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Collagen Fiber Diameter in the Rabbit Cornea After Collagen Crosslinking by Riboflavin/UVA

Wollensak, Gregor MD*; Wilsch, Michaela PHD†; Spoerl, Eberhard PHD*; Seiler, Theo MD, PHD‡

Basic Investigations

Objective: Collagen crosslinking of the cornea has been developed recently as a quasiconservative treatment of keratoconus. Biomechanical in vitro measurements have demonstrated a significant increase in biomechanical stiffness of the crosslinked cornea. The aim of the present study was to evaluate the effect of this new procedure on the collagen fiber diameter of the rabbit cornea.

Methods: The corneas of the right eyes of 10 New Zealand White albino rabbits were crosslinked by application of the photosensitizer riboflavin and exposure to UVA light (370 nm, 3 mW/cm2) for 30 minutes. The left fellow control eyes were either left untreated (rabbits 1–4), deepithelialized (rabbits 5–7), or deepithelialized and treated with riboflavin/dextran solution (rabbits 8–10) to exclude an influence of epithelial debridement or hydration changes on the fiber diameter. On ultrathin sections of samples from the anterior and posterior cornea, the collagen fiber diameter was measured semiautomatically with the help of morphometric computer software.

Results: In the anterior stroma, the collagen fiber diameter in the treated corneas was significantly increased by 12.2% (3.96 nm), and in the posterior stroma by 4.6% (1.63 nm), compared with the control fellow eyes. In the crosslinked eyes, the collagen fiber diameter was also significantly increased by, on average, 9.3% (3.1 nm) in the anterior compared with the posterior stroma within the same eye.

Conclusions: Collagen crosslinking using riboflavin and UVA leads to a significant increase in corneal collagen diameter. This alteration is the morphologic correlate of the crosslinking process leading to an increase in biomechanical stability. The crosslinking effect is strongest in the anterior half of the stroma because of the rapid decrease in UVA irradiance across the corneal stroma as a result of riboflavin-enhanced UVA absorption.

From the *Department of Ophthalmology, Universitätsklinikum CGC, Dresden, Germany; †Max-Planck-Institute for Molecular Cell Biology and Genetics, Dresden, Germany; and ‡Institut für Refraktive Chirurgie und Ophthalmo-Chirurgie (IROC), Zurich, Switzerland.

Received for publication June 11, 2003; revision received October 3, 2003; accepted October 7, 2003.

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

© 2004 Lippincott Williams & Wilkins, Inc.