Abstract
Purpose: :
To examine whether nuclear morphology could be used as a marker to classify ocular surface epithelial cells and to eventually identify epithelial stem cells.
Methods: :
For histology, nuclei were stained in a flat whole-mount specimen with DAPI. For an in vivo study, histone H2B-EGFP mice (Jackson Laboratory Stock 006069) were used to image epithelial nuclei. Nuclear outline of basal epithelial cells was manually drawn and digitized for further morphometric analyses with ImageJ. In some mice, a circular epithelial scrape injury was made in the limbus and nuclear morphology was determined in this area before injury and after the completion of the wound healing. As the abnormal limbus with abnormal stem cell distribution, eyes of Dstncorn1 mice were examined, with A/J mouse eyes serving as the normal control. Mitotic rates were determined by a systemic 24-hour continuous labeling with 5-ethynyl-2'-deoxyuridine.
Results: :
A preliminary study focused on four morphometric features: area, Feret's diameter (caliper length), circularity, and aspect ratio. These feature could distinguish a group of cells from the cornea, the limbus, and the conjunctiva when an average value of each population was compared. Similarly, when the data were plotted sequentially from low to high, a distribution curve of a population of cells (>1000 cells) was distinct among the cornea, the limbus and the conjunctiva. The curve also indicated that individual cells within a population could be distinguished. Nuclear area was positively correlated to Feret's diameter and circularity while negatively correlated to aspect ratio. Morphological patterns in a live eye were similar to those of a fixed eye. In eyes following epithelial scrape injury and subsequent healing, morphometric features were no longer limbal phenotype. In Dstncorn1 eyes, nuclear morphometry failed to distinguish limbal cells from corneal cells. Mitotic rates in the limbal area were lower immediately after completion of epithelial healing; they were higher than control in Dstncorn1 eyes. In both instances stem cell distribution is likely to be altered substantially compared with the normal eye.
Conclusions: :
Morphometric features of basal epithelial nuclei can distinguish a population of limbal cells from that of corneal cells and conjunctival cells. These features can also distinguish individual cells within the limbus. These results raise a possibility that morphometric parameters of nuclear shape, combined with other techniques, could assist in identification of individual stem cells in a living eye.
Keywords: cornea: epithelium • microscopy: light/fluorescence/immunohistochemistry • wound healing