Fig. 6. Silencing of HIF-1α in detached retinas increases cell death and decreases autophagy.

Retinal detachments were created in rats and siRNA against HIF-1α or Non-targeting controls were introduced into the subretinal space immediately following detachment. (A) Transcript expression for HIF-1α at 1 day post detachment evaluated using qRT-PCR. Ratios represent fold change in relation to attached control retinas in same animals. Error bars represent mean ± SEM, n=3 animals. p**<0.005. (B) Protein levels of HIF-1α, BNIP3, and LC3 evaluated by western blot analysis of retinal homogenates. Immunoblots shown are representative of three sets of animals. GAPDH served as a loading control. Densitometry ratios of the LC3-II/LC3-I bands are shown. Animals treated with siHIF-1α showed a significant decrease in LC3-II/LC3-I ratios in relation to animals treated with siControl (p**<0.005, n = 3 animals). (C) LC3 localization on cross-sections from rat retinas detached and treated with siRNA against HIF-1α or Non-targeting controls with immunohistochemical analysis. Arrows indicate punctate like structures. OS, outer segments; IS, inner segments; ONL, outer nuclear layer. (D) TUNEL staining of 3 day detached retinas treated with siRNA against HIF-1α or Non-targeting controls. Bar graph depicts number of TUNEL positive cells per 40X viewing field in the outer nuclear layer. Error bars represent mean ± SEM, n=3 animals with quantification of 9 non-overlapping fields of 300 µm per retina (p***<0.0005, Student’s t-test).