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M. Hovakimyan, O. Stachs, A. Wree, A. Krüger, A. Heisterkamp, R. Guthoff; Combined Confocal and Immunohistochemical Examination of Rabbit Cornea After Crosslinking. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3066.
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© ARVO (1962-2015); The Authors (2016-present)
Crosslinking of the cornea by application of Riboflavin (Vitamin B2) and UV-light is an innovative approach to increase the mechanical and biochemical stability of the stromal tissue. The better understanding of corneal wound healing is very important for evaluation of possible risks after treatment. In the presented study we performed a combined in vivo confocal laser scanning and histological investigation in rabbit corneae after crosslinking
12 New Zealand white rabbits were enrolled. Animal experiments were performed in compliance with the ARVO resolution on the use of animals in Ophthalmic and Vision Research. Before crosslinking confocal microscopy was used to examine all corneae. The right corneae were deepithelized and crosslinked using UV-light (wavelength 370 nm) exposition for 30 min, and 0.1% Riboflavin. The left eyes served as controls. Respectively, 6 animals were euthanized 3 days and 1 week after cosslinking. Confocal in vivo microscopy was performed in vivo before and after eutherisation. Thereafter the corneae were excised and processed for histology and immunohistochemistry.
Three days after crosslinking the epithelium was closed again, as shown by fluorescein application and in vivo confocal microscopy. Immunohistochemistry revealed some mitotic cells in the basal cell layer. A complete loss of cell nuclei in anterior and middle stroma, and only rare cell nuclei in posterior stroma could be shown.One week after crosslinking, the number of mitotic cells in epithelium was decreased. The anterior stroma was still lacking cell nuclei, whereas middle and posterior stroma were densely populated with cell nuclei with different morphology and reflectivity in confocal imaging. A large amount of these cells were mitotic, as shown by Ki-67 immunoreactivity.
The events, associated with wound healing at the first week after crosslinking have been shown by different approaches: in vivo confocal microscopy and histology. A good correlation between two methods could be shown, indicating a nearly complete epithelial regeneration by day 3, and a beginning regeneration in posterior and middle stroma by 1 week.
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