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Experimental Corticosteroid Myopathy.

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Experimental Corticosteroid Myopathy
By MICHAEL
G. SHEAHAN
AND PAUL
J. VIGNOS,
JR., M.D.
Triamcinolone treatment in rabbits using
1 mg./Kg. doses produces a differential
metabolic effect, with predominant involvement of glycolytic-type fibers of
muscle manifested by a significant
atrophy of type I1 fibers. A consequent
atrophy of muscle containing a large
T
of large doses of corticosteroids
in the treatment of various conditions,
such as connective tissue diseases, may
cause muscle weakness in
and
Although it has been suggested
that halogenated steroids produce the
greatest changes: studies comparing the
effects of various steroids have shown that
many steroid compounds will produce decreases in total body and individual muscle
weight in both dogs5 and mice.?
Histological studies on human biopsy
material have shown minimal and variable
nonspecific changes in the muscle fibers.
Histochemical staining methods have not
been applied in a systematic study of
steroid myopathy in human subjects because many patients with steroid-induced
myopathy also have a primary disease capable of causing histochemical alterations
in muscle.
Steroid myopathy in experimental animals is manifested by histological muscle
fiber changes ranging from no pathologic
change to fiber necrosis, regeneration, and
severe muscle atrophy. The type of fiber
atrophy has varied widely in past reports.
Generalized muscle fiber atrophy was reHE USE
Supported in part by a grant from the Muscular
Disease Society of Northeastern Ohio.
MICHAEL G. SHEAHAN:Fourth year medical
student, Case Western Reserve University School
of Medicine, Cleveland, Ohio. PAUL J. VIGNOS,
JR., M.D.: Associate Professor of Medicine, and
ARmmms
AND
proportion of type II fibers also occurs.
These changes take place before there
is any signscant alteration in size of
type I fibers, and before there is any
apparent loss of either glycolytic or oxidative enzyme activity.
ported by Faludi and co-workers in a histological study of mice treated with a variety of steroids, including 2 mg. of triamcinolone ( approximately 71 mg./ Kg. ) six
times a week for 4 weeks.? DAgostino and
Chiga, using both light and electron microscopy, reported primary involvement of
granular (type I, red) muscle fibers in
rabbits receiving 10 mg./Kg. of cortisone
for 12-14 days8 Smith has observed primarily agranular (type 11, white) muscle
fiber changes in histochemical studies of
rabbits receiving 6-10 mg./Kg. triamcinolone for 3-15 days6
The present study tries to document the
earliest changes in animal muscles associated with corticosteroid administration.
Triamcinolone was administered to rabbits
using dosage levels more comparable to
those used clinically. It was hoped that administration of amounts of corticosteroid
smaller than previously used might clarify
the primary type of muscle fiber involvement.
MATERIALS
AND METHODS
White, male New Zealand rabbits weighing
approximately 2 Kg. each were divided into test
Director, Arthritis Unit, University Hospitals o/
Cleuelund, Chelund, Ohio.
Reprint requests should be addressed'io Or.
Vignos at the University Hospitals of Cleveland,
2065 Adelbert Road, Clevelund, Ohio 44106.
RHEUMATISM, VOL. 12, No. 5 (OCTOBER
1969)
491
492
SHEAHAN AND VIGNOS
Table 1.-Effect
Animal no.
1
2
3
4
5
6
7
8
9
Mean
SD
P
of Corticosteroid on Muscle and Total Body Weight
Initial body wt., Kg.
Final body wt., Kg.
Control
Test
Control
2.27
1.84
2.09
2.05
1.88
2.18
2.18
2.39
2.58
2.16
0.23
2.47
1.90
2.02
2.03
1.92
2.07
2.36
2.28
2.67
2.19
0.26
>0.8
Test
2.66
2.62
2.27
1.87
2.58
2.38
2.25
1.93
2.43
1.80
2.58
1.68
2.63
2.08
3.16
2.20
3.27
2.80
2.65
2.15
0.35
0.38
<0.05
and pair-fed control groups. The test rabbits received an injection of 1 mg./Kg. triamcinolone
daily for 3 weeks. Injection was into the hamstring muscle group, alternating the side injected
each day. The test rabbits and the pair-fed controls were sacrificed after 3 weeks by an intravenous injection of 180 mg. pentobarbital sodium.
Muscles to be examined were immediately excised, weighed, and frozen in isopentane immersed
in liquid nitrogen. The entire left and right
soleus muscles were excised, and a defined section of each gastrocnemius muscle was utilized.
The portion of the gastrocnemius excised was that
part of the lateral head bounded by the peroneus
nerve, the vena saphena parva, and the plantaris
muscle. The weight of both soleus muscles was
expressed as per cent of total body weight at time
of sacrifice. The weight of the excised portion of
both gastrocnemius muscles was similarly expressed.
Frozen sections from the midportion of the
gastrocnemius and soleus muscles were cut at
-25OC in a cryostat and stained for fiber morphology using a modified trichrome reaction.9 Sections of each muscle were stained according to
reported histochemical methods for phosphorylase,lOJl succinic dehydrogenase,lZ NADH diaphorase,13J* and myofibrillar A”F’ase.15 Sections
of the appropriate control muscle were handled
in an identical manner.
Muscle fiber size was estimated by measuring
the diameters of more than 30 fibers of each
type ( I and 11) in at least five different high
power fields. Only fibers which appeared to be
cut in transverse section were measured. Maximum vertical and horizontal diameters of each
fiber were used to obtain an average maximum
diameter. Fibers on the edge of the section and
those contracted from the e n d o m y s i h were not
Gastrocnemius,
% body wt.
Control
Test
0.33
0.19
0.27
0.18
0.30
0.17
0.29
0.15
0.26
0.20
0.22
0.21
0.33
0.15
0.31
0.19
0.31
0.18
0.29
0.18
0.04
0.02
<0.001
Soleus
- % body &.
Control
Test
0.09
0.11
0.10
0.10
0.10
0.11
0.10
0.11
0.10
0.11
0.10
0.11
0.11
0.12
0.09
0.10
0.09
0.09
0.10
0.11
0.01
0.01
<0.05
measured. Although there are various ways to
measure muscle fiber thickness, the determination
of maximum fiber diameter was used in this study
to minimize any changes caused by fiber atrophy.
Since the atrophic fibers were found to assume
what has been termed an “angular” rather than
polygonal shape, the comparison of maximum diameters minimized the observed changes in fiber
transverse sectional area. All measurements were
made with an eyepiece micrometer calibrated with
a stage micrometer. Distinction of fiber type was
based on the ATPase (6 p sections incubated for
10 min.) or phosphorylase ( 16 p sections incubated for 10 min.) reactions. ATPase and
phosphorylase staining reactions give good
agreement in fiber diameter measurements. Only
obvious type I and I1 fibers were used.
RESULTS
There was a s i g d c a n t difference ( p <
0.05) in the terminal body weight of the
steroid-treated animals as compared to the
controls. There was no difference in initial
weights between the two groups (Table
1)There was a significant decrease in average weight of the gastrocnemius muscle in
the steroid-treated animals when compared to the controls (Table 1). Conversely, the average weight of the soleus
muscle from the steroid-injected animals
showed a slight but s i g d c a n t increase in
weight when compared to its control
(Table 1).
There was no difference in histologic ap-
493
EXPERIMENTAL CORTICOSTEROID MYOPATHY
Table 2.-Muscle
Animal no.
1
2
3
4
5
6
7
8
9
Mean
SD
P
_.__
Fiber Type Sizes in Control and Corticosteroid-Treated Rabbits
Gastrocnemius, p
Type I
TypeU Control
Test
Control
Test
42.0
39.0
45.9
41.5
41.2
49.9
49.8
45.0
48.1
44.7
4.0
>
__
46.7
42.3
49.0
41.1
39.2
43.5
40.1
37.8
44.0
42.6
3.6
0.2
57.7
46.3
53.3
40.0
65.8
40.8
54.4
34.1
58.9
41.8
58.8
39.1
68.4
35.7
64.0
39.6
66.1
44.8
60.8
40.2
5.4
3.9
<0.001
pearance between the test and control
muscles in the tissues stained with the
modified trichrome reaction except for the
appearance in the steroid-treated animals
of randomly distributed slightly angular
fibers, especially in the gastrocnemius
muscle. The angular-shaped fibers were
found to be histochemical type I1 fibers.
In the gastrocnemius, the average size
of the type I1 fiber in the steroid-treated
animals is significantly smaller than the average control type I1 fiber. Similar changes
are found in the soleus muscle, where the
average size of the type I1 fibers in the
steroid-treated animals is again significantly smaller than the average control
type I1 fiber. There was no statistical difference between the average size of the
injected and control type I fibers in
gastrocnemius or soleus (Table 2).
A sample photomicrograph of a control
gastrocnemius muscle showed that the
type I1 fibers in the control animal appeared to be larger than the type I fibers
(Figure 1A). In the steroid-treated animals, the type I1 fibers were .either the
same size as the type I fibers or smaller
(Figure 1B).
I n the control soleus muscle, the two
fiber types have a variable size relationship, although in general the two fiber
types appear to be of equal size (Figure
-
Soleus, p
Type I
Control
Test
63.8
52.7
62.2
59.0
63.6
68.4
67.9
58.0
56.0
61.3
5.3
57.3
53.7
66.3
57.1
42.9
59.7
54.9
50.4
63.9
56.2
7.0
>O.l
Type I1
C&&ol
64.0
49.7
67.7
64.2
68.1
55.3
72.6
66.3
65.0
63.7
7.0
<O.Ol
Test
49.2
46.0
50.7
46.2
45.1
47.3
51.7
58.0
71.2
51.7
8.3
2A). The soleus type I1 fibers of the steroid-injected animals generally appeared
somewhat smaller than the type I fibers
(Figure 2B). The difference in size between the two fiber types was not as striking as in the gastrocnemius, although actual
fiber measurement did show a significant
change.
The muscle from the steroid-treated animals did not show any decrease in enzyme
activity as judged by the four histochemical stains for oxidative and glycolytic enzyme activity. The quality, intensity, and
localization of all the stains used appeared
similar in the injected and control rabbit
muscles. The loss of phosphorylase activity
reported to occur with higher doses of triamcinolonee was not observed at the corticosteroid dosage levels used in this study.
DISCUSSION
Normal human and animal muscles are
composed of muscle fibers which can be
differentiated according to color, physiologic response to stimulus, and enzyme
content as judged by biochemical and histochemical technics. A number of muscle
fiber classifications have been proposed. In
this study, the histochemical classification
of Dubowitz and Pearse16 and Engel" has
been used. Type I fibers are red, slow in
response, and have predominantly oxidative
494
SHEAHAN AND VIGNOS
Fig. 1.-Control and corticosteroid-treated gastrocnemius muscle, ATPase reaction.
(A) Control muscle. (B) Steroid-treated muscle. Dark fibers are type I1 and light fibers
are type I. 126 X.
EXPERIMENTAL CORTICOSTEROID MYOPATHY
495
Fig. 2.-Control
and corticosteroid-treated soleus muscle, ATPase reaction. (A)
Control muscle. (B) Steroid-treated muscle. Dark fibers are type I1 and light fibers are
type I. 126 X.
496
metabolism. Type I1 fibers are white, fast,
and are predominantly glycolytic.
The results of this study indicate that
the primary change in experimental steroid
myopathy is a decrease in type I1 fiber
size. The change was seen in both the gastrocnemius and soleus muscles but was
more marked in the former.
Smiths and DAgostino and ChigaR reported an apparent reversal in the normal
size relationship between type I and I1
fibers in steroid-treated rabbit muscle. The
larger fibers in the control animals were
type 11, while the larger fibers in steroidtreated animals were type I. Fiber size
measurements were not given by these investigators. The reversal of the normal
fiber size relationship was interpreted by
Smith as an atrophy of type I1 fibers. This
interpretation is supported by the fiber
size measurements reported in our study.
DAgostino and Chiga suggested that the
change primarily involved an increase in
type I fiber size. No increase in type I
fiber size was found in our experiments.
Triamcinolone was used in both our study
and that of Smith, while D’Agostino and
Chiga used cortisone. Although it is possible that the different results may be explained by difference in steroids used,
other studies in experimental steroid myopathy make this unlikely. Faludi and coworkers report that seven different steroids
cause similar minimal changes in mouse
muscle.r
It has been suggested that steroid myopathy may be associated with an inhibition of muscle phosphorylase, resulting in
an increased dependence on the mitochondrial enzyme systems of the fibers for
energy production.6 Conversely, the mitochondrial oxidative phosphorylation system
has been implicated in steroid myopathy.s
Results of our study indicate that the decrease in size of the type I1 fibers occurs
before any decrease in phosphorylase, oxidative, or other enzyme activity can be de-
SHEAHAN AND VIGNOS
tected by the histochemical reactions used
in this study.
The ratio of type I to type I1 fibers
differs in the gastrocnemius and soleus
muscles as it does in other animal musThe gastrocnemius is composed
mainly of type I1 fibers, and the soleus is
composed almost entirely of type I fibers.
If the type I1 fibers are the principal fiber
type undergoing atrophy, one would expect
the gastrocnemius muscle to be more
atrophic than the soleus. A comparison of
the relative weight losses of the two muscles suggests that this is the case. Since
muscle weight was calculated as per cent
of total body weight, the weight of the
gastrocnemius muscle decreased out of proportion to total body weight in the steroidtreated animals to produce a relative
weight decrease in this muscle. The increase in weight of the soleus muscle in
the steroid-treated animals is interpreted
as a greater relative decrease in total body
weight than in soleus weight, due to the
high concentration of type I fibers in this
muscle.
The gastrocnemius and soleus muscles
were chosen for study since each represents a muscle composed predominantly of
one fiber type. Therefore, relative weight
changes should be more apparent if one
fiber type were preferentially affected
by steroid treatment. Furthermore, since
Smith reports that proximal muscles are
affected by triamcinolone to a greater extent than distal muscles,6 it seemed preferable to study distal muscles in the hope
that these muscles would show less severe alterations. This might make it easier
to distinguish early and possibly primary
alterations in muscle fiber types.
“Type I1 fiber atrophy” has been reported in a number of human muscle diseases. These include cachetic atrophy,
polymyositis, corticosteroid atrophy, subacute inclusion body encephalitis, and myasthenia gravk20 Patients with dissimilar
497
EXPERIMENTAL CORTICOSTEROID MYOPATHY
chronic disease also have type I1 fiber
atrophy.*l However, the majority of these
last patients were also taking corticosteroids. It is possible that type I1 is the more
vulnerable fiber type, and thus may be af-
fected preferentially by many different
disease processes acting through a common
pathway or through multiple metabolic derangements.
SUMMARIO
IN INTERLINGUA
Tractamento con triamcinolona in conilios in un dosage de 1 mg per kg produce un
effect0 metabolic differential con affection predominante de fibras muscular del typo
glycolytic, manifeste in un atrophia significative de fibras de typo 11. Occurre etiam un
consequente atrophia de musculo continente un alte proportion de fibras de typo 11.
Iste alterationes occurre ante que ulle significative alteration &mensional es marliteste
in fibras de typo I e ante omne apparente perdita de activitate enzymatic de typo
glycolytic o oxidative.
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Rapid examination of muscle tissue: an improved
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Cytochem. 6:208, 1958.
12. Nachlas, M. M., Tsou, K. C., DeSouza, E.,
Cheng, C. S., and Seligman, A. M.: Cytochemical
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use of a new p-nitrophenyl substituted ditetrazole. J. Histochem. Cytochem. 5:420, 1957.
13. Friede, R. L., Fleming, L. M., and Knoller,
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14. Scarpelli, D. G., Hess, R., and Pearse,
A. G. E.: Cytochemical localization of oxidative
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15. Padykuda, H. A., and Herman, E.: The
specificity of the histochemical method of adenosine triphosphatase. J. Histochem. Cytochem. 3:
170, 1955.
16. Dubowitz, V., and Pearse, A. G. E.: A
comparative histochemical study of oxidative enzyme and phosphorylase activity in skeletal muscle. Histochemie 2: 105, 1960.
17. Engel, W. K.: The essentiality of histoand cytochemical studies of skeletal muscle in
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18. Bullard, H. H.: Histological as related to
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1919.
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