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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.

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.