Fresh porcine eyes were obtained from an abattoir (Dorsey Meats, Woodsboro, MD) and soaked for 15 minutes in standard antibiotic solution containing 10,000 U/mL penicillin and 10,000 μg/mL streptomycin in balanced saline (BSS Plus; Alcon, Fort Worth, TX). Under sterile conditions, scleral segments free of RPE-choroid were dissected from the temporal equatorial side of the eye, which has the thinnest sclera and lacks perforating vessels,
48 51 and was stored at −80°C in optimal cutting temperature (OCT) compound (Tissue-Tek 4583; Sakura Finetek Inc., Torrance, CA). Under sterile conditions, the thawed sclera was sealed with an O-ring between the orbital and uveal compartments of a Ussing chamber (EasyMount; Physiologic Instruments, San Diego, CA). Temperature was maintained at 37°C with a circulating water bath, and 5% CO
2 and 95% O
2 were bubbled through the solution, to circulate and to maintain the pH. Leaks in our system were tested by applying hydrostatic pressure from the orbital side with culture medium. The minimum diameter of a scleral segment for a leak-free system was determined to be 8.5 mm, by adding Fl-Ova in balanced saline to the orbital chamber with the balanced saline alone in the uveal chamber. After a short time (<30 minutes) the sclera was removed, fixed, and sectioned for epifluorescence microscopy, to evaluate fluorescence in the borders of the O-ring area indicative of leaks.
Donor (5 mL of 50 μg/mL Fl-Ova in balanced saline, 10,000 U/mL penicillin, 10,000 μg/mL streptomycin, and complete miniprotease inhibitors [1 tablet per 10 mL of solution; Roche, Mannheim, Germany]) and receiving (5 mL balanced saline solution with supplements) solutions were loaded into the orbital and uveal chambers, respectively. The fluids from both compartments were sampled at 0, 12, 24, 30, 36, and 48 hours after incubation at 37°C. After each sampling, fresh donor and receiving solutions were reloaded to maintain a nearly identical concentration difference across the tissue at the start of each time interval and to minimize the effects of evaporation. Fl-Ova concentrations in each chamber, Cd (donor/orbital) and Cr (receiving/uveal), were measured by spectrofluorometry. From the known chamber volume (V), the total amount of ova (M) diffusing over the sampling time (t) was calculated and reported as a diffusion flux (R), in micrograms per hour, where M = Cr × V and R = M/t. The diffusion flux, R, can be related to the diffusion coefficient (D) by the equation: R = D × A × (Cd − Cr)/L, where A is the exposed area of the scleral tissue in the Ussing chamber and L is the tissue’s thickness, estimated to be 0.83 ± 0.2 mm from 10 scleral measurements with a Vernier caliper. For the present sample times, Cr was always less than 1% of Cd, and so Cr was omitted from the calculations.