EXPERIMENTAL

NEUROLOGY

49,

758-771

(1975)

Effects of Chronic Rotation and Fibers of Soleus and Plantaris WILLIAM Dcpartmcnt lIhersity

D.

MAKTIN

1 AND

Hypergravity on Muscle Muscles of the Rat EDWARD

H.

ROMONU

of Anatomy, Albevt B. Ckarldler Mcdicat Center, of Kentucky, Lr.zington, Krrztltcky 40506 Rcccivcd

July

17, 1975

Three lines of F3 Sprague-Dawley rats derived from a single mating were raised either outside the centrifuge at earth gravity (earth control), or under chronic rotation in the center of the centrifuge at 1.03s (rotation control), or in the rim of the centrifuge at 2g (rotation experimental). The rats were killed at 3 months of age, and serial sections of plantaris and soleus muscles were stained for succinic dehydrogenase activity, and for actomyosin ATPase activity following preincubation at pH 10.2. Muscle fibers in a cross-sectional unit area of soleus and both the superficial and deep regions of plantaris were identified as to fiber type according to an enzyme profile and counted. The proportion of each fiber type was calculated, and the diameter of 24 fibers of each type was measured. Analysis of variance was used to determine the effect of experimental treatment In soleus, the fiber population and sex on populations and diameters. shifted from 82% slow oxidative fibers in the controls to 100% slow oxidative fibers in the rotation experimentals in response to the stress of hypergravity. In plantaris, chronic rotation resulted in an increase in fast glycolytic fibers and a corresponding decrease in fast oxidative g;ycolytic fibers, in both the rotation control and experimental rats. The population changes were similar in both sexes. Muscle fiber diameters were similar in both controls, indicating no response to the stress of chronic rotation. A sexual dichotomy was noted in response to hypergravity, with muscle fiber diameters increasing in females, but decreasing in ma!cs. 1 Supported by General Research Support Grant RR05374, General Research Support Branch, Division of Research Facilities and Resources, NIH. The animals and centrifuges for this experiment were supplied by Dr. Charles Knapp, Wenner-Gren Research Laboratory, University of Kentucky, under Grant No. NGL-18-001 from the National Aeronautics and Space Administration. The authors thank Dr. John Haley, Department of Behavioral Sciences, University of Kentucky Medical Center. for assistance with the statistical analysis. The junior author is a third-year medical student.

Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

MUSCLE

759

FIBERS

INTRODUCTION Previous studies of the effect of chronic centrifugation on skeletal muscle have been limited to comparisons of the weight of selected muscles to either the total mass of the animal (4)) or to total body muscle mass (34). It is generally recognized that skeletal muscle is composed of three major muscle fiber types with typical histochemical (5, 28) and contraction-fatigue (2, 3, 7, 19) characteristics, and that gross muscles have varying proportions of each muscle fiber type (1). The adaptability of muscle fibers to stress has been demonstrated by analysis of muscles following the intermittent stress of various exercise regimens (6, 10, 11, 18)) or constant stress following ablation or tenotomy of synergistic muscles ( 13, 1.5, 20, 21). Chronic centrifugation offers the possibility of analyzing the effect of two constant stresses, chronic rotation and hypergravity, on skeletal muscle fibers of animals which are morphologically intact. Two antigravity muscles were chosen: Soleus muscle, which extends the talocrural joint by means of its attachment to the calcaneal process, and is primarily involved with the maintenance of posture; and plantaris muscle, which crosses the talocrural joint to serve as an origin for flexor digitorum brevis muscle, and is primarily involved in locomotion. The present study demonstrates that muscle fiber population shifts occur in soleus muscle in response to hypergravity, and in plantaris muscle in response to chronic rotation. Additionally, a sexual dichotomy was noted in response to hypergravity, with muscle fibers in the female increasing in diameter, while those of the male decreased in diameter. MATERIALS

AND

METHODS

Three experimental groups of 3-month-old Sprague-Dawley rats, each consisting of four males and four females, were selected from a population of rats of the third generation derived from a single pair mated in the chronic centrifuge facility of the Wenner-Gren Research Laboratory. All of the animals were fed and watered ad Zibitutrz, exposed to a 12 hr light/l2 hr dark cycle, and had an equal amount of cage space per animal. The experimental groups were : (a ) rotation experimental, housed from birth in the rim of a torus-shaped centrifuge and subjected to two stresses, continuous rotation at 30 rpm, and a gravitational force of 2y; (b ) rotation control, housed from birth in the center of the same centrifuge and subjected to a single stress, continuous rotation at 30 rpm but essentially at earth gravity (1.03~) ; and (c) earth control group, housed in the same room as the centrifuge at earth gravity without rotation. The animals were killed by an intraperitoneal injection of Nembutal, and body weights were recorded. The gastrocnemius. plantaris, ZUld

760

MARTIN

ocn tr S

e

AKD

ROMOND

m i

I

u s

Ll m

~

fLz!!J

FIG. 1. 1.4) Diagram of a cross-section through soleus and plantaris muscles to illustrate the regions which were sampled ; I = plantaris, superficial region ; II = plantaris, deep region; III = soleus. (B and C) Adjacent sections incubated for ATPase activity at pH 10.2 (B) and SDH activity (C) to illustrate the muscle fiber types; X238 ; fg = fast glycolytic ; fog = fast oxidative glycolytic ; so = slow oxidative. (D and E) Plantaris, at the top of the frame, and soleus muscles of an experimental animal which was subjected to hypergravity (D) and a control animal (E) ; ATPase, pH 10.2 ; X119. The muscle fiber population of soleus muscle has totally shifted to slow oxidative fihers in response to hypergravity.

soleus muscles were removed as a unit, kept moist 1 C until the muscles did not react to mechanical with talcum powder, and frozen in liquid nitrogen

with normal stimulation, (22).

Sections

saline at covered were

RIUS(‘LIr

761

1’1KEKS

cut from the muscle bellies at a thickness of 12 pm. Adjacent sections tvere incubated for succinic dehydrogenase activity (23 ) and adenosine triphosphatase activity following acid or basic preincubation ( 14). Soleus muscle, and the superficial and deep region of plantaris muscle (Fig. 1A) were photographed using Panatomic X film and printed at a final magnification of 100X. Muscle fibers in an area of 1 million pni” were classified according to the profile in Table 1. Fig. 1B. C. The proportion of each fiber type was calculated as a percentage of the total number of fibers counted in each region analyzed. Diagonals were drawn across the region, and the shortest diameter of 24 muscle fibers of each type which contacted or came near the diagonal was measured to the nearest (estimated ) 0.1 pm using a vernier caliper. A mean diameter was calculated for each muscle fiber type; and for graphical representation, the muscle fiber diameters were grouped at intervals of 5 pm, e.g., 15.019.9 pm (midpoint 17.5), etc. The muscle fiber proportions and mean muscle fiber diameters were statistically analyzed using a two-way analysis of variance to determine the effect of the experimental treatment and sex on each muscle fiber type within a muscle or muscle region. RESULTS In general, the body weights of the animals varied nith the esperimental treatment (Table 2 ) . Males were larger than females in each experimental group and underwent a greater change in body weight in response to chronic stress. When compared to the earth control group, a significant increase in body weight occurred in males and females of the rotation control group, and a significant decrease in body weight in the TABLE HISTOCHEMICAL Fiber

Fast

glycolytic

Slow

osidative

Fast

osidatiw

EXZVME

PROFILE

type

gl~col!Tic

1

USED TO CLASSIFV

MUSCLES

FIBERS

SI)H

Light (sparse particles) Intermediate (more numerous, particles) Dark (subsarcolcmmal particles)

IN THIS

STUDY

ATPaw ipH 10.2) Intermcdiatc Light cvrnl~

distributtd Ilark

accunlulation

of

762

MARTIN

AND

‘I-ABLE BODY

WEIGHT

ROMOND

2 IN GRAMS*~~~~~~~

RCc

EC* Male Female * Mean * EC = c KC = rl RE =

281.4 192.2

i f

f standard deviation. earth control. rotation control. rotation experimental.

12.4 12.8 Four

354.7 228.5

f f

REd 31.9 12.4

177.7 169.5

f f

6.2 28.8

in each group.

male rotation experimental animals; however, the weight decrease in rotation experimental females was not statistically significant. The mean muscle fiber diameter of all fiber types remained unchanged in the rotation controls (Figs. 2-4) despite the significant increase in body weight relative to the earth controls. In all groups, the muscle fibers of plantaris and soleus muscles presented a cormal microscopic appearance with no splitting of fibers or histochemical fiber type grouping. Po~Wation Chalzges. The proportion of each muscle fiber type was considerably different in the three regions which were analyzed (Table 3, earth control). The predominant fiber type in soleus was the slow oxidative fiber. In plantaris, the predominant fiber type was the fast oxidative glycolytic fiber, with the exception of the superficial region in the female where the proportion of fast oxiclative glycolytic and fast glycolytic fibers was approximately equal. The proportion of slow oxidative fibers was greater in the deep as compared to the superficial region of plantaris muscle in both sexes. In the experimental groups, the response of muscle fiber types to chronic stress differed in soleus and plantaris muscles. In soleus, chronic rotation resulted in no population change; however, hypergravity resulted in a significant shift to lOOr/c slow oxidative fibers with a corresponding decrease in the proportion of fast oxiclative glycolytic fibers (Tables 3, 3: Fig. lD, I?). In plantaris, chronic rotation resulted in a significant decrease in the proportion of fast oxidative glycolytic fibers and a corresponding increase in fast glycolytic fibers in the superficial region of the male, and in the deep region of both sexes (Tables 3, 4). A similar trend, which was not statistically significant, was noted in the superficial region of plantaris in the female in response to chronic rotation (Tables 3, 3). The proportion of slow oxidative fibers in plantaris remained unchanged by either hypergrayity or chronic rotation (Table 3).

MUSCLE

FIBERS

763

In the earth control group, muscle fibers of soleus muscle were larger in diameter than fibers of the same histochemical fiber type in plantaris (Figs. 3, 3). In soleus, the mean diameter of fast osidative glycolytic fibers was significantly smaller in the female (Table 5. Fig. 3), whereas in plantark, only fast glycolytic fibers exhibited a significant difference in fiber diameter between the male and female (Table 5, Fig. 3). In plantaris, the fzt glycolytic fibers were largest it1 diameter, Diauictrv

Clfangcs.

30.

DIAMETER

IN MICRONS

FIG. 2. Muscle fiber diameter distributions of slow oxidative fibers. The mear~ muscle fiber diameter in microns for each group is indicated in the upper right hand corner of each graph. EC = earth control ; RC = rotation control ; RE = rotation experimental.

764 FAST OXIDATIVE @g

GLYCOLYTlC

FIG. 3. Muscle ftber diameter distributions of fast oxidative glycolytic fibers. The mean muscle fiber diameter in microns for each group is indicated in the upper right h;nld corner of each graph. EC = earth control; RC = rotation control; RE = rotation experimental.

followed by the fast oxidative glycolytic fibers, with slow oxidative fibers having the smallest diameter (Figs. 2-4). The mean diameter of all three fiber types was larger in the deep than in the superficial region (Figs. 2-3). In general, the distributions of muscle fiber diameters in earth control and rotation control groups were similar, indicating no reaction to the stressof chronic rotation (Figs. 2-4 j . The response of muscle fiber types to hypergravity varied within the regions examined. In soleus muscle, the diameter of the slow oxidative fibers decreased significantly in the maIe and remained the same in the female, resulting in a significant sex-by-treatment interaction (Table 5, Fig. 2 ). In the deep region of plantaris. a similar trend toward a reduction of diameter in the male with no change of diameter in the female

hf USCLE

was noted

in all fiber

types;

765

~1HEHS

however.

only

the fast glycolytic

hibited a significant decrease in dianleter in the male sex-by-treatment interaction in response to hypergravity

fibers

ex-

and a significant (Table 5, Figs.

2-4). In the superficial region of plantaris, sexual dichotomy in response to hypergravity was noted. All three fiber types decreased in dianieter ii; the male and increased in diameter in the female resulting in a significant sex-by-treatment interaction (Table 5, Figs. 2-4 ).

EC . . . . . ..__. pc .----. RE .-.

FAST GLYCOLYTIC MALE

DIAMETER

IN

MICRONS

Muscle fiber diameter distributions of fast glycolytic fibers. The mean muscle fiber diameter in microns for each group is indicated in the upper right hand corner of each graph. EC = earth control ; RC = rotation control ; RE = rotation experimental. FIG.

4.

FIBER

glycolytic

superficial region deep region

region

a Four animals per group. EC approximately 200 muscle fibers.

Plantaris, Plantaris,

Fast

deep

Plantaris,

region

superhcial

glycolytic

superficial region deep region

POPULATIONS

Plantark,

Fast oxidative Soleus

Soleus Plantaris, Plantaris,

Slow oxidative

MUSCLE

= earth

EXPRESSED

1.1

1.1 0.5 2.2

control

; RC

38.9 f 2.8 17.6 f 4.5

56.5 =t 2.6 65.9 f 3.2

16.5 f

83.5 f 4.5 f 16.5 f

EC

RC

= rotation

f f

f

54.6

46.2 29.5

f

18.0 f 47.6

TABLE

4.0 1.0

1.4

3.9

1.7

1.7 1.1 1.8

3

RE

-

f f

f

f

; RE

45.9 25.1

59.6

50.5

EC

3.6 0.4 1.9

IN EACH

experimental.

49.2 f 1.4 19.4 i 3.4

15.7 f 3.6 47.6 f 1.6 63.0 f 3.2

84.3 i 2.9 f 17.6 i

OF FIBERS

= rotation

1.6 2.5

2.9

1.3

0.3 0.4

NUMBER

100.0 5.3 f 15.3 f

TOTAL

control

OF THE

Male

82.0 f 6.2 f 15.9 f

AS A PERCENTAGE

f

Sample

54.3 32.0

52.5

41.0

18.2

f f

f

f f

81.8 f 4.7 f 15.5 f

RC

Female

AREA

size for

2.8 3.3

2.9

4.8 2.5

4.8 1.0 0.9

RE

each

3.5

3.2 2.2

3.2

region

48.7 f 29.7 f

54.2 f

46.0 i

1.3 2.2

ERRORQ

100.0 5.3 f 16.0 f

STANDARD

was

s

2

s m 6

z 2 k-

?-

Muscle hypertrophy has been produced by various experimental procedures in animals. It is generally accepted that two types of hypertrophy occur, compensatory hypertrophy (13. 15, 20, 21) and exercise hypertrophy (10, 11. 21 ). Compensatory hypertrophy occurs following ablation, tenotomy. or denervation of synergistic tnuscles resulting in mechanical stretch and consequent hypertrophy of the remaining muscle (13. 29). Compensatorv . hypertrophy is transient in character with the peak hypertrophy occurring approximately 1 week after onset followed by a residual hypertrophy of lo-15F of control muscle weight (2Oj. Exercise hypertrophy has been more difficult to produce in animals. Previous studies of the effect of hypergravity on skeletal muscle, which could be considered a chronic exercise program, have yielded conflicting results. In the chicken, Burton et al. (4) noted hypertrophy of a hip

TABLE ANALYSIS

OF \TARIANCE

TABLE

4

FOR MUSCLE

Plantaris-sup

SloWoxidative Treatments(A) Sex (B) AXB Error Total Fast

oxidative

Treatments Sex (B) AXB Error Total Fast

FIRER

POPVLATIONV

Soleus

Plantaris-deep

df

MS

df --__-.

2 1 2 18 23

7.40 ns 7.04 ns 1.60 ns 2.95 3.40

2 1 2 18 23

df

MS

4.95 ns 1.35 ns 1.14 ns 11.86 9.87

2 1 2 18 23

757.54* 0.66 0.56 27.35 87.36

247X17+ 73.15 ns 5.80 ns 33.67 51.57

1 1 1 12 15

15.60 0.36 0.90 40.42 33.46

319.43* 54.60 ns 4.21 ns 37.07 59.53

MS

ns ns

glycolytic (A)

2 1 2 18 23

12.05* 251.55* 11.89 30.28 46.15

ns

2 1 2 18 23

2 1 2 18 23

75.99 ns 297.51* 29.99 ns 31.57 46.86

2 1 3 18 23

ns ns ns

glycolytic

Treatments Sex (B) AXB Error Total

(A)

* = significance less than 0.05 level. (2 ns = not statistically significant;

df = degrees

of freedom;

MS

= mean

square.

‘168

MARTIN

ANI)

ROMONII

TABLE 5 ANALYSIS

OF V~RIANCE'I‘AHLE

FOR

MUSCLE FIBER DIAMETERS*

Plantaris-sup

Plantaris-deep

Soleus

df

MS

9.37 ns 9.92 ns 81.89* 22.16 25.71

2 1 2 18 23

28.06 31.71 35.71 18.43 21.35

ns “S ns

2

2 18 23

6.62 ns 30.87 ns 157.15* 29.68 38.81

2 18 23

64.66 67.97 79.16 28.66 37.89

ns ns ns

2 1 2 18 23

21.21 ns 30.21 “S 416.06* 47.36 76.40

2 1 2 18 23

176.83* 130.71* 249.21* 29.96 66.18

df

MS

df

MS

2 1 2 18 23

100.19* 49.91 ns 146.78* 23.66 42.16

1

47.16 ns 460.63* 0.29 ns 24.99 53.86

Slow oxidative Treatments Sex (B) AxB Error Total Fast

oxidative

Treatments Sex 09 AXB Error Total Fast

(A)

2

1 2 18 23 glycolytic (A)

2

1

1

1 1 12 15

glycolytic

Treatments Sex (B) AxB Error Total * = significance a df = degrees

(A)

less than 0.05 level. of freedom; MS = mean

square;

ns = not

statistically

significant.

extensor and atrophy of a hip flexor muscle when muscle weight was compared to the weight of nonstressed control muscles. In the rat, Pitts, Bull, and Oyama (34) noticed no significant hypertrophy in selected antigravity muscles compared as a ratio of antigravity muscle weight to total muscle weight; however, only female rats were studied. In this study, exposure of female rats to hypergravity resulted in hypertrophy of all fiber types in the superficial region of plantaris muscle with no change in diameter in the other regions. The hypertrophy was of a magnitude which may not be detectable when compared to total body weight, but is consistent with reports of muscle fiber hypertrophy without increased muscle weight following various exercise regimens (9-l 1) . The selective hypertrophy of fiber types within muscle regions noted in this study suggests functional specialization within discrete gross muscles, and indicates that careful sampling of muscles is necessary for analysis of muscle fiber hypertrophy.

The influence of androgens on certain skeletal muscles is well documented (S. 12) ; however, limb muscles were considered to be independent of androgen levels since muscle nitrogen content and weight remained unchanged following castration (17). In general, the studies of exercise hypertrophy in the rat have been conducted on females, and are characterized by little gross muscle hypertrophy with some muscle fiber hypertrophy within regions of gross muscles (10, 11 ). In the male mouse, an exercise regimen of running resulted in a transient muscle fiber hypertrophy in tibialis anterior and biceps brachii muscles followed by slight hypertrophy when compared to muscles from nonexercised control animals (27). Consideration of these results suggests that the sexual differences in response to chronic centrifugation noted in this study may be a characteristic reaction to long-term stress. However, another possibility is that the rats used in his study may have undergone adaptive changes since they are members of the third generation to be constantly centrifuged. Further studies of the time course of change, and hormonal influence on muscle fiber diameters are necessary to answer this question. The adaptability of muscle fibers to experimental stress is well illustrated in the response of soleus and plantaris muscles to chronic centrifugation. Soleus muscle is considered to be a postural muscle, and the shift to a homogenous population of slow twitch contracting-slow fatiguing muscle fibers in response to hypergravity would appear to enhance this function. In plantaris muscle, which is primarily involved in locomotion, the population shift toward fast glycolytic fibers was in response to the stress of chronic rotation. This suggests that vestibular stimulation by chronic rotation may result in muscle fiber population changes, since other investigators noted that centrifugation affected synaptic structure in the lateral vestibular nucleus of the rat (16)) and specific changes in muscle fiber populations have been noted in response to programmed stimulation of muscle nerves in the cat (25). Other investigators have shown that the reaction of muscle fibers in rat varies with the experimental stress: e.g., the increased sarcoplasmic protein (10) and mumber of muscle fibers with high oxidative activity (6) in response to a repetitive low resistance exercise program of swimming; the increase in contractile protein in response to a forceful exercise program of weight lifting (11) ; and the increase in fibers with low myosin ATPase activity in compensatory hypertrophy resulting from inactivation of synergistic muscle (13, 15). The results of this study suggest that various factors must be considered when interpreting morphologic changes resulting from exposure to chonic stress. Sexual differences in response to hypergravity were noted in both body weight and changes in muscle fiber diameters. Muscle function as well as the experimental stress must be considered. since soleus

770

MARTIN

AND

KOMOND

and plantaris muscles differed in their response to experimental stress. Furthermore, in the specific case of the effects of chronic centrifugation, the results of this study on muscle fibers, and those of Smith (26) on bone development and growth indicate that the stress of chronic rotation in the absence of increased gravitational force must be considered in future studies. KEFEKENCES 1. AHIANO, fiber 2.

3.

4.

5. 6.

7.

8.

9. 10.

11. 12.

13. 14. 15. 16.

M. A., R. B. ARMSTRONG, and V. R. EDGERTON. 1973. Hindlimb muscle populations of five mammals. J. Histochcm Cytorhem. 21 : 51-55. BARNARD, R. J., V. R. EDGERTON, T. FURUKAWA, and J. B. PETER. 1971. Histochemical, biochemical, and contractile properties of red, white, and’ intermediate fibers. Amer. J. Physiol. 220: 410-414. BURKE, R. E., D. N. LEVINE, and F. F. ZAJAC, III. 1971. Mammalian motor units : Physiological-histochemical correlation in three types in cat gastrocnemius. Scicrtcc 174 : 709-712. BURTON, R. R., E. L. BESCH, S. J. SLUKA, and A. H. SMITH. 1967. Differential effects of chronic acceleration on skeletal murcles. J. -4ppl. Physiol. 213: 1193-119s. EDGERTON, V. R., and D. R. SIMPSON. 1969. The intermediate musc!e fiber of rats and guinea pigs. J. Hisfochrm. Cyforhcm. 17: 828-837. EDGERTON, V. R., L. GERCHMAN, and R. CARROW. 1969. Histochemical changes in rat skeletal muscle after exercise. E.rp. Ncurol. 24: 110-123. EDSTROM, L., and E. K~GELBURG. 1968. Histochemical composition, distribution of fibres, and fatigability of single motor units. J. Nc~rrol. Nr~troszlvg. Psychiat. 31: 424-433. GALAVAZI, G., and J. A. SZIRMAI. 1971. Cytomorphometry of skeletal muscles: The influence of age and testosterone on the rat M. levator ani. Z. Zellforsrh. 121: 507-530. GOLDSPINK, G. 1964. The combined effects of exercise and reduced food intake on skeletal muscle fibers J. Cc/l. Camp. Physiol. 21 : 118-122. GORDON, E. E., K. KOWALSKI, and M. FRITTS. 1967. Protein changes in yuadriceps muscle of rat with repetitive ‘exercises. Arch. Phys. Med. RehA. 48: 296-303. GORDON, E. E., Ii. KOWALSKI, and M. FRITTS. 1967. Protein changes in quadriceps muscle of rat with forceful exercises. Avch. Phys. Med. R&b. 48: 577-582. GUTMANN, E., V. HANZLIKOVA, and Z. LOJDA. 1970. Effect of androgens on histochemical fiber type. Differentiation in the temporal muscle of the guinea pig. Histochcwrie 24 : 287-291. GUTMANN, E., S. SCHIAFFANO, and V. HANZLIKOVA. 1971. Mechanism of compensatory hypertrophy in skeletal muscle of the rat. Erp. Nc~rol. 31: 451-464. GUTH, L., and F. J. SAMAHA. 1970. Procedure for the histochemical demonstration of actomyosin ATPase. Exp. Ncurol. 28: 365-367. GUTH, L., and H. YELLIN. 1971. The dynamic nature of so-called “fiber-types” of mammalian skeletal muscle. Enp. Newol. 31 : 277-300. JOHNSON, J. E., J. OYAMA, and W. R. MEHLER. 1975. Effects of centrifugation on the fine structure of the CNS : A study of the lateral vestibular nucleus oi the rat. Anaf. Rec. 181: 386.

17. KOTCIIAKIAN, C. 1)., C. TILLOTSOX, F. .L\c~TIN, E. IIOUCHEWTY, V. HAN;, and R. COALSON. 1956. The effect of castration on the weight and composition of the muscles of the guinea pig. Emfocviuology 58: 315. 18. KOWALSKI, Ii., E. E. GORDON, A. MARTINEZ, and J. ADAhfEK. 1969. Changes in enzyme activities of various muscle fiber types in rat induced by different exercises. J. Historhrw. Cytochcw. 17 : 601-607. 19. KIJC;ELBURG, E., and L. EDSTROM. 1968. Differential histochemical effects of muscle contractions on phosphorylase and glycogen in various types of fibres: relation to fatigue. J. Ncv~vol. Ncrrrosrrv!/. f’sychicrt. 31 : 415-423. 20. MA~KOVA, E., and P. HNIK. 1972. Time course of compensatory hypertrophy of slow and fast rat muscles in relation to age. Physiol. Hohc~rzosln~~. 21: 9-17. 21. MACKOVA, E., and P. HNIK. 1972. Compensatory hypertrophy induced by tenotomy of synergists is not true working hypertrophy. Physiol. Bolw~zos/u~~ 22: 43-49. 22. MOLINE, W. W., and G. G. GLENNER. 1964. Ultrarapid tissue freezing in liquid nitrogen. J. Histochcrrz. Cgfochew. 12 : 777-783. 23. PEARSE, A. G. E. 1961 Methods for succinate dehydrogenase using MTT, p. 910. ZIG “Histochemistry Theoretical and Applied.” 2nd ed. Little, Brown, Boston. 24. PITTS, G. C., L. S. BULL, and T. OYAMA. 1972. Effect of chronic centrifugation on body composition in the rat. .-lmr~. J. I’hysiol. 223: 1044-1048. 25. RILEY, D. A., and E. F. ALLIN. 1973. The effects of inactivity, programmed stimulation, and denervation on the histochemistry of skeletal muscle fiber types. E.rP. Ncrrrol. 40: 391-413. 26. ShUTIT, S. D. 1975. Effects of long term rotation and hypergravity on developing rat femurs. aJriot. Space Ewirorl. hfrd. 46: 248-253. 27. WALKER, M. G. 1966. The effect of exercise on skeletal muscle fihers. Cowp. Riockc~f. Physiol. 19 : 791-797. 28. YELLIN, H., and L. GUTH. 1970. The histochemical classification of muscle fihers. E.rp. Ncurol. 26: 424-432. 29. YELLIN, H. 1974. Changes in fiber types of the hypertrophy in denervated hemidiaphragm. Esp. Nmrol. 42 : 412-428.