背根神经节内注射和背根挤压伤为感官轴突再生模型
Summary
This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.
Abstract
Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.
Introduction
神经系统损伤后实现轴突再生是一项艰巨的任务1。研究轴突再生的中枢神经系统（CNS）的失败，研究人员利用神经损伤模型过多。由于中枢神经系统的地区不同，它使用一个适当的解剖模型来研究轴突再生是非常重要的。通过使用合适的模型，研究人员可以制定基于损伤的严重程度，感兴趣的神经元细胞类型的特定处理，并评估再生所需的脊髓束，而不是“一个对所有人”的治疗策略。
在脊髓损伤，例如，最衰弱的症状，从感觉和运动的损失造成的。感觉丧失是由在上行感觉通路造成的损害，而运动的损失是由该下行运动路径造成的损害。由于这些TW之间的蜂窝和解剖学上的差异Ø途径，许多有针对性的轴突再生的研究只专注于一个或另一个途径，与逻辑，即任一成功恢复将是巨大的利益给患者。在本文中，我们提出一种使用直接背根神经节（DRG）注射用病毒载体和在成年大鼠作为模型来研究感觉轴突再生的下颈脊髓并发背根挤压伤的协议。
DRG感觉神经元负责中继感官信息，如触觉和疼痛，从外围到中枢神经系统。在脊髓中的感觉神经元的轴突长突起作为一个很好的模型来研究长途轴突再生。此外，由于啮齿动物可以生存感觉传导通路的病变，如背根挤压伤以最小的福利并发症，研究人员可以研究中枢神经系统轴突再生，而无需完全损伤脊髓。一个四C5 – C8（宫颈升伊维尔基尼5 – 8）背根挤压伤已被证明是用于前爪传入神经阻滞2的有用模型。此外，背根挤压伤提供了一个“更清洁”的模型来研究轴突再生比直接脊髓损伤，因为它是由其他因素简单如胶质瘢痕的形成。
重编程为神经元再生状态使用病毒基因治疗已越来越多地被视为对许多神经系统疾病3有前途的治疗策略。研究表明携带生长促进蛋白的转基因可以实现鲁棒轴突再生与行为恢复4，5，6的腺相关病毒（AAV）载体的应用。在引发免疫应答和转导非分裂细胞，如神经元的能力AAV的表观低致病性，使它用于基因治疗的最佳载体。另外，重组AAV形式用于治疗。在这种形式中，它不能其病毒基因组整合到宿主基因组7，减少相对于其他的病毒载体，如慢病毒插入诱变的风险。这使得AAV基因治疗应用的安全选择。
作为一个包含DRG感觉神经元的细胞体，它是病毒的基因治疗管理的最合适的解剖目标，研究和/或促进感觉轴突再生。在比较不同的AAV血清型和慢病毒的研究中，AAV血清型5（AAV5）被证明是最有效的在当直接注射到DRG 8超过至少12周的时间过程转导的DRG神经元。此外，AAV可以实现40％以上的转导效率，转导所有DRG神经元亚型，如大直径神经丝200kDa的（NF200）阳性神经元和小直径降钙素基因相关肽（CGRP） -或同工凝集素B4（IB4）阳性神经元4,8。
由于DRG注射和背根挤压伤的手术过程是非常侵入性的细腻，我们认为，这篇文章将帮助新用户学习在一个非常有效的方式方法。在这篇文章中，我们将展示来自成年大鼠注射对照病毒AAV5-GFP（绿色荧光蛋白）到C6四个星期后有代表性的结果 – C7病种付费用并发C5 – C8背根挤压伤。这种模式特别适用于谁的研究人员正在研究利用病毒基因疗法来促进感觉轴突再生。
Protocol
Representative Results
Discussion
在本文中，我们提出了一种一步一步的指导，在成年大鼠的下颈脊髓进行DRG注射和背根挤压伤。由于这是一个非常侵入性和精细的外科手术，我们强烈建议所有潜在用户推进到活的动物在手术前获得足够的培训和实践。用户应该熟悉不仅与脊髓的解剖结构，而且还与周围的肌肉组织，椎体骨质结构，和血管。理想情况下，主管用户应该能够执行以最小的损伤的过程向周围的组织，通过而不诱导脊?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由从克里斯托弗和戴纳·里夫基金会，医学研究理事会，欧洲研究理事会ECMneuro和NHMRC剑桥生物医学研究中心的资助。我们想表达我们最深切的感谢海琳·梅勒范“T Spijker和贾斯泰纳·巴拉特在拍摄过程中的技术援助。我们要感谢伊丽莎白·莫洛内博士和乔斯特·弗哈根教授（荷兰神经科学研究所）在AAV生产协助。
Materials
| Fast Green FCF dye | Sigma-Aldrich | F7258 | For visualizing colorless solution. Recommended concentration: 1% |
| Cholera Toxin B subunit | List Biological Laboratories | 104 | For anterograde axonal tracing. Recommended concentration: 1% |
| IsoFlo | Zoetis | 115095 | Inhalation anesthetic (active ingredient: isoflurane) |
| Baytril 2.5% injectable | Bayer | 05032756093017 | Antibiotic (active ingredient: enrofloxacin). Manufacturer's recommended dosage: 10 mg/kg |
| Carprieve 5.0% w/v | Norbrook | 02000/4229 | Analgesic (active ingredient: carprofen). Manufacturer's recommended dosage: 4 mg/kg |
| Lacri-Lube | Allergan | PL 00426/0041 | Eye ointment |
| Olsen-Hegar Needle Holder | Fine Science Tools | FST 12502-12 | |
| Friedman Pearson Rongeur Curved 0.7mm Cup | Fine Science Tools | FST 16121-14 | |
| Bonn Micro Forceps | Fine Science Tools | FST 11083-07 | For performing dorsal root crush injury |
| Tissue Separating Scissors | Fine Science Tools | FST 14072-10 | |
| Fine Scissors | Fine Science Tools | FST 14058-11 | |
| Micro-Adson Forceps | Fine Science Tools | FST 11018-12 | |
| Goldstein Retractor | Fine Science Tools | FST 17003-03 | |
| Vannas Spring Scissors (straight) | Fine Science Tools | FST 15018-10 | |
| SURGIFOAM Absorbable Gelatin Sponge | Ethicon | 1972 | For bleeding control |
| Microliter Syringe RN701 (10 μl) | Hamilton | 80330 | |
| Custom-made Removable Needle (for DRG injection) | Hamilton | 7803-05 | 33 gauge, 38 mm, point style 3 |
| Custom-made Removable Needle (for CTB injection) | Hamilton | 7803-05 | 33 gauge, 10 mm, point style 3 |
| UltraMicroPump with SYS-Micro4 Controller | World Precision Instruments | UMP3-1 |
References
- Chew, D. J., Fawcett, J. W., Andrews, M. R. The challenges of long-distance axon regeneration in the injured CNS. Prog Brain Res. 201, 253-294 (2012).
- Wu, A., Lauschke, J. L., Morris, R., Waite, P. M. Characterization of rat forepaw function in two models of cervical dorsal root injury. J Neurotrauma. 26 (1), 17-29 (2009).
- Hocquemiller, M., Giersch, L., Audrain, M., Parker, S., Cartier, N. Adeno-Associated Virus-Based Gene Therapy for CNS Diseases. Hum Gene Ther. 27 (7), 478-496 (2016).
- Cheah, M., et al. Expression of an Activated Integrin Promotes Long-Distance Sensory Axon Regeneration in the Spinal Cord. J Neurosci. 36 (27), 7283-7297 (2016).
- Andrews, M. R., et al. Alpha9 integrin promotes neurite outgrowth on tenascin-C and enhances sensory axon regeneration. J Neurosci. 29 (17), 5546-5557 (2009).
- Tan, C. L., et al. Kindlin-1 enhances axon growth on inhibitory chondroitin sulfate proteoglycans and promotes sensory axon regeneration. J Neurosci. 32 (21), 7325-7335 (2012).
- McCarty, D. M., Young, S. M., Samulski, R. J. Integration of adeno-associated virus (AAV) and recombinant AAV vectors. Annu Rev Genet. 38, 819-845 (2004).
- Mason, M. R., et al. Comparison of AAV serotypes for gene delivery to dorsal root ganglion neurons. Mol Ther. 18 (4), 715-724 (2010).
- Hermens, W. T., et al. Purification of recombinant adeno-associated virus by iodixanol gradient ultracentrifugation allows rapid and reproducible preparation of vector stocks for gene transfer in the nervous system. Hum Gene Ther. 10 (11), 1885-1891 (1999).
- Kappagantula, S., et al. Neu3 sialidase-mediated ganglioside conversion is necessary for axon regeneration and is blocked in CNS axons. J Neurosci. 34 (7), 2477-2492 (2014).
- Alto, L. T., et al. Chemotropic guidance facilitates axonal regeneration and synapse formation after spinal cord injury. Nat Neurosci. 12 (9), 1106-1113 (2009).
- Bonner, J. F., et al. Grafted neural progenitors integrate and restore synaptic connectivity across the injured spinal cord. J Neurosci. 31 (12), 4675-4686 (2011).
- Wang, R., et al. Persistent restoration of sensory function by immediate or delayed systemic artemin after dorsal root injury. Nat Neurosci. 11 (4), 488-496 (2008).
- Wong, L. E., Gibson, M. E., Arnold, H. M., Pepinsky, B., Frank, E. Artemin promotes functional long-distance axonal regeneration to the brainstem after dorsal root crush. Proc Natl Acad Sci U S A. 112 (19), 6170-6175 (2015).
- Tanguy, Y., et al. Systemic AAVrh10 provides higher transgene expression than AAV9 in the brain and the spinal cord of neonatal mice. Front Mol Neurosci. 8, 36 (2015).
- Foust, K. D., Poirier, A., Pacak, C. A., Mandel, R. J., Flotte, T. R. Neonatal intraperitoneal or intravenous injections of recombinant adeno-associated virus type 8 transduce dorsal root ganglia and lower motor neurons. Hum Gene Ther. 19 (1), 61-70 (2008).
- Vulchanova, L., et al. Differential adeno-associated virus mediated gene transfer to sensory neurons following intrathecal delivery by direct lumbar puncture. Mol Pain. 6, 31 (2010).
- Gray, S. J., et al. Directed evolution of a novel adeno-associated virus (AAV) vector that crosses the seizure-compromised blood-brain barrier (BBB). Mol Ther. 18 (3), 570-578 (2010).
- Fagoe, N. D., Attwell, C. L., Kouwenhoven, D., Verhaagen, J., Mason, M. R. Overexpression of ATF3 or the combination of ATF3, c-Jun, STAT3 and Smad1 promotes regeneration of the central axon branch of sensory neurons but without synergistic effects. Hum Mol Genet. 24 (23), 6788-6800 (2015).
