Fig 2: Immunolabeling for Autophagy Using Autophagy Markers LC3B and ATG7(G–I and P–R) Immunostaining against LC3B and ATG7 was used to detect autophagy, which was mainly found in the rAAV-mTOR shRNA-treated group. (A–F and J–O) On the other hand, only background LC3B and weak, lesion-confined ATG7 expression were detected in the control (A–C and J–L) and rAAV-mTOR scrambled-treated groups (D–F and M–O). Scale bars, 100 µm. ONL, outer nuclear layer; RPE, retinal pigment epithelium.

Fig 3: Immunohistochemistry of Phalloidin and CD31 for Endothelial Cells Resulting from Laser-Induced CNVThe extensiveness of the CNV was measured via whole-mount phalloidin (red) and CD31 (yellow) immunostains. (A–I) Whereas the areas where CNV occurred were widespread for the control and scrambled shRNA-treated groups (A–F), CNV was substantially reduced for mice treated with mTOR shRNA (G–I). Bar graph shows CNV extent, as measured by pixels per lesion, using the Kruskal-Wallis test with post hoc analysis (J, mean ± SEM). Asterisk indicates statistically significant differences versus the control (*p < 0.05). Cross (†) indicates statistically significant differences versus the scrambled shRNA-treated group. Scale bars, 100 µm.

Fig 4: Characterization and retinal transduction of rAAV2-shmTOR-GFP. Schematic representation of rAAV2-shmTOR-GFP and rAAV2-shCon-GFP (A). ARPE-19 cells were infected with one of the experimental virus vectors, and western blotting performed thereafter showed that rAAV2-shmTOR-GFP treatment significantly reduced mTOR protein levels, whereas GFP expression correlated with virus amounts (B). β-actin was used here as a protein loading control. Compared with the normal control group, which did not undergo hyperoxic treatment, OIR model animals that were either mock-treated or injected with rAAV2-shCon-GFP exhibited elevated mTOR protein levels. This was almost completely abrogated in rats receiving rAAV2-shmTOR-GFP, which combined with GFP expression in vivo to reveal that the virus vectors successfully transduced the retinas of the rat OIR model (C). RT-qPCR showed markedly lower mTOR mRNA levels in retinas treated with the therapeutic virus vector, relative to rAAV2-shCon-GFP treatment (D). ITR, internal terminal repeat; GCL, ganglion cell layer; INL, inner nuclear layer (INL); ONL, outer nuclear layer.

Fig 5: Antiapoptotic effect of rAAV2-shmTOR-GFP, as determined by TUNEL assay. TUNEL-positive cells could not be found in the normal control group, which was not exposed to hyperoxic conditions, but were detected in transverse retinal sections taken from rats that were untreated, mock-treated, or injected with rAAV2-shCon-GFP. Significantly fewer apoptotic cells were found in the OIR model group treated with rAAV2-shmTOR-GFP (A), which was quantified (B; n = 5), indicating that mTOR inhibition by RNA interference has antiapoptotic activity.