title: Transepithelial Riboflavin Absorption in an Ex Vivo Rabbit Corneal Model.
abstract: OBJECTIVE
  To measure depth-specific riboflavin concentrations in corneal stroma using two-photon fluorescence microscopy and compare commercially available transepithelial corneal collagen cross-linking (CXL) protocols.
  
  
  METHODS
  Transepithelial CXL riboflavin preparations--MedioCross TE, Ribocross TE, Paracel plus VibeX Xtra, and iontophoresis with Ricrolin+--were applied to the corneal surface of fresh postmortem rabbit eyes in accordance with manufacturers' recommendations for clinical use. Riboflavin 0.1% (VibeX Rapid) was applied after corneal epithelial debridement as a positive control. After riboflavin application, eyes were snap frozen in liquid nitrogen. Corneal cross sections 35-μm thick were cut on a cryostat, mounted on a slide, and imaged by two-photon fluorescence microscopy. Mean (SD) concentrations were calculated from five globes tested for each protocol.
  
  
  RESULTS
  Peak riboflavin concentration of 0.09% (± 0.01) was observed within the most superficial stroma (stromal depth 0-10 μm) in positive controls (epithelium-off). At the same depth, peak stromal riboflavin concentrations for MedioCross TE, Ricrolin+, Paracel/Xtra, and Ribocross TE were 0.054% (± 0.01), 0.031% (0.003), 0.021% (± 0.001), and 0.015% (± 0.004), respectively. At a depth of 300 μm (within the demarcation zone commonly seen after corneal cross-linking), the stromal concentration in epithelium-off positive controls was 0.075% (± 0.006), while at the same depth MedioCross TE and Ricrolin+ achieved 0.018% (± 0.006) and 0.016% (0.002), respectively. None of the remaining transepithelial protocols achieved concentrations above 0.005% at this same 300-μm depth. Overall, MedioCross TE was the best-performing transepithelial formulation.
  
  
  CONCLUSIONS
  Corneal epithelium is a significant barrier to riboflavin absorption into the stroma. Existing commercial transepithelial CXL protocols achieve relatively low riboflavin concentrations in the anterior corneal stroma when compared to gold standard epithelium-off absorption. Reduced stromal riboflavin concentration may compromise the efficacy of riboflavin/ultraviolet corneal CXL.
authors.affiliation: Moorfields Eye Hospital London, United Kingdom.
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Gore
authors.affiliation: Keratoconus Research Institute, Department of Ophthalmology, St. Thomas' Hospital, London, United Kingdom.
authors.forename: David
authors.initials: D
authors.lastname: O'Brart
authors.affiliation: Department of Physics, Imperial College London, London, United Kingdom.
authors.forename: Paul
authors.initials: P
authors.lastname: French
authors.affiliation: Department of Physics, Imperial College London, London, United Kingdom 4Centre for Histopathology, London, United Kingdom.
authors.forename: Chris
authors.initials: C
authors.lastname: Dunsby
authors.affiliation: Moorfields Eye Hospital London, United Kingdom.
authors.forename: Bruce D
authors.initials: BD
authors.lastname: Allan
mesh_terms: D000818:Animals; D003319:Corneal Stroma / Q000378:metabolism* / Q000473:pathology; D004195:Disease Models, Animal; D019573:Epithelium, Corneal / Q000378:metabolism* / Q000473:pathology; D007640:Keratoconus / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D008856:Microscopy, Fluorescence; D009883:Ophthalmic Solutions; D011817:Rabbits; D012256:Riboflavin / Q000008:administration & dosage / Q000493:pharmacokinetics*; D014803:Vitamin B Complex / Q000008:administration & dosage / Q000493:pharmacokinetics
chemical_list: D009883:Ophthalmic Solutions; D014803:Vitamin B Complex; D012256:Riboflavin