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Int. J. Mol. Sci. 2020, 21, 2370 4 of 20 2.1. Decreased Mitochondria-Induced Apoptosis upon RL/NIRL Exposure To verify the effects of RL and NIRL on elements of the intrinsic mitochondria-induced apoptotic pathway in photoreceptors, Casp9, Bcl-2 and Bax protein expressions were analyzed. Upon BL irradiation protein expression of pro-apoptotic Bax and Caspase-9 was significantly increased in photoreceptor inner segments (IS) compared to non-irradiated controls, while Bcl-2 remained unchanged. Compared to only BL irradiated photoreceptors, the Bax and Caspase-9 protein levels decreased significantly after RL or NIRL treatment in mitochondria-rich IS. In contrast, anti-apoptotic Bcl-2 protein expression increased in IS upon RL or NIRL exposure (Figure 2A). Aside from the localization in the IS, Bcl-2 expression was observed in RL- or NIRL-treated retinas in retinal ganglion cells and in Müller cells (Figure 2A and Figure S3). In conformity with the immunohistochemical staining, analyzed western blots show a significantly lowered protein expression of Bax and Caspase-9 and increased protein expression of Bcl-2 after exposure to 810 nm NIRL and 670 nm RL (Figure 2B). Data indicate less mitochondria-induced apoptosis upon RL or NIRL treatment compared to BL damaged photoreceptors. Additionally, a terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay and Caspase-3 immunohistochemical staining support the observation of increased photoreceptor survival after RL or NIRL treatment (Figure S4). 2.2. Increased Respiration upon RL/NIRL Exposure To study the activity of oxidative phosphorylation (OXPHOS), we examined photoreceptors after RL and NIRL exposure regarding respiration. A histochemical assay on unfixed sections was performed to determine the activity and the localization of the OXPHOS complexes. Complex I (NADH-CoQ oxidoreductase) and II (Succinate dehydrogenase, SDH) were chosen, as these represent the starting points of the two OXPHOS pathways (complex I+III+IV or complex II+III+IV). While OXPHOS is closely associated with mitochondria, recently, functional extramitochondrial complexes have also been described within outer segments (OS) that are devoid of mitochondria. Thus, OXPHOS activity was expected in the mitochondria-rich IS as well as in the OS of photoreceptors [34]. Upon BL irradiation the activity of complex I decreased drastically by 0.40 in the mitochondria rich IS and by 0.42 in OS, respectively, related to normalized control. Complex II activity was reduced similarly by 0.40 in IS and by 0.52 in OS. In contrast to only BL exposed samples, the BL+RL and BL+NIRL treated photoreceptors restored their enzyme activity close to control levels (Figure 3A). Increased complex activities upon RL or NIRL exposure were observed in the mitochondria-rich IS but also in the OS. Upon 670 nm RL exposure Complex I activity increased in IS to 0.95 and in OS to 0.97. After 810 nm NIRL exposure, Complex I activity increased in IS to 0.94 and in OS to 0.88. Complex II activity was restored as well, being increased to 0.89 in IS and to 0.88 in OS upon 670 nm RL exposure and to 0.88 and to 0.86 after 810 nm NIRL exposure in IS and OS, respectively, related to controls. In addition to the histochemical investigation, OXPHOS levels were further analyzed by measuring oxygen consumption on isolated OS. The OXPHOS pathway I+III+IV was addressed by stimulation with fumarate and the OXPHOS pathway II+III+IV by adding succinate. RL or NIRL stimulation recovered the BL lowered oxygen consumption, indicating an increased flux through the electron transport chain (Figure 3B). Oxygen consumption increased by 84% for complex I and by 94% for complex II, after irradiation with 670 nm RL compared to non-irradiated control (Figure 3B).PDF Image | Photobiomodulation Mediates Neuroprotection against Retinal
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