Immunochemistry with 5-bromo-2-deoxyuridine for visualization of mitotic cells in the corneal epithelium

The purpose of this study was to determine whether the DNA of mitotic cells in the corneal and conjunctival epithelium can be labeled with 5-bromo-2-deoxyuridine (BrdU) immunostaining. Both corneas of four New Zealand white rabbits were deepithelialized in the center and the regenerated epithelium was evaluated for mitosis at 1, 3, 6, and 10 days. Unwounded corneas of three rabbits were labeled for baseline measurements. We administered the marker intravenously to all seven rabbits 15 h before scheduled killing. Immediately after killing, all of the globes were enucleated and histologic sections were prepared. In unwounded corneas, labeled cells were quantitated and the fraction of mitotic cells in the center of the cornea, in the periphery of the cornea, and in the conjunctiva were compared. In deepithelialized corneas, increase in mitosis in the central epithelium was quantitated. All of the unwounded eyes showed mitosis in the basal layer of both the corneal and conjunctival epithelium. In the center of the cornea 4.1 +/- 2.9% of the epithelial cells were labeled, in the corneal periphery 4.3 +/- 1.7% of the cells were labeled, and in the conjunctiva 4.1 +/- 1.9% of the epithelial cells were labeled, with a p value ranging from 0.84 to 0.99. In wounded corneas, when compared with cell counts in unwounded eyes, 52.6% of the epithelial cells were in mitosis on day 3, 13.9% were in mitosis on day 6, and by day 10 baseline values of 4.2% were obtained. We conclude that BrdU immunostaining is a safe, efficient, and less costly alternative to autoradiography for visualization of dividing corneal and conjunctival epithelium.