Neuroscience Letters, 25 (1981) 269-274 269 Elsevier/North-Holland Scientific Publishers Ltd.

R A P I D A T R O P H Y OF MOUSE SOLEUS MUSCLES AFTER T E N O T O M Y DEPENDS ON AN I N T A C T I N N E R V A T I O N

ELSPETH M. McLACHLAN

Department of Physiology, Monash University, Clayton, Victoria 3168 (Australia)

(Received May 6th, 1981; Accepted May 18th, 1981)

Changes in length and mass of mouse soleus muscles have been determined during the first 14 days after division of the Achilles tendon and/or the tibial nerve. Muscle atrophy and associated histological changes were detectable 24 h after tenotomy, and increased progressively over the first week. These changes were less marked in muscles which had also been denervated, and were rapidly reversed if the tendon became reattached. An attempt is made to distinguish the role of the nerve supply from the effects of reduced longitudinal tension in the production of atrophy after tenotomy.

Section of the tendon of a skeletal muscle is followed by muscle atrophy, that in soleus being more severe than in other muscles of the mammal ian hindlimb [1, 3, 4]. This type of a t rophy has been used as a model of disuse not involving t rauma to the nervous system, similar to that produced by artificial immobilization (e.g. refs. 3 and 8), and has generally been thought to be a consequence of muscle inactivity resulting f rom decreased autogenic reflex activation. In support to this idea, tonic electromyographic (EMG) activity has been reported to be absent [18] or significantly reduced [11] in tenotomized rabbit and cat soleus muscles within a day of operation, and reduced EMG and muscle tone have been observed in human muscles after partial rupture of the Achilles tendon [13]. Monosynaptic reflexes are apparently enhanced [12, 13], consistent with decreased muscle spindle activity [6]. However, little change in EMG was detectable in solei of freely-moving cats [16], while overall sensory outflow increases [10].

Tenotomy results in preferential a t rophy of slow-twitch muscle fibres in both soleus and gastrocnemius muscles of cats [4]. However, tenotomized rat soleus atrophies to at least the same extent [9] as does cat soleus [3, 16] although the former contains only 60-700/0 slow-twitch fibres in young adult animals [7] compared with almost 100070 in the cat [4]. It therefore seems possible that some other characteristic feature of soleus muscle is implicated in the production of fibre atrophy.

In the present experiments the onset o f the a t rophy after tenotomy has been examined in soleus muscles of albino Swiss mice and compared with that which follows denervation. The contribution of the nerve supply to the effects produced in tenotomized muscles has also been examined during this period by denervating the muscles at the time of tendon section.

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Under pentobarbital anaesthesia (30 mg/kg i.p.), about 1 mm of the Achilles tendon was removed and /or the tibial nerve was sectioned at mid-thigh level. At various times afterwards, the animals were sacrificed by cervical dislocation, and soleus muscles from both lesioned and contralateral sides were excised and dissected in vitro as free of connective tissue and tendon as possible. Muscle length was measured in situ (to the nearest 0.5 mm) with the ankle joint held flexed at 90 °. Wet muscle mass was determined with a torsion balance after firmly blotting the excised muscle dry with filter paper. Muscle length and mass were expressed in terms of the ratios of experimental to contralateral (control) muscles and mean values of these ratios determined for groups of at least 8 mice (C.V. usually < 10°70). Means are given + S.E.M. Statistical significance was determined by the unpaired t-test. In a few experiments, the excised muscles were also weighed after drying to constant weight; in all cases, the mean ratios of experimental to control mass were not significantly different from those determined wet (see refs. 3 and 9).

No evidence of compensatory hypertrophy of contralateral muscles was obtained as the mean ratios of unoperated soleus mass to whole body weight were the same for all experimental groups and for normal (unoperated) mice (see also ref. 1). In a group of sham-operated animals (in which 1 mm of the Achilles tendon was cleared

f rom its surroundings and briefly stretched), muscle mass ratios (mean 0.996 + 0.031, n = 8) were not different f rom unity or f rom those obtained for un-

operated animals (left:right, 1.005 + 0.015, n = 4). Another group of mice was sham-operated by exposing the sciatic nerve at mid-thigh level and dissecting the tibial from the common peroneal nerve; muscle mass ratios in these animals were again not different f rom unity (mean 1.012 _+ 0.017, n = 8). There were no diffe- rences in the results of the combined procedure if the muscles were denervated before or after tenomoty, so that neuronal traffic produced directly by cutting the tendon did not contribute to the muscle response examined.

The changes in muscle length and mass during the first week after tenotomy, denervation or a combination of these lesions are shown in Fig. 1, together with the data obtained after 14 days, by which time almost all the damaged tendons had re-

inserted to some degree (see below). Only a small ( < 5°70) reduction in muscle length occurred immediately on division

of the Achilles tendon (determined in acute experiments in which the whole muscle was exposed and cleared in situ before tendon division), but by 24 h later innervated muscles had become about 30°7o shorter (by reduction in sarcomere length [2]). The reduction was significantly less in muscles which had been denervated at the time of

tenotomy (see Fig. 1). The loss of weight which followed tenotomy alone was very rapid, operated

muscle mass being significantly reduced 24 h after tendon section (P < 0.01) and decreasing progressively over the first week by about 45%. Almost identical degrees of a t rophy have been reported after 3 and 7 days in rat soleus [9]. This change can be compared with the more gradual decline in muscle mass produced by denervation

271

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Fig. 1. Changes in length and mass of mouse soleus muscles after denervation (A), tenotomy ( o ) or a

combination of these procedures ((3). Points are the mean values for operated/contralateral muscles _+ S.E.M. At 14 days some tenotomized (11) and all denervated + tenotomized (IS]) muscles had become reattached. Significant differences between tenotomized and denervated plus tenotomized values are indicated *(P < 0.05), **(P < 0.01).

alone (only about 20°/o after one week). The rapid onset of atrophy after tendon section was prevented by simultaneous denervation of the muscle, so that muscle mass ratios were not significantly different from those for denervated muscles for the first few days. However this protection was temporary, and muscles which were both denervated and tenotomized had atrophied after 7 days to the same degree as muscles tenotomized alone.

Atrophy was equally rapidly reversed when the tendon began to reinsert on to the calcaneum by way of a connective tissue bridge. Manipulation of the ankle joint prior to dissecting out the muscle produced detectable movement of soleus (and gastrocnemius) in a few animals as early as 5 days after the operation and in almost all cases after 14 days. Although reinserted muscles were only slightly longer than tenotomized muscles (see Fig. 1), reinserted muscle mass relative to the contralateral side was significantly greater (P <0.01) than the ratios for those innervated muscles still unattached (reinserted mass ratios: 5 days, 0.813 + 0.051, n = 4; 7 days, 0 .774+ 0.031, n = 6; 14 days, 0 .700+ 0.031, n = 9). The same was true of the denervated plus tenotomized muscles which had reinserted (5 days, 0.950 + 0.019, n = 3; 7 days, 0.709 + 0.038, n = 5); after 14 days these muscles did not show any evidence of recovery of weight (because of progressive denervation atrophy) although they had all reinserted to some degree. This presumably resulted from the

272

shorter distance between the cut tendon and its point of attachment in denervated muscles, and indicates that innervation is not necessary for the repair of a damaged tendon. However any active shortening of an innervated tenotomized muscle would be expected to delay tendon repair.

Examination of transverse sections of tenotomized muscles showed that fibre diameter was increased l day after tenotomy (due to overall muscle shortening) but progressively decreased and was back to about control diameter by the 5th day. Increased nuclear staining between the muscle fibres indicative of fibroblast infiltration was detectable 1 day after tenotomy; this was less marked in denervated muscles although it was clearly present at 5 days. Reinserted muscles appeared normal. It seems that loss of muscle mass during the first week after tenotomy is associated primarily with a reduction in fibre volume and restoration of fibre cross- sectional area. Both decreased diameter and degenerative loss of soleus muscle fibres 2-3 weeks after tenotomy was associated with fibroblast infiltration [l 1, 14], but these changes were virtually absent if the muscles had been denervated. Whether or not fibre fragmentation or degeneration also occurs during the first week was not qualitatively obvious in the present material and requires further study.

If it is assumed that the trophic consequences of removing the nerve supply of a muscle simply add to the passive effect of reduced longitudinal tension produced by tendon section, it is possible to describe the relative contribution of the innervation to the atrophic changes observed. An analysis based on this assumption is illustrated in Fig. 2. It is evident that the presence of the nerve causes relatively little additional shortening of the muscle over that which occurs passively, although it is difficult to assess the extent to which tethering to the bulk of the tenotomized gastrocnemius limits active shortening. The direct effect of maintained decrease in length in reducing the number of sarcomeres [2, 17] may be responsible for this component of the atrophy. By contrast, the loss of muscle mass, and its rapid reversal upon reinsertion, are dependent to a greater extent on the presence of an intact innervation, particularly over the first few days. This 'active' component might even have continued to increase had reinsertion been prevented.

It is not clear from these experiments whether or not tenotomy is associated with relative neuromuscular inactivity. Certainly innervated muscles shortened more after tenotomy than did denervated ones (Fig. 1), implying some muscle activation had occurred. Degenerative changes in tenotomized rabbit and rat soleus are less marked in spinal animals [11, 15], except under some conditions of muscle stimulation, suggesting that it might rather be impulse activity in motoneurones which is responsible for these effects of tenotomy. Cat soleus muscles immobilized in a shortened position atrophy to the same degree whether or not impulse activity in motoneurones is prevented by cord section and deafferentation [5], but tenotomy atrophy is slightly less under these conditions [3]. In addition, dorsal root section substantially reduces tenotomy atrophy in rat soleus [9] suggesting some involvement of afferent axons. Perhaps reduction of muscle spindle activity (which

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Fig. 2. Calculated differences between effects of tenotomy and denervation on muscle length and mass, derived from data in Fig. 1. Fractional effect produced by the passive change in tension after tenotomy ((3, calculated by subtracting the effect of denervation alone from the response to denervation plus tenotomy) and by the additional active component dependent on intact innervation (O, calculated by subtracting the passive effects from those of tenotomy alone).

presumably occurs in both tenotomized and short immobilized muscles) contributes less in tenotomized muscles than other sensory changes such as the disruption or absence of tendon organ activity. In the presence of a modified sensory input to the motoneurone, it may be the altered pattern of electrical activity rather than its presence or absence which determines muscle fibre maintenance (see ref. 11).

I would like to thank Jandri Hoggins for preparing the histological material. This project is supported by the National Health and Medical Research Council and by a Monash University Special Research Grant.

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