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Epithelial remodeling after partial topography-guided normalization and high-fluence short-duration crosslinking (Athens protocol): Results up to 1 year

Kanellopoulos, Anastasios John MD*; Asimellis, George PhD

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Journal of Cataract and Refractive Surgery: October 2014 - Volume 40 - Issue 10 - p 1597-1602
doi: 10.1016/j.jcrs.2014.02.036
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We previously reported overall reduced corneal epithelial thickness in keratoconic eyes that were treated with (1) excimer laser debridement of the top 50 μm of the epithelium, (2) partial topography-guided excimer ablation, and (3) immediate high-fluence ultraviolet-A radiation (10 mW/cm2) and short-duration (10 minutes) corneal collagen crosslinking (CXL) with riboflavin in a procedure known as the Athens protocol.1–3 Our goal was to arrest the keratectasia progression4 and provide a less irregular anterior corneal surface. In l study,1 which was performed using high-frequency scanning ultrasound biomicroscopy (UBM), the epithelial thickness in a group of untreated keratoconic eyes was compared with that in a group of keratoconic eyes treated using the Athens protocol.

Epithelial thickness assessment has been facilitated by the development of anterior segment optical coherence tomography (AS-OCT).5 Although there are studies of AS-OCT epithelium measurement in the peer-reviewed literature, until recently and to our knowledge, the methodology and instrumentation mainly used an on-screen caliper tool6; thus, only local point–thickness measurements were reported. The recent availability of in vivo, 3-dimensional (3-D) corneal epithelial mapping by AS-OCT in clinical practice7 allows easy capture of optical images and high-speed measurements conferred by Fourier-domain signal processing.8,9

This study used this new clinical modality to evaluate the longitudinal postoperative changes in epithelial thickness distribution as well as the epithelial layer topographic variability in a large group of keratoconic cases treated using the Athens protocol. The results in these eyes were compared with those in untreated keratoconic eyes and in healthy control eyes.

Patients and methods

This observational comparative prospective study received approval by the Ethics Committee, Institute, and adhered to the tenets of the Declaration of Helsinki. All patients provided informed written consent at the time of the first clinical visit. Exclusion criteria were systemic disease, previous corneal surgery, history of chemical injury or delayed epithelial healing, and pregnancy or lactation.

Patient Enrollment and Surgical Technique

Group A

In Group A, eyes were treated for keratoconus with the Athens protocol. All procedures were performed by the same surgeon (A.J.K.) using an EX500 excimer laser10 (Alcon Surgical, Inc.) with topography-guided custom partial ablation. Figure 1 shows an example of treatment planning, the distribution of the ablation depth, a preoperative axial curvature map, a postoperative axial curvature map, and the difference in axial curvature map between preoperatively and postoperatively. Immediately after surface normalization, accelerated CXL was applied using the KXL System (Avedro, Inc.). The patients were followed for up to 1 year.

Figure 1
Figure 1:
A: Preoperative tomographic (anterior corneal instantaneous [tangential]) map obtained via Scheimpflug imaging. Keratometric power reported in diopters. B: Corresponding postoperative map. C: Treatment planning showing ablation depth (in μm) and topographic distribution obtained via the refractive platform software. D: Difference between postoperative sagittal curvature map (B) and preoperative sagittal curvature map (A) (N = nasal; T = temporal).

Group B

Group B comprised eyes with keratoconus that had not received surgical treatment. Inclusion criteria were a clinical diagnosis of progressive keratoconus (confirmed by a complete ophthalmologic evaluation), minimum age 17 years, and corneal thickness of at least 300 μm. The keratoconus diagnosis was further confirmed using the Wavelight Oculyzer II (Alcon Surgical, Inc.) and the Pentacam high-resolution Scheimpflug imaging camera11 (Oculus Optikgeräte GmbH).

Group C

Group C, the control group, comprised unoperated normal eyes with no current or past ocular pathology other than refractive error and no present irritation or dry-eye disorder, all of which were confirmed during a complete ophthalmologic evaluation. Contact lens wearers were excluded from this group.

Imaging Instrumentation

The RTVue-100 Fourier-domain AS-OCT system (Optovue, Inc.), running on analysis and report software version A6 (9.0.27), was used in the study. Data output included total corneal and epithelial thickness maps corresponding to a 6.0 mm diameter area. In all cases, to avoid potential artifacts (eg, due to eyedrop instillation), OCT imaging preceded the ocular clinical examination and was performed by the same trained investigator. The settings were as follows: L-Cam lens and 8 radial meridional B-scans per acquisition consisting of 1024 A-scans each with a 5 μm axial resolution. These 8 radial meridional scans, all acquired in less than 0.5 second, were used by the system software to produce by interpolation 3-D thickness maps. Images with quality greater than 30, determined using the signal strength index parameter, were considered in the study. The signal strength index parameter measures the average signal strength across the scan. Two consecutive individual acquisitions were obtained in each case (eye) to ensure data validity; the mean value of 2 was used in this study.

Data Collection and Statistical Analysis

In Group A, the postoperative measurements were performed at 1 and 6 months as well as at 1 year. Imaging in Group B and Group C was performed during the first clinical visit.

The main analysis report produced by the AS-OCT system displayed total corneal (reported as pachymetry) and epithelial 3-D thickness maps covering the 6.0 mm diameter area. Corneal pachymetry was assessed by the central corneal thickness (CCT) and minimum corneal thickness. Epithelial thickness assessment comprised the following measurements: pupil center, superior, inferior, minimum, maximum, mean, peripheral, topographic thickness variability, and epithelial thickness range. These data were collected as follows (Figure 2): Each thickness map was divided into 17 sections (2.0 mm diameter pupil center disk of 12.56 mm2 area; 8 sectors [octants] within the annulus between the 2.0 mm and 5.0 mm zones, each of 8.24 mm2 areas; and 8 sectors [octants] within the annulus of the 5.0 to 6.0 mm zones, each of 4.32 mm2 areas). For each of these sections, the mean epithelial thickness was displayed numerically in integer form with a minimum difference of 1 μm over the corresponding area.

Figure 2
Figure 2:
Comparative AS-OCT epithelial thickness (μm) 3-D maps shows an image from Group A taken 1 year postoperatively and an image from Group B (I = inferior; IN = inferior–nasal; IT = inferior–temporal; N = nasal; S = superior; SN = superior–nasal; ST = superior–temporal; T = temporal).

In this study, the reported center epithelium thickness was taken from the integer indication over the center 2.0 mm disk. The mean epithelial thickness was computed by the mean of all segments, and the peripheral epithelial thickness was computed by the mean of the thickness corresponding to 18 equispaced points along the 5.0 mm radius (data harvested by mouse-over indication over the epithelial thickness map). The superior, inferior, minimum, maximum, and topographic epithelial thickness variability (computed by the standard deviation [SD] of the 17 thickness values) were provided in tabular form by the software of the AS-OCT device (Figure 2). The thickness range was computed as follows: minimum epithelial thickness – maximum epithelial thickness.

Descriptive statistics, linear regression analysis to look for possible correlations, paired analysis t tests, and analysis of variance were performed using Minitab software (version 16.2.3, Minitab, Ltd.) and Origin Lab software (version 9, Originlab Corp.). Paired-analysis P values less than 0.05 were considered an indication of statistically significant results.


Table 1 shows the CCT, minimum corneal thickness, epithelial thicknesses, topographic thickness variability, and epithelial thickness range measured by AS-OCT in the 3 groups.

Table 1
Table 1:
Central corneal thickness (CCT), minimum corneal thickness, epithelial thicknesses, topographic thickness variability, and epithelial thickness range measured by the AS-OCT in the 3 groups. All units are in microns.

Group A (Athens protocol) comprised 175 eyes, 74 of women and 101 of men. The mean patient age at the time of surgery was 26.8 years ± 7.2 (SD) (range 18 to 48 years). There were 87 right eyes and 88 left eyes. The Athens protocol treatment was uneventful in all cases.

Group B (untreated keratoconic) comprised 193 eyes, 92 of women and 101 of men. The mean patient age at the time of examination was 31.1 ± 9.9 years (range 18.0 to 51.0 years). There were 91 right eyes and 102 left eyes.

Group C (control) comprised 160 eyes, 67 of women and 93 of men. The mean patient age at the time of examination was 35.45 ± 9.55 years (range 18.0 to 52.0 years). There were 74 right eyes and 86 left eyes.

Epithelial Thickness

In Group A, the difference in the center epithelial thickness between each postoperative timepoint was statistically significant (all P<.05). The difference in the mean center epithelial thickness (−4.31 μm; 95% confidence interval [CI], −6.31 to −2.30) between Group A 1 year after treatment and Group B at the time of examination was statistically significant (P<.05, 2-sample t test). The difference in the mean center epithelial thickness (−4.75 μm, 95% CI, −6.59 to −2.92) between Group A 1 year after treatment and Group C at the time of examination was also statistically significant (P<.05) (Figure 3).

Figure 3
Figure 3:
Mean and center epithelial thicknesses in the 3 groups. Error bars correspond to the SD (KCN = keratoconus, no treatment).

In Group A, the difference in topographic thickness variability between each postoperative timepoint was statistically significant (all P<.05). Figure 4 shows the epithelial thickness variability and range by group.

Figure 4
Figure 4:
Epithelial thickness variability and range in the 3 groups. Error bars correspond to the SD (KCN = keratoconus, no treatment).


Until recently, high-frequency UBM had been the gold standard for in vivo corneal epithelial 3-D imaging.12 The recent, rapid development and current high-speed imaging capabilities of AS-OCT13–15 have made acquisition of in vivo 3-D pachymetry corneal maps reliable and fast.16–19 Software refinement also enables clinical assessment of corneal asymmetry and focal thinning parameters for keratoconus classification.20 In addition, the higher axial resolution, increased accuracy, and finer image-processing capabilities of the current AS-OCT imaging systems have enabled, among other things, 3-D imaging of epithelial thickness.7

Epithelial thickness and irregularity indices (eg, center and mean epithelial thickness, epithelial thickness topographic irregularity, and thickness range) measured quantitatively with AS-OCT can serve as possible indicators of cornea instability, including ectasia and keratoconus.14 In this study, we evaluated these parameters with a Fourier-domain AS-OCT system in a large group of keratoconic patients who had combined treatment of excimer laser anterior surface normalization and simultaneous high-fluence accelerated CXL. This study adds new information based on its large group of treated keratoconic eyes and its comparison with untreated keratoconic eyes and healthy eyes. In addition, our study was performed with a commercially available AS-OCT system whose use may become more widespread in clinical settings.

Our findings confirm compensatory epithelial thickness changes previously described after various refractive corneal ablation procedures.21,22,A

The epithelial thickness and irregularity assessment in the Athens protocol–treated Group A suggests short-term variability in corneal thickness distribution between the third month and the sixth month.3 Specifically, in our study, the corneal and epithelial thickness distributions were characterized by large deviations that gradually became less irregular. The mean SD at the center epithelial thickness of 7.36 μm at 1 month gradually decreased to 6.80 at 3 months and to 4.57 μm at 1 year. The SD in Group B (untreated keratoconic) and in Group C (control) was 6.79 μm and 3.23 μm, respectively. In addition to fluctuating less between different eyes, the epithelial thickness in Group A progressed toward a reduced mean topographic variability and mean thickness range (from 5.43 μm and −24.69 μm to 4.64 μm and −19.94 μm, respectively). Both metrics were more regular than in Group B (5.77 μm and −21.83 μm, respectively). These results indicate that the epithelial thickness distribution was more uniform in the Athens protocol–treated group than in the untreated group of keratoconic eyes and had less overall thickness, as suggested by the reduced mean and center thickness values.

The findings in the current study agree with those in our previous study1; that is, although an overall thicker epithelium with large variations can be observed clinically and topographically in eyes with keratoconus, in eyes treated with CXL the variability in epithelium thickness and topographic thickness decreased by a statistically significant margin and was more uniform. We have theorized that epithelial hyperplasia in biomechanically unstable corneas (ie, increased epithelial regrowth activity) might be associated with a more elastic cornea.1 The laboratory and clinical findings of increased corneal rigidity after CXL are widely accepted,23–25 including in studies of accelerated high-fluence CXL.26

In conclusion, we present the results in a comprehensive study of the postoperative development of corneal epithelial thickness distribution after keratoconus management using combined anterior corneal normalization by topography-guided excimer ablation and accelerated CXL. The epithelial healing processes can be monitored by AS-OCT with ease in a clinical setting, expanding the clinical application of this technology. Our findings suggest less topographic variability and overall reduced epithelial thickness distribution in keratoconus eyes treated with CXL using the Athens protocol.

What Was Known

  • Postoperative epithelial remodeling after partial anterior surface normalization with an excimer laser and high-fluence CXL, assessed with high-frequency scanning UBM, results in reduced overall epithelial thickness and topographic variability.

What This Paper Adds

  • Detailed follow-up of Athens protocol–treated eyes up to 1 year confirmed previous ultrasound findings of the overall thinner and smoother epithelial thickness profiles compared with the profiles of untreated keratoconic eyes.


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Other Cited Material

A. Reinstein DZ, Aslanides IM, Patel S, Silverman RH, Coleman DJ, “Epithelial Lenticular Types of Human Cornea: Classification and Analysis of Influence on PRK,” poster presented at the annual meeting of the American Academy of Ophthalmology, Atlanta, Georgia, USA, October 1995. Abstract available in: Ophthalmology 1995; 102(suppl):S156
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