Morphological and immunohistochemical changes after corneal cross-linking

Cornea. 2013 Feb;32(2):111-7. doi: 10.1097/ICO.0b013e31824d701b.

Abstract

Purpose: 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.

MeSH terms

  • Actins / metabolism
  • Adolescent
  • Adult
  • Biomarkers / metabolism*
  • Collagen / metabolism*
  • Corneal Keratocytes / metabolism
  • Corneal Keratocytes / ultrastructure
  • Corneal Stroma / metabolism*
  • Cross-Linking Reagents / therapeutic use*
  • Female
  • Fluorescent Antibody Technique, Direct
  • Humans
  • Inhibitor of Apoptosis Proteins / metabolism
  • Keratoconus / metabolism*
  • Keratoconus / pathology*
  • Keratoconus / therapy
  • Keratoplasty, Penetrating
  • Male
  • Microscopy, Electron, Transmission
  • Middle Aged
  • Photosensitizing Agents / therapeutic use
  • Proto-Oncogene Proteins c-bcl-2 / metabolism
  • Riboflavin / therapeutic use
  • Survivin
  • Ultraviolet Rays
  • Young Adult
  • bcl-X Protein / metabolism

Substances

  • ACTA2 protein, human
  • Actins
  • BIRC5 protein, human
  • Biomarkers
  • Cross-Linking Reagents
  • Inhibitor of Apoptosis Proteins
  • Photosensitizing Agents
  • Proto-Oncogene Proteins c-bcl-2
  • Survivin
  • bcl-X Protein
  • Collagen
  • Riboflavin