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CD is a hallmark feature of anoxic depolarization and CSD. CD is characterized by a negative direct current (DC) potential shift and cessation of EEG activity. Anoxic depolarization develops rapidly after the initiation of hypoxia or global cerebral ischemia. Neonates are commonly subjected to various lengths of asphyxia or cerebral ischemia, which severely impairs cerebrovascular reactivity. Previous studies indicate that dilator responses are vulnerable to cerebral ischemia, head trauma, and CSD, whereas constrictor responses are more resistant. The newborn pig is an appropriate model for studying neonatal cerebral circulation, because developmental stage and vascular responses are very similar to those of human babies. In piglets, global cerebral ischemia has been shown to attenuate cerebrovascular responses to various vasodilatory stimuli including NMDA, arterial hypercapnia, CGRP, and aprikalim, but not forskolin(1–4). However, an impaired vasodilation to cAMP analogues has been found after traumatic brain injury in which transient CD also occurs(5).

The above-mentioned agents dilate cerebral arterioles by different mechanisms. NMDA-induced vasodilation is a neuronally mediated multistep process involving the activation of neuronal nitric oxide synthase(6,7). Hypercapnic vasodilation is dependent on prostanoid synthesis and requires intact endothelium in the newborn pig(8,9). CGRP, the activation of KATP by aprikalim, and the adenyl-cyclase activator forskolin directly affect vascular smooth muscle cells. The transient suppression of these physiologically relevant vasodilatory responses may be of importance in determining the final neurologic outcome of the ischemic insult.

The possible involvement of CD in the mechanism of diminished vascular responsiveness is currently unknown. Diminished cerebrovascular responses after CSD have been reported. In fact, both in the lissencephalic rat and the gyrencephalic cat, an abolition of the hypercapnic vasodilation lasting up to 10 h after a single CSD has been reported(10–13), although cerebral autoregulation was preserved(10). In contrast, no change in cerebrovascular reactivity to arterial hypercapnia, CGRP, or acetylcholine was observed in the rabbit(14). In CSD, the CD is accompanied by a large increase in regional cerebral blood flow and is followed by return to baseline or by prolonged hypoperfusion(10–14). The regulatory mechanisms by which these changes of vascular tone occur are still unclear but seem to involve competing influences of nitric oxide, CGRP, and prostanoids(15–19). In contrast with the reported reduced vascular responsiveness after CSD, repeated CSD are capable of producing similar increases in cerebral blood flow without attenuation of the vascular response in all species studied(13–15). Therefore, the possible role of CD in the attenuation of cerebrovascular responses during ischemia or CSD is still controversial.

The purpose of the present study was to investigate cerebral arteriolar dilator responses after inducing CD by direct application of KCl. Unlike in adult animals of several species, CSD is difficult to elicit in the piglet (personal observations). However, topical application of KCl offers an alternative means to study the intactness of cerebrovascular responsiveness to CD. We tested the hypothesis that CD reduces pial arteriolar dilation in response to NMDA, hypercapnia, CGRP, aprikalim, and forskolin.

METHODS

In these experiments, newborn piglets of either sex (1-7 d old, body weight 1-2 kg) were used. All procedures were approved by the Institution Animal Care and Use Committee. The animals were anesthetized with sodium thiopental (30-40 mg/kg i.p.) followed by intravenous injection of α-chloralose (75 mg/kg). Supplemental doses of α-chloralose were given to maintain a stable level of anesthesia. The right femoral artery and vein were catheterized to record blood pressure and to administer drugs and fluids, respectively. The piglets were intubated via tracheotomy and were artificially ventilated with room air. The ventilation rate (∼20/min) and tidal volume (∼20 mL) were adjusted to maintain arterial blood gas values and pH in the physiologic range. In the present study, the piglets had normal values for arterial pH (7.40 ± 0.03), PCO2 (29.5 ± 1 mm Hg), and PO2 (99 ± 3 mm Hg) (n = 27).

Body temperature was maintained at 37-38°C by a water-circulating heating pad. The head of the piglet was fixed in a stereotactic frame. The scalp was incised and removed along with the connective tissue over the calvaria. A circular (19 mm in diameter) craniotomy was made in the left parietal bone. The dura was cut and reflected over the skull. A stainless steel cranial window with three needle ports was placed into the craniotomy, sealed with bone wax, and cemented with cyanoacrylate ester and dental acrylic.

Extracellular DC potential was recorded in some animals to demonstrate CD. The DC potential was derived from the potential between a silver/silver-chloride electrode placed on the frontoparietal cortex 5 mm anterior to the cranial window and a grounded reference electrode placed in the subcutaneous connective tissue of the scalp, amplified (EXT-MC, Experimetria Ltd., Hungary), and stored with an on-line data acquisition software.

The closed window was filled with artificial cerebrospinal fluid (aCSF) warmed to 37°C and equilibrated with 6% O2 and 6.5% CO2 in balance N2 to give pH = 7.33, PCO2 = 46 mm Hg, and PO2 = 43 mm Hg. The aCSF consisted of (in mmol/L) NaCl 132, KCl 2.9, CaCl2 1.2, MgCl2 1.4, NaHCO3 24.6, urea 6.7, and glucose 3.7. Diameters of pial arterioles were measured using a microscope (Wild M36, Switzerland) equipped with a video camera (Panasonic, Japan) and a video micro scaler (IV-550, For-A-Co., Newton, MA). After surgery, the cranial window was gently perfused with aCSF until a stable baseline was obtained. At the end of the experiments, the animals were killed while anesthetized with an intravenous bolus of KCl.

Experimental protocol. We examined the responses of cerebral arterioles to NMDA (10-5, 5 × 10-5, 10-4 mol/L, n = 9), hypercapnia (5 or 10% inhaled CO2, n = 8), aprikalim (10-6, 10-5 mol/L, n = 8), CGRP (10-7, 10-6 mol/L, n = 8), and forskolin (10-6, 10-5 mol/L, n = 8) before and 1 h after 3 min of CD. These doses of drugs and CO2 were selected to yield intermediate and large changes in diameter. Whenever possible, we obtained data for two different drugs in each animal. With this protocol, we obtained arteriolar responses to aprikalim, forskolin, CGRP, and CO2 similar to those we have observed before when only one of these drugs was given. Further, NMDA was not given with any other drug. The drugs were dissolved in aCSF and administered topically through the injectable ports of the cranial window onto the brain surface with a single application. Arteriolar diameters were measured continuously for 5 min for each dose. Then the window was flushed with aCSF. The second substance was administered when the arteriolar diameters returned to baseline values. Hypercapnia was elicited by artificially ventilating the animal with a gas mixture (5 or 10% CO2, 21% O2, balance N2) for 5 min. Our previous results indicate that inhalation of 5 or 10% CO2 in air results in elevation of PCO2 to 45-50 and 70-75 mm Hg, respectively, with a simultaneous drop in pH(2). CD was achieved by single topical application of 1 mol/L KCl dissolved in aCSF for 3 min. After CD, the cranial window was infused several times with aCSF until arteriolar diameters returned to baseline.

Drugs. The drugs used in this study were NMDA, forskolin (both from Sigma Chemical Co.), aprikalim (Rhone-Poulenc-Rohrer), and CGRP (Research Biochemical International).

Statistics. Data are expressed as mean ± SEM. Data were analyzed using repeated measures ANOVA, followed by pair-wise comparisons using the Student-Newman-Keuls test where appropriate.

RESULTS

Mean arterial blood pressure was in normal range (57 ± 2 mm Hg, n = 27) and did not change by topical drug application or hypercapnia throughout the measurements. In all animals, topical application of 1 mol/L KCl resulted in a large reduction of diameters of pial arterioles from 105 ± 3 to 48 ± 4 µm (n = 27). Also, there was a negative deflection in the extracellular DC potential (maximum 5-7 mV) indicating CD (Fig. 1). After 3 min, the KCl was washed out by repeated infusion of aCSF, and arteriolar diameters returned to baseline within 30-40 min.

Figure 1
figure 1

CD induced by 1 mol/L KCl solution. On the tracing, negative DC potential shift can be observed during KCl application. The CD lasts approximately 3 min.

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Topical application of NMDA (n = 9) resulted in a dose-dependent arteriolar vasodilation (Fig. 2). The baseline diameters were very similar (102 ± 2 versus 99 ± 3 µm before and after CD). Only at the highest dose was the absolute change in diameter slightly (∼10%) decreased. However, the percent changes were intact (9 ± 1 versus 8 ± 1 at 10-5 mol/L, 19 ± 2 versus 18 ± 3 at 5 × 10-5 mol/L, and 29 ± 2 versus 26 ± 3 at 10-4 mol/L). Thus, the NMDA-induced vasodilation was not significantly affected by prior CD.

Figure 2
figure 2

Effect of CD on pial arteriolar dilation to NMDA (n = 9), hypercapnia (CO2, n = 8), and aprikalim (APK, n = 8). Baseline vascular diameters (b) were similar before and 1 h after 3 min of CD. All stimuli induced dose-dependent pial arteriolar vasodilation that was essentially unchanged after CD by 1 M KCl. Values are expressed as mean ± SEM.

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Inspiration of a gas mixture containing 5 or 10% CO2 (n = 8) resulted in a concentration-dependent cerebral vasodilation (Fig. 2); the baseline diameters were not significantly different (97 ± 3 versus 91 ± 4 before and after CD). The percent changes were 15 ± 2 versus 16 ± 4 at 5% and 27 ± 5 versus 27 ± 6 at 10% inspired CO2. In addition, the arteriolar dilation to hypercapnia was sustained and did not wane after CD similarly to the first challenge. We conclude that hypercapnic vasodilation remains intact at 1 h after topical KCl application.

CGRP (n = 8) caused a dose-dependent increase in arteriolar diameter; the response remained largely intact after 1 h of topical KCl application. Baseline diameters were 108 ± 5 versus 103 ± 4 µm before and after CD, and the percent changes were 15 ± 3 versus 16 ± 2 at 10-7 mol/L and 26 ± 4 versus 22 ± 3 at 10-6 mol/L. The aprikalim-induced vascular dilation (n = 8) was dose dependent and completely resistant to CD (Fig. 2). The baseline diameters were 102 ± 5 versus 103 ± 6 µm before and after CD. The percent changes for aprikalim were 21 ± 4 versus 18 ± 3 at 10-6 mol/L and 36 ± 5 versus 34 ± 5 at 10-5 mol/L. Similarly, there was no change in the responsiveness of pial arterioles to forskolin (n = 8). Baseline diameters were 101 ± 5 versus 96 ± 6 µm before and after CD, and percent changes were 16 ± 2 versus 16 ± 4 at 10-6 mol/L and 34 ± 5 versus 37 ± 7 at 10-5 mol/L.

DISCUSSION

The major finding from the present study is that cerebrovascular reactivity remains intact after CD in piglets. Although there may be a tendency for slight decreases in the vascular responses, especially at the highest doses (NMDA, hypercapnia, CGRP, but not aprikalim or forskolin), these changes were not significant. The overall picture indicates intact responsiveness to all dilator stimuli studied after CD.

Topical NMDA administration induces pial arteriolar vasodilation. This effect is indirect because cerebral vessels lack NMDA receptors(20–22). The activation of neuronal NMDA receptors leads to the synthesis of nitric oxide by neuronal nitric oxide synthase, resulting in a dilation of cerebral arterioles(6,7). This mechanism was shown to be vulnerable to even short periods of hypoxia, ischemia, or asphyxia(23–25). In contrast, NMDA-induced vasodilation proved to be resistant to CD, indicating that other mechanisms are responsible for the attenuation of the vascular response to NMDA after a short hypoxic/ischemic insult. Indeed, our results suggest that cortical neurons can be activated and show normal responses to external stimuli such as NMDA soon after recovery from depolarization.

Hypercapnic acidosis produces cerebral vasodilation by mechanisms that have not been identified. Extracellular acidosis probably plays a crucial role. In the newborn piglet, hypercapnic vasodilation is dependent on prostanoids and requires intact endothelium(8,9). Hypercapnic vasodilation is abolished at 1 h after global cerebral ischemia in piglets(2). Also, after a single CSD, a complete abolition of the response to hypercapnia was reported in the rat and the cat(10–12) but not in the rabbit(14). In the present study, CD in piglets did not have any effect on the pial arteriolar vasodilation to hypercapnia, suggesting that endothelial function and prostanoid synthesis are unaltered after transient depolarization. However, changes in pial arteriolar diameter and those of cortical blood flow may not be parallel to all vasoactive stimuli.

Aprikalim causes vasodilation by opening KATP channels on vascular smooth muscle cells. Activation of potassium channels is an important cellular mechanism in a number of vasodilatory substances regulating vascular tone. KATP channels are sensitive to ischemic stress(4). Our present results show that the response to aprikalim is unchanged after CD. Forskolin is an activator of the adenyl-cyclase-cAMP system and the Ca2+-sensitive K+ channels (KCa) as well. The sensitivity of KCa channels is reduced by experimental head trauma in piglets(5). But our results show intact vascular responses to forskolin, indicating that these regulatory pathways are also unaltered after CD. CGRP induces vasodilation by activating receptors on the vascular smooth muscle cells. The cellular mechanisms of vasodilation include the activation of adenyl-cyclase and the activation of KATP channels as well(4). CGRP-induced vasodilation is severely reduced after global cerebral ischemia(3). In the present study, we found no significant attenuation in the vascular responses to CGRP after CD. These results suggest that vascular smooth muscle cells are functioning and capable of responding to different stimuli after CD.

The discrepancy between the results of the present study and those of the literature(10–13) remains to be explained. The CD achieved in this study was similar to the time course and magnitude of normal CSD recorded in other species. However, CD by KCl is different from CSD because, during CD, not only the cerebral cortex but also the pial vasculature has been depolarized. By use of intravital microscopy, a transient hypoperfusion has been observed by the sluggish blood flow in the constricted arterioles or recorded by laser-Doppler flowmetry (unpublished observations). Thus, direct depolarization of the brain surface by KCl is a significant stress, whereas CSD is usually regarded as a major but transient and ultimately benign perturbation of brain blood flow and metabolism. The fact that we do not see any substantial change in the arteriolar response to either vasodilatory stimuli at 1 h after 3 min of CD makes a pronounced effect of a single CSD unlikely. Further, if blood flow and/or cerebral metabolism are already compromised, CD/CSD may be more deleterious to the integrity of cerebrovascular regulatory mechanisms.

In summary, our data indicate that CD by itself does not change the responsiveness of the cerebral vasculature to several stimuli. This finding may have implications in the pathophysiology of the reduced cerebrovascular reactivity after ischemic conditions.