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Effects of denervation and tenotomy on the gastrocnemius muscle in the frogA histologic and histochemical study.

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Effects of Denervation and Tenotomy on the
Gastrocnemius Muscle in the Frog: A
Histologic and Histochemical Study
HARVEY B. SARNAT,' JAY M. PORTNOY AND DAVID Y. K CHI
Departments of Neitrology and Pedratr~cs,Saint Louzs U7zzuersrty Schoot of
Medicine, St. Loz~zs,Missotiri 63104
ABSTRACT
The effects of denervation on the gastrocnemius muscle of the
frog were studied by histologic and histochemical methods. Thirteen Rnna
pipiens underwent unilateral sciatic neurotomy and were sacrificed weekly as
long as 46 days. Of the three normal populations of muscle fibers, the small
fibers underwent atrophy, the intermediate sized fibers remained unchanged i n
size, and the large fibers either did not change or underwent hypertrophy between 21 and 46 days. Necrosis of muscle fibers did not occur. Histochemical
stains showed persistence of the normal pattern after denervation. The small
fibers continued to have a high concentration of both oxidative and glycolytic
enzyme activity (NADH-TR, SDH, phosphorylase), and the large fibers continued
to have a low concentration of these enzymes. Depletion of glycogen stores was
seen with PAS. Hypertrophic muscle fibers had mostly subsarcolemmal nuclei
and few internal nuclei, suggesting that they may be physiologically tonic
rather than twitch fibers. Achilles tenotomy a t the time of denervation prevented
the hypertrophy of large fibers. Abnormal inclusions have been demonstrated in
mammalian muscle following tenotomy alone, but were not seen in the frog.
Skeletal muscle fibers of birds and mammals have characteristic histochemical
properties which form a basis for distinguishing two or more extrafusal muscle
fiber types (Dubowitz, '68; Brooke and
Engel, '69; Engel, '74; Karpati et al., '75;
Askanas and Engel, '75). The reciprocal
differences in oxidative and glycolytic enzyme activities are thought to reflect metabolic differences in energy-supplying systems for contractile function. Red or type I
muscle fibers presumably are chiefly aerobic
or oxidative, and white or type I1 muscle
fibers function mainly by anaerobic or
glycolytic pathways. A third type of muscle
fiber with intermediate histochemical reactions is found in many birds and mammals. In some species, such as the rat,
type I muscle fibers are smaller than those
of type 11, but in man they are of nearly
equal size.
Particular emphasis has been placed on
the myofibrillar myosin adenosine triphosphatase (ATPase) reaction in which type I1
fibers stain more darkly than do type I fibers at physiologic and alkaline pH ranges.
Preincubation of tissue sections at low pH
ranges reverses the ATPase staining propANAT. REC., 187: 335-346.
erties so that type I fibers are darker, and
also permits the identification of fiber subtypes. However, these subtypes do not exhibit a uniform pattern among different
species (Yellin and Guth, '70; Karpati and
Engel, '76). Because of these species-specific characteristics of fiber subtypes, distinguishing only the three major types of
muscle fibers is useful for comparative
purposes.
Fishes, amphibians, and reptiles also
have muscles composed of two or three
fiber types which are distinguished structurally by size and histochemical reactions.
These muscle fibers do not correspond to
the types seen in birds and mammals. In
amphibians, the smallest fibers have high
concentrations of oxidative enzymes as
well as of phosphorylase and glycogen, and
the largest fibers have least. A n intermediate sized fiber with intermediate histochemical quantities also is identified (Dubowitz and Pearse, '60, Ogata a n d Mori,
'64; Dubowitz, '68). Although three types
Received May 17, '76. Accepted Sept. 27, '76.
1 Reprint requests to present address: H . B. S., Section
o f Neurology, Princess Margaret Hospital for Children,
Perth, W.A. 6001, Australia.
335
336
HARVEY B . SARNAT, JAY M. PORTNOY A N D DAVID Y. K . CHI
of muscle fibers are thus recognized in
the frog on the basis of size and histochemical reactions, only two types are identified
by electrophysiologic methods (Peachey,
'6 1),
This investigation used light microscopy
and histochemical methods to study the
reaction of gastrocnemius muscle fibers to
denervation and to tenotomy in the frog.
The reaction of skeletal muscle to denervation appears to be similar in amphibians
and mammals, despite the differences in
histochemical fiber types.
MATERIALS A N D METHODS
Thirteen healthy frogs ( R a m pipiens)
from Wisconsin, each about 7 crn in length,
underwent unilateral sciatic neurotomy
under local anesthesia with 1% procaine
hydrochloride injected subcutaneously.
Neurotomy was accomplished by resecting
10-15 mm of the right sciatic nerve from
within the thigh. The femoral artery was
identified and avoided, and blood loss was
negligible. The frogs were killed in a closed
jar with chloroform, a t weekly intervals
for seven weeks following neurotomy. Just
before sacrifice, the lower limbs were examined for condition of the skin and to
determine if the denervated leg remained
flaccid and anesthetic. The circumference
of the calves was measured.
Right Achilles tenotomy was performed
in two additional frogs and they were killed
at 28 days. Two others underwent combined Achilles tenotomy and sciatic neurotomy, and were killed at 46 days. Three
frogs were killed as controls, immediately
upon arrival from the commercial supplier.
Two others were permitted to survive 46
days as chronic controls, without any surgical procedures, because the initially active frogs became inactive after a few days.
Immediately after sacrifice, the gastrocnemius muscles of both legs were resected
and rapidly frozen as described below.
Minimal shortening occurred in normal
muscles, and no contraction was observed
in denervated or tenotomized muscles. The
cut ends of the sciatic nerve were exposed
in frogs which had previously undergone
neurotomy, to verify that regeneration and
anastomosis had not occurred.
During the experiment, frogs were kept
in a 20-gallon glass aquarium with one
end raised 3 cm. The aquarium was filled
with sufficient water to cover the bottom
of the lower half. External lamps maintained a constant air temperature of 27°C
within the aquarium. The water was
changed every other day. Each frog was
fed a bolus of raw hamburger on alternate
days.
Muscle blocks were frozen in isopentane
(2-methylbutane) cooled to - 160" C in
liquid nitrogen. Cross-sections of 8 pm
were cut in a cryostat. Histochemical incubations were performed according to
standard methods used in processing human muscle biopsies (Dubowitz and Brooke,
'73). Stains included hematoxylin and eosin, modified Gomori trichrome, nicotinamide adenine dinucleotide tetrazolium
reductase (NADH-TR), succinic acid dehydrogenase (SDH), periodic acid-Schiff reaction (PAS), active and total phosphorylase, and myofibrillar ATPase preincubated
at pH 4.4, 4.7, 9.4, and 10.2. Of the latter
stain, pH 4.7 and 10.2 were found to be
the most useful and in some animals only
these two were carried out. Reactions were
performed at 4 C (refrigerator temperature), 20°C (room temperature), and 37°C
in some instances, with the preincubation
time kept constant at five minutes at acid
pH and 30 minutes at alkaline pH. Some
tissue blocks also were frozen by direct immersion in liquid nitrogen, as a control of
possible interference by isopentane with
the ATPase reaction. The PAS reaction
was controlled by prior diastase digestion
of other sections.
Muscle fibers were measured as the longest distance across the minor axis of each
fiber in cross-section. Only well preserved
fibers were measured; those with freeze
artifacts were excluded. One hundred gastrocnemius muscle fibers of each of the
three populations of fibers were measured
and tabulated in one animal each after 7,
21, 28, and 46 days denervation, i n a tenotomized frog, two animals which had undergone both procedures, and in two control frogs. Because the absolute size of
muscle fibers in individual animals was
partly related to the size of the frog, comparison of the denervated or tenotomized
muscle with its counterpart in the opposite
leg of each frog was considered a more
meaningful control than tabulating pooled
measurements from all animals. The muscles of animals not systematically mea-
DENERVATION O F FROG MUSCLE
sured in this way were examined for both
extremes of fiber size. The ratios of the
three fiber types were determined in all
frogs.
RESULTS
Gastrocnemius muscles of the normal
frogs were composed of a mixture of three
populations of muscle fibers in a mosaic
distribution. The three fiber types were
distinguished on the basis of both size and
histochemical properties. The smallest
muscle fibers ranged from 50-70 pm in
cross-sectional diameter, and had high
concentrations of PAS-glycogen,phosphorylase, and also of the oxidative enzymes
SDH and NADH-TR. The muscle fibers of
intermediate size were 70-1 10 pm and had
histochemical staining reactions intermediate in intensity between those of the
small and the large fibers. The fibers of
largest diameter were 130-170 pm, and
had minimal histochemically demonstrable
glycogen or oxidative enzyme activity or
phosphorylase activity. A histogram of the
three populations of different sized fibers
in a normal frog gastrocnemius muscle is
illustrated in figure 1. The small fibers
were most numerous and comprised about
50% of the total. Large fibers comprised
30 % , and intermediate fibers were 20 % .
Muscle fibers of all sizes had mostly internal nuclei, but occasional peripheral or
subsarcolemmal nuclei also were seen.
These peripheral nuclei were most commonly in the largest fibers, and a third of
the large fibers had nuclei limited exclu-
337
sively to the subsarcolemmal location. The
number of nuclei per 8 pm cross-section
was one to three in the small fibers, three
to four in the fibers of intermedate size,
and six to eight in the large fibers, regardless of internal or peripheral location. Internal nuclei often were arranged in a ring
or circle within the fiber.
Fibers of the tenotomized gastrocnemius
muscles did not contain nemaline rods, central cores, or other abnormal inclusions
shown with the trichrome or histochemical
stains used. No architectural or degenerative changes were seen in these muscle
fibers. Endomysial fibrosis did not occur.
No difference was seen in muscle fiber diameters between the tenotomized and opposite control gastrocnemii of these frogs.
A focus of interstitial inflammation was
found in one muscle after tenotomy.
Denervated limbs of all frogs remained
flaccid and anesthetic to pinprick. Decubiti or purulent drainage did not occur.
The circumference of denervated calves
after 21 days was as much as 50% smaller
than that of extended normal calves of the
opposite legs.
Denervated muscles showed no evidence
of fiber necrosis or architectural alterations. Target fibers were not demonstrated.
Selective atrophy of the small fibers and
hypertrophy of some of the large fibers began at about 14 days after denervation
and were progressive. No change in size
was seen in intermediate muscle fibers. A
histogram comparing the sizes of muscle
fibers in the control and denervated gas-
LEFT GASTROCNEM IUS
FIBER DIAMETER (p)
Fig. 1 Histogram of three populations of muscle fibers of different cross-sectional diameters
i n the normal gastrocnemius muscle of a control frog.
HARVEY B . SARNAT, JAY M. PORTNOY AND DAVID Y. K. CHI
RIGHT GASTROCNEMIUS
( no n - den e rvated )
4036 32 28 24 -
FROG D-23
Q
.. .. .....
.... ....
20 16 12 84FIBER DIAMETER (p)
Fig. 2 Histograms of diameters of muscle fibers in the (left) control gastrocnemius muscle
and (right) denervated gastrocnemius muscle after 46 days, of the same frog. This animal is
representative of the response to denervation seen after 14 days. Small fibers become atrophic
(t = 1.98, significant at the 97% level). No significant change is seen in intermediate fibers.
Some large fibers undergo hypertrophy (t = 2.53, significant at the 99% level). Standard deviation 55.
trocnemii of a representative frog, 46 days
after sciatic neurotomy, is illustrated in
figure 2. The minimal shortening of the
control muscle at the time of removal would
tend to minimize rather than exaggerate
the degree of hypertrophy of large fibers
in the denervated muscle. Most of the hypertrophic muscle fibers had predominantly
or exclusively subsarcolemmal nuclei and
few internal nuclei (fig. 3).
Denervated muscle fibers of all sizes retained their normal histochemical characteristics despite the changes in size (figs.
4, 5). Reversal of histochemical type, dedifferentiation, and type grouping did not
occur. A minor change was depletion of
glycogen stores from muscles which had
been denervated 14 days or more (fig. 6).
Diastase produced complete digestion of
the PASpositive material in both control
and denervated muscles. The proportion
of the three types of muscle fibers was unchanged in tenotomized or denervated muscles, indicating that fibers did not become
converted into other types.
Muscles which had been simultaneously
tenotomized and denervated showed similar atrophy of the small fibers as occurred
in muscles which had undergone simple
denervation, but no changes in size were
observed in intermediate and large fibers.
The results of the ATPase reactions
were somewhat variable. Incubation at
room temperature produced the best results. Sections cut from tissue frozen directly in liquid nitrogen did not differ from
those frozen in cooled isopentane. At preincubation pH 10.2, the small fibers were
selectively stained and the intermediate
and large fibers remained unstained, although some small fibers also failed to
stain (fig. 7). Preincubation at low pH
ranges usually yielded a uniform light
brown staining of muscle fibers of all sizes,
but in some sections the large fibers were
more intensely stained than the small and
intermediate sized fibers, and in other sections the small fibers were darkest. The
location of fibers within the muscle, either
in the center or near the periphery, did
not determine the quality of the ATPase
reaction. No difference was seen between
denervated and control muscles with
ATPase.
Analysis of variance and t-tests were
performed to determine if the changes in
c
LEFT GASTROCNEMIUS
L
(46Days post-left sciatic neurotomy)
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DENERVATION OF FROG MUSCLE
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muscle fiber size were statistically significant. Significant changes were found after
14 days’ denervation in the small and
large fibers, but not in the intermediate
fibers (fig. 2).
DISCUSSION
Mammalian and avian striated muscle
has two physiologic types of extrafusal fibers, slow twitch and rapid twitch. Fishes,
amphibians, and reptiles have a single
type of twitch muscle fiber, but in addition
possess a slow, tonically-contracting muscle fiber not found in mammals (Peachey,
’61), except perhaps as intrafusal muscle
spindle fibers (Boyd, ’61). These slow tonic
muscle fibers are found in even primitive
living vertebrates, such as myxinoids (Andersen et al., ’63). In the frog, the twitch
fibers are innervated by large-diameter
nerves which conduct at 8-40 mlsec, and
tonic muscle fibers are innervated by small
nerves which conduct at 2-8 m/sec (Kuffler and Vaughn Williams, ’53).Differences
in the neuromuscular junctions also are
found (Hess, ’60).
The dependence upon motor innervation
of mammalian muscle fibers to maintain
their physiologic and histochemical properties has been shown by the reversal of
those properties induced by denervation
and cross-reinnervation by a nerve orig-
I
I
I
I
\.
.
4--1 1-4
~
inally supplying another muscle of a different type (Buller et al., ’60; Close, ’65;
Romanul and Van der Muelen, ’67). A similar nerve-muscle relationship in which innervation determines physiologic properties of muscle fibers also occurs in amphibians, After cross-reinnervation of the
“tonus bundle” of tonic muscle fibers in the
iliofibularis muscle of the frog by the “fast”
sartorius nerve, the contraction due to prolonged action of acetylcholine was greatly
reduced in duration and became similar
to that of the normal sartorius muscle
(Miledi and Orkland, ’66). Ultrastructural
changes were not seen.
Reciprocal histochemical changes are
seen in pectoralis muscle fibers of the pigeon following denervation without subsequent reinnervation (Cherian et al., ’66).
This phenomenon occurs to only a limited
extent in mammals. Type I1 muscle fibers
of the acutely denervated hemidiaphragm
of the rat change their histochemical properties to resemble type I fibers (Nachmias
and Padykula, ’58; Khan et al., ’73). We
did not observe dedifferentiation or other
histochemical changes in frog muscle fibers after acute or chronic denervation as
long as 46 days, except for a partial depletion of glycogen stores.
Atrophy is the characteristic response of
muscle fibers to denervation in mammals,
340
HARVEY B . SARNAT, JAY M. PORTNOY AND DAVID Y. K . CHI
and type I1 fibers undergo atrophy more
promptly than do type I fibers (Feng and
Lu, '65). Post-denervation hypertrophy of
muscle fibers lasting several weeks also may
occur under some circumstances in many
birds and mammals (Stewart et al., '72;
Sola et al., '73), but this phenomenon has
not previously been reported i n amphibians. The extent of the hypertrophy appears
to be related mainly to the amount and duration of stretch (Stewart et al., '72). Tenotomy performed at the time of denervation
of the "slow" anterior latissimus dorsi muscle of the chicken resulted i n muscle fiber
atrophy only, in contrast to the hypertrophy of muscle fibers which followed denervation alone (Jirmanova and Zelena,
'70). Only atrophy followed simple denervation of the "fast" posterior latissimus
dorsi of the chicken. Neither atrophy nor
hypertrophy of muscle fibers occurred as
long as 171 days after denervation of the
hemidiaphragm of the marsupial quokka
(Stewart et al., '72), suggesting that factors other than stretch also may be important. The normally smaller type I fibers of
the rat diaphragm undergo hypertrophy
between the fourth and fourteenth postdenervation days, until there is uniformity
in size as well as in high oxidative enzyme
activity (Nachmias and Padykula, '58). In
man, infantile spinal muscular atrophy is
a denervative disease of motor neurons
which begins in fetal life or early infancy.
The majority of muscle fibers of both histochemical types undergo atrophy, but selective hypertrophy of some type I fibers
also occurs (Dubowitz and Brooke, '73).
Achilles tenotomy in the rat and cat was
followed in four to six weeks by the appearance of nemaline rods and central
cores in gastrocnemius muscle fibers, but
these inclusions did not appear if the muscle also was denervated (Engel et al., '66;
Shafiq et al.,'69). We were unable to demonstrate these inclusions after a comparable period of tenotomy in the gastrocnemius
muscle of the frog. Degenerative changes
occur in the red soleus muscle of the rabbit within three weeks after tenotomy, although are not seen in the white peroneus
longus muscle (McMinn and Vrobova, '62).
We did not observe muscle fiber necrosis
or fibrosis in the frog, except for a focus
of acute inflammation i n one muscle, which
probably represented infection.
A correlation has been shown relating
the slow tonic and the twitch muscle fibers
of the frog with ultrastructural differences
(Peachey and Huxley, '62; Page, '65). A
similar correlation has been made in the
snake (Hess, '65). The electron microscopic
differences involve the organization of myofibrils, the transverse tubular system. and
sarcoplasmic reticulum. Engel and Irwin
('67) found that i n the frog, the small and
intermediate sized muscle fibers, with
strong and intermediate histochemical reactions respectively, were both twitch fibers
physiologically. Slow tonic fibers were exclusively large, but some large fibers with
low enzymatic activity also were capable
of twitch responses. They also reported
that the tonic fibers exhibited a weak reaction for ATPase, and the twitch fibers
uniformly stained darkly. Tonic fibers were
distinguished from large twitch fibers histologically by the exclusively subsarcolemma1 location of nuclei i n the tonic fibers.
We did not find the ATPase reaction in
frog muscle to be of sufficient reliability
to make this distinction. We did observe,
however, that the hypertrophic fibers after
denervation had the fewest internal nuclei
and the largest number of subsarcolemma1 nuclei.
In conclusion, if the peripheral location
of nuclei in large fibers is indeed a valid
histologic marker of physiologically tonic
muscle fibers in the frog, the denervated
muscle fibers which hypertrophy are of the
tonic type. Fibers which atrophy or remain
unchanged in size are twitch fibers. All
muscle fibers retain their distinctive histochemical features for at least 46 days after
denerva tion.
LITERATURE CITED
Andersen, P., J . K. S . Jansen and Y. Llyning
I963 Slow and fast muscle fibers in the Atlanti hagfish ( M y x i n ~glutinosa). Acta Physiol.
Scandin., 57: 167-179.
Askanas, V . , and W . K. Engel 1975 Distinct
subtypes of type I fibers of human skeletal muscle. Neurology, 25: 879-887.
Boyd, I. A. 1961 The motor innervation of mammalian muscle spindles. J . Physiol., 159: 7-9.
Brooke, M., and W. K. Engel 1969 The histochemical analysis of human muscle biopsies with
regard to fiber types. Neurology, 19: 221-233.
Buller, A . J . , J. C. Eccles and R. M. Eccles 1960
Interactions between motoneurons and muscles
in respect of the characteristic speeds of their
responses. J. Physiol., 1 5 0 : 417439.
Cherian, K . M., F. D. Bokdawala, N . V. Vallyathan
DENERVATION OF FROG MUSCLE
and J. C. George 1966 Effects of denervation
on the red and white fibres of the pectoralis muscle of the pigeon. J. Neurol. Neurosurg. Psychiat.,
29 : 299-309.
Close, R. 1965 Effects of cross-union of motor
nerves to fast and slow skeletal muscles. Nature,
206: 8 3 1 4 3 2 .
Dubowitz, V. 1968 Developing and Diseased
Muscle. William Heinemann Publ. Co.,London.
Dubowitz, V., and M. H. Brooke 1973 Muscle
Biopsy: A Modern Approach. W. B. Saunders
Co., Philadelphia.
Dubowitz, V., and A. G. E. Pearse 1960 A comparative histochemical study of oxidative enzymes and phosphorylase activity in skeletal
muscle. Histochemie, 2: 105-1 17.
Engel, W. K. 1974 Fiber-type nomenclature of
human skeletal muscle for histochemical purposes. Neurology, 24: 344-348.
Engel, W. K., M. H. Brooke and P. G. Nelson
1966 Histochemical studies of denervated or
tenotomized cat muscle. Illustrating difficulties
in relating experimental animal conditions to
human neuromuscular diseases. Ann. N.Y. Acad.
Sci., 138: 160-185.
Engel, W. K., and R. L. Irwin 1967 A histochemical-physiological correlation of frog skeletalmusclefibers. Amer. J. Physiol.,213: 5 1 1 5 1 8 .
Feng, T. P., and D. X. Lu 1965 New lights on
the phenomenon of transient hypertrophy i n
the denervated diaphragm of the rat. Sci. Sin.,
14: 1772.
Hess, A. 1960 The structure of extrafusal muscle fibers i n the frog and their innervation studies
by the cholinesterase technique. Amer. J. h a t . ,
107: 129-151.
1965 The sarcoplasmic reticulum, the
T system, and the motor terminals of slow and
twitch muscle fibers in the garter snake. J. Cell
Biol., 26: 4 6 7 4 7 6 .
JirmanovA, I., and J. Zelena 1970 Effect of denervation and tenotomy on slow and fast muscles of the chicken. Z . Zellforsch., 106: 3 3 3 4 4 7 .
Karpati, G., A. A. =sen and S. Carpenter 1975
Subtypes of the histochemical type I muscle fibers. J. Histochem. Cytochem., 23: 89-91.
Karpati, G., and W. K. Engel 1976 Subtypes of
histochemical type I muscle fibers. Neurology,
26: 296 (letter to editor and reply).
Khan, M. A., B. A. Kakulas, P. G. Holt and 0. M.
Sola 1973 Denervation hypertrophy of rat
diaphragm: A histochemical study. In: Clinical
34 1
Studies in Myology, B. A. Kakulas, ed. Excerpta
Medica, Amsterdam, pp. 322-330.
Kuffler, S. W., and E. M . Vaughn Williams 1953
Small-nerve junctional potentials. The distribution of small motor nerves to frog skeletal muscle, and the membrane characteristics of the
fibres they innervate. J. Physiol., 121 : 289-317.
McMinn, R., and B. Vrobova 1962 Morphological changes in red and pale muscles following
tenotomy. Nature, 295 : 509.
Miledi, R., and P. Orkland 1966 Effect of a
“fast” nerve on “slow” muscle fibres in the frog.
Nature, 209: 717-718.
Nachmias, V. T., and H. A. Padykula 1958 A
histochemical study of normal and denervated
red and white muscles in the rat. J. Biophysic.
Biochem. Cytol., 4: 4 7 5 3 .
Ogata, T., and M. Mori 1964 Histochemical
study of oxidative enzymes in vertebrate muscles. J . Histochem. Cytochem., 12: 171-182.
Page, S. G. 1965 A comparison of the fine structure of frog slow and twitch muscle fibres. J.
Cell Biol., 26: 477-497.
Peachey, L . D. 1961 Structure and function of
slow striated muscle. In: Biophysics of Physiological and Pharmacological Actions. Amer. AsSOC. Adv. Sci., Washington, D.C., pp. 391-411.
Peachey, L. D., and A. F. Huxley 1962 Structural identification of twitch and slow striated
muscle fibres in the frog. J. Cell Biol., 13: 177180.
Romanul, F. C. A,, and J. P. Van der Meulen
1967 Slow and fast muscles after cross-innervation. Enzymatic and physiological changes.
Arch. Neurol., 17: 387-401.
Shafiq, S. A., M. A. Gorycki, S. A. Asiedu and A. T.
Milhorat 1969 Tenotomy. Effects on the fine
structure of the soleus of the rat. Arch. Neurol.,
20: 625-633.
Sola, 0. M., D. L. Christensen, M. A. Khan, B. A.
Kakulas and A. W. Martin 1973 Post-denervation muscle hypertrophy: A review with evaluation. In: Clinical Studies i n Myology. B. A.
Kakulas, ed. Excerpta Medica, Amsterdam, pp.
310-31 5 ,
Stewart, D. M., 0. M. Sola and A. W . Martin
1972 Hypertrophy as a response to denervation in skeletal muscle. Z . vergl. Physiol., 76:
146-167.
Yellin, H., and L. Guth 1970 The histochemical classification of muscle fibers. Exp. Neurol.,
26: 4 2 4 4 3 2 .
PLATE 1
EXPLANATION OF FIGURES
342
3
Gastrocnemius, 28 days post-denervation. The large fibers with fewest
internal nuclei are those which hypertrophy. Trichrome. Original magnification X 100.
4
Gastrocnemius, 42 days post-denervation. Oxidative enzyme activity
persists in the normal pattern, with the smallest fibers having the highest concentration and the large fibers having the lowest. NADH-TR.
Original magnification x 100.
DENERVATION OF FROG MUSCLE
Harvey B. Sarnat. Jay M. Portnoy and David Y. K. Chi
PLATE I
343
PLATE 2
EXPLANATION OF FIGURES
344
5
Gastrocnemius, 42 days post-denenation. Glycolytic enzyme activity
persists i n the normal pattern, similar to that of oxidative enzymes.
Total phosphorylase. Original magnification X 150.
6
Gastrocnemius, 46 days post-denenration, Distribution of glycogen
with the highest concentration in the small fibers persists, but glycogen
is depleted i n large and intermediate fibers. Glycogen was demonstrated in these fibers in the contralateral control gastrocnemius. PAS.
Original magnification X 100.
DENERVATION OF FROG MUSCLE
Harvey B . Sarnat, Jay M. Portnoy and David Y. K. Chi
PLATE 2
345
DENERVATION Oh FROG M US CL E
H a r v e y R Sarnat J a y M Portnay a n d David Y K C h i
EXPLANATION OF FIGURE
7
346
Gastrocnemius, 14 days post-denervation. ATPase stain preincubated
at p H 10.2 shows staining of some small fibers, but other small fibers
(arrow) and all those of intermediate and large size remain unstained.
PLATE 3
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