Method Article

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

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DOI:

10.3791/66097

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May 3rd, 2024

 ,  ,  ,  ,  ,  ,  ,  , 
1Department of Orthopedics, The First Affiliated Hospital, College of Medicine, Zhejiang University, 2College of Medicine, Zhejiang University, 3Department of Orthopedics, The Second Affiliated Hospital of Zhejiang Chinese Medical University, 4Department of Orthopedic Physical Therapy, Faculty of Physical Therapy, Nahda University in Beni Suef, 5Plastic & Cosmetic Center, The First Affiliated Hospital, College of Medicine, Zhejiang University

* These authors contributed equally

In This Article

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 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.

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 statistical difference (P < 0.05). Due to not meeting the experimental requirements of cell proliferation, the 20 kV/cm and 40 kV/cm groups were excluded in subsequent experiments (Figure 1).

Low-intensity pulsed electric fields promote the expression of S100β
S100β, as a specific marker protein of SCs, participates in various functions, including neurite extension and axonal proliferation14. Additionally, S100β protein has been found to act as a neurotrophic factor during development, and its expression increases in cases of nerve injuries. Previous studies have demonstrated that S100β can work with brain-derived neurotrophic factor (BDNF) to regulate different signal transduction cascades and contribute to neuronal maturation axon growth14,25. Under the microscope, scattered cytoplasmic S100β-positive cells in red were observed in cell crawling assays across all groups (Figure 2A). After three days of cultivation, the integrated optical density (IOD) of fluorescence in the 5 kV/cm group of RSC96 cells was significantly higher than that of the control group and the 10 kV/cm group, showing a significant statistical difference (***P < 0.001; ****P < 0.0001) (Figure 2B).

Low-intensity pulsed electric fields promote the expression of NF-H
NF-H interacts with other intermediate filaments to form a network and is a major component of the neuronal cytoskeleton. Under the microscope, scattered cytoplasmic NF-H-positive cells in red were observed in cell crawling assays across all groups (Figure 3A). After three days of cultivation, the integrated optical density (IOD) of fluorescence in the 5 kV/cm group of RSC96 cells was significantly higher than that of the control group and the 10 kV/cm group, showing a significant statistical difference (*P<0.05; **P < 0.01) (Figure 3B).

Low-intensity pulsed electric fields regulate the expression of GFAP
The expression of glial fibrillary acidic protein (GFAP) is one of the indicators of astrocyte activity. Astrocytes initially exhibit reactive proliferation following nerve injury, which has a protective effect in the early stages. However, excessive proliferation of glial cells can lead to the formation of glial scars, impeding the connectivity of neuronal fibers26. Previous research has demonstrated that genes typically expressed during axon growth are re-expressed, such as GFAP27. Under the microscope, scattered cytoplasmic GFAP-positive cells in red were observed in cell crawling assays across all groups (Figure 4A). After three days of cultivation, the integrated optical density (IOD) of fluorescence in the 5 kV/cm group of RSC96 cells was significantly lower than that of the control group and the 10 kV/cm group, showing a significant statistical difference (****P < 0.0001; ***P < 0.001) (Figure 4B).

Low-intensity pulsed electric fields promote the expression of Sox10
Sox10 is continuously expressed in SCs, a key transcription factor for peripheral nerve myelination13. Under the microscope, scattered cytoplasmic Sox10-positive cells in green were observed in cell crawling assays across all groups (Figure 5A). After 3 days of cultivation, the mean gray value in the 5 kV/cm group of RSC96 cells was significantly higher than that of the control group and the 10 kV/cm group, showing a significant statistical difference (*P < 0.05; *P < 0.05) (Figure 5B).

Low-intensity pulsed electric fields promote cell migration
Comparing the migration rates of RSC96 cells after 24 h of scratch assay, it was observed that the migration of RSC96 cells in the 5 kV/cm group was significantly accelerated compared to the control group and the 10 kV/cm group (Figure 6).

Cell proliferation bar graph; optical density (O.D.) values, varying electric fields, significant change.
Figure 1: CCK8 assay of cellular proliferation of RSC96 cells stimulated at 5 kV/cm, 10 kV/cm, 20 kV/cm and 40 kV/cm, and controls. The results represent the mean ± SD based on ≥3 replicates. NS not significant; *P < 0.05; **P < 0.01; **P < 0.01 Please click here to view a larger version of this figure.

Immunofluorescence microscopy of S-100β protein with DAPI staining and bar graph fluorescence results.
Figure 2: Expression of S100β in RSC96 cells stimulated at 5 kV/cm, 10 kV/cm, and controls. The results represent the mean ± SD based on ≥3 replicates. ***P < 0.001; ****P < 0.0001. Please click here to view a larger version of this figure.

Immunofluorescence microscopy, NF-H protein expression, electric field effect, fluorescence intensity.
Figure 3: Expression of NF-H in RSC96 cells stimulated at 5 kV/cm, 10 kV/cm, and controls. The results represent the mean ± SD based on ≥3 replicates. *P < 0.05; **P < 0.01. Please click here to view a larger version of this figure.

GFAP immunofluorescence assay results, microscopy images, and bar chart show fluorescence intensity.
Figure 4: Expression of GFAP in RSC96 cells stimulated at 5 kV/cm, 10 kV/cm, and controls. The results represent the mean ± SD based on ≥3 replicates. ****P < 0.0001; ***P < 0.001. Please click here to view a larger version of this figure.

Immunofluorescence microscope images of Sox10, DAPI staining, and merge under different conditions.
Figure 5: Expression of Sox10 in RSC96 cells stimulated at 5 kV/cm, 10 kV/cm, and controls. The results represent the mean ± SD based on ≥3 replicates. *P < 0.05; *P < 0.05. Please click here to view a larger version of this figure.

Cell migration analysis; scratch assay results; comparison at 0h and 24h; variable electric fields.
Figure 6: Cell scratching assay of RSC96 cells stimulated at 5 kV/cm, 10 kV/cm, and controls. The results represent the mean ± SD based on ≥3 replicates. **P < 0.01; ***P < 0.001. Statistical data is based on ordinary one-way ANOVA. Please click here to view a larger version of this figure.

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 sources and wires29. Moreover, a recent study has proved that the administration of perioperative lidocaine as an analgesic significantly diminishes the ES-related improvement in nerve regeneration25. These concerns may bring challenges to future studies in vivo.

We solely utilized nsPEF to stimulate RSC96 cells. The cells subjected to low-intensity stimulation exhibited noticeable changes. However, the duration of the stimulation effect induced by nsPEF is relatively short, approximately 3 days, according to experimental results. In the future, it may be considered to combine materials such as gold nanorods and nano hydrogel that can enhance the bioelectrical effects of pulse electric fields, thereby augmenting the bioelectrical effects of pulse electric fields and developing more targeted and efficient treatment approaches30,31.

A few vital considerations of the experimental operation are as follows. The CCK-8 experiment was performed first, through which the appropriate field strength and cell culture time after electrical stimulation were screened to save the subsequent experimental steps and practices. Under our instrument and experimental conditions, receiving five pulses of electrical signals is conducive to maximizing cellular changes. If the pulse frequency is too low, the electrical stimulation received by the cells is insufficient to activate proliferation and other responses. On the other hand, excessive frequency can lead to irreversible cellular damage. During the electrical stimulation, it is necessary to pay attention to the distance between the electrodes to maintain a proper position to ensure that the electrodes can be quickly separated after a predetermined number of electric shocks to prevent more electric shocks from affecting the experiment's results. After the completion of the electric shock, the instrument should be grounded in time to avoid damage to the instrument.

In this study, nsPEF with low field strength effectively promoted the proliferation, migration, synthesis, and secretion ability of relevant factors in SCs. Although the underlying mechanism still needs clarifying, this study provides a positive view of applying nsPEF to stimulate cell proliferation and migration. Such cell proliferation and migration induced by nsPEF are scarce19 in the current research fields. Moreover, many crucial factors are involved in the stimulation of SCs. This study assessed the specific marker protein and neurotrophic factor S100β, transcription factors Sox10, GFAP, and the neuronal cytoskeleton NF-H components. The alterations they undergo in nsPEF stimulation can assess the activity of SCs, the level of repair in peripheral nerves, and the prognosis in multiple dimensions. All outcomes demonstrated the significant stimulation of SCs and benign prognosis by nsPEF. In the future, we will conduct more studies on animal models and further investigate the effects of different intensities of nsPEF on SCs to screen out the optimal mode of pro-differentiation electric field stimulation.

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was funded by the National Key Scientific Instrument and Equipment Development Project (NO.82027803).

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Antifade mounting mediumWuhan Xavier Biotechnology Co., LTDG1401
Anti-GFAP Mouse mAbWuhan Xavier Biotechnology Co., LTDGB12100-100
Anti-Neurofilament heavy polypeptide Mouse mAbWuhan Xavier Biotechnology Co., LTDGB12144-100
Anti-S100 beta Mouse mAbWuhan Xavier Biotechnology Co., LTDGB14146-100
BSAWuhan Xavier Biotechnology Co., LTDGC305010
CoverslipJiangsu Shitai experimental equipment Co., LTD10212432C
CY3-labeled goat anti-mouse IgGWuhan Xavier Biotechnology Co., LTDGB21302
DAPI Staining ReagentWuhan Xavier Biotechnology Co., LTDG1012
Decolorizing shakerWuhan Xavier Biotechnology Co., LTDDS-2S100
High Voltage Power Supply for nsPEFMatsusada Precision Inc.AU-60P1.6-L
Histochemical penWuhan Xavier Biotechnology Co., LTDG6100
Membrane breaking liquidWuhan Xavier Biotechnology Co., LTDG1204
Microscope slideWuhan Xavier Biotechnology Co., LTDG6012
Palm centrifugeWuhan Xavier Biotechnology Co., LTDMS6000
PBS powderedWuhan Xavier Biotechnology Co., LTDG0002
PipetteWuhan Xavier Biotechnology Co., LTD
Positive fluorescence microscopeNikon, JapanNIKON ECLIPSE C1
Rabbit Anti-SOX10/AF488 Conjugated antibodyBeijing Bioss Biotechnology Co., LTDBS-20563R-AF488
RSC96 Schwann cellsWuhan Xavier Biotechnology Co., LTDSTCC30007G-1
scanister3DHISTECHPannoramic MIDI
Special cable for nsPEFTimes Microwave SystemsM17/78-RG217
Turbine mixerWuhan Xavier Biotechnology Co., LTDMV-100

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RSC96 CellsCell ProliferationCell MigrationCCK-8 AssayScratch AssayS100-Beta ExpressionSox10 Expression
Regulating Schwann Cell Growth by Nanosecond Pulsed Electric Field for Peripheral Nerve Regeneration In Vitro
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