A total of 25 male New Zealand big-eared rabbits, 4 to 5 months old, weighing 2.0 to 2.6kg, purchased from Shanghai Jiagan Biotechnology Co., Ltd., with animal production license numbered SCXK (H.) 2015-0005, were used in this study. The rabbits were fed in cages with automatic washing that were automatically washed once every 4 hours. The temperature and humidity of the feeding room were recorded, and the room ventilation was checked every morning and afternoon.
This study was approved by the Medical Ethics Committee of Jiading District Central Hospital. (No.: 2018K15), and was also in accordance with ARRIVE guidelines.
AS rabbit models
The experimental rabbits were grouped into five following groups: group A, the preoperative control group; group B, the week-4 after modeling group; group C, the week-8 after modeling group; group D, the week-12 after modeling group; and group E, the week-16 after modeling group. AS rabbit models were built via high-fat cholesterol feed and surgical balloon injury. Every day, all experimental rabbits were given 120g of high-fat cholesterol feed that contained 75.5% of basal rabbit feed, 10% of lard, 10% of yolk powder, 4% of cholesterol (of 95% purity), and 0.5% of cholate. After one week of adaptive feeding, the rabbits were subjected to balloon injury in the abdominal aorta. Before the surgery, the rabbits were anesthetized by intramuscular injection of 0.25/kg of suxinmian II (cetirizine hydrochloride injection, Jilin Shengda Animal Medicine Co., Ltd.); the hair from the left lower limb was removed, and the femoral artery was separated. During the surgery, a portable ultrasonic machine (M-Turbo, SonoSite, California) was used to guide the balloon catheter to 2cm above the level of the abdominal aortic, renal artery (of a feeding length of about 20cm). After that, the balloon was injected 7.5atm of air and dragged down to the bifurcation of the abdominal aorta and common iliac artery, which was repeated four times.
Preparation of ICAM-1-carrying targeted nanobubbles
ICAM-1-carrying targeted nanobubbles were prepared by the avidin-biotin binding method. An ultramix oscillator (Vortex-QL-861, at 2,000 oscillations/min and for 40 seconds) was used to activate ultrasonic bare bubbles labeled the avidin USphere (800ul/vial). After 40ul of Biotin/FITC-ICAM-1 antibodies were added into the vial of the bare bubbles, and the vial was fully suspended for 10 seconds in a scroll oscillator and then placed at room temperature for 30 minutes for incubation. After that, it was placed in a centrifuge to remove unbound antibodies. A fluorescence microscope (LAICA DM2000, Germany, 400x) was used to observe the binding of nanobubbles to ICAM-1, and a flow cytometer was used to measure the binding rate of Biotin/FITC-ICAM-1 to nanobubbles.
An ultrasonic diagnostic unit (Toshiba, Aplio400, Japan) was used at a probe transmission frequency of 11MHz, depth of 2cm, and frame rate of 45 frames/s. 2D ultrasonography was used to measure the thickness of the intima-media membrane at the site in the abdominal aorta that was the thickest. Color Doppler ultrasonography was used to observe blood filling in the lumen.
SonoVue ultrasonic contrast
The rabbits were anesthetized by intramuscular injection of 0.1ml/kg of suxinmian II (cetirizine hydrochloride injection, Jilin Shengda Animal Medicine Co., Ltd.), placed at the supine position on the operating bench; the hair was removed from the abdominal wall, and an indwelling needle (20G, Intima IITM, Suzhou Becton Dickinson Medical Devices Co., Ltd.) was placed in the left auricular vein. SonoVue (Bracco, Italy) was used as the ultrasonic contrast agent. An ultrasonic diagnostic unit (Toshiba, Aplio400, Japan) was used at a probe transmission frequency of 11MHz, depth of 2cm, a frame rate of 45 frames/s, and mechanical index of 0.08, with the focal depth below the posterior wall of the abdominal aorta. Before use, 5ml of 0.9% NaCl (China Otsuka Pharmaceutical Co., Ltd.) was injected into the bottle and shaken into microbubble suspension. After that, 0.1ml/kg of the contrast agent was extracted and quickly injected through the auricular vein, which was followed by an injection of 1ml of 0.9% NaCl.
ICAM-1-carrying targeted nano ultrasonic contrast
All experimental rabbits were given targeted nano ultrasonography under the same ultrasonic conditions as described above 1 hour after conventional ultrasonic contrast after being anesthetized in the same way as described above. Bolus injection of 0.3ml/kg of ICAM-1 targeted nanobubbles was quickly given to the rabbits at the site of the indwelling needle through the auricular vein. After that, the rabbits were immediately injected with 1ml of 0.9% NaCl to rinse the catheter, and continuous dynamic images were acquired in the contrast mode. Imaging of the target area was analyzed in the built-in software for the peak intensity (PI), time to peak (TTP), and area under curve (AUC) of contrast (Table 1).
Serum lipid test
After ultrasonic examinations, the rabbits’ hair around the site of the strongest heart beating was removed, disinfected with iodophor, and extracted 5ml of blood through intercostal cardiac puncture. The whole blood was centrifuged at 4℃ for 15 minutes for serum. Enzymes were used to measure the total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL), and low-density (LDL) in the serum samples.
The extracted abdominal aorta was sealed in 5% formalin solution and embedded in paraffin, which was then cross-sectioned for a transverse section of tissues of about 3μm in thickness. The section was then HE stained to observe the degree of atherosclerotic lesions, and Image-ProPlus 6.0 software (Media Cybernetics, Maryland) was used to measure and calculate the maximum plaque thickness.
The BCA protein concentration quantitative kit (Thermo, USA) was used to measure the expression of ICAM-1 in the abdominal aorta, and the image analysis system (Quantity One, Bio-Rad, California) was used to analyze the optical density of the target.
SPSS 19.0 (IBM, Armonk, NY, USA) was used for data analysis. Continuous variables were expressed as means ± standard deviation (SD). Independent samples t-test was performed for between-group comparisons, and one-way analysis of variance (ANOVA) followed by post-hoc LSD test for multiple-group comparisons. Pearson correlation analysis was used to analyze the correlation between CEUS parameters and pathological results. A P<0.05 was considered statistically significant.