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.
Keywords: static stretching, denervation, reinnervation, muscle atrophy, sciatic nerve freezing, soleus muscle
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 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.
Muscular loading including surgical ablation, exercise, and stretching produces muscular enlargement11)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 muscle15). 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 µm) 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<0.05.
Result
After the nerve freezing, the rats were ambulatory and dragged the foot of the frozen side. The loss of active movement on the ankle and toe joints was observed. The paralysis was alleviated and voluntary extension of digits was noticed approximately 3 weeks after the operation. No apparent difference was recognized in the activity between groups D and S.
The distance between the proximal margin of the lesion and the entrance of the sciatic nerve into the soleus muscle was 20.4 ± 1.1 mm in group D and 20.1 ± 0.8 mm in group S.
Changes in the sciatic nerve
The distal portion of the nerve to the frozen lesion was uniformly damaged and swollen. One week after the injury most myelin had clearly degenerated and the centers of myelin where axons had been located were densely stained. These findings were found up to 2 weeks after the injury (Fig. 1). A considerable number of macrophages, probably containing myelin debris, were observed. Central light areas indicated few axons. In contrast, 3 weeks after, the numerous thin formations of myelin emerged, surrounding the small light centers inside (Fig. 1). A significant difference between groups S and D could not be recognized.
Fig. 1.
Cross sections of the sciatic nerve after freezing, with 0.5% toluidin blue in 0.5% borate. A normal sciatic nerve (a) showed numerous instances of ovoid and ellipsoid myelin surrounding the light areas of axons at their centers. Most myelin and axons had degenerated 2 weeks after freezing (b). Numerous thin formations of myelin were observed with small light centers inside after 3 weeks (c). Bar=50 µm.
Changes in the wet weight and the cross-sectional areas of each fiber type in the soleus muscles
There was no significant difference between the left and right sides of the soleus muscle wet weights of normal 8-week-old rats. Because individual muscle wet weight was related to the body weight, the muscle to body weight ratio was employed. The ratios were significantly smaller than those of the contralateral control sides in each period (p<0.05, Table 1). In both groups D and S, the wet weight of the denervated soleus muscles progressively declined to a minimum 2 weeks after the injury and began to increase in the following week. A significant difference in the wet weights and the ratio of the soleus muscles to body weight between the groups D and S was recognized 2 weeks after the injury (p<0.05). However, after 3 weeks no significant difference in the weight of the rats was observed.
Table 1. The wet weight and the relative weight of the soleus muscle to the body before, 1, 2, and 3 weeks after the nerve freezing.
| Groups (Number of muscles) | Before freezing | 1 week | 2 weeks | 3 weeks | |
|---|---|---|---|---|---|
| Group D | MW (mg) | 52.7 ± 2.3*† | 37.0 ± 3.6*† | 62.1 ± 2.5*† | |
| (3) | MW/BW | 0.18 ± 0.13*† | 0.17 ± 0.02*† | 0.27 ± 0.02*† | |
| Group S | MW (mg) | 53.7 ± 2.3*† | 45.7 ± 1.5*#† | 63.7 ± 7.5* | |
| (3) | MW/BW | 0.28 ± 0.01*† | 0.27 ± 0.01*#† | 0.28 ± 0.03*† | |
| Contralateral side | MW (mg) | 71.7 ± 5.2 | 70.6 ± 4.6 | 74.2 ± 7.4 | 83.0 ± 5.9 |
| (6) | MW/BW | 0.39 ± 0.02 | 0.36 ± 0.02 | 0.35 ± 0.03 | 0.38 ± 0.02 |
Values are mean ± SD. MW: muscle wet weight. BW: body weight (g).
p<0.05 (compared with Contralateral side),
p<0.05 (compared with Group D),
p<0.05 (compared with before freezing).
The six contralateral muscles after freezing were the total of the uninjured side of Group D and S.
The changes in each fiber cross-sectional area are shown in Table 2. In both groups D and S, The cross-sectional area of muscle fiber had significantly decreased 2 weeks after the injury compared with before freezing and began to increase at 3 weeks.
Table 2. Total cross-sectional areas of soleus muscle type I and II fibers and the ratio of type II to total fiber number.
| Groups (Number of muscles) | Before freezing | 1 week | 2 weeks | 3 weeks | |
|---|---|---|---|---|---|
| FCA (µm2) | |||||
| Group D | Type I | 1485.6 ± 391.0* | 1285.3 ± 353.5*† | 1861.8 ± 534.9* | |
| (3) | Type II | 1095.0 ± 333.6 | 731.2 ± 332.4*† | 1184.3 ± 450.7* | |
| Type II fiber ratio(%) | 28.8 ± 6.1*† | 37.4 ± 14.7*† | 49.4 ± 6.7*† | ||
| FCA (µm2) | |||||
| Group S | Type I | 1808.4 ± 505.9 | 1765.2 ± 447.0*#† | 1841.7 ± 536.9* | |
| (3) | Type II | 1191.3 ± 266.8 | 1046.9 ± 262.8*† | 1270.2 ± 409.2* | |
| Type II fiber ratio(%) | 30.0 ± 4.3*† | 35.3 ± 8.5*† | 31.1 ± 1.5*#† | ||
| FCA (µm2) | |||||
| Contralateral side | Type I | 2185.8 ± 349.1 | 2043.2 ± 714.7 | 2632.8 ± 574.3 | 3117.6 ± 285.1† |
| (6) | Type II | 1506.6 ± 275.6 | 1470.3 ± 597.3 | 1856.1 ± 598.4 | 2189.8 ± 316.4† |
| Type II fiber ratio(%) | 11.7 ± 6.8 | 14.6 ± 3.9 | 11.7 ± 3.2 | 14.9 ± 2.8 | |
Values are mean ± SD. FCA; Fiber cross-sectional area.
p<0.05 (compared with Contralateral side),
p<0.05 (compared with Group D),
p<0.05 (compared with before freezing).
The significant difference in the type I fiber cross-sectional areas of denervated soleus muscles between groups D and S was recognized 2 weeks after the injury (p<0.05). However, after 3 weeks no significant difference in the weight of the rats was observed.
Conversion of the muscle fiber type
After 2 weeks, the percentage of the cross-sectional area in the denervated soleus muscles compared with the contralateral counterparts was 53.3 ± 8.7% in type I fibers and 41.0 ± 9.5% in type II fibers in group D, and 63.0 ± 19.7% in type I fibers and 42.1 ± 13.5% in type II fibers in group S. The type I fiber area of group D was intensely decreased but the difference was not significant. It suggested the conversion of the muscular from type I to II.
The ratios of the type II to total fiber numbers in the denervated soleus muscles were significantly higher than those in the normal rats and the contralateral uninjured side in each period (p<0.05, Table 2). In group D, the type II fiber ratios increased 3 weeks after the denervation, whereas type II fiber ratios in group S decreased after 3 weeks (Table 2).
Histochemical analysis of dennervated and the subsequent reinnervated soleus muscle
Small sized darkly stained type II and non-stained type I fibers with ATPase reaction were found in the soleus muscles 3 weeks after the nerve freezing in groups D (Fig. 2c) and S (Fig. 2b) compared with those in the normal control muscle (Fig. 2a). In both groups D and S the number of type II fibers was increased. Many moderately stained fibers were recognized in the group D, which were classified into type II.
Fig. 2.
Cross sections of the soleus muscle, stained with alkaline pretreated ATPase reaction. The number of darkly stained type II fibers increased in denervated and the subsequent reinnervated soleus muscles of both the stretching (b) and non-stretching (c) groups 3 weeks after freezing compared with that in the normal control (a). The size of type II fibers and non-stained type I fibers in both Fig 2b and 2c became small in comparison with that of the normal control. The type II fibers were significantly increased in both the stretching and non-stretching groups. Many moderately stained fibers were found, which were classified as type II fibers in the non-stretching group. Accordingly, in this group the number of type II fibers was significantly increased; however, these fibers were perhaps changing from type II to I. Bar = 100 µm.
Discussion
Freezing injury was selected as the method of denervation because this procedure uniformly and definitely damaged nerve fibers with reinnervation compared with other procedures such as nerve crush or transection with a suture10). Frozen sciatic nerves regenerated well and the histological changes of the soleus muscles caused by the denervation soon recovered along with the reinnervation.
The muscle wet weight and fiber cross-sectional areas were regenerated 2 to 3 weeks after the freezing. It was roughly estimated that it took approximately 3 weeks for the process of the sciatic nerve to attain to the soleus muscles for approximately 20 mm, which was compatible with the data reported previously9)10)18).
The denervation produced serious atrophy comparing the immobilization due to the muscle disuse caused by the paralysis and the tenotomy for the deprivation of trophic substances6)19)20). In both the short duration static stretching and non-stretching groups, the muscular wet weight and the cross-sectional area of type I and II fibers showed progressive decrease up to two weeks after the denervation, and the area of type I fibers were significantly smaller in the non-stretching group than in the stretching group. However, in 3 weeks the wet weight and the areas became equal. This suggested that the muscle atrophy ameliorated in both groups. The morphological responses of the muscle to the denervation were fiber type specific, although both type I and II fibers of the denervated soleus muscles elicited marked atrophy. The atrophy was significantly greater in type II fibers. These results were found in both the non-stretched and the short duration static stretched rats.
More than 75% of the normal soleus muscle of the rat is composed of type I fibers21)22). This value increases with age and changes by varying procedures8)23). A decrease of muscle activity generally facilitates transformation of fiber type from slow to fast of the muscle fiber composition8). The increase of type II fibers is observed not only in denervation but also in cast immobilization, unloading, and spinal cord injury24)25). In this study, no attempt was made to distinguish between subtypes of type II fibers due to technical difficulties. In the non-stretching groups, type II fibers increased 3 weeks after denervation as described using the sciatic nerve freezing model10), whereas the type II fiber ratios of the short duration static stretched groups decreased over 3 weeks. The ratio of type II to total fiber numbers was significantly increased in the non-stretching group more than in the stretching group. The histochemical analysis of the soleus muscle in this period showed many moderately stained fibers with ATPase reaction, which were classified as type II fibers. These moderately stained fibers were possibly type changing fibers from type II to I. The results suggested that short duration static stretching affected the reinnervated muscle fiber type composition.
Muscular rehabilitation for denervation-induced atrophy is not an easy task. Previously reported data indicated that rhythmic stretching and the associated increase in mechanical loading could attenuate the marked morphological adaptations that characterized the denervated skeletal muscle1). The early short duration stretching prevented the muscular atrophy of the denervated muscle26). Pachter and Eberstein27) observed that immobilization of the denervated muscle in a stretched position led to an increased postsynaptic area of junctional folds and clefts per nerve terminal. In contrast, Deschenes et al.7) stated that the mechanical stimuli were not sufficiently potent to ameliorate the muscle morphometric responses to the denervation. It was still hypothesized that the short duration stretching would ameliorate the changes in muscle morphometry induced by denervation and reinnervation. Whether these is a relation between the short duration static stretching and the nerve regeneration remains to be resolved. This study suggested an inhibiting effect of the stretching over a short period on the atrophy of the type I fibers in the denervated muscle. However, the recovery of the muscle atrophy occurred in association with the reinnervation. The nerve supply has an essential role in the morphological and probably functional recovery of the skeletal muscle. The denervation resulted in the reduction of the muscle wet weight and fiber size, and changes in fiber type composition. The mechanical stimuli provided by short duration static stretching could prevent the atrophy of the denervated muscle over a short period. We therefore presume that stretch training may be effective therapy for prevent the muscle atrophy by denervation in short period.
However, the reinnervation took the crucial role of recovering from the atrophy and of composing the integrity of the soleus muscles.
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