title: Collagen cross-linking using rose bengal and green light to increase corneal stiffness.
abstract: OBJECTIVE
  Photochemical cross-linking of corneal collagen is an evolving treatment for keratoconus and other ectatic disorders. We evaluated collagen cross-linking by rose bengal plus green light (RGX) in rabbit eyes and investigated factors important for clinical application.
  
  
  METHODS
  Rose bengal (RB, 0.1%) was applied to deepithelialized corneas of enucleated rabbit eyes for 2 minutes. The diffusion distance of RB into the stroma was measured by fluorescence microscopy on frozen sections. RB-stained corneas were exposed to green (532-nm) light for 3.3 to 9.9 minutes (50-150 J/cm(2)). Changes in the absorption spectrum during the irradiation were recorded. Corneal stiffness was measured by uniaxial tensiometry. The spatial distribution of the stromal elastic modulus was assessed by Brillouin microscopy. Viable keratocytes were counted on H&E-stained sections 24 hours posttreatment.
  
  
  RESULTS
  RB penetrated approximately 100 μm into the corneal stroma and absorbed >90% of the incident green light. RGX (150 J/cm(2)) increased stromal stiffness by 3.8-fold. The elastic modulus increased in the anterior approximately 120 μm of stroma. RB was partially photobleached during the 2-minute irradiation, but reapplication of RB blocked light transmission by >70%. Spectral measurements suggested that RGX initiated cross-linking by an oxygen-dependent mechanism. RGX did not decrease keratocyte viability.
  
  
  CONCLUSIONS
  RGX significantly increases cornea stiffness in a rapid treatment (≅12 minutes total time), does not cause toxicity to keratocytes and may be used to stiffen corneas thinner than 400 μm. Thus, RGX may provide an attractive approach to inhibit progression of keratoconus and other ectatic disorders.
authors.affiliation: Boston University Medical Sciences Program, Boston, MA, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: Cherfan
authors.forename: E Eri
authors.initials: EE
authors.lastname: Verter
authors.forename: Samir
authors.initials: S
authors.lastname: Melki
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Gisel
authors.forename: Francis J
authors.initials: FJ
authors.lastname: Doyle
authors.forename: Giuliano
authors.initials: G
authors.lastname: Scarcelli
authors.forename: Seok Hyun
authors.initials: SH
authors.lastname: Yun
authors.forename: Robert W
authors.initials: RW
authors.lastname: Redmond
authors.forename: Irene E
authors.initials: IE
authors.lastname: Kochevar
mesh_terms: D000818:Animals; D002452:Cell Count; D003094:Collagen / Q000378:metabolism*; D060527:Corneal Keratocytes / Q000166:cytology; D003319:Corneal Stroma / Q000187:drug effects* / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004548:Elasticity; D007640:Keratoconus / Q000188:drug therapy; D053844:Lasers, Solid-State; D008027:Light; D008856:Microscopy, Fluorescence; D017319:Photosensitizing Agents / Q000494:pharmacology*; D011817:Rabbits; D012395:Rose Bengal / Q000494:pharmacology*; D013718:Tensile Strength
chemical_list: D003432:Cross-Linking Reagents; D017319:Photosensitizing Agents; D012395:Rose Bengal; D003094:Collagen
keywords: collagen cross-linking; corneal stiffness; ectatic disorder; keratoconus; photochemistry