Original Article J. Phys. Ther. Sci. 14: 21–26, 2002 The Effect of Denervation and Subsequent Reinnervation on the Morphology of Rat Soleus Muscles

HARUTOSHI SAKAKIMA, PT, MS1), YOSHIHIRO YOSHIDA, MD1), NORIO MORIMOTO, MD1), KIYOHIRO SAKAE, MD1) 1)School of Health Sciences, Faculty of Medicine, Kagoshima University: 8–35–1 Sakuragaoka, Kagoshima 890-8506, Japan. TEL +81 99-275-6801, FAX +81 99-275-6804, E-mail [email protected]

Abstract. To investigate the effect of denervation and subsequent reinnervation on skeletal muscle, a histochemical study was performed on the soleus muscles of rats. Partial denervation was carried out by freezing the sciatic nerve locally, and the change in the nerve and the soleus muscles was examined for 5 weeks. The muscle fiber cross-sectional area of the denervated soleus muscles progressively declined to a minimum 2 weeks after the injury (type I fibers, 1209.1 ± 248.3 µm2; type II fibers, 802.4 ± 126.8 µm2) and began to reverse the decline at 3 weeks. The type II fiber ratios to total fiber of the denervated sides were consistently higher than the control levels, and muscle fibers stained in both acid preincubation and alkaline preincubation were observed. The proportion of type II fibers in the soleus muscles showed an increase and consequently a decrease with a short delay in response to denervation and consequent reinnervation. These data suggest that denervation elicits an alteration in fiber type composition and a reduction in fiber size. The increase of type II fibers seemed to occur in hybrid fibers containing both myosin heavy chains I and II at varying ratios in the same fibers. The reinnervation took the crucial role of recovering from atrophy and composing the integrity of the soleus muscles. However, the ability to generate muscle tension needs a much longer time to recover. This suggests a need to investigate interventions to facilitate the functional recovery of partially-denervated muscle. Key words: Partial denervation, Muscle fiber type, Soleus muscle atrophy.

(This article was submitted Oct. 9, 2001, and was accepted Nov. 12, 2001)

INTRODUCTION nerve injury, as nerve fibers proximal to the injury send out new sprouts that cross the lesion and Skeletal muscle atrophy occurs due to prolonged eventually reestablish their original connections. bed rest, plaster cast immobilization, tenotomy and One objective of rehabilitation treatment is to denervation. Among these conditions, denervation prevent the muscular atrophy associated with the produces the most serious atrophy1, 2), and results in degeneration of nerves. It is important for the profound alterations in the morphology of skeletal physical therapist to know the change in the muscle. These alterations include changes in fiber partially-denervated muscles, when physiotherapy type composition and fiber size3). is introduced. Clinically, peripheral nerve injury often occurs There are many reports on denervation of skeletal due to musculoskeletal injuries and compression by muscle. Most investigations have focused on the cast immobilization. The peripheral nerve system is effects of denervation, while relatively few studies capable of rapid and extensive reconstruction after have dealt with the regenerative processes. Bodine- 22 J. Phys. Ther. Sci. Vol. 14, No. 1, 2002

Fowler et al.4) observed the time course of muscle glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) atrophy and recovery after injuring the sciatic nerve and embedded in epoxy resin. of a rat with phenol, but the denervation was incomplete. It is reported that long-term denervated Histological and histochemical analysis muscles recover poorly, perhaps due to the The soleus muscles were mounted vertically on a deposition of interstitial collagen5). However, not cork plate in tragaganth gum jelly of appropriate enough attention has been paid to the time course of softness6) to obtain cross-sections. The mounted alterations in muscles after reinnervation. muscle was then frozen by immersion in isopentane Moreover, the change in muscle fiber ratio after solution cooled in liquid nitrogen. Transverse denervation remains controversial6–8). Therefore, sections (10 µm) were cut in a cryostat cooled to the aim of this study was basic research on the –20°C, and stained with hematoxylin and eosin for partially-denervated muscle. In this study, a frozen general observation. Cryosections were also sciatic nerve was induced in rats as an injury and the stained for the myosin adenosine triphosphatase resultant degenerative and regenerative changes in (ATPase, pH 10.5, 4.3) reaction according to Guth the soleus muscles were observed. and Samaha9), and then the muscle fibers were classified into type I or II fibers. The whole cross MATERIALS AND METHODS section of each soleus muscle stained by ATPase was photographed at a magnification of 20 for fiber- Animals and experimental protocols type composition. The central region of the soleus Thirty-six 8-week-old female Wistar rats muscles was photographed at a magnification of 50 weighting 170–188 g were used in this study. The and all the muscle fibers delineated by entire fiber rats were anesthetized by an intraperitoneal boundaries were measured for the cross-sectional injection of sodium pentobarbital (50 mg/kg). The areas. A random sample of 100–110 fibers from skin covering the right buttock was cut and the right each soleus muscle was analyzed. The proportion sciatic nerve was isolated. The nerve was frozen of the type II fiber area was expressed as a percent and thawed several times by contact with a stainless of the total fiber. The sciatic nerves were stained spatula 3 mm in diameter cooled by liquid nitrogen. with 0.5% toluidine blue in 0.5% borate and Care was taken not to injure other tissues. The observed by conventional light microscope. A nerve became white when frozen, and the proximal Power Macintosh 8,500 computer was employed margin of the frozen portion was loosely tied with a with NIH Image version 1.61 software (developed white thread for marking. The contralateral at the US National Institutes of Health and available hindlimbs were untreated and served as a control. on the Internet at http://rsp.info.nih.gov/nih-image/). Six normal 8-week-old rats were also used as controls, in particular to survey the ratio of the type Data analysis II to total fiber areas and numbers in the normal Statistical analysis were performed with a soleus muscles. Food and water were supplied ad Macintosh computer using the StatView version 4.5 libitum. The degree of paralysis in the hindlimbs of software (StatView). One-way analysis of variance the treated rats was checked every day. The animals (ANOVA) was used and when a significant F ratio were euthanatized via the inhalation of an overdose was found, post-hoc Fisher’s Protected Least of diethyl ether at 1, 2, 3, 4 or 5 weeks (6 rats in each Significant Differences (PLSD) test was performed group) after the nerve freezing. The soleus muscles on each variable. Significance was set at P<0.05. in both legs and the accompanying sciatic nerves of the frozen sides were removed en bloc. The RESULTS distance between the proximal margin of the lesion and the nerve entrance into the soleus muscle was After the nerve freezing, the rats were ambulatory measured. The soleus muscles were quickly and dragged the foot of the frozen side. A loss of removed, cleaned of fat and connective tissues and active movement on the ankle and toe joints was quickly frozen in isopentane chilled with liquid observed. The paralysis was alleviated and nitrogen. The whole muscles were then stored at voluntary extension of digits was noticed –70°C until analysis. The distal portion of the approximately 3 weeks after the operation. sciatic nerve to the lesion was immersed in 3% The distance between the proximal margin of the 23

Fig. 1. Cross section of sciatic nerve after freezing, stained by 0.5% toluidin blue in 0.5% borate. Bar=50 µm; a, normal sciatic nerve, shows numerous instances of ovoid and ellipsoid myelin surrounding light areas of axons; b, after 2 weeks, most myelin and axons were degenerated; c, after 3 weeks, numerous thin formations of myelin observed with small light centers inside.

Table 1. Total cross-sectional areas of soleus muscle type I and II fibers before and 1 to 5 weeks after nerve freezing

Groups Before (Number of freezing 1 week 2 weeks 3 weeks 4 weeks 5 weeks muscles) Experimental side (6) Type I 1442.4 ± 123.6*† 1209.1 ± 248.3*† 1789.9 ± 348.7* 1953.4 ± 235.2* 2122.3 ± 348.7* Type II 1190.5 ± 245.5† 802.4 ± 126.8*† 1224.6 ± 400.3*† 1276.8 ± 287.1* 1327.4 ± 358.7* Contralateral side (6) Type I 2361.8 ± 516.1 2453.6 ± 441.2 2782.3 ± 434.8 2873.0 ± 406.2† 2943.3 ± 383.7† 3102.0 ± 368.8† Type II 1711.2 ± 393.6 1609.7 ± 371.9 1814.1 ± 412.9 2031.8 ± 238.6 2551.6 ± 502.9† 2655.7 ± 478.7† Values are mean ± SD (µm2). *P<0.05 (compared with that of contralateral side), †P<0.05 (compared with that before freezing).

lesion and the entrance of the sciatic nerve into the Changes in the cross-sectional areas of each fiber soleus muscle was 20.1 ± 1.7 mm (mean ± SD). type in the soleus muscles The changes in each fiber cross-sectional area are Changes in the sciatic nerve shown in Table 1. The cross-sectional areas of The distal portion of the nerve to the frozen lesion muscle fiber had significantly decreased 2 weeks was uniformly damaged and swollen. One week after the injury compared with before freezing, and after the injury, most myelin had clearly began to increase at 3 weeks. Two weeks after the degenerated and the centers of myelin where axons injury, the relative cross-sectional area in the had been located were densely stained. These denervated soleus muscles compared with the findings were found up to 2 weeks after the injury contralateral counterparts was 50.3 ± 9.2% in type I (Fig. 1). A considerable number of macrophages, fibers and 39.0 ± 8.5% in type II fibers. probably containing myelin debris, were observed. Central light areas indicated few axons. In contrast, Changes in muscle fiber type ratio 3 weeks after the injury, numerous thin formations The ratios of type II to total fiber numbers in the of myelin emerged, surrounding the small light denervated soleus muscles were significantly higher centers inside (Fig. 1). than those in the normal rats and the contralateral uninjured side in each period (Table 2). Type II 24 J. Phys. Ther. Sci. Vol. 14, No. 1, 2002

Table 2. The ratio of type II fiber to total fiber number (% of fiber) Groups Before 1 week 2 weeks 3 weeks 4 weeks 5 weeks (Number of muscles) freezing Experimental side (6) 26.7 ± 8.4*† 36.4 ± 8.8*† 46.2 ± 8.7*† 33.4 ± 4.3*† 19.7 ± 4.5 Contralateral side (6) 11.7 ± 6.8 14.6 ± 3.9 11.7 ± 3.2 14.9 ± 3.2 10.6 ± 5.3 10.7 ± 3.3 Values are mean ± SD. *P<0.05 (compared with Contralateral side), †P<0.05 (compared with before freezing).

Fig. 2. Cross section of the soleus muscle at 3 weeks after nerve freezing, stained by alkaline pretreated ATPase reaction. Bar=100 µm; a, contralateral soleus muscle, darkly stained type II fibers are small in number; b, experimental side of same animal shown in Fig. 2a. Fiber cross-sectional areas are decreased in comparison with the contralateral side. Darkly stained type II fibers are significantly numerous. Note that muscle fibers with various intermediate densities are classified as type II fibers in this study. fibers increased and reached a maximum at 3 weeks showed a progressive decrease with time after after denervation and gradually decreased denervation4, 12). The denervation produced serious thereafter, but were still higher at 5 weeks (Fig. 2). atrophy compared to immobilization due to muscle disuse caused by paralysis and tenotomy in the DISCUSSION deprivation of trophic substances1, 13, 14). Type II muscle fibers are generally reported to undergo Freezing was chosen as the method of preferential atrophy after total denervation5, 6, 14) denervation since this method uniformly and and selective motor nerve cutting15). In contrast, definitively damages nerve fibers with Bodine-Fowler et al.4) stated that the slow fibers reinnervation more likely than in other procedures (type I) of rat soleus muscles showed more atrophy such as nerve crushing. The fiber cross-sectional than the fast fibers (type II). Tomanek and Lund16) areas were regenerated 2 to 3 weeks after freezing. and Herbison et al.1) found that type I fiber atrophy It was roughly estimated that it took approximately was equal to type II fiber atrophy. The 3 weeks for the sciatic nerve to contact the soleus morphological responses of the muscle to the muscles over approximately 20 mm, which was denervation were fiber type specific, although both compatible with data previously reported10–12). type I and II fibers of the denervated soleus muscles The cross-sectional area of type I and II fibers elicited marked atrophy. The atrophy was 25 significantly greater in type II fibers. composition. The changes evaluated in this study Following reinnervation, muscle cross-sectional are to a great extent reversible after short-term areas steadily increased. The reinnervation took the denervation, although some alterations such as fiber crucial role of recovering from the atrophy and size, fiber type ratio and contractile function may composing the integrity of the soleus muscles. need more time to approach normal values. 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