应用在位活体实时成像研究斑马鱼毛特纳运动神经再生和髓鞘再建

Axon and myelin sheath are important structures to maintain neuronal signaling transduction, and the myelin is produced by oligodendrocytes in the central nervous system (CNS). Axon damage and neurodegenerative diseases are characterized by axonal degeneration and demyeliantion. To explore the causes of CNS regeneration, demyelination/remyelination and search for countermeasures, we are going to use the zebrafish Mauthner cell axons (MAs) in the spinal cord as a model. In this proposal, by combining in-vivo time-lapse confocal imaging, two-photon axotomy, single-cell electroporation and neural axon or oligodendrocytes myelin transgenetically modified labeling approaches, the applicant aims to systematically examine the in vivo imaging of MAs regeneration and remyelinationafter injury. Through in vivo dynamic imaging of oligodendrocytes and innate immune inflammatory cells during the process of regeneration/remyelination, the applicant tries to explore the effects of the cell interactions between various cells on regeneration. Furthermore, by specifically labeling and imaging the subcellular structures in zebrafish Mauthner cell axons, we undertake to study their effects on regeneration/remyelination and the associated molecular mechanisms. This project may provide a new in vivo model for expand understanding mechanisms underlying the vertebrate CNS damage and repair as well as benefit to study the dynamic process of remyelination and axonal regeneration in the future.

轴突和髓鞘是维持神经信号传导的重要结构，神经损伤和退行性疾病都伴有脱髓鞘和轴突变性现象。为探索中枢神经系统（central nervous system, CNS）神经再生和髓鞘再建的机制，申请人拟以斑马鱼脊髓毛特纳运动神经元及其轴突（Mauthner axons, MAs）为模型，应用在位活体成像、双光子激光毁损、转基因和单细胞电转标记神经轴突和少突胶质细胞髓鞘等方法，开展毛特纳轴突损伤后再生，以及再生过程中髓鞘再建的可视化研究。并通过对轴突再生及髓鞘再建过程中少突胶质细胞和先天免疫炎症细胞的动态成像观察，探究各种细胞间的相互作用对神经损伤修复的影响。更进一步地，申请人拟利用单细胞电穿孔技术对斑马鱼毛特纳细胞内的亚细胞结构进行特异性标记、成像以探究其对轴突再生和髓鞘再建的影响从而揭示其分子机制。该项目将有助于拓展对脊椎动物中枢神经损伤修复的机制的深入了解。

通过本项目“应用在位活体实时成像研究斑马鱼毛特纳运动神经再生和髓鞘再建”的实施，我们提供一种新的以脊椎动物斑马鱼中枢神经系统损伤修复的单个神经元水平上可视化模型。提供了较为详细的毛特纳轴突损伤后再生过程中少突胶质细胞、先天免疫细胞对毛特纳细胞轴突再生和髓鞘再建的动态演化数据和实时观察细胞间相互作用的动态过程。成功建立了单细胞电穿孔的技术，对斑马鱼毛特纳细胞亚细胞结构进行特异性标记、成像及功能的调控。为揭示轴突再生的分子机制，促进轴突再生提供新的手段。丰富了斑马鱼中枢神经系统再生和再髓鞘化这一动态过程的认识。