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L. Chen, J. Zhao, T. Nagasaki; Role of Non-Limbal Epithelial Cells on Corneal Epithelial Homeostasis. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3375. doi: https://doi.org/.
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We have shown previously that central corneal epithelial cells of a young mouse, 6 weeks or younger, could sustain a normal, clear cornea for more than a year. In this study, we examined the same in adult mouse corneas, to determine the importance of central, non-limbal epithelial cells in maintenance of epithelial homeostasis.
Limbal epithelial cells were removed completely by mechanical scraping in ubiquitous GFP mice aged 11 to 26 weeks. Right eyes were used for injury and left eyes as untouched control. Corneal health was monitored for the life of the animals, up to two years, by observing transparency, vascularization, and centripetal epithelial cell movement (time-lapse GFP fluorescence microscopy). In some mice, a central epithelial scrape injury was made and healing rates were determined in both limbus-removed cornea and untouched cornea. Expression of keratin 12 (cornea marker) and keratin 15 (conjunctiva marker) was determined by whole-mount immunofluorescence.
A technique for complete removal of limbal epithelial cells was verified by thorough examination of DAPI-stained histological specimens that were prepared several hours after the injury. When the limbal epithelium was removed along a half of the circumference in 6 eyes, all corneas healed and remained clear and avascular. When the limbal epithelium was removed along the entire circumference in 20 eyes, 13 healed without incident and remained clear and avascular with normal centripetal epithelial cell movement. Three eye showed increased vasculature in the peripheral cornea, which persisted but did not grow into the central cornea. Four eyes developed a central scar and a bundle of blood vessels that grew radially from a small zone of the limbus to the scar. Healing rates of a central epithelial defect, examined more than 50 weeks after the limbal injury, were similar between the limbus-removed right eye and the untouched left eye. Histology showed that corneas that healed without incident were free of keratin 15.
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