In vivo imaging of Mauthner axon regeneration, remyelination and synapses re-establishment after laser axotomy in zebrafish larvae
In vivo imaging of Mauthner axon regeneration, remyelination and synapses re-establishment after laser axotomy in zebrafish larvae
复制标题
斑马鱼幼虫激光轴突切除术后 Mauthner 轴突再生、髓鞘再生和突触重建的体内成像
DOI：
10.1016/j.expneurol.2017.10.028
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发表时间：
2018-02-01
影响因子：
5.3
通讯作者：
Hu, Bing
中科院分区：
文献类型：
--
作者：
Hu, Bing-bing;Chen, Min;Hu, Bing
斑马鱼是研究中枢神经系统 (CNS) 轴突变性和再生的绝佳模型，因为我们可以在透明幼虫体内实时观察这些过程。先前的研究表明，机械性脊髓损伤后，莫特纳细胞（M-cell）轴突的再生能力很差。然而，与此结果不一致的是，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。通过使用蔡司 LSM 710 双光子显微镜，我们对 Tot-056 增强子捕获线幼虫中 GFP 标记的 M 细胞进行了特异性单侧轴切术。我们的结果表明，激光轴突切除术后 24 小时，远端轴突几乎完全退化。此后，近端轴突开始强劲再生，许多 M 细胞轴突在轴切术后 4 天几乎完全再生。此外，我们还观察到，当我们切断 Tg (mbp:EGFP-CAAX) 系幼虫中荧光染料标记的 M 细胞时，再生的轴突发生了髓鞘再生。此外，通过单 M 细胞与 Syp:EGFP 和 DsRed2 质粒的共电穿孔，我们观察到在激光损伤诱导的轴突重新延伸期间突触在体内重建，这表明去神经通路的重新支配。此外，我们进一步证明诺考达唑给药可以完全消除这种再生能力。这些结果共同表明，M 细胞轴突激光损伤的体内延时成像可能为理解 CNS 轴突再生的潜在细胞和分子机制提供强大的分析模型。
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 Tot-056 enhancer trap line larvae. Our results showed that distal axons almost degenerated completely at 24 h after laser axotomy. After that, the proximal axons initiated a robust regeneration and many of the M-cell axons almost regenerated fully at 4 days 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 timelapse 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.