The significance of photobiomodulation in formation of membrane potential of brain mitochondria in normoxia and after hypoxia in mice
Authors: Shchelchkova N. A. , Pchelin P. V. , Shkarupa D. N. , Vasyagina T. I. , Bavrina A. P. 
Published: May 2024

Photobiomodulation using low-intensity red light (LRL) is considered a safe, non-invasive, and cost-effective method that was proven to possess stimulating, restorative, and rejuvenating effects on body tissues. The therapeutic potential of photobiomodulation was demonstrated in various pathologies such as Alzheimer’s and Parkinson’s diseases and ischemic brain damage [1–3]. The potential photoacceptance of radiation by ETC’s complex IV (CIV) raises concern for the impact of LRL on mitochondria. However, ATP synthesis in mitochondria depends less on their functional state and more on the high electrical potential of coupled mitochondria. This study aimed to investigate the importance of photobiomodulation for the formation of brain mitochondria membrane potential in healthy mice and after hypoxia.
Male C57BL/6 mice were used in the study. The animals were divided into two groups: a healthy control group (n=20) and a group of animals exposed to simulated hypobaric hypoxia (n=20). Half of the control animals (n=10) and half of the animals subjected to hypoxia modeling (n=10) received a single transcranial exposure of LRL (Spectr LC-02, Russia), which had a wavelength of 650±30 nm, for 3 minutes. After 24 hours, the mitochondrial fraction of the left cerebral cortex of the brain was isolated. The resulting fraction was used to examine how the mitochondrial membrane potential (ΔmtMP) dynamically changes by employing the O2k-Fluorescence LED2 amperometric module of the Oroboros Oxygraph-2k respirometer (Oroboros Instruments, Austria) and the fluorescent dye tetramethylrhodamine methyl ester. The collected data were normalized for protein content using the Bradford method. Statistical analysis was conducted with GraphPad Prism 8 and Excel.
When investigating the impact of transcranial administration of LRL on ΔmtMP during CI-supported (CI, NADH-ubiquinone oxidoreductase) oxidative phosphorylation of the left cerebral cortex mitochondria in control animals, an increase of 18% was observed for the parameter. Further, a 40% increase was noted when studying CII-supported (CII, succinate dehydrogenase) oxidative phosphorylation compared to the untreated group. During the evaluation of basal respiration in the untreated control group, the measurement of ΔmtMP was 0.052±0.002 arb. units It was found that the transcranial application of LRL in mice caused a 2-fold increase of ΔmtMP (0.115±0.010 arb. units).
Simulation of hypobaric hypoxia results in a 20% decrease in ΔmtMP during CI-supported oxidative phosphorylation but has no effect on ΔmtMP during CII-supported oxidative phosphorylation. Basal respiration after hypoxia modeling showed a 33% decrease in ΔmtMP compared to control values (0.052±0.002 arb. units and 0.035±0.003 arb. units, respectively).
The transcranial administration of LRL following hypoxia modeling did not alter the dynamics of membrane potential during CI- and CII-supported oxidative phosphorylation, yet considerably amplified ΔmtMP when evaluating basal respiration.
The transcranial LRL irradiation stimulated the healthy control group, resulting in an increase in ΔmtMP for both CI- and CII-supported oxidative phosphorylation and basal respiration. This increase in coupling between oxidation and phosphorylation processes was observed. However, after hypoxia modeling, the photobiomodulation effect of LRL was only observable under basal respiration conditions. The effects of the LRL application align with findings from other studies that suggest an elevation in ΔmtMP and the creation of ATP resulting from the dissociation of NO and the binuclear center of CIV [4].