660-nm photobiomodulation modulates redox-metabolic responses after neuronal anoxia

Anoxia/reoxygenation injury is associated with oxidative imbalance, metabolic dysfunction, and activation of hypoxia-responsive pathways. We investigated whether 660 nm photobiomodulation (PBM) modulates these responses in an in vitro model of neuronal anoxia using murine hippocampal mHippoE-18 cells. As this study used an established cell line, ethical committee approval was not required. Cells were seeded at 5×10⁴ cells/well and subjected to anoxia in an atmosphere containing 95% N₂ and 5% CO₂, followed by 24 h of incubation. Immediately after anoxia, cells received 660 nm PBM for 60 s, at an irradiance of 50 mW/cm² and a fluence of 3 J/cm². Twenty-four hours after PBM, metabolic activity was assessed using resazurin, reactive oxygen species (ROS) were measured using H₂DCFDA, adherent cell biomass was evaluated by crystal violet staining, nuclear abundance was assessed by 4′,6-diamidino-2-phenylindole (DAPI) staining, and hypoxia-inducible factor 1-alpha (HIF-1α) expression was evaluated by immunofluorescence in independent experiments. To account for differences in cell abundance among groups, resazurin and ROS signals were additionally normalized to the mean nuclear abundance of DAPI-positive cells. Anoxia markedly reduced the abundance of DAPI-positive nuclei and increased metabolic and oxidative signals relative to the remaining cell population. PBM shifted both indices toward control levels: the resazurin/DAPI ratio decreased from 1.47 to 1.08, whereas the ROS/DAPI ratio decreased from 1.61 to 1.24, corresponding to reductions of approximately 27% and 23%, respectively. HIF-1α assessment further complemented the characterization of the hypoxic response and its potential modulation by PBM. Taken together, these findings indicate that PBM may attenuate the redox-metabolic overload associated with the post-anoxic period and modulate cellular mechanisms related to the hypoxic response.