title: Morphological and immunohistochemical changes after corneal cross-linking.
abstract: OBJECTIVE
  To present light and electron microscopic as well as immunohistochemical findings after corneal cross-linking (CXL).
  
  
  METHODS
  Six keratoconus corneas after CXL, 12 keratoconus corneas without CXL, and 7 normal corneas were examined by light microscopy, indirect immunohistochemistry using antibodies against proapoptotic BAX, antiapoptotic survivin, and BCL-2, and anti-smooth muscle actin and, in part, by transmission electron microscopy. Direct immunofluorescence with 4'6-diamidino-2-phenylindole was performed to analyze keratocytes/area in the anterior, middle, posterior, peripheral, and central corneal stroma.
  
  
  RESULTS
  The period between CXL and keratoplasty ranged from 5 to 30 months. All keratoconus corneas showed the typical histological changes. Increased proapoptotic BAX expression and/or antiapoptotic survivin expression were noticed in keratocytes and endothelium in 2 keratoconus specimens after CXL. Smooth muscle actin was only observed in subepithelial scar tissue of 2 keratoconus corneas without CXL. Keratoconus corneas after CXL revealed a significant reduction in keratocyte counts in the entire cornea (P = 0.003) compared with keratoconus corneas without CXL and normal corneas. This difference was because of a loss of keratocytes in the anterior (P = 0.014) and middle (P = 0.024) corneal stroma. Keratocytes in CXL corneas were reduced in the center (P = 0.028) and the periphery (P = 0.047).
  
  
  CONCLUSIONS
  CXL in human keratoconus can cause considerable morphologic corneal changes up to 30 months postoperatively. Especially noteworthy is a long-lasting, maybe permanent, keratocyte loss in the anterior and middle corneal stroma involving the central and peripheral cornea. As long-term corneal damage after CXL is of genuine concern, particular care should be taken to perform this procedure only in accordance with investigational protocols.
authors.affiliation: Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany. emessmer@med.uni-muenchen.de
authors.forename: Elisabeth M
authors.initials: EM
authors.lastname: Messmer
authors.forename: Peter
authors.initials: P
authors.lastname: Meyer
authors.forename: Martina C
authors.initials: MC
authors.lastname: Herwig
authors.forename: Karin U
authors.initials: KU
authors.lastname: Loeffler
authors.forename: Frank
authors.initials: F
authors.lastname: Schirra
authors.forename: Berthold
authors.initials: B
authors.lastname: Seitz
authors.forename: Michael
authors.initials: M
authors.lastname: Thiel
authors.forename: Thomas
authors.initials: T
authors.lastname: Reinhard
authors.forename: Anselm
authors.initials: A
authors.lastname: Kampik
authors.forename: Claudia
authors.initials: C
authors.lastname: Auw-Haedrich
mesh_terms: D000199:Actins / Q000378:metabolism; D000293:Adolescent; D000328:Adult; D015415:Biomarkers / Q000378:metabolism*; D003094:Collagen / Q000378:metabolism*; D060527:Corneal Keratocytes / Q000378:metabolism / Q000648:ultrastructure; D003319:Corneal Stroma / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000627:therapeutic use*; D005260:Female; D019085:Fluorescent Antibody Technique, Direct; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000378:metabolism; D007640:Keratoconus / Q000378:metabolism* / Q000473:pathology* / Q000628:therapy; D015948:Keratoplasty, Penetrating; D008297:Male; D046529:Microscopy, Electron, Transmission; D008875:Middle Aged; D017319:Photosensitizing Agents / Q000627:therapeutic use; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D012256:Riboflavin / Q000627:therapeutic use; D000077022:Survivin; D014466:Ultraviolet Rays; D055815:Young Adult; D051020:bcl-X Protein / Q000378:metabolism
chemical_list: C541116:ACTA2 protein, human; D000199:Actins; C107489:BIRC5 protein, human; D015415:Biomarkers; D003432:Cross-Linking Reagents; D051034:Inhibitor of Apoptosis Proteins; D017319:Photosensitizing Agents; D019253:Proto-Oncogene Proteins c-bcl-2; D000077022:Survivin; D051020:bcl-X Protein; D003094:Collagen; D012256:Riboflavin