Normal corneas (
n = 11; mean age, 40.2 years; range, 24–59) were received within 24 hours after death from the National Disease Research Interchange (Philadelphia, PA). KC corneas (
n = 11; mean age, 34.8 years; range, 25–46) were sent to the laboratory in MK (McCarey-Kaufman) medium on ice and were received within 24 hours after penetrating keratoplasty
(Table 1) . The diagnosis of KC in the participating patients was based on the presence of more than one of the following criteria: Munson’s sign, Rizzuti phenomenon, slit-lamp findings of stromal thinning, Vogt’s striae, Fleischer ring, and epithelial or subepithelial scarring. Retroillumination signs included scissoring on retinoscopy and the oil droplet sign (Charleux); photokeratoscopy signs were compression of mires inferotemporally, inferiorly or centrally; and videokeratography signs were localized increased surface power and/or inferior superior dioptric asymmetry. Informed consents were obtained from participants and the study was performed according to the tenets of the Declaration of Helsinki for research involving human subjects and human tissue. Normal and KC stromal cells were isolated and cultured as described previously.
31 When corneal stromal cells are cultured in 10% fetal bovine serum, they differentiate into fibroblasts.
32 33 Our corneal cells stained with vimentin (a marker for fibroblasts) but did not stain with α-smooth muscle actin (a marker for myofibroblasts). In addition, the morphology was consistent with fibroblasts and not corneal keratocytes. For individual experiments, third-passage fibroblasts were used. The cells were plated overnight in either 24-well plates (1 × 10
5) or 60-mm dishes (5 × 10
5), rinsed in Dulbecco’s phosphate-buffered saline containing calcium and magnesium (D-PBS; Mediatech Inc, Herndon, VA), and then incubated for 1 hour in D-PBS, (pH 7.0 or 5.0), with or without H
2O
2 (200 or 400 μM; Sigma-Aldrich, St. Louis, MO). Cultures were rinsed in D-PBS and then harvested for assays.