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 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 PNS^9,10. 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 research^17,18. 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 stimulation^21,22,23. 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.

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 incubate at 37 °C overnight.
4. The next day, observe the cell growth and density under a microscope.

2. Cell passage:

NOTE: If the cell density reaches 80%-90%, it is ready for passage.

1. Discard the culture medium and rinse the cells 1-2 times with phosphate-buffered saline (PBS) without calcium and magnesium ions.
2. Add 0.25% (w/v) trypsin-0.53 mM EDTA to the culture flask (1-2 mL for a T25 flask, 2-3 mL for a T75 flask) and incubate at 37 °C for 1-2 min.
3. Observe the cell detachment under a microscope. If most cells become round and detach, quickly return the flask to the working area, tap the flask gently, and add 3-4 mL of culture medium containing 10% FBS to stop the digestion.
4. Mix the contents, aspirate the solution, and centrifuge at 1000 x g for 5 min. Then, discard the supernatant and resuspend the cells by adding 1-2 mL of fresh culture medium and pipetting gently.
5. Transfer the cell suspension to a new T25 flask at a 1:2 ratio and add 7 mL of culture medium.

3. Operation of nsPEF device

1. Resuspend the RSC96 cells in 1 mL of DMEM culture medium and transfer them to colorimetric dishes with electrodes on both sides.
2. Turn on the power switch.
3. Adjust the parameters by rotating the knob on the instrument to alter the intensity of the electric field. The intensities set in this study are 5 kV/cm, 10 kV/cm, 20 kV/cm, and 40 kV/cm.
4. Carefully rotate the electrodes until sparks appear, allowing the cells to receive 5 pulses of nsPEF according to the preset field strength intensities before immediately separating the two electrodes. Following this treatment, take the experimental group cells treated with nsPEF and the untreated control group cells to perform sections 4-6, respectively, after a certain period of cell culture (1 day in this experiment).

4. Cell counting kit-8 (CCK-8) assay

1. Prepare a cell suspension of a particular concentration from RSC96 cells stimulated with electrical stimulation. Add 100 µL of the cell suspension to each well of a 96-well cell culture plate. Considering the requirements of the CCK-8 assay, control the total number of cells in the reagent kit between 1 x 10³ and 1 x 10⁶.
2. Take 10 µL of CCK-8 solution from the kit and add it to the 96-well cell culture plate. Incubate in a CO₂ incubator at 37 °C for an additional 30 min to 4 h.
3. Measure the absorbance. Use dual-wavelength measurement with a detection wavelength of 430-490 nm and a reference wavelength of 600-650 nm.
4. Determine the field strength intensity for subsequent experiments based on the experimental results of cell proliferation. Select cells with good proliferation for subsequent experiments.

5. Cell scratch assay

1. In each well of a six-well plate, seed 3 x 10⁵ cells with a total volume of 2 mL per well. Approximately 72 h later, the cells will cover the well. Conduct the test on the experimental and the control groups separately.
2. Use a pipette tip to draw a horizontal line at the bottom of the culture well. Ensure that the pipette tip is held vertically and try to avoid tilting it.
3. Aspirate the culture media and wash with PBS 2-3 times.
4. Add 2 mL of serum-free medium to each well.
5. Place the plate in the 37 °C incubator. Take pictures under a fourfold magnification of the inverted microscope at 0 h and 24 h to observe changes in cell migration.

6. Immunofluorescence

1. Cell permeabilization:
   1. After gently pipetting cell suspensions into culture dishes, draw circles with a histology pen at locations where cells are evenly distributed on the coverslip. Treat the control group and different experimental groups separately.
   2. Add 50-100 µL of permeabilization working solution (0.25-0.5% Triton X-100) and incubate at room temperature (RT) for 20 min. Wash three times with PBS for 5 min each time.
2. Serum Blocking: Add 3% BSA within the circles to cover the tissue uniformly. Incubate at RT for 30 min.
3. Primary antibody incubation: Gently remove the blocking solution and add the appropriately diluted primary antibody (mouse-derived, diluted at 1:300) to the cell wells. Place the cell culture plate in a humid box and incubate overnight at 4 °C.
4. Secondary antibody incubation: Place the cell plate on a shaker and wash it three times for 5 min each time. Add the corresponding secondary antibody (CY3-labeled goat anti-mouse IgG, diluted at 1:300) and incubate at RT for 50 min.
5. DAPI nuclear staining:
   1. Place the coverslip in PBS (pH 7.4) on a shaker and wash three times for 5 min each time.
   2. After gently drying the slide, add DAPI staining solution (2 µg/mL, 0.5 mL per circle) within the circles and incubate at RT for 10 min in a dark room.
6. Mounting: Place the coverslip in PBS (pH 7.4) on a shaker and wash three times for 5 min each time. After gently drying the slide, seal the coverslip with an anti-fading mounting medium for fluorescence.
7. Image acquisition: Excite DAPI at a wavelength of 330-380 nm and detect emission at 420 nm; excite at 465-495 nm and detect emission at 515-555 nm for AF488; excite at 510-560 nm and detect emission at 590 nm for CY3; excite at 608-648 nm and detect emission at 672-712 nm for CY5.

Results

Low-intensity pulsed electric fields stimulate cell proliferation
According to the CCK-8 assay, the proliferation rate of RSC96 in the 5 kV/cm group was significantly faster than that of the control group cells. However, as the parameters increased (20 kV/cm and 40 kV/cm), the proliferation rate was unstable, even lower than that of the control group. The cell proliferation rate of RSC96 cells in the 40 kV/cm group was significantly lower than the control and 5 kV/cm groups, showing a significant s...

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