在体内脊髓的鼠标线使用双光子显微镜成像
Summary
微创的协议，以稳定脊柱鼠标和执行重复<em>在体内</em>脊髓成像，利用双光子显微镜描述。这种方法结合了脊柱的稳定装置和麻醉方案，以尽量减少引起的呼吸运动和生产没有对齐或其他处理后的原始图像数据，需要。
Abstract
在体内成像已遗传工程小鼠表达荧光蛋白在特定的细胞类型 2-3显著扩大我们的生理和病理过程知识在众多的组织在 体内 4-7使用双光子显微镜 1 。在中枢神经系统（CNS）的研究，已被广泛的应用在体内成像在大脑中，这已经产生了众多新颖的细胞，如神经元，星形胶质细胞，小胶质细胞，在行为往往是意外的结果生理或病理条件下 8-17 。然而，大多是复杂的技术问题，有限的生活小鼠脊髓的研究在体内成像的实施。特别是，对脊髓的解剖毗邻的肺部和心脏产生显著运动伪影，使成像的生活脊髓一项艰巨的任务。 </P>
我们开发出一种新的方法，克服了稳定脊柱，减少呼吸道引起的运动，从而促进利用双光子显微镜的图像在体内小鼠脊髓的脊髓造影固有的局限性。这是通过深麻醉方法相结合的一个定制的脊柱稳定装置，导致呼吸道引起的运动的一个显着减少。这个视频协议显示了如何公开一个可以维持稳定的生理条件下过长时间保持组织损伤和出血最低生活脊髓的小面积。具有代表性的原始图像在高分辨率收购体内详细的小胶质细胞和血管之间的密切关系。一个timelapse序列显示了在生活小鼠脊髓小胶质细胞过程的动态行为。此外，一个相同的Z -帧的连续扫描显示S的出色的稳定性，这种方法可以实现图像和/或timelapse电影，不需要图像对齐收购后产生的栈。最后，我们表明这种方法可用于如何重新审视和重新映像在稍后的时间点脊髓同一地区，从而在体内持续的生理或病理过程的纵向研究。
Protocol
1。大厦的脊椎稳定装置订购Narishige STS的紧凑型脊髓夹钳和Narishige马6N头控股适配器。 自定义的设计和制造的不锈钢底板举行对齐Narishige的两个部分，使动物的头是支持，而其脊柱和尾巴夹紧。请记住，整个装置应适合在显微镜下的镜头，通常在降低显微镜阶段。 2。动物手术预热空气加热装置的显微镜室，并保持在37 ° C。 称量动物和麻醉?…
Discussion
这里介绍的方法， 可以在体内成像人口稠密荧光灯在使用双光子显微镜的麻醉小鼠的脊髓细胞结构稳定和重复性。所取得的稳定，是一个特制的脊椎稳定装置和麻醉方案，减少呼吸道引起的运动伪影的结果。脊椎稳定装置允许下的鼠体呼吸的空间，可使用市售的脊髓夹具和头部安装片（图1）。高重现性好，微创的方法是，始终没有广泛的图像后处理（图和视频1），可用于实验分析产生的?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
支持这项工作是由国家多发性硬化症协会授予RG4595A1 / T DD和美国国立卫生研究院/ NINDS补助NS051470，NS052189和NS066361 KA图和电影改编和/或重印达瓦洛斯等人 ，J神经科学方法。 2008年03月30日; 169（1）:1 – 7版权所有2008年从爱思唯尔的权限，。
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| Rhodamine B dextran | Invitrogen | D1841 | 70 kDa, diluted in ACSF (3% w/v) |
| Ketamine HCl | Bionichepharma | NDC No: 67457-001-10 | Injectable, 50mg/ml |
| Anased | Lloyd Labs | NADA No: 139-236 | Xylazine injectable, 20mg/ml |
| Acepromazine | Vedco | NADA No: 117-531 | Injectable,10mg/ml |
| Artificial tears ointment |
Phoenix pharmaceutical |
NDC No: 57319-760- 25 |
Lubricant |
| Betadine | Fisher | 19-061617 | |
| McPherson-Westcott Scissors |
World Precision Instruments |
555500S | Curved, blunt-tip scissors |
| Straight Forceps | World Precision Instruments |
555047FT | Toothed tip forceps |
| Small vessel cauterize | Fine Science Tools | 18000-00 | |
| Gelfoam | Pharmacia,Pfizer Inc. | Mixer Mill MM400 | |
| Compact spinal cord clamps |
Narishige | STS-A | |
| Head holding adaptor | Narishige | MA-6N | |
| Gelseal | Amersham Biosciences Corp. |
80-6421-43 | |
| Lactated Ringers | Baxter Healthcare | 2B8609 | |
| Buprenex | Reckit Benckiser Pharmaceuticals Inc. |
NDC No: 12496- 6757-1 |
Buprenorphine, injectable |
| Baytril | Bayer | NADA 140-913 | Enrofloxacin, antibacterial injectable 2.27% (20ml) |
| Heating pad – Large | Fine Science Tools | 21060-10 |
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Tags
In Vivo ImagingTwo-photon MicroscopyMouse Spinal CordFluorescent ProteinsSpecific Cell TypesPhysiological ProcessesPathological ProcessesCentral Nervous SystemBrain ImagingNeuronsAstrocytesMicrogliaTechnical ComplicationsSpinal Cord ImagingMovement ArtifactSpinal Stabilization DeviceDeep Anesthesia Protocol
Cite This Article
Davalos, D., Akassoglou, K. In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy. J. Vis. Exp. (59), e2760, doi:10.3791/2760 (2012).