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H. Wang, I.K. Sugino, M.A. Zarbin; Contribution of Cell Proliferation to Retinal Pigment Epithelium Wound Healing on Human Bruch's Membrane Explants . Invest. Ophthalmol. Vis. Sci. 2003;44(13):476.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine whether cell proliferation contributes to retinal pigment epithelium (RPE) resurfacing on the RPE basement membrane and inner collagenous layer (ICL) in aged human submacular Bruch's membrane explants. Methods: Debridements were created mechanically (using a nylon/plastic loop) in RPE/choroid/sclera explants obtained from paired donor eyes. Debridements exposing RPE basement membrane (RPEbm(+)-defects) were created in one eye, whereas debridements with the RPE basement membrane and superficial ICL removed exposing the deeper ICL (DICL-defects) were created in the fellow eye. The explants were followed for 10 days by visualizing RPE ingrowth with DAPI filters. At day-10, each explant was bisected after light fixation in 3.7% paraformaldehyde. To detect the proliferating RPE cells, half of the explant was treated with an antibody against Ki-67 nuclear antigen (monoclonal MIB-1, Molecular Probes) followed by incubation in Cy5 conjugated donkey anti-mouse IgG (Jackson Immuno Laboratories). The other half was used as a negative control with the primary antibody omitted. The specimens were then observed by confocal microscopy before further fixation in 1/2-strength Karnovsky's fixative. Thereafter the specimens were processed for scanning electron microscopy to confirm the debridement. Results: RPE cells at the original wound edge started to enlarge and migrate into the defect at day-2 to day-4. At day-10, all defects exhibited some degree of RPE resurfacing with better resurfacing seen in the RPEbm(+)-defect. In both types of defects, proliferating RPE cells were observed at the original wound edge, at the resurfacing leading edge, and in the zone between these two (transition zone). Most proliferating cells were present in the transition zone in both conditions. Conclusions: In addition to cell spreading and migration, cell proliferation contributes to RPE resurfacing on both RPE basement membrane and inner collagenous layer in human submacular Bruch's membrane explants.
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