The 22 human cadaveric eyes (11 pairs) used in this study were obtained from the Mid-America Tissue Bank (St. Louis, MO). The study protocol adhered to the tenets of the Declaration of Helsinki for research involving human subjects, including identifiable human tissue. The age of the donors ranged from 19 to 84 years. All donors were white. There were six males and three females; the tissue bank did not record the sex of two donors. Human cadaveric eyes were stored in a moist chamber after enucleation and transported to our laboratory. The time between death and tissue fixation ranged from 14 to 24 hours (average, 20 ± 3.5).
The globes were cleaned of extraocular tissue under a dissecting microscope. Eyes were included if there was no donor history of posterior segment disease and no visible signs of chorioretinal disease, including subretinal hemorrhage, extensive drusen, or irregular pigmentation of the macular RPE. An incision was made through the sclera 3 mm posterior to the limbus until the choroidal vessels were exposed.
18 19 Tenotomy scissors were introduced through this incision into the suprachoroidal space, and the incision was extended 360° circumferentially. Four radial relaxing incisions were made in the sclera, and the sclera was peeled posteriorly to expose the choroid. A circumferential choroidal incision was made along the ora serrata and extended into the subretinal space, and the RPE–choroid complex was separated gently from the neural retina. Eight radial incisions were made from the ora serrata toward the posterior pole and the RPE–choroid complex was flattened on an unlaminated, hydrophobic, polytetrafluoroethylene membrane (125–175 μm thickness, 0.5 μm pores; Millipore, Bedford, MA) with the RPE facing up. The flatmount was then fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 12 hours.
The RPE-choroid complex was mounted on a glass slide (
Fig. 1 , left) and the flatmount preparation was stained with a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) technique.
20 Briefly, samples were permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate solution on ice for 4 minutes. Cells were incubated with a mixture of fluorescein labeled nucleotides and terminal deoxynucleotidyl transferase (TdT) from calf thymus for 60 minutes. This enzyme catalyzes the polymerization of labeled nucleotides to free 3′-H terminals of DNA fragments. Fluorescence microscopy was used to visualize the apoptotic cells at the end of this period. Some of the flatmount preparations were also stained with propidium iodide to determine the chromatin condensation pattern within the TUNEL-positive cells.
The flatmount preparation was divided into four quadrants (superior temporal, inferior temporal, superior nasal, and inferior nasal) and four concentric regions centered on the fovea (zone 1, 0–1.5 mm radius; zone 2, 1.5–3.0 mm; zone 3, 3.0–12.5 mm; and zone 4, >12.5 mm;
Fig. 1 , right). The density of RPE cells (in cells per square millimeter) was determined by counting the number of cells within a 100 × 62.5-μm rectangle. Counts were performed in five areas per sector and the average count was used to determine the density. Cells that were intersected by the top and left sides of the rectangle were counted, whereas cells intersected by the right and bottom sides were not counted. For each zone we calculated the RD
50, defined as the donor’s age corresponding to 50% of the maximum change in density of RPE cells within a given zone. The apoptotic cells were visualized directly under a fluorescence microscope. Two observers masked to donor age determined the number of apoptotic cells in each zone and quadrant. The location of each apoptotic cell was marked with a pen on the reverse side of the hydrophobic membrane. The area of each section was determined with a planimeter (No. 620005; Keuffel & Esser Co., Morristown, NJ), and the density of RPE cells and the proportion of apoptotic RPE cells per 100,000 cells in each area was calculated. Regression analysis was used to determine the effect of aging on density and proportion of apoptotic cells in different zones. Best-fit curves were determined and data were analyzed for significance by ANOVA, with
P < 0.05 defined as statistically significant. Once a significant relationship between proportion of apoptotic cells and aging was established, we calculated the age corresponding to the proportion of apoptotic cells in the 5th and 50th percentiles (AP
5 and AP
50) in each zone. The proportion of apoptotic cells was compared between the young and old age groups and in different regions with a Student’s
t-test, and the results were considered significant at
P < 0.05.