In this study the influence of hydrogen peroxide (H O ) on the redox state, NADH protein binding, and mitochondrial membrane potential in Müller cells is investigated. Cultures of permanent human Müller cells MIO-M1 were exposed to H O in 75 µM and 150 µM concentration for two hours. Fluorescence emission spectra and lifetimes were measured by two-photon microscopy (excitation wavelength: 740 nm) at the mitochondria which were identified in the microscopic images by their fluorescence properties (spectra and intensity). Two hours of H O exposure did not impair viability of MIO-M1 cells in culture. Whereas the ratio of flavine- to NADH fluorescence intensity did not change under either H O concentration, the mean lifetime was significantly different between controls, not exposed to H O , and the 150 µM H O exposure (972 ± 63 ps vs. 1152 ± 64 ps, p = 0.014). One hour after cessation of the H O exposure, the value retuned to that of the control (983 ± 36 ps). A hyperpolarization of the mitochondrial membrane under 150 µM H O was found. These findings suggest a shift form free to protein-bound NADH in mitochondria as well as a hyperpolarization of their inner membrane which could be related to an impairment of Müller cell function despite their preserved viability. Exposure of human Müller cells to hydrogen peroxide for two hours results in a reversible change of protein binding of mitochondrial NADH upon unchanged redox ratio. The mitochondrial membrane potential is increased during exposure.

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