Corneal cross-linking (CXL) is an effective treatment to halt progression of progressive keratoconus.1 With extrafibrillar covalent bonds induced by photosensitizer riboflavin and ultraviolet A (UV-A) light of 370 nm, corneal Young's modulus can be increased by a factor of 4.5 in human eyes.2 However, there is increased risk of endothelial damage in thin corneas.3–6
Several modified CXL protocols have been developed to address thin corneas, which are frequently observed in advanced keratoconus. These modified protocols can be categorized into two groups: decreasing UV exposure or increasing stromal thickness. To decrease UV exposure, thicker riboflavin film or higher riboflavin concentration can lead to greater absorption of the UV light, thereby reducing efficacy and potential endothelial toxicity.7,8 Accelerated CXL protocols, with higher UV intensity for shorter irradiation time with the same total UV irradiation dose, can also halt the progression of keratoconus with potentially less depth of corneal penetration and therefore less significant endothelial cell loss.9 To increase stromal thickness, stroma can be swelled by hypo-osmolar riboflavin,10 covered by partial epithelium with customized epithelial debridement,11 or shielded by a riboflavin-soaked contact lens.12–14 Among these protocols, hypo-osmolar swelling has been found to be less efficacious,15,16 whereas contact lens–assisted CXL (CACXL) provides a straightforward option for CXL in thin corneas without requiring complicated equipment or precise manipulation. The safety of this protocol has been demonstrated recently.12,13 Ex vivo overall biomechanical properties evaluated with stress-strain tests have shown reduced efficacy,14 most probably due to a relative lack of oxygen in the deeper layers of the cornea,17 but depth-dependent stiffness distribution remains unknown.
In this study, a non-contact optical method, confocal Brillouin microscopy, was used to determine axial stiffness distribution before and after standard CXL and CACXL in porcine eyes, and these results were compared with traditional mechanical stress-strain evaluation to determine the comparative difference in both total and regional stiffening effects induced by each protocol.
Materials and Methods
The study was performed on 30 intact fresh porcine eyes that were kept in balanced salt solution during shipment and were used within 24 hours after collection. These 30 eyes were equally divided into two groups: standard CXL and CACXL. Pachymetry (Pachette 4; DGH Technology, Exton, PA) was performed on all eyes before experiments to guarantee similar hydration status for the two groups. For all eyes, adherent muscle and adipose tissue was detached without damaging the whole globe to allow for stable fixation, and epithelium was carefully removed with a crescent knife. Riboflavin solution was prepared by diluting riboflavin and dextran to 0.1% and 10% separately with 1X PBS. Unlike 20% dextran, which is generally used in standard CXL, hypo-osmolar riboflavin solution was used because of long-term disputes over the influence of the water content in Brillouin microscopy.18,19 A 10% dextran solution was used in previous Brillouin measurements to avoid corneal dehydration during CXL,20 which was also confirmed by Brillouin microscopy, so that the stiffening effect mainly derived from CXL and dehydration had little influence on it. Contact lenses without UV filters were selected for CACXL (Bausch & Lomb, made of hilafilcon B, hydration 59%, diameter 14.2 mm, PWR −0.25). A Brillouin scan was performed on all eyes before and after standard CXL or CACXL to make each eye serve as its own control.
CXL protocols are shown in Table 1 and Figure A (available in the online version of this article). For standard CXL, intact fresh porcine eyes were soaked by dropping the riboflavin solution on corneas directly every 2 minutes for 30 minutes. After soaking, eyes were placed under a UV-A light (CCL-365 Vario; MLase AG, Germering, Germany) and exposed to a power density of 3 mW/cm2 for 30 minutes. During exposure, riboflavin was added on corneas every 2 minutes.
Corneal cross-linking (CXL) protocols for standard CXL and contact lens–assisted CXL (CACXL). Hydrophilic contact lenses were used to make riboflavin solution penetrate through contact lenses and diffuse into the stroma. Thus, there was no need to lift contact lenses during ultraviolet (UV) irradiation.
A similar soaking procedure was conducted on eyes for CACXL. The difference was that the contact lens was immersed in the riboflavin solution for 30 minutes prior to UV-A irradiation. Then, the fully soaked contact lens was placed on the cornea to serve as a barrier between the UV-A light and the cornea. During irradiation, riboflavin solution was dropped on the contact lens directly every 2 minutes for 30 minutes.
Biomechanical Properties Measured by Brillouin Microscopy
Spontaneous Brillouin scattering derives from the interaction between light and acoustic phonons generated by inherent density fluctuation, providing direct information on the speed of sound and viscoelastic properties.21,22 The relation between the Brillouin shift Δf and the speed of sound vA can be expressed as:
where ρ is density, n is refractive index, λ is probing laser wavelength and M' is longitudinal modulus, which can represent the stiffness of the specimen.
To measure the optical frequency shift, a Brillouin spectrometer was built, using two virtually imaged phased arrays as the core dispersion components.23,24 A continuous-wave 532-nm laser (torus 532; Laser Quantum, Stockport, United Kingdom) served as the probing light. Laser treatment at 12 mW was delivered onto the specimen through a 40× objective lens with a numerical aperture of 0.6 (LUCPLFLN 40×; Olympus, Tokyo, Japan). The scattered light, collected by the same objective lens, was coupled into the Brillouin spectrometer and imaged by an EMCCD camera (iXon-L-897; Andor Technology, Belfast, United Kingdom) with an exposure time of 0.2 second. When calculating M', ρ/n2 was treated as a constant of 0.57 g/cm3 based on previous research.25–27 This estimation introduced a 0.3% uncertainty throughout the cornea.28–30
Brillouin Image Analysis
A cross-section of 200 µm (lateral) × 1,200 µm (axial) in the center of a cornea was selected for each scan to image Brillouin shifts as a function of depth. The step sizes in both directions are 10 µm. In Brillouin maps, red represented softness and blue rigidity. To compare depth-dependent stiffness variation after different CXL protocols, maximum increase ratios of Brillouin shifts were used. Moreover, the cross-sections were equally divided into three segments (anterior, middle, and posterior) and averaged percentage changes of longitudinal modulus in these three segments were calculated to indicate depth information.28,30
To verify CXL effect and support longitudinal modulus measured by Brillouin microscopy, mechanical stretching tests were operated on all corneas after CXL once Brillouin scan was completed, using a stress-strain extensometer (BT2-FZ0.5TS; Zwick/Roell, Kennesaw, GA) equipped with a 5 N capacity load cell. The stretching tests were not performed on untreated corneas because intact eyeballs were needed for CXL and the goal was to investigate CXL efficacy on the same cornea. A corneoscleral strip (5 mm width) was prepared centrally in the horizontal axis from each eye. Its thickness was determined by performing pachymetry on the intact eyeball immediately after standard CXL or CACXL. Two ends of each strip were clamped between two grips with an initial gap of 5 mm.
A preload of 0.2 N was applied on the strip first. After 30 seconds of hanging, three conditioning cycles were applied to realign fibril orientation and stabilize mechanical properties. In each cycle, distance between the two grips increased at a speed of 2 mm/min until a force of 2 N was reached. Then, a new stretching test was performed to measure Young's modulus. Forces and corresponding strains were recorded by the extensometer automatically. The thickness and width of the strip were used to convert force to stress. After the transformation, stress-strain dots were plotted and fitted by an exponential function σ = Aexp(B × ∊). Young's moduli, E, were calculated from the gradient of the exponential function at strains of 4%, 6%, 8%, and 10% separately.
Brillouin Images of Standard CXL and CACXL
The average corneal thickness was 1,092 ± 37 µm in the standard CXL group and 1,082 ± 28 µm in the CACXL group (P = .38). Representative Brillouin shifts for standard CXL and CACXL are shown in Figures 1–2. To demonstrate the stiffening effect of the CXL treatments, untreated corneas were scanned from the anterior to aqueous humor before CXL (Figure 1A and Figure 2A) and again after CXL (Figure 1B and Figure 2B). For better visualization of depth-dependent stiffness change, depth profiles were calculated from the cross-sectional images, Figures 1A–1B, by averaging over the transverse axis. Figure 1C shows the depth profiles of this representative sample before and after standard CXL. The abscissa represented geometric thickness, rescaled by thickness measured by the pachymeter, taking the refractive index into account. As corneas became stiffer after CXL, theoretically the speed of sound set in the pachymeter should be updated to a larger value to accurately measure corneas after CXL. However, in reality there was minimal effect on measurements based on our experimental results. The influence can be estimated in the following way. In light of the equation, the speed of sound is proportional to the Brillouin shift. Assuming the maximum frequency shift after CXL is fmax, the minimum shift after CXL is fmin, and the averaged shift before CXL is still f0, the variation, [(fmax+fmin)/2–f0]/f0, for the standard CXL group was 1.53% ± 0.44%, whereas that for the CACXL group was 1.18% ± 0.48%. Thus, the thickness error caused by the speed of sound (vA) was limited to approximately 1.5%. Taking the increase of the refractive index after CXL into account, the influence was likely even less. A horizontal shift was added to the depth profile after CXL to make the two depth profiles overlap in the posterior region for clearer comparison.
Representative Brillouin results for the standard corneal cross-linking (CXL) group. (a) Distribution of Brillouin shifts in an untreated eye. Aqueous humor is the bottom part of the color map. (b) Distribution of Brillouin shifts in the same eye after standard CXL. According to the equation, a higher Brillouin shift correlates to a larger longitudinal modulus. Increase of longitudinal modulus is elucidated with different colors. (c) Profiles of Brillouin shifts in (a) and (b) along the depth, averaged in lateral direction. The way to divide the cornea into three segments is also shown.
Representative Brillouin results for the contact lens–assisted corneal cross-linking (CACXL) group. (a) Distribution of Brillouin shifts for an untreated eye. Alhough thickness of this cornea is different from the one in Figure 2(a), similar stiffness distribution can be seen. (b) Distribution of Brillouin shifts for the same eye after CACXL. (c) Lateral averaged depth profiles of Brillouin shifts in (a) and (b).
Besides information on depth-dependent elasticity, Figure 1C also illustrates the method of dividing the cornea into three segments for depth analysis. In Figure 1C, the softer posterior part after CXL was confirmed by Brillouin microscopy, which was anticipated due to the use of the hypo-osmolar riboflavin solution designed to suppress the influence of dehydration on artifactual stiffening effects anteriorly. Compared with standard CXL, representative Brillouin shifts of CACXL are shown in Figure 2B.
After standard CXL, compared to pretreatment there was a significant increase in the longitudinal modulus in the anterior (2.56 vs 2.76 GPa, P < .001) and middle (2.55 vs 2.60 GPa, P < .001) regions, whereas there was no significant stiffening induced in the posterior region (2.48 vs 2.46 GPa, P = .096). After CACXL, compared to pretreatment there was a significant increase in the anterior (2.56 vs 2.70 GPa, P < .001) and middle (2.55 vs 2.59 GPa, P < .001) regions, whereas there was no significant stiffening induced in the posterior region (2.47 vs 2.46 GPa, P = .318).
When comparing techniques, similar stiffening profiles can be seen in standard CXL and CACXL, as shown in Figures 1–2. The difference existed in the maximum Brillouin shift. Increased ratios were used instead of absolute values for the maximum Brillouin shift, considering diversity and relative flat Brillouin distribution of untreated corneas. The averaged shift of the anterior region before CXL, f0, served as the denominator and difference between the maximum shift and the averaged shift, (fmax–f0), was used as the numerator when calculating the ratios. There was an increase of 5.74% ± 1.29% in the standard CXL group and 3.99% ± 0.83% in the CACXL group (P < .001).
To demonstrate the axial stiffening difference (from anterior to posterior stroma) between standard CXL and CACXL with statistical analysis, depth-averaged modulus in each of the anterior, middle, and posterior regions were calculated. The increase of mean modulus after standard CXL was 7.8% in the anterior, 1.7% in the middle, and −0.7% in the posterior regions. In comparison, the increase of mean modulus after CACXL was 5.5% in the anterior, 1.2% in the middle, and −0.4% in the posterior regions. A significant difference existed between techniques in the anterior one-third of the cornea (P < .001). No statistical differences were present between techniques in the middle or posterior regions, as shown in Figure 3. The negative variation in the posterior region demonstrated that hydration was induced in the posterior regions.
Percentage change of mean longitudinal modulus of the anterior, middle, and posterior for standard corneal cross-linking (CXL) (n = 15) versus contact lens–assisted corneal cross-linking (CACXL) (n = 15). ***P < .001.
Stress values at selected strain points were first averaged, then exponential functions were used to fit the stress-strain curves (Figure 4). The gradient of these curves indicates Young's modulus; corresponding results are listed in Table 2. Standard CXL provided a greater stiffening effect than CACXL at all selected strains (P < .001).
Stress-strain curves fitted by mean values at strains of 4%, 6%, 8%, and 10%. Standard corneal cross-linking (CXL) had greater stiffness than contact lens–assisted corneal cross-linking (CACXL) at all selected strains.
Young's Modulus in MPa Calculated at 4%, 6%, 8%, and 10% Strain
Based on Brillouin microscopy results in enucleated porcine eyes, a larger maximum increase of the Brillouin shift could be found in the standard CXL group, with CACXL achieving 70% of the total stiffening effect of standard CXL. Both techniques induced significant corneal stiffening in the anterior and middle corneal regions, whereas no stiffening was induced in the posterior region of the cornea for either technique. Stiffening differences between techniques only existed in the anterior one-third of the cornea, with CACXL achieving 71% of the stiffening effect of standard CXL in this region, whereas in the other two regions no statistical difference could be observed between techniques (Figure 3). Stretching tests verified that CACXL induced less stiffening effect than standard CXL (Figure 4).
The application of a contact lens did not appear to shift the CXL effect anteriorly, but rather blunted the CXL effect in the anterior and middle regions compared to the standard technique without a contact lens in place (Figures 1–2). This has potential clinical implications because the CXL effect seems to be less than standard CXL rather than having the same stiffening effect but having that effect be anteriorly shifted. If the goal is to provide similar overall stiffening for thin corneas, future alternative strategies for treating thin cornea still need to be evaluated. However, because the primary concern for thin corneas is endothelial protection, safety, a blunted response may be sufficient to protect the endothelium in thin corneas.
Due to the design of the current study, we were not able to evaluate the safety of CACXL. Endothelial safety of CACXL has been reported previously in human studies, with no significant effect on endothelial mosaic or endothelial cell count reported during 6 months of follow-up in two studies.12,13 Endothelial safety of CACXL has also been shown using hydroxypropyl methylcellulose–based riboflavin solutions, which tend to provide a significantly deeper demarcation line than dextran-based riboflavin solutions. In the study conducted by Malhotra et al.,31 endothelial cell count within 6 months of follow-up was not significantly different from baseline in both types of solutions.
Biomechanical efficacy of CACXL has been evaluated in mouse eyes using stress-strain extensometry. Kling et al.17 reported that the biomechanical effect of CACXL was reduced overall by approximately 30% when compared to standard CXL. This difference is most likely due to the essential role of oxygen in the CXL process.32 When applying a contact lens onto the cornea in CACXL, oxygen availability can be limited, considering the presence of riboflavin film over and under the contact lens and reduced oxygen solubility in liquid.14 Similarly, Wollensak et al.14 reported that, in porcine corneas, the biomechanical effect of the CACXL in the anterior 400 µm was approximately two-thirds that of standard CXL. Applying the non-destructive method of Brillouin microscopy, we obtained a similar outcome. Figure 3 showed that the stiffening result after CACXL is approximately 70% of the CXL, which was validated by mechanical extensometer as shown in Figure 4. Wollensak et al. also performed anterior segment optical coherence tomography to measure the thickness of riboflavin film over and under the contact lens during the CACXL treatment.14 Their result showed that a thick riboflavin film on the contact lens eliminated the CXL effect. They recommended that the efficacy of CACXL might be improved by reducing or eliminating the riboflavin film over the contact lens. In our study, we instilled riboflavin solution on the contact lens every 2 minutes during the UV irradiation time.
In contrast to previous results,14 we measured significant biomechanical changes with both Brillouin microscopy and extensometry in eyes that received frequent riboflavin drops during UV irradiation. Because the same contact lens, energy levels, and treatment times were used in both studies, the 10% dextran concentration in our study might have led to lower riboflavin film viscosity and thickness in comparison to 20% dextran in the previous study. CACXL in the in vivo clinical setting has showed clinical effectiveness with applying riboflavin with dextran 20% over and under contact lens every 3 minutes during 30 minutes of UV irradiation.12,13 Differences between the shape and size of the human and porcine cornea may have altered the contact lens fit, which might affect the riboflavin film over and under the contact lens.
Brillouin microscopy has two major advantages to traditional mechanical testing. As a non-invasive method for evaluation of the biomechanical properties, it allows for repeat, longitudinal measurements that are not possible with destructive testing methods. In the current study, all samples were measured at baseline and then again after CXL, which allowed each to serve as its own control. Brillouin imaging also provides a three-dimensional depth map of longitudinal modulus.21,22,28
There are limitations to our study. Hydration status affects Brillouin measurements, and this must be taken into account for in vitro experiments, especially when the corneal stroma is exposed for a long period of time such as this experiment. With the protection of the contact lens, evaporation is theoretically slower in the CACXL group, which could amplify the difference between CACXL and standard CXL. To reduce the influence of hydration status, frequency of riboflavin application was shortened to every 2 minutes and hypo-osmolar riboflavin solution was used. Based on results shown in Figure 3, CACXL and standard CXL had similar hydration status in the posterior corneal region, which confirmed that evaporation with subsequent dehydration was not a major factor in this study. Similar to previous studies, we found no stiffening effect in the posterior portion of the cornea with either CXL technique. This is not surprising, because it is unlikely the posterior corneal region can undergo significant CXL regardless of technique due to its ultrastructural differences compared to the anterior and middle corneal regions.33
Our data show that ex vivo analysis using Brillouin microscopy led to biomechanical results similar to those obtained in ex vivo stress-strain measurements.14 Moreover, Brillouin microscopy allowed us to analyze the biomechanical stiffening effect in a depth-dependent manner, which is not possible using stress-strain extensometry. Brillouin microscopy thus is an ideal investigation method to support and even replace some of the testing done under ex vivo conditions.
The stiffening effect of CACXL was approximately 70% of the standard CXL technique in porcine eyes as measured by Brillouin microscopy and confirmed through extensometry testing. There did not appear to be any major shift in depth of treatment but rather a blunted treatment response in the anterior corneal stromal region. It remains to be determined what aspects of the contact lens protocol, particularly related to contact lens used, oxygen diffusion capacity, and method of riboflavin application, could be altered to maximize the stiffening effect and minimize risk to the corneal endothelium.
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|Fluence (total) (J/cm2)||5.4||5.4|
|Riboflavin soak time & interval (minutes)||30 (q2)||30 (q2)|
|Treatment time (minutes)||30||30|
|Epithelial status||Off – 9-mm removal||Off – 9-mm removal|
|Chromophore||0.1% riboflavin||0.1% riboflavin|
|Light source||CCL-365 Vario, (MLase AG)||CCL-365 Vario, (MLase AG)|
|Protocol abbreviation in manuscript||CXL||CACXL|
Young's Modulus in MPa Calculated at 4%, 6%, 8%, and 10% Strain
|Method||4% Strain||6% Strain||8% Strain||10% Strain|
|Standard CXL||2.97 ± 0.40||3.61 ± 0.42||4.28 ± 0.47||5.00 ± 0.71|
|CACXL||1.53 ± 0.40||1.93 ± 0.47||2.40 ± 0.50||2.97 ± 0.57|
|P||< .001||< .001||< .001||< .001|