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J. Zhao, T. Nagasaki; Nodular structures in the corneal epithelium near the limbus of normal mouse eye. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1434.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:To characterize novel perilimbal epithelial structures in the normal mouse cornea. Methods: Flat whole–mounts of ocular surface from FVB and ICR mice were studied histologically. Various fluorescent markers were used to characterize perilimbal epithelial nodules. DNA synthesis was determined by metabolic labeling with BrdU. Developmental appearance of nodules was studied with FVB mice. TIE2–GFP mice, in which GFP expression was driven by a tie2 promoter, were used to examine tie2 expression. Results:Gross observation revealed no abnormal features in experimental eyes. DAPI nuclear staining of whole–mount ocular surface demonstrated distinct nuclear profiles between corneal basal epithelial cells and limbal basal epithelial cells, which enabled us to clearly define an epithelial boundary between the cornea and the limbus. The DAPI stain further revealed the presence of epithelial nodular structures at or just inside the cornea–limbus boundary along the entire circumference. Cells that make up the nodules could be distinguished from adjacent cells by: a) smaller cell size and higher cell density, b) brighter nuclear staining with DAPI or Hoechst 33342, in both a live and a fixed cornea, c) elevated expression of GFP in TIE2–GFP mice, d) lack of lamin A immunostaining, e) lack of, or infrequent, DNA synthesis, f) compromised plasma membranes as judged by penetration of ethidium homodimer–1, g) increased staining with wheat germ agglutinin or Helix pomatia lectin in some cells. Cells in the nodules resembled limbal epithelial cells more than corneal cells in two aspects: prominent nucleoli and weaker connexin 43 immunostaining. Developmentally, a sign of nodule formation was noted first at 3 weeks after birth, when a small ring–like structure of several cells emerged near the limbus. The number of nodules increased until the mouse reached maturity and their size varied widely from several to several dozens of cells. Some nodules formed a shape of a gourd with its broad base resting on the cornea–limbus boundary and a narrow neck extending toward the central cornea, perhaps an indication of centripetal movement. Along with the nodules, several goblet–like cells were identified at the cornea–limbus boundary. They appeared nearly identical to the conjunctival goblet cells, forming a cluster of several cells containing a small nucleus at a basal region and a large cytoplasm with vacuoles on top. Conclusions:Perilimbal epithelial nodules appear to represent aberrant differentiation of limbal stem cells or their progenies. Further analysis of these structures may assist understanding stem cell differentiation and homeostasis of ocular surface epithelium.
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