The corneal ectasia disorders, such as keratoconus, pellucid marginal degeneration, and postoperative corneal ectasia (keratectasia), share two main attributes: impairment of visual acuity and progressive corneal thinning and steepening that leads to asymmetric anterior and posterior corneal surfaces and irregular astigmatism.1,2 Variation in the thickness of the corneal epithelium can compensate to some extent and thereby mask underlying stromal topographic irregularities.3 For example, in eyes with mild to moderate keratoconus, the epithelium tends to be thinner over the apex of the corneal cone and thicker around the cone (called a “doughnut pattern”).4 Compared with the pattern seen in normal eyes, keratoconus is associated with thinner inferior and minimum corneal epithelial thickness and a larger difference between the minimum and the maximum epithelial thickness.4,5 This can make it difficult to assess the actual stromal irregularity by frontal surface corneal topography, especially in the early stages of the disease.3,4,6
Several methods, including very high-frequency ultra-sound, confocal microscopy, and optical coherence tomography (OCT), can be used to measure corneal epithelial thickness and provide better information about corneal ectasia.7–10 Spectral-domain OCT has the advantage of being a non-contact imaging method.11–15
Corneal cross-linking (CXL) has been used for more than a decade to halt or delay progression of corneal ectasia.16 The original Dresden protocol called for removal of the cor-neal epithelium to enhance penetration of the photoactivator (riboflavin) into the cornea. Epithelium-off CXL is widely used, so the profile changes associated with removal and regeneration of the corneal epithelium could be informative regarding changes in the underlying anterior stromal surface.3,17,18 The purpose of this study was to characterize the epithelium thickness profile before and after CXL in keratoconic eyes to determine whether epithelium-off CXL results in significant alteration of the epithelium thickness profile that could potentially influence interpretation of corneal topography images.
Patients and Methods
This epithelial-mapping study was a sub-study of a prospective randomized clinical trial, which found that different riboflavin dosing frequencies resulted in equivalent outcomes with epithelium-off CXL.19 An institutional review board (IRBCo, Inc., Buena Park, CA) approved the study and it was conducted according to the tenets of the Declaration of Helsinki. All participants provided written informed consent after the nature of the procedures had been explained.
The inclusion criteria were patients who were 10 years or older who had axial topography and slit-lamp findings characteristic of keratoconus and had demonstrated progression as defined by one or more of the following criteria within the previous 36 months: 1.00 diopter (D) or greater increase in the steepest keratometry value (or sim K); 1.00 D or greater increase in cylinder on subjective manifest refraction; 0.50 D or greater increase in myopia on subjective manifest refraction; and documented decrease in visual acuity associated with worsening irregular astigmatism and topographic features of ectasia. Contact lens wearers had to discontinue use prior to the screening and postoperative examinations for the following lengths of time: 3 days for soft lenses, 1 week for soft extended wear lenses, 2 weeks for soft toric lenses, and 3 weeks for rigid gas permeable lenses. Key exclusion criteria were previous ocular conditions that could predispose to complications, clinically significant corneal scarring, insufficient corneal thickness, a history of delayed epithelial healing, or pregnancy or lactation during the course of the study.
The CXL was performed per the standard Dresden protocol published by Wollensak et al. in 2003.20Table A (available in the online version of this article) summarizes the CXL methods used.
All patients had a complete ophthalmic examination preoperatively and at 3 and 6 months after the CXL procedure. The data collected included age, gender, uncorrected and corrected distance visual acuity, and maximum keratometry from Scheimpflug imaging (Pentacam; Oculus Optikgeräte, Wetzlar, Germany). The visual acuity measurements were converted from Snellen values to logMAR units for analysis.
In addition, anterior segment images were acquired with spectral-domain optical coherence tomography (RTVue 100; Optovue, Inc., Fremont, CA) as previously described5 and used to assess the corneal structure. The epithelial thickness profile across a 5-mm diameter centered over the pupil was evaluated with epithelial mapping software; this software was investigational at the time of the study and subsequently received marketing approval. Measurements taken from the epithelial map included the thinnest point, the thickest point, the difference between the minimum and maximum epithelial thickness, mean central epithelial thickness (within a 2-mm diameter), mean inferior epithelial thickness (2 to 5 mm inferiorly), and mean superior epithelial thickness (2 to 5 mm superiorly).
Data are expressed as mean ± standard deviation. Changes from baseline to 6 months after CXL were assessed with the paired t test, and correlations between different parameters were assessed with general linear methods. Analyses were performed with SAS software (version 9.4; SAS Institute, Cary, NC); all tests were two-sided and a P value of less than .05 was considered significant.
The study included 93 eyes from 93 patients who had progressive keratoconus and were treated with CXL; 80 eyes were classified as mild keratoconus (Scheimpflug flat keratometry reading < 51.00 D), 7 as moderate keratoconus (flat keratometry reading of 51.00 to 56.00 D or astigmatism ≥ 8.00 D) and 6 as severe keratoconus (flat keratometry reading > 56.00 D). The mean patient age at the time of CXL was 27 ± 11 years (range: 11 to 73 years); 76 patients (81%) were male and 17 (19%) were female.
Table B (available in the online version of this article) summarizes the maximum keratometry and visual acuity measurements recorded at the preoperative baseline examination and at 3 and 6 months after CXL. Between the baseline preoperative examination and the 6-month postoperative examination, the mean maximum keratometry reading decreased by 1.10 D (P < .0001). At baseline, the mean corrected distance visual acuity was 20/40 and the mean uncorrected distance visual acuity was 20/125; both improved by approximately half a line on the eye chart at 6 months after CXL (Table B).
Changes in Maximum Keratometry and Visual Acuity After CXL
Epithelial Changes Associated With CXL
The epithelial thickness profile was assessed across the central 5 mm of the cornea. Relative to the baseline reading, the mean epithelial thickness at the thinnest point increased modestly after CXL (from 41 to 42 μm) and the mean epithelial thickness at the thickest point decreased modestly (from 65 to 64 μm) (Table 1). Although neither of these changes were statistically significant, the net maximum–minimum epithelial thickness difference decreased significantly from 24 to 22 μm (P = .0023). The mean 3-mm central epithelial thickness increased from 51 ± 5.5 to 52 ± 5.6 μm (P = .042), whereas the mean inferior and superior epithelial thickness decreased by 1 μm within the area 2 to 5 mm from the corneal apex (P = .037 and .026, respectively). Figure A (available in the online version of this article) shows epithelial thickness maps of a representative case before and 6 months after CXL.
Epithelial Thickness Measurements (μm) Before and After CXL to Treat Keratoconus
Representative epithelial thickness maps obtained with spectral-domain optical coherence tomography show some regularization of the epithelial thickness profile across the central 5 mm of the cornea after epithelial removal and regeneration in association with corneal cross-linking. (A) Preoperative image. (B) Image of the same eye taken 6 months after corneal cross-linking.
At baseline, the difference between the minimum and maximum epithelial thickness readings within the central 5 mm of the cornea were strongly positively correlated with the maximum keratometry reading (R2 = 0.38, Figure 1). Linear regression analysis showed that an increase of 1.00 D in maximum keratometry was associated with an increase of 0.7 μm in the epithelial thickness difference (Figure 1C). At 6 months after CXL, the epithelial thickness difference also correlated strongly with the maximum keratometry reading (R2 = 0.59; meaning 59% of the variance in the epithelial thickness range was explained by the variance in maximum keratometry). The mean reduction of 2 μm in the epithelial maximum–minimum thickness range at 6 months after CXL was somewhat greater than would have been predicted based simply on the 1.10 D mean reduction in maximum keratometry after CXL, considering the linear regression correlation coefficient was 0.71 (Figure 1C).
In keratoconic eyes imaged prior to cross-linking, the maximum keratometry reading from Scheimpflug imaging was negatively correlated with the minimum corneal epithelial thickness (panel A), positively correlated with the maximum epithelial thickness (panel B), and positively correlated with the maximum–minimum epithelial thickness difference in the central 5 mm of the cornea (panel C), as evaluated with spectral-domain optical coherence tomography imaging and associated epithelial mapping software.
In this study, we evaluated changes in the epithelial thickness profile in the central 5 mm of the cornea after epithelial removal and regeneration associated with epithelium-off CXL in eyes with progressing keratoconus. We found that the epithelial maximum–minimum thickness range detected by SD-OCT was strongly positively correlated with Scheimpflug measurements of maximum keratometry at both baseline and 6 months after CXL. We also found that a modest reduction in maximum keratometry after CXL was associated with a modest reduction in the epithelial maximum–minimum thickness range. This modest reduction in epithelial masking could result in a slight underestimation of the true change in the maximum keratometry of the underlying stromal surface when comparing corneal tomography or topography images taken before and after CXL.
The epithelial thickness is more evenly distributed in the central 10 mm of the cornea in normal eyes than it is in eyes with keratoconus. Using ultrasonic biomicroscopy, Reinstein et al. found that the mean epithelial thickness was 53.4 ± 4.6 μm in normal eyes.21 The corneal epithelium was slightly thinner in the center and superiorly than inferiorly and peripherally, with minimal variations between the different regions of the surface.5,22–24 Rein-stein et al.4 also observed a so-called “doughnut pattern” on the eyes with keratoconus. The epithelium was thinner over the protruding area of the stroma, which usually coincided with the maximum elevation of the posterior surface of the cornea, and the epithelium was thicker in the surrounding mid-peripheral cornea. This finding is not pathognomonic of keratoconus, but was characteristic in the ectasia cases evaluated. The epithelial thickness variation tended to increase as keratoconus progressed. Our findings of a strong correlation between maximum keratometry and the epithelial maximum–minimum thickness difference support the findings by Reinstein et al.,3 who used different corneal topography and epithelial imaging devices (Atlas and Artemis ultrasound biomicroscopy).
Kanellopoulos and Asimellis likewise found with very high-frequency ultrasound (50 MHz) that epithelial thickness varied substantially in patients with keratoconus with a difference of up to 20 μm between various points of the same eye.25–27 They observed that often a thinner epithelium coincided with a thinner cornea. Their data comparing patients with keratoconus with controls suggested an overall thickening of the epithelium throughout the corneal surface in keratoconic eyes. This finding was described as a possible early keratoconus change and named “reactive” epithelial hyperplasia that was suspected to denote changes consistent with corneal biomechanical instability in keratoconus because the overall epithelial thickening dropped to subnormal in keratoconic eyes that had undergone CXL.25–27
Using SD-OCT, we measured a minimum epithelial thickness of 41.0 ± 6.1 μm in keratoconic eyes at baseline; this was similar to the minimum epithelial thickness measured by Rocha et al.22 in keratoconic eyes using the same imaging device (41.2 ± 6.5 μm). Rocha et al. found that the minimum epithelial thickness was significantly lower in keratoconic eyes than it was in normal eyes (50.45 ± 3.9 μm) or in eyes with ectasia after refractive surgery (46.5 ± 6.7 μm, P < .0001 between groups).
Our study is consistent with studies that suggest some epithelial regularization occurs after CXL (ie, the maximum–minimum epithelial thickness difference is reduced).17,28 Rocha et al.17 observed a decrease in peripheral epithelial thickness and a decrease in surface epithelial variability (as determined by the standard deviation and the maximum–minimum thickness difference) at 3 months after CXL. Likewise, we detected a modest decrease in the peripheral epithelial thickness and a modest reduction in the epithelial thickness range (maximum–minimum) from −24.2 ± 9.2 μm preoperatively to −22.1 ± 9.4 μm at 6 months after CXL (P = .0023). In contrast, the maximum–minimum epithelial thickness difference in normal eyes is approximately 8 μm,29 much lower than that seen in our keratoconic eyes before or after CXL.
The study strengths included the prospective nature of the measurements, the relatively large sample size (93 patients), and the sampling of only one eye per patient to ensure independent sampling. The principal study limitation was the inherently lower accuracy when imaging keratoconic eyes compared with normal eyes.
We confirmed that the epithelial thickness range is strongly positively correlated with maximum keratometry in keratoconic eyes. The epithelial thickness range decreased modestly after CXL, suggesting some epithelial profile regularization. This modest reduction in epithelial masking could cause one to slightly underestimate the true change in the maximum keratometry of the underlying stromal surface when comparing corneal tomography or topography images taken before and after CXL.
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Epithelial Thickness Measurements (μm) Before and After CXL to Treat Keratoconusa
|Time||Thinnest Point||Thickest Point||Thickest–Thinnest Difference||Central Thickness (2-mm Diameter)||Inferior Thickness (2 to 5 mm)||Superior Thickness (2 to 5 mm)|
|Before treatment||41.0 ± 6.1||65.1 ± 6.3||24.2 ± 9.2||50.8 ± 5.5||52.1 ± 4.0||56.5 ± 4.6|
|3 months postoperative||43.0 ± 5.0||64.8 ± 7.7||21.8 ± 9.5||51.9 ± 6.9||52.3 ± 4.4||56.0 ± 4.6|
|6 months postoperative||41.9 ± 6.7||64.1 ± 6.9||22.1 ± 9.4||51.9 ± 5.6||51.2 ± 4.7||55.5 ± 5.4|
|P (baseline vs 6 months)||.12||.067||.0023||.042||.037||.026|
|Treatment target||Keratoconus or post-refractive surgery ectasia|
|Fluence (total) (J/cm2)||5.4|
|Soak time and interval (minutes)||30 (q2)|
|Treatment time (minutes)||30|
|Chromophore||Riboflavin (Avedro, Waltham, MA)|
|Light source||UV-X, IROC, Zurich, Switzerland|
Changes in Maximum Keratometry and Visual Acuity After CXL
|Time||Maximum Keratometry (D)||CDVA (logMAR)||UDVA (logMAR)|
|Before treatment||58.90 ± 7.90||0.30 ± 0.21||0.82 ± 0.48|
|3 months postoperative||58.40 ± 8.10||0.31 ± 0.22||0.76 ± 0.39|
|6 months postoperative||57.80 ± 7.90||0.26 ± 0.17||0.76 ± 0.42|
|P (baseline vs 6 months)||< .0001||.011||.063|