Fig. 7: Fasting downregulates mTOR activity and upregulates autophagy in the retina and prevents RGC loss induced by ischemia/reperfusion injury.

Representative western blotting showing the downregulation of A ULK1 and B 4EBP1 phosphorylation in naive retina from mice subjected to 48, but not 24, hours fasting as compared with retinas from fed animals. In C, D, and E animals fasted for 48 h were subjected to retinal ischemia and killed after 24 h C or 7 days D, E. C Western blot analysis showing a significant increase of LC3II in both control and ischemic retinas from fasted animals as compared with fed. Significant decrease of the phosphorylated form of 4EBP1 (p-4EBP1) in the ischemic retinas of fasted mice as compared with fed was also reported. The results of the densitometric analysis of the autoradiographic bands reported in the graph show the comparison between the relative levels of the protein of interest in fasted vs fed animals. Values were normalized to loading control (actin). Data are shown as mean ± s.e.m. of 3–4 independent experiments for each experimental group. *P < 0.05, **P < 0.01 vs Fed (Student’s t test). c, control eye; I, ischemic eye; MW, molecular weight D Representative fluorescent photomicrographs of whole-mount ischemic and control retinas from fasted and fed animals. Fasting significantly increased the percentage of FluoroGold-labeled RGCs in the ischemic retinas as compared with fed animals. Images are representative of three independent experiments. Histogram in E reports the quantification of RGC survival under the different diet regimens. Thirty-two 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). Scale bar 75 μm