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

Date Published: June 23, 2017

Publisher: Public Library of Science

Author(s): Yirui Zhu, Peter S. Reinach, Hanlei Zhu, Qiufan Tan, Qinxiang Zheng, Jia Qu, Wei Chen, Dimitrios Karamichos.


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.

Partial Text

Corneal collagen cross-linking with riboflavin and UVA is the preferred treatment modality to strengthen biomechanical corneal properties and halt keratoconus progression as well as stabilize ectasia [1–3]. The standard CXL procedure firstly introduced by Wollensak et al [1] for patient use includes delivering UVA irradiation of 3 mW/cm2 for 30 min (i.e. 5.4 J/cm2) in combination with applying a 0.1% riboflavin solution as long as the corneal thickness is at least 400 μm [4]. On the other hand, the high-intensity (accelerated) CXL procedure applies the same dose as that used in the standard procedure by increasing the intensity even though UVA irradiance time is shortened. This modification increases patient comfort.

We determined the threshold UVA dose needed to induce collagen CXL as well as the optimal UVA dose range whose side effects are minimal and reversible in rat corneas. Such a characterization in this complementary novel model may provide insight into how to apply the high intensity CXL protocol to treat pathological conditions causing thinning of this tissue to even less than 200 μm in a clinical setting. We identified a threshold UVA dose of 0.54 J/cm2 because which represents the lowest dose for increasing collagen CXL. In associating the increases in enzymatic resistance with reversibility of damaging side effects on corneal integrity, we found that CXL using 9 mW/cm2 for 4 min (2.16 J/cm2) with 0.22% riboflavin is appropriate for obtaining a safe and effective CXL outcome. At UVA doses between 2.16 and 2.7 J/cm2, corneal transparency was maintained, while 3.24 J/cm2 is too high a dose because losses in central endothelial cell density were more extensive and irreversible. Therefore, a UVA dose of 2.7 J/cm2 is the maximum dose for inducing CXL without eliciting irreversible losses in endothelial cell density and stromal integrity in the rat.