detecting the expression of the neurotrophic and transcription factors in nanosecond pulsed electric field (nspef) stimulated rsc96 cells

用 nsPEF 刺激的雪旺细胞增强周围神经再生

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,&#160;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&#39;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.

作者聚焦：创新使用 nsPEF 促进周围神经再生

通过纳秒脉冲电场调节雪旺细胞生长以促进体外周围神经再生

Our research focuses on promoting peripheral nerve regeneration by nsPEF stimulated swan cells. We hope to develop a new way to activate swan cells efficiently as nerve regeneration is quite difficult in most cases. We innovatively use a nanosecond post electric field nsPEF to activate swan cells.We are delighted to find that nsPEF may be a preferred mode for peripheral nerve regeneration with minimal stimulation side effects. Besides our protocol provide a reliable basis for subsequent in depth and systematic research. Our laboratory will continually explore the application of nsPEF.In terms of nerve regeneration, we will build up animal models to promote in vivo testing and evaluate the safety of nsPEF in vivo. As there is limited evidence regarding the application of nsPEF in referral nerve regeneration, we hope we can make breakthroughs.

检测纳秒脉冲电场 （nsPEF） 刺激的 RSC96 细胞中神经营养因子和转录因子的表达

首先，在培养皿中取出电刺激的 RSC96 细胞。然后，使用组织学笔在细胞均匀分布在盖玻片上的位置画圆圈。向细胞中加入 50 至 100 微升透化工作溶液，并在室温下孵育 20 分钟。然后，用 PBS 洗涤细胞三次，每次 5 分钟。接下来，在圆圈内加入 3% BSA 以均匀覆盖组织，并在室温下孵育 30 分钟。然后，轻轻去除封闭溶液，并将适当稀释的一抗添加到细胞中。将细胞培养板置于潮湿的盒子中，并在 4 摄氏度下孵育过夜。然后，用 PBS 洗涤细胞 3 次，每次 5 分钟，同时连续摇晃。加入相应的二抗，在室温下孵育 50 分钟。在孵育结束时，将盖玻片放入 PBS 中并洗涤 3 次，每次摇晃 5 分钟。然后，让载玻片干燥，并将DAPI染色溶液添加到干燥的载玻片中。最后，用抗褪色封固剂密封干燥的盖玻片，以获得荧光。使用 Alexa Fluor 488、SCI3 和 SCI5 的特定激发和发射波长采集图像。显微镜分析显示，所有组中均有红色的细胞质 S-100 β 阳性细胞散射，与对照组和 10 千伏/厘米组相比，在 5 千伏/厘米组中观察到 S-100 β 荧光的积分光密度增加。在细胞爬行试验期间，在所有组中都观察到具有分散细胞质的GFAP和Sox10阳性细胞。然而，与对照组和10千伏特/厘米组相比，5千伏/厘米组的GFAP表达显着降低。5 千伏/厘米组的 RSC96 细胞的 Sox10 表达平均灰度值显着更高，表明 Sox10 表达增强。

detecting-expression-neurotrophic-transcription-factors-nanosecond

Research

JoVE Journal

Neuroscience

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

23 次观看.  Zhejiang University. 首先，在培养皿中取出电刺激的 RSC96 细胞。然后，使用组织学笔在细胞均匀分布在盖玻片上的位置画圆圈。向细胞中加入 50 至 100 微升透化工作溶液，并在室温下孵育 20 分钟。然后，用 PBS 洗涤细胞三次，每次 5 分钟。接下来，在圆圈内加入 3% BSA 以均匀覆盖组织，并在室温下孵育 30 分钟。然后，轻轻去除封闭溶液，并将适当稀释的一抗添加到细胞中。将细胞培?...

视频: 检测纳秒脉冲电场 （nsPEF） 刺激的 RSC96 细胞中神经营养因子和转录因子的表达

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

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.

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 ...

检测纳秒脉冲电场 （nsPEF） 刺激的 RSC96 细胞中神经营养因子和转录因子的表达

检测纳秒脉冲电场（nsPEF）刺激的 RSC96 细胞中神经营养因子和转录因子的表达