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Keratocyte Apoptosis After Corneal Collagen Cross-linking Using Riboflavin/UVA Treatment

Wollensak, Gregor MD; Spoerl, Eberhard PhD; Wilsch, Michaela PhD; Seiler, Theo MD, PhD

Basic Investigations

Purpose: Combined riboflavin/UVA treatment inducing collagen cross-links in the cornea has been shown to increase the biomechanical rigidity of the cornea and has been used successfully in the treatment of progressive keratoconus. The current study was undertaken to investigate the possible cytotoxic effect of combined riboflavin/UVA treatment on corneal keratocytes in vivo.

Methods: Thirty-four New Zealand white rabbits were treated with 0.1% riboflavin solution and surface UVA irradiances ranging from 0.75 to 4 mW/cm2 (1.35– 7.2 J/cm2) for 30 minutes. The animals were euthanized either 4 (n = 6) or 24 (n = 28) hours postoperatively. Four additional control eyes underwent epithelial debridement alone. The corneas of the enucleated eyes were evaluated in routine histologic sections. In addition, the TUNEL technique and transmission electron microscopy were used for the detection of keratocyte apoptosis.

Results: In the control eyes with corneal epithelial debridement only, apoptotic keratocytes were found in the anterior 50 μm of the corneal stroma 4 hours postoperatively. However, riboflavin/UVA-induced apoptosis was only visible in the rabbit eyes enucleated 24 hours postoperatively. In these eyes, we found apoptosis of keratocytes down to a variable stromal depth depending on the applied UVA irradiance. A cytotoxic UVA irradiance for keratocytes in the range of 0.5–0.7 mW/cm2 could be deduced.

Conclusions: Riboflavin/UVA treatment leads to a dose-dependent keratocyte damage that can be expected in human corneas down to a depth of 300 μm using a surface UVA dose of 5.4 J/cm2. Future studies should be done to examine the keratocyte repopulation and exclude possible adverse sequelae of keratocyte loss like stromal scarring or thinning.

Collagen cross-linking in the cornea using UVA and the photosensitizer riboflavin was developed by us recently for biomechanically strengthening the cornea. In a prospective clinical pilot study including 22 patients with progressive keratoconus and a follow-up of up to 4 years, the progression of keratoconus could be stopped in all treated eyes. Even regression with a reduction of the maximal keratometry readings by 2 diopters was achieved in 70% of patients. Corneal and lens transparency as well as endothelial cell density remained unchanged. 1 In stress-strain measurements of human, rabbit, and porcine cornea, 2–4 we could show a significantly increased biomechanical rigidity. After exposure to collagenases that play a role in both keratoconus and corneal ulceration, a significant delay in enzymatically induced tissue dissolution by 8 days was found in cross-linked corneas indicating an increased resistance to enzymatic digestion. 5 Other applications of the new cross-linking method lie in the treatment of corneal melting processes 6 and in the field of refractive surgery like prevention of postoperative keratectasia after LASIK for high myopes or regression of the refractive effect. 7

As part of an extensive safety evaluation of the new treatment, we have undertaken the following experimental study to assess the possible damage to the keratocytes after combined riboflavin/UVA treatment.

From the Department of Ophthalmology, Technical University of Dresden, Dresden, Germany (Drs. Wollensak and Spoerl); Max-Planck-Institute for Cell Biology and Genetics, Dresden, Germany (Dr. Wilsch); and the Department of Ophthalmology, University of Zurich, Zurich, Switzerland (Dr. Seiler).

Received for publication February 6, 2003;

revision received July 9, 2003; accepted August 15, 2003.

Reprints: Gregor Wollensak, MD, Wildentensteig 4, D-14195 Berlin, Germany (e-mail:

© 2004 Lippincott Williams & Wilkins, Inc.