Fourteen control human eyes from donors aged between 62 and 93 years (mean age, 75.4 years), with a postmortem time of up to 48 hours, were used (UK Transplant Support Service, Bristol Eye Bank, Bristol, UK). Informed consent for the use of the eyes for experimental studies was obtained, in accordance with the Declaration of Helsinki. Control donor eyes were obtained with the cornea removed for transplantation. The globes were dissected in a Petri dish lined with filter paper (Grade 50; Whatman, Maidstone, UK), moistened with Krebs’ bicarbonate buffer. The buffer composition was (mM): NaCl 118, glucose 6.6, NaHCO3 25, KCl 4.84, MgSO4 0.8, KH2PO4 1.2, CaCl2 1.8, 0.01% BSA. The posterior globe was inspected under a dissecting microscope for any evidence of subretinal or intraretinal blood, hard exudates, extensive drusen, irregular pigmentation of the RPE or any gross disease of the retina, and those exhibiting any abnormal appearance were discarded. The eyes were dissected at approximately the ora serrata to remove the remaining anterior portion, and then the globe was cut open to form a Maltese cross, the posterior segment being laid flat onto filter paper (Grade 50; Whatman). Vitreous was carefully removed, and the four quadrants (the arms of the cross) were removed and immersed in ice-cold Krebs’ buffer. Retina was then gently stripped from contact with the RPE and the RPE-choroid complex isolated from the sclera. Great care was taken when handling the aged tissue, as attachment of the RPE to Bruch’s membrane lessens with time after death. Samples were again carefully inspected under a dissecting microscope, and samples that had sustained damage to the RPE monolayer during dissection were discarded. The intact samples were used in the radiolabel uptake studies. In 6 of the 14 control donor eyes included in the study we could obtain only one intact trephine. In two eyes we obtained two intact trephines, in two eyes three intact trephines, and in four eyes four intact trephines.
Four melanoma-affected human eyes were used. The donors were 61, 68, 69, and 74 years of age. These eyes were processed within a few hours after enucleation with the patient under general anesthesia. The anterior segment was first removed and the globe hemisected so that the melanoma-containing region could be processed for histopathology. The other half was cut into two or three segments and used for the kinetic characterization of taurine transport.