Effect of Repeated Medium-Dose Red Laser Irradiation on Ocular Tissues in Guinea Pigs

Purpose: The purpose of this study was to investigate the effects of repeated red laser irradiation at a supraclinical dose on histology and apoptosis-related molecular markers in guinea pig ocular tissues. Methods: Guinea pigs (1 month old) were randomized into two groups: the repeated irradiation group and the control group. After pupil dilation, the repeated irradiation group received binocular red laser (650 nm, 10 mW) exposure for 3 minutes per session, 6 sessions daily, over 8 days. Ocular surface morphology was examined using slit lamp microscopy. Retinal histopathology was assessed by hematoxylin and eosin (H&E) staining, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, immunohistochemistry, and immunofluorescence. Protein and mRNA expression of apoptosis-related markers (Caspase-3, Caspase-9, Bax, and Bcl-2) were quantified by Western blotting and quantitative polymerase chain reaction. Results: Repeated red laser exposure-induced lesions localized to the retina temporal to optic nerve, characterized by outer nuclear layer (ONL) pyknosis, photoreceptor inner/outer segments degeneration, and retinal pigment epithelium (RPE) disruption. TUNEL staining revealed a significant increase in apoptotic cells within the ONL of irradiated eyes compared with controls (84.33 ± 54.32 vs. 5.06 ± 2.99 cells/section, P = 0.002). Analysis of apoptosis-related proteins revealed upregulation of Bax protein (P < 0.05), downregulation of Bcl-2 mRNA (P < 0.05), and an increased Bax/Bcl-2 mRNA ratio (P < 0.01). Pro-Caspase-9 protein levels decreased (P < 0.05), whereas Caspase-3 gene expression was elevated (P < 0.001). The RPE layer showed disrupted continuity with RPE65 expression reduced within 1000 µm of the optic nerve (1.00 ± 0.13 vs. 0.69 ± 0.07, P < 0.001). Conclusions: Through repeated red laser irradiation at supraclinical dose and frequencies, this study identified the vulnerable targets of red laser in the fundus, demonstrating damage to the ONL, photoreceptor inner/outer segments, and RPE, whereas no adverse effects were observed on anterior segment structures including the cornea and lens.