Fig. 3
Effects of GSK101 on retinal ganglion cell (RGC) apoptosis and survival. A TRPV4 activation leads to enhanced RGC apoptosis. A1–A3 4′,6-diamidino-2-phenylindole (DAPI) staining in saline-injected (control) (A1), GSK101-injected (GSK101) (A2), and HC-067 with GSK101 (HC) (A3) retinas at 1 week after injection in the regions at angle 0°. Images were acquired from whole flat-mounted retinal preparations. A4–A6, Counterstained images with TUNEL staining detection of RGC apoptosis (green). A7–A9, Merged images of corresponding TUNEL and DAPI images. Scale bar: 50 µm. B Bar chart summarizing mean numbers of TUNEL-positive cells in each retina under different conditions. n = 5 for all groups. C Pre-application of HC-067 reduces RGC apoptosis in COH retinas. C1–C3 DAPI staining in sham-operated (control) (C1), COH (C2), and HC-067 with COH (HC + COH) (C3) retinas at G2w. C4–C6 Counterstained images with TUNEL staining detection of RGC apoptosis (green). C7–C9 Merged images of corresponding TUNEL and DAPI images. Scale bar, 50 μm. D Bar chart summarizing mean numbers of TUNEL-positive cells in each retina under different conditions. n = 6 for all groups. *p < 0.05, **p < 0.01, and ***p < 0.001. E, F FluoroGold labeling images of surviving RGCs in flat-mounted retinas as shown in the central and peripheral panels, photographed at low magnification (20 ×). Fluorescence micrographs of flat-mounted retinas depicting FluoroGold-labeled RGCs in control (E1, E2), 1 week after GSK101 injection (E3, E4), 1 week after GSK101 plus HC-067 injection (E5, E6), 1 week after GSK101 plus R7050 injection (E7, E8), glaucoma 2 weeks (COH, F1, F2), HC-067 plus COH (F3, F4), and R7050 plus COH (F5, F6) retinas. Scale bar: 50 µm. G Schematic diagram showing different retinal regions. H Quantitative analysis of the density (cells/mm2) of surviving RGCs under different conditions in the central and peripheral regions