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A. J. Weis, J. N. Swanton, K. R. Huxlin, R. P. Phipps, H. B. Hindman; Central and Peripheral Corneal Keratocytes Respond Differently to Stimulation by Pro-Fibrotic TGF-β1. Invest. Ophthalmol. Vis. Sci. 2010;51(13):379.
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To determine if primary keratocytes isolated from central and peripheral regions of the cornea respond differently to transforming growth factor-beta-1 (TGF-β1) stimulation in vitro.
Six corneas were removed from eyes of post-mortem cats. Corneas were separated into central and peripheral regions using a 10 mm diameter trephine. Primary keratocytes were isolated from each region and cultured separately in serum-free defined medium under identical conditions. After 72 hr stimulation with 0, 0.1, 1.0, and 2.0 ng/mL TGF-β1, their responsiveness was characterized by protein expression (Western blots and immunohistochemistry for Thy-1, α-SMA, and fibronectin expression), entry into proliferative cell cycle stages (Ki-67 staining), and ability to close a mechanical wound.
At 0.1 ng/ml TGF-β1, peripherally derived cells expressed significantly more Thy-1, α-SMA, and fibronectin vs. centrally derived cells. Peripheral cells closed a mechanical wound faster with significantly greater percentage wound closure with 0.1 ng/ml TGF-β1 at 4 hours; 1.0 ng/ml TGF-β1 at 4, 8 and 12 hours; and 2.0 ng/ml TGF-β1 at 4 and 8 hours. Positive Ki-67 staining was significantly greater in centrally derived cells at early time-points (day 1 to day 6), but significantly greater in peripherally derived cells at later time-points (day 8 to day 13).
In felines, primary keratocytes isolated from central and peripheral regions of the cornea demonstrate a heterogeneous response to TGF-β1 stimulation with regard to proliferation, protein expression, and rate of wound closure. After TGF-β1 stimulation, keratocytes cultured from the peripheral cornea demonstrate delayed proliferation, greater protein expression, and a faster rate of wound closure vs. centrally derived cells. This has important implications in post-traumatic and post-surgical wound healing and visual outcomes.
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