We thank Prof. Dr. Ralf Gold and PD Dr. Gisa Ellrichmann for their advice and support.

All procedures were performed in accordance with the European Communities Council Directive for the care and use of laboratory animals.
1. Schwann cell culture
<ol>
	<li>Coating for Schwann cell culture
	<ol>
		<li>Coat the cell culture dishes under sterile conditions. Apply 2 mL of 0.01% poly-L-lysine (PLL) to two 60 mm tissue culture (TC) dishes each and incubate overnight at 4 &#176;C.</li>
		<li>Remove the PLL, wash the TC dishes 2x with distilled water, and incubate with 2 mL of 1 &#181;g/cm2 laminin overnight at 4 &#176;C. Wash the TC dishes 2x with aqua dest, and let the plates air-dry.</li>
	</ol>
	</li>
	<li>Medium preparation for Schwann cell culture
	<ol>
		<li>Prepare 50 mL of Schwann cell medium by adding 10% heat-inactivated fetal calf serum (FCS), 2 &#181;M forskolin, 10 nM neuregulin, and 50 &#181;g/mL gentamycin to Dulbecco&#8242;s Modified Eagle&#8242;s Medium (DMEM)/F-12 (high glucose) under sterile conditions.</li>
		<li>Prepare 70 mL of Leibovitz&#39;s L-15 medium with 50 &#181;g/mL gentamycin under sterile conditions.</li>
	</ol>
	</li>
	<li>Sciatic nerve preparation 
	NOTE: All sciatic nerve preparation steps are performed under a clean bench.
	<ol>
		<li>Prepare one 100 mm TC dish with 5 mL of ice-cold Dulbecco&#39;s phosphate-buffered saline (DPBS) without Ca2+ and Mg2+, one 100 mm TC dish with 5 mL of ice-cold Leibovitz&#39;s L-15 medium, and one 100 mm TC dish with 5 mL of ice-cold Leibovitz&#39;s L-15 medium and 50 &#181;g/mL gentamycin.</li>
		<li>Clean all the instruments by autoclaving. Spray the instruments and working area with 70% ethanol.</li>
		<li>Euthanize five 3-week-old male Sprague Dawley rats using CO2 inhalation and decapitation. Spray the rat&#39;s torso with 70% ethanol.</li>
		<li>Open the dorsal lower left limb with scissors and remove the biceps femoris muscle carefully. Loosen the sciatic nerve by smooth elevation with curved forceps, ensuring not to bruise the nerve.</li>
		<li>Hold the most proximal part of the nerve with the curved forceps to straighten the nerve, and clip the nerve as high as possible using scissors. Then, clip the nerve close to the sacral plexus and the paw with scissors. Repeat steps 1.3.4-1.3.5 for the right side. 
		NOTE: While opening the limb, take care not to bruise any blood vessels.</li>
		<li>Using forceps, put the left and right sciatic nerves into a 100 mm TC dish with ice-cold DPBS.</li>
	</ol>
	</li>
	<li>Sciatic nerve refurbishment
	<ol>
		<li>Use forceps to transfer all the nerves to a 100 mm TC dish with ice-cold Leibovitz&#39;s L-15 medium and 50 &#181;g/mL gentamycin. Continue using a stereomicroscope and remove fat, muscle, and blood vessels from the nerves with two pairs of fine forceps. Grab the nerves with forceps and transfer them to a 100 mm TC dish with ice-cold Leibovitz&#39;s L-15 medium.</li>
		<li>Identify the proximal and distal ends of the sciatic nerve. Remove the epineurium with one pair of fine forceps in a proximal to distal direction, while holding the proximal nerve end with the second pair of fine forceps.</li>
		<li>Transfer the purified nerves to a 100 mm TC dish with ice-cold Leibovitz&#39;s L-15 medium and 50 &#181;g/mL gentamycin. Tease the isolated nerve fascicles to separate and isolate single nerve fibers using two pairs of fine forceps.</li>
		<li>Transfer the nerve fibers to a 50 mL tube using a 10 mL serological pipette and take up as little medium as possible. Add 50 mL of Leibovitz&#39;s L-15 medium with 50 &#181;g/mL gentamycin to the nerve fibers and slew a few times in the 50 mL tube.</li>
	</ol>
	</li>
	<li>Enzymatic digestion of the sciatic nerve 
	&#8203;NOTE: The next steps (steps 1.5-1.8, 2.1, and 2.2) are performed under sterile conditions.
	<ol>
		<li>Prepare the enzymatic digestion solution containing 0.25% dispase II, 0.05% type I collagenase, and 50 &#181;g/mL gentamycin in 10 mL of DMEM (high glucose).</li>
		<li>Centrifuge the tube at 188 x g for 5 min at 4 &#176;C, remove the supernatant with a 25 mL serological pipette ,and transfer the pellet with the remaining Leibovitz&#39;s L-15 medium into a 60 mm TC dish using a 1,000 mL pipette.</li>
		<li>Rinse the 50 mL tube with 10 mL of the enzymatic digestion solution and add it to the dish containing the nerve fibers. Distribute the tissue in the dish carefully with the tip of a pipette to maximize the accessible surface for digestion.</li>
		<li>Incubate at 37 &#176;C and 5% CO2 for 18 h, and stop the digestion by adding 10 mL of 40% FCS in Hanks&#39; balanced salt solution, without Ca2 and Mg2+ (HBSS).</li>
	</ol>
	</li>
	<li>Cell separation
	<ol>
		<li>Transfer the digested nerves into a 50 mL tube using a serological pipette and centrifuge at 188 x g for 10 min at 4 &#176;C. Discard the supernatant and resuspend the pellet in 10 mL of DMEM containing 10% FCS and 50 &#181;g/mL gentamycin. Resuspend the pellet 20 times subsequently, using a 10 mL, 5 mL, 2 mL, 1 mL, and 200 &#181;L pipette tip.</li>
		<li>Filter the cell suspension through a 100 &#181;m cell strainer and centrifuge at 188 x g for 10 min at 4 &#176;C. Discard the supernatant and resuspend the pellet with 4 mL of DMEM containing 10% FCS and 50 &#181;g/mL gentamycin.</li>
		<li>Add 2 mL of the cell suspension to each of the two PLL- and laminin-coated 60 mm TC dishes, and incubate at 37 &#176;C and 5% CO2. Leave the plates untouched for 2 days in the incubator to protect the cells from mechanical stress and to support adherence.</li>
	</ol>
	</li>
	<li>Schwann cell differentiation
	<ol>
		<li>After 2 days, remove the medium and carefully rinse the plates 2x with DMEM (high glucose), 10% FCS, and 50 &#181;g/mL gentamycin. Afterward, add 2 mL of Schwann cell medium. Replace the Schwann cell medium every 2nd&#160;day, and observe the cell appearance and confluency using a microscope. 
		&#8203;NOTE: Schwann cells and DRG explants need to be prepared in a timely, coordinated manner for the coculture. Make sure a rat with embryos at E 13.5 is available for DRG preparation when the Schwann cells are close to a confluency of 80%.</li>
	</ol>
	</li>
	<li>Cell trypsinization and magnetic separation 
	NOTE: For exemplary pictures of different Schwann cell culture stages, see Supplementary Figure 1.
	<ol>
		<li>When the cells reach a confluency of about 80% (6-12 days of culture), carefully wash the plates 2x with 3 mL of DPBS and incubate with 2 mL of 0.05% Trypsin/EDTA (prewarmed to 37 &#176;C) for 3 min. When the cells detach from the plate bottom, inactivate digestion by the addition of 2 mL of DMEM with 10% FCS and 50 &#181;g/mL gentamycin. 
		NOTE: Stick to a trypsinization time of strictly 3 min, and proceed rapidly afterward.</li>
		<li>Resuspend the cell pellet in 2 mL of magnetic cell separation buffer containing DPBS with 0.5% bovine serum albumin (BSA) and 2 nM EDTA. Combine 10 &#181;L of the cell suspension with 10 &#181;L of trypan blue and count the cells using a staining chamber.</li>
		<li>Centrifuge the cell suspension at 188 x g for 10 min at 4 &#176;C and resuspend the cell pellet in 90 &#181;L of magnetic cell separation buffer per 1 x 107 cells. Add 10 &#181;L of Thy-1 microbeads per 1 x 107 cells. Resuspend the solution a few times and incubate for 15 min in the dark at 8 &#176;C.</li>
		<li>Add 2 mL of the magnetic cell separation buffer to the cell suspension and centrifuge at 300 x g for 10 min at 4 &#176;C. Discard the supernatant and resuspend the pellet in 500 &#181;L of magnetic cell separation buffer.</li>
		<li>Moisten the magnetic cell separation column with 1 mL of the magnetic cell separation buffer. Place the magnetic cell separation column in the magnetic cell separator. Apply the cells to the magnetic cell separation column. Collect the flow through and centrifuge at 300 x g at 4 &#176;C for 10 min. 
		&#8203;NOTE: Fibroblasts are positively selected and remain in the column, while Schwann cells pass the column. Fibroblasts can be collected with a stamp (e.g., as a negative control for Schwann cell staining protocols).</li>
		<li>Discard the supernatant and resuspend the pellet in 1 mL of the coculture medium (see step 3.1.1). Count the cells after staining with trypan blue and a staining chamber.</li>
	</ol>
	</li>
</ol>
2. DRG explant culture
<ol>
	<li>DRG growth medium preparation
	<ol>
		<li>Prepare DRG growth medium by adding 2% B27, 2% horse serum, 1% L-glutamine, 0.5% penicillin/streptomycin, and 10 ng/mL nerve growth factor (NGF) to the neurobasal medium. Store the growth medium at 4 &#176;C. 
		NOTE: The growth medium can be used for 2 days.</li>
	</ol>
	</li>
	<li>Coating for DRG explants
	<ol>
		<li>Incubate the coverslips in 70% ethanol for 1 h and place in the wells of 4-well dishes using curved forceps. After the ethanol has dried, apply 300 &#181;L of 0.2 mg/mL poly-D-lysine (PDL) per well and incubate overnight at 37 &#176;C and 5% CO2.</li>
		<li>Wash the coverslips 3x for 5 min each with DPBS consecutively. Take off the DPBS, apply 300 &#181;L of 1 &#181;g/mL laminin to the coverslips, and incubate overnight at 37 &#176;C and 5% CO2.</li>
		<li>After three washing steps with DPBS for 5 min, replace the DPBS with 190 &#181;L of DRG growth medium. Place the 4-well plates into the incubator at 37 &#176;C and 5% CO2.</li>
	</ol>
	</li>
	<li>DRG preparation 
	NOTE: Harvest the DRG of embryonic rats under a clean bench.
	<ol>
		<li>Before preparation, clean all the instruments with 70% ethanol. Fill 10 (two per embryo) 35 mm TC dishes with 2 mL of ice-cold HBSS per dish, and two 100 mm TC dishes with 5 mL of ice-cold HBSS per dish.</li>
		<li>Euthanize the pregnant rats (adult female Sprague Dawley rats, E13.5) by CO2 inhalation and decapitation.</li>
		<li>Spray the body with 70% ethanol and open the ventral torso of the rat. Carefully remove the uterus and place it into a 100 mm TC dish with ice-cold HBSS.</li>
		<li>Hold the uterus using curved forceps and open the uterus wall with fine forceps. Remove one amniotic sac and open it carefully by pinching a hole with fine forceps.</li>
		<li>Remove the embryo from the surrounding tissues, cut the umbilical cord, and decapitate the embryo using fine forceps. Place the torso into a 100 mm TC dish filled with HBSS using curved forceps and a spatula.</li>
		<li>Quickly remove all the embryos from the uterus and transfer them into one 100 mm TC dish filled with HBSS. If there are more than five embryos, prepare an additional 35 mm TC dish with 2 mL of HBSS, according to step 2.3.1.</li>
		<li>Gently place one embryo torso into a 35 mm TC dish filled with HBSS using a spatula and curved forceps. Under a stereomicroscope, open the dorsal part of the torso to divide the embryo into two halves using fine forceps and micro scissors. Turn one half to the side and identify the strand of DRG located in a line at the dorsal part of the embryo.</li>
		<li>Cut out the DRG as a whole strand using fine forceps and micro scissors. Place the DRG in a fresh 35 mm TC dish filled with 2 mL of HBSS, and separate a single DRG from the remaining tissue using fine forceps and micro scissors.</li>
	</ol>
	</li>
	<li>DRG cell culture
	<ol>
		<li>Take 4-well plates containing 190 &#181;L of DRG growth medium from the incubator to the clean bench where the DRG are to be prepared. Transfer a single DRG carefully into one well of a 4-well culture plate using fine forceps and a spatula. Place the DRG into the center of each well, since a central position is important for the attachment of the DRG.</li>
		<li>Work under sterile conditions from now on. Place the explant cultures in the incubator at 37 &#176;C and 5% CO2. The next day, add 50 &#181;L of the DRG growth medium carefully to each well using a 100 mL pipette.</li>
		<li>Observe DRG explant adherence and axon outgrowth using a microscope daily, and discard DRG explants that have detached from the coverslip or failed to outgrow axons on day 3 of culture. 
		&#8203;NOTE: DRG explants are very fragile in the first days of culture and need to be handled with care, especially when moving the plates in and out of the incubator when the medium is changed, or even when closing the incubator door. The precise volume of 190 &#181;L of medium per well is crucial to keep the DRG explants in place during the first day of culture. Loose DRG explants can be identified easily during the daily control, as they swim in the medium instead of adhering to the coverslip.</li>
	</ol>
	</li>
</ol>
3. Coculture
<ol>
	<li>Transfer of Schwann cells to the DRG explant culture
	<ol>
		<li>Prepare the coculture medium by adding 0.1% ascorbic acid to the DRG growth medium.</li>
		<li>On day 3 of the DRG explant culture, carefully replace the DRG growth medium with 250 &#181;L of the coculture medium containing 30,000 Schwann cells (from step 1.8) per well.</li>
		<li>Keep the coculture of the DRG explants and Schwann cells for up to 22 days. Replace 250 &#181;L of the coculture medium carefully every other day, and observe the appearance of the cells using a microscope. 
		NOTE: For an example of DRG axons and Schwann cells in the coculture in the first days of culture, see Figure 1.</li>
	</ol>
	</li>
	<li>Immunocytochemical staining 
	NOTE: Handle the coculture samples extremely carefully during the staining procedure, since they can be damaged easily.
	<ol>
		<li>To fix the cells on the coverslips, remove the medium slowly, wash 3x with DPBS carefully, and incubate in 4% paraformaldehyde (PFA) for 10 min. Replace the PFA with DPBS, and store the fixed cells at 4 &#176;C for up to 1 week. 
		CAUTION: When handling 4% PFA, wear the recommended personal protective equipment.</li>
		<li>Wash the coverslips 3x with DPBS for 5 min, then block with blocking solution (10% goat serum, 10% bovine serum albumin [BSA], 0.1% gelatin, and 0.05% Triton X-100) in DPBS for 1 h. Dilute the primary antibodies &#946;III-tubulin (1:7,500) and myelin basic protein (MBP) (1:750) in the blocking solution, and incubate overnight at 4 &#176;C.</li>
		<li>Wash the cells 3x with DPBS for 5 min. Use fluorescent dye-conjugated secondary antibodies in a dilution of 1:1,000 in blocking solution, and incubate for 2 h at room temperature (RT).</li>
		<li>Wash the cells 3x with DPBS for 5 min, and mount the cells on microscopic slides with fluorescence mounting medium, including 4&#8242;,6-diamidino-2-phenylindole (DAPI). Store the slides in the dark at 4 &#176;C until microscopic documentation. 
		CAUTION: When handling fluorescence mounting medium, wear the recommended personal protective equipment.</li>
	</ol>
	</li>
	<li>Analysis
	<ol>
		<li>Take pictures of eight defined regions surrounding the DRG explant in the center using an inverted microscope.</li>
		<li>Use an image analysis software application to quantify the areas of &#946;III-tubulin positive axons and myelination stained by MBP.</li>
		<li>Calculate the percentage of myelination as a ratio of the myelinated axons and non-myelinated axons.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Lee, K. H., Chung, K., Chung, J. M., Coggeshall, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+cell+body+size,+axon+size,+and+signal+conduction+velocity+for+individually+labelled+dorsal+root+ganglion+cells+in+the+cat.">Correlation of cell body size, axon size, and signal conduction velocity for individually labelled dorsal root ganglion cells in the cat.</a> The Journal of Comparative Neurology. 243 (3), 335-346 (1986).</li><li>Taveggia, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schwann+cells-axon+interaction+in+myelination.">Schwann cells-axon interaction in myelination.</a> Current Opinion in Neurobiology. 39, 24-29 (2016).</li><li>Gordon, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+nerve+regeneration+and+muscle+reinnervation.">Peripheral nerve regeneration and muscle reinnervation.</a> International Journal of Molecular Sciences. 21 (22), 8652 (2020).</li><li>Nocera, G., Jacob, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+Schwann+cell+plasticity+involved+in+peripheral+nerve+repair+after+injury.">Mechanisms of Schwann cell plasticity involved in peripheral nerve repair after injury.</a> Cellular and Molecular Life Sciences. 77 (20), 3977-3989 (2020).</li><li>Modrak, M., Talukder, M. A. H., Gurgenashvili, K., Noble, M., Elfar, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+nerve+injury+and+myelination:+Potential+therapeutic+strategies.">Peripheral nerve injury and myelination: Potential therapeutic strategies.</a> Journal of Neuroscience Research. 98 (5), 780-795 (2020).</li><li>Salzer, J. L., Bunge, R. P., Glaser, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+Schwann+cell+proliferation.+III.+Evidence+for+the+surface+localization+of+the+neurite+mitogen.">Studies of Schwann cell proliferation. III. Evidence for the surface localization of the neurite mitogen.</a> The Journal of Cell Biology. 84 (3), 767-778 (1980).</li><li>Wood, P. M., Bunge, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+sensory+axons+are+mitogenic+for+Schwann+cells.">Evidence that sensory axons are mitogenic for Schwann cells.</a> Nature. 256 (5519), 662-664 (1975).</li><li>Eldridge, C. F., Bunge, M. B., Bunge, R. P., Wood, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+axon-related+Schwann+cells+in+vitro.+I.+Ascorbic+acid+regulates+basal+lamina+assembly+and+myelin+formation.">Differentiation of axon-related Schwann cells in vitro. I. Ascorbic acid regulates basal lamina assembly and myelin formation.</a> The Journal of Cell Biology. 105 (2), 1023-1034 (1987).</li><li>Paivalainen, S., 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=Myelination+in+mouse+dorsal+root+ganglion/Schwann+cell+cocultures.">Myelination in mouse dorsal root ganglion/Schwann cell cocultures.</a> Molecular and Cellular Neuroscience. 37 (3), 568-578 (2008).</li><li>Clark, A. J., 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=Co-cultures+with+stem+cell-derived+human+sensory+neurons+reveal+regulators+of+peripheral+myelination.">Co-cultures with stem cell-derived human sensory neurons reveal regulators of peripheral myelination.</a> Brain. 140 (4), 898-913 (2017).</li><li>Taveggia, C., Bolino, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DRG+neuron/Schwann+cells+myelinating+cocultures.">DRG neuron/Schwann cells myelinating cocultures.</a> Methods in Molecular Biology. 1791, 115-129 (2018).</li><li>Andersen, N. D., Srinivas, S., Pinero, G., Monje, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+versatile+method+for+the+isolation,+purification+and+cryogenic+storage+of+Schwann+cells+from+adult+rodent+nerves.">A rapid and versatile method for the isolation, purification and cryogenic storage of Schwann cells from adult rodent nerves.</a> Scientific Reports. 6, 31781 (2016).</li><li>Pitarokoili, K., 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=Intrathecal+triamcinolone+acetonide+exerts+anti-inflammatory+effects+on+Lewis+rat+experimental+autoimmune+neuritis+and+direct+anti-oxidative+effects+on+Schwann+cells.">Intrathecal triamcinolone acetonide exerts anti-inflammatory effects on Lewis rat experimental autoimmune neuritis and direct anti-oxidative effects on Schwann cells.</a> Journal of Neuroinflammation. 16 (1), 58 (2019).</li><li>Gr&#252;ter, T., 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=Immunomodulatory+and+anti-oxidative+effect+of+the+direct+TRPV1+receptor+agonist+capsaicin+on+Schwann+cells.">Immunomodulatory and anti-oxidative effect of the direct TRPV1 receptor agonist capsaicin on Schwann cells.</a> Journal of Neuroinflammation. 17 (1), 145 (2020).</li><li>Lehmann, H. C., H&#246;ke, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schwann+cells+as+a+therapeutic+target+for+peripheral+neuropathies.">Schwann cells as a therapeutic target for peripheral neuropathies.</a> CNS &amp; Neurological Disorders - Drug Targets. 9 (6), 801-806 (2010).</li><li>Joshi, A. 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=Loss+of+Schwann+cell+plasticity+in+chronic+inflammatory+demyelinating+polyneuropathy+(CIDP).">Loss of Schwann cell plasticity in chronic inflammatory demyelinating polyneuropathy (CIDP).</a> Journal of Neuroinflammation. 13 (1), 255 (2016).</li><li>Klimas, 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=Dose-dependent+immunomodulatory+effects+of+bortezomib+in+experimental+autoimmune+neuritis.">Dose-dependent immunomodulatory effects of bortezomib in experimental autoimmune neuritis.</a> Brain Communications. 3 (4), (2021).</li><li>Szepanowski, F., 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=LPA1+signaling+drives+Schwann+cell+dedifferentiation+in+experimental+autoimmune+neuritis.">LPA1 signaling drives Schwann cell dedifferentiation in experimental autoimmune neuritis.</a> Journal of Neuroinflammation. 18 (1), 293 (2021).</li></ol>

The authors declare to have no conflicts of interest.

Here, we present a rapid and facile protocol for the generation of in vitro myelination by merging two separate cell type cultures, Schwann cells and dorsal root ganglion explants.
A critical step of the protocol is the cultivation of DRG explants, especially in the first days of culture. DRG are very fragile before a strong axonal network is built and must be handled very carefully, for example, when taken out of the incubator or during a change of medium. DRG that detach from the bo...

In the peripheral nervous system (PNS), rapid information transduction is mediated by myelin-enwrapped axons. The myelination of axons is essential to enable the fast propagation of electric impulses, since the conduction velocity of the nerve fibers correlates to the axon diameter and myelin thickness1. Sensory signaling from the periphery to the central nervous system (CNS) relies on the activation of first-order sensory neurons that reside in enlargements of the dorsal root, termed dorsal root ganglia (DRG). For the formation and maintenance of myelin, continuous communication between axons and Schwann cells, which are the myelinating Glia cells in the PNS, is mandatory2.
Many diseases of the PNS disturb the transduction of information by either primary axonal or demyelinating damage, resulting in hypesthesia or dysesthesia. First-order sensory neurons have the ability to regenerate to an extent after neuronal damage, by a complex interaction between the neuron and surrounding Schwann cells3. In this case, Schwann cells can undergo cellular reprogramming to clear axonal as well as myelin debris and promote axonal regeneration, resulting in remyelination4. Understanding the mechanisms of myelination in health and disease is important, in order to find possible treatment options for demyelinating disorders of the PNS. Myelin can also be damaged by acute neurotrauma, and approaches to promote myelination to advance functional recovery after peripheral nerve injury are under investigation5.
Our knowledge of peripheral myelination has benefited largely from myelinating cocultures of Schwann cells and sensory neurons. Since the first approaches were applied6,7,8, myelination has been studied intensely with the use of different coculture systems9,10,11. Here, we provide a rapid and facile protocol for robust in vitro myelination of dorsal root ganglion axons. The protocol for Schwann cell preparation is based on the protocol by Andersen et al.12, previously published in Pitarokoili et al.13. We use Schwann cells derived from juvenile rats and embryonic DRG explant cultures for the coculture, in which myelination occurs at around day 14. The goal of the method is to provide a system to investigate the formation of myelin as a result of direct axon-Schwann cell interaction, and to study modulators of PNS myelination. In comparison to dissociated neuronal cell cultures, DRG explants are more anatomically preserved and form long axonal processes. Quantification of the myelinated axon area provides a sufficient readout for myelination in the coculture. The method is a valuable tool to screen therapeutic compounds for their potential effect on PNS myelination, and can also be utilized in addition to in vivo studies in animal models14.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti-MBP, rabbit</td>
			<td>Novus Biologicals, Centannial, USA</td>
			<td>ABIN446360</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-&#223;III-tubulin, mouse&#160;</td>
			<td>Biolegend, San Diego, USA</td>
			<td>657402</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic acid&#160;</td>
			<td>Sigma Aldrich GmbH, Steinheim, Germany&#160;</td>
			<td>A4403-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>B27-supplement</td>
			<td>Thermo Fisher Scientific, Schwerte, Germany&#160;</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosphere Filter Tip, 100 &#181;L</td>
			<td>Sarstedt, N&#252;mbrecht, Germany&#160;</td>
			<td>70760212</td>
			<td></td>
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			<td>Biosphere Filter Tip, 1250 &#181;L</td>
			<td>Sarstedt, N&#252;mbrecht, Germany&#160;</td>
			<td>701186210</td>
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			<td>Biosphere Filter Tip, 20 &#181;L</td>
			<td>Sarstedt, N&#252;mbrecht, Germany&#160;</td>
			<td>701114210</td>
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		<tr>
			<td>Biosphere Filter Tip, 300 &#181;L</td>
			<td>Sarstedt, N&#252;mbrecht, Germany&#160;</td>
			<td>70765210</td>
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			<td>Bovine serum albumin</td>
			<td>Carl Roth, Karlsruhe, Germany&#160;</td>
			<td>8076.4</td>
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			<td>Cell strainer, 100 &#181;M</td>
			<td>BD Bioscience, Heidelberg, Germany</td>
			<td>352360</td>
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			<td>Centrifuge 5810-R</td>
			<td>Eppendorf AG, Hamburg, Germany</td>
			<td>5811000015</td>
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			<td>CO2 Incubator Heracell</td>
			<td>Heraeus Instruments, Hanau, Germany&#160;</td>
			<td>51017865</td>
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			<td>Coverslips 12 mm</td>
			<td>Carl Roth, Karlsruhe, Germany&#160;</td>
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			<td>Curved fine forceps&#160;</td>
			<td>Fine Science Tools GmbH, Heidelberg, Germany</td>
			<td>11370-42</td>
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			<td>DAPI fluoromount-G(R)</td>
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			<td>Dispase II</td>
			<td>Sigma Aldrich GmbH, Steinheim, Germany&#160;</td>
			<td>4942078001</td>
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			<td>Distilled water (Water Purification System)&#160;</td>
			<td>Millipore, Molsheim, France</td>
			<td>ZLXS5010Y</td>
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			<td>DMEM/F-12, GlutaMAX</td>
			<td>Thermo Fisher Scientific, Schwerte, Germany&#160;</td>
			<td>31331093</td>
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			<td>DPBS (no Ca2+ and no Mg2+)</td>
			<td>Sigma Aldrich GmbH, Steinheim, Germany&#160;</td>
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			<td>Ethanol&#160;</td>
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			<td>FCS</td>
			<td>Sigma Aldrich GmbH, Steinheim, Germany&#160;</td>
			<td>F7524</td>
			<td>FCS must be tested for Schwann cell culture</td>
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			<td>Fine forceps (Dumont #5)</td>
			<td>Fine Science Tools GmbH, Heidelberg, Germany</td>
			<td>11252-20</td>
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			<td>Forceps</td>
			<td>Fine Science Tools GmbH, Heidelberg, Germany</td>
			<td>11370-40</td>
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			<td>Forskolin</td>
			<td>Sigma Aldrich GmbH, Steinheim, Germany&#160;</td>
			<td>F6886-10MG</td>
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			<td>Gelatin</td>
			<td>Sigma Aldrich GmbH, Steinheim, Germany&#160;</td>
			<td>G1393-20ML</td>
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			<td>Gentamycin</td>
			<td>Thermo Fisher Scientific, Schwerte, Germany</td>
			<td>5710064</td>
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			<td>Goat anti-mouse IgG Alexa Fluor 488</td>
			<td>Thermo Fisher Scientific, Schwerte, Germany&#160;</td>
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			<td>Goat anti-rabbit IgG Alexa Fluor 568</td>
			<td>Thermo Fisher Scientific, Schwerte, Germany&#160;</td>
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			<td>HBSS (no Ca2+ and no Mg2+)&#160;</td>
			<td>Thermo Fisher Scientific, Schwerte, Germany&#160;</td>
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			<td>HERAcell Incubator</td>
			<td>Heraeus Instruments, Hanau, Germany&#160;</td>
			<td>51017865</td>
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			<td>Heraguard ECO 1.2</td>
			<td>Thermo Fisher Scientific, Schwerte, Germany&#160;</td>
			<td>51029882</td>
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			<td>Horse serum</td>
			<td>Pan-Biotech, Aidenbach, Germany</td>
			<td>P30-0712</td>
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			<td>Image J Software</td>
			<td>HIH, Bethesda, USA</td>
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			<td>Laminin</td>
			<td>Sigma Aldrich GmbH, Steinheim, Germany&#160;</td>
			<td>L2020-1MG</td>
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			<td>Leibovitz&#180;s L-15 Medium</td>
			<td>Thermo Fisher Scientific, Schwerte, Germany&#160;</td>
			<td>11415064</td>
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			<td>L-Glutamine 200 mM&#160;</td>
			<td>Thermo Fisher Scientific, Schwerte, Germany&#160;</td>
			<td>25030024</td>
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			<td>MACS Multistand&#160;</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, Germany</td>
			<td>130042303</td>
			<td></td>
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			<td>Microscissors</td>
			<td>Fine Science Tools GmbH, Heidelberg, Germany</td>
			<td>15000-08</td>
			<td></td>
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		<tr>
			<td>Microscope&#160;</td>
			<td>Motic, Wetzlar, Germany</td>
			<td>Motic BA 400</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Axio observer 7</td>
			<td>Zeiss, Oberkochen, Germany&#160;</td>
			<td>491917-0001-000</td>
			<td></td>
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		<tr>
			<td>Microscope slide</td>
			<td>VWR, Radnor, USA&#160;</td>
			<td>630-1985</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniMACS separator</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, Germany</td>
			<td>130091632</td>
			<td></td>
		</tr>
		<tr>
			<td>MS columns</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, Germany</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer counting chamber&#160;</td>
			<td>Assistant, Erlangen, Germany</td>
			<td>40441 &#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Neuregulin</td>
			<td>Peprotech, Rocky Hill, USA</td>
			<td>100-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium&#160;</td>
			<td>Thermo Fisher Scientific, Schwerte, Germany&#160;</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>NGF</td>
			<td>Sigma Aldrich GmbH, Steinheim, Germany&#160;</td>
			<td>N1408</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Biozol, Eching, Germany</td>
			<td>S-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunclon &#916; multidishes, 4 well</td>
			<td>Sigma Aldrich GmbH, Steinheim, Germany&#160;</td>
			<td>D6789</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Acros Organics, New Jersey, USA&#160;</td>
			<td>10342243</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermo Fisher Scientific, Schwerte, Germany&#160;</td>
			<td>15140-122</td>
			<td></td>
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		<tr>
			<td>Pipetboy</td>
			<td>Eppendorf AG, Hamburg, Germany</td>
			<td>4430000018&#160;</td>
			<td></td>
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			<td>Pipettes</td>
			<td>Eppendorf AG, Hamburg, Germany</td>
			<td>2231300004</td>
			<td></td>
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		<tr>
			<td>Poly-D-Lysin</td>
			<td>Sigma Aldrich GmbH, Steinheim, Germany&#160;</td>
			<td>P6407-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Lysin</td>
			<td>Sigma Aldrich GmbH, Steinheim, Germany&#160;</td>
			<td>P4707-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Reaction tubes, 15&#160;mL</td>
			<td>Sarstedt, N&#252;mbrecht, Germany&#160;</td>
			<td>62554502</td>
			<td></td>
		</tr>
		<tr>
			<td>Reaction tubes, 50&#160;mL</td>
			<td>Sarstedt, N&#252;mbrecht, Germany&#160;</td>
			<td>62547254</td>
			<td></td>
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		<tr>
			<td>Reaction vessels, 1.5 mL</td>
			<td>Sarstedt, N&#252;mbrecht, Germany&#160;</td>
			<td>72690001</td>
			<td></td>
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		<tr>
			<td>Safety Cabinet S2020 1.8</td>
			<td>Thermo Fisher Scientific, Schwerte, Germany&#160;</td>
			<td>51026640</td>
			<td></td>
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			<td>Scissors</td>
			<td>Fine Science Tools GmbH, Heidelberg, Germany</td>
			<td>14083-08</td>
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			<td>Serological pipette, 10 mL</td>
			<td>Sarstedt, N&#252;mbrecht, Germany&#160;</td>
			<td>861254025</td>
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			<td>Serological pipette, 25 mL</td>
			<td>Sarstedt, N&#252;mbrecht, Germany&#160;</td>
			<td>861685001</td>
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			<td>Serological pipette, 5 mL</td>
			<td>Sarstedt, N&#252;mbrecht, Germany&#160;</td>
			<td>861253001</td>
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			<td>Spatula</td>
			<td>Fine Science Tools GmbH, Heidelberg, Germany</td>
			<td>10094-13</td>
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			<td>Stereomicroscope Discovery.V8</td>
			<td>Zeiss, Oberkochen, Germany&#160;</td>
			<td>495015-0012-000&#160;</td>
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			<td>TC dish 100, cell +</td>
			<td>Sarstedt, N&#252;mbrecht, Germany&#160;</td>
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			<td>TC dish 35, cell +</td>
			<td>Sarstedt, N&#252;mbrecht, Germany&#160;</td>
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			<td>TC dish 60, cell +</td>
			<td>Sarstedt, N&#252;mbrecht, Germany&#160;</td>
			<td>833901300</td>
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			<td>Thy-1 Microbeads (MACS Kit)</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, Germany</td>
			<td>130-094-523</td>
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			<td>Triton X-100&#160;</td>
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			<td>Trypan Blue Solution 0.4%&#160;</td>
			<td>Thermo Fisher Scientific, Schwerte, Germany&#160;</td>
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in vitro myelination of peripheral axons in a coculture of rat dorsal root ganglion explants and schwann cells

The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, neurons and Schwann cells engage in a complex interaction to control the myelination of axons. Disturbances of this interaction and breakdown of the myelin sheath are hallmarks of inflammatory neuropathies and occur secondarily in neurodegenerative disorders. Here, we present a coculture model of dorsal root ganglion explants and Schwann cells, which develops a robust myelination of peripheral axons to investigate the process of myelination in the peripheral nervous system, study axon-Schwann cell interactions, and evaluate the potential effects of therapeutic agents on each cell type separately. Methodologically, dorsal root ganglions of embryonic rats (E13.5) were harvested, dissociated from their surrounding tissue, and cultured as whole explants for 3 days. Schwann cells were isolated from 3-week-old adult rats, and sciatic nerves were enzymatically digested. The resulting Schwann cells were purified by magnetic-activated cell sorting and cultured under neuregulin and forskolin-enriched conditions. After 3 days of dorsal root ganglion explant culture, 30,000 Schwann cells were added to one dorsal root ganglion explant in a medium containing ascorbic acid. The first signs of myelination were detected on day 10 of coculture, through scattered signals for myelin basic protein in immunocytochemical staining. From day 14 onward, myelin sheaths were formed and propagated along the axons. Myelination can be quantified by myelin basic protein staining as a ratio of the myelination area and axon area, to account for the differences in axonal density. This model provides experimental opportunities to study various aspects of peripheral myelination in vitro, which is crucial for understanding the pathology of and possible treatment opportunities for demyelination and neurodegeneration in inflammatory and neurodegenerative diseases of the peripheral nervous system.

Myelination in the coculture was assessed on days 10, 12, 14, 16, 18, and 20. The DRG explants and Schwann cells were stained for MBP, &#946;III-tubulin, and DAPI. The axonal network in the coculture was dense and did not change visibly in the time course of the observation. The first signs of myelin, in the form of small fragments, were detectable on day 10 and increased on day 12 (Figure 2). The MBP-positive areas increased over time until day 20 of culture. The myelination was quantified ...

Watch this Scientific Journal Video about In Vitro Myelination of Peripheral Axons in a Coculture of Rat Dorsal Root Ganglion Explants and Schwann Cells at JoVE.com

In Vitro Myelination of Peripheral Axons in a Coculture of Rat Dorsal Root Ganglion Explants and Schwann Cells

In the coculture system of dorsal root ganglia and Schwann cells, myelination of the peripheral nervous system can be studied. This model provides experimental opportunities to observe and quantify peripheral myelination and to study the effects of compounds of interest on the myelin sheath.

The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, ...

in-vitro-myelination-peripheral-axons-coculture-rat-dorsal-root

Research

JoVE Journal

Neuroscience

1.9K Views.  Ruhr-University Bochum, St. Josef Hospital. In the coculture system of dorsal root ganglia and Schwann cells, myelination of the peripheral nervous system can be studied. This model provides experimental opportunities to observe and quantify peripheral myelination and to study the effects of compounds of interest on the myelin sheath. 

Video: In Vitro Myelination of Peripheral Axons in a Coculture of Rat Dorsal Root Ganglion Explants and Schwann Cells

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

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 Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS). The dorsal root ganglia (DRG)/cancer cell model is a three dimensional (3D) in vitro model frequently used for studying the interaction between neural stroma and cancer cells. In this model, mouse or human cancer cell lines are grown in ECM adjacent to preparations of freshly dissociated cultured DRG. In this article, the DRG isolation protocol from mice, and implantation in petri dishes for co-culturing with pancreatic cancer cells are demonstrated. Five days after implantation, the cancer cells made contact with the DRG neurites. Later, these cells formed bridgeheads to facilitate more extensive polarized, neurotropic migration of cancer cells.

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...

An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to bridge the lesion site, properly organized axonal alignment is required. Since demyelination after nerve injury strongly impairs the conductive capacity of surviving axons, remyelination is critical for successful functioning of regenerated nerves. Previously, we demonstrated that mesenchymal stem cells (MSCs) aligned on a pre-stretch induced anisotropic surface because the cells can sense a larger effective stiffness in the stretched direction than in the perpendicular direction. We also showed that an anisotropic surface arising from a mechanical pre-stretched surface similarly affects alignment, as well as growth and myelination of axons. Here, we provide a detailed protocol for preparing a pre-stretched anisotropic surface, the isolation and culture of dorsal root ganglion (DRG) neurons on a pre-stretched surface, and show the myelination behavior of a co-culture of DRG neurons with Schwann cells (SCs) on a pre-stretched surface.

This protocol describes the isolation of dorsal root ganglion (DRG) neurons isolated from rats and the culture of DRG neurons on a static pre-stretched cell culture system to enhance axon alignment, with subsequent co-culture of Schwann Cells (SCs) to promote myelination.

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive targets for injection of novel therapeutics aimed at treating chronic pain. In small animal models, laminectomy has been used to facilitate DRG injection because it involves surgical removal of the vertebral bone surrounding each DRG. We demonstrate a technique for intraganglionic injection of lumbar DRG in a large animal species, namely, swine. Laminotomy is performed to allow direct access to DRG using standard neurosurgical techniques, instruments, and materials. Compared with more extensive bone removal via laminectomy, we implement laminotomy to conserve spinal anatomy while achieving sufficient DRG access. Intraoperative progress of DRG injection is monitored using a non-toxic dye. Following euthanasia on post-operative day 21, the success of injection is determined by histology for intraganglionic distribution of 4&#39;,6-diamidino-2-phenylindole (DAPI). We inject a biologically inactive solution to demonstrate the protocol. This method could be applied in future preclinical studies to target therapeutic solutions to DRG. Our methodology should facilitate testing the translatability of intraganglionic small animal paradigms in a large animal species. Additionally, this protocol may serve as a key resource for those planning preclinical studies of DRG injection in swine.

We describe a method for laminotomy in swine that provides access to lumbar dorsal root ganglia (DRG) for intraganglionic injection. Injection progress is monitored intraoperatively and histologically confirmed up to 21 days after surgery. This protocol could be used for future preclinical studies involving DRG injection.

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

Rapid Isolation of Dorsal Root Ganglion Macrophages

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after peripheral nerve injury. Peripheral monocytic cells, including macrophages, are known to respond to a tissue injury through phagocytosis, antigen presentation, and cytokine release. Emerging evidence has implicated the contribution of dorsal root ganglia macrophages to neuropathic pain development and axonal repair in the context of nerve injury. Rapidly phenotyping (or &#8220;rapid isolation of&#8221;) the response of dorsal root ganglia macrophages in the context of nerve injury is desired to identify the unknown neuroimmune factors. Here we demonstrate how our lab rapidly and effectively isolates macrophages from the dorsal root ganglia using an enzyme-free mechanical dissociation protocol. The samples are kept on ice throughout to limit cellular stress. This protocol is far less time consuming compared to the standard enzymatic protocol and has been routinely used for our Fluorescence-activated Cell Sorting analysis.

Here we present a mechanical dissociation protocol to rapidly isolate macrophages from the dorsal root ganglion for phenotyping and functional analysis.

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after ...