Duration of In Vitro Inhibitory Activity of Equine Serum against Equine Tear Film Matrix Metalloproteinases

Abstract

Abstract: : Purpose: Tear film proteinases are produced and released by corneal epithelial cells, stromal fibroblasts, leukocytes, and microbial pathogens. Excessive levels of tear film proteinases in ulcerative keratitis can lead to rapid degradation of stromal collagen and extracellular matrix (ECM) to cause corneal ?melting?. This is most pronounced in ulcerative keratitis of the horse. Two families of proteinases that affect the horse cornea include the matrix metalloproteinases (MMP) and the serine proteinases. Concentrations of MMP-9 and the serine proteinase neutrophil elastase (NE) are significantly elevated in ulcerated horse eyes. Tear film proteinases normally exist in balance with inhibitory factors that serve to prevent excessive degradation of collagen. The search for effective exogenous inhibitors of collagenase and other tear film proteinases is ongoing in an effort to inhibit MMP and NE activity in corneal ulceration. Autologous serum has been utilized topically for corneal ulcers with excessive proteinase activity. Binding of serum-derived alpha-2-macroglobulin to MMPs is extremely tenacious, rendering this circulating agent one of the strongest known inhibitors of MMPs. This experiment evaluated the amount and duration of in vitro inhibitory activity of autologous equine serum stored at various temperatures against MMPs. Methods: Gelatin zymography was used to isolate and determine MMPs from horse tears. Horse serum samples of -18°C (frozen), 4°C (cold), and 23°C (room temperature) were used to test the inhibition of tear film MMP activity at baseline, and at days 1-7. Gels were scanned and image analysis utilized to measure inhibitory activity. Results: There was no difference in MMP inhibition activity between serum at room temperature (-87.8% activity) and frozen serum (-90.9% activity). No difference in MMP inhibition between the serum kept at room temperature (-90.0 ± 5.2% activity) or in the cold (-90.6 ± 2.7% activity) was detected during the seven day trial. Inhibitory activity of serum towards tear film MMPs did not significantly change over the seven-day test period. Conclusions:We demonstrated high levels of in vitro inhibition of MMP activity from autologous horse serum. The in vitro inhibitory efficacy of serum towards equine tear film MMP did not diminish after one week at room temperature. Blood drawn into sterile containers containing no anticoagulants yields serum that can be used at room temperature or refrigerated until needed.

Keywords: cornea: clinical science • keratitis • wound healing