This study demonstrates that the non-invasive assessment of SPIO contrast agent uptake in vivo using high field MRI can be used as a marker of macrophage activity during the early stages of ANG II-induced AAA in apoE−/−mice. There was a strong correlation between MR T2WI signal susceptibility by SPIO and macrophage infiltration within the aneurysm wall. However, endogenous iron in the thrombi and hemosiderin in the vessel wall from hemorrhages in arterial dissections can also be visualized using high resolution MR.
MRI can provide anatomical, structural and functional characterization of the arterial wall 
. In this study, the 7.0T experimental MRI scanner offered adequate anatomic resolution for vascular morphology and signal characteristics, along with the location and size of the aneurysm. Adventitial remodeling and media rupture of the aneurysm were observed at 14 days following continuous ANG II infusion. Although in vivo
MRI imaging correlated well with the pathologic features of the disease, it did not have enough resolution to visualize the location of the medial wall rupture, partially due to the 1 mm slice thickness 
Interestingly, the presence of acute thrombus formation was observed in one mouse in the 1000 ng group, as shown in , resulting in occlusion of the abdominal aorta. Mural thrombus formation appeared hyperintense on T1-weighted images on MRI at day 15. Histological examination found fresh thrombi with platelets, inflammatory cells, and fibrin mesh. Therefore, high-resolution MRI can not only provide anatomical information, such as the location, caliber and length, of an aneurysm, but also depict pathophysiological changes associated with the aneurysm, such as inflammatory reactions and mural thrombosis.
Research has demonstrated that assessment of USPIO uptake with MRI can be used to detect focal hotspots of inflammation in asymptomatic AAA 
. Noninvasive imaging of macrophages may, therefore, yield valuable information about the pathogenesis of arterial aneurysm disease and predict the risk of aneurysm expansion and rupture 
. Previous studies have evaluated the use of intravenous USPIO and SPIO as MRI contrast agents for imaging macrophage activity in animal models of atherosclerosis and in human AAA 
AAA almost always relates to atherosclerosis, which is essentially a chronic inflammatory process, but the pathophysiology of AAA is quite different from that of non-aneurysmal atherosclerotic plaques. In AAA, inflammation is mostly confined to the media and adventitia of the aorta, whereas in atherosclerotic plaques, the inflammatory reaction is seen primarily in the intima 
. Atherosclerosis and AAAs display distinct histological features during initiation and progression, but have the common feature of macrophage infiltration throughout the progression of both disease processes 
is the smallest of SPIO commercially available in the authors' country. SPIO particles are quickly phagocytosed by macrophages in the body, which can be visualized using MRI as areas of signal loss caused by MRI susceptibility on T2WI. The dose of SPIO administered in the present study was determined based upon the previous report which was almost 10-fold greater than that used in clinical medicine 
. This was well tolerated in mice in our experimental conditions. SPIO can escape nonspecific uptake by the reticular-endothelial system (RES), which is engulfed by macrophages within vessels. Our results showed that the iron was mostly present within the shoulder and outer layer of the aneurysm vessel wall, and hypointense images on T2WI are associated with increased SPIO-laden macrophage. Most of the iron-laden macrophages were present in the region adjacent to the site of the external elastic lamina, although these areas were free of monocyte infiltration before the administration of ANG II and SPIO. Our results showed macrophage accumulation at the medial disruption areas, which was consistent with Daugherty's group who showed that there is macrophage accumulation in the media in response to AngII administration 
. However, it is unclear as to how the SPIO particles ultimately reach the aortic wall. It is assumed that SPIO migrates across the interendothelial junction of the vasa vasorum into the interstitium and is then engulfed by macrophages at sites of inflammation 
. Nevertheless, it remains unclear whether SPIO is phagocytosed while macrophages/monocytes are in the circulation. This study confirmed the location of iron-labeled cells in the aneurysm vessel wall through dual staining with Prussian blue and MAC-3 immunohistochemistry. Since Mac-3 is a marker of lysosomale activity associated with phagocytosis activity, we also used a tissue macrophage marker CD68 to confirm Prussian blue and macrophage double staining. Besides, controversy appears in the literature regarding the specific type of cell within the plaques that takes up the iron particles. Pande et al. showed that macrophages were the dominant cells for USPIO uptake; but some endothelial cells and smooth muscle cells were occasionally also noted to contain iron oxide particles 
. Trivedi et al. found Prussian blue staining appeared to colocalize to macrophages in the shoulder regions of the plaque and no colocalization with smooth muscle and/or endothelial cell 
. In the present study, double staining illustrates that SPIO colocalized with macrophages, but not with smooth muscles cells. These results demonstrate that iron-positive cells can be identified as macrophages.
It was observed that the SPIO-laden macrophages, which are abundant in the adventitial of the ANG II infusion groups, were minimal in atherosclerotic plaques in the saline-infusion group. Therefore, SPIO appears not suitable for the detection of plaques presumably because of its relatively large size which results in reduced transendothelial passage, tissue penetration, and easy uptake by the RES 
In a recently published article, SPIO-enhanced MRI visualizes leukocyte phagocytic activities in AAA patients 
. This important clinical study shows that MRI imaging allows in vivo
demonstration of SPIO uptake mainly localized at the luminal interfaces of the thrombi in high-risk AAAs. The uptake correlated with the abundance of leukocytes in the vessel luminal surface. While our study supports other published data by demonstrating that SPIO particles can be successfully used to monitor phagocytic activities in an AAA model of apoE−/−
mice, we also showed few Prussian-blue positive cells in the inner adventitia in three mice of the group with high dose ANG II infusion without SPIO administration. This is important as it can be hypothesized that iron in these cells may be free iron released from the lysed red blood cell during intramural hemorrhage. It has been demonstrated that ANG II-induced mice have diffuse accumulation of macrophages in the media and adventitia as well as in foci of hemorrhages and thrombi 
. Therefore, aneurysms with an acute intramural or mural thrombotic hemorrhage may cause the complicated MRI signals. It is known that fresh intraplaque hemorrhages produce isointensity or hypointensity on T2W/PD images 
. Based on the findings in this study, reconsideration may need to be given to the interpretation of findings published by Nchimi A et al 
, as the iron observed in the Prussian-blue-positive leukocytes may partially originate from RBC-degradation products. It is important to perform a comparative analysis both pre- and post-MR images following intravenous injection of SPIO 
. Furthermore, our study confirmed imaging of iron phagocytic activity could be enhanced by injections of SPIO 
To determine the origin of iron in Prussian-blue-positive macrophages, we further analyzed the SPIO-laden macrophages dependent on two points. We found that the number of Prussian blue-positive cells was much less in the mice without SPIO injection compared to the ones with SPIO injections. Therefore, imaging of iron phagocytic activity could be enhanced using intravenous injection of SPIO. Furthermore, the locations of hemosiderin-laden macrophages in non-SPIO injection group are associated with hemorrhages in the inner adventitia (). These results suggest the presence of endogenous iron from intramural hemorrhages.
This study focused on early inflammatory events in an ANG II-induced AAA in a mouse model. Following 14 days of ANG II infusion, activated macrophages released cytokines and proteolytic enzymes. MMP-9 has also been found in the aneurysm vessel wall which primarily colocalized with infiltrated macrophages. Since AAA progression and eventual aortic rupture depends on the activity of macrophage-derived MMP-9 
, developing an in vivo
imaging approach for MMPs may provide an alternative for visualization of inflammation associated with AAA. This method offers a method to indirectly assess plaque macrophage content and macrophage activity.
In summary, this study demonstrates that imaging of macrophage activity in the vessel wall with SPIO provides a valuable tool for studying aneurysm biology. But signal loss on T2WI in the aneurysm vessel wall is also partially influenced by endogenous hemosiderin iron from intramural hemorrhages and thrombi. Noninvasinve visualization of SPIO-laden macrophages within an aneurysm may provide physiological information other than size in assessing the risk of acute AAA rupture.