title: [Enzymatic evidence of the depth dependence of stiffening on riboflavin/UVA treated corneas].
abstract: OBJECTIVE
  It has been shown that the treatment of keratoconus with riboflavin/ultraviolet A (UVA) causes significant stiffening of the cornea due to cross-linking. The aim of this study was to evaluate how deep the mechanical stabilization after collagen cross-linking could be shown biochemically.
  
  
  METHODS
  Ten out of 20 enucleated porcine eyes were treated with riboflavin as a photosensitizer and UVA (370 nm, 3 mW/cm2, 30 min). The other 10 eyes served as controls. With a Microkeratom device, two flaps with a thickness of 200 microm and a diameter of 8 mm were cut off from each eye and put in a collagenase solution (NaCl plus collagenase A, 1:1). The surfaces of the flaps were measured digitally every day to characterize the dissolving behavior.
  
  
  RESULTS
  The resistance (regarding corneal collagen against enzymatic digestion) of the treated superficial flaps was considerably higher (p=0.001) compared to those that were cut secondarily and to the control flaps. But even the flaps from deeper layers showed a significant increase in resistance (p=0.02) compared with the untreated flaps. The half-life of the surfaces of the treated superficial flaps was 220 h; of those cut secondarily, it was 80 h. Both untreated flaps had a half-life of 50 h.
  
  
  CONCLUSIONS
  The biochemical study showed that the treatment of the cornea with riboflavin/UVA leads to significant collagen cross-linking not only in the anterior slice of 200 microm but also in the following 200 microm. This locally limited cross-linking effect may be explained by the absorption behavior for UVA of the riboflavin-treated cornea; 65% of UVA irradiation is absorbed in the first 200 microm and only 25-30% in the next 200 microm. Therefore, deeper-lying structures and especially the endothelium are not affected.
authors.affiliation: Universitäts-Augenklinik Carl Gustav Carus, Fetscherstrasse 74, 01307, Dresden, Germany. thomas.schilde@klinikum-nuernberg.de
authors.forename: T
authors.initials: T
authors.lastname: Schilde
authors.forename: M
authors.initials: M
authors.lastname: Kohlhaas
authors.forename: E
authors.initials: E
authors.lastname: Spoerl
authors.forename: L E
authors.initials: LE
authors.lastname: Pillunat
mesh_terms: D000818:Animals; D003094:Collagen / Q000187:drug effects / Q000378:metabolism*; D017364:Collagenases / Q000378:metabolism*; D003315:Cornea / Q000187:drug effects / Q000201:enzymology*; D003432:Cross-Linking Reagents; D007640:Keratoconus / Q000188:drug therapy / Q000628:therapy*; D008954:Models, Biological; D010777:Photochemistry; D017319:Photosensitizing Agents / Q000494:pharmacology / Q000627:therapeutic use*; D012256:Riboflavin / Q000494:pharmacology / Q000627:therapeutic use*; D013552:Swine; D013997:Time Factors; D014466:Ultraviolet Rays
chemical_list: D003432:Cross-Linking Reagents; D017319:Photosensitizing Agents; D003094:Collagen; D017364:Collagenases; D012256:Riboflavin