A preclinical ultrasound method for the assessment of vascular disease progression in murine models

Abstract

Introduction: The efficacy of preclinical ultrasound at providing a quantitative assessment of mouse models of vascular disease is relatively unknown. In this study, preclinical ultrasound was used in combination with a semi-automatic image processing method to track arterial distension alterations in mouse models of abdominal aortic aneurysm and atherosclerosis.

Methods: Longitudinal B-mode ultrasound images of the abdominal aorta were acquired using a preclinical ultrasound scanner. Arterial distension was assessed using a semi-automatic image processing algorithm to track vessel wall motion over the cardiac cycle. A standard, manual analysis method was applied for comparison.

Results: Mean arterial distension was significantly lower in abdominal aortic aneurysm mice between day 0 and day 7 post-onset of disease (p < 0.01) and between day 0 and day 14 (p < 0.001), while no difference was observed in sham control mice. Manual analysis detected a significant decrease (p < 0.05) between day 0 and day 14 only. Atherosclerotic mice showed alterations in arterial distension relating to genetic modification and diet. Arterial distension was significantly lower (p < 0.05) in Ldlr^-/- (++/--) mice fed high-fat western diet when compared with both wild type (++/++) mice and Ldlr^-/- (++/--) mice fed chow diet. The manual method did not detect a significant difference between these groups.

Conclusions: Arterial distension can be used as an early marker for the detection of arterial disease in murine models. The semi-automatic analysis method provided increased sensitivity to differences between experimental groups when compared to the manual analysis method.

Keywords: Vessel wall-motion; animal models; diagnostic imaging; high-frequency ultrasound; in vivo.

Figure 1.

Analysis of percent distension of the abdominal aorta in sham control mice (study 1). Results were obtained through manual diameter measurements (a) and semi-automatic measurement (b). Data represent Mean ± SEM. Unpaired t-test showed no significant difference for both methods (p > 0.05).

Figure 2.

Analysis of percent distension of the abdominal aorta in Angiotensin II mice (study 1). Results were obtained through manual diameter measurements (a) and semi-automatic method (b). Data represent Mean ± SEM. One way ANOVA with Tukey post-hoc test revealed significantly reduced distension at day 14 compared to baseline in (a) and at both days 7 and day 14 compared to baseline in (b).

Figure 3.

Representative images of abdominal aorta cross-sections after histological staining with haematoxylin eosin (H&E), Elastic van Gieson (EVG) and immunochemical staining for anti-alpha smooth muscle actin (α-SMA) in sham and angiotensin II (Ang II)-positive mice. H&E staining revealed thickening of the adventitial layer, EVG staining showed damage to the medial layer with fragmentation of the elastic lamina, α-SMA samples revealed loss of tight alignment of the elastic lamina and disorganisation of smooth muscle cell layers.

Figure 4.

Abdominal aorta percent distension for mice in atherosclerotic study (study 2) obtained through manual diameter measurements (a) and through semi-automatic method (b). Animals were grouped and compared regarding to their genotype and diet. Data represent Mean ± SEM. Significance was determined using one way ANOVA with Tukey post-hoc showed. *p < 0.05. Genotypes: wild type (++/++), group Ldlr^−/− (++/−−) and group Ldlr^−/−; GeneX^−/− (−−/−−).

Figure 5.

(a) Representative en face Oil Red O (ORO) staining showing atherosclerotic plaque distribution in the aortic root of mice fed chow diet (top) and western diet (bottom) in study 2. Quantitative analysis of the lesion area in mice aortic roots revealed a significant difference between chow (n = 13) and western (n = 7) diet (b). The positively stained arterial lesions area was measured and expressed as a percent of plaque coverage, relative to the total surface of aorta. Data shown as mean ± SD. Significance was tested using an unpaired Student’s t-test (p < 0.05).