Download Effects of Aging on Capillary Number and Luminal Size in Rat

Journal of Gerontology: BIOLOGICAL SCIENCES
2002, Vol. 57A, No. 12, B422–B427
Copyright 2002 by The Gerontological Society of America
Effects of Aging on Capillary Number and Luminal
Size in Rat Soleus and Plantaris Muscles
Yutaka Kano,1 Satoshi Shimegi,2 Hirotaka Furukawa,1 Hirokazu Matsudo,1 and Takudo Mizuta3
Departments of 1Applied Physics & Chemistry and 3Mechanical Engineering & Intelligent Systems, University of
Electro-Communications, Tokyo, Japan.
2Faculty of Health and Sport Sciences, Osaka University, Japan.
To clarify aging-related changes in the capillary network in skeletal muscle, we morphometrically examined the capillary supply to individual muscle fibers and capillary luminal size in
young (3-month-old) and old (19-month-old) male Wistar rats. All morphometric parameters for
capillary and muscle fiber were determined in the cross sections of the perfusion-fixed soleus
(SOL) and plantaris (PL) muscles. The range of fiber size was larger in the old muscles because
of hypertrophy and atrophy of fibers. However, the capillary supply to individual muscle fibers,
assessed as the mean of capillary contacts around a muscle fiber, did not change with aging in
SOL muscle (young rats 7.8 0.4 vs old rats 8.1 0.8) or PL muscle (young rats 6.4 0.3 vs old rats 7.0 0.9). The ratio of individual muscle fiber area to the number of capillary
contacts around a muscle fiber did not differ between young rats (SOL 361.7 76.0; PL 264.7 20.9) and old rats (SOL 350.2 61.3; PL 296.8 44.9). The mean capillary luminal diameter did not differ statistically in young and old rats (SOL, young rats 5.3 0.5 vs
old rats 5.1 0.1; PL, young rats 5.0 0.3 vs old rats 5.4 0.2). In conclusion, the relationship between capillary supply and muscle fiber size is similar for both young and old rats,
and the luminal size of each capillary was maintained with advancing age.
A
quantitative analysis of capillary supply to skeletal muscle is important for understanding the upper limit of the
capacity for delivery of oxygen and substrates to muscle cells.
The size of the capillary supply is determined by both the capillary number and the capillary luminal area. It has been well
documented that the number of capillaries is altered by several
factors, including development, aging, and alteration of muscle activity level such as exercise training and inactivation (1).
There is, however, a contradiction in animal studies for agingrelated change in the number of capillaries, including no
change (2,3), decrease (4,5), and increase (6). Human studies
using a biopsy technique also displayed an inconsistency on
that point, in which capillary supply was not influenced (7,8)
or decreased (9,10) with aging. In those studies, the common
parameters, the numerical capillary-to-fiber ratio and capillary
density, were used to describe muscle capillary number. Because these parameters depend on muscle fiber size, an inconsistency among studies for aging-related change in muscle
capillary number can be attributed to the different extent of
age-related muscle fiber atrophy. Therefore, we took the individual muscle fiber size into account for the analysis of capillary number supply in this study.
Capillary luminal diameter is an important factor in determining the functional potential for peripheral blood flows
and gas exchanges. Recently, we reported that capillary luminal size was decreased in atrophied skeletal muscles in
tail-suspended (hind-limb unweighting) rats (11). This finding leads to the hypothesis that the capillary lumen is
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smaller in the atrophied skeletal muscle of aged rats. However, to our knowledge, no study has examined this point.
The purpose of this study was to examine the age-related
changes of capillary number and luminal size in slow-twitch
(soleus) and fast-twitch (plantaris) muscles.
METHODS
Animals
The studies were performed on two groups of male
Wistar rats, consisting of young rats (3 months old; n 8)
and old rats (19 months old; n 7). All rats were housed in
a temperature-controlled room at 22 2C with a light–
dark cycle of 12 hours and were maintained on rat chow and
water ad libitum. All procedures performed in this study
conformed to the Guiding Principles for the Care and Use of
Animals in the Field of Physiological Sciences (published
by the Physiological Society of Japan).
Fixation and Preparation of Muscle
The rats were weighed and anesthetized with pentobarbital sodium (70 mg/kg1 of body mass). The abdominal
cavity was rapidly opened, and a catheter from a perfusion
apparatus was inserted into the abdominal aorta for perfusion fixation of the soleus (SOL) and plantaris (PL) muscles. The fixation procedure used has been described elsewhere (12). The SOL and PL muscles first were perfused
for 3 minutes with 0.1M phosphate buffer (pH 7.2) contain-
CAPILLARY SUPPLY IN AGED RAT MUSCLES
ing 2000 IU l1 of heparin, 2% procaine, and 0.3mM papaverine. Procaine and papaverine were used to minimize vascular resistance by preventing contraction of skeletal
muscle fibers and vascular smooth muscles. The vena cava
inferior was cut to provide outflow, and the pelvic limb vascular system was flushed with the phosphate buffer. The
muscles were then perfused for approximately 10 minutes
with a 2.5% glutaraldehyde and 2.0% paraformaldehyde mixture diluted 1:1 with 0.1M phosphate buffer (pH 7.2). After
fixation, the muscles were carefully dissected and then
weighted, and the midbelly region was cut transversely to
the long axis of the muscle. The block was postfixed in 1%
osmium tetroxide for 2 to 3 hours, dehydrated through a series
of increasing concentrations of ethanol, and embedded in Epon
812 (Quetol 812; Nishin-EM, Japan). The 1-m-thick sections were prepared by cutting the block in transverse orientation in relation to the muscle fiber axis in a microtome and
stained with 1% aqueous solution of Toluidine Blue.
Morphometric Analysis
The 1-m-thick sections were placed on the stage of a
microscope (Model E800, Nikon, Japan), and the microscopic field was projected to a monitor at a total magnification of 1400. The monitor resolution was calculated as
0.22 m/pixel from measurements made with a graticule. A
capillary was defined as any open vessel smaller than 10
m in diameter, as previously described (12). Each section
was subsampled at randomly selected points (105,000
m2). Capillary density (number/mm2), capillary-to-fiber
ratio, number of capillary contacts around a fiber, muscle fiber area per capillary contacts around a fiber, sharing factor
(the capillary contacts around a fiber/capillary-to-fiber ratio), capillary luminal diameter (in micrometers), and muscle fiber cross-sectional area were directly measured from
images of micrographs projected on a screen. NIH image
1.57 on a Macintosh PC 7300 was utilized to evaluate projected muscle transverse images. The capillary luminal diameter was measured only for capillaries with circular profiles (i.e., a difference between minimum and maximum
diameters of 20%) to eliminate capillaries not sectioned
transversely to the long axis of the vessel, on the basis of
previous studies (13,14). An average of 127 10 (SE) capillaries per sample was measured.
Statistical Procedures
The experimental data are all expressed as the mean SD. We used a two-way analysis of variance (age and muscle). Scheffé’s post hoc test was conducted to evaluate significant interaction effects. Values of p .05 were considered statistically significant.
RESULTS
Muscle Fiber
Light micrographs of transverse sections of SOL and PL
muscles in young and old rats are shown in Figure 1. Compared with that in young rats, the cross-sectional area of individual muscle fibers seems to be heterogeneous in old
rats. In the micrograph of PL muscle in old rats, many fibers
with large area are observed on the right-half side, and small
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and deformed fibers aggregate on the left-half side neighboring the large fibers.
Table 1 shows the body weight and muscle wet weight of
the SOL and PL in young and old rats. In old rats, muscle
weight demonstrated a significant increase (p .001), at
62.2% and 23.2% for SOL and PL muscles, respectively.
The relative muscle-to-body weight ratios significantly decreased in aged PL muscle (p .001), but did not differ between old and young in SOL muscle (Table 1).
The frequency distributions of the muscle fiber area collected in groups of 1000 m2 are given in Figure 2. We used
the coefficient of variation ( SD/mean) to quantify the heterogeneity of muscle fiber area. The coefficient of variation
differed significantly ( p .001) between young and old
rats (SOL, young rats 25.2 4.3% vs old rats 51.1 20.9%; PL, young rats 26.8 3.6% vs old rats 45.1 6.8%). The ranges of muscle fiber area in both SOL and PL
muscles were large in the old rats (SOL, 71.8–7878.1 m2;
PL, 279.4–4500.3 m2) compared with those in the young
rats (SOL, 548.5–5804.4 m2; PL, 295.4–2788.4 m2). In
addition, the proportion of muscle fibers with a small area
(under 1000 m2) increased in the old rats (SOL, 13.6 11.7%; PL, 13.9 8.4%), but this number was much
smaller in the young rats (SOL, 1.0 1.7%; PL, 4.0 4.3%). No significant interaction between age and muscle
was observed for the mean muscle fiber area.
Capillary Supply
Capillary morphometric data are presented in Table 2. No
significant interaction between age and muscle was observed
for capillary morphometric data, with the exception of the
capillary diameter ( p .01) and sharing factor ( p .05).
Capillary supply relative to whole muscle (capillary
density; capillary-to-fiber ratio).—Capillary density did
not differ between the young and old rats in either muscle.
However, a significantly ( p .001) increased capillary-tofiber ratio was indicated in the old rats compared with that
in the young rats (SOL, 20.4%; PL, 34.0%).
Capillary supply relative to muscle fiber (capillary
contacts; fiber area/capillary contact; sharing factor).—
Figure 3 shows that an increase in muscle fiber area is accompanied by an increase in the number of capillaries around a fiber. The number of mean capillary contacts around a fiber
was lower in PL muscle than in SOL muscle ( p .001).
However, fiber area/capillary contact was larger in SOL muscle than in PL muscle ( p .001). Aging increased the mean
capillary contacts around a fiber 3.8% in SOL muscle and
9.4% in PL muscle ( p .067). Consequently, fiber area/capillary contact did not differ between young and old rats.
A significant interaction effect between age and muscle
was observed for the capillary sharing factor. The capillary
sharing factor of old rats was lower than that of young rats
for SOL and PL muscles ( p .001).
Capillary Luminal Diameter
Frequency distributions for the capillary luminal area of
all groups are illustrated in Figure 4. There was no difference in the mean capillary luminal diameter between the two
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KANO ET AL.
Figure 1. Light micrographs of transversely sectioned perfusion-fixed muscle for soleus (SOL) and plantaris (PL) muscles in young and old
rats (bar 100 m).
age groups and between muscle types (Table 2). The range
and distribution of the capillary luminal size is similar for
both SOL and PL muscles.
DISCUSSION
There were two major findings in this present study.
First, muscle capillary supply is tightly coupled to individual muscle fiber size in both young and old rats, despite the
fact that the range and distribution of muscle fiber size is
larger in muscles of old rats. Second, the capillary luminal
diameter was maintained in old rats. To our knowledge, previous studies have made few quantitative analyses of capillary luminal size in old rats.
Muscle Fiber
We found no statistical difference in the mean muscles fiber cross-sectional area of PL and SOL muscles between
the young and old rats. Brown and Hasser (15) observed
that the mean muscle fiber area remained relatively constant
in rats until after the age of 28 months. Though a similar result was observed in 19-month-old rats in this study, a
change of muscle fiber area with aging must be considered
because it has been indicated that the range of the fiber
cross-sectional area was larger in the old muscles as a result
of an increased number of both hypertrophied and atrophied
fibers (16,17). Lexell and Taylor (16) suggested that the ap-
pearance of hypertrophied fibers was mainly the result of a
compensatory response to the reduction of the total number
of muscle fibers with aging. Furthermore, age-associated
selective muscle fiber atrophy occurred in type II fibers
rather than in type I fibers (16,18). The appearance of atrophied and hypertrophied fibers was observed in the muscles
of old rats, which is a characteristic feature of the early
stage of aging, so that fiber size was distributed in a wider
range in old animals than in young ones.
Capillary Supply
The relationship between the change of capillary supply
and aging was not clear. In human studies, muscle capillary
Table 1. Body and Muscle Weights for SOL and PL Muscles in
Young and Old Rats
Weight
Body (g)
Muscle (mg)
SOL
PL
Muscle:body (mg/g)
SOL
PL
Young
Old
351.1 14.2
584.9 72.2
113.7 7.4
287.6 18.1
184.4 24.7
354.4 34.8
0.32 0.02
0.82 0.04
0.32 0.04
0.60 0.08
Notes: Values are means SD. SOL soleus; PL plantaris.
CAPILLARY SUPPLY IN AGED RAT MUSCLES
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Figure 2. Histogram showing the distribution (%) of muscle fiber
cross-sectional area for soleus (SOL) and plantaris (PL) muscles in
young and old rats. Results are expressed as means SD.
supply has been reported to be maintained (7,8) or reduced
(9,10) with advancing age. In addition, results of animal studies conflict, with both maintained (2,3) and decreased (4,5)
capillary supply in relation to aging being reported. Recently,
Davidson and colleagues (6) reported that the capillary density and the capillary-to-fiber ratio in SOL and extensor digitorum longus muscles were significantly greater in older mice
than in young mice. The differences between these findings
may be due to methodological constraints with subjects. Coggan and coworkers (9) indicated that adaptation of muscle
capillarization to training appears to be very sensitive in old
subjects. Moreover, these results may depend on how the
capillary morphological data are expressed. Capillary density
and capillary-to-fiber ratio are widely used and accepted for
muscle capillary quantification. In many cases, however, capillary density does not reflect the change in capillary numbers; for example, an increase in capillary density may reflect
the atrophy of muscle fibers rather than capillary development, or there may be no change in capillary density if both
muscle fiber size and capillary supply number are reduced
with aging. Likewise, although the capillary-to-fiber ratio,
determined as the ratio of capillary number to muscle fiber
number/unit area, is relatively independent of muscle fiber
size, it does not adequately explain the change in the capillary-to-muscle fiber relationship of individual muscle fibers
(19). In this study, capillary density did not differ between the
young and old rats for either muscle type. However, a significantly increased capillary-to-fiber ratio was indicated in the
old rats compared with that in the young rats. In the present
study, these results might be explained by the difference in
degree of distributions of muscle fiber size between young
and old rats (Figure 2).
Figure 3. Diagram showing the relation between the number of
capillaries around a fiber and muscle fiber area. Each point represents the mean value of all fibers having a cross-sectional area within
a 1000-m2 interval. Results are expressed as means SD. SOL soleus; PL plantaris.
A more direct parameter in determining capillary supply
to individual muscle fiber is derived from counting the
number of capillary contacts around a fiber (20). The
present study showed that the mean of capillary contacts
around a fiber did not change with age, and that regardless
of the age (young or old rats) or the muscles (SOL or PL
muscle), large fibers are surrounded by more capillaries
than are small fibers (Figure 3). The relationship between
capillary supply and muscle fiber size is similar for both
slow-twitch (SOL) and fast-twitch (PL) muscles. Degens
and colleagues (21) indicated that the capillary supply to a
muscle fiber is determined by its size, and also the muscle
fiber type. In contrast, Ahmed and coworkers (22) indicated
that capillary supply is scaled according to muscle fiber
size, and is independent of muscle fiber type in human skeletal muscle. The results of the present study suggest that the
muscle capillary supply is tightly coupled to muscle fiber
size, rather than to fiber type. Furthermore, the present
study indicates that this coupling between capillary supply
and muscle fiber size is maintained in the skeletal muscle of
old rats.
Traditionally, capillary density and capillary-to-fiber ratio in muscle transverse sections have been used as a functional index of capillarization, although these parameters do
not always reflect the structural capacity for oxygen supply
to the muscle fiber (23,24). Chilibeck and colleagues (25)
reported that O2 uptake kinetics is more strongly related to
capillarization/fiber area (number of capillary contacts per
Table 2. Muscle Fiber and Capillary Morphometric Data for SOL and PL Muscles in Young and Old Rats
SOL
Data
Muscle fiber area (m2)
Cap. diameter (m2)
Cap. density (cap./mm2)
Cap. fiber ratio
Cap. contacts
Fiber area/cap. contact (m2)
Sharing factor
PL
Young
Old
Young
Old
Age
(p)
Muscle
(p)
Interaction
(p)
2755.2 682.7
5.3 0.5
1293.0 170.7
3.43 0.34
7.8 0.4
361.7 76.0
2.22 0.17
2729.0 575.7
5.1 0.3
1439.4 158.7
4.13 0.44
8.1 0.8
350.2 61.3
1.97 0.12
1594.1 91.0
5.0 0.3
1491.4 150.6
2.44 0.20
6.4 0.3
264.7 20.9
2.63 0.11
2022.0 353.7
5.4 0.2
1377.0 236.7
3.27 0.41
7.0 0.9
296.8 44.9
2.15 0.16
NS
NS
NS
.001
NS
NS
.001
.001
NS
NS
.001
.001
.01
.001
NS
.01
NS
NS
NS
NS
.05
Notes: Values are means SD. The p values refer to significant main effects identified by a two-way analysis of variance; NS not significant.
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KANO ET AL.
Figure 4. Histogram showing the distribution (%) of capillary luminal diameter (circular profiles only; i.e., 20% difference between minimum and maximum diameter) for soleus (SOL) and plantaris (PL) muscles in young and old rats. Results are expressed as
means SD.
fiber/fiber area) than to capillary density and capillary-tofiber ratio. Therefore, capillarization/fiber area is a better
parameter of O2 delivery to muscle fiber. In the present
study, SOL and PL muscle of old rats had a higher capillaryto-fiber ratio and sharing factor, but the fiber area/capillary
contact did not differ from that of young rats. As the capillary supply for a different fiber size (i.e., fiber area/capillary
contact) did not change with increased aging, the structural
capacity for O2 delivery to muscle fiber might be maintained in old rats.
Capillary Luminal Diameter
Some research has indicated that the size of capillary lumens in young rats is not homogeneous (11,12,26). Takahara and coworkers (26), using the microcorrosion casts
method, reported capillary diameters ranging from 3.1 m
to 13.2 m. Our previous studies, using the perfusion fixation method, indicated capillary diameters ranging from 2
m to 10 m. (11,12). The present data correspond well
with the values obtained from previous studies (Figure 4).
We have reported that capillary luminal size decreases in atrophied skeletal muscles of tail-suspended rats; that is, there
is reduced mechanical stress on hind limbs (11). The narrower capillaries in the atrophied muscle could be related to
an increase in blood-flow resistance. We hypothesized that
the capillary lumen is smaller in skeletal muscle of older
rats. The present results do not support this hypothesis. In
this study, the mean of capillary luminal diameter did not
statistically differ among the groups. In addition, the range
and distribution of the capillary luminal size are similar for
young and old rats. These data indicate that the capillary luminal area is stable with aging. These different findings between the tail-suspended and the older rats may be related to
the hemodynamics of locomotory muscles. McDonald and
coworkers (27) reported that blood flow to the antigravity
SOL muscle was reduced during hind-limb suspension. In
contrast, resting blood flow is maintained in skeletal muscle
of old rats (28). It has been suggested that capillary growth
is initiated by mechanical factors related to blood flow (29).
Therefore, it is likely that variable blood flow contributes to
the morphological changes of capillary lumens.
It was reported that resting blood flow is minimally affected by age, but blood flow during, or following, exercise
is generally reduced (30,31). Irion and colleagues (31) indicated that a reduction in exercise hyperemia contributes to
increased muscle fatigability in older male rats (24 months).
In humans, muscle blood-flow capacity was shown to decline with age in healthy subjects (32,33). The muscle
blood-flow capacity was mainly associated with both the
functional aspects, such as vasodilatory capacity in arteries
or arterioles, and the anatomical aspects, such as resistance
vessels or capillary supply. Several studies have demonstrated that the relaxation response to acetylcholine (ACh)
and nitric oxide (NO) in isolated blood vessels of rats decreases with aging (34–36). This finding suggests that the
function of the endothelium-dependent and endotheliumindependent vasodilation declines with age. However, an
age-related decrease in microvasculature is not a universal observation. Cook and colleagues (37) reported that the maximal diameter of arterioles did not differ between young rats
(12 months) and old rats (24 months). In this study, both
capillary numbers and luminal diameter were maintained in
skeletal muscle of old rats. Therefore, the age-related decrease in blood flow potential may be due to a decreased
functional resistance, rather than to morphological changes
in microvascular bed.
In conclusion, the relationship between capillary supply
and muscle fiber size is similar for both young and old rats,
and it is relatively independent of muscle type. Furthermore, the luminal size of each capillary was maintained
with advancing age.
Acknowledgments
This study was supported in part by Grant-in-Aid 10780013 for Scientific Research from the Japan Society for the Promotion of Science.
We gratefully acknowledge Dr. S. Kuno at the University of Tsukuba for
technical support.
Address correspondence to Dr. Y. Kano, PhD, Department of Applied
Physics and Chemistry, University of Electro-Communications, Chofu,
Tokyo 182-8585, Japan. E-mail: [email protected]
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Received April 23, 2002
Accepted September 3, 2002
Decision Editor: James R. Smith, PhD