Fig. 2: 8-AG treatment reduces apoptosis and protects photoreceptors in aged F344 rats.

The young (3 months), water-treated aged (24 months), or aged F344 rats treated with 8-AG for 8 weeks (24 months) were euthanized, and eyes were enucleated and prepared for immunohistochemistry or immunoblots. a–c are representative TUNEL-stained immunofluorescence images for young (a), water-treated aged (b), and 8-AG-treated aged rats (c), respectively, at the superior equatorial region of the retina sections, TUNEL⁺ cells are in green, and nuclei are stained with Hoechst33b342 in blue. Scale bar, 50 μm. d The Spidergrams of TUNEL⁺ cell numbers at different positions of the retinae. N = 3-4. e Immunoblots against the cleaved (top, 17, and 19 kDs) and intact caspase-3 (middle, 30 kDa) from rat retinal lysates. Ponceau stain (bottom) was used as the loading control. Each lane represents one rat retina. * Marks the excluded sample due to abnormal Ponceau stain. f. Bar plot of the band intensity ratio of cleaved: intact caspase-3. N = 7, 8, and 5 for young, water-treated aged, and 8-AG-treated aged F344 rat retinae. Data and error bars are means ± SDs. g–i Immunofluorescence images of rhodopsin (RHO, red) and Hoechst33342 for nucleus (blue) at superior central (SC) region of retinal cryosections of young (g), aged water-treated (h), and aged 8-AG-treated rats i. Scale bar, 50 μm. j The spidergram of RHO immunofluorescence intensity in the OS at different positions of the retinae. N = 8, 8, and 7 for young, water-treated aged, and 8-AG-treated-aged group, respectively. k Immunoblot of RHO from rat retinae. Each number represents one rat retina. Arrowheads show tetramer, dimer, and monomer of RHO from top to bottom, respectively. l Bar graph of RHO immunoblot intensity normalized by loading control. N = 8. m–o. Immunofluorescence images of peanut agglutinin (PNA, a marker of cones) and Hoechst33342 for nucleus (blue) at inferior central regions of retinal cryosections of young (m), aged water-treated (n) and aged 8-AG treated rats (o). Scale bar, 40 μm. N = 8. p The spidergram of PNA+ cone numbers at different positions of the retinae. Black circles, young rats; magenta squares, aged rats; blue triangles, aged rats treated with 8-AG. For spidergrams, data points and error bars are means and SEMs, and P values were calculated by two-way ANOVA. For the bar graph, column, and error bars are means and SDs, and P values were calculated by Kruskal–Wallis. Pink lines show P comparing the water-treated aged vs. young control, and blue lines show P comparing the 8-AG-treated aged vs. water-treated aged control.