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Immunofluorescence of Rabbit Corneas After Collagen Cross-Linking Treatment With Riboflavin and Ultraviolet A

Esquenazi, Salomon MD; He, Jiucheng MD, PhD; Li, Na PhD; Bazan, Haydee E P PhD

doi: 10.1097/ICO.0b013e3181bdf1cc
Basic Investigation

Purpose: To assess ultrastructural modifications in keratocytes and inflammatory cell response in rabbit corneas after riboflavin and ultraviolet A exposure using immunoflurescence microscopy.

Methods: Twenty adult New Zealand albino rabbits weighing 2.0-3.0 kg were used in this study. Two rabbits served as controls. The animals had their epithelia removed and were cross-linked with riboflavin 0.1% solution (10 mg riboflavin-5-phosphate in 10 mL of 20% dextran-T-500) applied every 3 minutes for 30 minutes, and exposed to ultraviolet A (360 nm, 3 mW/cm2) for 30 minutes. Four rabbits were humanely euthanized at each time point of 1, 3, and 11 days and at 3 and 5 weeks after the procedure. Immunohistochemistry studies of thin sections of each cornea were performed using terminal deoxynucleotyl transferase-mediated uridine triphosphate biotin nick-end labeling staining, alpha smooth muscle actin (α-SMA), CD-3, myeloperoxidase antibodies, and 4′,6-diamidino-2-phenylindole (DAPI) counterstaining. In another experiment, 6 additional rabbits were treated as above, and after 10 days of cross-linking, 5 μL of lipopolysaccharide endotoxin (1 μg/mL) was injected in the mid-stroma.

Results: Cross-linked corneas showed early stromal edema. By 5 weeks, complete resolution of the edema and a pronounced highly organized anterior 200-μm fluorescent zone was observed. Terminal deoxynucleotyl transferase mediated uridine triphosphate biotin nick-end labeling staining showed keratocyte death by both necrosis and apoptosis between days 1 and 3 after cross-linking. At day 1, the limbal area close to the cross-linking zone showed some inflammatory cells and α-SMA-positive cells, indicative of the presence of myofibroblasts. By day 3, some myofibroblasts had migrated to the area beneath the cross-linked stroma. Between days 3 and 5 weeks, there was an increase in α-SMA staining in the area surrounding the cross-linked stroma. The area of cross-linking remained acellular up to 5 weeks.

Conclusions: Collagen cross-linking results in early edema, keratocyte apoptosis, and necrosis, appearance of inflammatory cells in the surrounding area of treatment and transformation of surrounding keratocytes into myofibroblasts. Compaction of anterior stroma fibers, keratocyte loss, and displacement of cell nuclei including inflammatory cells may have clinical implications in the long-term risk of further corneal thinning in keratoconus and in the cross-linked corneal immune response.

From the *Department of Ophthalmology and Neuroscience Center of Excellence LSU Health Sciences Center, New Orleans, LA and also with Rand Eye Institute, Deerfield Beach, FL; and †Departments of Ophthalmology and Neuroscience Center of Excellence, LSU Health Sciences Center, New Orleans, LA.

Received for publication May 29, 2009; revision received June 19, 2009; accepted July 29, 2009.

Supported by the National Institute of Health Louisiana State University translational Centers of Biomedical Research Grant P20RR021970 (S.E.) and NEI R01EY04928 and R01EY06635 (H.E.P.B.).

Presented in part as a poster at the annual meeting of the Association for research in Vision and Ophthalmology, Fort Lauderdale, FL, May 2009.

The authors have no financial interest in any product mentioned in this article.

Reprints: Salomon Esquenazi, MD, LSU Eye Center, Louisiana State University, School of Medicine, 2020 Gravier Street, Suite D, 8 Floor, New Orleans, LA 70112 (e-mail:

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