Br J Ophthalmol. 2014 Feb;98(2):270-4. doi: 10.1136/bjophthalmol-2013-303816. Epub 2013 Nov 28.

Protection of corneal epithelial stem cells prevents ultraviolet A damage during corneal collagen cross-linking treatment for keratoconus.


Moore JE, Atkinson SD, Azar DT, Worthington J, Downes CS, Courtney DG, Moore CB.

School of Biomedical Sciences, University of Ulster, , Coleraine, Northern Ireland.



Background/Aims: Cross-linking of the cornea is usually carried out at a young age as a treatment to manage ectasia. The corneal limbal region contains delicate long-lived stem cells, which could potentially be deleteriously affected by Ultraviolet A (UV-A) radiation. Damage to these stem cells may not demonstrate as a clinical problem for many years subsequent to crosslinking treatment. UV-A radiation is known to have potential mutagenic effects upon mammalian DNA and can result in cancer.

Methods: Cultured corneal epithelial cells and ex vivo corneal tissue were treated with the standard clinical cross-linking protocol for UV-A irradiation. 8-hydroxydeoxyguansoine (8-OHdG) and cyclindependent kinase inhibitor genes (CDKN1A and CDKN2A) were assayed as markers of DNA damage using immunohistochemistry, ELISA and quantitative real time PCR.

Results: Staining of treated limbal tissue demonstrated the presence of 8-OHdG within p63 positive basal limbal cells. Levels of 8-OHdG and CDKN1A mRNA were found to be significantly increased in cultured corneal epithelial cells and limbal epithelial cells but no increase was demonstrated with the use of a polymethyl methylacrylate protective cover.

Conclusions: This study provides evidence that oxidative nuclear DNA damage can occur through cross-linking in layers of corneal epithelial cells at the limbus and that this can be easily prevented by covering the limbus.

KEYWORDS: Cornea; Stem Cells; Treatment Lasers; Vision

PMID: 24288393



Keratoconus is a disorder of the eye and results following changes within the cornea, which cause it to become thinner and more conical in shape. A relatively common procedure for the treatment of keratoconus is corneal collagen cross-linking; this procedure uses a vitamin B2 solution and ultra violet light to strengthen the collagen fibers in the eye by creating more cross-linking between them. Moore et al hypothesized that the delicate stem cells, which reside within the limbal region might be detrimentally affected by exposure to UV-A as a result of the cross-linking procedure.

This research study by Moore et al has shown the presence of 8-OHdG (a marker of oxidative nuclear DNA damage) within p63 positive basal limbal cells in the cornea. This study demonstrated that the ultra-violet light used to treat keratoconus causes a significant increase in 8-OHdG both within a corneal cell line and human donor corneas; indicating increased oxidative nuclear DNA damage. The placement of a Poly(methyl methacrylate) (PMMA) cover over the cells and human donor corneas gives a protective effect from the effects of UV-A treatment. Validation of the oxidative DNA damage was provided by assessment of DNA damage-inducible cyclin-dependent kinas inhibitor levels (CDKN1A).

This study provides evidence that oxidative nuclear DNA damage can result in corneal epithelial cells as a result of UV-A treatment and this may in turn decrease the proliferative ability of corneal epithelial stem cells; which could cause long-term damage, alter the regenerative capacity of the cornea and increase the risk of cancer.

SA Fig1

 Figure 1. Keratoconus is characterised by a thinning cornea which in turn causes development of a cone-like bulge.


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