Collagen cross-linking using rose bengal and green light to increase corneal stiffness

Invest Ophthalmol Vis Sci. 2013 May 13;54(5):3426-33. doi: 10.1167/iovs.12-11509.


Purpose: Photochemical cross-linking of corneal collagen is an evolving treatment for keratoconus and other ectatic disorders. We evaluated collagen cross-linking by rose bengal plus green light (RGX) in rabbit eyes and investigated factors important for clinical application.

Methods: Rose bengal (RB, 0.1%) was applied to deepithelialized corneas of enucleated rabbit eyes for 2 minutes. The diffusion distance of RB into the stroma was measured by fluorescence microscopy on frozen sections. RB-stained corneas were exposed to green (532-nm) light for 3.3 to 9.9 minutes (50-150 J/cm(2)). Changes in the absorption spectrum during the irradiation were recorded. Corneal stiffness was measured by uniaxial tensiometry. The spatial distribution of the stromal elastic modulus was assessed by Brillouin microscopy. Viable keratocytes were counted on H&E-stained sections 24 hours posttreatment.

Results: RB penetrated approximately 100 μm into the corneal stroma and absorbed >90% of the incident green light. RGX (150 J/cm(2)) increased stromal stiffness by 3.8-fold. The elastic modulus increased in the anterior approximately 120 μm of stroma. RB was partially photobleached during the 2-minute irradiation, but reapplication of RB blocked light transmission by >70%. Spectral measurements suggested that RGX initiated cross-linking by an oxygen-dependent mechanism. RGX did not decrease keratocyte viability.

Conclusions: RGX significantly increases cornea stiffness in a rapid treatment (≅12 minutes total time), does not cause toxicity to keratocytes and may be used to stiffen corneas thinner than 400 μm. Thus, RGX may provide an attractive approach to inhibit progression of keratoconus and other ectatic disorders.

Keywords: collagen cross-linking; corneal stiffness; ectatic disorder; keratoconus; photochemistry.

MeSH terms

  • Animals
  • Cell Count
  • Collagen / metabolism*
  • Corneal Keratocytes / cytology
  • Corneal Stroma / drug effects*
  • Corneal Stroma / metabolism
  • Cross-Linking Reagents / pharmacology*
  • Elasticity
  • Keratoconus / drug therapy
  • Lasers, Solid-State
  • Light
  • Microscopy, Fluorescence
  • Photosensitizing Agents / pharmacology*
  • Rabbits
  • Rose Bengal / pharmacology*
  • Tensile Strength


  • Cross-Linking Reagents
  • Photosensitizing Agents
  • Rose Bengal
  • Collagen