/. Embryol. exp. Morph. Vol. 36, 2, pp. 355-363, 1976
Printed in Great Britain
355
Developmental patterns of rat muscle
histochemistry
By RICHARD G. CURLESS 1 AND MARILYN B. NELSON 1
From the Department of Pediatrics, University of Arizona
SUMMARY
A developmental progression of histochemical fiber typing is demonstrated in normal rat
soleus muscle. By utilizing the acid lability of ATPase, the type II fibers are subdivided into
type IIA, IIB and IIC. No IIB fibers are present in the newborn or adult rat soleus. In the
1-day-old animal 90 % of the fibers can be differentiated into type I and II with all of the
type II's demonstrating 1IC characteristics. Only type I and IIC fibers are present until 18
days, when poorly differentiated IIA fibers first appear and gradually become the predominant type II fiber at the expense of IIC's.
Further analysis documents the sequence of disappearance of myotubes from up to 80 % of
all fibers in the 1-day-old to less than 1 % between 15 and 18 days. An analysis of the progression of fiber size according to type is presented.
INTRODUCTION
As muscle develops, the various fiber types differentiate, providing a useful
model for the study of control of differentiation. Dubowitz (1963a, b, 1965)
found that rat and muscle fibers did not fully differentiate until after birth.
Wirsen & Larsson (1964) described three populations of fibers in the fetal
mouse: primary, secondary, and tertiary, corresponding respectively to the white
intermediate and red fibers of adult muscle. It was suggested that these types
develop as three distinct populations.
In other animals patterns of muscle development have also been described.
Cooper, Cassens & Kastenschmidt (1970) pointed out that in pigs the first
appearance of differentiation was at one week of life, and at the same stage the
clumping of red fibers was also evident. In these animals intermediate and white
fibers were present by four weeks of age. Nystrom (1968) indicated that in the
kitten soleus multiple histochemical techniques failed to show fiber differentiation prior to 7-10 days except when utilizing myofibrillar ATPase. The ATPase
method clearly demonstrated two fibre populations at birth and three types by
21 days.
Dubowitz & Brooke (1973) pointed out that IIC fibers are rarely found in
normal 'mature' human muscle, but in neuromuscular disorders they often
represent as much as 15 % of the total fibers. In addition. Brooke (1973)
1
Authors' address: Department of Pediatrics, Pediatric Neurology Section, University of
Arizona, College of Medicine, Tucson, Arizona, 85724, U.S.A.
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R. G. CURLESS AND M. B. NELSON
indicated the presence of only types I and IIC fibers in preterm human infants,
suggesting the possibility that the IIC fiber is a precursor of the later appearing
IIA and IIB fiber types. Subsequently a large number of human muscle biopsies
(> 1000) have been analyzed by this system (Brooke, 1975; Stern, 1975 personal
communication).
Using a recently described method of infant needle biopsies (Curless & Nelson, 1975), we have been looking for abnormalities of muscle development in
hypotonic infants. From muscle tissue obtained in this manner, the fiber types
are counted and their morphology studied.
To study the effect of central nervous system lesions on muscle development
patterns, an animal model is required. The sequence of maturation in rat soleus
and gastrocnemius with respect to fiber typing and morphology has been studied
with a variety of techniques. Brooke, Williamson & Kaiser. (1971) gave a brief
report of developing patterns in the soleus and gastrocnemius of twelve postnatal rats and suggested that IIC fibers represent a primative histochemical
fiber capable of differentiation into types 11A & IIB. On this basis the young
rat was chosen as the animal model for correlation with the human infant.
The study reported here involves the postnatal patterns of fiber maturation
with emphasis on the pH-labile ATPase-typing system described by Brooke &
Kaiser (1970). Utilizing this system it is possible to separate fiber types into I,
II A, IIB, and IIC by observing sequential cross-sections of fibers stained for
ATPase at decreasing pH values. It was first necessary to establish the appearance
of normal developmental patterns utilizing the histochemical profile of fiber
types (I, II A, IIB, and IIC), and to determine the distribution and size of the
respective fibers.
MATERIAL AND METHODS
Thirty-three cage-reared Sprague-Dawley rats were used during the first
month of postnatal life. By twice daily examinations of pregnant females the
time of birth was known within 12 h. Previous studies indicated that an 'adult'
histochemical pattern could be expected by 4 weeks of age (Brooke et al. 1971).
The animals were anesthetized with Metofane, but kept alive until the procedure was complete. The ankle extensor muscles of both hind limbs were exposed and identified. Soleus was separated from gastrocnemius and plantaris
in vivo and all three muscles were removed intact by ligating their tendons of
origin and insertion. The tissue was promptly frozen in isopentane cooled to
- 1 7 0 °C in liquid nitrogen, and sliced in 8 ju,m sections using a cryostat at - 2 0 ° C .
After drying at room temperature the serial sections were stained with modified
Gomori trichrome and diphosphopyridine nucleotide-tetrazolium reductase
(NADH-TR) on the same day the tissue was sectioned (Engel & Cunningham,
1963).
Within 5 days of sectioning, the tissue was stained for myosin ATPase by the
pH-labile method described by Brooke & Kaiser (1970). Ideal and reproducible
Developmental patterns of rat muscle histochemistry
100
357
r
80 -
=
60 -
Fig. 1. Myotube frequency indicated as a percentage of all fibers. Note the rapid
decrement during the first 3 days of life with a gradual linear decline thereafter. The
circles indicate the upper limits for each age and the squares the lower limit. The
curves are approximations with the shaded area indicating the range.
results were obtained by pre-incubating the initial sections at 9-4 pH for 12 min
at 36 °C. Subsequent sections were pre-incubated at 4-5 and 4-25 respectively at
room temperature for 5 min. Then all three sections were incubated again at
36 °C for 12 min for the 9-4 pH sections and 36 °C for 15 min for the 4-5 and
4-25 pH's. By this method all the dark fibers at 9-4 were determined to be type
II. Those type II fibers which become pale following 4-5 pH pre-incubation are
called IIA. The pale type II fibers at a pre-incubation pH of 4-25 are the IIB's
and any remaining dark type II fibers are IIC's. In addition, the darkest fibers at
4-5 and 4-25 pH are type I.
Eleven animals represented a duplication study of similar age. Random microscopic fields from each soleus were examined and polaroid photographs were
taken with a microscope camera to permit fiber type counting and diameter
measurements. The size was determined by measuring the lesser fiber diameter
(defined as the maximum diameter across the lesser aspect of the muscle fiber). A
millimeter rule was used on a photograph of a 100-times magnified section of an
ATPase stain. The fibers were grouped according to measurements to the nearest
10 JLLM. The mean fiber diameter was calculated from a histogram of 200 fibers.
RESULTS
Morphological developmental patterns were analyzed with specific reference
to myotubes, spindles and internal nuclei. As indicated in Fig. 1, during the
first day of life 60-80 % of the fibers in any one field were in the myotube stage
358
R. G. CURLESS AND M. B. NELSON
100 r
80
o o 8
«=
60
-o
•Typc f
8-
Types II A + C
40
Type HA
20
Indeterminate
i
Type II C
i
15
20
25
30
Age (days)
Fig. 2. Fiber types determined by the pH-labile ATPase system shown as percentages
of total fibers counted for each age. Note the consistency of Type I percentage and the
reversal of IIC to IIA predominance between 17 and 23 days. At 17 days the type II's
are a mixture of IIA and IIC.
whereas by the third day only 15-30 % were in that stage. There were always
more myotubes along the periphery. Initially a small number of the myotubular
structures were horse-shoe shaped, but by 9 days of age all fibers appear round.
At 8 days the myotubes were primarily in the periphery of the fasicle and by 19
days less than one myotube per hundred could be found. After 8 days 90 % of
the myotubes stained darkly with acid pre-incubation (4-5 pH) ATPase technique, suggesting that these fibers were predominantly type I.
Muscle spindles were clearly evident in the 1-day-old animal. Initially the
intrafusal fibers were the same size as the individual extrafusal fibers. However,
by 8 days of age the entire spindle containing 3-5 fibers was the same diameter as
one large extrafusal fiber. In the 3-week-old animal the spindle diameter measured 25 jam, compared with the 30 fim diameter of most extrafusal fibers at this
age. The fiber density in the newborn animal was very low, with a large amount
of inter-fiber connective tissue. By 19 days the fibers were packed closely together, with connective tissue found only in small quantities in a perifascicular
distribution.
Study of the nuclei revealed a gradual migration from a central position to a
peripheral, subsarcolemmal location by 18 days. The young nuclei were round
and, in comparison to the fiber diameter, relatively large. By 19-20 days the
nuclei appeared flattened against the sarcolemmal membrane and represented a
small fraction of the mature fiber cross-sectional volume.
Histochemical analysis also revealed several clear milestones which are best
depicted by percentage and size according to fiber type (Figs. 2, 3). Type-I fibers
Developmental patterns of rat muscle histochemistry
100
359
r
80
60
Age (days)
Fig. 3. Fiber size increases with age regardless of the type. Note that IIC and IIA
fibers are the same size (the dark shaded area). Twenty-seven animals are in this
analysis, but the consistency of size at similar ages prevents this number of points
from appearing on the graph.
are striking in their consistency from the first day of life into adulthood. There is
no significant variation in the percentage of the total type Fs (53-65 %) between day 1 and day 29 (Fig. 2). The type-I fibers remain larger than the type
ll's, although by a lesser margin than in the younger animals (Fig. 3).
Fiber-type differentiation was evident in the 1-day-old animal but only at a
4-25 pH pre-incubation. The standard 9-4 pH failed to differentiate fiber types
at that age. Ten percent of these fibers were indeterminate in staining characteristics, but about 30 % type IIC's were clearly evident. Seven days was the first
indication of fiber typing with a 9-4 pH. No IIA fibers were found at this age.
At 18 days indeterminate type II fibers appeared which defied clear distinction between types IIA, B and C. Prior to that time the ATPase stain at pH 4-25
clearly demonstrated types I and IIC. However, this phase lasted only 2-3 days
and was followed by the first appearance of IIA fibers at 20-21 days (Fig. 2). By
29 days the majority of the type II fibers were IIA with less than 2 % IIC fibers.
This pattern of 65 % type I, 33 % type IIA and 2 % IIC represents the distribution found in adult rat soleus. No IIB fibers could be found at any stage of
development.
Oxidative enzyme activity, as indicated by NADH-TR staining, was not
helpful in identifying a developmental pattern in rat soleus. No type differentiation was seen regardless of the age of the animal. Dark staining in all fibers was
evident throughout the muscle. No changes were found at the critical ATPase
developmental reversal from IIC to IIA predominance between 18 and 21 days
of age.
360
R. G. CURLESS AND M. B. NELSON
Fig. 4. Shown are four examples of 9-4 pH ATPase sections of rat soleus. (A) 3day-old without type differentiation. Note spindle and myotubes (arrows) ±400.
(B) 11-day-old animal with clear type differentiation. Type II fibers (dark) are
smaller. Arrows indicate a myotube and a spindle x 400. (C) 15-day-old with infrequent myotubes (arrow) and spindle with two fiber types x 400. (D) 28-day-old
rat with rare myotubes. Arrow denotes a spindle x 176.
DISCUSSION
Studies on the developing cat indicate that during early developmental stages
the soleus muscle is slow contracting (Buller, Eccles & Eccles, 1960; Buller &
Lewis, 1964; Close, 1964). Guth and Samaha (1972) studied actomyosin ATPase
in three developing cats in biochemical and histochemical methods and found the
Developmental patterns of rat muscle histochemistry
361
majority of the fibers in the youngest animal to be low in specific ATPase
activity. The same fibers stained darkly at an alkaline pH and demonstrated
acid lability as expected with ATPase-rich fibers (type II). On the basis of this
study of one specimen it is possible that the histochemical method of analysis for
ATPase is not reflecting the quantity of ATPase present in the neonatal fibers.
However, recognizing the uncertainly of the basis for the histochemical change,
it is felt that documentation of a developmental pattern provides a basis for
further study of factors influencing development.
Several authors have commented upon the existence of postnatal muscle
developmental patterns in the newborn rat. Dubowtiz (1963 a, b, 1968) studied
multiple muscles by limb cross-sections and felt that soleus was all type I from
birth to maturity, Karpati & Engel (1967), utilizing the ATPase technique without acid pre-incubation, found no fiber type differentiation in the soleus of the
1-day-old rat, equal numbers of type I and II at 10 days and > 90 % type I in the
adult animal. Brooke et al. (1971), utilizing the successive acid pre-incubation
technique for ATPase stains, found type I and IIC fibers differentiated at 2 days
of age. During the third week of life some of the IIC fibers differentiated into IIA
fibers, and at 27 days more IIA fibers and less IIC fibers were noted.
The present study of development shows a consistent pattern in all of the areas
evaluated. It was found that the number of type I fibers remains stable throughout the developmental period and, in addition, this type makes up the majority
of the myotubular structures. The only obvious structural maturation in type I
fibers is an increase in diameter with increasing age. This lack of change was
emphasized by Engel & Karpati (1968), who suggested that as the lower motor
neuron unit changes in response to the developing central nervous system, the
type I neuron histochemically appears unaffected by this influence.
Type II fibers, however, seem very susceptible to maturational influence. They
are entirely IIC's in the first 17 days of life and undergo a dramatic conversion to
IIA fibers within a 3- to 4-day period. Less than 2 % IIC fibers remain after this
conversion and no change in percentage composition was found through adulthood. The likelihood that IIC and IIA fibers are similar or identical is supported
by the measurements of the fiber diameters (Fig. 3).
We found evidence of fiber-type differentiation in the 1-day-old animal, but
only with a 4.25 pH pre-incubation. The fact that the IIC fibers were uniformly
smaller than the type Fs supports the impression of two fiber types in the 1-dayold rat. In addition we have documented muscle spindles in 1- and 3-day-old
animals (Fig. 4), in contrast to previous observations that these structures were
not identified prior to 7 days of age (Engel & Karpati, 1968).
Oxidative enzyme techniques (NADH-TR) failed to reveal any change in
activity throughout the developmental phase. This is presumably a result of the
peculiarity of rat muscle types I and IIA for dark staining with oxidative enzyme
(Brooke & Kaiser, 1970). This is in contrast to human muscle, in which oxidative
stains reveal a checkerboard pattern in which the dark fibers are type I and a
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R. G. CURLESS AND M. B. NELSON
reciprocal light staining pattern is found with the ATPase (9-4 pH) method. We
were also unable to identify any differentiation in the oxidative staining pattern
of IIC fibers.
We have demonstrated an orderly progression of morphological and histochemical changes in rat soleus muscle. The most dramatic and abrupt change
involves the conversion of type IIC to IIA fibers between 18 and 21 days of age.
In addition fiber size, myotubes, nuclei and muscle spindles all show changes.
Supported in part by General Research Support Grant (GRSG) no. 5010-3150-62 and by a
grant from the American Academy of Cerebral Palsy.
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{Received 25 March 1976)