一种方法来提高背根神经节的对齐和髓鞘
Summary
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.
Abstract
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.
Introduction
在神经损伤，近端和远端神经断端经常由神经束的直接调整防止由于病灶部位1-2。通常情况下，轴突束是由轴突高度有序的排列和捆绑，形成连通的复杂网络。然而，神经再生是由于缺乏组织轴突3-4对准一个混乱的过程。因此，为了产生足够数量的再生该桥接病灶部位的轴突，有必要以诱导良好的组织轴突对齐。此外，伴随脱髓鞘由于在损伤部位的髓鞘细胞的死亡神经损伤。由于脱髓鞘强烈损害神经轴突尚存的导电能力，靶向治疗脱髓鞘或促进髓鞘是神经损伤后5功能恢复显著。因此，这个协议的目的是为了说明工程方法，解决这两个问题神经再生。
面各向异性，当沿着不同的轴测量，在材料的物理或机械性能被定义为一个差，已经应用影响细胞对准，生长和迁移6-7。除了地形，还有其他的方法来诱导各向异性。先前，我们研究了通过聚二甲基硅氧烷（PDMS）膜的机械静态预拉伸诱导的表面的各向异性。 “强加于大小变形超级”的理论预测，将细胞在拉伸方向感测有效刚度与垂直方向不同，并且在有效刚度这种差异是由于表面各向异性8。间充质干细胞（MSC）上的预拉伸的PDMS膜培养能够通过积极地拉动面，其结果，以感测的各向异性，在预拉伸方向9对齐。同样地，各向异性表面芳从一个机械预拉伸的表面伊辛影响对准，以及生长和髓鞘形成背根神经节的（DRG）轴突10。在这里，我们提供了一个静态预拉伸PDMS基板上诱导表面各向异性以增强轴突再生10的协议。
为了激发轴突对齐，用需要的模式拓扑特征，通过报道排列的纤维和渠道6,11-12提供联系指导，被证明有利于轴突排列11,13。但是，报道了通过拓扑特征，如纤维，频道和图案化诱导轴突对准技术，无法延长并增加轴突的厚度。与此相反，逐步机械拉伸导致更长和更厚的轴突与该拉伸14的幅度增大拉伸方向轴突对齐。然而， 在体内的结合有动力的电机装置不可行。与此相反，静态预拉伸引起的各向异性是不太复杂，并且可以更容易地并入未来支架设计用于体内应用。
在这个协议中，静态预拉伸细胞培养系统用于诱导表面各向异性无拓扑特征。预拉伸培养系统由PDMS膜，可拉伸帧和拉伸阶段，随后该膜被固定到框架和预定的拉伸幅度施加在拉伸阶段。预拉伸表面上培养了21天新鲜分离的DRG神经元中轴突对准和厚度进行监测。接着，雪旺细胞（SC）上的对准轴突为髓鞘监测共培养。通过采用预拉伸诱导表面各向异性，我们能够提高MSC和背根神经节9-10，分别对准细胞分化和轴突对齐增长。
Protocol
Representative Results
Discussion
为了诱导对预拉伸的表面轴突对准，存在两个关键步骤：1）的PDMS膜必须是平坦的和均匀的厚度;和2）的神经胶质细胞，必须从DRG中移除。混合的PDMS和交联剂并在烘箱中固化后，交联的PDMS凝胶应保持在平坦的工作台上，并仔细处理，以免任何倾斜。的PDMS膜的氧等离子处理之后，应当锁相环涂层6小时内，由于表面的等离子体处理后的亲水性（细胞附着必需）衰减随时间。因为在等离子体处理被施?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
笔者想感谢埃里克·瓦斯科对他的援助在PDMS基板的准备，墉王滨海马塔博士在密歇根大学实验室进行有益的建议和DRG隔离的培训博士和马克Tuszynski博士和博士为有益的建议和协议为SC隔离。W.玛丽·坎帕纳在加州大学圣地亚哥分校。这项研究是由美国国家科学基金会（CBET 0941055和CBET 1510895），美国国立卫生研究院（R21CA176854，R01GM089866和R01EB014986）的部分资助。
Materials
| Polydimethylsiloxane （PDMS) | Dow Corning | SYLGARD 184 | |
| Neurobasal Medium 1X | GibcoBRL | 21103-049 | |
| B27 Supplement 50X | GibcoBRL | 17504-044 | |
| Glutamax-I 100X | GibcoBRL | 35050-061 | |
| Albumax-I | GibcoBRL | 11020-021 | |
| Nerve Growth Factor-7S | Invitrogen | 13290-010 | |
| Penicillin-streptomycin | GibcoBRL | 15140-122 | |
| 0.05% Trypsin-EDTA/1mM EDTA | GibcoBRL | 25300-054 | |
| Poly-L-Lysine | Trevigen | 3438-100-01 | |
| Poly-D-Lysine | Sigma | p-6407 | |
| Fluoro-2 deoxy-uridine | Sigma | F0503 | |
| Uridine | Sigma | U3003 | |
| Hank’s Balanced Salt Solution (HBSS) | Invitrogen | 14170-112 | Isolation Buffer |
| Type I Collagenase | Worthington | LS004196 | |
| DMEM | Gibco | 11885 | |
| Heat inactivated Fetal Bovine Serum | Hyclone | SH30080.03 | |
| BPE | Clonetics | CC-4009 | |
| Forskolin | Calbiochem | 344270 | |
| Silicone chamber | Greiner bio-one | FlexiPERM ConA | |
| Plasma cleaning/etching system | March Instruments | PX-250 | |
| Anti-Thy 1.1 antibody | Sigma- Aldrich | M7898 | |
| Rabbit Complement | Sigma- Aldrich | S-7764 | |
| Standard growth medium | For 500 ml Neurobasal Medium 1X, add 10 ml of B-27 50X, 5 ml of Glutamax-I 100X, | ||
| 2.5 ml of Penicillin/Streptomycin (Penn/Strep), 1 ml of Albumax-I, and 1 μl of NGF– 7S (50 ug/ml). | |||
| FDU and Uridine stock solution | FDU 100mg in 10ml of ddw (10mg/ml), filter in the hood and divided in 500ul aliquots and store at -20 ºC. | ||
| Uridine 5g in 166.7ml of ddw (33mg/ml), filter in hood, divide in 200ul aliquots and store at -20 ºC. | |||
| Take 61.5ul of FDU (10mg/ml) and 20.5ul of Uridine(33mg/ml), and add 4918ul of ddw to a final stock concentration, | |||
| then divide in 1 ml aliquots and store at -20 ºC. |
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