Evaluation of Injured Axons Using Two-Photon Excited Fluorescence Microscopy after Spinal Cord Contusion Injury in YFP-H Line Mice.
Evaluation of Injured Axons Using Two-Photon Excited Fluorescence Microscopy after Spinal Cord Contusion Injury in YFP-H Line Mice.
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DOI：
10.3390/ijms160715785
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发表时间：
2015-07-13
影响因子：
5.6
通讯作者：
Imamura T
中科院分区：
文献类型：
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作者：
Horiuchi H;Oshima Y;Ogata T;Morino T;Matsuda S;Miura H;Imamura T
阐明受损轴突的变性过程对于开发用于治疗脊髓损伤的治疗模块是重要的。本研究的目的是建立一种实时观察活体动物脊髓挫伤后受损轴突的方法。我们在本研究中使用的YFP（黄色荧光蛋白）-H转基因小鼠在其神经纤维中表达荧光。通过IH（Infinite Horizon）撞击器对第11节椎骨处的脊髓进行挫伤，其施加50 kdyn的压力。在麻醉状态下观察期间再次暴露受损脊髓，然后采用双光子激发荧光显微镜进行观察，该显微镜可以观察到包括脊髓轴突在内的组织深部区域。损伤后轴突形态学变化不明显。伤后3天，轴突数量减少，残余轴突破碎。损伤后7天，受损组织中仅存在碎片。伤后观察期内未观察到后肢运动。尽管挫伤后立即出现后肢瘫痪，但受损轴突的形态学变性延迟。这种方法可能有助于阐明轴突变性的病理生理学和开发用于治疗脊髓损伤的治疗模块。
Elucidation of the process of degeneration of injured axons is important for the development of therapeutic modules for the treatment of spinal cord injuries. The aim of this study was to establish a method for time-lapse observation of injured axons in living animals after spinal cord contusion injury. YFP (yellow fluorescent protein)-H transgenic mice, which we used in this study, express fluorescence in their nerve fibers. Contusion damage to the spinal cord at the 11th vertebra was performed by IH (Infinite Horizon) impactor, which applied a pressure of 50 kdyn. The damaged spinal cords were re-exposed during the observation period under anesthesia, and then observed by two-photon excited fluorescence microscopy, which can observe deep regions of tissues including spinal cord axons. No significant morphological change of injured axons was observed immediately after injury. Three days after injury, the number of axons decreased, and residual axons were fragmented. Seven days after injury, only fragments were present in the damaged tissue. No hind-limb movement was observed during the observation period after injury. Despite the immediate paresis of hind-limbs following the contusion injury, the morphological degeneration of injured axons was delayed. This method may help clarification of pathophysiology of axon degeneration and development of therapeutic modules for the treatment of spinal cord injury.