Journal of Gerontology: BIOLOGICAL SCIENCES
2003, Vol. 58A, No. 10, 895–899
Copyright 2003 by The Gerontological Society of America
Effect of Age on Peripheral Vascular Response
to Transverse Aortic Banding in Mice
Yi-Heng Li, Anilkumar K. Reddy, Lyssa N. Ochoa, Thuy T. Pham, Craig J. Hartley,
Lloyd H. Michael, Mark L. Entman, and George E. Taffet
The DeBakey Heart Center, Huffington Center on Aging, and Sections of Geriatrics and Cardiovascular Sciences,
Department of Medicine, Baylor College of Medicine, Houston, Texas.
The placement of a ligature to constrict the transverse aorta has become a standard procedure to
induce cardiac hypertrophy in mice. Apart from cardiac response, there are adaptive changes in
the proximal and distal arterial system that function to maintain adequate peripheral perfusion.
The purpose of this study was to characterize the peripheral vascular response by measuring the
carotid blood flow using noninvasive Doppler methods, and to investigate the effect of aging on
the adequacy and timing of the response after aortic banding in mice. Five 16-month-old and 9
4-month-old male B6D2F1 mice underwent transverse aortic banding. Blood flow velocity was
measured with Doppler in the right and left carotid arteries (RCA and LCA) before, 1 day after,
and 7 days after, banding. Pulsatility index defined as (peak minimum)/mean velocity was used
to estimate local compliance and distal arterial resistance. The RCA/LCA mean velocity ratio was
lower and pulsatility index ratio was higher at 1 day after banding in older mice. However, at 7
days, the RCA/LCA mean velocity ratio and pulsatility index ratio were similar between the 2
age groups. Our data indicate that there is an age-related delay in the development of vascular
adaptations in carotid arteries after aortic banding. Older mice take a longer time for adaptation to
establish adequate and equal mean flow velocity in the carotid arteries.
C
ARDIAC hypertrophy is a common adaptative physiological response to increased demands on the heart.
One of the common methods to induce cardiac hypertrophy in mice is a pressure overload model produced by
constricting the transverse aorta in the arch between the
origins of the innominate artery and the left carotid arteries
(1). The model is thought to be successful because the innominate right subclavian and carotid arteries proximal to
the band are able to absorb the major portion of the energy
produced by cardiac contraction. Therefore, it is expected
that the placement of the aortic band will have profound
effects on the arteries as well as on the heart.
It is well recognized that cardiac hypertrophic response
following aortic constriction is diminished in aged animals (2–4). However, the effect of age on the capacity for
vascular adaptation to aortic banding is unknown, and this
might contribute to the actual loading sensed by the old
heart. There are progressive age-related morphological and
functional changes in arteries that could impact the development of adaptation. Endothelial cells become more
heterogenous in size and shape, which is associated with
a decreased production of the potent vasodilator nitric
oxide (5–7). In the media, there is thickening of the smooth
muscle layer, greater fragmentation of elastin, and increased
calcification (5–7). In normal subjects, there is a progressive
increase of cerebrovascular resistance with advancing age
(8,9). We hypothesized that these age-related vascular
changes might alter the peripheral vascular response to
aortic banding in mice.
To test our hypothesis, we focused on the adaptive
responses of the carotid arteries to transverse aortic banding in young and old mice. We used noninvasive Doppler
methods to study carotid blood flow velocity (or simply
‘‘velocity’’) changes after banding, and to investigate the
effect of aging on the arterial response to aortic banding in
mice. We measured peak, minimum, and mean velocities,
and calculated pulsatility index (¼ [peak minimum]/mean
velocity). While the velocity difference (peak minimum)
is an indicator of the compliance of the artery, the mean
velocity is an indicator of the resistance of the vasculature
distal to the site of measurement. Thus the pulsatility index
is an indicator of arterial compliance and resistance.
METHODS
Animal Preparation
Male B6D2F1 mice aged 16 months and 4 months were
obtained from National Institute on Aging colonies maintained by Harlan-Sprague-Dawley (Indianapolis, IN). The
B6D2F1 mice have a survival rate of 50% at 27 months of
age and can live up to a maximum age of 36 months (10).
They were caged in a dedicated specific pathogen-free room
and allowed to recover for 2 weeks after transport. They
were examined for obvious signs of disease or weight loss.
All mice were housed individually in the animal facility at
the Fondren-Brown vivarium of Baylor College of Medicine
approved by the American Association for Accreditation of
Laboratory Animal Care with the guidelines of the National
895
896
LI ET AL.
Institutes of Health Guide for the Care and Use of Laboratory Animals. Animals were kept in a room at controlled
temperature (248C) and lighting (14:10 hr light–dark cycle)
with free access to food and water. The diets of both age
groups consisted of normal chow.
Transverse aortic banding was performed in 4-month-old
mice (n ¼ 9) and 16-month-old mice (n ¼ 5) by using the
method described by Rockman and colleagues (1). In brief,
the mouse was anesthetized with pentobarbital sodium
given intraperitoneally (4 mg/ml, 10 ll/g body wt of mouse).
The animal was placed in the supine position and endotracheal intubation was performed. The endotracheal tube was
connected to a Harvard volume-cycled rodent ventilator
(Model 683; Harvard Apparatus, South Natick, MA) cycling
at »110 breaths/minute with volume sufficient to adequately
expand the lungs. Oxygen (100%) was supplied to the
inflow of the ventilator during operation. Under a microscope (Zeiss, Germany), the chest cavity was opened, and
the aortic arch was exposed after deflection of the thymus.
After the transverse aorta was isolated, a 7-0 nylon suture
was placed around the aorta between the origins of the
innominate and left carotid arteries. A 27-gauge (0.4-mm)
needle was placed against and tied to the aorta with the
suture. Then the needle was removed promptly to yield a
constriction with a diameter approximately equal to that of
the needle. In sham-operated mice (5 4-month-old mice
and 2 16-month-old mice), the suture was placed around the
aorta but not tightened. The sham-operated mice were
studied just to look at the stability of the measures and were
not used for any statistical comparisons. No effect of
opening and closing the chest on carotid flow was observed.
The chest wall was closed, and the mice were allowed to
recover from anesthesia under oxygen and a heat lamp to
maintain body temperature.
Doppler Studies
To evaluate the effect of aging on the changes of carotid
blood flow velocity after surgery, Doppler studies were performed in mice of both age groups, on both carotid arteries,
before (baseline) and at 1 day and 7 days after surgery. The
animals were anesthetized with an intraperitoneal injection
of pentobarbital sodium and taped supine to a temperaturecontrolled board. The board included electrocardiographic
electrodes placed under each limb and a heating pad under
the body. Body hair was clipped and the skin was moistened
with water to improve sound transmission. A 20 MHz Doppler probe was placed on the left and right sides of the neck
at a 458 angle to detect flow velocity in the left and right
carotid arteries. At each site, optimal Doppler flow velocity
signals were obtained by adjusting the position of the probe
and the Doppler range gate depth (1–3 mm) to obtain a
maximal velocity signal. We have verified in previous
studies (11,12) that consistent and reproducible signals are
obtainable from these sites in mice without image guidance.
The 20 MHz Doppler probe used in this study was
custom built in our laboratory. The probe consisted of
a 1-mm-diameter 20 MHz ultrasound crystal mounted at the
tip of a 2-mm-diameter, 10-cm-long stainless steel tube. The
probe was driven by a modular ultrasonic pulsed Doppler
instrument with a pulse repetition frequency of 125 kHz. A
computer-based Doppler signal processor (Indus Instruments, Houston, TX) was used to digitize, display, and store
the Doppler signals. The system provides a real-time
complex fast Fourier transform (FFT) display for operator
feedback during data acquisition. Typically, 2-second
segments of data were sampled and stored for later analysis.
During analysis, the maximum frequency (velocity) envelope was automatically calculated from the displayed FFT
spectrogram using the percentile method (13) by choosing
the frequency below which 95% of the total power in each
spectrum lies. Blood velocity (V) was calculated from the
Doppler shift frequency (f) using the Doppler equation (V
¼ c 3 f/2f0 3 cosh), where c is the speed of sound (1540
m/s) and f0 is the ultrasonic frequency (20 MHz). With the
probe position used for carotid blood flow velocity measurements, the Doppler angle (h) was 458, such that
cos(h) ¼ 0.71. The maximal, minimal, and mean velocities
of each signal were measured from the envelope waveform.
For estimation of the local compliance and distal vascular
resistance, the pulsatility index (14–16) was calculated for
the signals at both carotid arteries.
Data Analysis
The peak, minimum, and mean velocities were extracted
from the velocity signals of right and left carotid arteries
(RCA and LCA), and the pulsatility index of each signal
was calculated. Comparisons of parameters (peak velocity,
mean velocity, pulsatility index of RCA and LCA) were
made between baseline, 1 day, and 7 days within and across
groups. To minimize mouse-to-mouse variations, we also
calculated right-to-left ratios (RCA/LCA) of flow velocities
and pulsatility index of each mouse. The average of ratios
within each age group was then calculated. The values of the
parameters of both age groups are presented as means 6
SEM (standard error of mean). Within-group comparisons
were made by paired Student’s t test, and across-group
comparisons were made by unpaired Student’s t test by
using a p value ,.05 for significance.
RESULTS
Figure 1 shows the representative velocity signals from
the RCA and LCA before (baseline) and 1 day after aortic
banding in a 4-month old mouse. Prior to banding, the mice
had similar peak, minimum, and mean velocities in RCA
and LCA. After banding there was increased peak velocity
and, usually, negative minimum flow velocity (flow reversal) in the RCA. In the LCA, there was decreased peak flow
velocity and no negative minimal flow velocity after
banding. The mean velocity, however, was similar in both
vessels.
The body weights of 4-month old mice before and at
7 days after banding were 23.1 6 1.8 and 22.1 6 1.5,
respectively. The body weights of 16-month-old mice
before and at 7 days after banding were 32.4 6 1.6 and
30.5 6 2.3, respectively. The heart rates of 16-month-old
mice were consistently higher than 4-month-old mice. The
values for carotid blood flow velocities and pulsatility index
at baseline and after banding for mice of both age groups are
given in Table 1.
PERIPHERAL VASCULAR RESPONSE AFTER AORTIC BANDING IN AGED MICE
897
Figure 2. Right-to-left carotid peak velocity (Vpk) ratio at day 1 and day 7 in
both age groups with a baseline reference (dotted line).
peak and mean velocities of RCA and LCA were observed
between the groups at 7 days (Table 1).
The RCA/LCA ratios of the three parameters (peak
velocity, mean velocity, pulsatility index) are shown in
Figures 2, 3, and 4, respectively. At 1 day, the RCA/LCA
peak velocity ratio was similar (3.15 6 0.38 vs 3.69 6
0.42, p ¼ NS) between the 2 groups. However, the 16month-old mice had lower RCA/LCA mean velocity ratio
(0.78 6 0.14 vs 1.18 6 0.09, p , .05) and higher RCA/
LCA pulsatility index ratio (14.98 6 5.21 vs 5.71 6 0.96,
p , .05) than the 4-month-old mice (Figure 2). At 7 days, the
RCA/LCA peak flow velocity ratio (3.41 6 0.43 vs 3.63 6
0.40, p ¼ NS) was still the same between the 2 groups. Also,
the mean flow velocity ratio (1.05 6 0.15 vs 1.04 6 0.07,
p ¼ NS) and pulsatility index ratio (6.44 6 0.41 vs 6.07 6
0.77, p ¼ NS) also became similar between the 2 age groups
(Figure 3).
Figure 1. Representative recordings of Doppler velocity signals of the right
(right arrowhead) and left carotid (left arrowhead) arteries before (baseline) and
1-day after banding.
The following observations were made from comparisons
of parameters at 1 day and 7 days of each age group to their
respective baseline values. At 1 day, the mean velocity and
pulsatility index of RCA and LCA, and the peak velocity of
LCA of 4-month-old mice were significantly different from
their baseline values. However, by the seventh day, only the
peak velocity of RCA and LCA and the LCA pulsatility
index of 4-month-old mice were significantly different from
their baseline values. The mean velocities had returned to
prebanding levels. On the other hand, 16-month-old mice
had significant differences in the peak velocity of RCA and
LCA, and pulsatility index of LCA at 1 day when compared
with their baseline values. The above differences in 16month-old mice remained on the seventh day, at which time
the pulsatility index of RCA also had become significantly
different from the corresponding baseline value (Table 1).
The following observations were made from comparisons between the age groups at baseline, at 1 day, and at
7 days. At baseline and 1 day, no significant differences were
observed between the 2 groups in all parameters except in
LCA mean velocity. However significant differences in
DISCUSSION
Doppler examination is now a well-established method
for the assessment of circulatory physiology in patients.
Since mice have become important study subjects for human
diseases with the availability of various transgenic or mutant
models, we developed the Doppler system specifically used
in mice for cardiovascular phenotyping (12). The ability of
blood flow velocity, measured by Doppler ultrasound, to
serially monitor changes in carotid and middle cerebral
Table 1. Data From 4-Month-Old Mice (n ¼ 9) and 16-Month-Old Mice (n ¼ 5) Before (Baseline), at 1 Day, and
at 7 Days After Aortic Banding. All values are given as means 6 SE.
Baseline
Parameter
HR (beats/min)
RCA Vpk (cm/s)
RCA Vmean (cm/s)
RCA PI
LCA Vpk (cm/s)
LCA Vmean (cm/s)
LCA PI
4 Months
314
51.2
10.5
5.3
45.4
9.5
5.1
6
6
6
6
6
6
6
28
4.1
0.7
0.3
3.5
0.8
0.3
1 Day
16 Months
421
55.5
12.3
4.7
55.7
12.9
4.5
6
6
6
6
6
6
6
29 4.1
0.5
0.6
4.6
0.7 0.6
4 Months
322
64.3
8.0
10.1
19.5
6.9
2.2
6
6
6
6
6
6
6
25
5.6
0.4*
0.9*
3.4*
0.3*
0.5*
7 Days
16 Months
505
75.1
10.0
13.6
25.0
11.9
1.2
6
6
6
6
6
6
6
40 5.7*
3.1
3.0
3.3*
1.5 0.2*
4 Months
324
68.1
8.8
10.5
19.5
8.4
1.9
6
6
6
6
6
6
6
23
5.6*
1.0
1.0
1.4*
0.5
0.2*
Notes: All values are given as means 6 SEM (standard error of mean).
* p , .05 vs baseline (within-group comparison: paired Student’s t test).
p , .05 4-month vs 16-month (across-group comparison: unpaired Student’s t test).
HR ¼ heart rate; RCA ¼ right carotid artery; LCA ¼ left carotid artery; Vpk ¼ peak velocity; Vmean ¼ mean velocity; PI ¼ pulsatility index.
16 Months
497
102.0
13.4
10.8
31.2
13.0
1.7
6
6
6
6
6
6
6
35 5.7* 1.7 1.3*
3.0* 1.0 0.2*
898
LI ET AL.
Figure 3. Right-to-left carotid mean velocity (Vmean) ratio at day 1 and day 7
in both age groups with a baseline reference (dotted line).
artery blood flow has been used and validated in many
human studies (17–19). The pulsatility index (14–16), a
Doppler-derived parameter, has also been used extensively
as an expression of local vessel compliance and resistance to
blood flow of the vasculature distal to the site of measurement. If constant hemodynamic conditions are assumed,
changes of the pulsatility index may reflect corresponding
changes in local compliance and downstream vascular
resistance. Therefore, we used these Doppler methods to
evaluate the adaptation changes after transverse aortic
banding in mice. The results of the present study demonstrate that there were significant hemodynamic changes in
the carotid arteries after transverse aortic banding in mice,
and that there were age-related changes in the development
of the alterations.
In our study, the body weight and heart rate were different between the 4-month-old and the 16-month-old mice.
Although blood flow velocity in a given artery is independent of body weight across all mammals (11), it can be
influenced by physiological factors such as blood pressure
and cardiac output (20) and experimental factors such as
anesthesia and body temperature. To minimize any variation
in flow velocity due to the above-mentioned factors, we
calculated the right/left ratios of carotid flow velocity and
pulsatility index of each mouse and compared the groupaveraged ratios to determine the nature of the vascular
adaptations following aortic banding.
The results of the present study demonstrate that there are
significant hemodynamic changes in the carotid arteries after
transverse aortic banding in mice. In both 4-month-old and
16-month-old mice, the RCA/LCA peak flow velocity ratio
and pulsatility index ratio increased significantly after
banding when compared with baseline values. At 7 days
after banding, the mean flow velocity was still similar
between the RCA and LCA (ratio ;1) in both groups
(Figure 3), despite the significant right-to-left changes of
peak flow velocity (ratio ;3). These data indicate that, after
banding, the RCA replaces the aorta as the major source
of arterial compliance, resulting in a much higher RCA
peak velocity and pulsatility. Mice compensate by increasing downstream resistance in the RCA and decreasing it in
the LCA to maintain the normal mean flow and adequate
cerebral perfusion. The adaptation of the carotid artery by
Figure 4. Right-to-left carotid pulsatility index (PI) ratio at day 1 and day 7 in
both age groups with a baseline reference (dotted line).
vascular compliance change can be demonstrated by the sixfold increase of the RCA/LCA pulsatility index ratio at 7
days after banding when compared with the sham mice. At 7
days after banding, the severity of the stenosis produced was
similar in the 2 groups, as there was no difference in the
RCA/LCA peak flow velocity ratio. Given the similar RCA/
LCA ratios of mean flow velocity and pulsatility index in
both groups at 7 days, we concluded that ultimately there
was no significant age-related impairment of carotid adaptation response to transverse aortic banding in mice.
The adaptation process, however, was slower in older
mice. At 1 day after banding (Figure 2), the older mice had
a lower RCA/LCA mean flow velocity ratio and higher
RCA/LCA pulsatility index ratio when compared with the
younger mice. However, at 7 days, the older mice attained
the same level of vascular adaptation as the younger ones.
Our data indicated that older mice needed more time to
achieve the same vascular adaptation after aortic banding.
The slower carotid adaptation process after banding in
older mice may be due to the age-related arterial functional
changes resulting from the increase in arterial stiffness
and vascular tone with age (21). A previous study (22) has
shown that the a-adrenergic responses (vasoconstrictor) are
increased and the b-adrenergic responses (vasodilator) are
decreased with age in arteries. Additionally, the production
of nitric oxide from endothelium is decreased with aging.
When isolated from the aorta of old rats, the expression of
endothelial nitric oxide synthase mRNA in endothelial cells
was reduced (23). At 1 day after banding, it was most likely
that much more vasoconstriction occurred in the right
carotid artery of older mice, and resulted in a markedly
higher RCA/LCA pulsatility index ratio and lower mean
flow velocity ratio. However, at 7 days after banding, the
RCA/LCA pulsatility index ratio and mean flow velocity
ratio of older mice attained the same levels as those of
the younger mice at 1 day and 7 days. Therefore, these
age-related changes in the arterial system may delay the vascular adaptation observed in our study and may modify the
changes of pressure overload sensed by the old heart. Our
hypothesis pertaining to biochemical and molecular mechanisms of this phenomenon awaits confirmation.
PERIPHERAL VASCULAR RESPONSE AFTER AORTIC BANDING IN AGED MICE
Conclusion
Our data indicate that there is an age-related delay in
the development of vascular adaptations in carotid arteries
after aortic banding. Older mice take longer to adapt and
to establish adequate and equal mean flow velocity in both
carotid arteries.
ACKNOWLEDGMENTS
This work was supported by National Institutes of Health Grants AG17899 (G. E. Taffet) and HL-22512 (C. J. Hartley). Dr. Yi-Heng Li was
a visiting postdoctoral fellow supported by grants from the National
Science Council, Taipei, Taiwan, and the College of Medicine, National
Cheng Kung University, Tainan, Taiwan. The John A. Harford Foundation–AFAR student awards supported Ms. Ochoa’s work on this project.
The authors would like to thank James A. Brooks for his editorial review.
Address correspondence to George E. Taffet, MD, Huffington Center on
Aging, M-320 Baylor College of Medicine, One Baylor Plaza, Houston, TX
77030. E-mail: [email protected]
REFERENCES
1. Rockman HA, Knowlton KU, Ross J Jr, Chein KR. In vivo murine
cardiac hypertrophy: a novel model to identify genetic signaling
mechanisms that activate an adaptive physiological response. Circulation. 1993;87(suppl VII):14–21.
2. Isoyama S, Wei JY, Izumo S, Fort P, Schoen FJ, Grossman W. Effect
of age on the development of cardiac hypertrophy produced by aortic
constriction in the rat. Circ Res. 1987;61:337–345.
3. Boluyt MO, Opiteck JA, Esser KA, White TP. Cardiac adaptations to
aortic constriction in adult and aged rat. Am J Physiol. 1989;257:H643–
H648.
4. Isoyama S, Kuroha M, Sato F, Ito N, Takishima T. Aging effects on
myocardial hypertrophic response and coronary circulation in pressureoverload. Jpn Circ J. 1992;56:482–488.
5. Wei JY. Age and the cardiovascular system. N Engl J Med. 1992;
327:1735–1739.
6. Lakatta EG, Mitchell JH, Pomerance A, Rowe GG. Human aging:
changes in structure and function. J Am Coll Cardiol. 1987;10:42A–
47A.
7. Virmani R, Avolio AP, Mergner WJ. Effect of aging on aortic
morphology in populations with high and low prevalence of
hypertension and atherosclerosis. Am J Pathol. 1991;139:1119–1129.
8. Naritomi H, Meyer JS, Sakai F, Yamaguchi F, Shaw T. Effects of
advancing age on regional cerebral blood flow. Arch Neurol. 1979;
36:410–416.
899
9. Muller M, Schimrigk K. A comparative assessment of cerebral haemodynamics in the basilar artery and carotid territory by transcranial
Doppler sonography in normal subjects. Ultrasound Med Biol.
1994;20:677–687.
10. Forster MJ, Morris P, Sohal RS. Genotype and age influence the effect
of calorie intake on mortality in mice. FASEB J. 2003;17:690–692.
11. Hartley CJ, Reddy AK, Madala S, et al. Hemodynamic changes in
apolipoprotein E-knockout mice. Am J Physiol Heart Circ Physiol.
2000;279:H2326–H2334.
12. Hartley CJ, Taffet GE, Reddy AK, Entman ML, Michael LH. Noninvasive cardiovascular phenotyping in mice. Inst Lab Anim Res. 2002;
43:147–158.
13. Evans DH, McDicken WN. Doppler Ultrasound: Physics, Instrumentation and Signal Processing. 2nd Ed. New York: John Wiley & Sons;
2000:180.
14. Gosling RG, Dunbar G, King DH, et al. The quantitative analysis of
occlusive peripheral arterial disease by a non-intrusive ultrasonic technique. Angiology. 1971;22:52–55.
15. Lindegard KF. Indices of pulsatility. In: Newell DW, Aslid R, eds.
Transcranial Doppler. New York: Raven Press, 1992:67–82.
16. Evans DH, McDicken WN. Doppler Ultrasound: Physics, Instrumentation and Signal Processing. 2nd Ed. New York: John Wiley & Sons,
2000;203.
17. Bishop CCR, Powell S, Rutt D, Browse NL. Transcranial Doppler
measurement of middle cerebral artery blood flow velocity: a validation
study. Stroke. 1986;17:913–915.
18. Kirkham FJ, Padayachee TS, Parsons S, Seargeant LS, House FR,
Gosling RG. Transcranial measurement of blood velocities in the basal
cerebral arteries using pulsed Doppler ultrasound: velocity as an index
of flow. Ultrasound Med Biol. 1986;12:15–21.
19. Dahl A, Lindegaard KF, Russel D, et al. A comparison of trancranial
Doppler and cerebral blood flow studies to assess cerebral vasoreactivity. Stroke. 1992;23:15–19.
20. Berne RM, Levy MN. Cardiovascular Physiology. 8th Ed. St. Louis:
Mosby, Inc.; 2001:216–218.
21. Goyal. Changes with age in the aorta of man and mouse. Exp Gerontol.
1982;17:127–132.
22. Rudner XL, Berkowitz DE, Booth JV, et al. Subtype specific regulation
of human vascular a1-adrenergic receptors by vessel bed and age.
Circulation. 1999;100:2336–2343.
23. Kloss S, Bouloumie A, Mulsch A. Aging and chronic hypertension
decrease expression of rat aortic soluble guanylyl cyclase. Hypertension. 2000:35:43–47.
Received January 6, 2003
Accepted June 10, 2003
Decision Editor: James R. Smith, PhD