Chinese Academy of Sciences Key Laboratory of Brain Function and Disease, Hefei National Laboratory for Physical Sciences at the Microscale, School of Life Sciences, University of Science and Technology of China, No. 96 Jinzhai Road, Hefei, Anhui Province 230026, PR China.
Institute of Biomedical and Health Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, PR China.
Exp Neurol. 2018 Feb;300:67-73. doi: 10.1016/j.expneurol.2017.10.028. Epub 2017 Oct 29.
Zebrafish is an excellent model to study central nervous system (CNS) axonal degeneration and regeneration since we can observe these processes in vivo and in real time in transparent larvae. Previous studies have shown that Mauthner cell (M-cell) axon regenerates poorly after mechanical spinal cord injury. Inconsistent with this result, however, we have found that M-cell possesses a great capacity for axon regeneration after two-photon laser ablation. By using ZEISS LSM 710 two-photon microscope, we performed specific unilateral axotomy of GFP labeled M-cells in the Tol-056 enhancer trap line larvae. Our results showed that distal axons almost degenerated completely at 24h after laser axotomy. After that, the proximal axons initiated a robust regeneration and many of the M-cell axons almost regenerated fully at 4days post axotomy. Furthermore, we also visualized that regenerated axons were remyelinated when we severed fluorescent dye labeled M-cells in the Tg (mbp:EGFP-CAAX) line larvae. Moreover, by single M-cell co-electroporation with Syp:EGFP and DsRed2 plasmids we observed synapses re-establishment in vivo during laser injury-induced axon re-extension which suggested re-innervation of denervated pathways. In addition, we further demonstrated that nocodazole administration could completely abolish this regeneration capacity. These results together suggested that in vivo time-lapse imaging of M-cell axon laser injury may provide a powerful analytical model for understanding the underlying cellular and molecular mechanisms of the CNS axon regeneration.
斑马鱼是研究中枢神经系统（CNS）轴突变性和再生的优秀模型，因为我们可以在透明幼虫体内实时观察这些过程。以前的研究表明，机械性脊髓损伤后，Mauthner 细胞（M 细胞）轴突再生不良。然而，与这一结果不一致的是，我们发现 M 细胞在双光子激光消融后具有很强的轴突再生能力。通过使用 ZEISS LSM 710 双光子显微镜，我们在 Tol-056 增强子捕获线幼虫中对 GFP 标记的 M 细胞进行了特定的单侧轴突切断。我们的结果表明，激光轴突切断后 24 小时，远端轴突几乎完全退化。之后，近端轴突启动了强有力的再生，许多 M 细胞轴突在轴突切断后 4 天几乎完全再生。此外，当我们在 Tg（mbp：EGFP-CAAX）线幼虫中切断荧光染料标记的 M 细胞时，我们还观察到再生轴突被重新髓鞘化。此外，通过 Syp：EGFP 和 DsRed2 质粒的单个 M 细胞共转染，我们观察到在激光损伤诱导的轴突再延伸过程中体内突触的重建，这表明失神经途径的再神经支配。此外，我们进一步证明，用 nocodazole 处理可以完全消除这种再生能力。这些结果表明，对 M 细胞轴突激光损伤进行体内实时成像可能为理解中枢神经系统轴突再生的潜在细胞和分子机制提供一个强大的分析模型。
