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

Abstract

Purpose-To assess ultrastructural modifications in keratocytes and inflammatory cell response in rabbit corneas after riboflavin and ultraviolet A (UVA) exposure using immunoflurescence microscopy.Methods-Twenty adult New Zealand albino rabbits weighing 2.0 to 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 (10mgs riboflavin-5-phosphate in 10ml 20% dextran-T-500) applied every 3 minutes for 30 minutes, and exposed to UVA (360 nm, 3 mW/cm 2 ) 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.Immunohistochemestry studies of thin sections of each cornea were performed using TUNEL staining, Alpha smooth muscle actin (α-SMA), CD-3, myeloperoxidase (MPO) antibodies and DAPI counterstaining. In another experiment six additional rabbits were treated as above, and after 10 days of cross-linking, 5 μl of lipopolysaccharide (LPS) endotoxin (1μg/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. TUNEL staining showed keratocyte death by both necrosis and apoptosis between day 1 and 3 after cross-linking. At day 1 the limbal area close to the cross linking zone showed some inflammatory cells as well as 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 day 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

Intraoperative and Postoperative Corneal Thickness Change after Collagen Crosslinking Therapy

“…Both objective (14,15) and subjective (16) improvements were reported in eyes with keratoconus after CXL treatment, even in subjects under 18 (17). As collagen bonds are established at a depth of 250-350 µm in the anterior stroma, called the treatment zone (5,(18)(19)(20)(21), and for the protection of the endothelium, a minimum of 400 µm stromal thickness is suggested (5,22). However, according to other publications, a minimum of 330 µm of initial thickness is also sufficient for CXL after corneal swelling (23).…”

Effect of corneal epithelium on ultraviolet-A and riboflavin absorption

“…Immunofluorescence microscopy was proposed by Bottós et al (20) in 2008 as new approach to indirectly visualize the effect of collagen cross-linking, which was later validated by other authors (30) . Bottós et al (20) addressed the isolated effect of riboflavin on the collagen organization and corneal dehydration by applying 0,1% riboflavin solution to de-epithelialized corneas without UVA exposure.…”

UVA-Photoactivated Riboflavin Treatment of Vaginal Cells Derived from Pelvic Organ Prolapse Cases

“…Hence, the present study was carried out, and it demonstrated an almost twofold reduction in vaginal cell viability after UVA irradiation caused by increased necrosis. Indeed, increased apoptosis and necrosis after photopolymerization in other cell types such as keratocytes have been reported [27]. The deleterious effects of the long-wave UVA irradiation were due to lipid peroxidation and oxidative stress resulting from free radicals released by the endogenous target molecules.…”