THE ANATOMICAL RECORD 228:104-108 (1990)

Tenotomy-Induced Motor Endplate Alterations in Rat Soleus Muscle BRUCE R. PACHTER AND NEIL I. SPIELHOLZ Department of Rehabilitation Medicine, New York University Medical Center, New York, New York

ABSTRACT The effects of tenotomy on the ultrastructure of r a t soleus muscle motor endplates were examined both qualitatively and quantitatively. Rat soleus muscle was studied 2 weeks following tenotomy and compared with normal littermates. The motor endplates from the tenotomized muscles were found to exhibit both degenerative and regenerative changes. Degeneration consisted of postjunctional fold breakdown, exposed junctional folds, myelin-like bodies within the subjunctional sarcoplasm, and dense bodies within the Schwann cell cytoplasm. The regenerative changes consisted of several small nerve terminals occurring within the same primary synaptic cleft and several axons wrapped by the same Schwann cell. The results demonstrate that tenotomy induces denervation-like changes at endplates that lead to terminal sprouting within the neuromuscular junctional area and remodelling. Experimental tendon transection is often studied as a model of muscle disuse (see Talesara and Jasra, 1986). Tenotomized muscles have been examined electrophysiologically (Vrbova, 1962; Buller and Lewis, 1965), histologically (Eccles, 1944; Tomanek and Cooper, 1972; Baker, 1983), and histochemically (Engel et al., 1966; Talesara and Jasra, 1986). Vrbova (1963) has shown by EMG recordings that there was a dramatic decrease of postural activity in the soleus muscle, indicating a high degree of hypoactivity. Slow-twitch type I fibers have been found to be more susceptible to the effects of tenotomy t h a n the fast-twitch type I1 f i bers (Dias, 1979). Thus, while the experimental literature is replete with studies on tenotomized muscle, there is a paucity of information concerning the muscle’s motor endplate following tenotomy. Morphological examinations of the tenotomized endplate have been purely light microscopic in nature and the results contradictory. Osame et al. (1977) observed that the area of the soleplate of tenotomized rat muscles was larger than control muscles; in contrast, Dias (1979) observed that the soleplate area of tenotomized rabbit soleus was decreased in size compared with that of controls. Both studies were light microscopic in nature. Therefore, in light of the scarcity of data on the endplate following tenotomy, the present investigation was designed to assess qualitatively and quantitatively the fine structure of the neuromuscular junction and nerve terminal in tenotomized rat soleus muscle. MATERIALS AND METHODS

Six female Wistar rats (250-300 g) were divided into two equal groups. One group was anesthetized with sodium pentobarbital (36 mg/kg, i.p.) and chloral hydrate (160 mgikg). In each of the animals, a n incision along the posterior surface of the calf exposed the lower part of the triceps surae and all of the Achilles tendon down to the calcaneus. After freeing the Achilles ten(Q 1990 WILEY-LISS,

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don from surrounding tissue, i t and the plantaris tendon, which in the rat lies immediately medial to the true Achilles tendon, were cut about 5-7 mm proximal to their insertion into the calcaneus. The cut ends of the tendon were allowed to retract, and the skin was closed. The other group of animals served as controls. To break down any adhesions and prevent reattachment of the tendon in the tenotomized animals, the operated foot was dorsiflexed and plantarflexed a few times each day. After 2 weeks, the animals were anesthetized with ether and the soleus muscles from all animals were fixed in situ by 4% glutaraldehyde in phosphate buffer (0.1 M, pH 7.2) for 10-15 min and then removed. The muscles were then transferred to fresh cold 4% glutaraldehyde solution overnight, postfixed in 1% osmium tetroxide for 2 hr, dehydrated in graded alcohols, and embedded in Epon 812. The fixation technique was the same for both groups, so that any change brought about by the fixation was uniform for all muscles. The muscles were embedded flat and serially sectioned transversely a t 15 pm by a steel knife on a sliding microtome. The thick epon sections were cleared for light microscopy by curing a layer of Epon onto them within a sandwich of polystyrene film (Pachter et al., 1982) and the muscles surveyed by phase contrast for location of the endplate zones. Thick Epon sections containing endplates were mounted on a Beem capsule and ultrathin sections were cut, stained with lead citrate and uranyl acetate, and examined with a Zeiss EM-109 electron microscope. All endplates were photographed and analyzed

Received June 19, 1989; accepted December 8, 1989. Address reprint requests to B.R. Pachter, Department of Rehabilitation Medicine, New York University Medical Center, IRM RR-720, 400 East 34th Street, New York, NY 10016.

TENOTOMY EFFECTS ON MUSCLE ENDPLATE

Fig. I . Neuromuscular junction from control soleus muscle. A nerve terminal (NT) can be observed to lie within a cup-shaped depression of the muscle fiber surface. Contours of postsynaptic area (PS; clefts and folds) and of nerve terminal area are shown. x 15,000. Fig. 2. Neuromuscular junction from tenotomized soleus muscle.

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The postjunctional folds exhibit signs of degeneration (arrows). x 15,000. Fig. 3. Endplate from tenotomized muscle. Two large myelin-like figures (arrows) are shown in the junctional sarcoplasm. x 15,000.

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TABLE 1. Quantitative analvsis of endulate structures'

Control Tenotomized

Postsynaptic area (pm2)per Nerve terminal nerve terminal area (pm') 3.70 ? 0.25* 2.71 2 0.51** 2.98 ? 0.64

1.55 2 0.28

'Data are expressed as means & S.E. from a total of 63-66 endplates obtained from three animals. *Between control and tenotomized groups, F = 8.756, P < 0.004; intragroup difference, F = 2.312, P = nonsignificant. **Between control and tenotomized groups, F = 16.242, P < 0.001; intragroup difference, F = 1.170, P = nonsignificant.

a t a final magnification of x 15,000. No serial sections were taken. Thus all data are from different endplates. Electron micrographs representing motor endplates can be quantitatively analyzed according to stereological principles. Morphometric studies provide estimates of cell parameters. For each group of animals (three animals per group), morphometric analysis of a given endplate (21-22 endplates per animal) consisted of determining 1)the area of postsynaptic sarcoplasmic junctional folds and secondary clefts associated with a given nerve terminal (see Fig. 1)and 2) the nerve terminal area (see Fig. 1).The above areas were quantitatively assessed from electron micrographs (Engel and Santa, 1971) with a manually operated stylus point of a Numonics electron graphics digitizer having a builtin program for measuring areas. The statistical analysis of the EM data was based on a two-way repeated measures analysis of variance (ANOVA) that tested for differences between test conditions (control and tenotomized) and for within-group differences for each variable (postsynaptic area and nerve terminal area). The level of significance was established a t the 95% confidence interval ( P < 0.05). RESULTS

The ultrastructure of control rat soleus endplates is generally similar to that delineated by Santa and Engel (1973). Nerve terminals were observed to lie within a depression of the muscle fiber surface, which tends to be cup shaped (primary synaptic cleft). The muscle's sarcolemma in this region extended inward and formed a n elaborate system of both primary junctional folds and secondary clefts (Fig. 1). In tenotomized animals, the mean postsynaptic area of folds and clefts per nerve terminal was found to be significantly (P < 0.004) reduced as compared with controls (Table 1).Additionally, the mean nerve terminal area of tenotomized endplates was also significantly (P < 0.001) reduced when compared with control endplates (Table 1). The endplates from tenotomized muscles were found to exhibit a large number of ultrastructural alterations. The following changes in endplate structure were observed. 1) The junctional folds of some endplates exhibited degeneration (Fig. 2). 2) Myelin-like debris was observed in the subjunctional sarcoplasm (Fig. 3). 3) The Schwann cells often contained dense bodies (Fig. 4). 4) Large expanses of postjunctional folds with no nerve terminals were observed (Fig. 4).5) Several nerve terminal branches occurred within the same primary synaptic cleft and were separated from

one another by Schwann cell cytoplasm (Fig. 5 ) . 6 ) Several axons were wrapped by the same Schwann cell (Fig. 5). DISCUSSION

While the experimental literature is replete with studies on muscle following tenotomy, the present investigation to our knowledge is the only EM study to examine morphometrically and qualitatively the neuromuscular junction of tenotomized muscles. Our results demonstrate that tenotomy induces a number of alterations (both degenerative and regenerative) in the fine structure of soleus muscle endplates, indicating that neuromuscular junctions undergo structural remodelling in response to change in muscle activity. In the present study, the mean postsynaptic areas of folds and clefts per nerve terminal of tenotomized endplates were found to be significantly decreased a s compared with controls. Our electron microscopic morphometric data support the light microscopic measurements of Dias (1979), who observed t h a t the surface area of the motor endplates of rabbit soleus muscle was reduced after tenotomy. Various degenerative anomalies were observed in the tenotomized endplates. These changes consisted of degenerating postjunctional folds, whorled myelin-like structures in the subjunctional sarcoplasm as well as in Schwann cells, and regions of junctional folds unassociated with nerve terminals. In addition to the degenerative changes, the endplates from the tenotomized muscles also exhibited many reinnervational changes, often occurring on the same endplate. Regenerative changes included the presence of small nerve terminals isolated from one another by Schwann cell cytoplasm occupying the same primary synaptic cleft. Axonal sprouts were also seen enclosed in processes of Schwann cell cytoplasm and separated from the muscle's sarcolemma. These ultrastructural features are indicative of terminal axonal sprouting (Cardasis, 1983; Wernig and Herrera, 1987). Such terminal sprouting most probably serves to replace terminals as they are damaged or lost and thus acts to maintain the structural integrity of endplates. A number of studies have shown that reduction or abolition of muscle activity can also induce terminal sprouting (see Wernig and Herrera, 1987). In this regard, Vrbova (1963) and Karpati et al. (1972) showed by EMG recordings that there was a reduced motor unit activity in rabbit and r a t soleus muscle, respectively, following tenotomy, thus indicating a high degree of hypoactivity. Nerve sprouting has also been observed during aging and has been correlated to a decrease in muscle activity (Wernig and Herrera, 1987). In the present study, the mean nerve terminal area was also found to be reduced in endplates of tenotomized muscles a s compared with controls. Experimental tenotomy and limb immobilization in a shortened position are often studied as models of muscle disuse. Eisen et al. (1973) studied the effects of hypoactivity induced by skeletal fixation on the nerve fiber size in soleus muscle. They observed that such hypoactivity results in a significant reduction in the mean nerve fiber diameter of the soleus nerve. Additionally, To-

TENOTOMY EFFECTS ON MUSCLE ENDPLATE

Fig. 4. Tenotomized motor endplate exhibiting both degenerative and regenerative changes. A region of postjunctional folds unassociated with any terminal (thin arrow) is adjacent to a postsynaptic cleft containing several axonal sprouts (thick arrow). Dense bodies are apparent within the Schwann cell cytoplasm. X 15,000.

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Fig. 5. Neuromuscular junction from tenotomized muscle. Several nerve terminals (NT), which are separated from each other by Schwann cell cytoplasm, are shown within one primary synaptic cleft. In addition, several axon branches (arrows) are shown within a single Schwann cell. x 15,000.

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manek and Tipton (1967) found that tenectomy in the rat also resulted in a reduced nerve fiber diameter. In a tonic muscle like the soleus, the majority of the motor units are activated a s a function of postural maintenance. A tonic muscle is more dependent on neurotrophic andior neuroregulatory factors than is a phasic muscle (see Eisen et al., 1973). This greater dependence of muscle on nerve (or of nerve on muscle) could therefore account for the present results of decreased nerve terminal area as well as decreased postsynaptic area of folds and clefts with hypoactivity. Finally, the present results demonstrate that tenotomy induces both pre- and postsynaptic alterations in the neuromuscular junctional apparatus of soleus muscle fibers. Ultrastructural evidence of denervation-like changes and regeneration was observed to occur, thus suggesting that these endplates are in a state of synaptic remodelling. ACKNOWLEDGMENTS

The authors acknowledge the technical assistance of Ms. Barbara Zimmer. This study was supported by grant GO08300071 from the National Institute for Disability and Rehabilitation Research, United States Department of Education. LITERATURE CITED Baker, J.H. 1983 Segmental necrosis in tenotomized muscle fibers. Muscle Nerve, 6:29-39. Buller, A.J., and D.M. Lewis 1965 Some observations on the effects of tenotomy in the rabbit. J. Physiol., 178:326-342. Cardasis, C.A. 1983 Ultrastructural evidence of continued reorganization a t the aging (11-26 months) rat soleus neuromuscular junction. Anat. Rec., 207:399-415. Dias, P.L.R. 1979 Effects of tenotomy on the motor end plates of fast and slow twitch muscles of the rabbit. J . Anat., 129t399-404.

Eccles, J.C. 1944 Investigations on muscle atrophies arising from disuse and tenotomy. J . Physiol., 103t253-266. Eisen, A.A., S. Carpenter, G. Karpati, and A. Bellavance 1973 The effects of muscle hyper- and hypoactivity upon fibre diameters of intact and regenerating nerves. J . Neurol. Sci., 20:457-469. Engel, A.G., and T. Santa 1971 Histometric analysis of the ultrastructure of the neuromuscular junction in myasthenia gravis and in the myasthenic syndrome. Ann. N.Y. Acad. Sci., 183t46-63. Engel, W.K., M.H. Brooke, and P.G. Nelson 1966 Histochemical studies of denervated or tenotomised cat muscle. Illustrating difficulties in relating experimental animal conditions to human neuromuscular disease. Ann. N.Y. Acad. Sci., 138:160-185. Karpati, G., S. Carpenter, and A.A. Eisen 1972 Experimental corelike lesions and nemaline rods: A correlated morphological and physiological study. Arch. Neurol., 27:237-251. Osame, M., M. Kawabuchi, A. Igata, and H. Sugita 1977 Changes a t the neuromuscular junctions in the affected muscles of neostigmine-treated rats, tenotomized rats and a patient with nemaline myopathy. Acta Histochem. Cytochem., 10t70-80. Pachter, B.R., A. Eberstein, and J . Goodgold 1982 Electrical stimulation effect on denervated skeletal myofibers in rats: A light and electron microscopic study. Arch. Phys. Med. Rehab., 63: 427-430. Santa, T., and A.G. Engel 1973 Histometric analysis of neuromuscular junction ultrastructure in rat red, white, and intermediate fibers. In: New Developments in Electromyography and Clinical Neurophysiology. J.E. Desmedt, ed. Basel: Karger, pp. 41-54. Talesara, C.L., and P.K. Jasra 1986 Modifications in the histochemical and biochemical changes in tenotomized rat soleus by denervation. Pflugers Arch., 407t178-181. Tomanek, R.J., and R.R. Cooper 1972 Ultrastructural changes in tenotomized fast- and slow-twitch muscle fibers. J. Anat., 113: 409-424. Tomanek, R.J., and C.M. Tipton 1967 Influence of exercise and tenectomy on the morphology of a muscle nerve. Anat. Rec., 159:105112. Vrbova, G. 1962 Effect of tenotomy on the speed of contraction of fast and slow mammalian muscle. J . Physiol., 161t25-26P. Vrbova, G. 1963 Changes in motor reflexes produced by tenotomy. J . Physiol., I66t24 1-250. Wernig, A., and A.A. Herrera 1986 Sprouting and remodelling at the nerve-muscle junction. Prog. Neurobiol., 27:251-291.

Tenotomy-induced motor endplate alterations in rat soleus muscle.

The effects of tenotomy on the ultrastructure of rat soleus muscle motor endplates were examined both qualitatively and quantitatively. Rat soleus mus...
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