Understanding the mechanisms of photobiostimulation on mitochondrial bioenergetics: the challenges of respirometry with real-time irradiation at 635 nm

Abstract

Mitochondria are proposed to be the main target of photobiomodulation (PBM) at the cell level, where photon absorption would lead to therapeutic results. However, there is still plenty to be explored and understood about the effects of photons on mitochondria. This study investigates photon interactions at 635 nm on mitochondrial bioenergetics measuring mitochondrial respiration through high-resolution respirometry (HRR) techniques. Mice (C57BL/6 females) liver mitochondria were isolated and immediately irradiated with a LASER beam emitting at 635 nm while measuring mitochondrial respiration. The selected protocol for respiration analysis allowed the evaluation of oxidative phosphorylation, non-oxidative phosphorylation and the maximal capacity of electron transport chain. The following experimental conditions were applied: temperature (37 and 28 °C), sample concentration (0.5 and 0.25 mg.mL -1 ) and LASER power intensities (200 to 800 mW). At 37 °C and 0.5 mg.mL -1 , the effects were small, but indicated that higher powers might be more promising, and subsequent experiments followed with power intensities ranging from 600 to 800 mW. At 28 °C and 0.25 mg.mL -1 there was a photothermal influence of irradiation, interfering with the oxygen consumption measurements. These results entail the search for a way to correct such effect, aiming to bring the experimental conditions closer to those idealized for this study, that proposes to measure light effects in real time.