拉米内切手术和脊髓窗口植入小鼠
Summary
该协议描述将玻璃窗植入小鼠脊髓，以方便通过生命内显微镜进行可视化。
Abstract
该协议描述了一种用于小鼠脊髓体内成像的脊髓层膜切除术和玻璃窗植入方法。集成数字蒸发器用于以亚非卢兰的低流速实现稳定的麻醉平面。一根椎骨脊柱被切除，一个市售的封面玻璃被覆盖在一张薄的甘蔗床上。然后，使用组织粘合剂和牙科水泥将 3D 打印的塑料背板贴在相邻的椎脊上。稳定平台用于减少呼吸和心跳的运动伪影。这种快速和无夹紧的方法非常适合急性多光光荧光显微镜。在表达eGFP：Claudin-5——一种紧密结蛋白的转基因小鼠中，应用该技术在脊髓血管的双光子显微镜上，包括代表性数据。
Introduction
表达荧光蛋白的转基因动物模型与生命内显微镜结合，为解决生物学和病理生理学提供了一个强大的平台。为了将这些技术应用于脊髓，需要专门的协议来准备脊髓进行成像。其中一种策略是进行层切除术和脊髓窗口植入。理想的显微镜层切除术方案的主要特点包括保存原生组织结构和功能、成像场的稳定性、快速处理时间和结果的可重复性。一个特别的挑战是稳定成像场对呼吸和心跳引起的运动。据报道，为了实现这些目标，已经报告了多种前体和体内策略，以实现这些目标1、2、3、4、5。大多数体内方法涉及夹紧脊柱2，4的两侧，然后通常植入一个刚性金属装置3，4在手术期间的稳定性和下游成像应用。夹紧脊柱可能会损害血液流动，并诱导血脑屏障 （BBB） 蛋白质重塑。
这种方法的目的是使完整的脊髓可用于活小鼠的光学成像，同时尽量减少协议的侵入性，并改善结果。我们描述了单一层切除术和盖玻璃植入程序，与微创椭圆形塑料 3D 打印背板配对，仍然可实现坚固的机械稳定性。背板直接附着在前椎和后椎脊柱上，用牙科水泥。背板配有侧延伸臂，带螺钉孔，通过金属臂牢固地连接到显微镜级。这有效地将完整的前椎和后椎固定在显微镜阶段，为否则由呼吸和心跳引入的运动伪影提供机械阻力。该方法针对胸腔12级单椎骨的层切除术进行了优化，省略了在体内成像过程中用于稳定的替代策略中使用的钳子。该过程是快速的，每只鼠标大约需要 30 分钟。
该协议可用于研究BBB的疾病机制。BBB 是一个动态微血管系统，由内皮细胞、血管平滑肌、围细胞和星形细胞足过程组成，为中枢神经系统 （CNS） 提供高度选择性的环境。具有代表性的数据描述了该协议在转基因小鼠中的应用，该实验旨在表达增强的绿色荧光蛋白（eGFP）：Claudin-5，一种BBB紧密结蛋白。提供的背板打印文件也可以针对其他应用进行自定义。
Protocol
所有实验都遵循伊利诺伊大学芝加哥机构动物护理和使用委员会的协议。这是一个终端过程。 1. 试剂制备 准备人造脑脊髓液 （aCSF） 包含 125 mM NaCl、 5 mM KCl、 10 mM 葡萄糖、 10 mM HEPES、2 mM MgCl2+6H2O、2 mM CaCl2+2H2O 在 ddH2O. 无菌过滤器中冷冻，并冷冻在单独使用的脂肪中。使用前，在水浴中加热至39°C。 暖低熔点角蔗角 （2%…
Representative Results
植入的玻璃窗和生命内的双光子显微镜为评估中枢神经系统蛋白质的动态变化提供了一个有用的工具。BBB的功能完整性受紧密结蛋白7的表达、亚细胞定位和周转率的影响。先前的研究表明，紧密结蛋白在稳定状态8下进行快速和动态的重塑。目前描述的拉氏切除术和玻璃窗制剂已用于转基因eGFP：Claudin-5小鼠9，其中含有…
Discussion
此处描述的方法允许通过玻璃窗稳定成像小鼠的脊髓。该方法已应用于评估转基因eGFP：Claudin5+/-小鼠的BBB重塑，表达荧光BBB紧密结蛋白，但同样适用于脊髓中任何荧光蛋白或细胞的研究。
已经开发出了用于拉皮切除术和脊髓稳定的方法。所有协议都用于在成像和窗口实现期间稳定脊髓，以便直观地访问感兴趣的结构。切除的椎骨数量和可用协议的侵入性程度各不相同（例?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
S.E. Lutz 由国家促进转化科学中心、国家卫生研究院、格兰特 KL2TR002002 和伊利诺伊大学芝加哥医学院启动基金提供支持。西蒙·阿尔福德得到RO1 MH084874的支持。内容完全由作者负责，不一定代表NIH的官方观点。作者感谢哥伦比亚大学医学中心神经内科的Dritan Agalliu为Tg eGFP：Claudin-5小鼠，科学讨论，并深入了解手术协议和成像应用的发展。作者感谢加州大学欧文分校神经生物学和行为系的苏尼尔·甘地设计了立体定向装置和动物温度控制器的第一个原型，讨论了手术方案，以及双光子显微镜训练。作者还感谢史蒂夫·皮肯斯（W.Nuhsbaum，Inc.）协助定制手术立体显微镜，罗恩·利平斯基（鲸鱼制造）用于加工立体定向部件。
Materials
| 3D printer | Raise3D | Pro2 | For printing backplates |
| PLA 3D printing filament | Inland | PLA+-175-B | Black plastic 3D printing material |
| 3D CAD software | Dassault Systemes | Solidworks software | used to design 3D shapes |
| 3D printer software | Raise3D | Ideamaker software | software used to interface with the 3D printer |
| 3D printed oval backplate | custom | Stabilizing imaging field | |
| Surgical dissecting microscope | Leica | M205 C | Equipped with Leica FusionOptics, Planapo 0.63x M-series objective, and gliding stage |
| Microscope camera | Leica | MC170 | HD color camera for visualizing surgical field |
| Gliding stage | Leica | 10446301 | The gliding stage is constructed of two metal plates. The base plate is fixed. The upper plate slides on greased interface to allow rotational and linear movement. |
| Surgical station and stabilization fork | Whale Manufactoring | custom | Laminectomy |
| SomnoSuite low-flow isoflurane delivery unit | Kent Scientific | SS-01 | Surgical anesthesia administration with integrated digitial vaporizer |
| Stainless steel 1.5 inch mounting post | ThorLabs | P50/M | For mounting surgical station onto optical table for two-photon imaging |
| Counterbored Clamping Fork for 1.5" mounting Post | ThorLabs | PF175 | For stabilizing surgical station mount onto optical table for two-photon imaging |
| Ideal bone microdrill | Harvard apparatus | 72-6065 | Thinning bone for laminectomy |
| Water bath | Fisher Scientific | 15-462-10 | Warming saline |
| Cautery gun | FST | 18010-00 | Cauterizing minor bleeds |
| Heating pad | Benchmark | BF11222 | 1.9” x 4.5” silicone heater with 20” Teflon leads, 10W, 5V |
| K type thermocoupled rectal probe | Physitemp | RET3 | Measuring mouse body temperature |
| petroleum jelly | Sigma | 8009-03-8 | Lubricating rectal probe |
| Feedback-regulated thermal controller | custom | NA | Commercially available alternatives include the Physitemp TCAT series |
| PVA Surgical eye spears | Beaver-visitec international | 40400-8 | Absorbing blood |
| Electric trimmer | Wahl | 41590-0438 | Trimming mouse fur |
| Blade, #11 | FST | 14002-14 | Surgical tool |
| Forceps, #5 | FST | 11254-20 | Surgical tool |
| Forceps, #4 | FST | 14002-14 | Surgical tool |
| Titatnium toothed forceps | WPI | 555047FT | Surgical tool |
| Titanium Iris scissors | WPI | 555562S | Surgical tool |
| Vetbond tissue adhesive | 3M | 084-1469SB | Preparing tissue surface for dental acrylic |
| Ceramic mixing tray | Jack Richeson | 420716 | Mixing dental acrylic agent with accelerant |
| Orthojet dental acrylic | Lang Dental | 1520BLK, 1503BLK | Permanently bonding backplate to tissue |
| Small round cover glass, #1 thickness, 3 mm | Harvard apparatus | 64-0720 | optical window |
| NaCl | Fisher Scientific | 7647-14-5 | For aCSF |
| KCl | Fisher Scientific | 7447-40-7 | For aCSF |
| Glucose | Fisher Scientific | 50-99-7 | For aCSF |
| HEPES | Sigma | 7365-45-9 | For aCSF |
| MgCl2·6H2O | Fisher Scientific | 7791-18-6 | For aCSF |
| CaCl2·2H2O | Fisher Scientific | 10035-04-8 | For aCSF |
| Carprofen | Rimadyl | QM01AE91 | Analgesia |
| Bacteriostatic water | Henry Schein | 2587428 | Diluent for carprofen |
| Isoflurane | Henry Schein | 11695-6776-2 | Anesthesia |
| Lactated ringer solution | Baxter | 0338-0117-04 | Hydration for mouse |
| Agarose High EEO | Sigma | A9793 | gel point 34-37 degrees C |
| Opthalmic lubricating ointment | Akwa Tears | 68788-0697 | Prevent corneal drying |
| MOM Two-Photon Microscope | Sutter |
Riferimenti
- Weinger, J. G., et al. Two-photon imaging of cellular dynamics in the mouse spinal cord. Journal of Visualized Experiments. (96), (2015).
- Davalos, D., Akassoglou, K. In vivo imaging of the mouse spinal cord using two-photon microscopy. Journal of Visualized Experiments. (59), (2012).
- Farrar, M. J., Schaffer, C. B. A procedure for implanting a spinal chamber for longitudinal in vivo imaging of the mouse spinal cord. Journal of Visualized Experiments. (94), (2014).
- Fenrich, K. K., Weber, P., Rougon, G., Debarbieux, F. Implanting glass spinal cord windows in adult mice with experimental autoimmune encephalomyelitis. Journal of Visualized Experiments. (82), e50826 (2013).
- Figley, S. A., et al. A spinal cord window chamber model for in vivo longitudinal multimodal optical and acoustic imaging in a murine model. PLOS ONE. 8 (3), e58081 (2013).
- Helmchen, F., Denk, W. Deep tissue two-photon microscopy. Nature Methods. 2 (12), 932-940 (2005).
- Liebner, S., et al. Functional morphology of the blood-brain barrier in health and disease. Acta Neuropathologica. 135 (3), 311-336 (2018).
- Shen, L., Weber, C. R., Turner, J. R. The tight junction protein complex undergoes rapid and continuous molecular remodeling at steady state. Journal of Cell Biology. 181 (4), 683-695 (2008).
- Knowland, D., et al. Stepwise recruitment of transcellular and paracellular pathways underlies blood-brain barrier breakdown in stroke. Neuron. 82, 1-15 (2014).
- Lutz, S. E., et al. Caveolin1 Is Required for Th1 Cell Infiltration, but Not Tight Junction Remodeling, at the Blood-Brain Barrier in Autoimmune Neuroinflammation. Cell Reports. 21 (8), 2104-2117 (2017).
- Harrison, M., et al. Vertebral landmarks for the identification of spinal cord segments in the mouse. Neuroimage. 68, 22-29 (2013).
- Cupido, A., Catalin, B., Steffens, H., Kirchhoff, F., Bakota, L., Brandt, R. . Laser Scanning Microscopy and Quantitative Image Analysis of Neuronal Tissue. , 37-50 (2014).
- Sekiguchi, K. J., et al. Imaging large-scale cellular activity in spinal cord of freely behaving mice. Nature Communications. 7, 11450 (2016).
- Nadrigny, F., Le Meur, K., Schomburg, E. D., Safavi-Abbasi, S., Dibaj, P. Two-photon laser-scanning microscopy for single and repetitive imaging of dorsal and lateral spinal white matter in vivo. Physiological Research. 66 (3), 531-537 (2017).
- Adelsperger, A. R., Bigiarelli-Nogas, K. J., Toore, I., Goergen, C. J. Use of a Low-flow Digital Anesthesia System for Mice and Rats. Journal of Visualized Experiments. (115), (2016).
- Damen, F. W., Adelsperger, A. R., Wilson, K. E., Goergen, C. J. Comparison of Traditional and Integrated Digital Anesthetic Vaporizers. Journal of the American Association for Laboratory Animal Science. 54 (6), 756-762 (2015).
- Miyamoto, K., et al. Selective COX-2 inhibitor celecoxib prevents experimental autoimmune encephalomyelitis through COX-2-independent pathway. Brain. 129 (Pt 8), 1984-1992 (2006).
- Muthian, G., et al. COX-2 inhibitors modulate IL-12 signaling through JAK-STAT pathway leading to Th1 response in experimental allergic encephalomyelitis. Journal of Clinical Immunology. 26 (1), 73-85 (2006).
Citazione di questo articolo
Pietruczyk, E. A., Stephen, T. K., Alford, S., Lutz, S. E. Laminectomy and Spinal Cord Window Implantation in the Mouse. J. Vis. Exp. (152), e58330, doi:10.3791/58330 (2019).