Satellite Cell Proliferation and Myofiber Cross-Section Area Increase After Electrical Stimulation Following Sciatic Nerve Crush Injury in Rats
Introduction
Peripheral nerve injuries can lead to skeletal muscle denervation, resulting in irreversible atrophy characterized by a reduction in myofiber cross-sectional area (CSA) and strength. The loss of myonuclei is proportional to the reduction in myofiber CSA, maintaining a stable ratio known as the myonuclear domain (MND). Satellite cells, which are the primary source of new myonuclei for mature muscle fibers, play a crucial role in muscle regeneration. However, in denervated muscles, satellite cells often fail to proliferate adequately, leading to a progressive reduction in both the population and local density of these cells.
Electrical stimulation has been recommended as an effective therapy to prevent muscle atrophy following nerve injury. Previous studies have demonstrated that electrical stimulation can maintain muscle mass and force, and gradually restore myofiber CSA in denervated muscles. However, the underlying mechanisms of these therapeutic effects, particularly the role of satellite cell proliferation, have not been fully elucidated. This study aimed to evaluate the changes in satellite cell proliferation after electrical stimulation in nerve-injured rats and to determine whether these changes are related to the restoration of myofiber CSA.
Methods
Ethical approval for this study was obtained from the Committee on the Ethics of Animal Experiments of Peking University. Seventy-two adult male Sprague-Dawley rats were randomly divided into three groups: sham operation controls (Sham), sciatic nerve crush injury models (Injury), and sciatic nerve crush injury with daily electrical stimulation (Injury + Stim). The sciatic nerve crush injury was performed by exposing and crushing the left sciatic nerve 10 mm above the bifurcation for 30 seconds using a serrated clamp. Electrical stimulation was applied daily to the left gastrocnemius muscle of the Injury + Stim group using surface electrodes connected to a stimulator. The stimulation parameters included a pulse rate of 2 Hz, pulse width of 300 ms, voltage of 25 V, and current of 1 mA.
Animals were euthanized at 2, 4, and 6 weeks post-injury (wpi). Three days before euthanasia, rats were injected with 5-bromo-2’-deoxyuridine (BrdU) to label proliferating cells. The left gastrocnemius muscles were harvested for histological and molecular analyses, including hematoxylin-eosin (HE) staining, immunofluorescence, flow cytometry, and Western blotting. HE staining was used to measure myofiber CSA and myonuclei number per fiber. Immunofluorescence was performed to detect Pax7-positive satellite cells, while flow cytometry was used to assess BrdU-positive proliferating satellite cells. Western blotting was conducted to quantify Pax7 expression.
Results
The results showed that electrical stimulation significantly increased the percentage of Pax7-positive nuclei and BrdU-positive nuclei in the Injury + Stim group compared to the Injury group. At 6 wpi, the percentages of Pax7-positive nuclei and BrdU-positive nuclei in the stimulated muscles were 10.81 ± 0.56% and 34.29 ± 3.87%, respectively, compared to 2.58 ± 0.33% and 1.30 ± 0.09% in the non-stimulated muscles. The numbers of myonuclei per fiber and myofiber CSA were also significantly increased in the stimulated muscles. At 6 wpi, the myonuclei number per fiber and myofiber CSA in the Injury + Stim group were 2.19 ± 0.24 and 1906.86 ± 116.51 mm², respectively, compared to 1.10 ± 0.16 and 1095.1 ± 115.9 mm² in the Injury group.
Linear regression analysis revealed a significant positive correlation between the percentage of Pax7-positive nuclei and both myonuclei number per fiber (R² = 0.52) and myofiber CSA (R² = 0.60). These findings suggest that the enhanced proliferation of satellite cells induced by electrical stimulation is closely related to the increase in myonuclei number and myofiber CSA. However, there was no significant difference in the ratio of myofiber CSA to myonuclei number per fiber (MND) among the three groups, indicating that the MND remained stable despite the changes in myofiber CSA and myonuclei content.
Discussion
The findings of this study demonstrate that electrical stimulation promotes satellite cell proliferation in denervated muscles following sciatic nerve injury. The increased proliferation of satellite cells was associated with a higher number of myonuclei and a restoration of myofiber CSA, suggesting that electrical stimulation can mitigate muscle atrophy by enhancing satellite cell activity. The stable MND observed in all groups supports the myonuclear domain hypothesis, which posits that each myonucleus is responsible for a defined volume of cellular territory within a myofiber.
The mechanism by which electrical stimulation promotes satellite cell proliferation is not fully understood, but it may involve the activation of signaling pathways such as Wnt and PI3-Akt, which are known to regulate cell proliferation and myogenesis. Electrical stimulation has been shown to increase the expression of trophic factors such as neural cell adhesion molecule and vascular endothelial growth factor, which may also contribute to the enhanced proliferation of satellite cells.
One limitation of this study is that the apoptosis of satellite cells was not assessed. It is possible that electrical stimulation prevents apoptosis or autophagy of satellite cells, thereby maintaining their population in denervated muscles. Future studies should investigate the effects of electrical stimulation on satellite cell apoptosis and the signaling pathways involved in this process.
Conclusion
In conclusion, this study provides evidence that electrical stimulation enhances satellite cell proliferation in denervated muscles following sciatic nerve injury. The increased proliferation of satellite cells is associated with a higher number of myonuclei and a restoration of myofiber CSA, suggesting that electrical stimulation can effectively mitigate muscle atrophy by promoting muscle regeneration. These findings highlight the potential of electrical stimulation as a therapeutic intervention for preventing muscle atrophy in patients with peripheral nerve injuries.
doi.org/10.1097/CM9.0000000000000822
Was this helpful?
0 / 0