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In vivo confocal laser-scanning microscopy to characterize wound repair in rabbit corneas after collagen cross-linking


  • Conflict/competing interest: RF Guthoff is consultant to Heidelberg Engineering (Heidelberg, Germany). The other authors of this manuscript have no conflict of interest or commercial interest to declare.

  • Funding sources: The study was supported by the DFG (Transregio 37, Micro- and Nanosystems in Medicine – Reconstruction of Biological Functions).

Dr Marine Hovakimyan, Department of Ophthalmology, University of Rostock, Doberaner Strasse 140, D-18057 Rostock, Germany. Email:


Background:  Collagen cross-linking using the photosensitizer riboflavin combined with ultraviolet A light was developed to stiffen the cornea by increasing its mechanical and biochemical stability. Investigation of post-treatment events, such as wound healing, is important to evaluate possible risks and to optimize treatment protocols. This in vivo confocal laser-scanning microscopy study in rabbits was conducted to provide a quantitative and qualitative analysis of corneal wound repair over 16 weeks following collagen cross-linking.

Methods:  Six New Zealand White rabbits underwent riboflavin/ultraviolet A cross-linking. In vivo confocal laser-scanning microscopy using a Heidelberg Retina Tomograph equipped with a Rostock Cornea Module was performed preoperatively and at 2, 4, 8, 12 and 16 weeks postoperatively.

Results:  From 2 weeks onwards the epithelium demonstrated no abnormalities. Evidence of inflammation was visualized in the intermediate, basal cells and Bowman's membrane. Nerve fibre regeneration was first noted at 12 weeks. Keratocyte activation and hyperreflective extracellular matrix were observed consistently, but by 16 weeks keratocyte activation was diminished, and extracellular matrix resumed normal reflectivity. Cell density in the posterior stroma and endothelium regained preoperative values by 4 weeks, although anterior stroma keratocyte cell density was still reduced by about 10% at 16 weeks.

Conclusions:  Complete qualitative and quantitative characterization of corneal wound repair was achieved by in vivo confocal laser-scanning microscopy over 16 weeks following collagen cross-linking in rabbits. In terms of assessing the ever-increasing range of cross-linking protocols, in vivo confocal laser-scanning microscopy may contribute to minimizing the number of experimental animals, because multiple examinations of the same cases are possible over time.

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