title: Detection of subepithelial fibrosis associated with corneal stromal edema by second harmonic generation imaging microscopy.
abstract: OBJECTIVE
  Human corneas with or without stromal edema were examined by second harmonic generation (SHG) imaging microscopy to characterize stromal collagen organization.
  
  
  METHODS
  Tissue buttons from 31 corneas with stromal edema and 8 normal corneas were fixed, and 3-mm(2) blocks were cut and stained with phalloidin, to visualize the cytoskeleton. The blocks were examined by SHG imaging with a laser confocal microscope and a mode-locked titanium:sapphire femtosecond laser. Samples were scanned to a depth of 150 microm from the surface of Bowman's layer, and SHG forward- and backscatter signals were collected. Phalloidin staining was detected by conventional laser confocal microscopy. The three-dimensional structure of the anterior segment of the cornea was reconstructed from stacked SHG images.
  
  
  RESULTS
  Three-dimensional reconstruction of SHG signals showed adherence of interwoven collagen lamellae in the anterior stroma to Bowman's layer in both normal and edematous corneas. Abnormal SHG signals at the level of Bowman's layer were observed in edematous corneas; three-dimensional images revealed that these signals were actually localized above Bowman's layer and were indicative of subepithelial fibrosis. Phalloidin staining showed transdifferentiation of stromal cells into fibroblastic cells in edematous corneas. The incidence of subepithelial fibrosis or of fibroblastic cells increased beginning 12 months after the onset of clinical stromal edema.
  
  
  CONCLUSIONS
  SHG imaging of the anterior segment of edematous corneas revealed a normal appearance of interwoven collagen lamellae in the anterior stroma. The development of subepithelial fibrosis beginning 12 months after the onset of edema suggests that stromal edema may be a progressive disease.
authors.affiliation: Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan. morishig@yamaguchi-u.ac.jp.
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Morishige
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Yamada
authors.forename: Shinichiro
authors.initials: S
authors.lastname: Teranishi
authors.forename: Tai-ichiro
authors.initials: T
authors.lastname: Chikama
authors.forename: Teruo
authors.initials: T
authors.lastname: Nishida
authors.forename: Atsushi
authors.initials: A
authors.lastname: Takahara
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D003094:Collagen / Q000378:metabolism; D015715:Corneal Edema / Q000175:diagnosis* / Q000601:surgery; D003319:Corneal Stroma / Q000378:metabolism / Q000473:pathology*; D003599:Cytoskeleton / Q000378:metabolism / Q000473:pathology; D019573:Epithelium, Corneal / Q000473:pathology*; D005347:Fibroblasts / Q000473:pathology*; D005355:Fibrosis / Q000175:diagnosis; D005642:Fuchs' Endothelial Dystrophy / Q000473:pathology / Q000601:surgery; D006801:Humans; D007091:Image Processing, Computer-Assisted; D015948:Keratoplasty, Penetrating; D018613:Microscopy, Confocal; D008875:Middle Aged; D013997:Time Factors
chemical_list: D003094:Collagen