Fig. 8

Quantitative assessment of DLK-JNK activation after IA injury based on counts of p-JUN (+) RGCs. a-c. In whole-mount retinas dually stained for the RGC marker γ-synuclein (SNCG) (red) and the DLK-JNK marker p-JUN (green), there is induction of p-JUN in RGCs at day 1 (b) and attenuation of labeling by day 14 (c). No p-JUN (+) RGCS are seen in the sham scenario (a). Left-sided images have been acquired with green filter combination for red SNCG immunofluorescence, images at the center have been acquired with blue filter combination for green p-JUN immunofluorescence, and panels on the right are merged images in which double-labeled profiles appear orange. Note the extensive colocalization at day 1, based on the large number of orange profiles (b, right panel). Most p-JUN (+) nuclei at day 14 seem to belong to smaller or atrophic RGCs (c). d-e. Bar graphs of densities of p-JUN (+) RGCs in the retinas of sham and injured animals at 1, 3, 7, and 14 days post-injury (d), and of the ratios of densities of p-JUN (+) RGCs over densities of total RGCs from two representative time points, i.e. 1 and 14 days (e). Significant differences are indicated with asterisks. The comparison in (e) was done to ensure that reduction in numbers of double labeled profiles in (d) was not simply an artifact of the progressive death of RGCs. Data were analyzed with one-way ANOVA followed by Tukey’s post hoc test for (d) and with student’s t-test for (e). * p < 0.05. Scale bars: 25 μm