Assessment of mitochondrial dysfunction and epithelial-mesenchymal transition ofretinal pigment epithelial cells induced by nanosecond laser irradiation

Abstract

Background. Age-related macular degeneration (AMD) is a chronic retinal disease leading to irreversible central vision loss. Retinal pigment epithelium (RPE) dysfunction and death are central to AMD pathogenesis. The 2RT nanosecond laser is a promising treatment for early AMD, but its biological safety at the RPE cell level remains insufficiently studied. The aim. To assess nuclear DNA damage, mitochondrial activity and epithelial-mesenchymal transformation of human RPE cells after nanosecond laser exposure in an in vitro experiment.
Materials and methods. Primary RPE cell cultures were obtained from 20 human donor eyes (10 with blue and 10 with brown irises). Cells were exposed to a 2RT nanosecond laser (527 nm, 3 ns, 200 µm spot) with energy ranging from 0.10 to 0.60 mJ. DNA damage was assessed by comet assay at 24 hours. Mitochondrial activity was assessed by MTT assay at 24, 48, 72 hours and 7 days. Expression of EMT markers (vimentin, α-SMA, ZO-1, RPE-65) was assessed by immunocytochemistry at 7 days.
Results. For cells from blue-eyed donors, subthreshold regimens (0.10–0.35 mJ) did not induce DNA damage (tail DNA <5%), maintained mitochondrial activity at ≥90% of control and did not induce EMT. For cells from brown-eyed donors, the subthreshold range was narrower (0.10–0.30 mJ). At equal energy (0.40 mJ), DNA damage in brown-eyed donors was 2 times higher and mitochondrial activity reduction was more pronounced compared to blue-eyed donors.
Conclusion. Subthreshold 2RT laser regimens are biologically safe regarding DNA integrity, mitochondrial activity and absence of EMT induction. Cells from brown-eyed donors are more sensitive to laser exposure. A differentiated approach to laser energy selection based on RPE pigmentation is required.