Therapeutic potential of Pirfenidone for treating equine corneal scarring.

Abstract

OBJECTIVE: To evaluate the safety and efficacy of Pirfenidone (PFD) in the treatment of equine corneal fibrosis using an in vitro model. METHODS: Healthy donor equine corneas were collected and used to generate primary equine corneal fibroblasts (ECFs) by growing cultures in minimal essential medium supplemented with 10% fetal bovine serum. Equine corneal myofibroblasts (ECMs), used as a model of equine corneal fibrosis, were produced by growing ECF cultures in serum-free medium containing transforming growth factor &#x3b2;1 (1 ng/mL). Trypan blue viability assays and changes in ECF morphology were utilized to determine the optimal PFD dose for this in vitro model. Trypan blue viability, phase-contrast microscopy, and TUNEL assays were used to evaluate the cytotoxicity of PFD. Scratch and MTT assays were used to evaluate the effect of PFD on cellular migration and proliferation. Real-time PCR, immunoblot analysis, and immunocytochemistry were employed to determine the efficacy of PFD to inhibit ECM formation in vitro. RESULTS: Topical PFD application at 200 &#x3bc;g/mL successfully decreased &#x3b1;SMA expression when compared to the TGF&#x3b2;1 only treatment group (P < 0.01). PFD application &#x2264; 200 &#x3bc;g/mL did not affect ECF phenotype or cellular viability and did not result in significant cytotoxicity. CONCLUSIONS: Pirfenidone safely and effectively inhibits TGF&#x3b2;1-induced equine corneal fibrosis in vitro. In vivo studies are warranted.