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

Abstract

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&#xa0;nm, 10&#xa0;mW) exposure for 3&#xa0;minutes per session, 6 sessions daily, over 8&#xa0;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 &#xb1; 54.32 vs. 5.06 &#xb1; 2.99 cells/section, P&#xa0;=&#xa0;0.002). Analysis of apoptosis-related proteins revealed upregulation of Bax protein (P&#xa0;<&#xa0;0.05), downregulation of Bcl-2 mRNA (P&#xa0;<&#xa0;0.05), and an increased Bax/Bcl-2 mRNA ratio (P&#xa0;<&#xa0;0.01). Pro-Caspase-9 protein levels decreased (P&#xa0;<&#xa0;0.05), whereas Caspase-3 gene expression was elevated (P&#xa0;<&#xa0;0.001). The RPE layer showed disrupted continuity with RPE65 expression reduced within 1000&#xa0;&#xb5;m of the optic nerve (1.00 &#xb1; 0.13 vs. 0.69 &#xb1; 0.07, P&#xa0;<&#xa0;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.