Cell Mobility after Laser Axotomy Figure 2. Longitudinal changes in axonal morphology after laser axotomy. A. Cell before injury and a magnification of the axon shown in an inset. B. After laser exposure there was swelling of the distal axon adjacent to the injury site. C. 20 min after exposure an approximately 1 m separation became apparent between the ends of the axon proximal and distal to the injury site. D. The loss of membrane integrity was evidenced by bubbles and distal axon fragmentation. Scale bars: 10 mm. (See Supplemental Video S1 for the full series). doi:10.1371/journal.pone.0026832.g002 Cell motility response To measure the motility of cell bodies after axotomy more than 300 images from each of the 40 axotomized cells and 9 control cells were processed using a custom-made algorithm. The position of the cell body over time was used to compute the trajectory and instantaneous velocity (see section on image processing). As an example, Fig. 3 depicts the instantaneous velocity and trajectory of the cell shown in Supplemental Video S4. The horizontal line represents the baseline mean velocity of 9 control cells (30 nm/ min). The change in motility in response to axotomy could by divided into an initial and a late response. A dramatic increase in cell motility was typically observed immediately after exposure (Fig. 3A). In this example the cell tripled its baseline velocity approaching 100 nm/min after transection of its axon. This increase in motility was followed by a latency phase, lasting approximately 6 hr, where the velocity returned to baseline. This latency phase was consistently followed by a sustained migration away from their initial position. Despite the initial motility increase, cells moved close to the initial position during this early response. This is shown in the trajectory plot (Fig. 3B), where the points that correspond to the first 450 min are shown in black and it can be seen that it represented only a small fraction of the entire trajectory. We characterized the initial response by the maximum velocity the cell reached within the first 150 min and called it reaction velocity (vr). Moreover, we quantified the late response by computing the average velocity of the cell body during the time it velocity was higher than the basal velocity. That is, we did not compute the period of time in which cells were resting as illustrated in Supplemental video S4 and Fig. 3. After injury this cell remained in the same place for almost 500 minutes, after which it started to migrate away from the injury site. In this case the cell motility would be understimate if the velocity was averaged over the entire imaging session. Data are presented using notched box plots [33], where the distribution median is represented with a red line and the upper and lower quartiles are the extremes of the blue boxes (see Fig. 4). The box notches are useful to illustrate significant differences between distributions. If notches do not overlap, the medians are significantly different at approximately 95% confidence. Data are considered outliers if they do not fall within a normal distribution, i.e. outside the interval defined as q1zw(q3{q1) and q3zw(q3{q1), where q1 is the lower quartile, q3 the upper quartile and w is called whisker. The parameter w was set to 1.5, and assuming a normal distribution, this value leaves 99.3% of the data within that interval. The maximum and minimum measured values are connected with a dashed vertical line and outliers are represented by the + symbol. Protruded notches were used when the median was close to the upper or lower quartiles. RGC-5 soma motility and regenerative response The initial motility response (vr) to axonal injury was significantly higher (125677 nm/min vs. 35610 nm/min; p = 0.008) than in control cells (Fig. 4A). Cells that regenerated or replaced their injured axons (group A) had an intermediate vr between non-regenerating axotomized cells(group B) and control cells (110672 nm/min; p = 0.03 compared to control cells). The late motility response to injury followed the same pattern (Fig. 4B), with significantly higher velocities in the axotomized somas compared to controls (46615 nm/min vs. 2864 nm/min; p = 0.001). The late change in cell motility correlated with the regenerative response to injury: cells that regenerated their axons had a lower mean motility than cells that did not (54615 nm/min vs. 42614 nm/min; p = 0.015). These results demonstrate a PLoS ONE | www.plosone.org 3 November 2011 | Volume 6 | Issue 11 | e26832