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Am J Physiol Heart Circ Physiol 287: H331-H339, 2004. First published February 12, 2004; doi:10.1152/ajpheart.00831.2003
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Influence of the glia limitans on pial arteriolar relaxation in the rat

H. L. Xu, H. M. Koenig, S. Ye, D. L. Feinstein, and D. A. Pelligrino

Neuroanesthesia Research Laboratory, Department of Anesthesiology, University of Illinois, Chicago, Illinois 60607

Submitted 28 August 2003 ; accepted in final form 9 February 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We examined whether damage to the glia limitans (GL), via exposure to the gliotoxin L-{alpha}-aminoadipic acid (L-{alpha}AAA), alters hypercapnia-induced pial arteriolar dilation in vivo. Anesthetized female rats were prepared with closed cranial windows. Pial arteriolar diameters were measured using intravital microscopy. L-{alpha}AAA (2 mM) was injected into the space under the cranial windows 24 h before the study, and injury to the GL was confirmed by light microscopy. L-{alpha}AAA was associated with a reduction in pial arteriolar CO2 reactivity to 40–50% of the level seen in vehicle-treated controls, with no further reduction in the CO2 response after nitric oxide (NO) synthase (NOS) inhibition via N{omega}-nitro-L-arginine (L-NNA). Subsequent blockade of prostanoid synthesis, via indomethacin (Indo), reduced CO2 reactivity to 10–15% of normal. In vehicle-treated controls, L-NNA, followed by Indo, reduced the response to ~50% and then to 15–20% of the normocapnic value, respectively. On the other hand, L-{alpha}AAA had no effect on vascular responses to the endothelium-dependent vasodilator acetylcholine or the NO donor SNAP and did not alter cortical somatosensory evoked responses. This indicates an absence of any direct L-{alpha}AAA actions on pial arterioles or influence on neuronal transmission. Furthermore, L-{alpha}AAA did not alter the vasodilation elicited by topical application of an acidic artificial cerebrospinal fluid solution, suggesting that the GL influences the pial arteriolar relaxation elicited by hypercapnic, but not local extracellular (EC), acidosis. That differences exist in the mechanisms mediating hypercapnia- versus EC acidosis-induced pial arteriolar dilations was further exemplified by the finding that topical application of a neuronal NOS (nNOS)-selective blocker (ARR-17477) reduced the response to hypercapnia (by ~65%) but not the response to EC acidosis. Disruption of GL gap junctional communication, using an antisense oligodeoxynucleotide (ODN) connexin43 knockdown approach, was accompanied by a 33% lower CO2 reactivity versus missense ODN-treated controls. These results suggest that the GL contribution to the hypercapnic vascular response appears to involve the NO-dependent component rather than the prostanoid-dependent component and may involve gap junctional communication. We speculate that the GL may act to facilitate the spread, to pial vessels, of hypercapnia-induced vasodilating signals arising in the comparatively few scattered nNOS neurons that lie well beneath the GL.

connexin; gap junction; hypercapnia; L-{alpha}-aminoadipic acid; nitric oxide; prostanoids

BECAUSE OF THE INTIMATE ANATOMIC RELATIONSHIP between cerebral resistance vessels and astrocytes (19), it has been postulated that astrocytes play a vital role in the regulation of local perfusion in the brain (7). In fact, experimental evidence to that effect has been published (43). Small pial arterioles (<50 µm), although exhibiting a relative paucity of direct neural connections (8, 33), nevertheless appear to be regulated by extravascular influences (23, 38). For many years, investigators have utilized pial arterioles as surrogates in studies on the regulation of cerebral arteriolar function in vivo. In contrast to the radial ensheathment of astrocytes observed in parenchymal arterioles (5, 19), only one side of pial arterioles is in proximity to astrocytes. That is, pial vessels overlie a thick layer of astrocytic processes termed the glia limitans (GL). That "one-sided" association could account for the observation that the blood-brain barrier protein marker endothelial barrier antigen (EBA) in pial vascular endothelium is expressed only on the side of the vessel in contact with the GL (1).

In the present study, we examined whether the pial vessel/GL arrangement has any relevance to the reactivity of pial arterioles to vasodilating stimuli in vivo, particularly when the vascular responses are mediated by processes originating outside the vessel. Hypercapnia would appear to be one such stimulus. With few exceptions (28), evidence from multiple laboratories (9, 10, 29, 30, 37) points to neuronal nitric oxide (NO) synthase (nNOS)-generated NO playing major role in adult rodent CO2-induced cerebral vasodilation in vivo (including pial arterioles). However, nNOS-containing neurons are sparsely distributed in the rat cerebral cortex and are virtually absent from the outermost cortical layer (layer I) (34). Moreover, it is unlikely that other neuronal factors influence the hypercapnic response of pial arterioles, because nitroxidergic innervation of these arterioles does not extend to vessels smaller than ~100 µm in diameter (33). Furthermore, with only one exception (28), local blockade of neuronal transmission (with TTX) has not been found to alter hypercapnia-induced pial arteriolar dilation (23) or hypercapnic cerebral vasodilation in general (3, 42). Despite this, hypercapnia elicits a global dilation of pial arterioles, a response that is substantially diminished by selective nNOS blockade (29, 37).

Because of their unique association with neurons and pial vessels, we hypothesized, therefore, that astrocytes, via the GL, may act to transmit and amplify signals arising in comparatively few cells to the entirety of the pial arteriolar system. To that end, we determined whether injury of the GL [via topical exposure to the purported gliotoxin L-{alpha}-aminoadipic acid (L-{alpha}AAA)] alters hypercapnia-induced pial arteriolar dilation. On the basis of previous findings from our laboratory (40) showing that the gap junctional protein connexin43 (Cx43) is abundantly expressed in the GL, we postulated that Cx43-dependent gap junctional communication might serve to facilitate the spread of vasodilating signals along the GL, resulting in a more "global" influence on pial vessels. Thus we evaluated CO2 reactivity in rats where GL Cx43 expression was knocked down using an antisense oligodeoxynucleotide (ODN) strategy (40). In addition, previous studies have indicated that isocapnic local reductions in pH can dilate cerebral vessels in vivo and in vitro (2, 14, 16, 32). This led us to hypothesize that differences exist in the mechanisms mediating pial arteriolar relaxation elicited by hypercapnic acidosis versus local (extracellular) acidosis, to the extent that the vasodilation produced via topical application of an acidic artificial cerebrospinal fluid (aCSF) solution may not be influenced by the GL.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The experimental protocol was approved by the Institutional Animal Care and Use Committee. Age-matched female Sprague-Dawley rats (300–350 g) were used (supplied by Charles River; Wilmington, MA). Pial arteriolar reactivities were evaluated using a closed cranial window and intravital microscopy system (39). The windows were placed on the day preceding study using a procedure described in a previous paper from our laboratory (39). The rats were anesthetized with halothane, intubated, paralyzed with a short-acting muscle relaxant (vecuronium), and mechanically ventilated with a 0.8% halothane-70% N2O-30% O2 gas mixture. After window preparation, 300 µl of an aCSF solution that was either drug free (vehicle control) or containing L-{alpha}AAA (2 mM) was injected into the space under the cranial window. Pilot studies established that the pH values in drug-free and L-{alpha}AAA (2 mM)-containing, gas-equilibrated (20% O2-5% CO2-75% N2) aCSF solutions were quite similar, differing by <0.02 pH units. The cranial window access ports were then plugged, and the skin overlying the skull was sutured together. The animal was permitted to recover from anesthesia, extubated, and returned to its cage. On the day of study, after anesthesia induction with halothane and paralysis (curare), the rats were tracheotomized and mechanically ventilated. Bilateral femoral arterial and venous catheters, used for arterial blood gas measurement and drug infusion, were inserted under continuous anesthesia with 0.8% halothane-70% N2O-30% O2. After catheterization, the rat was placed in a head holder, and the cranial window inflow, outflow, and intracranial pressure monitoring cannulae were connected. Halothane was discontinued, and a 10 µg/kg fentanyl bolus was given intravenously. Anesthesia was maintained during the study with fentanyl (25 µg·kg–1·h–1 iv) and 70% N2O-30% O2. The space under the window was filled with aCSF (pH {approx}7.35–7.40) that was equilibrated with a gas mixture consisting of 20% O2-5% CO2-balance N2. The aCSF solution was suffused at 1.0 ml/min and controlled at 37°C. Body temperature was maintained at 37°C with a servo-controlled heating pad, and mean arterial pressure and intracranial pressure were monitored continuously during the experiment.

Vascular reactivity was assessed by measuring the diameters of pial arterioles (30–50 µm). The vessels were viewed by video microscopy, and measurements were made using a calibrated video microscaler (see Ref. 39). In all experiments, the initial diameter measurements were made >1 h posthalothane and after 40 min of drug-free aCSF suffusion. Two experimental series were evaluated. The first examined L-{alpha}AAA effects on hypercapnia-induced pial arteriolar dilation. For these experiments, the pial arteriolar response to 3-min hypercapnia [arterial PCO2 (PaCO2) {approx}65 mmHg] was measured. The results were expressed in terms of CO2 reactivity (percent diameter increase/mmHg PaCO2 change). After the return to normocapnia, pial arteriolar reactivity to the endothelium and NO-dependent vasodilator acetylcholine (ACh; 10 and 100 µM) was measured. Subsequently, pial arteriolar responses to suffusions of a NO donor [S-nitroso-N-acetyl penicillamine (SNAP); at 0.1 and 1.0 µM] were assessed. Diameter measurements from three separate pial arterioles were obtained (and averaged) after 5 min of suffusion at each concentration. The second series examined whether L-{alpha}AAA-induced GL damage affected pial arteriolar dilations elicited by topical application of an acidic aCSF solution. Thus, after a period of suffusion with a control aCSF solution and measurement of baseline diameters, the suffusate was replaced by a solution where the HCO3– content was reduced by 50% (more NaCl was added to maintain osmolality at normal levels). That solution was equilibrated with the "standard" gas mixture (containing 5% CO2). Pial arteriolar diameters were measured after 15 min. An additional set of experiments within this series was performed to examine whether differences exist in nNOS dependency when comparing hypercapnia- with acidic aCSF-induced responses. For these experiments, cranial windows were implanted 24 h before study (see earlier vehicle controls). The rats were sequentially exposed to 3 min of hypercapnia, 15 min of normocapnia, and 15 min of acidic aCSF. After the return to baseline suffusion (normal pH), the nNOS-selective inhibitor ARR-17477 (300 µM) was added to the suffusate. After 40 min, the above sequence was repeated.

Although the literature strongly indicates an absence of any direct neurotoxic actions of L-{alpha}AAA (except at very high concentrations; see, for example, Refs. 11 and 25), we nevertheless sought to examine whether 24-h exposure to topically applied L-{alpha}AAA affected cortical neuronal function. This was accomplished through monitoring cortical somatosensory evoked potentials (CSSEPs). As described in earlier publications from our laboratory, the CSSEPs were generated by needle electrodes placed subcutaneously in the whisker region (20). The stimulus current was 4.0 mA, with a duration of 0.1 ms and a frequency of 5.5 Hz. At least 500 consecutive responses were averaged. Recordings were taken from the contralateral cortex via stainless steel screws placed immediately adjacent to the region of the cortical surface exposed by the cranial window. Recordings were obtained on 2 consecutive days. The anesthesia and surgical preparation protocols were similar to those described above, with the exception that, on day 1, the rats were switched from halothane-N2O to fentanyl-N2O after surgery. The first series of recordings were made over 45–90 min after halothane discontinuation. Specifically, we measured the amplitude of the initial cortical wave complex [taken as the voltage difference (in µV) between the adjacent positive and negative wave peaks observed at 8–15 ms poststimulus]. This was immediately followed by an injection of 300 µl of vehicle or L-{alpha}AAA solution into the space under the cranial window. On the second day, the rats were anesthetized (see above) and femoral arterial and venous catheters were inserted. The animals were again prepared for CSSEP evaluations, with recording and stimulating electrodes placed in positions identical to those used on day 1. Recordings were made at 45–90 min posthalothane.

At the end of some experiments (5 rats from each group), brains were perfusion fixed with 4% paraformaldehyde, in PBS, introduced transcardially. The brains were then cut into blocks and placed into 4% paraformaldehyde and stored at 4°C until commencement of paraffin embedding. Eight-micrometer coronal sections were prepared from the paraffin-embedded blocks with the use of a microtome (39). The expression of the astrocytic marker protein glial fibrillary acidic protein (GFAP) or nNOS were assessed via immunofluorescence. For GFAP, the primary antibody used was a goat polyclonal (from Dako; diluted 250:1). For nNOS, we used a rabbit polyclonal antibody (from Transduction Laboratories; diluted 50:1). The secondary donkey anti-goat and donkey anti-rabbit antibodies (conjugated to Texas red or dichlorotriazinylaminofluorescein) were diluted 500:1.

An additional series of rats was utilized to evaluate whether Cx43-related gap junctional communication was involved in the pial arteriolar response to hypercapnia. We employed an antisense ODN strategy identical to the one described in a recent publication from our laboratory (40). Thus 300 µl of a solution containing either 5 µM Cx43 antisense (5'-GTCACCCATGTCTGG-3') or 5 µM Cx43 missense ODN (5'-CTGGACCAGTGCTCT-3') was injected into the space under the cranial window (see above) 24 h before pial arteriolar reactivity was analyzed. Six bases (3 at the 5'-end and 3 at the 3'-end) were phosphorothioated so as to minimize nuclease-mediated ODN breakdown. On the day of study, pial arteriolar responses to CO2 and SNAP were evaluated as described above. It was shown, in a recent publication from our laboratory, that Cx43 antisense (but not missense) ODN injection resulted in a substantial reduction in Cx43 expression in pial vessels (and the GL) underlying the cranial window (40).

In all experiments, arterial blood samples were taken at 30-min intervals for arterial PO2 (PaO2), PaCO2, and pH analysis using a Radiometer Copenhagen blood gas/pH analyzer (model ABL 520). PaO2 (≥100 mmHg), PaCO2 (30–40 mmHg), and pH ({approx}7.40) were maintained during the study.

All reagents were obtained from Sigma (St. Louis, MO) and dissolved in aCSF. Statistical comparisons of pial arterioles diameter values within groups were made using one-way ANOVA combined with a post hoc Tukey analysis. A level of P < 0.05 was considered significant in all statistical tests. Values are presented as means ± SE.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
In all experiments, arterial blood variables were within normal limits. That is, with the exception of brief periods of imposed hypercapnia, PaCO2, pH, and mean arterial blood pressure (MABP) in these groups did not show any significant differences when comparing initial and final values over the course of the experiments. Furthermore, no significant differences were observed when comparing PaCO2, pH, and MABP values at equivalent experimental time points in the L-{alpha}AAA and vehicle groups and in the Cx43 missense versus antisense ODN groups. The initial values measured in the above groups are presented in Table 1, along with initial pial arteriolar diameters. PaO2 values (not shown) were maintained above 100 mmHg in all rats studied.


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  		Table 1. Initial pial arteriolar diameters and arterial blood variables

 
Representative patterns of cortical GFAP expression at 24 h after vehicle or L-{alpha}AAA treatment are shown in Fig. 1. Note that in vehicle-treated brains (Fig. 1, A–C) expression of the astrocyte marker is highly concentrated in the glia limitans. On the other hand, in rats exposed to topical L-{alpha}AAA (Fig. 1, D–F) for 24 h, there was a reduction in the intensity of expression in the GL, with a loss of continuity (appearance of gaps). In addition, in the L-{alpha}AAA-treated brains, we often observed a fragmented expression of GFAP immediately below the cortical surface. We suspect that this represents "remnants" of dead or damaged astrocytes. However, we did not observe any overt signs of L-{alpha}AAA influence on astrocytes lying >100 µm below the cortical surface. These findings reveal that L-{alpha}AAA does indeed damage the GL.



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  		Fig. 1. Representative glial fibrillary acidic protein (GFAP) expression patterns in vehicle-treated (A–C) and L-{alpha}-aminoadipic acid (L-{alpha}AAA)-treated (D–F) rats. Each panel corresponds to a different rat. Note that, in normal (vehicle treated) brains, GFAP is highly concentrated in the glia limitans. However, in rats exposed to topical L-{alpha}AAA for 24 h, GFAP expression in the glia limitans takes on a patchy appearance, exhibiting a loss of continuity. Additionally, the L-{alpha}AAA-treated brains often exhibit a fragmented expression of GFAP near the brain surface. Scale bar = 50 µm.

 
Pial arteriolar responses to the NO donor SNAP in vehicle-treated and L-{alpha}AAA-treated rats are summarized in Fig. 2. The absence of any differences in vascular reactivity to SNAP in the two groups indicates that L-{alpha}AAA does not injure cerebral vascular smooth muscle cells and that damage to the GL does not alter smooth muscle function in pial arterioles. A similar lack of any L-{alpha}AAA-induced change was seen when measuring pial arteriolar reactivity to the endothelium- and NO-dependent vasodilating agonist ACh (Fig. 3). This suggests that L-{alpha}AAA does not damage vascular endothelium. The endothelium dependency of the arteriolar response to ACh in female rats has been demonstrated in previous publications from our laboratory (24).



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  		Fig. 2. Pial arteriolar responses to the nitric oxide (NO) donor S-nitroso-N-acetyl penicillamine (SNAP) in vehicle-treated (n = 5) and L-{alpha}AAA-treated (n = 6) rats. The absence of any change in vascular reactivity to SNAP indicates that L-{alpha}AAA does not affect pial vascular smooth muscle function. The absence of any effect of N{omega}-nitro-L-arginine (L-NNA; 1 mM, topical application) confirms that SNAP acts independently from endogenous generation of NO. Values are means ± SE.

 


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  		Fig. 3. Pial arteriolar responses to the endothelium-and NO-dependent vasodilating agonist acetylcholine (ACh) in vehicle-treated (n = 5) and L-{alpha}AAA-treated (n = 6) rats. The absence of any change in vascular reactivity to ACh indicates that L-{alpha}AAA is not toxic toward endothelial cells. The loss of ACh reactivity in the presence of L-NNA (1 mM, topical application) confirms, as expected (11), that ACh acts exclusively via endothelial NO synthase (eNOS)-derived NO. *P < 0.05 vs. initial value. Values are means ± SE.

 
No significant variations in the amplitudes of the CSSEP were noted in either group of animals when comparing results obtained on day 1, before vehicle or L-{alpha}AAA administration, to those obtained on the second day (Fig. 4). The lack of any apparent effects of L-{alpha}AAA on the primary cortical SSEP wave suggests that topically applied L-{alpha}AAA does not have any influence on neurons underlying the GL.



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  		Fig. 4. Cortical somatosensory evoked potential (CSSEP) amplitudes measured at 24 h after topical application of vehicle (n = 3) or L-{alpha}AAA (n = 4). The results are expressed as a percentage of the initial amplitude value measured immediately before vehicle or L-{alpha}AAA administration (25.2 ± 4.6 or 21.6 ± 2.0 µV, respectively). The amplitudes were taken as the potential difference between the positive and negative peaks of the primary cortical wave complex.

 
Pial arteriolar responses to hypercapnia in vehicle- and L-{alpha}AAA-treated animals are summarized in Fig. 5. The data is expressed as CO2 reactivity (percent pial arteriolar diameter change/mmHg increase in PaCO2). Note that, under control conditions, inhibition of NOS, with topically applied N{omega}-nitro-L-arginine (L-NNA; 1 mM), reduced the hypercapnic response to ~50% of baseline, with a further reduction to 15–20% of baseline in the presence of combined NOS and prostanoid synthesis inhibition. This is similar to findings reported in earlier publications (36, 38). On the other hand, in the presence of L-{alpha}AAA, the CO2 reactivity was 50–60% lower than that seen in vehicle-treated rats, with no addition change upon addition of the NOS inhibitor. However, the prostanoid-dependent component of the hypercapnic response was retained because the subsequent addition of indomethacin (Indo) reduced CO2 reactivity to 10–15% of the level seen in control rats. Earlier findings from our laboratory indicated that hypercapnia-induced pial arteriolar dilation occurred via an endothelium-independent, but nNOS-dependent, process (29, 36, 38). Similar to previously reported observations, immunohistochemical analysis revealed nNOS expression only in widely scattered cortical neurons but not in pial arterioles or astrocytes (Fig. 6). Furthermore, nNOS-positive neuronal cell bodies were located in the deeper cortical layers (see Ref. 34), at a considerable distance from the brain surface and pial vessels. Viewed at a higher magnification, the nNOS-positive neurons appeared to display numerous contacts with astrocytic processes (Fig. 6). No apparent differences in nNOS expression were noted when comparing vehicle- and L-{alpha}AAA-treated animals.



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  		Fig. 5. Pial arteriolar responses to hypercapnia (3-min exposure) in vehicle-treated (n = 5) and L-{alpha}AAA-treated (n = 6) animals. The data is expressed as CO2 reactivity [percent pial arteriolar diameter change/mmHg increase in arterial PCO2 (PaCO2)]. In the vehicle-treated group, the initial normocapnic and hypercapnic PaCO2 values were 38.3 ± 2.2 and 66.9 ± 2.5 mmHg, respectively, whereas during L-NNA (1 mM topical) and then L-NNA + indomethacin (Indo; 10 mg/kg iv), those values were 36.1 ± 4.2 and 66.2 ± 3.1 mmHg and 36.1 ± 3.4 and 66.3 ± 4.0 mmHg, respectively. In the L-{alpha}AAA-treated rats, the normocapnic and hypercapnic PaCO2 values were, respectively, 37.2 ± 4.6 and 69.6 ± 4.4 mmHg (initial), 33.7 ± 2.6 and 63.3 ± 4.1 mmHg (L-NNA), and 33.0 ± 1.5 and 64.2 ± 2.7 mmHg (L-NNA + Indo). *P < 0.05 vs. initial; {dagger}P < 0.05 vs. vehicle; ¶P < 0.05 vs. L-NNA. Values are means ± SE.

 


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  		Fig. 6. Immunohistochemical staining revealing neuronal NOS (nNOS) expression in sparsely scattered cortical neurons (arrowheads) located at a substantial distance from the cortical surface (arrows) in vehicle (A) and L-{alpha}AAA (B)-treated rats. At a higher magnification, the close association between nNOS-positive neurons (green) and astrocytes (GFAP staining in red) is revealed for both vehicle-treated (C) and L-{alpha}AAA-treated rats (D). Scale bars = 100 µm (A and B) and 20 µm (C and D).

 
In both vehicle-treated and L-{alpha}AAA-treated groups, switch over from suffusion of a pH normal (7.40 ± 0.03 and 7.40 ± 0.01, respectively) to a pH-reduced aCSF solution (6.96 ± 0.04 and 7.00 ± 0.03, respectively) was accompanied by a rather substantial pial arteriolar dilation. The pH sensitivities of pial arterioles (percent diameter change/0.1 pH unit reduction) were virtually identical in vehicle and L-{alpha}AAA-treated rats (Fig. 7). The nNOS dependency of the pial arteriolar response to hypercapnia vs acidic aCSF is demonstrated by the data summarized in Fig. 8. In confirmation of previous findings (29), topical application of the nNOS-selective inhibitor ARR-17477 (300 µM) reduced CO2 reactivity by ~65%. On the other hand, ARR-17477 had no effect on pH sensitivity.



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  		Fig. 7. Pial arteriolar responses to a reduction in suffusate pH (15-min exposure) in vehicle-treated (n = 4) and L-{alpha}AAA-treated (n = 4) animals. The data are expressed as H+ reactivity (percent pial arteriolar diameter change/0.1 pH unit reduction). Values are means ± SE.

 


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  		Fig. 8. Effects of topical application of the nNOS-selective blocker ARR-17477 (300 µM) on the pial arteriolar responses to a reduction in suffusate pH [A: expressed as H+ reactivity (percent pial arteriolar diameter change/0.1 pH unit reduction)] and to hypercapnia [B: expressed as CO2 reactivity (percent pial arteriolar diameter change/mmHg increase in PaCO2)]. The normocapnic and hypercapnic PaCO2 values before ARR-17477 suffusion (initial) were 33.3 ± 2.5 and 69.7 ± 4.6 mmHg, respectively. In the presence of ARR-17477, those values were 33.5 ± 0.9 and 76.2 ± 3.3 mmHg, respectively. Values are means ± SE; n = 3. *P < 0.05 vs. initial.

 
Downregulation of Cx43 expression in the GL, using an antisense ODN-based strategy (see Ref. 40), was accompanied by an approximately one-third lower pial arteriolar CO2 reactivity compared with results obtained in missense ODN-treated rats (Fig. 9A). No differences in the pial arteriolar responses to the NO donor SNAP, which acts directly on the vascular smooth muscle, were seen when comparing missense and antisense ODN-treated rats (Fig. 9B). These results imply that gap junctional communication, perhaps within the GL, plays some role in the pial arteriolar responses to hypercapnia.



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  		Fig. 9. A: effect of connexin43 (Cx43) downregulation in the glia limitans and neighboring tissue (see Ref. 40) on pial arteriolar CO2 reactivity. Cx43 knockdown was achieved using a topically applied antisense oligodeoxynucleotide (ODN; n = 9), with results compared with a control group given a missense ODN (n = 7). To demonstrate that gap junctional disruption selectively affects the hypercapnic response, pial arteriolar responses to the NO donor SNAP (B) were compared in the same missense and antisense ODN-treated rats. The normocapnic and hypercapnic PaCO2 values in the missense-treated group were 36.6 ± 1.9 and 63.7 ± 1.6 mmHg, respectively. In the antisense ODN-treated rats, those values were 34.6 ± 1.3 and 67.4 ± 2.3 mmHg, respectively. *P < 0.05 vs. missense. Values are means ± SE.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
There were several key findings in this study. First, 24-h exposure to a topically applied gliotoxin, L-{alpha}AAA, led to a reduction in brain surface expression of the astrocytic marker GFAP, indicating damage to the GL. Second, that injury was accompanied by a diminution in pial arteriolar CO2 reactivity without any changes in vascular reactivity to local extracellular acidosis, endothelium-dependent vasodilating function, general vascular smooth muscle responsiveness to vasodilating stimuli, or neural function. Third, gap junctions may be involved in hypercapnia-induced pial arteriolar dilation. The last was identified using an antisense ODN approach that essentially only affects cells at, or close to, the cortical surface. This suggests that astrocyte-to-astrocyte gap junctional communication within the GL may play a role in the vascular CO2 response. These findings, therefore, raise the possibility that astrocytes, including those comprising the GL, may act as a unit (syncytium) in conveying a signal, arising in cortical neurons far removed from the brain surface, to pial arterioles. This could explain how scattered nNOS-positive neurons in the cerebral cortex are capable of effecting hypercapnia-induced vasodilation over the entire pial vasculature. The apparent astrocytic contribution seems to involve the NO-related, but not the prostanoid-dependent, component of the CO2 response.

L-{alpha}AAA is a homolog of L-glutamic acid and an intermediate of lysine metabolism that can occur naturally in the brain (6). The mechanism behind the capacity for L-{alpha}AAA to selectively damage astrocytes, without affecting neurons or vascular cells, is not precisely known. One possibility relates to the observation that L-{alpha}AAA, at least in the cerebral cortex, is selectively taken up into astrocytes, perhaps via an astrocyte-specific glutamate transporter (25). The presence of L-{alpha}AAA may, in turn, lead to reductions in intracellular glutamate levels and cystine uptake (e.g., via interference with cystine/glutamate exchange or {gamma}-glutamyl transpeptidase activity), resulting in depletion of glutathione and, eventually, oxidative cell damage (25, 27). In the present study, the absence of any direct L-{alpha}AAA actions on pial vascular endothelium and smooth muscle function was evidenced by the observation that no changes in ACh or NO donor responses in pial arterioles were seen in the presence of L-{alpha}AAA.

We also attempted to examine whether L-{alpha}AAA had any neurotoxic effects. The presence of L-{alpha}AAA did not result in any measurable changes in the size of the primary cortical wave amplitude of the somatosensory evoked response. This indicates that topically applied L-{alpha}AAA did not affect neuronal function in the underlying tissue. However, we suspect that this is probably the result of L-{alpha}AAA acting superficially rather than reflecting an absence of direct L-{alpha}AAA actions toward neurons. Indeed, within the brain parenchyma, the importance of astrocytes in supporting neural transmission is well established and is consistent with the numerous sites of contact between these two cell types, especially in the vicinity of synapses (15). Thus damage to parenchymal astrocytes may have an impact on neural transmission within the cortex. On the other hand, the astrocytic processes comprising the GL have very limited contacts with neurons and their synapses. Accordingly, damage to this superficial layer is less likely to have an appreciable effect on synaptic function and CSSEP amplitudes. Yet, even if L-{alpha}AAA had penetrated into the neuropil, a direct neurotoxic action is not supported in the vast majority of relevant publications, spanning several decades (for example, Refs. 11, 18, 25, and 31). Furthermore, the neurons that are seemingly involved in the hypercapnic response are located at sites far removed from the brain surface and the influence of topical L-{alpha}AAA exposure. This was confirmed in the present study by the finding that nNOS neuron perikarya were seen only in the deeper cortical layers, although the present immunohistochemical procedure may not have been sufficiently sensitive to detect nNOS within the multiple processes extending from these neurons, perhaps toward the brain surface. Nevertheless, even in the unlikely scenario that some neurotoxicity could be elicited by L-{alpha}AAA, it seems improbable that damaging amounts of topically applied L-{alpha}AAA could reach nNOS-positive neurons.

We recently reported that Cx43 is 1) abundantly expressed at the brain surface in rats; 2) found in both vascular and nonvascular cells; and 3) is particularly concentrated in the GL (Ref. 40; see also Refs. 17 and 26). The expression of Cx43 is often linked to the presence of gap junctions between cells. Gap junctions between astrocytes are well described in the literature. On the other hand, heterocellular gap junctions, particularly between neurons and astrocytes, although having been identified in the central nervous system, are much less abundant (for a review, see Ref. 4). Although there is good evidence for the existence of gap junctional intercellular communication within cerebral vessels, including pial arterioles (41), gap junctions between astrocytes and cerebral blood vessels have not been identified. In consideration of the high abundance of Cx43 in the GL, the previous observation that the influence of topically applied Cx43 antisense ODN does not extend much beyond the GL (40), and the present finding that antisense ODN treatment is accompanied by a reduction in pial arteriolar CO2 reactivity, it is tempting to conclude that gap junctions between astrocytes of the GL participate in the CO2 response. However, the magnitude of the reduction in CO2 reactivity associated with ODN-induced Cx43 knockdown (~33%) was comparatively less than that seen with nNOS inhibitors [50–70% (Fig. 8); see also Refs. 29 and 37] or, in the present study, with L-{alpha}AAA (50–60%).

Whether those collective results actually can be used to provide an approximation of the contributions from GL gap junctions to hypercapnia-induced pial arteriolar dilations is uncertain. Alternative explanations include the possibility that the lesser effect of the antisense ODN reflects an incomplete knockdown of Cx43 protein (40) or that gap junctions composed of other connexins are involved. One might also consider contributions from gap junctions residing within the vessels themselves. However, there are two key observations that seem to speak against this. First, present and previous (40) findings have indicated that, with the exception of ovariectomized females, in vivo responses to vasodilators that act directly on pial arteriolar smooth muscle or through the endothelium, in rats, are not affected by Cx43 knockdown or blockers of gap junctional communication. Second, previous findings from our laboratory showed that the hypercapnic response in pial arterioles is not dependent on the endothelium and appears to be primarily controlled by extravascular influences (23, 38). On the other hand, the signal transduction mechanisms involved in hypercapnia-induced pial arteriolar responses at the cortical surface may be unique (e.g., in contrast to exogenous SNAP or ACh) in their dependence on intravascular gap junctions. One intriguing scenario is based on the anatomic relationship between cortical pial vessels and the GL, where extravascular signals arrive at pial arterioles through rather restricted (one-sided) contacts with the GL (1). Thus one cannot eliminate the possibility that gap junctions between pial vascular cells may act to amplify the vasodilating signal transmitted from those limited sites of contact, spreading that signal throughout the entire circumference of the arteriole, thereby enhancing the vasorelaxant response.

The findings related to pial arteriolar relaxations elicited by isocapnic reductions in suffusate pH are intriguing. Indeed, it suggests that hypercapnic acidosis and local isocapnic acidosis, while both promoting robust pial arteriolar dilations, are different with respect to the mechanisms involved. That is, evidence indicates that the former involves an integrated process that includes contributions from deep cortical nNOS-positive neurons, the GL, and gap junctions. On the other hand, the latter would appear to reflect direct actions of acidosis on the vessels themselves, perhaps a function of increased extracellular H+ acting on vascular smooth muscle, as suggested by the findings of Tian et al. (32).

In conclusion, the GL overlying the cerebral cortex appears to have an important influence on hypercapnia-induced pial arteriolar dilation in rats in vivo. Limited additional evidence suggested a contribution to that response from gap junctions that may reside within the GL. This implies that cortical astrocytes, including those comprising the GL, may act as a unit (syncytium) in transmitting and transducing NO-related signals arising in a limited number of scattered underlying neurons to all of the overlying pial arterioles. However, it should be emphasized that no attempts were made, in the present study, to examine whether microglial cells, which ordinarily only comprise a small fraction of the GL mass (12) but may upregulate after L-{alpha}AAA exposure (31), contribute to the CO2 response. Furthermore, the present findings do not provide any information as to the specific signal transduction elements involved in this apparent multicellular mechanism of hypercapnia-induced pial arteriolar relaxation. In attempting to characterize those elements in future studies, one should give consideration to permissive and obligatory functions of NO and vasodilating prostanoids (13, 36), the role of cyclic nucleotides (22), what factors traverse gap junctions, and even how signals from astrocytes are transmitted to pial arterioles in the absence of any apparent gap junctional connections. Finally, one cannot ignore evidence that hypercapnia/decreased pH can elicit vascular smooth muscle relaxation in isolated cerebral arteries and arterioles (2, 14, 32) and that applications of acidic solutions to the brain surface in vivo causes cerebral vasodilation independently from changes in PCO2 (present study and Ref. 16). These findings would seem to imply that CO2-induced vasodilation in vivo is related to an integration of local and perivascular influences. In any case, in view of the multiple redundant and overlapping influences that appear to modulate hypercapnic cerebral vasodilation, it should not be surprising that, in studies examining hypercapnia-induced vasodilation, different mechanisms may be revealed, depending on the experimental model employed (21, 22, 35). One implication of this is that perivascular inputs may supercede or modulate local influences to the extent that studies on isolated vessels may not provide an accurate picture of the mechanisms involved in hypercapnia-induced cerebral vasodilation.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported National Heart, Lung, and Blood Institute Grants HL-56162 and HL-52594.


   ACKNOWLEDGMENTS
 
The authors acknowledge the expert technical assistance of Dennis Riley.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. A. Pelligrino, Neuroanesthesia Research Laboratory, Dept. of Anesthesiology, Univ. of Illinois, 900 S. Ashland Ave., Molecular Biology Research Bldg., Rm. 4314, M/C513, Chicago, IL 60607 (E-mail: dpell{at}uic.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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