Name: in vitro myelination of peripheral axons in a coculture of rat dorsal root ganglion explants and schwann cells
Uploaded: 2023-02-10T12:50:00
Description: 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

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 wit...

<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>

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.

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		<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>
		</tr>
		<tr>
			<td>Biosphere Filter Tip, 1250 &#181;L</td>
			<td>Sarstedt, N&#252;mbrecht, Germany&#160;</td>
			<td>701186210</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosphere Filter Tip, 20 &#181;L</td>
			<td>Sarstedt, N&#252;mbrecht, Germany&#160;</td>
			<td>701114210</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosphere Filter Tip, 300 &#181;L</td>
			<td>Sarstedt, N&#252;mbrecht, Germany&#160;</td>
			<td>70765210</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Carl Roth, Karlsruhe, Germany&#160;</td>
			<td>8076.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer, 100 &#181;M</td>
			<td>BD Bioscience, Heidelberg, Germany</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5810-R</td>
			<td>Eppendorf AG, Hamburg, Germany</td>
			<td>5811000015</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator Heracell</td>
			<td>Heraeus Instruments, Hanau, Germany&#160;</td>
			<td>51017865</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips 12 mm</td>
			<td>Carl Roth, Karlsruhe, Germany&#160;</td>
			<td>P231.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved fine forceps&#160;</td>
			<td>Fine Science Tools GmbH, Heidelberg, Germany</td>
			<td>11370-42</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI fluoromount-G(R)</td>
			<td>Biozol, Eching, Germany</td>
			<td>SBA-0100-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Sigma Aldrich GmbH, Steinheim, Germany&#160;</td>
			<td>4942078001</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled water (Water Purification System)&#160;</td>
			<td>Millipore, Molsheim, France</td>
			<td>ZLXS5010Y</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12, GlutaMAX</td>
			<td>Thermo Fisher Scientific, Schwerte, Germany&#160;</td>
			<td>31331093</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS (no Ca2+ and no Mg2+)</td>
			<td>Sigma Aldrich GmbH, Steinheim, Germany&#160;</td>
			<td>D8537-6X500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol&#160;</td>
			<td>VWR, Radnor, USA&#160;</td>
			<td>1009862500</td>
			<td></td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>Sigma Aldrich GmbH, Steinheim, Germany&#160;</td>
			<td>F7524</td>
			<td>FCS must be tested for Schwann cell culture</td>
		</tr>
		<tr>
			<td>Fine forceps (Dumont #5)</td>
			<td>Fine Science Tools GmbH, Heidelberg, Germany</td>
			<td>11252-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools GmbH, Heidelberg, Germany</td>
			<td>11370-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Sigma Aldrich GmbH, Steinheim, Germany&#160;</td>
			<td>F6886-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma Aldrich GmbH, Steinheim, Germany&#160;</td>
			<td>G1393-20ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Thermo Fisher Scientific, Schwerte, Germany</td>
			<td>5710064</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG Alexa Fluor 488</td>
			<td>Thermo Fisher Scientific, Schwerte, Germany&#160;</td>
			<td>A11036</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG Alexa Fluor 568</td>
			<td>Thermo Fisher Scientific, Schwerte, Germany&#160;</td>
			<td>A11001</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS (no Ca2+ and no Mg2+)&#160;</td>
			<td>Thermo Fisher Scientific, Schwerte, Germany&#160;</td>
			<td>14170138</td>
			<td></td>
		</tr>
		<tr>
			<td>HERAcell Incubator</td>
			<td>Heraeus Instruments, Hanau, Germany&#160;</td>
			<td>51017865</td>
			<td></td>
		</tr>
		<tr>
			<td>Heraguard ECO 1.2</td>
			<td>Thermo Fisher Scientific, Schwerte, Germany&#160;</td>
			<td>51029882</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Pan-Biotech, Aidenbach, Germany</td>
			<td>P30-0712</td>
			<td></td>
		</tr>
		<tr>
			<td>Image J Software</td>
			<td>HIH, Bethesda, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma Aldrich GmbH, Steinheim, Germany&#160;</td>
			<td>L2020-1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#180;s L-15 Medium</td>
			<td>Thermo Fisher Scientific, Schwerte, Germany&#160;</td>
			<td>11415064</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine 200 mM&#160;</td>
			<td>Thermo Fisher Scientific, Schwerte, Germany&#160;</td>
			<td>25030024</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS Multistand&#160;</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, Germany</td>
			<td>130042303</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscissors</td>
			<td>Fine Science Tools GmbH, Heidelberg, Germany</td>
			<td>15000-08</td>
			<td></td>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr>
			<td>Pipetboy</td>
			<td>Eppendorf AG, Hamburg, Germany</td>
			<td>4430000018&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes</td>
			<td>Eppendorf AG, Hamburg, Germany</td>
			<td>2231300004</td>
			<td></td>
		</tr>
		<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>
		</tr>
		<tr>
			<td>Reaction vessels, 1.5 mL</td>
			<td>Sarstedt, N&#252;mbrecht, Germany&#160;</td>
			<td>72690001</td>
			<td></td>
		</tr>
		<tr>
			<td>Safety Cabinet S2020 1.8</td>
			<td>Thermo Fisher Scientific, Schwerte, Germany&#160;</td>
			<td>51026640</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools GmbH, Heidelberg, Germany</td>
			<td>14083-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette, 10 mL</td>
			<td>Sarstedt, N&#252;mbrecht, Germany&#160;</td>
			<td>861254025</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette, 25 mL</td>
			<td>Sarstedt, N&#252;mbrecht, Germany&#160;</td>
			<td>861685001</td>
			<td></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, ...

This model provides experimental opportunities to study various aspects of myelination of the peripheral nervous system in vitro. It enables myelin quantification and the examination of Axon-Schwann cell interactions. The co-culture develops a robust myelination from day 14 onward.The DRG explant cultures provide the advantage of intact structural architecture in comparison to dissociated neuronal cultures. This method is available to screen for tropotic compounds for inflammatory and neurodegenerative diseases of the peripheral nerve system. To begin, sterilize the work area in the euthanized rat's torso with 70%ethanol.Open the rat's dorsal lower left limb with scissors, and carefully remove the bicep's femorous muscle. Loosen the sciatic nerve by smooth elevation with curved forceps, ensuring not to bruise the nerve. Using forceps, Transfer the clipped nerves to a tissue culture dish with ice cold Leibovitz's L-15 medium with 50 micrograms per milliliter of gentamycin.Then, using a stereo microscope, remove the fat, muscle, and blood vessels from the nerves with two pairs of fine forceps. Identify the proximal and distal ends of the sciatic nerve and remove the epineurium with one pair of fine forceps in proximal to distal direction while holding the proximal nerve end with the second pair of fine forceps. After transferring the purified nerves to another tissue culture dish with ice cold Leibovitz's L-15 medium with Gentamycin, tease the isolated nerve fascicles to separate and isolate single nerve fibers using two pairs of fine forceps.Transfer the nerve fibers to a 50 milliliter tube using a 10 milliliter serological pipette, and take up as little medium as possible. Then add 50 milliliters of leibovitz's L 15 medium with gentamycin to the nerve fibers in slew a few times. Centrifuge the tube containing the nerve fibers at 188 G for five minutes at four degrees Celsius.After removing the supernatant, transfer the pellet with the remaining Leibovitz's L-15 medium into a 60 millimeter tissue culture dish. Rinse the 50 milliliter tube with 10 milliliters of the enzymatic digestion solution and add it to the nerve fibers dish. Distribute the nerve fibers in the dish carefully with the tip of a pipette.Incubate at 37 degrees Celsius and 5%carbon dioxide for 18 hours. And stop the enzymatic digestion by adding 10 milliliters at 40%FCS in HBSS. Transfer the digested nerves into a 50 milliliter tube to centrifuge at 188 G for 10 minutes at four degrees Celsius.After discarding the supernatant, re suspend the pellet in 10 milliliters of DMEM containing 10%FCS and 50 micrograms per milliliter of gentamycin. Next, filter the cell suspension through a 100 micrometer cell strainer. after centrifuging at 188 G for 10 minutes at four degrees Celsius, re suspend the pellet with four milliliters of DMEM containing FCS and gentamycin.Add two milliliters of the cell suspension to each of the two polylysine and laminin coated 60 millimeter tissue culture dishes. Incubate at 37 degrees Celsius and 5%carbon dioxide, leaving the plates untouched for two days in the incubator. Centrifuge the Schwann cell suspension at 188 G for 10 minutes at four degrees Celsius, and re suspend the cell pellet in 90 microliters of magnetic cell separation buffer per one times 10 to the seventh cells.Then add 10 microliters of ti1 micro beads per one times 10 to the seventh cells. Incubate the re suspended solution for 15 minutes in the dark at eight degrees Celsius. Next, add two milliliters in the magnetic cell separation buffer to the cell suspension.After centrifuging at 300 G for 10 minutes at four degrees Celsius, re suspend the pellet in 500 microliters of magnetic cell separation buffer. Place the magnetic cell separation column in the cell separator, and moisten the column with one milliliter of the magnetic cell separation buffer. Then apply the cells to it to collect the flow through for centrifugation.Carefully remove the uterus from a euthanized pregnant rat and place it into a 100 millimeter tissue culture dish with ice cold HBSS. Holding the uterus using curved forceps, open the uterus wall with fine forceps. Remove one amniotic sack and open it carefully by pinching a hole with fine forceps.Quickly remove all the embryos from the uterus and transfer them into a dish filled with HBSS. Gently, place one embryo torso into a 35 millimeter dish filled with HBSS. under a stereo microscope, Open the dorsal part of the torso to divide the embryo into two halves.Turn one half to the side and identify the strand of dorsal root ganglia, or DRG, located in the line at the dorsal part of the embryo. Cut out the DRG as a whole strand using fine forceps and micro scissors. After placing the DRG in a fresh dish filled with two milliliters of HBSS, Separate a single DRG from the remaining tissue using fine forceps and micro scissors.Take a 4 well plate containing 190 microliters of DRG growth medium in each well, and carefully transfer a single DRG into the center of each well using fine forceps and a spatula. Then place the DRG explant cultures in the incubator at 37 degrees Celsius and 5%carbon dioxide. The next day, carefully add 50 microliters of the DRG growth medium to each well and observe DRG explant adherence and axon outgrowth using a microscope daily.For co-culturing on day three of the DRG explant culture, carefully replace the DRG growth medium with 250 microliters of the co-culture medium containing 30, 000 schwann cells per well. For performing immunocytochemical staining, fix the cells on the cover slips by incubating the DPBS washed cells in 4%paraformaldehyde for 10 minutes. Take pictures of the immunocytochemically stained samples at eight defined regions surrounding the DRG explant in the center using an inverted microscope.The appearance of schwann cells with an elongated and spindle shaped morphology and the thin DRG axons in a co-culture is depicted here. Myelination in the co-culture was assessed on different days by staining the DRG explants and Schwann cells for myelin basic protein or MBP, beta three tubulin, and DAPI. The axonal network in the co-culture was dense, and did not change visibly in the time course of the observation.First signs of myelination were visible on days 10 and 12 of co-culture. From day 14, the MVP signal was more pronounced, and myelin-enwrapped axons were detected. The myelination increased with the co-culture time until day 20.The myelination was quantified as a ratio of the MVP and beta three tubulin positive areas. A significant increase in myelination was observed on days 18 and 20 compared to day 10. DRG explant cultures are fragile, and need to be handled carefully.For example, when taken out of the incubator or during medium change and fixation. Gene expression analysis of co-culture samples can be formed to investigate myelin in greater detail. Here, we detected the myelin markers PMP22, MAG, Oct6, Egr2, Olig1 on day 22.

Research

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Neuroscience

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