Corneal crosslinking (CXL) is a minimally invasive surgical treatment using riboflavin and ultraviolet-A (UVA) irradiation to stabilize the ectatic cornea and halt keratoconus progression.1,2 Wollensak et al.1 first described the standard CXL protocol, which involves removing the central 9.0 mm of the corneal epithelium, soaking the stromal surface with riboflavin 0.1% for 30 minutes, and then continuously irradiating it with 370 nm UVA irradiation with a fluence of 3 mW/cm2 for 30 minutes, delivering a dose of 5.4 J/cm2 for the treatment of keratoconus. Several clinical studies used this standard protocol and confirmed its efficacy and safety.3–5
The standard protocol has 1 disadvantage in that an extended period of exposure is required. Recently, several different modified crosslinking protocols were established to provide higher intensities for shorter times. They included using either a fixed dose of 9 mW/cm2 for 10 minutes, 18 mW/cm2 for 5 minutes, or 30 mW/cm2 for 3 minutes; the settings are based on the Bunsen-Roscoe law of reciprocity.6–8 Such modifications are designated as accelerated corneal CXL in which UVA is continuously delivering the same dose as that in the standard CXL protocol. However, with any of these changes, its efficacy could be limited by rapid development of oxygen depletion.9,10
Although the pulsed-light accelerated CXL procedure delivers the same UVA dose as that applied in the accelerated CXL protocol, its duration is twice as long. Recent studies found that providing oxygen replenishment with this procedure improves crosslinking efficacy11,12 because corneal stromal demarcation lines identified with both confocal microscopy and corneal optical coherence tomography (OCT) analysis were significantly deeper than those obtained with the continuous-light accelerated CXL protocol.12,13
To evaluate stromal CXL efficacy, the time required for enzymatic tissue dissolution is used as an index of its reorganization.14 In ex vivo porcine corneas having similar dry weights, a longer time was required to enzymatically digest corneas treated with the pulsed-light accelerated CXL than the continuous-light accelerated CXL protocol in which the UVA dose was 7.2 J/cm2 in both groups.15 This increased resistance to tissue dissolution in the pulsed-light accelerated CXL protocol is attributable to increased crosslinking, which hinders enzymatic access to its reactive sites within the stromal extracellular matrix. In contrast, another study demonstrated similar stiffening with the continuous-light accelerated CXL protocol (9 mW/cm2 for 10 minutes, 5.4 J/cm2) than that obtained with any of these different pulsed-light accelerated CXL protocols (ie, 30 mW/cm2 pulses for 8 minutes, 7.2 J/cm2; 100 mW/cm2 0.1 seconds-on, 0.9 seconds off pulses for 9 minutes, 5.4 J/cm2; 100 mW/cm2 0.01 seconds on, 0.99 seconds off pulses for 9 minutes, 0.54 J/cm2) based on a theoretical model using ex vivo porcine corneas.16 So far, there are no reports describing the changes in histological appearance and biomechanical strength obtained with the pulsed-light accelerated CXL protocol in rabbit corneas in vivo. The results of such studies using the pulsed-light accelerated CXL are expected to optimize treatment modalities.
We compared and contrasted the efficacy and selectivity of the same UVA irradiance intensity and dose in combination with riboflavin on CXL using either continuous-light or pulsed-light protocols in rabbit corneas.
MATERIALS AND METHODS
All animal protocols were approved by the Animal Care and Ethics Committee of Wenzhou Medical University, Wenzhou, China, and adhered to the Association for Research in Vison and Ophthalmology statement for the Use of Animals in Ophthalmic and Vision Research. The Japanese big ear white purebred rabbits (weight range 2 to 3 kg) were supplied by the Animal Breeding Unit of Wenzhou Medical University. All animals were healthy and free of ocular disease.
Fifty-Four Japanese white rabbits (2 to 3 kg) were included in the study and randomly divided into 2 groups of 27 rabbits each, namely, the continuous-light accelerated CXL group and pulsed-light accelerated CXL group. Anesthesia was administered by intravenous injection of pentobarbital sodium according to animal weight (100 mg/kg) and 0.5% proparacaine eyedrops were applied topically. After complete epithelial debridement using a single-edged razor blade, the rabbits were divided randomly and equally into the following groups:
Accelerated CXL with continuous-light using 9 mW/cm2 for 10 minutes (continuous-light accelerated CXL group). A riboflavin 0.1% solution containing 20% dextran T-500 was applied to the anterior corneal surface for 30 minutes before performing the procedure. Corneas were continuously exposed to 9 mW/cm2 UVA light for 10 minutes (total energy dose of 5.4 J/cm2) during which the riboflavin solution was reapplied at 5-minute intervals.
Accelerated CXL with pulsed on/off light using 9 mW/cm2 for 20 minutes (pulsed-light accelerated CXL group). A riboflavin 0.1% solution containing 20% dextran T-500 was applied to the anterior corneal surface for 30 minutes before being irradiated. Alternately, corneas were exposed to 9 mW/cm2 UVA in a pulsed irradiation mode for 1 second on and 1 second off for 20 minutes (total energy dose of 5.4 J/cm2).
Crosslinking was performed unilaterally in the animal's right eye, whereas the left eyes only received riboflavin solution for 30 minutes and served as the control group. Both irradiation protocols used a commercially available device (UV-360, New Vision, Inc.) with a 5 cm working distance and an 8.0 mm aperture. After a treatment, antibiotic eye drops (tobramycin) were applied 3 times daily until complete reepithelialization was achieved.
Anterior Segment Optical Coherence Tomography
Anterior segment OCT scanning was used to assess corneal characteristics after crosslinking. A custom-built, high-speed ultrahigh resolution spectral-domain OCT (SD-OCT) was used as described.17 Ultrahigh resolution SD-OCT scans of the cornea were performed postoperatively until a demarcation line became visible (6 rabbits in each group). Corneal stromal demarcation line depth was used to evaluate the extent of CXL.12,18–20 Central demarcation line depth was used to identify the short-term outcome parameters of the 2 CXL protocols. The demarcation line depth was measured centrally by 2 independent observers.
In Vivo Confocal Microscopy
One day and 1 week postoperatively, both corneas in each of 6 rabbits were examined by in vivo confocal laser scanning microscopy with the Heidelberg Retina Tomograph III (HRT III), Rostock Cornea Module (RCM) (Heidelberg Engineering, GmbH). The mean corneal thickness was calculated based on the depth difference between the most superficial and the deepest corneal structures. Endothelial cell densities (ECD) were evaluated using a built-in HRT III/RCM software program.
One day after CXL, 15 rabbits in each group were humanely killed by an intravenous injection of pentobarbital sodium overdose of 100 mg/kg body weight and both eyes were enucleated. A previously reported biomechanical inflation testing and inverse analysis were used.21–23 Briefly, the corneas were mounted on a custom-designed pressure chamber to perform an inflation test rig and a mechanical clamp stress–strain analysis. Details of the rig were described in a previous study.24 Mechanical clamps and cyanoacrylate adhesive glue 502 (Opalus, Foshan Opalus Adhesive Product Co., Ltd) were used to provide watertight edge connections for the specimens along their ring of scleral tissue. The pressure chamber was filled with a phosphate-buffered saline and connected to a syringe pump, the movement of which was controlled by custom-built Labview software. The central corneal thickness (CCT) was measured with an ultrasound pachymeter (SP-3000, Tomey Corp.) and corneal diameters in 4 directions were measured with a Vernier caliper. All corneas were first subjected to an initial inflation pressure of 2.0 mm Hg, the minimum required to achieve a fully inflated and smooth corneal surface. This was followed by 3 cycles of loading up to a maximum pressure of 30 mm Hg with a rate of 0.41 mm Hg/s, and unloading to precondition the tissue and stabilize its behavior before accepting results from the fourth cycle as representative of the cornea's biomechanical behavior.
As described in a previous study,21 experimentally obtained relationships between the posterior pressure and apical rise were converted into stress–strain behavior using mathematical shell analysis. To model the corneal mechanical behavior, the first order hyperelastic Ogden material model was used as follows:
where W is the strain energy per unit volume and material parameters μ and α represent the strain hardening exponent and the shear modulus, respectively. The -λ1, -λ2, and -λ3 represent the deviatoric principal stretches; λ1, λ2, λ3 are the principal stretches; and J = λ1λ2λ3. D is a compressibility parameter for which value is 0.081/μ in this study.25 The λ equals strain ε+1 and the stress, σ, is obtained by differentiating the strain energy. With the strain–stress relationship determined, the tangent modulus (Et, a direct measure of the tissue stiffness) at different stress levels was determined using the relationship Et = dσ/dε ≈ Δσ/Δε.21,26,27
Three rabbits from each group were enucleated 24 hours after having CXL and cornea buttons were excised. Each bisected corneal button was placed in formaldehyde 10.0% for approximately 24 to 48 hours and then embedded in paraffin. Hematoxylin–eosin (H&E) staining was performed on serial 4 μm thick sections and examined with a light microscope (Imager.Z1; Carl Zeiss Meditec AG).
Terminal Uridine Deoxynucleotidyl Nick End-Labeling Staining
A fluorescence-based terminal uridine deoxynucleotidyl nick end-labeling (TUNEL) assay was used to detect apoptotic DNA fragmentation (In Site Cell Death Detection Kit. Roche Applied Science) according to the manufacturer's recommendations (3 rabbits from each group). Counterstaining with 4′ 6-diamidino-2-phenylindole dihydrochloride (DAPI) (1:1000 dilution) was followed for 5 minutes. Sections were covered with anti-fade mounting medium (Life Technologies Corp.) and sealed with a cover slip for microscopic observation. The mean numbers of apoptotic cells in the TUNEL samples were calculated by counting the number of cells in 5 non-overlapping areas (0.1 mm × 0.1 mm) of 3 separate corneal sections, separated from each other by at least by 0.2 mm to avoid overlap. The columns in which counts were performed for TUNEL cells were randomly selected from the central cornea or peripheral cornea at the edge of each CXL-treated specimen.
Data are shown as mean measurements ± SEM for TUNEL, the central demarcation line depth, mean corneal thickness, and ECD. All statistical analysis was performed with SPSS software (version 19, IBM Corp.). The P values were determined using the Mann-Whitney U test and the Kruskal-Wallis test. A probability value of a P value less than 0.05 was considered significant.
Depth of Stromal Demarcation Line
Fourteen days after performing continuous-light accelerated CXL and pulsed-light accelerated CXL treatments, OCT identified a dense highly reflective linear zone in the stroma. Its demarcation line depths in the continuous-light accelerated CXL and pulsed-light accelerated CXL were 254.7 ± 47.4 μm and 341.1 ± 36.1 μm, respectively (Figure 1) (P < .01).
In the intact corneas, highly reflective and well-demarcated cell nuclei were visualized in the anterior stroma. One day and 7 days after inducing CXL, some keratocyte fragments were hyperreflective or had bright apoptotic bodies in pulsed-light accelerated CXL-treated corneas, whereas there were no cell structures left in the continuous-light accelerated CXL-treated stroma (Figure 2).
Corneal Thickness and Endothelial Cell Density
The mean corneal thicknesses in both CXL groups were similar to the controls before treatment. Stromal edema was apparent 1 day after treatment in both the continuous-light accelerated CXL-treated and pulsed-light accelerated CXL-treated groups based on increases in tissue thickness. After 1 day, the thickness was 95% and 75% greater than the control in the continuous-light accelerated CXL-treated and pulsed-light accelerated CXL-treated groups, respectively (P < .01) (Table 1). After 7 days, their thicknesses remained significantly greater than the controls, although the differences of both groups with their controls had decreased. In the continuous-light accelerated CXL group, thicknesses were 25 μm greater than that in the pulsed-light accelerated CXL group (P < .05). Initially, the mean ECD in the 2 different CXL-treated groups was significantly lower than that in the intact control endothelium (P < .01). One week later, the mean ECD in the pulsed-light accelerated CXL group increased slightly; however, the mean ECD in the continuous-light accelerated CXL group had not yet fully recovered because it was still significantly lower than its pretreatment value (P < .01) (Table 2).
Corneal stress–strain behavior evaluated with shell theory conformed to an exponential function that was determined to be a best fit of the mean value within each group in all cases. The mean stress at each strain level was obtained in each of the 3 groups. As Figure 3, A, shows, the stiffening is clearly a result of performing a CXL procedure. At the 0.03 to 0.04 strain levels, the pulsed-light accelerated CXL-treated corneas displayed a very definitive change in the stress–strain relationship for which the values were greater than those in the continuous-light accelerated CXL group. With both types of treatment, the increases were greater than those in the control group. These different stress–strain relationships suggest that the pulsed-light accelerated CXL-treated corneas became stiffer than the continuous-light accelerated CXL-treated corneas.
The tangent modulus–stress relationship also assessed the effectiveness of the 2 protocols on altering biomechanical properties. To make this determination, 5 stress levels (0.001 MPa, 0.002 MPa, 0.003 MPa, 0.004 MPa, and 0.005 MPa) were selected for analysis and comparison (Figure 3, B). The results agree with the stress–strain evaluation in that the increase in stiffness was greater in the pulsed-light accelerated CXL group than in the continuous-light accelerated CXL group.
Differences in H&E staining patterns between the 2 treatment groups are consistent with in vivo confocal assessments of structural alterations. Figure 4, A shows that an alteration of normal collagen architecture occurred after both types of CXL treatment. Normal collagen lamellar organization was lost in continuous-light accelerated CXL-treated corneas and a diffuse stromal edema was present mainly in the anterior and middle stroma immediately after treatment. In the pulsed-light accelerated CXL group, a few signs of keratocyte damage developed and there was less edema in the anterior stroma than in the continuous-light accelerated CXL group, which was confirmed with in vivo microscopy. In both CXL groups, the posterior structural stromal collagen configuration was conserved.
More stromal apoptotic cells were evident in both groups of treated corneas than in the controls (P < .001) (Figure 4, B and C, respectively). Such configurations were more preponderant in the continuous-light accelerated CXL group than in the pulsed-light accelerated CXL group (P < .05).
This study compared the effects of exposure to a fixed UVA dose of 5.4 J/cm2 using either a continuous-light or pulsed-light CXL protocol on efficacy and selectivity of CXL in rabbit corneas. Efficacy was evaluated based on determining the depths at which stromal demarcation lines were formed along with their effects on biomechanical strength. Selectivity was evaluated based on stromal histological changes, measurements of corneal thickness and changes in endothelial cell integrity and density. The pulsed-light accelerated CXL procedure was more efficacious than the continuous-light accelerated CXL procedure based on a deeper stromal demarcation line, greater changes in both the stress–strain relationship and tangent modulus. Regarding safety, corneal edema declined more rapidly; ECD recovery occurred sooner in the pulsed-light accelerated CXL than in the continuous-light accelerated CXL group.
Our results regarding stromal demarcation depths are consistent with 2 clinical studies in which the mean demarcation line was more superficial at only approximately 150 to 160 μm with the continuous-light accelerated CXL protocol after exposure to 30 mW/cm2 with a larger dose of 7.2 J/cm2 compared with approximately 210 μm obtained with the pulsed-light accelerated CXL protocol.13,28 At 14 days, we detected a stromal demarcation line that was 87 μm deeper with the pulsed-light accelerated CXL protocol than with the continuous-light accelerated CXL protocol. Considering that the depth of stromal demarcation line could be representative of crosslinking effectiveness. This difference suggests that the pulsed-light accelerated CXL protocol could be more effective than the continuous-light accelerated CXL protocol at inducing corneal stromal crosslinking in rabbits.
In porcine corneas, the pulsed-light accelerated CXL protocol increased crosslinking more than that with the continuous-light accelerated CXL protocol based on a greater enzymatic resistance to digestion in the pulsed CXL-treated group. In this study, the dose for both protocols was 7.2 J/cm2. With the pulsed-light accelerated CXL-treated group, 30 mW/cm2 was applied for 8 minutes at intervals of 10 seconds, whereas the continuous-light accelerated CXL group received 30 mW/cm2 for 4 minutes.15 However, this study did not directly determine whether the increases in CXL were associated with increases in biomechanical strength. Kling and Hafezi,16 in an ex vivo porcine study, compared standard CXL and continuous-light accelerated CXL with pulsed-light accelerated CXL with different intensities and doses based on the results obtained with applying a theoretical CXL model. They found higher efficacy with the standard CXL than with either the continuous-light accelerated CXL or the pulsed-light accelerated CXL protocols for which the efficacy of continuous-light accelerated CXL was similar to pulsed-light accelerated CXL. Standard CXL demonstrated a clear higher efficacy than accelerated CXL and pulsed CXL.
There is an association between increases in crosslinking and increases in corneal biomechanical strength based on the effects of the 2 procedures on stromal demarcation depths and stress–strain relationships and tangent modulus. The stress–strain and tangent modulus–stress relationships shown in Figure 3, A and B, respectively, indicate that the pulsed-light accelerated CXL and continuous-light accelerated CXL groups were significantly different from the control group. Because this difference was greater in the pulsed-light accelerated CXL group than that in the continuous-light accelerated CXL group, it suggests that the stiffness increase obtained with the pulsed-light accelerated CXL was greater than that with the continuous-light accelerated CXL protocol. This difference is suggestive of more corneal crosslinking in the pulsed-light accelerated CXL-treated group, which agrees with the deeper stromal demarcation line in the pulsed-light accelerated CXL group.
Oxygen is essential to drive the UVA–riboflavin CXL process and in a hypoxic condition crosslinking formation might be impaired.29 The CXL protocols delivering higher UVA intensity and shorter irradiation time result in decreased CXL efficacy within the most anterior stromal region.6,15 It is possible that reduced CXL efficacy with the continuous-light accelerated CXL protocols is caused by more rapid oxygen depletion compared with the more prolonged but less intense UVA exposure obtained with the standard CXL protocol.9 The possibility of oxygen depletion might be mitigated by the pulsed CXL protocol because oxygen levels are likely to increase during the intervening dark periods between light pulses.12,30 Furthermore, an ex vivo study found that the Bunsen-Roscoe reciprocity law is only valid for illumination intensities up to 40 to 50 mW/cm2 and illumination times lasting more than 2 minutes.20 This might explain why the pulsed CXL using higher intensity with 30 mW/cm2 and 100 mW/cm2 in the Kling and Hafezi16 study did not generate a better stiffening effect than that obtained with either the standard CXL protocol with 3 mW/cm2 or the accelerated CXL protocol with an intensity of 9 mW/cm2.
Transient declines in the ECD is a common complication of CXL treatment in rabbit corneas in which the thickness is less 400 μm, even though it does recover with different time courses.31,32 In the current study, losses in ECD were accompanied by massive edema after 1 day in both CXL groups. One week after treatment, in vivo confocal laser scanning of tissues treated with the pulsed-light accelerated CXL protocols showed that endothelial cell layer integrity and density recovered based on the reappearance of regular hexagonal cell-shaped arrays, whereas in the continuous-light accelerated CXL group, endothelial cell losses were still evident. Such recovery might have occurred because of stimulation of proliferation and migration of neighboring undamaged cells located outside the irradiated area.32
Some studies found that during 24 hours after exposure to the standard CXL protocol, the maximum cytotoxic damage occurred along with massive keratocyte apoptosis.33,34 Similar in this study, significant increases in keratocyte apoptosis occurred in the anterior stroma after either continuous-light accelerated CXL or pulsed-light accelerated CXL treatment. One and 7 days after CXL, in vivo confocal microscopy revealed remnants of fragmented keratocytes in the anterior stromal matrix that were identifiable as hyperreflective granular structures in the pulsed-light accelerated CXL group. These effects were confirmed by H&E stromal staining. In contrast, the prevalence of apoptotic cell figures at different stromal depths was unrelated to the depth of stromal demarcation lines. The CCT in both CXL-treated groups was significantly increased accompanied by declines in ECD. Although significant corneal thinning was observed at 7 days posttreatment, continuous-light accelerated CXL-treated tissues were thicker than pulsed-light accelerated CXL-treated corneas. These results point to the possibility that the continuous-light accelerated CXL is more injurious than the pulsed-light accelerated CXL protocol.
In conclusion, we found that CXL efficacy of a uniform UVA dosage is more effective and less injurious if it is delivered in a pulsed-light accelerated CXL protocol than in a continuous-light accelerated CXL protocol in rabbits. This assessment of efficacy is based on the depths of stromal demarcation lines along with differences in stress–strain relationship and tangent modulus. Even though both CXL procedures induced corneal edema and endothelial cell losses, the extent of recovery of endothelial cell integrity and density and thickness recovery was more rapid with the pulsed-light accelerated CXL protocol than with the continuous-light accelerated CXL. Further studies are warranted to determine whether these differences in CXL procedural efficacy and safety in rabbit corneas are translatable over the long-term to humans with ectatic disorders.
WHAT WAS KNOWN
- The pulsed-light accelerated CXL and continuous-light accelerated CXL procedures with UVA and riboflavin both increase resistance to enzymatic digestion.
- The stromal CXL demarcation line is significantly deeper in the pulsed-light accelerated CXL versus continuous-light accelerated CXL protocol. However, nothing was known about the relative safety and efficacy of biomechanical strength with these 2 CXL protocols.
WHAT THIS PAPER ADDS
- The greater changes in biomechanical properties obtained with the pulsed-light accelerated CXL protocol suggest that this protocol was more efficacious than the continuous-light accelerated CXL protocol.
- The enhanced stiffening effect coupled with its smaller disruptive changes in stromal architecture and corneal functional parameters suggest that the pulsed-light accelerated CXL protocol was more efficacious and less injurious than the continuous-light accelerated CXL protocol.
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Disclosures:None of the authors has a financial or proprietary interest in any material or method mentioned.