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Satellite cells and myonuclei in long-term denervated rat muscles.

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THE ANATOMICAL RECORD 243:430-437 (1995)
Satellite Cells and Myonuclei in Long-Term Denervated Rat Muscles
ANTONIO DE CASTRO RODRIGUES AND HENNING SCHMALBRUCH
Department of Medical Physiology, University of Copenhagen, The Panum Institute,
Copenhagen N , Denmark
ABSTRACT
Background The percentage of satellite cells rapidly decreases in aneurally regenerating soleus muscles of rat. Also denervation of
intact muscles causes fiber loss and regeneration, but the fate of satellite
cells is unknown; myonuclei have been suggested to undergo changes resembling those in apoptotic cells.
Methods: Rat soleus and extensor digitorum longus (EDL) muscles were
denervated at birth or at age 5 weeks and investigated after periods of up
to 38 weeks. At least 400 myonuclei in each muscle were assessed by electron microscopy, and satellite cell nuclei were counted. In situ nick translation and tailing were performed after 30 weeks denervation in order to
demonstrate DNA breaks associated with apoptosis.
Results: Myotubes indicating regeneration were prominent in the adult
denervated soleus and deep layers of EDL muscles after 7 weeks and in the
superficial parts of EDL muscle after 16 weeks. The percentage of satellite
cell nuclei slowly decreased to less than one fifth of normal after 20-30
weeks. Almost all satellite cells had vanished 10 weeks after neonatal denervation. Degenerating myonuclei in adult, but not in neonatally denervated muscles, remotely resembled apoptotic nuclei of lymphocytes, but no
evidence of DNA breaks was found.
Conclusion: Denervation of rat skeletal muscles causes, in addition to
fiber atrophy, loss of fibers with subsequent regeneration. Proliferation of
satellite cells under aneural conditions may lead to exhaustion of the satellite cell pool. This process is more rapid in growing than in adult muscles.
Myonuclei in denervated muscles do not show DNA breaks which can be
demonstrated by in situ nick translation. o 1995 Wiley-Liss, Inc.
Key words: Apoptosis, Denervation, Muscle regeneration, Satellite cells,
Skeletal muscle
Mononuclear satellite cells (Mauro, 1961) are myoblasts which proliferate and provide myonuclei during
growth (Moss and Leblond, 1971) and regeneration of
skeletal muscle fibers (for references see Schultz and
McCormick, 1994); satellite cells must also be involved
in the normal turn-over of myonuclei. The percentage
of satellite cell nuclei in relation to postmitotic myonuclei and the total number of satellite cells per mg
muscle is higher in frequently activated than in less
frequently activated muscles (Schmalbruch and Hellhammer, 1977; Gibson and Schultz, 1983).
Although fetal r a t muscles can develop without
nerve (Hughes and Ontell, 1992) and non-innervated
muscles regenerate initially very well (Whalen e t al.,
1990; Schmalbruch and Lewis, 1994), there is evidence
that the mitotic activity of satellite cells or myoblasts
is dependent on the direct influence of the motor nerve
or, indirectly, on contractile activity dependent on innervation. Embryonic chick muscles yield reduced proportions of clonable myoblasts if the muscles have been
denervated or curare-treated (Bonner, 1978,1980),and
neonatally denervated rat muscles contain fewer satellite cells than innervated muscles (Kelly, 1979;
0 1995 WILEY-LISS, INC.
Schmalbruch, 1990). Findings in adult muscles after
denervation vary: some authors found immediate (Ontell, 1974; McGeachie and Allbrook, 1978; Murray and
Robbins, 1982; McGeachie and Grounds, 1986) or delayed (after 30 days: Snow, 1983) proliferation of satellite cells while others failed to find consistent
changes within 10 weeks after denervation (Schmalbruch and Lewis, 1994). These discrepancies may be
due to different periods of denervation studied and may
also reflect differences in the denervation reactions of
different muscles (see Discussion). In addition to denervation atrophy, rat soleus muscles show fiber breakdown with subsequent proliferation of satellite cells
Received February 15, 1995; accepted July 1’7, 1995.
Address reprint requests to H. Schmalbruch, M.D., Department of
Medical Physiology, Division of Neurophysiology, University of
Copenhagen, The Panum Institute, Blegdamsvej 3c, DK 2200 Copenhagen N, Denmark.
Antonio de Castro Rodrigues’s present address is Department of
Anatomy, IBIUNESPIBotucatu, 18618-000 Rubiao Jr., Sao Paulo,
Brazil
SATELLITE CELL LOSS IN DENERVATED MUSCLES
and the formation of new fibers (Schmalbruch et al.,
1991a; Schmalbruch and Lewis, 1994).
The present paper describes a quantitative study of
the percentage of satellite cells in rat muscle denervated at birth or a t age 5 weeks and studied for up to 38
weeks. We also investigated myonuclei with respect to
DNA breaks which occur in apoptotic cells and which
may be responsible for fiber loss in denervated muscles
(Fidzianska et al., 1990; Fidzianska and Kaminska,
1991). The results indicate that the percentage of satellite cells declines after neonatal and adult denervation, and that there is no evidence of apoptotic DNA
breaks in myonuclei of adult muscles denervated for 30
weeks.
MATERIALS AND METHODS
The first series of rats studied were female Wistar
rats of 100-120 g body weight (age 5 weeks). They were
anesthetized with halothane, the right sciatic nerve
was cut at midthigh level, and a 5-10 mm long segment of the nerve was excised. The proximal nerve
stump was ligated, bent backwards, and sutured subcutaneously to the hamstring muscles in order to prevent reinnervation. Animals which were studied more
than 4 months after the operation were reoperated at
least every 3 months: the proximal nerve was isolated,
freshly sectioned, and ligated and sutured. In all, 31
rats were operated. The animals were again anesthetized with halothane after 5-38 weeks, and the hindlimbs were perfused via the abdominal aorta with
Ringer solution containing 1%procaine hydrochloride
and 5,000 U/1 heparin followed by 2.5% glutaraldehyde
in 0.1 M phosphate buffer. The denervated and contralateral soleus and extensor digitorum longus (EDL)
muscles were excised, postfixed with 1% osmium
tetroxide, and embedded in epoxy resin (Embed 812;
Electron Microscopy Sciences, Ft. Washington, PA) for
light and electron microscopy.
The second series of rats consisted of 20 newborn rats
(body weight 5.4-6.2 g), which were anesthetized by
hypothermia; the right sciatic nerve was cut at
midthigh level. No precautions were taken to prevent
reinnervation, because it is minimal due to axotomyinduced motoneuron loss (Schmalbruch, 1984, 1990).
Soleus and EDL muscles were fixed by vascular perfusion after 5 and 10 weeks, and prepared for light and
electron microscopy as described above.
Cross sections 1-3 pm thick passing through the
middle of the muscles were cut with dry glass knives,
stained with para-phenylenediamine, and viewed with
phase optics. All muscles of adult-denervated rats with
signs of reinnervation such as myelinated axons or
groups of unusually large fibers were discarded. None
of the muscles denervated for less than 10 weeks
showed such signs, but of the 22 rats which survived for
10 weeks or more, reinnervation of both muscles was
seen in 5 rats and reinnervation of the EDL alone in 6
rats. Neonatally denervated muscles showed either no
reinnervation, or there was a well-delineated group of
at most 100 large fibers which obviously were reinnervated (Schmalbruch, 1990).Satellite cells were counted
only in regions containing small presumably denervated fibers, although the few nuclei of the large fibers
would probably not have affected the results.
Thin sections for electron microscopy were cut from
431
the same blocks, collected on 300-mesh grids, and
stained with uranyl acetate and lead citrate. One section covering 30-50 mesh-holes was selected under the
electron microscope (PHILIPS CM 10) at low magnification. A chart of the section was drawn, and the holes
C‘fields”)covered by the section numbered. Myonuclei
and satellite cell nuclei in each field were counted at
high magnification. Myonuclei were nuclei within cells
containing myofilaments; satellite cells were identified
by their position beneath the basal lamina of a muscle
fiber, they showed only one nucleus and no myofilaments. Serial sections were cut a t a distance of at least
20 pm in order to avoid counting the same nuclei twice.
An area containing at least 400 myonuclei was investigated for each muscle. In 4 rats, the sections were
assessed independently by two observers; the percentages of satellite cells did not differ by more than 0.66%.
Non-denervated contralateral muscles served as controls. The lengths of the nuclei of muscle fibers and
satellite cells were not determined, but longitudinal
sections did not disclose systematic gross differences
which might have affected the counts.
The possibility of nuclear changes as in apoptotic cells
was investigated by assessing DNA breaks in myonuclei by in situ nick translation or tailing (Gold et al.,
1993; Oberhammer et al., 1994). Two adult rats denervated for 30 weeks were perfused with 4% paraformaldehyde and the muscles were embedded in paraffin.
Cross and longitudinal sections 5 pm thick were
mounted on coated glass slides. Identically prepared
sections of the atrophic thymus of pmn mice (Schmalbruch et al., 1991b)which show pronounced apoptosis in
lymphoid organs served as positive controls. Deparaffinized sections were incubated at 37°C for 1 h with
digoxigenin-DNA labeling mixture and either DNA
polymerase I (Kornberg polymerase) in nick translation
buffer in order to label single-strand DNA breaks or
terminal deoxynucleotidyl transferase in tailing buffer
in order to label double-strand DNA breaks (all reagents from Boehringer Mannheim, Germany). Digoxigenin was visualized with alkaline-phosphatase-labeled anti-digoxigenin (Boehringer) and Fast Blue BB
(Sigma Chemical Co., St. Louis, MO).
The animal experiments were approved by the Supervisory Committee of the Danish Ministry of Justice.
The rats were kept in the Animal Department of the
Panum Institute, Copenhagen, Denmark, in plastic
cages at a 12 h light-dark cycle and had free access to
food and water.
The declining percentage of satellite cells after denervation was described by exponential or linear equations fitted by the least square method; the software
was commercial (Kaleidagraph 2.0, Synergy Software,
Reading, PA).
RESULTS
Adult Denewated Muscles
General morphology
Denervation atrophy was more pronounced in the
soleus than in the EDL muscle (Al-Amood and Lewis,
1989).The size of the fibers in the soleus muscle did not
vary between different parts of the muscle before and
after denervation, while the fiber size in the EDL varied greatly in normal muscle. The superficial part
432
A.D.C. RODRIGUES AND H. SCHMALBRUCH
Fig. 1. Rat soleus (a) and deep EDL (b)muscle, 8 and 7 weeks after denervation at age 5 weeks. Epoxy
section for light microscopy, p-phenylenediamine; phase optics. The muscle fibers are atrophic, most have
subsarcolemmal nuclei (unstained),but several myotube-like fibers with central nuclei (arrows) are seen
in both muscles. One muscle spindle containing 6 (rather than 4 as in normal soleus muscle) intrafusal
fibers is seen in a. The perfused blood vessels are ballooned. Bar: 20 pm.
which comprised about two-thirds of the muscle was
dominated by large fibers poor in mitochondria, and
the deep part of the muscle consisted mainly of small
fibers rich in mitochondria. The regional differences in
the EDL initially became more pronounced after denervation, but these regional differences had vanished
after 15-17 weeks. Soleus muscle fibers and fibers of
the deep EDL presented with degenerative changes
such as disordered myofibrils and sarcolemmal folds
due to atrophy after about 12 weeks, whereas the structure of the superficial EDL fibers was more or less normal until 16 weeks after denervation. Myotube-like fibers with central nuclei began to occur in the soleus
and deep EDL muscle after about 7 weeks (Fig. 1)and
in the superficial EDL after 16 weeks. After 38 weeks,
the majority of fibers in both muscles had central nuclei; most of these myotube-like fibers showed signs of
atrophy a t that time (Fig. 2a,b). The interstitial space
between the muscle fibers became wider with increasing atrophy and contained collagen and fat cells in soleus muscles denervated for more than 9 weeks. Muscle
spindles in r a t muscles normally contain 4 intrafusal
fibers, but already 7 weeks after denervation all spindles of the soleus contained a n increased number of
fibers (Fig. la); spindles with more than 4 fibers were
found in the EDL after 19 weeks.
Satellite cells
The percentage of satellite in innervated soleus and
EDL muscles declined with age. The counts in muscles
of young rats roughly corresponded to those of Gibson
and Schultz (1983) in rats aged 1 month. The decline,
however, was steeper than suggested by the findings of
Gibson and Schultz (1983) in rats aged 12 months. The
percentages in rats of about the same age varied considerably (Fig. 31, and no systematic differences between contralateral muscles of operated rats and muscles of non-operated rats were observed.
Satellite cells became more prominent shortly after
denervation, and many could unambiguously be identified by light microscopy. This was mainly due to the
fact that the amount of cytoplasm increased. After 5
weeks, two satellite cells might be found closely attached to each other and to the same muscle fiber, or a
cell which contained two nuclei on the same cross section and which presumably was a myotube was in the
position of a satellite cell (Snow, 1983). This was more
often seen in the soleus than in the EDL. The satellite
cells in normal soleus muscles were usually positioned
close to a capillary (Schmalbruch and Hellhammer,
1977); this was not the case in atrophic muscles after
more than 9 weeks denervation, possibly because of the
rearrangement of the capillary system in atrophic muscles.
The percentage of satellite cells in both muscles decreased after denervation. EDL muscles showed a normal or increased percentage of satellite cells during the
first weeks after denervation, and even after 20 weeks
the percentage in some muscles was normal. The decrease was, however, distinct after more than 15
SATELLITE CELL LOSS IN DENERVATED MUSCLES
Fig. 2. a: Rat soleus muscle 19 weeks after denervation at age 5
weeks. Electron micrograph. The muscle fibers contain central nuclei
some of which are indented. One fiber shows a degenerating myonucleus (straight arrow) (at higher magnification shown in c ) together
with a normal looking nucleus. Curved arrow: Satellite cell. b: Rat
EDL muscle 38 weeks after denervation at age 5 weeks. Electron
micrograph (same magnification as a). All fibers resemble myotubes
433
undergoing atrophy. The endomysial connective tissue has increased.
c,d Degenerating myonuclei in rat soleus muscles 19 weeks after
denervation at age 5 weeks. The chromatin has condensed and the
nuclear shape is highly irregular. The perinuclear cistern is distended
and contains glycogen granules (arrows). Bars: a,b (in b), 10 km; c,d
(in c ) , 1 km.
A.D.C. RODRIGUES AND H. SCHMALBRUCH
434
2 1 2 4
.I
,"
EDL+SOLEUS
O
SOLEUS
denervated at birth
---
0
0
10
20
40
30
50
60
121
10
20
30
age (weeks)
40
5
6
1
age (weeis)
9
10
11
Fig. 4. Diagram showing the percentage of satellite cells in neonatally denervated soleus and EDL muscles at age 5 and 10 weeks (soleus: open symbols; EDL: filled symbols). The linear regression (y =
7.47-0.73 x , R = 0.75, P < 0.01) was calculated for the pooled data.
The percentage at 5 weeks is less than half of normal (see Fig. 3);
satellite cells are virtually absent at age 10 weeks.
EDL
0
4
0
50
60
Fig. 3. Diagrams showing the decrease of the percentage of satellite
cells with age in normal rat muscles (open symbols, broken lines) and
in muscles denervated at age 5 weeks (filled symbols, solid lines).
Exponential regressions could be fitted (normal soleus: y =
9 . 8 4 . ~ O' Z x , R = 0.69, P < 0.01; normal EDL: y = 9 . 4 6 ~ 0' 4 x , R =
0.88, P < 0.001; denervated soleus; y = 22.35.e-0 l o x
R,= 0.95, P <
0.001; denervated EDL: y = 22.22.e-0'9", R = 0.70, P < 0.01). The
crosses connected by stippled lines are data from Gibson and Schultz
(1983) and show the percentages at 1 and 12 months. The percentage
of satellite cells in the EDL apparently increases after denervation
and then decreases; some data are still above normal even at age 25
weeks. The onset of the decrease in the denervated soleus muscle is
earlier than in the EDL, and the eventual decrease is more distinct.
weeks. In the soleus muscle, the first counts after denervation were normal, and the decrease apparently
started after 5-6 weeks. The counts in denervated
EDL muscles varied more than counts in denervated
soleus muscles. The relative decrease was smaller in
EDL than in soleus muscles. The decline of the percentage of satellite cells in normal rats and after denervation could be described as exponential (Fig. 3).
Myonuclei
The myonuclei in normal soleus and EDL muscles of
adult rats were situated below the sarcolemma; a few
internal myonuclei were observed close to myotendinous junctions and in the endplate zone. During the
first weeks after denervation. the fibers atroDhied and
the myonuclei maintained their peripheraf position.
Between such unambiguously atrophic fibers, however,
there was, with time, a n increasing proportion of myo-
tube-like fibers rich in sarcoplasm and with one central
nucleus on a cross section. These fibers also showed
signs of atrophy and degeneration a t later stages; cross
sections might reveal several internal nuclei in highly
atrophic fibers (Figs. 1, 2a,b). From week 7 on, more
and more myonuclei in the soleus muscle became
crenated and showed very dense chromatin. The perinuclear cistern was dilated and contained glycogen
granules, probably because the membrane was defective. The same fiber sometimes contained relatively
normal-looking myonuclei together with dense and
crenated myonuclei (Fig. 2). These changes in the appearance of myonuclei were much less pronounced in
the EDL than in the soleus muscle; crenated and dense
myonuclei were not found in the EDI, before 19 weeks
after denervation. The morphological changes of myonuclei in long-term denervated muscles (Fig. 2c,d) had
a remote resemblance to the morphological changes of
nuclei in lymphocytes undergoing apoptosis although
typical apoptotic bodies (Wyllie et al., 1980) were not
seen. We never saw unambiguously fragmented myonuclei.
Apoptosis is associated with DNA cleavage (Wyllie,
1980; Cohen, 1991). We therefore performed in situ
nick translation and tailing on soleus and EDL muscles
denervated for 30 weeks in order to visualize single- or
double-stranded DNA breaks. Identically prepared and
processed sections of the atrophic thymus of the mouse
mutant pmn (Schmalbruch et al., 1991b)) which contains a n abundance of apoptotic cells, served as controls. Apoptotic cells in the mouse thymus were heavily
stained after 20 min incubation with Fast Blue BB,
whereas no staining a t all was seen in muscles. Incubation was then continued for 90 min until almost all
nuclei of the connective tissue showed unspecific staining. Nevertheless, myonuclei never showed reactions
comparable to that of apoptotic lymphocytes (Fig. 5).
Neonatally Denervated Muscles
The morphology of rat soleus muscles 4 weeks after
neonatal dener&on
has been described (Schmal-
SATELLITE CELL LOSS IN DENERVATED MUSCLES
435
Fig. 5. Thymus of pmn mouse (a)and cross section of rat soleus muscle 30 weeks after denervation at
age 5 weeks (b).In situ nick tailing, Fast Blue BB, no counterstain. In a, numerous apoptotic nuclei react
intensely. In b, only unspecific staining of connective tissue cells is seen although the section shown in
b was overstained (90 min as compared to 20 min in a). The tissue was identically fixed and embedded,
and both sections were processed simultaneously and photographed under identical optical conditions.
Bar: 20 bm.
bruch, 19901, and the present study did not reveal differences between soleus and EDL. In brief, the denervated but not the normal muscles contained many
myotube-like fibers with one central nucleus. Most
clusters of developing muscle fibers which are still
present at birth had broken up, and the fibers had detached. The fibers apparently failed to grow, and the
fiber diameters were about the same as at birth
(Schmalbruch, 1990). After 10 weeks, the muscles consisted mostly of fat and dense fibrotic tissue, and the
number of fibers had decreased. Almost all fibers were
small atrophic myotubes which tended to be grouped
around blood vessels and never formed clusters surrounded by a common basal lamina. Some myonuclei
were crenated, but nuclei with condensed chromatin
and distended perinuclear cistern as in adult long-term
denervated muscles (Fig. 2c,d) were not seen. Occasionally, thin fibers were in close contact with macrophages, or a macrophage had invaded the basal lamina
of a degenerating fiber; the basal lamina was incomplete and cellular processes ensheathed the degenerating muscle fiber. Also these fibers did not have condensed nuclei. The percentage of satellite cells in
neonatally denervated muscles at age 5 weeks was less
than half as compared to normal muscles a t the same
age; it tended to be higher in the EDL than in the
soleus muscle. After 10 weeks, satellite cells were prac-
tically lacking in both muscles (Fig. 4).The majority of
muscle fibers measured 3-5 pm in diameter at that
time, but some fibers still measured up to 10 p,m. The
few satellite cells that were seen in these muscles were
only on the larger fibers.
DISCUSSION
The present study demonstrates that the incidence of
satellite cells decreases in rat soleus and EDL muscles
after denervation. This decrease is steeper than the
age-related decrease in normal muscles. The initial reaction in adult EDL after denervation is an increase of
the percentage of satellite cells, and the decrease as
compared to the normal decrease is smaller than in the
soleus. The decrease in the soleus muscle may also
have a brief delay (Fig. 3). The decline is more rapid in
neonatally denervated (Fig. 4) than in adult denervated muscles. Myotubes indicating myogenesis after
fiber breakdown occur first in the adult denervated soleus but later also in the EDL; eventually, both muscles consist of myotube-like fibers only (as in soleus)
(Fig. 2a,b). Neonatally denervated muscles consist of
myotubes, possibly because maturation has been arrested.
For proliferation, satellite cells depend on innervation or innervation-mediated muscle activity; their
percentages are normal in regenerated and reinner-
436
A.D.C. RODRIGUES AND H. SCHMALBRUCH
vated muscles (Schultz, 1984) but rapidly decline during aneural regeneration (Schmalbruch and Lewis,
1994). The events in a denervated muscle might be
described as prolonged and non-synchronous fiber
breakdown and aneural regeneration (Anzil and
Wernig, 1989; Schmalbruch et al., 1991a; Schmalbruch
and Lewis, 1994); this also affects intrafusal fibers
which increase in number (Schroder, 1974) (Fig. la).
The decline of the percentages of satellite cells is
slower in denervated adult muscles than in aneurally
regenerating muscles, possibly because the exhaustion
of the satellite cell pool in a non-innervated muscle
(Anzil and Wernig, 1989) relates to the formation of
new myonuclei. This may also account for the rapid
decline after neonatal denervation: myogenic cells of
embryonic rat muscles do not proliferate after denervation, and at the same time the pool of myoblasts is
exhausted by the formation of secondary myotubes
(Ross et al., 1987). It is conceivable that the poor recovery of muscle force when surgical repair of a motor
nerve in a patient has been unduly delayed, not only
reflects impeded axonal regeneration through the fibrotic endoneurium, but it may also be due t o loss of
satellite cells when the muscle has remained denervated for a long period of time. In rats, satellite cells
are rapidly lost after neonatal denervation, and rat soleus muscles are only sparsely reinnervated when a
nerve that has not previously been injured is transplanted 4 weeks after birth (Schmalbruch, 1990).
The percentage of satellite cells depends on the total
numbers of satellite cells and myonuclei. The total
number of satellite cells in normal soleus muscles increases slightly between 1and 12 months, but the percentage decreases because the number of myonuclei
increases (Gibson and Schultz, 1983). In our material,
the normal decrease of the percentage was steeper than
that reported by Gibson and Schultz (1983) for rats
aged 1 and 12 months (Fig. 3). This difference may
reflect different growth rates of the rats used, but it
was probably not due to inactivity (Darr and Schultz,
1989) after contralateral denervation, because the
counts were not lower than those of Gibson and Schultz
(1983) in contralateral muscles of young rats. One
might argue that the decreased percentage after denervation was due to an increased number of myonuclei.
This is, however, unlikely for adult denervated muscles
which undergo pronounced atrophy, and it is certainly
not the case in neonatally denervated muscles which
contain only scattered muscle fibers. On the contrary,
the loss of satellite cells may be larger than suggested
by the decreasing percentages, because the number of
myonuclei may be decreasing as well.
The number of nuclei analyzed in each muscle (at
least 400)was obviously large enough to demonstrate
the decrease of the percentage of satellite cells. Despite
their low incidence in aged rats, the results after longterm denervation did not overlap with those for normal
muscles; the difference between normal and neonatally
denervated muscles was obvious, because virtually no
satellite cells were found after 10 weeks (Figs. 3, 4).
The absolute number of myonuclei could not be determined in the present study, but circumstantial evidence suggests that it decreases after denervation.
Normal soleus muscle fibers of adult rats have a crosssectional area of 3,000-5,000 p,m2 (Schmalbruch,
1990), the mean length of the myonuclei is 13.4 pm,
and there are 76 per mm fiber length (Schmalbruch
and Hellhammer, 1977). This agrees with the fact that
a fiber cross section in the electron microscope on the
average contains one myonucleus (own unpublished
data). Ten weeks after denervation, the cross-sectional
area of the muscle fibers has declined to 26 pm2, i.e., to
less than 1%of normal, while their number has not
changed (about 3,000) during this period (Schmalbruch
and Lewis, 1994). Muscles denervated for 10 weeks or
more show less than one myonucleus per fiber in a
given sectional plane: one-third to one-half of the fibers
are without nucleus and the remaining; ones rarely contain more than one myonucleus (Schmalbruch et al.,
1991a; Schmalbruch and Lewis, 1994; Fig. 2).
The extent of muscle fiber breakdown and regeneration after denervation differs in different muscles and
relates to the rate of atrophy. Atrophy is faster and
there are more histological signs of regeneration in rat
soleus than in rat EDL, and in rat than in guinea pig
soleus (Al-Amoodand Lewis, 1989; this paper; Lewis et
al., manuscript submitted). Also the relative loss of satellite cells tends to be larger in the soleus than in the
EDL (Fig. 3).
Fidzianska et al. (1990) and Fidzianska and Kaminska (1991) found degenerating myonuclei with dense
chromatin in muscles of a patient with infantile spinal
muscular atrophy and in neonatally injured rat muscles, and proposed that immature denervated muscle
fibers die by means of apoptosis (for references see Cohen, 1991). We did not detect myonuclei with condensed chromatin after neonatal denervation. In adult
denervated muscles, however, degenerating myonuclei
(Fig. 2c,d) had a remote resemblance to nuclei of apoptotic lymphocytes (Wyllie et al., 1980). Typical apoptotic bodies were lacking. DNA “laddering,” indicating
cleavage of DNA during synchronized apoptosis in cell
culture (Wyllie, 1980; Cohen, 19911, cannot be demonstrated in tissue in which the majority of nuclei is obviously normal. Our attempts to demonstrate DNA
breaks by in situ tailing and nick translation failed in
muscles, although the methods produced intense staining of apoptotic lymphocytes in cont,rol sections from
the atrophic thymus of a mouse mutant (Fig. 4). This
suggests that DNA breaks do not occur in degenerating
mynuclei of rat muscles denervated for 30 weeks.
Apoptosis without DNA breaks has been induced experimentally in cell cultures (Cohen et al., 1992;
Schulze-Osthoff et al., 1994), but it is unknown
whether it occurs under natural conditions.
ACKNOWLEDGMENTS
The authors thank Dr. D.M. Lewis, Bristol, for comments on the manuscript, and Marianne Bjaerg and
Bettina Bay Jensen for expert technical help. Financial
support was provided by the Danish MRC and Aage
Haensch Fond. A.C.R. was on leave of absence from
UNESP, Botucatu, SP, Brazil, and supported by a
travel grant from CNPq Brazil (Proc. 201704/93-5-NV).
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