title: Penetration depth of corneal cross-linking with riboflavin and UV-A (CXL) in horses and rabbits.
abstract: 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.
  
  
  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.
authors.affiliation: Augen Vet, Lindenthalguertel 83, 50935, Cologne, Germany.|Division of Ophthalmology, Equine Department, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse, 260, 8057, Zurich, Switzerland.|Center for Clinical Studies, Vetsuisse Faculty, University of Zurich Winterthurerstrasse, 260, 8057, Zurich, Switzerland.
authors.forename: Nicolin S
authors.initials: NS
authors.lastname: Gallhoefer
authors.affiliation: Division of Ophthalmology, Equine Department, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse, 260, 8057, Zurich, Switzerland.
authors.forename: Bernhard M
authors.initials: BM
authors.lastname: Spiess
authors.affiliation: Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich Winterthurerstrasse, 268, 8057, Zurich, Switzerland.
authors.forename: Franco
authors.initials: F
authors.lastname: Guscetti
authors.affiliation: Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich Winterthurerstrasse, 268, 8057, Zurich, Switzerland.
authors.forename: Monika
authors.initials: M
authors.lastname: Hilbe
authors.affiliation: Division of Veterinary Epidemiology, Vetsuisse Faculty, University of Zurich Winterthurerstrasse, 260, 8057, Zurich, Switzerland.
authors.forename: Sonja
authors.initials: S
authors.lastname: Hartnack
authors.affiliation: Laboratory for Ocular Cell Biology, University of Geneva Rue Alcide-Jentzer, 22, CH-1211, Geneva 14, Switzerland.|Department of Ophthalmology, University of Southern California, 1450 San Pablo St., Los Angeles, CA 90033, USA.|The ELZA Institute AG, Webereistrasse 2, 8953 Dietikon, Zurich, Switzerland.
authors.forename: Farhad
authors.initials: F
authors.lastname: Hafezi
authors.affiliation: Division of Ophthalmology, Equine Department, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse, 260, 8057, Zurich, Switzerland.
authors.forename: Simon A
authors.initials: SA
authors.lastname: Pot
mesh_terms: D000818:Animals; D015415:Biomarkers; D016923:Cell Death; D003315:Cornea / Q000502:physiology*; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D006736:Horses; D017319:Photosensitizing Agents / Q000494:pharmacology*; D011817:Rabbits; D012256:Riboflavin / Q000494:pharmacology*; D046509:Tissue Culture Techniques; D014466:Ultraviolet Rays
chemical_list: D015415:Biomarkers; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D017319:Photosensitizing Agents; D012256:Riboflavin
keywords: CXL; fluorescent biomarkers; horse; infectious keratitis; penetration depth; rabbit