RT Journal Article
A1 Hollyfield, J. G.
A1 Rayborn, M. E.
A1 Shadrach, K. G.
A1 Yang, X.
A1 Li, Y.
A1 Salomon, R. G.
A1 Perez, V.
T1  Correlation Between RPE Pathology and CEP Antibody Titer in Mice Immunized With CEP-MSA
JF Investigative Ophthalmology & Visual Science
JO Invest. Ophthalmol. Vis. Sci.
YR 2008
VO 49
IS 13
SP 474
OP 474
SN 1552-5783
AB    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.   
RD 4/13/2021