Relevant muscle- and species-specific differences may be found in the reaction of muscles to hindlimb suspension. This problem has been studied in 5 rabbits following a one-week hindlimb suspension, and in 5 ground-based controls. The soleus and the tibialis were prepared for light and transmission electron microscopy. In suspension the animals occasionally extended and flexed the hindlimbs, but, when standing still, their hindfeet were plantar-flexed to an angle of 180”. In this position the length of the soleus was determined to be 35% less than in controls, whereas that of the tibialis was 30% more. Histologically, the tibialis fibers usually exhibited a preserved sarcomeric pattern, whereas soleus fibers displayed a regular sequence of areas of shortened sarcomeres, alternating with areas of myofibrillar disruption. These findings demonstrated that hindlimb suspension induces a focal breakdown of the soleus myofibrils, probably dependent on the reduced longitudinal tension of the suspended soleus and its phasic contractions against no load. It is conceivable that similar factors could also be responsible for soleus muscle atrophy induced by hypogravity as well as by other clinical conditions during which a stressful plantar flexion of the feet occurs against no load. Key words: soleus muscle hindlimb suspension myofibrillar disruption mitochondria loss MUSCLE & NERVE 14:358-369 1991
MYOFIBRILLAR DISRUPTION IN THE RABBIT SOLEUS MUSCLE AFTER ONE-WEEK ARCHINTO P. ANZIL, MD, GIUSEPPE SANCESARIO, MD, ROBERTO MASSA, MD, and GlORGlO BERNARDI, MD
After prolonged space fli hts, astronauts suffer from various symptoms2322g,37 which can be considered characteristic of a chronic space adaptation syndrome, as opposed to the acute one which mainly encompasses motion sickness and fluid accumulation in the upper part of the body.34 Throughout the flight astronauts display postural
From the Department of Pathology, SUNY-HSCB, Brooklyn, New York (Dr. Anzil). and the Institute of Neurology, 2nd University of Rome, Italy (Drs. Sancesario, Massa, and Bernardi). Acknowledgments: The authors thank Susanne Luh for her technical assistance and Roberta Losacco and Dr. Lucinda Byatt for reviewing the linguistic style They also thank Professors George Karpati (McGill University, Montreal) and Sergio Villaschi (Anatomia Patologica. 2nd University, Rome) for their constructive comments. A P A and G.S. were associated with the Max Planck Institute for Psychiatry in Planegg-Martinsried (FRG) during the collation of the results This study was supported in part by a grant from the Consiglio Nazionale delle Ricerche (project no. PSN85-083, 87-048) Address reprint requests to Dr G Sancesario Institute of Neurology, 2nd University of Rome, Via 0. Raimondo 8. 001 73 Rome, Italy. Accepted for publication April 29, 1990 CCC 0148-639X1911040358-012 0 1991 John Wiley & Sons, Inc.
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changes7t4‘ and experience reduced muscular stren th, while leg muscle atrophy becomes evident!,43 In spite of strong in-flight exercise such muscle changes cannot be totally prevented.37 Ethical restriction on the use of muscle biopsies from crew members has prevented the direct study of hypogravity-induced muscle atrophy. In rats, prolonged space flights induce marked atrophy in the soleus muscle (tonic plantar flexor), as opposed to slight or none in the gastrocnemius (fast plantar flexor), tibialis anterior, and extensor digitorum longus (fast d o r s i f l e x ~ r s ) . ~ ~The ’~~~~.”~ pattern of hypogravity-induced muscle atrophy in rats seems to be comparable, at least in part, to that observed after hindlimb suspension in the same species as well as in other rodents, such as mice and harnsters.8,1 !,15,16,27.3 1.32,36,38.4k41 Therefore, this simple and inexpensive groundbased model has been increasingly utilized to reproduce the effects of weightlessness on antigravity muscles in mammals. So far, morphological studies using this model have concentrated on histochemical changes showing, in the soleus, an al-
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tered fiber-type distribution and a reduced crosssectional area. In general terms, prolonged hypokinesia and reduced load-bearing activity have been considered as the primary factors responsible for hypogravity-induced as well as for suspension-induced muscle atrophy. 14*' 7,2 1,28339 However, it would be important to study whether, in animals with different hindlimb anatomofunctional characteristics, there are relevant species-specific differences in the response of a muscle to weightlessness. The rabbit was selected for our study because it has a peculiar type of locomotion. I t is a jumper whose hindlimbs are much longer and more strongly developed than its forelimbs. Furthermore, the rabbit soleus muscle may be very sensitive to experimental manipulation. For instance, following tenotomy, unlike the rat soleus, the rabbit soleus is affected by longterm structural abnormalities including fiber loss and fatty tissue substitution. 1 2 4 3 1 3 2 2 9 In the present work, the fine structure of the rabbit soleus and tibialis anterior was examined after the animals had been held suspended for one week. The soleus underwent a process of myofibrillar disruption, probably dependent on biomechanical conditions specifically determined by hindlimb suspension. ANIMALS AND METHODS
The experiments were performed on 10 young male New Zealand rabbits (1073 to 1140 g of b.w.) obtained from M.I.L. Morini (Keggio E., Italy). The animals were fed on a standard laboratory diet and given tap water ad libitum; greens and carrots were added twice a week. After 7 days of laboratory acclimatization, the rabbits were randomly divided into 2 groups and were used for control or suspension experiments. Animal experimentation was conducted in accordance with the institution's guide for the care and use of laboratory animals. Pilot studies have shown that a head-down suspension (at about 30") promptly causes breathing distress in the rabbit, probably because, in this position, the voluminous intestines move toward and press on and block the diaphragm. Furthermore, unlike rats, a rabbit cannot be suspended from its tail, which is too small for this purpose. A horizontal suspension" of the animal was preferable using an apparatus similar to that described by Musacchia et al.32 In brief, 5 nonanesthetized rabbits were dressed in a denim harness, the head and the legs extending through the openings. Using this harness the animals were sus-
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pended horizontally from a hook so that, while the forelimbs stood on a wooden support, the hindlimbs dangled down freely. The animals could move their forequarters through an arc of about 100" along the wooden tray on which food and water were provided. The following clinical parameters were assessed in ground-based controls and in suspended animals: body weight, food intake, and motor behavior. Body weight was assessed before and after the period of suspension. Food intake was measured daily. The behavior of the animals was also observed daily for 30 minutes in the morning and for an additional 30 minutes in the afternoon. In particular the angles of the ankle and knee joints were recorded using a goniometer placed laterally on the leg. To evaluate more precisely the effect of prolonged suspension on hindlimb posture, a lateral x-ray of the body was taken in control animals on the ground and in the suspended group just after initiating suspension and immediately before terminating it. Throughout these procedures, care was taken to avoid touching or inducing startling reactions in the animals. A third group of animals ( n = 2; 1100 to 1140 g b.w.) was used to determine the length o f t h e soleus and tibialis in relation to the ankle joint angle. These animals were anesthetized and the limbs dissected. The length of the soleus and tibialis muscles was measured directly after holding the ankle joint angle in flexion at 35" and in extension at 180", as had been determined previously in control and suspended animals, respectively. After 1 week the animals were removed from the suspension harness, and were freely allowed to assume standing posture on the ground for about 2 minutes in order to assess the reversibility of any postural changes which had taken place during suspension. The animals were then anesthetized (ketamine 20 mg/kg IM, pentobarbital 50 mg/kg IP) and their hindlimbs were maintained in the position they had assumed during suspension, holding the ankle joint angle in full plantar flexion. The abdomen was opened, a cannula was inserted in the abdominal aorta, and the hindquarters were perfused with 4% glutaraldehyde solution in 0.1 mol/L phosphate buffer, pH 7.4. The soleus and tibialis anterior were removed and kept in cold perfusate overnight. Under a stereomicroscope, several samples of approximately 2 x 2 mm were cut from the midbelly region of the muscles. The pieces were washed in buffer and postfixed for 90 minutes in
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1% buffered osmium tetroxide (at 20"C), dehydrated in graded alcohols, carried into propylene oxide, and embedded in Epon 812. The samples were properly positioned and cut in transverse or longitudinal sections on a LKB ultramicrotome. Two micron thick sections were stained with p-paraphenylenediamine. l8 They were viewed and photographed in a phase contrast Leitz automatic photomicroscope. Thin (silver) sections were stained on the grids with uranyl acetate and lead citrate, and examined in a Zeiss EM10 electron microscope. The unpaired t test was used to compare food consumption and body weight in control and suspended animals; at least 100 fibers were evaluated from each muscle in longitudinal and transverse sections, using phase and electron microscopy. The statistical significance of morphological data was not checked as alterations in the sarcoplasmic structure were consistent in all the suspended rabbit solei, and as none of the control animals displayed comparable myofibrillar damage. RESULTS
Docile rabbits did not require anesthesia to be put in a suspension harness. However, throughout the experiment, they occasionally performed fast forward- backward oscillations of the hindlimbs when trying to reach the support on which their forelimbs stood. In particular, hindlimbs were rapidly shortened and lengthened by multijoint flexion and extension without supporting the body mass against the force of gravity. Such intense motor activity only lasted a few minutes each time, followed by long intervals of rest. Although postural attitudes varied somewhat throughout the experiment, when the animals were at rest they assumed a characteristic posture with the head thrust forward, and the hindfeet in full plantar flexion directed toward the unreachable cage floor (Fig. 1). Thus, the ankle and the knee angles, which measured 35" to 40" in the control animals at rest on the cage floor, increased to 90" to 100" in the suspended animals during the first day, and later reached a peak of about 180" in both hindlimbs with negligible variations from animal to animal until the end of the suspension. In addition to these postural changes, by the second to third day, the hindfeet of suspended animals became swollen with venous stasis and dependent edema. The swelling seemed to involve only the subcutaneous tissue of the hindfeet without evident extension, upon histological examinaGeneral Observations.
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C FIGURE 1. Schematic drawing performed from radiographs, showing the posture of rabbit on the ground (C) and after suspension for few minutes (A) or for one week (B). Note the overall extension of the hindlimbs in (b).
tion, to the deeper soft tissue and, in particular, to the endomysial spaces of the soleus and tibialis anterior muscles. However, some minor congestion in the venous system of the suspended hindlimb muscles cannot be ruled out with certainty. 58 The food intake per animal was 617 (mean rt_ SD) g/week in the suspended animals, 41 g/week in the controls ( P < 0.05). and 713 Mean body weight was also less in the suspended animals ( 1208 k 1 14 g) than in the controls ( 1310 k 155 g), although the difference was not statistically significant. Upon terminating the suspension period, the animals appeared healthy and promptly reassumed a normal posture once they touched the cage floor. The length of the soleus and tibialis muscles, determined in the third group of animals, showed opposite changes in relation to the ankle joint angles. When the ankle and knee joints were rotated from 35" to 180", as was observed in the suspended animals, the length of the soleus belly was shortened by 35%, passing from 4.3 cm to 2.8 cm; at the same time, the tibialis belly was stretched by 30%, passing from 4.6 to 6.0 cm.
*
*
In the controls, tibialis large muscle fibers with few mitochondria predominate (Fig. 2a). After suspension, the fibers were rounded in form and contained swollen mitochondria (Fig. 2b). In longitudinal sections, the fibers displayed a linear contour; the sarcomeric pattern was evident in control as well as in suspended animals, although the myofibrils were frequently out of alignment in the latter (Fig. 3). Morphological Evaluations.
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FIGURE 2. Epon-embeddedsemi-thin sections stained with paraphenylenediamine. Tibialis muscle fibers in control (a) and in one-week suspended animals (b) (bar = 40 pm).
Centrally located myonuclei occurred in 0.5% of the control fibers and in 1% to 4% of the suspended fibers. In the control rabbit soleus, muscle fibers showed no great variation in size and shape and were typically rich in mitochondria. After suspension, muscle fibers showed conspicuous and extensive changes, whereas intramuscular blood vessels and nerve branches appeared normal. In semithin cross-sections most of the muscle fibers were round and contained large irregularly shaped areas of disorganized sarcoplasm (Figs. 4a and b). The nuclei appeared to be empty vesicles with prominent nucleoli; they were located centrally in 0.5% of the control fibers and in 0.5% to 3% of the suspended fibers. N o evident changes in the endomysial spaces were seen. In longitudinal semi-thin sections, the fibers had a wavy contour and a multifocal loss of cross-striations arranged in series and alternating with areas of preserved
Hindlimb Suspension Damages Soleus
sarcomeres along the observable course of the myofibers (Figs. 5a and b). Each area of myofibrillar disruption involved the space of 3 to 7 sarcomeres and appeared as a sinuous linear or Y-shaped band, extending across the width of the fiber. The surrounding myofibrils were contracted and were often not aligned (Figs. 5b and 6). The ultrastructural study of areas of myofibrillar disruption showed bundles of filaments which were either relatively intact or disorderly massed, sparse dots resembling remnants of Z band material, disoriented elements of sarcoplasmic reticulum, and T tubules which appeared swollen at some points (Figs. 6, 7, and 9). It is noteworthy that invariably, just inside the disrupted areas, mitochondria had disappeared without leaving any residual trace. The transitional sarcomeres from a disrupted area to an adjacent preserved area, either displayed fragmented Z lines or did not display one at all, whereas the relative A bands were still intact (Fig.
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FIGURE 3. Longitudinal cut section of tibialis fibers after one-week suspension. Epon-embedded semi-thin section stained with paraphenylenediamine. Part of a muscle fascicle: the sarcomeres appear out of alignment in all fibers. A central nucleus is evident in the bottom fiber (bar = 40 pm).
6). Exceptionally, in some fibers (less than 0.1%) a few consecutive sarcomeres appeared devoid of Z lines and mitochondria, while the myofilaments were well arranged (data not shown). In the subsarcolemmal region and in that surrounding or adjoining the nuclei, so-called sarcoplasmic masses of criss-crossing filamentous structures were often found, associated with underlying areas of myofibrillar disruption (Fig. 8). I n the preserved areas, the sarcomeres were generally characterized by prominent Z discs, very short I bands, and unremarkable A bands (Fig. 6). In these areas, the intermyofibrillar mitochondria were longitudinally oriented and gathered into clumps; they were sinuous and more electron-dense than normal (Fig. 6). The plasma membrane was thrown into short folds decorated with multiple endocytotic vesicles (Fig. 9). N o changes in the basal lamina were apparent. Nuclei were euchromatic with a narrow rim of marginal heterochromatin. DISCUSSION
T h e results of this study show, for the first time, that suspension causes marked myofibrillar alterations in the rabbit soleus. By contrast, the muscle fibers of the suspended tibialis usually display a preserved sarcomeric pattern. The types of lesions in the soleus seem distinguishable per se from contracture artifacts due to
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faulty preparatory technique. Characteristically, the soleus fibers show a regular sequence of areas of myofibrillar destruction and of shortened sarcomeres containing abnormally oriented mitochondria. Such microscopic changes show evidence that a considerable decrease in the number and in the length of the sarcomeres takes place in the suspended soleus. Overstretched or slightly stretched sarcomeres with large I bands were never observed in the soleus samples, neither were changes referring to a process of A-band separation that frequently occurs in ischemic myopathy or after lengthening contraction^.^^'^^ The essential characteristics of the process of myofibrillar disruption are: a wavy contour of the fibers, focal disarray of the myofilaments, loss of the Z bands, and mitochondria1 depletion. Such structural abnormalities appear to be closely associated as they were all manifest after one week of suspension. The mechanism by which myofibrils are damaged is not clear. The occurrence of isolated Z-band lesions without filament disarray has a very low incidence after a one-week suspension, thus it does not warrant the suggestion of a temporal sequence in the process leading to myofibrillar changes. On the other hand, the finding of mitochondria loss may be of particular interest because of its frequency in the suspended soleus and of its
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FIGURE 4. Cross-cut sections of rabbit soleus after one-week suspension. Epon-embedded semi-thin sections stained with paraphenylenediamine. (a) Part of a muscle fascicle: irregular patches of myofibrillar disorganization are clearly visible (arrow) in all fibers; vesicular nuclei are located mostly under the sarcolemma (bar = 40 km). (b) In this higher magnification the same findings (arrow) are shown to better advantage (bar = 10 pm).
possible correlation with the stage of myofibrillar d i ~ r u p t i o n .Kuncl ~~ and Meltzer2’ have demonstrated that different variants in the process of myofibrillar disruption are associated with
Hindlimb Suspension Damages Soleus
clumped mitochondria early in the time course and with the disappearance of mitochondria 24 hours later. ‘Therefore, it is possible to suggest that myofibrillar changes may develop slowly in
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FIGURE 5. Longitudinally cut sections of rabbit soleus after one-week suspension. Epon-embedded semi-thin sections stained with paraphenylenediamine. (a) Fibers display a wavy contour (bar = 40 Fm). (b) View of a segment of a single muscle fiber: areas of blurred myofibrils running a diagonal course across the width of the fiber (bar = 10 pm).
the soleus during the suspension period and long before the animals are returned to the cage floor. This assumption is also supported by the association, in the suspended soleus, between areas of myofibrillar disruption devoid of mitochondria and the presence of sarcoplasmic masses, which
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are thought to be the result of an advanced stage of sarcoplasmic damage. Although myofibrillar lesions in the soleus seem to be directly caused by suspension, such structural changes could be intensified by abruptly returning the animals to their normal postures on
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the ground. When this occurs, the soleus, which adapts to a shorter length in suspension, is probably unduly stretched and the myofibrils are severely distorted when the hindlimbs bear the animal's weight again. Further research on animals suspended for various periods of time and killed immediately after the end of the suspension must be carried out to study this aspect in greater detail. The type of pathology described in the soleus, although characteristic, is not specific. T o some extent, similar changes may be experimentally reproduced with repetitive lengthening contractions, isometric contractions induced by drugs (emetine, phencyclidine, etc), limb immobilization and tenotomy, and may be present in a variety of neuromuscular diseases. 1,9712*13,24,33A decrease in the number of mitochondria, with a concomitant re-
duction of their volume, has also been described It is in the soleus of rats after space noteworthy that immobilization causes a marked (up to 4-fold) increase in the calcium uptake of the soleus." Had those ionic changes occurred in the soleus of suspended rabbits, probably in a more extensive form due to the subtle changes present in the sarcoplasmic membrane, one might hypothesize that they could compromise calcium homeostasis when calcium is released from the sarcoplasmic reticulum of active m y ~ f i b e r s . ~ , ~ ~ Thus, the sequence of contraction- relaxation would have been impaired, while increased proteolytic activities would have lead to focal lysis of the Z band and mitochondria. The extent of sarcomere loss described in the rabbit soleus is consistent with the drastic decrease in muscle mass by more than 50% detected in the
FIGURE 6. The following (Figs. 6 through 9) are electron micrographs of rabbit soleus, cut longitudinally, after one-week suspension. Uranyl acetate and lead citrate staining. An area of myofibrillar disruption is shown (center-bottom of the picture): mitochondria and Z lines are missing from this segment of the muscle cell. In the adjacent areas the myofibrils are contracted, while mitochondria appear twisted and electron dense (bar = 1 pm).
Hindlimb Suspension Damages Soleus
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FIGURE 7. Detail of an area of focal myofibrillar disruption: glycogen granules (double arrow), abnormally oriented triads (arrow), and sometimes swollen T tubules (arrowhead) appear among the masses of myofilaments (bar = 0.5 pm).
rat soleus mostly durin.g the first week of hindlimb suspension. l 6 However, contrary to the rat, the adaptation of the rabbit soleus to suspension progresses to structural myofibrillar damage. We believe that the process of myofibrillar disruption in the suspended soleus is probably more dependent on the presence of biomechanical factors associated with the horizontal hindlimb suspension of the rabbit, rather than on the absence of both the weight loading function and locomotion activity. Suspension conditions affect the contractile activity of the flexor and extensor muscles of the hindlimbs in different ways.44 During suspension, the flexor muscles are continuously engaged in acting against gravitational force, however, they cannot efficiently counteract the drooping of the hindlimbs and the full plantar flexion of the ankle for long. Such is the posture
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usually assumed by rabbits during horizontal suspension, with the result that the length of the ankle extensor and flexor muscles may be inverted. Furthermore, in suspension, when the freely hanging limbs are phasically extended, the resistance of body mass does not oppose the contraction of the extensor muscles. Therefore, in the absence of hindlimb loading, the rabbit soleus may undergo the negative effects of both chronic shortening and contraction episodes against no load. These conditions may be crucial because (a) phasic stimuli without a load may be detrimental to the structural integrity of the rabbit soleus muscle3' and (b) chronic shortening has been reported to cause a progressive degenerative process in rat soleus and gastrocnemius.' Furthermore, recent reports have demonstrated that passive tension of the soleus, and not just muscle activation, appears
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FIGURE 8. Part of a superficial sarcoplasmic mass, continuous with an underneath area of myofibrillar disruption (bar = 1 km).
to play an important role in preventing or decreasing muscle atrophy induced by limb immobilization.' 1,26 The segmental myofibrillar loss discussed above may represent a cytoarchitectural remodeling of the fibers trying to adapt to the shorter length of the soleus muscle kept in suspension. The nuclei of the soleus myofibers, which are generally euchromatic with prominent nucleoli, seem to be reactive and probably elicit a compensatory process for the breakdown of the myofibrils. In conclusion, important muscle- and speciesspecific differences are found in the reaction of a muscle to simulated hypogravity. T h e biomechanical conditions under which a muscle is activated may play a major role in determining its cytoarchitectural remodeling. In this study, myofibrillar loss in the soleus of the horizontally suspended rabbit was demonstrated to be associated with sustained shortening of the muscle coupled with epi-
Hindlimb Suspension Damages Soleus
sodes of contraction against no load. Consequently, damage to the soleus may not only be caused by the conditions of hypokinesialhypodynamia that obviously affect the suspended hindlimbs. This hypothesis may explain why strong inflight exercises cannot fully prevent hypogravityinduced muscle atrophy in astronauts. In crewmembers phasic and tonic soleus muscle activities against no load may also occur, and these represent detrimental factors to muscle's structural integrity. This last assumption has therapeutic consequences; it is conceivable that the application of a correct passive tension to the antigravity muscles during space flights may contribute to the prevention of the muscle damage caused by hypogravity. Further studies should investigate whether similar phasic and tonic activities against no load could also occur in the soleus of patients with central nervous disorders and stressful plantar flexion of the foot.
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FIGURE 9. Changes of sarcolemma, overlying an area of myofibrillar disruption (bar
=
0.5 pn).
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MYOFIBRILLAR DISRUPTION IN THE RABBIT SOLEUS MUSCLE AFTER ONE-WEEK ARCHINTO P. ANZIL, MD, GIUSEPPE SANCESARIO, MD, ROBERTO MASSA, MD, and GlORGlO BERNARDI, MD
After prolonged space fli hts, astronauts suffer from various symptoms2322g,37 which can be considered characteristic of a chronic space adaptation syndrome, as opposed to the acute one which mainly encompasses motion sickness and fluid accumulation in the upper part of the body.34 Throughout the flight astronauts display postural
From the Department of Pathology, SUNY-HSCB, Brooklyn, New York (Dr. Anzil). and the Institute of Neurology, 2nd University of Rome, Italy (Drs. Sancesario, Massa, and Bernardi). Acknowledgments: The authors thank Susanne Luh for her technical assistance and Roberta Losacco and Dr. Lucinda Byatt for reviewing the linguistic style They also thank Professors George Karpati (McGill University, Montreal) and Sergio Villaschi (Anatomia Patologica. 2nd University, Rome) for their constructive comments. A P A and G.S. were associated with the Max Planck Institute for Psychiatry in Planegg-Martinsried (FRG) during the collation of the results This study was supported in part by a grant from the Consiglio Nazionale delle Ricerche (project no. PSN85-083, 87-048) Address reprint requests to Dr G Sancesario Institute of Neurology, 2nd University of Rome, Via 0. Raimondo 8. 001 73 Rome, Italy. Accepted for publication April 29, 1990 CCC 0148-639X1911040358-012 0 1991 John Wiley & Sons, Inc.
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changes7t4‘ and experience reduced muscular stren th, while leg muscle atrophy becomes evident!,43 In spite of strong in-flight exercise such muscle changes cannot be totally prevented.37 Ethical restriction on the use of muscle biopsies from crew members has prevented the direct study of hypogravity-induced muscle atrophy. In rats, prolonged space flights induce marked atrophy in the soleus muscle (tonic plantar flexor), as opposed to slight or none in the gastrocnemius (fast plantar flexor), tibialis anterior, and extensor digitorum longus (fast d o r s i f l e x ~ r s ) . ~ ~The ’~~~~.”~ pattern of hypogravity-induced muscle atrophy in rats seems to be comparable, at least in part, to that observed after hindlimb suspension in the same species as well as in other rodents, such as mice and harnsters.8,1 !,15,16,27.3 1.32,36,38.4k41 Therefore, this simple and inexpensive groundbased model has been increasingly utilized to reproduce the effects of weightlessness on antigravity muscles in mammals. So far, morphological studies using this model have concentrated on histochemical changes showing, in the soleus, an al-
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tered fiber-type distribution and a reduced crosssectional area. In general terms, prolonged hypokinesia and reduced load-bearing activity have been considered as the primary factors responsible for hypogravity-induced as well as for suspension-induced muscle atrophy. 14*' 7,2 1,28339 However, it would be important to study whether, in animals with different hindlimb anatomofunctional characteristics, there are relevant species-specific differences in the response of a muscle to weightlessness. The rabbit was selected for our study because it has a peculiar type of locomotion. I t is a jumper whose hindlimbs are much longer and more strongly developed than its forelimbs. Furthermore, the rabbit soleus muscle may be very sensitive to experimental manipulation. For instance, following tenotomy, unlike the rat soleus, the rabbit soleus is affected by longterm structural abnormalities including fiber loss and fatty tissue substitution. 1 2 4 3 1 3 2 2 9 In the present work, the fine structure of the rabbit soleus and tibialis anterior was examined after the animals had been held suspended for one week. The soleus underwent a process of myofibrillar disruption, probably dependent on biomechanical conditions specifically determined by hindlimb suspension. ANIMALS AND METHODS
The experiments were performed on 10 young male New Zealand rabbits (1073 to 1140 g of b.w.) obtained from M.I.L. Morini (Keggio E., Italy). The animals were fed on a standard laboratory diet and given tap water ad libitum; greens and carrots were added twice a week. After 7 days of laboratory acclimatization, the rabbits were randomly divided into 2 groups and were used for control or suspension experiments. Animal experimentation was conducted in accordance with the institution's guide for the care and use of laboratory animals. Pilot studies have shown that a head-down suspension (at about 30") promptly causes breathing distress in the rabbit, probably because, in this position, the voluminous intestines move toward and press on and block the diaphragm. Furthermore, unlike rats, a rabbit cannot be suspended from its tail, which is too small for this purpose. A horizontal suspension" of the animal was preferable using an apparatus similar to that described by Musacchia et al.32 In brief, 5 nonanesthetized rabbits were dressed in a denim harness, the head and the legs extending through the openings. Using this harness the animals were sus-
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pended horizontally from a hook so that, while the forelimbs stood on a wooden support, the hindlimbs dangled down freely. The animals could move their forequarters through an arc of about 100" along the wooden tray on which food and water were provided. The following clinical parameters were assessed in ground-based controls and in suspended animals: body weight, food intake, and motor behavior. Body weight was assessed before and after the period of suspension. Food intake was measured daily. The behavior of the animals was also observed daily for 30 minutes in the morning and for an additional 30 minutes in the afternoon. In particular the angles of the ankle and knee joints were recorded using a goniometer placed laterally on the leg. To evaluate more precisely the effect of prolonged suspension on hindlimb posture, a lateral x-ray of the body was taken in control animals on the ground and in the suspended group just after initiating suspension and immediately before terminating it. Throughout these procedures, care was taken to avoid touching or inducing startling reactions in the animals. A third group of animals ( n = 2; 1100 to 1140 g b.w.) was used to determine the length o f t h e soleus and tibialis in relation to the ankle joint angle. These animals were anesthetized and the limbs dissected. The length of the soleus and tibialis muscles was measured directly after holding the ankle joint angle in flexion at 35" and in extension at 180", as had been determined previously in control and suspended animals, respectively. After 1 week the animals were removed from the suspension harness, and were freely allowed to assume standing posture on the ground for about 2 minutes in order to assess the reversibility of any postural changes which had taken place during suspension. The animals were then anesthetized (ketamine 20 mg/kg IM, pentobarbital 50 mg/kg IP) and their hindlimbs were maintained in the position they had assumed during suspension, holding the ankle joint angle in full plantar flexion. The abdomen was opened, a cannula was inserted in the abdominal aorta, and the hindquarters were perfused with 4% glutaraldehyde solution in 0.1 mol/L phosphate buffer, pH 7.4. The soleus and tibialis anterior were removed and kept in cold perfusate overnight. Under a stereomicroscope, several samples of approximately 2 x 2 mm were cut from the midbelly region of the muscles. The pieces were washed in buffer and postfixed for 90 minutes in
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1% buffered osmium tetroxide (at 20"C), dehydrated in graded alcohols, carried into propylene oxide, and embedded in Epon 812. The samples were properly positioned and cut in transverse or longitudinal sections on a LKB ultramicrotome. Two micron thick sections were stained with p-paraphenylenediamine. l8 They were viewed and photographed in a phase contrast Leitz automatic photomicroscope. Thin (silver) sections were stained on the grids with uranyl acetate and lead citrate, and examined in a Zeiss EM10 electron microscope. The unpaired t test was used to compare food consumption and body weight in control and suspended animals; at least 100 fibers were evaluated from each muscle in longitudinal and transverse sections, using phase and electron microscopy. The statistical significance of morphological data was not checked as alterations in the sarcoplasmic structure were consistent in all the suspended rabbit solei, and as none of the control animals displayed comparable myofibrillar damage. RESULTS
Docile rabbits did not require anesthesia to be put in a suspension harness. However, throughout the experiment, they occasionally performed fast forward- backward oscillations of the hindlimbs when trying to reach the support on which their forelimbs stood. In particular, hindlimbs were rapidly shortened and lengthened by multijoint flexion and extension without supporting the body mass against the force of gravity. Such intense motor activity only lasted a few minutes each time, followed by long intervals of rest. Although postural attitudes varied somewhat throughout the experiment, when the animals were at rest they assumed a characteristic posture with the head thrust forward, and the hindfeet in full plantar flexion directed toward the unreachable cage floor (Fig. 1). Thus, the ankle and the knee angles, which measured 35" to 40" in the control animals at rest on the cage floor, increased to 90" to 100" in the suspended animals during the first day, and later reached a peak of about 180" in both hindlimbs with negligible variations from animal to animal until the end of the suspension. In addition to these postural changes, by the second to third day, the hindfeet of suspended animals became swollen with venous stasis and dependent edema. The swelling seemed to involve only the subcutaneous tissue of the hindfeet without evident extension, upon histological examinaGeneral Observations.
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C FIGURE 1. Schematic drawing performed from radiographs, showing the posture of rabbit on the ground (C) and after suspension for few minutes (A) or for one week (B). Note the overall extension of the hindlimbs in (b).
tion, to the deeper soft tissue and, in particular, to the endomysial spaces of the soleus and tibialis anterior muscles. However, some minor congestion in the venous system of the suspended hindlimb muscles cannot be ruled out with certainty. 58 The food intake per animal was 617 (mean rt_ SD) g/week in the suspended animals, 41 g/week in the controls ( P < 0.05). and 713 Mean body weight was also less in the suspended animals ( 1208 k 1 14 g) than in the controls ( 1310 k 155 g), although the difference was not statistically significant. Upon terminating the suspension period, the animals appeared healthy and promptly reassumed a normal posture once they touched the cage floor. The length of the soleus and tibialis muscles, determined in the third group of animals, showed opposite changes in relation to the ankle joint angles. When the ankle and knee joints were rotated from 35" to 180", as was observed in the suspended animals, the length of the soleus belly was shortened by 35%, passing from 4.3 cm to 2.8 cm; at the same time, the tibialis belly was stretched by 30%, passing from 4.6 to 6.0 cm.
*
*
In the controls, tibialis large muscle fibers with few mitochondria predominate (Fig. 2a). After suspension, the fibers were rounded in form and contained swollen mitochondria (Fig. 2b). In longitudinal sections, the fibers displayed a linear contour; the sarcomeric pattern was evident in control as well as in suspended animals, although the myofibrils were frequently out of alignment in the latter (Fig. 3). Morphological Evaluations.
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FIGURE 2. Epon-embeddedsemi-thin sections stained with paraphenylenediamine. Tibialis muscle fibers in control (a) and in one-week suspended animals (b) (bar = 40 pm).
Centrally located myonuclei occurred in 0.5% of the control fibers and in 1% to 4% of the suspended fibers. In the control rabbit soleus, muscle fibers showed no great variation in size and shape and were typically rich in mitochondria. After suspension, muscle fibers showed conspicuous and extensive changes, whereas intramuscular blood vessels and nerve branches appeared normal. In semithin cross-sections most of the muscle fibers were round and contained large irregularly shaped areas of disorganized sarcoplasm (Figs. 4a and b). The nuclei appeared to be empty vesicles with prominent nucleoli; they were located centrally in 0.5% of the control fibers and in 0.5% to 3% of the suspended fibers. N o evident changes in the endomysial spaces were seen. In longitudinal semi-thin sections, the fibers had a wavy contour and a multifocal loss of cross-striations arranged in series and alternating with areas of preserved
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sarcomeres along the observable course of the myofibers (Figs. 5a and b). Each area of myofibrillar disruption involved the space of 3 to 7 sarcomeres and appeared as a sinuous linear or Y-shaped band, extending across the width of the fiber. The surrounding myofibrils were contracted and were often not aligned (Figs. 5b and 6). The ultrastructural study of areas of myofibrillar disruption showed bundles of filaments which were either relatively intact or disorderly massed, sparse dots resembling remnants of Z band material, disoriented elements of sarcoplasmic reticulum, and T tubules which appeared swollen at some points (Figs. 6, 7, and 9). It is noteworthy that invariably, just inside the disrupted areas, mitochondria had disappeared without leaving any residual trace. The transitional sarcomeres from a disrupted area to an adjacent preserved area, either displayed fragmented Z lines or did not display one at all, whereas the relative A bands were still intact (Fig.
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FIGURE 3. Longitudinal cut section of tibialis fibers after one-week suspension. Epon-embedded semi-thin section stained with paraphenylenediamine. Part of a muscle fascicle: the sarcomeres appear out of alignment in all fibers. A central nucleus is evident in the bottom fiber (bar = 40 pm).
6). Exceptionally, in some fibers (less than 0.1%) a few consecutive sarcomeres appeared devoid of Z lines and mitochondria, while the myofilaments were well arranged (data not shown). In the subsarcolemmal region and in that surrounding or adjoining the nuclei, so-called sarcoplasmic masses of criss-crossing filamentous structures were often found, associated with underlying areas of myofibrillar disruption (Fig. 8). I n the preserved areas, the sarcomeres were generally characterized by prominent Z discs, very short I bands, and unremarkable A bands (Fig. 6). In these areas, the intermyofibrillar mitochondria were longitudinally oriented and gathered into clumps; they were sinuous and more electron-dense than normal (Fig. 6). The plasma membrane was thrown into short folds decorated with multiple endocytotic vesicles (Fig. 9). N o changes in the basal lamina were apparent. Nuclei were euchromatic with a narrow rim of marginal heterochromatin. DISCUSSION
T h e results of this study show, for the first time, that suspension causes marked myofibrillar alterations in the rabbit soleus. By contrast, the muscle fibers of the suspended tibialis usually display a preserved sarcomeric pattern. The types of lesions in the soleus seem distinguishable per se from contracture artifacts due to
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faulty preparatory technique. Characteristically, the soleus fibers show a regular sequence of areas of myofibrillar destruction and of shortened sarcomeres containing abnormally oriented mitochondria. Such microscopic changes show evidence that a considerable decrease in the number and in the length of the sarcomeres takes place in the suspended soleus. Overstretched or slightly stretched sarcomeres with large I bands were never observed in the soleus samples, neither were changes referring to a process of A-band separation that frequently occurs in ischemic myopathy or after lengthening contraction^.^^'^^ The essential characteristics of the process of myofibrillar disruption are: a wavy contour of the fibers, focal disarray of the myofilaments, loss of the Z bands, and mitochondria1 depletion. Such structural abnormalities appear to be closely associated as they were all manifest after one week of suspension. The mechanism by which myofibrils are damaged is not clear. The occurrence of isolated Z-band lesions without filament disarray has a very low incidence after a one-week suspension, thus it does not warrant the suggestion of a temporal sequence in the process leading to myofibrillar changes. On the other hand, the finding of mitochondria loss may be of particular interest because of its frequency in the suspended soleus and of its
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FIGURE 4. Cross-cut sections of rabbit soleus after one-week suspension. Epon-embedded semi-thin sections stained with paraphenylenediamine. (a) Part of a muscle fascicle: irregular patches of myofibrillar disorganization are clearly visible (arrow) in all fibers; vesicular nuclei are located mostly under the sarcolemma (bar = 40 km). (b) In this higher magnification the same findings (arrow) are shown to better advantage (bar = 10 pm).
possible correlation with the stage of myofibrillar d i ~ r u p t i o n .Kuncl ~~ and Meltzer2’ have demonstrated that different variants in the process of myofibrillar disruption are associated with
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clumped mitochondria early in the time course and with the disappearance of mitochondria 24 hours later. ‘Therefore, it is possible to suggest that myofibrillar changes may develop slowly in
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FIGURE 5. Longitudinally cut sections of rabbit soleus after one-week suspension. Epon-embedded semi-thin sections stained with paraphenylenediamine. (a) Fibers display a wavy contour (bar = 40 Fm). (b) View of a segment of a single muscle fiber: areas of blurred myofibrils running a diagonal course across the width of the fiber (bar = 10 pm).
the soleus during the suspension period and long before the animals are returned to the cage floor. This assumption is also supported by the association, in the suspended soleus, between areas of myofibrillar disruption devoid of mitochondria and the presence of sarcoplasmic masses, which
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are thought to be the result of an advanced stage of sarcoplasmic damage. Although myofibrillar lesions in the soleus seem to be directly caused by suspension, such structural changes could be intensified by abruptly returning the animals to their normal postures on
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the ground. When this occurs, the soleus, which adapts to a shorter length in suspension, is probably unduly stretched and the myofibrils are severely distorted when the hindlimbs bear the animal's weight again. Further research on animals suspended for various periods of time and killed immediately after the end of the suspension must be carried out to study this aspect in greater detail. The type of pathology described in the soleus, although characteristic, is not specific. T o some extent, similar changes may be experimentally reproduced with repetitive lengthening contractions, isometric contractions induced by drugs (emetine, phencyclidine, etc), limb immobilization and tenotomy, and may be present in a variety of neuromuscular diseases. 1,9712*13,24,33A decrease in the number of mitochondria, with a concomitant re-
duction of their volume, has also been described It is in the soleus of rats after space noteworthy that immobilization causes a marked (up to 4-fold) increase in the calcium uptake of the soleus." Had those ionic changes occurred in the soleus of suspended rabbits, probably in a more extensive form due to the subtle changes present in the sarcoplasmic membrane, one might hypothesize that they could compromise calcium homeostasis when calcium is released from the sarcoplasmic reticulum of active m y ~ f i b e r s . ~ , ~ ~ Thus, the sequence of contraction- relaxation would have been impaired, while increased proteolytic activities would have lead to focal lysis of the Z band and mitochondria. The extent of sarcomere loss described in the rabbit soleus is consistent with the drastic decrease in muscle mass by more than 50% detected in the
FIGURE 6. The following (Figs. 6 through 9) are electron micrographs of rabbit soleus, cut longitudinally, after one-week suspension. Uranyl acetate and lead citrate staining. An area of myofibrillar disruption is shown (center-bottom of the picture): mitochondria and Z lines are missing from this segment of the muscle cell. In the adjacent areas the myofibrils are contracted, while mitochondria appear twisted and electron dense (bar = 1 pm).
Hindlimb Suspension Damages Soleus
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FIGURE 7. Detail of an area of focal myofibrillar disruption: glycogen granules (double arrow), abnormally oriented triads (arrow), and sometimes swollen T tubules (arrowhead) appear among the masses of myofilaments (bar = 0.5 pm).
rat soleus mostly durin.g the first week of hindlimb suspension. l 6 However, contrary to the rat, the adaptation of the rabbit soleus to suspension progresses to structural myofibrillar damage. We believe that the process of myofibrillar disruption in the suspended soleus is probably more dependent on the presence of biomechanical factors associated with the horizontal hindlimb suspension of the rabbit, rather than on the absence of both the weight loading function and locomotion activity. Suspension conditions affect the contractile activity of the flexor and extensor muscles of the hindlimbs in different ways.44 During suspension, the flexor muscles are continuously engaged in acting against gravitational force, however, they cannot efficiently counteract the drooping of the hindlimbs and the full plantar flexion of the ankle for long. Such is the posture
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usually assumed by rabbits during horizontal suspension, with the result that the length of the ankle extensor and flexor muscles may be inverted. Furthermore, in suspension, when the freely hanging limbs are phasically extended, the resistance of body mass does not oppose the contraction of the extensor muscles. Therefore, in the absence of hindlimb loading, the rabbit soleus may undergo the negative effects of both chronic shortening and contraction episodes against no load. These conditions may be crucial because (a) phasic stimuli without a load may be detrimental to the structural integrity of the rabbit soleus muscle3' and (b) chronic shortening has been reported to cause a progressive degenerative process in rat soleus and gastrocnemius.' Furthermore, recent reports have demonstrated that passive tension of the soleus, and not just muscle activation, appears
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FIGURE 8. Part of a superficial sarcoplasmic mass, continuous with an underneath area of myofibrillar disruption (bar = 1 km).
to play an important role in preventing or decreasing muscle atrophy induced by limb immobilization.' 1,26 The segmental myofibrillar loss discussed above may represent a cytoarchitectural remodeling of the fibers trying to adapt to the shorter length of the soleus muscle kept in suspension. The nuclei of the soleus myofibers, which are generally euchromatic with prominent nucleoli, seem to be reactive and probably elicit a compensatory process for the breakdown of the myofibrils. In conclusion, important muscle- and speciesspecific differences are found in the reaction of a muscle to simulated hypogravity. T h e biomechanical conditions under which a muscle is activated may play a major role in determining its cytoarchitectural remodeling. In this study, myofibrillar loss in the soleus of the horizontally suspended rabbit was demonstrated to be associated with sustained shortening of the muscle coupled with epi-
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sodes of contraction against no load. Consequently, damage to the soleus may not only be caused by the conditions of hypokinesialhypodynamia that obviously affect the suspended hindlimbs. This hypothesis may explain why strong inflight exercises cannot fully prevent hypogravityinduced muscle atrophy in astronauts. In crewmembers phasic and tonic soleus muscle activities against no load may also occur, and these represent detrimental factors to muscle's structural integrity. This last assumption has therapeutic consequences; it is conceivable that the application of a correct passive tension to the antigravity muscles during space flights may contribute to the prevention of the muscle damage caused by hypogravity. Further studies should investigate whether similar phasic and tonic activities against no load could also occur in the soleus of patients with central nervous disorders and stressful plantar flexion of the foot.
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FIGURE 9. Changes of sarcolemma, overlying an area of myofibrillar disruption (bar
=
0.5 pn).
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