Fenrich Keith K, Weber Pascal, Rougon Genevieve, Debarbieux Franck
Developmental Biology Institute of Marseille-Luminy (IBDML), Aix Marseille University.
J Vis Exp. 2013 Dec 21(82):e50826. doi: 10.3791/50826.
Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as multiple sclerosis. Here we present a low-cost and reproducible glass window implantation protocol that is suitable for intravital microscopy and studying the dynamics of spinal cord cytoarchitecture with subcellular resolution in live adult mice with EAE. Briefly, we surgically expose the vertebrae T12-L2 and construct a chamber around the exposed vertebrae using a combination of cyanoacrylate and dental cement. A laminectomy is performed from T13 to L1, and a thin layer of transparent silicone elastomer is applied to the dorsal surface of the exposed spinal cord. A modified glass cover slip is implanted over the exposed spinal cord taking care that the glass does not directly contact the spinal cord. To reduce the infiltration of inflammatory cells between the window and spinal cord, anti-inflammatory treatment is administered every 2 days (as recommended by ethics committee) for the first 10 days after implantation. EAE is induced only 2-3 weeks after the cessation of anti-inflammatory treatment. Using this approach we successfully induced EAE in 87% of animals with implanted windows and, using Thy1-CFP-23 mice (blue axons in dorsal spinal cord), quantified axonal loss throughout EAE progression. Taken together, this protocol may be useful for studying the recruitment of various cell populations as well as their interaction dynamics, with subcellular resolution and for extended periods of time. This intravital imaging modality represents a valuable tool for developing therapeutic strategies to treat autoimmune demyelinating diseases such as EAE.
成年啮齿动物的实验性自身免疫性脑脊髓炎（EAE）是研究诸如多发性硬化症等自身免疫性脱髓鞘疾病的标准实验模型。在此，我们介绍一种低成本且可重复的玻璃窗植入方案，该方案适用于活体显微镜检查，并能在患有EAE的成年活体小鼠中以亚细胞分辨率研究脊髓细胞结构的动态变化。简而言之，我们通过手术暴露T12 - L2椎骨，并使用氰基丙烯酸酯和牙科粘结剂组合在暴露的椎骨周围构建一个腔室。从T13到L1进行椎板切除术，并在暴露的脊髓背表面涂抹一层薄的透明硅橡胶弹性体。将一块改良的玻璃盖玻片植入暴露的脊髓上方，注意玻璃不要直接接触脊髓。为减少窗口与脊髓之间炎性细胞的浸润，在植入后的前10天每2天进行一次抗炎治疗（按照伦理委员会的建议）。仅在抗炎治疗停止2 - 3周后诱导EAE。使用这种方法，我们在87%带有植入窗口的动物中成功诱导出EAE，并使用Thy1 - CFP - 23小鼠（脊髓背侧的蓝色轴突），在整个EAE进展过程中对轴突损失进行了量化。综上所述，该方案对于以亚细胞分辨率并在较长时间段内研究各种细胞群的募集及其相互作用动态可能是有用的。这种活体成像方式是开发治疗诸如EAE等自身免疫性脱髓鞘疾病的治疗策略的宝贵工具。
