Laser-based single-axon transection for high-content axon injury and regeneration studies.

Darío Kunik,Santiago Costantino,Tsuneyuki Ozaki,Carolyne Dion,Leonard A Levin,Nic D Leipzig

doi:10.1371/journal.pone.0026832

Abstract

The investigation of the regenerative response of the neurons to axonal injury is essential to the development of new axoprotective therapies. Here we study the retinal neuronal RGC-5 cell line after laser transection, demonstrating that the ability of these cells to initiate a regenerative response correlates with axon length and cell motility after injury. We show that low energy picosecond laser pulses can achieve transection of unlabeled single axons in vitro and precisely induce damage with micron precision. We established the conditions to achieve axon transection, and characterized RGC-5 axon regeneration and cell body response using time-lapse microscopy. We developed an algorithm to analyze cell trajectories and established correlations between cell motility after injury, axon length, and the initiation of the regeneration response. The characterization of the motile response of axotomized RGC-5 cells showed that cells that were capable of repair or regrowth of damaged axons migrated more slowly than cells that could not. Moreover, we established that RGC-5 cells with long axons could not recover their injured axons, and such cells were much more motile. The platform we describe allows highly controlled axonal damage with subcellular resolution and the performance of high-content screening in cell cultures.

Highlights

Significant effort has been devoted to elucidating the pathways that lead to neuronal dysfunction and death in diseases as disparate as Alzheimers disease, trauma, multiple sclerosis, peripheral neuropathies, and glaucoma [1,2,3,4]
We demonstrated how a semi-automated picosecond laserbased platform for axonal injury can be used to study the biology of the axon response to transection
RGC-5 cells with transected axons showed a change in motility as a response to injury, and this motility change correlated with the ability of RGC-5 cells to regenerate their transected axons (Fig. 4)

Summary

Introduction

Significant effort has been devoted to elucidating the pathways that lead to neuronal dysfunction and death in diseases as disparate as Alzheimers disease, trauma, multiple sclerosis, peripheral neuropathies, and glaucoma [1,2,3,4]. It has become evident that cell bodies (somas) and axons follow divergent degeneration pathways, with axonal injury or degeneration often preceding cell death [1,5,6]. Improved techniques for studying the responses of somas and axons to injury would help in the development of therapies for these otherwise irreversible neuronal diseases. Femtosecond laser pulses were demonstrated to cut single axons in vivo [15,16,17,18]. Most in vitro axoprotection studies have been carried out by cutting axons with scalpels, needles, or glass pipettes [19–

Methods

Results

Conclusion