Transauricular balloon angioplasty in rabbit thoracic aorta: a novel model of experimental restenosis

Abstract

Background: The aim of this study was to demonstrate a percutaneous transauricular method of balloon angioplasty in high-cholesterol fed rabbits, as an innovative atherosclerosis model.

Methods: Twenty male New Zealand rabbits were randomly divided into two groups of ten animals, as follows: atherogenic diet plus balloon angioplasty (group A) and atherogenic diet alone (group B). Balloon angioplasty was performed in the descending thoracic aorta through percutaneous catheterization of the auricular artery. Eight additional animals fed regular diet were served as long term control. At the end of 9 week period, rabbits were euthanized and thoracic aortas were isolated for histological, immunohistochemical and biochemical analysis.

Results: Atherogenic diet induced severe hypercholesterolemia in both group A and B (2802 ± 188.59 and 4423 ± 493.39 mg/dl respectively) compared to the control animals (55.5 ± 11.82 mg/dl; P < 0.001). Group A atherosclerotic lesions appeared to be more advanced histologically (20% type IV and 80% type V) compared to group B lesions (50% type III and 50% type IV). Group A compared to group B atherosclerotic lesions demonstrated similar percentage of macrophages (79.5 ± 9.56% versus 84 ± 12.2%; P = 0.869), more smooth muscle cells (61 ± 14.10% versus 40.5 ± 17.07; P = 0.027), increased intima/media ratio (1.20 ± 0.50 versus 0.62 ± 0.13; P = 0.015) despite the similar degree of intimal hyperplasia (9768 ± 1826.79 μm² versus 12205 ± 8789.23 μm²; P = 0.796), and further significant lumen deterioration (23722 ± 4508.11 versus 41967 ± 20344.61 μm²; P = 0.05) and total vessel area reduction (42350 ± 5819.70 versus 73190 ± 38902.79 μm²; P = 0.022). Group A and B animals revealed similar nitrated protein percentage (P = NS), but significantly higher protein nitration compared to control group (P < 0.01; P < 0.01, respectively). No deaths or systemic complications were reported.

Conclusion: Transauricular balloon angioplasty constitutes a safe, minimally invasive and highly successful model of induced atherosclerosis in hyperlipidaemic rabbits.

Figure 1

Masson’s trichrome and Van Gieson staining in descending thoracic aortas. a1. Great amount of collagen tissue deposition, and a2. Severe elastic fiber disruption and disorientation in balloon injured atherosclerotic thoracic aortas. b1. Slight collagen tissue increment and b2. Focal fragmentation and disorientation of elastic tissue in non-injured atherosclerotic thoracic aortas. c1. Absence of fibrosis, and c2. normal elastic fiber orientation in control thoracic aortas.

Figure 2

RAM-11 and HHF-35 positive cell distribution in three groups. a. No significant difference in RAM-11 cell percentage was observed between angioplasty induced and spontaneous atherosclerotic lesions: a P = non significant, Bonferroni’s post hoc ANOVA analysis vs Group B; b P < 0.001, Bonferroni’s post hoc ANOVA analysis vs contol; c P < 0.001, Bonferroni’s post hoc ANOVA analysis vs control. b. Significant increase in HHF-35 cells was noticed in angioplasty induced atherosclerotic lesions: d P < 0.05, Tamhane’s post hoc ANOVA analysis vs Group B; e P < 0.001, Tamhane’s post hoc ANOVA analysis vs control; f P < 0.001, Tamhane’s post hoc ANOVA analysis vs control.

Figure 3

Ram-11 and HHF-35 staining in thoracic aortas. a1. Plethora RAM-11 positive foam cells, and a2. significant amounts of positive for a-actin SMCs are observed in angioplasty induced atherosclerotic lesions. b1. Significant foam cell formation, and b2. several a-actin positive SMCs between foam cells in spontaneous atherosclerotic lesions. c1. Lack of foam cell deposition, and c2. Normal presence of HHF-35 positive SMCs in the media of control aortas.

Figure 4

Comparison of neointimal area, intima/media ratio, lumen area and total vessel area between groups. a. Atherogenic diet and balloon angioplasty induced significant intimal hyperplasia in hyperlipidaemic rabbits: a P = non significant, Tamhane’s post hoc ANOVA analysis vs Group B; b P < 0.001, Tamhane’s post hoc ANOVA analysis vs control; c P < 0.05, Tamhane’s post hoc ANOVA analysis vs control. b. A significant increase in intima/media ratio was observed in angioplasty induced atherosclerotic lesions: d P < 0.05, Tamhane’s post hoc ANOVA analysis vs Group A; e P < 0.001, Tamhane’s post hoc ANOVA analysis vs Control; f P < 0.001, Tamhane’s post hoc ANOVA analysis vs Group A. c. Angioplasty resulted in a significant reduction in lumen of injured thoracic aortas: j P = 0.05, Tamhane’s post hoc ANOVA analysis vs Group B; k P < 0.001, Tamhane’s post hoc ANOVA analysis vs control; l P = non significant, Tamhane’s post hoc ANOVA analysis vs control. d. Injured thoracic aortas demonstrated a significant decrease in total vessel area consistent with constrictive remodeling occurrence: g P < 0.05, Bonferroni’s post hoc ANOVA analysis vs Group B; h P = non significant, Bonferroni’s post hoc ANOVA analysis vs control; i P = non significant, Bonferroni’s post hoc ANOVA analysis vs control.

Figure 5

Tyrosine nitration of proteins is not increased by the transauricular balloon angioplasty. a. Western Blot analysis for 3′-nitrotyrosine (upper panel) and β-actin (lower panel) in total protein lysates of descending aortas. b. The protein amounts were quantified by densitometric analysis of the corresponding bands and the ratio of nitrated proteins/β-actin was calculated for each lane. Results are expressed as mean ± S.E.M. of the percentage change of the amounts of 3′-nitrotyrosine compared with control animals. n.s, not statistically significant.

Figure 6

Intraarterial transauricular access in the rabbit thoracic aorta. a. Direct percutaneous catheterization of the central auricular artery with a 22-gauge intravenous catheter, b. Repeated tract dilations of the peripheral transauricular artery with the introduction of a 4-F vascular sheath, c. Insertion of an 6.0-mm/40- mm (Cordis) peripheral dilatation balloon catheter over the guide wire, d-e Roadmap image of the auricular artery and the common carotid artery.

Figure 7

Balloon angioplasty of descending thoracic aorta. a. Roadmap images of the rabbit’s carotid artery and the thoracic aorta, b. Insertion of the guide wire into the descending thoracic aorta, c. Insertion of a 6.0-mm/40-mm peripheral dilatation balloon catheter over the guide wire and placement to the level of descending thoracic aorta d. Inflation of the balloon and injury of the descending aorta below the orifice of subclavian artery.