Purpose
Distal electrical stimulation (Estim) on interphase of neuromuscular junction has been validated as an effective and innovative approach for denervated muscle injury from our previous publication. Electrical pulse on affected cells has direct influence on intracellular environment and protein translation. However, the underlying mechanism and regulatory microenvironment are still unexplored. The main purpose of this study is to explore the regulatory role of satellite cell and the molecular mechanism at the neuromuscular junction of denervated muscle by distal Estim.

Materials and Methods
Critical gap (10mm) injury at sciatic nerve of adult SD rat will be created, followed by immediate distal Estim. Longterm functional recovery was verified at 6 weeks. To further investigate the regulatory mechanism, the denerved gastrocnemius muscle was harvest to analyze the microenvironment of neuromuscular junction (NMJ) by single cell RNA sequence (scRNAseq).

Results
Distal Estim facilitate functional recovery of peripheral nerve regeneration after 6 weeks, in terms of gait function, muscle weight, NMJ regeneration. With scRNAseq, we identified that the satellite cell cluster has the highest number of differential expressing gene, indicating that the cell type was the most significantly affected by the electric stimulation treatment. As the quantitative analysis showing, Myod1 and Myog was up-regulated the most in Estim group, indicating that satellite cell was activated to proliferate. On top of that, the gene set enrichment analysis reveals the genes involved in vessel remodeling pathway having a significantly higher expression level in E-stim group than control group, such as Aqp1, Myog, Myod1, Pax7, Cdh5, are known as associated with angiogenesis and vasculature development pathway.

Conclusion
Distal Estim is an effective modality to facilitate neuromuscular regeneration by regulating satellite cell activation at NMJ of denerved muscle. In addition, we had also identified the genetic regulatory network within activated satellite cells.