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Corneal stromal demarcation line after 4 protocols of corneal crosslinking in keratoconus determined with anterior segment optical coherence tomography
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ARTICLE
Corneal stromal demarcation line after 4 protocols of corneal crosslinking in keratoconus determined with anterior segment optical coherence tomography Leopoldo Spadea, MD, Lucia Di Genova, MD, Emanuele Tonti, MD
Purpose: To use anterior segment optical coherence tomography (AS-OCT) to compare corneal stromal demarcation line depth after 4 treatment protocols of corneal crosslinking (CXL).
Setting: Eye Clinic, Sapienza University of Rome, Terracina (Latina), Italy. Design: Prospective case series. Methods: Patients with progressive keratoconus were delegated to one of the following CXL treatments: (1) conventional epithelium (epi)-off 3 mW/cm2 according to the standard Dresden protocol (CCXL group), (2) accelerated epi-off 10 mW/cm2 (A-CXL group), (3) transepithelial epi-on 3 mW/cm2 (TE-CXL group), or (4) transepithelial epi-on by iontophoresis 10 mW/cm2 (I-CXL group). Two independent observers measured the corneal stromal demarcation line using AS-OCT.
identified on AS-OCT scans in 109 eyes (90.8%). One month after the treatment, the mean stromal demarcation line depth was 275.05 mm G 41.83 (SD) in the C-CXL group, 279.35 G 33.07 mm in the A-CXL group, 132.60 G 22.14 mm in the TE-CXL group, and 235.40 G 37.08 mm in the I-CXL group. The difference in stromal demarcation line depth was not statistically significant between the C-CXL and A-CXL group, but it was statistically significant (P < .05) between the epi-off and epi-on CXL groups and between the 2 epi-on groups, where the demarcation line was significantly deeper in the I-CXL group than in the TE-CXL group.
Conclusion: The corneal stromal demarcation line was significantly deeper after epi-off 30-minute standard CXL treatment and after epi-off 9-minute accelerated CXL with high-intensity ultraviolet-A irradiation.
Results: The study comprised 70 patients (120 eyes, 30 eyes in each group). The corneal stromal demarcation line was
K
eratoconus is a progressive bilateral multifactorial disorder involving complex interactions of both genetic and environmental factors and is characterized by ectasia of the central or paracentral regions of the cornea. This process results in irregular astigmatism and impaired vision.1 The incidence of keratoconus is 1.3 to 22.3 new cases per 100 000 people every year, with a prevalence of 1 in 2000 and with nearly 135 000 cases in the United States.2,3 Keratoconus is associated with atopic disease, connective tissue disorders, and Down syndrome; and patients whose ethnicity derives from the Middle East and Asia might also carry an increased risk for the condition.4,5 Corneal crosslinking (CXL) is a surgical technique used to increase corneal biomechanical strength. Corneal crosslinking relies on the use of riboflavin as a photosensitizer
J Cataract Refract Surg 2018; 44:596–602 Q 2018 ASCRS and ESCRS
and ultraviolet-A (UVA) light to promote the formation of intrafibrillar and interfibrillar covalent bonds by photosensitized oxidation.6 This technique can delay the progression of corneal steepening, potentially stabilizing the cornea, and improve refractive error by reducing the irregular astigmatism caused by the biochemical instability of the cornea. The major indication for CXL is to inhibit the progression of corneal ectasia secondary to conditions such as keratoconus, pellucid marginal degeneration, or iatrogenic corneal ectasias.7,8 Histopathological studies after CXL show keratocyte apoptosis limited to the anterior stroma after only 24 hours, with an increased density of the extracellular matrix in the stroma.9 This leads to the formation of a “demarcation line,” a transition zone between crosslinked stroma and untreated tissue that can be appreciated on slitlamp evaluation
Submitted: September 6, 2017 | Final revision submitted: February 13, 2018 | Accepted: February 13, 2018 From the Department of Biotechnology and Medical-Surgical Sciences, Sapienza University of Rome, Terracina (Latina), Italy. Corresponding author: Leopoldo Spadea, MD, Via Benozzo Gozzoli 34, 00142 Rome, Italy. Email: [email protected]. Q 2018 ASCRS and ESCRS Published by Elsevier Inc.
0886-3350/$ - see frontmatter https://doi.org/10.1016/j.jcrs.2018.02.017
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as early as 2 weeks after the intervention.10 The present study analyses the depth of the demarcation line for 4 different protocols of CXL. PATIENTS AND METHODS This prospective interventional study was approved by the ethics committee of the Department of Biotechnology and MedicalSurgical Science, Sapienza University of Rome, and was comprised of patients affected by progressive keratoconus who had CXL between June 2013 and March 2016 at the Eye Clinic of Sapienza, University of Rome, Terracina (Latina), Italy. All patients were presented with the risks and benefits of the procedures and provided an informed written consent. The study included eyes with progressive keratoconus of patients with no other systemic or ocular disease. Inclusion criterion was keratoconus with documented progression in the past 12 months. Progression was defined as an increase in maximum keratometry (K) of 1.00 diopter (D) or more in the previous 12 months and patient-reported deterioration of visual acuity (excluding other possible non-corneal-related reasons for deterioration). Exclusion criteria were corneal opacity, previous herpetic keratitis, previous ocular surgery, active ocular infection, autoimmune disease, diabetes, pregnancy, and lactation. The clinical diagnosis of keratoconus was facilitated by Scheimpflug camera–Placido corneal topography (Sirius, Costruzione Strumenti Oftalmici). Diagnostic criteria parameters were mean K, topographic astigmatism, thinnest pachymetry, corneal volume, maximum K, and significant higher-order aberrations levels. Furthermore, clinical indicators such as irregular astigmatism, reduced corrected distance visual acuity (CDVA), reduced contrast sensitivity, and microscopic signs at slitlamp evaluation such as apical protrusion with corneal thinning and scarring, Vogt striae, and Fleischer ring were considered. Patients with progressive keratoconus were assigned to one of the following CXL techniques: conventional epithelium-off (epioff) 3 mW/cm2 (C-CXL group), accelerated epi-off 10 mW/cm2 (A-CXL group), transepithelial epithelium-on (epi-on) 3 mW/cm2 (TE-CXL group), or transepithelial epi-on iontophoresis 10 mW/ cm2 (I-CXL group) (Table 1). The entire population was assigned into epi-off or epi-on protocol based on corneal pachymetry value: epi-off CXL groups’ inclusion criteria were corneal thicknesses greater than 400 mm at the thinnest point; in contrast, all the eyes with pachymetric values less than 400 mm at the thinnest point were enrolled in the epi-on CXL group. Subsequently in epi-off groups, patients were randomly assigned to 1 of the 2 treatment protocols (conventional or accelerated); likewise, in epi-on groups patients were randomly assigned to the
transepithelial or iontophoresis CXL technique group. All surgical techniques were performed under sterile conditions by the same surgeon (L.S.). One drop of pilocarpine 1.0% was instilled 30 minutes preoperatively to constrict the pupil and reduce UVA irradiation to the lens and retina. Preoperatively, oxybuprocaine 0.4% drops, were instilled every 5 minutes for 15 minutes as topical anesthesia. The aiming beam was focused on the central cornea while the patient fixated on a pulsating green light. The 4 surgical techniques that were used in the different groups were as follows: Conventional epi-off 3 mW/cm2 After epithelium removal by using a blunt metal spatula to mechanically scrape off a 9.0 mm diameter area, the cornea was soaked for 15 minutes in riboflavin 0.1% solution (Ricrolin) and then exposed to a solid-state UVA illuminator (CBM Vega X-linker device, Costruzione Strumenti Oftalmici) for 30 minutes, during which the riboflavin solution was applied every 3 minutes. The UVA illuminator was calibrated to 3.0 mW/cm2 of surface irradiance (5.4 J/cm2 surface dose) using a UV light meter at the specified working distance. Accelerated epi-off 10mW/cm2 After epithelium removal by using a blunt metal spatula to mechanically scrape off a 9.0 mm diameter area, the cornea was soaked for 15 minutes in riboflavin 0.1% solution and then exposed to a solid-state UVA illuminator. Then, the corneas were irradiated by the device calibrated to 10 mW/cm2 (5.4 J/cm2 surface dose) and UVA irradiation was applied on the central 9.0 mm of the cornea for 9 minutes. During this time, the riboflavin solution was applied to the cornea every 3 minutes. Transepithelial epi-on 3mW/cm2 Without any deepithelialization, riboflavin 0.1% solution in 15% dextran T500 containing sodium ethylenediaminetetraacetic acid (EDTA) 0.01% and trometamol (Ricrolin TE) was instilled every 10 minutes for 2 hours. An eyelid speculum was inserted and riboflavin was instilled over the cornea every 3 minutes for 15 minutes. Ultraviolet-A irradiation was delivered using a solid-state UVA illuminator. The device was calibrated to 3.0 mW/cm2 of surface irradiance (5.4 J/cm2 surface dose) using an UV light meter at the specified working distance. Ultraviolet-A irradiation was applied to the central 9.0 mm of the cornea for 30 minutes and during this time, the riboflavin solution was instilled every 5 minutes. Transepithelial epi-on by Iontophoresis 10mW/cm2 In the I-CXL group, an 8.0 mm wide iontophoretic application device was placed on the corneal surface using an annular suction ring. The device was filled with approximately 0.5 mL solution riboflavin 0.1%, sodium EDTA 0.01%, and trometamol from the open proximal side until the electrode (stainless steel mesh) was covered.
Table 1. Patient characteristics at baseline. Parameter
C-CXL
A-CXL
TE-CXL
I-CXL
Number of eyes (patients) Age (y) Mean G SD Range Sex (F/M) Corneal thickness (mm) Mean G SD Range Epithelial thickness (mm) Mean G SD Range
30 (19)
30 (18)
30 (17)
30 (16)
31.08 G 6.60 18, 42 9/10
26.70 G 7.60 13, 46 6/12
37.00 G 5.60 32, 44 10/7
36.50 G 7.60 27, 44 8/8
436.14 G 38.8 402, 520
469.34 G 40.30 407, 542
389.20 G 29.03 369, 438
409.20 G 59.12 340, 497
43.12 G 4.50 34, 52
42.90 G 4.50 35, 55
37.75 G 7.50 33, 49
36.80 G 11.18 22, 48
A-CXL Z accelerated epithelium-off 10 mW/cm2; C-CXL Z conventional epithelium-off 3 mW/cm2; I-CXL Z transepithelial epithelium-on by iontophoresis 10 mW/cm2; TE-CXL Z transepithelial epithelium-on 3 mW/cm2
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The device was connected to the constant current generator (I-ON XL, Sooft Italia S.p.A.) for 5 minutes set at 1 mA (the total dose of 5 mA/5 min was monitored by the generator). Then, it was exposed to the solid-state UVA illuminator for 9 minutes, irradiating an area of 9.0 mm in diameter (energy delivered 10 mW/ cm2). During this time, the riboflavin solution was applied on the cornea every 5 minutes. Postoperatively, in all 4 groups, eyes were rinsed with a balanced salt solution and a therapeutic bandage soft contact lens was applied. An antibiotic regimen of ofloxacin drops and flurbiprofen drops was administered 4 times a day for 1 week. Successively, topical corticosteroid (clobetasone butyrate 0.1%) drops were administered for 1 month and gradually tapered. Patients received a comprehensive eye examination before and at 1, 3, 6, and 12 months after the surgery. During each visit, patients had slitlamp evaluations and anterior segment optical coherence tomography (AS-OCT) (RTVue, Optovue, Inc.). By using AS-OCT, the stromal demarcation line was detected within an enhanced image of the cornea on the horizontal meridian.11 The image was captured when the corneal reflex was visible and demarcation line depth was measured using the caliper tool option provided by the software. Two independent examiners (L.D.G., E.T.) analyzed the depth of the demarcation line in the center of the cornea from the epithelial side to the hyperreflective line into the stroma, comparing all cases after 1 month at the same postoperative period. To increase the accuracy of the analysis, measures of the demarcation line were scored into 3 groups as follows: (1) demarcation line not identifiable, (2) demarcation line visible but not clearly measurable, (3) clearly visible and measurable demarcation line. Only images included in the third group were included in the study. The demarcation line depth was also compared with the preoperative corneal pachymetry to assess the percentage volume of the treated cornea by multiplying the demarcation line depth value by a factor of 100 and then dividing the obtained result by the overall corneal pachymetry data in each individual case. Statistical analysis was performed using SPSS software (SPSS, Inc.). Data are shown as means G SD, and range. The nonparametric Mann-Whitney U test was used for statistical analysis. A P value less than 0.05 was considered statistically significant.
RESULTS The study comprised 120 eyes of 70 patients with progressive keratoconus. There were 30 eyes in the C-CXL group, 30 eyes in the A-CXL group, 30 eyes in the TE-CXL group, and 30 eyes in the I-CXL group (Table 1). None of the treated patients presented adverse events during the follow-up period. Most characteristics at baseline were similar between epi-off groups and between epion groups. The mean corneal thickness was different in the 4 groups according to the clinical indications for treatment (Table 1). It was not possible to observe the corneal demarcation line in 5 eyes (16.6%) treated with transepithelial epi-on and in 6 eyes (20.0%) treated with transepithelial epi-on by iontophoresis CXL. The demarcation line was clearly visible and measurable (score 3) in all the remaining cases (Figure 1). There was always a statistically significant agreement (P ! .05) between the measurements of the 2 independent observers. At the first follow-up, 1 month after the treatment, the mean depth of the stromal demarcation line in C-CXL and A-CXL groups was 275.05 mm G 41.8 and 279.34 G 33.06 mm, respectively, and in the TE-CXL and Volume 44 Issue 5 May 2018
Figure 1. High-resolution AS-OCT scan visualizing the corneal stromal demarcation line 1 month after CXL. Top left: Conventional epioff 3 mW/cm2. Central corneal demarcation line (yellow arrows) is visible to a depth of 271 mm. Top right: Accelerated epi-off 10 mW/cm2. The demarcation line (yellow arrows) is clearly visible to a depth of 315 mm. Bottom left: Transepithelial epi-on by iontophoresis 10 mW/cm2. The demarcation line (yellow arrows) is visible with a characteristic honeycomb pattern to a depth of 191 mm. Bottom right: Transepithelial epi-on 3 mW/cm2. The demarcation line (yellow arrows) is fairly visible to a depth of 110 mm (ASOCT Z anterior segment optical coherence tomography; CXL Z corneal crosslinking; epi-off Z epithelium-off; epi-on Z epithelium-on).
I-CXL groups, it was 132.60 G 22.14 mm and 235.40 G 37.08 mm, respectively (Figure 2). The demarcation line depth remained stable at the 1-year follow-up on the analysis of the 2 examiners; no patients were lost to follow-up. These findings evidenced that the demarcation line is statistically significantly deeper using epi-off techniques than with the epi-on techniques. The difference in demarcation line depth between C-CXL and ACXL groups was not statistically significant; however, it was statistically significant between TE-CXL and I-CXL groups (P ! .05) (Table 2). This study also related the demarcation line depth to the preoperative corneal pachymetry to assess the percentage volume of the treated cornea (Figure 3). Concerning this datum, no statistically significant difference was found between the treated cornea
Figure 2. The demarcation line depth (mm) in 4 CXL protocols in keratoconus determined with AS-OCT (A-CXL Z accelerated epithelium-off 10 mW/cm2 corneal crosslinking; AS-OCT Z anterior segment optical coherence tomography; C-CXL Z conventional epithelium-off 3 mW/cm2 corneal crosslinking; CXL Z corneal crosslinking; I-CXL Z transepithelial epithelium-on by iontophoresis 10 mW/cm2 corneal crosslinking; TE-CXL Z transepithelial epithelium-on 3 mW/cm2 corneal crosslinking).
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Table 2. Statistically significant differences in demarcation line values. P Value
Mean ± SD
Parameter DL depth (mm) DL (%) DL-epi (%)
1) C-CXL
2) A-CXL
3) TE-CXL
4) I-CXL
275.05 G 41.80 279.34 G 33.06 132.60 G 22.14 235.40 G 37.08 63.34 G 8.55 59.62 G 6.66 34.41 G 7.50 57.58 G 4.96 7.41 G 9.70 65.71 G 7.31 38.00 G 8.03 63.27 G 6.12
1 Vs. 2
1 Vs. 3
1 Vs. 4
2 Vs. 3
2 Vs. 4
3 Vs. 4
.661 .065 .111
.001* .001* .001*
.05† .105 .063
.001* .001* .001*
.05† .502 .506
.05† .05† .05†
A-CXL Z accelerated epithelium-off 10 mW/cm2; C-CXL Z conventional epithelium-off 3 mW/cm2; DL Z demarcation line; DL (%) Z percentage of the volume of the cornea treated evidenced by the demarcation line; DL-Epi (%) Z percentage of the volume of the cornea treated, excluding the preoperative epithelial thickness, evidenced by the demarcation line; I-CXL Z transepithelial epithelium-on by iontophoresis 10 mW/cm2; TE-CXL Z transepithelial epithelium-on 3 mW/cm2 *Statistically significant difference between the 2 groups (P ! .001) † Statistically significant difference between the 2 groups (P ! .05)
volume of each of the 2 epi-off groups (C-CXL and A-CXL), whereas a statistically significant difference was noted in comparison to the TE-CXL group (P ! .001). Moreover, a statistically significant difference was found between the TE-CXL group and the I-CXL group (P ! .05) (Table 2). In contrast to the different depth of the demarcation line, the volume of treated cornea did not differ statistically significantly between the C-CXL and A-CXL groups and between the 2 epi-off groups and the iontophoretic I-CXL group. To further assess the treated volume of the cornea, the preoperative epithelial thickness (evaluated by using the AS-OCT) was also excluded from the pachymetry measurements. However, the volume’s values of the treated cornea obtained excluding the preoperative epithelial thickness did not show consistent differences with those recorded with total pachymetry (Figure 4). Moreover, the epithelial thickness did not change significantly from the baseline to the last follow-up. DISCUSSION The effectiveness and safety of conventional CXL procedure, also known as the Dresden protocol, is widely established
Figure 3. Percentage of the volume of the cornea treated evidenced by the demarcation line in 4 CXL protocols in keratoconus determined with AS-OCT (A-CXL Z accelerated epithelium-off 10 mW/cm2 corneal crosslinking; AS-OCT Z anterior segment optical coherence tomography; C-CXL Z conventional epithelium-off 3 mW/cm2 corneal crosslinking; CXL Z corneal crosslinking; I-CXL Z transepithelial epithelium-on by iontophoresis 10 mW/cm2 corneal crosslinking; TE-CXL Z transepithelial epithelium-on 3 mW/cm2 corneal crosslinking).
and confirmed by several studies.12–15 The interaction between riboflavin 0.1% molecules absorbed in corneal tissue and UVA light rays irradiated at 3 mW/cm2 for 30 minutes (5.4 J/cm2 energy dose) releases reactive oxygen species that promote the formation of molecular bridges between and within collagen fibers.16 In the past few years, to minimize UV exposure duration, complication rate, patient discomfort, and possible adverse events, attention has focused on the possibility of creating new CXL protocols.17 The accelerated high-flow CXL, based on the principle of photochemical reciprocity, also known as the Bunsen-Roscoe law, is able to minimize the UV exposure and the patient discomfort associated with the duration of the CXL procedure.18 An adequate amount of riboflavin corneal absorption is required for achieving an effective CXL. Thus, the epithelium debridement in conventional CXL has the double goal of overcoming the barrier formed by corneal epithelium tight junctions that would limit the penetration of macromolecule as Vitamin B2 and avoid the absorption of UV rays by the epithelium.19 Different strategies have been proposed to enhance transepithelial riboflavin penetration, such as increasing riboflavin imbibition time and new riboflavin solution
Figure 4. Percentage of the volume of the cornea treated, excluding the preoperative epithelial thickness, evidenced by the demarcation line in 4 CXL protocols in keratoconus determined with AS-OCT (ACXL Z accelerated epithelium-off 10 mW/cm2 corneal crosslinking; AS-OCT Z anterior segment optical coherence tomography; CCXL Z conventional epithelium-off 3 mW/cm2 corneal crosslinking; CXL Z corneal crosslinking; I-CXL Z transepithelial epithelium-on by iontophoresis 10 mW/cm2 corneal crosslinking; TE-CXL Z transepithelial epithelium-on 3 mW/cm2 corneal crosslinking).
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formulations to facilitate its absorption. Therefore, several topical drugs, including benzalkonium chloride and EDTA have been used with good results to enhance epithelial penetration, especially in ultrathin keratoconic corneas (pachymetry less than 400 mm), to avoid CXL cytotoxic effect on the corneal endothelium, crystalline lens, and other intraocular tissues.20,21 Ocular iontophoresis is a well-known approach used in most recent CXL protocols, primarily introduced to enhance the penetration of the photosensitizer within the stroma. It has been shown that a 5-minute protocol of iontophoresis achieves a riboflavin concentration in the corneal stroma sufficient for CXL treatment, with the advantage of shortening the imbibition time while preserving the integrity of corneal epithelium.22 Moreover, an ex vivo study confirmed the effectiveness of iontophoresis imbibition in obtaining an adequate riboflavin concentration within the stroma and the induction of a dose-dependent keratocyte damage.23 One of the most debated points of CXL is to find new reliable parameters to measure the actual effectiveness of the intervention. Nowadays, the interpretation of the depth of the demarcation line is considered an indirect measurement of CXL penetration within the stroma and, therefore, an indirect treatment indicator of effectiveness. The debate has focused on the correlation between treatment effectiveness and depth of the corneal stromal demarcation line; however, there is still much to understand about the real modification of corneal collagen structure after the photochemical CXL reaction.24 In the present study, conventional and accelerated epi-off CXL protocols, characterized by the removal of the corneal epithelium, did not differ in the depth of the demarcation line and therefore in terms of keratocyte apoptosis in the anterior stroma. Different studies have reported a shallower demarcation line depth with accelerated epi-off protocol when compared with the conventional epi-off protocol.25,26 Authors have suggested that the Bunsen-Roscoe law of reciprocity might not directly apply to CXL in living corneal tissue. Thus, an increased total energy dose should probably be applied to the keratoconic cornea to achieve a treatment effect comparable to the already proven effective conventional epi-off CXL technique.26 Subsequently, Kymionis et al.27 have modified the accelerated epi-off protocol increasing the total surface energy dose, achieving similar demarcation line depth when compared with the conventional epi-off CXL technique, although further safety studies of the long-term effects are required. In this direction, in epi-on protocols, attention should be focused on iontophoresis-assisted imbibition versus chemical enhancers mediated transepithelial techniques rather than on CXL fluency. The difference in demarcation line depth between epi-off techniques versus epi-on protocols is secondary to the presence of 2 factors: the shielding effect of the intact epithelium and the reduced penetration into the stroma of the riboflavin during the imbibition phase. Thus, the corneal stromal demarcation line was significantly deeper after the conventional epi-off techniques when compared with the epi-on transepithelial techniques. Volume 44 Issue 5 May 2018
The iontophoresis CXL protocol, as reported in the literature, improves penetration of riboflavin through the intact corneal epithelium during the imbibition phase.23 For this reason, iontophoresis CXL presents a greater penetration into the stroma and therefore a greater CXL efficacy and deeper demarcation line than the epi-on transepithelial technique, more similar to the epi-off protocols. The difference in depth of the demarcation line between the epi-off (conventional epi-off CXL and accelerated epi-off) approach and the epi-on protocols might be linked to the shielding effect of the intact corneal epithelium of the latter technique, which reduces UVA light penetration. Some authors further suggest removing the epithelium-associated mucins to reduce epithelium absorption, thus improving the bioavailability of riboflavin in the stroma in the iontophoresis approach.28 When considering the pachymetric preoperative values and the treated volume of the overall cornea to quantify the penetration of treatment through the corneal stroma, the treatment depth is significantly higher in transepithelial epi-on by iontophoresis CXL than in transepithelial epi-on CXL. It is interesting to note that although the conventional epi-off CXL and accelerated epi-off protocols reach significantly greater demarcation line depth values than the transepithelial epi-on by iontophoresis CXL protocol, the relative amount of treated cornea volume is comparable when the preoperative corneal thickness is considered. Results about epithelial patterns confirm that in keratoconus, the corneal epithelium acts as a masking agent. In the area of the cone usually, the epithelial layer is thinner than in the other sides of the cornea.29 A better understanding of the effectiveness of CXL treatment in corneas of different thickness can assist the clinician in choosing the appropriate CXL technique for each selected case. The literature demonstrates that a shallower crosslinking treatment will have a less stabilizing effect.30,31 In this direction, assessing how much the treatment goes deeply into each cornea can serve as an index of effectiveness in stabilizing keratoconus and iatrogenic keratectasia. The measurement of the percentage depth of the demarcation line related to the overall corneal pachymetry values could be noteworthy, such that even to different absolute values of demarcation, line depth could correspond to similarly treated volume of the cornea. Laboratory studies showed how the efficacy of standard CXL is inversely related to pachymetric data; the stiffening effect after CXL decreased by 4.1% per 100 mm of corneal thickness.32 The latest corneal stiffness measurement methods are required to correlate the demarcation line depth to efficacy of the crosslinking custom approach. Recent techniques have been developed to measure the intrinsic corneal stiffness using spectral analysis of scattered light within the cornea. This interaction provides an elastic modulus map of the studied tissue with a biomechanical differentiating feature.33 It would be interesting, although there are no studies at present, to use this device for in vivo corneal stiffness measurements to correlate demarcation line depth with the effectiveness in each CXL
CORNEAL STROMAL DEMARCATION LINE AFTER 4 CXL PROTOCOLS
protocol, finding a minimum cutoff in demarcation line depth expressed in relation to the corneal thickness of the single patient. Hence, the need for enhanced custom CXL treatment in the future. To our knowledge, this is the first clinical study evaluating corneal stromal demarcation line depth with 4 treatment protocols of CXL.
WHAT WAS KNOWN Epithelium-off and epi-on CXL protocols have different efficacy and safety profiles in the treatment of corneal ectasias. The demarcation line is considered an indication of the depth of CXL treatment into the stroma.
WHAT THIS PAPER ADDS This was the first study to evaluate demarcation line depth with 4 different CXL treatment protocols. The demarcation line was significantly deeper using epi-off techniques than with the epi-on techniques. The difference in demarcation line depth was not statistically significant between C-CXL and A-CXL; however, it was statistically significant between TE-CXL and I-CXL.
REFERENCES 1. Vazirani J, Basu S. Keratoconus: current perspectives. Clin Ophthalmol 2013; 7:2019–2030. Available at: https://www.ncbi.nlm.nih.gov/pmc /articles/PMC3798205/pdf/opth-7-2019.pdf. Accessed March 19, 2018 2. Rabinowitz YS. Keratoconus. Surv Ophthalmol 1998; 42:297–319. Available at: http://www.keratoconus.com/resources/MajorCReview-Kerato conus.pdf. Accessed March 19, 2018 3. Zadnik K, Barr JT, Edrington TB, Everett DF, Jameson M, McMahon TT, Shin JA, Sterling JL, Wagner H, Gordon MO, the Collaborative Longitudinal Evaluation of Keratoconus (CLEK) Study Group. Baseline findings in the Collaborative Longitudinal Evaluation of Keratoconus (CLEK) Study. Invest Ophthalmol Vis Sci 1998; 39:2537–2546. Available at: http://iovs.arvojour nals.org/article.aspx?articleidZ2161642. Accessed March 19, 2018 4. Gordon-Shaag A, Millodot M, Shneor E, Liu Y. The genetic and environmental factors for keratoconus. Biomed Res Int 2015; article ID:795738. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4449900 /pdf/BMRI2015-795738.pdf. Accessed March 19, 2018 5. Woodward MA, Blachley TS, Stein JD. The association between sociodemographic factors, common systemic diseases, and keratoconus; an analysis of a nationwide health care claims database. Ophthalmology 2016; 123:457–465.e2. Available at: https://www.ncbi.nlm.nih.gov/pmc/arti cles/PMC4766030/pdf/nihms733251.pdf. Accessed March 19, 2018 6. Spoerl E, Huhle M, Seiler T. Induction of cross-links in corneal tissue. Exp Eye Res 1998; 66:97–103 7. Wollensak G, Spoerl E, Seiler T. Riboflavin/ultraviolet-A–induced collagen crosslinking for the treatment of keratoconus. Am J Ophthalmol 2003; 135:620–627. Available at: http://iogen.fi/files/2011/10/Wollensak_et_al_Ke ratoconus_2003.pdf. Accessed March 19, 2018 8. Spadea L. Corneal collagen cross-linking with riboflavin and UVA irradiation in pellucid marginal degeneration. J Refract Surg 2010; 26:375–377 9. Wollensak G, Spoerl E, Wilsch M, Seiler T. Keratocyte apoptosis after corneal collagen cross-linking using riboflavin/UVA treatment. Cornea 2004; 23:43–49. Available at: http://s499648585.mialojamiento.es/biblio teca/CROSS-LINKING/Wollensaak%20et%20al%20Keratocyte%20Apop tosis%202004.pdf. Accessed March 19, 2018 10. Seiler T, Hafezi F. Corneal cross-linking-induced stromal demarcation line. Cornea 2006; 25:1057–1059 11. Kanellopoulos AJ, Asimellis G. Introduction of quantitative and qualitative cornea optical coherence tomography findings induced by collagen cross-linking for keratoconus: a novel effect measurement benchmark. Clin Ophthalmol 2013; 7:329–335. Available at: http://www.ncbi.nlm.nih .gov/pmc/articles/PMC3577010/pdf/opth-7-329.pdf. Accessed March 19, 2018
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12. Raiskup-Wolf F, Hoyer A, Spoerl E, Pillunat LE. Collagen crosslinking with riboflavin and ultraviolet-A light in keratoconus: long-term results. J Cataract Refract Surg 2008; 34:796–801 13. 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 14. Wittig-Silva C, Whiting M, Lamoureux E, Lindsay RG, Sullivan LJ, Snibson GR. A randomized controlled trial of corneal collagen crosslinking in progressive keratoconus: preliminary results. J Refract Surg 2008; 24:S720–S725 15. Caporossi A, Mazzotta C, Baiocchi S, Caporossi T, Denaro R. Age-related long-term functional results after riboflavin UV A corneal cross-linking. J Ophthalmol 2011; article ID: 608041. Available at: https://www.ncbi.nlm .nih.gov/pmc/articles/PMC3151503/pdf/JOP2011-608041.pdf. Accessed March 19, 2018 16. Spoerl E, Seiler T. Techniques for stiffening the cornea. J Refract Surg 1999; 15:711–713 17. Ziaei M, Barsam A, Shamie N, Vroman D, Kim T, Donnenfeld ED, €ell J, Holland EJ, Kanellopoulos J, Mah FS, Randleman JB, Daya S, Gu for the ASCRS Cornea Clinical Committee. Reshaping procedures for the surgical management of corneal ectasia. J Cataract Refract Surg 2015; 41:842–872. Available at: https://pdfs.semanticscholar.org/ca8d/2671b 5df568ed9b175cfa7de97fe03b7e819.pdf. Accessed March 19, 2018 18. Gatzioufas Z, Richoz O, Brugnoli E, Hafezi F. Safety profile of highfluence corneal collagen cross-linking for progressive keratoconus: preliminary results from a prospective cohort study. J Refract Surg 2013; 29:846–848 ri L, No gra di A, Hopp B, Bor Z. UV absorbance of the human 19. Kolozsva cornea in the 240- to 400-nm range. Invest Ophthalmol Vis Sci 2002; 43:2165–2168. Available at: http://iovs.arvojournals.org/article.aspx?arti cleidZ2123733. Accessed March 19, 2018 20. Kissner A, Spoerl E, Jung R, Spekl K, Pillunat LE, Raiskup F. Pharmacological modification of the epithelial permeability by benzalkonium chloride in UVA/riboflavin corneal collagen cross-linking. Curr Eye Res 2010; 35:715–721 21. Spadea L, Mencucci R. Transepithelial corneal collagen cross-linking in ultrathin keratoconic corneas. Clin Ophthalmol 2012; 6:1785–1792. Available at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3497455/pdf/opth-6-1 785.pdf. Accessed March 19, 2018 22. Raiskup F, Spoerl E. Corneal crosslinking with riboflavin and ultraviolet A. I. Principles. Ocul Surf 2013; 11:65–74 23. Mencucci R, Ambrosini S, Paladini I, Favuzza E, Boccalini C, Raugei G, Vannelli GB, Marini M. Early effects of corneal collagen cross-linking by iontophoresis in ex vivo human corneas. Graefes Arch Clin Exp Ophthalmol 2015; 253:277–286 24. Spadea L, Tonti E, Vingolo E. Corneal stromal demarcation line after collagen cross-linking in corneal ectatic diseases: a review of the literature. Clin Ophthalmol 2016; 10:1803–1810. Available at: https://www.ncbi.nlm .nih.gov/pmc/articles/PMC5034907/pdf/opth-10-1803.pdf. Accessed March 19, 2018 25. Kymionis GD, Tsoulnaras KI, Grentzelos MA, Plaka AD, Mikropoulos DG, Liakopoulos DA, Tsakalis NG, Pallikaris IG. Corneal stroma demarcation line after standard and high-intensity collagen crosslinking determined with anterior segment optical coherence tomography. J Cataract Refract Surg 2014; 40:736–740 26. Brittingham S, Tappeiner C, Frueh BE. Corneal cross-linking in keratoconus using the standard and rapid treatment protocol: Differences in demarcation line and 12-month outcomes. Invest Ophthalmol Vis Sci 2014; 55:8371–8376. Available at: http://iovs.arvojournals.org/article.aspx?arti cleidZ2212704. Accessed March 19, 2018 27. Kymionis GD, Tsoulnaras KI, Grentzelos MA, Liakopoulos DA, Tsakalis NG, Blazaki SV, Paraskevopoulos TA, Tsilimbaris MK. Evaluation of corneal stromal demarcation line depth following standard and a modifiedaccelerated collagen cross-linking protocol. Am J Ophthalmol 2014; 158:671–675 28. Lombardo M, Serrao S, Raffa P, Rosati M, Lombardo G. Novel technique of transepithelial corneal cross-linking using iontophoresis in progressive keratoconus. J Ophthalmol 2016; article ID:7472542. Available at: https: //www.ncbi.nlm.nih.gov/pmc/articles/PMC5002487/pdf/JOPH2016-747 2542.pdf. Accessed March 19, 2018 29. Kymionis GD, Grentzelos MA, Kounis GA, Diakonis VF, Limnopoulou AN, Panagopoulou SI. Combined transepithelial phototherapeutic keratectomy and corneal collagen cross-linking for progressive keratoconus. Ophthalmology 2012; 119:1777–1784
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30. Caporossi A, Mazzotta C, Paradiso AL, Baiocchi S, Marigliani D, Caporossi T. Transepithelial corneal collagen crosslinking for progressive keratoconus: 24-month clinical results. J Cataract Refract Surg 2013; 39:1157–1163 31. Moramarco A, Iovieno A, Sartori A, Fontana L. Corneal stromal demarcation line after accelerated crosslinking using continuous and pulsed light. J Cataract Refract Surg 2015; 41:2546–2551 32. Kling S, Hafezi F. An algorithm to predict the biomechanical stiffening effect in corneal cross-linking. J Refract Surg 2017; 33:128–136. Available at: http://www.elza-institute.com/wp-content/uploads/2017/08/Algorithm 2017.pdf. Accessed March 19, 2018 33. Scarcelli G, Besner S, Pineda R, Kalout P, Yun SH. In vivo biomechanical mapping of normal and keratoconus corneas. JAMA Ophthalmol 2015; 133:480–482. Available at: http://jamanetwork.com/journals/jamaophthal mology/fullarticle/2089674. Accessed March 19, 2018
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Disclosures: None of the authors has a financial or proprietary interest in any material or method mentioned.
First author: Leopoldo Spadea, MD Department of Biotechnology and Medical-Surgical Sciences, Sapienza University of Rome, Terracina (Latina), Italy
ARTICLE
Corneal stromal demarcation line after 4 protocols of corneal crosslinking in keratoconus determined with anterior segment optical coherence tomography Leopoldo Spadea, MD, Lucia Di Genova, MD, Emanuele Tonti, MD
Purpose: To use anterior segment optical coherence tomography (AS-OCT) to compare corneal stromal demarcation line depth after 4 treatment protocols of corneal crosslinking (CXL).
Setting: Eye Clinic, Sapienza University of Rome, Terracina (Latina), Italy. Design: Prospective case series. Methods: Patients with progressive keratoconus were delegated to one of the following CXL treatments: (1) conventional epithelium (epi)-off 3 mW/cm2 according to the standard Dresden protocol (CCXL group), (2) accelerated epi-off 10 mW/cm2 (A-CXL group), (3) transepithelial epi-on 3 mW/cm2 (TE-CXL group), or (4) transepithelial epi-on by iontophoresis 10 mW/cm2 (I-CXL group). Two independent observers measured the corneal stromal demarcation line using AS-OCT.
identified on AS-OCT scans in 109 eyes (90.8%). One month after the treatment, the mean stromal demarcation line depth was 275.05 mm G 41.83 (SD) in the C-CXL group, 279.35 G 33.07 mm in the A-CXL group, 132.60 G 22.14 mm in the TE-CXL group, and 235.40 G 37.08 mm in the I-CXL group. The difference in stromal demarcation line depth was not statistically significant between the C-CXL and A-CXL group, but it was statistically significant (P < .05) between the epi-off and epi-on CXL groups and between the 2 epi-on groups, where the demarcation line was significantly deeper in the I-CXL group than in the TE-CXL group.
Conclusion: The corneal stromal demarcation line was significantly deeper after epi-off 30-minute standard CXL treatment and after epi-off 9-minute accelerated CXL with high-intensity ultraviolet-A irradiation.
Results: The study comprised 70 patients (120 eyes, 30 eyes in each group). The corneal stromal demarcation line was
K
eratoconus is a progressive bilateral multifactorial disorder involving complex interactions of both genetic and environmental factors and is characterized by ectasia of the central or paracentral regions of the cornea. This process results in irregular astigmatism and impaired vision.1 The incidence of keratoconus is 1.3 to 22.3 new cases per 100 000 people every year, with a prevalence of 1 in 2000 and with nearly 135 000 cases in the United States.2,3 Keratoconus is associated with atopic disease, connective tissue disorders, and Down syndrome; and patients whose ethnicity derives from the Middle East and Asia might also carry an increased risk for the condition.4,5 Corneal crosslinking (CXL) is a surgical technique used to increase corneal biomechanical strength. Corneal crosslinking relies on the use of riboflavin as a photosensitizer
J Cataract Refract Surg 2018; 44:596–602 Q 2018 ASCRS and ESCRS
and ultraviolet-A (UVA) light to promote the formation of intrafibrillar and interfibrillar covalent bonds by photosensitized oxidation.6 This technique can delay the progression of corneal steepening, potentially stabilizing the cornea, and improve refractive error by reducing the irregular astigmatism caused by the biochemical instability of the cornea. The major indication for CXL is to inhibit the progression of corneal ectasia secondary to conditions such as keratoconus, pellucid marginal degeneration, or iatrogenic corneal ectasias.7,8 Histopathological studies after CXL show keratocyte apoptosis limited to the anterior stroma after only 24 hours, with an increased density of the extracellular matrix in the stroma.9 This leads to the formation of a “demarcation line,” a transition zone between crosslinked stroma and untreated tissue that can be appreciated on slitlamp evaluation
Submitted: September 6, 2017 | Final revision submitted: February 13, 2018 | Accepted: February 13, 2018 From the Department of Biotechnology and Medical-Surgical Sciences, Sapienza University of Rome, Terracina (Latina), Italy. Corresponding author: Leopoldo Spadea, MD, Via Benozzo Gozzoli 34, 00142 Rome, Italy. Email: [email protected]. Q 2018 ASCRS and ESCRS Published by Elsevier Inc.
0886-3350/$ - see frontmatter https://doi.org/10.1016/j.jcrs.2018.02.017
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as early as 2 weeks after the intervention.10 The present study analyses the depth of the demarcation line for 4 different protocols of CXL. PATIENTS AND METHODS This prospective interventional study was approved by the ethics committee of the Department of Biotechnology and MedicalSurgical Science, Sapienza University of Rome, and was comprised of patients affected by progressive keratoconus who had CXL between June 2013 and March 2016 at the Eye Clinic of Sapienza, University of Rome, Terracina (Latina), Italy. All patients were presented with the risks and benefits of the procedures and provided an informed written consent. The study included eyes with progressive keratoconus of patients with no other systemic or ocular disease. Inclusion criterion was keratoconus with documented progression in the past 12 months. Progression was defined as an increase in maximum keratometry (K) of 1.00 diopter (D) or more in the previous 12 months and patient-reported deterioration of visual acuity (excluding other possible non-corneal-related reasons for deterioration). Exclusion criteria were corneal opacity, previous herpetic keratitis, previous ocular surgery, active ocular infection, autoimmune disease, diabetes, pregnancy, and lactation. The clinical diagnosis of keratoconus was facilitated by Scheimpflug camera–Placido corneal topography (Sirius, Costruzione Strumenti Oftalmici). Diagnostic criteria parameters were mean K, topographic astigmatism, thinnest pachymetry, corneal volume, maximum K, and significant higher-order aberrations levels. Furthermore, clinical indicators such as irregular astigmatism, reduced corrected distance visual acuity (CDVA), reduced contrast sensitivity, and microscopic signs at slitlamp evaluation such as apical protrusion with corneal thinning and scarring, Vogt striae, and Fleischer ring were considered. Patients with progressive keratoconus were assigned to one of the following CXL techniques: conventional epithelium-off (epioff) 3 mW/cm2 (C-CXL group), accelerated epi-off 10 mW/cm2 (A-CXL group), transepithelial epithelium-on (epi-on) 3 mW/cm2 (TE-CXL group), or transepithelial epi-on iontophoresis 10 mW/ cm2 (I-CXL group) (Table 1). The entire population was assigned into epi-off or epi-on protocol based on corneal pachymetry value: epi-off CXL groups’ inclusion criteria were corneal thicknesses greater than 400 mm at the thinnest point; in contrast, all the eyes with pachymetric values less than 400 mm at the thinnest point were enrolled in the epi-on CXL group. Subsequently in epi-off groups, patients were randomly assigned to 1 of the 2 treatment protocols (conventional or accelerated); likewise, in epi-on groups patients were randomly assigned to the
transepithelial or iontophoresis CXL technique group. All surgical techniques were performed under sterile conditions by the same surgeon (L.S.). One drop of pilocarpine 1.0% was instilled 30 minutes preoperatively to constrict the pupil and reduce UVA irradiation to the lens and retina. Preoperatively, oxybuprocaine 0.4% drops, were instilled every 5 minutes for 15 minutes as topical anesthesia. The aiming beam was focused on the central cornea while the patient fixated on a pulsating green light. The 4 surgical techniques that were used in the different groups were as follows: Conventional epi-off 3 mW/cm2 After epithelium removal by using a blunt metal spatula to mechanically scrape off a 9.0 mm diameter area, the cornea was soaked for 15 minutes in riboflavin 0.1% solution (Ricrolin) and then exposed to a solid-state UVA illuminator (CBM Vega X-linker device, Costruzione Strumenti Oftalmici) for 30 minutes, during which the riboflavin solution was applied every 3 minutes. The UVA illuminator was calibrated to 3.0 mW/cm2 of surface irradiance (5.4 J/cm2 surface dose) using a UV light meter at the specified working distance. Accelerated epi-off 10mW/cm2 After epithelium removal by using a blunt metal spatula to mechanically scrape off a 9.0 mm diameter area, the cornea was soaked for 15 minutes in riboflavin 0.1% solution and then exposed to a solid-state UVA illuminator. Then, the corneas were irradiated by the device calibrated to 10 mW/cm2 (5.4 J/cm2 surface dose) and UVA irradiation was applied on the central 9.0 mm of the cornea for 9 minutes. During this time, the riboflavin solution was applied to the cornea every 3 minutes. Transepithelial epi-on 3mW/cm2 Without any deepithelialization, riboflavin 0.1% solution in 15% dextran T500 containing sodium ethylenediaminetetraacetic acid (EDTA) 0.01% and trometamol (Ricrolin TE) was instilled every 10 minutes for 2 hours. An eyelid speculum was inserted and riboflavin was instilled over the cornea every 3 minutes for 15 minutes. Ultraviolet-A irradiation was delivered using a solid-state UVA illuminator. The device was calibrated to 3.0 mW/cm2 of surface irradiance (5.4 J/cm2 surface dose) using an UV light meter at the specified working distance. Ultraviolet-A irradiation was applied to the central 9.0 mm of the cornea for 30 minutes and during this time, the riboflavin solution was instilled every 5 minutes. Transepithelial epi-on by Iontophoresis 10mW/cm2 In the I-CXL group, an 8.0 mm wide iontophoretic application device was placed on the corneal surface using an annular suction ring. The device was filled with approximately 0.5 mL solution riboflavin 0.1%, sodium EDTA 0.01%, and trometamol from the open proximal side until the electrode (stainless steel mesh) was covered.
Table 1. Patient characteristics at baseline. Parameter
C-CXL
A-CXL
TE-CXL
I-CXL
Number of eyes (patients) Age (y) Mean G SD Range Sex (F/M) Corneal thickness (mm) Mean G SD Range Epithelial thickness (mm) Mean G SD Range
30 (19)
30 (18)
30 (17)
30 (16)
31.08 G 6.60 18, 42 9/10
26.70 G 7.60 13, 46 6/12
37.00 G 5.60 32, 44 10/7
36.50 G 7.60 27, 44 8/8
436.14 G 38.8 402, 520
469.34 G 40.30 407, 542
389.20 G 29.03 369, 438
409.20 G 59.12 340, 497
43.12 G 4.50 34, 52
42.90 G 4.50 35, 55
37.75 G 7.50 33, 49
36.80 G 11.18 22, 48
A-CXL Z accelerated epithelium-off 10 mW/cm2; C-CXL Z conventional epithelium-off 3 mW/cm2; I-CXL Z transepithelial epithelium-on by iontophoresis 10 mW/cm2; TE-CXL Z transepithelial epithelium-on 3 mW/cm2
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The device was connected to the constant current generator (I-ON XL, Sooft Italia S.p.A.) for 5 minutes set at 1 mA (the total dose of 5 mA/5 min was monitored by the generator). Then, it was exposed to the solid-state UVA illuminator for 9 minutes, irradiating an area of 9.0 mm in diameter (energy delivered 10 mW/ cm2). During this time, the riboflavin solution was applied on the cornea every 5 minutes. Postoperatively, in all 4 groups, eyes were rinsed with a balanced salt solution and a therapeutic bandage soft contact lens was applied. An antibiotic regimen of ofloxacin drops and flurbiprofen drops was administered 4 times a day for 1 week. Successively, topical corticosteroid (clobetasone butyrate 0.1%) drops were administered for 1 month and gradually tapered. Patients received a comprehensive eye examination before and at 1, 3, 6, and 12 months after the surgery. During each visit, patients had slitlamp evaluations and anterior segment optical coherence tomography (AS-OCT) (RTVue, Optovue, Inc.). By using AS-OCT, the stromal demarcation line was detected within an enhanced image of the cornea on the horizontal meridian.11 The image was captured when the corneal reflex was visible and demarcation line depth was measured using the caliper tool option provided by the software. Two independent examiners (L.D.G., E.T.) analyzed the depth of the demarcation line in the center of the cornea from the epithelial side to the hyperreflective line into the stroma, comparing all cases after 1 month at the same postoperative period. To increase the accuracy of the analysis, measures of the demarcation line were scored into 3 groups as follows: (1) demarcation line not identifiable, (2) demarcation line visible but not clearly measurable, (3) clearly visible and measurable demarcation line. Only images included in the third group were included in the study. The demarcation line depth was also compared with the preoperative corneal pachymetry to assess the percentage volume of the treated cornea by multiplying the demarcation line depth value by a factor of 100 and then dividing the obtained result by the overall corneal pachymetry data in each individual case. Statistical analysis was performed using SPSS software (SPSS, Inc.). Data are shown as means G SD, and range. The nonparametric Mann-Whitney U test was used for statistical analysis. A P value less than 0.05 was considered statistically significant.
RESULTS The study comprised 120 eyes of 70 patients with progressive keratoconus. There were 30 eyes in the C-CXL group, 30 eyes in the A-CXL group, 30 eyes in the TE-CXL group, and 30 eyes in the I-CXL group (Table 1). None of the treated patients presented adverse events during the follow-up period. Most characteristics at baseline were similar between epi-off groups and between epion groups. The mean corneal thickness was different in the 4 groups according to the clinical indications for treatment (Table 1). It was not possible to observe the corneal demarcation line in 5 eyes (16.6%) treated with transepithelial epi-on and in 6 eyes (20.0%) treated with transepithelial epi-on by iontophoresis CXL. The demarcation line was clearly visible and measurable (score 3) in all the remaining cases (Figure 1). There was always a statistically significant agreement (P ! .05) between the measurements of the 2 independent observers. At the first follow-up, 1 month after the treatment, the mean depth of the stromal demarcation line in C-CXL and A-CXL groups was 275.05 mm G 41.8 and 279.34 G 33.06 mm, respectively, and in the TE-CXL and Volume 44 Issue 5 May 2018
Figure 1. High-resolution AS-OCT scan visualizing the corneal stromal demarcation line 1 month after CXL. Top left: Conventional epioff 3 mW/cm2. Central corneal demarcation line (yellow arrows) is visible to a depth of 271 mm. Top right: Accelerated epi-off 10 mW/cm2. The demarcation line (yellow arrows) is clearly visible to a depth of 315 mm. Bottom left: Transepithelial epi-on by iontophoresis 10 mW/cm2. The demarcation line (yellow arrows) is visible with a characteristic honeycomb pattern to a depth of 191 mm. Bottom right: Transepithelial epi-on 3 mW/cm2. The demarcation line (yellow arrows) is fairly visible to a depth of 110 mm (ASOCT Z anterior segment optical coherence tomography; CXL Z corneal crosslinking; epi-off Z epithelium-off; epi-on Z epithelium-on).
I-CXL groups, it was 132.60 G 22.14 mm and 235.40 G 37.08 mm, respectively (Figure 2). The demarcation line depth remained stable at the 1-year follow-up on the analysis of the 2 examiners; no patients were lost to follow-up. These findings evidenced that the demarcation line is statistically significantly deeper using epi-off techniques than with the epi-on techniques. The difference in demarcation line depth between C-CXL and ACXL groups was not statistically significant; however, it was statistically significant between TE-CXL and I-CXL groups (P ! .05) (Table 2). This study also related the demarcation line depth to the preoperative corneal pachymetry to assess the percentage volume of the treated cornea (Figure 3). Concerning this datum, no statistically significant difference was found between the treated cornea
Figure 2. The demarcation line depth (mm) in 4 CXL protocols in keratoconus determined with AS-OCT (A-CXL Z accelerated epithelium-off 10 mW/cm2 corneal crosslinking; AS-OCT Z anterior segment optical coherence tomography; C-CXL Z conventional epithelium-off 3 mW/cm2 corneal crosslinking; CXL Z corneal crosslinking; I-CXL Z transepithelial epithelium-on by iontophoresis 10 mW/cm2 corneal crosslinking; TE-CXL Z transepithelial epithelium-on 3 mW/cm2 corneal crosslinking).
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Table 2. Statistically significant differences in demarcation line values. P Value
Mean ± SD
Parameter DL depth (mm) DL (%) DL-epi (%)
1) C-CXL
2) A-CXL
3) TE-CXL
4) I-CXL
275.05 G 41.80 279.34 G 33.06 132.60 G 22.14 235.40 G 37.08 63.34 G 8.55 59.62 G 6.66 34.41 G 7.50 57.58 G 4.96 7.41 G 9.70 65.71 G 7.31 38.00 G 8.03 63.27 G 6.12
1 Vs. 2
1 Vs. 3
1 Vs. 4
2 Vs. 3
2 Vs. 4
3 Vs. 4
.661 .065 .111
.001* .001* .001*
.05† .105 .063
.001* .001* .001*
.05† .502 .506
.05† .05† .05†
A-CXL Z accelerated epithelium-off 10 mW/cm2; C-CXL Z conventional epithelium-off 3 mW/cm2; DL Z demarcation line; DL (%) Z percentage of the volume of the cornea treated evidenced by the demarcation line; DL-Epi (%) Z percentage of the volume of the cornea treated, excluding the preoperative epithelial thickness, evidenced by the demarcation line; I-CXL Z transepithelial epithelium-on by iontophoresis 10 mW/cm2; TE-CXL Z transepithelial epithelium-on 3 mW/cm2 *Statistically significant difference between the 2 groups (P ! .001) † Statistically significant difference between the 2 groups (P ! .05)
volume of each of the 2 epi-off groups (C-CXL and A-CXL), whereas a statistically significant difference was noted in comparison to the TE-CXL group (P ! .001). Moreover, a statistically significant difference was found between the TE-CXL group and the I-CXL group (P ! .05) (Table 2). In contrast to the different depth of the demarcation line, the volume of treated cornea did not differ statistically significantly between the C-CXL and A-CXL groups and between the 2 epi-off groups and the iontophoretic I-CXL group. To further assess the treated volume of the cornea, the preoperative epithelial thickness (evaluated by using the AS-OCT) was also excluded from the pachymetry measurements. However, the volume’s values of the treated cornea obtained excluding the preoperative epithelial thickness did not show consistent differences with those recorded with total pachymetry (Figure 4). Moreover, the epithelial thickness did not change significantly from the baseline to the last follow-up. DISCUSSION The effectiveness and safety of conventional CXL procedure, also known as the Dresden protocol, is widely established
Figure 3. Percentage of the volume of the cornea treated evidenced by the demarcation line in 4 CXL protocols in keratoconus determined with AS-OCT (A-CXL Z accelerated epithelium-off 10 mW/cm2 corneal crosslinking; AS-OCT Z anterior segment optical coherence tomography; C-CXL Z conventional epithelium-off 3 mW/cm2 corneal crosslinking; CXL Z corneal crosslinking; I-CXL Z transepithelial epithelium-on by iontophoresis 10 mW/cm2 corneal crosslinking; TE-CXL Z transepithelial epithelium-on 3 mW/cm2 corneal crosslinking).
and confirmed by several studies.12–15 The interaction between riboflavin 0.1% molecules absorbed in corneal tissue and UVA light rays irradiated at 3 mW/cm2 for 30 minutes (5.4 J/cm2 energy dose) releases reactive oxygen species that promote the formation of molecular bridges between and within collagen fibers.16 In the past few years, to minimize UV exposure duration, complication rate, patient discomfort, and possible adverse events, attention has focused on the possibility of creating new CXL protocols.17 The accelerated high-flow CXL, based on the principle of photochemical reciprocity, also known as the Bunsen-Roscoe law, is able to minimize the UV exposure and the patient discomfort associated with the duration of the CXL procedure.18 An adequate amount of riboflavin corneal absorption is required for achieving an effective CXL. Thus, the epithelium debridement in conventional CXL has the double goal of overcoming the barrier formed by corneal epithelium tight junctions that would limit the penetration of macromolecule as Vitamin B2 and avoid the absorption of UV rays by the epithelium.19 Different strategies have been proposed to enhance transepithelial riboflavin penetration, such as increasing riboflavin imbibition time and new riboflavin solution
Figure 4. Percentage of the volume of the cornea treated, excluding the preoperative epithelial thickness, evidenced by the demarcation line in 4 CXL protocols in keratoconus determined with AS-OCT (ACXL Z accelerated epithelium-off 10 mW/cm2 corneal crosslinking; AS-OCT Z anterior segment optical coherence tomography; CCXL Z conventional epithelium-off 3 mW/cm2 corneal crosslinking; CXL Z corneal crosslinking; I-CXL Z transepithelial epithelium-on by iontophoresis 10 mW/cm2 corneal crosslinking; TE-CXL Z transepithelial epithelium-on 3 mW/cm2 corneal crosslinking).
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formulations to facilitate its absorption. Therefore, several topical drugs, including benzalkonium chloride and EDTA have been used with good results to enhance epithelial penetration, especially in ultrathin keratoconic corneas (pachymetry less than 400 mm), to avoid CXL cytotoxic effect on the corneal endothelium, crystalline lens, and other intraocular tissues.20,21 Ocular iontophoresis is a well-known approach used in most recent CXL protocols, primarily introduced to enhance the penetration of the photosensitizer within the stroma. It has been shown that a 5-minute protocol of iontophoresis achieves a riboflavin concentration in the corneal stroma sufficient for CXL treatment, with the advantage of shortening the imbibition time while preserving the integrity of corneal epithelium.22 Moreover, an ex vivo study confirmed the effectiveness of iontophoresis imbibition in obtaining an adequate riboflavin concentration within the stroma and the induction of a dose-dependent keratocyte damage.23 One of the most debated points of CXL is to find new reliable parameters to measure the actual effectiveness of the intervention. Nowadays, the interpretation of the depth of the demarcation line is considered an indirect measurement of CXL penetration within the stroma and, therefore, an indirect treatment indicator of effectiveness. The debate has focused on the correlation between treatment effectiveness and depth of the corneal stromal demarcation line; however, there is still much to understand about the real modification of corneal collagen structure after the photochemical CXL reaction.24 In the present study, conventional and accelerated epi-off CXL protocols, characterized by the removal of the corneal epithelium, did not differ in the depth of the demarcation line and therefore in terms of keratocyte apoptosis in the anterior stroma. Different studies have reported a shallower demarcation line depth with accelerated epi-off protocol when compared with the conventional epi-off protocol.25,26 Authors have suggested that the Bunsen-Roscoe law of reciprocity might not directly apply to CXL in living corneal tissue. Thus, an increased total energy dose should probably be applied to the keratoconic cornea to achieve a treatment effect comparable to the already proven effective conventional epi-off CXL technique.26 Subsequently, Kymionis et al.27 have modified the accelerated epi-off protocol increasing the total surface energy dose, achieving similar demarcation line depth when compared with the conventional epi-off CXL technique, although further safety studies of the long-term effects are required. In this direction, in epi-on protocols, attention should be focused on iontophoresis-assisted imbibition versus chemical enhancers mediated transepithelial techniques rather than on CXL fluency. The difference in demarcation line depth between epi-off techniques versus epi-on protocols is secondary to the presence of 2 factors: the shielding effect of the intact epithelium and the reduced penetration into the stroma of the riboflavin during the imbibition phase. Thus, the corneal stromal demarcation line was significantly deeper after the conventional epi-off techniques when compared with the epi-on transepithelial techniques. Volume 44 Issue 5 May 2018
The iontophoresis CXL protocol, as reported in the literature, improves penetration of riboflavin through the intact corneal epithelium during the imbibition phase.23 For this reason, iontophoresis CXL presents a greater penetration into the stroma and therefore a greater CXL efficacy and deeper demarcation line than the epi-on transepithelial technique, more similar to the epi-off protocols. The difference in depth of the demarcation line between the epi-off (conventional epi-off CXL and accelerated epi-off) approach and the epi-on protocols might be linked to the shielding effect of the intact corneal epithelium of the latter technique, which reduces UVA light penetration. Some authors further suggest removing the epithelium-associated mucins to reduce epithelium absorption, thus improving the bioavailability of riboflavin in the stroma in the iontophoresis approach.28 When considering the pachymetric preoperative values and the treated volume of the overall cornea to quantify the penetration of treatment through the corneal stroma, the treatment depth is significantly higher in transepithelial epi-on by iontophoresis CXL than in transepithelial epi-on CXL. It is interesting to note that although the conventional epi-off CXL and accelerated epi-off protocols reach significantly greater demarcation line depth values than the transepithelial epi-on by iontophoresis CXL protocol, the relative amount of treated cornea volume is comparable when the preoperative corneal thickness is considered. Results about epithelial patterns confirm that in keratoconus, the corneal epithelium acts as a masking agent. In the area of the cone usually, the epithelial layer is thinner than in the other sides of the cornea.29 A better understanding of the effectiveness of CXL treatment in corneas of different thickness can assist the clinician in choosing the appropriate CXL technique for each selected case. The literature demonstrates that a shallower crosslinking treatment will have a less stabilizing effect.30,31 In this direction, assessing how much the treatment goes deeply into each cornea can serve as an index of effectiveness in stabilizing keratoconus and iatrogenic keratectasia. The measurement of the percentage depth of the demarcation line related to the overall corneal pachymetry values could be noteworthy, such that even to different absolute values of demarcation, line depth could correspond to similarly treated volume of the cornea. Laboratory studies showed how the efficacy of standard CXL is inversely related to pachymetric data; the stiffening effect after CXL decreased by 4.1% per 100 mm of corneal thickness.32 The latest corneal stiffness measurement methods are required to correlate the demarcation line depth to efficacy of the crosslinking custom approach. Recent techniques have been developed to measure the intrinsic corneal stiffness using spectral analysis of scattered light within the cornea. This interaction provides an elastic modulus map of the studied tissue with a biomechanical differentiating feature.33 It would be interesting, although there are no studies at present, to use this device for in vivo corneal stiffness measurements to correlate demarcation line depth with the effectiveness in each CXL
CORNEAL STROMAL DEMARCATION LINE AFTER 4 CXL PROTOCOLS
protocol, finding a minimum cutoff in demarcation line depth expressed in relation to the corneal thickness of the single patient. Hence, the need for enhanced custom CXL treatment in the future. To our knowledge, this is the first clinical study evaluating corneal stromal demarcation line depth with 4 treatment protocols of CXL.
WHAT WAS KNOWN Epithelium-off and epi-on CXL protocols have different efficacy and safety profiles in the treatment of corneal ectasias. The demarcation line is considered an indication of the depth of CXL treatment into the stroma.
WHAT THIS PAPER ADDS This was the first study to evaluate demarcation line depth with 4 different CXL treatment protocols. The demarcation line was significantly deeper using epi-off techniques than with the epi-on techniques. The difference in demarcation line depth was not statistically significant between C-CXL and A-CXL; however, it was statistically significant between TE-CXL and I-CXL.
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30. Caporossi A, Mazzotta C, Paradiso AL, Baiocchi S, Marigliani D, Caporossi T. Transepithelial corneal collagen crosslinking for progressive keratoconus: 24-month clinical results. J Cataract Refract Surg 2013; 39:1157–1163 31. Moramarco A, Iovieno A, Sartori A, Fontana L. Corneal stromal demarcation line after accelerated crosslinking using continuous and pulsed light. J Cataract Refract Surg 2015; 41:2546–2551 32. Kling S, Hafezi F. An algorithm to predict the biomechanical stiffening effect in corneal cross-linking. J Refract Surg 2017; 33:128–136. Available at: http://www.elza-institute.com/wp-content/uploads/2017/08/Algorithm 2017.pdf. Accessed March 19, 2018 33. Scarcelli G, Besner S, Pineda R, Kalout P, Yun SH. In vivo biomechanical mapping of normal and keratoconus corneas. JAMA Ophthalmol 2015; 133:480–482. Available at: http://jamanetwork.com/journals/jamaophthal mology/fullarticle/2089674. Accessed March 19, 2018
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Disclosures: None of the authors has a financial or proprietary interest in any material or method mentioned.
First author: Leopoldo Spadea, MD Department of Biotechnology and Medical-Surgical Sciences, Sapienza University of Rome, Terracina (Latina), Italy