Fluorescence spectroscopy for non-invasive measurement of mechanical stiffness after photo-crosslinking of rabbit cornea

Abstract

Background and Objectives: Keratoconus is a disease characterized by progressive steepening and thinning of the cornea, altering visual acuity and sometimes potentiating the need for corneal transplant if the disease progresses.1–3 Corneal crosslinking, a procedure that uses topical riboflavin and UV light to increase the stiffness of the cornea through the creation of collagen crosslinks was recently approved by the FDA for use in the U.S. The objective of the present study was to investigate whether endogenous collagen fluorescence changes following treatment can be correlated to alterations in the stiffness of the cornea, thereby guiding treatment parameters. Study Design and Results: 78 ex-vivo rabbit eyes divided into three groups: riboflavin solution plus UV irradiation, dextran solution plus UV irradiation, and riboflavin solution only. An additional group of eyes received no treatment. The epithelium was removed from each sample and topical riboflavin was applied. Eyes were irradiated with a 365 nm black ray UV lamp for various treatment times, ranging from half the clinical treatment time to three times the length. Mechanical testing was performed to determine the force/displacement relationship for the various treatment times. Fluorescence spectral changes following treatment corresponded with changes in stiffness. In particular, a decrease in the value of fluorescence intensity at 290/340 nm excitation/emission wavelengths corresponded to an increase in corneal stiffness following treatment. It may be possible to use fluorescence spectral changes of endogenous corneal crosslinks to evaluate mechanical stiffness changes non-invasively.