Transepithelial corneal cross-linking assisted by two continuous cycles of iontophoresis for progressive keratoconus in adults: retrospective 5-year analysis

Keratoconus (KC) has long been considered as a progressive, non-inflammatory corneal thinning and ectasia with reduced biomechanical stability, which may lead to severe visual impairment in young and even pediatric patients. In very severe cases, lamellar or penetrating corneal transplantation is the final therapeutic option to regain vision. In recent years, corneal collagen crosslinking (CXL) has been recognized as a safe and effective treatment to delay or halt further progression of KC and can reduce the need of keratoplasty [1]. During CXL, riboflavin interacts with ultraviolet-A light to create crosslinking of protein fibrils followed by formation of interchain disulfide bonds, thus arresting the progression of corneal ectasia by increasing the biomechanical stability of the cornea. CXL has been considered as one of the standard treatments of progressive keratoconus worldwide.

Various protocols [1] of CXL have been extensively investigated and applicated. CXL using classic Dresden’s protocol with epithelium removal (conventional CXL) showed long-term efficacy of stabilization and improvement for KC. However, postoperative complications using standard epi-off protocol [2‐5], such as corneal haze, sterile corneal infiltrates, recurrent erosion syndrome, have been reported and should be taken into consideration. To reduce the risk of postoperative complications, epithelium-on(epi-on) protocols assisted by iontophoretic delivery or transepithelial riboflavin were brought into sight and were considered by some researchers to be a better choice.

Iontophoresis, in which an electrical gradient is used to drive negatively charged riboflavin molecules across the intact epithelium, may further enhance riboflavin penetration in transepithelial CXL. Laboratory and clinical studies [6‐9] of iontophoresis have been encouraging, demonstrating increased transepithelial penetration of riboflavin and improvement of corneal biomechanics. Nevertheless, most studies showed inferior results of standard protocol of iontophoresis when compared to epi-off protocol [10‐13]. The limited depth of riboflavin penetration and lower concentration of riboflavin in the corneal stroma were the critical inadequacies of standard iontophoresis protocol [14]. To overcome this shortcoming, transepithelial CXL assisted by two continuous cycles of iontophoresis [15] was taken into consideration, and some study showed better short-term outcome than that by standard iontophoresis [15]. Theoretically, two continuous cycles of standard iontophoresis (enhance iontophoresis) allowed time for riboflavin to penetrate and diffuse more posteriorly. However, the long-term effect of this modified iontophoretic protocol remained unclear.

This study aimed to compare the long-term efficacy of enhanced iontophoresis-assisted transepithelial corneal crosslinking (EI-CXL) and conventional corneal crosslinking (C-CXL) in adults with progressive keratoconus, as well as the characteristics in visual acuity, corneal topography, and morphological alteration.

In the past decade, although some study demonstrated that iontophoresis-assisted CXL could achieve the same clinical result as standard CXL [21], most evidences have demonstrated inferior results of standard protocol of transepithelial CXL when compared to the conventional epi-off protocol. However, our data indicated that EI-CXL achieved by doubling the iontophoresis cycle displayed approximate long-term(5 years) efficacy with C-CXL in halting the progression of keratoconus in adults. This modified transepithelial protocol might be a good alternative in the management of progressive keratoconus. To our knowledge, our study was the first to demonstrate the long-term effects of transepithelial CXL assisted by iontophoresis for 10 min.

Riboflavin is water soluble and negatively charged at physiological pH. In transepithelial CXL, iontophoresis can be applied effectively to enhance riboflavin penetration. Existing recommendations for iontophoresis in transepithelial CXL utilize 1 mA for 5 min with a 0.1% riboflavin solution. Standard iontophoresis allowed riboflavin imbibition with one-half the concentrations of the C-CXL technique in a rabbit model [8]. Variables in protocols of iontophoresis are able to obtain improved riboflavin penetration. For example, the use of the cationic surfactant benzalkonium chloride has been shown with percutaneous iontophoresis to have a synergistic effect on the transport of anions [6]. An ex vivo study utilized two cycles of iontophoresis each followed by a 5-min soak period to allow time for riboflavin to diffuse more posteriorly, and the stromal riboflavin concentrations were found to be similar to epithelium-off controls [22]. Transepithelial CXL assisted by two continuous cycles of iontophoresis also showed better short-term outcome than that by standard iontophoresis [15]. In our study, no significant differences of the long-term refractive and visual outcome were found between C-CXL and EI-CXL group. The cessation of keratoconus progression with up to 5 years follow-up demonstrated the potential of this modified iontophoretic protocol.

The depth of the acellular zone of anterior corneal stroma after CXL has been correlated with the effectiveness of the CXL treatment [23‐25]. The limited penetration depth of riboflavin is one of the critical inadequacies of transepithelial CXL with standard iontophoresis protocol. The penetration depth ranged from 100 to 240 μm in most clinical studies [25‐29]. In our previous study [30], the penetration depth of transepithelial CXL using the same iontophoretic device for 5 min was about 133 μm from the corneal surface. The penetration depths in these iontophoretic protocols were all shallower when compared to that in conventional CXL. The huge range of different penetration depth among these reports might be contributed to the variety of iontophoretic devices, riboflavin concentration, and UV radiation parameters used in the CXL surgery. In a series of laboratory investigation, it has been shown that by increasing riboflavin concentration, iontophoresis application times, and allowing short periods of time for riboflavin, which in initially deposited only into the epithelium and anterior stroma, to diffuse deeper into the stroma, concentrations of up to 80% of that achieved with epithelium-off application with a homogeneous distribution throughout the stroma can be achieved [26]. Apparently increased penetration depth could be achieved by repeated iontophoresis. In this study, IVCM showed an average penetration depth of 251 μm in EI-CXL, which achieved an increase of almost 90% of that achieved with iontophoresis using riboflavin 0.1% and 0.1 mA for 5 min in our previous standard iontophoresis (133 μm). The penetration depth in EI-CXL was also far deeper than those achieved with any of the transepithelial CXL protocols using chemical enhancers. With such improved transepithelial penetration depth, EI-CXL achieved approximate results to C-CXL during the follow-up period of 5 years. However, it should be noted that the penetration depth was still more superficial in EI-CXL group than that of C-CXL group.

The statistical analysis showed no differences in the tomographic and refractive parameters between group during the 5-year follow-up (Table 2). Although improvement of K_max, K2, and SE were observed after surgery within both groups, no significant improvement of BCVA was observed in either group of this study. In addition, the refractive data of spherical, cylinder diopter, spherical equivalent, and K_max showed higher absolute value in the EI-CXL group at 5 years, even though no statistical difference was found. Furthermore, there was a slight increase of corneal stromal cell density and loss of hyperreflective needle-shaped reflection at 5 years after EI-CXL (Fig. 2e), while that in C-CXL group remained almost unchanged (Fig. 2j). This subtle difference of representative IVCM images noted at 5 years after surgery implied that the corneal collagen turnover might happen earlier in EI-CXL group. The earlier turnover of corneal collagen, the more superficial demarcation line in combination with tapering refractive improvement over time after EI-CXL might indicate an inferior efficacy of the EI-CXL protocol when compared with the C-CXL protocol. Further follow-up is needed to determine whether the corneal collagen turnover may induce loss of EI-CXL effect duration with new corneal instability or keratoconus progression in 5 to 10 years after EI-CXL.

The main limitations of our study include the retrospective study design, lack of randomization, and a relatively small sample size that may reduce the power to further interpretation. As a regional eye center in China, our hospital treated a large number of keratoconus patients who lived far away from our hospital. Most of these patients could not guarantee the regular follow-up, increasing the difficulty to conduct a prospective study. In addition, other factors such as age < 14 years, too thin cornea, and K_max > 60.0 D further cut down the final number of included cases. Furthermore, the treatment protocol was selected and decided by individual patients, depending on their self-estimated tolerance to postoperative pain and acceptance to potential weak effect of epi-on CXL. Besides, patients with more severe keratoconus might prefer C-CXL in order to gain possible better outcome. The lack of randomization inevitably led to selection bias.

In conclusion, transepithelial CXL assisted by enhanced iontophoresis achieved improved stromal penetration depth. Although less than that achieved by conventional epithelium-off protocol, its considerable stromal riboflavin penetration as well as long-term tomographic and refractive improvement, without obvious postoperative complications, demonstrated the potential of EI-CXL to be a better transepithelial protocol. Randomized multicenter clinical trials are needed to further determine the long-term efficacy level.