Arthur Hammer

Corneal biomechanical properties at different corneal cross-linking (CXL) irradiances.

PURPOSE New corneal cross-linking (CXL) devices are capable of using higher UV-A light irradiances than used in original CXL protocols. The Bunsen-Roscoe law states that a photochemical reaction should stay constant if the delivered total energy is kept constant; however, little clinical data are available to support this hypothesis. METHODS We investigated the biomechanical properties of four groups (n = 50 each) of porcine corneas. Three groups were exposed to riboflavin 0.1 % and UV-A irradiation of equal total energy (3 mW/cm(2) for 30 minutes, 9 mW/cm(2) for 10 minutes, and 18 mW/cm(2) for 5 minutes). Controls were exposed to riboflavin 0.1% without irradiation. Youngs modulus of 5-mm wide corneal strips was used as an indicator of corneal stiffness. RESULTS We observed a decreased stiffening effect with increasing UV-A intensity. Youngs modulus at 10% strain showed significant differences between 3 mW/cm(2) and 9 mW/cm(2) (P = 0.002), 3 mW/cm(2) and 18 mW/cm(2) (P = 0.0002), 3 mW/cm(2) and the control group (P < 0.0001), and 9 mW/cm(2) and the control group (P = 0.015). There was no difference between 18 mW/cm(2) and the control group (P = 0.064) and between 9 mW/cm(2) and 18 mW/cm(2) (P = 0.503). CONCLUSIONS The biomechanical effect of CXL decreased significantly when using high irradiance/short irradiation time settings. Intrastromal oxygen diffusion capacity and increased oxygen consumption associated with higher irradiances may be a limiting factor leading to reduced treatment efficiency. Our results regarding the efficiency of high-irradiance collagen cross-linking (CXL) raise concerns about the clinical efficiency of the new high-irradiance CXL devices already used in clinical practice without proper validation.

Antibacterial efficacy of accelerated photoactivated chromophore for keratitis-corneal collagen cross-linking (PACK-CXL).

PURPOSE To investigate whether optimized photoactivated chromophore for keratitis-corneal collagen cross-linking (PACK-CXL) treatment settings allow accelerating treatment while maintaining antibacterial efficacy. METHODS Staphylococcus aureus and Pseudomonas aeruginosa strains were irradiated with ultraviolet-A light of equal fluence but different intensity settings (18 mW/cm² for 5 minutes and 36 mW/cm² for 2.5 minutes). The killing rate was determined by comparing the number of colony-forming units between cross-linked specimens and non-irradiated controls. The potential additional effect of 0.001% benzalkonium chloride was also investigated. RESULTS The killing rates for Staphylococcus aureus were 92.5% ± 5.5% (5 minutes at 18 mW/cm²) and 94.4% ± 2.9% (2.5 minutes at 36 mW/cm²). For Pseudomonas aeruginosa, the killing rates were 93.2% ± 8.3% (5 minutes at 18 mW/cm²) and 92.9% ± 5.0% (2.5 minutes at 36 mW/cm²). The presence of benzalkonium chloride in the riboflavin solution did not increase the killing rate significantly. CONCLUSIONS The antibacterial efficacy of PACK-CXL follows the Bunsen-Roscoe law of reciprocity and can be maintained even when the irradiation intensity is considerably increased. These optimized settings may allow a shortened treatment time in the future for PACK-CXL and thus help facilitate the transition from the operating room to the slit lamp for treatment.

Increased Biomechanical Efficacy of Corneal Cross-linking in Thin Corneas Due to Higher Oxygen Availability.

PURPOSE To compare the currently available ultraviolet-A (UV-A) corneal cross-linking (CXL) treatment protocols for thin corneas with respect to oxygen, UV fluence, and osmotic pressure. METHODS Freshly enucleated murine (n = 16) and porcine (n = 16) eyes were used. The dependency on oxygen and the amount of UV absorption were evaluated using different CXL protocols, including standard CXL, contact lens-assisted CXL (caCXL), and CXL after corneal swelling. The CXL protocol was adapted from the treatment parameters of the human cornea to fit the thickness of murine and porcine corneas. Immediately after CXL, the corneas were subjected to biomechanical testing, including preconditioning, stress relaxation at 0.6 MPa, and stress-strain extensiometry. A two-element Prony series was fitted to the relaxation curves for viscoelastic characterization. RESULTS Standard CXL was most efficient; prior corneal swelling reduced the long-term modulus by 6% and caCXL by 15% to 20%. Oxygen reduction decreased the long-term modulus G∞ by 14% to 15% and the instantaneous modulus G0 by 2% to 5%, and increased the short-term modulus G2 by 22% to 31%. Reducing the amount of absorbed UV energy decreased the long-term modulus G∞ by 5% to 34%, the instantaneous modulus G0 by 7% to 29%, and the short-term modulus G2 by 17% to 20%. The amount of absorbed UV light was more important in porcine than in murine corneas. CONCLUSIONS The higher oxygen availability in thin corneas potentially increases the overall efficacy of riboflavin UV-A CXL compared to corneas of standard thickness. Clinical protocols for thin corneas should be revised to implement these findings.

The Effect of Standard and High-Fluence Corneal Cross-Linking (CXL) on Cornea and Limbus

PURPOSE When treating peripheral ectatic disease-like pellucid marginal degeneration (PMD), corneal cross-linking with UV-A and riboflavin (CXL) must be applied eccentrically to the periphery of the lower cornea, partly irradiating the corneal limbus. Here, we investigated the effect of standard and double-standard fluence corneal cross-linking with riboflavin and UV-A (CXL) on cornea and corneal limbus in the rabbit eye in vivo. METHODS Epithelium-off CXL was performed in male New Zealand White rabbits with two irradiation diameters (7 mm central cornea, 13 mm cornea and limbus), using standard fluence (5.4 J/cm(2)) and double-standard fluence (10.8 J/cm(2)) settings. Controls were subjected to epithelial removal and riboflavin instillation, but were not irradiated with UV-A. Following CXL, animals were examined daily until complete closure of the epithelium, and at 7, 14, 21, and 28 days. Animals were killed and a corneoscleral button was excised and processed for light microscopy and immunohistochemistry. RESULTS For both irradiation diameters and fluences tested, no signs of endothelial damage or limbal vessel thrombosis were observed, and time to re-epithelialization was similar to untreated controls. Histological and immunohistochemical analysis revealed no differences in the p63 putative stem cell marker expression pattern. CONCLUSIONS Even when using fluence twice as high as the one used in current clinical CXL settings, circumferential UV-A irradiation of the corneal limbus does not alter the regenerative capacity of the limbal epithelial cells, and the expression pattern of the putative stem cell marker p63 remains unchanged. This suggests that eccentric CXL may be performed safely in PMD.

Establishing Corneal Cross-Linking With Riboflavin and UV-A in the Mouse Cornea In Vivo: Biomechanical Analysis

PURPOSE To establish corneal cross-linking (CXL) with riboflavin and UV-A in in the mouse cornea in vivo and to develop tools to measure the biomechanical changes observed. METHODS A total of 55 male C57BL/6 wild-type mice (aged 5 weeks) were divided into 14 groups. Standard CXL parameters were adapted to the anatomy of the mouse cornea, and riboflavin concentration (0.1%-0.5%) and fluence series (0.09-5.4 J/cm²) were performed on the assumption of the endothelial damage thresholds. Untreated and riboflavin only corneas were used as controls. Animals were killed at 30 minutes and at 1 month after CXL. Corneas were harvested. Two-dimensional (2D) biomechanical testing was performed using a customized corneal holder in a commercially available stress-strain extensometer/indenter. Both elastic and viscoelastic analyses were performed. Statistical inference was performed using t-tests and specific mathematical models fitted to the experimental stress-strain and stress-relaxation data. Adjusted P values by the method of Benjamini and Hochberg are reported. RESULTS For all CXL treatment groups, stress-relaxation showed significant differences (P < 0.0001) after 120 seconds of constant strain application, with cross-linked corneas maintaining a higher stress (441 ± 40 kPa) when compared with controls (337 ± 39 kPa). Stress-strain analysis confirmed these findings but was less sensitive to CXL-induced changes: at 0.5% of strain, cross-linked corneas remained at higher stress (778 ± 111 kPa) when compared with controls (659 ± 121 kPa). CONCLUSIONS Cross-linking was induced in the mouse cornea in vivo, and its biomechanical effect successfully measured. This could create opportunities to study molecular pathways of CXL in transgenic mice.

Determination of the excimer laser ablation rate in previously cross-linked corneas

PURPOSE To evaluate the need for and quantify the extent of nomogram adjustments to compensate for potential changes in the amount of effective corneal stroma ablated in previously cross-linked corneas. METHODS Ex vivo porcine corneas were divided into two groups (the corneal cross-linking [CXL] group, n = 30; and the control group, n = 3): these experimental corneas underwent CXL including deepithelialization, instillation of riboflavin solution for 25 minutes, and ultraviolet-A irradiation at 9 mW/cm2 for 10 minutes. The control group was deepithelialized only. Four consecutive excimer laser ablations of 50 pm each were performed (AMARIS 750S; SCHWIND eye-tech-solutions, Kleinostheim Germany), and stromal bed thickness was measured with a built-in optical coherence pachymeter. To determine the potential influence of riboflavin, a third group (the riboflavin group, n = 12) underwent deepithelialization and instillation of riboflavin, but no ultraviolet-A irradiation. RESULTS The mean individual ablation depth across the four ablations was significantly smaller in cross-linked corneas (-17%) when compared to untreated control corneas (P < .001). A consistent reduction of 12% was observed via a cumulative analysis when assessing the relative isolated effect of CXL on the ablation rate. There was no significant effect from riboflavin in the deeper ablations, except for the first ablation (68.6 + 1.1 mm [range: 1 to 50 pm]). This may be due to a measurement error in pachymetric readings due to the thin film of riboflavin on the surface that resists even extensive rinsing. CONCLUSIONS CXL reduces the corneal ablation depth of excimer lasers in the anterior 200 pm of the porcine cornea by approximately 12%. Further clinical studies are needed to validate these findings in human corneas.

A constant-force technique to measure corneal biomechanical changes after collagen cross-linking.

Purpose To introduce a constant-force technique for the analysis of corneal biomechanical changes induced after collagen cross-linking (CXL) that is better adapted to the natural loading in the eye than previous methods. Methods For the biomechanical testing, a total of 50 freshly enucleated eyes were obtained and subdivided in groups of 5 eyes each. A Zwicki-Line Testing Machine was used to analyze the strain of 11 mm long and 5 mm wide porcine corneal strips, with and without CXL. Before material testing, the corneal tissues were pre-stressed with 0.02 N until force stabilization. Standard strip extensiometry was performed as control technique. For the constant-force technique, tissue elongation (Δ strain, %) was analyzed for 180 seconds while different constant forces (0.25 N, 0.5 N, 1 N, 5 N) were applied. Results Using a constant force of 0.5 N, we observed a significant difference in Δstrain between 0.26±0.01% in controls and 0.12±0.03% in the CXL-treated group (p = 0.003) over baseline. Similarly, using a constant force of 1 N, Δstrain was 0.31±0.03% in controls and 0.19±0.02% after CXL treatment (p = 0.008). No significant differences were observed between CXL-treated groups and controls with 0.25 N or 5 N constant forces. Standard stress-strain extensiometry failed to show significant differences between CXL-treated groups and controls at all percentages of strains tested. Conclusion We propose a constant-force technique to measure corneal biomechanics in a more physiologic way. When compared to standard stress-strain extensiometry, the constant-force technique provides less variability and thus reaches significant results with a lower sample number.

Corneal Cross-Linking with Riboflavin and UV-A in the Mouse Cornea in Vivo: Morphological, Biochemical, and Physiological Analysis

Purpose To morphologically, biochemically, and physiologically characterize corneal cross-linking with riboflavin and UV-A light (CXL) in a newly established in vivo murine model. Methods C57BL/6 wild-type mice (N = 67) were treated with various CXL protocols, with modification of the following parameters: total energy (fluence) used, duration of UV-A irradiation, continuous versus pulsed irradiation, and CXL under hypoxic conditions (contact lens). Corneas were evaluated biomicroscopically, histologically, and using optical coherence tomography. Conformational collagen changes were evaluated via changes in the speed of enzymatic digestion. Results A fluence of 5.4 J/cm2 induced scar formation, while fluences of < 0.18 J/cm2 induced neovascularization. Fluences between 1.62 and 2.7 J/cm2 reduced epithelial thickness, but maintained a transparent cornea after 1 month. Pulsed UV irradiation inhibited neovascularization, but favored scar formation. Changes in the speed of enzymatic digestion suggest that CXL in mice, when compared to humans, requires less UV-A energy than the difference in corneal thickness between the species would suggest. Conclusions We demonstrated the in vivo response of very strong and very weak CXL and identified the best suited range of UV fluence in murine corneas. The presented murine CXL model may be helpful in future research addressing cellular and molecular pathways associated to CXL treatment. Translational Relevance Adverse tissue reactions following CXL treatment were observed, if the administered UV energy was out of the treatment window—raising concern about novel CXL treatment protocols that have not been previously validated in an experimental setting.

Repeated Cross-linking After a Short Time Does Not Provide Any Additional Biomechanical Stiffness in the Mouse Cornea In Vivo

PURPOSE To study whether repeated collagen cross-linking (CXL) performed in vivo in mice shows an additive effect on mechanical corneal stiffness. METHODS In this experimental study, epithelium-off CXL was performed in a total of 18 eyes from male C57BL/6 mice, with 0.27%-riboflavin solution applied for 20 minutes, followed by ultraviolet-A (UVA) irradiation (365 nm, 9mW/cm2) for 2:50 minutes (fluence 1.53 J/cm2). CXL was performed as either a single (1×CXL) or a repeated (2×CXL) treatment. Un-irradiated corneas served as controls. In the 2×CXL group, the procedure was performed on day 1 and day 4 to ensure complete reepithelialization between sessions. Biomechanical analysis was performed on day 7. Corneas were harvested with a small scleral ring and mounted on a customized two-dimensional flap holder. The biomechanical measurement consisted of three parts: (1) pre-conditioned during three cycles from 0.04 to 0.4 N, (2) stress relaxation during 120 seconds following 0.4 N force application, and (3) stress-strain curve until break. RESULTS After the relaxation period of 120 seconds, highly significant differences (P < .001) were found between the controls and both 1×CXL corneas and 2×CXL corneas. No significant difference (P = .70) was detected between the 1×CXL and 2×CXL groups. The stress remaining after relaxation was 355 ± 25.2 kPa in the control group, 457 ± 34.1 kPa in the 1×CXL group, and 463 ± 22.2 kPa in the 2×CXL group. No significant differences in the stress-strain curves were found between the conditions. CONCLUSIONS Repeated CXL 3 days after the first procedure does not further increase corneal stiffness in mice in vivo. [J Refract Surg. 2017;33(1):56-60.].

Analysis of Riboflavin Compounds in the Rabbit Cornea In Vivo.

ABSTRACT Purpose: To investigate the composition and concentration of individual riboflavin compounds in the corneal stroma in vivo after soaking with various commercially available riboflavin formulations. Methods: Experiments were performed in 26 rabbit corneas in vivo: 24 corneas were soaked with riboflavin formulations for 30 minutes or with 0.9% NaCl for control (n = 2). After treatment, corneas were excised and prepared for ultra-high-pressure liquid chromatography (UHPLC) analysis. Additionally, computational chemical analysis of riboflavin compounds and keratan sulfate were performed. Results: The amount of riboflavin and riboflavin phosphate isomers in cornea decreased by a factor of 10 to 100, when compared to the amount in riboflavin formulations. In particular, we found an inverse relationship in the ratio of riboflavin to riboflavin phosphate isomer concentration between formulations and cornea. The electronegativity and ionization potential of riboflavin and phosphate isomers are different. Conclusions: The inverse relationship observed might be explained by a stronger electronegativity of the phosphate isomers, leading to a stronger repulsion by corneal proteoglycans. Indicating the individual concentration of riboflavin compounds in formulations is more representative than the total riboflavin concentration. Riboflavin formulations and CXL protocols might be improved considering the differences in diffusion and ionization potentials of the different riboflavin compounds.