Enzymatic, clinical and histologic evaluation of corneal tissues in experimental fungal keratitis in rabbits

Mycotic keratitis, being frequently refractive to most of the currently ava ilable antifungal therapy. continues to pose a therapeutic challenge to the clinician. in keratitis of infectious etiology stromal dissolution may be brought about by a combination of agent and host factors. An understanding of the source and nature of corneal tissue damage is essential For evolving more effective therapeutic modalities in the treatment of fungal keratitis . In the present study, nle hare characterized the extracellular proteases produced in vitro by corneal fungal pathogens namely the Aspergillus flavus and Fusarium solani when collagen was provided as the sole nitrogen source . In addition, fungal infected rabbit corneas were investigated for proteol ytic activities and nature of inflammatory reaction. Gelatin zymography det ected protease bands with molecular mass ranging from 100 to 200 kDa in the culture extracts of A. flavus and a single major band of molecular mass ap proximately 200 kDa in the culture extracts of F. solani. A basal proteolyt ic activity of mass 65 kDa was visualized in all uninfected and infected ra bbit corneal extracts. infected corneas in addition revealed the presence o f additional proteolytic species of mass 92 and 200 kDa. The enzyme inhibit ory profile suggested that fungal cultures in vitro contained predominantly serine protease activity and to a lesser extent metalloprotease activity. However, Fungal infected corneal homogenates showed the presence of metallo proteinase activity alone, the enzymatic activities entirely being sensitiv e to ethylene diamine tetra acetate (EDTA), a metalloprotease inhibitor. In terestingly, the serine proteolytic activity detected in Fungal cultures in vitro was not present in the fungal infected corneas in vivo. However, the possible role of Fungal serine proteases in the activation of corneal matr ix metalloproteinases (MMPs) cannot be ruled out. Based on the criteria of molecular mass, proteolytic activity in the presence of calcium at neutral pH, and sensitivity to inhibition by a metalloprotease inhibitor, the 65 an d 92 kDa gelatinases were identified as MMP 2 and MMP 9, respectively. The expression of 92 and 200 kDa gelatinases correlated positive ly with the am ount of polymorphonuclear cells present in the infected tissues. Activated resident corneal cells or inflammatory cells may largely contribute to the increased proteolytic activities in fungal infected corneas resulting in ti ssue matrix degradation in fungal keratitis. (C) 2001 Academic Press.