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Volumetric assessment of arteriosclerotic plaque burden in ApoE-KO mice using high resolution MR microscopy
Courtney Whalen1, Alexandra Caamano2, Floyd Mattie1, Lukas Neuberger3, Neil Kuan-Hsun Huang1, Catharine Ross1, Rita Castro1,4, and Thomas Neuberger5,6

1Nutritional Sciences, The Pennsylvania State University, University Park, PA, United States, 2The Pennsylvania State University, University Park, PA, United States, 3State College Are High School, State College, PA, United States, 4University of Lisbon, Lisbon, Portugal, 5Huck Institute of the Life Science, University Park, PA, United States, 6Biomedical Engineering, The Pennsylvania State University, University Park, PA, United States

Synopsis

There is a need to address current knowledge gaps in atherosclerosis' etiology to develop successful strategies to reduce mortality due to this disease. In this study, we investigated the feasibility of using 14T MR microscopy to measure the extent of atherosclerosis in apoE-KOmice on diet studies. Results from the ex-vivo 3D MR microimaging of the aortic atherosclerotic plaques with a resolution of 10x10x15µm3 are presented. A contrast agent was used to increase the sensitivity of the 14-tesla system and provide the resolution necessary to assess in detail the wall vessel changes associated with the atherosclerotic process.

INTRODUCTION

Cardiovascular disease (CVD)causes one in three (approximately 800,000) deaths reported each year in the United States with costs estimated in 2017, to more than $316 billion1. The main cause of CVD is atherosclerosis, a chronic condition in which arteries harden through the build-up, in the vessel wall, of plaques that are abundant in lipids. ApoE knockout (KO) mice are an established model of atherosclerosis that accumulate plaques throughout the aorta, similarly to humans2. In these mice, the plaque burden is traditionally measured by serial sectioning of the aorta and subsequent histopathologic analyses to score and measure atherosclerotic lesions3. Alternatively, en-face preparations, using lipid staining of the luminal side of the entire aorta, are also used, providing a wider spatial view of the plaque. Nevertheless, these traditional methods are labor-intensive, time-consuming and rely on subjective quantification measures. Moreover, and most importantly, the assessment of plaque burden is confined to 2D analyses 3 thus lacking information on plaque thickness. In this study, we report the usefulness and feasibility of 14T MRI to the 3D volumetric assessment of the atherosclerotic plaque burden in aortas from ApoE KO mice.

METHODS

Aortas were obtained from 23-week-old apoE-KO mice or controls after 16 weeks under different diets. Following perfusion with PBS and 10% neutral buffered formalin (10% NBF), aortas were fixed overnight in 10% NBF. Surrounding tissue was removed, the aortas were washed in PBS, and transferred to a 4mm inner diameter tube containing PBS, 0.25% Magnevist, and 0.25% sodium azide. The measured T1 and T2 times for the PBS solution were 153ms and 121ms respectively. All MRI experiments were conducted on an Agilent 14 tesla micro imaging system using a home-built saddle coil (inner diameter 7mm). Standard three-dimensional gradient echo imaging (imaging time ~ 50h) yielded a resolution of 10x10x15 microns. All data was reconstructed using Matlab (The Mathworks, Inc., Natick, MA). By zero filling the data by a factor of two, the final pixel resolution was 5x5x7.5 microns. Data segmentation was performed using Avizo 9.5 (Thermo Fisher Scientific, USA).

For cryosectioning aortas were pinned into an aligned position and embedded in Optimal Cutting Temperature (OCT) compound. After slicing (12µm thickness) sections were stained for both hematoxylin and Oil Red-O, or probed for histone targets using antibodies.

RESULTS

Histologic images of aortic sections from an apoE-KO mouse and the corresponding 14T-MRI slices are shown in Figure 1. Similarities in endothelial cell layer and elastin rings in the wall texture between histology and the MRI image are indicated by the red arrows. Ex-vivo visualization of the arteriosclerotic plaque in mice aortas was obtained either by traditional histological analyses (Figure 2) or by 14T-MRI microscopy (Figures 3 and 4). The three-dimensional MRI data set allowed for a volume quantification of the observable plaques (Figure 4). Lastly, a representative image of immune histological analysis of a previously scanned aorta is shown (Figure 5) proving that Magnevist does not interfere with the staining.

DISCUSSION

As shown in figure 1, our MRI setup provided enough resolution to assess the aortic wall vessel structure with similar details as the classical histological analysis.

Figure 2 illustrates the results obtained with traditional histological analyses in which Oil Red-O was used to stain lipids to assess plaque burden. Figure 2A and 2B display single cross-sections of plaques. Sampled serially these images can be used to estimate atherosclerotic burden, but will not allow to calculate an accurate volume of the observed plaques. Figure 2B depicts a standard en face preparation of an intact aorta stained with Oil Red-O to allow assessing the plaque burden of the entire aorta. Again, a volume measurement of the plaques is not feasible. Both techniques are time-consuming, laborious, and destructive, precluding the use of the tissue for further studies.

As demonstrated in figures 3 and 4, 14T-MR microscopy allowed imaging of atherosclerotic plaques with sufficient resolution to render 3D models of the entire vessel. The volumes of the different plaques in the whole aorta were calculated (figure 4C) allowing the quantification of the atherosclerotic burden. As this technique is non-destructive, the imaged mouse aortas could be used for other downstream applications, including immune histological analysis (figure 5), thus maximizing the utility of each sample.

Besides the presented very high-resolution MR micro imaging with the extraordinary scan time, datasets with a lower resolution were also acquired (data not shown). These datasets had a very high signal to noise ratio (resolution of 15x15x25µm3) but reduced the imaging time to an overnight scan. A plaque quantification was still possible, some details in the aorta wall were lost.

CONCLUSION

We have demonstrated the usefulness and feasibility of 14T MRI in the 3D volumetric assessment of the atherosclerotic plaque burden in aortas from apoE-KO mice.

Acknowledgements

This research was supported, in part, by the Huck Institutes of the Life Sciences.

References

1. Benjamin EJ et al. Heart Disease and Stroke Statistics-2017 Update: A Report from the American Heart Association. Circulation. 2017;136(10):e196.

2. Getz GS, Reardon CA. ApoE knockout and knockin mice: the history of their contribution to the understanding of atherogenesis. J Lipid Res. 2016;57(5):758-66.

3. Mohanta S, Yin C, Weber C, Hu D, Habenicht AJ. Aorta Atherosclerosis Lesion Analysis in Hyperlipidemic Mice Bio Protoc. 2016;6(11):e1833.

Figures

Figure 1. Comparison of histology and MRI slices of the same area of the same descending aorta of an apoE-KO mouse. (A) Histological and MRI images of branch point between the descending aorta and an intercostal artery. Arrows point to the observed similarities in endothelial cell layer texture between histology and the MRI image. (B) Histological and MRI images of a further inferior branch point between the descending aorta and an intercostal artery. Arrow highlight the similar observation of elastin rings in the wall of the descending aorta.

Figure 2. Histology demonstrates the accumulation of plaque in an apoE-KO mouse. Oil Red and Hematoxylin stained histological slices of (A) plaques accumulating on the descending aorta; (B) plaques accumulating at the branch point of an intercostal artery from the descending aorta; (C) a whole dissected aorta from an apoE-KO mouse (left) and a wild-type control mouse (right).

Figure 3. 14T-MRI imaging and visualization of plaque accumulation in the aorta of an apoE-KO mouse. (A) Image of the plaque in the aortic arch. (B) Image of the ascending aorta and the branch to the brachiocephalic artery. (C) Image of plaque accumulation along the wall of the descending aorta

Figure 4. Three-dimensional aorta reconstruction allows for calculation of plaque volumes. (A) Image of the three-dimensional reconstruction from the segmentation of the aortic arch. With different plaques highlighted in different colors. (B) Image of the three-dimensional reconstruction from the segmentation of the aorta. With different plaques highlighted in different colors. (C) Table of plaque volumes.

Figure 5. Subsequent use of an imaged aorta to immunohistochemistry studies. After MRI imaging the aorta can be embedded in Optimal Cutting Temperature (OCT) compound, sliced, and used for immunohistochemistry analysis. This tissue was incubated in a 1:500 dilution of an antibody targeting a specific methylation mark (trimethylation of histone 3 lysine 27).

Proc. Intl. Soc. Mag. Reson. Med. 27 (2019)
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