生产和轴突隔离从感觉神经元的生化分析使用多孔过滤器
Summary
This article describes a neuronal culture system that can be used to obtain large quantities of pure axonal samples for biochemical and immunocytochemical analyses. This technique will allow an improved understanding of normal axonal physiology and signaling pathways leading to neurodegeneration.
Abstract
Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is required to shape functional neuronal circuits. The protocol described here explains how to use cell culture inserts bearing a porous membrane (filter) to obtain large amounts of pure axonal preparations suitable for examination by conventional biochemical or immunocytochemical techniques. The functionality of these filter inserts will be demonstrated with models of developmental pruning and Wallerian degeneration, using explants of embryonic dorsal root ganglion. Axonal integrity and function is compromised in a wide variety of neurodegenerative pathologies. Indeed, it is now clear that axonal dysfunction appears much earlier in the course of the disease than neuronal soma loss in several neurodegenerative diseases, indicating that axonal-specific processes are primarily targeted in these disorders. By obtaining pure axonal samples for analysis by molecular and biochemical techniques, this technique has the potential to shed new light into mechanisms regulating the physiology and pathophysiology of axons. This in turn will have an impact in our understanding of the processes that drive degenerative diseases of the nervous system.
Introduction
神经元的轴突舱显示了很大程度的躯体树突状隔间功能独立的。在投射神经元，轴突包含了大部分神经蛋白1,2。轴突的生理和病理生理的研究已经获得了在神经科学界的势头，因为最近的研究表明，早期轴突功能障碍似乎是神经退行性疾病和神经精神障碍3的 一个共同特征。这很可能是一个更好地了解正常和病理条件下轴突独家机制将揭示早期事件驱动功能障碍灯。
本协议描述了如何使用培养插入轴承的多孔膜（过滤器），以获得大量的纯粹性轴索材料，适用于生化和免疫组化分析。使用过滤器的插入，研究轴突生物学最早是由管家和实施同事4，并进一步向它的当前配置开发的特维斯和他的同事5。该技术现在处于当前使用通过研究轴突和树突生物学的不同方面，在每种情况下轻微的修改，以适应研究神经细胞的群体，所执行的处理和生物化学分析的类型，使用6-9。本协议不打算以适应这样的各种应用程序的所有备选方案，而是提供了一个简单的方法，是适用于大多数应用。特别是，这里所描述的协议使用三重涂层，最大限度地提高产量轴索用于生化分析和是研究文献中描述的轴突变性，其它涂层最合适的生产小于缩回和退化速度从营养因子剥夺9时轴突。
在此过程中，神经元细胞体保持隔离之上的过滤器的wh异亮氨酸的轴突穿过的孔，并沿其底表面上生长。纯轴突（免费神经胶质细胞和神经元细胞体污染物）在过滤器的底部表面生长，可用于采集生化分析或可固定在原位 ，并通过免疫细胞化学技术检测9。该过程依赖于使用胚胎感觉神经元从背根神经节（DRG）。胚胎背根节被广泛地用于研究轴突生物学，因为来自这些细胞的神经突起进行体外快速和强劲增长时，在神经生长因子（NGF）保持，并且当失去这个因素，因为它们迅速地进行变性。此外，DRG神经元缺乏树突，以便所有通过这种方法收集的突起是纯粹的轴突。
过滤器滤芯代表相比，用于研究轴突其他方法有很大的优势。研究依赖于使用显微镜来区分发生在从那些在胞体的过程xons提供有限的生化信息。作为另一个例子，条块培养系统（ 例如 Campenot室10或微流体装置11）是用于成像的方法和差分处理细胞区室的有用的，但仅提供少量的轴突材料制成的，排除了使用这些技术对于需要生化分析相对大量的样品。此外，它们的使用需要显著培训，以及专业的设备，他们是费时。常用于植隔离轴突另一种方法是轴突样品采集之前手动删除植中心（含神经元胞体）。虽然这可以产生大量的轴突富集制剂，在该制剂的轴突可以笼罩在神经胶质细胞和来自6个孔中（如一个最小的实验的一个例子）快速除去20外植体的体连续是费时和可行性的限制。
与此相反，这里提出的方法产生，然后可以通过几乎任何生化技术，通常被用于分析全细胞溶胞产物，如蛋白质印迹，免疫沉淀6，northern印迹5质谱6，RNA纯化进行分析丰富和纯轴索制剂7,12,13，等等。此外，该协议可以用于免疫荧光（IF）的技术，因为它可以固定轴突在过滤器的底侧上生长由中频进一步加以研究。这种方法大大方便了轴突的具体过程的分析，因为没有细胞体中的最后准备。这是一个显著的优势，因为从细胞体高免疫荧光信号往往破坏检查和较弱的信号由薄的结构，如神经轴突起源的分析。
两个应用程序的培养方法，研究轴突变性ARE中所述;发育修剪和损伤引起的退行性变的模型的模型（通常被称为Wallerian变性）。发育修剪的造型是基于一个事实，即在体 内的感觉神经元竞争限制目标源性神经生长因子和金额那些未能得到足够的神经营养支持简并14。这种现象可以通过从培养基中，其中，因为发生在体内 ，启动轴突变性，然后进行细胞体破坏吸NGF来模拟在胚胎背根神经节培养物。建模Wallerian变性，过滤器与细胞体的顶侧被刮掉。这已经增长到过滤器成为从细胞体物理上分离并随后经受谁最先报道的现象在1850年16称为Wallerian变性15的快速和刻板退化过程，A.沃勒命名后的底侧的轴突。
Protocol
Representative Results
Discussion
重要参数适应这种技术时要考虑到包括神经元群，将待研究的衬底，所述过滤器的孔径大小和表面积。所有这些参数会影响所获得的神经突制剂的特异性，质量和数量，并且必须由最终用户仔细考虑。在此协议中提出的例子，使用DRG神经元具有延伸仅轴突，从而给出纯的（特定的）轴突制剂的优点。
胚胎病种付费的轴突生长是非常快的比较，从中枢神经系统的神经元，使轴?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This project was supported by grant MOP62827 from the Canadian Institutes of Health Research.
Materials
| CD-1 mouse, E13 timed pregnancy | Charles River | In house timed pregnancies can be done by mating mice and checking for vaginal plugs (Day E0) | |
| Falcon Cell Culture Inserts | Corning | 353102 | 1 µm pore size, fits 6-well receiving plate |
| Falcon Cell Culture Companion Plates | Corning | 353502 | Receiving plates for inserts |
| Poly-D-Lysine | Sigma-Aldrich | P6407 | Used at 1 mg/ml in water. Can re-use once. |
| Laminin | Sigma-Aldrich | L2020 | Used at 10 ug/ml in water. Take 100X stock from -80 °C and thaw overnight at 4 °C |
| PureCol Bovine Collagen Solution | Advanced Biomatrix | 5005-B | |
| Leibovitz's L-15 Medium | Invitrogen | 11415-064 | Can be replaced by DMEM. |
| Neurobasal Medium | Invitrogen | 21103-049 | |
| B-27 Serum-Free Supplement | Invitrogen | 17504-044 | |
| 5-Fluoro-2′-deoxyuridine (FDU) | Sigma-Aldrich | F0503 | |
| NGF (2.5S, beta subunit) | Cedarlane | CLMCNET-001 | |
| Anti-NGF Antibody | Prepared in-house | – | As described in: Acheson, A., Barker, P.A., Alderson, R.F., Miller, F.D. & Murphy, R.A. Detection of brain-derived neurotrophic factor-like activity in fibroblasts and Schwann cells: inhibition by antibodies to NGF. Neuron.7, 265-75, (1991) |
| Alomone Labs | AN-240 | Commercial option | |
| Anti-Tubulin Antibody | Millipore | MAB5564 | |
| Alexa Fluor 488 Goat Anti-Mouse Antibody | Invitrogen | A-11029 | |
| DRG culture medium | |||
| Neurobasal Medium 2 % B27 Neurobasal Supplement 2 mM l-Glutamine 1 % Penicillin-Streptomysin 10 µM FDU 15 ng/ml NGF (bottom compartment only) |
|||
| 1X Laemmli Sample Buffer | |||
| 10 % SDS 0.5 M DTT 0.5 M Tris (pH 6.8) 5 % Glycerol 0.01 % (w/v) Bromophenol blue |
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