Abstract
Purpose::
To determine the effects of EDTA, which disrupts corneal endothelial cell contact inhibition, on cell cycle progression and endothelial cell density (ECD) in the central and peripheral cornea.
Methods::
Human donor corneoscleral rims (3 pairs, donor ages: 15, 49 and 56 years) were obtained from the National Disease Research Interchange (Philadelphia, PA). Each corneoscleral rim was divided into 4 or 8 pie-shaped wedges which were pre-incubated at 37°C for 1 day in Medium-199 with fetal bovine serum, growth factors, and gentamicin. Samples were treated for 1 hour with EDTA 0.5, 2.5 and 5.0 mM or control medium. Samples were then either processed for ZO-1 immunolocalization or, to examine cell cycle progression and ECD, were returned to culture for 2 days before immunolocalizing for Ki-67 and staining with propidium iodide or DAPI. Images were obtained from 3 adjacent 20X-objective fields (regions 1, 2 and 3), centered between the cut edges and extending radially from the corneal center (region 1, closest to the cut edges at the tip of the wedge) to the periphery (region 3, farthest from the cut edges). Cell nuclei and Ki-67 positive cells were counted in a 0.2 mm2 area of each region, and the percentage of Ki-67 positive (%Ki-67+) cells and ECD were calculated.
Results::
Confocal microscopy evaluating ZO-1 localization showed no disruption of endothelial cell contact with control and 0.5 mM EDTA treatment but showed progressively greater disruption with 2.5 and 5.0 mM EDTA. Cell contact disruption was uniform in each region. EDTA did not increase %Ki-67+ cells (1.5 ± 1.4%, 2.1 ± 3.5%, 1.0 ± 1.2%, and 2.0 ± 2.8% with control, 0.5 mM, 2.5 mM, and 5.0 mM EDTA, respectively) and had little effect on ECD (2379 ± 634 cells/mm2, 2579 ± 810 cells/mm2, 2931 ± 561 cells/mm2, and 2578 ± 821 cells/mm2 with control, 0.5 mM, 2.5 mM, and 5.0 mM EDTA, respectively). Regardless of treatment, region 1 had lower ECD and higher %Ki-67+ cells than region 3 (ECD in regions 1 and 3: 2157 ± 667 cells/mm2 and 2897 ± 653 cells/mm2, p=0.015; %Ki-67+ in regions 1 and 3: 4.1 ± 4.7% and 0.5 ± 0.9%, p=0.039). Regions with no Ki-67 positive cells had greater ECD (3270 ± 494 cells/mm2) than those with any Ki-67 positive cells (2188 ± 549; p<0.001). The Ki-67 positive cells noted at the cut edge of all samples served as an internal positive control.
Conclusions::
EDTA causes disruption of endothelial cell contact but does not appear to promote endothelial cell proliferation. Regardless of treatment, greatest cell cycle progression is noted in region 1, which is closest to the cut edges of the sample. This region also has lower ECD, which might be attributed to cell loss or migration associated with the cut edges of the sample.
Keywords: cornea: endothelium • cornea: basic science