Skeletal muscle injury is one of the most common injuries in sports-related traumas.
The standard therapeutic methods for treating skeletal muscle injuries such as muscle fragmentation generally use conservative therapy that mainly involves resting of the damaged site. However, recovery can take over several months depending on the extent of skeletal muscle damage (Garret, 1990; Huard et al., 2002; Lehto and Jarvinen, 1991; Peterson and Renstrom, 2001). This is a serious problem for athletes eager for an early return. Even though significant clinical efforts are being made to improve the current treatment of skeletal muscle injuries, it is not easy to establish the treatment of accelerated recovery.
The regenerative process of injured skeletal muscle is highly coordinated and complex phenomena. Skeletal muscle-specific stem cells, so-called muscle satellite cells, are well known to be responsible for repair and regeneration of adult skeletal muscle tissues (Best and Hunter, 2000; Grounds, 1999; Hill et al., 2003; Saito and Nonaka, 1994; Seale and Rudnicki, 2000). Injured skeletal muscles in genetically developed mice lacking muscle satellite cells exhibited a severe depression of regenerative potential (Seale et al., 2000a). However, the regulatory mechanisms for the regenerative potential of injured skeletal muscle are still unclear. It has been reported that the proliferative stimuli for satellite cells is stimulated by extracellular stimuli, such as mechanical (Morioka et al., 2008) and heat (Kojima et al., 2007) stresses, resulting that such stresses facilitated the regeneration of injured rat tibialis anterior and mouse soleus muscle, respectively. On the other hand, unloading reportedly impairs the regenerative potential of atrophied soleus muscle (Matsuba et al., 2009). These observations suggest that the proliferative potential of injured skeletal muscle might be sensitive to extracellular stimuli.
Microcurrent electrical neuromuscular stimulation (MENS) was developed as a physical therapy modality delivering current in the microampere range. It has been reported that MENS has several physiological effects such as pain relief (Larner and Kirsch, 1981) and facilitation of tissue repair including tendon injuries (Nessler and Mass, 1987; Owoeye et al., 1987), skin ulcers (Gault and Gatens, 1976; Wolcott et al., 1969), wounds (Byl et al., 1994; Huckfeldt et al., 2007), bedsores (Carley and Wainpel, 1985) and ligament injuries (Miyazaki et al., 2007). Recently, we have confirmed that the regrowth of unloading-associated atrophied mouse soleus muscle is stimulated by MENS (Ohno et al., 2013). Previous studies (Curtis et al., 2010; Lambert et al., 2002) have suggested that MENS facilitates a repair of injured skeletal muscle and shortens the recovery period. However, it is still unclear whether MENS has stimulating effects on activation of regenerative potential in injured skeletal muscle.
The purpose of this study was to investigate the effect of MENS on the regeneration process of injured skeletal muscle and to investigate whether satellite cells in injured skeletal muscle are activated by MENS. Evidences from this study suggest that MENS shortens the recovery period through the activation of satellite cells in injured skeletal muscle.
Animals and grouping
All experimental procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health (Bethesda, MD) and were approved by the Animal Use Committee of Toyohashi SOZO University. All procedures adhered to the American College of Sports Medicine animal standards.
Male C57BL/6J mice, aged 7 weeks old, were used (n = 30). Mice were randomly divided into two groups: (1) cardiotoxin (CTX)-injected (CX, n = 15) and (2) CTX-injected with MENS treatment (MX, n = 15) groups. Five mice of both groups were housed in a home cage (20 x 31 cm and 13.5 cm height) in a clean animal room controlled at approximately 23[degrees]C and at 55% humidity with a 12/12 hours light-dark cycle. Solid diet and water were provided ad libitum.
Initiation of necrosis-regeneration cycle
In mice of both groups, 0.1 ml CTX (10 [micro]M in physiological saline (PS); Sigma, St.Louis, MO, USA) of Naja naja atra venom was injected into the proximal, middle and distal part of the left tibialis anterior (TA) muscle, using a 27-gauge needle under anesthesia with sodium pentobarbital (50 mg x [kg.sup.-1] body weight, i.p.). Injection of CTX was performed carefully to avoid the damage in the nerves, blood vessels (Conteaux et al., 1988; Fletcher and Jiang, 1993). This treatment causes the initiation of necrosis-regeneration cycle (Conteaux et al., 1988; Fletcher and Jiang, 1993), and used as a muscle injury model in previous studies (Kojima et al., 2007, Morioka et al., 2008).
Forty eight hours after the CTX-injection, the left hindlimb of mice in MX group were treated with MENS (10 [micro]A, 0.3 Hz, 250 msec) by using an electical stimulator (Trio300, Ito Co., Ltd., Tokyo) for 60 min a day and 3 days a week for 3 weeks under anesthesia with i.p. injection of sodium pentobarbital (50 mg x [kg.sup.-1]). Mice in CX group were also anesthetized for 60 min without MENS treatment. Before MENS treatment, the epilation of mouse hindlimb was performed using a commercial hair remover for human. Then, two electrodes were placed on the distal anterior side of the knee joint and the proximal anterior side of ankle joint, respectively. In the present study, no muscle contraction in hindlimb of mice was observed during MENS. The condition for MENS was set by accounting the body size of the experimental animal and the duration of MENS treatment compared with humans which are clinically treated with MENS (Morinaga, 2007).
TA muscles of left hindlimb in both groups were dissected 1, 2 and 3 weeks after the CTX-injection under anethsesia. TA muscles was trimmed of excess fat and connective tissues, weighted, frozen in liquid nitrogen, and stored at -80[degrees]C until analyses.
Histochemical and immunohistochemical analysis
TA muscles were cross-sectionally cut into halves at the middle of the long axis. Serial transverse cryosections (7 pm thick) of the distal region of frozen TA muscles cut at -20[degrees]C and mounted glass slides. The sections were air-dried and stained to evaluate the pathological stages by using hematoxylin and eosin (H&E) and the profiles of Pax7-positive nuclei by using the standard immunohistochemical technique (Kojima et al., 2007), respectively. General pathological observations including centrally located myofiber nuclei were based on H&E staining. The cross-sectional area (CSA) of muscle fibers was also analyzed.
Briefly, monoclonal anti-Pax7 antibody (Developmental Studies Hybridoma Bank, Iowa, IA, USA) was used for the detection of muscle satellite cells (Asakura et al., 2002; Hawke and Garry, 2001; Morgan and Partridge, 2003; Seale et al., 2000b; 2001). Cross sections were fixed with...