Resistance exercise effects on aging skeletal muscle in rats.

Resistance Exercise Effects on Aging Skeletal Muscle in Rats A need exists for therapeutic exercise, particularly resistance exercise, for older men and women to alleviate the debilitating effects of bed rest and surgery and, for the healthy older adult, to enhance functional capacity particularly for activities of daily living. Physical therapists routinely prescribe resistance exercise for older patients; however, a paucity of data exists to support the use of resistance exercise for the geriatric population.

Afew investigators have introduced resistance exercise to humans at older ages and report moderate improvements in muscle strength. Larsson put five men (mean age = 59.2 years) on a quadriceps femoris muscle strengthening program twice a week for 15 weeks and found a 7.5% increase in isometric torque capability.[1] In addition to the muscle strength gains, Larsson found a reversal of age-associated quadriceps femoris muscle atrophy postexercise. Moritani and deVries also reported an increase in muscle strength following a three-times-a-week strengthening program for the elbow flexors.[2] The male subjects in this eight-week study averaged 69.6 years of age. I also have found improvements in knee flexion and extension and ankle plantar flexion and dorsiflexion torque in 60- to 70-year-old men and women following a three-month low-intensity activity program. Isometric muscle strength increases ranged from 8% to 13% (unpublished research).

In each of these studies, the subjects were relatively young, which provided little insight as to how an 80- to 90-year-old individual might react to muscle strengthening activity. Aniansson and colleagues did use older subjects (63-84 years of age) for their study of exercise effects on muscle strenth measures.[3] Unfortunately, the authors did not indicate the proprotion of strength improvements (6%-13%) made by the 60- to 70-year-old participants versus those made by the 70- to 80-year-old or 80- to 90-year-old participants.

Liemohn suggests that humans become less adaptable to muscle strength training with advancing years.[4] Liemohn's exercise participants ranged in age from 42 to 83 years. Fifty-two men, all Veterans Administration Medical Center residents, participated in a six-week strengthening program for the elbow flexors and extensors and knee flexors and extensors. Subjects in the 71- to 80-year-old age group had no significant improvements in strength. Subjects in the 41- to 50-year-old age group, however, significantly increased strength in six of eight categories. Fewer improvements occurred as subjects got older, suggesting decreased strength trainability with advancing age.[4]

Studies of exercise and aging using animals have yielded conflicting results. Stebbins et al introduced treadmill exercise to rats 21 months of age and found, after a five-month running program, that exercised animals had fewer small-diameter fibers in the gastrocnemius muscle than age-matched controls.[5] This results suggests that the exercise helped maintain muscle fiber integrity in the aging gastronemius muscle. Tomanek and Woo also reported favorable results from their study of compensatory hypertrophy in rats.[6] They denervated the gastrocnemius muscle of 45-day-old and 19-month-old rats to induce compensatory hypertrophy of the plantaris muscle. After 15 weeks, which included 12 to 13 weeks of treadmill walking, fiber diameter increases for young and old animals were comparable (12.6% vs 13.1%, respectively). Results from these two studies imply a positive adaptation of an aging muscle to physical activity.

Conversely, Drahota and Gutmann found no compensatory hypertrophy in muscles from old rats when compared with your rats.[7] Added resistance was imposed on the extensor digitorum longus muscle in 1-month-old and 2-year-old rats. Two weeks later, the muscles were removed, and muscle nitrogen assessed. Total muscle nitrogen in the young rats increased 29.7%, whereas no change occurred in the older animals, suggesting an inability of old muscle tissue to respond to an exercise stimulus.

The short length of exercise time used by some investigators may explain why their human or animal subjects failed to show improvements in strength or muscle enlargement with exercise. Drahota and Gutmann[7] did not observe compensatory hypertrophy in their old rats, whereas Tomanek and Woo[6] found compensatory hypertrophy in their old rats to be comparable to that occurring in their young animals. The difference might be that Drahota and Gutmann[7] followed their animals for only twoo weeks; Tomanek and Woo[6] followed theirs for 15 weeks. Liemohn exercised his older male subjects for 5 weeks.[4] Perhaps, if the exercise period had been 10 to 15 weeks, significant improvements might have been found for the oldest group, which showed little or no change in muscle strength.

Some investigators have concluded that exercise is detrimental, if introduced after a certain age.[8-10] These researchers suggest that a "threshold of aging" exists after which the introduction of exercise becomes harmful. Steinhagen-Thiesen and colleagues put mice 6, 22, and 27 months of age on a five-week treadmill-running program. Mice 6 and 22 months of age showed an increase in hind-limb muscle weight in response to the exercise, whereas the 27-month-old animals lost muscle weight.[8] Silbermann and co-investigators found a higher proportion of muscle fibers displaying central nuclei, atrophy, and lipofuscin in 27-month-old rats that treadmill exercised for 10 weeks when compared with age-matched controls.[9] McCafferty and Edington conducted a 10-month treadmill exercise program for rats beginning at ages 3, 5, 10, 15, and 20 months. The younger animals gained muscle weight with the exercise. The 15-month-old rats had no increase in muscle weight, but the oldest animals (aged 20 months) showed a decrease in muscle wet weight.[10] The question of why the exercise was harmful was not addressed by the investigators in these studies.

Based on previous investigations, there was a question of whether muscle strengthening exercise for humans or animals well advanced in age was appropriate. Consequently, the purpose of this study was to determine whether a resistance exercise program could be beneficial when initiated at progressively older ages in rats. The hypothesis was that resistance exercise can promote skeletal muscle cell growth as evidenced by an increased in muscle weight and fiber size in an aging or aged rat. The null hypothesis was that the exercises given would have no effect on skeletal muscle in old rats. The alternate hypothesis, that the resistance exercise as given would be harmful, was also considered.

Method

Animals

Sixty female Sprague-Dawley rats were ordered from a rat colony.(1) These retired breeders were about 10 months of age when purchased and remained at the breeding farm until age-appropriate. When the rats were age-appropriate (18, 21, 24, and 27 months), they were delivered to the university (15 in each shipment) and housed in a vivarium where temperature (22[degrees]C) and hours of light and darkness were controlled (12/12). Purina rat chow and water were available ad libitum.

After arrival, the rats were assigned randomly to control (n = 5) or exercise (n = 10) groups. All animals were handled twice daily and given peanut butter. All animals were weighed biweekly (Tab. 1).

Rats in this study ranged in age from 21 to 30 months at the end of the exercise program. A 21-month-old rat is at the threshold of old age, being roughly equivalent to a 60-year-old human. A 30-month-old rat is quite advanced in age, comparable to an 80-year-old person.[11] The average half-life for a female Sprague-Dawley rat colony is 30 months (ie, half of the rats in a colony will have died before age 30 months).[12]

Rats were chosen for study for three reasons: 1) Variables such as diet, stress level, and environment can be controlled; 2) exercise and aging effects for rats and humans are similar[13]; and 3) the rat life span is relatively short, and aging effects occur more rapidly than in humans.

Surgery

Two weeks before the experimental period began, each rat underwent surgery for removal of the left palmaris longuns (PL) muscle. This small muscle was removed to permit study of skeletal muscle both before and after the exercise program. The controls permitted study of an aging muscle that was not exercised. The PL muscle was selected because of its small size and because its elimination did not hamper a rat's ability to use its wrist flexors effectively (personal observations).

Before initiating this study, a pilot study of 15 rats was conducted to determine 1) which wrist flexor could be removed without severely reducing the muscle mass available and 2) whether bilateral PL muscle comparisons, based on weight and fiber-type distribution, could be made. The validity of using bilateral comparisons was established by Gollnick et al for some rat lower extremity muscles.[14] For this investigation, the pilot study verified that the PL muscle was present in all animals and that bilateral variations in weight and fiber-type distribution were not significant.

The selection of a wrist flexor muscle for study was made after watching rats' climbing behavior and after dissecting 15 arms and forearms. When rats pull themselves up onto a higher surface, they pronate their forearms, grasp, and pull; wrist flexion motion is obvious. Involvement of the PL muscle in the chin-up activity, however, cannot be verified because the muscle is too small for electromyographic study.

To remove the PL muscle, each rat was first anesthetized with halothane. The skin over the forearm flexor muscle was shaved, and an incision was made with a sterile scalpel from the wrist to approximately three quarters of the distance to the elbow. The PL muscle was isolated, removed, weighed, and frozen in liquid nitrogen. The rat forearm was immediately stitched, and the anesthesia turned off. Each surgery took seven to eight minutes to complete, and no rats were lost during this procedure. Typically, rats were walking normally and using their front paws to hold food within half an hour of the surgery. All wounds healed well, without infection.

After the three-month exercise program was over, rats were killed with an intraperitoneal injection of sodium pentobarbital. The right PL muscle was removed at this time and frozen using the same procedure described above.

Exercise Protocol

Each exercised rat performed resisted chin-ups (forearms pronated) five days a week. To accomplish the task, each rat was suspended over the edge of a table; the animal was holding onto the lip of the tabletop with both paws but with the elbows extended (Fig. 1). For one chin-up to be included in the count, rats had to pull themselves up onto the tabletop through full range motion with maximum resistance, which was manually applied through the tail (ie, each time the rat pulled up I pulled down). Resistance was graded throughout the ROM less at the beginning and end of the motion and more in the midranges as in propr ioceptive neuromuscular facilitation. It was not possible to quantify the amount of resistance tolerated by each animal.

The first two or three days rats were trained to perform chin-ups repetitively; resistance was added after the third day. Rats performed 10 repetitions, rested for three to five minutes, and then performed another set of 10 repetitions. Three sets of 10 repetitions were performed every morning and afternoon, for a total of 60 repetitions a day. Animals were given peanut butter as a reward when all repetitions were performed. One to two weeks of exercise were required before rats were capable of performing all 60 repetitions. Resistance was added to tolerance during the three-month exercise period.

Differences in ability to tolerate resistance did not seem to differ among groups; rather, differences were observed among animals. The length of time required to reach maximum tolerable resistance appeared greater for older animals. For the 21- and 24-month-old rats, resistance was gradually increased for a period of six to seven weeks; after that period of time, the rats seemed unable to tolerate additional resistance. Older rats took longer, eight to nine weeks, to reach a plateau after which no additional resistance could be tolerated (subjectively determined). Whether rats were incapable of tolerating added resistance or simply unwilling to respond to additional challenge could not be discerned.

To obtain a relative index of performance capacity at the end of the exercise protocol, each rat was required to perform a maximally resisted chin-up on a postage scale. During the chin-up, the postage meter gave a fleeting readout in grams that was indicative of the amount of resistance each rat was handling. These data were not very precise, but reflected that rats were lifting approximately 1-1/3 to 1-3/4 times their body weight during each chin-up. Rats began the exercise program at ages 18, 21, 24, and 27 months. At completion of the program, the rats' ages were 21, 24, 27, and 30 months, respectively.

Tissue Preparation

Within two or three minutes after removal, each PL muscle was fixed at an in-situ length, coated with talcum powder, frozen in liquid nitrogen, and placed in a freezer at -80[degrees]C. During tissue processing, each muscle was placed in a cryostat (-20[degrees]C), mounted on a chuck with OCT[R] (ornithine carbamoyltransferase), and sectioned transversely at 8 [mu]. Serial sections were stained with adenosine triphosphatase (ATPase) (pH 9.4, 4.65, 4.3),[15] nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR),[16] and hematoxylin and eosin (H&E)[17] for determination of muscle fiber types and routine morphology.

Data Management

Photomicrographs (100 X) were taken of each PL muscle in cross-section. Photographed tissue sections had been stained with ATPase at a pH of 9.4, which permitted visualization of muscle fiber types I and II. Subclassification of type II fibers into types IIa and IIb was not possible in almost half of the older control animals. Thus, complete data could be obtained only for fiber types I and II. Using a Hewlett-Packard Model HP-85 digitizer(2) and a Model 9111A graphics tablet,(2) area measurements of an average of 55 type I (almost all of the type I fibers present in the PL muscle) and 175 type II fibers (one third to one half of the total number present) were made for each animal. Fibers to be measured were selected randomly. Digitizing is a highly reliable method of area measurement, accounting for [is less than or equal to] 1% of the variability associated with area measurements (personal observation).

Data Analysis

A one-way analysis of variance (ANOVA) was performed to determine significant differences (p [is less than] .05) in PL muscle wet weight over time (age) before and after the three-month period of intervention. The ANOVA was also used to detect significant differences over time in muscle fiber area measurements for the control and exercised animals. A two-way ANOVA was used to determine differences between the control and exercised rats for body weight, PL muscle wet weight, and fiber area measurements at each age. A Scheffe post hoc test was performed if significance was apparent. Wet weights for PL muscles removed before the three-month intervention period were compared with wet weights for PL muscles removed after the three-month period using a paired (two-tailed) t test. The paired t test was also used to compare are measurement for fiber types I and II in PL muscles removed before and after the three-month intervention period for both control and exercised rats.

Results

Results are based on data obtained for 34 exercised rats and 20 controls. Three rats died within 48 hours after their arrival at the animal facility, 2 rats refused to exercise and were eliminated from study, and 1 animal became so ill (appeared to have a stroke) that it was removed from the exercise protocol. Thus, the exercise groups by age were as follows: 21 months ( = 10), 24 months (n = 9), 27 months (n = 8), and 30 months (n = 7).

Body Weight

Most control rats gained body weight during the period they were followed. Rats performing the chin-up protocol lost body weight during the three-month exercise period. The weight loss for exercised rats average 13 g (Tab. 2). Thirty-month-old exercised rats had had significantly lower body weights postexercise when a comparison with age-matched control was made postexercise.

Muscle Wet Weight

The control animals' PL muscle weight remained essentially unchanged when the muscles removed before the three-month period that the animals were followed were compared with the muscles removed after the three-month period (Tab. 3). Three of four groups of exercised rats showed an increase in muscle weight postexercise (range = 20%-50%); only the 50% increase was significant (p [is less than] .01) for the 24-month-old animals (Tab. 3). The increases in PL muscle weight for exercised rates occurred despite a concomitant loss of body weight during the three-month exercise period.

As animals aged. PL muscle weights tended to decrease. The loss of muscle weight with age was not statistically significant with one exception; the pre-exercise PL muscle wet weight for the 30-month-old exercised rats was significantly less than that of the 21-month-old exercised animals (Tab. 3).

The increase in wet weight was variable, with fewer of the oldest animals showing an increase. Seven of the 21-month-old rats, 7 of the 24-month-old rats, 2 of the 27-month-old rats, and 5 of the 30-month-old rats showed an increase in wet weight of 5 mg or more when pre-exercise values were compared with those obtained postexercise. Eight of the exercised rats (23%) had muscle weights at least 5 mg less than pre-exercise values, and 5 showed no change in muscle wet weight postexercise.

Muscle Fiber Area

Type I muscle fiber area did not change with advancing age in either the control or the exercised animals. The variability in type I fiber size was considerable. A trend toward type I fiber enlargement with exercise was seen, but a significant increase in type I fiber area occurred with exercise only for the 27-month-old exercised rats (Tab. 4). Type I fibers represent between 1.5% and 6% of the total in the rat PL muscle.

No significant aging effect was found for either control or exercised rats based on type II muscle fiber area measurements (Tab. 5). Comparisons of left and right type II PL muscle fibers (ie, comparisons of muscles removed before and after the three-month period of study) did not reveal a significant decrease in fiber area with aging in the control animals. The resistance exercise program resulted in a significant type II muscle fiber size increase for three of the four exercise groups (Tab. 5, Fig. 2). Fiber size increases ranged from 10% for the 27-month-old rats to 46% for the 24-month-old animals (p [is less than] .01). Twenty-two of the exercised rats (65%) had an increase in type II fiber size of 200 [micrometer]m.sup.2 or more (average variability among controls). Eleven animals had muscle fiber areas that were unchanged with exercise. In contrast to values for muscle wet weight only, 1 exercised rat showed a decline in type II muscle fiber area.

Histological Observations

There was no evidence to suggest that the exercise as given did any harm to the muscles studied. Degenerating and regenerating fibers, loss of striations, groups of fibers with central or pyknotic nuclei, loss of cytoplasmic staining, and large numbers of macrophages (indicators of muscle damage) were not seen in muscle cross-sections from rats that performed resistance exercise.

Within the tissues studied, occasional encircled fibers and fibers with central nuclei were found in both groups. A tendency for increased intramuscular fat with advancing age was seen in cross-sections from both control and exercised rats.

Discussion

This study provided evidence to support the introduction of resistance exercise for aging or aged rats. The primary hypothesis of this study, that resistance exercise could result in muscle cell growth, even in old age, was supported because three of the four exercise groups had significant increases in type II muscle fiber size. Limited support for the null hypothesis also was found because some animals showed little or no skeletal muscle change with the exercises given. Minimal support was obtained for the hypothesis that exercise in old age is harmful.

Three of the four exercise groups showed significant increases in muscle fiber size with the exercise protocol. Why the 27-month-old group failed to show significant change is unclear. One possibility is that the exercise intensity was not sufficient for the 27-month-old group of animals. Although rats in all groups tolerated more and more resistance during the exercise period, perhaps rats in the 27-month-old group did not work up to their full capacity. Whether rats were working as hard as possible could not be discerned. An alternate explanation for the lack of improvement is that the 27-month-old animals were not capable of adding muscle protein in response to resistance exercise. There may be, for some older organisms, a point in the aging process, regardless of chronological age, after which muscle hypertrophy cannot occur.[4,10] Evidence to favor this possibility is that not all animals in each group gained muscle weight or fiber area. Possibly, increases in muscle strength for some animals were due to neural factors, as suggested by Moritani and deVries.[2]

A larger proportion of animals in the 21- and 24-month-old groups showed increases in muscle weight and type II fiber area than rats in the 27- and 30-month-old groups. Additionally, the magnitude of type II muscle fiber enlargement was greater for the two younger groups. The 24-month-old rats showed the largest increase (46%), followed by the 21-month-old (33%) and finally the 30- and 27-month-old animals (27{ and 10{, respectively). These findings tend to support the concept of a more limited response to exercise with advancing age.[2,4,7] Exercise intensity for rats in this study, however, was unknown. The question remains that if exercise intensity could have been made identical for all animals, would a similar outcome (improved strenth, muscle hypertrophy) have been achieved for all groups of animals?

One of the limitations of the study was that involvement of the PL muscle in the chin-up protocol could not be verified. The lack of weight increase for controls in contrast to the 20% to 50% increase in muscle weight for three of the four groups of exercised rats suggests that the PL muscle was being used by the majority of animals during exercise. Future studies of this nature should incorporate a method to verify muscle use.

Another limitation to this study was that the manual resistance used could not be quantified. There is no doubt in the investigator's mind that the application of resistance was consistent for each animal and graded according to individual ability. Application of manual resistance to a rat is really no different in technique than the application of manual resistance to a human patient using PNF. In neither case can resistance be quantified, but lack of objectivity does not diminish the basic goodness of the manual resistance concept: maximal resistance throughout the entire arc of motion.

Another factor, length of the exercise period, emerged as being important in this study. Younger rats usually reached a plateau (ie, no additional resistance was tolerated) six to eight weeks into the exercise protocol, whereas the older rats required eight to nine weeks to reach a point where no further resistance could be added. One question that remains is: If the exercise period had been longer than three months, could additional resistance have been tolerated?

It is possible that the variable data obtained for muscle weight and size were the result of limited exercise performance. Animals did not tolerate additional resistance beyond six to nine weeks, which could be the result of inability to further improve exercise performance or could reflect animal unwillingness to respond to greater demand. The subjective impression of the investigator is that animals were working as hard as they could. This impression is based on perceived consistency of performance on a day-to-day and week-to-week basis. Further investigation is needed of the concept of an end point in performance capacity for old rats (and humans).

Changes in fiber size with exercise were observed primarily in the type II fibers as opposed to the type I fibers. This result may be due to 1) the small proportion of type I fibers with fewer data points to measure and 2) fiber recruitment order. Evidence to suggest that type II fibers are preferentially recruited before type I fibers during heavy resistance activity was presented by Gollnick et al.[18]

Although significant improvements in muscle weight and fiber area occurred, whether the change for 30-month-old rats should be considered hypertrophy is questionable. The 27% increase in type II fiber size for the oldest group resulted in fibers that were comparable in size to those of the 21-month-old animals before they started the chin-up protocol. Perhaps the change in muscle size for the oldest exercised rats should be considered a reversal of atrophy.

All control groups except the 21-month-old group gained body weight during the three-month period they were followed; all exercise groups lost body weight. Thus, exercised animals that gained muscle weight did so even though a concomitant loss of body weight was occurring. The possibility exists that exercised rats would have shown greater increases in muscle size in response to exercise if they had not lost body weight. Rats typically gain body weight continuously during their lifetime unless ill or unless they reach a premorbid state.

A general trend toward reduced muscle weight and fiber area was seen with advancing age in both control and exercised rats. This finding is consistent with that of others who have demonstrated a decline in muscle mass and fiber area, particularly type II fibers, with aging.[19-21]

Results from this study do not support the findings of Steinhagen-Thiesen et al [8] and others [9, 10] who suggest that exercise in old age is harmful to skeletal muscle. These investigators subjected rats and mice to a program of forced running on a treadmill. Results from this study may indicate that the beneficial aspects of physical activity are more likely to be observed in older organisms when volitional exercise is used.

One question that must be addressed is, how applicable are the results from this rat study to humans? The rat is often used as a model to study the effects of a particular perturbation on skeletal muscle (and other systems). Numerous investigators have demonstrated that changes that occur within rat skeletal muscle with aging and exercise are in a direction similar to those that occur within human skeletal muscle with age and exercise. [22-25] Thus, it may be inferred that what occurred with resistance exercise for rats in this study (increased muscle weight and fiber area) is likely to occur in the skeletal muscle of aging humans. Direct extrapolation of these results to humans, however, is not justified.

Further study of exercise effects on elderly humans, particularly for those in their 8th, 9th, and 10th decades of life, is needed. Future study of exercise effects on skeletal muscle in an aging or aged organism should also include data such as total muscle protein and biochemical essays of oxidative and glycolytic enzymes.

Summary

Thre of four groups of aging or aged rats showed significant increases in type II muscle fiber area subsequent to a three-month resistance exercise program. In tissues from exercised animals, no evidence of harm to the muscle studied was found.

(*1) Sasco, Inc, PO Box 9556, O'Fallon, MO 63366

(*2) Hewlett-Packard Desktop Computer Div, 3404 E Harmony Rd, Fort Collins, CO 80525.

References

[1] Larsson L: Physical training effects on muscle morphology in sedentary males at different ages. Med Sci Sports Exerc 14:203-206, 1982

[2] Moritani T, deVries HA: Potential for gross muscle hypertrophy in older men. J. Gerontol 35:672-682, 1980

[3] Aniansson A, Ljundberg P, Rundgren A, et al: Effect of a training programme for pensioners on condition and strength. Arch Gerontol Geriatr 3:229-241, 1984

[4] Liemohn WP: Strength and aging: An exploratory study. Intl J Aging Hum Dev 6:347-357, 1975

[5] Stebbins CL, Schultz E, Smith R, et al: Effects of chronic exercise during aging on muscle and end-plate morphology in rats. J Appl Physiol: Respirat Environ Exercise Physiol 58:45-51, 1985

[6] Tomanek RJ, Woo YK: Compensatory hypertrophy of the plantaris muscle in relation to age. J. Gerontol 25:23-29, 1972

[7] Drahota Z, Gutmann E: The effect of age on compensatory and "post-functional hypertrophy" in cross-striated muscle. Gerontologia 6:81-90, 1962

[8] Steinhagen-Thiesen E, Reznik A, Hiltz H: Negative adaptation to physical training in senile mice. Mech Ageing Dev 12:231-236, 1980

[9] Silbermann M, Finkelbrand S, Weiss A, et al: Morphometric analysis of aging skeletal muscle following endurance training. Muscle Nerve 6:136-142, 1983

[10] McCafferty WB, Edington DW: Skeletal muscle and organ weights of aged and trained male rats. Gerontologia 20:44-50, 1974

[11] Gutmann E, Hanzlikova V: Basic mechanisms of aging in the neuromuscular system. Mech Ageing Dev 1:327-349, 1972

[12] The Sprague-Dawley Rat. Wilmington, MA, Charles River Breeding Laboratories, Inc, 1978

[13] Caccia M, Harris JB, Johnson MA: Morphology and physiology of skeletal muscle in aging rodents. Muscle Nerve 2:202-212, 1979

[14] Gollnick PD, Timson BF, Moore RL, et al: Muscular enlargement and number of fibers in skeletal muscles of rats. J Appl Physiol: Respirat Environ Exercise Physiol 50:936-943, 1981

[15] Brooke MH, Kaiser KK: Three "myosin adenosine triphosphatase" systems: The nature of their pH liability and sulfhydryl dependence. J Histochem Cytochem 18:670-672, 1970

[16] Novikoff AB, Schir W, Drucker J: Mitochondrial localization of oxidative enzymes: Staining results with two tetrazolium salts. J Celi Biol 9:47-61, 1961

[17] Lillie RD, Fullmer HM: Histopathologic Technic and Practical Histochemistry, ed 4. New York, NY, McGraw-Hill Inc, 1977, pp 26-43

[18] Gollnick PD, Piehl K, Saltin B: Selective glycogen depletion pattern in human muscle fibers after exercise of varying intensity and at varying pedal rates. J Physiol (Lond) 241:45-57, 1974

[19] Froklis VV, Martynenko OA, Zamostyan VP: Aging of the neuromuscular apparatus. Gerontology 22:244-279, 1976

[20] Aniansson A, Hedberg M, Henning G-B, et al: Muscle morphology, enzymatic activity, and muscle strength in elderly men: A follow-up study. Muscle Nerve 9:585-591, 1986

[21] Fujisawa K: Some observations on the skeletal musculature of aged rats: I. Histological ob ervations. J Neurol Sci 22:353-366, 1974

[22] Saltin B, Gollnick PD: Skeletal muscle adaptibility: Significance for metabolism and performance. In Handbook of Physiology: Section 10. Skeletal Muscle. Bethesda, MD, American Physiological Society, 1983, pp 555-631

[23] Inokuchi S, Ishikawa H, Iwamoto S, et al: Age-related changes in the histochemical composition of the rectus abdominis muscle of the adult human. Hum Biol 47:231-249, 1975

[24] Bass A, Gutmann E, Hanzlikova V: Biochemical and histochemical changes in energy supply-enzyme pattern of muscles of the rat during old age. Gerontologia 21:31-45, 1975

[25] Gollnick PD, Armstrong RG, Saltin B, et al: Effect of training on enzyme activity and fiber composition of human skeletal muscle. J Appl Physiol 34:107-111, 1973

M Brown, PhD, PT, is Assistant Professor, Program in Physical Therapy, School of Medicine, Washington University, PO Box 8083, 660 S Euclid Ave, St Louis, MO 63110 (USA).

This project was made possible by a grant from the Foundation for Physical Therapy.

This article was submitted September 23, 1987; was with the author for revision for 17 weeks; and was accepted August 15, 1988.