Name: Author Spotlight: Innovative Use of nsPEF to Boost Peripheral Nerve Regeneration
Uploaded: 2024-05-03T13:30:00
Description: Read this Scientific Article Protocol about Author Spotlight: Innovative Use of nsPEF to Boost Peripheral Nerve Regeneration at JoVE.com

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

1. Thawing of cryopreserved RSC96 cells
<ol>
	<li>Thaw the cryovial containing 1 mL of cell suspension by rapidly shaking it in a 37 &#176;C water bath, and then add it to a centrifuge tube containing 4-6 mL of complete culture medium and mix well.</li>
	<li>Centrifuge at 1000 x g for 3-5 min, discard the supernatant and resuspend the cells in 3 mL of complete culture medium.</li>
	<li>Add the cell suspension to a culture flask (or dish) containing 6-8 mL of complete culture medium and incubat...

Enhancing Peripheral Nerve Regeneration with nsPEF-Stimulated Schwann Cells

<ol>
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B., Dun, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migrating+Schwann+cells+direct+axon+regeneration+within+the+peripheral+nerve+bridge.">Migrating Schwann cells direct axon regeneration within the peripheral nerve bridge.</a> Glia. 69 (2), 235-254 (2021).</li><li>Zhang, Y., Zhao, Q., Chen, Q., Xu, L., Yi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+control+of+peripheral+nerve+regeneration.">Transcriptional control of peripheral nerve regeneration.</a> Mol Neurobiol. 60 (1), 329-341 (2023).</li><li>Nishi, M., Kawata, M., Azmitia, E. 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E., N&#250;&#241;ez, G., Villanelo, F., Perez-Acle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanosecond+pulsed+electric+field+(nsPEF):+Opening+the+biotechnological+Pandora's+box.">Nanosecond pulsed electric field (nsPEF): Opening the biotechnological Pandora's box.</a> Int J Mol Sci. 23 (11), 6158 (2022).</li><li>Nuccitelli, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First-in-human+trial+of+nanoelectroablation+therapy+for+basal+cell+carcinoma:+proof+of+method.">First-in-human trial of nanoelectroablation therapy for basal cell carcinoma: proof of method.</a> Exp Dermatol. 23 (2), 135-137 (2014).</li><li>Nuccitelli, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-thermal+nanoelectroablation+of+UV-induced+murine+melanomas+stimulates+an+immune+response.">Non-thermal nanoelectroablation of UV-induced murine melanomas stimulates an immune response.</a> Pigment Cell Melanoma Res. 25 (5), 618-629 (2012).</li><li>Carr, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+nanosecond+pulsed+electric+field+(nsPEF)+can+affect+membrane+permeabilization+and+cellular+viability+in+a+3D+spheroids+tumor+model.">A nanosecond pulsed electric field (nsPEF) can affect membrane permeabilization and cellular viability in a 3D spheroids tumor model.</a> Bioelectrochemistry. 141, 107839 (2021).</li><li>Hornef, J., Edelblute, C. 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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...

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.

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			<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>
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		<tr>
			<td>Membrane breaking liquid</td>
			<td>Wuhan Xavier Biotechnology Co., LTD</td>
			<td>G1204</td>
			<td></td>
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		<tr>
			<td>Microscope slide</td>
			<td>Wuhan Xavier Biotechnology Co., LTD</td>
			<td>G6012</td>
			<td></td>
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		<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>
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			<td>scanister</td>
			<td>3DHISTECH</td>
			<td>Pannoramic MIDI</td>
			<td></td>
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			<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>
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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.

Author Spotlight: Innovative Use of nsPEF to Boost Peripheral Nerve Regeneration

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

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...

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

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 ...

Unsere Forschung konzentriert sich auf die Förderung der peripheren Nervenregeneration durch nsPEF-stimulierte Schwanenzellen. Wir hoffen, einen neuen Weg zu entwickeln, um Schwanenzellen effizient zu aktivieren, da die Nervenregeneration in den meisten Fällen recht schwierig ist. Wir verwenden auf innovative Weise ein Nanosekunden-postelektrisches Feld nsPEF, um Schwanenzellen zu aktivieren.Wir freuen uns, dass nsPEF ein bevorzugter Modus für die periphere Nervenregeneration mit minimalen Stimulationsnebenwirkungen sein kann. Darüber hinaus bietet unser Protokoll eine verlässliche Grundlage für die anschließende vertiefte und systematische Forschung. Unser Labor wird die Anwendung von nsPEF kontinuierlich untersuchen.Im Hinblick auf die Nervenregeneration werden wir Tiermodelle aufbauen, um In-vivo-Tests zu fördern und die Sicherheit von nsPEF in vivo zu bewerten. Da es nur begrenzte Evidenz für die Anwendung von nsPEF bei der Regeneration von Überweisungsnerven gibt, hoffen wir, dass wir Durchbrüche erzielen können.