在哺乳动物的中枢神经系统视神经横断后的实验操作方法
Summary
视神经横断是一个成人中枢神经系统损伤的广泛使用的模型。这种模式是执行的全球目标的视网膜或直接目标受伤的视网膜神经节细胞神经元群的实验操作的理想选择。
Abstract
视网膜神经节细胞（RGC的）是中枢神经系统的神经元，输出的视觉信息从视网膜到大脑，通过视神经。视神经眼轨道内可以访问，并完全切断（axotomized），切割研资局在整个人口的轴突。视神经横断重现模型在1-4成人中枢神经系统的细胞凋亡的神经细胞死亡。这种模式是特别有吸引力，因为眼睛的玻璃体腔行为作为药物输送到视网膜的胶囊，允许通过人工注射的实验操作。通过玻璃体液体化学品的扩散，确保他们的行为后，整个研资局人口。病毒载体，质粒或短干扰RNAs（siRNAs），也可以传递到玻璃体腔感染或转染视网膜细胞 5-12 。腺相关病毒（AAV）载体的高向性是有利于目标的视网膜神经节细胞，感染率接近90％的细胞注射部位6，7，13-15附近。此外，视网膜神经节细胞，可以选择性地转，运用到自己的目标上丘 10的siRNA，质粒或病毒载体的削减 16-19视神经或注射载体结束。这使研究人员能够在受伤的神经元群细胞凋亡的机制研究没有其他旁观者神经元或神经胶质细胞周围的混杂影响。研资局凋亡有一个特点的时间进程，使细胞死亡是推迟3-4天postaxotomy后，这些细胞迅速退化。这提供了一个针对在细胞凋亡有关的途径的实验操作的窗口。操作直接针对视网膜神经节细胞从横断视神经残端后，立即切割神经干切断时间。相比之下，通过人工途径传递物质，它们可以被注射，手术前或在手术后第3天前开始axotomized RGC的凋亡。在本文中，我们展示了视神经横断后的实验操作的几种方法。
Protocol
1。外科技术使用无菌技术操作，并按照您的特定机构的动物使用协议，应进行实验。活体组织接触到的工具和材料（解决方案，测试物质，示踪剂，针等）必须是无菌的，以防止感染动物福利和研究的潜在的负面影响和不利影响。 2。麻醉老鼠将被麻醉的兽医异氟醚蒸发器系统。使用医疗级氧气在0.8 L / min的异氟醚气体蒸发的速度。放置在附加麻醉中的动…
Discussion
视神经横断是一个成人中枢神经系统神经细胞凋亡的高度重复性的模型。在这个手稿中表现出的实验操作许可证伤后RGC的凋亡机制的研究。
全球定位视网膜，眼内注射是有用的。这个过程需要一定的实践，关键是不要伤害了的玻璃吸管尖端的镜头。晶状体的损害已经造成生长因子的释放，改变细胞的存活和再生20，21。同样重要的是要小心地插入和退出提示的方向平?…
Declarações
The authors have nothing to disclose.
Acknowledgements
PDK是支持由CIHR经营授予（澳门币86523）
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Stereotaxic Frame | Stoelting, Kopf, WPI | |||
| Rat Gas Mask | Stoelting, Kopf, WPI | |||
| Anesthesia System | VetEquip | 901806 | ||
| Isoflurane (PrAErrane) | Baxter Corp | DIN 02225875 | ||
| Surgical Microscope | WPI, Zeiss, Leica | |||
| Alcaine Eye Drops | Alcon | |||
| Tears Naturale P.M. | Alcon | |||
| Fine tip Dumont forceps | Fine Science Tools | 11252-00 | ||
| 10 μl Hamilton Syringe (1701RN; 26s/2”/2) | Hamilton Syringe Co. | 80030 | ||
| 1/16 inch Compression Fittings | Hamilton Syringe Co. | 55751-01 | ||
| 1/16 inch OD, 0.010 inch ID, PEEK Tubing | Supelco, Bellefonte, PA | Z226661 | ||
| Dual RN Glass Coupler | Hamilton Syringe Co. | 55752-01 | ||
| Mineral Oil Priming Kit: includes syringe, needles, rubber septa | Hamilton Syringe Co. | PRMKIT |
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Tags
Optic Nerve TransectionExperimental ManipulationsMammalian CNSRetinal Ganglion CellsOptic NerveAxotomizedApoptotic Neuronal Cell DeathVitreous ChamberDrug DeliveryIntraocular InjectionsViral VectorsPlasmidsSiRNAsAdeno-Associated Virus (AAV) VectorsRGCs TargetingSuperior ColliculusApoptotic Mechanisms
Citar este artigo
D’Onofrio, P. M., Magharious, M. M., Koeberle, P. D. Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS. J. Vis. Exp. (51), e2261, doi:10.3791/2261 (2011).