Mechanisms of Ageing and Development, 58 (1990) 21--35 Elsevier Scientific Publishers Ireland Ltd.

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A COMPARISON OF SKELETAL MUSCLE M O R P H O L O G Y W I T H TRAINING BETWEEN YOUNG AND OLD FISCHER 344 RATS

MARY L. M I T C H E L L , W I L L I A M C. BYRNES and ROBERT S. M A Z Z E O Department of Kinesiology, University of Colorado, Boulder, CO 80309 (U.S.A.) (Received July 12th, 1990) (Revision received September 10th, 1990)

SUMMARY

Muscle mass, fiber area, total fiber number, fiber population and capillarity were assessed in 6- and 25-month-old animals trained by treadmill running at 75°70 mean maximal capacity for 10 weeks. Serial cross-sections were stained for ATPase activity to differentiate fiber types and expose capillaries. Aging and training effects were demonstrated in maximal running speed and endurance running time. Soleus muscle mass increased in the aged, however, soleus and EDL total fiber number were declining. When adjusted for muscle weight, a significant reduction existed in fiber number for the aged soleus. Fiber area increased in the old soleus compared to young. All changes in the EDL were specific to the deep region. While there was a tendency for the capillaries/fiber and fiber area to be higher with training in the Type I fibers of the soleus, it was only significant for the young animals. Thus, while the majority of variables that showed a training effect did so across both age groups, this was not the case for vascularity. Age-associated changes in muscle morphology appear to be both muscle and fiber specific.

Key words: Exercise; Aging; Histochemistry; Capillarization

INTRODUCTION

During middle and old age, a prominent loss in working capacity in mammalian skeletal muscle is characterized by a decline in muscular strength [1--5]. Research documents decreases in muscle mass with increasing age, consequently, the relationCorrespondence to : Robert S. Mazzeo, Ph.D., Department of Kinesiology, Box 354, University of Colorado, Boulder, CO 80309, U.S.A.

0047-6374/91/$03.50 Printed and Published in Ireland

© 1991 Elsevier Scientific Publishers Ireland Ltd.

22 ship between losses in muscular strength and mass is a critical issue in aging studies [1,2,4,5]. The decline in muscle mass and muscle strength associated with the aging process may be a consequence of the decrease in fiber area a n d / o r fiber number. Aging studies with humans and animals have reported a decrease in the cross-sectional area of the muscle fibers [1,2,6--9] as well as the number of fibers [1,6,8,9-13]. Although it has been established that fiber area decreases in the fast-twitch muscle fibers (Type II) with increasing age, losses within the slow-twitch (Type I) population remain controversial [2,8,14 16]. Total fiber number has been shown to remain constant throughout life, uniformly decline or selectively decrease with age. Several investigations employing number counts report that a constant number of muscle fibers exists throughout life [2,6,17], while others claim a comparable agerelated decline between representative fast-twitch and slow-twitch muscles [9,11,18]. Other studies support a selective decline specific to the fast-twitch muscle fibers [ 11]. Studies concerned with age-associated fiber population changes include three theories; (1) a transformation from one type to another, (2) atrophy of specific muscle fibers and (3) a relatively constant muscle fiber population [1,3,10,14,19,20]. Vascularization directly influences functional capacity, therefore, changes in capillarization is another important factor of aging. Measurement of capillary density and the number of capillaries in relation to each fiber area (Type I, Type lla and Type lib), show no definitive changes with age [1]. Unfortunately, little is known regarding the influence of endurance training on vascularization of skeletal muscle in aged populations. Attempts are being made to understand which functional losses in skeletal muscle are specific to the aging process and which changes occur as a result of disease a n d / or inactivity. It is generally accepted that endurance training improves functional capacity during old age [1,2,21--24]. However, the underlying mechanisms responsible for enhanced physical capacity in elderly individuals may differ from those found in their younger counterparts. The purpose of this study was to examine morphological age-related changes in skeletal muscle of young and old Fischer 344 rats and to determine if endurance training attenuates any age-associated declines in skeletal muscle. METHODS

Animals Thirty female Fischer 344 rats were obtained from the National Institute of Aging at the ages of 3 and 22 months. The animals were individually housed in a climate (22°C) and light cycle controlled (12 h light, 12 h dark) room. They were provided purina rodent chow and water ad libitum.

Training Based on an initial graded-exercise test (GXT) the animals were pair-matched into

23 trained (TR) and untrained (UN) groups. Training consisted of treadmill running at 75°70 mean maximal capacity with a constant gradient df 15°70, 1 h/day, 5 days/week for a period of 10 weeks. Initially, the animals ran 10 min at 26.8 and 16.1 m / m i n for the young and old animals, respectively. Running time was increased 5 m i n / d a y until they reached 1 h/day. Thereafter, the time was held constant while the speed was increased 3 m / m i n every 2 weeks to maintain an intensity of 75%. This increase was based on previous research documented to improve VO 2 max and running capacity in rats of similar ages [25]. To familiarize the sedentary animals with treadmill running, the control groups ran 10 min/week. At the end of the training session a second G X T and an endurance test were performed. The endurance test was run at 75% of the initial maximal running speed for each age group and exhaustion was recognized as the animals loss of the righting reflex.

Dissection Each group was killed at least 24 h after their endurance test. Prior to dissection, animals were anesthetized with Nembutal (50 m g / k g body wt.). The skin was removed from the hind leg followed by the removal of the soleus and the extensor digitorum longus (EDL), at their origin and insertion. Immediately following removal, excess connective tissue was dissected free. The muscle was then weighed and frozen in isopentane, cooled in liquid nitrogen. Samples were stored at - 6 0 ° C until histochemica! analysis. Water content of muscle was determined from wet and dry weights.

Histology Muscle samples were allowed to equilibrate at - 20°C inside a Tissue-Tek cryostat before mounting and cutting. Transverse serial sections, 10 pm thick, were collected with glass coverslips and dried at r o o m temperature prior to staining. In order to differentiate fiber types and to expose the capillaries serial cross-sections were stained for ATPase activity with the technique of Brooke and Kaiser at p H 4.3, 4.5 and 4.7 [26] and the technique of Tunell and Hart [27], respectively.

Computer analysis The Zidas Image Analysis System (Carl Zeiss) was used to calculate total fiber number, fiber area, the percentage of each fiber type (Type I, Type IIa and Type IIb), the number of capillaries/fiber and the number of capillaries/fiber area for each muscle. Superficial and deep regions of the EDL were analyzed separately.

Statistics Data are reported as mean _ S.E. Mean differences between the young and old as well as the trained and untrained groups were analyzed with a 2 x 2 fixed effect analysis of variance. Significance was defined as P < 0.05.

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RESULTS

Body weight Over the 10-week training period total body weight gradually declined in both TR and UN old animals, while body weight steadily increased in both young groups. The old animals lost an average of 38.0 ___ 6.4 g and 28.0 _.+ 5.6 g for the TR and UN groups, while the young TR animals gained 56.6 __. 4.8 g and the young UN gained 48.6 _+ 2.3 g. Mean body weight at the end of the training session is shown in Table I. The old animals weighed significantly more than the young with no differences between TR and UN animals in either age group.

Training effects This training regimen has been previously documented to increase maximal oxygen consumption and running capacity in female Fischer 344 rats of similar ages [25]. The training effects in the present study are comparable to those previously reported. Differences in maximal running speed and endurance time indicate an aging as well as a training effect. Peak running speed improved significantly with training by 63070 and 82°/0 for the young and old animals, respectively. Endurance time followed a similar pattern as maximal running speed where the young groups endurance capacities were significantly greater than the old groups and the TR animals ran significantly longer than the UN for both age groups. The young and old TR animals improved their endurance time by 987070 and 955°70 over the UN groups.

TABLE I MEAN TOTAL BODY AND MUSCLE WEIGHT

Body wt. (g)

Muscle wt. (rag) Soleus

Muscle wt./body wt. (rag~g) EDL

Soleus

EDL

86.3 ± 1.3

9 0 . 4 ± 1.4

0.45 ± 0.01

0 . 4 6 ± 0.01

95.5 ± 5.2

7 0.47 ± 0 . 0 2 8

7 0 . 4 4 ± 0.01

0.38 ± 0.02* 7 0 . 4 0 ± 0.02* 6

0.33 ± 0.02* 7 0.35 ± 0.02* 6

Young UN

196.0 ± 1.6

n TR n

7 203.0 ± 4.2 8

7 8

7 89.3 __- 3.3 8

2 7 1 . 7 ± 4.0* 7 2 5 6 . 4 ± 5.9* 8

102.7 ± 2.9* 7 104.5 ± 4.0* 6

90.3 ± 4.5 7 91.7 ± 4 . 0 6

8

Old UN n TR n

Values are mean ± S.E. *Significantly different from y o u n g animals ( P

A comparison of skeletal muscle morphology with training between young and old Fischer 344 rats.

Muscle mass, fiber area, total fiber number, fiber population and capillarity were assessed in 6- and 25-month-old animals trained by treadmill runnin...
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