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.
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.
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.
1. Thawing of cryopreserved RSC96 cells
2. Cell passage:
NOTE: If the cell density reaches 80%-90%, it is ready for passage.
3. Operation of nsPEF device
4. Cell counting kit-8 (CCK-8) assay
5. Cell scratch assay
6. Immunofluorescence
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).
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.
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.
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.
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.
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.
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.
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.
The authors have nothing to disclose.
This work was funded by the National Key Scientific Instrument and Equipment Development Project (NO.82027803).
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 |