Laser-Based Single-Axon Transection for High-Content Axon Injury and Regeneration Studies

DARÍO KUNIK; CAROLYNE DION; TSUNEYUKI OZAKI; LEONARD A. LEVIN; SANTIAGO COSTANTINO

PLOS ONE

PUBLIC LIBRARY SCIENCE

Lugar: San Francisco; Año: 2011 vol. 6 p. 26832 - 26832

1932-6203

The investigation of the regenerative response of the neurons to axonal injury is essential to the development of newaxoprotective therapies. Here we study the retinal neuronal RGC-5 cell line after laser transection, demonstrating that theability of these cells to initiate a regenerative response correlates with axon length and cell motility after injury. We showthat low energy picosecond laser pulses can achieve transection of unlabeled single axons in vitro and precisely inducedamage with micron precision. We established the conditions to achieve axon transection, and characterized RGC-5 axonregeneration and cell body response using time-lapse microscopy. We developed an algorithm to analyze cell trajectoriesand 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 orregrowth of damaged axons migrated more slowly than cells that could not. Moreover, we established that RGC-5 cells withlong axons could not recover their injured axons, and such cells were much more motile. The platform we describe allowshighly controlled axonal damage with subcellular resolution and the performance of high-content screening in cell cultures.