Fig. 1. Modulation and distribution of LC3 following retinal ischemia/reperfusion injury.

Mice were subjected to retinal ischemia in the right eye (I) for 60 min and killed after 0, 1, 6, or 24 h. For each animal, retina from contralateral eye was used as control. A Immunoblot showing the time-dependent modulation of LC3 expression in whole retinal lysates at the indicated time of reperfusion (Rep time). Histograms represent the densitometric analysis of the bands expressed as B LC3I and C LC3II normalized to loading control (actin). Dashed lines indicate the baseline expression of the protein in non-ischemic retinas set to 1. Data are reported as mean ± s.e.m. (4–6 independent experiments for each group). ^#P < 0.05 vs control non-ischemic retina (Student’s t test); *P < 0.05 vs 1 and 24 h of reperfusion (ANOVA followed by Tukey–Kramer multiple comparisons test); ***P < 0.001 vs 0 h of reperfusion (Student’s t test). C, control non-ischemic retina; I, ischemic retina; MW, molecular weight; Short Exp, short exposure; Long Exp, longer exposure. D Confocal images showing the upregulation of endogenous fluorescence in retinas of GFP-LC3 transgenic mice subjected to ischemia and killed after 6 h of reperfusion. The inserts are higher magnification photomicrographs showing the signal distribution in GCL and IPL. E colocalization of GFP-LC3 signal with the RGC marker TUJ1 (red) in ischemic retinas reperfused for 6 h. F Representative retinal tissue sections from GFP-LC3 transgenic mice showing the partial colocalization of lysosomal marker LAMP-2 (red) with GFP-LC3-positive round-shaped vesicles (white arrowhead) at the ganglion cell layer (GCL) of the ischemic retina. Images are representative of three animals per experimental conditions. Frozen tissue sections were prepared as described in the methods and nuclei counterstained with DAPI (blue). GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars D 50 μm, E 47.62 μm F 50 μm. G Immunoblot showing the ex vivo analysis of autophagic flux in retinas subjected to ischemia (I, Isch) followed by 6 h of reperfusion as compared with non-ischemic retinas (C, Ctr). Samples from individual retinas were split in half and incubated for 2 h in medium with (NH₄Cl/Leu) or without (vehicle) ammonium chloride (NH₄Cl, 20 mM) and leupeptin (Leu, 200 μM) to inhibit lysosomal enzymatic activity. Histograms show the densitometric analysis of the bands normalized on internal control (actin). Data are reported as mean ± s.e.m. of three independent experiments. *P < 0.05, **P < 0.01 (Student’s t test)