In vitro and ex vivo therapeutic strategies for equine corneal fibrosis

Abstract

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Purpose: The aims were to (1) To explore the impact of equine corneal fibroblast (ECF) to myofibroblast (ECM) differentiation by altering the expression of the Smad genes either individually or in combination and (2) develop an ex vivo model of the equine cornea in which potential therapeutics could be tested. Methods: In study 1, equine corneal fibroblasts were incubated for 72 hours +/- Transforming Growth Factor-1 and plasmids which either silenced expression of Smad genes 2, 3, and 4 or in combination with plasmids that over expressed Smad gene 7. Immunofluorescence and qRTP-CR techniques quantified levels of equine corneal fibroblasts (ECF) differentiation to equine corneal myofibroblasts (ECM) by measuring alpha smooth muscle actin (SMA), a known marker of ECM transdifferentiation via immunofluorescence and qRT-PCR. In study 2, fresh equine corneas were harvested and randomly assigned to one of two organ culture systems, (a) immersion condition organ culture system (IC) or (b) air/liquid interface organ culture system (ALC) for 7 days. Degree of corneal opacity, cell viability and structural change was evaluated using serial daily gross photography, histology and TUNEL assay. Results: In study 1, silencing of pro-fibrotic Smad genes had similar effects in the reduction of ECM trandifferentiation. Combination gene utilizing Smad 2 and Smad 7 did not have a significant reduction in ECM trandifferentiation compared to Smad 7 individually. In study 2, ALC maintained values similar to control eyes on all parameters evaluated. Conclusion: Combination gene therapy did not prove to have an additive effect on the degree of inhibition of ECM transdifferentiation. While the ALC model system is a viable option to maintain a healthy equine cornea in an ex-vivo setting for wound healing studies.