Figure 3. Quantification of microglial response to injury in acute brain slices.

Slices from CX₃CR1^GFP/+ mice were imaged with confocal microscopy for 20 min before induction of injury and 20 min after injury, and 2D projections of the optical stacks at each time point were used for analysis by tracking moving objects larger than 2 μm in diameter. To confirm the involvement of P2Y₁₂ receptors in the response to injury, the slices were treated with 2 μM of the selective P2Y₁₂ receptor antagonist clopidogrel or DMSO vehicle. A, The image shows all tracks detected with Imaris for a baseline (left) and an injury (right) recording from a representative DMSO-treated slice; both panels are on the same scale. B, All tracks for a baseline (left) and an injury (right) recording from a representative clopidogrel-treated slice. The tracks are color-coded according to their duration; tracks with longer duration move in the direction of the injury. Scale bar: 5 μm. C-E, The average displacement of all moving objects (C), the average velocity of movement (D), and the fraction of tracks with longer than 5 μm displacement (E) were calculated for baseline (BL) recordings before injury and after the induction of the injury in the same slice. Numbers of slices for each condition are shown in parentheses. Statistics: two-way repeated measures ANOVA and Tukey’s post hoc test. *, p < 0.05.