Hyaluronan Facilitates Corneal Epithelial Wound Healing in Diabetic Rats

Abstract

We investigated the effect of hyaluronan on corneal epithelial wound healing in rats affected by diabetes. Furthermore, because hyaluronan is thought to affect corneal epithelial wound healing through the mechanism of binding of hyaluronan to provisional fibronectin in the wounded area, we compared the localization of fibronectin immunohistochemically during corneal epithelial wound healing in diabetic and non-diabetic rats. Streptozotocin was used to induce diabetes in half the rats. Two weeks after treatment, the whole corneal epithelium of diabetic and untreated rats was debrided. The rats were divided into groups (seven or eight rats per group), and hyaluronan eye drops at concentrations of 0.03, 0.1, or 0.3%, chondroitin sulfate (3%), or phosphate buffered saline (PBS) was given in eye drops 6 times a day for 4 days, starting immediately after debridement. The area of the corneal epithelial wound was measured immediately after debridement and at 12, 18, 24, 30, 48, 72, and 96 hours afterwards. Although the healing process was similar in non-diabetic and diabetic rats, the healing rate in diabetic rats was slower than that in normal controls. In both diabetic and non-diabetic rats, hyaluronan increased the healing rate in a dose-dependent manner; the difference was significant compared with the PBS-treated group, at hyaluronan doses of 0.1% and 0.3%. However, chondroitin sulfate did not affect corneal epithelial wound closure, regardless of whether the rats were diabetic or not; the healing rates were identical to those of PBS-treated diabetic and non-diabetic controls. In both diabetic and non-diabetic corneas, fibronectin was localized in the corneal subepithelial region, and in streaks between collagen fibers of the stroma. One day after debridement, a layer of fibronectin immunofluorescence was clearly visible on the surface of the denuded stroma. As healing progressed staining of fibronectin diminished at the interface between the new epithelium and the stroma. These changes in localization of fibronectin during corneal epithelial wound healing were similar in both diabetic and non-diabetic rats. Our results demonstrate that hyaluronan facilitates corneal epithelial wound healing in diabetic rats, and suggest that one possible mechanism of its stimulatory effect lies in its binding to a provisional fibronectin matrix, in both diabetic and non-diabetic rats.