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Effects of passive exercise on neurogenic atrophy in rat skeletal muscle
EXPERIMENTAL
NEUROLOGY
90,467-470
(1985)
RESEARCH
NOTE
Effects of Passive Exercise on Neurogenic in Rat Skeletal Muscle
Atrophy
B.R.PACHTERANDA.EBERSTEIN' Department of Rehabilitation Medicine, New York Universil), Medical Center. New York, New York 10016 Received April 2, 1985; revision received June 28. I985 Passiveexercise treatment for 23 days produced a retardation of type II muscle fiber atrophy in denervated extensor digitorum longus muscle of rat compared with denervated-nontreated animals. The type I muscle fibers of both denervated groups were similar to that of Control rats. 0 1985 Academic Press, Inc.
After denervation induced by nerve transection, various alterations occur in muscle. For example, the electrical properties of the muscle’s membrane become altered (lo), the muscle fibers become highly sensitive to acetylcholine over their entire length instead of only at the end-plate due to the extrajunctional spread of acetylcholine receptors (1 ), the fiber’s organelles undergo alterations (3), there is a decrease in the mitochondrial oxidative capacity of the component muscle fibers (II), the neuromuscular junctions begin to degenerate ( 16), and the fibers become atrophic (3). In rehabilitation medicine, passive exercise or stretch is often utilized as a therapeutic modality to retard the development of contractures, decrease fibrosis, as well as enhancing blood flow to the muscle (9, 15). Although passive exercise is extensively used in rehabilitation, there is a paucity of studies that have evaluated its actual benefit. Abbreviation: EDL-extensor digitorum longus. ’ This research was supported by grant GOO83OOO71 from the National Institute of Handicapped Research, U.S. Department of Education, Washington, DC., and from the National Easter Seal Research Foundation. The authors acknowledge the technical assistance of Carin Colbjornsen and Ruth Johnston 467 0014-4886/85 $3.00 Copynghl 0 1985 by Academic Press. Inc All rights of reproductmn I” any form reserved.
468
PACHTER
AND
EBERSTEIN
In a recent study evaluating passive movement on denervated rabbit limb muscle, Petajan et al. (14) reported no statistically significant difference in the mean fiber diameter between denervated-nontreated and denervated-exercised muscles. The authors acknowledged that the passive movement they employed imposed a mechanical strain on the denervated muscle fibers which resulted in many structural alterations of the muscle fibers as well as endomysial fibrosis. It would appear that such a mechanical strain might also induce additional necrosis and muscle atrophy. In view of this uncertainty, we studied the effect of passive exercise on rat muscle denervated by cutting the sciatic nerve and evaluated its effects on the component myofibers. A total of 12 female Wistar rats (2 to 3 months of age) were used in this study. The right hind limb of eight animals was denervated under ether anesthesia by excision of a 0.5-cm segment of the sciatic nerve just proximal to the bifurcation of the tibia1 and peroneal nerves. The cut end of the proximal portion of the nerve was capped with Silastic tubing to prevent reinnervation. The denervated animals were divided into two groups according to the treatment received. One group of four rats received passive exercise treatment, and another group of four rats was not treated and served as denervated controls; the remaining four animals were not denervated nor exercised and served as normal controls. Passive exercise treatment began 4 h after surgery and was carried out in the conscious animal. Each rat was confined in a restrainer cage with its denervated foot secured with tape to a device designed to immobilize the ankle but allow flexion of the toes. This was accomplished by a cut-away “boot” to support the ankle with a hinged “sole” to permit toe flexion and extenison. The hinge was forced up (toes extended) by a cam shaft coupled to a small DC motor and forced down (toes flexed) by a spring attached to the underside of the hinge. The extensor digitorum longus (EDL) muscle was thus stretched 3 mm, 20 times per minute, one hour per day per rat for a total of 23 days. The duration of passive exercise in this study was similar to that used in a prior investigation utilizing electrical stimulation on denervated EDL muscle (13). After 23 days of passive exercise treatment, the EDL muscles from the two denervated groups were exposed and the proximal end of the sciatic nerve was stimulated to ascertain if any reinnervation had occurred. No response was observed in any of the experimental animals. The EDL muscles from all groups were then removed with a U-shape muscle biopsy clamp and quickfrozen in isopentane cooled with liquid nitrogen. Cross sections (10 pm) from the muscle belly (middle third of muscle) were cut in a cryostat at -30°C. To differentiate muscle fiber types, the histochemical method for myofibrillar ATPase in the modification of Guth and Samaha (8) was used, and types I and II identified. To quantitate the degree of change in fiber size, a Numonics electronic graphics digitizer was used to measure cross-sectional areas of at least 100
NEUROGENIC
ATROPHY
IN RAT
469
muscle fibers per muscle from photomicrographs of ATPase-stained sections. The significance between the groups was determined by Student’s t test. All P values below 0.05 were considered significant. The results of fiber sizes are summarized in Table 1. It is apparent from Table 1 that there was no significant difference for each experimental group as regards the type I fiber’s cross-sectional area 23 days postdenervation. The type II fibers from both denervated groups atrophied compared with controls; however, the type II fibers from the denervated-exercised group were significantly larger (P < 0.01) than those type II fibers in the denervated-nonexercised group. We did not observe in either experimental group any evidence of structural alterations or endomysial fibrosis. Petajan er al. (14) reported no statistically significant benefit for rabbit muscles that received passive exercise compared with nontreated denervated muscles after 21 days of treatment. They also observed greater structural alterations and endomysial fibrosis in the treated muscles. In contrast, our exercise protocol did not produce any of the pathologic changes nor endomysial fibrosis described by Petajan et al. (14). In addition, our data show that passive exercise is highly beneficial in retarding denervation atrophy in the type II muscle fibers. This is the predominate fiber population in rat EDL muscle. The type I muscle fibers, as seen in the present study, were unaffected by denervation, at least to 23 days postsurgery. This is consistent with the findings of Niederle and Mayr (12). It has been suggested that the pronounced resistance of type I fibers to denervation atrophy may be due to their minor degree of neurotrophic dependence (2). Passive exercise has been shown to reduce joint contractures and edema as well as enhance the circulation (9, 15). The possibility exists that passive exercise might slow protein degradation in the muscle, thereby retarding the process of atrophy. Muscle activity (6) and increased work demands (4, 5) are known to stimulate protein synthesis and inhibit protein breakdown. PasTABLE I Type I and Type II Fibers in Control, Denervated-Nontreated, and Denervated-Exercised Rat Extensor Digitorum Lmgus Muscles” Control Fiber areas Type1 Type 11
1132 +_273 2020 f 550
Denervated-nontreated 1092 f 191 833 + 95
Denervated-exercised 1051 rt_245 1148 + 145
0 Values are X k SD pm*. Measurements were made in cross sections from middle third of muscle length in four rats per group. There was a significant difference (P < 0.01) between the denervated-nontreated and denervated-excised type II fibers.
470
PACHTER
AND
EBERSTEIN
sive stretch was reported (5) to inhibit protein breakdown in innervated muscle and was shown by Goldspink (7) to cause an increased accumulation of proteins and promote muscle growth in denervated muscle. Our data demonstrate that passive exercise in muscle without an intact nerve supply can retard muscle fiber atrophy at least to 23 days postdenervation. Presumably, it does so by inhibiting protein degradation. REFERENCES 1. AXELSSON, J., AND S. THESLEFF. 1959. Study of supersensitivity in denervated mammalian skeletal muscle. J. Physiol. (London) 147: 178-193. 2. BAJUSZ,E. 1964. “Red” skeletal muscle fibers: relative independence of neural control. Science 145: 938-939.
ENGEL, A. G., AND H. H. STONNINGTON. 1974. Morphological effects of denervation of muscle: quantitative ultrastructural study. Ann. N. Y. Acad. Sci. 228: 68-88. 4. GOLDBERG, A. L.. C. JABLECKI, AND J. B. LA. 1974. Effect of use and disuse on amino acid transport and protein turnover in muscle. Ann. N. Y. Acad. Sci. 228: 190-20 1. 5. GOLDBERG, A. L., J. D. ETLINGER, D. F. GOLDSPINK, ANDC. JABLECKI. 1975. Mechanism of work-induced hypertrophy of skeletal muscle. Med. Sci. Sports 7: 248-26 1. 6. GOLDSPINK, D. J. 1977. The influence of activity on muscle size and protein turnover. J. 3.
Physiol.
(London)
264: 283-296.
7. GOLDSPINK, D. J. 1978. The influence of passive stretch on the growth and protein turnover of the denervated extensor digitorum longus muscle. Biochem. J. 174: 595-602. 8. GUTH. L., AND F. J. SAMAHA. 1970. A modification of the myofibrillar ATPase stain. Exp. Neural.
28: 365-367.
9. KOTTKE, F. J.. D. L. PAULEY, AND R. A. PTAK. 1966. Rationale for prolonged stretching for correction of shortening of connective tissue. Arch. Phys. Med. Rehabil. 47: 345-352. 10. L@MO, T. 1976. Role of activity in control of membrane and contractile properties of skeletal muscle. Pages 289-321 in S. THESLEFF, Ed., Motor Innervation of Muscle. Academic Press, New York/London. 1I. NEMETH, P. M. 1982. Electrical stimulation of denervated muscle prevents decreases in oxidative enzymes. Muscle Nerve 5: 134- 139. 12. NIEDERLE, B., AND R. MAYR. 1978. Course of denervation atrophy in type 1 and type II fibers of rat extensor digitorum longus muscle. Anal. Embryo/. 153: 9-2 I. 13. PACHTER, B. R., A. EBERSTEIN,AND J. GOODGOLD. 1982. Electrical stimulation effect on denervated skeletal myofibers in rats: a light and electron microscopic study. Arch. Pys. Med.
Rehabil.
63: 427-430.
14. PETAJAN, J. H., G. SONGSTER, AND D. MCNEIL. 1981. Effects of passive movement on neurogenic atrophy in rabbit limb muscles. Exp. Neurol. 71: 92-103. 15. POLLOCK, L. J., A. J. ARIEF’F,I. C. SHERMAN, AND M. SCHILLER. 1950. The effect of massage and passive movement upon the residuals of experimentally produced section of the sciatic nerves of the cat. Arch. Phys. Med. 31: 265-276. 16. PULLIAM, D. L.. AND E. W. APRIL. 1979. Degenerative changes at the neuromuscular junctions of red, white and intermediate muscle fibers. J. Neurol. Sci. 43: 205-222.
NEUROLOGY
90,467-470
(1985)
RESEARCH
NOTE
Effects of Passive Exercise on Neurogenic in Rat Skeletal Muscle
Atrophy
B.R.PACHTERANDA.EBERSTEIN' Department of Rehabilitation Medicine, New York Universil), Medical Center. New York, New York 10016 Received April 2, 1985; revision received June 28. I985 Passiveexercise treatment for 23 days produced a retardation of type II muscle fiber atrophy in denervated extensor digitorum longus muscle of rat compared with denervated-nontreated animals. The type I muscle fibers of both denervated groups were similar to that of Control rats. 0 1985 Academic Press, Inc.
After denervation induced by nerve transection, various alterations occur in muscle. For example, the electrical properties of the muscle’s membrane become altered (lo), the muscle fibers become highly sensitive to acetylcholine over their entire length instead of only at the end-plate due to the extrajunctional spread of acetylcholine receptors (1 ), the fiber’s organelles undergo alterations (3), there is a decrease in the mitochondrial oxidative capacity of the component muscle fibers (II), the neuromuscular junctions begin to degenerate ( 16), and the fibers become atrophic (3). In rehabilitation medicine, passive exercise or stretch is often utilized as a therapeutic modality to retard the development of contractures, decrease fibrosis, as well as enhancing blood flow to the muscle (9, 15). Although passive exercise is extensively used in rehabilitation, there is a paucity of studies that have evaluated its actual benefit. Abbreviation: EDL-extensor digitorum longus. ’ This research was supported by grant GOO83OOO71 from the National Institute of Handicapped Research, U.S. Department of Education, Washington, DC., and from the National Easter Seal Research Foundation. The authors acknowledge the technical assistance of Carin Colbjornsen and Ruth Johnston 467 0014-4886/85 $3.00 Copynghl 0 1985 by Academic Press. Inc All rights of reproductmn I” any form reserved.
468
PACHTER
AND
EBERSTEIN
In a recent study evaluating passive movement on denervated rabbit limb muscle, Petajan et al. (14) reported no statistically significant difference in the mean fiber diameter between denervated-nontreated and denervated-exercised muscles. The authors acknowledged that the passive movement they employed imposed a mechanical strain on the denervated muscle fibers which resulted in many structural alterations of the muscle fibers as well as endomysial fibrosis. It would appear that such a mechanical strain might also induce additional necrosis and muscle atrophy. In view of this uncertainty, we studied the effect of passive exercise on rat muscle denervated by cutting the sciatic nerve and evaluated its effects on the component myofibers. A total of 12 female Wistar rats (2 to 3 months of age) were used in this study. The right hind limb of eight animals was denervated under ether anesthesia by excision of a 0.5-cm segment of the sciatic nerve just proximal to the bifurcation of the tibia1 and peroneal nerves. The cut end of the proximal portion of the nerve was capped with Silastic tubing to prevent reinnervation. The denervated animals were divided into two groups according to the treatment received. One group of four rats received passive exercise treatment, and another group of four rats was not treated and served as denervated controls; the remaining four animals were not denervated nor exercised and served as normal controls. Passive exercise treatment began 4 h after surgery and was carried out in the conscious animal. Each rat was confined in a restrainer cage with its denervated foot secured with tape to a device designed to immobilize the ankle but allow flexion of the toes. This was accomplished by a cut-away “boot” to support the ankle with a hinged “sole” to permit toe flexion and extenison. The hinge was forced up (toes extended) by a cam shaft coupled to a small DC motor and forced down (toes flexed) by a spring attached to the underside of the hinge. The extensor digitorum longus (EDL) muscle was thus stretched 3 mm, 20 times per minute, one hour per day per rat for a total of 23 days. The duration of passive exercise in this study was similar to that used in a prior investigation utilizing electrical stimulation on denervated EDL muscle (13). After 23 days of passive exercise treatment, the EDL muscles from the two denervated groups were exposed and the proximal end of the sciatic nerve was stimulated to ascertain if any reinnervation had occurred. No response was observed in any of the experimental animals. The EDL muscles from all groups were then removed with a U-shape muscle biopsy clamp and quickfrozen in isopentane cooled with liquid nitrogen. Cross sections (10 pm) from the muscle belly (middle third of muscle) were cut in a cryostat at -30°C. To differentiate muscle fiber types, the histochemical method for myofibrillar ATPase in the modification of Guth and Samaha (8) was used, and types I and II identified. To quantitate the degree of change in fiber size, a Numonics electronic graphics digitizer was used to measure cross-sectional areas of at least 100
NEUROGENIC
ATROPHY
IN RAT
469
muscle fibers per muscle from photomicrographs of ATPase-stained sections. The significance between the groups was determined by Student’s t test. All P values below 0.05 were considered significant. The results of fiber sizes are summarized in Table 1. It is apparent from Table 1 that there was no significant difference for each experimental group as regards the type I fiber’s cross-sectional area 23 days postdenervation. The type II fibers from both denervated groups atrophied compared with controls; however, the type II fibers from the denervated-exercised group were significantly larger (P < 0.01) than those type II fibers in the denervated-nonexercised group. We did not observe in either experimental group any evidence of structural alterations or endomysial fibrosis. Petajan er al. (14) reported no statistically significant benefit for rabbit muscles that received passive exercise compared with nontreated denervated muscles after 21 days of treatment. They also observed greater structural alterations and endomysial fibrosis in the treated muscles. In contrast, our exercise protocol did not produce any of the pathologic changes nor endomysial fibrosis described by Petajan et al. (14). In addition, our data show that passive exercise is highly beneficial in retarding denervation atrophy in the type II muscle fibers. This is the predominate fiber population in rat EDL muscle. The type I muscle fibers, as seen in the present study, were unaffected by denervation, at least to 23 days postsurgery. This is consistent with the findings of Niederle and Mayr (12). It has been suggested that the pronounced resistance of type I fibers to denervation atrophy may be due to their minor degree of neurotrophic dependence (2). Passive exercise has been shown to reduce joint contractures and edema as well as enhance the circulation (9, 15). The possibility exists that passive exercise might slow protein degradation in the muscle, thereby retarding the process of atrophy. Muscle activity (6) and increased work demands (4, 5) are known to stimulate protein synthesis and inhibit protein breakdown. PasTABLE I Type I and Type II Fibers in Control, Denervated-Nontreated, and Denervated-Exercised Rat Extensor Digitorum Lmgus Muscles” Control Fiber areas Type1 Type 11
1132 +_273 2020 f 550
Denervated-nontreated 1092 f 191 833 + 95
Denervated-exercised 1051 rt_245 1148 + 145
0 Values are X k SD pm*. Measurements were made in cross sections from middle third of muscle length in four rats per group. There was a significant difference (P < 0.01) between the denervated-nontreated and denervated-excised type II fibers.
470
PACHTER
AND
EBERSTEIN
sive stretch was reported (5) to inhibit protein breakdown in innervated muscle and was shown by Goldspink (7) to cause an increased accumulation of proteins and promote muscle growth in denervated muscle. Our data demonstrate that passive exercise in muscle without an intact nerve supply can retard muscle fiber atrophy at least to 23 days postdenervation. Presumably, it does so by inhibiting protein degradation. REFERENCES 1. AXELSSON, J., AND S. THESLEFF. 1959. Study of supersensitivity in denervated mammalian skeletal muscle. J. Physiol. (London) 147: 178-193. 2. BAJUSZ,E. 1964. “Red” skeletal muscle fibers: relative independence of neural control. Science 145: 938-939.
ENGEL, A. G., AND H. H. STONNINGTON. 1974. Morphological effects of denervation of muscle: quantitative ultrastructural study. Ann. N. Y. Acad. Sci. 228: 68-88. 4. GOLDBERG, A. L.. C. JABLECKI, AND J. B. LA. 1974. Effect of use and disuse on amino acid transport and protein turnover in muscle. Ann. N. Y. Acad. Sci. 228: 190-20 1. 5. GOLDBERG, A. L., J. D. ETLINGER, D. F. GOLDSPINK, ANDC. JABLECKI. 1975. Mechanism of work-induced hypertrophy of skeletal muscle. Med. Sci. Sports 7: 248-26 1. 6. GOLDSPINK, D. J. 1977. The influence of activity on muscle size and protein turnover. J. 3.
Physiol.
(London)
264: 283-296.
7. GOLDSPINK, D. J. 1978. The influence of passive stretch on the growth and protein turnover of the denervated extensor digitorum longus muscle. Biochem. J. 174: 595-602. 8. GUTH. L., AND F. J. SAMAHA. 1970. A modification of the myofibrillar ATPase stain. Exp. Neural.
28: 365-367.
9. KOTTKE, F. J.. D. L. PAULEY, AND R. A. PTAK. 1966. Rationale for prolonged stretching for correction of shortening of connective tissue. Arch. Phys. Med. Rehabil. 47: 345-352. 10. L@MO, T. 1976. Role of activity in control of membrane and contractile properties of skeletal muscle. Pages 289-321 in S. THESLEFF, Ed., Motor Innervation of Muscle. Academic Press, New York/London. 1I. NEMETH, P. M. 1982. Electrical stimulation of denervated muscle prevents decreases in oxidative enzymes. Muscle Nerve 5: 134- 139. 12. NIEDERLE, B., AND R. MAYR. 1978. Course of denervation atrophy in type 1 and type II fibers of rat extensor digitorum longus muscle. Anal. Embryo/. 153: 9-2 I. 13. PACHTER, B. R., A. EBERSTEIN,AND J. GOODGOLD. 1982. Electrical stimulation effect on denervated skeletal myofibers in rats: a light and electron microscopic study. Arch. Pys. Med.
Rehabil.
63: 427-430.
14. PETAJAN, J. H., G. SONGSTER, AND D. MCNEIL. 1981. Effects of passive movement on neurogenic atrophy in rabbit limb muscles. Exp. Neurol. 71: 92-103. 15. POLLOCK, L. J., A. J. ARIEF’F,I. C. SHERMAN, AND M. SCHILLER. 1950. The effect of massage and passive movement upon the residuals of experimentally produced section of the sciatic nerves of the cat. Arch. Phys. Med. 31: 265-276. 16. PULLIAM, D. L.. AND E. W. APRIL. 1979. Degenerative changes at the neuromuscular junctions of red, white and intermediate muscle fibers. J. Neurol. Sci. 43: 205-222.