REPORT
The Effect of Short Duration Static Stretching and Reinnervation on the Recovery of Denervated Soleus Muscle in the Rat Harutoshi SAKAKIMA1 and Yoshihiro YOSHIDA1 1
School of Health Sciences, Faculty of Medicine, Kagoshima University, 8–35–1, Sakuragaoka, Kagoshima 890-8506, Japan
Abstract. Denervation elicits profound alterations in the morphometry of the skeletal muscle. There is evidence that the increased mechanical load placed upon the muscle via rhythmic stretching attenuates denervation induced alterations in muscle morphology. To investigate the effect of short duration static stretching (40 min/day) for denervated and reinnervated muscle, a histochemical study was performed on the soleus muscle of the rat. Twenty-one eight-week-old female Wistar rats were used. Partial denervation was carried out by locally freezing the proximal root of the sciatic nerve innervating the soleus muscle. Contralateral hindlimbs were untreated and served as control. Axonal degeneration was evident within the sciatic nerve following freezing, although variable amounts of damage were observed and thin nerve fibers were observed at 3 weeks. No clear difference of morphological change of the sciatic nerve was observed in the short duration static stretching groups (group S) or the non-stretching groups (group D). The wet weight of the denervated soleus muscles progressively declined to a minimum at 2 weeks after injury (group D, 50.8 ± 8.9%; group S, 61.3 ± 4.2%) and began to reverse in the following 3 weeks. Muscle wet weight for short duration static stretching groups similarly decreased and began to reverse in the following 3 weeks. The muscle fiber cross-sectional area also similarly changed with the muscle wet weight. The type II fiber ratios of the denervated sides were consistently higher than the control levels. In non-stretching groups, type II fibers had increased by 3 weeks after denervation (49.4%), whereas type II fiber ratios of the short duration static stretching groups decreased after 3 weeks (31.3%). These data suggests that mechanical stimuli provided by short duration static stretching can prevent the atrophy of the denervated muscle over a short period. In addition, it was indicated that short duration static stretching affected the reinnervated muscle fiber type composition. However, the reinnervation took the crucial role of recovering from the atrophy and composing the integrity of the soleus muscles. Key words: static stretching, denervation, reinnervation, muscle atrophy, sciatic nerve freezing, soleus muscle (J Jpn Phys Ther Assoc 5: 13–18, 2002)
The reduced stretch and decreased contractile activity in
prolonged bed rest, plaster cast immobilization, tenotomy and denervation cause rapid skeletal muscle atrophy1-5). Among these conditions, denervation produces the most serious atrophy6), and results in profound alterations in the morphometry of the skeletal muscle. These alterations include changes in fiber type composition and fiber size7). The denervation of the soleus muscle elicited an increase in Received: July 30, 2001 Accepted: January 12, 2002 Correspondence to: Harutoshi Sakakima, School of Health Sciences, Faculty of Medicine, Kagoshima University, 8–35–1, Sakuragaoka, Kagoshima 890-8506, Japan
the proportion of type II to total fibers, concomitant with a decrease in that of type I fibers7)8). The peripheral nervous system is capable of rapid and extensive reconstruction after nerve injury9). After nerve injury by freezing, the proximal portion of the injured nerve fibers send out new sprouts that cross the lesion and eventually reestablish their original connections10). The axonal extension has an essential relation with the morphological and functional integrity of the skeletal muscle10). One of the major goals in rehabilitation medicine is to prevent the muscular atrophy associated with the degeneration of nerves, and to promote their recovery along with the regeneration of the nerves.
Sakakima, et al.
14
Muscular loading including surgical ablation, exercise, and stretching produces muscular enlargement 11)12) . Stretching or increased tension on muscles is a major component contributing to skeletal muscular mass increase12). Stretching is a more effective stimulation to increase the protein synthesis in the muscular tissues than muscular contraction induced by electrical stimulus13) . Williams14) reported that in mouse soleus muscle a 30 min daily stretch produced only 9% loss of muscle weight compared with a 48% loss for continuous immobilization in the shortened position for 2 weeks. The passively muscle stretching for a 15 min prevented the increase in the connective tissue on immobilized muscle 15) . The short duration static stretching is more feasible than long-time stretching by the immobilization. There are few clinical or experimental reports on the effects of stretching on denervated and reinnervated muscles. In this study, the effect of short duration static stretching on denervated and reinnervated soleus muscles was studied.
Materials and Methods Animals and experimental protocols Twenty-one eight-week-old female Wistar rats weighing 197.2 ± 4.6 g (mean ± standard deviation) were obtained from Japan SLC Inc. (Hamamatsu, Japan). The following experimental protocol was approved by the ethical board of the Institute of Laboratory Animal Sciences of Kagoshima University. The rats were anesthetized by an intraperitoneal injection of sodium pentobarbital (50 mg/kg). The skin covering the right buttock was cut and the right sciatic nerve was isolated. The nerve was frozen and thawed several times by contact with a stainless spatula 3 mm in diameter cooled by liquid nitrogen. Care was taken not to injure the other tissues. The nerve became white when frozen, and the proximal margin of the frozen portion was loosely tied with a white thread for marking. Contralateral hindlimbs were untreated and served as a type of control. Three normal 8 week-old rats were also used as controls, in particular to survey the ratio of the type II to total fiber sizes and numbers in the normal soleus muscles. Food and water were supplied ad libitum. The degree of paralysis in the hindlimbs of the treated rats was checked every day. The rats were randomly assigned to 2 groups. In the first group the rats received the short duration static stretching treatment, which began 1 day after the injury and the bilateral soleus muscles were maximally stretched using nonelastic tape to maintain the dorsiflex posture of the ankle joints under arousal for 40 min light period a day, 6 times a week (group S, n=9). The stretching duration was decided referring to the report of Williams14). In the second
group the rats were not stretched and were served as denervated controls (group D, n=9). The animals were euthanized via the inhalation of an overdose of diethyl ether 1, 2, and 3 weeks (3 rats in each group) after the nerve freezing. The soleus muscles in both legs and the accompanying sciatic nerves of the frozen sides were removed en bloc. The distance between the proximal margin of the lesion and the nerve entrance into the soleus muscle was measured. The soleus muscles were quickly removed, cleaned of fat and connective tissues and quickly frozen in isopentane chilled with liquid nitrogen. The whole muscles were then stored at –70°C until analysis. The distal portion of the sciatic nerve to the lesion was immersed in 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) and embedded in epoxy resin. Histological and histochemical analysis The soleus muscles were mounted vertically on a cork plate in tragaganth gum jelly of appropriate softness16) to obtain cross-sections. The mounted muscle was then frozen by immersion in isopentane solution cooled in liquid nitrogen. Transverse sections (10 µ m) were cut in a cryostat cooled to – 20°C and stained with hematoxylin and eosin for the general observation. Cryosections were also stained for myosin adenosine triphosphatase (ATPase, pH 10.5, 4.3) reaction according to Guth and Samaha17) with some modifications, and then the muscle fibers were classified into type I or II fibers. The whole cross section of each soleus muscle stained by ATPase were photographed at a magnification of 20 for fiber-type composition. The central region of the soleus muscles was photographed at a magnification of 50 and all the muscle fibers delineated by entire fiber boundaries were measured for the cross sectional areas. A random sample of 100–110 fibers from each soleus muscle was analyzed. The proportion of the type II fiber area was expressed as a percent of that of the total fiber. The sciatic nerves were stained with 0.5% toluidine blue in 0.5% borate and observed by conventional light microscope. A Power Macintosh 8500 computer was employed with the NIH Image version 1.61 software (developed at the US National Institutes of Health and available on the Internet at http://rsp.info.nih.gov/nihimage/). Data analysis Statistical analyses were performed with a Macintosh computer using the StatView version 4.5 software (StatView). One-way analysis of variance (ANOVA) was used and when a significant F ratio was found, post-hoc Fisher’s protected least significant differences (PLSD) test was performed on each variable. Student’s t-test was employed when the data of groups D and S were compared. Significance was set at p
The Effect of Short Duration Static Stretching and Reinnervation on the Recovery of Denervated Soleus Muscle in the Rat Harutoshi SAKAKIMA1 and Yoshihiro YOSHIDA1 1
School of Health Sciences, Faculty of Medicine, Kagoshima University, 8–35–1, Sakuragaoka, Kagoshima 890-8506, Japan
Abstract. Denervation elicits profound alterations in the morphometry of the skeletal muscle. There is evidence that the increased mechanical load placed upon the muscle via rhythmic stretching attenuates denervation induced alterations in muscle morphology. To investigate the effect of short duration static stretching (40 min/day) for denervated and reinnervated muscle, a histochemical study was performed on the soleus muscle of the rat. Twenty-one eight-week-old female Wistar rats were used. Partial denervation was carried out by locally freezing the proximal root of the sciatic nerve innervating the soleus muscle. Contralateral hindlimbs were untreated and served as control. Axonal degeneration was evident within the sciatic nerve following freezing, although variable amounts of damage were observed and thin nerve fibers were observed at 3 weeks. No clear difference of morphological change of the sciatic nerve was observed in the short duration static stretching groups (group S) or the non-stretching groups (group D). The wet weight of the denervated soleus muscles progressively declined to a minimum at 2 weeks after injury (group D, 50.8 ± 8.9%; group S, 61.3 ± 4.2%) and began to reverse in the following 3 weeks. Muscle wet weight for short duration static stretching groups similarly decreased and began to reverse in the following 3 weeks. The muscle fiber cross-sectional area also similarly changed with the muscle wet weight. The type II fiber ratios of the denervated sides were consistently higher than the control levels. In non-stretching groups, type II fibers had increased by 3 weeks after denervation (49.4%), whereas type II fiber ratios of the short duration static stretching groups decreased after 3 weeks (31.3%). These data suggests that mechanical stimuli provided by short duration static stretching can prevent the atrophy of the denervated muscle over a short period. In addition, it was indicated that short duration static stretching affected the reinnervated muscle fiber type composition. However, the reinnervation took the crucial role of recovering from the atrophy and composing the integrity of the soleus muscles. Key words: static stretching, denervation, reinnervation, muscle atrophy, sciatic nerve freezing, soleus muscle (J Jpn Phys Ther Assoc 5: 13–18, 2002)
The reduced stretch and decreased contractile activity in
prolonged bed rest, plaster cast immobilization, tenotomy and denervation cause rapid skeletal muscle atrophy1-5). Among these conditions, denervation produces the most serious atrophy6), and results in profound alterations in the morphometry of the skeletal muscle. These alterations include changes in fiber type composition and fiber size7). The denervation of the soleus muscle elicited an increase in Received: July 30, 2001 Accepted: January 12, 2002 Correspondence to: Harutoshi Sakakima, School of Health Sciences, Faculty of Medicine, Kagoshima University, 8–35–1, Sakuragaoka, Kagoshima 890-8506, Japan
the proportion of type II to total fibers, concomitant with a decrease in that of type I fibers7)8). The peripheral nervous system is capable of rapid and extensive reconstruction after nerve injury9). After nerve injury by freezing, the proximal portion of the injured nerve fibers send out new sprouts that cross the lesion and eventually reestablish their original connections10). The axonal extension has an essential relation with the morphological and functional integrity of the skeletal muscle10). One of the major goals in rehabilitation medicine is to prevent the muscular atrophy associated with the degeneration of nerves, and to promote their recovery along with the regeneration of the nerves.
Sakakima, et al.
14
Muscular loading including surgical ablation, exercise, and stretching produces muscular enlargement 11)12) . Stretching or increased tension on muscles is a major component contributing to skeletal muscular mass increase12). Stretching is a more effective stimulation to increase the protein synthesis in the muscular tissues than muscular contraction induced by electrical stimulus13) . Williams14) reported that in mouse soleus muscle a 30 min daily stretch produced only 9% loss of muscle weight compared with a 48% loss for continuous immobilization in the shortened position for 2 weeks. The passively muscle stretching for a 15 min prevented the increase in the connective tissue on immobilized muscle 15) . The short duration static stretching is more feasible than long-time stretching by the immobilization. There are few clinical or experimental reports on the effects of stretching on denervated and reinnervated muscles. In this study, the effect of short duration static stretching on denervated and reinnervated soleus muscles was studied.
Materials and Methods Animals and experimental protocols Twenty-one eight-week-old female Wistar rats weighing 197.2 ± 4.6 g (mean ± standard deviation) were obtained from Japan SLC Inc. (Hamamatsu, Japan). The following experimental protocol was approved by the ethical board of the Institute of Laboratory Animal Sciences of Kagoshima University. The rats were anesthetized by an intraperitoneal injection of sodium pentobarbital (50 mg/kg). The skin covering the right buttock was cut and the right sciatic nerve was isolated. The nerve was frozen and thawed several times by contact with a stainless spatula 3 mm in diameter cooled by liquid nitrogen. Care was taken not to injure the other tissues. The nerve became white when frozen, and the proximal margin of the frozen portion was loosely tied with a white thread for marking. Contralateral hindlimbs were untreated and served as a type of control. Three normal 8 week-old rats were also used as controls, in particular to survey the ratio of the type II to total fiber sizes and numbers in the normal soleus muscles. Food and water were supplied ad libitum. The degree of paralysis in the hindlimbs of the treated rats was checked every day. The rats were randomly assigned to 2 groups. In the first group the rats received the short duration static stretching treatment, which began 1 day after the injury and the bilateral soleus muscles were maximally stretched using nonelastic tape to maintain the dorsiflex posture of the ankle joints under arousal for 40 min light period a day, 6 times a week (group S, n=9). The stretching duration was decided referring to the report of Williams14). In the second
group the rats were not stretched and were served as denervated controls (group D, n=9). The animals were euthanized via the inhalation of an overdose of diethyl ether 1, 2, and 3 weeks (3 rats in each group) after the nerve freezing. The soleus muscles in both legs and the accompanying sciatic nerves of the frozen sides were removed en bloc. The distance between the proximal margin of the lesion and the nerve entrance into the soleus muscle was measured. The soleus muscles were quickly removed, cleaned of fat and connective tissues and quickly frozen in isopentane chilled with liquid nitrogen. The whole muscles were then stored at –70°C until analysis. The distal portion of the sciatic nerve to the lesion was immersed in 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) and embedded in epoxy resin. Histological and histochemical analysis The soleus muscles were mounted vertically on a cork plate in tragaganth gum jelly of appropriate softness16) to obtain cross-sections. The mounted muscle was then frozen by immersion in isopentane solution cooled in liquid nitrogen. Transverse sections (10 µ m) were cut in a cryostat cooled to – 20°C and stained with hematoxylin and eosin for the general observation. Cryosections were also stained for myosin adenosine triphosphatase (ATPase, pH 10.5, 4.3) reaction according to Guth and Samaha17) with some modifications, and then the muscle fibers were classified into type I or II fibers. The whole cross section of each soleus muscle stained by ATPase were photographed at a magnification of 20 for fiber-type composition. The central region of the soleus muscles was photographed at a magnification of 50 and all the muscle fibers delineated by entire fiber boundaries were measured for the cross sectional areas. A random sample of 100–110 fibers from each soleus muscle was analyzed. The proportion of the type II fiber area was expressed as a percent of that of the total fiber. The sciatic nerves were stained with 0.5% toluidine blue in 0.5% borate and observed by conventional light microscope. A Power Macintosh 8500 computer was employed with the NIH Image version 1.61 software (developed at the US National Institutes of Health and available on the Internet at http://rsp.info.nih.gov/nihimage/). Data analysis Statistical analyses were performed with a Macintosh computer using the StatView version 4.5 software (StatView). One-way analysis of variance (ANOVA) was used and when a significant F ratio was found, post-hoc Fisher’s protected least significant differences (PLSD) test was performed on each variable. Student’s t-test was employed when the data of groups D and S were compared. Significance was set at p
