Figure 5. Effects of axon length on regeneration and motility. A. The regenerative response of cells depended on axon length. Cells with longer axons were less likely to reconnect or regrow their axons after axotomy. B. Correlation between cell motility and axon length. Cell with longer axons showed a higher motility after axotomy (R = 0.3 for linear regression). doi:10.1371/journal.pone.0026832.g005 much more likely to migrate toward the transection site (p = 0.0002). These results are in agreement with recent results [30] obtained with goldfish RGC cells, as we observed a migration of the whole cell to the injury site, presumably as a result of the secretion of specific molecules at the injury site. Our experiments show that both attraction and repulsion can be triggered depending to the level of damage. Conclusions We demonstrated how a semi-automated picosecond laser- based platform for axonal injury can be used to study the biology of the axon response to transection. We established correlations between cell motility after injury, axon length, and the initiation of the regeneration response. 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). Cells that were capable of repair or regrowth of the damaged axon migrated more slowly Cell Mobility after Laser Axotomy than cells that were not able to repair the damage. Moreover, we established that cells with long axons could barely recover their injured axons (Fig. 5A), and such cells showed a much higher motility (Fig. 5B). Afetr We also found that cells that either reconnected or regrew prAfetrocesses migrated towards the injury site, and cells that were not able to reconnect or regrow moved away from the injury site (Fig. 6). Taken together, these results suggest that RGC-5 cell motility was related to the level of injury and also that the liberation of chemoattractant molecules from the injury site depend on the injury level. It is tempting to speculate that the cell classification that was observed reflects the damage that was induced. Thus, RGC-5 cells that were slightly injured repaired their axon; RGC-5 cells that were more severely injured and were not able to reconnect their axons, replaced them with new processes; and cells that were dramatically injured could not self-repair even if the integrity of isolated axons was preserved. This analysis suggests that RGC-5 cells with long axons are less capable of reconnecting or replacing an injured process. This behavior could be explained by a macromolecule or organelle transport phenomena toward the injured axon, with longer neurites needing more time to be reconnected. However we also observed that long processes appear to survive longer than short processes, implying that this behavior cannot be simply explainded in terms of molecular transport toward the injury site and distal axon. Further experiment work is requiered to elucidate why shorter axons are more likely to be reconnected or replaced by injured cells. Our results using the RGC-5 line are encouraging and demonstrate that laser-assisted transection can be used in primary cultures to help answer useful questions about axon protection and recovery after injury. The platform we described allowes the induction of highly controlled axonal damage with subcellular resolution and the the experiments demonstrate how low-energy picosecond pulses can be utilized for intracellular surgery. These lasers are useful to induce high-precision neuronal axotomy, avoiding collateral tissue damage, and yielding clean transection without pulling or tearing the axonal or somal membrane. Axon recovery and regeneration after injury was observed, showing that the damage induced by the laser was well localized. This platform can be used for improving our understanding of the molecular mechanisms of the axon response to injury. It laso allows novel high-content screens for neuroprotective and axoprotective molecules which could potentially lead to therapies for diseases associated with axonal injury. Materials and Methods Cell Culture RGC-5 cells were kindly provided by Neeraj Agarwal, Ph.D. [34] and cultured as previously described [31,32]. Cells at passage 9 to 20 were inoculated in Dulbecco’s modified Eagle medium (DMEM) at 1000 cells/cm2 on poly-D-lysine-coated glass-bottom dishes. Cells were then incubated at 37uC in 5% CO2/air for 6 hours to allow cells to attach to the dish surface. Once cells were adherent, staurosporine (316 nM) was added to induce cell differentiation. After 4 hours, culture media was replaced by DMEM modified for long-term imaging by replacing the sodium bicarbonate with HEPES (10 mM). Dishes were then moved to the microscope for imaging and laser axotomy. See Supplemental Materials File S1 for details on reagent and media preparation. Microscope and laser injury platform An inverted microscope (Olympus IX-71, Tokyo, Japan) equipped with a motorized two-axis stage (Thorlabs MAX201, PLoS ONE | www.plosone.org 5 November 2011 | Volume 6 | Issue 11 | e26832