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 |
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
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
3-chain are involved with the shear stress-induced constrictions.
cerebrovascular circulation; endothelium; integrins; cremaster muscle arteriole; calcium; fura 2
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INTRODUCTION |
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 3-subunit, are involved with
the response to shear stress in the rat MCA.
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METHODS |
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.
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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)
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(1)
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where D was the distance to achieve laminar flow,
r was the vessel radius, û was the average
velocity, and 
was the kinematic viscosity (viscosity/density).
û was calculated according to the equation
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(2)
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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)
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(3)
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where t was shear stress (in dyn/cm2) and
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. was calculated by using the above measurements after rearranging Poiseuille's law. The working equation was
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(4)
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where L was the length. , 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)](/content/vol280/issue5/fulltext/H2011/img005.gif)
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(5)
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where [Ca2+]i was the intracellular
Ca2+ concentration in the vascular smooth muscle, 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. , 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
3-integrin, and a control protein were purchased from
PharMingen (San Diego, CA). Because F-11 is a mouse IgG1
(
-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
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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).
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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)
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(6)
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where d was the vessel diameter and 
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.
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RESULTS |
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.
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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).
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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 105 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.
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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.
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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.
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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 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.
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|
Studies presented in Fig. 11 show the
results of F-11, an antibody against 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 (-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 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.
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|
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.
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| |
DISCUSSION |
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 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 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 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 3-chain,
constricted the rat MCA (Fig. 11). A subset of monoclonal antibodies
with epitopes on the 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
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
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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ABSTRACT
INTRODUCTION
