Identifying Vulnerable Atherosclerotic Plaque in Rabbits Using DMSA-USPIO Enhanced Magnetic Resonance Imaging to Investigate the Effect of Atorvastatin

Abstract

Background: Rupture of an atherosclerotic plaque is the primary cause of acute cardiovascular and cerebrovascular syndromes. Early and non-invasive detection of vulnerable atherosclerotic plaques (VP) would be significant in preventing some aspects of these syndromes. As a new contrast agent, dimercaptosuccinic acid (DMSA) modified ultra-small super paramagnetic iron oxide (USPIO) was synthesized and used to identify VP and rupture plaque by magnetic resonance imaging (MRI).

Methods: Atherosclerosis was induced in male New Zealand White rabbits by feeding a high cholesterol diet (n = 30). Group A with atherosclerosis plaque (n = 10) were controls. VP was established in groups B (n = 10) and C (n = 10) using balloon-induced endothelial injury of the abdominal aorta. Adenovirus-carrying p53 genes were injected into the aortic segments rich in plaques after 8 weeks. Group C was treated with atorvastatin for 8 weeks. Sixteen weeks later, all rabbits underwent pharmacological triggering, and imaging were taken daily for 5 d after DMSA-USPIO infusion. At the first day and before being killed, serum MMP-9, sCD40L, and other lipid indicators were measured.

Results: DMSA-USPIO particles accumulated in VP and rupture plaques. Rupture plaques appeared as areas of hyper-intensity on DMSA-USPIO enhanced MRI, especially T2*-weighted sequences, with a signal strength peaking at 96 h. The group given atorvastatin showed few DMSA-USPIO particles and had lower levels of serum indicators. MMP-9 and sCD40L levels in group B were significantly higher than in the other 2 groups (P <0.05).

Conclusion: After successfully establishing a VP model in rabbits, DMSA-USPIO was used to enhance MRI for clear identification of plaque inflammation and rupture. Rupture plaques were detectable in this way probably due to an activating inflammatory process. Atorvastatin reduced the inflammatory response and stabilizing VP possibly by decreasing MMP-9 and sCD40L levels.

Fig 1. SNR on MRI.

SNR was determined, where SI (signal intensity) is the average signal strength of the abdominal aorta wall, and SD is the standard deviation of the signal intensity in the background of the scan.

Fig 2. Image changing signal in multi-contrast MRI sequences of DMSA-USPIO enhanced.

Image of plaques in T1WI and PDW showing a slightly higher signal and an equal signal in T2WI.

Fig 3. Images (A) and SNR change(B) of the 3 groups (a, b, c) between 0 and 96h after injecting USPIO.

Both had signal loss at 96h compared with 0h. VP in group B (A-b) had variable changing signal. *Group B gave the peak of negative enhancement at 96 h.

Fig 4. SNR trend of images (4-A) of MRI in T2*WI and chart (4-B) of group B before, and at 24, 48, 72, 96, 120h after DMSA-USPIO infusion.

Maximal signal loss (yellow arrow) occurs at 96 h. The SNR trend chart shows that maximal signal loss occurs at 96 h for T2*WI in group B.

Fig 5. Pre and post times of rupture plaque after DMSA-USPIO infused in T2WI.

Acute mixed hemorrhage showed variable signal loss of post rupture plaque compared with pre rupture plaque.

Fig 6. Three kinds of histology staining of sections from the 3 groups.

H&E: The walls are significantly thickened in group A and B.The lumen of group C shows average thickening comparatively and the wall of plaque was thickest in group B. Intraplaque hemorrhage could be seen in group B. Large foam cells and collagen fibers are visible in group B compared with another two groups. Masson staining: hyperplasia of the collagen fibers in group B is more obvious than in another two groups. Prussian blue staining: iron particles have accumulated in macrophages in groups A and B, especially the latter; group C had very few iron particles.

Fig 7. Electron microscopy of the 3 groups (a, b, c).

Lysosomes and macrophages (yellow arrows) are more common in group B than groups A and C. Large fat was deposited in group B (red arrows). Iron particles (yellow arrows) are apparent in macrophages. In contrast, few Iron particles are seen in group C.

Fig 8. The ROC curve for SNR_mean to predict VP of group B at 96h.

Area under the curve (AUC) was 0.908 (P = 0.000).The cutoff value was 23.9.