Long-term reiention of regenerative capability after denervation of skeletal muscle and dependency of late differentiation on innervation.код для вставкиСкачать
THE ANATOMICAL RECORD 220:429-434 (1988) Long-Term Retention of Regenerative Capability After Denervation of Skeletal Muscle, and Dependency of Late Differentiation on Innervation ADARSH K. GULATI Department ofdnatomy, Medical College of Georgia, Augusta, GA 30912 ABSTRACT The present study examines the influence of denervation on the regenerative ability of skeletal muscle in rats. Muscle denervation was achieved by transecting and ligating the cut ends of the sciatic nerve. Four to 48 weeks after denervation, the extensor digitorum longus (EDL) muscle was autotransplanted to induce muscle regeneration. The transplanted EDL muscles were examined at 1-12 weeks. Normal (i.e., no prior denervation) EDL muscle autotransplants were also examined for comparison. Denervation resulted in progressive atrophy of muscle, marked by a reduction in the size of myofibers and an increase in endomysialperimysial connective tissue. In spite of these alterations, typical events of muscle regeneration were invariably observed after transplantation. Initial myofiber degeneration amd subsequent regeneration of myotubes occurred in a manner similar to normal muscle transplants. However, only a partial maturation of myotubes was observed in denervated muscles. These results show that extended denervation does not abolish the capability for muscle regeneration. The precursor myosatellite cells, proposed to be responsible for muscle regeneration, retain their regenerative potential after denervation. It is concluded, however, that the presence of intact innervation is crucial for the terminal differentiation and maturation of regenerating muscle. It is well known that mammalian skeletal muscle can regenerate with substantial functional recovery following an injury (Carlson, 1978; Allbrook, 1981). An extensively used model for studying various aspects of this regenerative response is the autotransplantation of extensor digitorum longus (EDL) muscle in rats (Carlson and Gutmann, 1975; Hansen-Smith and Carlson, 1979; Gulati, 1986, 1987a). A precise cascade of events that follows transplantation includes degeneration of a majority of myofibers, activation and proliferation of precursor myosatellite cells, differentiation of myoblasts, fusion to form myotubes, and eventual maturation into myofibers. Denervation of skeletal muscle causes extensive changes in its morphological, biochemical, and physiological characteristics. The main reason for these alterations, commonly referred to as “atrophic,” is the interruption of neuron-muscle interaction (Sellin et al., 1980; Kabara and Tweedle, 1981; Jakubiec-Puka et al., 1981; Salonen et al., 1985; Czyzewski et al., 1985).These drastic changes in skeletal muscle after denervation are likely to be’reflected in the ability of skeletal muscle to regenerate after injury. In the present study, the effect of denervation on muscle regeneration in rats was examined. The results reveal that skeletal muscle is capable of regeneration but not maturation after an extended denervation. A preliminary report of this work has been published elsewhere in abstract form (Gulati, 1987b). 0 1988 ALAN R. LISS, INC. MATERIALS AND METHODS A total of 56 male Fischer rats weighing 200-250 gm were used in this study. Animals were prepared by a two-phase surgical procedure for study of the effect of denervation on regeneration. For the first phase, each rat was anesthetized with chloral hydrate (40 mg/100 gm body weight, i.p.), and both hind legs were denervated by complete transection of the sciatic nerve in the upper thigh. In order to achieve extended denervation, cut ends of the sciatic nerve were tightly ligated with a 4-0 silk suture. The surgical wound was then closed and the animals were allowed to recover. The second surgical phase was carried out at 4, 12, 24, and 48 weeks after denervation. All rats were reanesthetized and the EDL muscle was autotransplanted to induce muscle.regeneration according to a procedure described earlier (Gulati, 1986; 1987a). Briefly, it consisted of cutting the proximal tendon close to the knee, lifting the muscle from its bed completely, and transplanting it back at the same site by suturing the cut tendon. Four muscles from different animals were examined at 1,2,4, and 24 weeks after transplantation for each of the denervation intervals. Normal (i.e., no prior denervation) EDL muscle autotransplants were similarly examined at each time Received August 3, 1987; accepted November 4, 1987. 430 A.K. GULATI interval for comparison. In addition, the tibialis anterior muscle, which was not transplanted and lies adjacent to the EDL muscle, was checked to confirm denervation in each animal. For morphological analysis, the transplanted EDL muscles were removed and divided into proximal and distal halves. One piece was immediately frozen in liquid nitrogen, and the other was immersion-fixed in 3% glutaraldehyde in cacodylate buffer (0.1 M, 4"C, pH 7.4). The frozen muscle was sectioned in a cryostat and sections stained with periodic acid-Schiff (PAS)/hematoxylin for light microscope analysis. The second piece after overnight fixation in 3% glutaraldehyde was postfixed in 2% osmium tetroxide, dehydrated, and embedded in epon. Thin sections were cut from selected blocks, mounted on grids, stained with uranyl acetate and lead citrate, and examined with a Phillips 400 electron microscope. RESULTS The normal EDL muscle consists of myofibers of varying diameters and staining intensity. Individual myofibers have a thin pericellular endomysium and groups of myofibers are enclosed by a thicker perimysium (Fig. 1). The various regeneration steps after transplantation of normal EDL muscle were identical to those described earlier (Carlson and Gutmann, 1975; Gulati, 1986; 1987a). In 7-day transplants of normal muscle, a myogenic zone composed of myoblasts and small myotubes was seen between the outer zone of original surviving myofibers and the inner zone of degenerating myofibers (Fig. 2). The myotubes grew larger and at 4 weeks the entire muscle consisted of polygonal myofibers. The regenerated myofibers possessed prominent central nuclei (Fig. 3). A thin endomysium and perimysium similar to that in normal muscle was also evident (compare Figs. 1and 3). Progressive atrophy of skeletal muscle was seen with increased duration of denervation. Myofibers became smaller, their differential staining disappeared, and a thickening of endomysium as well as perimysium was typically observed (Figs. 4 and 7). In spite of these atrophic changes, transplantation of short- and longterm denervated muscles invariably resulted in the initial degeneration of a majority of myofibers and subsequent regeneration of myotubes. These myotubes, however, remained small and failed to mature into myofibers in the absence of innervation. It should be pointed out that degenerating and regenerating myofibers in various transplants were easily distinguishable on the basis of well-established morphological features described in earlier studies (Carlson, 1973; Hansen-Smith and Carlson, 1979; Allbrook, 1981; Gulati, 1986, 1987a). The degenerating myofibers possessed pyknotic nuclei located in the peripheral region. The cytoplasmic organelles and the sarcolemma were in various stages of disintegration and phagocytosis by macrophages. On the other hand, the smaller regenerating myotubes and myofibers possessed vesicular nuclei, invariably located in the central region. Well-organized cytoplasmic organelles and myofilaments were also observed in regenerating muscle cells. A summary of results from various groups is given in Table 1. However, to demonstrate these findings morphologically, results from 12 and 48 week denervation groups are discussed below. Many myotubes along with a thin layer of outer surviving myofibers were seen in 4-week transplants with 12-week prior denervation (Fig. 5). The regenerated myotubes were small and round compared to nondenervated normal transplants (compare Figs. 5 and 3). As no attempt was made to restore innervation of the regenerated muscle in the present study, the muscle regenerFig. 1. Cross section of a normal EDL muscle. Myofibers of different sizes and staining intensity are present. The arrow points to the perimysium. PAS-hematoxylin, x 160. Fig. 2. Cross section of a 1-week autotransplanted normal (i.e., no prior denervation) EDL muscle. Three distinct zones are visible: a peripheral zone of surviving myofibers (S), a myogenic zone (M) consisting of myoblasts and small myotubes, and an inner zone of degenerating ischemic myofibers (D). PAS-hematoxylin, x 160. Fig. 3. Cross section of a 4-week regenerated normal EDL muscle. The original surviving myofiber zone (S) and regenerated myofiber zone (R) are seen. Regenerated myofibers are polygonal and possess prominent centrally located nuclei (arrowheads). PAS-hematoxylin, x 160. Fig. 4. Cross section of a 12-week denervated muscle. Typical features of denervation atrophy, marked by thickening of endomysium and perimysium (arrow) as well as reduction of myofiber size are seen. PAShematoxylin, x 160. Fig. 5. Cross section of a 4-week autotransplanted muscle denervated 12 weeks earlier. A zone of original surviving myofibers (S) and a zone of regeneration (R)is visible. The regenerated myotubes are small and of rounded appearance as compared to normal nondenervated transplants (compare to Fig. 3).PAS-hematoxylin, x 160. Fig. 6. Cross section of a 12-week denervated EDL muscle, examined 24 weeks after transplantation. The overall size of muscle is much reduced and extensive collagenous connective tissue (arrows) surrounds the myotubes. Many large blood vessels (V) are also seen. PAShematoxylin, x 160. TABLE 1. Relative extent of regeneration and recovery of denervated muscle transplants Semiquantitative presence in the muscle transplants' Denervation interval (weeks before transplantation) 0 4 12 24 48 Total number of animals 8 12 12 12 12 'Semiquantitative analysis: + + + to - indicate maximal presence to absence. Undifferentiated myoblasts - + ++ ++ Myotubes Myofibers ++ +++ +++ +++ ++ - +++ + - Connective tissue + ++ ++ +++ +++ MUSCLE REGENERATION AFTER DENERVATION Figs. 1-6. 431 432 A.K. GULATI Figs. 7-12. 433 MUSCLE REGENERATION AFTER DENERVATION ate continued to undergo progressive atrophy of the regenerated myotubes. The overall size of muscles was reduced and extensive collagenous connective tissue was observed throughout the muscle regenerate (Fig. 6). In muscles denervated for 48 weeks and then transplanted, the presence of regenerated myotubes was clearly evident at all times after transplantation (Figs. 8-10]. Again extensive connective tissue matrix was observed throughout the muscle regenerate. Electron microscope observations confirmed these results. Many small myotubes with large vesicular central nuclei and cytoplasmic myocontractile filaments were seen (Figs. 11and 12). A basal lamina and collagenous matrix surrounded the individual myotubes. In addition, undifferentiated cells, also with vesicular nuclei but lacking myofilaments (probably immature myoblasts), were occasionally observed adjacent to the myotubes in these muscles (Fig. 12). In all the muscles analyzed patent blood vessels were observed (Figs. 6,7, and 121, implying that these muscles remain vascularized even after extended denervation. No nerves were observed in any of the muscles examined, confirming denervation. DISCUSSION Myosatellite cells present in the skeletal muscle are considered myogenic stem cells that function in the repair of injured muscle fibers (Snow, 1979; Campion, 1984). Short-term interruption of nerve supply has been shown to result in an increase in the number of myosatellite cells within the target muscle (Ontell, 1974; Snow, 1983). The present results provide evidence that precursor myosatellite cells persist, remain functional, and initiate regenerative repair after long-term denervation. Carlson and Gumann (1975)have described the effect of 4-week denervation on muscle regeneration after autotransplantation. They reported that short-term dener- Fig. 7. Cross section of a 48-week denervated muscle showing typical atrophic changes. Muscle cells are small and thickening of endomysial and perimysial (arrows) connective tissue are seen. Blood vessels N ) are also seen. PAS-hematoxylin, x 160. Fig. 8. Cross section of a 4-week autotransplanted muscle, denervated 48 weeks earlier. A zone of surviving myofibers (S) and a zone of regenerated myotubes (R) are again evident. Extensive connective tissue (arrow) surrounds the small, rounded myotubes (compare with Figs. 3 and 5). PAS-hematoxylin, x 160. Fig. 9. Cross section of a 48-week denervated EDL muscle, examined 24 weeks after transplantation. Extensive connective tissue matrix (heavy arrows) surrounds the small myotubes. The curved long arrow points to a myotube, also shown in Figure 10 at a higher magnification. PAS-hematoxylin, x 160. Fig. 10. A higher magnification of transplant in Figure 9. Regenerated myotubes (heavy arrows and curved arrow) interspersed within the connective tissue matrix (C) are seen. PAS-hematoxylin, X640. Fig. 11. Electron micrograph of a 48-week denervated EDL muscle examined 24 weeks after transplantation (as in Fig. 10). Regenerated myotubes (M) with well-formed contractile myofibrils are seen. Bundles of collagen fibers (C) are seen around the myotubes. X3,995. Fig. 12. Electron micrograph of a different region of muscle in Figure 11. Two undifferentiated cells (one labeled, U), with many mitochondria in the cytoplasm are seen along with regenerated myotube (M). Also note the presence of a small blood vessel (V) and collagen fibers (C). ~ 5 , 1 7 5 . vated grafts underwent a more rapid degeneration and rapid regeneration compared to the nondenervated normal EDL transplants. A similar rapid myofiber degeneration and subsequent regeneration, although not monitored carefully, was also observed in the present study. Muscles regenerating in the presence of innervation recover their morphological, histochemical, and physiological features (Carlson, 1973; Hall-Craggs, 1978; Allbrook, 1981). When muscle injury is combined with denervation, myotubes do regenerate, even after longterm denervation, but their growth and maturation fail to occur. Iannaccone et al. (1982) also observed partial maturation of human skeletal muscle grown in vitro under aneural conditions. They suggested that lack of nutritional, hormonal, or neural factors results in the arrest of myotube maturation. In the present experimental paradigm denervated muscle transplants were vascularized, implying that neural factors rather than nutritional or hormonal factors are important in the terminal maturation of myofibers. Expression of myosin isoforms in regenerating muscle have also been shown to be regulated by innervation. The embryonic isoform of myosin accumulates in such muscles in the absence of nerves (Cararo et al., 1981, 1983). Taken together, these observations mean that muscle regeneration is a multistep phenomenon regulated by different factors, and the terminal maturation of myofibers is dependent on neural activity. It remains to be determined whether long-denervated muscle regenerates permit functional reinnervation with differentiation of myofibers. This is quite plausible, since reinnervation and restoration of muscle receptors has been demonstrated recently in atrophied muscle after denervation (Hansen-Smith, 1986; Barker et al., 1986; Brunetti et al., 1987). ACKNOWLEDGMENTS The author thanks Drs. Dale E. Bockman and Nidhi K. Gulati for helpful comments, Mrs. Brenda Headrick for technical assistance, and Ms. Sandra Dunn for preparation of the manuscript. LITERATURE CITED Allbrook, D. (1981) Skeletal muscle regeneration. Muscle nerve, 4:234245. Barker, D., J.J.A. Scott, and M.J. Stacey (1986) Reinnervation and recovery of cat muscle receptors after long-term denervation. Exp. Neurol., 94:184-202. Brunetti, O., C. Carobi, and U. Pazzaglia (1987) Influence of atrophy on the eficiencv of muscle reinnervation. Exp. Neurol., 963248252. Campion, D.R. (1984) The muscle satellite cell: A review. Int. Rev. Cytol., 873225-251. Carlson, B.M. (1973) The regeneration of skeleton muscle-A review. Am. J. Anat., 137:119-150. Carlson, B.M. 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