Multimodal safety assessment of repeated 650-nm red-light irradiation in chick retina.

PURPOSE: To systematically evaluate the dose-dependent ocular safety profile and biological mechanisms of 650 nm repeated red-light (RL) therapy using the chick model. METHODS: Chicks received monocular 650 nm red-light exposure. Dosimetry was strictly defined as corneal irradiance (power density measured at the corneal surface) under two regimens: a dose-escalation regimen (1, 5, 10, 15 mW/cm2 for 7 days) and a mid-term (5 mW/cm2 for 30 days) regimens (n = 7 per group). Safety assessments included axial length measurements, in vivo macroscopic imaging via swept-source optical coherence tomography and angiography (SS-OCTA), in vivo full-field electroretinography (ffERG), high-resolution ex vivo cellular imaging via full-field OCT (ffOCT), histology/TUNEL assay and untargeted metabolomic profiling coupled with computational toxicology screening. RESULTS: Physiological axial growth was maintained across all irradiances. Retinal architecture, photoreceptor integrity and retinal ganglion cell survival remained intact, with no apoptotic signaling detected. SS-OCTA revealed a significant dose-dependent increase in the choroidal vessel volume per unit area (CVV/a) without accompanying changes in choroidal thickness or vascular index, indicating physiological vascular modulation rather than structural changes. Dual mode of ffOCT demonstrated preserved photoreceptor and retinal ganglion cell layer densities with stable intracellular metabolic motility, ruling out subcellular damage or metabolic stress. ffERG parameters remained stable across all treatment groups. Metabolomic profiling indicated adaptive shifts toward anabolic and bioenergetic pathways, while computational screening detected no enrichment of photochemical or redox-active risk signatures with dose escalation. CONCLUSIONS: The chick retina demonstrates a wide biological safety margin for 650 nm repeated red-light exposure, tolerating both supraphysiological irradiance and extended treatment duration without structural, functional or molecular evidence of photochemical injury. These preclinical findings provide evidence evaluating the biological safety margin of repeated 650 nm red light therapy in the retina.