Expression analysis of wound–related genes in impaired corneal wound healing induced by a dry condition.

Abstract

Abstract: : Purpose: We previously reported that a severe dry condition impairs the wound healing system and accelerates apoptotic epithelial defect in a rat cornea (IOVS 2003 44:4682–4688). To better understand the molecular pathophysiology of the recovery delayed mechanism, expression levels of ocular surface wound–related genes under dry conditions were analyzed by comparison with those under a normal environment. Methods: Eight–week–old male rats were placed in a dry–conditioned room(23±2ºC, relative humidity 25±2% and constant 2–4 m/s air flow) to enhance tear evaporation and were maintained for 12 hours, just after scraping the central region of corneal epithelium(0.4 mm²) as a trigger for corneal erosion. As controls, rats wounded in the same area were kept in a normal room(23±2ºC, relative humidity 60±5%) to allow complete corneal recovery. To obtain specimens, rats treated under dry and normal conditions were sacrificed 0, 3, 6, 12 hours after wounding and total RNA was extracted from the whole cornea and the bulbar conjunctiva respectively. Semi –quantitative Real–time RT–PCR analysis was carried out to compare mRNA levels of wound–related genes such as IL–1α, IL–1ß, IL–6, TNFα, EGF, HGF, TGFß, IFNγ, ICAM–1, Integrinα1, Fibronectin and MMP–9. Results: The corneal mRNA expressions of IL–1ß, IL–6 and Fibronectin significantly increased after 3 to 12 hours under dry conditions compared with those under normal conditions (about 5–fold respectively), while levels of the other mRNAs expressed were approximately the same. In the conjunctiva, IL–1α, IL–6 and ICAM–1 expressions were remarkably upregulated under dry conditions. In both the dry and normal environment, corneal IL–1α and IL–6 mRNA levels peaked at 3 and 6 hours respectively, but the Fibronectin mRNA gradually increased especially under the dry conditions following the expression of those inflammatory cytokines. Consequently, the Fibronectin mRNA level was elevated in response to the area of epithelial defect under dry conditions. Conclusions: Under dry conditions, systematic regulation of wound healing–related genes at the RNA level was clearly detected on the ocular surface regardless of the resulting in corneal erosion.

Keywords: wound healing • gene/expression • cornea: tears/tear film/dry eye