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Am J Physiol Heart Circ Physiol 274: H982-H989, 1998;
0363-6135/98 $5.00
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Vol. 274, Issue 3, H982-H989, March 1998

Nitric oxide from neuronal NOS plays critical role in cerebral capillary flow response to hypoxia

Antal G. Hudetz, Hui Shen, and John P. Kampine

Departments of Anesthesiology and Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

We investigated, using a direct, intravital microscopic technique, whether nitric oxide (NO) from neuronal nitric oxide synthase (nNOS) plays a role in the cerebral capillary flow response to acute hypoxia. Erythrocyte flow in subsurface capillaries of the frontoparietal cortex of adult Sprague-Dawley rats was visualized using epifluorescence videomicroscopy after fluorescent labeling of red blood cells (RBC) in tracer concentrations. The velocity of labeled RBC in individual capillaries was measured off-line using an image analysis system. Hypoxia was produced by lowering the inspired O2 concentration to 15% for 5 min in control animals and in those pretreated with the selective nNOS inhibitor 7-nitroindazole (7-NI; 20 mg/kg ip). In the control group, hypoxia increased RBC velocity by 34 ± 8%. In the group treated with 7-NI, this response was reversed to a statistically significant 8 ± 3% decrease. This paradoxical response to hypoxia after 7-NI was observed in nearly all capillaries. 7-NI itself decreased the baseline RBC velocity by 12 ± 4%. The cerebral hyperemic response to hypoxia was also assessed with the laser Doppler flow (LDF) technique. In control animals, hypoxia produced a 33 ± 6% increase in LDF, similar to the increase in RBC velocity. After 7-NI treatment, the response to hypoxia was moderately attenuated but still significant at a 19 ± 2% increase in LDF. These results support the role of NO from nNOS in the cerebral hyperemic response to hypoxia. They imply that 7-NI interfered with a physiological mechanism that was fundamental to cerebral capillary flow regulation and provide direct evidence that cerebral capillary perfusion may be dissociated from a concurrent change in regional tissue perfusion as reflected by LDF. In conclusion, NO from nNOS contributes to the maintenance of RBC flow in cerebral capillaries and plays a critically important role in the selective regulation of cerebral capillary flow during hypoxia.

7-nitroindazole; microcirculation; cranial window; laser Doppler; vasodilation; hyperemia; cerebral blood flow

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

ACUTE HYPOXIA INDUCES cerebral vasodilation and a pronounced increase in cerebral blood flow (CBF). The increase in CBF is reflected in an increase in the rate of perfusion of the cerebral capillary network. Until recently, a major issue of CBF regulation has been whether capillaries were recruited during hypoxia and other hyperemic states. This question has now been largely settled (12, 14), and the focus of interest has been shifted toward hyperemia-associated changes in heterogeneity of perfusion and the possibility of physiological regulation of cerebral capillary flow (13).

Hypoxic vasodilation in the cerebral circulation has been ascribed to multiple mechanisms (35) including direct vascular, neuronal, and tissue metabolic effects and a number of potential mediators such as adenosine, prostaglandins, and potassium and hydrogen ions. Recently, nitric oxide (NO) has been demonstrated to play a major role in cerebral vasodilation to various physiological stimuli, for example, hypercapnia (34, 36, 39, 47) and functional activation (4, 17, 29, 47). The role of NO in the hyperemic response to hypoxia has been controversial, however. The majority of earlier studies (7, 18, 23, 26, 36) demonstrated no effect of NO synthase (NOS) inhibition on the hypoxia-induced increase in CBF. On the other hand, several investigators demonstrated a role for NO in the hypoxic dilation of pial arteries (3, 19, 37). In addition, Reid and co-workers (39) suggested a role for NO in hypoxia-induced increase in local CBF.

A complicating factor in assessing the current state of knowledge of hypoxic vasodilation is that the relative importance of NO and other parenchymal mediators may be different in different segments in the cerebrovascular tree (35). For example, hypoxia directly dilates large cerebral vessels, but this effect diminishes in small resistance vessels (35), which are under the principal influence of parenchymal control. Neuronal activation dilates the intracerebral vasculature without an effect on pial arteries (2). NO in the brain is constitutively produced by both endothelial NOS and neuronal NOS (nNOS). NO from nNOS appears to play the principal role in the CBF response to functional activation (4), which may also accompany cerebral vasodilation to hypoxia (37, 40). The role of NO specifically from nNOS in the hypoxic cerebrovascular response has not been elucidated. Finally, the cellular mechanisms regulating the intracerebral microcirculation during hypoxia are poorly understood. In particular, no information is available on whether NO or any of the other postulated mediators would directly influence the pattern of cerebral capillary perfusion.

We have recently perfected an intravital videomicroscopic technique that can be applied to directly study the physiological responses of erythrocyte flow in the cerebral capillary network (14, 15). This technique has enabled us to examine the characteristics of hypoxia- and hypercapnia-induced increases in cerebral capillary perfusion (12). We have also demonstrated the significant role of NO in the maintenance of steady- state capillary perfusion in normoxia (6) but have not studied this phenomenon during hypoxia.

The purpose of the present work was to examine, using intravital microscopy, whether NO from nNOS plays a role in the cerebral capillary flow response to acute hypoxia. The selective nNOS inhibitor 7-nitroindazole (7-NI) was used to test whether NO from nNOS was required for the hypoxia-induced hyperemic response in cerebrocortical capillaries.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

The experiments were performed on 27 male Sprague-Dawley rats weighing 281-426 g. The animals were anesthetized with pentobarbital sodium (60 mg/kg) and tracheotomized, and femoral arterial and venous lines were placed for the measurement of arterial blood pressure, blood gases, and pH and for the infusion of drugs. The head was secured in a stereotaxic apparatus, and a closed cranial window was installed over the right parietal cortex using techniques described previously (14). To aid the visualization of flow in subsurface capillaries, red blood cells (RBC) were labeled in vitro with fluorescein isothiocyanate and injected intravenously in tracer quantities into the circulation. Capillaries were identified by the presence of single-file RBC flow (implying vessel diameter <5 µm) and their organization into an anastomosing network of relatively short, tortuous vascular segments. The capillary circulation was viewed and video recorded at ×125 optical magnification, using a ×40 ultralong-working-distance (8 mm) microscope objective lens. A 100-W mercury vapor lamp was used for epi-illumination. Video recording of the microcirculation was limited to 1-min periods. Microscope field and focus adjustments before video recording were performed at reduced illumination intensity. A heat-intensity filter and a 455-nm high-pass filter were used to block infrared and ultraviolet irradiation of the tissue.

During the experimental period, the animals were paralyzed (gallamine, 80 mg) and artificially ventilated. Under control conditions, the animals were ventilated with a 30% O2-70% N2 mixture. Anesthesia was maintained with pentobarbital sodium infusion (7-10 mg/h). Arterial blood pressure, end-tidal carbon dioxide (ETCO2), and inspired oxygen concentration were continuously monitored. ETCO2 was maintained at 35 mmHg. Arterial blood gas tensions and pH were checked regularly.

Hypoxia was produced by lowering the concentration of inspired O2 to 15%. Airway O2 concentration stabilized at this level within 2 min. An additional 2-min period was allowed for circulatory adjustments to take place, and then the microcirculation was video recorded for 1 min. A control recording was performed immediately before the hypoxic challenge. During the hypoxic period, the arterial blood pressure was stabilized by an intravenous infusion of the alpha 1-agonist methoxamine at a rate of 0.4-2.5 mg/h. The preferential alpha 1-adrenergic agonists intravenously have no direct effect on the cerebral circulation (42).

A separate group of rats (n = 7) was pretreated with intraperitoneal administration of the nNOS inhibitor 7-NI at 20 mg/kg. This dose of 7-NI results in 70% inhibition of brain NOS catalytic activity (34). The response to hypoxia was tested 45 min after 7-NI treatment, using the same protocol as in the control group (n = 7). The capillary circulation was video recorded before and after 7-NI administration and during the hypoxic challenge. In three additional animals, the capillary flow response to the NO donor (Z)-1{N-methyl-N-[6(N-methylammoniohexyl)amino]}diazen-1-ium-1,2-diolate (MAHMA-NONOate; 10-4 M) was tested by injecting the drug under the cranial window.

In two additional groups of animals, the CBF response to hypoxia was assessed by the laser-Doppler flow (LDF) measurement technique. A multichannel digital flowmeter with 1-mm-diameter probes was used (Oxford Optronix, Oxford, UK). This flowmeter uses near-infrared light of 780 nm, resulting in perfusion values unaffected by the concentrations of oxy- vs. deoxyhemoglobin. The LDF probes were carefully positioned using a micromanipulator over cortical areas devoid of large pial vessels. One group of animals (n = 6) was treated with the same dose of 7-NI for 45 min. The control animals (n = 7) were treated with peanut oil, the vehicle for 7-NI.

RBC velocity in individual capillaries was measured off-line during video playback by frame-to-frame image tracking (15). The velocity was measured in four to five capillaries in each network contained within a field size of 380 µm × 280 µm. RBC velocity was measured at ~3-s intervals, and the obtained velocities were averaged over the 1-min recording period. RBC velocity in individual capillaries was first averaged across each network, and then these values were averaged to obtain the group mean and standard error.

Statistical testing of the effect of hypoxia on RBC velocity and on LDF within each experimental group was done using the paired t-test. Because LDF does not reflect absolute flow, LDF values were expressed in percentage of baseline before treatment. The effect of 7-NI on the hypoxic response of RBC velocity or LDF was tested by comparing the percent change in measured values with and without treatment, using the unpaired t-test. The effect of 7-NI on baseline RBC velocity was tested with the paired t-test. Statistical significance was assumed at P < 0.05.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Under resting conditions, rapid movement of labeled erythrocytes through networks of convoluted, tortuous capillaries lying ~50 µm beneath the level of pial surface vessels was observed. Although the rate of perfusion varied from capillary to capillary, erythrocyte flow was essentially continuous in almost all microvessels. In a few capillaries, RBC perfusion was intermittent, showing brief periods (from 30 ms to seconds) of flow stagnation. Transient changes in capillary perfusion were limited to <10% of all visible vessels. Figure 1 shows a capillary network as seen on the video monitor as an example.


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  		Fig. 1.   Fluorescence videomicroscopic images of cortical microcirculation as seen on video monitor. Fluorescently labeled red blood cells (RBC) appear as bright streaks rather than dots because of their high velocity. To capture the passage of labeled cells in several capillaries, 5 snapshots of same vascular field were made and are displayed here. Bottom right panel, reconstruction of network of capillaries in which cells traveled. Scale bar, 50 µm.

Table 1 shows systemic arterial pressure, blood gas, pH, and hematocrit values obtained during normoxia and hypoxia in four experimental groups. There was no significant change in any of these parameters except the arterial PO2, which was decreased during hypoxia. All other parameters were in the normal physiological range, and there were no significant intergroup differences.

                              
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  		Table 1.   Mean arterial blood pressure, blood gas, pH, and hemoglobin values in 4 experimental groups

In the control group, RBC velocity increased during hypoxia from 0.64 ± 0.06 to 0.85 ± 0.09 mm/s, indicating a 34 ± 8% increase (P < 0.001). RBC velocity increased in each capillary.

After treatment with the NOS inhibitor 7-NI, normoxic baseline RBC velocity decreased in 27 of 28 capillaries. Figure 2 depicts the effect of 7-NI in each experiment. The average decrease in RBC velocity after 7-NI treatment was 12 ± 4% (P < 0.05). This observation suggests that NO from nNOS played an important role in the maintenance of resting capillary flow.


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  		Fig. 2.   Effect of selective neuronal nitric oxide synthase (nNOS) inhibitor 7-nitroindazole (7-NI) on cerebral capillary RBC velocity. Each symbol represents average RBC velocity from a different experiment. 7-NI decreased baseline RBC velocity in each experiment.

In the animals treated with 7-NI, hypoxia failed to increase RBC velocity. Instead, hypoxia produced a small but significant decrease (8 ± 3%, P < 0.05) in RBC velocity. This paradox response was observed in 24 of 28 capillaries. Figure 3 summarizes the mean changes in RBC velocity from both experimental groups.


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  		Fig. 3.   Effect of hypoxia on cerebral capillary RBC velocity with and without pretreatment with nNOS inhibitor 7-NI. Hypoxia produced a significant increase in RBC velocity in control animals. 7-NI reversed hypoxic response to a small but significant decrease in RBC velocity. Open bars, normoxia; solid bars, hypoxia. Values are means ± SE. * P < 0.001 vs. normoxia control; dagger  P < 0.05 vs. normoxia 7-NI.

To illustrate the change in RBC velocity distribution in individual capillaries, frequency histograms of the data were constructed. Figure 4 shows that hypoxia widened and shifted the distribution of measured velocities to the right. The range of velocities in normoxia was 0.44 to 0.99 (SD 0.15) mm/s, and in hypoxia it was 0.47 to 1.43 (SD 0.24) mm/s. After 7-NI treatment, the velocity range was left-shifted at 0.37 to 0.88 (SD 0.13) mm/s, and during hypoxia it was reduced to 0.38 to 0.78 (SD 0.12) mm/s. The distribution means were identical to the values graphed in Fig. 3.


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  		Fig. 4.   Frequency distribution of RBC velocity in cerebrocortical capillaries during normoxia (A and C) and hypoxia (B and D) in 2 groups of experiments, control (A and B) and 7-NI treated (C and D). In control group, hypoxia widened and shifted distribution of measured velocities to the right. After 7-NI treatment, hypoxic velocities remained substantially lower than in control group.

To demonstrate that exogenous NO had the ability to increase capillary perfusion after 7-NI treatment, we performed experiments with topical injections of the rapid-acting NO donor, MAHMA NONOate. Figure 5 shows that repeated injections of the NO donor (10-4 M) under the cranial window produced sizable, transient increases in capillary RBC velocity.


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  		Fig. 5.   Effect of repeated injections of NO donor MAHMA-NONOate under cranial window of 7-NI-treated animals. Arrows, times of injection. The NO donor produced large, transient increases in capillary RBC velocity.

The cerebral hyperemic response to hypoxia was also assessed using laser-Doppler flowmetry. In the control group, hypoxia produced a 33 ± 6% (P < 0.001) increase in LDF, similar to the increase in RBC velocity. In the animals receiving 7-NI treatment, this response was smaller but still substantial at 19 ± 2% (P < 0.001). Thus 7-NI attenuated, but did not abolish, the hypoxic LDF response. The degree of attenuation of the hypoxic response was 42% and was statistically significant (P < 0.005). The LDF results are summarized in Fig. 6.


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  		Fig. 6.   Cerebral blood flow response to hypoxia as assessed by laser-Doppler flow (LDF) with and without pretreatment with 7-NI. Open bars, normoxia; solid bars, hypoxia. 7-NI moderately attenuated hyperemic response to hypoxia. Values are means ± SE. * P < 0.001 vs. normoxia; dagger  P < 0.005 vs. change in control group.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Hypoxic cerebral vasodilation and NO. The main goal of this study was to examine the possibility that NO from nNOS plays a role in the response of cerebral capillary circulation to acute hypoxia. Previous studies that addressed the role of NO in the cerebrovascular hypoxic response in vivo have focused on changes in regional CBF or pial vessel diameter and used various nonselective inhibitors of NOS such as NG-nitro-L-arginine methyl ester (L-NAME) or NG-monomethyl-L-arginine (L-NMMA). The effect of the NOS inhibitor 7-NI in the cerebral hypoxic response has not been investigated before. In the brain, NO is constitutively produced by both endothelial NOS and nNOS. The abundance and activity of these isoforms varies with the cerebral region (44). When administered intraperitoneally in vivo, 7-NI acts as a specific inhibitor of nNOS (22, 30). Therefore, the use of 7-NI helps to delineate whether NO produced specifically by nNOS was involved in the cerebrovascular response to hypoxia.

The majority of former studies using nonselective NOS inhibitors concluded that systemic NOS inhibition did not influence the CBF response to acute hypoxia (7, 18, 23, 26, 36). This view has been challenged by at least three recent studies. First, Reid and co-workers (39) found that after an intracortical administration of the NOS inhibitor L-NMMA, the hypoxic response of local CBF was attenuated by 60-70%. Second, Pelligrino et al. (37) and Ishimura et al. (19) found that L-NAME applied in topical superfusion to the brain surface significantly attenuated the dilation of pial arteries to hypoxia. Hypoxia also produced an increase in guanosine 3',5'-cyclic monophosphate (cGMP) concentration in the cerebrospinal fluid (3, 9), suggesting that NO production was increased during hypoxia.

Our present findings support the role of NO, particularly of NO from nNOS, in the CBF response to hypoxia. Although the reason for controversy among the previous studies remains unclear, it appears that intracortical or topical (but not intravenous) application of nonselective NOS inhibitors mimicked the more specific inhibitory effect of 7-NI on the hypoxic response. The importance of the route of administration has been suggested by the observation that intravenous L-arginine was unable to reverse the inhibitory effect of intracortical L-NMMA on the hypoxic response (39). It is also possible that secondary effects of systemic NOS inhibition, e.g., a right shift in hemoglobin saturation (24), confounded the results of earlier studies.

In this study, an intravenous infusion of the alpha 1-agonist methoxamine was used to stabilize arterial blood pressure during the hypoxic challenge. We considered the possibility that methoxamine may have influenced the results by attenuating the hypoxic vasodilation, especially after NOS inhibition (45), i.e., after treatment with 7-NI. Although adrenergic agents given intravenously normally do not influence the cerebral vessels (42), they may do so after disruption of the blood-brain barrier, e.g., after prolonged hypoxia or anoxia. We found no evidence, however, that moderate hypoxia lasting for a few minutes would reproduce this effect. In fact, acute hypoxic hypoxia, elicited by an inhalation of 7% O2, does not alter blood-brain barrier permeability (5). In our study, fractional inspired O2 concentration was decreased to 15% for 5 min. Thus the effect of methoxamine on cerebral vessels in vivo was probably negligible during both normoxia and hypoxia. Furthermore, the concentration and infusion rate of methoxamine required to stabilize the arterial blood pressure during hypoxia was not different in control and after 7-NI treatment. Even if methoxamine gained access to cerebrovascular smooth muscle during hypoxia, this would not explain why 7-NI influenced the hypoxic capillary flow response so much more than the LDF response (see Capillary flow versus LDF).

A limitation of the experimental protocol used in this study was that the capillary flow response was tested with and without 7-NI treatment in separate animal groups. This experimental design has lower statistical power than the within-group design in which the response is tested before and after treatment in the same animal. On the other hand, the effect of 7-NI cannot be reversed, and therefore a desirable postcontrol measurement was not feasible in the same experiment. Because the hypoxic response was remarkably consistent within each experimental group, the between-group comparison should provide an adequate assessment of the effect of 7-NI on this response.

Capillary flow versus LDF. The major finding of this study is that 7-NI completely abolished and, moreover, reversed the capillary RBC flow response to acute hypoxia. Although the hypoxic response of cerebrocortical LDF was also attenuated, the dramatic inhibitory effect of 7-NI on the hypoxia-induced change in capillary flow was not reproduced by LDF. This finding suggests that 7-NI interfered with a microvascular phenomenon that was not fully reflected by a change in regional CBF as assessed by the LDF technique. The interpretation of this surprising finding requires both methodological and physiological consideration.

First, one has to consider the differences between the microscopic flow measurement and LDF in terms of the sampled tissue volume, distribution, and the microvessels contained. LDF reflects average, nondirectional flux of RBC (velocity × tissue concentration) in a tissue volume containing several hundred microvessels including arterioles, capillaries, and venules. The contribution of various microvessels to the LDF signal is determined by the velocity of flow (both high and near-zero velocities are excluded) and the fraction of total blood volume contained by the particular vascular segment. Most importantly, LDF does not distinguish between capillary flow and shunt flow. In contrast, the videomicroscopic measurement was restricted to true capillaries within the top 70-µm layer of the cerebral cortex. Because the LDF signal power is exponentially weighted toward surface vessels, it is difficult to resolve whether there was a significant difference in the effective depth of measurement of LDF versus videomicroscopy and whether it contributed to the observed difference in the results. To examine this question further, we have recently acquired an LDF probe with small fiber separation (130 µm) and used this "microprobe" to test the response to hypoxia before and after 7-NI treatment. The depth of measurement with the new probe, estimated from the banana-shaped photon migration pattern in cerebral tissue, is about one-fifth of the larger probe used in this study and is comparable to the imaging depth of fluorescence microscopy. Results from preliminary experiments (n = 4 rats) show that the hypoxic response assessed with this new microprobe is similar (27 ± 3%) to that recorded with the larger probe and that the LDF response after 7-NI treatment (21 ± 2%) is moderately attenuated. Thus the measurement depth below the cortical surface is unlikely to be the major factor responsible for a difference between the microscopic flow and LDF measurements.

In the control experiments, the relative increases in capillary flow and in LDF during hypoxia were similar, suggesting that under normal physiological conditions, RBC flow in single capillaries was representative of microregional RBC flow as assessed by the LDF technique. After 7-NI treatment, however, LDF but not capillary flow increased during hypoxia. This observation suggests that if LDF reflected a true increase in RBC flow, then the extra RBC flow must have been channeled through cerebral microvessels other than the capillaries observed by videomicroscopy, e.g., through arteriovenous shunts or thoroughfare channels. Thoroughfare channels or precapillary arteriovenous anastomoses have been observed in the cerebral gray matter of the rat (31), and shunting of 7- to 8-µm microspheres through precapillary anastomoses has been established (25). We recently demonstrated that during autoregulatory vasodilation, RBC flow in the cerebral microcirculation may be differentially altered in two groups of capillaries, i.e., true (exchange) capillaries and thoroughfare channels (13). We suggested that blood flow through the exchange capillaries may be selectively regulated via flow redistribution without a change in net regional blood flow. The present results raise the possibility that a similar phenomenon may occur in hypoxia. The results also provide direct evidence for the first time that cerebral capillary perfusion may be dissociated from a concurrent change in regional CBF as reflected by LDF. Moreover, the implications of this observation are probably not limited to LDF. Clearly, all regional flow measurements yield indexes of average tissue perfusion, by RBC, plasma, or both, and none of the regional blood flow measurement techniques are truly selective to capillary flow. Thus a redistribution of blood flow within the capillary network could go undetected by these techniques.

Physiological regulation of cerebral capillary flow. The remarkable effect of 7-NI on the capillary RBC flow response to hypoxia suggests that 7-NI interfered with a mechanism that was fundamental to cerebral capillary flow regulation. This effect was only partially reflected in regional RBC flow (LDF), which is also controlled at upstream arterial sites and by multiple mechanisms, particularly by adenosine (27, 37). However, NO from nNOS proved to be of critical importance for the control of capillary RBC flow. On the basis of these results, we postulate that NO from nNOS may be involved in a selective, neuronal regulation of capillary RBC flow in hypoxia.

The involvement of a neuronal mechanism in the cerebral hypoxic response has support in the literature. Reis et al. (40) suggested the presence of hypoxia-sensing neurons in the rostral ventrolateral medulla that regulate, via intrinsic, polysynaptic pathways, cortical CBF without affecting cortical glucose utilization. The additional role of cortical "vasodilator neurons" has also been raised (40). Recently, Pelligrino et al. (37) suggested that hypoxic dilation of pial arterioles was dependent on neuronal activation that involved the N-methyl-D-aspartate (NMDA) receptor/NO pathway. In that study, hypoxic vasodilation was blocked by the topical application of tetrodotoxin or the NMDA receptor antagonist MK-801. Recent studies have also established that NO from nNOS is of principal importance in neuronal activation-induced cerebral hyperemia. Experiments in endothelial NOS gene-deficient mice have convincingly demonstrated that NO from nNOS played the major role in the cerebrocortical blood flow response to vibrissal stimulation (4). 7-NI has been shown to block cortical CBF increases to chemical stimulation by NMDA (47) or kainate (29). 7-NI also attenuated cerebellar cortical hyperemia-elicited parallel fiber stimulation (17). Our hypothesis of neuronal NO-dependent regulation of cerebral capillary flow is consistent with these findings.

The cellular site and mode of action of NO in the regulation of cerebral capillary flow are as yet unknown. The sources of NO from nNOS are multiple and may be involved to a variable degree in different vasodilator responses. During hypoxia, NO may be released from NOS-containing neurons and dendrites situated in close association with intracerebral arterioles and capillaries (38, 16), from microvascular endothelial cells (10), or from astrocytes (32). NO could also act as a neurotransmitter or neuromodulator proximal to the site of vasodilation. Because 7-NI blocks nNOS throughout the brain, it is not possible to conclude whether NO from local or central sources is involved in the hypoxic response. However, selective regulation of capillary flow would be more consistent with local rather than central action of NO. A recently proposed regulatory pathway based on the NO/ascorbic acid cycle represents an interesting hypothesis compatible with local regulation of cerebral capillary flow during hypoxia (28).

Regardless of the site of its production, NO from nNOS appears to be critically important for a component of the cerebral hypoxic response that specifically affects or regulates capillary RBC flow. Our current hypothesis is that during hypoxia, RBC flow increases more in the exchange capillaries than in the thoroughfare channels and that this response is mediated or supported by NO from nNOS. In other words, in the absence of neuronal NO, hypoxia still increases regional blood flow, but it fails to increase RBC flow in the exchange capillaries.

If blood flow through cerebral capillaries is selectively regulated, then this regulatory mechanism must involve contractile cells associated with the terminal arterioles, thoroughfare channels, or the capillaries themselves. Nakai et al. (33) described precapillary sphincters as ringlike impressions in microvascular casts at the orifice of intracerebral arterioles. Cerebral capillaries are heavily invested with pericytes (21), and the ability of retinal pericytes to contract and relax in vitro is well established (41). Pericytes are relaxed by sodium nitroprusside via a cGMP-dependent mechanism (11). At the extreme, capillary endothelial cells themselves could contract and relax, as recently demonstrated in cultured brain vascular endothelial cells (43).

A selective increase in capillary flow in hypoxia could be brought about by essentially two different ways: 1) a dilation or decrease in resistance to flow of the exchange capillaries, and 2) a constriction or increase in resistance of the postulated thoroughfare channels. It has been shown that purified nNOS activity declines with a decrease in O2 concentration in the physiological range (1). This O2 dependence of nNOS activity would be consistent with hypoxia-induced closure of thoroughfare channels and the consequent redistribution of flow to the exchange capillaries. However, this hypothesis leaves unexplained why baseline RBC velocity in the exchange capillaries decreases after nNOS inhibition with 7-NI. Alternatively, tissue hypoxia may be sensed by respiratory cytochromes, heme proteins, or neuronal plasma membrane K+ channels (20). Signals generated by tissue hypoxia in capillary endothelial cells or in neuronal and glial processes in apposition to the capillary wall could be communicated within the capillary endothelium (8) to precapillary contractile cells. Woolsey and co-workers (46) proposed that during functional activation of the somatosensory (barrel) cortex, capillary endothelial cells may sense and integrate parenchymal metabolic signals and communicate these signals upstream to facilitate the coordinated dilation of penetrating arterioles that supply a common microvascular module.

In conclusion, the present results suggest that NO from nNOS is involved in the maintenance of RBC flow in cerebral capillaries and plays a critical role in the cerebral capillary flow response to hypoxia. Capillary RBC flow in hypoxia may be specifically regulated by a neuronal mechanism independent of regional RBC flow.

    ACKNOWLEDGEMENTS

The technical assistance from James D. Wood, the help in data analysis from Bharat B. Biswal, and the secretarial assistance from Anita Tredeau and Angela Barnes are greatly appreciated.

    FOOTNOTES

This work was supported by grants from the American Heart Association (GIA-95009340) and the National Science Foundation (BES-9411631).

Address for reprint requests: A. G. Hudetz, Dept. of Anesthesiology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226.

Received 27 June 1997; accepted in final form 17 November 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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AJP Heart Circ Physiol 274(3):H982-H989
0363-6135/98 $5.00 Copyright © 1998 the American Physiological Society


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