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. 2021 Dec;11(6):1241-1252.
doi: 10.21037/cdt-21-291.

A novel animal model for vulnerable atherosclerotic plaque: dehydrated ethanol lavage in the carotid artery of rabbits fed a Western diet

Ruochi Zhao #  1 , Hongyan Liu #  2 , Shangshi Zhang  3 , Qi Lu  4 , Xiaohong Fei  1 , Honglin Zhou  1 , Junsong Liu  1 , Honghua Ye  1 , Xiaomin Chen  1 , Hanbin Cui  1
Affiliations

A novel animal model for vulnerable atherosclerotic plaque: dehydrated ethanol lavage in the carotid artery of rabbits fed a Western diet

Ruochi Zhao et al. Cardiovasc Diagn Ther. 2021 Dec.

Abstract

Background: The study of unstable atherosclerotic plaques is limited by the absence of ideal animal models to reproduce the plaque instability observed in humans. In this study, we attempted to develop a novel animal model for vulnerable atherosclerotic plaques using dehydrated ethanol lavage in rabbits fed a Western diet (WD).

Methods: A total of 30 New Zealand White (NZW) rabbits were randomized to 5 groups, including a control group with or without WD, a balloon injury with WD group, and an ethanol injury with or without WD group. Operations were conducted using the right common carotid artery as the target vessel. All animals were followed up for 3 months unless a vascular event occurred. Blood samples and carotid artery specimens were ultimately collected for analysis of atherogenesis.

Results: Compared to rabbits in which lesions were induced by balloon injury, those subjected to an ethanol lavage with high cholesterol diet showed progressive atherosclerotic lesions in all carotid artery segments, which were characterized by greater plaque burden, smaller minimum lumen area (MLA), and increased vulnerability as indicated by abundant macrophages, scattered smooth muscle cell (SMC) composition, higher matrix metalloproteinase-9 (MMP-9) expression in plaques, thinner fibrous cap thickness, and higher possibility of stroke event (50% vs. 0%). Meanwhile, the serum interleukin-1β (IL-1β) and monocyte chemoattractant protein-1 (MCP-1) levels in the ethanol injury group with a high-cholesterol diet were significantly higher than those in the balloon injury group after 3 months (all P<0.01).

Conclusions: We successfully established a novel animal model for vulnerable atherosclerosis by ethanol exposure of the carotid segment that has a higher predictive value for the probability of ischemic events than the balloon injury model. Therefore, it may represent a promising animal model for investigating new therapeutic approaches, novel imaging modalities, and underlying mechanisms for vulnerable atherosclerotic plaque.

Keywords: Animal model; endothelial cell (EC) injury; inflammatory marker; vulnerable atherosclerotic plaque.

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Conflict of interest statement

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://dx.doi.org/10.21037/cdt-21-291). HC reports that the study was supported by grant of Zhejiang Provincial Natural Science Foundation, China (grant No. LY12H02001) and Zhejiang Provincial Key Research Project (grant No. 2021C03096). HY reports that the study was partly supported by Ningbo Social Development Research Project (Grant No. 2011C51003 to HY). The other authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1
CCK8 assay of human coronary artery ECs. Left and right panels represent OD value and survival rate of EC, respectively, after treatment of HCAEC with dehydrated ethanol for 10 seconds. Cell proliferation was represented as cell quantity. Data are presented as mean±SD (n=6). EC, endothelial cell; OD, optical density; HCAEC, human coronary artery EC.
Figure 2
Figure 2
Serum lipid profile and inflammatory markers. (A) Serum lipid level measurements at three months, including TC, HDL-C, LDL-C and TG. Normal diet groups in figure contain group A and E, while western diet groups contain group B, C and D. Panels B, C, and D represent serum hs-CRP (B), IL-1β (C) and MCP-1 (D) measurements at end of observation, respectively. *, P<0.05; **, P<0.01; ***, P<0.001 vs. normal control (group A) or counterpart group. TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; TG, triglycerides.
Figure 3
Figure 3
Morphological characteristics of atherosclerotic plaque stained with hematoxylin and eosin. Upper panels show a low-power view of the atherosclerotic plaque development in the carotid artery in rabbits with normal chaw diet (A), on Western-type diet (B), underwent balloon injury with Western-type diet (C), underwent dehydrated ethanol exposure with Western diet (D), and dehydrated ethanol exposure with normal chaw diet (E), respectively. ×40. (F-J) show a high-power view of the region outlined by the box in A, B, C, D and E, respectively (×200). Scale bar = 200 µm (A-E) and 50 µm (F-J).
Figure 4
Figure 4
Cellular composition in the atherosclerotic plaque stained with immunohistochemical method. (A-C) show the presence of endothelial cells labeled by antibody against vWF in carotid arteries of rabbits fed a regular chow diet (group A), balloon injury followed by a Western diet (group C), and dehydrated ethanol exposure followed by a Western diet (group D), respectively (×40). (E-G) show the presence of smooth muscle cells labeled by antibody against α-SMC in carotid arteries of rabbits in groups A, C, and D, respectively (×40). (I-K) show the presence of macrophages labeled by antibody against CD68 in carotid arteries of rabbits in groups A, C, and D, respectively (×40). (M-O) show the content of MMP-9 labeled by antibody against MMP-9 in carotid arteries of rabbits in groups A, C, and D, respectively (×40). (D,H,L,P) show the results of statistical analysis (using the software Image-Pro Plus 6.0) for the mean density of staining against vWF, α-SMC, CD68 and MMP-9 in carotid arteries, respectively. The dark red, green and grey columns in (D,H,L,P) represent the tissue staining density in rabbits of groups A, C and D, respectively. Comparisons were based on the data of 3 adjacent replicates stained with most significant atherosclerotic lesion per specimen (n=18/group). Carotid arteries from the rabbits fed a Western diet (group B) and undergone ethanol injury with normal chaw diet (group E) are not presented here, because only fatty streak rather than advanced atherosclerotic lesion was detected, for which immunohistochemical staining was not performed. Scale bar = 200 µm. vWF, von Willebrand factor; SMC, smooth muscle cell.
Figure 5
Figure 5
Fibrous cap thickness in relation to the injury type (A,B) show the fibrous cap thickness (indicated by red arrows) and plaque burden in atherosclerotic plaque induced by balloon injury with western-type diet (group C) and ethanol exposure with western-type diet (group D), respectively. (Movat pentachrome staining, ×40). The latter was characterized by more severe plaque load, thinner fibrous cap and large NC. Panel (C) illustrates the comparison of minimal thickness of fibrous cap in rabbits undergone balloon injury (group C) and ethanol exposure (group D) (n=18 replicates/group). Since the advanced atherosclerotic plaque did not develop in groups A, B and E, their data related to cap thickness was not provided here.
Figure 6
Figure 6
Angiographic and histological features of the atherosclerotic plaque in the rabbit undergone stroke event. (A) shows clinical signs of stroke, displaying head tilt and ipsilateral limb paralysis. (B) shows severe stenosis in the right carotid artery illustrated by CTA examination. (C1-C5) show the atherosclerotic lesions detected by histological staining (H&E stain, ×40) in the corresponding plane of (B) (indicated by arrows). The arrowheads in (C) indicate the intra-plaque neovascularization occurred in lesions induced by ethanol exposure. RC and LC in panel B represent right and left carotid artery, respectively. Left carotid artery did not receive surgical procedure which shows normal manifestation. CTA, computer tomographic angiogram.
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