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Introduction
Severe injuries in skeletal muscle result in muscle weakness that delays recovery, which exposes patients to neuromuscular complications that increase hospital stay and mortality (1). In muscular dystrophy, an imbalance between muscle damage and muscle repair through stem-cell mediated regeneration is thought to contribute to progressive decline in muscle function (2).
Microcurrent therapy (MCT) is a novel treatment for soft tissue injury, where the MCT showed better therapeutic compliance than traditional therapy (3). Regeneration therapy device which produces a microcurrent range was used for treatment of chronic wounds and ulcers associated with chronic diseases as diabetes mellitus (4). Recently, high therapeutic efficiency of cell-stimulation with low-intensity electric current was proved in inflammatory conditions (5).
Satellite cells (Scs) have been distinguished by immunostaining and expression of CD34 surface receptors including (6). These cells are activated in response to injury and undergo cell division (7). Recently, it was confirmed that the regenerative capacity of skeletal muscle tissue resides in satellite cells, the quiescent adult stem cells (8).
The present work aimed at investigating the relation between microcurrent therapy and local stem cells in regeneration of induced skeletal muscle injury in albino rat.
Materials and Methods
The study was conducted at the Animal House of Kasr El Aini, Faculty of Medicine, Cairo University, according to the guide for the care and use of laboratory animals.
Experimental design
Twenty six male albino rats weighing 150∼200 g were housed in a temperature and light-controlled room, with free access to food and water. The animals were classified into the following groups, which were kept in separate cages:
Sham group (S Group): Included 6 rats receiving anaesthesia but not undergoing injury of gastrocnemius-soleus muscle. S1 subgroup 3 rats sacrificed 2 weeks later. S2 subgroup 3 rats sacrificed 4 weeks later.
Injury group (I Group): Included 10 rats subjected to soleus muscle injury without any treatment procedures, other than the routine care of the wound. Betadine was applied to the wound site and rinsing with normal saline was performed at the site of injury on daily base. To induce anesthesia, ketamine hydrochloride (Ketalar, (Parke Davis Barcelona, Spain) (35 mg/kg) was injected into the gluteus maximus muscle of the animal. Using aseptic techniques, a 3 cm longitudinal skin incision was made along the leg. Then an incision (1 cm in length) was made perpendicular to the orientation of the muscle. The fascia and skin were sutured (9). Daily aseptic dressing was applied on the wound. The animals were subdivided into 2 subgroups:
Microcurrent group (M Group): Included 10 rats subjected to soleus muscle injury by the same protocol as in I group. Micro-current electric stimulator, model EMSI-4250 (made in Taiwan) was used for the treatment. Intensity was 100 microampere (10) and the frequency 10 Hz. Clip electrodes were used and microcurrent therapy was applied 3 sessions/week (11). The negative lead (1.0×1.0 cm) was placed over the muscle injury site, while the positive lead was placed proximally on the thigh region of the same side. The microcurrent was applied for 20 minutes in each session (12). The animals were subdivided into 2 subgroups:
The animals were sacrificed by a lethal dose of ether. Soleus muscle was exposed and muscle specimens were placed in 10% formol saline. 5 microns thick sections were prepared and subjected to the following studies.
Hematoxylin and eosin (13)
Immunohistochemical (IHC) Study
Anti-alpha smooth muscle actin (α-SMA) immuno-staining: The marker for this antibody stains smooth muscle cells in vessel walls, gut wall, myometrium and myoepithelial cells. Anti-alpha smooth muscle actin antibody (Rabbit polyclonal antibody) (ab5694) was used at a concentration 0.5∼2 μg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol. Glioma tissue sections were used as positive control specimens. Cellular localization is the cytoplasm. On the other hand, one of the muscle sections was used as a negative control by passing the step of applying the primary antibody (14).
CD34 immunostaining is the marker for hematopoietic progenitor cells, small vessel endothelium (15) and satellite cells of skeletal muscle (16) of a variety of tissues. CD34 goat polyclonal Ab (Sigma-Aldrich Chemie Corporation laboratories, Germany, catalogue ID SAB4300690). The sections were treated with CD34, at 5∼15 μg/ml ready to use at room temperature. Cellular localization is the cell membrane. Tonsil sections were used as positive control specimens. On the other hand, one of the muscle sections was used as a negative control by passing the step of applying the primary antibody.
Morphometric study
Using Leica Qwin 500 LTD computer assisted image analysis system (Cambridge, United Kingdom), the area of atypical muscle fibers was estimated in 10 low power fields (LPF) in control and experimental groups using interactive measurements menu. The area% of α SMA positive (+ve) cells and CD34 +ve cells were measured in 10 high power fields using binary mode.
Statistical analysis
Quantitative data were summarized as means and standard deviations and compared using one-way analysis-of variance (ANOVA). Any significant ANOVA was followed by Bonferroni post-hoc test to detect which pairs of groups caused the significant difference. p-values <0.05 were considered statistically significant. Calculations were made on SPSS 9,0 software (17).
Results
Histological results
Skeletal muscle sections of S group showed longitudinal muscle fibers (Fig. 1A) that exhibited oval pale nuclei and transverse striations in the sarcoplasm (Fig. 1B). In subgroup I1 (Sacrifice 2weeks following muscle injury) the injured area demonstrated atypical fibers widely separated by infiltrating cells. Most fibers contained dark nuclei. Distended capillaries were noted (Fig. 2A). The fibers revealed partial loss of striations and some fibers recruited strong acidophilic sarcoplasm with focal vacuolations on close observation (Fig. 2B). In subgroup I2 (Sacrifice 4 weeks following muscle injury), atypical fibers appeared separated by few infiltrating cells. Some fibers exhibited dark nuclei (Fig. 3A). Partial loss of striations was found in some fibers on close observation (Fig. 3B).
In subgroup M1 (Sacrifice 2weeks following injury and MCT), few atypical fibers were found. Other fibers exhibited multiple flat nuclei, some of these nuclei were centrally located (Fig. 4A). Striations appeared in some areas of the sarcoplasm on close observation (Fig. 4B). In subgroup M2 (Sacrifice 3 weeks following injury and MCT), few atypical fibers were seen surrounded by multiple typical fibers, some of which were observed with centrally located nuclei (Fig. 5A). Multiple fibers recruited striations in most areas of the sarcoplasm on close observation (Fig. 5B).
Immunohistochemical results
Skeletal muscle sections of control rats demonstrated few alpha smooth muscle actin (α SMA) +ve spindle cells in between the muscle fibers (Fig. 6A). In subgroup I1 +ve spindle cells were found in the lining of blood vessels existing between atypical fibers (Fig. 6B). In subgroup I2 few +ve spindle cells were detected among some fibers with partial loss of striations (Fig. 6C). In subgroup M1 some +ve spindle cells were evident among some muscle fibers with centrally located nuclei (Fig. 6D). Subgroup M2 showed multiple +ve spindle cells among muscle fibers with striations in most areas of the sarcoplasm (Fig. 6E).
Skeletal muscle sections of control rats revealed few CD34 +ve spindle cells at the periphery of the fibers (Fig. 7A). In subgroup I1, occasional +ve spindle and oval cells appeared around atypical fibers (Fig. 7B). In subgroup I2, some fields demonstrated few +ve spindle cells among muscle fibers (Fig. 7C). In subgroup M1 multiple +ve spindle cells were obvious at the periphery of muscle fibers (Fig. 7D). In subgroup M2 less numerous +ve spindle cells appeared at the periphery of the fibers (Fig. 7E).
Morphometric results
A significant (p<0.05) decrease in the mean area of atypical fibers was estimated in subgroup M1 compared to the injury subgroups I1 and I2. In addition, a significant (p<0.05) decrease was found in subgroup M2 compared to M1 (Table 1).
A significant (p<0.05) increase in the mean area% of alpha SMA+ve immunostaining was detected in subgroup M1 compared to the injury subgroups I1 and I2. In addition, a significant (p<0.05) increase was found in subgroup M2 compared to M1 (Table 1).
A significant (p<0.05) increase in the mean area% of CD34+ve immunostaining was estimated in subgroup M2 compared to the injury subgroups I1 and I2. In addition, a significant (p<0.05) increase was found in subgroup M1 compared to M2 (Table 1).
Discussion
The current study demonstrated modulating effect of MCT on induced skeletal musle injury, which was associated with satellite cell existence in albino rat. This was evidenced by histological, immunohistochemical and morphometric studies.
In subgroup I1 (Sacrifice 2 weeks following muscle injury) the injured area demonstrated atypical fibers widely separated by infiltrating cells and distended capillaries. In accordance, it was stated that thermal injury of skeletal muscle of rat revealed greater interfiber distances and substantially increased amount of connective tissue (1). It was added that within the first week of traumatic muscle injury, granulated tissue and acute inflammation were found (9).
Most fibers contained dark nuclei, indicating apoptosis. The fibers revealed partial loss of striations and some fibers recruited strong acidophilic sarcoplasm with focal vacuolations on close observation. Crawford et al. (18) related injury of DNA to reactive oxygen species. In addition, similar degenerative cytoplasmic changes were described following muscle injury (2).
In subgroup I2 (Sacrifice 4 weeks following muscle injury), atypical fibers appeared separated by few infiltrating cells. Some fibers exhibited dark nuclei and partial loss of striations. The previous results indicated mild regression of degenerative changes. Concomitantly, it was documented that mononuclear cells can develop into a variety of different muscle cell lineages including myoblasts, satellite cells, and muscle derived stem cells (19).
In subgroup M1 (Sacrifice 2 weeks following injury and MCT), few atypical fibers were found. Other fibers exhibited multiple flat nuclei, some of these nuclei were centrally located. Striations appeared in some areas of the sarcoplasm. It was postulated that MCT limits muscle damage and inflammation, known as delayed onset muscle soreness in cases of injury (12). It was reported that neuro-muscular conductivity improved in response to MCT following muscle injury (20). It was recorded that Scs are the adult skeletal muscle stem cells that are quiescent, but during muscle regeneration proliferate and generate distinct daughter cells by segregating template DNA strands to the stem cell (21).
In subgroup M2 (Sacrifice 4 weeks following injury and MCT), few atypical fibers were seen surrounded by multiple typical fibers, some of which were observed with centrally located nuclei. Multiple fibers recruited striations in most areas of the sarcoplasm. These findings indicated progressive improvement of structural changes by MCT, proved by a significant decrease in mean area of atypical fibers. It was mentioned that Scs are located beneath the basal lamina of adult muscle fibers and are normally arrested in G0 of the cell cycle (22). They can be activated in response to stimuli, initiating a regenerative process, restoring the normal architecture of muscle within 2 weeks. This was confirmed by Francois et al. (23) who stated that survival of the satellite cells is a critical requirement for efficient muscle reconstitution following injury.
The regeneration capacity was assessed in the present work using α SMA. In subgroup I2 few +ve spindle cells were detected among some fibers with partial loss of striations. In subgroup M1 the +ve spindle cells were more evident and in subgroup M2 the +ve cells became multiple among the muscle fibers. This was confirmed by a significant increase in the mean area% of α SMA +ve cells. It was postulated that activation of muscle precursor cells is an important determinant for the efficiency of muscle regeneration. It was added that the main source of muscle precursor cells are satellite cells, which proliferate and migrate to the injured site (24).
In subgroup I1, occasional CD34+ve spindle and oval cells appeared around atypical fibers. Subgroup I2 demonstrated few +ve spindle cells among muscle fibers. On the other hand, in subgroup M1 multiple +ve spindle cells were obvious at the periphery of muscle fibers. While in subgroup M2 the +ve spindle cells appeared less. This was evidenced by a significant increase in the mean area% of CD34 +ve cells in microcurrent group. It was documented that Scs within the adult skeletal muscle are an enriched population of CD34+ve cells (16). It was proved that Scs are responsible for the regenerative potential of skeletal muscle (25). Alfaro et al. (26) considered that CD34 beyond being a stem cell marker, may play an important function in modulating stem cell activity. On the other hand, Ieronimakis et al. (6) noted a minority of satellite cells lacking CD34 to be described. Local SCs were documented as a promising treatment option for various diseases (27). In addition, histological examination suggested the therapeutic potential of local pluripotent cells in the treatment of ischemic diseases (28).
It can be commented that a definite therapeutic effect of the microcurrent was found on induced skeletal muscle injury. This effect was proved to be related to satellite cell activation.
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