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PM Ladage, WM Petroll, JP G Bergmanson, JV Jester, HD Cavanagh; 3-D Location of BrdU-labeled Epithelial Cells Following Overnight RGP Lens Wear in Rabbit Cornea . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3079.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To study three-dimensionally the effects of overnight RGP lens wear on the proliferative patterns of rabbit corneal epithelial cells. Methods: Five NZW rabbits were unilaterally fitted with a medium oxygen transmissible RGP contact lens (CL) (Dk/t=27); the contra-lateral eye served as control. 24 hours later, 5-bromo-deoxyuridine (BrdU, 200mg/kg) in PBS was injected IV at 9:00 AM to label dividing corneal epithelial cells. Animals were sacrificed 24-hours post injection and whole-mount corneas were immunostained with anti-BrdU antibodies and counterstained with Propidium Iodide. To assess the spatial location and distribution of BrdU-labeled cells in the central corneal epithelium, four 3-D images of the full-thickness epithelium were obtained by laser confocal microscopy (238 x 238 x 35-45µm). In addition, epifluorescence microscopy was used to collect sequential images from the superior to inferior limbus to determine the regional proliferation rates of both groups. Results: The mean proliferation rate of the central basal corneal epithelium in the control was 108±39 BrdU-labeled cells/0.578mm2 while the CL wearing group was significantly lower with 36±21 BrdU-labeled cells/0.578mm2 (-66%, P=0.019, paired T-test) . 24-hours after BrdU-injection, 70% of all BrdU- labeled cells in the control group were localized in the basal cell layer versus 72% for the CL group (NS). Approximately 91.6% (control) and 92.5% (CL) of all BrdU-labeled cells were organized in horizontal pairs while 4.4% (control) and 3.9% (CL) showed a vertical orientation (NS). Only 3.9 (control) and 3.5% (CL) of the BrdU-cells were not in pairs (NS). Conclusions: Previously we have shown that short-term wear of low and hyper Dk/t RGP lenses suppresses central epithelial proliferation by respectively 80% (Dk/t=10) and 40% (Dk/t=97). These findings combined with the result of this study suggest an inverse relationship between oxygen lens transmissibility and suppression of the proliferation rate in the central corneal epithelium. Orientation (horizontal vs. vertical) of BrdU-labeled cells was the same in both control and CL group; and, the ratio of cells in the basal cell versus supra-basal cell layers was also identical in both groups. Proliferation inhibition during CL wear appears to be related to a decrease in cells entering the cell cycle or passing the G1 checkpoint of the cell cycle; if the proliferation suppression was related to a delayed S-phase more single labeled cells in the CL group as compared to control should have been found.
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