MRI study of atherosclerotic plaque progression using ultrasmall superparamagnetic iron oxide in Watanabe heritable hyperlipidemic rabbits

Abstract

Objective: The purpose of this study was to evaluate plaque progression by using MRI with ultrasmall superparamagnetic iron oxide (USPIO) and by histopathological studies.

Methods: We divided 12 Watanabe heritable hyperlipidemic (WHHL) rabbits into 4 groups based on their age (3, 9, 14 and 26 months) and injected them intravenously with 0.8 mmol (Fe) kg(-1) of USPIO (size, 32 nm; concentration, 15 mg dl(-1)). On the fifth post-injection day, they were again given an intravenous injection with 40 μmol kg(-1) of the same USPIO, and MR angiography (MRA) was performed. The signal-to-noise ratio (SNR) in regions of interest in the wall of the upper abdominal aorta was calculated on coronal images. Specimens from the same level of the aorta were subjected to iron staining and RAM-11 immunostaining and used for histopathological study. For statistical analysis of the MRA and histopathological findings, we used analysis of variance [Tukey's honest significant difference (HSD) test].

Results: In 9-month-old rabbits, the SNR was significantly lower than in rabbits of the other ages (p < 0.01), and the area of RAM-11 (DAKO Corporation, Glostrup, Denmark) and iron uptake in the aortic wall was significantly larger (RAM-11, p < 0.01; iron, p < 0.05). These areas were the smallest in 3-month-old rabbits.

Conclusion: Histopathologically, the number of macrophages was the greatest in 9-month-old rabbits. Our findings indicate that the SNR on MRI scans reflects the number of macrophages in the aortic wall of WHHL rabbits.

Advances in knowledge: USPIO-enhanced MRI visualized the accumulation of macrophages in early atherosclerotic plaques of WHHL rabbits in the course of natural progression.

Figure 1.

Curved planar reconstruction of ultrasmall superparamagnetic iron oxide-enhanced MR images obtained under a three-dimensional fast spoiled gradient echo protocol. The aortas of four Watanabe heritable hyperlipidemic rabbits aged 3 (a), 9 (b), 14 (c) and 26 months (d) are shown. The aortic wall was relatively smooth in 3-month-old rabbits. Some irregularities are apparent in 14- and 26-month-old rabbits (c, d). The insets show regions of interest placed in the aortic wall.

Figure 2.

Axial MR images of the abdominal aorta in rabbits obtained after ultrasmall superparamagnetic iron oxide injection. The white lines indicate the measurement of lengths of the vessel wall area. The aortas of four Watanabe heritable hyperlipidemic rabbits aged 3 (a), 9 (b), 14 (c), and 26 months (d) are shown. Note that the area of signal loss of the aortic wall in 9-month-old rabbits is relatively diffuse and in 14- and 26-month-old rabbits is relatively spotty.

Figure 3.

Signal-to-noise ratio (SNR) in the aortic wall of rabbits injected with ultrasmall superparamagnetic iron oxides. SNR was significantly lower in 9-month-old rabbits than in rabbits of the other age groups (*p < 0.01).

Figure 4.

Histology of the abdominal aorta of four rabbits aged 3 (a, e), 9 (b, f), 14 (c, g) and 26 months (d, h). Prussian blue (a–d, blue) and RAM-11 (DAKO Corporation, Glostrup, Denmark) staining (e–h, red) at identical sites. The localization of iron coincided with the accumulation of macrophages in rabbits of all ages. Note intimal hyperplasia and moderate ultrasmall superparamagnetic iron oxide (USPIO) uptake in the neointima of the 3-month-old rabbits (a, e). In 9-month-old animals, there is greater USPIO uptake and progressive intimal thickening (b, f). The USPIO uptake and the number of macrophages were lower in 14-month-old rabbits (c, g). RAM-11 staining and the uptake of USPIOs were seen in small areas in 26-month-old rabbits (d, h). Intimal thickening progressed with increasing age. Bars = 100 μm (magnification, ×200).

Figure 5.

The area of iron uptake in the aortic wall of rabbits injected with ultrasmall superparamagnetic iron oxides was significantly larger in 9-month-old rabbits than in the other age groups (*p < 0.05; **p < 0.01).

Figure 6.

The RAM-11-positive areas (DAKO Corporation, Glostrup, Denmark) in the aortic wall of rabbits injected with ultrasmall superparamagnetic iron oxides were significantly larger in 9-month-old rabbits than in the other age groups (*p < 0.01).