Severe injuries to the skeletal muscle result in muscle weakness, which delays recovery, exposes patients to neuromuscular complications that increase hospital stay, and increase the probability of 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 method for soft-tissue injury; MCT showed better therapeutic compliance compared with traditional therapy 3. A regeneration therapy device that produces a microcurrent range was used for treatment of chronic wounds and ulcers associated with chronic diseases such as diabetes mellitus 4. Recently, cell stimulation with a low-intensity electric current was proved to have high therapeutic efficiency in inflammatory conditions 5.
Satellite cells (Scs) have been distinguished by immunostaining and expression of CD34 surface receptors 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 Scs, the quiescent adult stem cells 8.
The present work aimed at investigating the possible relation between MCT and Scs in regeneration of induced skeletal muscle injury in albino rats.
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.
Twenty-four male albino rats weighing 150–200 g were housed in a room under controlled conditions of temperature and light with free access to food and water. The animals were classified into the following groups and kept in separate cages:
Control group (group I)
This group included four rats not subjected to injury of the gastrocnemius–soleus muscle.
Experimental group (group II)
This group included 20 rats and was subdivided into two subgroups: Injury subgroup (SubgroupIIa): 10 rats subjected to gastrocnemius-soleus muscle injury. without any treatment procedures, other than the routine care of the wound. 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 3cm longitudinal skin incision was made along the leg. Then an incision (1cm in length) was made perpendicular to the orientation of the muscle. The fascia and skin were sutured 9. Betadine was applied to the wound site and rinsing with normal saline was performed at the site of injury on daily base. Daily aseptic dressing was applied on the wound. The animals were subdivided into two subgroups:
- Subgroup IIa1: this group comprised five rats sacrificed 1 week after the day of injury.
- Subgroup IIa2: this group comprised five rats sacrificed 3 weeks after the day of injury.
Injury and microcurrent therapy subgroup (subgroup IIb)
Subgroup IIb included 10 rats subjected to gastrocnemius–soleus muscle injury by the same protocol as in subgroup IIa. A microcurrent electric stimulator, model EMSI-4250, Electrostim Medical Services Inc. (Taiwan), was used for the treatment. Intensity was 100 microamperes 10 and the frequency was 10 Hz. Clip electrodes were used and MCT was applied at 3 sessions/week 11. The negative lead (1.0×1.0 cm) was placed over the muscle injury site, whereas the positive lead was placed proximally on the thigh region of the same side. The microcurrent was applied for 20 min during each session 12. The animals were subdivided into two subgroups:
- Subgroup IIb1: this group comprised five rats sacrificed 1 week after the day of injury.
- Subgroup IIb2: this group comprised five rats sacrificed 3 weeks after the day of injury.
The animals were sacrificed by a lethal dose of ether. The gastrocnemius–soleus muscle was exposed and muscle specimens were placed in 10% formol saline. Five-micron-thick sections were prepared and subjected to the following studies:
- Anti-α-smooth muscle actin (α-SMA) immunostaining: the marker for this antibody stains smooth muscle cells in vessel walls, the gut wall, the myometrium, and myoepithelial cells. Anti-α-SMA antibody (rabbit polyclonal antibody) (ab5694) was used at a concentration of 0.5–2 µg/ml. Heat-mediated antigen retrieval should be performed before commencing IHC staining. Glioma tissue sections were used as positive control specimens. Cellular localization was in the cytoplasm. In contrast, one of the muscle sections was used as a negative control by omitting the application of the primary antibody 14.
- CD34 immunostaining is the marker for hematopoietic progenitor cells, small vessel endothelium 15, and Scs of the skeletal muscle 16 of a variety of tissues. CD34 goat polyclonal Ab (catalogue ID SAB4300690; Sigma-Aldrich Chemie Corporation laboratories, Taufkirchen, Germany). 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.
Using Leica Qwin 500 Ltd image analysis, (Cambridge, UK), the area of atypical muscle fibers was estimated in 10 low-power fields in the control and experimental groups using interactive measurements. The area% of α-SMA-positive cells and CD34-positive cells was measured in 10 high-power fields using binary mode.
Quantitative data were summarized as means and SDs and compared using one-way analysis of variance. Any significant analysis of variance was followed by the Bonferroni post-hoc test to detect which pairs of groups caused the significant difference. P-values less than 0.05 were considered statistically significant. Calculations were made using SPSS 9.0 software, (New York, USA) 17.
Skeletal muscle sections of control rats showed longitudinal muscle fibers with minimal connective tissue (CT) in between (Fig. 1) that exhibited pale oval nuclei and transverse striations in the sarcoplasm (Fig. 2).
In subgroup IIa1 (sacrificed 1 week after muscle injury) the injured area showed atypical fibers widely separated by infiltrated cells and CT cells. Most fibers contained dark nuclei. Distended capillaries were noted (Fig. 3). Some fibers revealed partial loss of striations, and other fibers recruited strong acidophilic sarcoplasm with focal vacuolations on close observation (Fig. 4). In subgroup IIa2 (sacrificed 3 weeks after muscle injury), atypical closely packed fibers appeared separated by a few infiltrating cells and CT cells. Some fibers exhibited dark nuclei (Fig. 5). Partial loss of striations was found in some fibers on close observation (Fig. 6).
In subgroup IIb1 (sacrificed 1 week after injury and MCT), a few deeply acidophilic fibers were found. Other fibers exhibited multiple flat nuclei; some of these nuclei were centrally located (Fig. 7). Striations appeared in some areas of the sarcoplasm on close observation (Fig. 8). In subgroup IIb2 (sacrificed 3 weeks after injury and MCT), a few deeply acidophilic fibers were seen surrounded by multiple typical fibers, some of which were observed with centrally located nuclei (Fig. 9). Multiple fibers recruited striations in most areas of the sarcoplasm on close observation (Fig. 10).
Skeletal muscle sections of control rats showed few α-SMA-positive spindle cells in between the muscle fibers (Fig. 11). In subgroup IIa1 positive spindle cells were found in the lining of blood vessels existing between atypical fibers (Fig. 12). In subgroup IIa2 a few positive spindle cells were detected among some fibers with partial loss of striations (Fig. 13). In subgroup IIb1 some positive spindle cells were evident among some muscle fibers with centrally located nuclei (Fig. 14). Subgroup IIb2 showed multiple positive spindle cells among muscle fibers with striations in most areas of the sarcoplasm (Fig. 15).
Skeletal muscle sections of control rats revealed a few CD34-positive spindle cells at the periphery of the fibers (Fig. 16). In subgroup IIa1, occasional positive spindle and oval cells appeared around atypical fibers (Fig. 17). In subgroup IIa2, some fields demonstrated a few positive spindle cells among muscle fibers (Fig. 18). In subgroup IIb1 multiple positive spindle cells were obvious at the periphery of muscle fibers (Fig. 19). In subgroup IIb2 some positive spindle cells appeared at the periphery of the fibers (Fig. 20).
A significant (P<0.05) decrease in the mean area of atypical fibers was estimated in subgroup IIb1 compared with the injury subgroups IIa1 and IIa2. In addition, a significant (P<0.05) decrease was found in subgroup IIb2 compared with IIb1 (Table 1).
A significant (P<0.05) increase in the mean area% of α-SMA-positive immunostaining was detected in subgroup IIb1 compared with the injury subgroups IIa1 and IIa2. In addition, a significant (P<0.05) increase was found in subgroup IIb2 compared with IIb1 (Table 1).
A significant (P<0.05) increase in the mean area% of CD34-positive immunostaining was estimated in subgroup IIb2 compared with the injury subgroups IIa1 and IIa2. In addition, a significant (P<0.05) increase was found in subgroup IIb1 compared with IIb2 (Table 1).
The current study demonstrated the modulating effect of MCT on induced skeletal muscle injury, which was associated with Sc existence in albino rats. This was evidenced by histological, immunohistochemical, and morphometric studies.
In subgroup IIa1 (Sacrifice 1week following muscle injury) the injured area demonstrated atypical fibers widely separated by infiltrating cells and distended capillaries. In accordance,some authors stated that thermal injury of gastrocnemius muscle of rat revealed greater interfiber distances and substantially increased amount of connective tissue 1. Other investigators 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. Some authors related injury of DNA to reactive oxygen species 18. In addition, some investigators described similar degenerative cytoplasmic changes after muscle injury 2.
In subgroup IIa2 (sacrificed 3 weeks after muscle injury), atypical fibers appeared separated by a few infiltrating cells. Some fibers exhibited dark nuclei and partial loss of striations. The previous results indicated mild regression of degenerative changes. Concomitantly, some workers documented that mononuclear cells can develop into a variety of different muscle cell lineages, including myoblasts, Scs, and muscle-derived stem cells 19.
In subgroup IIb1 (sacrificed 1 week after injury and MCT), a few atypical fibers were found. Other fibers exhibited multiple flat nuclei, some of which were centrally located. Striations appeared in some areas of the sarcoplasm. Some authors postulated that MCT limits muscle damage and inflammation, known as delayed-onset muscle soreness in cases of injury 12. Other investigators reported improved neuromuscular conductivity in response to MCT following muscle injury 20. Some workers 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 IIb2 (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. 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. Some authors 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 some workers who stated that survival of the satellite cells is a critical requirement for efficient muscle reconstitution following injury 23.
The regenerative capacity was assessed in the present work using α-SMA. In subgroup IIa2 a few positive spindle cells were detected among some fibers with partial loss of striations. In subgroup IIb1 positive spindle cells were more evident, and in subgroup IIb2 positive cells multiplied among the muscle fibers. This was confirmed by a significant increase in the mean area% of α-SMA-positive cells. Some authors 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 Scs, which proliferate and migrate to the injured site 24.
In subgroup IIa1, occasional CD34+ve spindle and oval cells appeared around atypical fibers. Subgroup IIa2 demonstrated few +ve spindle cells among muscle fibers. On the other hand, in subgroup IIb1 multiple +ve spindle cells were obvious at the periphery of muscle fibers. While in subgroup IIb2 the +ve spindle cells appeared less. This was evidenced by a significant increase in the mean area% of CD34 +ve cells in mirourrent group. Some investigators documented Scs within adult skeletal muscle as an enriched population of CD34+ve cells 16. Some authors proved that Scs are responsible for the regenerative potential of skeletal muscle 25. Some investigators considered that CD34 beyond being a stem cell marker, may play an important function in modulating stem cell activity 26. On the other hand, workers noted a minority of satellite cells lacking CD34 has been described 6.
Conflicts of interest
There is no conflict of interest to declare.
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