ROS levels of retinal neurons before and after SOD treatment in 8 and 20 wk db/m and db/db mice.

<p>In situ ROS generation was determined in freshly prepared retinal sections by immunohistochemical staining for dihydroethidium (DHE). The quantitatively analyze was performed by using an image analysis software (Adobe Photoshop CS5, CA). The thickness of each retinal section was 10 µm. Micrographs of retinal sections that were stained with DHE: <b>Panel A:</b> a: 8 wk db/m; b: 20 wk db/m; c: 20 wk db/m, 2-hour SOD treatment; d: 20 wk db/m 5-hour SOD treatment; e: 8 wk db/db; f: 20 wk db/db; g: 20 wk db/db, 2-hour SOD treatment; h: 20 wk db/db, 5-hour SOD treatment. ONL: outer nucleus layer; OPL: outer plexiform layer; INL: inner nucleus layer; GCL: ganglion cell layer. <b>Panel B:</b> quantitative analysis of retinal ROS levels. The fluorescent intensity of DHE labeled neurons was quantified (means±SEM, n = 4). In comparison with db/m mice, the intracellular ROS generation was significantly increased in retinas of 8 and 20 wk db/db mice (8 wk: *p<0.05; 20 wk: ***p<0.0001, one-way ANOVA). <b>Panel C:</b> SOD treatment for 2- and 5- hour significantly reduced the ROS levels in 20 wk db/db mice retina (2 hours: **p<0.001; 5 hours: ***p<0.0001, one-way ANOVA). Scale bar: 20 µm. Other conventions are as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030343#pone-0030343-g002" target="_blank">Figures 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030343#pone-0030343-g003" target="_blank">3</a>.