Decreased UV-filtering ability of corneal epithelium may be the cause of stromal pathologies in keratoconus

Abstract

Purpose : Etiology of Keratoconus (KC) is not clear. Ultraviolet (UV) light is one of the factors implicated in the etiology of KC. This study tests the hypothesis that UV light absorbance and antioxidant mechanisms of corneal epithelium may play an important role in the etiology of KC by using an experimental model of epithelial UV exposure.

Methods : Corneal epithelial cells were obtained from patients applied for corneal cross-linking or excimer laser. Cells were incubated in 12 well plates in Keratinocyte Serum Free Medium for 24 hours until they attached the surface and formed a monolayer. Cells from KC patients were divided into 3 groups: UV-A-KC, UV-B-KC, and No-UV-KC. Cells from normal eyes which underwent excimer laser also divided into 3 groups: UV-A-N, UV-B-N, and No-UV-N. Each group is composed of 6 samples. UV intensity was set as 2000 mJ/cm² for UV-A and 150 mJ/cm² for UV-B application. After UV application, cells were incubated for another 24 hours. Then following enzyme activities were measured: Glutathion Peroxidase (GPX), Glutathione Reductase (GR), Superoxide Dismutase (SOD), Catalase (CAT), Aldehyde Dehydrogenase (ALDH), Nitric-Oxide Synthetase (NOS). Levels of antioxidant molecules were measured: Glutathione (GSH), NAD/NADH, Uric Acid, Ferritin, Ascorbic Acid, Hydrogen Peroxide (H₂O₂), Nitric-Oxide (NO), Malondialdehyde (MDA), Nitrothyrosine-3 (NT), and Total Anti-oxidant Capacity (TAC). Local Ethics Committee approval was obtained. Statistical Analysis was performed by SPSS 18.0 software. Statistical significance test within groups were done by Kruskal-Wallis test. Comparison of groups was done by Mann-Whitney U test.

Results : ALDH, TAC, NOS, NAD/NADH levels were lower in No-UV-KC group compared to No-UV-N group (p<0.05). G6PD and NO levels were higher in No-UV-KC group compared to No-UV-N group (p<0.05). G6PD increased after UV-B application in both KC and N groups (p<0.05). Ferritin decrased after UV-A and UV-B application in N group (p<0.05), but not in KC group (p>0.05). UV absorption of cells were measured and was found to be significantly lower in KC group compared to N group (p<0.05).

Conclusions : Decreased UV filtering ability combined with weaker antioxidant defense may cause chronic low dose UV and oxidant exposure of Bowman's membrane and anterior corneal stroma, which may cause clinical findings of KC.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.