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.

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.

[Keratoconus treatment by corneal cross-linking (CLX)]. / Prise en charge du kératocône par cross-linking du collagène cornéen.

Keratoconus is a disease of the cornea that usually begins during puberty and progressively weakens its biomechanical structure. Keratoconic eyes show a conic shape and progressive thinning, both leading to irregular astigmatism and reduced vision that cannot be corrected by glasses. In early cases, special contact lens can partly compensate for the visual loss while they do not stop disease progression. Until recently, the only treatment option was a corneal transplant. In 1999, a technique called corneal collagen cross-linking (CXL) was used in human corneas suffering from keratoconus for the first time. CXL uses a process called photopolymerization to halt the progression of keratoconus with an efficacy of more than 95%. Today our challenge is to screen and identify patients early enough to offer a treatment on time before irreversible vision loss develops.

A Novel Role for Corneal Pachymetry in Planning Cataract Surgery by Determining Changes in Spherical Equivalent Resulting from a Previous LASIK Treatment.

Objectives: To provide a metric to differentiate between hyperopic and myopic ablation of a prior LASIK treatment based on the corneal pachymetry profile after laser vision correction (LVC). Methods: Pachymetry data were retrospectively recovered from patients who had previous LASIK for refractive purposes between 2019 and 2020. Patients with any corneal disorder were excluded. Ablation spherical equivalent was predicted from the central to semiperipheral corneal thickness (CPT) ratio, both values were provided by using the Pentacam user interface software (UI), and values were computed from extracted raw pachymetry data. Results: Data of 157 eyes of 81 patients were collected, of which data were analysed for 73 eyes of 73 patients to avoid concurrence of measurements in both eyes per subject (42% female; mean age 40.9; SD 12.8). The CPT ratio cutoff for distinction between myopic and hyperopic LASIK was 0.86 for Pentacam UI data. Sensitivity and specificity were 0.7 and 0.95, respectively. Accuracy increased with computation of the CPT ratio based on extracted raw data with sensitivity and specificity of 0.87 and 0.99, respectively. There was a marked linear correlation between the CPT ratio and the ablation spherical equivalent (R2 = 0.93). Conclusions: CPT ratio cutoffs can correctly classify if a cornea previously had a hyperopic versus myopic LASIK surgery and estimate the ablation spherical equivalent of such treatment. This could prove useful for increased accuracy of intraocular lens (IOL) calculations for patients with no historical data of their prior LVC surgery at the time of cataract surgery planning.

Case Report: Multimodal Imaging of Toxic Retinopathies Related to Human Immunodeficiency Virus Antiretroviral Therapies: Maculopathy vs. Peripheral Retinopathy. Report of Two Cases and Review of the Literature.

Purpose: We report two patients with toxic retinopathy from either ritonavir or didanosine and reviewed the literature on the topics. We provide an overview of the retinal toxicity of these two antiretroviral drugs in human immunodeficiency virus-positive patients. Methods: First, we performed a retrospective study of the medical charts of two patients examined by us, one with ritonavir maculopathy and one with didanosine peripheral retinopathy. Secondly, we searched the world literature for similar cases through PubMed and Google Scholar, using the terms "HIV," "AIDS," "ritonavir," "didanosine," "maculopathy," "retinopathy," "visual loss," and "toxicity" to retrieve the appropriate literature on the subject. Results: Patient 1: A 49-year-old woman complained of progressive central visual loss over the past 12 months. History disclosed ongoing ritonavir therapy for the past 11 years. Ritonavir maculopathy was diagnosed, and visual loss increased relentlessly despite cessation of treatment. Patient 2: A 55-year-old man complained of slowly progressive peripheral visual field constriction for the past 5 years. History disclosed didanosine therapy for 13 years, however, stopped 4 years before the onset of visual symptoms. No alteration of therapy was offered to patient 2 as didanosine therapy was interrupted 9 years previously. Since 2011, 11 cases of ritonavir maculopathy have been reported in the literature. Relentless worsening of vision was reported in 3/7 patients despite cessation of ritonavir therapy. Didonasine peripheral retinopathy was first described in 1992, and a total of 24 patients have been reported since. Relentlessly progressive peripheral retinopathy was diagnosed despite the previous cessation of therapy in 14 patients. Conclusion: Ritonavir causes a slowly progressive atrophic maculopathy, and didanosine toxicity results in a relentlessly progressing peripheral atrophic retinopathy. The relentless progression of both toxic retinopathies reflects permanent alterations of the retinal metabolism by these medications. Both ritonavir and didanosine toxic retinopathies are rare events, but their clinical presentation is highly specific.

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.

Differential Gene Transcription of Extracellular Matrix Components in Response to In Vivo Corneal Crosslinking (CXL) in Rabbit Corneas.

PURPOSE: We studied changes in gene transcription after corneal crosslinking (CXL) in the rabbit cornea in vivo and identified potential molecular signaling pathways. METHODS: A total of 15 corneas of eight male New-Zealand-White rabbits were de-epithelialized and equally divided into five groups. Group 1 served as an untreated control. Groups 2 to 5 were soaked with 0.1% riboflavin for 20 minutes, which in Groups 3 to 5 was followed by UV-A irradiation at a fluence of 5.4 J/cm2. Ultraviolet A (UVA) irradiation was delivered at 3 mW/cm2 for 30 minutes (Group 3, standard CXL protocol), 9 mW/cm2 for 10 minutes (Group 4, accelerated), and 18 mW/cm2 for 5 minutes (Group 5, accelerated). At 1 week after treatment, corneal buttons were obtained; mRNA was extracted and subjected to cDNA sequencing (RNA-seq). RESULTS: A total of 297 differentially transcribed genes were identified after CXL treatment. CXL downregulated extracellular matrix components (collagen types 1A1, 1A2, 6A2, 11A1, keratocan, fibromodulin) and upregulated glycan biosynthesis and proteoglycan glycosylation (GALNT 3, 7, and 8, B3GALT2). Also, CXL activated pathways related to protein crosslinking (transglutaminase 2 and 6). In 9.1% of the significantly different genes, CXL at 3 mW/cm2 (Group 1) induced a more distinct change in gene transcription than the accelerated CXL protocols, which induced a lower biomechanical stiffening effect. CONCLUSIONS: Several target genes have been identified that might be related to the biomechanical stability and shape of the cornea. Stiffening-dependent differential gene transcription suggests the activation of mechano-sensitive pathways. TRANSLATIONAL RELEVANCE: A better understanding of the molecular mechanisms behind CXL will permit an optimization and individualization of the clinical treatment protocol.

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.

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.

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.

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.

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.

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 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. Young's 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. Young's 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.

The Biomechanical Effect of Corneal Collagen Cross-Linking (CXL) With Riboflavin and UV-A is Oxygen Dependent.

PURPOSE: In an attempt to reduce treatment time in corneal collagen cross-linking (CXL) with riboflavin and ultraviolet-A (UV-A), recent protocol modifications include shorter irradiation times at higher fluence, while maintaining constant total applied energy (Bunsen-Roscoe law of reciprocity). While such parameter changes might produce similar biological results within a certain range, the limits of reciprocity are unknown. Limitations in the corneal oxygen diffusion capacity and its potential impact on the efficacy of CXL, raise concerns regarding the efficiency of high-fluence CXL, and also of transepithelial CXL. METHODS: Porcine corneas were treated with an epithelium-off CXL at a fluence of 9 mW/cm2 under two different atmospheres: one with a regular oxygen content (21%) and another in a helium-supplemented, low-oxygen environment (<0.1%). Untreated corneas served as controls (n = 20 each). Five-millimeter corneal stripes were prepared and biomechanical stiffness was measured using an extensometer. RESULTS: Corneas cross-linked under normal oxygen levels showed a significant increase in biomechanical stability (14.36 MPa ± 2.69 SD), whereas corneas treated similarly, but in a low-oxygen atmosphere showed a Young's modulus similar to untreated controls (11.72 MPa ± 2.77 SD). CONCLUSIONS: The biomechanical effect of CXL seems to be oxygen dependent. This dependency will be of particular importance in high-fluence and transepithelial CXL and will most likely require major protocol modifications to maintain the efficiency of the method. TRANSLATIONAL RELEVANCE: The oxygen dependency of CXL shown here raises concerns about the effectiveness of high-fluence and transepithelial CXL. Both methods were introduced to clinical ophthalmology without thorough validation.