In this study we have demonstrated that corneal stromal riboflavin penetration in iontophoretic transepithelial CXL can be improved by increasing soak time, solution concentration, and iontophoretic dosage.
The corneal epithelium has been previously shown to be a significant barrier to riboflavin penetration into the stroma.
9,29,30 This has been confirmed by a series of experiments using spectrophotometry as an indirect measure of stromal riboflavin concentration. Using our TPF methodology, which can directly quantify riboflavin concentration through the entire depth of the cornea,
13,28,31 we previously confirmed these findings, demonstrating very limited riboflavin diffusion through an intact epithelium with any of the several current commercially available transepithelial riboflavin formulations and protocols.
Riboflavin is water soluble and negatively charged at physiological pH, and laboratory studies suggest that iontophoresis can be applied effectively to enhance riboflavin penetration in transepithelial CXL. Several groups have reported similar increases in corneal biomechanics
19,22,23 in animal models as compared with epithelium-off CXL. Preliminary clinical studies have also been encouraging, with reported cessation of disease progression with up to 15 months follow-up and improvements in keratometric and visual parameters.
24–26 However, the relative efficacy of this technique compared to epithelium-off CXL remains to be determined especially over longer-term follow-up, and current randomized prospective studies comparing the two techniques (
clinicaltrials.gov.uk NCT02117999, NCT01868620, ISRCT 04451470) have yet to be reported.
Existing recommendations for iontophoresis in transepithelial CXL utilize 1 mA for 5 minutes with a 0.1% wt/vol riboflavin solution. Improved riboflavin penetration can be obtained by modifying these parameters. Stromal riboflavin concentrations using protocol A here were similar to epithelium-off controls (
P = 0.63) (
Fig. 4;
Table 3). This protocol utilized a solution of 0.25% wt/vol riboflavin with BAC and the St. Thomas'/Cardiff Iontophoresis protocol. This protocol was derived in collaborative pilot experimentation at St Thomas' Hospital and Cardiff University using spectrophotometry, and comprises two cycles of iontophoresis each followed by a 5-minute soak period to allow time for riboflavin to diffuse more posteriorly. The use of the cationic surfactant BAC has been shown with percutaneous iontophoresis to have a synergistic effect on the transport of anions.
22 We similarly observed this synergistic effect with higher stromal concentrations achieved with protocol A (0.25% wt/vol riboflavin, BAC) compared to protocol C (0.25% wt/vol riboflavin, no BAC) (
Fig. 4;
Table 3). A further variable in iontophoresis relates to the zeta potential and electrophoretic mobility of different riboflavin solutions that may, in part, explain our results. Although beyond the scope of this study, this warrants further investigation in the future.
A key advantage of our TPF imaging method, as compared to whole-tissue analysis (high-performance liquid chromatography, HPLC;
19,32–34 spectrophotometry
9,29,30), is the ability to quantify riboflavin concentration within the epithelium. We have previously demonstrated in transepithelial protocols (without rinsing the cornea) that epithelial riboflavin concentration often exceeds that in the underlying stroma.
13 Analyzing a full-thickness specimen, including a riboflavin-loaded epithelium and precorneal film, will lead to significant overestimation of corneal stromal riboflavin concentrations.
13 Clinically, riboflavin loading in the epithelium may reduce CXL efficacy by attenuating ultraviolet light transmission to the stroma. It may also result in an “arc-eye” type of reaction, particularly where riboflavin preparations including BAC are used, accounting for the epithelial defects seen in up to two-thirds of patients after transepithelial CXL using MedioCross TE.
35 The ideal transepithelial protocol would load the stroma with riboflavin while leaving the epithelium relatively clear. To this end, in this study we rinsed the surface of the globe after iontophoresis in a bid to wash out some of the riboflavin from within the epithelium.
Figures 3 and
4 and
Table 3 show that this approach did not work, since there was no significant difference in fluorescence between the epithelium and the subjacent stroma. Extended rinsing (5 minutes) of the ocular surface in protocols A and B (MedioCross TE) also reduced corneal stromal concentrations of riboflavin up to an approximate depth of 200 μm. Stromal riboflavin elution was less evident with non-BAC-containing solutions (protocols C and D), where the epithelial tight junctions were still probably largely intact. Although iontophoresis is not commercially promoted for use with BAC-containing solutions, this stromal washout effect with prolonged rinsing of the ocular surface may compromise tissue cross-linking within the anterior cornea.
It is important to note that iontophoresis was ineffective with riboflavin formulations containing saline (protocols G and H) that produced minimal riboflavin penetration and some stromal swelling.
There are a number of limitations to this study. Firstly, results in ex vivo rabbit corneas, although a better anatomical match to human corneal epithelial thickness than porcine eyes, may be affected by postmortem changes in epithelial layer integrity. Despite steps to minimize this as described in the Methods section, some epithelial degradation and enhanced permeability are likely, and our results may overestimate stromal riboflavin absorption in clinical transepithelial CXL. Secondly, migration of riboflavin within the snap-frozen tissue would have started as soon as the section thawed on the slide, resulting in an underestimation of stromal riboflavin concentrations. Finally, imaging through two media of different refractive indices (oil and water either side of the coverslip) may have increased optical aberrations as the laser light passes through. Any induced aberrations may have resulted in a small absolute loss of signal; but since this same method was employed for all imaged samples, no relative change in signal between samples should be present. Given these limitations, we are unable to guarantee an absolute concentration from these TPF data alone.
In conclusion, this study confirms that transepithelial riboflavin penetration can be improved by increasing soak time, riboflavin concentration in the soak preparation, and iontophoretic dosage. While the optimum tissue concentration for effective CXL is unknown, protocol C (0.25% wt/vol riboflavin [BAC-free], St. Thomas'/Cardiff Iontophoresis protocol) achieves more than 50% greater stromal penetration compared to the standard iontophoresis protocol, as well as far higher concentrations than we have seen with non-iontophoresis transepithelial protocols.
24 Although less than that achieved epithelium-off, its considerable stromal riboflavin penetration, without relying on epithelial-toxic additives, may represent the best transepithelial technique to date. Clinical trials will determine whether this level of stromal riboflavin penetration produces effective CXL.