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 incubate at 37 &#176;C overnight.</li>
	<li>The next day, observe the cell growth and density under a microscope.</li>
</ol>
2. Cell passage:
NOTE: If the cell density reaches 80%-90%, it is ready for passage.
<ol>
	<li>Discard the culture medium and rinse the cells 1-2 times with phosphate-buffered saline (PBS) without calcium and magnesium ions.</li>
	<li>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 &#176;C for 1-2 min.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Transfer the cell suspension to a new T25 flask at a 1:2 ratio and add 7 mL of culture medium.</li>
</ol>
3. Operation of nsPEF device
<ol>
	<li>Resuspend the RSC96 cells in 1 mL of DMEM culture medium and transfer them to colorimetric dishes with electrodes on both sides.</li>
	<li>Turn on the power switch.</li>
	<li>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.</li>
	<li>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).</li>
</ol>
4. Cell counting kit-8 (CCK-8) assay
<ol>
	<li>Prepare a cell suspension of a particular concentration from RSC96 cells stimulated with electrical stimulation. Add 100 &#181;L 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.</li>
	<li>Take 10 &#181;L of CCK-8 solution from the kit and add it to the 96-well cell culture plate. Incubate in a CO2 incubator at 37 &#176;C for an additional 30 min to 4 h.</li>
	<li>Measure the absorbance. Use dual-wavelength measurement with a detection wavelength of 430-490 nm and a reference wavelength of 600-650 nm.</li>
	<li>Determine the field strength intensity for subsequent experiments based on the experimental results of cell proliferation. Select cells with good proliferation for subsequent experiments.</li>
</ol>
5. Cell scratch assay
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Aspirate the culture media and wash with PBS 2-3 times.</li>
	<li>Add 2 mL of serum-free medium to each well.</li>
	<li>Place the plate in the 37 &#176;C incubator. Take pictures under a fourfold magnification of the inverted microscope at 0 h and 24 h to observe changes in cell migration.</li>
</ol>
6. Immunofluorescence
<ol>
	<li>Cell permeabilization:
	<ol>
		<li>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.</li>
		<li>Add 50-100 &#181;L 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.</li>
	</ol>
	</li>
	<li>Serum Blocking: Add 3% BSA within the circles to cover the tissue uniformly. Incubate at RT for 30 min.</li>
	<li>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 &#176;C.</li>
	<li>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.</li>
	<li>DAPI nuclear staining:
	<ol>
		<li>Place the coverslip in PBS (pH 7.4) on a shaker and wash three times for 5 min each time.</li>
		<li>After gently drying the slide, add DAPI staining solution (2 &#181;g/mL, 0.5 mL per circle) within the circles and incubate at RT for 10 min in a dark room.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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.</li>
</ol>

Enhancing Peripheral Nerve Regeneration with nsPEF-Stimulated Schwann Cells

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The authors have nothing to disclose.

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 S100&#946;, 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.

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.

<table><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

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.

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 &#60; 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 S100&#946; 
S100&#946;, as a specific marker protein of SCs, participates in various functions, including neurite extension and axonal proliferation14. Additionally, S100&#946; 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 S100&#946; 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 S100&#946;-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 &#60; 0.001; ****P &#60; 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&#160;than that of the control group and the 10 kV/cm group, showing a significant statistical difference (*P&#60;0.05; **P &#60; 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 &#60; 0.0001; ***P &#60; 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 &#60; 0.05; *P &#60; 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).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66097/66097fig01.jpg" /> 
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 &#177; SD based on &#8805;3 replicates. NS not significant; *P &#60; 0.05; **P &#60; 0.01; **P &#60; 0.01 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66097/66097fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66097/66097fig02.jpg" /> 
Figure 2: Expression of S100&#946; in RSC96 cells stimulated at 5 kV/cm, 10 kV/cm, and controls. The results represent the mean &#177; SD based on &#8805;3 replicates. ***P &#60; 0.001; ****P &#60; 0.0001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66097/66097fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66097/66097fig03.jpg" /> 
Figure 3: Expression of NF-H in RSC96 cells stimulated at 5 kV/cm, 10 kV/cm, and controls. The results represent the mean &#177; SD based on &#8805;3 replicates. *P &#60; 0.05; **P &#60; 0.01. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66097/66097fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66097/66097fig04.jpg" /> 
Figure 4: Expression of GFAP in RSC96 cells stimulated at 5 kV/cm, 10 kV/cm, and controls. The results represent the mean &#177; SD based on &#8805;3 replicates. ****P &#60; 0.0001; ***P &#60; 0.001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66097/66097fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66097/66097fig05.jpg" /> 
Figure 5: Expression of Sox10 in RSC96 cells stimulated at 5 kV/cm, 10 kV/cm, and controls. The results represent the mean &#177; SD based on &#8805;3 replicates. *P &#60; 0.05; *P &#60; 0.05. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66097/66097fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66097/66097fig06.jpg" /> 
Figure 6: Cell scratching assay of RSC96 cells stimulated at 5 kV/cm, 10 kV/cm, and controls. The results represent the mean &#177; SD based on &#8805;3 replicates. **P &#60; 0.01; ***P &#60; 0.001. Statistical data is based on ordinary one-way ANOVA. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66097/66097fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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

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.

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Neuroscience

653 Views.  Zhejiang University. Ons onderzoek richt zich op het bevorderen van perifere zenuwregeneratie door nsPEF gestimuleerde zwanencellen. We hopen een nieuwe manier te ontwikkelen om zwanencellen efficiënt te activeren, aangezien zenuwregeneratie in de meeste gevallen vrij moeilijk is. We maken op innovatieve wijze gebruik van een nanoseconde post elektrisch veld nsPEF om zwanencellen te activeren.We zijn verheugd te ontdekken dat nsPEF een voorkeursmodus kan zijn voor perifere zenuwregeneratie met minimale st...

Video: Auteur in de schijnwerpers: innovatief gebruik van nsPEF om de regeneratie van perifere zenuwen te stimuleren

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

Bioengineering

Analyzing Murine Schwann Cell Development Along Growing Axons

The development of peripheral nerves is an intriguing process. Neurons send out axons to innervate specific targets, which in humans are often more than 100 cm away from the soma of the neuron. Neuronal survival during development depends on target-derived growth factors but also on the support of Schwann cells (SCs). To this end SC ensheath axons from the region of the neuronal soma (or the transition from central to peripheral nervous system) to the synapse or neuromuscular junction. Schwann cells are derivatives of the neural crest and migrate as precursors along emerging axons until the entire axon is covered with SCs. This shows the importance of SC migration for the development of the peripheral nervous system and underlines the necessity to investigate this process. In order to analyze SC development, a setup is needed which next to the SCs also includes their physiological substrate for migration, the axon. Due to intrauterine development in vivo time-lapse imaging, however, is not feasible in placental vertebrates like mouse (mus musculus). To circumvent this, we adapted the superior cervical ganglion (SCG) explant technique. Upon treatment with nerve growth factor (NGF) SCG explants extend axons, followed by SC precursors migrating along the axons from the ganglion to the periphery. The beauty of this system is that the SC are derived from a pool of endogenous SC and that they migrate along their own physiological axons which are growing at the same time. This system is especially intriguing, because the SC development along axons can be analyzed by time-lapse imaging, opening further possibilities to gain insights into SC migration.

Here we describe a Schwann cell (SC) migration assay in which SCs are able to develop along extending axons.

The  development of peripheral nerves is an intriguing process. Neurons send out  axons to innervate specific targets, which in humans are often more than ...

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) are structures containing the sensory neurons of the peripheral nervous system. When dissociated, they can be co-cultured with SC-like adipose-derived stem cells (ASC), providing a valuable model to study in vitro nerve regeneration and myelination, mimicking the in vivo environment at the injury site.

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the major glial cells of the PNS, play a vital role in promoting PNS regeneration by dedifferentiating into a regenerative cell phenotype following injury. However, the dedifferentiated state of SCs is challenging to maintain through the time-period needed for regeneration and is impacted by changes in the surrounding extracellular matrix (ECM). Therefore, determining the complex interplay between SCs and differing ECM to provide cues of regenerative potential of SCs is essential. To address this, a strategy was created where different ECM proteins were adsorbed onto a tunable polydimethylsiloxane (PDMS) substrate which provided a platform where stiffness and protein composition can be modulated. SCs were seeded onto the tunable substrates and critical cellular functions representing the dynamics of SC phenotype were measured. To illustrate the interplay between SC protein expression and cellular morphology, differing seeding densities of SCs in addition to individual microcontact printed cellular patterns were utilized and characterized by immunofluorescence staining and western blot. Results showed that cells with a smaller spreading area and higher extent of cellular elongation promoted higher levels of SC regenerative phenotypic markers. This methodology not only begins to unravel the significant relationship between the ECM and cellular function of SCs, but also provides guidelines for the future optimization of biomaterials in peripheral nerve repair.

This methodology aims to illustrate the mechanisms by which extracellular matrix cues such as substrate stiffness, protein composition and cell morphology regulate Schwann cell (SC) phenotype.

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the ...

Electric-Field-Induced Neural Precursor Cell Differentiation in Microfluidic Devices

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell culture system that was used for a long-term cell differentiation study using microscopy. The microfluidic system consists of the following major components: an optically transparent electrotactic chip, a transparent indium-tin-oxide (ITO) heater, a culture media-filling pump, an electrical power supply, a high-frequency power amplifier, an EF multiplexer, a programmable X-Y-Z motorized stage, and an inverted phase-contrast microscope equipped with a digital camera. The microfluidic system is beneficial in simplifying the overall experimental setup and, in turn, the reagent and sample consumption. This work involves the differentiation of neural stem and progenitor cells (NPCs) induced by direct current (DC) pulse stimulation. In the stem cell maintenance medium, the mouse NPCs (mNPCs) differentiated into neurons, astrocytes, and oligodendrocytes after the DC pulse stimulation. The results suggest that simple DC pulse treatment could control the fate of mNPCs and could be used to develop therapeutic strategies for nervous system disorders. The system can be used for cell culture in multiple channels, for long-term EF stimulation, for cell morphological observation, and for automatic time-lapse image acquisition. This microfluidic system not only shortens the required experimental time, but also increases the accuracy of control on the microenvironment.

In this study, we present a protocol for the differentiation of neural stem and progenitor cells (NPCs) solely induced by direct current (DC) pulse stimulation in a microfluidic system.

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell ...

Finite Element Modelling of a Cellular Electric Microenvironment

Clinical studies show electrical stimulation (ES) to be a potential therapy for the healing and regeneration of various tissues. Understanding the mechanisms of cell response when exposed to electrical fields can therefore guide the optimization of clinical applications. In vitro experiments aim to help uncover those, offering the advantage of wider input and output ranges that can be ethically and effectively assessed. However, the advancements in in vitro experiments are difficult to reproduce directly in clinical settings. Mainly, that is because the ES devices used in vitro differ significantly from the ones suitable for patient use, and the path from the electrodes to the targeted cells is different. Translating the in vitro results into in vivo procedures is therefore not straightforward. We emphasize that the cellular microenvironment's structure and physical properties play a determining role in the actual experimental testing conditions and suggest that measures of charge distribution can be used to bridge the gap between in vitro and in vivo. Considering this, we show how in silico finite element modelling (FEM) can be used to describe the cellular microenvironment and the changes generated by electric field (EF) exposure. We highlight how the EF couples with geometric structure to determine charge distribution. We then show the impact of time dependent inputs on charge movement. Finally, we demonstrate the relevance of our new in silico model methodology using two case studies: (i) in vitro fibrous Poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT-PSS) scaffolds and (ii) in vivo collagen in extracellular matrix (ECM).

This paper presents a strategy for building finite element models of fibrous conductive materials exposed to an electric field (EF). The models can be used to estimate the electrical input that cells seeded in such materials receive and assess the impact of changing the scaffold's constituent material properties, structure or orientation.

Clinical studies show electrical stimulation (ES) to be a potential therapy for the healing and regeneration of various tissues. Understanding the ...

Transplantation of Schwann Cells Inside PVDF-TrFE Conduits to Bridge Transected Rat Spinal Cord Stumps to Promote Axon Regeneration Across the Gap

Among various models for spinal cord injury in rats, the contusion model is the most often used because it is the most common type of human spinal cord injury. The complete transection model, although not as clinically relevant as the contusion model, is the most rigorous method to evaluate axon regeneration. In the contusion model, it is difficult to distinguish regenerated from sprouted or spared axons due to the presence of remaining tissue post injury. In the complete transection model, a bridging method is necessary to fill the gap and create continuity from the rostral to the caudal stumps in order to evaluate the effectiveness of the treatments. A reliable bridging surgery is essential to test outcome measures by reducing the variability due to the surgical method. The protocols described here are used to prepare Schwann cells (SCs) and conduits prior to transplantation, complete transection of the spinal cord at thoracic level 8 (T8), insert the conduit, and transplant SCs into the conduit. This approach also uses in situ gelling of an injectable basement membrane matrix with SC transplantation that allows improved axon growth across the rostral and caudal interfaces with the host tissue.

This article describes a technique to insert a hollow conduit between the spinal cord stumps after complete transection and fill with Schwann cells (SCs) and injectable basement membrane matrix in order to bridge and promote axon regeneration across the gap.

Among various models for spinal cord injury in rats, the contusion model is the most often used because it is the most common type of human spinal cord ...

Medicine

Purification of Fibroblasts and Schwann Cells from Sensory and Motor Nerves in Vitro

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and motor phenotypes involved in different patterns of neurotrophic factor gene expression and other biological processes, affecting nerve regeneration. The present study has established a protocol to obtain highly purified rat sensory and motor SCs and fibroblasts more rapidly. The ventral root (motor nerve) and the dorsal root (sensory nerve) of neonatal rats (7-days-old) were dissociated and the cells were cultured for 4-5 days, followed by isolation of sensory and motor fibroblasts and SCs by combining differential digestion and differential adherence methods sequentially. The results of immunocytochemistry and flow cytometry analyses showed that the purity of the sensory and motor SCs and fibroblasts were &#62;90%. This protocol can be used to obtain a large number of sensory and motor fibroblasts/SCs more rapidly, contributing to the exploration of sensory and motor nerve regeneration.

Here, we present a method to purify fibroblasts and Schwann cells from sensory and motor nerves in vitro.

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and ...

Implantation and Control of Wireless, Battery-free Systems for Peripheral Nerve Interfacing

Peripheral nerve interfaces are frequently used in experimental neuroscience and regenerative medicine for a wide variety of applications. Such interfaces can be sensors, actuators, or both. Traditional methods of peripheral nerve interfacing must either tether to an external system or rely on battery power that limits the time frame for operation. With recent developments of wireless, battery-free, and fully implantable peripheral nerve interfaces, a new class of devices can offer capabilities that match or exceed those of their wired or battery-powered precursors. This paper describes methods to (i) surgically implant and (ii) wirelessly power and control this system in adult rats. The sciatic and phrenic nerve models were selected as examples to highlight the versatility of this approach. The paper shows how the peripheral nerve interface can evoke compound muscle action potentials (CMAPs), deliver a therapeutic electrical stimulation protocol, and incorporate a conduit for the repair of peripheral nerve injury. Such devices offer expanded treatment options for single-dose or repeated dose therapeutic stimulation and can be adapted to a variety of nerve locations.

This is a protocol for the surgical implantation and operation of a wirelessly powered interface for peripheral nerves. We demonstrate the utility of this approach with examples from nerve stimulators placed on either the rat sciatic or phrenic nerve.

Peripheral nerve interfaces are frequently used in experimental neuroscience and regenerative medicine for a wide variety of applications. Such interfaces ...

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

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