The recovery of long-term denervated rat muscles after Marcaine treatment and grafting

Abstract

Disruption of the nerve supply results in the rapid loss of mass and contractile force in skeletal muscles. These losses are reversible to a high degree in short-term denervated muscles with grafting and nerve implantation. However, return is much poorer in long-term denervated muscles. This study examined the basis for the differences in the recovery of non-denervated and 7-month denervated rat extensor digitorum longus (EDL) muscles after grafting and nerve implantation. We found that the level of recovery is related to the ability of muscle fibers to degenerate and regenerate after grafting. Fibres within long-term denervated muscles do not degenerate and regenerate as well as those within muscles which are not denervated prior to grafting. The functional recovery of the denervated muscles is significantly improved when their fibers are induced to degenerate with the myotoxic anesthetic, Marcaine. Degeneration of these fibers is followed by massive regeneration. The finding that denervated muscles are capable of being restored to a significant level by inducing regeneration may be useful in the clinical treatment of denervated muscles.

Introduction

Denervation results in a rapid and profound loss of mass- (Gutmann and Hnik, 1962; Gulati, 1990; Carlson et al., in press) and force-generating ability (Carlson et al., in press) in skeletal muscles. Short-term denervated muscles recover well after reinnervation, but after longer periods of denervation, variously estimated at 6 to 18 months in humans (Bateman, 1962; Anderl, 1977) and 4 to 7 months in rats (Carlson et al., in press), denervated muscles do not become completely restored even when motor nerves regenerate into the muscles.

In clinical practice, the strategy for returning function to a denervated region is to first allow spontaneous reinnervation of the denervated muscles and if this fails, to replace the atrophied tissue with new muscle tissue in the form of muscle grafts (Thompson, 1971; Harii, 1991; Frey and Giovanoli, 1995). Muscle transplantation to restore function, however, is not without problems (see Frey and Giovanoli, 1995). The transferred muscle may be too bulky, thus necessitating secondary reduction surgery, and the muscle may have a different fiber composition or angle of pennation from that of the original muscles. These factors affect not only the functional capacity of the transplanted muscles but also the cosmetic acceptability of the transfer, especially in the face.

Few attempts have been made to devise experimental means of restoring the denervated muscles themselves because of the assumption that these muscles are incapable of being restored after they atrophy (Thompson, 1971; Harii, 1991; Frey and Giovanoli, 1995). Gulati and Carlson have used grafting to test various aspects of recovery in denervated muscles. Gulati (1990)found that the cross-sectional area of denervated muscle fibers in rats increased after the muscles were grafted into innervated sites. Carlson et al., in pressfound that grafting improved the functional capacity of denervated rat muscles. However, functional return in long-term denervated muscles was poor compared with that in muscles denervated for short periods of time prior to grafting (Carlson and Faulkner, 1993; Carlson et al., in press).

There are several possible explanations for the poor recovery of long-term denervated muscles after they are grafted. One is that these muscles do not become reinnervated as well as normal or short-term denervated muscles after grafting. Nerves may not be able to reach motor endplates due to the fibrosis that takes place within the synaptic clefts (Satio and Zacks, 1969) and interstitial regions (Schmalbruch et al., 1991) during denervation. Alternatively, denervated muscle fibers may become reinnervated after grafting but the number of fibers may be much smaller than that in normal muscles because of muscle fiber attrition (Anzil and Wernig, 1989; Schmalbruch et al., 1991) or loss of satellite cells (Lu and Carlson, 1993; Viguie and Carlson, 1994).

One of the more provocative explanations for the poor recovery of long-term denervated muscles after they are grafted is that they do not degenerate and regenerate as well as grafts of normal or short-term denervated muscles. Carlson (1976)found that 15–20% of the muscle fibers in grafts of 14 day denervated muscles survive the grafting process compared with 2–5% in grafts of normal muscles (Carlson, 1976). The present study found that 80–95% of fibers in 7-month denervated extensor digitorum longus (EDL) muscles survived the grafting process and, therefore, did not regenerate when grafted.

The primary question in this study was whether or not induced degeneration and regeneration with grafting could improve the functional capacity of denervated muscles over grafting alone. To answer this, denervated and control, non-denervated muscles were treated with Marcaine before grafting. The EDL muscle was chosen for this experiment because its long tendons at both the origin and insertion make it ideal for grafting and for performing muscle physiology. Marcaine, a widely used local anesthetic (Vandermeulen et al., 1995; Wong et al., 1995), is extremely myotoxic (Carlson, 1976; Grim et al., 1988; Itagaki et al., 1995). Treatment with Marcaine causes damage to muscle fibers, myonuclei, and capillaries with no apparent damage to the satellite cells of the muscle (Hall-Craggs, 1980). Marcaine-induced necrosis is followed by muscle fiber regeneration (Benoit and Belt, 1970; Carlson et al., 1992). This study found that, without Marcaine treatment, few fibers in 7-month denervated EDL muscles regenerated after grafting. With Marcaine treatment, virtually fibers degenerated and then regenerated. The force generating capacity of the Marcaine-treated denervated muscle grafts was significantly higher than that of non-treated grafts.