Corneal cross-linking (CXL) is a photochemical reaction introduced at Dresden University to treat progressive keratoconus via an increase in corneal stiffness.1 However, the pathogenesis of keratoconus remains unclear. It has been suggested that the primary event is the loss and/or slippage of collagen fibrils and changes to the extracellular matrix in the corneal stroma.2 These alterations induce a focal biomechanical instability of the corneal stroma with consequent changes in the cornea's anatomical and topographic architecture.3
CXL treatment appears to lead to the creation of covalent cross-links in the cornea with increasing biomechanical stiffness, as suggested by several in vitro studies.4–6 Conversely, the in vivo evaluation of CXL in the first postoperative months has been a complex issue due to the well-known decrease of corneal thickness and increase in curvature.7–10 The demarcation line, which is a white line visible with anterior segment optical coherence tomography (AS-OCT), has been proposed to be a good indicator of the efficacy of the procedure in the first months,11,12 but it is not present in all protocols.12,13 The ideal way of judging the outcome of CXL would be to directly assess its stiffening effect.
The Corvis ST (Oculus Optikgeräte GmbH; Wetzlar, Germany) is an ultra-high speed Scheimpflug camera coupled with a non-contact tonometer, and uses the acquired images to produce estimates of intraocular pressure (IOP) and dynamic corneal response parameters (DCRs).14,15 The DCR indices frequently used are the inverse integrated concave radius (1/R), deformation amplitude ratio (DA Ratio), and stiffness parameter applanation 1 (SP-A1). The 1/R and DA Ratio are particularly useful measures of corneal biomechanics because they are less dependent on IOP,16 which can slightly increase after CXL due to the use of steroids.17
Several studies have demonstrated the role of these DCRs in assessing the in vivo biomechanical properties of the cornea14,16,18 and in the diagnosis and treatment of patients with keratoconus.19–21 In particular, changes in these DCRs may be evident before corneal shape modifications have occurred.22
The aim of this study was to investigate whether there was an association between the stiffening effect following CXL evident by the change in DCRs and the demarcation line depth.
Patients and Methods
In this prospective, single center, clinical study, we included 66 eyes of 66 patients that were treated with CXL at the Royal Liverpool University Hospital. The inclusion criteria for CXL were a documented progression of keratoconus based on a change in the curvature within the cone area of at least 1.00 diopter (D) on instantaneous map and or a thinning of more than 20 µm in minimum corneal thickness measured with Pentacam (Oculus Optikgeräte GmbH) at least 3 months apart.9,23 Exclusion criteria were previous herpetic keratitis, dry eye, severe corneal infection, and simultaneous ocular or systemic autoimmune disease. Additional exclusion criteria were pregnancy or breastfeeding, the presence of central or paracentral opacities, and the use of rigid contact lenses for more than 4 weeks before the baseline evaluation. Approval of the Royal Liverpool University Hospital Internal Review Board ethics committee was obtained and the study was conducted in accordance with the standards set in the 1964 Declaration of Helsinki, revised in 2000.
We previously described the CXL protocol used in our center.22,24 Briefly, it involves removal of the corneal epithelium with 20% alcohol followed by 15 minutes of 0.1% riboflavin (VibeX Rapid; Simovision, Overijse, Netherlands). Subsequently, corneas are irradiated with ultraviolet-A 365-nm light using Avedro's KXL machine (Avedro Inc., Waltham, MA) at an irradiance of 6 mW/cm2 for 15 minutes, delivering a total of 5.4 J/cm2. Postoperative care includes chloramphenicol ointment until complete epithelial healing followed by dexamethasone drops (Table 1).
At baseline (day of CXL) and 1 month after CXL, corrected distance visual acuity (CDVA), slit-lamp biomicroscopy, biomechanical IOP15 (bIOP), dilated funduscopy, DCRs provided by the Corvis ST, and AS-OCT using CASIA SS-1000 (Tomey Corporation, Nagoya, Japan) were assessed. The bIOP was derived by finite element simulations that take into account the influence of CCT, age, and DCRs.15,25
The outcome measures were the changes in the DCRs following CXL, specifically 1/R, DA Ratio, and SP-A1. A summary of parameters provided by the Corvis ST used in the study is presented in Table A (available in the online version of this article).
Corvis ST Parameters
AS-OCT was performed independently by two corneal fellows (RV and AT). The image was captured when the corneal reflex was visible and the demarcation line identified as an enhanced line in the stroma. After detection of the demarcation line, its depth was measured using the caliper tool provided by the manufacturer. The demarcation line depth was measured by placing the caliper tool in the posterior edge of the hyperreflective line in the central cornea using the same magnification.
Statistical analysis was performed using the SPSS statistical software (version 24.1; IBM Corporation, Armonk, NY) and Microsoft Excel 2016 software (Microsoft Corporation, Redmond, WA). Data are described as mean ± standard deviation. A paired t test was used to test the change in 1/R, SP-A1, and DA Ratio after CXL. The Wilcoxon signed-rank test was used to evaluate the change in bIOP.
To assess the association between demarcation line depth and stiffening effect, 1/R was used as a main outcome measure to avoid multiple tests. Additionally, instead of using the demarcation line depth, the ratio with postoperative pachymetry was employed to take into account the thickness of the cornea, which is known to affect corneal stiffness.26
A general linear model was employed with change in 1/R as the dependent variable, sex and laterality as fixed factors, and age, demarcation line ratio, change in corneal thickness, preoperative 1/R, and preoperative maximum keratometry as covariates. A P value of less than .05 was considered significant.
Sixty-six eyes of 66 patients (50 men and 16 women, 34 left eyes [51.5%], age 24 ± 6 years) were evaluated. No patients were lost to follow-up. There was no significant difference between the preoperative (56.65 ± 7.91 D) and postoperative (57.10 ± 8.10 D) Pentacam-derived maximum keratometry/steepest point (P = .72). The preoperative and postoperative thinnest corneal thicknesses were 489.8 ± 34.67 and 466.36 ± 34.01 µm, respectively (P < .01). There was a significant difference between preoperative (13.3 ± 2.34 mm Hg) and postoperative (17.06 ± 4.43 mm Hg) bIOP (P < .001).
DCRS After CXL
The DCRs after CXL displayed an increase in corneal stiffness evident by a significant rise in the SP-A1 (P =.002) and a significant decrease in 1/R and DA Ratio (P < .001 for 1/R and P = .005 for DA Ratio) (Table 2, Figure 1).
DCR Parameters Before and After CXL
Distribution of the change (delta) of inverse radius.
Table 2 shows the details of each of the DCRs before and after CXL.
Demarcation Line Depth and Corneal Stiffening
The mean demarcation line depth was 207 ± 82 µm (range: 0 to 389 µm). There were no significant associations between the change in 1/R (1/R used as main outcome measure of the stiffening effect) and the demarcation line ratio (P = .46), and no significant associations with age (P = .33), sex (P = .11), preoperative maximum keratometry (P = .10), or laterality (P = .82). This is evident in Figure 2, which shows a plot between the change in 1/R, demarcation line ratio, and postoperative pachymetry (demarcation line ratio R2 = 0.002 and P = .75, Figure 2).
Scatter plot of the ratio between demarcation line (DLratio) and postoperative pachymetry (x axis) and change (delta) of the inverse radius (y axis) (R2 = .002 and P = .75).
There was a significant association between the preoperative 1/R and the change in 1/R (P < .0001). This is evident in Figure 3, which shows a plot between the change in 1/R and the preoperative 1/R; stiffer corneas (lower values of 1/R) were less affected than less stiff corneas (R2 = 0.23, P < .0001). There was also a small but significant association (P = .02, R2 = .083) between the change in 1/R and the change in corneal thickness.
Linear, logarithmic, and inverse curve analysis between the change (delta) of inverse radius and the preoperative inverse radius (R2 = .23, P < .01). Black line shows no change (delta = 0) and the red lines show ±1.96 standard deviations from the mean change in inverse radius.
The possibility of in vivo evaluation of corneal biomechanics after CXL has been debated in the past decade. The first instrument available on the market, the Ocular Response Analyzer (ORA; Reichert Ophthalmic Instruments, Depew, NY), was not able to demonstrate, in a repeatable way, the stiffening effect in vivo.27,28
The Corvis ST that was later introduced differs from the ORA because, through the high-speed Scheimpflug camera, it is capable of obtaining images throughout the deformation phases. Because of this advantage, the DCRs of the Corvis ST have been shown to provide information that is useful for the evaluation of corneal biomechanics in patients with keratoconus.16,19,20,27 We recently reported that these changes in corneal biomechanics following CXL are measurable before corneal shape modifications are evident.22
It has been suggested that the demarcation line provides an indirect measure to evaluate the effect of CXL, with the assumption that the “deeper the demarcation line is, the better the CXL effect.”11
Given this background, the aim of our study was to evaluate whether there is any correlation between the demarcation line depth and the stiffening effect after CXL evaluated by changes in the DCRs. We used the change in the inverse 1/R as a measure of the corneal stiffening effect following CXL.16,22 The demarcation line ratio was used instead of demarcation line depth to control for changes in corneal thickness, which has an effect on corneal stiffness. Using these parameters, we were not able to demonstrate any significant association between demarcation line ratio and change in corneal stiffness following CXL measured as a change in 1/R.
An important finding was the significant association between the change in 1/R and the preoperative 1/R, suggesting that stiffer corneas (lower values of 1/R) were less affected than less stiff corneas. This finding might explain the variability in the stiffening effect following CXL as evident in Figure 1 and could lead to the hypothesis that some keratoconic corneas, even if they are progressing, are already relatively stiff and may not stiffen further following CXL. More studies will be needed to evaluate this finding. Although the stiffening effect was demonstrated by a significant increase of SP-A1 and a significant decrease of 1/R and DA Ratio, not all corneas stiffened after CXL.
Our results suggest that although demarcation line is associated with CXL, its location does not appear to be related to corneal stiffening. A recent study showed no significant correlation between the demarcation line depth and the change in keratometry values after 12 months of follow-up.29 Furthermore, iontophoresis transepithelial CXL has been shown to reduce the progression of keratoconus and does not induce any demarcation line.12,30 It is unclear whether the demarcation line reflects the depth of the treatment or a wound healing effect.11
Our findings need to be interpreted with caution. The DCRs are indices that are correlated with biomechanical changes; they do not measure them directly. An interesting option would be to assess the correlation between the increase of Brillouin modulus31 and the demarcation line depth.
Further limitations of this study were the relatively small number of patients and the short-term follow-up. With a longer follow-up, it would be possible to detect whether the change in DCRs following CXL is associated with the improvement in corneal shape indices (such as maximum keratometry). Another study is currently in progress to assess the correlation between change in corneal biomechanics after CXL and the flattening effect at 12 months.
In the current study, we can be confident (80% power) that we would have detected a high (80%) association between the change in 1/R and demarcation line ratio if it existed. However, the sample size and hence the power of this study would be insufficient to be confident that a lower association might not have been detected. The clinical significance of the latter would seem minimal, given that there was an extremely low (R2 = .002) and non-significant association.
Despite these limitations, our results would indicate that there is no significant association between the change in stiffening induced by CXL and the demarcation line depth.
- Wollensak G, Spoerl E, Seiler T. Riboflavin/ultraviolet-a-induced collagen crosslinking for the treatment of keratoconus. Am J Ophthalmol. 2003;135: 620–627. doi:10.1016/S0002-9394(02)02220-1 [CrossRef]
- Meek KM, Tuft SJ, Huang Y, et al. Changes in collagen orientation and distribution in keratoconus corneas. Invest Ophthalmol Vis Sci. 2005;46:1948–1956. doi:10.1167/iovs.04-1253 [CrossRef]
- Roberts CJ. Biomechanics in keratoconus. In: Barbara A, ed. Textbook of Keratoconus: New Insights. New Delhi: Jaypee Brothers Medical Publishers; 2012:29–32. doi:10.5005/jp/books/11483_5 [CrossRef]
- Spoerl E, Wollensak G, Seiler T. Increased resistance of cross-linked cornea against enzymatic digestion. Curr Eye Res. 2004;29:35–40. doi:10.1080/02713680490513182 [CrossRef]
- Schumacher S, Mrochen M, Wernli J, Bueeler M, Seiler T. Optimization model for UV-riboflavin corneal cross-linking. Invest Ophthalmol Vis Sci. 2012;53:762–769. doi:10.1167/iovs.11-8059 [CrossRef]
- Wollensak G, Iomdina E. Biomechanical and histological changes after corneal crosslinking with and without epithelial debridement. J Cataract Refract Surg. 2009;35:540–546. doi:10.1016/j.jcrs.2008.11.036 [CrossRef]
- Caporossi A, Mazzotta C, Baiocchi S, Caporossi T. Long-term results of riboflavin ultraviolet a corneal collagen cross-linking for keratoconus in Italy: the Siena Eye Cross Study. Am J Ophthalmol. 2010;149:585–593. doi:10.1016/j.ajo.2009.10.021 [CrossRef]
- Raiskup-Wolf F, Hoyer A, Spoerl E, Pillunat LE. Collagen cross-linking with riboflavin and ultraviolet-A light in keratoconus: long-term results. J Cataract Refract Surg. 2008;34:796–801. doi:10.1016/j.jcrs.2007.12.039 [CrossRef]
- Vinciguerra R, Romano MR, Camesasca FI, et al. Corneal cross-linking as a treatment for keratoconus: four-year morphologic and clinical outcomes with respect to patient age. Ophthalmology. 2013;120:908–916. doi:10.1016/j.ophtha.2012.10.023 [CrossRef]
- Vinciguerra P, Mencucci R, Romano V, et al. Imaging mass spectrometry by matrix-assisted laser desorption/ionization and stress-strain measurements in iontophoresis transepithelial corneal collagen cross-linking. Biomed Res Int. 2014;2014:404587. doi:10.1155/2014/404587 [CrossRef]
- Spadea L, Tonti E, Vingolo EM. Corneal stromal demarcation line after collagen cross-linking in corneal ectatic diseases: a review of the literature. Clin Ophthalmol. 2016;10:1803–1810. doi:10.2147/OPTH.S117372 [CrossRef]
- Vinciguerra P, Romano V, Rosetta P, et al. Transepithelial iontophoresis versus standard corneal collagen cross-linking: 1-year results of a prospective clinical study. J Refract Surg. 2016;32:672–678. doi:10.3928/1081597X-20160629-02 [CrossRef]
- Vinciguerra P, Randleman JB, Romano V, et al. Transepithelial iontophoresis corneal collagen cross-linking for progressive keratoconus: initial clinical outcomes. J Refract Surg. 2014;.30:746–753. doi:10.3928/1081597X-20141021-06 [CrossRef]
- Ambrósio R Jr, Lopes B, Faria-Correia F, et al. Ectasia detection by the assessment of corneal biomechanics. Cornea. 2016;35:e18–e20. doi:10.1097/ICO.0000000000000875 [CrossRef]
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- Kanellopoulos AJ, Cruz EM, Ang RE, Asimellis G. Higher incidence of steroid-induced ocular hypertension in keratoconus. Eye Vis (Lond). 2016;3:4. doi:10.1186/s40662-016-0035-9 [CrossRef]
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|Fluence (total) (mJ/cm2)||5.4|
|Soak time and interval (minutes)||15|
|Treatment time (minutes)||15|
|Chromophore carrier||Hydroxypropyl methylcellu-lose (HPMC)|
|Chromophore osmolarity||Slightly hypotonic|
|Light source||UV-A KXL system (Avedro Inc, Waltham, MA)|
|Irradiation mode (interval)||Continuous|
|Abbreviation in the manuscript||CXL|
DCR Parameters Before and After CXL
|Parameter||Mean ± SD||P|
|Stiffness Parameter A1||.002|
| Preoperative||60.3 ± 18.8|
| Postoperative||66.5 ± 19.1|
|Deformation Amplitude Ratio||.005|
| Preoperative||6.01 ± 1.32|
| Postoperative||5.63 ± 1.20|
|Inverse Integrated Concave Radius||< .0001|
| Preoperative||11.92 ± 2.36|
| Postoperative||11.0 ± 2.13|
Corvis ST Parameters
|Deformation Amplitude Ratio (DA Ratio)||The deformation amplitude measured at 1 or 2 mm from the center of the cornea in the nasal-temporal region.|
|Inverse Integrated Concave Radius||The inverse function of the radius of curvature at the highest concavity state (1/R) is plotted and the integrated sum is calculated between the first and second applanation events.|
|Biomechanical IOP||An estimate of corrected intraocular pressure (IOP) that is able to compensate for central corneal thickness, age, and biomechanics.|
|Stiffness Parameter at A1||Resultant pressure (adjusted IOP at A1 minus bIOP) divided by deflection amplitude at applanation 1 or highest concavity. Therefore, stiffness is measured by force divided by displacement.|