Laser-Based Single-Axon Transection for High-Content Axon Injury and Regeneration Studies Darı´o Kunik1,2, Carolyne Dion1,3, Tsuneyuki Ozaki1,3, Leonard A. Levin1,4,5, Santiago Costantino1,2,4* 1 Centre de Recherche de l’Hoˆ pital Maisonneuve-Rosemont, Montre´al, Quebec, Canada, 2 Institut de Ge´nie Biome´dical, Universite´ de Montre´al, Montre´al, Quebec, Canada, 3 Energie Materiaux et Communications, Institut National de la Recherche Scientifique, Quebec City, Quebec, Canada, 4 De´partement d’Ophtalmologie Universite´ de Montre´al, Montre´al, Quebec, Canada, 5 Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, Wisconsin, United States of America 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. Citation: Kunik D, Dion C, Ozaki T, Levin LA, Costantino S (2011) Laser-Based Single-Axon Transection for High-Content Axon Injury and Regeneration Studies. PLoS ONE 6(11): e26832. doi:10.1371/journal.pone.0026832 Editor: Nic D. Leipzig, The University of Akron, United States of America Received April 26, 2011; Accepted October 5, 2011; Published November 2, 2011 Copyright: 2011 Kunik et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by grants from the Fondation de l’Hoˆ pital Maisonneuve-Rosemont to SC, the National Sciences and Engineering Research Council (NSERC) and the Fonds Qube´be´cois de la Recherche sur la Nature et les Technologies (FQRNT) grants to SC and TO, the Canadian Institutes of Health Research (CIHR) to LAL and SC, and the National Institutes of Health (NIH) R21 EY017970 to LAL. DK is a recipient of a HMR postdoctoral fellowships and CD of a NSERCf postdoctoral fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: santiago.costantino@umontreal.ca 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 [1–4]. In recent years, 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]. After injury, isolated axons undergo Wallerian and retrograde degeneration, which are active axon degeneration processes independent of apoptosis and relevant to different diseases [3,7] Although there is an excellent understanding of soma death programs and multiple methods for inhibiting soma death are available the pathways for axon degeneration and preventing axon loss are as yet poorly understood. Improved techniques for studying the responses of somas and axons to injury would therefore help in the development of therapies for these otherwise irreversible neuronal diseases. Laser-based technologies have been shown to be powerful yet precise tools to manipulate organelles, chromosomes and micro- tubules [8–14]. Recently femtosecond laser pulses were demon- strated to cut single axons in vivo [15–18]. Despite the success of optical techniques in-vivo, this approach has rarely been used in in-vitro to explore axonal injury models that were otherwise inaccessible. Most in vitro axoprotection studies have been carried out by cutting axons with scalpels, needles, or glass pipettes [19– 23], and only recently single-axon transection has been demon- strated using a nanoknife [24]. However all these mechanical approaches share the disadvantage of tearing and pulling transected membranes during the cut process and this would interfere with the regeneration ability of cells [16]. Ultrafast laser- based transections have the advantage of producing rapid clean cuts without tearing and pulling transected membranes. In addition, they are easy to automate [12,14,25,26]. Recently, 180 ps visible (532 nm) laser pulses were used to cut axon bundles in vitro [27]. Hellman et al. showed that high-energy (600–800 mJ) single pulses are enough to transect axon bundles in cultured dorsal root ganglion cells. These authors demonstrated that optical transection is also compatible with protein-patterned substrates, a useful technique for micro-manipulating the cell environment [28,29]. Nanosecond pulses were also utilized to study the axon response to laser exposure [30], showing evidence of chemoat- tractant molecules secreted at the injury site. The growth cones of the injured cells, as well as the ones of adjacent cells turned and migrated toward the injury site. Here we demonstrate that low energy picosecond laser pulses can achieve transection of unlabeled single axons in vitro with micron resolution. Using as a model a cell line (RGC-5) that when exposed to a low concentrations of staurosporine becomes differentiated and develops a neuronal morphology [31,32], we established the conditions to achieve axon transection and characterized RGC-5 axon regeneration and cell body response to injury using time-lapse PLoS ONE | www.plosone.org 1 November 2011 | Volume 6 | Issue 11 | e26832