Quantitative assessment of UVA-riboflavin corneal cross-linking using nonlinear optical microscopy

Abstract

Purpose: Corneal collagen cross-linking (CXL) by the use of riboflavin and ultraviolet-A light (UVA) is a promising and novel treatment for keratoconus and other ectatic disorders. Since CXL results in enhanced corneal stiffness, this study tested the hypothesis that CXL-induced stiffening would be proportional to the collagen autofluorescence intensity measured with nonlinear optical (NLO) microscopy.

Methods: Rabbit eyes (n = 50) were separated into five groups including: (1) epithelium intact; (2) epithelium removed; (3) epithelium removed and soaked in riboflavin, (4) epithelium removed and soaked in riboflavin, with 15 minutes of UVA exposure; and (5) epithelium removed and soaked in riboflavin, with 30 minutes of UVA exposure. Corneal stiffness was quantified by measuring the force required to displace the cornea 500 μm. Corneas were then fixed in paraformaldehyde and sectioned, and the collagen autofluorescence over the 400- to 450-nm spectrum was recorded.

Results: There was no significant difference in corneal stiffness among the three control groups. Corneal stiffness was significantly and dose dependently increased after UVA (P < 0.0005). Autofluorescence was detected only within the anterior stroma of the UVA-treated groups, with no significant difference in the depth of autofluorescence between different UVA exposure levels. The signal intensity was also significantly increased with longer UVA exposure (P < 0.001). Comparing corneal stiffness with autofluorescence intensity revealed a significant correlation between these values (R(2) = 0.654; P < 0.0001).

Conclusions: The results of this study indicate a significant correlation between corneal stiffening and the intensity of collagen autofluorescence after CXL. This finding suggests that the efficacy of CXL in patients could be monitored by assessing collagen autofluorescence.

Figure 1.

(a) Indentation force measurement apparatus was composed of an x–y translation stage supporting the cornea holder (expanded view), which clamped the cornea between two metal plates with an O-ring. Corneal stiffness was measured with a force transducer with 250-μm diameter indenting probe attached to a z-axis translation stage. (b) After measuring stiffness, the corneas were fixed, bisected through the central cornea, embedded in agarose, and vibratome sectioned to obtain a 300-μm-thick central corneal slice. (c) The central regions of the vibratome sections were scanned with nonlinear optical microscopy.

Figure 2.

Force-displacement plots for the five treatment groups (n = 10/group) showing average ± SE for displacements through 500 μm in 50-μm steps.

Figure 3.

Average corneal stiffness (+SD) for each group showing no significant difference (NS) between the control groups, and significantly increased stiffness between the UVA-exposed and control groups.

Figure 4.

(a) Collagen autofluorescence image (400–450 nm) of control corneas treated with riboflavin. Corneas were fixed and washed to remove excess riboflavin. (b) Collagen autofluorescence image (400–450 nm) of a riboflavin-soaked and 30-minute UVA-exposed cornea showing increased fluorescence in the anterior corneal stroma after removal of the riboflavin. Note the presence of cellular autofluorescence in the corneal stroma and corneal endothelium (arrows). (c) Emission spectra of nonlinear optical signals generated by 760-nm femtosecond laser light from riboflavin and corneas after UVA-induced CXL. Riboflavin showed peak fluorescence at 521 nm, whereas CXL corneas showed peak fluorescence at 425 nm. Bar, 100 μm.

Figure 5.

Collagen autofluorescence (400–450 nm) image taken from (a) a control, riboflavin-treated cornea, (b) a 15-minute UVA-exposed cornea, and (c) a 30-minute UVA exposed cornea. Note the presence of autofluorescence in the anterior stroma after UVA exposure (b, c) and the increase intensity after 30-minute UVA exposure (c). To quantify the collagen autofluorescence, the average intensity along each plane (y) was measured and plotted as a function of depth (x). Bar, 100 μm.

Figure 6.

Representative smoothed collagen autofluorescence signals for (a) control, riboflavin-treated, (b) 15-minute UVA-exposed, and (c) 30-minute UVA-exposed corneas starting at the anterior cornea (ANT) and extending to the posterior cornea (POS). Autofluorescence intensity was quantified by first determining the average plus one SD of the background fluorescence level (gray line) within 150 μm of the posterior cornea (stable region, between points b and c). Intensity values above this level were assumed to represent the area of CXL (CXL region), and the area under the curve were integrated to represent the fluorescence increased by CXL (CXL region, between ANT and point a).

Figure 7.

Plot of collagen autofluorescence within the anterior 100 μm of cornea (CAF₁₀₀) and the total autofluorescence signal (CAF_total) for corneas exposed to UVA for 15 or 30 minutes (R² = 0.86; P < 0.001) between the CAF₁₀₀ and CAF_total and that 30 minutes of UVA exposure generated significantly more collagen autofluorescence than did 15 minutes of UVA exposure (P < 0.001).

Figure 8.

Linear regression analysis of collagen autofluorescence and corneal stiffness between control corneas treated with riboflavin alone and corneas treated with UVA for 15 or 30 minutes. Note that there was a significant correlation, with increasing corneal stiffness associated with increasing collagen autofluorescence (P < 0.001).