Name: dorsal root ganglion injection and dorsal root crush injury as a model for sensory axon regeneration
Uploaded: 2017-05-03T14:00:00
Description: Watch this Scientific Journal Video about Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration at JoVE.com

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。

以下所有动物的程序是按照英国动物（科学程序）法案1986年如果不熟悉这些程序进行的，请向当地/国家法规检查并启动协议前寻求兽医的意见。 1.选择动物的合适的应变注：背根挤压伤引起的轰动和爪的传入神经阻滞的损失。前爪传入神经阻滞常见的不良影响包括过度修饰，自残，和爪自残。 <ol><li>获得动物的程序。 注：对于大鼠，更具活性的菌株，例如利斯?...

<ol>
	<li>Chew, D. J., Fawcett, J. W., Andrews, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+challenges+of+long-distance+axon+regeneration+in+the+injured+CNS.">The challenges of long-distance axon regeneration in the injured CNS.</a> Prog Brain Res. 201, 253-294 (2012).</li><li>Wu, A., Lauschke, J. L., Morris, R., Waite, P. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+adeno-associated+virus+(AAV)+and+recombinant+AAV+vectors.">Integration of adeno-associated virus (AAV) and recombinant AAV vectors.</a> Annu Rev Genet. 38, 819-845 (2004).</li><li>Mason, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+AAV+serotypes+for+gene+delivery+to+dorsal+root+ganglion+neurons.">Comparison of AAV serotypes for gene delivery to dorsal root ganglion neurons.</a> Mol Ther. 18 (4), 715-724 (2010).</li><li>Hermens, W. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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.</a> Hum Gene Ther. 10 (11), 1885-1891 (1999).</li><li>Kappagantula, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neu3+sialidase-mediated+ganglioside+conversion+is+necessary+for+axon+regeneration+and+is+blocked+in+CNS+axons.">Neu3 sialidase-mediated ganglioside conversion is necessary for axon regeneration and is blocked in CNS axons.</a> J Neurosci. 34 (7), 2477-2492 (2014).</li><li>Alto, L. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotropic+guidance+facilitates+axonal+regeneration+and+synapse+formation+after+spinal+cord+injury.">Chemotropic guidance facilitates axonal regeneration and synapse formation after spinal cord injury.</a> Nat Neurosci. 12 (9), 1106-1113 (2009).</li><li>Bonner, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grafted+neural+progenitors+integrate+and+restore+synaptic+connectivity+across+the+injured+spinal+cord.">Grafted neural progenitors integrate and restore synaptic connectivity across the injured spinal cord.</a> J Neurosci. 31 (12), 4675-4686 (2011).</li><li>Wang, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+restoration+of+sensory+function+by+immediate+or+delayed+systemic+artemin+after+dorsal+root+injury.">Persistent restoration of sensory function by immediate or delayed systemic artemin after dorsal root injury.</a> Nat Neurosci. 11 (4), 488-496 (2008).</li><li>Wong, L. E., Gibson, M. E., Arnold, H. M., Pepinsky, B., Frank, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artemin+promotes+functional+long-distance+axonal+regeneration+to+the+brainstem+after+dorsal+root+crush.">Artemin promotes functional long-distance axonal regeneration to the brainstem after dorsal root crush.</a> Proc Natl Acad Sci U S A. 112 (19), 6170-6175 (2015).</li><li>Tanguy, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+AAVrh10+provides+higher+transgene+expression+than+AAV9+in+the+brain+and+the+spinal+cord+of+neonatal+mice.">Systemic AAVrh10 provides higher transgene expression than AAV9 in the brain and the spinal cord of neonatal mice.</a> Front Mol Neurosci. 8, 36 (2015).</li><li>Foust, K. D., Poirier, A., Pacak, C. A., Mandel, R. J., Flotte, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+intraperitoneal+or+intravenous+injections+of+recombinant+adeno-associated+virus+type+8+transduce+dorsal+root+ganglia+and+lower+motor+neurons.">Neonatal intraperitoneal or intravenous injections of recombinant adeno-associated virus type 8 transduce dorsal root ganglia and lower motor neurons.</a> Hum Gene Ther. 19 (1), 61-70 (2008).</li><li>Vulchanova, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+adeno-associated+virus+mediated+gene+transfer+to+sensory+neurons+following+intrathecal+delivery+by+direct+lumbar+puncture.">Differential adeno-associated virus mediated gene transfer to sensory neurons following intrathecal delivery by direct lumbar puncture.</a> Mol Pain. 6, 31 (2010).</li><li>Gray, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+evolution+of+a+novel+adeno-associated+virus+(AAV)+vector+that+crosses+the+seizure-compromised+blood-brain+barrier+(BBB).">Directed evolution of a novel adeno-associated virus (AAV) vector that crosses the seizure-compromised blood-brain barrier (BBB).</a> Mol Ther. 18 (3), 570-578 (2010).</li><li>Fagoe, N. D., Attwell, C. L., Kouwenhoven, D., Verhaagen, J., Mason, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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.</a> Hum Mol Genet. 24 (23), 6788-6800 (2015).</li></ol>

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?...

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。

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	<tbody>
		<tr>
			<td>Fast Green FCF dye</td>
			<td>Sigma-Aldrich</td>
			<td>F7258</td>
			<td>For visualizing colorless solution. Recommended concentration: 1%</td>
		</tr>
		<tr>
			<td>Cholera Toxin B subunit</td>
			<td>List Biological Laboratories</td>
			<td>104</td>
			<td>For anterograde axonal tracing. Recommended concentration: 1%</td>
		</tr>
		<tr>
			<td>IsoFlo</td>
			<td>Zoetis</td>
			<td>115095</td>
			<td>Inhalation anesthetic (active ingredient: isoflurane)</td>
		</tr>
		<tr>
			<td>Baytril 2.5% injectable</td>
			<td>Bayer</td>
			<td>05032756093017</td>
			<td>Antibiotic (active ingredient: enrofloxacin). Manufacturer&#39;s recommended dosage: 10 mg/kg</td>
		</tr>
		<tr>
			<td>Carprieve 5.0% w/v</td>
			<td>Norbrook</td>
			<td>02000/4229</td>
			<td>Analgesic (active ingredient: carprofen). Manufacturer&#39;s recommended dosage: 4 mg/kg</td>
		</tr>
		<tr>
			<td>Lacri-Lube</td>
			<td>Allergan</td>
			<td>PL 00426/0041</td>
			<td>Eye ointment</td>
		</tr>
		<tr>
			<td>Olsen-Hegar Needle Holder</td>
			<td>Fine Science Tools</td>
			<td>FST 12502-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Friedman Pearson Rongeur Curved 0.7mm Cup</td>
			<td>Fine Science Tools</td>
			<td>FST 16121-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Bonn Micro Forceps</td>
			<td>Fine Science Tools</td>
			<td>FST 11083-07</td>
			<td>For performing dorsal root crush injury</td>
		</tr>
		<tr>
			<td>Tissue Separating Scissors</td>
			<td>Fine Science Tools</td>
			<td>FST 14072-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors</td>
			<td>Fine Science Tools</td>
			<td>FST 14058-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Adson Forceps</td>
			<td>Fine Science Tools</td>
			<td>FST 11018-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Goldstein Retractor</td>
			<td>Fine Science Tools</td>
			<td>FST 17003-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors (straight)</td>
			<td>Fine Science Tools</td>
			<td>FST 15018-10</td>
			<td></td>
		</tr>
		<tr>
			<td>SURGIFOAM Absorbable Gelatin Sponge</td>
			<td>Ethicon</td>
			<td>1972</td>
			<td>For bleeding control</td>
		</tr>
		<tr>
			<td>Microliter Syringe RN701 (10 &#956;l)</td>
			<td>Hamilton</td>
			<td>80330</td>
			<td></td>
		</tr>
		<tr>
			<td>Custom-made Removable Needle (for DRG injection)</td>
			<td>Hamilton</td>
			<td>7803-05</td>
			<td>33 gauge, 38 mm, point style 3</td>
		</tr>
		<tr>
			<td>Custom-made Removable Needle (for CTB injection)</td>
			<td>Hamilton</td>
			<td>7803-05</td>
			<td>33 gauge, 10 mm, point style 3</td>
		</tr>
		<tr>
			<td>UltraMicroPump with SYS-Micro4 Controller</td>
			<td>World Precision Instruments</td>
			<td>UMP3-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

dorsal root ganglion injection and dorsal root crush injury as a model for sensory axon regeneration

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.

作为代表性，呈现与所连接的DRG的横向脊髓部分直接显示该协议的有效性在转导DRG神经元和注射对照病毒，AAV5-GFP后过四星期跟踪在脊髓感觉轴突，入C7 DRG而不背根挤压伤（ 图1A）。在这两个背柱和脊髓背角神经轴突表达GFP（ 图1B），以及注入的DRG（ 图1C）中的细胞体和轴突。所述楔形核的额外的解剖分析，在脑干中的感觉轴突终端?...

Watch this Scientific Journal Video about Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration at JoVE.com

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

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.

背根神经节内注射和背根挤压伤为感官轴突再生模型

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Research

JoVE Journal

Neuroscience

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

染色质免疫沉淀从以下轴索损伤的背根神经节组织

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after ...

科研

神经科学

Live Imaging of Dorsal Root Axons after Rhizotomy

实时成像背根轴突后根切断术

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

培养成年的背根神经节神经元轴突再生的遗传研究

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

在癌神经侵袭的体外模型：背根神经节型号

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...

An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons

一种方法来提高背根神经节的对齐和髓鞘

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to ...

Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine

Laminotomy 对猪腰背根神经节通路和注射的治疗

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

应用流式细胞仪分析单侧坐骨神经和背根神经节的免疫细胞

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

背根神经节分离与原代培养对神经递质释放的研究

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

哺乳动物轴突再生: 成年小鼠背根神经节体内电穿孔的研究

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

Rapid Isolation of Dorsal Root Ganglion Macrophages

多尾根结鱼巨噬细胞的快速隔离

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after ...