Contrast Ultrasound Imaging of the Aorta Does Not Affect Progression of Atherosclerosis or Cardiovascular Biomarkers in ApoE-/- Mice

Abstract

Objectives: Ultrasound contrast agents (UCAs) enhance cardiovascular ultrasound imaging. Adverse biological effects have occurred after administration of UCAs, and more research is needed for a comprehensive understanding of the risks involved. We used the ApoE(-/-) mouse model of atherosclerosis to characterize the effects of ultrasound and UCAs on atherosclerosis and plasma biomarkers.

Methods: Male ApoE(-/-) mice (8 weeks old; n = 24) were intravenously infused with a UCA (2 × 10(10) Definity microbubbles per hour; Lantheus Medical Imaging, North Billerica, MA) and exposed to 2.8-MHz center frequency ultrasound (10 Hz pulse repetition frequency, 1.4 microseconds pulse duration, 2 minutes exposure duration, and 2 sites) at 1 of 3 derated peak rarefactional pressure amplitudes (0, 1.9, or 3.8 MPa), and then consumed either a chow or Western diet for 4 weeks (n = 4 per group). Blood plasma samples were collected before ultrasound exposure and at 2 and 4 weeks after exposure and assayed for total cholesterol and von Willebrand Factor (vWF). A pathologist measured atheroma thickness in formalin-fixed, hematoxylin-eosin-stained transverse aorta sections and scored them for severity of atherosclerosis.

Results: Plasma total cholesterol initially averaged 286 mg/dL in the Western diet group and increased to 861 mg/dL after 4 weeks on the diet (P < .0001). Total cholesterol did not increase significantly in the chow diet group. Plasma vWF increased after 2 weeks on the Western diet (P < .0001). Atheroma thickness was greater in animals consuming the Western diet than in chow-fed animals (P < .05). Ultrasound had no significant effect on plasma total cholesterol, plasma vWF, or atheroma thickness.

Conclusions: Contrast ultrasound did not increase the severity of atherosclerosis or alter cardiovascular biomarkers in the ApoE(-/-) mouse model.

Keywords: atherosclerosis; biomarkers; cardiovascular disease; contrast agents; endothelium; microbubbles; ultrasound.

Figure 1

Study design. Baseline blood samples were obtained from 24 male ApoE−/− mice. The mice then received the UCA and were exposed to ultrasound at 0, 1.9, or 3.8 MPa in situ PRPA, with n = 8 per group. Half of the mice continued to consume the chow diet, and half were switched to the Western diet. Subsequent blood samples were obtained at 2 and 4 weeks. Mice were euthanized at 4 weeks, and aortas were obtained for analysis.

Figure 2

Plasma cholesterol (A) and vWF (B). Plasma total cholesterol was analyzed using an enzymatic colorimetric kit. Human control sera were included in each assay for quality control. Plasma vWF was determined using an in-house validated sandwich enzyme-linked immunosorbent assay. Bars indicate SEM; n = 3 or 4 per group per time point.

Figure 3

Aortic histologic analysis. The aortic arch and descending aorta were fixed briefly in situ with 10% NBF and then carefully excised and fixed in 10% NBF for an additional 24 hours, embedded in paraffin, sectioned, and stained with hematoxylin-eosin (A). A pathologist blinded to the exposure conditions measured atheroma thickness with an ocular micrometer (B) and assigned an atherosclerosis score between 0 and 5 using the American Heart Association classification scheme for human atherosclerotic lesions (C). Bars indicate SEM (n = 3–4 per group; *P < .05).