Fig. 3.

Aqp4-AS1 silencing protects against diabetes mellitus-induced retinal vascular dysfunction. (a) qRT-PCR assays were conducted to compare Aqp4-AS1 levels in non-diabetic retinas and diabetic retinas after 2-month, 4-month, and 6-month induction of diabetes (n = 6). (b) Diabetic C57BL/6J mice (2-month-old, male) received an intravitreous injection of scrambled (Scr) shRNA, Aqp4-AS1 shRNA, or left untreated (Ctrl). Six months after the induction of diabetes, qRT-PCRs were conducted to detect Aqp4-AS1 levels (n = 6). (c) Retinal trypsin digestion was performed to detect acellular capillaries. Acellular capillaries were quantified in 30 random fields per retina and averaged. Red arrows indicated acellular capillaries (n = 6). Scale bar, 10 μm. (d) Mice were infused with Evans blue dye for 2 h. The fluorescence signal of flat-mounted retina was detected using a fluorescence microscope. A representative image was shown. Meanwhile, the quantification of Evans blue leakage was conducted (n = 6). Scale bar, 200 μm. (e) qRT-PCR assays were conducted to determine the relative levels of VEGF, Ang-1, IL-1β, IL-6, and ICAM-1 mRNA (n = 6). (f) ELISA assays were conducted to detect the amount of VEGF, Ang-1, IL-1β, IL-6, and ICAM-1 in retinal lysates (n = 6). *P < 0.05 versus Ctrl group; ^#P < 0.05 DR+Aqp4-AS1 shRNA versus DR+Scr shRNA or DR. The significant difference was evaluated by the Kruskal-Wallis test followed by the post hoc Bonferroni test.