实时成像背根轴突后根切断术
1Temple University,Shriners Hospitals Pediatric Research Center and Department of Anatomy and Cell Biology, 2Medical Research Service,Department of Veterans Affairs Hospital, 3Department of Neurobiology and Anatomy,Drexel University College of Medicine, 4Shriners Hospitals Pediatric Research Center and Department of Anatomy and Cell Biology,Temple University School of Medicine
Summary
一个<em>在体内</em>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。
Abstract
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。
Protocol
1。显微镜设置和成像准备我们的成像成立了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。 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。 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）。 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。…
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Name of the reagent | Company | Catalogue number | Comments |
|---|---|---|---|
| H line thy1-YFP (2-4 months old, either sex) | Jackson Laboratory (Bar Harbor, ME) | 003782 | |
| Xylazine (AnaSed injection, sterile solution) | Lloyd Laboratories, (Shenandoah, LA) | 4811 | 8 mg/kg |
| Ketamine (Ketamine hydrochloride injection, USP) | Hospira, Inc. (Lake Forest, IL) | 2051 | 120 mg/kg |
| Buprenorphine (Buprenex injectable) (0.05 mg/kg) | Reckitt Benckiser Pharmaceuticals Inc.(Richmond, VA) | 7571 | |
| Small animal hair clippers | Oster Professional, (McMinnville, TN) | 76059-030 | |
| Hair removal lotion | Church & Dwight Co (Princeton, NJ) | NAIR with Baby Oil | |
| Gauze sponges | Fisher Scientific, (Pittsburgh, PA) | 22-362-173 | |
| Cotton-tipped swabs | Fisher Scientific, (Pittsburgh, PA) | 14-960-3Q | |
| 1 mL syringes | Becton, Dickson and Company Franklin Lakes, NJ) | 309602 | |
| Subcutaneous (Sub-Q) needles, 26ga. | Becton, Dickson and Company (Franklin Lakes, NJ) | 305115 | |
| Spring scissors and forceps | Fine Science Tools, (Foster City, CA) | ||
| 2.5-mm curved rongeurs | Fine Science Tools, (Foster City, CA) | 16221-14 | |
| Lactated Ringer’s Injection USP | B. Braun Medical Inc., (Irvine, CA) | BBR-L7502 | |
| Sterile saline solution | APP Pharmaceuticals, (Schaumburg, IL) | 918610 | |
| Thin synthetic matrix membrane (Biobrane) | Bertek Pharmaceuticals, (Morgantown, WV) | 62794-096-251 | |
| Artificial dura | Gore Preclude MVP Dura Substitute, W.L. Gore and Associates, (Flagstaff, AZ) | 1MVP40 | |
| 5-0 silk sutures | Ethicon, Inc. (Somerville, NJ) | K-580 | |
| Wound clips | Perfect – Ets Bruneau, (Burnea, France) | A75 | |
| Fluorescent stereomicroscope | Leica Microsystems, (Wetzlar, Germany) | MZ16 | |
| CCD camera | Hamamatsu, (Bridgewater, NJ) | ORCA-Rx2 | |
| Temperature Controller | World Precision Instruments (Sarasota, FL) | ATC 1000 | |
| Metamorph software | Molecular Devices, (Sunnyvale, CA) | ||
| Photoshop | Adobe Systems, San Jose, CA |
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Tags
Live ImagingDorsal Root AxonsRhizotomySpinal Root InjuriesChronic PainSensory LossRegenerationDorsal Root Entry Zone (DREZ)Molecular EventsCellular EventsNerve InjuryPostmortem AnalysesAxon RegenerationReal-time EventsLiving AnimalNeurons Serially In VivoOpticsMouse TransgenicsThy1-GFP Transgenic MiceFluorescent ColorsIn Vivo ImagingMuscleBrainPhysiological MechanismsStatic AnalysesSpinal Cord Imaging
Cite This Article
Skuba, A., Himes, B. T., Son, Y. Live Imaging of Dorsal Root Axons after Rhizotomy. J. Vis. Exp. (55), e3126, doi:10.3791/3126 (2011).