Quantitative immunohistochemical detection of oxidized low density lipoprotein in the rabbit arterial wall

Quantitative immunohistochemical techniques were developed for mapping low density lipoprotein (LDL) oxidation within arterial tissue. Antibodies were raised by immunizing rabbits with Cu(2+)-oxidized rabbit LDL. ELISAs showed that they reacted strongly with oxidized rabbit LDL, weakly with other oxidized lipoproteins, and not at all with native LDL. Using optimized histological procedures, the antibodies were applied to sections of calibration gels containing LDL at various concentrations and levels of oxidation, and to sections of aortas from normal and heritable hyperlipidemic rabbits. Binding was measured with a rhodamine-labeled secondary antibody and carefully calibrated techniques of digital imaging fluorescence microscopy. Values obtained using a nonspecific primary antibody were subtracted. Specific binding to calibration sections increased linearly with respect to the concentration of oxidized LDL and the duration of its exposure to Cu2+, approximately linearly with respect to its modified lysine content, and nonlinearly with respect to its relative electrophoretic mobility. Specific staining was detected in sections of aortas from heritable hyperlipidemic but not normal rabbits. In the former, it was higher in the intima than in the media and was greater downstream than upstream of intercostal branch ostia; the average level was lower in those branches with the least intimal thickening but the difference between upstream and downstream regions was larger. These results correlate with the known pattern of lipid deposition in hyperlipidemic rabbit aortas. A small but significant amount of specific staining was observed in sections which were devoid of intimal thickening, which is consistent with LDL oxidation occurring prior to disease or during its earliest stages.