Thymosin beta 4 Expression In Corneal Wound Healing

Abstract

Abstract: : Purpose: The purpose of these studies was to localize and to quantify the protein levels of thymosin beta 4 (Tß₄) in the corneas of young vs old mice corneas after wounding. Methods:Corneas from young (6–8 weeks of age) and old (12–13 months of age) ICR mice were debrided with a razor blade to create a 2 mm size epithelial defect. A time course study was performed to examine the rates of corneal healing in the young vs old mice. Animals (n=10/group/time point) were sacrificed at various time points (0, 6, 24 and 48 hours) after wounding and the corneal buttons were excised and assayed by ELISA (ALPCO, New Hampshire) for Tß₄ quantification. In addition, whole eyes (n=3/group/time point) from 0, 24, and 48 hours time points were harvested for corneal immunohistochemical (IHC) analysis of Tß₄. Results: Young corneas re–epithelialized by 20 hours after wounding, 4 hours earlier than those in the aged mice. Normal, unwounded corneas from the young and aged animals demonstrated similar Tß₄ protein levels (2.5 µg/ml cornea). After wounding, the old and young animals both exhibited increases in Tß₄ protein levels compared to their respective normal controls but the aged corneal Tß₄ expression levels were markedly blunted compared to those in the young. Immediately after wounding, young corneal Tß₄ levels were almost 2.5 times more than the aged. These levels peaked at 24 hours when young corneas measured over three–fold more Tß₄ than the aged (17.5 µg/ml and 5 µg/ml cornea, respectively) and they decreased at 48 hours (12.5 µg/ml and 4 µg/ml cornea, respectively). The IHC staining in the young corneas was more intense and regular in the epithelial nuclei and at the leading wound edges compared to the aged. The aged corneas localized Tß₄ more to the epithelial and endothelial basement membrane regions layers. While IHC localized Tß₄ to the leading epithelial edges of the wounds in both groups, staining was more intense in the young corneas. Conclusions: This is the first study to demonstrate the presence of Tß₄ in corneas after wounding. In this epithelial wound model, young and aged corneas differ in the rates of healing and in quantity and staining patterns of Tß₄. Since Tß₄ is known to promote corneal wound healing, the findings herein may suggest a possible relationship between corneal Tß₄ levels and rates of re–epithelialization.

Keywords: cornea: epithelium • wound healing • gene/expression