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J. G. Hollyfield, M. E. Rayborn, K. G. Shadrach, X. Yang, Y. Li, R. G. Salomon, V. Perez; Correlation Between RPE Pathology and CEP Antibody Titer in Mice Immunized With CEP-MSA. Invest. Ophthalmol. Vis. Sci. 2008;49(13):474.
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To define the relationship between antibody titer and retinal pigment epithelium (RPE) pathology in a mouse model for age-related macular degeneration (AMD) immunized with carboxyethylpyrrole (CEP).
3 month old C57Bl/6 mice were immunized with CEP-MSA or MSA controls in complete Freund’s adjuvant (CFA) at day 0, followed by challenge at day 10 in incomplete Freund’s adjuvant (IFA). To generate a strong immunological response and confirm that CEP-MSA is immunogenic, animals received three immunizations during a 3 month period, the final given 10 days before the mice were sacrificed. Direct ELISA for the detection of CEP-antibody was performed in 96-well plates coated with CEP-BSA (100 µl/well) at 1:1000 dilution in PBS and incubated at 37 °C for 1 hr, using 1% BSA solution as a blank control.Eyes were fixed in 2% glutaraldehyde, 2% formaldehyde (freshly prepared from paraformaldehyde) in 0.1 M cacodylate buffer, pH 7.2 at 4°C overnight, then post-fixed in 1 % osmium tetroxide, dehydrated in graded ethanol, to propylene oxide and then transferred to a 1:1 mixture of a plastic resin mixture containing Polybed 812 and Araldite 502 with polymerizer. After polymerization, 5-10, 1µm plastic sections were cut, mounted on microscope slides and stained with toluidine blue.
Histopathology of mice recovered at 3 months following immunization as described above showed variable pathologies. Each sample was placed in one of the following categories based on the average number of lesions present per section: Normal: Two of the six naïve mice studied had one and three lysed RPE cells respectively, and one of these also contained a single pyknotic RPE cell. Since these were naïve mice with no immunization, these lesions were considered normal background variability. When 4 or fewer lesions were found, the tissue was scored as normal. Minor Pathology: This category contained from 5 to 10 separate areas of RPE or outer retinal pathology in each section studied. ModeratePathology: This category contained 11 to 15 separate sites of RPE or outer retinal pathology. Severe Pathology: Mice in this category have 16 or more sites of RPE pathology in each section. Four eyes in this group had lesions in the RPE over 20-70 µm long. We then plotted the degree of pathology against the antibody titer of the animals in these groupings and found a correlation coefficient of 0.97.
These data demonstrate a close relationship between the CEP-antibody titer and the severity of outer retina pathology.
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