Distal electrical stimulation enhances neuromuscular reinnervation and satellite cell differentiation for functional recovery

Abstract

Background: Peripheral nerve injuries lead to significant motor deficits, with limited treatment options for full functional recovery. Distal electrical stimulation (E-stim) has shown promise in promoting neuromuscular reinnervation, though its mechanisms are not yet fully understood. This study aims to investigate the regulatory effects of distal E-stim on neuromuscular junction (NMJ) reinnervation and Satellite cell activity in denervated muscle injury.

Methods: Using a sciatic nerve critical gap model in Sprague-Dawley rats (8-week-old, random sex), we applied distal E-stim and assessed neuromuscular and functional recovery through histological, biochemical, and functional evaluations over six weeks. The Sciatic Function Index (SFI) was measured at baseline and at subsequent time points post-injury. We quantified muscle mass, NMJ morphology, and neurotransmitter levels (acetylcholine and acetylcholinesterase), and analyzed muscle fiber electrophysiology using single-muscle electromyography to assess denervated muscle autoelectricity. Additionally, single-cell RNA sequencing was performed to examine gene expression in Satellite cells.

Results: Distal E-stim significantly enhanced neuromuscular reinnervation, as evidenced by improved SFI scores, increased muscle mass, and reduced muscle atrophy. Histological analysis showed larger muscle fiber cross-sectional areas and enhanced NMJ structure. Elevated levels of acetylcholine and acetylcholinesterase, along with reduced fibrillation potentials in muscle fibers, further indicated preserved NMJ function. Single-cell RNA sequencing revealed upregulation of genes associated with muscle differentiation and angiogenesis in Satellite cell clusters, suggesting that distal E-stim fosters a regenerative environment.

Conclusions: Our findings demonstrate that distal E-stim promotes functional recovery through NMJ preservation and Satellite cell differentiation, offering novel insights into molecular mechanisms that may enhance electroceutical therapies for peripheral nerve injuries. Further research could optimize E-stim protocols to maximize clinical benefits for patients with neuromuscular impairments.

Keywords: Denervation muscle injury; Electrical stimulation; Neuromuscular regeneration; Peripheral nerve regeneration; Satellite cell.

Fig. 1

Distal electric stimulation improves functional recoveries. (A) Schematic illustration depicting electric stimulation applied to the distal stump of the nerve gap in a SD rat model. The “Naive” group is defined as having both hind legs intact. In the “Control” group, the left side underwent transection surgery with ligation using a 10 mm conduit, while the right side remained intact. In the “E-stim” group, the left side underwent nerve transection surgery with ligation using a 10 mm conduit, followed by a single instance of electrical stimulation; the right side remained intact. (B, C) A higher level of toe spreading was observed in the E-stim group. Sciatic function index data obtain at postoperative weeks1, 2 and 6. Each group comprised 3 independent animals, compared to the Naïve group. (n = 3 per timepoint. Data was presented with mean standard deviation. * indicated p < 0.05; ** indicated p < 0.01, *** indicated p < 0.001). (D, E) Gastrocnemius muscles were harvested from both hind legs of the Control group and the E-stim group at postoperative weeks 1, 2, and 6 following nerve transection. Muscle weight (MW) preservation rate was calculated by normalizing the left gastrocnemius muscle weight to that of the contralateral muscle. Scale bar = 1 cm. (n = 5 per timepoint. Data was presented with mean standard deviation. * indicated p < 0.05; ** indicated p < 0.01, *** indicated p < 0.001)

Fig. 2

Distal electric stimulation ameliorates atrophy and abnormal autoelectricity of denervated muscle fiber. (A, B) Hematoxylin and Eosin staining of the cross-section of gastrocnemius muscle in each experimental group. The average surface area per fiber was calculated by three randomly picked myofiber under high power field of independent animal in each group at postoperative weeks 2 and 6. (n = 5, employing one-way ANOVA with Tukey multiple comparison analysis. * indicated p < 0.05; ** indicated p < 0.01, *** indicated p < 0.001, **** indicated p < 0.0001). (C, D) Electromyography recordings measured the frequency of muscle fibrillation in 100 micro second of five independent animals in each experimental group at postoperative days 6, 9, and 14 following nerve transection. The findings indicate a lower frequency of muscle fibrillation in the E-stim group compared to the Control group. (n = 5, employing t-tests analysis. * indicated p < 0.05; ** indicated p < 0.01, and *** indicated p < 0.001)

Fig. 3

Histologic structure, neurotransmitter regulation and associated molecular regulation in neuromuscular junction. (A) Schematic illustration of the acetylcholine releasing at NMJ. (B, C) Immunofluorescent stain of the neuromuscular junction at week 1 post-injury in each experimental group, illustrating the density of double staining for pre- (NF200, green color) and postsynaptic (alpha-bungarotoxin, red color) markers each group. The accompanying bar chart on the right presents statistical indices of density and significance, revealing that the NMJ count in each experimental group. Scale bar = 50 μm. (D) Acetylcholine levels were quantified using ELISA array to assess ACh expression in muscle samples innervated by transected sciatic nerves in the Control and E-stim groups. The relative expression level is defined by the index normalized to the healthy contralateral side (right side) of the same rat. (E) Acetylcholinesterase expression level in the Control and E-stim groups. The expression level is normalized to the total protein expression of the sample. (F) Comparison of expression levels of neuron physical function-associated molecules between the E-stim and Control groups. (n = 5 per timepoint. Data was presented with mean standard deviation. Statistical analysis was conducted using Prism software, employing t-tests and one-way ANOVA with Tukey multiple comparison analysis. * indicated p < 0.05; ** indicated p < 0.01, *** indicated p < 0.001, **** indicated p < 0.0001)

Fig. 4

Single cell RNA sequencing in denervated muscle. (A) UMAP visualization conducted using the R package Seurat. Samples were mapped based on quality control criteria, including cells with nFeature RNA counts between 600 and 3000, while maintaining mitochondrial RNA levels below 40%. Three sample groups were merged: Naive (n = 2, total cell count = 11253), Control (n = 2, total cell count = 11780), and E-stim (n = 2, total cell count = 14280). (B) Feature plots of biomarkers for annotation. The color gradient represents the expression level of genes across the global UMAP, utilizing known housekeeping genes specific to different cell types. (C) Simplified GO enrichment heatmap processed using the R package simplifyEnrichment. The word cloud on the right side summarizes the frequency of functions modulated within the GO database. The size of characters represents keywords of functions, linearly correlated with the level of regulation. (D) Volcano plot illustrating all differentially expressed genes between the E-stim group and the Control group, plotted using the R package ggplot2 with screening criteria including a log2 fold change > 0.5d a p-value < 0.05. (E) Analysis of accumulated differentially expressed genes among each cluster, applying criteria of a log2 fold change > 0.5 and a p-value < 0.05. The highest count and percentage of differentially expressed genes is observed in the Satellite cell cluster, indicating that Satellite cells exhibit the most pronounced response to electrical stimulation

Fig. 5

Gene ontology (GO) analysis of Satellite cell cluster by distal E-stim. (A) Dot plot depicting up and down-regulated GO terms within the E-stim Satellite cell cluster relative to their level in the Control Satellite cell. The size of the dots indicates the number of genes involved in a Gene Ontology (GO) category, while the color represents the significance of regulation. (B) Volcano plot illustrating the differential expression of genes between the E-stim Satellite cell and Control Satellite cell groups. Screening criteria included a log2 fold change > 1 and a p-value < 0.05. (C) Heatmap displaying Satellite cells across the Naive, Control, and E-stim groups. Genes were selected based on the criteria outlined in plot B. Genes highly modulated by electrical stimulation post-nerve transection are also prominently expressed in the Naive group, serving as a baseline for Satellite cell activity. (D) Network visualization depicting interactions among differentially expressed genes within the E-stim Satellite cell cluster. The size of the dots representing GO function names corresponds to the number of genes involved, while the color gradient of dots with gene codes indicates the log fold change of specific genes. (E) Dot plot illustrating the RNA expression level of highly modulated genes within the Satellite cell cluster. The size of the dots indicates the ratio of cells within the Satellite cell cluster expressing a particular gene. The color gradient represents the average expression level compared with the standardized gene expression across all cells within the Satellite cell population

Fig. 6

Satellite cell differentiation in denervated muscle by distal E-stim. (A) Ridge plot displaying the RNA expression levels of Pax7, MyoD, and Myogenin in Satellite cell clusters from different experimental groups three days post-nerve transection. Pax7 serves as a biomarker of quiescent Satellite cells, while activation of Satellite cells involves co-expression of Pax7 with MyoD. Myogenin is a transcription factor that regulates myocyte fusion during development. (B, C) Immunofluorescent staining of muscle tissue samples taken three days post-injury. The left column illustrates Pax7 distribution in myofibers (Red: Pax7/Green: Laminin/Blue: DAPI), with no significant differences noted in expression among the three groups. The middle column depicts MyoD-expressing cells in myofibers (Red: MyoD/Green: Laminin/Blue: DAPI), indicating activation of Satellite cells. The highest expression of MyoD is observed in the E-stim group. The column on the right displays myogenin-expressing cells in myofibers (Red: Myogenin/Blue: DAPI). The Naive group exhibits the highest level of myogenin expression, consistent with its unharmed sciatic nerve and intact neuromuscular function. Additionally, the E-stim group demonstrates a higher density of myogenin-expressing cells compared to the Control group. (n = 3–4 per group. Data was presented with mean standard deviation. Statistical analysis was conducted using Prism software, employing one-way ANOVA with Tukey multiple comparison analysis. * indicated p < 0.05; ** indicated p < 0.01, *** indicated p < 0.001, **** indicated p < 0.0001)