Abstract
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.
Highlights
The capacity of neurons, especially those in the adult mammalian central nervous system, to regenerate after injury is limited, leading to permanent deficits in the nervous system[1, 2]
We studied four elements of the axonal regeneration properties of the anterior lateral microtubule cells (ALM) and the posterior lateral microtubule cells (PLM): axonal regrowth, axonal reconnection, axonal fusion, and regrowth lengths
While the early design of tapered channel geometry ensured the high-degree immobilization[22], we found that the arbitrary orientation of the worms was unfavorable for axotomy and post-surgical imaging[36]
Summary
The capacity of neurons, especially those in the adult mammalian central nervous system, to regenerate after injury is limited, leading to permanent deficits in the nervous system[1, 2]. In another attempt of on-chip laser surgery, a single-layer multi-trap microfluidic device has been developed and used to ablate single synapses[32] This simple chip used a tapered channel geometry to immobilize the animals, post-surgery housing and imaging were limited to a couple of hours due to the lack of a separate housing and feeding areas. The tapered geometry led the animals to rotate on the anterior-posterior axis plane of their bodies away from the glass interface, as the channel width becomes smaller than its height[36] This body orientation requires the laser beam to go through the cover glass and a significant portion of the animal body, leading to unsuccessful axotomies and low-contrast imaging of the lateral neurons
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