T Senoo1, Y Obara, N C Joyce. 1. Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, MA 02114, USA.
Abstract
PURPOSE: To determine whether it is possible to induce proliferation in the endothelium of older donor corneas by treatment of the intact monolayer with EDTA. METHODS: Corneas from donors 52 to 75 years of age were obtained from an eye bank and were usually cut in quarters to increase sample size. The effect of EDTA dose (0.02-2.0 mg/ml) and incubation time (6, 30, and 60 minutes) on endothelial cell-cell contacts was evaluated by staining for ZO-1, a cell junction marker. Cell death was tested by a commercial live-dead assay. Corneal pieces were incubated for 0, 24, 48, or 60 hours in culture medium (M-199, 10% fetal bovine serum, 10 ng/ml epidermal growth factor, 20 ng/ml fibroblast growth factor) before EDTA treatment. After treatment, pieces were incubated in the same medium for 24, 48, 72, or 96 hours to permit cell cycle entry. Tissue was fixed, stained for Ki67 (a marker for late G1-phase through the M-phase), and mounted in medium containing propidium iodide to visualize all nuclei. Confocal images were evaluated by computer (Image software; NIH, Bethesda, MD) to count Ki67-positive and propidium iodide-stained cells. RESULTS: EDTA released corneal endothelial cell-cell contacts in a dose- and time-dependent manner. At doses and incubation times tested, EDTA did not induce significant cell death. Preincubation in culture medium for 24 hours was needed for endothelial cells to efficiently initiate proliferation in response to EDTA. The endothelium of corneas incubated in mitogen-containing medium for up to 108 hours without EDTA treatment did not stain for Ki67. EDTA at 2.0 mg/ml for 60 minutes appeared optimal and stimulated 16% to 18% of the cells to proliferate. Ki67-positive mitotic figures were visible 48 hours after exposure to EDTA. Formation of daughter cells was visible after double-staining for Ki67 and ZO-1. CONCLUSIONS: EDTA released cells from contact inhibition and promoted proliferation in corneal endothelium from older donors. The authors hypothesize that corneal endothelium from older individuals divide in situ when exposed to positive growth factors under conditions in which cells have been transiently released from contact inhibition.
PURPOSE: To determine whether it is possible to induce proliferation in the endothelium of older donor corneas by treatment of the intact monolayer with EDTA. METHODS: Corneas from donors 52 to 75 years of age were obtained from an eye bank and were usually cut in quarters to increase sample size. The effect of EDTA dose (0.02-2.0 mg/ml) and incubation time (6, 30, and 60 minutes) on endothelial cell-cell contacts was evaluated by staining for ZO-1, a cell junction marker. Cell death was tested by a commercial live-dead assay. Corneal pieces were incubated for 0, 24, 48, or 60 hours in culture medium (M-199, 10% fetal bovine serum, 10 ng/ml epidermal growth factor, 20 ng/ml fibroblast growth factor) before EDTA treatment. After treatment, pieces were incubated in the same medium for 24, 48, 72, or 96 hours to permit cell cycle entry. Tissue was fixed, stained for Ki67 (a marker for late G1-phase through the M-phase), and mounted in medium containing propidium iodide to visualize all nuclei. Confocal images were evaluated by computer (Image software; NIH, Bethesda, MD) to count Ki67-positive and propidium iodide-stained cells. RESULTS:EDTA released corneal endothelial cell-cell contacts in a dose- and time-dependent manner. At doses and incubation times tested, EDTA did not induce significant cell death. Preincubation in culture medium for 24 hours was needed for endothelial cells to efficiently initiate proliferation in response to EDTA. The endothelium of corneas incubated in mitogen-containing medium for up to 108 hours without EDTA treatment did not stain for Ki67. EDTA at 2.0 mg/ml for 60 minutes appeared optimal and stimulated 16% to 18% of the cells to proliferate. Ki67-positive mitotic figures were visible 48 hours after exposure to EDTA. Formation of daughter cells was visible after double-staining for Ki67 and ZO-1. CONCLUSIONS:EDTA released cells from contact inhibition and promoted proliferation in corneal endothelium from older donors. The authors hypothesize that corneal endothelium from older individuals divide in situ when exposed to positive growth factors under conditions in which cells have been transiently released from contact inhibition.
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