Each year, millions of people are affected by nerve injuries involving both the peripheral nervous system (PNS) and the central nervous system (CNS)¹. Studies have demonstrated that the axonal repair capacity of the CNS is quite limited after nerve injuries, while the PNS shows enhanced capacity due to the significant plasticity of SCs². Nevertheless, achieving complete regeneration after peripheral nerve injuries remains arduous and continues to pose a significant challenge to human health³^,⁴. Nowadays, autografts have remained a common treatment despite the drawbacks of donor site morbidity and limited availability⁵. This situation has prompted researchers to explore alternative therapies, including materials⁶, molecular factors⁷, and electrical stimulation (ES). As a factor promoting axonal growth and nerve regeneration⁸, choosing an appropriate method of ES and exploring the relationship between ES and SCs become essential.

SCs are the main glial cells of the PNS, playing a crucial role in the regeneration of the PNS⁹^,¹⁰. Following peripheral nerve injuries, SCs undergo rapid activation, extensive reprogramming², and transition from a myelin-forming state to a growth-supportive morphology to conduct the regeneration of the nerve². A substantial proliferation of SCs occurs at the distal end of the injured nerve, while SCs of the distal stump undergo proliferation and elongation to form Bungner's band, which are necessary to guide axons to grow towards the target organ¹¹. Moreover, SCs from the proximal and distal nerve stumps migrate into the nerve bridge to form SC cords promoting axon regeneration¹². Furthermore, previous studies have demonstrated that the synthesis and secretion of relevant factors related to SCs change in cases of peripheral nerve regeneration, including transcriptional factors¹³, neurotrophic factors¹⁴, and myelination regulators¹³. This also provides indicators for assessing the activity of SCs. Based on these, the promotion of SC proliferation, migration, synthesis, and secretion of relevant factors have been extensively investigated for improving peripheral nerve regeneration¹⁵.

Previous studies have demonstrated the possibility of using ES for nerve regeneration¹. A widely accepted explanation is that ES can induce depolarization of cell membranes, alter membrane potential, and affect membrane protein functions by changing the charge distributions on these biomolecules¹. However, widely applied Intense PEF may cause severe pain, involuntary muscle contractions, and heart fibrillation⁸. It also increases creatine kinase (CK) activity, decreases muscle strength, and induces the development of delayed onset muscle soreness (DOMS)¹⁶. nsPEF is an emerging technique that stimulates test subjects with high-voltage electric fields within a nanosecond pulse duration, and it is gradually being used in cellular-level research¹⁷^,¹⁸. Previous studies have reported that the possible rationale of nsPEF promoting cell proliferation and organelle activity is the formation of membrane nanopores and the activation of ionic channels, which leads to an increase in cytoplasmic Ca²⁺ concentration¹⁹. nsPEF utilizes pulse power technology to charge the cell membrane, producing pulses characterized by short duration, rapid rise time, high power, and low energy density²⁰. These characteristics suggest that nsPEF may be a preferred mode with minimal stimulation side effects⁸. Furthermore, nsPEF offers advantages such as minimally invasive procedures, reversibility, adjustability, and non-destructiveness to neural tissues compared to surgical interventions. One mainstream research direction of nsPEF in the biomedical field is its application for tumor tissue ablation using high-energy electric field stimulation²¹^,²²^,²³. Some research results indicate that 12-nsPEF can stimulate peripheral nerves without causing damage²⁴. However, at present, there is limited evidence regarding the application of nsPEF in the field of nerve regeneration. Moreover, stimulating SCs using nsPEF is a pioneering attempt, contributing to further in vivo and clinical research. This study explores whether nsPEF stimulation of SCs can promote nerve regeneration and provide a reliable basis for subsequent in-depth and systematic research.

Detecting the Expression of the Neurotrophic and Transcription Factors in Nanosecond Pulsed Electric Field (nsPEF) Stimulated RSC96 Cells