A study of stromal riboflavin absorption in ex vivo porcine corneas using new and existing delivery protocols for corneal cross-linking

Abstract

Purpose: To indirectly measure stromal riboflavin penetration using commercially available riboflavin solutions and new and existing epithelium-off, trans-epithelial and iontophoresis-assisted delivery protocols.

Methods: Forty porcine eyes were divided into eight groups. Group 1: Ricrolin applied to the de-epithelialised cornea for 30 min; Group 2: epithelium-intact, no treatment; Groups 3-5: epithelium-intact, 30-min application of Ricrolin TE, Mediocross TE or ParaCel/Vibex, respectively. Group 6: epithelium-intact, Ricrolin(+) iontophoresis-assisted delivery for 5 min; Group 7: epithelium-intact, Ricrolin(+) iontophoresis-assisted delivery for 5 min with a 20-min riboflavin soak; and Group 8: epithelium-intact, Ricrolin(+) iontophoresis-assisted delivery for 5 min, 15-min soak and another 5 min of iontophoresis. After a saline wash, light transmission spectra were obtained from each cornea, before and after epithelial removal.

Results: Corneas in groups 1 and 8 showed a distinct riboflavin absorption peak between 400 and 520 nm. The optical density of the corneas in groups 3-7 did not differ significantly from that of the untreated corneas (group 2).

Conclusions: A modification to the standard iontophoresis trans-epithelial technique resulted in successful penetration of riboflavin into the stroma and appears to offer the most promise for epithelium-on cross-linking.

Keywords: cornea; cross-linking; iontophoresis; spectrophotometry.

Figure 1

Iontophoresis riboflavin delivery system modified for use in ex vivo eyes.

Figure 2

Sample holder used for spectrophotometry studies. After mounting the cornea on the sample platform, the anterior clamp is positioned to form a tight seal around the tissue. Silicon oil is then injected on either side of the cornea, first into the posterior chamber of the holder (A), to maintain the natural curvature of the cornea, and then into the anterior chamber (B).

Figure 3

An average optical density spectrum from riboflavin‐treated corneas. (A) The region of absorption due to riboflavin (between 400 and 520 nm) is highlighted in grey. By isolating the riboflavin peak and fitting and subtracting a power law background (dashed line) arising from the optical density of the cornea itself (B), the absorption due to the presence of riboflavin within the tissue could be assessed (C) and compared between treatment groups.

Figure 4

Post‐treatment corneal thickness (±SD).

Figure 5

Photographs of treated and untreated corneas captured before (Epi‐on) and after (Epi‐off) epithelium removal. All treatment abbreviations are detailed in Table 1.

Figure 6

The averaged optical density of untreated corneas (epi‐on, no Tx) and corneas treated with a range of riboflavin delivery protocols (as detailed in Table 1) are shown immediately post‐treatment (A) and after post‐treatment removal of the epithelium (B).