The effects of inactivity, programmed stimulation, and denervation on the histochemistry of skeletal muscle fiber types

EXPERIMENTAL

NEUROLOGY

391-413 (1973)

40,

The Effects of Inactivity, Programmed Denervation on the Histochemistry Muscle Fiber Types D. A.

Laboratory Stroke, ment

of Neurochemistry,

RILEY

AND

E. F. ALLIN

Stimulation, of Skeletal

and

l

National Institute

of Neurological Diseases afld U.S. Public Health Service, Depavtof Health, Education, and Welfare, Bethesda, Maryland 20014, and Department of Anatomy, University of Wisconsin, Madison, Wisconsin 53706

National Institutes

of Health,

Received

February

28,1973

The role of nerve impulse pattern in determining the histochemical characteristics of muscle fibers was investigated in cats. Motor neuron activity was eliminated by transecting the spinal cord between segments S2 and S3 and cutting all dorsal rootlets caudal to this level. In some of these cats, caudal nerves to the tail muscles were stimulated supramaximally for 1 mo in patterns approximating the output of either phasic or tonic motor neurons. Intertransverse muscles of the tail were examined histochemically for activities of a mitochondrial oxidative enzyme, a glycolytic enzyme, and myofibrillar ATPase. Three fiber types, red, white, and intermediate, were defined in control muscles on the basis of histochemical staining intensities and myofibril size and shape. Red fibers have high oxidative, low glycolytic, and low ATPase activities and wide, polygonal fibrils. White fibers are at the opposite extreme in all of these features and have more narrow, rectangular fibrils. Intermediate fibers are intermediate in all of these features except that their ATPase activity is high like that of the white fibers, but is more sensitive to high pH and formaldehyde. Specific arrangement of these fiber types within muscle fasciculi facilitated their identification in experimental muscles. The red fibers tend to be in the deeper parts of fasciculi ; white fibers tend to be at the periphery; and intermediate fibers are found in both locations. In the nonstimulated muscles, prolonged disuse brought about a progressive decrease of oxidative and glycolytic enzymatic activities. Tonic stimulation increased oxidative activity and decreased glycolytic activity. Phasic stimulation resulted in high glycolytic activity, while oxidative activity was decreased. Following prolonged disuse and both programs of stimulation, myofibrillar ATPase 1 The authors thank the many people at the University of Wisconsin and NIH who helped complete this study. Part of this work was supported by NIH Training Grant No. 2TOl-GM00723-11 USPHS Grant HD 00277. 391 Copyright All rights

Q 1973 by Academic Press, Inc. of reproduction in any form reserved.

392

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ALLIN

activity remained unchanged in white and red fibers but decreased somewhat in varying numbers of intermediate fibers. There was preferential atrophy of white fibers in the disused muscles but they achieved nearly control size following tonic or phasic stimulation. Thus, the patterns of impulse activation exert a major influence on the oxidative and glycolytic metabolic properties of muscle fibers and fiber cross-sectional area. However, since myofibrillar ATPase activity remained relatively unaltered, this characteristic may be controlled by some mechanism other than impulse transmission.

INTRODUCTION Histochemical methods have demonstrated that the constituent fibers of most skeletal muscles exhibit a variety of metabolic properties (3, 49). Within a given fiber various combinations of oxidative, glycolytic, and contractile enzymatic activities are found; however, all of the fibers of a single motor unit (i.e., all fibers innervated by the same alpha motor neuron) have virtually the samecombination of enzymatic activities (7, 8). Cross-reinnervation studies have revealed that these histochemical properties are neurally regulated (19, 26, 38). When a “white” muscle composed predominately of fibers of high glycolytic, high myofibrillar ATPase, and low oxidative activities is reinnervated by the nerve from a “red” muscle with fibers mostly of the opposite characteristics, the white muscle is converted to a red muscle. A red muscle can be converted to a white muscle by reinnervation with the nerve of the white muscle. Since (most) neurons of red musclesfire tonically, while those of white muscles generally fire phasically (5,6, 31)) the question can be asked whether these patterns of activation determine the histochemical properties of the muscle fibers. Various exercise regimens can affect muscle fiber energy metabolism in different ways (12, 24). Since there is presently no satisfactory way to predict accurately the activity patterns of single motor units during exercise, there is no way of knowing with certainty which aspects of the exercise are responsible for the observed changes. Attempts have been made to use programmed electrical stimulation to activate all fibers in a muscle in a uniform and defined manner (11, 36, 37, 41, 43). This type of “exercise” generally resulted in significant physiological or metabolic changes. Two problems in the design of such experiments is that stimulation is generally superimposed upon voluntary activity and that stimulation of the afferents may provoke reflex effects, thus making it impossible to know the actual program of activity. To avoid these complications, we chose to inactivate completely the muscles of the cat’s tail by a combination of spinal cord transection (removing

voluntary

input

to the

caudal

cord)

and

section

of all

dorsal

rootlets below the transection (removing reflex input to the cord). This

MUSCLE

HISTOCHEMISTRY

393

isolation procedure produces chronically quiescent muscles without denervation (9, 10, 28, 46, 47). The tail muscles were then activated either tonically or phasically by controlled electrical stimulation of their motor nerves, producing uniform activation of all muscle fibers. These stimulation programs produced distinctive histochemical appearances which were different from those of control muscles. Preliminary reports of this study have been published ( 1,Z). MATERIAL

AND

METHODS

Operative Procedures. Adult male and female cats (1.5-5.5 kg) were anesthetized with sodium pentobarbital (40 mg/kg ip) and prepared for sterile surgery. A laminectomy of vertebrae L7 and Sl was performed and the dura was incised down the midline. With the aid of a dissecting microscope, all dorsal rootlets caudal to cord segment Sl were cut and the filum terminale transected to destroy any stray afferents which it might contain. The spinal cord was transected between segments Sl and S2 by picking out cord substance with watchmaker’s forceps and iridectomy scissors in about a l-mm wide transverse section of the cord. This procedure was aided by suction. A blunt, spade.-shaped instrument was then pressed against the inside wall of the dura to sever all remaining nerve tracts. The ventral spinal artery was preserved to prevent necrosis of the spinal cord and the dorsal vein of the cord was also preserved whenever possible. The dura and skin incisions were sutured and the animals given bicillin (lSO,OOO-300,000 units, im.). Postoperatively, all cats had good hind-limb control. Complete removal of the spinal cord caudal to Sl was done in one cat (no. 18). Two other denervations (cats 19, 20) resulted from unsuccessful isolation attempts in which the ventral artery was accidently cut. The caudal cords had completely dissolved by about 2 mo. In a fourth cat (no. 21)) denervation was produced by transecting the caudal nerves in the sacral neural canal. Chronic stimulation was given the caudal nerves via either implanted wire electrodes (six cats) or superficial electrodes (six cats) encircling the base of the tail. After dorsal exposure of the sacrum, straight platinum wire (0.2 mm diam.) electrodes were implanted through openings made with a dental drill in the arch of the third sacral vertebra. The electrodes were installed either one just above and one below, or both above the caudal nerves (about 8 mm apart) and fixed in place by acrylic resin. A flexible multistranded, Teflon-coated stainless steel wire (0.5 mm diam) was run under the skin from each electrode to an acrylic plug-in mound anchored by vitallium screws to an iliac blade. This mound protroded

394

RILEY

AND

ALLIN

through an opening in the skin, the edges of which were sutured to it. In other cats, the wires were run subcutaneously to a flat plug-in device anchored by encircling tape to the dorsal surface of the base of the tail. Stimulation Procedures. The maximal current required for full activation of the muscles was determined by gradually increasing the current through the implanted electrodes until the height of the summated nerve and muscle action potential, as recorded with surface electrodes distally on the tail, no longer increased with an increase in current. Symmetrical biphasic pulses of 1 msec duration were used for stimulation. Maximum current ranged from 1 to 3 ma. Supramaximal current used for actual chronic activation was set 30-80% above maximum, but to preclude nerve damage no cat was given more than 4.5 ma. Initial resistances between the electrodes in different cats varied from 3370 to 6215 ohms. During the 1-mo period of stimulation, the greatest single increase in electrode resistance was 1400 ohms ; it was attributable in this case to bone growth beneath the dorsal electrodes. Current levels were checked weekly and voltage adjustments were made to maintain supramaximal levels. In other cats, percutaneous stimulation of the caudal nerves was accomplished by two electrodes which were reapplied daily in the same location, around the base of the tail. These electrodes, 8 mm wide, were made of a flexible, electrically conducting woven material (kindly supplied by United Aircraft Corporation), which was pressed permanently onto a strip of waterproof adhesive tape. The tape kept the material from unraveling, and prevented drying out of a conducting paste (EKG-Sol) applied on the material to lower the resistance between electrode and skin. The bands were placed about 1 cm apart, separated by a strip of tape encircling the tail at the level of an intervertebral joint, and completely encircled the tail over an area from which hair was removed. Strips of tape were applied with sufficient tension to hold the electrodes in position without producing edema. Maximal current was not determined for the encircling band electrodes in every cat; rather, a constant voltage of 40 v peak to peak of the biphasic pulse was given to all cats. In one normal anesthetized cat, 40 v (16-18 ma) was determined to activate fully muscles all down the tail, on the basis of electromyographic recording and visual inspection. This voltage was not exceeded during chronic stimulation for fear of burning the skin. Two programs of stimulation, tonic (10 Hz, given in 40 set trains, repeated every 200 set) and phasic (50 Hz, given in 0.8 set trains, repeated every 200 set) were given an average of 6 hr daily for 1 mo. Stimulation was commenced immediately, 1 mo, or 4 mo following the isolation surgery. Six days were used to build up to the full program. This was accomplished for the tonic program by increases of 6 set/day in

MUSCLE

HTSTOCHEMISTRY

395

train duration and for the phasic program by increases of 1 hr/day in the length of daily stimulation. During stimulation, each cat was put in a humane semimobile harness. Adjustment to the harness was successful in all cases and all cats eventually exceeded their preoperative weight. ExPerimental Design. Table 1 shows the times after surgery at which intertransverse muscles were biopsied, when particular animals were stimulated, and by what program. In addition to the 13 animals biopsied the day of surgery (isolations and denervations), there were 17 other normal animals that supplied control muscles. In this paper, the term “isolated muscles” is used as an abbreviation for “caudal intertransversarii of cats subjected to isolation of the caudal spinal cord and not stimulated chronically.” “Stimulated muscles” are those which received stimulation chronically at some time following the isolation procedure. Both isolated and stimulated muscles, it will be noted, are derived from cats on which cord isolation has been performed. A single animal provided both isolated and stimulated muscles, when chronic stimulation was commenced following a period of inactivity after isolation surgery (e.g., cat 8, Table 1). Histochemistry and Morflhology. There are generally 12 serially homologous pairs of intertransverse muscles suitable for examination ; these were biopsied sequentially in a distal to proximal direction to eliminate any chance of nerve or vascular damage to the remaining muscles. Control and experimental muscles were examined histochemically for activities of a mitochondrial oxidation enzyme (NADH diaphorase). a glycolytic enzyme (phosphorylase) , and the contractile protein (myofibrillar ATPase) . The diaphorase procedure (30) using nitro blue tetrazolium was employed. Sections were pretreated in ice-cold 100% acetone to remove and thus avoid staining of triglyceride droplets (33). Phosphorylase activity was demonstrated by the method of Eranko and Palkama (14). Myofibrillar ATPase activity was demonstrated using the method of Niles, Chayen, Cunningham, and Bitensky (32) with and without prior fixation of the sections in 2.5% buffered formaldehyde (pH 7.2) at either 4 C or room temperature. Some control muscle sections were preincubated in alkali (IS). The excised muscles were held at rest length, rapidly frozen in liquid nitrogen, and stored in sealed glass tubes kept in a cold-box filled with dry ice. In this way the muscles from one cat (which had been sequentially biopsied over a period of up to 5.5 mo) could be sectioned simultaneously and stained on the same slide. A cryostat microtome was used to cut 10 ~lfn sections at -20 C. One or more sections of control muscle was always included on the slide with the experimental muscles.

RILEY

396

AND

ALLIN

TABLE SCHEDULE

Months Cat

0

1

1 2

1

OF INDIVIDUAL

2

after

CATS surgery

3

5

4

6

X X 10 Hz

3

B..

X

50 Hz 4

B ..

X 50 Hz

5

B ..

X 10 Hz

6 7

B .. B

B

x

B

B

x

10 Hz B..

8

B

X

B

X

10 Hz B ..

9

1 Hz 10 11 12

B .. B

X B

X X 10 Hz

13

B

B..

X 10 Hz

14

B

. ..

B

X 10 Hz

1.5

B

.*.

B

X 50 Hz

16

B

B..

X 50 Hz

17 18 19 20 21

B B

.. .

B

X

X X

B

B

X B

X

a All cats had isolation surgery, except 18, 19, 20, and 21 which had denervation surgery. B indicates time at which one or more muscles were biopsied; X indicates terminal biopsy and perfusion of the cat; 1 Hz, indicates stimulation at this frequency for 1 mo; 10 Hz, As for 1 Hz; 50 Hz, As for 1 Hz; dotted line indicates that the full program of stimulation was not given during a 6 day “build up” period.

To measure fiber circumference in control muscles, clear plastic sheets were placed over photographs (X 330) of muscle sections stained for ATPase activity after formaldehyde fixation. Dots were marked with a

MUSCLE

fine point felt pen 2 fiber to estimate its ence in contact with fibers were measured

397

HISTOCHEMISTRY

mm apart around the circumference of an individual total circumference and the portion of the circumferthe perimysial interfascicular septum. One hundred in each muscle of five control cats. RESULTS

Control Muscles. Consecutive sections of three control muscles, each from a different cat, were stained for diaphorase, phosphorylase, or myofibrillar ATPase activities. The diaphorase and phosphorylase activities of single fibers were estimated visually as low, moderate, or high, even though there appeared to be a continuum of staining intensities for both enzymes. The relative scales for rating these intensities had to be expanded or contracted to accommodate for the differences in absolute range between muscles. In unfixed sections, ATPase activity of single fibers was either high or low. This bimodality of activity was found in 20 additional control muscles, each from a different cat, however two other control muscles treated in the same way had some fibers of moderate activity. In unfixed sections stained for ATPase activity, myofibril shape was similar in all fibers. When sections were fixed in formaldehyde before incubation, differences in fibril shape were preserved and served as an additional feature to distinguish fibers. Three fiber types, red, white, and intermediate, were defined by grouping fibers according to the combinations of staining intensities and myofibril shape (Table 2). This terminology was modeled after that of Ogata and Mori (34, 35). Red fibers have high diaphorase, low phosphorylase, and TABLE HISTOCHEMICAL

Fiber

AND MORPHOLOGICAL AND EXPERIMENTAL

type

Control

Red

White Intermediate

2 CHARACTERISTICS MUSCLE FIBERS”

Nonstim.

OF CONTROL

Tonic

stim.

Phasic

stim.

1

3

\I:

1 1

1 2

1 1

\V W

1

1

2

W

1

2

2

W

3

3

1

N

3

2

2

M

3 2 3

1 1 1

1 1 1

N M M

3 2 3

2 1 1

3 3 3

N M M

3 2 3

3 2 2

1 2 2

N M M

- C

a Profile sequence (left to right) : myofibrillar ATPase activity, phosphorylase activity, diaphorase activity, and myofibril pattern (N = narrow, M = medium, and W = wide). Highest enzymatic activity is scored 3. Tonic stim. = 10 Hz, in 40 set bursts every 200 sec. Phasic stim. = 50 Hz, in 0.8 set bursts every 200 sec. Both programs of stimulation were given 6 hr/day for 1 mo. Nonstim. = S-mo isolated muscle.

398

RILEY

AND

ALLIN

FIG. 1. Cross sections of control muscles : A, NADH diaphorase activity; B. phosphorylase activity; C. myofibrillar ATPase activity, without fixation; D. myofibrillar ATPase activity after 5 min fixation in formaldehyde at 4 C. A-D are serial and X 290. Fixation decreased the activity in the intermediate fibers (I) while the activities of the white (W) and red (R) fibers were little changed (compare C and D). This result was also produced by alkaline preincubation. E and F are serial and X 830. E. Myofibrillar ATPase activity after 5 min fixation at 26 C, photographed with phase optics to demonstrate differences in fibril shape between red (R), white (W), and intermediate (I) fibers. Aligment of fibril profiles is common in white fibers (arrow). F. diaphorase activity; peripheral accumulation of stain (p) was only present in red fibers.

MUSCLE

HISTOCHEMISTRY

399

low ATPase activities and large polygonal fibrils (Fig. IA-C, E). White fibers have low diaphorase, high phosphorylase, and high ATPase activities and small, often rectangular, fibrils which tend to align in rows (Fig. lAC, E). The diaphorase and phosphorylase activities of intermediate fibers are usually between those of red and white fibers. Their ATPase activity is high like that of white fibers, although relatively more labile at pH 10.4 or following formaldehyde fixation (Fig. 1D). Intermediate fibers have moderately large polygona fibrils with frequent sarcoplasmic invaginations. In the three control muscles examined here, there were an average of 42% (range 40-46%) white, 21% (19-230/O) intermediate, and 37% (3141%) red fibers. The cytochemical distribution of the reaction product of diaphorase activity differs among fiber types (Fig. 1F). In red fibers, wide and dense lines of stain completely encircled all fibrils with a continuous network. White fibers were characterized by faint thin lines of stain and wider and more intense lines which run across the fiber and, upon intersecting, form small clumps of stain. Within the intermediate fibers there were more dense and thick lines of stain than faint and thin lines. The faint lines in the intermediate and white fibers outlined single fibrils, thick lines segregated groups of fibrils. In control muscles, the white and intermediate fibers rarely had subsarcolemmal accumulations of stain, although red fibers occasionally had small peripheral accumulations (IFig. lA, F). The histochemical method for phosphorylase demonstrates the ability of the enzyme to convert glucose-l-phosphate’ to glycogen ; the glycogen is then stained by iodine. The intensity of the stain is directly proportional to the amount of glycogen produced and the color of the stain reflects the chain lengths of glycogen (44). A blue color indicates a longer chain length than does a red color. In control muscles, white and intermediate fibers are stained blue, whereas red fibers had a red tinge. In all three fiber types, phosphorylase activity was located primarily between fibrils, but some intramyofibrillar staining was present (Fig. lB, 3C). Glycogen in unincubated sections did not stain, either in control or experimental muscles, presumably because of its very highly branched structure. Within all fibers, ATPase activity was localized to the myofibrils. Staining was also present in the endothelial cells of capillaries, but this never interfered with the interpretation of intracellular staining. White fibers had a larger cross-sectional area than either red or intermediate fibers in the same control muscle. This was determined by measuring 58, 66, and 70 fibers in three control cats, respectively. Student’s f test showed the average cross-sectional area of white fibers (2340 pm2 * 167 SE) to be significantly larger than that of either red (977 pm2 *

400

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FIG. 2. A. Control muscle stained for diaphorase activity (X 65). Arrows lie on the perimysial septum surrounding a fasciculus in which red (R), white (W), and intermediate (I) fibers are differentially distributed. B. A phasically stimulated muscle stained for diaphorase activity (X 350). White fibers are paIe, while red and intermediate both stain comparably to control intermediate fibers. C. A muscle isolated for 5 mo, stained for diaphorase activity (X 250). Enough cytochemical staining pattern remains to distinguish white and intermediate fibers from red fibers, but not white fibers from intermediate fibers. D. Serial to C, and stained for myofibrillar ATPase activity without fixation (X 250). Moderately staining fibers (m) are present. E. A tonically stimulated muscle stained for diaphorase activity (X 250).

MUSCLE

HISTOCHEMISTRY

401

156) or intermediate 1370 ~*fn~ +- 167). The difference in size between red and intermediate fibers was not statistically significant. Fibers of the same type varied in size from animal to animal, but within an animal proportional differences between types were relatively constant. Fiber types appeared to be differentially distributed in the fasciculi. White fibers were more frequently peripherally located, abutting the perimysial interfascicular septum, whereas red fibers were more often found toward the center of a fasciculus, and intermediate fibers occurred about equally in both positions (Fig. 2A) . This impression was verified by measuring the amount of each fiber’s circumference in contact with the septum and expressing this as a percentage of the total circumference of the fiber. The averaged values of 100 fibers from each of 5 control cats were: red 13% (range ll-15%), intermediate 26% (23-30%), and white 37% (34-440/o). Isolated Muscles. Following isolation, no motor unit electrical activity was recordable in the intertransverse muscles with surface or needle electromyographic electrodes. Positive identification of fiber types was possible after one month of inactivity because very little histochemical or morphological change had occurred. The cytochemical distribution of diaphorase activity had undergone a slight change in all fibers, as evidenced by discontinuities in the lines of staining and frequent clumping of stain. After 2 or more months types were still identifiable (Table 2) though with less certainty, by characteristics which continued to correlate as expected from control data (location within fasciculi, myofibrillar ATPase activity, myofibril shape). Since there was no obvious loss of fibers, their intrafascicular location remained the most reliable external means of identification, in the sense of assessing the overall picture, not identification of individual fibers. At 2 mo, diaphorase activity had decreased noticeably in red fibers and by 5 mo, all fibers exhibited low diaphorase activity. The remaining activity was largely in clumps, especially in the white and intermediate fibers which could no longer be discriminated on the basis of either diaphorase intensity or cytochemical pattern. However, enough pattern remained to allow distinction of white and intermediate fibers from red fibers (Fig. 2C). All fibers showed a greater decline in phosphorylase than in diaphorase activity at 1 mo. The degree of loss of phosphorylase activity and irRed fibers are recognizable by cytochemical stain distribution. White fibers and intermediate fibers are distinct from red fibers but they cannot be readily distinguished without checking the serial section. (F). Peripheral accumulation of stain is present in white and intermediate fibers. F. In this unfixed section stained for myofibrillar ATPase activity, intermediate fibers stain moderately (X 250).

402

RILEY

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FIG. 3. Cross sections: A. NADH diaphorase activity in a muscle denervated for 2.5 mo (X 310). All fibers are pale staining and have a similar cytochemical distribution of stain. Intensity is difficult to score because of clumping of the stain. B. Serial to A and stained for myofibrillar ATPase activity without fixation (X 310). Moderately staining fibers (m) are present and darkly staining fibers are atrophied. C. A control muscle stained for phosphorylase activity (X 310). Red fibers (R) stain lightly and white fibers (W) stain darkly, while staining in the intermediate fibers (I) is usually between that of the red and white fibers. D. A muscle isolated for 4

MUSCLE

HISTOCHEMISTRY

403

regularity in thickness of the lines of stain (clumping) was directly proportional to the original activity; i.e., white fibers showed the greatest loss and red fibers the least. As the period of disuse progressed to 3 mo, not only did activity gradually decrease, but staining became redder in all fibers. At 4 and 5 mo, when staining was pale in most fibers, there was moderate staining in many of the fibers of the red type, which represented an increased activity in these fibers. At this time, they did not stain pale pink as in control red fibers, but very red. Some fibers of either the white or intermediate type showed a moderate degree of purple staining (Fig. 3D). In muscles isolated for 1-5 mo, the extremes of ATPase activity (high and low) in unfixed sections are virtually unchanged from that in comparable fibers of control sections incubated on the same slide. However, in the muscles of two cats 1 mo after isolation surgery and five cats from periods 2 to 5 mo, there were fibers with moderate ATPase activity (#Fig. 2D). Moderately staining fibers were more frequently found the longer the period of time since isolation surgery. Once they were found in an animal, subsequent biopsies also always revealed their presence (Table 3). The time of their first appearance varied greatly from cat to cat, in some being present as early as 1 mo, while other animals had none even after 5 mo (Table 3). Moderately staining fibers were dispersed equally throughout the muscle, and most had fibrils of the shape characteristic of the normal intermediate fiber type. In two cats biopsied at 1 mo, when diaphorase staining intensities were essentially unchanged, the fibers of moderate ATPase activity also had moderate diaphorase activity, like that of intermediate fibers in control muscles. The emergence of moderately staining fibers is probably due to a decrease in the ATPase activity of intermediate fibers. Little or no atrophy was detectable in isolated muscles at 1 mo, but by 2 mo selective atrophy of white fibers was evident. White and intermediate fibers appeared more angular, while red fibers tended to be rounder than normal, with little or no reduction in size (Fig. 2D). The amount of mo, stained for phosphorylase activity (X 260). While most fibers are pale staining, some red fibers (R) and occasional white or intermediate fibers (x) stain moderately. E. A tonically stimulated muscle stained for phosphorylase activity (X 315). Compared to staining intensities of controls, intensity in red fibers is unchanged, that of intermediate fibers is near the red fiber level, while that of white fibers is near the intermediate fiber levels. F. A phasically stimulated muscle stained for phosphorylase activity (X 295). Compared to controls, staining intensity is higher in most red fibers and unchanged in white and intermediate fibers. G. and H. Both are muscles from cat 16, incubated on the the same slide for myofibrillar ATPase activity without fixation (X 195). G. A muscle isolated for 4 mo, showing fiber atrophy. H. A phasically stimulated muscle showing fiber regrowth.

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AND

ALLIN

TABLE THE

PRESENCE

OR ABSENCE

ATPASE

OF FIBERS

ACTIVITY Months

Cat No. 3 6 8 9 2 7 17 1.5 10 13 16 11 12

Nonstim.

Tonic

Phasic

stim.

stim.

after

isolation 2

+ + 0 0

+

MYOFIBRILLAR

SECTIONY or denervation 3

Den.

18 19 20

surgery

4

5

6

0 0

0 0 0

+ + +

0 0 0

0 0 0

0 +

0 0 + + 0 +

16 17 10

controls

OF MODERATE

IN UNFIXED

1

3 6 8 9 13 1.5

1 Hz

Other

0

3

0 +

+ 0

0

+ + 0 +

(15)

(2)

a Key to Table 3. Nonstim., muscles not chronically stimulated following isolation surgery; Tonic stim., muscles stimulated by the lo-Hz program; Phasic stim., muscles stimulated by the SO-Hz program ; 1 Hz, muscle stimulated by the l-Hz program; Den., muscles denervated; Other controls, muscles biopsied from normal cats not used in the present experiment; parentheses enclose the number of cats from which muscles were taken; +, presence of moderately staining fibers (4-19% of the fiber population); 0, absence of moderately staining fibers.

MUSCLE

HISTOCHEMISTRY

405

atrophy of white and intermediate fibers varied in the same muscle. Atrophy was most pronounced in muscles isolated for 5 mo. With one exception out of 12 cats, the most atrophic fibers had high ATPase activity. The exception was a single muscle, which at 3 mo showed many small angular fibers of low ATPase activity. Denervated Muscles. Fiber types were identifiable in denervated muscles up to about 2 mo, after which time degenerative changes had so distorted diaphorase and phosphorylase staining patterns and intensities, and fibril shape, that identification became uncertain. Location of fiber types within fasciculi was unreliable because fibers had apparently been lost as a result of degeneration. When denervated fibers were traced serially in cross section, some of them had focal regions of degeneration which were characterized by a lack of fibrils and of enzymatic activities. Similar regions of disruption were infrequent in isolated muscles and rare in control muscles. Changes in diaphorase and phosphorylase activities were similar to those seen in isolated muscles, except that they occurred earlier and appeared to progress more rapidly. In fibers denervated for 1 mo, there was diaphorase staining over fibrils and marked clumping of stain between fibrils. By 3 mo, all fibers had a low level of activity and the pattern of staining was so disorganized that all fibers looked similar (Fig. 3A). Differences of myofibril shape between fibers were no longer evident at this time ; most fibers had large fibrils with indistinct borders. At the end of the first month of denervation, phosphorylase activity had decreased more than in a muscle isolated for 1 mo. The decrease in activity was greatest in the white and intermediate fibers, in which activity was originally the highest. Disorganization of the staining network progressively increased. Coincident with the earlier loss of activity was an earlier appearance of the shift of iodine staining from blue to red. The extremes of ATPase activity (high and low) were virtually unchanged, except in degenerating regions. In unfixed sections, there were no fibers of moderate ATPase activity in muscles denervated for 1 mo, but they were present from 2 mo onward (Fig. 3B). Since fiber typing criteria were largely not usable, it was not possible to decide whether or not these were intermediate fibers. Denervation caused progressive atrophy similar to that of inactivity, though at a faster rate. At 1 mo, white fibers showed a greater decrease in size than intermediate or red fibers. At longer periods, the fibers of high ATPase activity were the most atrophic (Fig. 3B). Similar differential atrophy has been observed in other denervation studies (17, 27).

406

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Stinzulated Muscles. The histochemical appearance of muscles activated by the same program of stimulation was similar whether the stimulation was begun immediately, 1, or 4 mo following surgery. Fiber types remained identifiable in stimulated muscles by their location within fasciculi, myofibrillar ATPase activity, and myofibril shape (Table 2). Tonic stimulation produced large changes in diaphorase activity, as evidenced by enhanced staining of white and intermediate fibers, which now stained as intensely as control red fibers. Red fibers showed less staining than control fibers (Fig. ZE). Within the red, white, and intermediate fibers, cytochemical distribution of diaphorase staining around fibrils largely retained its original characteristics; i.e., in red fibers individual fibrils were surrounded by dense lines of stain, whereas in the white and intermediate fibers dense lines of stain, now wider and more intense, surrounded groups of fibrils (#Fig. ZE). There was a more marked subsarcolemmal staining in the white and intermediate fibers than occurred in control red fibers. Phosphorylase activity was less in the white and intermediate fibers, but was at control levels in red fibers after tonic stimulation (Fig. 3E). The cytochemical distribution of stain within fiber types was similar to that of comparable control fibers. Diaphorase activities of white and intermediate fibers were at control levels following phasic stimulation, but activity in red fibers was lower (Fig. 2B). This program of stimulation had the greatest effect on phosphorylase activity, which was at normal levels in the white and intermediate fibers and was elevated in red fibers (Fig. 3F). After stimulation in either the tonic or phasic mode, the color of the iodine-staining and the cytochemical distribution of the reaction products of phosphorylase and diaphorase activities in all fibers was similar to controls. Following tonic or phasic stimulation, ATPase activity in unfixed sections remained high in white fibers and low in red fibers. However, five of nine stimulated cats had fibers of moderate ATPase activity (Fig. ZF, Table 3). This was about the same percentage as for isolated muscles. In both the tonically and phasically stimulated groups, there were an equal number of cats with and without moderates. As was true in isolated muscles, most of these fibers had myofibrils characteristic of intermediate fibers in control muscles. In those animals in which stimulation was begun 4 mo after isolation surgery, the average cross-sectional area of the atrophic white fibers was estimated to have increased two- or threefold. Neither program or activation appeared to be superior to the other in promoting this growth. Most red and intermediate fibers were comparable in size to corresponding types in control muscles, but since they had atrophied less after isolation, their regrowth was less than that of white fibers (Fig. 3H).

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DISCUSSION In the present experiment, isolated muscles were examined to establish the effects of zero activity (i.e., total muscle disuse). To assume that the changes in the isolated muscles were solely the result of inactivity is probably too simplistic, because isolation of the cord from all incoming impulses simultaneously eliminated any possible incoming transneuronal “trophic” influences (16) which may secondarily affect the muscle. To approach this issue and to provide a check on the results of isolation, other means of producing inactivity have to be devised, possibly a longterm anesthetic nerve block (29). Stimulated muscles were compared with isolated muscles taken the same number of months following isolation, to enable us to distinguish changes which were the result of a specific program of activation from those of isolation alone. Alteration in fiber characteristics after denervation was also studied at comparable time periods, because if unwanted denervation occurred in isolated or stimulated muscles it had to be recognized. There is always a chance of motor neuron death after spinal cord surgery (46) and nerve fibers may have been damaged during chronic electrical stimulation. However, no evidence of damage caused by stimulation was found and the number, size, and Nissl staining of motor neurons remained normal (A. H. Martin, unpublished observations). This study demonstrated that the pattern of contractile activity largely determines the levels of phosphorylase and diaphorase activities within muscle fibers. Tonic stimulation resulted in high oxidative and low glycolytic enzymatic activities, while phasic stimulation produced the opposite histochemical changes. In order to relate these changes to specific aspects of the pattern of impulse activation, one must consider the similarities and differences between the tonic and phasic programs of stimulation that were used. Both programs had identical higher-order periodicity (6 hr/day) , number of impulse trains (lOS/day), and interval from the onset of one train to the onset of the next (200 set). However, they differed in impulse frequency (10 and 50 Hz), train duration (40 and 0.8 set), cumulative impulse total (tonic tenfold greater than phasic), and number of pulses per train (400 and 40). Other tonic and phasic programs will have to be investigated in which these variables are set equal, in order to better define the controlling aspects. It seems likely that a phasic program of 50 Hz in which the total number of impulses is equal to that of the tonic program will also increase oxidative activity, since in the bat cricothyroid muscle, a high contractile frequency (Hz) and high oxidative activity (judging from mitochondrial content) are clearly compatible (39). One

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cat was given chronic low frequency stimulation (1 Hz,2 continuously, 12 hr/day, for 1 mo) in which the total number of impulses was equal to that of the tonic program. Since this program (1 HZ) also resulted in high oxidative activity, it may be the total number of impulses that determines oxidative activity. The alterations in the level of activity of these enzymes could have resulted from changes in enzyme concentration, from changes in the nature of the enzyme, or both. For example, red iodine-staining of glycogen is characteristic of highly branched polysaccharides (short chains of glucose subunits) and blue staining is indicative of long unbranched chains of glucose subunits (44). The color shift from blue to red (iodine-staining of glycogen produced in the phosphorylase histochemical reaction) following prolonged disuse and the subsequent return from red to blue staining after chronic stimulation indicated that the nature of the glycogen branching enzyme (amylo 1,4-1,6 transglucosylase) was changing. Although ethanol was added to the incubation medium to inhibit the activity of the branching enzyme (45), this inhibitor may not have worked on the “altered” enzyme of isolated muscles. Different branching enzymes probably occur naturally ; most fibers of the gastrocnemius muscle of the rat normally stain blue while soleus fibers stain red (40). However, Romanul (40) interpreted the red staining of soleus fibers as due to the production of unbranched oligosaccharides. The decreased ATPase activity of some intermediate fibers in isolated muscles was probably not due to a decrease in enzyme (actomyosin) concentration, because enzyme quantity and fiber volume decreased essentially at the same rate, and staining over the myofibrils themselves was reduced. If atrophy were responsible, intensely stained white fibers (which showed the greatest atrophy) would have shown the greatest decrease in ATPase activity. Gradual replacement of existing ATPase by an enzyme less active or more alkali labile is consistent with the higher incidence of moderately staining fibers at later times than at 1 mo, and the existence of more than one level of moderate staining. Slow replacement is compatible with the probable slow rate of myosin turnover (15). Since “emergence” of moderate staining occurred sporadically and in all experimental groups with no obvious differential frequency, its explanation is unclear. The myofibrillar ATPase activity and the myofibrillar shape of red and white iibers appeared unchanged in stimulated muscles. However, the period of chronic stimulation (1 mo) may have been too short to produce a detectable histochemical change in myofibrillar ATPase, since cross-reinnervated 2 As with the other two programs of stimulation, an initial 6-day period was used to build up to the full program. In this case, the duration of stimulation was increased about 2 hr/day.

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muscles of the cat show better conversion of myosin and myofibrillar ATPase activities at 12 n-10 than at 6 mo (4). On the other hand, a large amount of growth occurred in fibers (especially white fibers) stimulated after 4 mo of isolation ; synthesis of myofibrillar proteins must have been correspondingly large. Even though this growth occurred in response to two different programs of stimulation, high and low ATPase activitites were similar to those in control muscles. Thus, ATPase activity appears not to be controlled by the pattern of activation alone. Myofibrillar ATPase activity of rat and cat hind limb muscles can be changed without altering their original innervation, simply by removal of synergistic muscles (21, 22, 42, 48) or by inactivation of antagonistic muscles (20, 25). These procedures are likely to modify the pattern of muscle use and the amount of tension actively and passively generated in the muscle. The authors were unable to assess whether the reported changes in ATPase activity resulted from the conversion of ATPase activity of the original fibers or from the formation of new fibers with a different ATPase. However, the fiber atrophy and apparent fiber splitting observed in some of the illustrations (20. 21) is suggestive of a turnover of the fiber population. In the present study, there was no evidence of significant fiber loss or fiber formation in response to inactivity or chronic stimulation, nor significant alteration of ATPase activity in the majority of fibers. Since myofibrillar ATPase activity of a muscle can be changed by cross-reinnervation, removal of its synergists, or inactivation of its antagonists, ATPase activity appears to be controlled by influences other than (or in combination with) nerve impulses, such as trophic factors (16) or the amount of tension developed in the muscle (23). The present experiment supports the hypothesis of a sustaining (trophic) influence of nerve on muscle. Even though both isolation and denervation resulted in removal of neural activation of contractile activity, only denervation led to marked muscle degeneration (disorganization or necrosis as distinct from simple atrophy). The continued health of the muscle fiber was thus dependent on intact motor innervation, but not solely on impulse transmission. Tower (47) reported that long-term inactivation of puppy hind limb muscles by a lumbosacral cord isolation resulted in eventual replacement of muscle fibers by fibrous tissue, suggesting that nerve impulses were essential for survival. However, she failed to preserve the vascular supply to the cord and apparently overexposed the cord during laminectomy, causing it to become flattened. Furthermore, paralyzed hind limbs tend to be sat upon by the animals, thereby impairing blood perfusion though the muscles. Since these deleterious conditions

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were avoided in the present study, this probably accounted for the prolonged survival of inactive muscle fibers without their fibrous replacement. If neural control of fiber type characteristics is complete, and is mediated entirely by impulse transmission, eventual uniformity of all fibers would be brought about by chronic activation in any uniform pattern (although the initial effects might differ, depending on the previous history of activation). Well before the achievement of complete uniformity, there would be a clear shift toward homogeneity in all features. This was not observed for myofibrillar ATPase, myofibril shape, or cytochemical stain distribution. If our premises are sound, there would appear to be another sort of control exerted over these qualities, which is not mediated by impulses. Should even a single feature be under impulse-independent control, then identical impulse patterns would not necessarily produce homogeneity of the features. For instance, if the nature of the myosin ATPase were the sole characteristic not modulated by impulses, then because the energy budget for contraction would differ in fibers of different ATPase activity, identical patterns of impulses would impose unequal demands on the ATP-generating machinery. Therefore, the adaptive change in this machinery would differ in degree and perhaps even in direction, depending on the particular impulse rhythm. That some such combination of controls is involved for most of the features studied is strongly suggested by the observed differences among fiber types in their responses to identical activation. In the case of fiber cross-sectional area, for example, the originally larger white fibers came to be smaller than the red fibers after 5 mo of inactivity, and after both phasic and tonic stimulation the white fibers were larger. In contrast to our findings, Klinkerfuss and Haugh (28) reported the presence of many atrophied fibers of high oxidative enzymatic activity in the cat gastrocnemius, after 1 mo of isolation disuse. Differences in the muscles or in the experimental circumstances must be assumed. Since virtually all of the muscle fibers of a single motor unit are histochemically identical and uniformly activated by their motor neuron (7, 8, 13) but motor neurons differ in their impulse output (6), we can now say that the coexistence in a single muscle of fibers with diverse histochemical and morphological characteristics (i.e., fiber types) is partly due to differences in the pattern of use of motor units. However, since uniform activation of all fibers in a histochemically diverse muscle did not result in complete uniformity of fiber type characteristics, control of fiber type differentiation is probably not determined exclusively by the pattern of impulse activation.

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40. ROMANUL, F. C. A. 1964. Enzymes in muscle. I. Histochemical studies of enzymes in individual muscle fibers. Arch. Neural. 11: 355-368. 41. SALMONS, S., and G. VRBOVK. 1969. The influence of activity on some contractile characteristics of mammalian fast and slow muscles. J. Physiol. 201: 535-549. 42. SCHIAFFINO, S., and S. PIEROBON BORMIOLI. 1973. Adaptive changes in developing rat skeletal muscle in response to functional overload. E.--p. Neural. 40 : 126137. 43, SRETER, F. A., I. GERGELY, S. SALMONS, and F. ROMANUL. 1973. Synthesis by fast muscle of myosin light chains characteristic of slow muscle in response to long-term stimulation. Nature (Lorzdon) 241: 17-19. 44. SWANSON, M. A. 1948. Studies on structure of polysaccharides: IV. Relation of iodine color to structure. J. Biol. Gem. 172 : 825-837. 45. TAKEUCHI, T., and H. KURIAKI. 1955. Histochemical detection of phosphorylase in animal tissues. 1. Histochern. Cytochem. 3 : 153-160. 46. TOWER, S. A. 1937. Function and structure in the chronically isolated lumbosacral spinal cord cord of the dog. J. Camp. Neur. 67 : 109-131. 47. TOWER, S. S. 1937. “Trophic” control of non-nervous tissues by the nervous system: A study of muscle and bone innervated from an isolated and quiescent region of spinal cord. J. Comfi. New. 67: 241-267. 48. WETZEL, M. C., R. L. GERLACH, L. Z. STERN, and L. K. HANNAPEL. 1973. Behavior and histochemistry of functionally isolated cat ankle extensors. Exp. Neurol. 39 : 223-233. 49. YELLIN, H. 1972. Differences in histochemical attributes between diaphragm and hindleg muscles of the rat. Anat. Rec. 173 : 333340.