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Albarracin et al. BMC Neuroscience 2013, 14:125 http://www.biomedcentral.com/1471-2202/14/125 Page 4 of 14 (A) (B) (C) Figure 2 Quantitative assessment of the effects of 670 nm light on the ONL thickness of hyperoxic mice. The average thickness of the ONL was measured along the retinas of the nontreated (NT), hyperoxia-exposed (solid colored lines) and 670 nm-treated (Tr), hyperoxia-exposed mice (broken colored lines) from (A) 0d, 3d, 7d and (B) 10d and 14d groups. The retinas from 0d groups (NT, solid black line and Tr, broken black line) served as controls. (C) At 14d, the ONL of the Tr group (broken green line) in the inferior region was significantly thicker than the NT retinas (solid green line). *Statistically significant difference (p < 0.001) compared to the 670 nm light-treated retinas of the same time point. (Figure 7B, green; Figure 8A). In 10dTr retinas, the photo- receptor IS were less disrupted (Figure 7E, green) and showed levels of Cox Va immunoreactivity that were com- parable with retinas at 7d (Figure 8A). After 14d exposure to hyperoxia, photoreceptor IS were shortened in NT animals, but retained good morphology in Tr retinas. However, levels of Cox Va immunoreactivity in the 14dTr animals were similar to those in the 10d groups (Figure 8A). Conversely, to determine the level of Cox Va expressed by surviving photoreceptors in the hotspot area following hyperoxic exposure of the nontreated and 670 nm-treated groups, Cox Va immunostaining in the IS was normalised to the number of photoreceptor nuclei. There was a correlation observed between the population of photoreceptor nuclei and the level of Cox Va expressed in the IS. The level of Cox Va labelling is proportional to the number of photoreceptors in early timepoints (results not shown) and in retinas with prolonged hyperoxic ex- posure, except for retinas from 10dNT group (Figure 9). In the 14dNT retinas, the reduction of photoreceptor nu- clei density (Figure 9, black bar) correlated with the re- duction of the level of Cox Va expression in the IS (Figure 9, gray bar). By contrast, the number of photo- receptor nuclei and the level of Cox Va immunoreactivity in all 670 nm-treated groups were close to control base- line levels (0dNT, non-hyperoxic, nontreated). Oxidative stress status (acrolein) Baseline immunoreactivity to acrolein was determined from control retinas (0d; Figure 8B, red). A 3-fold increase in acrolein immunoreactivity above baseline was observed in 3dNT retinas (Figure 8B, black bar), increasing to 20- fold at 7d, 36-fold at 10dNT and 37-fold in 14dNT. In contrast, there was no significant increase of acrolein im- munoreactivity in 3dTr retinas above baseline (Figure 8B, red); after 7 days of exposure to hyperoxia there was a 20-fold increase in acrolein immunoreactivity above base- line, comparable with 7dNT retinas. However, acrolein immunoreactivity was maintained at this level at the later timepoints in the Tr groups (10dTr and 14dTr). Gene expression Expression of Hmox-1, C3 and Fgf-2 genes were analysed by quantitative RT-PCR to monitor the hyperoxia- induced stress responses of the NT and Tr retinas. Hmox-1 There was no significant change in Hmox-1 gene expres- sion after 3 days of exposure to hyperoxia, in either NT (black bar) or Tr (red bar) animals (Figure 10A). By 7 days exposure, there was a 2 to 2.75-fold up-regulation in Hmox-1 expression in both 7dNT and 7dTr animals (dif- ference not significant). By 10 days, there was a 4-foldPDF Image | 670 nm light mitigates oxygen-induced degeneration 2013
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