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Long-term reiention of regenerative capability after denervation of skeletal muscle and dependency of late differentiation on innervation.

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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.
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. "
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