我们感谢阿兰泰斯勒博士评论和社论帮助。这项工作是由NIH NS062320支持。

1。显微镜设置和成像准备<ol><li>我们的成像成立了Leica MZ16较快的快门和MetaMorph软件控制冷却的CCD相机的荧光体视显微镜。 </li><li>准备一个恒温控制加热垫，并调整到32.5 ° C，以保持动物的体温期间和手术后的输出。 </li><li>暖无菌林格氏液，或到32.5 ° C，在提前在手术过程中脊髓灌溉的人工脑脊液（学联）。 </li><li>甲苯噻嗪（8毫克/公斤），氯胺酮（120毫克/公斤）的鸡尾酒腹腔注射麻醉动物。 </li><li>剃须与小动物快船队的上背部和传播的一个小水滴在剃光面积脱毛乳液与棉拭子。几分钟后，删除应用的乳液，使用70％乙醇浸泡纱布海绵。 </li></ol> 2。椎板切除手术暴露L5背根<ol><li>将加热的（32.5 ° C）垫的动物，并用70％乙醇浸泡棉签消毒皮肤。 </li><li>在明亮的体视显微镜视场照明，执行正中切口（2 - 3厘米），背部皮肤。如果有必要，使用棉拭子止血。 </li><li>反映脊髓肌肉公开基础腰椎。 </li><li>揭露右路半椎板使用小rongeurs的L3 - S1脊髓段。 4-6腰椎和骶脊髓段部分椎板切除暴露创建卸下背右髋关节的髂骨水平L5椎体部分（L5背根神经节的位置）到L2椎体（2 rostrally到最后一根肋骨，椎骨尾鳍）。与温暖的无菌林格氏液灌注腔。 </li><li>支持垫（轧棉纱布）上的位置，拉平脊椎骨的动物。使用回缩挂钩的暴露面积扩大。 </li><li>此时（约30分钟后，第一次腹腔注射麻醉，应皮下注射的一种补充（0.5X），以保持动物完全麻醉。此外，麻醉气体（2-4％0.5L/min氧气异氟醚），可用于重复麻醉成像会议期间超过1小时。 </li></ol> 3。根切断术/背根粉碎<ol><li>切换到荧光激发想像YFP的标记（+）轴突。 </li><li>使用一个Sub - Q（26ga.）针的尖端，执行一个小切口覆L5背根（DR）的硬脑膜。多次与林格氏液灌注，和干净的棉拭子轻轻。 </li><li>确定被压扁的网站，并插入罚款镊子的一侧（杜蒙＃5）subdurally。 </li><li>轻轻地但坚决关闭镊子，控股10秒L5根内侧部分，然后轻轻松开镊子。 </li><li>生理溶液反复冲洗干净的棉签拭子轻轻。 </li></ol> 4。图像采集和运算程序<ol><li>获取多个图像整个暴露面积，包括粉碎站点前，右后粉碎在低和高放大倍率的DREZ。 </li><li>图像采集单一快照或多个数据流的10至20帧，在30个收购 - 40毫秒的曝光时间。对焦的图像，然后选定，并概述蒙太奇以后使用Photoshop创建。 </li><li>为了最大限度地减少疤痕形成，紧紧申请了一块薄合成基质膜（Biobrane），其次是人工硬脑膜，暴露脊髓上。可以肯定的裁片以适应恰恰暴露脊髓窗口，使它们附着在脊髓。 </li><li>用无菌5-0缝线关闭肌肉和关闭伤口剪辑正中切口。 </li><li>注入林格氏液（0.3至0.5毫升皮下注射），和管理丁丙诺啡术后镇痛（0.05毫克/公斤）皮下注射，每2天12小时。 </li><li>保持一个加热垫（34 ° -35℃），直至收回的动物。 </li></ol> 5。重复成像<ol><li>麻醉动物和删除的伤口剪辑和缝合。 </li><li>取出人工硬脑膜和薄合成基质膜的补丁，并保持在无菌林格氏液管供以后重复使用。 </li><li>轻轻地取出任何一个Sub - Q的弯针和细镊子尖端积累的结缔组织瘢痕。与温暖的林格氏液灌注经常。 </li><li>重新暴露手术视野，包括粉碎站点和DREZ，搬迁YFP +前几届会议成像轴突，重复第4节中描述的程序。 </li></ol> 6。代表性的成果： 我们观察到，而他们没有一个横断损伤后再生，几乎所有YFP +的网站通过轴突增长粉碎后3天伤（图1）11。通常情况下，粉碎后的第二天，我们观察到死亡的回近端残端神经轴突变性和碎片/相同轴突远端粉碎，这证实，轴突已经适当损坏变性（例如，图1; 3和第5天） 。另外还有几个标准适用明确区分，也难幸免，或已经从伤病中恢复的轴突再生的轴突。这些包括以下内容：（1）再生轴突显示一个的的YFP的非荧光部分扩张+的粉碎网站由于近端和远端变性（对比的未标记的差距缩小由于荧光细胞质加气站的粉碎网站轴突如果轴突存活的损伤）;（2）再生轴突薄得多，明亮的荧光，更比幸存下来的损伤的轴突起伏;（3）再生突起更薄，更昏暗比轴突退化的荧光片段的荧光，通过他们延长;（4）对比尚存或幸免轴突再生轴突停止在DREZ;（5）对比尚存或幸免轴突再生轴突不会出现Ranvier节点。图1显示了四个后立即粉碎肤浅YFP +轴突（A1彩色箭头）。粉碎后3天，所有四个轴突延长一个单一的神经突起的生长，通过粉碎网站（A2）。五天后粉碎，突起保持稳定，并没有从这些或其他的近端轴突（A3）的额外增长。 再生突起，越过粉碎网站通过一个退化的轴突（即，endoneurial管）更厚和更明亮的的荧光片段拉长，并早在4天到达的DREZ粉碎后（约3mm / 2 天 ） 11。重复这些轴突和他们的技巧两个星期（图2）每两三天成像透露，他们并没有增长向前或收回，但仍然不动。唯一显着的变化是肿胀的提示和一些轴突的轴。因此，这些意见的DREZ表现出惊人的快速和慢性的再生轴突的固定。 <img src="/files/ftp_upload/3126/3126fig1.jpg" alt="图1"/> 图1：重复成像L5 DR YFP的轴突在背根粉碎网站超过5天。被粉碎的L5根内侧部分（红色箭头）和天0，3，粉碎后5的影像。在右面板（A1 - A3的）的区域被放大的美眉。 <img src="/files/ftp_upload/3126/3126fig2.jpg" alt="图2"/> 图2：轴突重复成像在抵达DREZ L5根粉碎后超过20天的每天4，三个轴突（彩色箭头）抵达的DREZ 。这些轴突的尖端保持在相同的位置，并在随后的成像会议上7天，9，13，15和 20类似的外观。轴突尖端相对其他轴突提示和标志性建筑（星号）的位置被用来确定轴突成像会话之间的蠕动。

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没有利益冲突的声明。

成像DR直接在活老鼠再生，是特别具有挑战性的，因为它需要大量的背椎板切除，监察多个微创手术和麻醉的程序，在随后的成像会话广域轴突的生长。几种策略帮助克服这些挑战。首先，成功的成像要求减少麻醉和出血时间，降低小鼠的死亡率（约25％），并通过细致的术后护理。死亡率也减少了使用，允许快速的摄像头的速度快速采集多个图像，无需插管或呼吸中断设置。上外露的电源线防止...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td> H线thy1 - YFP（2-4个月左右，雌雄不限） </td><td>杰克逊实验室（酒吧港，ME） </td><td> 003782 </td><td></td></tr><tr><td>甲苯噻嗪（AnaSed注射剂，无菌溶液） </td><td>劳埃德的实验室，（雪兰，LA） </td><td> 4811 </td><td> 8毫克/公斤</td></tr><tr><td>氯胺酮（盐酸氯胺酮注射液，USP） </td><td> Hospira公司，公司（森林湖，IL） </td><td> 2051 </td><td> 120毫克/公斤</td></tr><tr><td>丁丙诺啡（Buprenex注射）（0.05毫克/公斤） </td><td> Reckitt Benckiser的制药公司（里士满，弗吉尼亚州） </td><td> 7571 </td><td></td></tr><tr><td>小动物电推剪</td><td>奥斯特专业（麦克明维尔，TN） </td><td> 76059-030 </td><td></td></tr><tr><td>脱毛乳液</td><td>教会德怀特合作（普林斯顿，新泽西州） </td><td> NAIR与婴儿油</td><td></td></tr><tr><td>纱布海绵</td><td>费舍尔科学，（宾夕法尼亚州匹兹堡） </td><td> 22-362-173 </td><td></td></tr><tr><td>棉拭子</td><td>费舍尔科学，（宾夕法尼亚州匹兹堡） </td><td> 14 - 960 - 3Q </td><td></td></tr><tr><td> 1毫升的注射器</td><td>流式细胞，迪克森和公司，新泽西州富兰克林湖） </td><td> 309602 </td><td></td></tr><tr><td>皮下（子Q）的针头，26ga。 </td><td>流式细胞，迪克森公司（新泽西州富兰克林湖，） </td><td> 305115 </td><td></td></tr><tr><td>剪刀和镊子春</td><td>精细的科学工具，（福斯特城，加利福尼亚） </td><td></td><td></td></tr><tr><td> 2.5毫米的弧形rongeurs </td><td>精细的科学工具，（福斯特城，加利福尼亚） </td><td> 16221-14 </td><td></td></tr><tr><td>乳酸钠林格注射药典</td><td>贝朗医疗公司（加利福尼亚州欧文） </td><td> BBR - L7502 </td><td></td></tr><tr><td>无菌生理盐水溶液</td><td> APP制药（Schaumburg的IL） </td><td> 918610 </td><td></td></tr><tr><td>薄膜合成的基质膜（Biobrane） </td><td> Bertek制药（摩根，西弗吉尼亚州） </td><td> 62794-096-251 </td><td></td></tr><tr><td>人工硬脑膜</td><td>戈尔排除MVP杜拉替补，戈尔和联营公司（弗拉格斯塔夫，AZ） </td><td> 1MVP40 </td><td></td></tr><tr><td> 5-0丝线缝合</td><td>爱惜康公司（新泽西州萨默维尔，） </td><td> K - 580 </td><td></td></tr><tr><td>伤口剪辑</td><td>完美 - ETS布鲁诺（Burnea，法国） </td><td> A75 </td><td></td></tr><tr><td>荧光体视显微镜</td><td>徕卡显微系统，（韦茨拉尔，德国） </td><td> MZ16 </td><td></td></tr><tr><td> CCD相机</td><td>滨松市（新泽西州Bridgewater的） </td><td> ORCA - RX2 </td><td></td></tr><tr><td>温度控制器</td><td>世界精密仪器公司（佛罗里达州萨拉索塔） </td><td>空管1000 </td><td></td></tr><tr><td> MetaMorph软件</td><td>分子器件，（加利福尼亚州桑尼维尔，） </td><td></td><td></td></tr><tr><td> Photoshop中</td><td> Adobe系统公司，加利福尼亚州圣何塞， </td><td></td><td></td></tr></tbody></table>

live imaging of dorsal root axons after rhizotomy

根脊髓受伤受伤的初级感觉神经轴突无法再生进入脊髓，导致慢性疼痛和永久的感觉丧失。到脊髓的背根（DR）的轴突再生，防止背根进入区（DREZ），CNS和PNS之间的接口。我们理解的分子和细胞活动，防止再生DREZ，是不完整的部分，因为已经从事后分析，推导出了复杂的变化与神经损伤有关。细胞的动态过程，如轴突再生，是最好的研究与技术，捕捉每一个活生生的动物的多个观测的实时事件。我们体内连续监测神经元的能力已大大增加，由于在光学和转基因小鼠的革命性的创新。 thy1 GFP转基因小鼠的几行，在神经细胞亚群的基因在不同的荧光色标记，允许单个神经元 在体内1成像。这些老鼠已经广泛使用在体内的肌肉2-4和大脑5-7成像，到生理机制提供了新颖的，不可能解决静态分析的见解。最近才开始的生活脊髓中的神经元的成像研究。李奇曼和他的同事们第一次证明了跟踪受伤的背柱（DC）的轴突， 宽视场显微镜8,9了其可行性。多光子深深定位的直流轴突在体内成像，小胶质细胞和血管也已经完成 10 。在过去的几年中，我们已率先应用在活体成像监测的DR采用宽视场显微镜和H thy1 - YFP小鼠轴突再生。这些研究，使我们以一个新颖的假说为什么DR轴突再生防止脊髓内的11。 在H线thy1 - YFP的小鼠，不同的YFP +轴突是表面上的位置，可以同时监控几个轴突。我们已经了解到，博士轴突到达在DREZ更好的成像比在脊髓型颈椎病，腰。在本报告中，我们描述了，我们已经找到有用的，以确保成功的长期和反复轴突再生DR成像的几种策略。这些措施包括消除重复插管和呼吸中断的方法，最大限度地减少手术相关的压力和瘢痕形成，并获得稳定的图像分辨率高，没有光毒性。

Watch this Scientific Journal Video about Live Imaging of Dorsal Root Axons after Rhizotomy at JoVE.com

Live Imaging of Dorsal Root Axons after Rhizotomy

一个<em&gt;在体内</em&gt;成像监测初级感觉神经轴突，背根粉碎后的协议描述。该程序利用宽视场荧光显微镜和thy1 - YFP转基因小鼠，并允许超过4厘米的期票，与中枢神经系统的接口轴突的相互作用反复轴突再生的成像。

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. ...

live-imaging-of-dorsal-root-axons-after-rhizotomy

Research

JoVE Journal

Neuroscience

13.8K 次观看.  Shriners Hospitals Pediatric Research Center and Department of Anatomy and Cell Biology. 一个在体内成像监测初级感觉神经轴突，背根粉碎后的协议描述。该程序利用宽视场荧光显微镜和thy1 - YFP转基因小鼠，并允许超过4厘米的期票，与中枢神经系统的接口轴突的相互作用反复轴突再生的成像。

视频: Live Imaging of Dorsal Root Axons after Rhizotomy

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

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 injury (Teng et al., 2006). It is recognized that transcriptional programs essential for neurite and axonal outgrowth are 
reactivated upon injury in the PNS (Makwana et al., 2005). However the tools available to analyze neuronal gene regulation in vivo are limited and 
often challenging.
The dorsal root ganglia (DRG) offer an excellent injury model system because both the CNS and PNS are innervated by a 
bifurcated axon originating from the same soma. The ganglia represent a discrete collection of cell bodies where all transcriptional events occur, 
and thus provide a clearly defined region of transcriptional activity that can be easily and reproducibly removed from the animal. Injury of nerve 
fibers in the PNS (e.g. sciatic nerve), where axonal regeneration does occur, should reveal a set of transcriptional programs that are distinct from 
those responding to a similar injury in the CNS, where regeneration does not take place (e.g. spinal cord). Sites for transcription factor binding, 
histone and DNA modification resulting from injury to either PNS or CNS can be characterized using chromatin immunoprecipitation (ChIP). 
Here, we describe a ChIP protocol using fixed mouse DRG tissue following axonal injury. This powerful combination provides a means for characterizing the pro-regeneration chromatin environment necessary for promoting axonal regeneration.

We present a method for chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury. The approach can be used to identify specific transcription factor binding sites and epigenetic modification of histone and DNA important for the regeneration of injured axons in both the peripheral and central nervous system.

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

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

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

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 ...

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

尼古丁诱导的钙信号和神经递质的释放了沿着腹侧海马轴突实时成像

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

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

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 neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS). The dorsal root ganglia (DRG)/cancer cell model is a three dimensional (3D) in vitro model frequently used for studying the interaction between neural stroma and cancer cells. In this model, mouse or human cancer cell lines are grown in ECM adjacent to preparations of freshly dissociated cultured DRG. In this article, the DRG isolation protocol from mice, and implantation in petri dishes for co-culturing with pancreatic cancer cells are demonstrated. Five days after implantation, the cancer cells made contact with the DRG neurites. Later, these cells formed bridgeheads to facilitate more extensive polarized, neurotropic migration of cancer cells.

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

This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

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 ...

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.

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 ...

Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive targets for injection of novel therapeutics aimed at treating chronic pain. In small animal models, laminectomy has been used to facilitate DRG injection because it involves surgical removal of the vertebral bone surrounding each DRG. We demonstrate a technique for intraganglionic injection of lumbar DRG in a large animal species, namely, swine. Laminotomy is performed to allow direct access to DRG using standard neurosurgical techniques, instruments, and materials. Compared with more extensive bone removal via laminectomy, we implement laminotomy to conserve spinal anatomy while achieving sufficient DRG access. Intraoperative progress of DRG injection is monitored using a non-toxic dye. Following euthanasia on post-operative day 21, the success of injection is determined by histology for intraganglionic distribution of 4&#39;,6-diamidino-2-phenylindole (DAPI). We inject a biologically inactive solution to demonstrate the protocol. This method could be applied in future preclinical studies to target therapeutic solutions to DRG. Our methodology should facilitate testing the translatability of intraganglionic small animal paradigms in a large animal species. Additionally, this protocol may serve as a key resource for those planning preclinical studies of DRG injection in swine.

We describe a method for laminotomy in swine that provides access to lumbar dorsal root ganglia (DRG) for intraganglionic injection. Injection progress is monitored intraoperatively and histologically confirmed up to 21 days after surgery. This protocol could be used for future preclinical studies involving DRG injection.

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 ...

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral tissues, such as skin, muscle and visceral organs, as well as the spinal dorsal horn of the central nervous system. Sensory neurons transmit somatic sensation, including touch, pain, thermal, and proprioceptive sensations. Therefore, DRG primary cultures are widely used to study the cellular mechanisms of nociception, physiological functions of sensory neurons, and neural development. The cultured neurons can be applied in studies involving electrophysiology, signal transduction, neurotransmitter release, or calcium imaging. With DRG primary cultures, scientists may culture dissociated DRG neurons to monitor biochemical changes in single or multiple cells, overcoming many of the limitations associated with in vivo experiments. Compared to commercially available DRG-hybridoma cell lines or immortalized DRG neuronal cell lines, the composition and properties of the primary cells are much more similar to sensory neurons in tissue. However, due to the limited number of cultured DRG primary cells that can be isolated from a single animal, it is difficult to perform high-throughput screens for drug targeting studies. In the current article, procedures for DRG collection and culture are described. In addition, we demonstrate the treatment of cultured DRG cells with an agonist of neuropeptide FF receptor type 2 (NPFFR2) to induce the release of peptide neurotransmitters (calcitonin gene-related peptide (CRGP) and substance P (SP)).

Dorsal root ganglia (DRG) primary cultures are frequently used to study physiological functions or pathology-related events in sensory neurons. Here, we demonstrate the use of lumbar DRG cultures to detect the release of neurotransmitters after neuropeptide FF receptor type 2 stimulation with a selective agonist.

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 ...

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its highly-organized subcellular architecture, we recently developed a new imaging modality, named spectral reflectometry (SpeRe), which enables unprecedented label-free nanoscale imaging of the live myelinated axons in situ. The underlying principle is to obtain nanostructural information by analyzing the reflectance spectrum of the multilayered subcellular structure. In this article, we describe a detailed step-by-step protocol for performing a basic SpeRe imaging of the nervous tissues using a commercial confocal microscopic system, equipped with a white-light laser and a tunable filter. We cover the procedures of sample preparation, acquisition of spectral data, and image processing for obtaining nanostructural information.

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

Here, we present a step-by-step protocol for imaging myelinated axons in a fixed brain slice using a label-free nanoscale imaging technique based on spectral reflectometry.

髓轴突原位光谱 Reflectometric 显微术

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its ...

Rapid Isolation of Dorsal Root Ganglion Macrophages

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after peripheral nerve injury. Peripheral monocytic cells, including macrophages, are known to respond to a tissue injury through phagocytosis, antigen presentation, and cytokine release. Emerging evidence has implicated the contribution of dorsal root ganglia macrophages to neuropathic pain development and axonal repair in the context of nerve injury. Rapidly phenotyping (or &#8220;rapid isolation of&#8221;) the response of dorsal root ganglia macrophages in the context of nerve injury is desired to identify the unknown neuroimmune factors. Here we demonstrate how our lab rapidly and effectively isolates macrophages from the dorsal root ganglia using an enzyme-free mechanical dissociation protocol. The samples are kept on ice throughout to limit cellular stress. This protocol is far less time consuming compared to the standard enzymatic protocol and has been routinely used for our Fluorescence-activated Cell Sorting analysis.

Here we present a mechanical dissociation protocol to rapidly isolate macrophages from the dorsal root ganglion for phenotyping and functional analysis.

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 ...

In Vitro Myelination of Peripheral Axons in a Coculture of Rat Dorsal Root Ganglion Explants and Schwann Cells

The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, neurons and Schwann cells engage in a complex interaction to control the myelination of axons. Disturbances of this interaction and breakdown of the myelin sheath are hallmarks of inflammatory neuropathies and occur secondarily in neurodegenerative disorders. Here, we present a coculture model of dorsal root ganglion explants and Schwann cells, which develops a robust myelination of peripheral axons to investigate the process of myelination in the peripheral nervous system, study axon-Schwann cell interactions, and evaluate the potential effects of therapeutic agents on each cell type separately. Methodologically, dorsal root ganglions of embryonic rats (E13.5) were harvested, dissociated from their surrounding tissue, and cultured as whole explants for 3 days. Schwann cells were isolated from 3-week-old adult rats, and sciatic nerves were enzymatically digested. The resulting Schwann cells were purified by magnetic-activated cell sorting and cultured under neuregulin and forskolin-enriched conditions. After 3 days of dorsal root ganglion explant culture, 30,000 Schwann cells were added to one dorsal root ganglion explant in a medium containing ascorbic acid. The first signs of myelination were detected on day 10 of coculture, through scattered signals for myelin basic protein in immunocytochemical staining. From day 14 onward, myelin sheaths were formed and propagated along the axons. Myelination can be quantified by myelin basic protein staining as a ratio of the myelination area and axon area, to account for the differences in axonal density. This model provides experimental opportunities to study various aspects of peripheral myelination in vitro, which is crucial for understanding the pathology of and possible treatment opportunities for demyelination and neurodegeneration in inflammatory and neurodegenerative diseases of the peripheral nervous system.

In the coculture system of dorsal root ganglia and Schwann cells, myelination of the peripheral nervous system can be studied. This model provides experimental opportunities to observe and quantify peripheral myelination and to study the effects of compounds of interest on the myelin sheath.

大鼠背根神经节外植体和雪旺细胞共培养中外周轴突的体外髓鞘形成

The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, ...