Neuropathology and Applied Neurobiology 1979, 5, 85-101
F I N E STRUCTURE O F MUSCLE I N HUMAN D I S U S E ATROPHY: SIGNIFICANCE OF PROXIMAL MUSCLE I N V O L V E M E N T I N MUSCLE D I S O R D E R S *
DARAB K . DASTUR, BOMI M . GAGRAT AND DAYA K . MANGHANI The Neuropathology Unit, Post- Graduate Research Laboratories, Grant Medical College and J.J. Group of Hospitals; and Nerve-Muscle Research Cell, Bombay Hospital, Bombay, India Accepted for publication 25 May 1978
Dastur D.K., Gagrat B.M. & Manghani D.K. (1979) Neuropathology and Applied Neurobiology 5, 85-101 Fine structure of muscle in human disuse atrophy: significance of proximal muscle involvement in muscle disorders The universal occurrence of weakness of skeletal musculature o n disuse, however produced, and the paucity of published reports o n the fine structural changes in human disuse atrophy, prompted the present investigation. The quadriceps muscle of a leg immobilized i n plaster cast (for fracture) and of the opposite non-immobilized limb was biopsied in four adult males, after periods of immobilization from 50 to 75 days. These 8 muscle specimens were examined for histopathological changes, and muscle fibre diameters were measured by micrometry from paraffin sections. The histograms revealed a larger proportion of small fibres ( < 2 0 pm) and a smaller proportion of large fibres (> 40pm) i n the immobilized limb compared t o the opposite. Thus, light microscopy showed only atrophic changes. This was confirmed by electronmicroscopy, where atrophy of few t o several muscle fibres was seen i n the form of loss of myofibrils, collapse and folding of the basement membrane and prominence of glycogen or muscle nuclei. The atrophic change was more severeintheimmobilized limbs, but it was also noticeable in all the non-immobilized limbs. Degenerative changes, especially disorganization and breakdown of myofibrils, and fragmentation of plasma membrane, were also seen i n occasional atrophied muscle fibres, again more frequently in the immobilized limb. Lipofuscin was often found accumulated in muscle fibres and occasionally in endothelial cells of intramuscular blood vessels ; the latter showed prominent pinocytotic vesicles or thickened basement membrane. It is concluded that both atrophy and degeneration of fibres of proximal muscles can occur as non-specific consequences of disuse of t h e limb in man, that degeneration is a later and more severe change, that muscles even of the non-immobilized leg are subjected to disuse atrophy during bed-rest, and that the proximal muscles in man seem t o have a natural susceptibility t o atrophy and degeneration in any muscular disorder.
* Part of this material was presented at the XIth World Congress of Neurology i n h s t e r d a m , September 1977. 0305-1846/78/0400-0085$02.00
0 1979 Blackwell Scientific Publications
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Introduction There is extensive literature on t h e ‘disuse atrophy’ of different muscle groups produced by a variety of techniques, but most of the reports are on experimental animals. I n the classic histopathological account of experimental disuse atrophy, Tower (1937) found the essential change t o be the gradual reduction i n size of all muscle fibres, with reduction of both sarcoplasm and myofibrils, but the cross striations remained well preserved over a long period of time. She did not, however, find any degenerative changes. Accounts of this condition i n human muscles are limited t o passing references in the clinical literature t o the wasting which follows immobilization of limb (Sunderland, 1968). The great difficulty of obtaining suitable human material for assessing histological changes is obvious. An earlier investigation from this Unit on cases of disuse atrophy of skeletal muscles reported histological and histochemical features (Patel, Razzak & Dastur, 1969). To the best of our knowledge, ultrastructural studies on atrophy of human muscles have not hitherto been reported. A further purpose of this investigation was also to record the changes occurring i n the non-immobilized limbs of such patients. The implications of even moderate degrees of disuse of muscles i n man appear t o be far greater than has been realized so far. I n view of our recent observation of generalized atrophy, partly on the basis of disuse, in the proximal muscles of patients with nutritional osteomalacia (Dastur, Gagrat, Wadia, Desai & Bharucha, 1975), it was also intended t o to see whether disuse from immobilization would result in only non-specific atrophy of muscle fibres or any other changes.
Materials and methods Muscle biopsies could be obtained from only 4 patients from the Department of Orthopaedic Surgery of our Hospitals, men between 38 and 50 years (Table 1)who had sustained closed traumatic uncomplicated fractures of the femur in 3 cases and of the upper ends of tibia and fibula in one. They were immobilized i n plaster or by skeletal traction for a period of 7 to 8 weeks and, in addition, one patient by skin traction for 12 days. The muscular strength of the involved limbs could not be tested due to pain. None of the patients had any neuromuscular disorder, evidence of severe malnutrition, or any generalized disease. Obtaining such patients with plastering adequate t o immobilize the quadriceps muscle, and who were prepared for biopsy from both limbs, was difficult and only 4 patients, who gave willing permission, could be so investigated over 14 years. Immediately o n removal of the plaster cast or traction, both t h e quadriceps muscles were biopsied under local anaesthesia. Part of each specimen was fixed in formaldehyde solution for the preparation of paraffin sections and stained with haematoxylin and eosin. Muscle fibre diameters (smallest) were measured using stage and eyepiece micrometry. Histograms of muscle fibre sizes were plotted. Using Student’s t test, statistical comparison was made between t h e mean fibre diameters of immobilized and non-immobilized limbs, and between the latter two and the mean fibre diameter of quadriceps muscle obtained earlier from control subjects admitted to t h e orthopaedic wards who were without weakness or immobilization of the lower limbs. While Araldite sections showed less shrinkage and more compactly arranged fibres, they could not be used for measuring fibre sizes as the number of fibres included i n such sections was often inadequate. For electron microscopy, a portion of the freshly biopsied muscle from the patient was fixed i n cold
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4% glutaraldehyde i n Millonig’s phosphate buffer. The tissue was cut into small pieces with proper orientation, treated with 1%,OsO, i n Millonig’s buffer, dehydrated i n a graded series of alcohols followed by propylene oxide, embedded i n Araldite and cut on a Porter-Blum ultramicrotome. Semithin (1pm thick) sections were first examined and thin sections were cut and stained with uranyl acetate and lead citrate, and examined and photographed in a Philips 200 electron microscope.
Results QUANTITATIVE HISTOLOGY
Plastic sections and paraffin sections of muscles from the immobilized limbs of all the four patients showed ill-defined groups of small fibres lying amidst normal looking
b
< 20 u m
v)
=
54%
F
I
-
13 (A)
< 20 urn > 40 prn
= =
78.5% 3.0%
20
?!
e
10
0
10
30
50 10 30 Fibre diameter ( pm)
50
Figure 1. a H-595 (A): Quadriceps muscle from immobilized limb; compactly arranged muscle fibres of varying size, including small atrophic fibres, especially i n the fascicle on the right (arrows). Blood vessels i n the interfascicular space appear normal. 1 pm plastic section, Toluidine blue. x100. b Histograms of quadriceps muscle fibre diameters, from the immobilized limb of two of the patients. Note the large number of small fibres and the very small number of large fibres (above 40 pm); the normal bimodal pattern is lost i n case 1-13 (A).
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b
I
0
10
30
50
-
13 (6)
70 10 30 Fibre diameter ( p m)
< 20 p
=
> 40 p
=
50
16.5% 35.0%
70
Figure 2. a H-595 (B): Quadriceps muscle from non-immobilized limb (cf. Figure 1). Small angular atrophic fibres are fewer t h a n i n the opposite limb. 1 pm plastic section, Toluidine blue. x 250. b Histograms of quadriceps muscle fibre diameters from non-immobilized limb of the patients i n Figure l b , showing a smaller number of smaller fibres in 1-13 (B), and a larger number of large fibres i n both.
fibres (Figure la). The muscle fibres from the opposite non-immobilized limb also showed a similar but lesser change (Figure 2a). These atrophied fibres, particularly from immobilized muscles, were frequently angulated, occasionally rounded, with relatively increased nuclei. No muscle from either limb showed any evidence of hypertrophied fibres, necrosis, phagocytosis or regeneration, evidence of inflammation or increase in endomysial connective tissue or fat, or of changes in blood vessels. Thus. no myopathic changes were found on light microscopy in muscles from either limb. The average muscle fibre diameter in paraffin sections from the four immobilized limbs was 24.24 pm and that from the non-immobilized limbs was 30.17 pm, both being considerably less than the mean fibre diameter (46.63 pm) of normal quadriceps muscle
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Table 1. Muscle fibre diameters i n patients with disuse atrophy statistical comparison with control subjects NP/no.
H-595 H-693 H-709 1-13
Age (years)
of
45 50 40 38
Duration immobilization (days) 75 50 56 56
Mean muscle fibre diameter ( p m ) : immobilized non-immobilized limb limb 22.4 35.7 18.90 20.0
29.0 29.3 32.4 30.0
PATIENTS WITH DISUSED LIMBS
(n = 4 ; mean age =43)
Mean? s.d. CONTROLS ( n = 8; meanage = 43)
I 24.24 ? 8.7---30.17 46.63 ? 10.6
2.9
1 J *
M e a n ? s.d.
t =statistically * =statistically
not significant highly significant ( P < 0.001). s.d. =standard deviation
(Table 1). However, the mean muscle fibre diameter for the immobilized limbs was not statistically significantly different from that for the non-immobilized limbs. There was no relationship between the duration of immobilization and the average fibre diameter (Table 1). Fibre size spectra from the immobilized limbs (Figure lb) showed a high proportion of small fibres ( < 20 pm), especially when compared to the non-immobilized limb (Figure 2b) of the same subject. One patient (H/693) was unique in showing a more severe non-specific atrophy in the non-immobilized limb than in the the immobilized (Figure 3). A suggestion of bimodality was seen in the histograms of fibre sizes from both limbs of case H/595 (Figures l b & 2b): a descending step-ladder pattern is seen from the small t o the large fibres in the immobilized limbs of cases H-709 and 1-13 (Figures 3 & lb).
FINE STRUCTURAL CHANGES I N THE MUSCLES FROM IMMOBILIZED LIMBS
The most frequent change in the immobilized muscles was atrophy of fibres of varying severity present in all four cases. The overall impression was of a moderate atrophic change, as evidenced by small atrophic fibres with compactly arranged myofibrils, intact sarcomeres and more or less loosened basement membrane (Figures 4 & 5a). With more severe atrophy, the loosely lying basement membrane was thrown into deep folds, at times with an accordion-like appearance, and a t other times like long tails of empty basement membrane (Figure 5b). The complete atrophy of a fibre with
H - 693 (A)
20
- 709 < 20 (A) Lim 1 40 prn
= =
< 20 k i r n
68.7% 4.2%
=
13.6%
1 40 p m = 32.09%
10
- 0 v)
P
,
H
-
< 20 11 = 16.8% > 40 p = 33.6%
693 (B) < 20 11 = 26.4% > 40 11 = 16.05%
10
0
10
30
50
70 10 Fibre diameter (prn)
30
50
70
Figure 3. Histograms of quadriceps muscle fibre diameters from the immobilized (upper histograms) and non-immobilized (lower histograms) limbs of two patients. Note that while the immobilized muscle of case H-709 is more affected (like the other two cases illustrated in Figures l b & 2b), t h e non-immobilized muscle is more affected and shows fewer large and more small fibres, in case H-693.
Figure 4. H-709: Two moderately well preserved muscle fibres with only minimal atrophy as indicated by the scalloped basement membrane ; in the centre is collapsed empty basementmembrane, except for parts of plasma membrane (p.m.), of a totally atrophied muscle fibre. Uranyl acetate and lead citrate. x 6300.
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total disappearance of its myofibrillar and sarcoplasmic content was indicated just by the presence of a n empty collapsed basement membrane (central structure in Figure 4). Three of the four immobilized muscles showed some degenerative changes in addition to atrophy, different stages and forms of which are seen in Figures 5b, 6a and b, and 7. In the early stages the atrophic fibres showed patchy loss or degeneration of myofibrils and depletion of sarcoplasmic reticulum or mitochondria. Increased number of nuclei, central or peripheral (Figure 6a), or a large nucleolus (Figure 5b, and fibre on left in Figure 6a) were seen in the degenerating fibres. Later, the myofibrillar architecture became disorganized or the myofilaments actually disrupted (fibre on right in Figures 6a & 6b). The most advanced degeneration was represented by a very small fibre almost devoid of myofibrils, myofilaments and organelles, and containing mainly glycogen (Figure 7). In such atrophied and degenerating fibres thickening of the basement membrane, fusion with the plasma membrane, which appeared thickened or fragmented (Figure 7), was observed. In one specimen (H-709A) a different type of atrophy was seen represented merely by smallness of fibre size (around 5 pm), without any loosening or even scalloping of the basement membrane. Rarely, macrophages containing phagosomes, vacuoles and granular bodies were encountered (Figure 7). While there was generally increased collagen between the
Figure 6 . H-709 (A) a: Parts of two muscle fibres; on t h e left, atrophic and bearing lipofuscin deposits near the single nucleus; on the right, showing disorganized degenerated myofibrils with scattered myofilaments, multiple nuclei, one of them possibly dividing (arrows) and with two separate nucleoli (? evidence of regeneration).
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Figure 6. b Another severely degenerated fibre with only few scattered myofilaments remaining. In the centre is a dense pyknotic nucleus with a nuclear membrane only along one side (arrow).On the other side are laminated myelin figures from tissue breakdown. a x 8300; b x 32 750.
fibres, a t times flocculent material, possibly proteinaceous oedema fluid, was also seen around degenerating fibres (Figures 5b & 7), and in the vicinity of small blood vessels with thickened basement membrane (Figure 7). Rarely the latter was reduplicated (Figure 5a). The tight junctions of vascular endothelial cells were everywhere well maintained. Pinocytotic vesicles often appeared increased in endothelial cells of blood vessels of both limbs (Figure 7). Lipofuscin pigment was found in both muscle fibres (Figures 3b, 5b & 6a), and endothelial cells of blood vessels. FINE STRUCTURAL CHANGES I N MUSCLES FROM NON-IMMOBILIZED LIMBS
Muscle from the side opposite to the immobilized limb has been termed the ‘nonimmobilized limb’ muscle and not ‘control’, because fine structural changes were seen here too. Like the immobilized muscle, the non-immobilized muscles also showed atrophy in varying forms and severity, but less frequently. In these, the myofibrillar architecture remained intact and appeared normal. However, muscle fibre degeneration was also seen i n some of the fibres and this again appeared to commence with increase i n
Figure 7. H-693 (A): Atrophied degenerating fibre (upper) containing only glycogen and a few effete organelles. Basement membrane is thickened and can be distinguished from the plasma membrane only a t arrows. Note thickened nuclear membrane. To the right mid-zone is part of a macrophage with a membrane-bound phagosome ( x ) , mitochondria and granules. Below is part of a small blood vessel with an intact tight junction (arrow) between the two endothelial cells t h a t contain many pinocytotic vesicles. The pericyte outside also shows pinocytotic vesicles. The vascular basement membrane is also thickened and homogeneous. Note flocculent material in the interstices. x 18 600.
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size of nucleoli and nuclei and multiplication of the latter, and small spots of degeneration in some of the I-bands (Figure 8a). More advanced degeneration of myofibrils accompanied by prominence of glycogen and mitochondria within the fibres, and of fibroblasts outside (Figure 8b) were seen. Generally the triads and sarcoplasmic reticulum were depleted as i n the immobilized limbs, but in one muscle the T-tubules were prominent (Figure 8b). Deposition of lipofuscin was seen under the basement membrane of some fibres, but its quantity was much less than the excessive amounts seen in the immobilized muscles. The venules and capillaries again showed homogeneous thickening of their basement membranes. In summary, three types of atrophic fibres were encountered in the muscles of both the limbs, although more on the immobilized side. The first showed merely a reduction in the fibre size with preservation of the normal appearances of all the constituents including myofibrils, basement membrane and plasma membrane. The second type was the most frequently encountered; it showed focal loss of myofibrils, with preservation of the remainder, and a n intact, but loosely folded basement membrane. The third type of atrophic fibre showed, in addition, degenerative changes of the remaining myofibrils and, at times, of the sarcoplasmic constituents. Thus, the degeneration appeared to be a late and more severe change was supervening upon atrophy of the fibres. Overt muscle fibre necrosis and phagocytosis were not observed.
Discussion CLINICO-PATHOLOGICAL ASPECTS
In the present material, quantitative histology revealed that the average muscle fibre diameter in both of the limbs of the four patients was substantially reduced as compared to controls. The generalized smallness of fibre size without any myopathic change or group atrophy in the immobilized limb as compared to the non-immobilized, emphasized simple atrophy due to disuse in muscles previously well-developed. Sunderland & Laverack (1969) described various histological changes in human muscles removed at necropsy from two individuals with permanent tenotomy of 1 and 20 years’ duration respectively. Histological examination showed various degrees of non-uniform atrophy of muscle fibres with only occasional areas of degeneration. These findings are in accord with those of Chor & Dolkart (1936) who concluded that ‘disuse atrophy consists primarily of a uniform reduction of the bulk of each muscle cell. . . ., This type of atrophy was associated with very simple structural changes. Stillwell, McLarren & Gersten (1967) also reported a diffuse non-neurogenic atrophy with prolonged immobilization of the lower extremity. Tomlinson, Walton & Rebeiz (1969) found group atrophy related to inactivity and not to age, even young comatose patients showing it. More recently the effect of weightlessness on muscle has been studied in astronauts returning from space. Even in the short interval of 14 days decrease in neuromuscular function was encountered, this being very severe when there was no opportunity to
Figure 8 . See page 97 for legend.
Human muscle in disuse atrophy
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carry out exercise. In reporting this, Bourne (1973) commented that the effect of weightlessness in space is similar to that of confinement to bed rest, both procedures producing loss of muscle substance, of mineral salts from bones, and of water from the body. It is interesting that significant weakness and possible wasting of the lower limb muscles developed i n young adult males, i n an extremely fit condition at the start of the voyage, on weightless confinement to the small space capsule. Viewed in this context, the disuse atrophy observed by us in the non-immobilized limb of our four patients need not be surprising as that leg was also rested in bed along with the immobilized leg. The greater atrophy and degeneration in such a limb than in the immobilized, as in case H-693, is difficult t o explain, but this was the oldest patient of all (Table 1) and perhaps he moved about very little in bed. At the same time, all patients with legs i n plaster casts are urged to contract the muscles in the cast, with no particular instruction about exercising the rest of the limbs. MUSCLE CHANGES WITH SPECIAL REFERENCE TO F I N E STRUCTURE
I n the current ultrastructural study a fairly severe degree of atrophy was seen in the muscles of the immobilized limb. All four cases showed many small fibres, a t times with collapsed basement membrane and prominent muscle nuclei, and degenerative changes in some of the muscle fibres. The non-immobilized muscles also showed similar changes though less frequently. The muscle of cats experimentally immobilized i n plaster casts underwent a more or less well-defined sequence of degenerative changes in which many fibres remained as sarcotubes covered by basement membrane and containing precipitated protein and fragments of sarcolemma (Cooper, 1972). Walker, Schrodt & Truong (1965) reported two ultrastructural types of lesion after tenotomy of r a t muscle. I n one there was disorientation and loss of myofibrils and dense staining and abnormal branching of sarcoplasmic reticulum (SR). In the other the myofibrils were better preserved and the SR was depleted. In the present human study the triads were not readily encountered and the T-tubes rarely appeared prominent i n degenerating muscle fibres, the brunt of the damage being borne by the myofibrils. Experimental tenotomy results not only in disuse atrophy, but also i n a total and sudden loss of tone in the muscles concerned. In this artificial situation the more tonic type I muscle fibres are likely t o be affected more. This was, in fact, observed by Resnik, Engel & Nelson (1968) and by Shafiq, Gorycki, Asiedu & Milhorat (1969) i n their tenotomized animals which showed degeneration of Z-discs of type I fibres. Such a predilection for type I fibres was not seen during immobilization in a plaster cast of the human limb (Patel, Razzak & Dastur, 1969), probably because these muscles Figure 8. Non-immobilized limb muscles. a H-709 (B): A fibre showing early atrophy and splitting. Note one large folded (?-splitting) nucleus and part of a small nucleus at the split. Note early degeneration of some of the I-bands (arrows). b H-693 (B): More advanced degeneration of a fibre; a few myofibrils remaining, loss of distinction of A- and I-bands, and sarcoplasm filled with glycogen, mitochondria and T-tubules (upper arrow) without sarcoplasmic reticulum. The lower fibre shows lamellated profiles (lower arrow) of possibly degenerated mitochondria. a x 6900; b 21 650.
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are not totally deprived of their tone. On the other hand, histochemical examination of human muscle under disuse, such as during prolonged bed-rest or i n arthritis of the hip joint (Bundschu, Suchenwirth & D’Avis, 1973), shows a preferential atrophy of type I1 fibres. Tenotomy does not seem to provide a n ideal model for the study of disuse atrophy in man and, as Guth (1968) commented, there is a hazard with all these experimental procedures in the interpretation of findings. The disuse atrophy that we found was very reminiscent of that seen i n the muscles of osteomalacic patients with a ‘nutritional’ or a ‘mixed’ aetiology (Dastur, Gagrat, Wadia, Desai & Bharucha, 1975). We reported ultrastructural evidence that most osteomalacic muscle weakness was not ‘myopathic’, but a non-specific atrophy occurring partly due t o disuse resulting from restricted movement of the legs and partly consequent on the additional malnutrition. The patients with ‘mixed’ aetiology (such as those associated with hyperparathyroidism or hyperthyroidism) showed many degenerative changes as well. It now appears that degeneration may occasionally occur in muscle fibres already atrophied by disuse through bed-rest ;i n the present study degenerative changes were encountered almost exclusively in atrophied, small fibres. After castration to produce disuse atrophy of the androgen-sensitive levator ani muscle of rats, the principal change observed was a reduction in the diameter of the myofibrils caused by loss of myofilaments at the periphery (Gori, Pellagrino & Pollera, 1967). Innervation and vasculature are unaffected by this procedure, and the structure of the neuromuscular junction also remains practically intact (Gutmann, Hanzlikova & Sobotkova, 1969). The castration method of producing disuse atrophy also results in a proportionate decrease of sarcoplasmic and contractile proteins (Bass, Gutmann, Hanzlikova, Hajek & Syrovy, 1969). The ultrastructural and chemical investigations of Gutmann & Hanzlikova (1973) showed disuse atrophy t o be accompanied by preservation of the integrity of the axon and of acetylcholine synthesis and release, but with a disproportionate decrease in the amount of protein fractions. This finding is consistent with the myofibrillar loss in an otherwise intact muscle fibre, such as that observed in the immobilized limbs of our patients. There was a general paucity of mitochondria i n the atrophied and degenerating fibres of our muscle specimens, and only infrequently their prominence a t sites of myofibrillar loss. Bergmann & Afifi (1969) failed t o notice any mitochondrial abnormality after tenotomy of the rabbit soleus muscle. However, they reported a n over 50% reduction in size in the average myofibril and of the average muscle fibre; they attributed these changes to the hydrolytic enzymes i n the intermyofibrillar space. Lipofuscin was frequently found by us i n subsarcolemmal phagolysosomes or i n endothelial cells of blood vessels just as it was in the weakened muscles i n osteomalacia (Dastur et aZ.,1975). Experimental muscle atrophy arising from disuse is said to be associated with a rise in lysosomal hydrolase activity, particularly of ,B-glucosidase (Max, Mayer & Vogelsang, 1971). Max (1972) postulated that mitochondrial dysfunction i n the form of a defective respiratory control may be of significance in the initiation of disuse atrophy in the rat. Here one might recall the homogeneous thickening of the basement membrane of muscle fibres and of small blood vessels in some
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of the muscles of the present study and i n osteomalacic muscle (Dastur et al., 1975). Similar diffuse ground-glass-like thickening of the vascular basement membrane has been seen i n brain oedema due to tumour or infection (e.g. Figure 7 of Dastur & Dave, 1977), and probably from anoxia during anoxic-ischaemic cerebral lesions i n the rat (Hills, 1964). IMPLICATIONS O F PRESENT FINDINGS TO HUMAN MUSCLE DISORDERS
The involvement, both clinically and pathologically (and especially on fine structural examination) of proximal muscles in disuse atrophy in the present and in other studies on man is interesting and important. We have also been impressed by atrophic and degenerative changes in proximal muscles in metabolic disorders, such as osteomalacia (Dastur et al., 1975), and in malnutrition in children (Dastur, Daver & Manghani, unpublished observations). Furthermore, in human muscular dystrophies, the ‘classical’ disorders producing proximal muscle weakness and wasting, the electron microscope changes of both atrophy and degeneration are similar to those seen in the present material, although of greater severity and extent (Dastur & Razzak, 1974). The three types of atrophy i n disuse noted above are almost identical t o the three types of atrophic fibres reported earlier by Milhorat, Shafiq & Goldstone (1966) i n human dystrophies. The degenerative changes i n the myofibrils, the sarcoplasm and even the plasma membrane, as also the increase in nuclear size and the relative prominence of glycogen or mitochondria in fibres undergoing degeneration during dystrophy, have now been encountered in disuse atrophy, osteomalacia and malnutrition. Even in spinal muscular (denervation) atrophy, particularly the juvenile or infantile varieties, where proximal muscles are involved, the atrophy and degeneration are comparable to those described here (Roth, Graziana, Terry & Scheinberg, 1965; Roy, Dubowitz & Wolman, 1971; Dastur & Razzak, 1974). Therefore, these morphologic changes, though doubtless important, appear to be non-specific. It suggests that the proximal limb muscles of man may be naturally susceptible t o different kinds of ‘noxa’ and insults, acquired or inherited. It is of interest in this connection that even in young active laboratory rats, the thigh muscles show less isotopic calcium (carrier-free 45CaC12)uptake than the abdominal or back muscles (Gagrat, Dastur & Raghavendran, 1973): calcium in sarcoplasmic reticulum is essential, of course, for normal muscle activity. Perhaps even a mild inadequacy of vascular supply or of innervation may precipitate structural changes in, and then functional insufficiency of, these muscles. Where the aetiological factor can be removed, as in disuse or malnutrition, recovery takes place; where it cannot be removed, as in the inherited dystrophies, the muscle damage continues.
Acknowledgements Grateful acknowledgement is due t o the Tata Institute of Fundamental Research, Bombay, for the generous use of their electron microscope; and to Mr N. Solanki of
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Darab K. Dastur, Bomi M. Gagrat and Daya K. Manghani
this Unit for the photographic dark-room work. Thanks are due to Dr J. C. N. Joshipura, Honorary Orthopaedic Surgeon, J.J. Hospitals, and his Registrar, Dr D. T. Rajpopat, for making available the muscle biopsies. Part of this work was conducted during a research grant from the Muscular Dystrophy Association of Western Australia. Perhaps our greatest thanks go to the four patients who volunteered for this study and permitted two muscle biopsies each.
References BASSA., GUTMANN E., HANZLIKOVA V., HAJEKI. & SYROVY L. (1969) The effect of castration and denervation upon the contraction properties and metabolism of the levator ani muscle of t h e rat. Physiologia Bohemoslovaca 18, 177. Quoted by Gutmann E. & Hanzlikova V. (1973) BERGMANN R.A. & AFIFI A.K. (1969) The structure of the rabbit soleus muscle and t h e structural alterations resulting from tenotomy. Johns Hopkins Medical Journal 124, 119-131 BOURNEG.H. (1973) The effects of weightlessness o n muscle. In Clinical Studies in Myology. Part 11, Proceedings of the IInd International Congress o n Muscle Diseases, pp. 115-123, ed. B.A. Kakulas. Excerpta Medica International Congress Series No. 295, Perth, Western Australia BUNDSCHU H.D., SUCHENWIRTH, R. & D’AVIS W. (1973) Histochemical changes in disuse atrophy of human skeletal muscle. In Basic Research in Myology, Part I, Proceedings of the IInd International Congress o n Muscle Diseases, pp. 108-112, ed. B. A. Kakulas, Excerpta Medica International Congress Series No. 294, Perth, Western Australia R.E. (1936) A study of simple disuse atrophy i n the monkey. American Journal of CHORE. & DOLKART Physiology 117, 626-630 COOPERR.R. (1972) Alterations during immobilisation and regeneration of skeletal muscle in cats. Journal of Bone Joint Surgery 54A, 919-953 DASTURD.K. & RAZZAKZ.A. (1974) Electronmicroscopic comparison of muscular dystrophies and atrophies. Proceedings of the IIIrd International Congress on Muscle Diseases, p. 93. Newcastleupon-Tyne, Excerpta Medica International Congress Series No. 334. DASTURD.K. & DAVEU.P. (1977) Ultrastructural basis of the vasculopathy i n and around brain tuberculomas. Possible significance of altered basement membrane. American Journal of Pathology 89, 35-50 DASTURD.K., GAGRATB.M., WADIAN.H., DESAIM.M. & BHARUCHA E.P. (1975) Nature of muscular change in osteomalacia: Light and electronmicroscope observations. Journal of Pathology 117, 211-228 GAGRAT B.M., DASTURD.K. & RAGHAVENDRAN K.V. (1973) Distribution of 45CaC1, in the rat, with special reference t o CNS, muscle and bone. IRCS International Research Communications System, (73-12), 16-7-24 C. & POLLERA M. (1967) The castration atrophy of the dorsal bulbocavernous GORI Z., PELLAGRINO muscle of r a t : a n electronmicroscopic study. Experimental a n d Molecular Pathology 6, 172-198 GUTHL. (1968) ‘Tropic’ influences of nerve on muscle. Physiological Reviews 48, 645-687 E., HANZLIKOVA V. & SOBOTKOVA M. (1969) Effect of castration on motor end-plate structure GUTMANN and function of the levator ani muscle of the rat. Physiologica Bohemoslovaca 18, 482. Quoted by Gutmann E. & Hanzlikova V. (1973) GUTMANN E. & HANZLIKOVA V. (1973) Specific and non-specific changes i n muscle atrophy and hypertrophy. In Clinical Studies in Myology, Part 11, Proceedings of the IInd International Congress o n Muscle Diseases, pp. 124-135, ed. B. A. Kakulas. Excerpta Medica International Congress Series, No. 295, Perth, Western Australia HILLSC.P. (1964) Ultrastructural changes in t h e capillary bed of t h e rat cerebral cortex in anoxic ischaemic brain lesions. American Journal of Pathology 44, 531-551
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MAX S.R. (1972) Disuse atrophy of skeletal muscle: loss of functional activity of mitochondria. Biochemistry and Biophysics Research Communications 46, 1394-1395 MAX S.R., MAYERR.F. & VOGELSANG L. (1971) Lysosomes and disuse atrophy of skeletal muscle. Archives of Biochemistry 146, 227-232 MILHORAT A.T., SHAFIQS.A. & GOLDSTONE L. (1966) Changes i n muscle structure i n dystrophic patients, carriers and normal siblings seen by electronmicroscopy: correlation with levels of serum creatine phosphokinase (CPK). Annals of the New York Academy of Science 138, 246-292 PATEL A.N., RAZZAK Z.A. & DASTURD.K. (1969) Disuse atrophy of human skeletal muscle. Archives of Neurology (Chicago) 20, 413421 RESNICKJ.S., ENGELW.K. & NELSONP.G. (1968) Changes i n Z-disc of skeletal muscle induced by tenotomy. Neurology (Minneapolis) 18, 737-740 ROTHS., GRAZIANI L.J., TERRYR.D. & SCHEINBERG L.C. (1965) Muscle fine structure in the KugelbergWelander syndrome (chronic spinal muscular atrophy). Journal of Neuropathology and Experimental Neurology 24, 444454 ROYS., DUBOWITZ V. & WOLMAN L. (1971) Ultrastructure ofmuscle in infantile spinal muscular atrophy. Journal of the Neurological Sciences 12, 219-232 SHAFIQS.A., GORYCKI M.A., ASIEDUS.A. & MILHORAT A.T. (1969) Tenotomy: effect on fine structure of soleus of rat. Archives of Neurology (Chicago) 20, 625-633 STILLWALL D.M., MCLARREN G.L. & GERSTEN J.W. (1967) Atrophy of quadriceps muscle due to immobilisation of the lower extremity. Archives of Physical Medicine 48, 289-295 SUNDERLAND S. (1968) Changes in striated muscle due to disuse. In Nerve and Nerve Injuries, pp. 302311. E. & S. Livingstone, Edinburgh & London SUNDERLAND S. & LAVERACK J.O. (1969) Changes in human muscles after permanent tenotomy. Journal of Neurology, Neurosurgery and Psychiatry 22, 167-174 TOMLINSON B.E., WALTONJ.N. & REBERGJ.J. (1969) Effects of ageing and cachexia upon skeletal muscle: a histopathological study. Journal of the Neurological Sciences 9, 321-346 TOWERS.S. (1937) Trophic control of non-nervous tissues by the nervous system: a study of muscle and bone innervated from a n isolated and quiescent region of the spinal cord. Journal of Comparative Neurology 67, 241-268 WALKER S.M., SCHRODT G.R. & TRUONC X.T. (1965) Electronmicroscope study of sarcoplasmic reticulum and myofilaments of tonotomized rat muscle. American Journal of Physical Medicine 44, 176-192
B
F I N E STRUCTURE O F MUSCLE I N HUMAN D I S U S E ATROPHY: SIGNIFICANCE OF PROXIMAL MUSCLE I N V O L V E M E N T I N MUSCLE D I S O R D E R S *
DARAB K . DASTUR, BOMI M . GAGRAT AND DAYA K . MANGHANI The Neuropathology Unit, Post- Graduate Research Laboratories, Grant Medical College and J.J. Group of Hospitals; and Nerve-Muscle Research Cell, Bombay Hospital, Bombay, India Accepted for publication 25 May 1978
Dastur D.K., Gagrat B.M. & Manghani D.K. (1979) Neuropathology and Applied Neurobiology 5, 85-101 Fine structure of muscle in human disuse atrophy: significance of proximal muscle involvement in muscle disorders The universal occurrence of weakness of skeletal musculature o n disuse, however produced, and the paucity of published reports o n the fine structural changes in human disuse atrophy, prompted the present investigation. The quadriceps muscle of a leg immobilized i n plaster cast (for fracture) and of the opposite non-immobilized limb was biopsied in four adult males, after periods of immobilization from 50 to 75 days. These 8 muscle specimens were examined for histopathological changes, and muscle fibre diameters were measured by micrometry from paraffin sections. The histograms revealed a larger proportion of small fibres ( < 2 0 pm) and a smaller proportion of large fibres (> 40pm) i n the immobilized limb compared t o the opposite. Thus, light microscopy showed only atrophic changes. This was confirmed by electronmicroscopy, where atrophy of few t o several muscle fibres was seen i n the form of loss of myofibrils, collapse and folding of the basement membrane and prominence of glycogen or muscle nuclei. The atrophic change was more severeintheimmobilized limbs, but it was also noticeable in all the non-immobilized limbs. Degenerative changes, especially disorganization and breakdown of myofibrils, and fragmentation of plasma membrane, were also seen i n occasional atrophied muscle fibres, again more frequently in the immobilized limb. Lipofuscin was often found accumulated in muscle fibres and occasionally in endothelial cells of intramuscular blood vessels ; the latter showed prominent pinocytotic vesicles or thickened basement membrane. It is concluded that both atrophy and degeneration of fibres of proximal muscles can occur as non-specific consequences of disuse of t h e limb in man, that degeneration is a later and more severe change, that muscles even of the non-immobilized leg are subjected to disuse atrophy during bed-rest, and that the proximal muscles in man seem t o have a natural susceptibility t o atrophy and degeneration in any muscular disorder.
* Part of this material was presented at the XIth World Congress of Neurology i n h s t e r d a m , September 1977. 0305-1846/78/0400-0085$02.00
0 1979 Blackwell Scientific Publications
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Introduction There is extensive literature on t h e ‘disuse atrophy’ of different muscle groups produced by a variety of techniques, but most of the reports are on experimental animals. I n the classic histopathological account of experimental disuse atrophy, Tower (1937) found the essential change t o be the gradual reduction i n size of all muscle fibres, with reduction of both sarcoplasm and myofibrils, but the cross striations remained well preserved over a long period of time. She did not, however, find any degenerative changes. Accounts of this condition i n human muscles are limited t o passing references in the clinical literature t o the wasting which follows immobilization of limb (Sunderland, 1968). The great difficulty of obtaining suitable human material for assessing histological changes is obvious. An earlier investigation from this Unit on cases of disuse atrophy of skeletal muscles reported histological and histochemical features (Patel, Razzak & Dastur, 1969). To the best of our knowledge, ultrastructural studies on atrophy of human muscles have not hitherto been reported. A further purpose of this investigation was also to record the changes occurring i n the non-immobilized limbs of such patients. The implications of even moderate degrees of disuse of muscles i n man appear t o be far greater than has been realized so far. I n view of our recent observation of generalized atrophy, partly on the basis of disuse, in the proximal muscles of patients with nutritional osteomalacia (Dastur, Gagrat, Wadia, Desai & Bharucha, 1975), it was also intended t o to see whether disuse from immobilization would result in only non-specific atrophy of muscle fibres or any other changes.
Materials and methods Muscle biopsies could be obtained from only 4 patients from the Department of Orthopaedic Surgery of our Hospitals, men between 38 and 50 years (Table 1)who had sustained closed traumatic uncomplicated fractures of the femur in 3 cases and of the upper ends of tibia and fibula in one. They were immobilized i n plaster or by skeletal traction for a period of 7 to 8 weeks and, in addition, one patient by skin traction for 12 days. The muscular strength of the involved limbs could not be tested due to pain. None of the patients had any neuromuscular disorder, evidence of severe malnutrition, or any generalized disease. Obtaining such patients with plastering adequate t o immobilize the quadriceps muscle, and who were prepared for biopsy from both limbs, was difficult and only 4 patients, who gave willing permission, could be so investigated over 14 years. Immediately o n removal of the plaster cast or traction, both t h e quadriceps muscles were biopsied under local anaesthesia. Part of each specimen was fixed in formaldehyde solution for the preparation of paraffin sections and stained with haematoxylin and eosin. Muscle fibre diameters (smallest) were measured using stage and eyepiece micrometry. Histograms of muscle fibre sizes were plotted. Using Student’s t test, statistical comparison was made between t h e mean fibre diameters of immobilized and non-immobilized limbs, and between the latter two and the mean fibre diameter of quadriceps muscle obtained earlier from control subjects admitted to t h e orthopaedic wards who were without weakness or immobilization of the lower limbs. While Araldite sections showed less shrinkage and more compactly arranged fibres, they could not be used for measuring fibre sizes as the number of fibres included i n such sections was often inadequate. For electron microscopy, a portion of the freshly biopsied muscle from the patient was fixed i n cold
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4% glutaraldehyde i n Millonig’s phosphate buffer. The tissue was cut into small pieces with proper orientation, treated with 1%,OsO, i n Millonig’s buffer, dehydrated i n a graded series of alcohols followed by propylene oxide, embedded i n Araldite and cut on a Porter-Blum ultramicrotome. Semithin (1pm thick) sections were first examined and thin sections were cut and stained with uranyl acetate and lead citrate, and examined and photographed in a Philips 200 electron microscope.
Results QUANTITATIVE HISTOLOGY
Plastic sections and paraffin sections of muscles from the immobilized limbs of all the four patients showed ill-defined groups of small fibres lying amidst normal looking
b
< 20 u m
v)
=
54%
F
I
-
13 (A)
< 20 urn > 40 prn
= =
78.5% 3.0%
20
?!
e
10
0
10
30
50 10 30 Fibre diameter ( pm)
50
Figure 1. a H-595 (A): Quadriceps muscle from immobilized limb; compactly arranged muscle fibres of varying size, including small atrophic fibres, especially i n the fascicle on the right (arrows). Blood vessels i n the interfascicular space appear normal. 1 pm plastic section, Toluidine blue. x100. b Histograms of quadriceps muscle fibre diameters, from the immobilized limb of two of the patients. Note the large number of small fibres and the very small number of large fibres (above 40 pm); the normal bimodal pattern is lost i n case 1-13 (A).
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b
I
0
10
30
50
-
13 (6)
70 10 30 Fibre diameter ( p m)
< 20 p
=
> 40 p
=
50
16.5% 35.0%
70
Figure 2. a H-595 (B): Quadriceps muscle from non-immobilized limb (cf. Figure 1). Small angular atrophic fibres are fewer t h a n i n the opposite limb. 1 pm plastic section, Toluidine blue. x 250. b Histograms of quadriceps muscle fibre diameters from non-immobilized limb of the patients i n Figure l b , showing a smaller number of smaller fibres in 1-13 (B), and a larger number of large fibres i n both.
fibres (Figure la). The muscle fibres from the opposite non-immobilized limb also showed a similar but lesser change (Figure 2a). These atrophied fibres, particularly from immobilized muscles, were frequently angulated, occasionally rounded, with relatively increased nuclei. No muscle from either limb showed any evidence of hypertrophied fibres, necrosis, phagocytosis or regeneration, evidence of inflammation or increase in endomysial connective tissue or fat, or of changes in blood vessels. Thus. no myopathic changes were found on light microscopy in muscles from either limb. The average muscle fibre diameter in paraffin sections from the four immobilized limbs was 24.24 pm and that from the non-immobilized limbs was 30.17 pm, both being considerably less than the mean fibre diameter (46.63 pm) of normal quadriceps muscle
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Table 1. Muscle fibre diameters i n patients with disuse atrophy statistical comparison with control subjects NP/no.
H-595 H-693 H-709 1-13
Age (years)
of
45 50 40 38
Duration immobilization (days) 75 50 56 56
Mean muscle fibre diameter ( p m ) : immobilized non-immobilized limb limb 22.4 35.7 18.90 20.0
29.0 29.3 32.4 30.0
PATIENTS WITH DISUSED LIMBS
(n = 4 ; mean age =43)
Mean? s.d. CONTROLS ( n = 8; meanage = 43)
I 24.24 ? 8.7---30.17 46.63 ? 10.6
2.9
1 J *
M e a n ? s.d.
t =statistically * =statistically
not significant highly significant ( P < 0.001). s.d. =standard deviation
(Table 1). However, the mean muscle fibre diameter for the immobilized limbs was not statistically significantly different from that for the non-immobilized limbs. There was no relationship between the duration of immobilization and the average fibre diameter (Table 1). Fibre size spectra from the immobilized limbs (Figure lb) showed a high proportion of small fibres ( < 20 pm), especially when compared to the non-immobilized limb (Figure 2b) of the same subject. One patient (H/693) was unique in showing a more severe non-specific atrophy in the non-immobilized limb than in the the immobilized (Figure 3). A suggestion of bimodality was seen in the histograms of fibre sizes from both limbs of case H/595 (Figures l b & 2b): a descending step-ladder pattern is seen from the small t o the large fibres in the immobilized limbs of cases H-709 and 1-13 (Figures 3 & lb).
FINE STRUCTURAL CHANGES I N THE MUSCLES FROM IMMOBILIZED LIMBS
The most frequent change in the immobilized muscles was atrophy of fibres of varying severity present in all four cases. The overall impression was of a moderate atrophic change, as evidenced by small atrophic fibres with compactly arranged myofibrils, intact sarcomeres and more or less loosened basement membrane (Figures 4 & 5a). With more severe atrophy, the loosely lying basement membrane was thrown into deep folds, at times with an accordion-like appearance, and a t other times like long tails of empty basement membrane (Figure 5b). The complete atrophy of a fibre with
H - 693 (A)
20
- 709 < 20 (A) Lim 1 40 prn
= =
< 20 k i r n
68.7% 4.2%
=
13.6%
1 40 p m = 32.09%
10
- 0 v)
P
,
H
-
< 20 11 = 16.8% > 40 p = 33.6%
693 (B) < 20 11 = 26.4% > 40 11 = 16.05%
10
0
10
30
50
70 10 Fibre diameter (prn)
30
50
70
Figure 3. Histograms of quadriceps muscle fibre diameters from the immobilized (upper histograms) and non-immobilized (lower histograms) limbs of two patients. Note that while the immobilized muscle of case H-709 is more affected (like the other two cases illustrated in Figures l b & 2b), t h e non-immobilized muscle is more affected and shows fewer large and more small fibres, in case H-693.
Figure 4. H-709: Two moderately well preserved muscle fibres with only minimal atrophy as indicated by the scalloped basement membrane ; in the centre is collapsed empty basementmembrane, except for parts of plasma membrane (p.m.), of a totally atrophied muscle fibre. Uranyl acetate and lead citrate. x 6300.
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total disappearance of its myofibrillar and sarcoplasmic content was indicated just by the presence of a n empty collapsed basement membrane (central structure in Figure 4). Three of the four immobilized muscles showed some degenerative changes in addition to atrophy, different stages and forms of which are seen in Figures 5b, 6a and b, and 7. In the early stages the atrophic fibres showed patchy loss or degeneration of myofibrils and depletion of sarcoplasmic reticulum or mitochondria. Increased number of nuclei, central or peripheral (Figure 6a), or a large nucleolus (Figure 5b, and fibre on left in Figure 6a) were seen in the degenerating fibres. Later, the myofibrillar architecture became disorganized or the myofilaments actually disrupted (fibre on right in Figures 6a & 6b). The most advanced degeneration was represented by a very small fibre almost devoid of myofibrils, myofilaments and organelles, and containing mainly glycogen (Figure 7). In such atrophied and degenerating fibres thickening of the basement membrane, fusion with the plasma membrane, which appeared thickened or fragmented (Figure 7), was observed. In one specimen (H-709A) a different type of atrophy was seen represented merely by smallness of fibre size (around 5 pm), without any loosening or even scalloping of the basement membrane. Rarely, macrophages containing phagosomes, vacuoles and granular bodies were encountered (Figure 7). While there was generally increased collagen between the
Figure 6 . H-709 (A) a: Parts of two muscle fibres; on t h e left, atrophic and bearing lipofuscin deposits near the single nucleus; on the right, showing disorganized degenerated myofibrils with scattered myofilaments, multiple nuclei, one of them possibly dividing (arrows) and with two separate nucleoli (? evidence of regeneration).
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Figure 6. b Another severely degenerated fibre with only few scattered myofilaments remaining. In the centre is a dense pyknotic nucleus with a nuclear membrane only along one side (arrow).On the other side are laminated myelin figures from tissue breakdown. a x 8300; b x 32 750.
fibres, a t times flocculent material, possibly proteinaceous oedema fluid, was also seen around degenerating fibres (Figures 5b & 7), and in the vicinity of small blood vessels with thickened basement membrane (Figure 7). Rarely the latter was reduplicated (Figure 5a). The tight junctions of vascular endothelial cells were everywhere well maintained. Pinocytotic vesicles often appeared increased in endothelial cells of blood vessels of both limbs (Figure 7). Lipofuscin pigment was found in both muscle fibres (Figures 3b, 5b & 6a), and endothelial cells of blood vessels. FINE STRUCTURAL CHANGES I N MUSCLES FROM NON-IMMOBILIZED LIMBS
Muscle from the side opposite to the immobilized limb has been termed the ‘nonimmobilized limb’ muscle and not ‘control’, because fine structural changes were seen here too. Like the immobilized muscle, the non-immobilized muscles also showed atrophy in varying forms and severity, but less frequently. In these, the myofibrillar architecture remained intact and appeared normal. However, muscle fibre degeneration was also seen i n some of the fibres and this again appeared to commence with increase i n
Figure 7. H-693 (A): Atrophied degenerating fibre (upper) containing only glycogen and a few effete organelles. Basement membrane is thickened and can be distinguished from the plasma membrane only a t arrows. Note thickened nuclear membrane. To the right mid-zone is part of a macrophage with a membrane-bound phagosome ( x ) , mitochondria and granules. Below is part of a small blood vessel with an intact tight junction (arrow) between the two endothelial cells t h a t contain many pinocytotic vesicles. The pericyte outside also shows pinocytotic vesicles. The vascular basement membrane is also thickened and homogeneous. Note flocculent material in the interstices. x 18 600.
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size of nucleoli and nuclei and multiplication of the latter, and small spots of degeneration in some of the I-bands (Figure 8a). More advanced degeneration of myofibrils accompanied by prominence of glycogen and mitochondria within the fibres, and of fibroblasts outside (Figure 8b) were seen. Generally the triads and sarcoplasmic reticulum were depleted as i n the immobilized limbs, but in one muscle the T-tubules were prominent (Figure 8b). Deposition of lipofuscin was seen under the basement membrane of some fibres, but its quantity was much less than the excessive amounts seen in the immobilized muscles. The venules and capillaries again showed homogeneous thickening of their basement membranes. In summary, three types of atrophic fibres were encountered in the muscles of both the limbs, although more on the immobilized side. The first showed merely a reduction in the fibre size with preservation of the normal appearances of all the constituents including myofibrils, basement membrane and plasma membrane. The second type was the most frequently encountered; it showed focal loss of myofibrils, with preservation of the remainder, and a n intact, but loosely folded basement membrane. The third type of atrophic fibre showed, in addition, degenerative changes of the remaining myofibrils and, at times, of the sarcoplasmic constituents. Thus, the degeneration appeared to be a late and more severe change was supervening upon atrophy of the fibres. Overt muscle fibre necrosis and phagocytosis were not observed.
Discussion CLINICO-PATHOLOGICAL ASPECTS
In the present material, quantitative histology revealed that the average muscle fibre diameter in both of the limbs of the four patients was substantially reduced as compared to controls. The generalized smallness of fibre size without any myopathic change or group atrophy in the immobilized limb as compared to the non-immobilized, emphasized simple atrophy due to disuse in muscles previously well-developed. Sunderland & Laverack (1969) described various histological changes in human muscles removed at necropsy from two individuals with permanent tenotomy of 1 and 20 years’ duration respectively. Histological examination showed various degrees of non-uniform atrophy of muscle fibres with only occasional areas of degeneration. These findings are in accord with those of Chor & Dolkart (1936) who concluded that ‘disuse atrophy consists primarily of a uniform reduction of the bulk of each muscle cell. . . ., This type of atrophy was associated with very simple structural changes. Stillwell, McLarren & Gersten (1967) also reported a diffuse non-neurogenic atrophy with prolonged immobilization of the lower extremity. Tomlinson, Walton & Rebeiz (1969) found group atrophy related to inactivity and not to age, even young comatose patients showing it. More recently the effect of weightlessness on muscle has been studied in astronauts returning from space. Even in the short interval of 14 days decrease in neuromuscular function was encountered, this being very severe when there was no opportunity to
Figure 8 . See page 97 for legend.
Human muscle in disuse atrophy
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carry out exercise. In reporting this, Bourne (1973) commented that the effect of weightlessness in space is similar to that of confinement to bed rest, both procedures producing loss of muscle substance, of mineral salts from bones, and of water from the body. It is interesting that significant weakness and possible wasting of the lower limb muscles developed i n young adult males, i n an extremely fit condition at the start of the voyage, on weightless confinement to the small space capsule. Viewed in this context, the disuse atrophy observed by us in the non-immobilized limb of our four patients need not be surprising as that leg was also rested in bed along with the immobilized leg. The greater atrophy and degeneration in such a limb than in the immobilized, as in case H-693, is difficult t o explain, but this was the oldest patient of all (Table 1) and perhaps he moved about very little in bed. At the same time, all patients with legs i n plaster casts are urged to contract the muscles in the cast, with no particular instruction about exercising the rest of the limbs. MUSCLE CHANGES WITH SPECIAL REFERENCE TO F I N E STRUCTURE
I n the current ultrastructural study a fairly severe degree of atrophy was seen in the muscles of the immobilized limb. All four cases showed many small fibres, a t times with collapsed basement membrane and prominent muscle nuclei, and degenerative changes in some of the muscle fibres. The non-immobilized muscles also showed similar changes though less frequently. The muscle of cats experimentally immobilized i n plaster casts underwent a more or less well-defined sequence of degenerative changes in which many fibres remained as sarcotubes covered by basement membrane and containing precipitated protein and fragments of sarcolemma (Cooper, 1972). Walker, Schrodt & Truong (1965) reported two ultrastructural types of lesion after tenotomy of r a t muscle. I n one there was disorientation and loss of myofibrils and dense staining and abnormal branching of sarcoplasmic reticulum (SR). In the other the myofibrils were better preserved and the SR was depleted. In the present human study the triads were not readily encountered and the T-tubes rarely appeared prominent i n degenerating muscle fibres, the brunt of the damage being borne by the myofibrils. Experimental tenotomy results not only in disuse atrophy, but also i n a total and sudden loss of tone in the muscles concerned. In this artificial situation the more tonic type I muscle fibres are likely t o be affected more. This was, in fact, observed by Resnik, Engel & Nelson (1968) and by Shafiq, Gorycki, Asiedu & Milhorat (1969) i n their tenotomized animals which showed degeneration of Z-discs of type I fibres. Such a predilection for type I fibres was not seen during immobilization in a plaster cast of the human limb (Patel, Razzak & Dastur, 1969), probably because these muscles Figure 8. Non-immobilized limb muscles. a H-709 (B): A fibre showing early atrophy and splitting. Note one large folded (?-splitting) nucleus and part of a small nucleus at the split. Note early degeneration of some of the I-bands (arrows). b H-693 (B): More advanced degeneration of a fibre; a few myofibrils remaining, loss of distinction of A- and I-bands, and sarcoplasm filled with glycogen, mitochondria and T-tubules (upper arrow) without sarcoplasmic reticulum. The lower fibre shows lamellated profiles (lower arrow) of possibly degenerated mitochondria. a x 6900; b 21 650.
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are not totally deprived of their tone. On the other hand, histochemical examination of human muscle under disuse, such as during prolonged bed-rest or i n arthritis of the hip joint (Bundschu, Suchenwirth & D’Avis, 1973), shows a preferential atrophy of type I1 fibres. Tenotomy does not seem to provide a n ideal model for the study of disuse atrophy in man and, as Guth (1968) commented, there is a hazard with all these experimental procedures in the interpretation of findings. The disuse atrophy that we found was very reminiscent of that seen i n the muscles of osteomalacic patients with a ‘nutritional’ or a ‘mixed’ aetiology (Dastur, Gagrat, Wadia, Desai & Bharucha, 1975). We reported ultrastructural evidence that most osteomalacic muscle weakness was not ‘myopathic’, but a non-specific atrophy occurring partly due t o disuse resulting from restricted movement of the legs and partly consequent on the additional malnutrition. The patients with ‘mixed’ aetiology (such as those associated with hyperparathyroidism or hyperthyroidism) showed many degenerative changes as well. It now appears that degeneration may occasionally occur in muscle fibres already atrophied by disuse through bed-rest ;i n the present study degenerative changes were encountered almost exclusively in atrophied, small fibres. After castration to produce disuse atrophy of the androgen-sensitive levator ani muscle of rats, the principal change observed was a reduction in the diameter of the myofibrils caused by loss of myofilaments at the periphery (Gori, Pellagrino & Pollera, 1967). Innervation and vasculature are unaffected by this procedure, and the structure of the neuromuscular junction also remains practically intact (Gutmann, Hanzlikova & Sobotkova, 1969). The castration method of producing disuse atrophy also results in a proportionate decrease of sarcoplasmic and contractile proteins (Bass, Gutmann, Hanzlikova, Hajek & Syrovy, 1969). The ultrastructural and chemical investigations of Gutmann & Hanzlikova (1973) showed disuse atrophy t o be accompanied by preservation of the integrity of the axon and of acetylcholine synthesis and release, but with a disproportionate decrease in the amount of protein fractions. This finding is consistent with the myofibrillar loss in an otherwise intact muscle fibre, such as that observed in the immobilized limbs of our patients. There was a general paucity of mitochondria i n the atrophied and degenerating fibres of our muscle specimens, and only infrequently their prominence a t sites of myofibrillar loss. Bergmann & Afifi (1969) failed t o notice any mitochondrial abnormality after tenotomy of the rabbit soleus muscle. However, they reported a n over 50% reduction in size in the average myofibril and of the average muscle fibre; they attributed these changes to the hydrolytic enzymes i n the intermyofibrillar space. Lipofuscin was frequently found by us i n subsarcolemmal phagolysosomes or i n endothelial cells of blood vessels just as it was in the weakened muscles i n osteomalacia (Dastur et aZ.,1975). Experimental muscle atrophy arising from disuse is said to be associated with a rise in lysosomal hydrolase activity, particularly of ,B-glucosidase (Max, Mayer & Vogelsang, 1971). Max (1972) postulated that mitochondrial dysfunction i n the form of a defective respiratory control may be of significance in the initiation of disuse atrophy in the rat. Here one might recall the homogeneous thickening of the basement membrane of muscle fibres and of small blood vessels in some
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of the muscles of the present study and i n osteomalacic muscle (Dastur et al., 1975). Similar diffuse ground-glass-like thickening of the vascular basement membrane has been seen i n brain oedema due to tumour or infection (e.g. Figure 7 of Dastur & Dave, 1977), and probably from anoxia during anoxic-ischaemic cerebral lesions i n the rat (Hills, 1964). IMPLICATIONS O F PRESENT FINDINGS TO HUMAN MUSCLE DISORDERS
The involvement, both clinically and pathologically (and especially on fine structural examination) of proximal muscles in disuse atrophy in the present and in other studies on man is interesting and important. We have also been impressed by atrophic and degenerative changes in proximal muscles in metabolic disorders, such as osteomalacia (Dastur et al., 1975), and in malnutrition in children (Dastur, Daver & Manghani, unpublished observations). Furthermore, in human muscular dystrophies, the ‘classical’ disorders producing proximal muscle weakness and wasting, the electron microscope changes of both atrophy and degeneration are similar to those seen in the present material, although of greater severity and extent (Dastur & Razzak, 1974). The three types of atrophy i n disuse noted above are almost identical t o the three types of atrophic fibres reported earlier by Milhorat, Shafiq & Goldstone (1966) i n human dystrophies. The degenerative changes i n the myofibrils, the sarcoplasm and even the plasma membrane, as also the increase in nuclear size and the relative prominence of glycogen or mitochondria in fibres undergoing degeneration during dystrophy, have now been encountered in disuse atrophy, osteomalacia and malnutrition. Even in spinal muscular (denervation) atrophy, particularly the juvenile or infantile varieties, where proximal muscles are involved, the atrophy and degeneration are comparable to those described here (Roth, Graziana, Terry & Scheinberg, 1965; Roy, Dubowitz & Wolman, 1971; Dastur & Razzak, 1974). Therefore, these morphologic changes, though doubtless important, appear to be non-specific. It suggests that the proximal limb muscles of man may be naturally susceptible t o different kinds of ‘noxa’ and insults, acquired or inherited. It is of interest in this connection that even in young active laboratory rats, the thigh muscles show less isotopic calcium (carrier-free 45CaC12)uptake than the abdominal or back muscles (Gagrat, Dastur & Raghavendran, 1973): calcium in sarcoplasmic reticulum is essential, of course, for normal muscle activity. Perhaps even a mild inadequacy of vascular supply or of innervation may precipitate structural changes in, and then functional insufficiency of, these muscles. Where the aetiological factor can be removed, as in disuse or malnutrition, recovery takes place; where it cannot be removed, as in the inherited dystrophies, the muscle damage continues.
Acknowledgements Grateful acknowledgement is due t o the Tata Institute of Fundamental Research, Bombay, for the generous use of their electron microscope; and to Mr N. Solanki of
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Darab K. Dastur, Bomi M. Gagrat and Daya K. Manghani
this Unit for the photographic dark-room work. Thanks are due to Dr J. C. N. Joshipura, Honorary Orthopaedic Surgeon, J.J. Hospitals, and his Registrar, Dr D. T. Rajpopat, for making available the muscle biopsies. Part of this work was conducted during a research grant from the Muscular Dystrophy Association of Western Australia. Perhaps our greatest thanks go to the four patients who volunteered for this study and permitted two muscle biopsies each.
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B
