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Am J Physiol Heart Circ Physiol 280: H2011-H2022, 2001;
0363-6135/01 $5.00
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Vol. 280, Issue 5, H2011-H2022, May 2001

Effects of luminal shear stress on cerebral arteries and arterioles

Robert M. Bryan Jr.1,2,3, Sean P. Marrelli1, Marie L. Steenberg1, Lisa A. Schildmeyer1, and T. David Johnson1

Departments of 1 Anesthesiology, 2 Molecular Physiology and Biophysics, and 3 Division of Cardiovascular Sciences, Department of Medicine, Baylor College of Medicine, Houston, Texas 77030


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The effect of luminal shear stress was studied in cerebral arteries and arterioles. Middle cerebral arteries (MCA) and penetrating arterioles (PA) were isolated from male Long-Evans rats, mounted in a tissue bath, and pressurized. After the development of spontaneous tone, inside diameters were 186 ± 5 µm (n = 28) for MCA and 65 ± 3 µm (n = 37) for PA. MCA and PA constricted ~20% with increasing flow. Flow-induced constriction persisted in MCA and PA after removal of the endothelium. After removal of the endothelium, the luminal application of a polypeptide containing the Arg-Gly-Asp amino acid sequence (inhibitor of integrin attachment) abolished the flow-induced constriction. Similarly, an antibody specific for the beta 3-chain of the integrin complex significantly inhibited the flow-induced constriction. The shear stress-induced constriction was accompanied by an increase in vascular smooth muscle Ca2+. For example, a shear stress of 20 dyn/cm2 constricted MCA 8% (n = 5) and increased Ca2+ from 209 ± 17 to 262 ± 29 nM (n = 5). We conclude that isolated cerebral arteries and arterioles from the rat constrict to increased shear stress. Because the endothelium is not necessary for the response, the shear forces must be transmitted across the endothelium, presumably by the cytoskeletal matrix, to elicit constriction. Integrins containing the beta 3-chain are involved with the shear stress-induced constrictions.

cerebrovascular circulation; endothelium; integrins; cremaster muscle arteriole; calcium; fura 2


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ALTHOUGH THERE ARE EXCEPTIONS, it is generally considered that peripheral vessels dilate in response to an increased luminal shear stress (24, 25, 28, 29). However, in the cerebral circulation there is controversy as to whether luminal shear stress dilates or constricts the cerebral vessels (3, 4, 10, 12, 14, 31, 37, 41, 44). The controversy and confusion could be due, at least in part, to the following: 1) the different species studied, 2) the different vessel segments studied (larger arteries, smaller arteries, or arterioles), 3) the different techniques used by the investigators (vessel rings vs. pressurized vessel segments), and/or 4) the technical problems and potential artifacts involved with shear stress studies.

The first goal of the present study was to establish a model for the study of shear stress by using a peripheral vessel, the cremaster muscle arteriole (CMA). It has been established that the CMA dilates with increased shear stress (25). If we could reproduce this well-established response in the CMA, then the validity of our results obtained in cerebral vessels, by using the same model, would be substantially strengthened. Second, we sought to determine the effects of increased flow (or shear stress) in two different vessel segments in the cerebrovascular tree, the middle cerebral artery [MCA; inside diameter (ID) ~190 µm] and the penetrating arteriole (PA) (ID ~60 µm). It is possible that responses to shear stress might be different at different segments along the cerebral vascular tree. After determining that cerebral arteries and arterioles constrict to increased flow or shear stress, we tested the following two hypotheses: 1) intact endothelia are required for the shear stress response in the rat MCA, and 2) that integrins, specifically an integrin containing the beta 3-subunit, are involved with the response to shear stress in the rat MCA.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Harvesting and mounting vessels. The Animal Protocol Review committee at Baylor College of Medicine approved the experimental protocol. Male Long-Evans rats (250-350 g) were anesthetized with 3% isoflurane and decapitated. The brain was immediately removed and placed in cold (4°C) physiological saline solution (PSS). With the aid of a dissecting microscope, MCA and PA were carefully harvested (5, 15, 46). In addition to the cerebral vessels, CMA were harvested (25). Sections of the three vessel groups were mounted in a vessel chamber (5, 15, 25, 46). Micropipettes were inserted into both ends of each vessel and the vessel was secured with nylon ties. The vessels were bathed in PSS, which was equilibrated with a gas composed of 20% O2-5% CO2, balance N2. The pH of the bath was ~7.40, PCO2 ~35 mmHg, and PO2 ~130 mmHg (5). The bath was maintained at 37°C for cerebral vessels and 33°C for CMA (5, 25). In one study using MCA, MOPS buffer was used instead of the bicarbonate buffer (see Drugs and reagents for composition of buffers). The MOPS buffer was allowed to equilibrate with room air and had a pH of ~7.40.

Luminal pressure was set by raising reservoirs to the appropriate height above the vessels (Fig. 1) (5), generally 80 mmHg for MCA and 60 mmHg for PA and CMA. These pressures were near the pressures experienced in vivo for each vessel type. Flow through the lumen of the vessels was produced by a variable speed syringe pump (model 22, Harvard Apparatus; South Natick, MA). Pressure transducers on either side of the vessel chamber provided a measurement of perfusion pressure (see P1 and P2 in Fig. 1). Before the vessel was mounted, the resistance of the tubing and micropipettes on either side of the vessel was measured. From the resistances of the micropipettes, an algorithm (see Methods development for validation of algorithm) was used to determine the upstream pressure and downstream pressure (P1 and P2, respectively) to obtain the desired luminal pressure of the vessel. After flow with the syringe pump was initiated, the input reservoir was clamped (see Fig. 1, left) and the output reservoir was lowered to maintain the desired luminal pressure in the vessel. With each subsequent increase in luminal flow, the output reservoir was appropriately lowered.


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  		Fig. 1.   Diagram of the system for studying flow and shear stress in middle cerebral arteries (MCA), cerebral penetrating arterioles (PA), and cremaster muscle arterioles (CMA). P1, upstream pressure transducer; P2, downstream pressure transducer, both for measuring perfusion pressure across the vessel.

In some MCA, all PA, and all CMA, a servo-null pressure system (1) was used to directly measure luminal pressure in the vessel. For the servo-null pressure measurement, a micropipette (tip diameter ~2-3 µm) was inserted through the vessel wall (see Fig. 1). The luminal pressure of the vessel was displayed on digital readout.

The vessels were magnified with an inverted microscope equipped with a video camera and monitor. ID of the vessels was measured manually from the video screen or from the videotape made at the time of the study. In some cases, the outside diameter was continuously measured from the videotape by using image analysis (see Fig. 4).

After mounting and pressurizing was completed, the vessels of all groups developed spontaneous tone. MCA and PA constricted ~18-20% and CMA constricted ~50% of the maximum diameter (determined in Ca2+-free buffer) over the course of 1 h. Experimental protocols were not initiated until the vessel diameters were stable over a period of 15 min.

The length of vessel used was dependent on the luminal flow desired for a particular study. The length of vessel that was necessary to achieve laminar flow was determined according to the following equation (18)
D=0.115·r<SUP>2</SUP>·<A><AC>u</AC><AC>ˆ</AC></A>/&ggr; (1)
where D was the distance to achieve laminar flow, r was the vessel radius, û was the average velocity, and gamma  was the kinematic viscosity (viscosity/density). û was calculated according to the equation
<A><AC>u</AC><AC>ˆ</AC></A>=Q<IT>/&pgr;·r<SUP>2</SUP></IT> (2)
where Q was flow through the lumen and r was the inside radius. The length necessary to establish laminar flow was not dependent on the vessel radius because r2 for the calculated distance was in both the numerator and denominator when the above equations were combined. The maximum flow for PA and CMA was 40 µl/min and required ~25 µm to achieve laminar flow. The length of PA and CMA were at least 500 µm. For MCA, a maximum flow of 300-500 µl/min was used for some studies (see Figs. 5, 6, 8, and 9) and ~100 µl/min for other studies (see Figs. 10-12). For flows of 500 and 100 µl/min, distances of 436 and 87 µm, respectively, were needed to establish laminar flow. The length of the MCA ranged from 280 to 1,200 µm; only the longer MCA were used with the higher rates of flow.

Shear stress was calculated using the following equation (25, 30, 41)
t=4&eegr;Q<IT>/&pgr;r<SUP>3</SUP></IT> (3)
where t was shear stress (in dyn/cm2) and eta  was viscosity.

In initial studies, vessel diameter was measured as flow was changed in predetermined steps (Figs. 3-9). In other studies, we attempted to set a flow to produce a given shear stress (Figs. 10-12).

The presence of intact endothelium in cerebral vessels was verified by luminal administration of ATP, an agonist for P2Y2 receptors. ATP dilates cerebral arteries and arterioles via an endothelium-dependent mechanism involving nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) for the MCA and EDHF for the PA (46-48). The endothelium was removed by passing air through the lumen of the vessel as previously described (5, 47, 48). Absence of dilation to luminally applied ATP indicated that the endothelium had been successfully removed. Vessels denuded of endothelium dilated to the NO donors, S-nitroso-N-acetylpenicillamine (SNAP) or (Z)-1-{N-methyl-N-[6-(N-methylammoniohexyl)amino]}diazen-1-ium-1,2-diolate (MAHMA NONOate), indicating that the vascular smooth muscle was intact.

In one study, albumin (0.5%) was added to the luminal perfusate. In another study, dextran (2, 4, and 6%, molecular wt = 65,000) was added to the luminal perfusates to increase the viscosity. Viscosities of the PSS solutions, alone and after addition of albumin or dextran, were determined by perfusing the PSS solution through a polyethylene tube of known length and radius. The flow rate of the PSS solution was controlled by a variable speed infusion pump (model 22, Harvard Apparatus), the temperature was maintained at 37°C with the use of a water bath, and the input and output pressures (Pi and Po, respectively) across the tubing were measured with pressure transducers. eta  was calculated by using the above measurements after rearranging Poiseuille's law. The working equation was
&eegr;=&pgr;(P<SUB>i</SUB><IT>−</IT>P<SUB>o</SUB>)<IT>r<SUP>4</SUP>/8</IT>Q<IT>L</IT> (4)
where L was the length. eta , Pi, and Po are defined above. Viscosities at 37°C were calculated (in cP) as 1.06 for PSS, 1.6 for PSS with 2% dextran, 2.6 for PSS with 4% dextran, 3.9 for PSS with 6% dextran, and 1.13 for PSS with 0.5% albumin.

Measurement of vascular smooth muscle Ca2+ using fura 2. Ca2+ concentrations in the cytoplasm of vascular smooth muscle and vessel diameter were simultaneously measured as previously described (32). Briefly, fura 2-acetoxymethyl ester (AM) (1 µM final concentration) was added to the extraluminal bath. After 10-15 min exposure, the vessel was washed to remove extracellular fura 2-AM, and an additional 30 min was allowed for intracellular de-esterification of fura 2-AM to fura 2. For Ca2+ measurements, the vessels were illuminated with excitation light alternating between wavelengths of 340 and 380 nm, with the use of a xenon arc lamp, appropriate filters, and a filter changer. In addition, red light from a separate lamp was used in a transmission mode to illuminate the vessels for diameter measurements. The light was collected with a quartz objective (Nikon ×10, numerical aperture; NA = 0.5) and subsequently split and filtered with a dichoric mirror. The red light was diverted to a charge-coupled device for diameter measurements and the remainder was diverted to a photomultiplier after passing through a 510-nm narrow band-pass filter. Intensities of the 510-nm fluorescence light were used to quantitate intracellular Ca2+ according to the following equation
[Ca<SUP><IT>2+</IT></SUP>]<SUB>i</SUB><IT>=&bgr;×</IT>(R<IT>−</IT>R<SUB>min</SUB>)<IT>×K</IT><SUB>d</SUB><IT>/</IT>(R<SUB>max</SUB><IT>−</IT>R) (5)
where [Ca2+]i was the intracellular Ca2+ concentration in the vascular smooth muscle, beta  was the ratio of the 380 nm fluorescence intensity for Ca2+-unbound fura 2 over Ca2+-bound fura 2, R was the ratio the of light intensity at 510 nm when excited at 340 nm to the intensity when excited at 380 nm (340:380 ratio) at a given condition (i.e., shear stress), Rmin was the 340:380 in the absence of [Ca2+]i, Rmax was 340:380 when [Ca2+]i was sufficiently high to saturate fura 2, and the dissociation constant, Kd, was 282 nm. beta , Rmin, and Rmax were determined in a separate group of vessels as previously described (32).

Drugs and reagents. ATP, serotonin, and dextran (molecular wt = 65,000) were purchased from Sigma (St. Louis, MO). SNAP was purchased from Research Biochemicals (Natick, MA). MAHMA NONOate was purchased from Alexis Biochemicals (San Diego, CA). Bovine blood albumin was purchased from USB (Cleveland, OH). The integrin blocker, Gly-Arg-Gly-Asp-Asn-Pro (GRGDNP) peptide, and the inactive control peptide, Gly-Arg-Gly-Glu-Ser-Pro (GRGESP) were purchased from Life Technologies (Rockville, MD). Monoclonal anti-CD61 (F-11), specific for beta 3-integrin, and a control protein were purchased from PharMingen (San Diego, CA). Because F-11 is a mouse IgG1 (kappa -isoform), a nonreactive mouse IgG of the same isoform was used as a control peptide. Fura 2-AM (50 µg) was purchased from TefLabs (Austin, TX) and dissolved in 75 µl of DMSO (containing 14% pluronic). MAHMA NONOate was dissolved in 0.01 M NaOH; all other reagents and drugs were dissolved in distilled water.

The bicarbonate PSS was composed of (in mM) 119 NaCl, 24 NaHCO3, 4.7 KCl, 1.18 KH2PO4, 1.17 MgSO4, 1.6 CaCl2, 5.5 glucose, and 0.026 EDTA (5). The MOPS buffer was composed of (in mM) 145 NaCl, 1.2 NaH2PO4, 4.7 KCl, 1.17 MgSO4, 1.6 CaCl2, 5.5 glucose, 3 MOPS, 2 pyruvate, and 0.02 EDTA.

Statistical analysis. All of the data are presented as means ± SE. For statistical analysis, the one- or two-way repeated measures ANOVA was used with a post hoc Student-Newman-Keuls test for comparison (where appropriate) of individual groups and individual data points. The acceptable level of significance was defined as P < 0.05.

Methods development. In initial studies, we identified two potential problems with experiments where luminal flow was altered. Either of these problems was capable of introducing significant artifacts into the experimental data if not properly controlled. The problems were differences in pH between the luminal perfusate and extraluminal bath, and uncontrolled pressure changes in the vessel that occurred with changes in flow.

The first artifact involves differences in pH between the luminal perfusate and extraluminal bath. Depending on whether the luminal perfusate was more acidic or more basic than the extraluminal bath, hydrogen ion delivery or hydrogen ion removal would be increased, respectively, as the luminal flow was increased. Because cerebral vessel diameter is sensitive to pH (9), any change in pH could be interpreted erroneously as a flow- or shear stress-induced dilation. This problem is particularly significant with bicarbonate buffers where pH is dependent on the PCO2 of the buffer solution. Passing the luminal perfusate through gas-permeable Silastic tubing (0.025 in. ID × 0.065 in. outer diameter; OD, 52 cm long; model 602-175, Dow Corning), which was coiled in the extraluminal buffer, before entering the vessel lumen (Fig. 1) allowed for equilibration of the luminal perfusate with the buffer in the extraluminal bath. The Silastic tubing also added surface area for the temperature of the luminal perfusate to equilibrate with the extraluminal bath.

In an initial study, the pH and PCO2 of the luminal perfusate were collected after passing through the gas-permeable Silastic tubing and compared with the buffer in the extraluminal bath. No significant differences (i.e., equilibration) between luminal and extraluminal PCO2 and pH existed with flow rates up to 1,000 µl/min, a value double the rate of flow used in any experiment (Table 1).

                              
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  		Table 1.   Comparison of pH and PCO2 in the PSS perfused through the lumen of the vessels (at different rates of flow) to the PSS in the extraluminal bath

The second problem involved uncontrolled pressure changes in the vessels that could occur with changes in flow. Over a specific pressure range, cerebral vessels exhibit a myogenic response (15); that is, MCA and PA constrict with increased pressure and dilate with decreased pressure. A change in diameter due to the myogenic response, if luminal pressure were not precisely controlled, could be interpreted erroneously as a flow-induced effect. On the basis of input and output resistances of the micropipette and tubing in the system, an algorithm was developed to predict input and output pressures (P1 and P2, respectively, in Fig. 1) for a given perfusion pressure across the system. The accuracy of the algorithm was tested by directly measuring the luminal pressure when luminal flow was increased. Figure 2A shows the actual luminal pressure, measured using the servo-null method, plotted as a function of perfusion pressure (P1 and P2 in Fig. 1) in MCA after the application of the algorithm. The target pressure for the study was 80 mmHg. Perfusion pressures across the system for flows of 10, 20, 50, 100, 200 µl/min were 2.8 ± 0.3 mmHg, 4.7 ± 0.5, 9.9 ± 0.7, 22 ± 3, and 49 ± 7, respectively (n = 7 for each group). Our data indicates that the luminal pressure could be controlled within narrow limits using the algorithm when perfusion pressure increased up to and in excess of 50 mmHg.


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  		Fig. 2.   A: pressure in isolated MCA (measured directly by the servo-null method) as a function of the perfusion pressure across the artery and micropipettes in the vessel preparation (see Fig. 1). Output reservoir (right side of Fig. 1) was adjusted as luminal flow was increased to maintain a target pressure of 80 mmHg in the vessel lumen. On the basis of resistances of the individual pipettes, an algorithm was used to calculate P1 and P2 necessary to maintain the target pressure in the lumen. Dotted horizontal line (80 mmHg), the target pressure, and the individual points represent the pressure measured directly. Data were derived from 6 different MCA. Symbols represent different flows. B: percent error in the diameter of MCA due to deviations from the target pressure of 80 mmHg. Data are on the basis of studies of the myogenic response in cerebral vessels (15).

The error in diameter due to the myogenic response of MCA is shown in Fig. 2B. Deviation of luminal pressure from the target (80 mmHg) to 75 mmHg, for example, would produce only a 1% error in diameter (Fig. 2B). Thus the algorithm used for the studies maintained relatively tight control of the luminal pressure. Inaccuracies of the algorithm would produce only minor artifacts in the measured diameter. The algorithm was not as accurate when used with the smaller CMA and PA due to the greater perfusion pressures across the system. Thus the servo-null method was used to directly measure luminal pressures for all CMA and PA.

The Reynolds number, a prediction of whether laminar or turbulent flow exists, was calculated using the following equation (2)
N<SUB>R</SUB><IT>=<A><AC>u</AC><AC>ˆ</AC></A>d&rgr;/&eegr;</IT> (6)
where d was the vessel diameter and rho  was the density. NR was a dimensionless number; a value <2,000 predicts laminar flow, a value between 2,000 and 3,000 predicts a transition to turbulent flow, and a value above 3,000 predicts turbulent flow (2). The calculated Reynolds number for any vessel (MCA, PA, or CMA) at the highest rate of flow was <200; in most cases it was <100. It can be seen that conditions for turbulent flow were never approached in this study.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Figure 3A shows that CMA (~80 µm ID) dilated to increased flow through the lumen (P < 0.001 using repeated-measures ANOVA). In Fig. 3B the diameter change was plotted as a function of shear stress (Eq. 3 for calculations). If no dilation of the CMA had occurred with flow, then the shear stress would have been ~160 dyn/cm2 at a flow of 40 µl/min. However, because the CMA dilated with increased flow, the shear stress at 40 µl/min was actually only 50 dyn/cm2. These results confirm previous studies (25) in the CMA and demonstrate the validity of our methods for studying flow and shear stress in isolated vessels.


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  		Fig. 3.   A: effects of increasing flow on the diameter of CMA (n = 6). B: diameters of the CMA plotted as a function of shear stress. *P < 0.001 compared with zero flow.

In contrast to CMA, MCA constricted to increased luminal flow. A typical response in an individual MCA as luminal flow was increased from 0 to 300 µl/min is shown in Fig. 4. The OD is reported in Fig. 4, whereas in the other figures the ID is reported. The image analysis system used to continuously measure diameter best tracked the OD. A flow of 90 µl/min is most consistent with normal physiological shear stress (20 dyn/cm2) for the vessel shown in Fig. 4. Mean ID of MCA as a function of flow at luminal pressures of 40, 60, 80, and 100 mmHg are shown in Fig. 5. Note that flow-induced constrictions occurred at all pressures studied (P < 0.004 for all pressures using repeated measures ANOVA, n = 7 for each pressure). On stopping flow the diameters of the MCA dilated to near the original diameter. Figure 6, A-D, shows the ID when the data was plotted as a function of shear stress. The major constriction occurred at shear stresses between 0 and 50 dyn/cm2, a range that includes normal physiological shear stresses (26). The luminal application of 10-5 M ATP, an agonist that dilates through an endothelium-dependent mechanism (47), dilated the MCA 29 ± 5% (n = 7), indicating that the endothelium was functional. Serotonin (10 µM) constricted MCA with and without luminal flow (150 µl/min) by 25 ± 1% (n = 18) and 25 ± 2% (n = 6), respectively. The addition of 15 mM KCl to the extraluminal bath dilated MCA with (150 µl/min) and without luminal flow by 22 ± 3 and 18 ± 3%, respectively (n = 13 for each group, P = 0.37). MCA dilate or constrict to various drugs or conditions in the absence or presence of luminal flow and do so over a range of luminal flows including 500 µl/min (5, 22, 15, 16, 23, 33, and authors' observations).


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  		Fig. 4.   Outside diameter of an individual MCA when luminal shear stress was changed by altering luminal flow. The luminal pressure was held constant at 80 mmHg.



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  		Fig. 5.   Effects of luminal flow on the inside diameter of MCA at different luminal pressures (n = 7 for each pressure). *P < 0.05 compared with zero flow for each corresponding group.



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  		Fig. 6.   Inside diameter (ID) of rat MCA plotted as a function of shear stress at different luminal pressures (n = 7 for each group). Shear stress was increased by increasing luminal flow. Flow was measured as 40 mmHg (A), 60 mmHg (B), 80 mmHg (C), and 100 mmHg (D).

In a separate study, the flow-induced constrictions were compared in a group of MCA (n = 4) using the algorithm (see METHODS) and a group where the servo-null technique was used to maintain the pressure at 80 mmHg (n = 4). The response of the two groups at rates of flow between 0 and 400 µl/min was almost identical (data not shown). Thus puncturing the vessel wall with the servo-null micropipette did not influence the constrictor response to increased flow.

The addition of albumin (0.5%) to the luminal perfusate (n = 14) had no significant effect on the flow-induced constriction (data not shown). Additionally, substitution of the MOPS buffer for the bicarbonate buffer in the luminal perfusate and extraluminal bath (n = 4) had no significant effects on the constrictor response to luminal flow for shear stresses up to 200 dyn/cm2 (data not shown).

Similar to the MCA, PA (~70 µm ID) significantly constricted when luminal flow was increased from 0 to 40 µl/min. The constrictor response to flow at luminal pressures of 40 and 60 mmHg was highly significant (P = 0.001 and P = 0.00003, respectively, by using repeated-measures ANOVA, n = 10 for each group). A luminal pressure of 60 mmHg for the PA is considered near the normal physiological pressure. At a luminal pressure of 80 mmHg, the constrictor response was near but did not reach statistical significance (P = 0.07, n = 10). Figure 7 shows the responses for luminal pressures of 40, 60, or 80 mmHg when plotted as a function of shear stress (n = 10 for each pressure). When expressed as percent change in diameter, the flow-induced constriction was similar for MCA and PA. The luminal application of 10-5 M ATP to the PA dilated the vessels 13 ± 4% (n = 7) indicating that the endothelium was intact. PA with (25 µl/min) and without luminal flow constricted 38 ± 8 and 41 ± 7%, respectively, when pH was increased from 7.4 to 7.7. Conversely, the same PA dilated 10 ± 5 and 9 ± 4%, respectively when pH was decreased to 7.1. PA dilate or constrict to various conditions in the absence or presence of luminal flow (15, 17, 47, and unpublished observations).


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  		Fig. 7.   ID of rat PA plotted as a function of shear stress at different luminal pressures (n = 10 for each group). Shear stress was increased by increasing luminal flow.

The flow-induced constrictions persisted after removal of the endothelium in MCA (P < 0.001) and PA (P < 0.001). Figure 8, A and B, shows the absolute ID of MCA and PA plotted as a function of shear stress when endothelium was intact or after removal by passing air through the lumen. The luminal pressures for MCA and PA for the studies described in Fig. 8 were 80 and 60 mmHg, respectively. Note that removal of the endothelium significantly constricted both MCA and PA. The absence of dilation to the luminal application of ATP confirmed the removal of the endothelium. Figure 8, C and D, shows the same data when plotted as percent change in diameter of MCA and PA, respectively.


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  		Fig. 8.   ID of rat MCA (A) and PA (B) plotted as a function of shear stress in control vessels and after removal of the endothelium. C and D show results for MCA and PA, respectively, when the diameters were plotted as % change from the diameter at 0 dyn/cm2 (no flow). Shear stress was increased by increasing luminal flow. n = 7 and 11 for intact and denuded MCA, respectively; n = 14 and 5 for intact and denuded PA, respectively.

Figure 9 shows diameter changes in MCA (luminal pressure of 80 mmHg) when the shear stress was changed by either increasing flow through the lumen or by increasing the viscosity of the PSS at a constant flow of 20 µl/min. Viscosity was increased by the addition of dextran (molecular wt = 65,000) to the luminal perfusate. The viscosity of the PSS with 0, 2, 4, and 6% dextran was calculated to be 1.06, 1.6, 2.6, and 3.6 cP. The constriction of the MCA to increasing dextran in the luminal perfusate was highly significant (P < 0.0001 using repeated measures ANOVA). Constrictions to shear stress were similar regardless of whether the shear stress was increased by increasing flow or by increasing viscosity at a constant flow. These results conclusively demonstrate that it was shear stress, and not some other aspect of increased flow, that was responsible for the constriction of the MCA.


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  		Fig. 9.   ID of rat MCA when shear stress was increased by increasing luminal flow (n = 10) or by increasing viscosity (n = 6) at a constant flow of 20 µl/min. Dextran was added to the physiological saline solution (PSS) to increase the viscosity.

The hypothesis that integrin binding was involved with the shear stress-induced constrictions in MCA was tested using two blockers of integrin binding, an Arg-Gly-Asp (RGD) containing peptide and an antibody specific for the beta 3-integrin. Because of the expense of these antagonists, the studies were conducted in the following manner. First, the endothelium was removed, because it did not have to be present for the shear stress-induced constriction (Fig. 8), and the antagonists were administered luminally. Removal of the endothelium would ensure that the antagonist could get past the barrier formed by the tight junctions between endothelial cells. Second, only one shear stress, 50 dyn/cm2, was studied instead of a range of shear stresses as in previous experiments.

Figure 10 shows the effects of a blocker of integrin binding, an RGD-containing peptide, and an inactive control peptide on the constriction produced by shear stress (luminal pressure of 80 mmHg). The amino acid sequence of the active blocker was GREDNP and the sequence of the inactive peptide was GRGESP. The inactive peptide had no effect on the constriction produced by changing the shear stress from 0 to 50 dyn/cm2 (Fig. 10A, n = 6). On the other hand, the active RGD-containing peptide completely inhibited the constriction to the shear stress of 50 dyn/cm2 (Fig. 10B, n = 6). After the RGD peptide was washed out, the constriction to the shear stress was restored. The presence of the RGD peptide did not affect the constrictor response to serotonin (n = 10, data not shown). Thus the RGD peptide did not produce a general inhibition to constriction.


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  		Fig. 10.   Effects of an inactive peptide (A) and an Arg-Gly-Asp (RGD) containing peptide (B) (inhibitor of integrin binding) on the diameter of rat MCA when shear stress was increased from 0 to 50 dyn/cm2 (n = 6 for each group). Endothelium was removed and the peptide was administered in the luminal PSS. *P < 0.05 compared with corresponding diameter at zero shear stress. Shear stress was increased by increasing luminal flow.

Studies presented in Fig. 11 show the results of F-11, an antibody against beta 3-integrin, on the shear stress-induced constrictions. Application of the F-11 peptide apparently acted as an antagonist because it constricted the MCA not having luminal flow (Fig. 11B). In addition to constricting the MCA, the antibody also inhibited the shear stress-induced constriction. After the wash, the response could be restored. Because the F-11 peptide constricted the MCA, we used an NO donor, SNAP, to restore the original diameter before applying luminal shear stress. Under these conditions, there was a significant constriction to shear stress in the presence of F-11; however, the response was markedly attenuated compared with the control response (P < 0.001) or after wash (P = 0.005). The presence of a nonreactive mouse IgG1 (kappa -isoform), control peptide, did not affect the shear stress-induced constriction. The presence of the F-11 peptide did not affect the constrictor response to serotonin or the dilator response to 15 mM of KCl (n = 4, data not shown).


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  		Fig. 11.   Effects of an inactive control peptide (A) and F-11 (B), an antibody to the beta 3-integrin, on the diameter of rat MCA when shear stress was increased from 0 to 50 dyn/cm2 (n = 5 for each group). The endothelium was removed and the peptide was administered in the luminal PSS. Because the F-11 peptide constricted the MCA without luminal flow, S-nitroso-N-acetylpenicillamine (SNAP), an NO donor, was added to dilate vessels to near their original diameter without flow (C). Shear stress was increased by increasing luminal flow. *P < 0.05 compared with corresponding diameter at zero shear stress. **P < 0.05 compared with the control with zero shear stress.

Figure 12 shows mean MCA diameter and VSM [Ca2+]i measured simultaneously from five MCA when the shear stress was increased to ~50 dyn/cm2. Shear stress significantly decreased MCA diameter (P = 0.008) and increased [Ca2+]i (P = 0.003). For example, mean MCA diameter during no-flow condition was 212 ± 7 µm and decreased to 194 ± 5 µm when shear stress was increased to 20 dyn/cm2. In the same vessels, [Ca2+]i increased from 209 ± 17 to 262 ± 29 nM (n = 5). Figure 12 also shows diameter and [Ca2+]i when K+ in the extracellular bath was increased to 15 and 60 mM. Increases of K+ to 15 mM activate inwardly rectifier K+ channels and dilate cerebral vessels (23); K+ concentrations of 60 mM depolarize and constrict vessels. The dilations elicited by activating the inward rectifier K+ channels dilated the MCA to near maximum (252 ± 15 µm after 15 mM KCl compared with 267 ± 11 µm after removal of Ca2+) and significantly decreased [Ca2+]i to 128 nM. At 60 mM KCl, the vessels constricted and [Ca2+]i increased to 470 nM.


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  		Fig. 12.   Mean MCA diameter and vascular smooth muscle [Ca2+]i measured simultaneously from 5 MCA when the shear stress was increased. Shear stress significantly decreased MCA diameter (P = 0.008) and increased intracellular Ca2+ concentration ([Ca2+]i) (P = 0.003). Also shown are diameter and [Ca2+]i when K+ in the extracellular bath was increased to 15 and 60 mM. Increase of K+ to 15 mM activates inward rectifier K+ channels and dilates cerebral vessels (23); K+ concentrations of 60 mM depolarize and constrict vessels. *P < 0.05 compared with corresponding value at zero shear stress.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We report three significant findings in the present study. First, luminal flow constricted rat MCA and PA. Second, endothelium is not required for flow to constrict MCA and PA. Third, integrin binding, specifically an integrin containing the beta 3-subunit, was involved with the shear stress-induced constriction in MCA.

Luminal flow constricted rat MCA and PA. This represents the first time that the response to flow was tested in different segments along the cerebrovascular tree in a single study. Our results demonstrate that luminal flow constricted rat MCA and PA, and the response occurred over pressures ranging from 40 to 80 mmHg in PA and from 40 to 100 mmHg in MCA (Figs. 4-7). The majority of the constriction occurred between 0 and 50 dyn/cm2 in both MCA and PA. During normal physiological conditions shear stress is considered to be between 11 and 60 dyn/cm2; however, during stenotic conditions shear stresses can reach levels in excess of several hundred dynes per square centimeter (26).

With the use of identical techniques we demonstrated that rat CMA dilated in response to increased luminal flow (Fig. 3). This flow-induced dilation, which is consistent with previous results (25), adds validity to our methods and support to our results in cerebral vessels. Technical problems and associated artifacts cannot account for constrictor responses in cerebral vessels. Thus we conclude that rat MCA and PA constrict in response to luminal flow. In the MCA, this response is due to increased shear stress and not some other aspect associated with increased flow. We can draw this conclusion with a high degree of certainty, because the MCA constricted in a similar manner when the shear stress was increased by either altering flow or by altering viscosity at a constant flow (Fig. 9).

A summary of the literature reveals approximately a dozen published papers from four laboratories dealing with the effects of luminal flow on diameter or tone of cerebral vessels. Luminal flow depolarized the vascular smooth muscle and constricted cat MCA (~680 µm) (31). The flow-induced constriction occurred at pressures of 70 and 100 mmHg. The same laboratory reported that flow constricted cerebral arteries (~500 µm) isolated from 2- to 14-day-old piglets at lower rates of flow, but at higher rates, the cerebral vessels dilated back to near the original diameter through an NO-related mechanism (41). The constrictor component of the flow response did not occur when the flow through the lumen was pulsatile (42). Ngai and Winn (37) reported that PA (~50 µm, pressurized to 60 mmHg) isolated from rat dilated at a flow of 10 µl/min via NO release and constricted toward the original baseline at greater rates of flow. Studies (4, 12, 13, 43) of rabbit cerebral arteries (ranging from ~120 to 250 µm in diameter) indicate either a flow-induced constriction or flow-induced dilation with the response possibly being dependent on the vessel tone or luminal pressure. The flow-induced dilation in the rabbit arteries was reported to have both an endothelium-dependent and an endothelium independent component (43, 44) or was reported to be completely endothelium independent (13). The response in the rabbit is somewhat inconsistent, due possibly to the different arteries studied (MCA, MCA branches, or posterior cerebral arteries) and the different methods used (wire mounted or pressurized) (3, 4, 12-14, 43, 44). Fujii et al. (10, 11) reported that a flow-mediated dilation occurred in vivo in the rat basilar artery (250-300 µm) when either one or both carotid arteries were occluded. The dilation was not produced by the release of NO or cyclooxygenase metabolites from the endothelium.

Our results in the rat are most consistent with those reported for the cat (31). We show that the rat MCA and PA constrict to increases in luminal flow and that the response was independent of luminal pressure over a range of pressures (Figs. 4-7). Like cat cerebral vessels, the flow-induced constriction persisted even after removal of the endothelium in MCA and PA (Fig. 8).

Of note are the differences between the present study and those by Ngai and Winn (37) in rat PA. Ngai and Winn (37) reported that flows of 5 and 10 µl/min through the lumen produced dilations of 5 and 15%, respectively. At higher rates of flow, the vessels began to constrict to near the original diameter before flow was initiated.

Although there are differences between our study and those of Ngai and Winn (37), we cannot fault their results for technical reasons. Ngai and Winn (37) apparently gave careful attention to the problems associated with the study of flow in isolated vessels. At present we cannot explain the differences between our study and those by Ngai and Winn (37).

Endothelium is not required for the flow to constrict MCA and PA. Flow-induced constrictions persisted after removal of the endothelium in rat MCA and PA (Fig. 8) and cat MCA (31). This observation may not seem logical on initial consideration because the cells receiving the mechanical stimuli were not required for the response to occur. We speculate that the forces at the luminal surface of the endothelium are transmitted through the cytoskeletal matrix to mechanoreceptors on the extraluminal side of the endothelium (8). Although the shear stress-induced constriction occurs in the presence or absence of endothelium, the endothelium does influence the response by attenuating the constrictor response to luminal shear stress (unpublished observations).

Integrin binding, specifically beta 3-integrin, involved in shear stress-induced constriction in MCA. The extracellular matrix is mechanically linked to the cytoskeleton and nucleus through a complex structural system (19, 27). Integrins, a class of adhesion proteins, bridge the extracellular matrix to cytoplasmic actin filaments and in doing so are capable of activating several classical signaling pathways (19, 27). Although the integrins recognize the RGD sequence in the matrix ligand, different integrins are capable of distinguishing between different RGD-containing proteins of the extracellular matrix (19, 27). Of interest, recent studies (7, 34, 39, 45) demonstrate that integrins have effects on vascular tone by altering [Ca2+]i and Ca2+ currents in vascular smooth muscle. Furthermore, integrin signaling was shown to be involved with flow-induced dilations in the isolated coronary arteriole (35) and flow-induced constriction in cat MCA (31). We have extended these findings and now report that integrins also play an important role with shear stress-induced constrictions in the rat MCA (Fig. 10). Furthermore, we have provided evidence that a beta 3-integrin likely participates in the constrictor response to shear stress (Fig. 11). Of interest is the observation that F-11, an antibody to the beta 3-chain, constricted the rat MCA (Fig. 11). A subset of monoclonal antibodies with epitopes on the beta 3-subunit is known to lock the integrin complex in an active form and trigger a signal response (19). Although the constrictor response to F-11 makes interpretation more difficult, our results are, nevertheless, consistent with the idea that an integrin containing the beta 3-subunit has a key role in shear stress-induced constrictions.

With increasing rates of shear stress, Ca2+ in vascular smooth muscle increased 60-70 nM (Fig. 12). An increase in this magnitude is sufficient to account for constriction associated with the shear stress. Presumably, shear stress increased cytoplasmic Ca2+ by altering integrin binding.

Upstream dilations and the role of shear stress in the cerebral circulation. Resistance arteries and arterioles hundreds to thousands of micrometers upstream from an activated area must dilate to maximize circulatory control (20, 40). The brain is no exception and apparently abides by this general principle. For example, dilations (10-40%) have been reported in vivo in upstream pial arterioles of the rat after stimulation of the somatosensory cortex by whisker stimulation (6) or by electrical stimulation of the sciatic nerve (36, 38). In the cerebellum of the rat, stimulation of the parallel fibers produced 10% dilation in upstream arterioles supplying the activated folium (21).

Given that upstream dilations do occur in the cerebral circulation, how can our results, showing shear stress-induced constrictions, be reconciled with the upstream dilations reported in vivo? We hypothesize that luminal shear stress has a different role in the cerebral circulation than it does in much of the peripheral circulation. For a vessel to dilate, it must be partially constricted or, to state it in another way, it must have tone. There are several mechanisms, including intrinsic properties of the vascular smooth muscle (15) and vasoconstrictor agents that produce tone in arteries and arterioles. In the cerebral circulation of the rat, we hypothesize that a steady-state shear stress is an additional mechanism for the arteries and arterioles to develop and maintain tone. Thus upstream dilations in the cerebral circulation must depend on a mechanism other than a steady shear stress on the vessel wall. Consistent with this idea, Ngai and Winn (38) reported that upstream dilations in vivo occurred after stimulation of the somatosensory cortex without a change in wall shear rate. Thus an increased shear stress does not appear to drive the upstream dilation in the cerebral circulation. Another mechanism must be considered for the response.

We further hypothesize that shear stress-induced constriction is a means whereby blood volume and intracranial pressure are tightly regulated in the brain. Within the cranial cavity, there are many components including various cell types, blood, and cerebrospinal fluid. An increase in the volume of any one component will increase the intracranial pressure, given no compensation from the other components. Dilations, which increase blood volume, will tend to increase intracranial pressure and, thus decrease the perfusion pressure in the brain. In the periphery, where organs and tissues are not contained within a rigid structure, there is more freedom to dilate and increase blood volume. Because the brain has to contend with this unique problem, regulation of blood volume and intracranial pressure becomes a major issue. Therefore, the brain must have tighter control over dilator mechanisms than peripheral vessels. We speculate, therefore, that flow or shear stress-induced constrictions are a means to tightly regulate and govern changes in blood volume and intracranial pressure in the brain.

In summary, we report that rat MCA and PA constrict to increased luminal flow. In the MCA, at least, this flow-induced response is due to shear stress on the luminal wall of the vessel. Although endothelia are the direct recipient of the shear forces, they are not necessary for the shear stress-induced constriction. Finally, binding of a beta 3-integrin has a major role in the shear stress-induced constriction.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Neurological Disorders and Stroke Grant RO1-NS-37250.


    FOOTNOTES

Address for reprint requests and other correspondence: R. M. Bryan, Jr., Dept. of Anesthesiology, Baylor College of Medicine, 1 Baylor Plaza, Suite 434D, Houston TX 77030 (E-mail: rbryan{at}bcm.tmc.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.

Received 29 August 2000; accepted in final form 17 November 2000.


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DISCUSSION
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