Age and topographical comparison of telomere lengths in human corneal endothelial cells

Purpose: Human corneal endothelium exhibits both age-related and topographical differences in relative proliferative capacity and in senescence characteristics. The purpose of these studies was to compare telomere lengths in human corneal endothelial cells (HCEC) from the central and peripheral areas of corneas from young and older donors to determine whether these changes may be due to replicative senescence or to stress-induced premature senescence. Methods: Pairs of corneas from five young (<30 years old) and six older donors (>65 years old) were separated into central and peripheral areas using a 9.5 mm diameter trephine to remove scleral tissue and a 6.0 mm diameter trephine to mark the central-peripheral boundary. One of the pair of corneas was cut into quarters and stained with a peptide nucleic acid (PNA)/fluorescein isothiocyanate (PNA/FITC) probe that specifically binds to telomere repeats. HCEC from the central (0 - 6.0 mm) and peripheral areas (6.0 - 9.5 mm) were isolated from the second cornea, mounted on slides by Cytospin, and stained with the PNA/ FITC probe. Fluorescence confocal microscopy was used to obtain digital images. The average FITC intensity of nuclei was compared between the central and peripheral areas within and between the two age groups. Cc1185 and 1301 cells were analyzed as controls. Student's unpaired t-test was used to determine the statistical significance of the data. Results: Average FITC intensity from the central endothelium was 205.8±4.2 (younger) and 194.2±10.5 (older) and from the peripheral endothelium was 208.1:±9.3 (younger) and 195.9±lO.8 (older). Average intensity of single cells isolated from central endothelium was 113.9±31.1 (younger) and 107.9±26.1 (older) and from the periphery was 109.9±12.0 (younger) and 106.9±32.4 (older). Average FITC intensity of Ccl185 cells and 1301 cells was 50.5±5.0 and 206.9±19.4, respectively. Comparison of the results indicates no statistically significant difference between the central and peripheral areas within each group or between the young and older age group. Conclusions: Results indicate that the age-related and topographical reduction in relative proliferative capacity and senescence characteristics observed in HCEC are not due to replicative senescence caused by critically short telomeres but implicate stress-induced premature senescence as a cause of these clinically important changes.