Fifteen human donor eyes (4 male and 11 female) were dissected to obtain full-thickness scleral specimens. Eyes were obtained from the U. K. Tissue Transplant Service (Bristol, UK), transported from the eye bank on ice, and stored at 4°C in the laboratory. The study was undertaken in accordance with the guidelines of the Declaration of Helsinki for research involving human tissue. The mean donor age was 60 years, with a range of 39 to 84 years. Those with known systemic conditions with ocular manifestations were excluded.
Sections of full-thickness sclera measuring approximately 8 by 9 mm were dissected from the equatorial superotemporal regions in all donor eyes. Areas under the rectus muscles and involving the vortex veins were avoided. Specimens with any suggestion of damage were excluded. An ophthalmic surgeon (OAA) carefully removed the episclera and choroid, with forceps, spring scissors, and a dissecting microscope. A 1-mm wide scleral section was removed from the edge of each specimen, with a razor blade. This section was used to determine scleral thickness, as described later.
The specimens were placed in a modified Ussing chamber
6 with a 6-mm aperture. The screws that hold each half of the chamber together were tightened with a torque-range screwdriver (RS Components, Corby, UK), by applying the minimum force necessary to prevent leakage around the specimen (approximately 30 cN · m
−1). The integrity of tissue clamped in this type of device has been demonstrated.
5 7 8 9 Histology of clamped sclera has shown tissue compression to extend for a minimal distance into the central aperture, with the collagen fibrils remaining intact.
5 Both hemichambers were filled with 750 μL phosphate-buffered saline (PBS; sodium chloride 137 mM, potassium chloride 2.7 mM, phosphate buffer 10 mM, pH 7.4 at 25°C; Sigma, Poole, UK), with penicillin 100,000 U · L
−1, streptomycin 100 mg · L
−1, and amphotericin B 250 μg · L
−1 (Sigma-Aldrich, St. Louis, MO). In addition 0.412 mM fluorescein isothiocyanate (FITC) labeled bovine serum albumin (Sigma-Aldrich) was placed into the hemichamber facing the internal scleral surface. During both the setup and subsequent fluid removal stages, care was taken to keep the fluid volume in each hemichamber identical, to prevent the risk of harmful hydrostatic pressure gradients developing across the sample. Evaporation was minimized by sealing the tops of the chambers with insulation tape. Each tape was pierced to prevent a pressure gradient developing across the sample. Small plastic-encased magnetic stirrers were inserted into the bottom of each hemichamber. Experiments were conducted at 25°C, with the specimens protected from ambient illumination.
After a mean (±SD) of 11.11 ± 1.32 hours (
t 1), 250 μL of fluid was simultaneously removed from both chambers. This process was repeated at a mean at 22.93 ± 1.66 hours (
t 2). Any potential effect of leaching of glycosaminoglycans was minimized by limitation of the experimental duration to no more than 24 hours.
10 Two samples were taken to eliminate any measurement error induced by a delay in the appearance of FITC-labeled bovine albumin in the second chamber, after commencing the experiment. Pilot studies, removing three samples over the same time period, had demonstrated the rate of increase in FITC-labeled albumin in the second chamber to be linear (
R 2 = 0.99) and to occur after a significant time delay. The sample times were chosen as they occurred during the linear portion of diffusion, irrespective of the age of the donor. The maximum absorbance of FITC-albumin was at 490 nm and therefore this frequency was used for spectrophotometric quantification of each sample (UV-160; Shimadzu, Kyoto, Japan). The concentration of FITC-labeled bovine albumin was then calculated from predetermined standard curves.