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Circulation Research. 1995;77:832-840

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(Circulation Research. 1995;77:832-840.)
© 1995 American Heart Association, Inc.

Articles

Modulation of Cerebral Arteriolar Diameter by Intraluminal Flow and Pressure

Al C. Ngai, H. Richard Winn

From the Department of Neurological Surgery, University of Washington School of Medicine, Seattle.


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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Abstract We determined whether cerebral arterioles in vitro adjust their diameters in response to changes in intraluminal flow rate and pressure. Intracerebral arterioles (38- to 55-µm diameter) were isolated from Sprague-Dawley rats and cannulated with a perfusion system that permitted separate control of intraluminal pressure and flow rates. Increasing pressure at 0 flow, in 20 mm Hg steps from 20 to 100 mm Hg, resulted in myogenic constriction, which was greatest at 60 mm Hg ({approx}20%). Increasing flow rate at a constant pressure of 60 mm Hg elicited a biphasic response. At flow rates of up to 10 µL/min, the arterioles dilated by up to 14.5±2.2% of their control diameter. At higher (>10 µL/min) flow rates, however, a progressive restoration of resting diameter was observed. Application of the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA, 0.1 mmol/L) caused a 15.4±1.7% decrease in control diameter (at 60 mm Hg, zero flow). Although L-NMMA did not affect the responses to increases in pressure or to vasodilators (adenosine and pH 6.8 buffer), it abolished the dilator responses to flow rate increases and to acetylcholine. In contrast, inhibition of prostaglandin synthesis by indomethacin (10 µmol/L) had no effect on flow-induced dilation. These results show that changes in intraluminal flow rates and pressure can independently influence cerebral arteriolar tone and suggest that the flow-induced dilator responses of cerebral arterioles are mediated by an arginine metabolite, such as nitric oxide.


Key Words: shear stress • flow velocity • cerebral circulation • nitric oxide • myogenic response


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
As blood flows through the lumen of a vessel, it exerts a frictional drag on the endothelial cells that line the vascular wall. Until recently, little was known about the physiological role and effect of this frictional force, or shear stress, in the vascular system. The discovery that endothelial cells can respond to external stimuli by releasing an endothelium-derived relaxing factor,1 now identified as nitric oxide (NO)2 or a closely related compound,3 provided impetus for studies on shear-induced responses of blood vessels. Subsequently, an increase in intraluminal flow was found to induce relaxation of vessels in a wide variety of vascular beds.4 5

In the brain, flow-induced dilation has been demonstrated at the arterial level.6 7 8 Whether cerebral arterioles also possess flow sensitivity is unknown. Such flow sensitivity, however, has important functional implications. Because increased functional activity in the brain is accompanied by a local increase in parenchymal blood flow,9 10 subsequent flow-mediated dilation of upstream penetrating arterioles as well as pial arterioles may facilitate tissue blood flow by restoring downstream perfusion pressure.11 Flow-induced dilation is also potentially important in establishing collateral flow when there is local occlusion, for example, by microvascular embolism.

In various arteries12 13 and arterioles,14 15 flow-induced dilation required the integrity of the endothelium. Cultured bovine aortic endothelial cells released a nitrovasodilator identified as NO in response to shear stress stimuli.16 17 L-Arginine analogues, which competitively inhibit NO synthase (NOS), the enzyme that catalyzes the formation of NO from L-arginine,18 blunted the flow-induced response.19 20 21 Furthermore, the inhibition was reversed by L-arginine, thus implicating NO as the mediator of flow-induced dilation. In rat cremaster muscle arterioles, however, indomethacin but not arginine analogues abolished flow-induced dilator responses,15 22 suggesting that prostaglandins may be a mediator of flow-induced dilation in some tissues.

In addition to shear stress, changes in intravascular pressure may also modulate vascular tone. Blood vessels react to an increase or decrease in transmural pressure by constriction and dilation, respectively.23 This ability to respond to pressure was originally proposed by Bayliss24 to reside in vascular smooth muscle cells and was therefore termed a myogenic response. Myogenic responses have been well documented in both arteries25 26 and arterioles27 28 29 of the cerebral circulation.

In the present study, we determined the effects of increases in flow velocity on intracerebral arterioles with an in vitro method that allowed flow-induced changes in vessel diameter to be evaluated while holding constant other variables, such as pressure and metabolism. For comparison, we also investigated the responses of intracerebral arteries to changes in intraluminal pressure. In addition, we explored the mechanism of flow-induced dilation by testing for the possible involvement of both NO and prostaglandins.


*    Materials and Methods
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 up arrowIntroduction
 *Materials and Methods
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 down arrowDiscussion
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Vessel Isolation and Cannulation
A total of 32 male Sprague-Dawley rats, weighing between 350 and 400 g, were anesthetized with pentobarbital (50 mg/kg IP) and decapitated. The brain was rapidly removed from the skull, immersed in buffered saline solution containing 1% dialyzed bovine serum albumin, and cooled to 4°C. A piece of cerebral cortex {approx}2 mm thick containing the first portion of the middle cerebral artery was dissected from the brain. The pia mater and its attached penetrating intracerebral arterioles were then separated from the parenchyma, and a segment of an intracerebral arteriole {approx}0.5 to 1 mm in length was severed from the pia and transferred to a temperature-controlled vessel chamber (1-mL volume) mounted on the stage of an inverted microscope.

The isolated vessel was cannulated at both ends by using a system of concentric glass pipettes consisting of a perfusion pipette within a holding pipette, as described previously.30 31 The pipettes were shaped with a microforge (Stoelting Co) according to a design that minimized flow resistance.32 The holding pipette was narrowed near its tip to form a constriction 40 to 45 µm wide, whereas the perfusion pipette was tapered to a tip of 25- to 30-µm diameter. The downstream end of the vessel was connected to a manometer used to set intraluminal pressure via an outflow reservoir (Fig 1Down). The upstream end was connected to a microinfusion pump for intraluminal fluid perfusion. A fluid reservoir pressurized by the manometer could be inserted into the fluid path between the vessel and the pump by means of a three-way liquid switch. Upstream pressure was monitored with a pressure transducer. The cannulated vessel was observed on a video monitor via a video camera, and lumen diameter was measured with a video dimension analyzer.



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  		Figure 1. Schematic diagram of experimental setup.

Fluid resistance in the perfusion apparatus resided for the most part in the perfusion pipette, which was fabricated from glass tubes of 1.0-mm internal diameter. The rest of the system consisted mainly of PE-50 tubing (internal diameter, 58 mm) that was considerably larger in diameter. We measured the back pressure generated by flow in either the upstream or downstream apparatus by connecting the perfusion pump to each side separately (at points A and B in Fig 1Up) and found it to critically depend on pipette tip diameter. Thus, the balance of upstream and downstream resistances could be primarily achieved by using perfusion pipettes of similar tip diameter and shape and secondarily fine-tuned by matching the lengths of the PE-50 tubing. With such a procedure, the pressure heads generated by a flow rate of 10 µL/min at either upstream (point A, Fig 1Up) or downstream (point B) sites did not differ by >2 mm Hg between each of five pairs of matched pipettes. With a vessel in place in such a "matched resistance" system, the increase in upstream pressure generated by flow could be countered by an equal reduction of downstream pressure, such that intravascular pressure at the middle of the vessel remained unchanged. Thus, independent control of flow and pressure could be achieved. The rise in pressure gradient generated by increasing flow rates was documented in 10 experiments. As shown in Fig 2Down, there was a linear increase of {approx}2.5 mm Hg/µL · min-1, indicating steady nonturbulent flow in the system up to 25 µL/min. However, the data do reveal greater deviation from linearity at the higher flow rates.



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  		Figure 2. Graph showing the relation between pressure gradient and flow generated by the infusion pump in the experimental apparatus after vessel cannulation. Data were obtained from 10 preparations. The line drawn through the data is the regression line, and the regression equation is shown in the inset. r indicates the correlation coefficient.

After cannulation, intraluminal pressure was set at 60 mm Hg. Before tone development, "passive" vessel diameter was measured. Treatment with a supramaximal concentration of a potent vasodilator (1 mmol/L sodium nitroprusside) at the end of five experiments verified that passive diameter indeed represented the maximally dilated state. The bath solution was next changed to one without albumin, and bath temperature was raised to 37°C. The vessel was perfused at a rate of 2 µL/min. After an equilibration period of {approx}40 minutes, during which time the bath solution was changed once every 5 to 10 minutes, viable arterioles developed vasomotion and spontaneously contracted. Control diameter was measured after the development of steady tone. Reactivity of the vessels was assessed by changing the extraluminal pH from 7.3 to 6.8 and from 7.3 to 7.6. Vessels with weak pH response (<15% dilation or constriction) were excluded from data collection. In addition, responses to endothelium-dependent (acetylcholine) and -independent (adenosine) vasoactive agents were evaluated. Because extraluminal acetylcholine had little effect on intracerebral arterioles,31 33 acetylcholine was applied by intraluminal infusion, whereas adenosine was applied extraluminally.31 The time course of tone development was also found to be an important indicator of vascular viability. Only vessels that displayed contractions within 5 minutes after the onset of bath warming generated satisfactory tone and responsiveness to vasoactive agents such as H+ ion, adenosine, and acetylcholine.

Acetylcholine was dissolved in buffered saline containing 1% albumin for intraluminal perfusion. Before the start of each solution change, the upstream end of the vessel was opened to the pressurized fluid reservoir, which offers less flow resistance than the cannulated arteriole. Rapid solution change could thus be accomplished at a high flow rate (30 µL/min for 5 minutes) with minimal (<5 mm Hg) increase in upstream pressure. Since the perfusate conduits occupied a volume of 20 µL, total replacement could be achieved within 5 minutes. Flow to the reservoir was then switched off, and the vessel was perfused with the new solution at 2 µL/min for 5 to 10 minutes until a steady vessel diameter was measured.

Protocols
After the development of spontaneous tone at 60 mm Hg, the relation between pressure and vessel diameter was studied under zero-flow conditions. Pressure was initially decreased to 20 mm Hg and then raised in 20-mm Hg steps from 20 to 100 mm Hg. This pressure range corresponds to the autoregulatory range of similarly sized pial arterioles in vivo.34 At each step, vessel diameter was monitored for 5 minutes to determine steady state diameter. Pressure was then reset to 60 mm Hg, and preheated saline (pH 7.3) containing 1% albumin was infused into the vessel by means of the infusion pump. The relation between flow rate and vessel diameter was determined by increasing flow from 0 to 25 µL/min in 5-µL/min increments. Each step increase in flow rate led to a rise in upstream pressure, and downstream pressure was adjusted (within 20 seconds) so that upstream and downstream pressure changed by an equal and opposite magnitude, thus maintaining intravascular pressure constant at 60 mm Hg. The vessel was allowed to adjust to the new flow rate for 10 minutes, and steady state diameter was measured.

Pharmacological Treatments
To test the involvement of NO in the pressure and flow response of intracerebral arterioles, the above procedures were repeated in the presence of the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA). Vessels were treated with L-NMMA (0.1 mmol/L) for 30 minutes until a new steady state diameter was reached. This was followed by reassessment of the pressure- and flow-diameter relations. When response alteration by L-NMMA was observed, L-NMMA was replaced by L-arginine (1 mmol/L), and the protocol was repeated in an effort to reverse the effects of L-NMMA.

Because cyclooxygenase reaction products such as prostaglandins have been implicated as a mediator of flow-mediated dilation, flow-diameter relations were also determined in the present study before and after vessel treatment with indomethacin (10 µmol/L).

In a separate series of experiments, the efficacy and specificity of NO blockade by L-NMMA was evaluated by determining the response of intracerebral arterioles to acetylcholine (1 µmol/L), adenosine (1 µmol/L), and H+ ion (pH 6.8) before and after L-NMMA (0.1 mmol/L) treatment. Acetylcholine was applied by intraluminal perfusion, whereas adenosine and pH 6.8 solutions were applied extraluminally.

Drugs and Solutions
The composition of the buffered saline solution is as follows (mmol/L): NaCl 144.0, KCl 3.0, CaCl2 2.5, MgSO4 1.5, glucose 5.0, pyruvate 2.0, EDTA 0.02, MOPS 2.0, and NaH2PO4 1.2. L-NMMA, sodium nitroprusside, and adenosine were dissolved directly into this buffered solution. Indomethacin was dissolved in a 0.1 mol/L NaHCO3/ethanol mixture (3:1 [vol/vol]) to produce a concentration of 1 mmol/L. L-Arginine was made up in saline to yield a stock concentration of 10 mmol/L. Subsequent dilutions were made with buffered saline, and pH was adjusted to 7.3. Intraluminally applied saline solutions contained 1% bovine albumin. All drugs were obtained from Sigma Chemical Co.

Data Analysis
All data are expressed as mean±SEM. Only one vessel was studied from each animal. For comparison of responses to vasoactive agents, internal vessel diameters were normalized as percentage of control diameters (steady state diameters measured before treatment at 60 mm Hg intraluminal pressure and 37°C). Statistical analyses were performed with SYSTAT software (SYSTAT Inc). One-way ANOVA with repeated measures was used to determine if responses to pressure or flow were statistically significant. Comparisons of vascular diameters and responses between two groups (eg, control and L-NMMA–treated groups) were made by performing repeated-measures ANOVA on the paired differences between the two groups. Differences in the responses to pressure or flow between two groups were determined with the F test. Differences in diameters between two groups were determined by testing whether the sum of the differences in diameters was significantly different from zero. Mean values were compared using paired Student's t tests. The Bonferroni correction was applied when there were multiple groups. A value of P<.05 was considered significant.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Isolated intracerebral arterioles (n=32) developed myogenic tone at 37°C and 60 mm Hg and constricted to 55% to 70% (mean, 65.8±1.0%) of their passive diameters. Vessel diameter after spontaneous tone development at 37°C ranged from 38 to 55 µm. As shown in the TableDown, these vessels were responsive to vasoactive stimuli including pH, adenosine, and acetylcholine. As is characteristic of cerebral vessels, intracerebral arterioles exhibited marked sensitivity to pH changes. Constriction was observed when extraluminal pH was raised from 7.3 to 7.6, and dilation ensued when pH was lowered to 6.8. In contrast, intraluminal infusion of pH 6.8 buffer (n=5) had no significant dilator effect (data not shown).


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  		Table 1. Response of Intracerebral Arterioles to Vasoactive Agents

Effects of Pressure and Flow on Arteriolar Diameter
The steady state response of intracerebral arterioles (n=8) to stepwise increases in intraluminal pressure under zero-flow conditions is shown in Fig 3Down. Changes in diameter were overall statistically significant (P<.01, ANOVA). In response to a step increase in pressure from 20 to 40 mm Hg, the vessels constricted by 16% and in most cases required 1 to 2 minutes to attain steady state diameter. The next step increase in pressure (from 40 to 60 mm Hg) induced a smaller constrictor response (5%). Further increase in pressure did not enhance tone but instead led to a progressive increase in vessel diameter. Elevating pressure to or beyond 100 mm Hg in a few experiments resulted in failure to regain control diameter, probably because of irreversible damage caused by forced distension.



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  		Figure 3. Left, Graph showing the steady state diameter of intracerebral arterioles as a function of intravascular pressure in the absence of flow (n=8). Right, The change in steady state diameter of intracerebral arterioles as a function of intraluminal flow rate at constant intravascular pressure of 60 mm Hg (n=12). Values are mean±SEM. *P<.05, #P<.01 vs diameter at pressure of 20 mm Hg (left) or at flow of 0 µL/min (right), respectively.

The steady state response to flow increase at constant intravascular pressure was examined in an additional four vessels (total n=12). After each step increase in flow rate, pressure was adjusted to maintain a constant intravascular pressure of 60 mm Hg. As shown in Fig 3Up, the flow-induced response also exhibited a biphasic profile. Vasodilation was observed at the slower flow rates of 5 and 10 µL/min. The diameter increases of 4.9±1.6% at 5 µL/min and 14.5±2.2% at 10 µL/min were statistically significant. Further increase in flow did not cause diameter increase but instead led to a progressive decrease in vessel diameter. ANOVA indicated significant differences in diameter overall (P<.01).

Effects of L-NMMA and Indomethacin on Pressure and Flow-Induced Response
In control conditions (no flow, 60 mm Hg intravascular pressure), intracerebral arterioles (n=12) constricted by an average of 15.4±1.7% after incubation in L-NMMA. However, L-NMMA did not appear to affect the pressure-diameter relation (n=5, Fig 4Down). There was no significant difference between pressure-induced responses before and after L-NMMA treatment, as indicated by ANOVA of the paired differences between control and L-NMMA–treated groups. The pressure-induced response, calculated as the percent reduction in vessel diameter from 20 to 60 mm Hg intravascular pressure, was not significantly affected by L-NMMA (20±2% before L-NMMA versus 22±1% after L-NMMA application). Thus, NO inhibition did not appear to influence the response of intracerebral arterioles to pressure.



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  		Figure 4. Graph showing relation between steady state diameters of intracerebral arterioles and intraluminal pressure before (control) and after application of 0.1 mmol/L NG-monomethyl-L-arginine (L-NMMA) (n=5 arterioles). Values are mean±SEM. *P<.05, #P<.01 vs corresponding diameters at 20 mm Hg. {delta}Significant difference in diameters between control and L-NMMA–treated groups (P<.01). L-NMMA did not significantly alter pressure-induced responses.

On the other hand, application of L-NMMA completely abolished (P<.05 versus control) the dilator response (14.9±3.1% at 10 µL/min) of arterioles (n=7) to flow increase (Fig 5Down). Application of L-arginine caused the vessels to regain control diameter within {approx}5 minutes and appeared to induce a slight dilation (5.6±4.6% increase of control diameter), although the diameter change was not statistically significant. L-Arginine restored the ability of the vessels (P<.05 versus control) to dilate in response to flow (11.4±1.6% at 10 µL/min). There was no difference between the flow-diameter relations in the control condition and after L-arginine treatment.



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  		Figure 5. Graph showing relation between steady state diameters of intracerebral arterioles and intraluminal flow rate before (control) and after application of 0.1 mmol/L NG-monomethyl-L-arginine (L-NMMA) and after reversal of nitric oxide synthase inhibition with L-arginine (1 mmol/L) (n=7 arterioles). Values are mean±SEM. Intravascular pressure was kept constant at 60 mm Hg. *P<.05, #P<.01 vs corresponding diameters at flow of 0 µL/min. {delta}Significant difference in diameters between control or L-arginine–treated groups and L-NMMA–treated groups (P<.01). Flow-induced dilation was abolished by L-NMMA and restored by L-arginine.

In a separate series of experiments (n=5), the effect of flow increase on vessel diameter was evaluated before and after inhibition of prostaglandin synthesis by indomethacin (Fig 6Down). Indomethacin alone led to 13.6±3.7% constriction of intracerebral arterioles in the absence of flow but did not affect flow-induced dilation. In untreated vessels, flow increase caused a peak dilation of 13.4±2.0%, whereas peak dilator response was 12.2±1.9% after indomethacin treatment. These results suggested that prostaglandins did not mediate flow-induced dilation of intracerebral arterioles. Furthermore, because both indomethacin and L-NMMA constricted intracerebral arterioles to a similar extent, these experiments provided evidence that the inhibition of flow-induced dilation by L-NMMA could not be attributed to an increase in vascular tone.



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  		Figure 6. Graph showing relation between the diameter of intracerebral arterioles and flow rate before (control) and after treatment with 10 µmol/L indomethacin (n=5 arterioles). Intravascular pressure was maintained at 60 mm Hg. Values are mean±SEM. *P<.05 vs corresponding diameters at flow of 0 µl/min. {delta}Significant difference between diameters of control and indomethacin-treated groups (P<.05). Indomethacin had no effect on arteriolar response to flow.

Pharmacological Blockade by L-NMMA
As shown in Fig 7Down, intraluminal acetylcholine (1 µmol/L) dilated intracerebral arterioles significantly by 14.7±2.2%. L-NMMA abolished this response. In contrast, the more potent dilator effects of adenosine (1 µmol/L) and reduced pH (6.8) were not significantly affected by L-NMMA.



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  		Figure 7. Bar graphs comparing the dilator response of intracerebral arterioles to acetylcholine (1 µmol/L), pH 6.8 buffer, and adenosine (1 µmol/L) before (hatched bars) and after (open bars) incubation of the arterioles with 0.1 mmol/L NG-monomethyl-L-arginine (L-NMMA). Values are mean±SEM. Acetylcholine was infused intraluminally, whereas both adenosine and pH 6.8 buffer were applied extraluminally (for each vasodilator agent, n=5). % Dilation indicates change in vessel diameter expressed as percentage of control diameter. *Significantly different (P<.05) from response before L-NMMA treatment.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
The present study provided the first description of the response profile of brain arterioles to increases in intraluminal flow. Infusion of buffered saline elicited flow rate–dependent dilation. The peak dilation of 15% was observed at a flow rate of 10 µL/min. However, faster flow rates did not evoke further relaxation but instead led to the restoration of resting diameter. The latter response is unlikely to be mediated by a pressure-sensitive myogenic mechanism, because luminal pressure was maintained constant at 60 mm Hg. On the other hand, blockade of the flow-induced response by the NOS inhibitor L-NMMA suggested that an arginine metabolite, such as NO, may mediate flow-induced dilation in cerebral parenchymal arterioles.

Methodology and Vascular Reactivity
A major concern in studies involving isolated arterioles is whether the vessels might have incurred injury during the isolation and cannulation procedure. Another potential problem is that isolated arterioles removed from their normal environment may lose responsiveness to vasoactive stimuli. Therefore, it is of paramount importance to establish the viability of isolated arterioles and to ensure that their physiological responsiveness was not compromised by the in vitro methodology.

Previous studies have established several criteria19 30 for arteriolar reactivity in vitro. These requirements are development of spontaneous myogenic tone at 37°C, vasomotion (rhythmic activity), and responsiveness to vasoactive agents. Intracerebral arterioles in the present study fulfilled such criteria. These vessels spontaneously constricted to 55% to 70% of their passive diameter as the bathing medium was warmed to 37°C. As shown in Fig 3Up, these vessels exhibited marked myogenic responses within the intravascular pressure range of 20 to 60 mm Hg, relaxing when pressure was lowered and constricting when pressure was raised. Furthermore, rhythmic contractions were consistently observed during tone development and persisted throughout the experiment. The degree of tone generation in vessels in the present study matched that achieved in previous studies.28 29 33

We assessed the responsivity of intracerebral arterioles to three vasoactive agents: pH, acetylcholine, and adenosine. The in vivo responses of pial arterioles to changes in perivascular pH have previously been examined in cats.35 Superfusion of pH 6.8 cerebrospinal fluid induced a dilation of 18%, whereas pH 8 led to 15% constriction. Responses of intracerebral arterioles to similar pH values in the present study exceeded (TableUp) the pH reactivity of pial arterioles in vivo. The high pH responsivity of rat parenchymal arterioles agreed with values from previous work28 29 33 using a similar rat preparation and may therefore be characteristic of intracerebral arterioles in the rat. The response of rat pial arterioles superfused in situ with 1 µmol/L adenosine was between 17% and 21%.36 37 These in vivo responses were also lower than the 26% dilation obtained in the present study, but the difference may be attributed to the lack of parenchymal uptake and metabolism of adenosine in isolated vessels. In contrast to both adenosine and pH responses, isolated intracerebral arterioles were relatively unresponsive to extraluminally applied acetylcholine. However, intraluminal acetylcholine in the present study induced 12% dilation, which is comparable to the in vivo dilator effect of acetylcholine observed in rat pial arterioles.38 39

In the present study, intracerebral arteriole segments were cannulated and sealed from the external medium so that the vessel lumen could be pressurized and perfused. This method allowed the independent control of pressure and flow. The dissociation of pressure changes from flow stimuli was important because of the marked pressure-induced myogenic responses observed in the present study that may confound interpretation of the response to flow alone. A limitation of the present study is that intraluminal pressure was not directly measured. Inasmuch as upstream and downstream resistances were matched, it was assumed that pressure in the middle portion of the arteriole remained fixed at 60 mm Hg despite an increase in flow, by balancing the increase in upstream pressure with an equal decrease in downstream pressure. Nevertheless, local variations in pressure may occur, for instance, when there is regional turbulence, which may also cause rapid fluctuations in wall shear rate. Two observations suggest that intravascular pressure remained steady during high flow rates. First, the diameters of three passive, maximally dilated arterioles were measured during perfusion at rates of up to 20 µL/min while midpoint pressure was maintained at 40 mm Hg (which was chosen because the passive arteriole was relatively compliant at this pressure). No change in vessel diameter was observed, indicating a lack of change in intravascular pressure. Second, as shown in Fig 2Up, perfusion pressure increased linearly up to a flow rate of 25 µL/min, suggesting that flow conditions in the arterioles remained laminar even at the higher flow rates.

Flow-Induced Response
Flow-induced responses have been investigated in both arteries and arterioles.4 5 Although in most studies the response was dilation, flow-induced constriction has also been observed.40 41 In the present study, the response of intracerebral arterioles to increasing flow rate was biphasic. At lower flow rates, the response was dilation, whereas at higher flow rates (>10 µL/min) the vessels progressively restored their resting (no-flow) diameter. There is precedent for such a biphasic response, however, as Hoogerwerf et al40 had described a similar response profile to increases in flow in rabbit femoral arteries, which displayed an initial marked dilation, followed by constriction toward initial diameter at progressively higher flow rates.

With regard to the cerebral circulation, Garcia-Roldan and Bevan6 reported that both constrictor and dilator responses can be evoked by a flow stimulus (20 µL/min) in cannulated pial arteries (>200-µm diameter) from the rabbit. The direction of the response appeared to be pressure dependent. When intraluminal pressure was high (90 mm Hg), flow-induced constriction was observed, but when pressure was low (30 mm Hg), the response to the same rate of flow was dilation. Subsequently, Gaw and Bevan8 showed that middle cerebral artery segments (200- to 250-µm diameter) from the rabbit relaxed in response to an increase in intraluminal flow rate. In an in vivo study in the rat, Fujii et al7 observed by means of a cranial window the response of the basilar artery to occlusion of the common carotid artery. During occlusion, an increase in flow velocity was followed by dilation of the basilar artery.

The present study extended the above observations to the arteriolar level. The maximal dilation of intracerebral arterioles, elicited by an increase in flow rate from zero to 15 µL/min, was 15%. For comparison, isolated rabbit pial arteries exhibited an 8% dilation at a flow rate of 20 µL/min,6 whereas rat basilar arteries in vivo dilated by as much as 29% in response to a 203% increase in flow velocity.7 No data on flow-induced dilation are available for comparison in cerebral arterioles. However, arterioles from other vascular beds exhibited greater flow sensitivity. For example, flow induced a maximal 29% increase in the diameter of pig coronary arterioles19 and a 46% dilation in rat cremaster muscle arterioles.15

In arteries and arterioles from a wide variety of vascular beds, flow-induced dilation requires an intact endothelium and probably involves NO release by the endothelium.4 18 In the present study, the dilator responses of intracerebral arterioles to increases in intraluminal flow rate were inhibited by L-NMMA. Moreover, these vessels recovered their ability to dilate after treatment with the NOS substrate L-arginine. The present results therefore suggest that an arginine metabolite, such as NO, is the mediator of flow-mediated dilation in cerebral arterioles. Previous studies on the mechanism of flow-induced dilation in the cerebral vasculature, however, have yielded disparate results. Gaw and Bevan8 showed that NOS inhibitors attenuated, but did not abolish, the relaxation response of rabbit middle cerebral artery segments to an increase in intraluminal flow. Furthermore, neither aspirin nor indomethacin affected the relaxation response. In contrast, Fujii et al7 showed that shear-induced dilation of the basilar artery was unaffected by a host of pharmacological agents, including indomethacin and L-NMMA. Although the lack of effect of indomethacin on the flow-induced dilator response of cerebral arterioles in the present study is consistent with the above findings, the observation that L-NMMA completely abolished the dilator response is not. This discrepancy may be related to differences in vessel size or location (intraparenchymal versus extraparenchymal) or to differences in experimental procedures. The present study used a cannulated and pressurized preparation, which is considered to be more physiological than the isometric vessel ring technique used by Gaw and Bevan. The study of Fujii et al, on the other hand, was conducted in vivo. In such a preparation, it is often difficult to completely rule out the participation of other mechanisms of vascular communication, such as neurogenic or propagated dilator responses.42

The mechanism underlying the biphasic flow response, ie, the decrease in diameter at high (>10 µL/min) flow rates, is unknown. Such a response may represent a homeostatic mechanism that acts to prevent harmful rises in capillary perfusion pressure resulting from large flow increases. Because intraluminal pressure was maintained constant at 60 mm Hg, the constriction observed after the vessel had reached its maximally dilated state was unlikely to be a myogenic response. On the other hand, the possibility that flow-induced constriction may contribute to this response should be considered. Bevan and Joyce41 suggested that flow-induced changes in vascular tone may represent the net result of competition between flow-dependent constrictor and dilator responses. One may therefore speculate that flow-induced constriction may dominate over dilation at higher flow rates. In support of this hypothesis is the observation by Kuo et al43 that pig coronary venules constricted slightly (2% to 4%) at high flow rates. In the same study, venules responded to flow by constriction after endothelium denudation, suggesting that smooth muscle cells may contract in response to shear stress and that this flow-induced constriction could be unmasked by removing the dilator effect of endothelial cells. In the present study, however, NO blockade by L-NMMA abolished the dilator response but did not result in net constriction. This observation therefore suggests that intracerebral arterioles did not constrict in response to flow stimuli or that the blockade of flow-mediated dilation was incomplete, either because of incomplete NOS inhibition or because of the existence of an NO-independent mechanism of flow-induced dilation.

Little is known about the normal in vivo range of blood flow rates in cerebral arterioles. Such information, however, may impart functional significance to the flow-response curve (Fig 3Up) of intracerebral arterioles in the present study. Flow rate in a cerebral arteriole may be estimated, assuming a circular vascular profile, with the following formula: Q={pi}D2/4, where Q is flow rate, is mean velocity, and D is vessel diameter. In a preliminary study44 we have measured red blood cell velocity in pial arterioles in the rat with the dual-slit technique.45 The average control centerline velocity (equal to 1.6 times ) of pial arterioles (mean diameter, 44 µm) similar in size to intracerebral arterioles in the present study is 18.5 mm/s. This value is compatible with data provided by Ma et al46 in the rat (21.0±2.3 mm/s in 49.3±1.5-µm pial arterioles). Calculation with the above equation yields a "resting" flow rate of {approx}1 µL/min. Taking viscosity of blood ({approx}2 cp in rat arterioles)47 as three times that of saline (0.7 cp at 37°C), blood flow at 1 µL/min would produce a level of wall shear stress (40 dyne/cm2) in intracerebral arterioles similar to that produced by saline flow of 3 µL/min in the present in vitro system. Thus, our diameter-flow data (Fig 3Up) indicate that intracerebral arterioles under resting conditions in vivo are capable of significant dilation in response to an increase in intraluminal flow rate. On the other hand, cerebral arterioles during normal functional activity are unlikely to be exposed to the high shear forces caused by saline flow of >10 µL/min. Such a pronounced increase in flow rate, however, may occur in extreme conditions of enhanced activity, such as during seizures.48

Pressure-Induced Responses
The in vitro responses of brain parenchymal arterioles to changes in intravascular pressure have been examined in two previous studies in the rat. Dacey and Duling28 found that arterioles of 19-µm diameter constricted by 8% as pressure was raised from 20 to 60 mm Hg, whereas in larger (44-µm) arterioles Takayasu and Dacey29 found a 12% increase in diameter as pressure was lowered from 60 to 20 mm Hg. In the present study, the same change in pressure resulted in a 21% change in diameter. The greater myogenic response observed in the present study may be attributed to the extra care taken to preserve vessel viability and responsivity in the present experiment. A comparison with the two studies mentioned above revealed that arterioles in the present study exhibited a greater response to pH and other vasoactive agents.

On the other hand, it is difficult to assess the effect of transmural pressure per se on cerebral vessels in vivo, because a reduction in blood pressure may have metabolic consequences. Bohlen and Harper27 used a method that allowed them to raise transmural pressure without affecting perfusion pressure. They placed craniotomized rats in a sealed box with only the head exposed to atmospheric pressure. Changing the pressure in the box altered both arterial and venous pressure by equal amounts, thus maintaining constant perfusion pressure in the cerebral arterioles. Since the pial arterioles were exposed to atmospheric pressure, such maneuvers changed the transmural pressure in these vessels. With such a technique, Bohlen and Harper found that pial arterioles (30- to 60-µm diameter) constricted by 16% when ambient pressure was reduced by 16 mm Hg from control (50 mm Hg) but dilated by 6% when pressure was raised by 10 mm Hg. Their data also showed a 22% change in diameter when transmural pressure was changed from 60 to {approx}40 mm Hg. These changes are comparable to those observed in the present in vitro study.

Ever since the first description of the myogenic response by Bayliss,24 the site of mechanoreception in the vessel wall to pressure has been attributed to the vascular smooth muscle.23 More recently, however, there is dispute as to whether the endothelium may act as the sensor in contractile responses to pressure.49 Furthermore, transmural pressure inhibited NO release from human endothelial cells,50 suggesting that endothelium-derived NO may take part in pressure-induced constriction. If NO is involved in the myogenic response of intracerebral arterioles to pressure, then inhibition of NO formation by arginine analogues should attenuate the constrictor response to pressure. In the present study, however, neither the response profile nor the degree of constriction to stepwise increases in intraluminal pressure was altered after treatment with L-NMMA (0.1 mmol/L), which completely abolished the dilator response to acetylcholine. The present study thus suggested that NO was not involved in pressure-induced responses of intracerebral arterioles.

Action of L-NMMA
NOS immunoreactivity has been localized in the endothelium but not in the smooth muscle of rat cerebral arteries,51 and a constitutive form of NOS in cerebral endothelium has been cloned and characterized.52 Local or systemic application of arginine analogues caused constriction of cerebral vessels,53 suggesting that NO may be chronically released in cerebral vessels. Consistent with these findings, the present experiment demonstrated that intracerebral arterioles were remarkably sensitive to L-NMMA inhibition, constricting by 15% even under no-flow conditions.

In the brain, both selective destruction of the endothelium and arginine analogues abolished the relaxation of arteries and arterioles in response to acetylcholine.53 Consistent with these data, L-NMMA completely inhibited acetylcholine-induced dilation of intracerebral arterioles in the present study. In contrast, the action of adenosine, an endothelium-independent dilator of cerebral vessels,54 was not altered by L-NMMA. This lack of effect of L-NMMA on adenosine-induced dilation of intracerebral arterioles is consonant with findings in cat pial arterioles in vivo.55 L-NMMA also had no effect on the dilator response of intracerebral arterioles to pH 6.8 buffer, suggesting that NO is not involved in the dilator response of cerebral arterioles to hydrogen ion. This observation is consistent with a recent finding in isolated rat cerebral arteries56 but is at variance with the in vivo finding that NOS inhibition markedly attenuated the increase in cortical blood flow elicited by extracellular acidosis in the rat.57

Summary
Rat penetrating arterioles were perfused while maintaining intravascular pressure constant. Increasing flow rates elicited a biphasic response. At flow rates up to 10 µL/min, progressive dilation (up to 15% of control diameter) was observed. At higher flow rates, the vessels did not dilate further but instead constricted until resting diameter was restored at 20 µL/min. The vessels also exhibited myogenic constriction from 20 to 60 mm Hg intravascular pressure in the absence of flow. The dilator response to flow increase was abolished by the NOS inhibitor L-NMMA but was unaffected by indomethacin. These results thus suggest that NO or a related L-arginine metabolite, but not prostaglandin, mediates flow-induced dilation in rat cerebral arterioles. On the other hand, NO was not involved in the myogenic response of these arterioles to pressure changes.


*    Acknowledgments
 
This study was supported by grants NS07144, NS21076, and NS30305 from the National Institutes of Health.


*    Footnotes
 
Reprint requests to H. Richard Winn, MD, Department of Neurological Surgery, Box 359766, Harborview Medical Center, 325 Ninth Ave, Seattle, WA 98104.

Received February 2, 1995; accepted June 22, 1995.


*    References
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 *References
 

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