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

我们感谢Ralf Gold教授和PD Dr. Gisa Ellrichmann的建议和支持。

所有程序均按照欧洲共同体理事会关于实验动物护理和使用指令执行。
1. 雪旺细胞培养
<ol><li>雪旺细胞培养包衣<ol><li>在无菌条件下涂覆细胞培养皿。将 2 mL 的 0.01% 聚-L-赖氨酸 （PLL） 分别施用到两个 60 mm 组织培养 （TC） 培养皿中，并在 4 °C 下孵育过夜。</li>
		<li>取出PLL，用蒸馏水清洗TC培养皿2x，并在4°C下用2mL的1μg/ cm2 层粘连蛋白孵育过夜。 用水?...

<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>

在这里，我们提出了一种快速简便的方案，用于通过合并两种独立的细胞类型培养物，雪旺细胞和背根神经节外植体来产生 体外 髓鞘。
该方案的一个关键步骤是培养DRG外植体，特别是在培养的第一天。DRG在建立强大的轴突网络之前非常脆弱，必须非常小心地处理，例如，当从培养箱中取出或在更换培养基时。从井底分离并被发现在介质中游泳的DRG表明栽培不成功。对...

在周围神经系统（PNS）中，快速信息转导由髓磷脂包裹的轴突介导。轴突的髓鞘形成对于电脉冲的快速传播至关重要，因为神经纤维的传导速度与轴突直径和髓鞘厚度相关1.从外围到中枢神经系统（CNS）的感觉信号依赖于位于背根增大中的一级感觉神经元的激活，称为背根神经节（DRG）。为了形成和维持髓磷脂，轴突和雪旺细胞（PNS中的髓鞘胶质细胞）之间的连续通信是强制性的2。
PNS的许多疾病会干扰原发性轴突或脱髓鞘损伤的信息转导，导致感觉迟钝或感觉迟钝。一级感觉神经元具有在神经元损伤后通过神经元与周围雪旺细胞之间的复杂相互作用再生一定程度的能力3。在这种情况下，雪旺细胞可以进行细胞重编程以清除轴突和髓鞘碎片并促进轴突再生，从而导致髓鞘再生4。了解髓鞘形成在健康和疾病中的机制很重要，以便找到PNS脱髓鞘疾病的可能治疗方案。髓磷脂也可能因急性神经创伤而受损，促进髓鞘形成以促进周围神经损伤后功能恢复的方法正在研究中5。
我们对外周髓鞘形成的了解在很大程度上受益于雪旺细胞和感觉神经元的髓鞘共培养。自从应用第一种方法6，7，8以来，使用不同的共培养系统对髓鞘形成进行了深入研究9，10，11。在这里，我们为背根神经节轴突的稳健体外髓鞘形成提供了一种快速简便的方案。雪旺细胞制备的方案基于Andersen等人12的方案，该方案先前发表在Pitarokoili等人13上。我们使用来自幼年大鼠的雪旺细胞和胚胎DRG外植体培养物进行共培养，其中髓鞘形成发生在第14天左右。该方法的目标是提供一个系统来研究由于直接轴突 - 雪旺细胞相互作用而导致髓磷脂的形成，并研究PNS髓鞘形成的调节剂。与解离的神经元细胞培养物相比，DRG外植体在解剖学上保存得更多，并形成长轴突过程。有髓鞘轴突区域的定量为共培养中的髓鞘形成提供了足够的读数。该方法是筛选治疗性化合物对PNS髓鞘形成的潜在影响的宝贵工具，并且还可以用于动物模型中的体内研究14。

<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>
		</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>
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			<td>2231300004</td>
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in vitro myelination of peripheral axons in a coculture of rat dorsal root ganglion explants and schwann cells

髓鞘形成过程对于在神经系统中实现快速和充分的信号转导至关重要。在周围神经系统中，神经元和雪旺细胞进行复杂的相互作用以控制轴突的髓鞘形成。这种相互作用的紊乱和髓鞘的分解是炎症性神经病的标志，继发于神经退行性疾病。在这里，我们提出了背根神经节外植体和雪旺细胞的共培养模型，该模型开发了强大的外周轴突髓鞘形成，以研究周围神经系统中的髓鞘形成过程，研究轴突 - 雪旺细胞相互作用，并分别评估治疗剂对每种细胞类型的潜在影响。方法学上，收获胚胎大鼠（E13.5）的背根神经节，从其周围组织中分离，并作为整个外植体培养3天。从3周龄的成年大鼠中分离雪旺细胞，并对坐骨神经进行酶消化。所得雪旺细胞通过磁激活细胞分选纯化，并在神经调节素和毛喉素富集条件下培养。背根神经节外植体培养3天后，在含有抗坏血酸的培养基中将30，000个雪旺细胞加入到一个背根神经节外植体中。在共培养的第10天，通过免疫细胞化学染色中髓鞘碱性蛋白的散射信号检测到髓鞘形成的最初迹象。从第14天开始，髓鞘形成并沿轴突繁殖。髓鞘形成可以通过髓鞘碱性蛋白染色作为髓鞘形成面积和轴突面积的比率来量化，以解释轴突密度的差异。该模型为体外研究 外周髓鞘形成的各个方面提供了实验机会，这对于理解周围神经系统炎症和神经退行性疾病中脱髓鞘和神经变性的病理学和可能的治疗机会至关重要。

在第10、12、14、16、18和20天评估共培养中的髓鞘形成。对DRG外植体和雪旺细胞进行MBP、βIII-微管蛋白和DAPI染色。共培养中的轴突网络是密集的，并且在观察的时间过程中没有明显变化。髓磷脂的最初迹象，以小碎片的形式，在第10天可检测到，并在第12天增加（图2）。MBP阳性区域随着时间的推移而增加，直到培养的第20天。将髓鞘化量化为MBP和βIII-微管蛋白阳性区域的比率?...

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

在背根神经节和雪旺细胞的共培养系统中，可以研究周围神经系统的髓鞘形成。该模型为观察和量化外周髓鞘形成以及研究目标化合物对髓鞘的影响提供了实验机会。

大鼠背根神经节外植体和雪旺细胞共培养中外周轴突的体外髓鞘形成

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

该模型为体外研究周围神经系统髓鞘形成的各个方面提供了实验机会。它能够进行髓磷脂定量和轴突-雪旺细胞相互作用的检查。共培养从第14天开始形成强大的髓鞘形成。与解离的神经元培养物相比，DRG外植体培养物具有完整的结构结构优势。该方法可用于筛选周围神经系统炎症和神经退行性疾病的热带化合物。首先，用70%乙醇对安乐死大鼠躯干的工作区域进行消毒。用剪刀打开大鼠的左下肢，小心地去除二头肌的股骨肌肉。用弯曲的镊子平滑抬高坐骨神经，确保不会瘀伤神经。使用镊子，将夹紧的神经转移到带有冰冷的Leibovitz的L-15培养基的组织培养皿中，每毫升庆大霉素为50微克。然后，使用体视显微镜，用两对细镊子从神经中取出脂肪、肌肉和血管。识别坐骨神经的近端和远端，用一对细钳在近端到远端方向切除外神经，同时用第二对细钳固定近端神经末梢。将纯化的神经转移到另一个组织培养皿中后，用冰冷的Leibovitz的L-15培养基和庆大霉素，挑逗分离的神经束，以使用两对细镊子分离和分离单个神经纤维。使用10毫升血清移液管将神经纤维转移到50毫升管中，并尽可能少地吸收培养基。然后将50毫升含有庆大霉素的莱博维茨L 15培养基加入神经纤维中几次。将含有神经纤维的管在 188 G 下在 4 摄氏度下离心五分钟。除去上清液后，将沉淀与剩余的Leibovitz's L-15培养基转移到60毫米组织培养皿中。用10毫升酶消化溶液冲洗50毫升管，并将其添加到神经纤维培养皿中。用移液管的尖端小心地分布培养皿中的神经纤维。在 37 摄氏度和 5% 二氧化碳下孵育 18 小时。并通过在HBSS中加入10毫升40%FCS来停止酶消化。将消化的神经转移到50毫升的管中，在4摄氏度下以188G离心10分钟。弃去上清液后，将沉淀重新悬浮在含有10%FCS和每毫升庆大霉素50微克的10毫升DMEM中。接下来，通过100微米的细胞过滤器过滤细胞悬液。在4摄氏度下以188 G离心10分钟后，用4毫升含有FCS和庆大霉素的DMEM重新悬浮沉淀。将两毫升细胞悬液添加到两个聚赖氨酸和层粘连蛋白包被的60毫米组织培养皿中。在 37 摄氏度和 5% 二氧化碳下孵育，将板在培养箱中保持两天不变。在4摄氏度下以188G离心雪旺细胞悬液10分钟，然后将细胞沉淀重新悬浮在90微升磁性细胞分离缓冲液中，每1次10至第七个细胞。然后向第七个细胞中加入10微升ti1微珠，每1乘以10。将重悬溶液在黑暗中以8摄氏度孵育15分钟。接下来，在磁性细胞分离缓冲液中加入两毫升到细胞悬液中。在4摄氏度下以300G离心10分钟后，将沉淀重新悬浮在500微升磁性细胞分离缓冲液中。将磁性细胞分离柱放入细胞分离器中，并用一毫升磁性细胞分离缓冲液润湿色谱柱。然后将细胞施加到其上以收集流过以进行离心。小心地从安乐死的怀孕大鼠身上取出子宫，并将其放入带有冰冷HBSS的100毫米组织培养皿中。用弯曲的镊子握住子宫，用细镊子打开子宫壁。取出一个羊膜袋，用细镊子捏一个孔小心地打开。快速从子宫中取出所有胚胎，并将它们转移到装满HBSS的培养皿中。轻轻地，将一个胚胎躯干放入装满HBSS的35毫米培养皿中。在体视显微镜下，打开躯干的背侧部分，将胚胎分成两半。将一半转向一侧，识别位于胚胎背侧部分线中的背根神经节或DRG链。用细镊子和微型剪刀将DRG整根线剪掉。将DRG放入装有两毫升HBSS的新鲜培养皿中后，使用细镊子和微型剪刀将单个DRG与剩余的组织分开。在每个孔中取一个含有190微升DRG生长培养基的4孔板，并使用细镊子和刮刀小心地将单个DRG转移到每个孔的中心。然后将DRG外植体培养物置于37摄氏度和5%二氧化碳的培养箱中。第二天，小心地向每个孔中加入50微升DRG生长培养基，并每天使用显微镜观察DRG外植体粘附和轴突生长。为了在DRG外植体培养的第三天共培养，小心地用250微升共培养基替换DRG生长培养基，每孔含有30， 000个雪旺细胞。为了进行免疫细胞化学染色，通过将DPBS洗涤的细胞在4%多聚甲醛中孵育10分钟来将细胞固定在盖玻片上。使用倒置显微镜在中心DRG外植体周围的八个特定区域拍摄免疫细胞化学染色样品的照片。这里描述了具有细长和纺锤形形态的雪旺细胞的外观以及共培养中的细DRG轴突。通过对DRG外植体和雪旺细胞的髓鞘碱性蛋白或MBP、β三微管蛋白和DAPI染色，在不同日期评估共培养中的髓鞘形成。共培养中的轴突网络是密集的，并且在观察的时间过程中没有明显变化。髓鞘形成的最初迹象在共培养的第10天和第12天可见。从第14天开始，MVP信号更加明显，并且检测到髓鞘包裹的轴突。髓鞘形成随着共培养时间的增加而增加，直到第20天。髓鞘形成被量化为MVP和β三个微管蛋白阳性区域的比率。与第10天相比，在第18天和第20天观察到髓鞘形成显着增加。DRG外植体培养物很脆弱，需要小心处理。例如，当从培养箱中取出或在更换培养基和固定期间。可以形成共培养样品的基因表达分析，以更详细地研究髓磷脂。在这里，我们在第22天检测到髓鞘标志物PMP22，MAG，Oct6，Egr2，Olig1。

Research

JoVE Journal

Neuroscience

Live Imaging of Dorsal Root Axons after Rhizotomy

实时成像背根轴突后根切断术

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

科研

神经科学

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

培养成年的背根神经节神经元轴突再生的遗传研究

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

背根神经节神经元和区分脂肪干细胞：一种<em&gt;在体外</em&gt;共培养模型来研究周围神经再生

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

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

在癌神经侵袭的体外模型：背根神经节型号

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

一种方法来提高背根神经节的对齐和髓鞘

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

背根神经节内注射和背根挤压伤为感官轴突再生模型

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

Laminotomy 对猪腰背根神经节通路和注射的治疗

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

应用流式细胞仪分析单侧坐骨神经和背根神经节的免疫细胞

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

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

哺乳动物轴突再生: 成年小鼠背根神经节体内电穿孔的研究

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

多尾根结鱼巨噬细胞的快速隔离

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