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The Effect of Riboflavin–UV-A Treatment on Corneal Limbal Epithelial Cells—A Study on Human Cadaver Eyes

Vimalin, Jeyalatha MS, MLT; Gupta, Nidhi DNB; Jambulingam, Malathi PhD; Padmanabhan, Prema MS; Madhavan, Hajib N. MD, PhD

doi: 10.1097/ICO.0b013e3182498902
Basic Investigation

Purpose: To determine the effect of riboflavin–UV-A treatment on the corneal limbal epithelial cells during a corneal collagen cross-linking (CXL) procedure.

Methods: Thirty freshly enucleated human cadaveric eyeballs were subjected to a CXL procedure, mimicking the clinical protocol. During the UV-A exposure, one half of the limbus (sector A) was left unprotected, whereas the other half (sector B) was covered by a metal shield. Limbal biopsies from both sectors before and after the procedure were analyzed. Each strip of tissue was divided into 3 segments, for cell count of viable cells, for cultivation on human amniotic membrane (HAM), and for stem cell and differentiated corneal epithelial cell marker studies using reverse transcriptase–polymerase chain reaction.

Results: Compared with the cell count before CXL, there was a statistically significant drop in the mean number of viable cells after CXL in sector A but not in sector B. Biopsies from both sectors before CXL and from sector B after CXL showed good growth on HAM. Biopsies from sector A after CXL showed no growth on HAM. The putative stem cell marker ABCG2 was absent in all samples and p63 was absent in 3 of 10 samples taken from sector A after CXL. All markers were present in all samples from sector B after CXL.

Conclusions: Riboflavin–UV-A treatment can result in damage to limbal epithelial cells, particularly the stem cells. Covering the limbal region with a metal shield effectively prevents this damage.

*L and T Microbiology Research Centre, Vision Research Foundation

Department of Cornea and Refractive Surgery, Medical Research Foundation, Sankara Nethralaya, Chennai, India.

Reprints: Prema Padmanabhan, Department of Cornea and Refractive Surgery, Medical Research Foundation, Sankara Nethralaya, 41 College Rd, Chennai 600 006, India (

Supported by the Department of Science and Technology (DST; 105-2008-P).

The authors state that they have no proprietary interest in the products named in this article.

Received October 18, 2011

Accepted January 3, 2012

Copyright © 2012 Wolters Kluwer Health, Inc. All rights reserved.