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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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 雪旺细胞培养包衣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。 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?…
Representative Results
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?…
Discussion
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…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢Ralf Gold教授和PD Dr. Gisa Ellrichmann的建议和支持。
Materials
| Anti-MBP, rabbit | Novus Biologicals, Centannial, USA | ABIN446360 | |
| Anti-ßIII-tubulin, mouse | Biolegend, San Diego, USA | 657402 | |
| Ascorbic acid | Sigma Aldrich GmbH, Steinheim, Germany | A4403-100MG | |
| B27-supplement | Thermo Fisher Scientific, Schwerte, Germany | 17504-044 | |
| Biosphere Filter Tip, 100 µL | Sarstedt, Nümbrecht, Germany | 70760212 | |
| Biosphere Filter Tip, 1250 µL | Sarstedt, Nümbrecht, Germany | 701186210 | |
| Biosphere Filter Tip, 20 µL | Sarstedt, Nümbrecht, Germany | 701114210 | |
| Biosphere Filter Tip, 300 µL | Sarstedt, Nümbrecht, Germany | 70765210 | |
| Bovine serum albumin | Carl Roth, Karlsruhe, Germany | 8076.4 | |
| Cell strainer, 100 µM | BD Bioscience, Heidelberg, Germany | 352360 | |
| Centrifuge 5810-R | Eppendorf AG, Hamburg, Germany | 5811000015 | |
| CO2 Incubator Heracell | Heraeus Instruments, Hanau, Germany | 51017865 | |
| Coverslips 12 mm | Carl Roth, Karlsruhe, Germany | P231.1 | |
| Curved fine forceps | Fine Science Tools GmbH, Heidelberg, Germany | 11370-42 | |
| DAPI fluoromount-G(R) | Biozol, Eching, Germany | SBA-0100-20 | |
| Dispase II | Sigma Aldrich GmbH, Steinheim, Germany | 4942078001 | |
| Distilled water (Water Purification System) | Millipore, Molsheim, France | ZLXS5010Y | |
| DMEM/F-12, GlutaMAX | Thermo Fisher Scientific, Schwerte, Germany | 31331093 | |
| DPBS (no Ca2+ and no Mg2+) | Sigma Aldrich GmbH, Steinheim, Germany | D8537-6X500ML | |
| Ethanol | VWR, Radnor, USA | 1009862500 | |
| FCS | Sigma Aldrich GmbH, Steinheim, Germany | F7524 | FCS must be tested for Schwann cell culture |
| Fine forceps (Dumont #5) | Fine Science Tools GmbH, Heidelberg, Germany | 11252-20 | |
| Forceps | Fine Science Tools GmbH, Heidelberg, Germany | 11370-40 | |
| Forskolin | Sigma Aldrich GmbH, Steinheim, Germany | F6886-10MG | |
| Gelatin | Sigma Aldrich GmbH, Steinheim, Germany | G1393-20ML | |
| Gentamycin | Thermo Fisher Scientific, Schwerte, Germany | 5710064 | |
| Goat anti-mouse IgG Alexa Fluor 488 | Thermo Fisher Scientific, Schwerte, Germany | A11036 | |
| Goat anti-rabbit IgG Alexa Fluor 568 | Thermo Fisher Scientific, Schwerte, Germany | A11001 | |
| HBSS (no Ca2+ and no Mg2+) | Thermo Fisher Scientific, Schwerte, Germany | 14170138 | |
| HERAcell Incubator | Heraeus Instruments, Hanau, Germany | 51017865 | |
| Heraguard ECO 1.2 | Thermo Fisher Scientific, Schwerte, Germany | 51029882 | |
| Horse serum | Pan-Biotech, Aidenbach, Germany | P30-0712 | |
| Image J Software | HIH, Bethesda, USA | ||
| Laminin | Sigma Aldrich GmbH, Steinheim, Germany | L2020-1MG | |
| Leibovitz´s L-15 Medium | Thermo Fisher Scientific, Schwerte, Germany | 11415064 | |
| L-Glutamine 200 mM | Thermo Fisher Scientific, Schwerte, Germany | 25030024 | |
| MACS Multistand | Miltenyi Biotec, Bergisch Gladbach, Germany | 130042303 | |
| Microscissors | Fine Science Tools GmbH, Heidelberg, Germany | 15000-08 | |
| Microscope | Motic, Wetzlar, Germany | Motic BA 400 | |
| Microscope Axio observer 7 | Zeiss, Oberkochen, Germany | 491917-0001-000 | |
| Microscope slide | VWR, Radnor, USA | 630-1985 | |
| MiniMACS separator | Miltenyi Biotec, Bergisch Gladbach, Germany | 130091632 | |
| MS columns | Miltenyi Biotec, Bergisch Gladbach, Germany | 130-042-201 | |
| Neubauer counting chamber | Assistant, Erlangen, Germany | 40441 | |
| Neuregulin | Peprotech, Rocky Hill, USA | 100-03 | |
| Neurobasal medium | Thermo Fisher Scientific, Schwerte, Germany | 21103049 | |
| NGF | Sigma Aldrich GmbH, Steinheim, Germany | N1408 | |
| Normal goat serum | Biozol, Eching, Germany | S-1000 | |
| Nunclon Δ multidishes, 4 well | Sigma Aldrich GmbH, Steinheim, Germany | D6789 | |
| Paraformaldehyde | Acros Organics, New Jersey, USA | 10342243 | |
| Penicillin/Streptomycin | Thermo Fisher Scientific, Schwerte, Germany | 15140-122 | |
| Pipetboy | Eppendorf AG, Hamburg, Germany | 4430000018 | |
| Pipettes | Eppendorf AG, Hamburg, Germany | 2231300004 | |
| Poly-D-Lysin | Sigma Aldrich GmbH, Steinheim, Germany | P6407-5MG | |
| Poly-L-Lysin | Sigma Aldrich GmbH, Steinheim, Germany | P4707-50ML | |
| Reaction tubes, 15 mL | Sarstedt, Nümbrecht, Germany | 62554502 | |
| Reaction tubes, 50 mL | Sarstedt, Nümbrecht, Germany | 62547254 | |
| Reaction vessels, 1.5 mL | Sarstedt, Nümbrecht, Germany | 72690001 | |
| Safety Cabinet S2020 1.8 | Thermo Fisher Scientific, Schwerte, Germany | 51026640 | |
| Scissors | Fine Science Tools GmbH, Heidelberg, Germany | 14083-08 | |
| Serological pipette, 10 mL | Sarstedt, Nümbrecht, Germany | 861254025 | |
| Serological pipette, 25 mL | Sarstedt, Nümbrecht, Germany | 861685001 | |
| Serological pipette, 5 mL | Sarstedt, Nümbrecht, Germany | 861253001 | |
| Spatula | Fine Science Tools GmbH, Heidelberg, Germany | 10094-13 | |
| Stereomicroscope Discovery.V8 | Zeiss, Oberkochen, Germany | 495015-0012-000 | |
| Surgical scissors | Fine Science Tools GmbH, Heidelberg, Germany | 14007-14 | |
| TC dish 100, cell + | Sarstedt, Nümbrecht, Germany | 833902300 | |
| TC dish 35, cell + | Sarstedt, Nümbrecht, Germany | 833900300 | |
| TC dish 60, cell + | Sarstedt, Nümbrecht, Germany | 833901300 | |
| Thy-1 Microbeads (MACS Kit) | Miltenyi Biotec, Bergisch Gladbach, Germany | 130-094-523 | |
| Triton X-100 | Sigma Aldrich GmbH, Steinheim, Germany | X100-500ML | |
| Trypan Blue Solution 0.4% | Thermo Fisher Scientific, Schwerte, Germany | 15250061 | |
| Trypsin (2.5%), no phenol red | Thermo Fisher Scientific, Schwerte, Germany | 15090-046 | |
| Trypsin-EDTA (0.05%), phenol red | Thermo Fisher Scientific, Schwerte, Germany | 25300-054 | |
| Type I Collagenase | Sigma Aldrich GmbH, Steinheim, Germany | C1639 | |
| Water bath type 1008 | GFL, Burgwedel, Germany | 4285 |
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Citar este artigo
Blusch, A., Sgodzai, M., Rilke, N., Motte, J., König, J., Pitarokoili, K., Grüter, T. In Vitro Myelination of Peripheral Axons in a Coculture of Rat Dorsal Root Ganglion Explants and Schwann Cells. J. Vis. Exp. (192), e64768, doi:10.3791/64768 (2023).