Mechanisms of corneal tissue cross-linking in response to treatment with topical riboflavin and long-wavelength ultraviolet radiation (UVA).
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.
McCall
A Scott
AS
Division of Biology, Kansas State University, Manhattan, Kansas 66506-4901, USA. smccall@ksu.edu
Kraft
Stefan
S
Edelhauser
Henry F
HF
Kidder
George W
GW
Lundquist
Richard R
RR
Bradshaw
Helen E
HE
Dedeic
Zinaida
Z
Dionne
Megan J C
MJ
Clement
Ethan M
EM
Conrad
Gary W
GW
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
D017319:Photosensitizing Agents; D026082:Singlet Oxygen; D003094:Collagen; D019810:Sodium Azide; D017666:Deuterium Oxide; D003931:Diacetyl; D012256:Riboflavin