title: Establishing Corneal Cross-Linking With Riboflavin and UV-A in the Mouse Cornea In Vivo: Biomechanical Analysis.
abstract: OBJECTIVE
  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.
authors.affiliation: Laboratory for Ocular Cell Biology, University of Geneva, Geneva, Switzerland.
authors.forename: Arthur
authors.initials: A
authors.lastname: Hammer
authors.affiliation: Laboratory for Ocular Cell Biology, University of Geneva, Geneva, Switzerland 2Center for Applied Biotechnology and Molecular Medicine (CABMM), University of Zurich, Zurich, Switzerland.
authors.forename: Sabine
authors.initials: S
authors.lastname: Kling
authors.affiliation: Research Center for Statistics, University of Geneva, Geneva, Switzerland.
authors.forename: Marc-Olivier
authors.initials: MO
authors.lastname: Boldi
authors.affiliation: Laboratory for Ocular Cell Biology, University of Geneva, Geneva, Switzerland.
authors.forename: Olivier
authors.initials: O
authors.lastname: Richoz
authors.affiliation: Laboratory for Ocular Cell Biology, University of Geneva, Geneva, Switzerland.
authors.forename: David
authors.initials: D
authors.lastname: Tabibian
authors.affiliation: Department of Ophthalmology, Emory University, Atlanta, Georgia, United States.
authors.forename: J Bradley
authors.initials: JB
authors.lastname: Randleman
authors.affiliation: Center for Applied Biotechnology and Molecular Medicine (CABMM), University of Zurich, Zurich, Switzerland 5Department of Ophthalmology, University of Southern California, Los Angeles, California, United States 6ELZA Institute, Dietikon/Zurich, Switzerlan.
authors.forename: Farhad
authors.initials: F
authors.lastname: Hafezi
mesh_terms: D000818:Animals; D001696:Biomechanical Phenomena; D003315:Cornea / Q000187:drug effects / Q000503:physiopathology* / Q000528:radiation effects; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004195:Disease Models, Animal; D007640:Keratoconus / Q000188:drug therapy / Q000503:physiopathology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D017319:Photosensitizing Agents / Q000494:pharmacology; D012256:Riboflavin / Q000494:pharmacology*; D014466:Ultraviolet Rays
chemical_list: D003432:Cross-Linking Reagents; D017319:Photosensitizing Agents; D012256:Riboflavin