Regulating Schwann Cell Growth by Nanosecond Pulsed Electric Field for Peripheral Nerve Regeneration In Vitro

Summary

Here, we present a protocol for applying nanosecond pulse electric field (nsPEF) to stimulate Schwann cells in vitro. The synthesis and secretion ability of relevant factors and cell behavior changes validated the successful stimulation using nsPEF. The study gives a positive view of the peripheral nerve regeneration method.

Abstract

Schwann cells (SCs) are myelinating cells of the peripheral nervous system, playing a crucial role in peripheral nerve regeneration. Nanosecond Pulse Electric Field (nsPEF) is an emerging method applicable in nerve electrical stimulation that has been demonstrated to be effective in stimulating cell proliferation and other biological processes. Aiming to assess whether SCs undergo significant changes under nsPEF and help explore the potential for new peripheral nerve regeneration methods, cultured RSC96 cells were subjected to nsPEF stimulation at 5 kV and 10 kV, followed by continued cultivation for 3-4 days. Subsequently, some relevant factors expressed by SCs were assessed to demonstrate the successful stimulation, including the specific marker protein, neurotrophic factor, transcription factor, and myelination regulator. The representative results showed that nsPEF significantly enhanced the proliferation and migration of SCs and the ability to synthesize relevant factors that contribute positively to the regeneration of peripheral nerves. Simultaneously, lower expression of GFAP indicated the benign prognosis of peripheral nerve injuries. All these outcomes show that nsPEF has great potential as an efficient treatment method for peripheral nerve injuries by stimulating SCs.

Introduction

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 health^3,4. Nowadays, autografts have remained a common treatment despite the dra....

Protocol

1. Thawing of cryopreserved RSC96 cells

1. Thaw the cryovial containing 1 mL of cell suspension by rapidly shaking it in a 37 °C water bath, and then add it to a centrifuge tube containing 4-6 mL of complete culture medium and mix well.
2. Centrifuge at 1000 x g for 3-5 min, discard the supernatant and resuspend the cells in 3 mL of complete culture medium.
3. Add the cell suspension to a culture flask (or dish) containing 6-8 mL of complete culture medium and incubat.......

Discussion

In recent years, the application of nsPEF has experienced boosting growth, as reported. nsPEF has a highly targeted effect on only the desired area, providing enough energy to treat without causing additional thermal damage, making it safer for the human body²⁸. These characteristics give it promising translational prospects in tumor treatment and nerve regeneration. However, some studies have proposed some limitations of nsPEF. Compared with materials research, ES is constrained by external power.......

Materials

Name	Company	Catalog Number	Comments
Antifade mounting medium	Wuhan Xavier Biotechnology Co., LTD	G1401
Anti-GFAP Mouse mAb	Wuhan Xavier Biotechnology Co., LTD	GB12100-100
Anti-Neurofilament heavy polypeptide Mouse mAb	Wuhan Xavier Biotechnology Co., LTD	GB12144-100
Anti-S100 beta Mouse mAb	Wuhan Xavier Biotechnology Co., LTD	GB14146-100
BSA	Wuhan Xavier Biotechnology Co., LTD	GC305010
Coverslip	Jiangsu Shitai experimental equipment Co., LTD	10212432C
CY3-labeled goat anti-mouse IgG	Wuhan Xavier Biotechnology Co., LTD	GB21302
DAPI Staining Reagent	Wuhan Xavier Biotechnology Co., LTD	G1012
Decolorizing shaker	Wuhan Xavier Biotechnology Co., LTD	DS-2S100
High Voltage Power Supply for nsPEF	Matsusada Precision Inc.	AU-60P1.6-L
Histochemical pen	Wuhan Xavier Biotechnology Co., LTD	G6100
Membrane breaking liquid	Wuhan Xavier Biotechnology Co., LTD	G1204
Microscope slide	Wuhan Xavier Biotechnology Co., LTD	G6012
Palm centrifuge	Wuhan Xavier Biotechnology Co., LTD	MS6000
PBS powdered	Wuhan Xavier Biotechnology Co., LTD	G0002
Pipette	Wuhan Xavier Biotechnology Co., LTD
Positive fluorescence microscope	Nikon, Japan	NIKON ECLIPSE C1
Rabbit Anti-SOX10/AF488 Conjugated antibody	Beijing Bioss Biotechnology Co., LTD	BS-20563R-AF488
RSC96 Schwann cells	Wuhan Xavier Biotechnology Co., LTD	STCC30007G-1
scanister	3DHISTECH	Pannoramic MIDI
Special cable for nsPEF	Times Microwave Systems	M17/78-RG217
Turbine mixer	Wuhan Xavier Biotechnology Co., LTD	MV-100