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R. Joseph, R.R. Pfister, C.I. Sommers; Expression of K3 Keratin in Alkali Treated Rabbit Corneas . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2755.
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In 2002 there were 17.4% chemical injuries of the eye with the majority being alkali–based compounds. In our established rabbit model of corneal injury we have previously described a phenomenon characterized by the migration of corneal epithelium over the severely alkali–injured corneal stroma up to 72 hours, but then the leading edge loses its adhesion at 84 hours and rolls back like a rug at 96 hours after alkali exposure. This phenomenon is observed even in the presence of normal stem cell source in the cornea. However, no molecular explanation for the persistence of this epithelial defect has been advanced.
Rabbits were divided into 2 groups of 30 each. In group 1, the cornea was exposed to 1N NaOH in an 11 mm well for 35 seconds. In group 2, the central cornea was marked with a 11 mm trephine, then abraded. Rabbits were randomly assigned to 5 time periods to be sacrificed at 48 hours, 72 hours, 84 hours, 96 hours and 114 hours post injury. At the designated time, 6 rabbits from each group were killed and the globe removed. The cornea was trephined, and the tissue layers separated for analysis. The epithelial proteins were isolated and extracted in buffer containing 3 M urea and non–ionic detergents. The insoluble fraction was dissolved in presence of 1% SDS and then separated on 10% SDS polyacrylamide gel. Peptide mass finger printing of the coomassie blue–stained bands were performed by MALDI–TOF mass spectrometry.
MALDI–TOF analysis demonstrated the gradual loss of two specific proteins, keratin 3 and keratin 12 in alkali–treated corneas 84 hours after the treatment when compared to 48 hours after injury. But the keratin 3 and keratin 12 was present in normal and abraded corneas during 48 hours and 84 hours after injury. Keratin 3 and keratin 12 are cornea specific keratins, and are known to be responsible for the integrity of the epithelium. The time course of the loss of the keratins is consistent with the shedding off the corneal epithelium.
Keratins and other cytoskeletal proteins participate in the formation of desmosomes and hemidesomes. The K3/K12 keratin pair is one of the major constituents of differentiated and stratified corneal epithelium. Alkali treatment resulted in the loss of K3/K12 keratin pairs after 84 hours of alkali treatment. This would possibly play a role in the loss of corneal integrity and also in the detachment of epithelium from the basement membrane.
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