concanavalin-a and Eye-Injuries

The aim of this study was to establish a clinically relevant short-term animal model of dry eye with utility in identifying compounds with potential therapeutic efficacy.. Rabbit lacrimal glands were injected with the T-cell mitogen Concanavalin A (Con A) and inflammation, tear function, and corneal epithelial cell integrity were subsequently assessed. The inflammatory response was characterized by quantifying biochemical markers of inflammation ex vivo and by confirming inflammatory cell influx by histology. Matrix metalloproteinase-9 (MMP-9) and proinflammatory cytokines IL-1beta, IL-8, and TGF-beta1 were quantified in tissue extracts. Tear function was monitored by measuring tear fluorescein clearance and tear breakup time (TBUT). Corneal epithelial cell integrity was determined by quantifying the uptake of methylene blue dye following the exposure of rabbits to a low-humidity environment. The anti-inflammatory corticosteroid, dexamethasone, was administered topically as indicated for each study.. Histopathologic evaluation of lacrimal glands injected with Con A revealed a pronounced inflammatory process characterized by lymphocytic infiltration, multifocal necrosis, and fibroplasia. Elevated levels of MMP-9 and cytokines IL-1beta, IL-8, and TGF-beta1 were detected in the lacrimal gland and cornea. Inflammation of the rabbit lacrimal gland following an injection of Con A significantly reduced tear clearance and TBUT and increased susceptibility to desiccation-induced corneal damage. Dexamethasone was prophylactically and therapeutically effective in this inflammation model of dry eye, restoring tear function and inhibiting corneal injury following topical ocular application.. Characteristics of this rabbit lacrimal gland inflammation model of dry eye are consistent with the current understanding of dry eye as a local ocular surface inflammatory response to abnormal tear volume and composition. These results suggest that this rabbit model of dry eye may be employed to assess the therapeutic efficacy of mechanistically diverse agents on clinically relevant signs of ocular surface disease. These methods were strategically developed to be applicable for advancing drug discovery for a broad spectrum of dry eye patients.

Topics: Animals; Anti-Inflammatory Agents; Concanavalin A; Cornea; Corneal Injuries; Cytokines; Dacryocystitis; Desiccation; Dexamethasone; Disease Models, Animal; Dry Eye Syndromes; Eye Injuries; Lacrimal Apparatus; Rabbits; Tears

Acanthamoeba keratitis is a sight-threatening corneal infection. It is known that: (i) more amoebae bind to the surface of injured corneas than to the normal corneal surface and (ii) mannose-containing glycoproteins (GPs) possess binding sites for Acanthamoeba. The present study was undertaken to determine whether subtle corneal surface injury exposes mannose-GPs and whether more amoebae bind to the mannose-GPs of injured corneas than to those of normal corneas.. Corneal cup assays were developed to determine whether corneal surface injury exposes binding sites for a mannose/glucose-specific lectin, succinylated-concanavalin A (s-ConA). To determine whether injury exposes mannose-GPs, corneal surface proteins were biotinylated, biotin-labeled mannose-GPs were allowed to bind to s-ConA-agarose beads and were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE). Amoeba binding to mannose-GPs of corneal epithelia was analyzed by PAGE-blot overlay assays.. S-ConA binding site density was 2.4 times greater on the injured corneal surface than on the surface of normal corneas. Based on the analysis of the s-ConA-bound, biotin-labeled corneal surface proteins, approximately 5.2 times greater amounts of mannose-GPs were present on the surface of injured corneas than on the normal corneal surface. PAGE-blot overlay assays of s-ConA bound GPs of unlabeled corneal epithelia revealed that, on a per mg total cell protein basis, injured corneal epithelium contained 1.8 times greater amounts of Acanthamoeba-reactive mannose-GPs than normal corneal epithelium.. Subtle corneal injury exposes mannose-GPs on the surface of injured corneas. The newly exposed GPs may serve to provide additional attachment sites for the amoebae. This, in turn, could render the cornea susceptible to the infection.

Topics: Acanthamoeba; Adhesiveness; Animals; Binding Sites; Concanavalin A; Electrophoresis, Polyacrylamide Gel; Epithelium, Corneal; Eye Injuries; Immunoenzyme Techniques; Mannose; Membrane Glycoproteins; Rabbits