INTRODUCTION

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THE MECHANISMS CONTRIBUTING to hypoglycemia-induced cerebral vasodilation remain unsettled. To a large degree, the lack of consistency among published reports may be a reflection of multiple mechanisms being involved in the hypoglycemic response of cerebral vessels. Based on studies employing pharmacological blockers, three prominent vasodilating stimuli have been identified variously as contributing to hypoglycemic cerebrovasodilation. These are adenosine, nitric oxide (NO), and -adrenoceptor (-AR) activation (8, 12, 14, 15, 23, 27). Which mechanism predominates may depend on a number of factors, including animal age, hypoglycemic severity, species, state of consciousness (i.e., anesthetized or awake), brain region, and/or vessel segment (i.e., artery vs. arteriole). Moreover, the possibility exists that interactions among the above vasodilating pathways may occur to the extent that the effects of selective blockers may overlap or blocking more than one of the pathways may be required to attenuate the response.

In the present study, we addressed the hypothesis that hypoglycemia-induced cerebral arteriolar dilation may involve interactions among NO- and -AR-related mechanisms. The rationale behind this hypothesis derives from a number of published observations. First, hypoglycemic "stress" is associated with increased activity of central and peripheral catecholaminergic pathways. Several intracerebral sites may be involved in this hypoglycemic effect, including the locus coereleus (LC), hypothalamus, and medullary centers (3, 18, 20, 21). Activation of neurons within those structures can lead to initiation of sympathoadrenal activation and, ultimately, -AR activation. Second, NO generated via the activation of the neuronal (n) nitric oxide synthase (NOS) isoform has been reported to suppress the activity of sympathoexcitatory neurons arising in some of those structures (10, 28, 34-36). Third, there is evidence that hypoglycemia can increase excitatory amino acid (EAA) release and receptor activity (4, 19). EAA receptor activation is a phenomenon that is often associated with an increased nNOS activity (see Ref. 16). Thus we considered the possibility that blocking brain nNOS activity may produce two competing influences on hypoglycemia-induced arteriolar dilation-increased -AR activation but diminished perivascular NO production. The end result might be no change in the hypoglycemic response.

Using rats equipped with closed cranial windows, we examined the effects of a -AR blocker, an nNOS inhibitor, or a combination of the two agents (given systemically) on pial arteriolar diameter changes in rats subjected to fixed levels of "moderate" (i.e., precoma) hypoglycemia (plasma glucose = 1.2-1.6 mM). We also sought to examine whether such interactions occurred locally, within, or in the vicinity of cerebral arterioles as opposed to occurring at an intracerebral site that, in turn, transmits vasodilating signals to distal arterioles. To that end, systemic administration of a -AR antagonist was combined with local application of an aqueous-soluble nNOS-selective inhibitor. The results of those experiments indicated that hypoglycemic pial arteriolar dilation was suppressed only in the presence of combined systemic, but not local, administration of the two blockers. This not only suggested that the arteriolar response involves an interplay between the two pathways but also that the source of the vasodilating signal arises distally and presumably is transmitted via a neural pathway to the pial arteriole. The latter possibility was supported in an additional group of rats given the nerve action potential blocker TTX via topical application.

	METHODS

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The study protocol was approved by the Institutional Animal Care and Use Committee. Sprague-Dawley rats, 250-350 g, were used. All rats were fasted overnight before study. Anesthesia was induced with halothane, and the rat was paralyzed (curare), tracheotomized, and mechanically ventilated. Surgical anesthesia for insertion of bilateral femoral arterial and venous catheters consisted of 0.8% halothane-70% N₂O-30% O₂. After catheterization, the animal was placed in a head holder, and the skull was exposed to permit placement of a closed cranial window. The cranial window design and surgical implantation were described in detail in previous publications (31, 33). The 11-mm-diameter acrylic windows were placed over a 10-mm craniotomy and were fixed to the skull with cyanoacrylate gel. The windows were equipped with three ports [inflow, outflow, and intracranial pressure (ICP) monitoring]. After window placement, the halothane was discontinued, and a loading dose of intravenous fentanyl was given (10 µg/kg). Anesthesia during the study was intravenous fentanyl (25 µg · kg¹ · h¹) plus ventilation with 70% N₂O-30% O₂. Cannulas were secured in the inflow, outflow, and ICP-monitoring ports of the cranial window, and the space under the window was filled with artificial cerebrospinal fluid (aCSF). The composition of the aCSF is provided elsewhere (17, 33). The aCSF was suffused at 0.5 ml/min and was maintained at a temperature of 37°C, a PCO₂ of 40-45 mmHg, PO₂ of 50-60 mmHg, and pH  7.35. The ICP was controlled at 5-10 mmHg by adjusting the height of the outflow cannula. The reactivities of 25-50 µm pial arterioles on the exposed cortical surface were assessed via measurement of diameter changes. A microscope (Nikon) and color video camera (Sony) arrangement was equipped with an epi-illumination, dark field system (Fryer, Huntley, IL). The vessels were displayed on a video monitor, and diameter measurements were made using a calibrated video microscaler (Optech).

In all experiments, initial diameter measurements were made after a 30-min period of cortical suffusion with drug-free aCSF (initiated at 1 h posthalothane). The rats were divided into groups based on subsequent experimental manipulations. We examined the effects of -AR blockade with propranolol (Pro, 1.5 mg/kg iv, n = 4), nNOS inhibition with 7-nitroindazole (7-NI, 40 mg/kg ip, n = 6), or combined Pro + 7-NI (n = 5) on pial arteriolar diameter changes in rats subjected to hypoglycemia (plasma glucose = 1.2-1.6 mM). The 7-NI was suspended in corn oil (32). Two additional groups were included. In one group, Pro (1.5 mg/kg iv) was combined with the aqueous-soluble nNOS-selective inhibitor ARR-17477 (300 µM via cortical suffusion, n = 5). The other group served as a vehicle-treated control (n = 6). The controls actually consisted of two groups (n = 3 each). In one group, used as a time control for 7-NI, the rats received 1.0 ml of corn oil intraperitoneally. In the other, saline was given intravenously at a time point equivalent to the introduction of Pro. Because the diameter changes accompanying hypoglycemia were identical in these two control groups, the results were combined and are presented as a single group. Because of solubility problems, 7-NI is unsuitable for topical applications in aqueous media. Therefore, ARR-17477 was used as the agent for producing local nNOS inhibition. The nNOS selectivities of 7-NI and ARR-17477, at the doses used in this study, were documented in earlier reports (24, 32) and in preliminary experiments. In the latter case, the pial arteriolar dilations produced by topical application of the endothelial NOS-dependent vasodilator ACh were unaffected by 45-60 min of ARR-17477 (300 µM) suffusion or at 30-120 min after intraperitoneal 7-NI (40 mg/kg). We used the well-documented capacity of nNOS-selective inhibition to attenuate cerebrovascular CO₂ reactivity (29, 31, 32) to judge whether systemic administration of 7-NI, on the one hand, and topical application of ARR-17477, on the other, yielded similar magnitude reductions in brain nNOS activity (at least in the vicinity of the pial circulation). Thus, at the start of all experiments, the reactivities of pial arterioles to hypercapnia (arterial PO₂ = 60-65 mmHg) were assessed. A second CO₂ response was performed 30 min after 7-NI (in the 7-NI and Pro + 7-NI groups) or 45 min after ARR-17477 (in the Pro + ARR-17477 group) or vehicle (in the Pro or control groups). A final series of experiments (n = 5) was used to examine whether the hypoglycemia-induced pial arteriolar dilation was dependent on signals transmitted along neuronal pathways. To that end, the sodium channel blocker TTX (1 µM) was suffused (25). In these experiments, the TTX was introduced ~60 min before insulin, and the CO₂ reactivity assessments were replaced by 5-min suffusions of the NO donor S-nitrosopenicillamine (SNAP, 1 µM), applied before and ~45 min after introduction of TTX. The SNAP was employed to confirm the absence of any direct actions of TTX on pial arteriolar smooth muscle function.

The experimental time lines for all groups are represented diagrammatically in Fig. 1. Insulin (5 IU) was injected intravenously at 20 min after the second hypercapnic period (or SNAP suffusion) or at 10 min after administration of Pro. That dose of insulin was sufficient to reduce plasma glucose in the 1.2-1.6 mM range within a period of ~45 min. When plasma glucose reached ~1.5 mM, a slow infusion (50-100 µl/min) of 0.5% dextrose was initiated to maintain plasma glucose within the 1.2-1.6 mM range for a minimum period of 20 min. During that period, plasma was analyzed every 3-5 min to ensure that the glucose level remained within the 1.2-1.6 mM range.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Experimental time line. Arrows denote the time points where blood samples were taken for analysis of plasma glucose content ([Glu]p); pial arteriolar diameter (diam) measurements were obtained; a hypercapnic challenge (CO2 reactiv) was imposed; and the study drugs 7-nitroindazole (7-NI), ARR-17477 (ARR), propranolol (Pro), insulin, or vehicle (veh) were introduced. It should be noted that, over the final 20-min period of plasma glucose clamping, blood samples were obtained every 3-5 min to confirm that plasma glucose indeed remained within the 1.2-1.6 mM range.

Mean arterial blood pressure (MABP) was continuously monitored, and rectal temperature was servocontrolled at 37°C. Arterial blood samples were taken at 30- to 60-min intervals for measurement of PO₂, PCO₂, pH, and plasma glucose. Additional arterial samples, for analysis of plasma glucose only, were obtained immediately before insulin injection and starting at 15-20 min postinsulin at 5-min intervals. The blood gas/pH analyses were performed on a Radiometer-Copenhagen (model ABL) blood gas/pH analyzer, and the glucose analysis was performed on a Yellow Springs Instruments 2300 STAT glucose analyzer (Yellow Springs, OH). At the termination of the experiments, the rats were killed with a halothane overdose.

The SNAP and Pro were obtained from Sigma (St. Louis, MO). 7-NI was from ICN Biologics (Aurora, OH). TTX was obtained from Research Biochemicals (Natick, MA), and ARR-17477 was a gift from Astra Arcus (Rochester, NY). For comparisons of pial arteriolar diameter changes within a given experiment, a repeated-measures ANOVA with a post hoc Tukey analysis was used. For statistical comparisons of diameter changes between groups, we employed a one-way ANOVA with a post hoc Tukey analysis. All values are reported as means ± SE. Statistical significance was taken at the P < 0.05 level.

	RESULTS

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Arterial blood variables. The key arterial blood variables measured at the start and near the end of each experiment are summarized in Table 1. With the exception of periods of imposed hypercapnia, no significant variations in PO₂, PCO₂, pH, or MABP were observed in any of the groups over the course of each experiment. It should be noted, however, that MABP was maintained at control levels via blood replacement (0.5-1 ml) from a donor rat after Pro administration. Under normal circumstances, MABP would be expected to fall in the presence of Pro.

Pial arteriolar diameter changes. The initial pial arteriolar diameters measured in rats given vehicle only, 7-NI only, Pro only, combined 7-NI + Pro, ARR-17477, and TTX were 32.9 ± 0.9, 42.0 ± 3.7, 32.1 ± 0.6, 34.2 ± 0.9, 28.1 ± 1.0, and 39.6 ± 5.3 µm, respectively. The diameter changes (relative to the initial value) measured at 45-60 min after introduction of vehicle, 7-NI, ARR-17477, or TTX are summarized in Fig. 2. In the Pro, 7-NI + Pro, and the ARR-17477 + Pro groups, the changes given in Fig. 2 were derived from measurements made just before Pro administration. A significant reduction in diameter (P < 0.05 vs. baseline) was seen in rats treated with 7-NI (7 and 10% in the 7-NI and 7-NI + Pro groups, respectively). A similar reduction was observed in the rats given ARR-17477 topically (7%). The nNOS inhibitor-associated diameter changes were not, however, significantly different from the diameter change (2.5%) measured in the vehicle control group. TTX administration was accompanied by a more pronounced vasoconstriction (14 ± 4.2% diameter change, P < 0.05 vs. initial value). That value was also significantly different from the change measured in the control group. The stability of the TTX effect was indicated by data showing that the diameter reduction at 15 min of TTX suffusion (12.4 ± 2.4%) was statistically similar to that seen at 45 min. The long-term stability of the TTX effect was supported in two additional rats, used as time controls, where the TTX suffusion was extended for 1 h under normoglycemic conditions. No further changes were observed beyond those measured at 45 min (data not shown). Addition of Pro was accompanied by no significant change in diameter in any of the groups where the drug was administered. That is, relative to the diameter measured at the time of Pro administration, the diameter changes at 15 min post-Pro (and 5 min before insulin administration) were 3.4 ± 2.9% (Pro alone), 0.7 ± 0.7% (7-NI + Pro), and 1.2 ± 1.1% (ARR-17477 + Pro).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Baseline pial arteriolar diameter changes measured at 45-60 min after administration of the neuronal nitric oxide synthase (nNOS) inhibitors 7-NI (ip) or ARR-17477 (topically), Pro alone (iv), or TTX (topically). The vehicle time control change is provided for comparison. The changes shown for the 7-NI + Pro and ARR-17477 + Pro groups were taken from measurements made just before injection of Pro. Subsequent administration of Pro produced no further changes in diameters in those groups (see text). Values are means ± SE. *P < 0.05 vs. baseline (initial) diameter value.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Relationship between pial arteriolar diameter changes and plasma glucose in control rats.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Effects of nNOS inhibition, via 7-NI or ARR-17477, on pial arteriolar CO2 reactivities. Data are expressed as a percentage of the initial CO2 reactivities. Initial values (in %diameter increase/mmHg PCO2 change; means ± SE) are shown in each bar. In the Pro-treated groups, the second hypercapnic challenge was imposed before Pro administration. *P < 0.05 vs. initial CO2 reactivity value. Data expressed as means ± SE.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Effects of global nNOS blockade, with ip 7-NI, either alone or in combination with iv Pro-induced global -adrenoceptor blockade, iv Pro alone, topical nNOS blockade with ARR-17477 combined with iv Pro, and topical blockade of Na+ channels (nerve action potentials) with TTX on hypoglycemia-induced changes in pial arteriolar diameters (expressed as %change from baseline). Average plasma glucose concentrations (in mmol/l) in each group, measured over the final ~20 min of each experiment, are given in the bars (means ± SE). *P < 0.05 vs. vehicle control. Values are means ± SE.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The results of these experiments addressing mechanisms of hypoglycemia-induced pial arteriolar dilation can be summarized as follows. First, neither global nNOS nor -AR blockade, when performed in separate experiments, induced a significant reduction in the vasodilating response. Second, combined systemic administration of nNOS + -AR blockers was accompanied by a substantial repression of hypoglycemia-induced pial arteriolar relaxation. Third, topical application of an nNOS blocker, when combined with a systemically administered -AR antagonist, did not significantly alter hypoglycemic cerebral vasodilation. Fourth, local application of an agent that prevents impulse conduction along nerves (TTX) was associated with a substantial attenuation of the pial arteriolar response to hypoglycemia.

These results suggest that -ARs and nNOS-derived NO contribute to hypoglycemia-induced pial arteriolar dilation in a complex and interactive manner. Moreover, the interaction does not appear to occur locally within the pial circulation. We suspect that these results may reflect a capacity for NO to repress sympathetic neuronal activity. Thus, when NOS is inhibited, any stress, like hypoglycemia, is going to elicit a more profound sympathetic activation.

When comparing present results with previous experimental findings, one must bear in mind that, in the majority of the reports published to date regarding the effects of hypoglycemia on cerebral hemodynamics, cerebral blood flow (CBF) was measured. Therefore, some restraint should be observed when attempting to equate the pial arteriolar dilations of the present study with the hypoglycemia-induced increases in CBF seen in earlier reports. In one such study, in adult rats, Hollinger and Bryan (12) reported that the component of increased sympathetic activity responsible for hypoglycemic cerebral hyperemia is -AR activation. However, the sympathetic/-AR activation component of that hypoglycemia-induced CBF increase appears to reside within intracerebral structures and not in the periphery. That is, in a follow-up study, Bryan and co-workers (5) reported that, although hypoglycemia elicits an increased release of catecholamines into the circulation, plasma catecholamine levels measured before and after hypoglycemia did not correlate with the CBF changes. Therefore, results to date, including those of the present study, imply a role for intracerebral sympathetic activation in hypoglycemia-induced cerebral vasodilation. More specifically, the activation seems to involve -ARs localized in regions containing nNOS but separate from responding arterioles (at least pial arterioles).

A number of brain regions have been identified in which both nNOS and -ARs are expressed. These include the rostral ventrolateral medulla (RVLM), the LC, and the hypothalamus (22, 34). More importantly, and with relevance to the present study, NO, presumably via cGMP, has been reported to reduce the activity of sympathetic neurons in those regions. That action may be direct (i.e., via cGMP-dependent protein kinase-mediated phosphorylations; see Ref. 34) or indirect, through the increased release of inhibitory neurotransmitters (e.g., GABA; see Ref. 37). Also, NO/cGMP-associated repression of sympathetic neuronal activity may involve actions on the -AR itself (1). Hypoglycemia has been linked to increased noradrenergic activity in the regions listed earlier (20, 21). Hypoglycemia has also been reported to increase cerebral EAA release (4, 6) and to enhance N-methyl-D-aspartate (NMDA) receptor binding (19). Activation of NMDA receptors in neurons containing nNOS will increase NO production (7). Moreover, NMDA receptor activation has been linked to increased NO generation in the RVLM, LC, and hypothalamus (2, 30). Therefore, we might speculate that, while nNOS inhibition might potentiate hypoglycemia-induced arteriolar relaxation by increasing -AR stimulation and activity, that effect may be counteracted by the reduction in NO production. The combination of increased -AR stimulation and reduced NO generation could explain why we did not see an increased response in the presence of a nNOS inhibitor alone.

The absence of any influence of Pro, by itself, is somewhat more difficult to explain. It is unlikely that this relates to a "disinhibition" mechanism akin to that suggested earlier in relation to NO influences on sympathetic/-AR activity. For example, the literature indicates that cerebral -AR activation leads to enhanced EAA release (11, 26), which, in turn, would be expected to increase nNOS activity. The explanation may actually be simpler. That is, the capacity for NO to reduce -AR function in hypoglycemia may be of such a magnitude so as to render the arteriolar response insensitive to -AR blockade. As long as sufficient NO continues to be generated, vasodilation will be supported during hypoglycemia. Thus only when both systems are blocked will a loss of arteriolar reactivity be observed.

Although the present investigation suggests a rather novel mechanism regarding hypoglycemia-induced dilation of rat pial arterioles in vivo, it does not resolve the controversy raised by the divergent findings in the literature. Thus adenosine receptor blockade has been reported to attenuate the vasodilating response in anesthetized newborn pigs (27) and adult rats (23) under conditions of severe hypoglycemia and loss of spontaneous EEG activity (plasma glucose <1 mM), but not moderate hypoglycemia (plasma glucose 1-3 mM; see Ref. 23). On the other hand, in awake adult rats, there is evidence to indicate a role for adenosine in the vasodilation accompanying moderate (plasma glucose = 2-3 mM) hypoglycemia (14). A similar level of disagreement exists concerning the participation of NO in the cerebrovascular response to hypoglycemia. Nonselective inhibition of NOS was found to reduce the vasodilation accompanying hypoglycemia in the anesthetized piglet (15) and in the awake goat (8) but not the awake rat (13). Activation of -ARs was found to play a major and regionally selective role in the CBF increases accompanying hypoglycemia in the awake rat (12) but not in the unanesthetized goat (9). Some of the lack of agreement in these reports may be a function of species and animal age as well as the selectivities of the antagonists used [e.g., caffeine is a poorly selective adenosine antagonist (14), whereas 8-phenyltheophylline derivatives are highly selective (23, 27)]. Another, and perhaps more likely, explanation for differences in experimental findings relates to the level of hypoglycemia studied. Thus, even in the moderate hypoglycemia range (1-3 mM), similar to findings in rats exposed to another stressor-hypoxia (25), different factors may contribute depending on whether the plasma glucose lies at the upper or lower end of that range. One must also give consideration to the prospect that combinations of stressors, like immobilization plus hypoglycemia (e.g., Refs. 8, 9, 12-14), as opposed to hypoglycemia imposed in the presence of anesthesia, may alter the relative contributions from NO-, -AR-, or adenosine-related pathways. On the other hand, the presence of anesthesia may limit the magnitude of the hypoglycemia-related increase in neuronal activity. That could influence whether and to what extent nNOS or -AR blockers alter hypoglycemic vasodilation. That is, one cannot ignore the possibility that the roles of NO and -AR activation in promoting pial arteriolar dilation more directly relate to a capacity to enhance the increased neuronal activity initiated by hypoglycemia rather than to direct effects on the vessels. In that case, an anesthesia-induced limitation on the hypoglycemia-related increase in neuronal activity could minimize the individual effects of the pharmacological inhibitors used.

In conclusion, the present findings point to a mechanism of hypoglycemia-induced cerebral (pial) arteriolar dilation that is complex and interactive. Our data suggested a process involving the combined effects of nNOS-derived NO and -ARs. The results further implied a mechanism whereby the interaction between these two pathways occurs at a site (or sites) not in the immediate vicinity of the arterioles and where the vasodilating signal is transmitted via neural pathways.