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Int. J. Mol. Sci. 2020, 21, 2370 8 of 20 heatmap demonstrating their fold change. The overlapping DEGs show strong evidence that especially different genes of crystallin classes are altered by BL and RL/NIRL treatment. Upon RL or NIRL exposure crystallin genes were upregulated 1.1–2.2-fold compared to BL irradiated photoreceptors. Of particular interest were αA-crystallinand αB-crystallin(cryaa and cryab), for which distinct retinal functions have been identified, for example anti-apoptotic and anti-oxidative functions [35,36]. Figure 4. NIRL and RL induce reduced ROS production in BL irradiated cells. (A) Representative images of CM-H2DCFDA labeled unfixed retinal vibratome sections. Staining demonstrates ROS production in form of hydrogen peroxide (H2O2), peroxynitrite anions (ONOO-), hydroxyl radicals (HO.) or peroxide radicals (ROO.). Retinal layers: outer segments [OS], inner segments [IS], outer nuclear layer [ONL]. The graph displays fluorescence intensity of IS and OS normalized to Control (IS), demonstrating ROS production of n = 4 experiments ± SEM. Retinas were cultivated for 40 min (Control: 40 untreated, BL: 30 min BL + 10 min post-cultivation, BL+NIRL 810 nm: 30 min BL + 10 min 810 nm RL, BL+RL 670 nm: 30 min BL + 10 min 810 nm NIRL) for early event analysis. (B,C) Late event analysis after 9 h of cultivation. Eyes were treated for 6 h with BL, following 10 min NIRL or RL and post-cultivated for another 3 h. (B) Representative images of Nox2 and Nox4 immunohistochemical staining in retinal sections. N = 3 experiments. (C) Western blot analysis of isolated OS using antibodies against Nox4 and β-Actin as loading control. Protein level was quantified using ImageJ. N = 6 experiments ± SEM. * p < 0.05, ** p < 0.01 compared with BL.PDF Image | Photobiomodulation Mediates Neuroprotection against Retinal
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