Acta Physiol Scand 1992, 146, 281-282
Muscle fibre number following hindlimb im mobiI izat ion Y. OISHI, A. ISHIHARA" and S. K A T S U T A t Faculty of General Education, Kumamoto University, Kumamoto 860, * College of Liberal Arts and Sciences, Kyoto University, Kyoto 606, and t Institute of Health and Sport Sciences, University of Tsukuba, Ibaraki 305, Japan It is widely accepted that immobilization induces a decrease in the percentage of slow-twitch oxidative (SO) fibres and an increase in that of fast-twitch oxidative glycolytic (FOG) fibres in skeletal muscles. The decreased percentage of SO fibres is considered to be due to a selective loss of SO fibres using the rat soleus muscle (Booth & Kelso 1973) or a type shift of fibres from SO to FOG using the rat vastus intermedius muscle (Boyes & Johnston 1979). In those studies, however, muscle fibre number was determined only using counts from histochemical muscle transverse sections. This study investigated changes in total fibre number of the rat soleus muscle following hindlimb immobilization, by the direct fibre number counting method. Fourteen 9-week-old male Wistar rats, weighing between 270 and 320 g, were used in this study. The left hindlimb of rats was casted with plaster under pentobarbital sodium anaesthesia. All casts were checked so that ankle and knee were fixed at their resting angles. The right contralateral limb was used as the control. Animals were maintained on a 12 h light-dark cycle at a room temperature of 2 2 k 2 "C and food and water were provided ad libitum. After 6 weeks of immobilization, both left and right soleus muscles were dissected. The soleus muscle fibre number was studied from seven rats, while fibre type composition and fibre size of the soleus muscle were examined in the second set of seven rats. Muscle fibre number was determined by the nitric acid digestion method described by Gollnick et al. (1981). All the single fibres were counted directly under a dissecting microscope. Serial frozen sections of the soleus muscle were cut transversely in a cryostat and stained for myosin ATPase (Padykula & Herman 1955), a-GPD (Wattenberg & Leong 1960) and SDH (Nachlas et al. 1957). All the fibres on the whole muscle cross-section
were identified as SO or FOG (Peter et a!. 1972). Fibre area was determined on SDH-stained sections using a digitizer connected to a personal computer. Areas of more than 40 fibres of each type were measured. The immobilized and the contralateral control limbs were compared using Student's t-tests. A decrease in the total muscle fibre number was found by immobilization (Table 1). Immobilization induced a decrease in the percentage of SO fibres and an increase in that of FOG fibres. An atrophy of both SO and FOG fibres was found by immobilization (Table 2 and Fig. 1). This study observed the 13.7% decrease in the total fibre number of the immobilized soleus muscle. However, individual number of the decreased SO or FOG fibres by immobilization could not be determined. Immobilization finally caused an increase in the percentage of FOG fibres in the soleus muscle. Type shift of fibres from FOG to SO in the soleus muscle occurs during normal development (Kugelberg 1976). The increased FOG fibres in the soleus muscle of the immobilized limb may be due to inhibition in type shift of fibres from FOG to SO which occurs during normal development because the percentage of SO fibres in the soleus muscle is related to the body weight (Kugelberg 1976) and gain in body weight during development was inhibited by immobilization in this study. On the other hand, type shift of fibres from SO to FOG by immobilization is also considered. Further studies are needed to clarify the mechanism of the increased FOG fibres by immobilization. Table 1. Total fibre number of the soleus muscle from control and immobilized limbs of seven rats obtained by the direct fibre counting method Control
Received 12 February 1992, accepted 9 July 1992. Key roords : histochemistry, immobilization, muscle fibre number, rat, soleus muscle. Correspondence : Y. Oishi, Faculty of General Education, Kumamoto University, Kumamoto 860, Japan.
Body weight (g) Muscle weight (mg) Total fibre number
Immobilized
362.7 & 37.0 152.8f 16.1 83.8f20.8+ 2903 f 115 2504 f236"
Mean values & SD. * P < 0.01 compared with the control.
28 1
282
Y . Oishi et al.
T a b l e 2. Fibre type composition and fibre size of the soleus muscle from control and immobilized limbs of seven rats obtained from histochemical transverse section Control Body aeight (g) Muscle weight (mg)
385 4k 35.3 160.7k22.9
so Fibre t?pe ( O n ) Fibre area b m ' ) Mean values+SD.
Immobilized
*
93.4 2.2 4.546 li:350
113.319.5" FOG
so
FOG
6.6 3199 748
78.4-t 10.3" 2400 i469"
21.6" 1979 k 455"
" P < 0.01 compared with the control.
Fig. 1. Transverse sections of the soleus muscle of the control (A) and immobilized (B) limbs, stained for adenosine triphosphatase after alkaline preincubation. Dark and light fibres show fasttwitch oxidative glycolytic and slow-twitch oxidative fibres, respectively. Bar indicates 100 pm.
REFERENCES BOOTH,F.K'. & KELSO,J.R. 1973. Effect of hind-limb immobilization on contractile and histochemical properties of skeletal muscle. Pjugers .4rrh 342, 231-238. BOYES, G . & JOHNSTON,L . 1979. Iluscle fibre composition of rat vastus intermedius following immobilisation at different muscle lengths. Pfiigevs -4rrh 381, 195-200. GOLLNICK,P.D., TIMSON,B.F., MOORE,R.L. s( RIEDY,.M.1981. Muscular enlargement and number of fibres in skeletal muscles of rats. 3 Appl Physiql 50: 936943. KUGELBERG, E. 1976. Adaptive transformation of rat soleus motor units during growth. 3.Yeirro/ Sci 27, 269-289.
M., TsoU, K., DESOUZA,E., CHENG,C. & SELIGMAN, '4. 1957. Cytochemical demonstration of succinic dehydrogenase by the use of a new p nitrophenyl substituted ditetrazole. 3 Histochem Cj'rorhem 5, 420-436. PADYKUL.4, H A . & HERMAN, E. 1955. T h e specificity of the histochemical method for adenosine triphosphatase. 3 Historhem Cytochem 3, 17c-195. PETER, J.B., BARNARD,R.J., EDGERTON,V.R., GILLESPIE, C.A. & STEMPEL,K.E. 1972. Metabolic profiles of three fiber types of skeletal muscle in guinea-pigs and rabbits. Biochemistry 11, 26272633. WATTENBERG, L.W. & LEONG, J.L. 1960. Effects of coenzyme Qo and menadione on succinic dehydrogenase activity as measured by tetrazolium salt reduction. 3 Histochem Cytochem 8, 296-303. YACHLAS.
Muscle fibre number following hindlimb im mobiI izat ion Y. OISHI, A. ISHIHARA" and S. K A T S U T A t Faculty of General Education, Kumamoto University, Kumamoto 860, * College of Liberal Arts and Sciences, Kyoto University, Kyoto 606, and t Institute of Health and Sport Sciences, University of Tsukuba, Ibaraki 305, Japan It is widely accepted that immobilization induces a decrease in the percentage of slow-twitch oxidative (SO) fibres and an increase in that of fast-twitch oxidative glycolytic (FOG) fibres in skeletal muscles. The decreased percentage of SO fibres is considered to be due to a selective loss of SO fibres using the rat soleus muscle (Booth & Kelso 1973) or a type shift of fibres from SO to FOG using the rat vastus intermedius muscle (Boyes & Johnston 1979). In those studies, however, muscle fibre number was determined only using counts from histochemical muscle transverse sections. This study investigated changes in total fibre number of the rat soleus muscle following hindlimb immobilization, by the direct fibre number counting method. Fourteen 9-week-old male Wistar rats, weighing between 270 and 320 g, were used in this study. The left hindlimb of rats was casted with plaster under pentobarbital sodium anaesthesia. All casts were checked so that ankle and knee were fixed at their resting angles. The right contralateral limb was used as the control. Animals were maintained on a 12 h light-dark cycle at a room temperature of 2 2 k 2 "C and food and water were provided ad libitum. After 6 weeks of immobilization, both left and right soleus muscles were dissected. The soleus muscle fibre number was studied from seven rats, while fibre type composition and fibre size of the soleus muscle were examined in the second set of seven rats. Muscle fibre number was determined by the nitric acid digestion method described by Gollnick et al. (1981). All the single fibres were counted directly under a dissecting microscope. Serial frozen sections of the soleus muscle were cut transversely in a cryostat and stained for myosin ATPase (Padykula & Herman 1955), a-GPD (Wattenberg & Leong 1960) and SDH (Nachlas et al. 1957). All the fibres on the whole muscle cross-section
were identified as SO or FOG (Peter et a!. 1972). Fibre area was determined on SDH-stained sections using a digitizer connected to a personal computer. Areas of more than 40 fibres of each type were measured. The immobilized and the contralateral control limbs were compared using Student's t-tests. A decrease in the total muscle fibre number was found by immobilization (Table 1). Immobilization induced a decrease in the percentage of SO fibres and an increase in that of FOG fibres. An atrophy of both SO and FOG fibres was found by immobilization (Table 2 and Fig. 1). This study observed the 13.7% decrease in the total fibre number of the immobilized soleus muscle. However, individual number of the decreased SO or FOG fibres by immobilization could not be determined. Immobilization finally caused an increase in the percentage of FOG fibres in the soleus muscle. Type shift of fibres from FOG to SO in the soleus muscle occurs during normal development (Kugelberg 1976). The increased FOG fibres in the soleus muscle of the immobilized limb may be due to inhibition in type shift of fibres from FOG to SO which occurs during normal development because the percentage of SO fibres in the soleus muscle is related to the body weight (Kugelberg 1976) and gain in body weight during development was inhibited by immobilization in this study. On the other hand, type shift of fibres from SO to FOG by immobilization is also considered. Further studies are needed to clarify the mechanism of the increased FOG fibres by immobilization. Table 1. Total fibre number of the soleus muscle from control and immobilized limbs of seven rats obtained by the direct fibre counting method Control
Received 12 February 1992, accepted 9 July 1992. Key roords : histochemistry, immobilization, muscle fibre number, rat, soleus muscle. Correspondence : Y. Oishi, Faculty of General Education, Kumamoto University, Kumamoto 860, Japan.
Body weight (g) Muscle weight (mg) Total fibre number
Immobilized
362.7 & 37.0 152.8f 16.1 83.8f20.8+ 2903 f 115 2504 f236"
Mean values & SD. * P < 0.01 compared with the control.
28 1
282
Y . Oishi et al.
T a b l e 2. Fibre type composition and fibre size of the soleus muscle from control and immobilized limbs of seven rats obtained from histochemical transverse section Control Body aeight (g) Muscle weight (mg)
385 4k 35.3 160.7k22.9
so Fibre t?pe ( O n ) Fibre area b m ' ) Mean values+SD.
Immobilized
*
93.4 2.2 4.546 li:350
113.319.5" FOG
so
FOG
6.6 3199 748
78.4-t 10.3" 2400 i469"
21.6" 1979 k 455"
" P < 0.01 compared with the control.
Fig. 1. Transverse sections of the soleus muscle of the control (A) and immobilized (B) limbs, stained for adenosine triphosphatase after alkaline preincubation. Dark and light fibres show fasttwitch oxidative glycolytic and slow-twitch oxidative fibres, respectively. Bar indicates 100 pm.
REFERENCES BOOTH,F.K'. & KELSO,J.R. 1973. Effect of hind-limb immobilization on contractile and histochemical properties of skeletal muscle. Pjugers .4rrh 342, 231-238. BOYES, G . & JOHNSTON,L . 1979. Iluscle fibre composition of rat vastus intermedius following immobilisation at different muscle lengths. Pfiigevs -4rrh 381, 195-200. GOLLNICK,P.D., TIMSON,B.F., MOORE,R.L. s( RIEDY,.M.1981. Muscular enlargement and number of fibres in skeletal muscles of rats. 3 Appl Physiql 50: 936943. KUGELBERG, E. 1976. Adaptive transformation of rat soleus motor units during growth. 3.Yeirro/ Sci 27, 269-289.
M., TsoU, K., DESOUZA,E., CHENG,C. & SELIGMAN, '4. 1957. Cytochemical demonstration of succinic dehydrogenase by the use of a new p nitrophenyl substituted ditetrazole. 3 Histochem Cj'rorhem 5, 420-436. PADYKUL.4, H A . & HERMAN, E. 1955. T h e specificity of the histochemical method for adenosine triphosphatase. 3 Historhem Cytochem 3, 17c-195. PETER, J.B., BARNARD,R.J., EDGERTON,V.R., GILLESPIE, C.A. & STEMPEL,K.E. 1972. Metabolic profiles of three fiber types of skeletal muscle in guinea-pigs and rabbits. Biochemistry 11, 26272633. WATTENBERG, L.W. & LEONG, J.L. 1960. Effects of coenzyme Qo and menadione on succinic dehydrogenase activity as measured by tetrazolium salt reduction. 3 Histochem Cytochem 8, 296-303. YACHLAS.
