For each animal, a single cross section was examined, and low-power transmission electron micrographs (i.e., magnification, ×7200) were made of the entire section, from the perioptic to the ciliary body portion (approximately 10 micrographs). Then, one representative high-power micrograph (i.e., magnification, ×25,000) was made from each low-power section by an individual unaware of the experimental conditions and used for semiquantitative scoring. The high-power micrographs were graded by two independent examiners for the presence and severity of BLD. A severity score of 0 to 15 points was determined for each section by summation of the median scores of all the micrographs from a section on each of four different categories of abnormalities (from 0–3 points for each): continuity of BLD (score: 0, no BLD16 months old; 1, occasional BLD16 months old i.e., focal nodule; 2, BLD16 months old, extending under fewer than two RPE cells; and 3, BLD16 months old extending under two or more RPE cells); maximum thickness of BLD (score: 0, no BLD; 1, flat BLD16 months old; 2, deposits thickness <25% of RPE cell cross-sectional thickness; and 3, deposit thickness ≥25% of RPE cell cross-sectional thickness); nature of deposit content (score: 0, no BLD16 months old; 1, homogeneous BLD16 months old; 2, any banded structures within BLD16 months old; and 3, three or more banded structures within BLD16 months old); presence of BrM abnormalities (score: 0, no abnormalities; 1, collagenous thickening, no deposit; 2, thickening with circular profiles or nonspecific debris; and 3, presence of basal linear deposits represented as banded structures, granular material or membranous debris); and assessment of other choriocapillaris endothelial damage or invasion (score 0, no alterations; 1, loss of fenestrations; 2, loss of fenestrations and thickening; and 3, choriocapillaris invasion into BrM). BrM thickness was also directly measured in three different standardized locations in each image and then averaged to provide a mean score for that micrograph. The mean of 10 micrographs was used to assign and “average” BrM thickness for an individual specimen.
Groups were compared by determining the mean and standard deviations. A t-test was used for statistical analysis of differences. In addition, the frequency of BLD was determined using two different criteria. “Any BLD” was defined as the presence of any discrete focal nodule of homogeneous material of intermediate electron density between the RPE cell membrane and BrM in at least one micrograph within a section. “Moderate BLD” was defined as the presence, in at least three micrographs, of the following: continuous BLD extending under two or more cells, deposit thickness equal to or greater than 25% of RPE cell cross-sectional thickness, or the presence of any banded structures within the BLD. Differences in the relative frequency were tested using χ2 test (χ2).