-arginine methyl ester|
|NOS||=||nitric oxide synthase|
This study was supported by grants HL-30260, HL-46558, and HL-50587 from the National Institutes of Health.
- Received January 30, 1997.
- Revision received March 13, 1997.
- Accepted March 26, 1997.
- Copyright © 1997 by American Heart Association
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