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The number of nuclei in adult rat muscles with special reference to satellite cells.

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The Number of Nuclei in Adult Rat Muscles with Special
Reference to Satellite Cells
Institute of Neurophyswlogy, University of Copenhagen,DK-2100Copenhagen 0,Denmark
The number and the size of different populations of nuclei were
studied in skeletal muscles and in the diaphragm of male Wistar rats of 200-250
g weight. Nuclei on cross-sections were counted and classified by electron microscopy, their incidence was corrected for their different lengths, and the number of
nuclei per mm3 of muscle was determined by light microscopy. The total number
of nuclei per mm3was 5 lo4in the superficial part of the anterior tibial muscle,
it was 10 lo4 in the soleus, and it was 15 lo4in the diaphragm. Half of the nuclei were localized inside muscle fibres. The incidence of satellite cell nuclei on
cross sections was 4% of muscle nuclei in the anterior tibial muscle, and 8%in
soleus and diaphragm. The number of satellite cells per mm3 muscle were 900,
4,900, and 5,300 in these muscles.
More than half of the satellite cells were closely associated with a capillary. In
the anterior tibial muscle, and in 1-pm sections no satellite cells could be identified by light microscopy. In the soleus muscle and in the diaphragm, satellite
cells were more rich in cytoplasm and many were visible in the light microscope.
Satellite cells of muscle fibres are considered to be myogenic stem cells (for references Mauro et al., '70). In the subclavius
muscle of rats i t has been demonstrated, that
their number remains constant from one day
to six months after birth, whereas their incidence decreases with age, because the number
of myonuclei increases (Hellmuth and Allbrook, '73).After denervation, during muscle
hypertrophy (Aloisi et al., '63;Ontell, '74) and
during regeneration (Church, '70) the number
of satellite cells increases by mitotic division
(Shafiq e t al., '68).
In denervation and regeneration experiments frequently the diaphragm and hindlimb muscles of rats are used. Nevertheless,
the incidence of satellite cells in the diaphragm is unknown, for leg muscles data are
given only in two reports (Aloisi et al., '73;
Ontell, '74). Therefore we have determined
the incidence and the number of different nuclei in a fast and a slow leg muscle, and in the
diaphragm of adult rats.
Six male Wistar rats of 200-250 g weight
(age 7 to 9 weeks) were, under [email protected], fixed by perfusion with glutaraldeANAT. REC., 189: 169-176.
hyde via the abdominal aorta, either orthograde (3 rats) to fix the leg muscles, or retrograde (3 rats) to fix the diaphragm. Muscle
contracture was avoided by procaine (for
method Schmalbruch, '71). During orthograde
perfusion the right foot was kept in plantarflexion and the left one in dorsiflexion to
stretch the extensor or flexor muscles, respectively. The soleus muscle, the superficial part
of the anterior tibial muscle, and the costal
region of the right diaphragm were excised,
postfixed in osmium tetroxide and embedded
in Epon. Thus, leg muscles were taken from
three animals and the diaphragm from three
other animals for electron microscopy. Crosssections were stained with lead citrate, and
from one section of a series all nuclei were
photographed a t a magnification of X 10,000,
and typed from the negatives. When additional sections from the same block were studied, the distance between the sections was 20
p m or more to avoid serial sections of the
same nuclei. Only mononuclear cells enclosed
within the basal lamina compartment of a
muscle fibre, and separated from the muscle
fibre by a membrane-bound gap 20-50 nm wide
were classified as satellite cells.
Received Nov. 10, '76. Accepted April 14, '77.
Nomenclature. In the following, the nuclei
of muscle fibres are called myonuclei, whereas
myonuclei plus satellite cell nuclei corresponding to the nuclei of muscle fibres as to be
seen by light microscopy are called muscle nuclei. All other nuclei as those of interstitial
cells, of cells from blood vessels, of Schwann
cells and of blood cells are denoted as nonmuscle nuclei.
For light microscopy, longitudinal sections
1 p m thick were stained by the method described by Ontell ('74) to identify satellite
cells. From these sections, the length of myonuclei of satellite cells, and of non-muscle nuclei were measured with a screw-micrometer
eye-piece (objective Leitz Apo 40, n.a. 0.9).In
six nonadjacent fibres the length of ten sarcomeres each were measured, because t h e
length of myonuclei varies with the sarcomere length.
To determine the number of nuclei per mm3
muscle, from one rat of 210 g weight the three
muscles to be studied were stretched in an adjustable holder to 130%equilibrium length
(2.8-2.9 p m sarcomere length) and without
fixation frozen in liquid nitrogen. Cross-sections 10 pm thick were cut in a cryostat and
stained with haematoxylin-eosin. The number
of muscle nuclei per cross-section of a single
fibre was determined and the number of fibres
per mm2 cross-sectional area calculated from
the area occupied by 500 to 600 fibres.
The number of muscle nuclei in a segment
of a single fibre is given by the equation x =
where z is the number of muscle nuclei
Their incidence is N
= . I!
Because L
When n, is replaced by n, =
lm * x + d. x
is obtained.
This equation can be transformed into the
formula given above. With respect to the correction factor the formula is identical with
that introduced by Agduhr ('41)and its later
modifications (for references Haug, '67).
From the number of nuclei per mm fibrelength and the number of fibres per mm2 crosssectional area the number of muscle nuclei per
mm3 muscle was obtained. From these values
and from the incidence of other nuclei, the size
of the populations of different nuclei could be
calculated (for correction for different lengths
of nuclei, see below).
The results from different animals were
pooled, and the dfferences in the incidence of
satellite cells tested by the Chi2 test (Croxton, '53).
Sources of error
Missed satellite cells
This error cannot be excluded when a light
microscopical technique is used; but it may occur as well when nuclei are assessed by electron microscopy, in particular when no photographs are taken. Though every doubtful
satellite cell can be identified on the screen at
high magnification, its nucleus may resemble
lm +d
a myonucleus and the cytoplasm may be so
contained in a fibre segment of the length L, scarce, that the microscope is not switched to
Nis the number of nuclei per fibre cross-sec- the higher magnification. In a few instances,
tion, 1, is the mean length of muscle nuclei, we have on plates found satellite cells which
and d is the thickness of the section. This had been photographed as myonuclei.
equation takes into account, that in most
cases a nucleus cut into serial sections will oc- Nuclei counted twice
From all micrographs with satellite cells,
cur in - + 1sections, because it penetrates the
prints were made a t x 30,000,and compared.
first or the last section or both incompletely. For other nuclei, each 30 consecutively taken
Only in rare cases the number of sections micrographs were simultaneously placed on a
showing the same nucleus will be -.1
light plate and checked for nuclei photod
A fibre segment with a length L containing graphed twice.
x nuclei with a mean length of 1, can be cut Doubtful satellite cells
into n = sections: the number of sections
The basal lamina of the myofibre may as a
double leaflet penetrate a part of the gap beshowing a nucleus will be
tween satellite cell and muscle fibre (Muir,
n, = - + x.
'70; Schmalbruch, '76). A section passing only
through this part would show a cell separated
from the muscle fibre and surrounded by a
basal lamina. Then this cell resembles a
pericyte belonging to a capillary which is not
in the section. In cases of doubt, such cells
were classified as pericytes.
percentage (S') of satellite cells in relation to
the population of muscle nuclei ( = 100%)was
obtained: S' = 100
The error
caused by the thickness of the section was neglected, because it was less than 1%of the nuclear length. The same formula was used to correct the incidence of muscle nuclei with respect to that of non-muscle nuclei.
The probability of a nucleus to be cut by a
cross section depends on its length perpendicular to the section. In other muscles satellite
cell nuclei have been shown to be 30% to 50%
in table 1. The incishorter than myonuclei (Church, '70; Muir,
satellite cells
'70; Trupin, '76). This error was corrected by
was smaller in the superficial part of the anterior tibial muscle than in the soleus muscle
s.4n ,where Sand Mare the
the formula - = M
(p < 0.05; Chi2 = 4.44) and in the diaphragm
true numbers, s and rn the counted numbers, (p < 0.05; Chi2 = 4.57). The true incidences of
and 1, and lmare the mean lengths of nuclei of satellite cells were 4%, 11%and 8%in anterior
satellite cells and of myonuclei, respectively. tibial and soleus muscles, and in the diaThe formula was transformed so that the the phragm, respectively. For the anterior tibial
M. soleus
M. tibialis ant.
superficial part
Total number of nuclei
Muscle nuclei (%oftotal)
Satellite cell nuclei
(%ofmuscle nuclei)
Fibrocytenuclei (%oftotal)
Endothelial cell nuclei
(%of total)
Pericyte nuclei (% of total)
Other (91 of total)
403 (56%)
329 (67%))
32 (7.9%)
112 (16%)
13 (4.0%)
54 (11%)
41 (7.8%)
139 (13!!)
126 (18%)
26 (4%)
51 (74)
71 (14%)
19 (4x)
19 (4%)
60 (25%)
56 (5%)
55 (5%)
Sarcomere lengths
Nuclear lengths
( % of mean)
Satellite cell nuclei
Non-muscle nuclei
2.8 wm
13.4pm (25%)
9.6pm (27%)
8.3pm (29%)
16.Osm (21%)
11.5wm (28%)
9 . 9 r m (32%)
Corrected incidence
Muscle nuclei (of total)
Non-musclenuclei (of total)
Satellite cell nuclei
(of muscle nuclei)
Myofibres per mmz
Nuclei per fibre
Muscle nuclei per mm myofibre
per mm3muscle
Satellite cells per mm3muscle
Total number of nuclei per mm3muscle
8.2 wm (35%)
523 (50%)
5 310
4 920
A: Number of nuclei in different rat muscles as counted in electron micrographs of cross-sections. Each count is pooled fmm three animals.
B: Sarcomere lengths and lengths of nuclei as measured by light microscopy in longitudinal sections from the same specimens as used in A.
C: Percentage of different nuclei corrected for different nuclear lengths.
D: Number of fibres per mmz cross-sectional area and number of nuclei per fibre as counted in cryostat sections 10 pm thick.
E: Number of nuclei per mm fibre length, and nuclei per mm3 muscle as calculated from the data given in C and D.
* Satellite cells could not be identified by light microscopy, and the incidence could not be corrected.
muscle no correction for the different lengths
of myonuclei and satellite cells nuclei was possible, because by light microscopy no satellite
cells could be identified in this muscle. In
three rats the results for the two leg muscles
were consistent: in the soleus muscles the incidences of satellite cells on cross-sections
were 8.1%,10.4%,and 7.1%in the anterior tibial muscles they were 3.7%,4.4%, and 4.3%.In
the diaphragm which was studied in three
other rats, the incidences were 11.0%, 9.9%
and 3.7%. We cannot explain the low number
of satellite cells in third rat. It is unlikely to
be accidental, because in each muscle 300 to
400 nuclei were assessed.
The total number of nuclei per mm3 muscle
in the soleus muscles was twice and in the diaphragm three times as large as in the anterior
tibial muscle. From that, and from the incidence of satellite cells, it could be calculated,
that the soleus muscle and the diaphragm contained five t o six times as many satellite cells
per mm3 muscle as the anterior tibial muscle.
In the anterior tibial muscle the nuclei of
satellite cells were surrounded by a narrow
rim of cytoplasm (fig. 1). In the other muscles,
satellite cells were more rich in cytoplasm. It
was filled with ribosomes and short fragments
of rough endoplasmic reticulum (fig. 2). Glycogen granules were never found. With respect
to other cytoplasmic constituents the satellite
cell conformed to descriptions in previous
reports (for references Muir, ’70).
Satellite cells were often found in close association with a myonucleus, the same section
passing through both nuclei or through a myonucleus and a cytoplasmic projection of a satellite cell. This confirmed that satellite cells
and myonuclei are frequently “paired” (Ontell, ’74).In 48 of 80 micrographs showing a
satellite cell nucleus a capillary was adjacent
to the satellite cell.
The study presented confirms, that on cross
sections satellite cells occur more frequently
in slow than in fast muscles (Aloisi e t al., ’73).
The soleus muscle contained five to six times
as many satellite cells per mm3 tissue as the
superficial part of the anterior tibial muscle
(table 1).With respect to the number of satellite cells the diaphragm was similar to the
soleus muscle. The greater number of satellite
cells was due in part to the higher incidence of
satellite cells, and in part to the larger number of nuclei. Whereas soleus muscle and dia-
phragm consist mostly of fibres rich in mitochondria, the superficial part of the anterior
tibial muscle contains predominantly “white”
The uncorrected incidence of satellite cells
was higher than in previous reports, though
the precautions taken in this study ascertained that the counts were rather too low
than too high. In Wistar rats of 150 g weight
Aloisi e t al. (’73) found 1.7% in the extensor
digitorum longus and 4.7% in the soleus muscle. In the soleus muscle of 200-g and 800-g
Wistar rats Ontell (‘74) found by light microscopy 3.6% and 1.2%, respectively. Some of
these differences may be explained by results
of Hellmuth and Allbrook (‘73): in the subclavius muscle of 5-week old rats the incidence of satellite cells as determined by electron microscopy was 9.4%,in 10-week old rats
it was 4.6%;later it remained constant. In our
laboratory, male Wistar rats reach a weight of
110 g after five weeks and of 250 g after nine
weeks. Hence the rats used by Aloisi et al.
(’73), the 200-g rats of Ontell (’741, and those
used in this study were of a n age when the incidence of satellite cells still declines. Comparison of different data is difficult, when the
age is unknown, because the rate of growth
may vary from laboratory to laboratory. On
the other hand, it cannot be excluded, that, at
least, in the light microscope study (Ontell,
’741, satellite cells have been overlooked.
The estimates of the total number of nuclei
were rather crude. Nevertheless, the result
for the anterior tibial muscle (4.5 lo4 per
mm3, i.e., roughly per mg) was close to the
value obtained biochemically for the gastrocnemius muscle from the total amount of
DNA. In 35-day and 80-day old rats Enesco
and Leblond (’62) found 5 lo4 nuclei per mg
muscle. Histochemically, the anterior tibial
muscle is similar to the gastrocnemius muscle. Applying the same method as in this
study to l - p m Epon sections, Krogh (unpublished) found in nine 300-g Sprague-Dawley
rats in the soleus muscle 4.1 X lo4 (S.D. 5 X
lo3) muscle nuclei per mm3 muscle. This value
is close to that in table 1.
The difference in the number of nuclei and
hence also in the number of endothelial cells
between soleus and anterior tibial muscle is
reflected in the number of capillaries. When
the number of endothelial cells and pericytes
are calculated from the data given in table 1,
the soleus muscle contains two-and-a-half
times as many as the anterior tibial muscle.
In the soleus muscle, 3.8 times as many capillaries per mm2 cross-sectional area have been
found (2,180 and 575, respectively) (Schmalbruch, ’71).
This work was done with the support of
grants from the Danish Medical Research
Council and the Danish Association against
Rheumatic Diseases.
Agduhr, M. 1941 A contribution to the technique of determining the number of nerve cells per volume unit of
the tissue. Anat. Rec., 80: 191-202.
Aloisi, M., J. Mussini and S. Schiaffino 1973 Activation of
muscle nuclei in denervation and hypertrophy. In: Basic
Research in Myology. B. A. Kakulas, ed. Excerpta Medica, Amsterdam, pp. 338-345.
Church, J.C. T. 1970 Cell populations in skeletal muscle
after regeneration. J.Embryol. exp. Morph., 23: 531-537.
Croxton, F. E. 1953 Elementary Statistics. Dover, New
York, pp. 1-376.
Enesco, M., and C. P. Leblond 1962 Increase in cell number
as a factor in the growth of the organs and tissues of the
young male rat. J. Embryol. exp. Morph., 10: 530-562.
Haug, H. 1967 Probleme und Methoden der Struktur-
zahlung im Schnittpraparat. In: Quantitative Methoden
in der Morphologie. E. R. Weibel, and H. Elias, eds.
Springer, Berlin-Heidelberg-New York, pp. 58-78.
Hellmuth, A. E., and D. Allbrook 1973 Satellite cells as the
stem cells of skeletal muscle. In: Basic Research in
Myology. B. A. Kakulas, ed. Excerpta Medica, Amsterdam, pp. 343-345.
Mauro, A., S. A. Shafiq and A. T. Milhorat 1970 Regeneration of striated muscle, and myogenesis. Excerpta Medica, Amsterdam, pp. 1-299.
Muir, A. R. 1970 The structure and distribution of satellite cells In: Regeneration of striated muscle, and myogenesis. A. Mauro, S. A. Shafiq and A. T. Milhorat, eds.
Excerpta Medica, Amsterdam, pp. 91-100.
Ontell. M. 1974 Muscle satellite cells: A validated technique for light microscopic identification and a quantitative study of changes in their population following denervation. Anat. Rec., 178: 211-228.
Schmalbruch, H. 1971 “Rote” Muskelfasern. Z. Zellforsch., 119: 120-146.
1976 Muscle fibre splitting and regeneration in
diseased human muscle. Neuropathology and Applied
Neurobiology, 2: 3-19.
Shafiq, S. A,, M. A. Gorycki and A. Mauro 1968 Mitosis
during postnatal growth in skeletal and cardiac muscle
of the rat. J.Anat. (London), 103: 135-141.
Trupin, G. L. 1976 The satellite cells of normal anuran
skeletal muscle. Develop. Biol., 50: 517-524.
1 Anterior tibia1 muscle, rat. Satellite cell and myonucleus close to a capillary. The cytoplasm of the satellite cell is scarce, and i t is unlikely that this cell can be identified
by light microscopy. X 22,500.
2 Diaphragm, rat. Satellite cell. The cell is more rich in cytoplasm than that shown in 1.
Note large mitochondria in the myofibre. X 22,500.
H. Schmalbruch and U. Hellhammer
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adults, muscle, nuclei, satellite, references, number, rat, special, cells
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