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Am J Physiol Heart Circ Physiol 293: H2193-H2201, 2007. First published July 13, 2007; doi:10.1152/ajpheart.00190.2007
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Cerebral microvascular dilation during hypotension and decreased oxygen tension: a role for nNOS

Holly D. Bauser-Heaton and H. Glenn Bohlen

Department of Cellular and Integrative Physiology, Indiana University Medical School, Indianapolis, Indiana

Submitted 13 February 2007 ; accepted in final form 6 July 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Endothelial (eNOS) and neuronal nitric oxide synthase (nNOS) are implicated as important contributors to cerebral vascular regulation through nitric oxide (NO). However, direct in vivo measurements of NO in the brain have not been used to dissect their relative roles, particularly as related to oxygenation of brain tissue. We found that, in vivo, rat cerebral arterioles had increased NO concentration ([NO]) and diameter at reduced periarteriolar oxygen tension (PO2) when either bath oxygen tension or arterial pressure was decreased. Using these protocols with highly selective blockade of nNOS, we tested the hypothesis that brain tissue nNOS could donate NO to the arterioles at rest and during periods of reduced perivascular oxygen tension, such as during hypotension or reduced local availability of oxygen. The decline in periarteriolar PO2 by bath manipulation increased [NO] and vessel diameter comparable with responses at similarly decreased PO2 during hypotension. To determine whether the nNOS provided much of the vascular wall NO, nNOS was locally suppressed with the highly selective inhibitor N-(4S)-(4-amino-5-[aminoethyl]aminopentyl)-N'-nitroguanidine. After blockade, resting [NO], PO2, and diameters decreased, and the increase in [NO] during reduced PO2 or hypotension was completely absent. However, flow-mediated dilation during occlusion of a collateral arteriole did remain intact after nNOS blockade and the vessel wall [NO] increased to ~80% of normal. Therefore, nNOS predominantly increased NO during decreased periarteriolar oxygen tension, such as that during hypotension, but eNOS was the dominant source of NO for flow shear mechanisms.

nitric oxide; brain; arterioles; in vivo; microelectrode; neuronal nitric oxide synthase

SUPPRESSION OF NITRIC OXIDE (NO) formation by an intravascular arginine analog decreases cerebral blood flow by 10–40% (17, 18, 40, 42). This indicates that a source of NO actively influences cerebral resistance vessels at rest. Furthermore, Jones et al. (25) and Kajita et al. (26) found that, during hypotension, the lower limit of brain blood flow autoregulation was increased to ~90 mmHg in the presence of nitroarginine compared with ~65 mmHg in the natural state. The observations of reduced resting blood flow and impaired flow autoregulation indicate that NO regulation has important roles in both maintaining brain resting blood flow and dilatory responses to hypotension. These various observations would predict that, when the various NO synthase (NOS) isoforms are inhibited, even a mild reduction in arterial blood pressure would compromise brain blood flow and tissue oxygenation.

Exactly how NO mechanisms are involved in brain blood flow regulation are difficult to predict but may involve feedback processes linked to oxygen. In studies of the small intestinal vasculature, we have found that arterioles were sensitive to small reductions in oxygen tension and responded with substantial increases in NO concentration ([NO]) (36, 53). In the case of the small intestine, both tissue and perivascular oxygen tension declined. However, cerebral autoregulation is known to be quite competent to preserve tissue oxygen tension during hypotension. In a prior study, Rubin and Bohlen (45) found that tissue PO2 was essentially constant during hypotension down to mean arterial pressures of ~60 mmHg in rats, and the present study supported this earlier finding. However, studies of oxygen tension in the walls of cerebral arterioles during hypotension have not been investigated during in vivo conditions. That there is an interaction of oxygen availability and NO in the brain vasculature is known from multiple studies of systemic hypoxia. Without exception, these studies (2, 27, 54) have shown that severe systemic hypoxia at normocapnia is associated with a large increase in blood flow; the increase in blood flow essentially does not occur if NOSs are blocked. Furthermore, blockade of neural NOS (nNOS) appeared to be more important than the influence of endothelial NOS (eNOS) (23). However, 7-nitroindazole (7-NI) predominantly used in the past to block nNOS is only marginally more specific for nNOS than eNOS; half-maximal concentrations for inhibition (IC50) between nNOS and eNOS are 0.71 and 0.8 µM (19, 20, 28, 34, 46, 48a, 48b). Consequently, the form of NOS that determines resting and altered NO during reduced oxygen tension and hypotension is not entirely clear.

Our hypothesis was that both vessel wall and brain tissue nNOS could donate NO to the arterioles at rest and during periods of reduced perivascular oxygen tension, such as during hypotension or reduced local availability of oxygen. Whether endothelial cells have a significant population of functional nNOS is controversial. nNOS has been found in limited amounts in adult cerebral endothelial cells (32, 52). Our concern was that brain tissue has extensive nNOS as well as substantial vascular eNOS and that the dominating source of NO could come from tissue or microvessels. To determine to what extent the perivascular NO was of nNOS or eNOS origin, we used a highly specific nNOS blocker (20). Rather than use total body NOS inhibition, as has been done in some past studies of the role of NO in cerebral regulation (9, 17, 18, 23, 27, 33, 40, 42, 43, 46), we applied the nNOS blocker only to those arterioles in which we could measure the [NO] with NO-sensitive microelectrodes. To evaluate NO responses at the arteriolar wall, highly spatially accurate measurements with <10 µm outer diameter NO microelectrodes were used that are sensitive to NO only at their open tip (3, 5, 36). Furthermore, in the same preparations and vessel, we correlated changes in perivascular PO2 during hypotension and local reductions in oxygen availability to the [NO] responses before and after nNOS blockade. The combination of PO2 and [NO] measurements with highly specific nNOS inhibition allowed us to investigate whether a local reduction in oxygen tension at a normal arterial pressure could cause an increase in vessel wall [NO] similar to that during hypotension through a nNOS mechanism.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
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The techniques and procedures used with animals were approved by the Indiana University School of Medicine Institutional Animal Care and Use Committee.

Surgical preparation of the cerebral cortex. Adult male Sprague-Dawley rats of 300–400 g (Harlan Sprague Dawley, Indianapolis, IN) were anesthetized with thiopental sodium (200 mg/kg; Abbott, Chicago, IL) subcutaneously injected over four sites along the upper and lower thighs. Additional anesthetic (20% original dosage) was administered intraperitoneally if needed. Body temperature was maintained at 37°C with an underbody heating pad held at 35–36°C. The trachea was intubated (PE-240), and rats were ventilated (Harvard Apparatus, Holliston, MA) at the conscious rate of 70 breaths/min. Tidal volume was determined on the basis of weight (values given by Harvard Apparatus) and adjusted as needed to maintain percent saturation of hemoglobin with oxygen ~95% (ear or paw measurement). When measured, end tidal carbon dioxide tension was typically 38–42 mmHg. The femoral artery was cannulated (PE-50) to monitor arterial blood pressure. Rats were given saline (~0.5 ml·100 g–1·h–1) to compensate for fluid loss. Only those animals with a constant blood pressure ≥100 mmHg were used.

After securing the head of the animal in a stereotaxic holder and following bilateral exposure of the parietal bones, the bones were removed by drilling inside the suture lines using a hand-held, high-speed drill (Fine Scientific Tools, Foster City, CA). Bone wax (Surgical Specialties, Reading, PA) was used to minimize bleeding. The dura mater was dried with air, and thrombin crystals (Sigma, St. Louis, MO) were applied to any bleeding areas. The dura mater was opened with a 30-gauge needle and very fine forcep tips inserted between brain tissue and dura mater. After dura removal, bicarbonate-buffered solution equilibrated with 5% O2-5% CO2-90% N2 (37°C) was suffused over the brain throughout the remainder of the experiment at 5 ml/min (21, 45). The temperature of the fluid above the brain was held at 37°C by passing ~900 ml/min heated distilled water through the bath support and the flowing heated artificial cerebrospinal fluid layer (5 ml/min) over the brain. The PO2 of the fluid ~200 µm above the brain surface was routinely 40–50 mmHg, as measured with oxygen microelectrodes.

Observation of cerebral microvasculature. Cortical microvessels 20–60 µm in inner diameter (2A) were observed with an X20 Nikon water-immersion lens (Model BHMJ; Olympus, Hyde Park, NY). The images were collected using a Video Scope camera (model CCD 200E; Videoscope International, Washington, DC) and stored and analyzed using an image analysis system (Image 1, Universal Imaging, West Chester, PA); VHS recording of the microscope video images provided continuous timing imaging of the vascular responses. Image analysis dimensional calibrations in the X and Y directions were made with a stage micrometer marked in 10- and 100-µm units.

Perivascular NO measurements. [NO] was measured using microelectrodes made according to methods previously described by this laboratory (3, 5, 36). Tips of the microelectrode were sharpened to an outer diameter of ~10 µm or less. The slight recess formed electrolytically in the microelectrode tip was electroplated with Nafion (Aldrich, Milwaukee, WI) at +0.7 V to eliminate the undesired detection of negatively charged elements, such as tyrosine and nitrate, and to act as a physical barrier to proteins reaching the charged surfaces. Unless the Nafion is electroplated it becomes very fragile when wet, and tissue penetration dislodges the membrane. This is not a problem after Nafion is electroplated.

NO microelectrodes were polarized relative to a silver-silver chloride electrode to the voltage most sensitive to [NO] (+0.7 or +0.9 V) by a Keithley model 6517A electrometer (Cleveland, OH). The microelectrodes operated in the 3- to 10-pA range with a typical increase in current of 3–5 pA per 1,000 nM of NO. Calibration of the microelectrode was done prior to experimentation in 37°C saline by measurement of the electrical current response of the electrode to 0, 600, and 1,200 nM [NO] using NO-N2 gases of various compositions (Matheson, Joliet, IL). To obtain a zero [NO] current during experiments, the microelectrode tip was placed 200 µm above tissue; further elevation of the microelectrode tip above tissue did not lower the current measured. Electronic drift of the electrode was calculated over time, and a virtual baseline for any tissue measurement time point was determined. The virtual baseline was subtracted from the measured NO current and the resulting value multiplied by the calibration factor to determine the vessel or tissue [NO] in nanomoles. During the in vivo measurement, the tip of the microelectrode was placed against the wall of the microvessel and moved as needed to maintain contact as the vessel diameter changed during perturbations. Only the open tip of the microelectrode can sense NO. Every pharmaceutical agent tested was exposed to electrodes in the test chamber to determine whether at the functional concentration there was an electrode interaction. None of the agents used had an effect. In addition, the NO electrodes are completely insensitive to oxygen up to a PO2 >140 mmHg.

Perivascular O2 measurements. Perivascular O2 was measured using an oxygen-sensitive microelectrode polarized at –0.7 V relative to a ground. The oxygen microelectrodes are made as NO microelectrodes described above with an outer diameter tip of 10 µm and coated with Nafion, as done for NO microelectrodes (Aldrich). The measurements were managed using a Keithley model 6517A electrometer. Each oxygen-sensitive electrode was calibrated prior to experimentation in response to 0, 40, and 144 mmHg PO2 in 37°C heated saline. A zero-oxygen tension during experiments was obtained by occluding a small arteriole with the microelectrode tip. Local oxygen use by tissue quickly eliminated oxygen availability within 30–45 s. As was done for NO measurements, the open tip of the oxygen measurement microelectrode was placed against the wall of the microvessel and moved as needed to maintain contact as the vessel diameter changed during perturbations. PO2 microelectrodes were insensitive to the concentrations of pharmaceutical agents used and did not react to 1,200 nM NO.

For simultaneous use of the NO and PO2 microelectrodes, the polarization voltage was provided by batteries in the microelectrode input to the Keithley electrometer, and either a common or separate ground electrodes were used. Interaction of the microelectrodes was tested by separate calibration for NO and PO2 and then simultaneous calibrations for each species of gas. The microelectrodes did not interact in a pure nitrogen-equilibrated saline environment nor during calibration for either NO or O2 when used in tandem configurations. NO microelectrodes insensitive to PO2 ≤144 mmHg (Atmospheric, Indianapolis, IN) and, likewise, PO2 microelectrodes were insensitive to NO ≤1,200 nM, which is a much higher concentration than arteriolar wall [NO].

Determination of percent saturation. Percent saturation was measured while using a Nonin model 9847V pulse oximeter (Plymouth, MN) located on the ear or fore paw pad of the rat. The fore paw pad generally has lower percent saturation of hemoglobin than the ear vasculature by 4–5 percent saturation units.

Effect of induced hypotension on perivascular and tissue NO and PO2. Perivascular NO and oxygen were measured as the arterial pressure was lowered by removal of blood through the femoral catheter. After each millliter of blood was removed, the animal was allowed to stabilize to a lower arterial pressure before data were collected. Blood pressure and the NO and PO2 measurement systems were monitored and recorded via Powerlab software, an analog-to-digital system (AD Instruments, Colorado Springs, CO).

Determination of source of oxygen loss during hypotension. To determine the primary location of oxygen losses from small arteries and arterioles during hypotension, perivascular oxygen tension was measured in successive branching arterioles at rest and during various degrees of hypotension. The PO2 of collecting venules was also measured to evaluate tissue PO2 with the oxygen microelectrode during hypotension. We specifically used the smallest collecting venules as representative of blood just leaving the brain capillaries. The featureless surface of the brain made measurements of tissue PO2 difficult due to motion of the brain as the blood pressure was altered. Therefore, small venules provided a defined target for microelectrode placement, and their oxygen tension should be representative of tissue oxygenation.

Effect of lowered bath oxygen tension on the generation of NO. To determine the effect of reduced oxygen availability on the generation of NO, the bath oxygen saturation was changed from 90% N2-5% CO2-5% O2 to 95% N2-5% CO2-0% O2. This lowered the bath oxygen tension from 40–45 to 10–15 mmHg over the surface of the brain to simulate a reduction in available oxygen near cortical surface microvessels while maintaining mean arterial blood pressure. The periarteriolar NO and PO2 were measured simultaneously using microelectrodes placed next to the vessel wall at both high and low oxygen tensions. The diameter of the vessel well was also recorded using image analysis.

Involvement of nNOS in cerebral microcirculation regulation. To evaluate the effects of nNOS on vascular response to reduced oxygen tension and hypotension, the vessel diameter and NO were measured using image analysis and a microelectrode, respectively, before and after blockade of nNOS. To initially test for nNOS-dependent responses the vessels were exposed to 5 µM glutamate, and then vessels were allowed to recover. Glutamate receptors of neurons, astrocytes, and glial cells are known to activate nNOS through stimulation of N-methyl-D-aspartate (NMDA) receptors (35). NMDA receptor allows nNOS to uncouple from its scaffolding protein and begin NO generation (1, 35). Previous studies (48, 50) have shown that a concentration of 100 µM glutamate is toxic to neurons. On the basis of all of these findings, we tested various concentrations of glutamate to stimulate NMDA receptors. We found that, although 10 µM caused near-maximum vasodilation, the vessel did not consistently fully recover vascular tone after removal of 10 µM glutamate. During application of 5 µM, adequate vessel dilation occurred and a large increase in [NO] developed and was followed by full recovery upon removal of the glutamate. Our evidence of nNOS suppression is that the vasodilation and substantial increase in [NO] in response to 5 µM glutamate during natural conditions is virtually absent following NMDA receptor blockade with 50 µM N-(4S)-(4-amino-5-[aminoethyl]aminopentyl)-N'-nitroguanidine (N4S) in the same arterioles.

To determine whether nNOS blockade influenced eNOS function, NO and dilatory responses to increased blood flow were evaluated. As the vessel of interest was monitored, its nearest collateral arteriole of equivalent size was nontraumatically occluded as far upstream as possible. This maneuver forced the vessel observed to perfuse ~50% more tissue than normal. After obtaining normal responses, nNOS was blocked and the occlusion study repeated as vessel diameter and [NO] were measured.

Statistical methods. One- and two-way analyses of variance for repeated measures were used to test for differences in groups, and a Tukey least significant difference test was used for specific comparisons. Statistica Software (Statsoft, Tulsa, OK) was used for the various tests.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Arteriolar responses to glutamate. Glutamate at 5 µM was used to activate the NMDA receptor used by nNOS-containing neurons and possibly endothelial cells to allow nNOS to uncouple from its scaffolding protein and begin NO generation. Glutamate induced rapid and large increases in [NO] and arteriolar diameter that were used to test the efficacy of NMDA blockade by 50 µM N4S applied for 30 min before the level of blockade was tested. N4S prevents the NMDA receptor from allowing uncoupling of nNOS from its scaffolding protein. In effect, nNOS cannot be activated through its primary activation mechanism. At control conditions following application of 5 µm of glutamate, NO and diameter increased 133.1 ± 8.5 and 121.6 ± 4.1% (Fig. 1), respectively, of control. After nNOS blockade, resting NO concentration and arteriolar diameter were reduced to 82.6 ± 4.7 and 90.5 ± 3.6%, respectively, of control. Application of 5 µm of glutamate following blockade resulted in essentially no change in NO and diameter, and the measured values were 80.0 ± 6.01 and 87.1 ± 8.21%, respectively. These data indicate that N4S adequately blocked the overall process of nNOS activation and subsequent generation of NO by nNOS. After these measurements associated with nNOS blockade were completed, 1 mM NG-nitro-L-arginine methyl ester (L-NAME) was added to the bathing medium. This approach was used to determine the lowest possible [NO] with combined N4S and L-NAME blockade and associated vasoconstriction. The [NO] declined to 62.1 ± 5.1% of the original control, and the diameter decreased to 71.4 ± 0.9%.


Figure 1
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  		Fig. 1. Arteriolar response to glutamate before and after neuronal nitric oxide synthase (nNOS) blockade. Seven experiments are included to show adequate blockade of nNOS by eliminating both nitric oxide (NO) and dilatory responses to 5 µM glutamate stimulation of the N-methyl-D-aspartate (NMDA) receptor. *Statistical significance compared with control; #statistical significance compared with glutamate in the absence of N-(4S)-(4-amino-5-[aminoethyl]aminopentyl)-N'-nitroguanidine (N4S). L-NAME, NG-nitro-L-arginine methyl ester.

 
To determine whether the nNOS blocker used interfered with eNOS functions, we evaluated NO and dilatory responses to increased blood flow, as described in METHODS. The data in Fig. 2 show that dilation to increased flow was essentially normal after nNOS blockade. The resting [NO] was reduced by nNOS blockade, but the increase in [NO] during collateral occlusion was 82% of that during natural conditions. This indicated that eNOS was functionally acceptable after N4S blockade of nNOS.


Figure 2
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  		Fig. 2. Determination of nNOS blockade on ability of in vivo cerebral arterioles to demonstrate flow-mediated dilation in 5 arterioles of 5 rats. The dilation and NO concentration of the chosen arteriole was measured at rest and during occlusion of a collateral arteriole. The amount of tissue the vessel must perfuse was increased ~50% by the occlusion of the collateral arteriole, and the NO concentration increased and the vessel dilated. The measurements were repeated after nNOS was blocked, and the same arterioles dilated essentially normally during collateral arteriole occlusion. However, their resting NO concentration was decreased, but the increase in NO concentration during collateral occlusion was ~80% of normal, although at lower absolute concentrations. *Significant (P < 0.05) change from normal resting conditions.

 
Effect of hemorrhagic hypotension on inner diameter, periarteriolar NO, and O2. The data for all measurements in Fig. 3 were obtained by recording NO and PO2 on the arteriolar wall and recording the vessel diameter as the arterial pressure was lowered by hemorrhage. For this study, we used the largest arterioles available in each preparation. For these vessels, the resting diameter was 46.3 ± 2.8 µm, the resting NO was 660 ± 106 nM, and the resting perivascular oxygen tissue was 79.9 ± 5.1 mmHg. To measure NO and PO2 on all arterioles with diameters less than ~60 mm, the microelectrode tip had to pierce the overlying pial membrane and a small amount of overlying tissue to reach the side of the vessel wall. In effect, the vessels were covered and embedded in the pial surface layer of the brain.


Figure 3
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  		Fig. 3. The relationship between periarteriolar oxygen, NO, and inner diameter during hypotension caused by hemorrhage under control conditions (solid lines and bullet) and after nNOS blockade (dashed lines and {circ}) in the same 6 rats. The periarteriolar oxygen tension decreased during hypotension, but both the NO concentration and inner vessel diameter increased. In the same 6 animals, the measurements were repeated after nNOS blockade with N4S. After blockade (dashed lines and {circ}) the NO concentration was lower at rest, which is defined as arterial pressures of 100–110 mmHg, and failed to increase during hypotension. Perivascular oxygen tension was also lower by about one-third after nNOS blockade at all arterial pressures. Inner vessel diameter decreased after nNOS blockade, but the vessels were able to dilate, although not normally, during hypotension.

 
During hemorrhagic hypotension, periarteriolar oxygen tension decreased, [NO] simultaneously increased, and arteriolar diameter increased, as shown for six animals in Fig. 3. The pressure range of 100–110 mmHg was used as the control reference for all measurements. For a given change in arterial pressure, a new steady state for all three measurements was generally achieved within 50–60 s after the arterial pressure was changed, but we allowed >5 min to make sure the responses were stable. The decline in oxygen tension and elevation of [NO] were significant at a 10-mmHg drop in mean arterial pressure to the 90- to 100-mmHg range.

After nNOS blockade, repeated measurements on exactly the same sites along the vessel indicated a lowered resting oxygen tension by ~30% over the entire arterial pressure range, a 22% depressed [NO] at all arterial pressures, and no indication of increased [NO] with hypotension. N4S caused constriction of 3–5% of every vessel studied, and this small amount of constriction persisted over the entire range of arterial pressures. Although the constriction was small, it was important because the perivascular oxygen tension decreased an average of 26 mmHg, or about one-third at the arterial pressure range of 100–110 mmHg, and this lowered oxygen tension persisted for all of the lowered arterial pressures.

Source of periarteriolar oxygen loss during hypotension. We routinely measured percent saturation of hemoglobin in the forelimb paw pad rather than in the ear because access to the ears was limited by the bath-heating device. The paw pad is not ideal because it typically has a lower hemoglobin saturation than the ear by 4–5 percent saturation units, and during severe hypotension poor perfusion of the paw did artifactually yield low percent saturation data in some cases. To further evaluate this issue with a better perfused tissue in five animals, we used the ear vasculature and repeated bouts of hypotension as needed to obtain a pressure profile. At resting conditions with a mean arterial pressure of 103.7 ± 1.1 mmHg, the percent saturation was 94.2 ± 1.5%. At the lowest arterial pressures reached in a given animal of 55.9 ± 0.9 mmHg, the percent saturation was 92.6 ± 1.7%. There was no statistical significance between these values or any of those recorded at intervening arterial pressures.

Periarteriolar oxygen tension declined from larger to small arterioles at rest because all vessels were losing oxygen to the tissue (Fig. 4). In Fig. 4, solid lines are used for the largest arterioles and the next generation arteriole is represented by a dashed line. The same symbols on these lines are used for consecutive arterioles. Note in Fig. 4 that at the resting arterial pressure, which is the rightward end points of the lines in Fig. 4, the oxygen tension in large arterioles is greater than that in the intermediate diameter branch arterioles they immediately perfused. When hypotension was induced, the oxygen tension in both the large and intermediate diameter arterioles decreased. The observation of a decline in oxygen tension in large arterioles during hypotension indicated that oxygen was lost further upstream in the vasculature, such as in the small arteries or the very large arterioles.


Figure 4
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  		Fig. 4. Oxygen losses along large and intermediate arterioles were evaluated by measuring perivascular oxygen tension in the largest arteriole available and its next branch order during normal and hypotensive conditions. Solid lines represent larger arterioles and dashed lines are the smaller vessels, with the same symbols on these lines used for consecutive arterioles in a given animal. There was a substantial reduction of oxygen in the largest arterioles during hypotension that likely occurred in part in even larger upstream vessels. The loss of oxygen from larger vessels in turn lowered the oxygen tension, reaching smaller arterioles at rest and during hypotension. These data suggest that all resistance vessels experience decreased oxygen tension during hypotension. The data are based on 3 pairs of arterioles from 3 animals.

 
Investigation of collecting venule oxygen tension during hypotension demonstrated that oxygen tension remained relatively constant during hypotension down to ~60 mmHg, as shown by data from five rats in Fig. 5. The data demonstrate that capillary bed tissue oxygenation was maintained by vasodilation of the arterioles to quite low arterial pressures (Fig. 3).


Figure 5
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  		Fig. 5. Collecting venule PO2 as an indicator of tissue PO2 was measured during hypotension. The venules used were the smallest venules on the surface of the brain that received only blood flow from deeper cortical tissues. Five separate examples from 5 rats indicated adequate oxygenation of cerebral tissue during hypotension. Tissue oxygenation was maintained even at arterial pressures of 55–65 mmHg.

 
Arteriolar responses to locally reduced oxygen tension. A reduction in bath oxygen tension from 5% O2 to 0% O2 caused an increase in NO production and simultaneously a decline in periarteriolar oxygen tension. Examples of the perivascular oxygen tension, [NO], and inner diameter responses in six rats are shown in Fig. 6, A and B; the same vessel is represented in both figures by the same line format. A reduction in periarteriolar oxygen tension as small as 10 mmHg increased the [NO] at that vessel wall as much as 50% in some vessels (Fig. 6A). Some vessels appear more responsive in their increase in [NO] to a decline in oxygen tension than others. However, every vessel demonstrated a substantial increase in NO when oxygen tension was decreased and every vessel dilated. The various increases in [NO] at reduced bath oxygen tension coincided with a simultaneous 15–20% increase in vessel diameter (Fig. 6B). Dilation of this magnitude could increase cerebral blood flow by 75–100% if all vessels exhibited this response.


Figure 6
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  		Fig. 6. A: perivascular NO concentration ([NO]) and PO2 were measured at rest and when the bath oxygen tension was lowered to reduce the tissue oxygen content. Even 10–20 mmHg reductions in periarteriolar PO2 caused a 20–50% increase in periarteriolar NO production. B: the same line pattern in A and B presents the same vessel. The data in B represent the diameter/periarteriolar PO2 relationships. Inner diameter increased as periarteriolar oxygen tension decreased. A reduction in bath oxygen tension to lower periarteriolar PO2 caused a 10–20% increase in inner vessel diameter. The data set is based on 6 arterioles studied in 6 rats.

 
Arteriolar responses following nNOS blockade. Following nNOS blockade by N4S, the normal increase in NO production and arteriolar vasodilation in response to lowered bath oxygen tension, as shown in Fig. 7, was no longer generated. These results are based on studies in six rats. The NO concentration following nNOS blockade dropped to 75.6 ± 7.7% of control at a bath oxygen tension of 40–45 mmHg, which for this purpose is termed high oxygen in Fig. 7, and remained at 75.8 ± 9.6% of control during reduced oxygen tension, or low oxygen. The inner arteriolar diameter did not increase as a result of exposure to decreased oxygen tension after N4S blockade (Fig. 7B).


Figure 7
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  		Fig. 7. A: [NO] responses. B: the diameter responses to reduced bath and perivascular PO2 before and following blockade of nNOS by N4S. After nNOS blockade, reduction of oxygen availability in the tissue bath did not cause the normal increase in periarteriolar [NO] and vessel diameter. These data are based on evaluations of individual vessels in 6 rats. *Statistical significance compared with control; #statistical significance compared with low oxygen conditions absent of drug.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Many different mechanisms likely contribute to cerebral autoregulation and have been extensively reviewed (8, 15, 29, 49). The available data for a possible role of NO are based exclusively on what does not happen in vascular regulation during hypotension after various NOSs are suppressed. Therefore, we chose to measure in vivo perivascular and tissue NO and PO2 to evaluate the hypothesis that nNOS could provide NO to the arterioles at rest and during periods of reduced perivascular oxygen tension, such as during hypotension or reduced local availability of oxygen. With the advent of highly specific nNOS inhibitors, removal of this form of NO generation allowed us to evaluate whether nNOS or eNOS best accounted for the NO measured at the arteriolar wall. In the interpretation of our data, it is important to point out that the animals were anesthetized with thiopental, and, as with barbiturate anesthetics in general, the brain metabolic rate should be reduced on the basis of studies by other investigators (38). However, several investigators (14, 37) have reported that, if hypotension during deep anesthesia was avoided, the decline in metabolism was minimized. We maintained blood pressure by careful support of body temperature, intravascular fluid support, and mechanically assisted ventilation to maintain oxygenation and normocapnia in normal ranges. We chose thiopental over halothane because halothane anesthesia is associated with dilation of cerebral vessels through a NO mechanism linked to nNOS (14). We will demonstrate well-maintained nNOS mechanisms under thiopental anesthesia. However, all forms of anesthesia lower the cerebral metabolic rate, and as such we have likely underestimated the role of oxygen mechanisms, although our results support an important linkage of oxygen availability and nNOS activity to influence arteriolar dilation.

When hypotension was induced, periarteriolar [NO] increased, whereas oxygen tension around the arteriole decreased (Fig. 3), but venular oxygen tension was almost constant (Fig. 5). The reduction of periarteriolar oxygen and maintenance of tissue oxygenation estimated from venular oxygen tension seemed paradoxical. We first determined that the decline in periarteriolar oxygen tension was not due to a decrease in systemic oxygenation during hypotension (RESULTS). Given that arterial blood oxygen content was essentially the same at normal and reduced arterial pressure (RESULTS), a reduction in perivascular oxygen tension around larger arterioles simply indicated that oxygen was lost from upstream vessels to the tissue. As shown in Fig. 4, there was also a decrease in periarteriolar oxygen tension from the large arterioles to smaller arterioles both at resting arterial pressures and during hypotension. The loss of oxygen from the arterioles was expected because tissue oxygen tension was consistently below 20 mmHg, with most values about 15 mmHg, as we have previously shown (47). The loss of oxygen to tissue by arterioles during resting conditions was first proven by Duling and colleagues (11, 12) and has been confirmed (4, 13, 41) in very divergent vascular beds. We propose that, because the vessel diameter was increased during hypotension, the surface area of the vessel obviously increased and would facilitate oxygen exchange from the arterioles to tissue. Furthermore, the increase in vessel diameter would cause the velocity of blood cells to decrease at a relatively constant blood flow, allowing more time for oxygen losses as blood moves slower through the resistance vasculature. However, oxygen tension and therefore oxygen content in the arterioles did not fall sufficiently to compromise oxygenation at the tissue level. As shown in Fig. 5, the vascular oxygen supply, judged by venular oxygen tension in small collecting venules, was sufficient to maintain capillary bed oxygenation to quite low arterial pressures. These observations confirm our earlier evaluation in the rat brain (16) that tissue oxygen tension is well preserved during hemorrhagic hypotension. However, a modest to even large decrease in periarteriolar oxygen tension during hypotension could be an error signal used in regulation of vascular tone.

A decline in oxygen tension within resistance vessels has been implicated in cerebral vascular control through a NO mechanism based on the studies of Armstead (2) and Kirkeby et al. (27). Following blockade of all forms of NOS by systemic administration of L-NAME, the cerebral vasculature did not dilate in response to systemic hypoxia. The hypoxia in these referenced studies was extreme because systemic oxygen partial pressures of 30–50 mmHg were used. Although such large reductions in oxygenation would be of clinical concern in emergencies, our studies shown in Figs. 6 and 7 investigated the response of the cerebral vasculature to reductions in oxygen tension comparable with those that occurred around cerebral arterioles during moderate hypotension (Fig. 3). Rather than reduce whole body oxygenation, only the microvasculature viewed was subjected to a decrease in oxygen tension. As shown in Figs. 6 and 7 for separate groups of animals, a 10- to 30-mmHg decrease in periarteriolar oxygen tension by bath oxygen tension manipulation caused a 40–50% increase in [NO]. By comparison, in Fig. 3, a 10- to 30-mmHg reduction in arteriolar wall oxygen tension during hypotension also caused a 20–50% increase in [NO]. Therefore, whether periarteriolar oxygen tension was reduced by hypotension or localized reductions, similar increases in [NO] occurred.

To place the cerebral vascular [NO] responses of the brain vasculature in perspective at reduced oxygen tension, they are approximately equivalent to those of rat intestinal arterioles during similar localized reductions in periarteriolar PO2 (36, 53). What is markedly different is that in the intestinal vasculature eNOS was the dominant source of increased [NO] at lowered oxygen tension, but nNOS will be shown to be the dominant source for the brain vasculature. In Fig. 7, the data indicated that blockade of nNOS with N4S eliminated both vasodilation and increased [NO] in response to locally decreased oxygen tension. Furthermore, as shown in Fig. 3, the [NO] did not increase during hypotension. Multiple studies (24–26, 43) have concluded that blockade of eNOS and nNOS through nonspecific arginine analogs or nNOS by 7-NI interfered with cerebral vascular dilatory responses to both hypotension and hypoxia. All previously published studies implicating nNOS as a major source of NO have been done with 7-NI and propose a role for nNOS of neurons, glial cells, and astrocytes in cerebral regulation of blood flow (9, 23, 31, 44, 51). Although we will ultimately agree with the findings of these past studies, there is a technical issue for the use of 7-NI. The selectivity of 7-NI for nNOS over eNOS is only slightly different. The IC50 between nNOS and eNOS are 0.71 and 0.8 µM (19, 28, 34, 46, 48a). This small difference in IC50 values does not provide good confidence that eNOS would be functional in the presence of 7-NI. The nNOS blocker we chose, N4S, has a 2,100-fold selectivity over eNOS and 70-fold selectivity over inducible NOS (1, 2, 20, 22). To evaluate the nNOS blockade with N4S, the dilatory and [NO] responses to 5 µM glutamate were compared before and after blockade (Fig. 1). Glutamate stimulates the NMDA receptor found on neurons and activates nNOS through an influx of calcium into the neurons and neuronal supporting cells (35). It is possible that glutamate receptors have actions other than NO generation when NMDA receptor is activated. Leffler et al. (30) and Parfenova et al (39) have shown that, in newborn pigs, glutamate activation of the NMDA receptor can also cause the release of carbon monoxide. However, Domoki et al. (10), also using the in vivo brain of newborn pigs, reported that NO from nNOS origin was the dominant vasodilator released by glutamate stimulation. In our experience after N4S blockade, neither vasodilation nor increased [NO] occurred in response to glutamate (Fig. 1). This would argue that in young adult rats, a well-developed dilatory mechanism involves NMDA receptors linked to nNOS. We were also concerned that N4S might have effects during in vivo conditions on the functions of eNOS. To evaluate this possibility, the responses of vascular wall [NO] and diameter to increased blood flow were evaluated (Fig. 2). nNOS blockade had little effect on the dilation to increased blood flow but did lower the resting and high flow [NO]. However, the increase in [NO] during high flow following nNOS blockade was 82% of that during natural conditions. This observation predicts that eNOS was highly functional after blockade of nNOS with N4S.

The regulation of the cerebral arterioles after nNOS blockade was dysfunctional in terms of supplying oxygen to tissue, yet the vessels did retain the ability to dilate during hypotension. As shown in Fig. 3, nNOS blockade caused a small constriction at rest (MAP 100–110 mmHg) that persisted, as the arterioles did in fact dilate to reduced arterial pressures. The small amount of constriction was important because nNOS blockade lowered the resting periarteriolar PO2 by about one-third, and this decline persisted at all hypotensive arterial pressures. In addition, the [NO] was lowered by nNOS blockade and demonstrated no appreciable increase during hypotension compared with about 40–50% increase in [NO] at arterial pressures below 90 mmHg under natural conditions. These various observations indicate that the NO influence of nNOS is particularly important to minimizing the decline in brain oxygen supply during hypotension, but there are other well-developed mechanisms to partially preserve vasodilation.

Although nNOS has important regulatory functions in the cerebral circulation, it is not the major source of perivascular NO. Note in Figs. 1, 2, 3, and 7 that the resting [NO] decreased about 20–25% after nNOS blockade; each figure represents studies of a different set of animals. We believe nNOS is very suppressed because glutamate did not cause either dilation or increased [NO]. This means that about 80% of the NO remaining is from either eNOS or possibly released from NO complexed with blood proteins. As shown in the studies presented by Fig. 2, eNOS is very functional after N4S blockade of nNOS and is likely generating a substantial portion of the [NO] after nNOS blockade. On the basis of these observations, we expected the [NO] to increase via eNOS mechanisms to some extent during hypotension after N4S blockade. However, the [NO] did not increase even with severe hypotension following nNOS blockade, as shown in Fig. 3. These observations predict that nNOS is almost exclusively used to raise perivascular NO during hypotension and that eNOS has little influence. However, although the arterioles were constricted at rest after N4S, they were able to dilate during hypotension, as shown in Fig. 3, although the [NO] did not increase. This remaining dilation could be from many non-NO mechanisms that other investigators have found associated with cerebral autoregulation and have been reviewed in depth (1, 33, 47). However, the much lower than normal vascular wall oxygen tension during hypotension following nNOS blockade shown in Fig. 3 and loss of cerebral vascular responses to reduced oxygen tension in Fig. 7 illustrate how important nNOS contributions are to resting cerebral vascular tone and its ability to respond to both hypotension and reduced oxygen availability.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Grant HL-20605 from the National Heart, Lung, and Blood Institute.


   ACKNOWLEDGMENTS
 
We express our appreciation for Dr. Donald Buerk on methodologies used to simultaneously measure nitric oxide and oxygen tension. We thank Dr. Jin Song for surgical assistance in the studies on hemorrhagic hypotension.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. G. Bohlen, Dept. of Cellular and Integrative Physiology, Indiana University Medical School, 635 Barnhill Drive, Indianapolis, IN 46202 (e-mail: gbohlen{at}iupui.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
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 ABSTRACT
 METHODS
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 DISCUSSION
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