Am J Physiol Heart Circ Physiol 286: H388–H393, 2004.
First published September 25, 2003; 10.1152/ajpheart.00683.2003.
Chronic intermittent hypoxia impairs endothelium-dependent dilation
in rat cerebral and skeletal muscle resistance arteries
Shane A. Phillips,1 E. B. Olson,2 Barbara J. Morgan,3 and Julian H. Lombard1
1
Department of Physiology, Medical College of Wisconsin, Milwaukee 53226; and Departments of 2Population Health
Sciences and 3Orthopedics and Rehabilitation, University of Wisconsin, Madison, Wisconsin 53706
Submitted 28 July 2003; accepted in final form 16 September 2003
served in patients with sleep-disordered breathing (5, 16) and
in rats exposed to intermittent hypoxia, concurrent with an
elevation in arterial pressure (30).
Impaired endothelial function in the vessels that lie immediately proximal to the microcirculation could increase vascular resistance and promote the development of hypertension.
On the other hand, it has been hypothesized that elevated blood
pressure could produce endothelial dysfunction (20). Impaired
relaxation or paradoxical vasoconstriction in response to ACh
and blunted responses to physiological dilator stimuli such as
reduced oxygen availability and increased shear stress have
been observed in several experimental models of hypertension
(13, 14, 20); however, it is not clear whether this endothelial
dysfunction is a cause or a consequence of the elevation in
arterial pressure.
To our knowledge, no studies have evaluated the effect of
intermittent hypoxia per se on endothelial function in the
absence of hypertension. Therefore, the goal of the present
study was to test the hypothesis that chronic intermittent
hypoxia (CIH), applied for a relatively brief duration that does
not result in a sustained increase in arterial pressure, impairs
responses to vasodilator stimuli in resistance arteries of the
cerebral and skeletal muscle circulations.
MATERIALS AND METHODS
ACUTE EPISODES OF SLEEP-DISORDERED BREATHING produce hypoxemia, hypercapnia, and arousal from sleep. Recent studies have
demonstrated that sleep-disordered breathing in humans is
associated with the presence of hypertension (23, 32) and other
cardiovascular diseases such as congestive heart failure and
stroke (7, 25). Intermittent airway obstructions and episodic
hypoxia (both hallmarks of sleep-disordered breathing) cause
sustained elevations of blood pressure in dog and rat models (4,
8, 9). The key component responsible for raising blood pressure in these experimental models appears to be hypoxia rather
than hypercapnia or sleep disruption, because the addition of
CO2 does not augment the hypertensive effects of intermittent
hypoxia (9) and repetitive arousal from sleep does not cause
sustained increases in blood pressure (4). The mechanisms by
which intermittent hypoxia causes hypertension are not known,
although one potential contributor is endothelial dysfunction.
Attenuated endothelium-dependent vasodilation has been ob-
Animals. Age-matched, male Sprague-Dawley rats (Harlan Teklad;
Madison, WI) weighing 250–400 g at the time of arrival were used for
all experiments. Each rat was fed standard rat chow (Purina) and
provided drinking water ad libitum during exposure to either intermittent normoxia or hypoxia. All rats were housed in an animal care
facility at the University of Wisconsin-Madison, which is approved by
the American Association for the Accreditation of Laboratory Animal
Care, and all protocols were approved by the Medical School’s
Animal Care and Use Committee. On the day of the study, rats
exposed to normoxic and hypoxic conditions were weighed and
anesthetized with an injection of pentobarbital sodium (50 mg/kg ip,
Abbott Laboratories; Chicago, IL), and a carotid artery was cannulated with polyethylene tubing for arterial pressure measurement
before the isolation of cerebral and skeletal muscle resistance arteries
(see Preparation of isolated vessels).
Hypoxic exposure. Rats were exposed to CIH for 12 h/day (from
1800 hours to 0600 hours) for 14 days. In the hypoxia chamber, rats
were housed three per cage, in accordance with space recommendations set forth in the National Institutes of Health Guide for the Care
and Use of Laboratory Animals (NIH Pub. No. 85-23, Revised 1985).
Oxygen concentrations in the chamber were monitored using a heated
zirconium sensor (Fujikura America; Pittsburgh, PA) connected to
solenoid valves that controlled the flow of oxygen and nitrogen. The
Address for reprint requests and other correspondence: J. H. Lombard, Dept.
of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd.,
Milwaukee, WI 53226 (E-mail: [email protected]).
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.
acetylcholine; gracilis artery; middle cerebral artery; vascular reactivity
H388
0363-6135/04 $5.00 Copyright © 2004 the American Physiological Society
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Phillips, Shane A., E. B. Olson, Barbara J. Morgan, and Julian
H. Lombard. Chronic intermittent hypoxia impairs endotheliumdependent dilation in rat cerebral and skeletal muscle resistance
arteries. Am J Physiol Heart Circ Physiol 286: H388–H393, 2004.
First published September 25, 2003; 10.1152/ajpheart.00683.2003.—
The goal of the present study was to evaluate the effects of relatively
short-term chronic intermittent hypoxia (CIH) on endothelial function
of resistance vessels in the skeletal muscle and cerebral circulations.
Sprague-Dawley rats were exposed to 14 days of CIH (10% fraction
of inspired oxygen for 1 min at 4-min intervals, 12 h/day, n ⫽ 6).
Control rats (n ⫽ 6) were housed under normoxic conditions. After 14
days, resistance arteries of the gracilis muscle (GA) and middle
cerebral arteries (MCA) were isolated and cannulated with micropipettes, perfused and superfused with physiological salt solution, and
equilibrated with 21% O2-5% CO2 in a heated chamber. The arteries
were pressurized to 90 mmHg, and vessel diameters were measured
via a video micrometer before and after exposure to ACh (10⫺7–10⫺4
M), sodium nitroprusside (10⫺6 M), and acute reduction of PO2 in the
perfusate/superfusate (from 140 to 40 mmHg). ACh-induced dilations
of GA and MCA from animals exposed to CIH were greatly attenuated, whereas responses to nitroprusside were similar to controls.
Dilations of both GA and MCA in response to acute reductions in PO2
were virtually abolished in animals exposed to CIH compared with
controls. These findings suggest that exposure to CIH reduces the
bioavailability of nitric oxide in the cerebral and skeletal muscle
circulations and severely blunts vasodilator responsiveness to acute
hypoxia.
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INTERMITTENT HYPOXIA IMPAIRS ENDOTHELIAL FUNCTION
valves were operated by a microprocessor-controlled timer. This
system was set to provide hypoxic exposures at 4-min intervals. The
first minute of each exposure was transitional, with nitrogen flushed
into the chamber at a rate sufficient to achieve a fraction of inspired
oxygen (FIO2) of 0.10 within 60 s and to maintain this level of FIO2 for
an additional minute. Oxygen was then introduced at a rate sufficient
to achieve a FIO2 of 0.209 within 30 s and to maintain this level of FIO2
for the remainder of the 4-min interval. Daily checks of chamber
oxygen concentrations during hypoxia and normoxia were made using
a TED60T oxygen sensor (Teledyne; City of Industry, CA). Figure 1
provides an illustration, obtained via a mass spectrometer, of the FIO2
profile produced by our intermittent hypoxia protocol. The temperature of the chamber was maintained at 22 ⫾ 1°C, and the relative
humidity was maintained between 30 and 70%.
Normoxic exposure. Three rats were maintained in standard housing conditions of the animal care facility, whereas three rats were
maintained for 14 days under normoxic conditions in the hypoxia
chamber, where they were subjected to the same noises and airflow
perturbations experienced by the hypoxia-exposed rats.
Preparation of isolated vessels. The small muscular branch of the
femoral artery supplying the gracilis muscle was freed from surrounding tissue and allowed to equilibrate in situ for 30 min with the
application of warm physiological salt solution (PSS). After the
equilibration period, the artery was carefully excised. The anesthetized rats were then decapitated, and the brain was quickly removed
and transferred to a Lucite-coated petri dish for removal of the middle
cerebral artery (MCA). The MCA and the gracilis artery were located
using a dissecting microscope (Leica; Buffalo, NY) and carefully
isolated to separate the vessel from the surrounding parenchymal
tissue. Both vessels were immediately immersed in warmed PSS
bubbled with 21% O2-5% CO2-74% N2. The PSS used in these
experiments had the following ionic composition (in mM): 119.0
NaCl, 4.7 KCl, 1.6 CaCl2, 1.18 NaH2PO4, 1.17 MgSO4, 24.0
NaHCO3, 5.5 dextrose, and 0.03 EDTA. The arteries were handled by
the adhering connective tissue only to avoid damage to the endothelium and smooth muscle layers.
The proximal and distal ends of the MCA and gracilis artery were
cannulated with glass micropipettes (100–150 ␮m, FHC; Brunswick,
ME) and secured to the pipettes using 10-0 nylon sutures in a
superfusion-perfusion chamber (10, 21). The vessels were stretched to
the in situ length, and side branches were singly ligated with small
strands teased from a 6-0 silk suture (Ethicon; Somerville, NJ) to
ensure optimal pressurization. The inflow pipette was connected to a
reservoir perfusion system that allowed the intraluminal pressure and
luminal gas concentration to be controlled. Vessel diameter was
AJP-Heart Circ Physiol • VOL
RESULTS
Mean arterial pressure, body weight, and vessel diameter.
Table 1 summarizes data describing the rats exposed to normoxia or intermittent hypoxia for 14 days. Although resting
diameter (Table 1) and maximum diameter of the vessels in
Table 1. Characteristics of rats exposed to normoxia or CIH
Group
Normoxia
CIH
n
Age, wk
Body
Weight, g
MAP,
mmHg
6 11.5⫾0.1 397⫾34.0 127⫾5.5
6 11.8⫾0.1 406⫾14.0 126⫾8.5
Resting Diameter, ␮m
Gracilis artery
MCA
147⫾8.0
134⫾9.9
145⫾13.9
132⫾5.3
Values are means ⫾ SE; n, total number of normoxic control or chronic
intermittent hypoxia (CIH) rats used in the study. Numbers of vessels for
individual studies are indicated in the figures summarizing the results of those
experiments. The characteristics of rats exposed to normoxia or CIH include
body weight, mean arterial pressure (MAP), and the internal diameters of the
isolated gracilis arteries and middle cerebral arteries (MCA) in physiological
salt solution.
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Fig. 1. Representative trace of the atmospheric changes that occurred during
exposure to intermittent hypoxia. Percent O2 was determined by continuous
sampling from the chamber using a mass spectrometer. Intermittent hypoxia
was achieved by flushing nitrogen into the chamber until the O2 concentration
reached 10%. Hypoxia was maintained for 1 min, after which oxygen was
flushed into the chamber to reestablish normoxia, as described in MATERIALS
AND METHODS.
measured using television microscopy and a video micrometer. Any
vessel that did not exhibit significant levels of active tone (as evidenced by a substantial increase in resting diameter upon exposure to
Ca2⫹-free PSS) was not used in this study. The level of resting tone
in the vessel was calculated as follows: T ⫽ [(⌬D ⫻ D⫺1
max) ⫻ 100],
where T is resting tone (in %), ⌬D is the diameter increase in the
maximally relaxed vessel, and Dmax represents the maximum diameter
of the vessel at that pressure.
Response to reduced PO2. After the initial control period in PSS
equilibrated with 21% O2, the responses of gracilis arteries and MCA
to reduced PO2 were assessed in each group. PO2 reduction was
achieved by simultaneous perfusion and superfusion of the arteries for
25 min with PSS equilibrated with 0% O2-5% CO2-95% N2 as
previously described (10). Under these conditions, control values for
PO2 during 21% O2 perfusion and superfusion are ⬃140 Torr, whereas
equilibration of the PSS reservoirs with 0% O2 reduces both the
luminal and extraluminal PO2 to 35–45 Torr (10). After exposure of
the vessels to hypoxia, the perfusate and superfusate were reequilibrated with 21% O2 for 20 min, and recovery from reduced PO2 was
verified by measuring the vessel diameter.
Response to vasodilator agents and Ca2⫹-free solution. The responses of resistance arteries to the endothelium-dependent vasodilator ACh (10⫺7–10⫺4 M) and the nitric oxide (NO) donor sodium
nitroprusside (10⫺6 M, Sigma; St. Louis, MO) were assessed in
cerebral and skeletal muscle vessels in each group of rats. When these
drugs were administered, the superfusion was stopped in the bath, the
vessel was pressurized by clamping the outflow pipette, and an
appropriate amount of drug was added to the tissue chamber to
achieve the final desired concentration in the superfusion solution.
Vessel diameter was monitored continuously and was measured at the
point of its maximum value after the addition of the dilator agent.
After the response of the arteries to the various vasodilator stimuli
had been determined, vessel diameter was determined after the vessels
were maximally dilated with Ca2⫹-free relaxing solution containing
the following constituents (in mM): 92.0 NaCl, 4.7 KCl, 1.17
MgSO4 䡠7H2O, 20.0 MgCl䡠6H2O, 1.18 NaH2PO4, 24.0 NaHCO3,
0.026 EDTA, 2.0 EGTA, and 5.5 dextrose.
Statistical analysis. All data are expressed as means ⫾ SE. Responses to incremental doses of ACh were analyzed with two-way
repeated-measures ANOVA. Significant differences between the individual means after two-way ANOVA were determined using a
Student-Newman-Keuls post hoc test. Differences in the responses to
hypoxia, nitroprusside, and maximal dilation in calcium-free solution
between groups were determined utilizing an unpaired t-test.
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INTERMITTENT HYPOXIA IMPAIRS ENDOTHELIAL FUNCTION
Ca2⫹-free solution tended to be reduced in arteries of animals
receiving hypoxic exposure, the difference between animals
exposed to normoxia (gracilis: 178 ⫾ 6.0 ␮m; MCA: 192 ⫾
13.4 ␮m) and those exposed to CIH (gracilis: 169 ⫾ 11.9 ␮m;
MCA: 164 ⫾ 8.6 ␮m) was not statistically significant. In
addition, there was no significant difference in body weight or
mean arterial pressure between groups.
Response of resistance arteries to ACh and sodium nitroprusside. Figures 2 and 3 summarize the response of resistance
arteries to ACh in animals exposed to normoxia or intermittent
hypoxia. In these experiments, cerebral and skeletal muscle
arteries from animals maintained under normoxic conditions
for 14 days demonstrated a pronounced dilation to incremental
doses of ACh (10⫺7–10⫺4 M), similar to previous results (10).
There were no significant differences in the responses to ACh
(10⫺5 M) in vessels of animals exposed to standard normoxic
conditions (gracilis: 17 ⫾ 1.7 ␮m; MCA: 19 ⫾ 1.3 ␮m) or
housed under normoxic conditions in the hypoxia chamber
(gracilis: 24 ⫾ 2.0 ␮m; MCA: 21 ⫾ 0.5 ␮m). However,
Fig. 3. Response to 10⫺7–10⫺4 M ACh in middle cerebral arteries of rats
exposed to normoxia and CIH for 14 days (n ⫽ 6 rats/group). Data are
presented as mean changes (in ␮m) ⫾ SE from the diameter measured before
application of ACh. *Significant difference from the response of vessels from
normoxic control animals, P ⬍ 0.05.
AJP-Heart Circ Physiol • VOL
Fig. 4. Responses to sodium nitroprusside (10⫺6 M) in isolated gracilis
arteries (A) and middle cerebral arteries (B) from animals exposed to normoxia
or CIH for 14 days (n ⫽ 5–6). Data are presented as means ⫾ SE. There was
no significant difference in the response of vessels to nitroprusside in normoxic
control animals and animals exposed to CIH.
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Fig. 2. Response to10⫺7–10⫺4 M ACh in gracilis arteries of rats exposed to
normoxia or chronic intermittent hypoxia (CIH) for 14 days (n ⫽ 6 rats/group).
Data are presented as mean changes in diameter (in ␮m) ⫾ SE from control
measured before the application of ACh. *Significant difference from the
response of vessels from normoxic control animals, P ⬍ 0.05.
dilation of skeletal muscle and cerebral arteries to ACh was
significantly reduced in animals exposed to intermittent hypoxia for 14 days (Figs. 2 and 3). The responses of skeletal
muscle and cerebral resistance arteries from rats administered
normoxia and intermittent hypoxia to the NO donor sodium
nitroprusside are summarized in Fig. 4. In contrast to vessel
responses to ACh, exposure to CIH had no effect on the
vascular relaxation in response to sodium nitroprusside in
either skeletal muscle or cerebral resistance arteries.
Responses of resistance arteries to acute reductions in PO2.
Figure 5 summarizes the responses of skeletal muscle (A) and
cerebral resistance arteries (B) to acute hypoxia in rats exposed
to normoxia or intermittent hypoxia for 14 days. Arteries from
rats exposed to normoxia demonstrated a significant increase in
diameter in response to reduced PO2, as previously reported in
both skeletal muscle (10) and cerebral resistance arteries (21).
However, gracilis arteries (Fig. 5A) and MCA (Fig. 5B) from
rats receiving intermittent hypoxia for 14 days failed to dilate
in response to an acute reduction in perfusate and superfusate
PO2 from ⬃140 to ⬃35–45 Torr. As in the case of ACh, there
was no significant difference in the vasodilator responses to
acute hypoxia in vessels from animals exposed to standard
normoxic conditions (gracilis: 16 ⫾ 1.2 ␮m; MCA: 15 ⫾ 2.5
␮m) or housed under normoxic conditions in the hypoxia
INTERMITTENT HYPOXIA IMPAIRS ENDOTHELIAL FUNCTION
H391
Fig. 5. Responses to simultaneous reduction of perfusate and superfusate O2
concentration from 21% to 0% O2 in isolated gracilis arteries (A) and middle
cerebral arteries (B) of animals exposed to normoxia or CIH for 14 days (n ⫽
5–6). Data are presented as mean changes in diameter from the 21% O2 control
value (in ␮m) ⫾ SE. *Significant difference from the response of arteries from
normoxic control animals, P ⬍ 0.05.
chamber (gracilis: 25 ⫾ 1.3 ␮m; MCA: 13 ⫾ 1.2 ␮m).
However, gracilis arteries (Fig. 5A) and MCA (Fig. 5B) from
rats receiving intermittent hypoxia for 14 days failed to dilate
in response to an acute reduction in perfusate and superfusate
PO2 from ⬃140 to ⬃35–45 Torr.
Maximal relaxation in calcium-free PSS. Figure 6 shows the
responses to calcium-free PSS in skeletal muscle (A) and
cerebral resistance arteries (B) from rats administered normoxia or intermittent hypoxia for 14 days. Maximal dilation of
both vessel types was unaltered by exposure to intermittent
hypoxia. There was also no significant difference in the resting
active tone of gracilis arteries from rats administered normoxia
(%tone: 16 ⫾ 2.9, n ⫽ 5) and intermittent hypoxia (%tone:
23 ⫾ 2.8, n ⫽ 6) or in the resting active tone of MCA from
animals administered normoxia (%tone: 23 ⫾ 7.2, n ⫽ 5) or
intermittent hypoxia (%tone: 19 ⫾ 3.5, n ⫽ 5).
DISCUSSION
In the present study, exposure to CIH for 14 days significantly impaired endothelium-dependent vasodilator responses
to ACh (Figs. 2 and 3) and acute hypoxia (Fig. 5) in resistance
arteries from the skeletal muscle and cerebral vascular beds but
did not alter vessel responses to the NO donor sodium nitroprusside (Fig. 4) or maximal dilation of the arteries in Ca2⫹AJP-Heart Circ Physiol • VOL
Fig. 6. Maximal dilation of gracilis (A) and middle cerebral (B) arteries (n ⫽
4–5) in Ca2⫹-free physiological salt solution. Data are presented as changes in
mean internal diameter (in ␮m) ⫾ SE. There were no significant differences in
the response of vessels from animals exposed to normoxia versus CIH.
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free PSS (Fig. 6). These observations suggest that impaired
vasodilator responses in resistance arteries are due to altered
mechanisms of endothelium-dependent vascular relaxation after exposure to intermittent hypoxia rather than altered sensitivity to NO or structural impairments in the arterial wall that
restrict the vessels’ ability to increase their diameter in response to dilator stimuli. This impairment of endothelial function was evident before the development of hypertension in this
experimental model (known to raise blood pressure), because
mean arterial pressures were nearly identical in the hypoxiaand normoxia-exposed rats (Table 1).
Previous studies of animal models indicate that the primary
prohypertensive component of CIH is transient blood oxygen
desaturation (4, 9, 26, 31) rather than the hypercapnia and sleep
disruption that occurs during sleep-disordered breathing (3, 4).
The present findings, obtained in a model that produces hypocapnic hypoxia, suggest that hypoxia is also the primary
stimulus that impairs endothelial function, because rats exposed to the same noise and cage environment during normoxia did not demonstrate similar alterations in their responses
to dilator stimuli.
The sensitivity of blood vessels to vasoactive stimuli has
been extensively studied during hypertension and sleep-disordered breathing (2, 16, 22, 28). For example, a previous study
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INTERMITTENT HYPOXIA IMPAIRS ENDOTHELIAL FUNCTION
AJP-Heart Circ Physiol • VOL
anisms that mediate hypoxic dilation of resistance arteries,
independent of elevations in arterial blood pressure.
Although the mechanism by which intermittent hypoxia
causes impairments in the ability of resistance arteries to
respond to endothelium-dependent vasodilator stimuli is unknown, other investigators have determined that elevated levels of reactive oxygen species deplete vascular NO during
hypertension and exposure to sustained hypoxia (11, 19, 27,
31). Coupled with studies suggesting that patients with obstructive sleep apnea have increased superoxide generation
(24), additional studies investigating the role of reactive oxygen species in the vasculature of this model of sleep-disordered
breathing seem to be warranted. Future studies of the effect of
CIH on vascular control mechanisms may be important in
determining factors that cause individuals to develop hypertension as a consequence of sleep-disordered breathing. It is
possible that endothelial dysfunction arising from CIH may
impair blood flow regulation and compromise tissue perfusion
and oxygen delivery during stresses such as hemorrhage,
exercise, and episodes of hypoxemia caused by sleep-disordered breathing, even in the absence of a significant elevation
of arterial blood pressure.
GRANTS
This study was supported by National Heart, Lung, and Blood Grants
HL-65289, HL-29857, and HL-72920.
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Effect of CIH on maximal relaxation of skeletal muscle and
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impaired ability of arteries to respond to vasodilator stimuli in
hypertension.
Effect of CIH on acute hypoxia-induced dilation in skeletal
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isolated cerebral and skeletal muscle resistance arteries from
normoxic rats demonstrated a significant increase in diameter
in response to a simultaneous reduction in superfusion and
perfusion PO2 (Fig. 5A), similar to observations reported in
previous studies (10, 20). However, dilator responses to acute
hypoxia were completely abolished in arteries from rats subjected to CIH (Fig. 5B). This alteration in the ability of arteries
to dilate in response to hypoxia may be important in the
development and progression of hypertension caused by sleepdisordered breathing. The finding that gracilis arteries and
MCA fail to dilate during acute exposure to hypoxia is important because it demonstrates that short-term exposure to intermittent hypoxia can lead to alterations in the signaling mech-
INTERMITTENT HYPOXIA IMPAIRS ENDOTHELIAL FUNCTION
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