Colocalization of Hyaluronan and Water in Rabbit Corneas After Photorefractive Keratectomy by Specific Staining for Hyaluronan and by Quantitative Microradiography

Abstract

Endogenous hyaluronan (HA) has previously been shown to be a part of corneal wound healing after photorefractive keratectomy (PRK). HA is known to bind considerable amounts of water. This study aimed to show whether HA colocalized with water in the wound area after PRK. Five New Zealand White rabbits were subjected to PRK in one eye each. After 4 weeks, the rabbits were killed and the corneas were freeze-sectioned. The freeze-dried sections were exposed to soft x-rays generated at 3 kV and the microradiographs were evaluated densitometrically. The same sections were then stained for the presence of HA using the HA-binding region of bovine nasal cartilage proteoglycans linked to biotin. The microradiographs showed, in a zone underlying the epithelium, that the water content was increased. In the corresponding zone, the cornea stained positive for HA. HA that is reactively formed in the corneal wound after PRK colocalized to the hydrated area of the corresponding location as revealed by quantitative microradiography. The findings suggest that HA causes local shifts in water content in the corneal wound and thereby also local shifts in transparency.