The Experimental Study Of Targeted Contrast Ultrasonography On Atherosclerosis Of Abdominal Aorta In Rabbits

Objective This study was designed to prepare the specific targeted ultrasound contrast agent (TUCA) and to test its value in early diagnosis in atherosclerosis. The perfluorocarbon-filled albumin microbubbles were firstly mixed with anti-ICAM-1 antibody for conjugation. Then it was administrated intravenously in vivo to test its targeting ability with injured endothelium and atheromatous plaques of abdominal aorta in order to provide some theoretical evidences for the use of TUCA in blood vessel disease. Methods 1. Perfluorocarbon-filled albumin microbubbles were prepared by sonication using a commercial available sonicator. Then it was mixed with anti-ICAM-1 monoclonal antibody and glutaraldehyde to form TUCA. Immunofluorescence was applied to confirm the conjugation. 2. Sixteen New Zealand rabbits were fed with cholesterol diet four weeks to establish the animal model of endothelium dysfunction. Then the rabbits were randomly divided into two groups. Non-specific targeted microbubbles were administrated to the group one. Specific targeted microbubbles conjugated anti-ICAM-1 monoclonal antibody were administrated to the group two. The dosage of contrast agent was 0.02 ml/kg. Contrast second harmonic imaging was applied to record the time to peak concentration and the duration time for both of the specific targeted and non-specific targeted microbubbles on normal and on injured endothelium of abdominal aorta. The pathological of endothelial cells and the albumin microbubbles adhered to the surface of endothelial were evaluated by electron microscope. Immunofluorescence were performed in which the distribution of conjugated or non-conjugated microbubbles were observed under microscopy. 3. Atherosclerosis model was set up by feeding high cholesterol diet in 26 rabbits after twelve weeks. The rabbits were then randomly divided into 3 groups. Non-targeted microbubbles, targeted microbubbles and both of the microbubbles were injected intravenously into the group one, the group two and the group three, respectively. The dosage of the targeted microbubbles and the non-targeted ones was 0.02 ml/kg equally. The contrast imaging of the abdominal aorta was digitally recorded. The video intensity of endoderm and atheromatous plaques of the three groups were analysed. Rabbits of the group one and two were killed after contrast. Aortic specimens were harvested for HE staining, immunochemistry staining, and immunofluorescence studies respectively. Pathological changes of AS were evaluated by HE slice. Immunochemistry and immunofluorescence were used to detect the sticky degree both about the targeted albumin microbubbles and the non-targeted ones to aorta vessel. Results 1. The diameter of TUCA ranged from 2 to 5 μm, and the concentration was 6×109 /ml, almost the same as the non-targeted ones. The conjugation was further approved by immunofluoroscopy. 2. The imaging duration time and the time to peak concentration for the targeted and the non-targeted microbubbles had no difference on normal endothelium(P>0.05). The anti-ICAM-1 conjugated microbubbles persisted significantly longer while the time to peak concentration was much shorter in the group two on the injured endothelium than that of the group one (P<0.01). The microbubbles attached to the injured endothelial cells were mostly the targeted ones. The endoderm fluorescent intensity of the group two was more than that of the group one. 3. A significant visual grayscale enhancement could be detected in the endoderm and atheromatous plaques after contrast(P<0.01). The targeted albumin microbubbles produced more significantly video intensity enhancement of endoderm and atheromatous plaques than the non-targeted ones did(P<0.05). Pathological changes of AS had been detected by histological slice. Immunochemistry and immunofluorescence disclosed that the adhesive number of targeted microbubbles to the inflamed tissue was more than that of the non-targeted ones. Conclusions 1. The anti-ICAM-1 antibody conjugated albumin microbubbles could be prepared by using glutaraldehyde as a couplant. The conjugation is confirmed by the study as a technically feasible and practical method.2. The anti-ICAM-1 conjugated microbubbles which have been shown to bind injured endothelial cells can be used for assessing endothelial function. 3. Intravenous injection of anti-ICAM-1 conjugated microbubbles can enhance the grayscale imaging of injured endoderm and atheromatous plaques which is effective for the improvement of video intensity.