Detection of Atherosclerotic Plaques in the Rabbit Aorta Using Ultrasound Microbubbles Conjugated to Interleukin-18 Antibodies

Abstract

BackgroundThe purpose of the study was to investigate the ability of microbubbles (MBs) targeting interleukin-18 (IL-18) to detect plaques in a rabbit atherosclerotic plaque model.Material/MethodsA rabbit atherosclerotic plaque model was established. The locations of the atherosclerotic plaques were verified by two-dimensional scanning and color Doppler flow imaging. An IL-18 antibody was conjugated to naked MBs (MBc) using the biotin-streptavidin conjugation method, resulting in the formation of MBIL-18. MBc and MBIL-18 were then used for contrast-enhanced ultrasound (CEUS) studies. The locations of CD34 and IL-18 within the plaques were determined by immunohistochemistry, and IL-18 expression levels in the plaques were determined by Western blot analysis. The relationships between IL-18 expression and the contrast intensity of the 2 MBs were analyzed.ResultsMBc and MBIL-18 were both uniformly dispersed. Fluorescence microscopy and flow cytometry revealed that IL-18 was successfully conjugated to MBs. CEUS images showed that the intensity of the MBIL-18 signal was substantially enhanced and prolonged compared with that of the MBc signal. Immunohistochemistry showed that CD34 expression was significantly increased in the plaques and that IL-18 was mainly located in the inner parts and base of the atherosclerotic plaques. Western blot analysis revealed that IL-18 expression was higher in the plaque regions. Correlation analysis showed that IL-18 expression was correlated with the contrast intensity of MBIL-18 (r=0.903, P<0.05) but not with MBc (r=0.540, P>0.05).ConclusionsMBs targeting IL-18 may be a novel, noninvasive method of diagnosing atherosclerotic plaques.

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“…The slides were stained with hematoxylin and eosin (H&E) and cd34 (ZM-0046, 1:200, ZSGB-BIO, Beijing, china) for histopathological and immunohistochemical analyses using standard techniques and reagents. The sample was exposed to an immunospecific primary antibody diluted in 5% goat serum overnight at 4˚C, then to a goat-anti-mouse secondary antibody (PV-6002, ZSGB-BIO) for 1 h at room temperature (35,36). Measurements were performed with an optical microscope (DP73; Olympus, Tokyo, Japan; x4 to x40 magnification) that was attached to a video camera and connected to a computer equipped with image analysis software (cellSens Standard software, Olympus).…”