Fig. 6: Rapamycin promotes autophagy and increases RGC survival following retinal ischemia/reperfusion.

A Rapamycin treatment schedule. Rapamycin (10 mg/Kg) or vehicle were injected i.p. once a day for 6 consecutive days; ischemia was induced the fifth day and mice killed after 24 h for the biochemical analysis B, C, D or 7 days for evaluation of RGC survival E, F. Western blotting analysis of B ULK and C 4EBP1 phosphorylation levels was performed to indirectly check the inhibition of mTOR activity in the retina of rapamycin-treated mice. p-ULK (S757) and p4EBP (T37/46) were reduced in control and ischemic retinas from rapamycin-treated mice as compared with both control and ischemic retinas of vehicle-treated mice. D Rapamycin reduced basal p62 expression in non-ischemic retina as compared with vehicle-treated control and prevented the accumulation of p62 in the ischemic retinas at 24 h of reperfusion. Histograms represent the densitometric analysis of the bands normalized by the internal loading control (actin). Data are reported as mean ± s.e.m. of three independent experiments for each group. *P < 0.05, **P < 0.01, ***P < 0.001 (ANOVA followed by Tukey–Kramer for multiple comparisons test). C, control non-ischemic retina; I, ischemic retina; MW, molecular weight. E Representative fluorescent photomicrographs of whole-mount ischemic and control retinas from vehicle and rapamycin-treated mice. Systemic treatment with rapamycin significantly increased the percentage of FluoroGold-labeled RGCs in the ischemic retinas as compared with vehicle-treated animals. Images are representative of three independent experiments. Scale bar 75 μm. F Histogram reports the result of RGC count. Twenty images per retina were acquired and the total number of labeled cells in the ischemic retina I was compared with contralateral, non-ischemic retina C and expressed as percentage of RGC survival. Results are reported as mean ± s.e.m. of three independent experiments. *P < 0.05 (Student’s t test)