title: Correlation Between Multimodal Microscopy, Tissue Morphology, and Enzymatic Resistance in Riboflavin-UVA Cross-Linked Human Corneas.
abstract: OBJECTIVE
  To explore the utility of multimodal microscopy as a noninvasive tool to assess corneal collagen cross-linking (CXL) efficacy, we investigated the correlation between riboflavin (RF) axial profile, second harmonic generation (SHG) imaging, and histological/biochemical changes of human corneas after RF-ultraviolet A (UVA)-catalyzed CXL.
  
  
  METHODS
  De-epithelialized human corneoscleral tissues were imaged by confocal and multiphoton microscopy to study RF tissue diffusion profile and SHG-based roughness index (Rq) after CXL. We installed 0.1% RF for 5, 10, and 20 minutes, respectively, followed by UVA irradiation, while dextran drug vehicle-treated corneas served as controls. Masson's trichrome staining and collagenase digestion assay were employed to assess ultrastructural modifications of collagen lamellae and bioenzymatic strength following RF-UVA CXL.
  
  
  RESULTS
  Stromal absorption of RF was significantly higher in 20 minutes compared with 5- and 10-minute drug instillations. The roughness index of SHG images was reduced after RF-UVA CXL at all RF instillation time points compared with dextran controls. Interestingly, correlation between axial profiles of RF dosage and Rq index was only observed in 10- and 20-minute RF instillations (R(2) = 0.13 and 0.28, respectively, all P < 0.05), but not in the 5-minute group. Masson's trichrome staining revealed collagen fibril compaction in cross-linked corneas in an RF dose-dependent manner. Collagenase digestion assay showed significantly increased biochemical strength by higher RF doses in cross-linked corneas.
  
  
  CONCLUSIONS
  Intrastromal RF distribution profiles correlated with histological and functional property changes in RF-UVA cross-linked corneas. A riboflavin-defined threshold further determined the sensitivity of SHG imaging as a noninvasive imaging modality to assess the efficacy of RF-UVA CXL.
authors.affiliation: Department of Ophthalmology and Optometry Medical University of Vienna, Vienna, Austria.
authors.forename: Maria
authors.initials: M
authors.lastname: Laggner
authors.affiliation: Department of Ophthalmology and Optometry Medical University of Vienna, Vienna, Austria.
authors.forename: Andreas
authors.initials: A
authors.lastname: Pollreisz
authors.affiliation: Department of Ophthalmology and Optometry Medical University of Vienna, Vienna, Austria.
authors.forename: Gerald
authors.initials: G
authors.lastname: Schmidinger
authors.affiliation: Department of Rheumatology, Medical University of Vienna, Vienna, Austria.
authors.forename: Ruth A
authors.initials: RA
authors.lastname: Byrne
authors.affiliation: Department of Rheumatology, Medical University of Vienna, Vienna, Austria.
authors.forename: Clemens
authors.initials: C
authors.lastname: Scheinecker
authors.affiliation: Department of Ophthalmology and Optometry Medical University of Vienna, Vienna, Austria.
authors.forename: Ursula
authors.initials: U
authors.lastname: Schmidt-Erfurth
authors.affiliation: Department of Ophthalmology and Optometry Medical University of Vienna, Vienna, Austria.
authors.forename: Ying-Ting
authors.initials: YT
authors.lastname: Chen
mesh_terms: D000704:Analysis of Variance; D003094:Collagen / Q000378:metabolism; D003319:Corneal Stroma / Q000187:drug effects / Q000378:metabolism / Q000473:pathology / Q000528:radiation effects; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D006801:Humans; D008853:Microscopy / Q000379:methods*; D064847:Multimodal Imaging; D017319:Photosensitizing Agents / Q000494:pharmacology*; D012256:Riboflavin / Q000378:metabolism*; D014466:Ultraviolet Rays / Q000009:adverse effects*
chemical_list: D003432:Cross-Linking Reagents; D017319:Photosensitizing Agents; D003094:Collagen; D012256:Riboflavin