/ . Embryol. exp. Morph. Vol. 23, 2, pp. 531-37, 1970
531
Printed in Great Britain
Cell populations in skeletal muscle after regeneration
By J. C. T. C H U R C H 1
From the Department of Anatomy, Makerere University College,
Kampala, Uganda
Much is known of the processes involved in the regeneration of skeletal
muscle after injury. Yet in most accounts, the results are expressed in general or
relative terms. Thus Adams, Denny-Brown & Pearson (1962) state that provided
the architecture of the muscle survives, the reconstruction of 'considerable
lengths' of muscle fibre is feasible. Wright (1963) makes a plea for the application of quantitation, with the choice of 'some muscle that is sufficiently small for
counting and measuring techniques to be reasonably applicable...' The web of
the fruit bat Eidolon helvum (Kerr) contains a number of small muscles which
have proved very suitable for the detailed study, in light and electron microscopy, of muscle regeneration (Church & Noronha, 1965; Church, Noronha &
Allbrook, 1966) (Fig. 1 A-D), and for the quantitation of recovering lesions
(Church, 1968).
Satellite cells (Mauro, 1961), which are possibly the main source of myoblasts in regenerating muscle, have often been observed, yet again their numbers have only been expressed in general terms. Shafiq & Gorycki (1965) see
them 'much more commonly' in the injured area. Lee (1965) indicates that
similar cells are seen 'more frequently' in denervated than in normal muscle.
Laguens (1963) sees a 'large number' of satellite cells in human dystrophic
muscle. Shafiq, Gorycki & Milhorat (1967) report that they are 'commonly
seen' in human dystrophic and polymyositic muscle, where they are 'more
numerous' at the foci of regeneration.
The pleomorphism of the cells in healing muscle during the first few weeks
after injury renders the certain identification of any given cell, and therefore
accurate quantitation, very difficult. However, in crush lesions to bat web
muscles, despite extensive damage to every fibre in the injured segment of muscle,
regeneration is practically complete by 4 weeks, so that quantitation is thereafter
feasible. The accurate localization of lesions in vivo permits repeated injury of
the same site. In this report, the quantitation is given for lesions after single and
double injury.
1
Author's address: Department of Anatomy, The Medical School, P.O. Box 7072,
Kampala, Uganda, East Africa.
34-2
•
532
J. C. T. C H U R C H
FIGURE 1
(A) A light photomicrograph of normal bat web muscle, in transverse section,
showing numbers of muscle fibres (m/), nerve fascicles (n), and a satellite cell (s),
hardly distinguishable at this magnification, x 450.
(B) A light photomicrograph, magnified from Fig. 1 A, showing the satellite cell (s),
an endomycial fibroblast (ƒ), a myonucleus (m) and a capillary (c). x 1700.
(C) An electron photomicrograph of the cells in Fig. 1B, showing the satellite cell (s),
fibroblast (ƒ), myonucleus (m) and a pericyte (p) adjacent to the capillary (c). x 4500.
(D) An electron photomicrograph, enlarged from Fig. 1 C, showing the satellite cell
(s) lying under the muscle fibre basement membrane (bm), with its inner plasma
membrane lying adjacent to that of the syncytium (pp), x 21500.
Skeletal muscle
533
regeneration
METHODS
Small crush lesions were made in the webs of adult fruit bats, as described
elsewhere (Church et al. 1966). One lesion was fixed 3 months after injury. The
other lesion was allowed to recover for 4 weeks, then crushed again, and fixed
after a further 4 weeks. Material was prepared, and examined in light and electron microscopy, as previously described. Serial transverse sections about 2/i
thick were cut at intervals of 250 JLL along the muscle. In this way, at least four
of the serial sections could be expected to pass through the previously crushed
region of the muscle. High power light microscopy revealed the satellite cells in
these preparations with sufficient clarity for them to be distinguished from other
cells. Using a squared grid, counts were made of muscle fibres, myonuclei,
satellite cells and endomycial fibroblasts. A few longitudinal sections were made,
so that the mean length of myonuclei and satellite cell nuclei could be determined, from which their relative frequency of appearance in transverse section
Table 1. The number of muscle fibres, myonuclei, satellite cells and fibroblasts,
as counted in serial transverse sections (1-21) at 250 ^ intervals in a segment of
muscle crushed 13 weeks previously
Satellite cell/myonuclei ratios are followed by corrected satellite cell counts, and corrected ratios.
T.S.
No.
No. of
fibres
No. of
myonuclei
No. of
satellite
cells
No. of
fibroblasts
Sat. cell/
myonuclei
ratio (%)
Satellite
cells
x 1-3
Corrected
Sat.
cell/
myonuclei
ratio (%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
643
660
647
673
687
671
683
701
704
704
692
704
690
693
683
695
696
669
674
686
680
251
250
270
277
237
272
254
257
251
252
262
283
263
194
208
298
233
242
224
245
262
42
26
33
26
24
33
24
25
20
38
41
42
54
36
28
29
31
31
20
28
23
37
33
33
35
35
32
66
75
57
65
103
73
68
81
59
64
47
46
50
31
38
16-7
10-4
12-2
9-4
101
121
9-4
9-7
80
15 1
15-6
14 8
20-5
18-6
13-5
9-7
13-3
12-8
8-9
114
8-8
54-6
33-8
42-9
33-8
31-2
42-9
31-2
32-5
26 0
49-4
53-3
54-6
70-2
46-8
36-4
37-7
40-3
40-3
26 0
36-4
29-9
21-8
13-5
15 9
140
13-2
15-8
12-3
12-6
10-4
19 6
20-3
19 3
26-7
241
17-5
12-7
17-3
16-7
11 6
14-9
11-4
534
J. C. T. CHURCH
could be calculated. Light microscopy was correlated with electron microscopy,
to establish further the identity of cells being counted.
Table 2. The numbers of muscle fibres, myonuclei, satellite cells and fibroblasts,
as counted in serial transverse sections (1-15) at 250 ju, intervals in a segment of
muscle crushed twice, 8 weeks and 4 weeks previously
Satellite cell/myonuclei ratios are followed by corrected satellite cell counts, and
corrected ratios.
T.S.
No.
No. of
fibres
No. of
myonuclei
No. of
satellite
cells
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
550
568
577
577
594
575
579
560
588
572
591
573
575
578
594
217
224
237
202
257
312
294
266
221
195
228
214
219
245
233
22
27
26
45
75
75
66
70
67
30
31
27
31
26
36
No. of
fibroblasts
Sat. cell/
myonuclei
ratio (%)
32
39
31
46
100
115
110
106
99
63
51
28
41
39
41
10 0
121
110
22-3
29-2
24 0
22-4
26-3
30-3
15-4
13-6
12-6
14-2
10 6
15-5
Corrected
Satellite Sat. cell/
cells
myonuclei
x 1-3
ratio (%)
28-6
351
33-8
58-5
97-5
97-5
85-8
91 0
87-1
390
40-3
35 1
40-3
33-8
46-8
13-2
15-7
14-3
29 0
37-9
31 3
29-2
34-2
39-4
20 0
17-7
16-4
18-4
13-8
20 1
RESULTS
Results of the quantitation are given in Tables 1 and 2, and in Figs. 2 and 3.
Muscle fibre number in both lesions remains virtually unchanged in both undamaged and previously damaged regions. In the first lesion, seen 13 weeks
after injury, there is some variation of the number of myonuclei in the region of
the lesion, but without any overall loss. By contrast, the satellite cells show a
definite increase in the lesion. This is mirrored by an increase in the fibroblasts
over a distance of about 3 mm, straddling the injured region.
In the second lesion, crushed twice, the myonuclei show a definite increase in
the injured segment of muscle, and this is mirrored by a dramatic increase in the
satellite cells and the fibroblasts, rising to 2-3 times the normal numbers. It
was noted in this lesion that though the axons had regrown along the damaged
Schwann cell tubes, myelination stopped abruptly at a point about 1 mm
proximal to the lesion, from which it is concluded that the nodes of Ranvier are
at least 1 mm apart in these nerves, this being comparable to those of other
species (Bloom & Fawcett, 1962).
In longitudinal sections, satellite cell nuclei varied in length from 5-5 to
Skeletal muscle regeneration
535
100 jti, and myonuclei varied from 100 to 12-5 /A. On calculation of the means,
myonuclei were about 30 % longer than satellite cell nuclei, so that satellite
cell counts, as seen in transverse section, were multiplied by a factor of 1-3
to obtain a more accurate ratio.
Fibres
600 Fibres
Satellite cells
120 h
0
T.S
Fibroblasts
T.S. 1 2 3 4 5 6 7 8 9
12
3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 20 21
Nerve
101112131415
Lesion
Lesion
Fig. 3
Fig. 2
Fig. 2. A histogram of the number of muscle fibres, myonuclei, satellite cells and
fibroblasts seen in serial transverse sections (1-21), cut at 250 ji intervals in a segment of muscle crushed 13 weeks previously. The lesion occupied the region from
sections 11-14.
Fig. 3. A histogram of the number of muscle fibres, myonuclei, satellite cells and
fibroblasts seen in serial transverse sections (1-15), cut at 250 /* intervals in a segment of muscle crushed twice, 8 weeks and 4 weeks previously. The lesion occupied
the region from sections 5-9.
DISCUSSION
The crush lesions to these muscles, made in this way, are severe enough to
cause gross damage to the sarcoplasm and its contained myonuclei, though
with little disturbance of connective tissue architecture, and no disruption of
basement membranes: conditions which would favour complete regeneration,
if this is potentially possible. There is, in fact, complete restitution of all injured
Myelin
536
J. C. T. C H U R C H
elements, going on to an increased cellular response, particularly after double
injury. As myonuclei have not been diluted by the process of regeneration, as
might be shown by reduction in numbers in or either side of the injured region,
and as they are incapable of mitosis (Okazaki & Holtzer, 1966), they must have
been derived from cells dividing locally or coming in to the lesion from elsewhere. Whatever the source of these cells, the response is adequate to replace
the myonuclei not only to normal but to greater than normal numbers.
The satellite cells also increase to greater than normal numbers. These cells
could be a separate cell line, with function as yet unknown, which would not
provide myoblasts, but which would, in a similar way to fibroblasts and other
cells, respond to injury by proliferation. Evidence is now accumulating that
satellite cells are capable of giving rise to myoblasts following injury (Shafiq &
Gorycki, 1965; Church et al. 1966). Walker (1963) also showed that following
a second injury, muscle fibres were derived from cells originating from within
previously injured fibres. The inference from the present study is that satellite
cells not only are capable of providing myoblasts to make up a deficiency in
myonuclei and syncytial sarcoplasm, but also set aside further satellite cells so
that the process is repeatable. Tt would be of some interest to follow the longterm recovery of these lesions, to see if the satellite cell population returns to
normal numbers.
The proliferation of the fibroblasts can be equated with their response to any
injury. Connective tissue cells do not contribute to the regeneration of skeletal
muscle fibres (Walker, 1963).
SUMMARY
1. These results are consistent with those of previous studies, and show that
skeletal muscle fibres have a remarkable potential for regeneration.
2. The recovery pattern is here expressed as a complete numerical restoration
of the formed elements of the muscle fibres, with, if anything, an increased
cellular response in the 1-3 month period following injury.
3. Skeletal muscle can withstand repeated injury of this type, and still recover
completely.
RÉSUMÉ
Populations cellulaires dans le muscle squelettique
après régénération
1. Ces résultats concordent avec ceux d'études précédentes et montrent que
les fibres musculaires squelettiques ont un remarquable pouvoir de régénération.
2. Le tableau de la récupération peut être exprimé sous forme d'une restauration numérique complète des fibres musculaires avec, éventuellement en surplus,
une réponse cellulaire accrue pendant la période de 1-3 mois.
3. Les muscles squelettiques peuvent supporter des lésions répétées de ce
type et encore pouvoir récupérer complètement.
Skeletal muscle regeneration
537
This work formed part of a thesis accepted for the degree of M.D. of Cambridge University. 1 am grateful to Professor R. W. Haines and Professor A. R. Muir for assistance
and criticism in the preparation of this paper. The electron microscope was donated by
the Wellcome Trust, and maintained with grants from the Ministry of Overseas Development (U.K.). A Makerere College research grant covered the expenses of the experimental
work.
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(Manuscript received 13 May 1969)