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D. L. Harris, C. Zhu, N. C. Joyce; Detection of Oxidative DNA Damage in Human Corneal Endothelium. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2703.
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To determine whether oxidative DNA damage increases in an age-dependent manner in human corneal endothelial cells (HCEC).
Human corneas from donors younger than 30 years old and older than 50 years old were obtained from the NDRI and formed 2 age comparison groups. Some corneas were separated into central and peripheral areas using 6.0 and 9.0 mm diameter punches, respectively. Others were used for primary culture of the entire endothelium rather than specific topographic areas. Cells were cultured following published protocols. Immunolocalization studies were performed with an antibody that specifically binds 8-hydroxy-2’-deoxyguanosine (8-OHdG), a known marker of oxidative DNA damage. Negative controls used secondary antibody only. Fluorescence confocal microscopy visualized positive staining. Single laser images taken at the same relative tissue depth were compared in the ex vivo corneas. Conventional fluorescence microscopy visualized staining in cultured cells.
In ex vivo corneas, differences in both age and topographical localization were noted. Central endothelium from older donors appeared more intensely stained than that of young donors, although relative intensity differed from cell to cell. Many cells in the central region from older donors showed a compact, intense nuclear staining, indicating oxidative damage to nuclear DNA. In other cells there was relatively equal intensity in both the nucleus and cytoplasm. The cytoplasmic pattern suggested staining of mitochondrial DNA. Positive staining was also found in peripheral cells from both age-groups, but overall relative intensity appeared higher in HCEC from older donors. In peripheral cells, cytoplasmic staining appeared more intense than nuclear staining, suggesting a higher level of mitochondrial vs. nuclear DNA damage. In cultured cells from young donors, the majority of positive staining was observed in the perinuclear cytoplasm. In cells from older donors, nuclei were intensely positive, while the cytoplasm appeared less intense.
Overall, positive staining for 8-OHdG was observed in HCEC, both in ex vivo corneas and in culture. Comparison of staining intensity suggests an age-related increase in total DNA damage. Cytoplasmic staining suggests the presence of oxidative damage to mitochondrial DNA, particularly in peripheral endothelium in both age-groups. The intense nuclear stain in central endothelium from older donors strongly suggests higher levels of oxidative damage to nuclear DNA in these cells.
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