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.

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 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 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.

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/cm 2 ) 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 stressrelaxation 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 6 40 kPa) when compared with controls (337 6 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 6 111 kPa) when compared with controls (659 6 121 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.