Fig. 4

RFC1 protein increased after retinal ischemia, and the overexpression of RFC1 before ischemia by LV salvaged decreased collagen-4 and occludin levels. A The representative Western blotting image from control (n = 5) and ischemic (n = 3) retinas. Housekeeping gene β-Tubulin III was used as loading control. B The graph shows the relative density measurements of bands which were calculated by proportioning to loading control. One-hour permanent retinal ischemia increased relative RFC1 protein levels compared to control. C Retinal ischemia significantly augmented the percentage of mean grey value of RFC1 by 114.5% (n = 3). Representative confocal images of control or ischemic retinal microvessels stained by anti-RFC1 antibody (red). Significant immunosignal increase in ischemic microvessels was observed compared to controls. Also, ischemic microvessels demonstrated expected characteristics such as constrictions and protruding pericyte bodies (asterisk). Of note, ischemic pericyte bodies showed denser RFC1 immunosignal (white arrows). D, E In contrast, retinal ischemia decreased collagen-4 by 42.07% (n = 3), occludin by 50.94% (n = 3). F, G In addition, microvessels treated with LV-RFC1, 10 days before ischemia showed 176% increase in collagen-4 (n = 3) and 60.9% in occludin (n = 3) immunosignal compared to Control-LV delivered ones indicating RFC1 overexpression before ischemia might retrieve loss of collagen-4 and occludin in ischemia. (*p ≤ 0.05). Nuclei were labelled with Hoechst 33258 (blue) in all images. Data are mean ± S.E.M. Mann-Whitney U; Scale bars= 10 μm