本项工作由国家重点科学仪器设备发展专项（NO.82027803）资助。

1. 冻存 RSC96 细胞的解冻
<ol><li>通过在37°C水浴中快速摇动解冻含有1mL细胞悬液的冻存管，然后将其加入含有4-6mL完全培养基的离心管中并充分混合。</li>
	<li>以1000× g 离心3-5分钟，弃去上清液并将细胞重悬于3mL完全培养基中。</li>
	<li>将细胞悬浮液加入含有6-8mL完全培养基的培养瓶（或培养皿）中，并在37°C下孵育过夜。</li>
	<li>第二天，在显微镜下观察细胞生长和密度。</li>
</ol>2.细胞传代：
注意：如果细胞密度达到80%-90%，则准备传代。
<ol><li>丢弃培养基，用不含钙离子和镁离子的磷酸盐缓冲盐水 （PBS） 冲洗细胞 1-2 次。</li>
	<li>向培养瓶中加入0.25%（w / v）胰蛋白酶-0.53mM EDTA（T25烧瓶为1-2mL，T75烧瓶为2-3mL），并在37°C下孵育1-2分钟。</li>
	<li>在显微镜下观察细胞脱离。如果大多数细胞变圆并脱落，请迅速将烧瓶放回工作区域，轻轻敲击烧瓶，然后加入 3-4 mL 含有 10% FBS 的培养基以停止消化。</li>
	<li>混合内容物，吸出溶液，并以1000× g 离心5分钟。然后，丢弃上清液并通过加入 1-2 mL 新鲜培养基并轻轻移液来重悬细胞。</li>
	<li>将细胞悬液以 1：2 的比例转移到新的 T25 烧瓶中，并加入 7 mL 培养基。</li>
</ol>3. nsPEF设备的操作
<ol><li>将 RSC96 细胞重悬于 1 mL DMEM 培养基中，并将它们转移到两侧都有电极的比色培养皿中。</li>
	<li>打开电源开关。</li>
	<li>通过旋转仪器上的旋钮来调整参数以改变电场的强度。本研究中设定的强度为 5 kV/cm、10 kV/cm、20 kV/cm 和 40 kV/cm。</li>
	<li>小心地旋转电极，直到出现火花，让电池根据预设的场强强度接收 5 个 nsPEF 脉冲，然后立即分离两个电极。在这种处理之后，取用nsPEF处理的实验组细胞和未处理的对照组细胞，在一定时期的细胞培养（本实验为1天）后分别进行第4-6节。</li>
</ol>4. 细胞计数试剂盒-8 （CCK-8） 检测
<ol><li>从用电刺激刺激的 RSC96 细胞中制备特定浓度的细胞悬液。将 100 μL 细胞悬液添加到 96 孔细胞培养板的每个孔中。考虑到 CCK-8 测定的要求，将试剂盒中的细胞总数控制在 1 x 103 和 1 x 106 之间。</li>
	<li>从试剂盒中取出 10 μL CCK-8 溶液，并将其添加到 96 孔细胞培养板中。在37°C的CO2 培养箱中再孵育30分钟至4小时。</li>
	<li>测量吸光度。使用双波长测量，检测波长为 430-490 nm，参考波长为 600-650 nm。</li>
	<li>根据细胞增殖的实验结果确定后续实验的场强强度。选择增殖良好的细胞进行后续实验。</li>
</ol>5. 细胞划痕测定
<ol><li>在六孔板的每个孔中，接种 3 x 105 个细胞，每孔总体积为 2 mL。大约72小时后，细胞将覆盖孔。分别对实验组和对照组进行试验。</li>
	<li>使用移液器吸头在培养孔的底部画一条水平线。确保移液器吸头垂直握住，并尽量避免倾斜。</li>
	<li>吸出培养基并用PBS洗涤2-3次。</li>
	<li>向每个孔中加入 2 mL 无血清培养基。</li>
	<li>将板置于37°C培养箱中。在0小时和24小时倒置显微镜的四倍放大下拍照，观察细胞迁移的变化。</li>
</ol>6. 免疫荧光
<ol><li>细胞通透性：<ol><li>轻轻地将细胞悬浮液移液到培养皿中后，用组织学笔在细胞均匀分布在盖玻片上的位置画圆圈。对照组和不同实验组分别治疗。</li>
		<li>加入 50-100 μL 透化工作溶液 （0.25-0.5% Triton X-100），并在室温 （RT） 下孵育 20 分钟。用PBS洗涤3次，每次5分钟。</li>
	</ol></li>
	<li>血清阻断：在圆圈内添加 3% BSA，以均匀覆盖组织。在室温下孵育 30 分钟。</li>
	<li>一抗孵育：轻轻去除封闭溶液，并将适当稀释的一抗（小鼠来源，以 1：300 稀释）加入细胞孔中。将细胞培养板置于潮湿的盒子中，并在4°C下孵育过夜。</li>
	<li>二抗孵育：将细胞板放在振荡器上，洗涤 3 次，每次 5 分钟。加入相应的二抗（CY3 标记的山羊抗小鼠 IgG，以 1：300 稀释）并在室温下孵育 50 分钟。</li>
	<li>DAPI核染色：<ol><li>将盖玻片放入PBS（pH 7.4）中，放在振荡器上，洗涤3次，每次5分钟。</li>
		<li>轻轻干燥载玻片后，在圆圈内加入DAPI染色溶液（2μg/ mL，每圈0.5mL），并在室温下在暗室中孵育10分钟。</li>
	</ol></li>
	<li>安装：将盖玻片放入PBS（pH 7.4）中，放在振荡器上，洗涤3次，每次5分钟。轻轻干燥载玻片后，用抗褪色封固剂密封盖玻片以获得荧光。</li>
	<li>图像采集：在330-380nm的波长处激发DAPI，并在420nm处检测发射;AF488 在 465-495 nm 处激发并检测 515-555 nm 处的发射;在 510-560 nm 处激发，在 590 nm 处检测 CY3 的发射;在 608-648 nm 处激发，并在 672-712 nm 处检测 CY5 的发射。</li>
</ol>

用 nsPEF 刺激的雪旺细胞增强周围神经再生

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作者没有什么可透露的。

据报道，近年来，nsPEF的应用经历了推动增长。nsPEF 仅对所需区域具有高度针对性的影响，提供足够的能量进行治疗而不会造成额外的热损伤，使其对人体更安全28.这些特性使其在肿瘤治疗和神经再生方面具有广阔的转化前景。然而，一些研究提出了nsPEF的一些局限性。与材料研究相比，ES受到外部电源和电线29的约束。此外，最近的一项研究证明，围手术期使?...

每年有数百万人受到涉及周围神经系统 （PNS） 和中枢神经系统 （CNS） 的神经损伤的影响1。研究表明，神经损伤后中枢神经系统的轴突修复能力相当有限，而 PNS 由于 SCs2 的显着可塑性而显示出增强的能力。然而，在周围神经损伤后实现完全再生仍然艰巨，并继续对人类健康构成重大挑战 3,4。如今，尽管存在供体部位发病率高和可用性有限的缺点，但自体移植物仍然是一种常见的治疗方法5.这种情况促使研究人员探索替代疗法，包括材料6、分子因子7 和电刺激 （ES）。作为促进轴突生长和神经再生的一个因素8，选择合适的 ES 方法并探索 ES 和 SCs 之间的关系变得至关重要。
SCs 是 PNS 的主要神经胶质细胞，在 PNS 9,10 的再生中起着至关重要的作用。周围神经损伤后，SC 经历快速激活、广泛的重编程2，并从髓鞘形成状态过渡到生长支持形态以进行神经再生2。SC 的大量增殖发生在损伤神经的远端，而远端残端的 SC 经历增殖和伸长以形成 Bungner 带，这是引导轴突向靶器官生长所必需的11。此外，来自近端和远端神经残端的 SC 迁移到神经桥中形成 SC 索，促进轴突再生12。此外，先前的研究表明，在周围神经再生的情况下，与SCs相关的因子的合成和分泌会发生变化，包括转录因子13、神经营养因子14和髓鞘形成调节因子13。基于这些，促进SC增殖、迁移、合成和相关因子的分泌，在改善周围神经再生方面已被广泛研究15。
先前的研究表明了使用 ES 进行神经再生的可能性1。一个被广泛接受的解释是，ES 可以通过改变这些生物分子上的电荷分布来诱导细胞膜的去极化，改变膜电位并影响膜蛋白的功能1。然而，广泛应用的强烈 PEF 可能会导致剧烈疼痛、不自主肌肉收缩和心脏颤动8.它还会增加肌酸激酶 （CK） 活性，降低肌肉力量，并诱导迟发性肌肉酸痛 （DOMS）16 的发展。nsPEF 是一种新兴技术，它在纳秒脉冲持续时间内用高压电场刺激测试对象，并逐渐用于细胞水平的研究17,18。先前的研究表明，nsPEF促进细胞增殖和细胞器活性的可能原理是膜纳米孔的形成和离子通道的激活，这导致细胞质Ca2+浓度的增加19。nsPEF 利用脉冲功率技术对细胞膜进行充电，产生的脉冲具有持续时间短、上升时间快、功率高、能量密度低等特点20.这些特征表明，nsPEF 可能是一种首选模式，刺激副作用最小8。此外，与手术干预相比，nsPEF 具有微创手术、可逆性、可调节性和对神经组织的非破坏性等优势。nsPEF在生物医学领域的一个主流研究方向是其在利用高能电场刺激进行肿瘤组织消融方面的应用21,22,23。一些研究结果表明，12-nsPEF 可以刺激周围神经而不会造成损伤24.然而，目前，关于nsPEF在神经再生领域的应用证据有限。此外，使用 nsPEF 刺激 SC 是一项开创性的尝试，有助于进一步的体内和临床研究。本研究探讨了nsPEF刺激SCs是否能促进神经再生，为后续的深入系统研究提供可靠依据。

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

regulating schwann cell growth by nanosecond pulsed electric field for peripheral nerve regeneration in vitro

雪旺细胞 （SC） 是周围神经系统的髓鞘化细胞，在周围神经再生中起着至关重要的作用。纳秒脉冲电场 （nsPEF） 是一种适用于神经电刺激的新兴方法，已被证明可有效刺激细胞增殖和其他生物过程。为了评估 SC 在 nsPEF 下是否发生显着变化并帮助探索新的周围神经再生方法的潜力，将培养的 RSC96 细胞在 5 kV 和 10 kV 下进行 nsPEF 刺激，然后继续培养 3-4 天。随后，评估了SCs表达的一些相关因子，以证明刺激成功，包括特异性标记蛋白、神经营养因子、转录因子和髓鞘形成调节因子。代表性结果表明，nsPEF显著增强了SCs的增殖和迁移，并增强了合成对周围神经再生有积极贡献的相关因子的能力。同时，GFAP表达较低提示周围神经损伤的良性预后。所有这些结果表明，nsPEF作为一种有效的治疗方法，通过刺激SCs治疗周围神经损伤具有巨大的潜力。

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.

低强度脉冲电场刺激细胞增殖 CCK-8实验显示，5 kV/cm组细胞中RSC96的增殖速率明显快于对照组细胞。然而，随着参数的增加（20 kV/cm和40 kV/cm），增殖率不稳定，甚至低于对照组。40 kV/cm组RSC96细胞增殖率明显低于对照组和5 kV/cm组，差异有统计学意义（P < 0.05）。由于不满足细胞增殖的实验要求，在后续实验中排除了20 kV/cm和40 kV/cm组（图1）。
<p class="jove_content"...

Read this Scientific Article Protocol about Author Spotlight: Innovative Use of nsPEF to Boost Peripheral Nerve Regeneration at JoVE.com

在这里，我们提出了一种应用纳秒脉冲电场（nsPEF）在 体外刺激雪旺细胞的方案。相关因子的合成和分泌能力以及细胞行为变化验证了nsPEF刺激的成功性。该研究对周围神经再生方法给出了积极的看法。

Schwann cells (SCs) are myelinating cells of the peripheral nervous system, playing a crucial role in peripheral nerve regeneration. Nanosecond Pulse ...

我们的研究重点是通过nsPEF刺激的天鹅细胞促进周围神经再生。我们希望开发一种新方法来有效地激活天鹅细胞，因为在大多数情况下，神经再生是相当困难的。我们创新性地使用纳秒后电场nsPEF来激活天鹅细胞。我们很高兴地发现，nsPEF可能是周围神经再生的首选模式，同时刺激副作用最小。此外，我们的协议为后续的深入和系统的研究提供了可靠的基础。我们的实验室将不断探索nsPEF的应用。在神经再生方面，我们将建立动物模型，以促进体内试验，并评估nsPEF在体内的安全性。由于关于nsPEF在转诊神经再生中的应用的证据有限，我们希望我们能取得突破。

regulating-schwann-cell-growth-nanosecond-pulsed-electric-field-for

Research

JoVE Journal

Neuroscience

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

409 次观看.  Zhejiang University. 我们的研究重点是通过nsPEF刺激的天鹅细胞促进周围神经再生。我们希望开发一种新方法来有效地激活天鹅细胞，因为在大多数情况下，神经再生是相当困难的。我们创新性地使用纳秒后电场nsPEF来激活天鹅细胞。我们很高兴地发现，nsPEF可能是周围神经再生的首选模式，同时刺激副作用最小。此外，我们的协议为后续的深入和系统的研究提供了可靠的基础。我们的实验?...

视频: 作者聚焦：创新使用 nsPEF 促进周围神经再生

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.