Human donor eyes were obtained from the Iowa Lions Eye Bank (Iowa City, IA) and were preserved within 8 hours of death after informed consent of the donor families. The use of human donor eyes conformed to the Declaration of Helsinki. Eyes were assigned to either early AMD or age-matched control donors based on chart review. Eyes with advanced AMD (geographic atrophy or choroidal neovascularization), identified in the chart, on gross examination of the eyes, or histologically, were omitted from the study. Eyes were designated as early AMD if they had been diagnosed as being notable for macular drusen and/or RPE changes; that is, they were AREDS grades 2 or 3.
1 The age of donors ranged from 60 to 100 years. The mean age of AMD donors was 84.8 ± 7.9 years, and the mean age of controls was 81 ± 8.9 years.
Either a 6-mm or bisected 8-mm punch centered on the fovea of each donor was collected and fixed in 4% paraformaldehyde in 10 mM phosphate-buffered saline, before sucrose cryoprotection and cryostat sectioning, using methods described previously.
18 Sections of 45 maculas from 42 donors (
n = 24 unaffected,
n = 21 clinically diagnosed with early-stage AMD) were collected. Lectin histochemistry using
Ulex europaeus agglutinin-I (UEA-I; Vector Laboratories, Burlingame, CA) was performed as described previously,
19 and sections were also labeled with either anti-CD45 (2.5 μg/mL; BD Pharmingen, San Diego, CA) to assess leukocytes or anti–C5b-9 terminal complement complex (1.5 μg/mL; Dako, Carpinteria, CA) to image sub-RPE deposits as described previously.
20 Sections were counterstained with diamidino-phenol-indole (DAPI; Molecular Probes, Eugene, OR), and triple-labeled sections were photographed using the ×20 objective of a fluorescence microscope (BX41; Olympus, Center Valley, PA) and a digital camera (SPOT RT; Diagnostic Instruments, Inc., Sterling Heights, MI). Nonoverlapping photomicrographs spanning the section (over a mean length of 549 μm/donor) were collected. Examples of this labeling are depicted in
Figures 1A and
1B. All images were collected with identical camera settings.