Corneal collagen crosslinking: new hope

  Laser Eye Institute, Halle, Germany
  
   Corneal collagen crosslinking treatment (CXL) of progressive keratoconus using the chromophore riboflavin and UVA was introduced by Wollensak et al. in 2003 [1, 2]. Crosslinking is an old and widespread technique. Both chemical and physical crosslinking methods are being used in many areas of modern technology like in hardening filling materials in dentistry, preserving biological tissue specimens in pathology or also in the leather and automobile industry. The success of the corneal crosslinking method is based on the induced increase of corneal biomechanical rigidity by about 300 % and the anterior localization of the effect leaving the endothelium intact. In both animal and human studies, a long-term effect of the treatment has been shown. Studies in rabbits have revealed apoptosis of keratocytes after CXL down to 300 μm stromal depth. These findings were later confirmed by confocal microscopy in patients [3]. In both in vivo and in vitro studies, the cytotoxicity threshold for keratocytes was determined to be 0.5 mW/cm². The endothelial cells are more sensitive and have a cytotoxic threshold of 0.36 mW/cm². Therefore, the minimal stromal thickness must be 400 μm to prevent endothelial apoptosis. The post-CXL wound healing process including the stromal repopulation with new keratocytes is completed after 2 — 3 months. As a morphological correlate of the intrafibrillar crosslinking effect, an increase in collagen fiber diameter of the anterior stroma by 12.2 % was found and as a biochemical correlate a collagen polymer band of at least 1000 kDa and multiple proteoglycan polymer bands in sizes from 100–150 kDa were identified on electrophoresis. In hydration experiments, the absence of stromal swelling in the anterior 250 μm due to interfibrillar crosslinks was demonstrated. The hydrothermal shrinkage temperature was increased from 70 °C to 75 °C in the anterior stroma after CXL. The resistance against collagenase digestion was increased from six to fifteeen days. Microbiologically, a significant bactericidal effect was described against various bacteria. Also, the resistance against collagenase digestion was increased from six to fifteen days. The first and main application of CXL is the treatment of progressive keratoconus because in keratoconus the biomechanical stability of the cornea is reduced significantly.
  
  Methods. CXL is a photodynamic crosslinking method. In CXL, the absorption of UVA light is potentiated by the photosensitizer riboflavin at its absorption maximum of 370 nm so that 90 % of UVA is absorbed in the corneal stroma. Thereby, the biomechanical effect is maximal and the risk for the endothelium, lens and retina minimal. So-called reactive oxygen species (ROS) are created by the UVA inducing covalent intraand interfibrillar collagen and proteoglycan crosslinks [2; Fig. 1].
  A temperature effect is excluded because there is only a minimal temperature rise by 2 °C at the corneal surface. In standard CXL, the epithelium is debrided in the central 8 mm of the cornea after topical anesthesia with proparacaine eye drops. Corneal pachymetry is performed to ensure a stromal thickness of 400 μm. A 0.1 % riboflavin-phosphate solution with 20 % dextran T-500 is applied every three minutes starting 15 minutes before the irradiation. UVA light of 370 nm is applied for thirty minutes with a surface UVA irradiance of 3 mW/cm² using an array of UVA diodes in their focussing distance. After the crosslinking procedure, antibiotic ointment like ofloxacine is applied for 3 to 5 days until epithelial closure. Other postoperative regimens include the application of non-steroidal antiinflammatory drugs (NSAID's), antibiotic drops and the use of a bandage contact lens. In the transepithelial treatment variant called C3-R, benzalkoniumchloride containing anesthetic drops are applied for 30 minutes before the irradiation loosening the tight junctions of the epithelium. However, using C3-R the biomechanical effect is significantly reduced compared to CXL due to inhomogeneous and reduced diffusion of riboflavin [4]. A continuing problem in CXL treatment are corneas thinner than 400 μm because of the risk for the endothelium. There are three different approaches for such cases: Leaving the epithelium intact in the areas of stromal thinning (pachymetry guided epithelial debridement), swelling the stroma with the help of a hypoosmolar riboflavin solution or using transepithelial C3-R. H owever, all of these methods result in a weaker overall biomechanical crosslinking-effect.
  
  Results. The first study on crosslinking treatment of keratoconus was performed in 2003 by Wollensak [1]. In this 3-year study, 22 patients with progressive keratoconus were treated with riboflavin and UVA. In all treated eyes, the progression of keratoconus was at least stopped («freezing»). In 16 there also was a slight reversal and flattening of the keratoconus by 2 diopters. Best corrected visual acuity improved slightly in 15 eyes. The 6-year-long term follow-up data of this study including 241 treated eyes again demonstrated a decrease of keratectasia by up to 4.34 dpt after 3 years, a slight increase in visual acuity and improved contact lens tolerance [5]. Two eyes had to be crosslinked twice due to continued progression. These patients had steroid treatment for neurodermitis, however. Other treatment failures were reported in pregnant patients. Apart from that, neither progression of keratectasia nor serious complications have been observed in the Dresden study so far. In CXL, the early crosslinking effect stops the progression or keratectasia («freezing») while the reduction of keratectasia and a «push-up» effect on the cone in about 50 % of the patients is caused in the following months by the wound contraction during wound healing («remodeling »). The main indication of CXL is the treatment of progressive keratoconus. CXL for keratoconus is increasingly combined with refractive corrections using PRK or intracorneal ring —insertion. Besides the paradigm keratoconus, CXL treatment is now being evaluated in various other clinical indications like post-LASIK keratectasia [6], pellucid marginal degeneration, bullous keratopathy, ulceration and severe keratitis. However, in infectious keratitis the use of crosslinking is only reasonable in infections restricted to the anterior third of the stroma. In various clinical studies, post-CXL endothelial damage could be excluded using confocal microscopy, lens damage by Scheimpflug-Pentacam exams and retinal damage by optical coherence tomography. Typically, an anterior demarcation area can be seen on anterior optical coherence tompography and increased stromal collagen reflectivity and tissue compaction on confocal microscopy. Apart from a temporary glare for 2 to 3 months post-CXL due to honeycomb edema-induced light scattering in the anterior stroma only rare, single cases with serious side effects have been described many of which are due to the incorrect use of a postoperative bandage contact lens. Reported side effects are peripheral sterile infiltrates with stromal thinning, calcific band keratopathy, severe keratitis due to E. coli, Acanthameba or Streptococcus infection with perforation, reactivation of a herpes infection, diffuse lamellar keratitis in CXL after LASIK and stromal scarring or perforation after CXL in thin corneas.
  
  Conclusions. CXL is a safe and efficient procedure to stabilize the cornea. Preoperative pachymetry is mandatory to avoid damage to the endothelium. The use of postoperative NSAID's and a bandage lens should not be routine after CXL due to the risk of severe infection. Collagen-crosslinking treatment has become the treatment of first choice for progressive keratoconus and post-LASIK ectasia and is entering the mainstream use. CXL can be combined with refractive corrections like intracorneal rings or PRK to further reduce the optical distortions. The usefulness of CXL for other indications like pellucid marginal degeneration, severe keratitis, ulceration, flap stabilization or corneal edema is being assessed in current studies. There is hope that the need for keratoplasty in various corneal diseases can be diminished significantly in the future thanks to corneal collagen crosslinking treatment.