Mitomycin C: a promising agent for the treatment of canine corneal scarring

Objective  To evaluate the safety and efficacy of mitomycin C (MMC) in prevention of canine corneal scarring.

Methods  With an in vitro approach using healthy canine corneas, cultures of primary canine corneal fibroblasts or myofibroblasts were generated. Primary canine corneal fibroblasts were obtained by growing corneal buttons in minimal essential medium supplemented with 10% fetal bovine serum. Canine corneal myofibroblasts were produced by growing cultures in serum-free medium containing transforming growth factor β1 (1 ng/mL). Trypan blue assay and phase-contrast microscopy were used to evaluate the toxicity of three doses of MMC (0.002%, 0.02% and 0.04%). Real-time PCR, immunoblot, and immunocytochemistry techniques were used to determine MMC efficacy to inhibit markers of canine corneal scarring.

Results  A single 2-min treatment of 0.02% or less MMC did not alter canine corneal fibroblast or keratocyte phenotype, viability, or growth. The 0.02% dose substantially reduced myofibroblast formation (up to 67%; P < 0.001), as measured by the change in RNA and protein expression of fibrosis biomarkers (α-smooth muscle actin and F-actin).

Conclusion  This in vitro study suggests that a single 2-min 0.02% MMC treatment to the canine corneal keratocytes is safe and may be useful in decreasing canine corneal fibrous metaplasia. In vivo studies are warranted.

Regulation of scleral cell contraction by transforming growth factor-β and stress competing roles in myopic growth

Reduced extracellular matrix accumulation in the sclera of myopic eyes leads to increased ocular extensibility and is related to reduced levels of scleral transforming growth factor-β (TGF-β). The current study investigated the impact of this extracellular environment on scleral cell phenotype and cellular biomechanical characteristics. Scleral cell phenotype was investigated in vivo in a mammalian model of myopia using the myofibroblast marker, α-smooth muscle actin (α-SMA). In eyes developing myopia α-SMA levels were increased, suggesting increased numbers of contractile myofibroblasts, and decreased in eyes recovering from myopia. To understand the factors regulating this change in scleral phenotype, the competing roles of TGF-β and mechanical stress were investigated in scleral cells cultured in three-dimensional collagen gels. All three mammalian isoforms of TGF-β altered scleral cell phenotype to produce highly contractile, α-SMA-expressing myofibroblasts (TGF-β3 > TGF-β2 > TGF-β1). Exposure of cells to the reduced levels of TGF-β found in the sclera in myopia produced decreased cell-mediated contraction and reduced α-SMA expression. These findings are contrary to the in vivo gene expression data. However, when cells were exposed to both the increased stress and the reduced levels of TGF-β found in myopia, increased α-SMA expression was observed, replicating in vivo findings. These results show that although reduced scleral TGF-β is a major contributor to the extracellular matrix remodeling in the myopic eye, it is the resulting increase in scleral stress that dominates the competing TGF-β effect, inducing increased α-SMA expression and, hence, producing a larger population of contractile cells in the myopic eye.