Sabine Kling

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.

Stromal Demarcation Line in Pulsed Versus Continuous Light Accelerated Corneal Cross-linking for Keratoconus.

PURPOSE To compare the stromal demarcation line depth in pulsed versus continuous corneal cross-linking (CXL) for keratoconus. METHODS Seventy eyes underwent epithelium-off cross-linking, with 0.1% riboflavin applied during 10 minutes prior to ultraviolet irradiation at 30 mW/cm2. Thirty-six eyes received pulsed irradiation (1 second on, 1 second off) for 8 minutes and 34 eyes underwent continuous irradiation for 4 minutes. Total fluence was 7.2 J/cm2 for both groups. Patients were evaluated at 3 months after the procedure. RESULTS A significantly deeper stromal demarcation line was observed in the pulsed group compared to the continuous group (201.11 ± 27.76 vs. 159.88 ± 20.86 µm; P < .001). CONCLUSIONS The pulsed corneal cross-linking protocol induced a significantly deeper stromal demarcation line when compared to the 4 minutes of highly accelerated continuous CXL protocol. Neither CXL protocol induced a shallower demarcation line comparable to less accelerated CXL protocols previously reported.

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.

Corneal biomechanics – a review

In recent years, the interest in corneal biomechanics has strongly increased. The material properties of the cornea determine its shape and therefore play an important role in corneal ectasia and related pathologies. This review addresses the molecular origin of biomechanical properties, models for their description, methods for their characterisation, techniques for their modification, and computational simulation approaches.

An Algorithm to Predict the Biomechanical Stiffening Effect in Corneal Cross-linking

PURPOSE To develop an algorithm to predict the stiffening effect of CXL and to verify the accuracy with results obtained from experimental measurements. METHODS The algorithm considers different variables: the reaction kinetics of riboflavin diffusion and riboflavin photodegradation to determine the effective riboflavin concentration in different stromal layers; the oxygen diffusion and ultraviolet (UV) absorption to determine the amount of reactive oxygen species as a function of time and stromal depth. For the experimental comparison, corneas were deepithelialized, followed by riboflavin instillation for 30 minutes and UV irradiation. Different pulsed and continuous-light conditions were analyzed with irradiances ranging from 3 to 100 mW/cm2 and irradiation times from 8 to 30 minutes. Stress-relaxation measurements were performed in fresh-enucleated porcine (n = 66) and rabbit (n = 2) eyes directly after treatment, using a load of 0.6 MPa. RESULTS A clear linear relationship was observed between the concentration of newly induced cross-links and the experimentally observed stiffening factor (R2 = 0.9432). An additional 1 mol/m3 of cross-links increased the mechanical stress resistance of the cornea by 50.4%. The efficacy of standard CXL in murine, lapine, and porcine corneas was inversely related to corneal thickness. The stiffening effect after CXL decreased by 4.1% per 100 µm (R2 = 0.9961). CONCLUSIONS The proposed model, supported by data in porcine, murine, and lapine corneas, suggests a possibility of also predicting the biomechanical CXL efficacy in human corneas. The biomechanical efficacy of CXL may be increased by prolonged UV irradiation at reduced irradiances or by a higher oxygen pressure in the environment. Pulsed CXL does not accelerate CXL or increase its efficacy when compared to standard CXL of the same irradiation duration. This model might be used to calculate customized irradiation settings for high-risk cases, but also topography-guided CXL treatments. [J Refract Surg. 2017;33(2):128-136.].

Biomechanical Differences Between Femtosecond Lenticule Extraction (FLEx) and Small Incision Lenticule Extraction (SmILE) Tested by 2D-Extensometry in Ex Vivo Porcine Eyes

Purpose To evaluate the biomechanical stability of ex vivo porcine corneas after femtosecond lenticule extraction (FLEx) and small incision lenticule extraction (SmILE) refractive surgeries. Methods Forty-five porcine eyes were equally divided into three groups: Groups 1 and 2 were treated with FLEx and SmILE procedure, respectively. Group 3 served as control. A refractive correction of -14 diopters (D) with a 7-mm zone using either a 160-μm flap (FLEx) or a 160-μm cap (SmILE) was performed. For two-dimensional (2D) elastic and viscoelastic biomechanical characterization, two testing cycles (preconditioning stress-strain curve from 1.27 to 12.5 N, stress-relaxation at 12.5 N during 120 seconds) were conducted. Youngs modulus and Prony constants were calculated. Results At 0.8% of strain, FLEx (370 ± 36 kPa) could resist a significantly lower stress than SmILE (392 ± 19 kPa, P = 0.046) and the control group (402 ± 30 kPa, P = 0.013). Also, FLEx (46.1 ± 4.5 MPa) had a significantly lower Youngs modulus than the control group (50.2 ± 3.4 MPa, P = 0.008). The Youngs modulus of SmILE (48.6 ± 2.5 MPa) had values situated between untreated corneas and FLEx-treated corneas. When compared to untreated controls, the stress resistance decreased by 8.0% with FLEx and 2.5% with SmILE; Youngs modulus decreased by 5.1% with FLEx and 1.04% with SmILE. With a cap-based procedure, both anterior cap and stromal bed carry the intraocular pressure, while in a flap-based procedure, only the stromal bed does. Conclusions Compared to flap-based procedures like FLEx, the cap-based technique SmILE can be considered superior in terms of biomechanical stability, when measured experimentally in ex vivo porcine corneas.

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.

Biomechanical Weakening of Different Re-treatment Options After Small Incision Lenticule Extraction (SMILE).

PURPOSE To determine the corneal weakening induced by different re-treatment options after small incision lenticule extraction (SMILE) and investigate the potential of corneal cross-linking (CXL) to reestablish the original corneal stress resistance. METHODS A total of 96 freshly enucleated porcine corneas were used. The initial refractive correction was defined to be -11.00 diopters (D) and the required enhancement to be -3.00 D. Three different re-treatment options were analyzed: -3D Re-SMILE, -3D photorefractive keratectomy (PRK) on top of the SMILE cap, and cap-to-flap conversion and -3D excimer ablation on the stromal bed (LASIK). The control condition did not receive any treatment. Subsequently, accelerated CXL (9 mW/cm2, 10 min) was performed in two groups with currently common enhancement techniques: following cap-to-flap conversion (-3D LASIK enhancement) and in controls. Biomechanical properties were measured with stress-strain extensometry ranging from 1.27 to 12.5 N. RESULTS The Re-SMILE and PRK enhancement did not significantly reduce the overall elastic modulus of the cornea compared to controls (24.7 ± 2.23 and 22.7 ± 2.61 versus 23.8 ± 3.35 MPa, P ≥ .176), whereas LASIK enhancement did (22.2 ± 3.37 MPa, P = .048). CXL treatment significantly increased the elastic modulus compared to all non-cross-linked conditions (P ≤.001). Refractive surgery decreased the overall elastic modulus by 7%, whereas CXL increased it by 20%. CONCLUSIONS In enhancement, the corneal biomechanical integrity is less affected with both Re-SMILE and PRK enhancement. Corneal weakening through laser refractive surgery is small compared to the stiffening effect after CXL. [J Refract Surg. 2017;33(3):193-198.].

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.