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Guzel Bikbova, Azat Khalimov, Gyulli Kazakbaeva, Mukharram Bikbov; Molecular mechanisms of corneal collagen crosslinking. Invest. Ophthalmol. Vis. Sci. 2018;59(9):522. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the mechanisms of ultrastructural transformation of cornea during collagen crosslinking.
148 adult SD rats and 164 Grey Shinshilla rabbits were divided into five groups: 1 - intact, 2 - UV irradiation (370 nm, 3 mW/cm2 for 10 min – rats, 30 min – rabbits) without riboflavin (control), 3 - UV with isotonic solution of riboflavin, 4 - UV with riboflavin solution 0.1% + Dextran T-500 20%, 5 - UV with riboflavin solution 0.1% + Hydroxylpropylmethylcellulose 1.0%. IHC analysis had been done by indirect immunoperoxidase staining using monoclonal and polyclonal antibodies for caspase-3 and -8, Bcl-2, p53, TGFβ1, FGF-1. Additionally, free radical oxidation (FRO) and production of reactive oxygen species (ROS) assessed by ferrous iron-induced (FI) and luminol-dependent (LD) chemiluminescence (CL). Statistical analyses were performed by one-way ANOVA.
The number of caspase-3 and -8 immunopositive cells in rats corneal stroma were increased in all groups that were treated with UV on the 3d day after procedure mainly in anterior and midstroma. By 3 months caspase-8 immunopositive cells increased exept group 2, where tissue destruction had been observed. Caspase-3 immunospositive cells were decreasing with time in all groups. Bcl-2 immunopositivity had been increased slightly in 2 group, and significantlyin 3-5 groups on the 3d day then by 3 month decreased by half. TGFβ1 immunopositivity had been observed in all groups treated with UV, however FGF-1 was slightly increased by 3d day and had not been found after 3 months. The dynamics of the changes (2d group) in the parameters of the LD CL in the anterior chamber aqueous of the rabbits showed a 5-fold increase in spontaneous luminosity (p<0.01) and a 6-fold increase in the luminescence (p<0.01) from the first hour after UV without riboflavin, followed by gradual (14 days) decrease. Similar but less intensive changes had been observed in UV treated groups with riboflavin. Activation of FRO processes in rats was noted in all tissues of the eyeball.
Caspase-3 production in the early period indicates the induction of the caspase cascade in keratocytes in response to corneal damage caused by its deepithelialization and exposure to UV radiation. The key pathogenetic factor of UV exposure to the cornea is the induction of free radicals with the activation of FRO processes, which are compensated to a certain extent by the presence of riboflavin.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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