1 Journal of Refractive Surgery • Vol. xx, No. x, 2010 ORIGINAL ARTICLE Corneal Wound Healing After Ultraviolet-A/ Riboflavin Collagen Cross-linking: A Rabbit Study Marcella Q. Salomão, MD; Shyam S. Chaurasia, PhD; Abhijit Sinha-Roy, PhD; Renato Ambrósio, Jr, MD, PhD; Andrew Esposito, MD; Ricardo Sepulveda, MD; Vandana Agrawal, MS; Steven E. Wilson, MD From Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio (Salomão, Chaurasia, Sinha-Roy, Esposito, Sepulveda, Agrawal, Wilson); Singapore Eye Research Institute, Singapore (Chaurasia); and Instituto de Olhos Renato Ambrosio, Rio de Janeiro, Brazil (Ambrósio). Supported in part by US Public Health Service grants EY010056 and EY015638 from the National Eye Institute and Research to Prevent Blindness, New York, New York. The authors have no commercial or proprietary interest in the materials pre- sented herein. Correspondence: Steven E. Wilson, MD, Cole Eye Institute, I-32, Cleveland Clinic, 9500 Euclid Ave, Cleveland, OH 44195. Tel: 216.444.3871; E-mail: wilsons4@ccf.org Received: April 19, 2010; Accepted: October 25, 2010 Posted online: December 1, 2010 ABSTRACT PURPOSE: To investigate corneal wound healing follow- ing ultraviolet-A (UVA)/riboflavin corneal collagen cross- linking (CXL) in rabbit corneas. METHODS: Thirty-six rabbits were enrolled in the study. Animals were divided into three treatment groups and corneas were analyzed at 24 hours and 4 weeks post- operatively. Thus, each group had 6 rabbits at each time point. Treatment groups were: 1) standard UVA+ribofla- vin CXL, 2) UVA alone, and 3) riboflavin alone. One eye of each rabbit served as an untreated control eye. TUNEL assay was performed to detect stromal cell apoptosis. Immunocytochemistry was performed to detect the in- flammatory marker CD11b expressed in monocytes and the alpha-smooth muscle actin (SMA) marker expressed in myofibroblasts. RESULTS: At 24 hours, corneas from the UVA+ribo- flavin CXL group had significantly more apoptosis than the UVA alone and riboflavin alone groups. Eyes from all three groups had significantly more inflammatory cell influx into the cornea than unwounded controls. Four weeks after the procedure, many corneas in the UVA+riboflavin CXL group had mild haze, but very few SMA-positive myofibroblasts could be detected in the central cornea. CONCLUSIONS: Riboflavin+UVA CXL triggers more anterior keratocyte apoptosis than corneal scrape with UVA alone or riboflavin alone. Inflammation moni- tored by the monocyte marker CD11b was present, but not statistically different among the three groups. Very little myofibroblast generation could be detected after UVA+riboflavin CXL, indicating that the mild stromal haze associated with this procedure is nor- mally related to transient corneal fibroblast generation rather than more persistent haze due to generation of myofibroblasts. [J Refract Surg. 2010;xx(x):xxx-xxx.] doi:10.3928/1081597X-20101201-02 O ver the past decade, a technique to generate col- lagen cross-linking (CXL) within the cornea, using the photosensitizer riboflavin (vitamin B2) and ultraviolet-A (UVA) (370 nm) light, similar to photopolymer- ization in polymers, has been advanced to attempt to stabilize the cornea in patients with keratoconus and patients at risk for a keratoconus-like ectasia related to surgery. 1-3 Collagen CXL remains under study in numerous clinical trials and has gained widespread acceptance as a treatment to slow progres- sion of keratoconus and ectasia. 1 The aim of this treatment is to increase intra- and interfi- brillar covalent bonds by photosensitized oxidation to coun- teract progressive corneal thinning associated with ectatic disorders of the cornea and, consequently, to promote bio- mechanical stabilization of the cornea. One study demon- strated stabilization of the corneal shape after CXL and also regression of corneal steepening by 2.00 diopters (D) in 70% of treated eyes. 1 In addition, stress–strain measurements have been performed in human and animal corneas after this pro- cedure. 4-6 These studies demonstrated a significant increase in biomechanical strength and mechanical rigidity (stiffening effect) after the treatment. The riboflavin/UVA CXL treatment commonly involves epithelial removal followed by administration of the drug and UVA light that trigger a wound-healing response in the corneal epithelium and stroma. Our purpose in this experimental study was to assess the effects of riboflavin/UVA treatment