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Int. J. Mol. Sci. 2020, 21, 2370 3 of 20 favorable as it combines the efficiency and control common to in vitro techniques with close imitation of the in vivo environment. Ex vivo models are common experimental designs to examine retinal tissue regeneration with a highly controlled setting, e.g., by the delivery of equal amounts of light to the retina by a constant distance [29,31,32]. We chose blue light (BL) irradiation as a damaging model of irradiated photoreceptors and analyzed cellular and molecular action of RL or NIRL after post-treatment [29,30]. The exposure to intense artificial light (e.g., white LED) with enriched emission spectrum in blue radiations and blue light is a risk factor causing photochemical damage. BL leads to oxidative stress in cells, at first observed in the photoreceptor cells. Due to their high content of mitochondria, they are especially susceptible to oxidative stress. Pathological effects of light-damaged photoreceptors are the structural degeneration of outer segments (OS), oxidative stress, lipid oxidation and cell death [29,30,33]. Altogether, we report for the first time a complex pattern of beneficial neuroprotective effects by red and near-infrared light on BL-damaged photoreceptors that are valuable for transferring experimental RL/NIRL therapy into clinical applications. 2. Results An established ex vivo BL retina damaging model that mimics photochemical damaging was used to evaluate the cellular and molecular mechanisms of RL and NIRL [29,30]. In order to be able to use ex vivo cultivation as a model of damage, it had to be ensured beforehand that cultivation itself does not cause significant morphological retinal damage. The issue was addressed by HE-staining of cultivated retina, resulting in an optimal total cultivation time of 9 h (Figure S1). This blue light model is especially characterized by oxidatively stressed photoreceptors [29,30]. Figure S2 in the supplement shows apoptotic cells in untreated and blue light damaged retinas, confirming that photoreceptors are the first cell type where oxidative stress due to blue light irradiation occurs, whereas retinal cells in the ganglion cell layer (GCL) and inner nuclear layer (INL) are not affected. To examine the impact of RL and NIRL stimulation on BL-irradiated photoreceptors, eyes were damaged with blue light followed by RL/NIRL treatment. Eyes were grouped regarding their exposure to light (Figure 1). Figure 1. Schematic set up of mouse eyes cultivation with light irradiation. (A) Eyes were grouped regarding their exposure to light as following; Control: Non-light-irradiated eyes; BL: Only blue light irradiated eyes; BL+NIRL 810 nm: blue light irradiation + near-infrared light postexposure 810 nm diode laser; BL+RL 670 nm: blue light irradiation + red light postexposure 670 nm LED. (B) Early event analysis after 40 min of cultivation. Eyes were treated for 0.5 h with BL, following 10 min NIRL or RL and direct analysis after light treatment. Only reactive oxygen species (ROS) analysis using CM-H2DCFDA vital dye was performed after 40 min of cultivation. (C) Late event analysis was performed 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.PDF Image | Photobiomodulation Mediates Neuroprotection against Retinal
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