title: Mechanisms of corneal tissue cross-linking in response to treatment with topical riboflavin and long-wavelength ultraviolet radiation (UVA).
abstract: OBJECTIVE
  Treatment of de-epithelialized human corneas with riboflavin (RF) + long-wavelength ultraviolet light (UVA; RFUVA) increases corneal stroma tensile strength significantly. RFUVA treatment retards the progression of keratoconus, perhaps by cross-linking of collagen molecules, but exact molecular mechanisms remain unknown. Research described here tested possible chemical mechanisms of cross-linking.
  
  
  METHODS
  Corneas of rabbits and spiny dogfish sharks were de-epithelialized mechanically, subjected to various chemical pretreatments, exposed to RFUVA, and then subjected to destructive tensile stress measurements. Tensile strength was quantified with a digital force gauge to measure degree of tissue cross-linking.
  
  
  RESULTS
  For both rabbit and shark corneas, RFUVA treatment causes significant cross-linking by mechanism(s) that can be blocked by the presence of sodium azide. Conversely, such cross-linking is greatly enhanced in the presence of deuterium oxide (D(2)O), even when RF is present at only one tenth the currently used clinical concentrations. Blocking carbonyl groups preexisting in the stroma with 2,4-dinitrophenylhydrazide or hydroxylamine blocks essentially all corneal cross-linking. In contrast, blocking free amine groups preexisting in the stroma with acetic anhydride or ethyl acetimidate does not affect RFUVA corneal cross-linking. When both carbonyl groups are blocked and singlet oxygen is quenched, no RFUVA cross-linking occurs, indicating the absence of other cross-linking mechanisms.
  
  
  CONCLUSIONS
  RFUVA catalyzes cross-linking reactions that require production of singlet oxygen ((1)O(2)), whose half-life is extended by D(2)O. Carbonyl-based cross-linking reactions dominate in the corneal stroma, but other possible reaction schemes are proposed. The use of D(2)O as solution media for RF would enable concentration decreases or significant strength enhancement in treated corneas.
authors: Division of Biology, Kansas State University, Manhattan, Kansas 66506-4901, USA. smccall@ksu.edu
authors: A Scott
authors: AS
authors: McCall
authors: Stefan
authors: S
authors: Kraft
authors: Henry F
authors: HF
authors: Edelhauser
authors: George W
authors: GW
authors: Kidder
authors: Richard R
authors: RR
authors: Lundquist
authors: Helen E
authors: HE
authors: Bradshaw
authors: Zinaida
authors: Z
authors: Dedeic
authors: Megan J C
authors: MJ
authors: Dionne
authors: Ethan M
authors: EM
authors: Clement
authors: Gary W
authors: GW
authors: Conrad
mesh_terms: D000287:Administration, Topical; D000818:Animals; D003094:Collagen / Q000378:metabolism*; D003319:Corneal Stroma / Q000187:drug effects* / Q000378:metabolism; D017666:Deuterium Oxide / Q000494:pharmacology; D003931:Diacetyl / Q000032:analysis; D006207:Half-Life; D009682:Magnetic Resonance Spectroscopy; D010778:Photochemotherapy; D017319:Photosensitizing Agents / Q000008:administration & dosage / Q000494:pharmacology*; D011817:Rabbits; D012256:Riboflavin / Q000008:administration & dosage / Q000494:pharmacology*; D026082:Singlet Oxygen / Q000378:metabolism; D019810:Sodium Azide / Q000494:pharmacology; D048251:Squalus acanthias; D013718:Tensile Strength; D014466:Ultraviolet Rays
chemical_list: D017319:Photosensitizing Agents; D026082:Singlet Oxygen; D003094:Collagen; D019810:Sodium Azide; D017666:Deuterium Oxide; D003931:Diacetyl; D012256:Riboflavin