Mechanisms of corneal tissue cross-linking in response to treatment with topical riboflavin and long-wavelength ultraviolet radiation (UVA)

Abstract

Purpose: Treatment of de-epithelialized human corneas with riboflavin (RF) + long-wavelength ultraviolet light (UVA; RFUVA) increases corneal stroma tensile strength significantly. RFUVA treatment retards the progression of keratoconus, perhaps by cross-linking of collagen molecules, but exact molecular mechanisms remain unknown. Research described here tested possible chemical mechanisms of cross-linking.

Methods: Corneas of rabbits and spiny dogfish sharks were de-epithelialized mechanically, subjected to various chemical pretreatments, exposed to RFUVA, and then subjected to destructive tensile stress measurements. Tensile strength was quantified with a digital force gauge to measure degree of tissue cross-linking.

Results: For both rabbit and shark corneas, RFUVA treatment causes significant cross-linking by mechanism(s) that can be blocked by the presence of sodium azide. Conversely, such cross-linking is greatly enhanced in the presence of deuterium oxide (D(2)O), even when RF is present at only one tenth the currently used clinical concentrations. Blocking carbonyl groups preexisting in the stroma with 2,4-dinitrophenylhydrazide or hydroxylamine blocks essentially all corneal cross-linking. In contrast, blocking free amine groups preexisting in the stroma with acetic anhydride or ethyl acetimidate does not affect RFUVA corneal cross-linking. When both carbonyl groups are blocked and singlet oxygen is quenched, no RFUVA cross-linking occurs, indicating the absence of other cross-linking mechanisms.

Conclusions: RFUVA catalyzes cross-linking reactions that require production of singlet oxygen ((1)O(2)), whose half-life is extended by D(2)O. Carbonyl-based cross-linking reactions dominate in the corneal stroma, but other possible reaction schemes are proposed. The use of D(2)O as solution media for RF would enable concentration decreases or significant strength enhancement in treated corneas.

Figure 1.

(A) Tissue manipulations for experimentation and tension measurements. Chemical treatments used for capping carbonyl groups in tissue or for capping free amine groups, both before the standard RFUVA treatment, are shown. (B) Example of the tension versus time profile recorded while destructive tension was applied to a strip of corneal tissue. All strips displayed plots of this shape. Destructive tension was the maximum tension exerted just before the strip broke. Strips broke within the central region of the cornea. Two such 2-mm strips were assayed from each cornea, with four corneas assayed per treatment group; thus, eight strips were assayed per treatment group (total, n = 8).

Figure 2.

Mean destructive tension for control and oxygen-manipulated corneas. Effects of azide and deuterium oxide on destructive tension values for corneal strips. (A) Rabbit. (B) Shark. Mean destructive tension for nontreated saline control corneas and for RFUVA-treated corneas are shown in this figure and in Figures 4 to 7 as horizontal dashed lines to provide a convenient visual gauge of the degree to which each chemical treatment affects the degree of cross-linking with respect to those standard values. A 2-fold increase in azide concentration still caused the same degree of inhibition. A 10-fold decrease in RF concentration from clinical values still caused significantly greater stimulation of cross-linking when it was dissolved in D₂O. Each group, n = 8. *P < 0.01; **P < 0.05.

Figure 3.

Available carbonyl groups in corneas were labeled with fluorescent marker Alexa Fluor 488 hydrazide versus saline-treated controls at pH 7.5 in water. (A1–A3) Rabbit corneas. (B1–B3) Shark corneas. (A1, B1) One cornea of each pair was ligated with carbonyl marker Alexa Fluor 488 hydrazide; the other cornea was a saline-treated control. Bright-field illumination only. (A2, B2) Fluorescence and bright-field illumination. (A3, B3) Fluorescence illumination only. Scale bar, 5 mm.

Figure 4.

Mean destructive tension for carbonyl-capped corneas. Effects of acidic methanol control treatments and subsequent carbonyl group capping with either DNPH or hydroxylamine, both dissolved in acidic methanol, before RFUVA treatment. (A) Rabbit, *P < 0.001. (B) Shark, **P < 0.05. Each group, n = 8. (C) Available carbonyl groups in corneas were labeled with fluorescent marker Alexa Fluor 488 hydrazide before and after carbonyl capping reaction. (A, AA) Rabbit acidic methanol control corneas labeled with Alexa Fluor 488 hydrazide. (B, BB) Rabbit corneas carbonyl capped with hydroxylamine before labeling with Alexa Fluor 488 hydrazide. Degree of labeling is diminished by capping carbonyl groups. (C, CC) Shark acidic methanol control corneas labeled with Alexa Fluor 488 hydrazide. (D, DD) Shark corneas carbonyl capped with hydroxylamine before labeling with Alexa Fluor 488 hydrazide. Degree of labeling is diminished by capping carbonyl groups. Scale bar, 5 mm.

Figure 5.

Mean destructive tension for amine-capped corneas. Effects of blocking tissue amine groups. (A) Rabbit, with ethyl acetimidate in pH 9.2 aqueous pyrophosphate buffer; *P < 0.05. (B) Shark, with acetic anhydride in pyridine or with ethyl acetimidate in pH 9.2 aqueous pyrophosphate buffer; *P < 0.05. Each group, n = 8.

Figure 6.

Mean destructive tension for ¹O₂-quenched and carbonyl-capped corneas. Effects of both blocking tissue carbonyl groups with hydroxylamine in methanol and quenching ¹O₂ by the presence of azide during RFUVA. All cross-linking is blocked. (A) Rabbit. (B) Shark. Each group, n = 8.

Figure 7.

Mean destructive tension for solvent-treated rabbit corneas. Effects of common organic solvents DMF, pyridine, and acetic anhydride in pyridine on mean destructive tension of rabbit corneas. Each group, n = 8.

Figure 8.

(A) Proposed mechanisms by which RF can induce cross-linking of collagen molecules in the presence of UVA. Pathway (a) is singlet oxygen-dependent, which produces imidazolone (compound 1). This short-lived intermediate can then react with an uncapped nucleophile (Nu). Pathway (b) invokes endogenous carbonyls (allysine) as a nucleophile in a subsidiary ¹O₂-dependent pathway. Pathway (c) suggests that a self-activation product of RF, 2,3-butanedione (compound 2) could be formed on excitation of the RF, yielding an additional ¹O₂ -dependent pathway that would react strongly with endogenous carbonyls. (B) Experimental gating of the putative reaction mechanisms. X represents the point along each of the three pathways at which the reactions would be blocked in those corneas treated with azide or with any of the carbonyl-capping protocols shown in Figure 1A.