Quantitative and qualitative estimation of atherosclerotic plaque burden in vivo at 7T MRI using Gadospin F in comparison to en face preparation evaluated in ApoE KO mice

Abstract

Background: The aim of the study was to quantify atherosclerotic plaque burden by volumetric assessment and T1 relaxivity measurement at 7T MRI using Gadospin F (GDF) in comparison to en face based measurements.

Methods and results: 9-weeks old ApoE-/- (n = 5 for each group) and wildtype mice (n = 5) were set on high fat diet (HFD). Progression group received MRI at 9, 13, 17 and 21 weeks after HFD initiation. Regression group was reswitched to chow diet (CD) after 13 weeks HFD and monitored with MRI for 12 weeks. MRI was performed before and two hours after iv injection of GDF (100 μmol/kg) at 7T (Clinscan, Bruker) acquiring a 3D inversion recovery gradient echo sequence and T1 Mapping using Saturation Recovery sequences. Subsequently, aortas were prepared for en face analysis using confocal microscopy. Total plaque volume (TPV) and T1 relaxivity were estimated using ImageJ (V. 1.44p, NIH, USA). 2D and 3D en face analysis showed a strong and exponential increase of plaque burden over time, while plaque burden in regression group was less pronounced. Correspondent in vivo MRI measurements revealed a more linear increase of TPV and T1 relaxivity for regression group. A significant correlation was observed between 2D and 3D en face analysis (r = 0.79; p<0.001) as well as between 2D / 3D en face analysis and MRI (r = 0.79; p<0.001; r = 0.85; p<0.001) and delta R1 (r = 0.79; p<0.001; r = 0.69; p<0.01).

Conclusion: GDF-enhanced in vivo MRI is a powerful non-invasive imaging technique in mice allowing for reliable estimation of atherosclerotic plaque burden, monitoring of disease progression and regression in preclinical studies.

Fig 1. Study design.

For atherosclerotic plaque progression analysis ApoE^-/- mice (n = 20; progression group) were set on high fat diet (HFD) at 9 weeks of age. Mice were than grouped (n = 5 for each time point) and MRI performed after 9, 13, 17 and 21 weeks on HFD. En face preparation was performed immediately after MRI. For atherosclerotic plaque regression analysis ApoE^-/- mice (n = 5, regressiony group) were also set on HFD at 9 weeks of age. First MRI examination was performed 13 weeks after starting the HFD. At this time point diet was switched back to chow diet (CD) and MRI analysis was repeated at the age of 28 and 34 weeks before en face preparation was done.

Fig 2. Progression and regression of atherosclerosis adapted from en face preparation.

En face preparation of the aortic arch showing the plaque progression over time. The regression group, which was reswitched to chow diet (CD) 13 weeks after starting high fat diet (HFD) shows a slower increase of total plaque area (TPA). No atherosclerotic lesion was detectable in wildtype mice. (n.s.—non significant, *p<0.05, **p<0.01).

Fig 3. 3D plaque volume analysis based on en face preparation.

(A) The total plaque volume (TPV) estimated by 3D confocal microscopy analysis. Shown is an animal of the progression (30 weeks of age) (A) and regression group (34 weeks of age) (B) with different plaque burden. Plaque progression over time can be estimated by TPV (C). The regression group, which was reswitched to chow diet 13 weeks after starting HFD shows a smaller TPV. (n.s.—non significant, *p<0.05, **p<0.01).

Fig 4. Progression and regression of atherosclerosis at 7T MRI.

MRI in vivo showed a transversal slice orientation through the ascending aorta. Progression of atherosclerotic lesion (arrow) in ApoE^-/- mice can be visualized over time. Calculated TPV showed a more linear increase in case of mouse group reswitching to chow diet (CD) 13 weeks after starting HFD. Interobserver analysis showed a strong and significant correlation of r = 0.71; p<0.001. No plaque was detectable in wildtype mice. (n.s.—non significant, *p<0.05, **p<0.01).

Fig 5. Correlation between plaque size based on MRI and en face analysis.

Graphs show correlation between total plaque area (TPA) and total plaque volume (TPV) based on en face and MRI analysis. Correlation coefficients and p-values are given for 2D en face versus 3D en face (A), MRI versus 2D en face (B) and MRI versus 3D en face (C). Strongest correlation of r = 0.85; p<0.001 is detectable for the estimated TPV at MRI and 3D en face.

Fig 6. Quantitative MRI analysis using T1 mapping in atherosclerotic plaque.

Signal enhancement due to the GDF uptake into the atherosclerotic lesion in one mouse of the progression group (30 weeks of age) (A) and in comparison one mouse of the regression group (34 weeks of age) (B) with corresponding T1 mapping. In case of progression group T1 relaxation time decreased stronger in comparison to the regression group indicated by the yellow color. The estimated delta R1 revealed a stronger and more exponential acceleration for ApoE^-/- mice, which remained on HFD (C). (n.s.—non significant, *p<0.05, **p<0.01).

Fig 7. Correlation between T1 relaxivity and plaque size.

Graphs show correlation coefficients and p-values for delta R1 versus total plaque area (TPA) (A) and delta R1 versus total plaque volume (TPV) (B) based on en face analysis. Strongest correlation of r = 0.79; p<0.001 is detectable for the estimated delta R1 at MRI and TPA based on 2D en face.