Fig. 5

Antioxidants (AO) protect OTCs from phototoxic damage during chronic photostimulation with blue (450 nm) and green (505 nm) light. (a) Propidium iodide (PI) staining was used to detect phototoxic damage or cell death in OTCs after 14 h of strong photostimulation (100 pulses/minute at 12.5 Hz) using 450 nm or 505 nm LEDs. For the stimulation, cultures were placed inside a custom-built stimulation box that was controlled by a Raspberry Pi computer. Each individual membrane insert containing ChR2-transduced OTCs was illuminated with an LED module from below. (b, d) PI-positive cells were detected in the granule cell layer (GCL), hilus, and the CA areas of stimulated OTCs. (c, e) The addition of AO to the incubation medium dramatically reduced the amount of cell damage or death. (f) A quantification of the PI signal showed that cell damage was most pronounced in the 450 nm condition compared to the unstimulated control (two-way robust ANOVA with post hoc Brunner-Munzel tests, P = 0.020) as well as compared with cultures that were stimulated with 505 nm LEDs (P = 0.020). Furthermore, OTCs that were stimulated with either wavelength and treated with AO contained a significantly lower number of PI-positive cells compared with stimulated cultures that were not incubated with AO (450 nm vs. 450 nm + AO: P = 0.016; 505 nm vs. 505 nm + AO: P = 0.043). Contr.: n = 5; Contr. + AO: n = 6; 450 nm: n = 5; 450 nm + AO: n = 8; 505 nm: n = 5; 505 nm + AO: n = 11. n represents number of OTCs. Bars represent means ± SEM. *P < 0.05.