Electrical and Computer Engineering, The University of Texas at Austin, Austin, TX, 78712, USA.
Mechanical Engineering, The University of Texas at Austin, Austin, TX, 78712, USA.
Sci Rep. 2017 Aug 29;7(1):9837. doi: 10.1038/s41598-017-10302-4.
Several sophisticated microfluidic devices have recently been proposed for femtosecond laser axotomy in the nematode C. elegans for immobilization of the animals for surgery to overcome time-consuming and labor-intensive manual processes. However, nerve regeneration studies require long-term recovery of the animals and multiple imaging sessions to observe the regeneration capabilities of their axons post-injury. Here we present a simple, multi-trap device, consisting of a single PDMS (polydimethylsiloxane) layer, which can immobilize up to 20 animals at the favorable orientation for optical access needed for precise laser surgery and high-resolution imaging. The new device, named "worm hospital" allows us to perform the entire nerve regeneration studies, including on-chip axotomy, post-surgery housing for recovery, and post-recovery imaging all on one microfluidic chip. Utilizing the worm hospital and analysis of mutants, we observed that most but not all neurodevelopmental genes in the Wnt/Frizzled pathway are important for regeneration of the two touch receptor neurons ALM and PLM. Using our new chip, we observed that the cwn-2 and cfz-2 mutations significantly reduced the reconnection possibilities of both neurons without any significant reduction in the regrowth lengths of the severed axons. We observed a similar regeneration phenotype with cwn-1 mutation in ALM neurons only.
最近已经提出了几种复杂的微流控设备，用于秀丽隐杆线虫的飞秒激光轴突切断，以固定动物进行手术，从而克服耗时且劳动密集型的手动过程。然而，神经再生研究需要动物的长期恢复和多次成像会话，以观察其轴突受伤后的再生能力。在这里，我们提出了一种简单的多阱设备，由单个 PDMS（聚二甲基硅氧烷）层组成，可以将多达 20 个动物固定在合适的位置，以便进行精确的激光手术和高分辨率成像所需的光学访问。新设备名为“worm hospital”，可让我们在一个微流控芯片上执行整个神经再生研究，包括在芯片上进行轴突切断、手术后的恢复、以及恢复后的成像。利用 worm hospital 和突变体分析，我们观察到 Wnt/Frizzled 通路中的大多数（但不是全部）神经发育基因对于两个触觉感受器神经元 ALM 和 PLM 的再生是重要的。使用我们的新芯片，我们观察到 cwn-2 和 cfz-2 突变显著降低了这两个神经元的重新连接可能性，而对切断的轴突的再生长度没有任何显著影响。我们仅在 ALM 神经元中观察到 cwn-1 突变的类似再生表型。
