Faculty Bibliography

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.

OBJECTIVE:CXL penetration depth is an important variable influencing clinical treatment effect and safety. The purposes of this study were to determine the penetration depth of CXL in rabbit and equine corneas in epithelium-on and epithelium-off procedures and to assess an ex vivo fluorescent biomarker staining assay for objective assessment of CXL penetration depth. PROCEDURES/METHODS:CXL treatment was performed according to a standardized protocol on 21 and 17 rabbit eyes and on 12 and 10 equine eyes with and without debridement, respectively. Control corneas were treated similarly, but not exposed to CXL. Hemicorneas were stained with either phalloidin and DAPI to visualize intracellular F-actin and nuclei, or with hematoxylin and eosin. Loss of actin staining was measured and compared between groups. RESULTS:Epithelium-off CXL caused a median actin cytoskeleton loss with a demarcation at 274 μm in rabbits and 173 μm in horses. In non-CXL-treated controls, we observed a median actin cytoskeleton loss with a demarcation at 134 μm in rabbits and 149 μm in horses. No effect was detected in the epithelium-on procedure. CONCLUSIONS:CXL penetration depth, as determined by a novel ex vivo fluorescent assay, shows clear differences between species. A distinct effect was observed following epithelium-off CXL treatment in the anterior stroma of rabbits, but no different effect was observed in horses in comparison with nontreated controls. Different protocols need to be established to effectively treat equine patients with infectious corneal disease.

PURPOSE/OBJECTIVE:To assess the efficacy of a modified high concentration riboflavin solution containing benzalkonium chloride 0.01% for transepithelial corneal cross-linking (CXL). METHODS:In this prospective, interventional multicenter cohort study, 26 eyes of 26 patients with documented progressive keratoconus who underwent transepithelial CXL were included. Follow-up at 6 and 12 months postoperatively included slit-lamp examination, uncorrected and corrected distance visual acuity (logMAR), maximum keratometry (Kmax), and corneal pachymetry (corneal thinnest point) as determined by Scheimpflug imaging. Statistical analysis was performed using repeated measures analysis of variance and the Friedman test for parametric and non-parametric data, respectively. P values less than .05 were considered significant. RESULTS:Kmax did not change significantly at postoperative months 6 and 12. Changes in corneal thinnest point did not change postoperatively over 12 months. Uncorrected and corrected distance visual acuity did not change postoperatively. Progression (defined by an increase in Kmax greater than 1.00 diopter occurred in 46% of eyes at 12 months. Corneal epithelial defects were observed in 46% of the patients and marked punctate corneal epitheliopathy/loose epithelium in 23% of the patients in the immediate postoperative period. No corneal infection, sterile infiltrates, or haze were observed. CONCLUSIONS:Transepithelial CXL with an enhanced riboflavin solution did not effectively halt progression of keratoconus. Significant epithelium damage was evident in the immediate postoperative period. [J Refract Surg. 2016;32(6):372-377.].

PURPOSE/OBJECTIVE: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.

BACKGROUND:Corneal cross-linking (CXL) using ultraviolet light-A (UV-A) and riboflavin is a technique developed in the 1990's to treat corneal ectatic disorders such as keratoconus. It soon became the new gold standard in multiple countries around the world to halt the progression of this disorder, with good long-term outcomes in keratometry reading and visual acuity. The original Dresden treatment protocol was also later on used to stabilize iatrogenic corneal ectasia appearing after laser-assisted in situ keratomileusis (LASIK) and photorefractive keratectomy (PRK). CXL efficiently strengthened the cornea but was also shown to kill most of the keratocytes within the corneal stroma, later on repopulated by those cells. REVIEW/METHODS:Ultraviolet-light has long been known for its microbicidal effect, and thus CXL postulated to be able to sterilize the cornea from infectious pathogens. This cytotoxic effect led to the first clinical trials using CXL to treat advanced infectious melting corneal keratitis. Patients treated with this technique showed, in the majority of cases, a stabilization of the melting process and were able to avoid emergent à chaud keratoplasty. Following those primary favorable results, CXL was used to treat beginning bacterial keratitis as a first-line treatment without any adjunctive antibiotics with positive results for most patients. In order to distinguish the use of CXL for infectious keratitis treatment from its use for corneal ectatic disorders, a new term was proposed at the 9th CXL congress in Dublin to rename its use in infections as photoactivated chromophore for infectious keratitis -corneal collagen cross-linking (PACK-CXL). CONCLUSION/CONCLUSIONS:PACK-CXL is now more frequently used to treat infections from various infectious origins. The original Dresden protocol is still used for this purpose. Careful modifications of this protocol could improve the efficiency of this technique in specific clinical situations regarding certain types of pathogens.

PURPOSE/OBJECTIVE: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.

PURPOSE/OBJECTIVE:To compare the long-term outcomes of accelerated and standard corneal cross-linking protocols in the treatment of progressive keratoconus. DESIGN/METHODS:Prospective randomized clinical trial. METHODS:Thirty-one eyes with keratoconus were treated with an accelerated protocol (18 mW/cm(2), 5 min) and all contralateral eyes were treated with the standard method (3 mW/cm(2), 30 min) using the same overall fluence of 5.4 J/cm(2). RESULTS:At 18 months after the procedure, the standard group showed significant improvement in spherical equivalent (P < .05), K-readings (P < .05), Q value (P < .05), index of surface variance (P < .05), and keratoconus index (P = .008) and decline in central corneal thickness (P < .05), but no significant change in visual acuity, corneal hysteresis, corneal resistance factor, P2 area, or endothelial cell density. In the accelerated group, central corneal thickness was the only parameter with statistically significant change. However, neither of these parameters showed significant differences between the standard and the 18 mW/cm(2) accelerated protocol, except K-reading (P = .059) and index surface variance (P = .034). CONCLUSION/CONCLUSIONS:An accelerated cross-linking protocol, using 18 mW/cm(2) for 5 minutes, shows a comparable outcome and safety profile when compared to the standard protocol, but better corneal flattening is achieved with the standard method than the accelerated method. Overall, both methods stop the disease progression similarly. This study will continue to examine more long-term results.