INTRODUCTION

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THE MUSCULAR DYSTROPHIES are genetically determined disorders with progressive degeneration of skeletal muscle fibers. Duchenne muscular dystrophy is due to the lack of the muscle dystrophin (14), a subsarcolemmal protein that links F-actin to a group of transsarcolemmal glycoproteins anchored to merosine, a compound of the extracellular matrix (4). Duchenne muscular dystrophy patients show a loss of skeletal muscle fibers that is not counterbalanced by muscle fiber regeneration, and fibrosis progressively replaces the degenerated myofibers (1). The muscles of mdx mice, a spontaneous mutant of the C57Bl/10 strain (designated below as C57) (2) also lack dystrophin because of a mutation that inserts a stop codon in exon 1 of the dystrophin gene (28). At the postnatal stage, hindlimb muscles are histologically normal (31). At the time of weaning, an acute round of myofiber degeneration-regeneration occurs, probably due to the increase in muscle activity (22), but no dystrophic phenotype is evident until 15 mo of age. Old mdx mice exhibit more severe dystrophic changes (18).

The mechanisms of the myofiber degeneration involved in dystrophin-deficient muscle remain speculative. The relationship between degeneration and contraction in mdx muscle has been assessed by a number of studies, the results of which are somewhat divergent. In recording chambers, the passive lengthening of both the diaphragm and the extensor digitorum longus (EDL) muscles during contraction damaged the myofibers (21, 25) and reduced the maximal tetanic force of EDL muscles (12) but was ineffective in the release of intracellular creatine kinase (19). In vivo, more controversial effects were described, although all experiments described the fact that mdx skeletal muscle fibers are more easily damaged (3, 5-7, 9-12, 27, 32). The long-term overload of EDL by removing the synergistic tibialis anterior muscle (TA) led to a progressive weakness and increased the areas of degeneration in mdx mice of various ages (5, 12). Susceptibility to necrosis was also noticed in the TA muscle after eccentric exercise induced by the repeated stimulation of the sciatic nerve (32). In another study on lengthened TA muscles (27), the stimulation of the peroneal nerve performed 12 days after contractions resulted in similar TA force loss in the adult mdx and C57 mice. After spontaneous nocturnal wheel running, EDL muscles presented a decrease in their strength (3) but an increase in the force output of the plantaris muscle (10) and an improved resistance of skeletal muscles to fatigability (10). Intentional wheel running also slowed the progression of dystrophy in the diaphragm (7) but not in association with the ₂-agonist clenbuterol (6). Finally, endurance swimming was claimed to have beneficial effects in EDL and soleus (Sol) contractile properties (9), and this effect was enhanced by clenbuterol treatment (11).

Up until now, three studies have suggested that mdx muscle degeneration is not inevitable. Wheat kernel ingestion was claimed to protect the progression of muscle weakness in mdx mice (15). The percentage of centronucleated myofibers was significantly reduced in plantaris muscles by 20-45 days postoperatively when the lumbosacral plexus was unilaterally avulsed or the thoracic spinal cord was sectioned in 15- to 18-day-old mdx mice (16). A single injection of the tetanus toxin in the posterior compartment of 3-wk-old mdx mice prevented the occurrence of centronucleated fibers in Sol muscle for 4 wk (20). To exemplify this later result, we hypothesized that the immobilization of one hindlimb in mdx mice done before the occurrence of the first round of degeneration would delay the onset of necrosis as a tetanus paralysis did. However, since the length at which a muscle is immobilized plays a role in its characteristics (30), we immobilized the right hindlimb of mdx and wild C57 mice in positions devoted to stretching or shortening of the Sol and EDL muscles. At the age of 35 days, sedentary mdx mice usually exhibit 50% centronucleated fibers in Sol muscle and 30% in EDL muscle (20). In mice with a hindlimb immobilized from the age of 21 days, the percentage of centronucleated fibers remained <10% 14 days later in both muscles, whatever their length of fixation.

	MATERIALS AND METHODS

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Animals and experimental protocol. Twenty-one-day-old male mice from the mdx and C57 inbred colonies of the Faculté de Médecine Saint-Antoine (Paris, France) were housed in plastic cages in a room kept at a constant temperature (21°C) with a natural night-day light cycle. They were fed with a commercial cube diet (A03 UAR, Villemoisson, France) and water ad libitum. Twenty-three mdx mice and thirteen C57 mice were used in this study. All protocols were conducted according to the Guide for the Care and Use of Laboratory Animals [DHEW Publication No. (NIH) 85-23, Revised 1985, Office of Science and Health Reports, DRR/NIH, Bethesda, MD 20892]. All surgical procedures were performed under chloral hydrate anesthesia (3.5%, 0.3 ml ip). The animals from the same litter were distributed randomly into three experimental groups. Nine mdx mice were sham operated in the first group, to be used as controls for spontaneous degeneration-regeneration. In the second group (mdx n = 7, C57 n = 7), the right hindlimb was immobilized in a position shortening Sol and stretching EDL muscles. In the third group (mdx n = 7, C57 n = 6), the right hindlimb was immobilized in a position stretching Sol and shortening EDL muscles. Muscles were examined 14 days later, when mice were 35 days old. Anesthetized animals were killed by cervical dislocation.

Immobilization procedure. To preserve the growth of the limb in these very young mice and to ensure a near-normal behavior of the animals, we immobilized the right hindlimb by applying a lightweight splint made from a glazed iron wire (diameter: 0.6 mm), with an open ring at the proximal extremity to be rolled around the thighbone and firmly fastened by mean of a pair of pliers. Several strips (width: 4 mm) of glazed malleable metal, meant to wrap the leg and the foot, were soldered at the distal part. The size of each splint was adjusted to the morphology of each mouse. Figure 1, A and B, shows X-ray photographs of adult mice illustrating good opacity of bones. Drawings of the two types of splints used in this study and the resulting length of the muscles are shown in Fig. 1, C and D. The first type (Fig. 1C) was developed to shorten Sol and stretch EDL muscles. The length of the wire was adjusted to flex the leg on the thigh, and a U-shaped piece of metal at the distal extremity put the foot in an extended position, with the flexed digits glued to the foot sole. The second type of splint (Fig. 1D) was committed to stretch the Sol and to shorten the EDL muscles. The wire was not straight but bent angular to maintain the leg extended at the thigh, and the foot was flexed on the leg and glued to a metallic shoe. Both splints were adjusted to the leg by means of a flange. The animals were anesthetized, and the right hindlimb was shaved. A small incision was made on the skin at the lateral side of the midthigh, and the muscles were spread to uncover the thighbone. Then the ring of the proximal part of the splint was secured around the bone, and sulfanilamide powder was applied. The leg and the foot were wrapped with the metal splint strips. Finally, the skin was sutured at the midthigh with prolene 5/0, and mice were kept in a warm place until they started to recover from anesthesia and were returned to normal conditions.

View larger version (90K): [in this window] [in a new window]   Fig. 1.   Immobilization of right leg in mice. A and B: X-ray photographs of the 2 types of splints and related position of hindlimb bones. C and D: drawing of the corresponding length of leg muscles.

Quantitative histology. Fourteen days after the onset of immobilization, the animals were anesthetized with chloral hydrate, and the sciatic nerve was stimulated by using a train of supramaximal square pulses through a stainless steel needle inserted at the sciatic notch. The contraction of the muscles located in the anterior and posterior compartment was checked visually to detect possible denervation of these muscles as a result of the presence of the splint. This was never observed. Then, Sol and EDL were excised bilaterally, mounted onto a cork piece with tragacanth gum, and frozen in isopentan chilled by liquid nitrogen. Cross sections of each muscle (10 µm thick) were cut with a cryostat in the middle part of the muscle belly (22, 32). The slides were processed by hematoxylin and eosin staining. To assess muscle degeneration-regeneration, a single section taken at the midpoint of the muscle was selected (22), and the muscle fibers of the cross section were counted by hand on microphotographs (magnification: ×100). The fibers showing peripheral nuclei did not endure degeneration and were classified as surviving fibers, and those presenting centrally placed nuclei and a ring of basophilic cytoplasm (diameter 7 µm) were considered as regenerating fibers. Nuclei with no discernible surrounding cytoplasm were discarded. The ratio of regenerating to surviving myofibers was calculated. Moreover, the whole cross-sectional area of the muscles was assessed by planimetry. Then, the mean cross-sectional area of the fibers was evaluated by the ratio of the whole muscle surface to the total number of myofibers. Results are given as the SD around the mean for each group. Comparisons between the means were made by using a two-sided alternate t-test assuming Gaussian populations with different SDs. Results were considered significant for P = 0.05. For both Sol and EDL muscles, comparisons were done 1) between the immobilized (shortened or stretched) or the sham-operated side and the intact side of the same animal; 2) between stretched and shortened muscles; and 3) between these muscles and the sham-operated ones. Comparisons were also done among the intact sides of the three experimental groups and between the mdx and C57 results for immobilized muscles.

	RESULTS

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Effects of immobilization in C57 mice. The locomotion of the mice with the right hindlimb immobilized was not impaired, and the behavior of the animals seemed normal. As shown in Fig. 2, A and B, the total number of myofibers after immobilization of Sol and EDL muscles in C57 was the same in treated and contralateral muscles. However, a 14-day period of Sol or EDL immobilization generated a muscle fiber atrophy in mice, as it was previously described in other mammals (23, 30). The immobilization of Sol muscle in the shortened position showed a significantly smaller fiber cross-sectional area than that of the contralateral muscle (P = 0.02). This effect was enhanced in EDL muscle immobilized in the stretched position (P = 0.006) (Fig. 2, C and D). No centrally nucleated fibers were detectable. These results indicate that the method used to immobilize the hindlimb did not injure the leg muscles. Therefore, we assumed that any modification occurring during mdx muscle degeneration-regeneration resulted from immobilization itself and not from the surgical procedure.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Effects of hindlimb immobilization in 3-wk-old C57Bl/10 mice on total number (No) (A and B) and the cross-sectional area of myofibers (C and D) in Sol (A and C) and EDL (B and D) muscles in shortened or stretched position. Values are means ± SD. * P < 0.05, ** P < 0.01. Nos. of animals: Sol shortened = 7, Sol stretched = 6, EDL shortened = 6, EDL stretched = 7.

Effects of Sol immobilization in mdx mice. In sham-operated animals, the total number of Sol myofibers and their cross-sectional areas were the same on both sides (Fig. 3, B and D). The centronucleation of the myofibers was significantly more prominent (P = 0.04) in the nonoperated side (Fig. 3F). This asymmetry of muscle degeneration-regeneration was previously observed in sedentary 35-day-old mdx mice (31). In operated animals, Sol muscles of the free left hindlimb showed the same percentage of centronucleated fibers (50.5 ± 8.5%) than those in control animals (Fig. 3, E and F). This suggests that the use of the left limb muscles was not increased to compensate for the contralateral immobilization. Immobilization did not modify the number of myofibers but reduced their cross-sectional area (Fig. 3, A and C). The atrophy reached 23% when Sol muscle was stretched (not significant) and 33% when Sol muscle was shortened (P = 0.02). The difference between the stretched and shortened muscles was not significant. This suggests that the atrophy results more from immobilization than from the length of the muscle. These percentages of atrophy were similar to those of C57 mice. However, intact muscles in mdx strain exhibited a cross-sectional area near 40% greater than that of C57 mice (P < 0.05). The most striking feature resulting from immobilization was the reduced number of myofibers that presented the first round of necrosis regeneration. Only 8.4 ± 6.5% centronucleated fibers were observable when Sol muscle was stretched (contralateral: 46.6 ± 10.3% centronucleated fibers) and 1.2 ± 1.6% (contralateral: 50.4 ± 16.7%) when Sol muscle was shortened. Differences between the two sides were highly significant (P = 0.0008) both in the stretched and shortened Sol positions (Fig. 3E). The difference between shortened muscles (1.2 ± 1.6%) and stretched ones (8.4 ± 6.5%) was also significant (P < 0.05).

View larger version (37K): [in this window] [in a new window]   Fig. 3.   Effects of 14-day immobilization of Sol muscle in 35 day-old mdx mice on total no. of myofibers (A), their cross-sectional area (C), and percentage of centronucleation of myofibers (E) in relation to length. Values from sham-operated controls are given in B, D, and F. Values are means ± SD. * P < 0.05, *** P < 0.001. Nos. of animals: Sol shortened = 7, Sol stretched = 7, controls = 9.

Effects of EDL immobilization in mdx mice. In EDL muscle, the modifications resulting from immobilization were similar to those observed in Sol muscle. In sham-operated animals, the number of myofibers was smaller (P = 0.05) in the right than in the left leg (Fig. 4B). The percentage of centronucleated fibers (30%) was the same on both sides (Fig. 4F). In immobilized mdx mice, the number of myofibers was not different, regardless of the length of the fixation (Fig. 4A). As in Sol muscles, atrophy was present in immobilized EDL. The myofiber cross-sectional area was smaller in stretched (34%, P = 0.005) than in shortened EDL muscles (22%, P = 0.03) (Fig. 4C). The difference between the two groups was not significant. The cross-sectional area was also identical in intact C57 and intact mdx muscles. As in Sol muscles, the immobilization prevented the occurrence of the first round of degeneration-regeneration (Fig. 4E). In shortened EDL, 3.3 ± 3.1% centronucleated fibers were counted (contralateral: 34.7 ± 11.1%, P = 0.002) and in stretched EDL 0.5 ± 0.5% (contralateral: 32.9 ± 17.5%, P = 0.002). Figure 5 illustrates the histological aspects of immobilized and contralateral Sol and EDL muscles.

View larger version (33K): [in this window] [in a new window]   Fig. 4.   Effects of 14-day immobilization of EDL muscle in 35 day-old mdx mice on total no. of myofibers (A), their cross-sectional area (C), and percentage of centronucleation of myofibers (E) in relation to length. Values from sham-operated controls are given in B, D, and F. Values are means ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. Nos. of animals: EDL shortened = 7, EDL stretched = 7, controls = 9.

View larger version (151K): [in this window] [in a new window]   Fig. 5.   Morphological patterns observed in Sol (A-D) and EDL (E-H) muscles of immobilized (A, C, E, and G) and contralateral (B, D, F, and H) hindlimb in 35 day-old mdx mice. Stretched muscles: A and E. Shortened muscles: C and G. Hematoxylin and eosin staining. Magnification ×250.

	DISCUSSION

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There are three major findings from this study. First, the immobilization of one hindlimb in young mdx mice prevented the initial occurrence of necrosis in most of the Sol and EDL myofibers of this limb. Second, a 14-day period of immobilization lead to a noticeable reduction of the myofibers' cross-sectional area but not of their number in immobilized mdx muscles. Additionally, the length at which a muscle was immobilized played a role in the occurrence of centronucleation and atrophy of its myofibers.

The survival of postnatal perinucleated myofibers after the immobilization of leg muscles in mdx mice at the age of 3 wk is the outstanding result of this study. Two previous works (16, 20) showed that disuse could alleviate muscle degeneration in young mdx mice. First, when the lumbosacral plexus was unilaterally avulsed or the thoracic spinal cord was sectioned in 15- to 18-day-old mdx mice, the percentage of centronucleated myofibers observed in plantaris muscles by 20 or 45 days postoperatively was significantly reduced compared with controls (16). Second, when 21-day-old mdx mice were injected unilaterally in the leg with the tetanus toxin, the percentage of centronucleated myofibers observed in Sol and EDL muscles by 1-8 wk postoperatively was also significantly reduced (20). An absence of neural activation characterizes these two experimental models of muscle disuse. In contrast, the limb immobilization by mechanical fixation preserves end-plate activity and reflex functions (8) despite the occurrence of a few ultrastructural changes observable in end plates of immobilized muscles (23). In rat Sol muscle, the degree of spontaneous electromyographic activity depends on the position of limb immobilization. Ten days after immobilization, a dramatic reduction of tonic muscular activity could be observed in shortened Sol muscle, which shifted to phasic activity. Only slight modifications are observable in the stretched position (8, 13). In the same experiments, TA exhibited rare phasic activity regardless of its length of immobilization (13). These observations of EMG activity can explain the percentage of centronucleated fibers observed in this study. Sol muscle, which contains ~30% type I myofibers in mice (24), in the stretched position exhibited the highest level of the centronucleation, probably resulting from tonic activity during immobilization. The putative phasic activity of shortened muscles and of stretched EDL (which includes only type II fibers) gave a very low percentage of centronucleation. The deleterious effect of eccentric and isometric contractions (as observed during immobilization) on the survival of mdx myofibers probably results from abnormalities at the myotendinous junction, which is the site of tensile failure in normal muscle fibers (17). The lack of dystrophin at this structural level seems to lead to holes in the cell membrane, to a reduction of digitlike processes, and to costameric markings from 4 wk of age (26). However, the relationship between these structural abnormalities and the load distribution of mechanical stress in dystrophinopathies remain to be determined (17).

Beside the occurrence of degeneration-regeneration, immobilization also affected the cross-sectional area of the Sol and EDL myofibers but not their total number in C57 and mdx strains. This observation is of importance in interpreting the reduced percentage of centronucleated myofibers as a beneficial effect. A constant number of fibers eliminates the possibility that immobilization deletes regeneration following degeneration. If this had been the case, the number of fibers with centrally placed nuclei would have decreased, increasing the percentage (but not the number) of normal fibers with peripheral nuclei. The constant number of myofibers associated with a decrease in the cross-sectional area of these myofibers shows an atrophic process that appeared in C57 as well in mdx mice, resulting from immobilization but not from the absence of dystrophin. It was previously shown that sedentary mdx mice exhibited muscle hypertrophy from 35 days of age, with wider mean cross-sectional areas of their Sol and EDL myofibers compared with those of C57 mice (see Ref. 31). However, such hypertrophy did not counterbalance atrophy due to hindlimb immobilization. We keep in mind that our evaluation of the cross-sectional area using the ratio of the area of the whole muscle to the number of fibers includes in the calculation the area filled in by connective tissue, blood vessels, and mononucleated cells as muscular fibroblasts. We assume that, before 3 wk of age, mdx muscle did not present a dystrophic accumulation of fat and connective tissue and that fibroblasts and blood vessels are similar in C57 and in mdx strains. Sol muscle atrophy was more pronounced after immobilization in the shortened position as did EDL muscle atrophy after immobilization in a stretched position, as described previously in rat muscles (29). This suggests that dystrophin is not involved in the relationship between muscle atrophy and length of immobilization.

In conclusion, the occurrence of the first round of degeneration-regeneration in mdx Sol and EDL myofibers was prevented by mechanical immobilization of one hindlimb in mice from the age of 21 to 35 days. The present study demonstrates that, even with the neural input intact, immobilization of the limb prevents the dystrophic changes. This provides the evidence that it is the actual contraction of the muscle itself that leads to muscle necrosis when dystrophin is missing. However, the early atrophy resulting from muscle immobilization and the the need for respiratory muscles to be permanently active rule out any therapeutic implications of these results to slow the development of human dystrophinopathies.