Experimental protocol illustrating the serial magnetic resonance imaging (MRI) and the contrast agent injections performed in Watanabe and control rabbits. MION-47 = monocrystalline iron-oxide nanoparticle.

Watanabe rabbits exhibited lipid-rich plaque formation (A) with a high density of macrophages (acid phosphatase staining in panel B). The high density of macrophages in the atherosclerotic plaque could be confirmed by RAM-11 immunostaining (macrophages stained green by RAM-11 and cell nuclei stained blue by 4',6-diamidino-2-phenylindole-dihydrochloride [DAPI], panels C and D). No plaque formation was observed in control rabbits (E to H).

Representative black-blood turbo field-echo imaging (A, B, and D) and black-blood turbo spin-echo imaging baseline images (C, E, and F) of the aortic wall in Watanabe (A to C) and control rabbits (D to F). Watanabe rabbits exhibited increased wall thickness in the thoracic (*p < 0.001 vs. control rabbits) and in the abdominal aorta (p < 0.001 vs. control rabbits) (G). Wall thickness measured with magnetic resonance imaging (MRI) correlated closely with histology on matched slices (H) (asymptotic concordance of 0.73, p < 0.0001).

Conventional T₁-weighted magnetic resonance angiography (MRA) in a Watanabe rabbit at baseline (A), on day 0 (B), on day 1 (C), and on day 6 (D) after the injection of superparamagnetic nanoparticles are shown. Injection of the contrast agent resulted in decreased intravascular signal, due to T₂*-shortening of the blood. Using inversion recovery with ON-resonant water suppression (IRON), blood, fat, and muscle were homogenously suppressed at baseline (E). Monocrystalline iron-oxide nanoparticle (MION) 47 injection contributed to strong intravascular OFF-resonance enhancement on day 0 (F) and day 1 (G), which approached baseline at later times (H and I), allowing for better judgment of the aortic wall.

On conventional magnetic resonance (MR) images, subtle areas of signal hypointensity (negative contrast) were detected in the aorta (solid arrows and overlaid regions of interest in panels B and D) of hyperlipidemic rabbits after MION-47 injection compared with baseline images (A and C). On IRON images, tissue was homogeneously suppressed at baseline (E and G), while positive signal was seen after MION-47 injection (solid arrows in panels F and H). The positive signal corresponds to iron deposition in matched slices on histology (fast nuclear red staining in panels I and K and combined acid phosphatase and Prussian blue staining in panels J and L). The dotted red lines on the MR images correspond to the cross section of the vessel. In both Watanabe and control rabbits, positive signals were present in paraspinal ribs (dotted arrows in panels F, M, and O) and in lymph nodes (arrowheads in panel O). As expected, normal rabbits showed no plaque on histology (N and P). Abbreviations as in Figure 4.

(A) Normalized enhancement ratio (NER) increased in areas of plaque in Watanabe rabbits on day 3 (*p < 0.001 vs. baseline) and further increased on day 6 (p < 0.05 vs. day 3) only in the hyperlipidemic group. (B) The NER of the aortic wall on day 6 correlated with macrophage density in atherosclerotic plaques on matched slices (p < 0.001 by clustered regression analysis, taking into account the repeated measures performed in the same rabbit). (C) A cutoff value of NER = 1.27 provided sensitivity of 91% and specificity of 89% (area under the curve [AUC]: 0.97, standard error [SE]: 0.02, 95% confidence interval: 0.87 to 0.99, p < 0.001) for the detection of macrophage-rich atherosclerotic plaque. The dashed curves represent the 95% confidence bounds of the receiver-operating characteristics curve. IRON = inversion recovery with ON-resonant water suppression.