Mechanism of Action of Riboflavin + Ultraviolet Radiation Treatment in Corneal Strengthening

Abstract

Purpose: : Chronic degenerative central cornea thinning, Keratoconus, is halted by application of 0.1 wt% Riboflavin (2.65 mM Vitamin B₂) (RF) solution + Ultraviolet Radiation: 365 nm, 3mW/cm² (UVA). This clinical treatment may increase corneal component cross-linking, but the fundamental mechanism of the (RF+UVA) effect is not understood.

Methods: : Mechanistic studies were done to determine if singlet oxygen (¹O₂) is a reactive intermediate and to identify reactive cross-linking moieties in corneas. ¹O₂ was modulated via addition of sodium azide to clinical solutions or preparation of RF solutions in deuterium oxide (D₂O). Modulation of reactive groups in corneas was accomplished via whole cornea pretreatment with ethyl acetimidate, hydroxylamine, 2,4-dinitrophenylhydrazone (DNPH), or acetic anyhydride. Experiments were performed on corneas of dogfish sharks (Squalus acanthius) and rabbits (New Zealand White) to quantitatively compare the effects of the (RF+UVA) protocol across species.

Results: : Azide severely limited corneal strengthening at concentrations as low as 1.325 mM. Conversely, RF solutions made in D₂O produced strengthening equivalent to control clinical treatment at 1/10th the clinical RF concentration, whereas clinical RF concentrations in D₂O produced corneas 3-fold stronger than clinically treated controls, positively indicating involvement of ¹O₂. Acetic anhydride and ethyl acetimidate selectively react with free amino groups, yet pretreatment with either chemical alone still allowed statistically significant increases in corneal strength to occur in response to subsequent clinical (RF+UVA) treatment, strongly indicating that the cross-linking reaction is independent of free amine availability. In contrast, pretreatment with hydroxylamine or DNPH arrested subsequent (RF+UVA) cross-linking in both species, indicating endogenous aldehydes are necessary for the strengthening reaction.

Conclusions: : Strong evidence for ¹O₂ as a rate-limiting reactive intermediate and aldehyde availability as a cross-linking target was experimentally verified in whole corneas. This understanding of the underlying reaction requirements should allow optimization of this protocol to make it more effective clinically.

Keywords: keratoconus • cornea: clinical science • cornea: basic science