Figure 3
SC effects on RGC survival after pONT can be evaluated using an algorithm on whole retinal mounts. RGCs in the whole retina were labelled with Brn-3a antibody and imaged by confocal fluorescence microscopy. (a) Normal retina, (b) pONT retina, (c) pONT+SC/DONS retina, (d) pONT+SC/IVT retina, and (a1, b1, c1 and d1) represent corresponding areas of high magnification images at 21 days following pONT. A substantial loss of RGCs was noted in the pONT only retina (b and b1), compared with the normal retina (a and a1), and SC/DONS treatment (c and c1) clearly reduced RGC loss. This increase in RGC survival was also seen in SC/IVT-treated eyes (d and d1). The number of RGCs in retinal whole mounts was analysed using our newly established algorithm in ImageJ. To validate the algorithm, RGCs in 0.92 mm2 retinal sections were manually counted by four trained individuals. (e) Correlation of algorithm output to average manual counting fitted a straight line with a correction factor based on a previous study30 (Pearson’s correlation coefficient=0.9860, P<0.0001, R2=0.9722). (f) Bland–Altman plot shows good agreement between algorithm and manual counts after correction. Scale bars: 50 μm
