In Vitro and In Vivo Comparative Analysis of Muscle Regenerative Processes Induced by Different Microcurrent Waveforms in Skeletal Muscle Atrophy

Objective: To evaluate the regenerative effects of various microcurrent waveforms in cast-induced gastrocnemius muscle atrophy in rabbits, with comprehensive in vitro and in vivo analyses. Methods: After two weeks of enforced hindlimb immobilization via casting, twenty-four rabbits, four studied groups were formed. Each group received a different treatment for two weeks: Group 1 (control) was given sham microcurrent; Group 2 was treated with a square waveform microcurrent; Group 3 with a sine waveform; and Group 4 with a tri-angular waveform. Treatments were administered daily for one hour. We assessed calf circumference, muscle thickness via ultrasound, tibial nerve CMAP, muscle fiber CSA, and protein expressions through Western blot analysis. Results: Notably, the sine waveform group (Group 3) showed significantly enhanced recovery across all measured parameters (p < 0.05). This group exhibited superior im-provements in muscle thickness, CMAP amplitude, and fiber CSA. Detailed immuno-histochemical analysis showed a marked rise in the expression of cellular proliferation and angiogenesis markers such as BrdU, PCNA, VEGF, and PECAM-1. Furthermore, western blot analysis highlighted a robust upregulation in myogenic regulatory factors including MyoD and Myogenin. It also showed a reduction in inflammatory and apop-totic markers such as TNF-α, NF-κB, cleaved caspase-3, and a notable decrease in stress response proteins p-CHK1 and p-CHK2. Conclusion: Sine waveform microcurrent stimulation was the most potent in promoting muscle regeneration in both dexamethasone-induced C2C12 myoblasts and cast-induced muscle atrophy, highlighting its significant therapeutic potential. Further studies are warranted to refine clinical application parameters.