High-intensity corneal collagen crosslinking with riboflavin and UVA in rat cornea

<div><p>Corneal collagen cross-linking (CXL) halts human corneal ectasias progression by increasing stromal mechanical stiffness. Although some reports describe that this procedure is effective in dealing with some infectious and immunologic corneal thinning diseases, there is a need for more animal models whose corneal thickness more closely resemble those occurring in these patients. To meet this need, we describe here high-intensity protocols that are safe and effective for obtaining CXL in rat corneas. Initially, a range of potentially effective UVA doses were evaluated based on their effectiveness in increasing tissue enzymatic resistance to dissolution. At UVA doses higher than a threshold level of 0.54 J/cm², resistance to enzymatic digestion increased relative to that in non-irradiated corneas. Based on the theoretical threshold CXL dose, a CXL regimen was established in which the UVA tissue irradiance was 9 mW/cm², which was delivered at doses of either 2.16, 2.7 or 3.24 J/cm². Their dose dependent effects were evaluated on ocular surface morphological integrity, keratocyte apoptotic frequency, tissue thickness and endothelial cell layer density. Doses of 2.16 and 2.7 J/cm² transiently decreased normal corneal transparency and increased thickness. These effects were fully reversed after 14 days. In contrast, 3.24 J/cm² had more irreversible side effects. Three days after treatment, apoptotic frequency in the CXL-2.16 group was lower than that at higher doses. Endothelial cell losses remained evident only in the CXL-3.24 group at 42 days posttreatment. Stromal fiber thickening was evident in all the CXL-treated groups. We determined both the threshold UVA dose using the high-intensity CXL procedure and identified an effective dose range that provides optimal CXL with minimal transient side effects in the rat cornea. These results may help to provide insight into how to improve the CXL outcome in patients afflicted with a severe corneal thinning disease.</p></div

CXL-induced apoptosis and keratocyte density.

<p>(A) Apoptosis of the rat keratocytes (green, positive TUNEL staining) are present in the CXL-treated areas and control cornea on day 3. The control cornea had a normal DAPI staining pattern. In the cross-linked corneas, some keratocyte were apoptotic. Apparent endothelial layer damage was detected in the CXL-3.24 group. (B) Significant difference was evident between keratocyte density in CXL-treated and control corneas on day 3. The keratocyte counts significantly decreased in the CXL-2.7 and 3.24 groups compared with the CXL-2.16 group (### p < 0.01 versus CXL-treated groups, *** p < 0.001 versus control group, respectively; Data are presented as Mean ± SEM, n = 3. scale bars = 50 μm).</p

Anterior segment optical coherence tomography.

<p>UHR-OCT scan visualized the anterior segment integrity at 7 days posttreatment. It appeared normal except for mild corneal edema after receiving doses of either 2.16 or 2.7 J/cm². Significant corneal edema and disrupted epithelial intactness and discontinuity in the central area developed in the CXL-3.24 group (n = 6, Scale bar = 500 μm).</p

UVA doses evaluated for efficacy and safety.

<p>UVA doses evaluated for efficacy and safety.</p

CXL-induced changes in stromal and endothelium ultrastructure on day 7.

<p>The stroma had a compacted collagenous matrix (Left arrow) containing degenerative changes in keratocytes across the stroma in all CXL-treated groups (Middle arrow). No such defects were detected in the posterior stroma and endothelium of the CXL-2.16 group (n = 3).</p

Time dependence of central endothelial cell density recovery after different CXL treatments.

<p>Time dependence of central endothelial cell density recovery after different CXL treatments.</p

Identification of a threshold UVA dose mediating CXL.

<p>Identification of a threshold UVA dose mediating CXL.</p

Differential effects of CXL treatment on clinical signs.

<p>(A) Representative slit lamp images showing differences between the 4 different groups on day 7 after CXL-treatment. (B) Time dependent changes in inflammatory index among the 4 groups from day 1 to 42 (Data are presented as Mean ±(SEM, CXL groups versus control group; n = 6, **p < 0.01).</p

Dependence of enzymatic dissolution time on UVA dose.

<p>Dependence of enzymatic dissolution time on UVA dose.</p

Primers used for quantitative RT-PCR.

<p>Primers used for quantitative RT-PCR.</p