细胞动力学在小鼠脊髓双光子成像
Summary
成像的小鼠脊髓一种新的体外准备。该协议允许对活细胞相互作用的整个脊髓双光子成像。
Abstract
Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.
Introduction
小鼠模型脱髓鞘，包括实验的自身免疫性脑脊髓炎（EAE）和颅内感染neuroadapted小鼠肝炎病毒（MHV），是优秀的工具来研究分子途径和与疾病相关的细胞相互作用。他们带领和支持FDA的批准有效性医药疗法，主要针对戒烟自身免疫和炎症1。然而，一旦内源性髓鞘再生失败时，目前批准的疗法不能有效地修复脱髓鞘病变在中枢神经系统。因此，修复为重点的疗法在这个阶段的疾病是慢性症状和改善生活质量的减轻是至关重要的。最近，神经前体细胞（筹备）已经走到了前列作为一个潜在的再生治疗方法针对炎症和脱髓鞘的领域。一些研究突出的NPC诱导endogeno的能力我们髓鞘再生，并直接在髓鞘2-8参与。由于筹备参与直接的髓鞘再生，就必须了解他们的动力学和相互作用移植后的内源性细胞。移植后，NPC的腹侧迁移到脑白质损伤区，然后吻侧和尾侧相对于移植部位5,9。迁移的动力学不同的应对环境线索; NPC的移植到未受损脊髓具有比的NPC移植到受损脊髓6更大的流速。后一个洄游期间，传送的NPC广泛增殖，以更高的速率在 受损脊髓一个相对于一个完整的脊髓6。最后，大多数的NPC分化成少突胶质细胞，并开始直接髓鞘4,6,9。
脱髓鞘病变是复杂的，并且可以包括小区中的一个的各阶段不同的人口ctivation。例如，有源多发性硬化症（MS）病变可以包括活化的T细胞，小胶质细胞M1和M1巨噬细胞，但是慢性无声的MS病变可以包括主要由具有少量炎症细胞10-13活性星形细胞的显著人口。由于效应细胞的多样性，双光子（2P）成像脱髓鞘的小鼠模型是一个非常有用的工具，以帮助了解病灶内局部细胞的相互作用。在MS和许多广泛使用的MS研究模型，大部分病灶位于脊髓，无法进入活体2P成像的区域的腹侧由于损伤深度和脊髓的高脂质含量。绕过病变内的这些问题，并研究细胞-细胞相互作用沿腹侧脊髓我们已开发出一种简单的体外 2P成像制剂6。
本研究遵循了以前的方法的出版物，这表明该过程对于移栽增强型绿色荧光蛋白（EGFP）的NPC -expressing到小鼠的脊髓以下MHV诱导脱髓鞘14 JHMV应变。五周龄小鼠感染JHMV和移植EGFP-的NPC在胸9级后第14天感染。这里介绍的协议提供了有关如何提取脊髓，作出体外琼脂糖准备，并具有增强的黄色荧光蛋白（EYFP）-expressing轴突图像移植EGFP-NPC互动的详细步骤。小鼠表达EYFP下神经元特异性大鼠Thy1启动子在此过程15被使用。只有一些轴突表达EYFP，使其可用于单个成像轴突。在这里，我们将展示7天移植后取出脊髓;然而，脊髓可以在任何时间点移植后进行萃取。虽然我们显示的NPC的相互作用受损轴突，我们的协议可以结合使用遗传荧光标记其它类型的细胞，调查了许多在整个小鼠脊髓发生细胞相互作用的。
Protocol
Representative Results
Discussion
实时2P成像完好的组织要求调查NPC动力学和相互作用下移植到脱髓鞘小鼠脊髓。活2P成像通常用于确定在活的小鼠上脊髓背侧细胞动力学，并已经被用于研究背侧脱髓鞘在脱髓鞘疾病17-19。但是，因为移植的NPC迁移到腹侧白质，使用2P显微镜其位于太深图像在原位， 离体的准备是必要的。这一方法已被用于在我们的实验室确定的NPC移植到受损的和未受损脊髓6的能动性和?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
This work was supported in part by National Institutes of Health (NIH) Grants R01 GM-41514 (to M.D.C.), R39 GM-048071 (to I.P.), and R01 NS-074987 (to T.E.L.) and the National Multiple Sclerosis Society (NMSS) Collaborative Center Research Award CA1058-A-8 (to C.M.W., T.E.L. and M.D.C.), NMSS Grant RG4925, NIH Training Grant T32-AI-060573 (to M.L.G.), NMSS Postdoctoral Fellowship FG 1960-A-1 (to J.G.W.), and funding from the George E. Hewitt Foundation for Medical Research (M.P.M.).
Materials
| Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
| Isoflurane, USP | Piramal Critical Care, Inc | N/A | |
| Fine scissors | Fine Science Tools | 14060-09 | sharp |
| scalpel blade #10 | Fine Science Tools | 10010-00 | |
| scalpel handle | Fine Science Tools | 10003-12 | |
| Luer rongeurs | Fine Science Tools | 16001-15 | |
| Graefe forceps | Fine Science Tools | 11052-10 | |
| Vannas scissors | Fine Science Tools | 15615-08 | |
| scalpel blade #11 | Fine Science Tools | 10011-00 | |
| RPMI-1640 | Gibco | 12-115F | |
| agarose, low gelling temperature | Sigma | A9414-25G | |
| Parafilm | Fisher Scientific | 13-374-12 | |
| Vetbond (tissue adhesive) | 3M | 1469SB | |
| 22 mm square cover slip | Fisher Scientific | 12-547 | |
| 25x dipping objective, 1.1 NA | Nikon | CFI Apo LWD 25XW | |
| Single inline solution heater | Warner Instruments | 64-0102 | |
| 520 nm single-edge dichroic beam splitter | Semrock | FF520-Di02-25×36 | Brightline |
| 560 nm single-edge dichroic beam splitter | Semrock | FF560-FDi01-25×36 | Brightline |
| photomultiplier tubes | Hamamatsu | R928 | |
| C/L variable-speed tubing pump | Masterflex | 77122-22 | |
| digital thermometer | Comar Instruments | 3501 | |
| Chameleon Ultra Ti:Sapphire laser | Coherent | N/A | |
| Slidebook 6 software | 3i | N/A | |
| Imaris 7.7 software | Bitplane | N/A |
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