A simplified murine intimal hyperplasia model founded on a focal carotid stenosis

Abstract

Murine models offer a powerful tool for unraveling the mechanisms of intimal hyperplasia and vascular remodeling, although their technical complexity increases experimental variability and limits widespread application. We describe a simple and clinically relevant mouse model of arterial intimal hyperplasia and remodeling. Focal left carotid artery (LCA) stenosis was created by placing 9-0 nylon suture around the artery using an external 35-gauge mandrel needle (middle or distal location), which was then removed. The effect of adjunctive diet-induced obesity was defined. Flowmetry, wall strain analyses, biomicroscopy, and histology were completed. LCA blood flow sharply decreased by ∼85%, followed by a responsive right carotid artery increase of ∼71%. Circumferential strain decreased by ∼2.1% proximal to the stenosis in both dietary groups. At 28 days, morphologic adaptations included proximal LCA intimal hyperplasia, which was exacerbated by diet-induced obesity. The proximal and distal LCA underwent outward and negative inward remodeling, respectively, in the mid-focal stenosis (remodeling indexes, 1.10 and 0.53). A simple, defined common carotid focal stenosis yields reproducible murine intimal hyperplasia and substantial differentials in arterial wall adaptations. This model offers a tool for investigating mechanisms of hemodynamically driven intimal hyperplasia and arterial wall remodeling.

Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1

Schematic of the mouse mid-focal stenosis and distal-focal stenosis models. A: Left carotid artery. B: A 9-0 nylon suture is tied around the common carotid artery and a 35-gauge blunt needle mandrel; then the needle mandrel is removed. C: Mid-focal (2.5 mm proximal to bifurcation) stenosis model. D: Distal-focal (1 mm proximal to bifurcation) stenosis model.

Figure 2

Microscopic analyses in the mid-focal stenosis model. Representative vascular endothelial cell, vascular smooth muscle cell (VSMC), macrophage, and collagen-stained images of mid-focal stenosis in carotid arteries in C57BL6 mice (standard chow) at day 28. The sections were taken at the 600- to 1200-μm (proximal to its focal stenosis) locations. Red line represents the internal elastic lamina. LCCA, left common carotid artery; RCCA, right common carotid artery. Scale bars = 50 μm.

Figure 3

Microscopic analyses in distal-focal stenosis model. Representative vascular endothelial cell (sections taken at 1200 μm proximal to the focal stenosis), vascular smooth muscle cell (1400 μm), macrophage (600 μm), and collagen (1800 μm) stained images of distal focal stenosis in carotid arteries in C57BL6 mice fed standard chow or DIO, at day 28. Red line depicts internal elastic lamina. Scale bars = 50 μm.

Figure 4

Mid-focal stenosis morphometry. A: Ideal luminal, intimal, and medial areas relative to the mid-focal stenosis (x axis). Straight line represents an average of 5 locations in the sham-operated animals (n = 4). Sham-operated animals had zero intima area, lumen area (0.08 ± 0.01 mm2), and media area (0.02 ± 0.002 mm2). Intimal hyperplasia was observed primarily in LCA proximal to the focal stenosis. B: Area within external elastic lamina showing negative remodeling in the carotid artery distal to the mid-focal stenosis. Sham-operated animal area within external elastic lamina was 0.10 ± 0.01 mm2.

Figure 5

Representative Masson’s trichrome-stained images of mid- and distal-focal stenosis models. A: Mid-focal stenosis of a carotid artery at intervals proximal (left) and distal (right) to mid-focal stenosis in animals fed standard chow. B: Distal-focal stenosis of a carotid artery proximal to its focal stenosis in animals fed standard chow. C: Distal-focal stenosis of a carotid artery proximal to its focal stenosis in mice with diet-induced obesity. The specimens were analyzed at 4 weeks after surgery. Scale bars = 50 μm.

Figure 6

Distal focal stenosis morphometry with dietary perturbations. Morphometric analysis at 28 days after distal-common carotid focal stenosis. A: Intimal area relative to distance proximal to the focal stenosis. Straight line represents an average of 10 locations from the sham-operated animals (n = 6). Sham-operated animals had absolute values of intimal area of essentially zero. The DIO chow accentuated the intimal hyperplasic response (P < 0.001). B: Medial thickening was observed in both dietary groups compared with sham-operated control mice [DIO (0.03 ± 0.003) versus standard chow (0.03 ± 0.001) mm2]. C: Subtle differences (P < 0.01) in the area within the external elastic lamina (reflective of total wall remodeling) were observed between the 2 dietary groups. In sham-operated animals, the area within the external elastic lamina was 0.11 ± 0.01 mm2 in animals with DIO, and 0.10 ± 0.004 mm2 in the group fed standard chow, respectively.

Figure 7

Blood flow, wall shear stress, and circumferential strain analyses in distal-focal stenosis model. A: Peak and mean blood flow before and after focal stenosis. B: Peak and mean arterial wall shear stress at baseline and after creation of the distal focal stenosis. C: Circumferential strain in the proximal common carotid artery decreased immediately after creation of the distal focal stenosis in animals fed standard chow or a high-fat diet. *P < 0.05 between RCCA-Baseline and RCCA-Post, **P < 0.001 between LCCA-Baseline and LCCA-Post.

Figure 8

Comparison of ultrasound measurements and morphometry. A and B: Carotid artery dimensions (B-mode and M-mode). Data were recorded 1 mm proximal to the mid-focal stenosis in systolic and diastolic phases. C: A similar pattern of arterial lumen size was observed between the 2 methods.

Figure 9

Duplex ultrasonography. A and B: Representative longitudinal color Doppler images of proximal and distal LCA after mid-focal stenosis creation at days 4 and 27. Duplex ultrasonography confirmed the luminal narrowing with flow acceleration at the stenosis and otherwise laminar flow. Arrows indicate the focal stenosis site. C and D: Pulsed-wave mode velocity at proximal and distal LCA after mid-focal stenosis; data were obtained 1 mm proximal to the focal stenosis at day 27. E and F: Peak and mean blood flow proximal and distal relative to the mid-LCA focal stenosis and in the contralateral right carotid artery at days 4 and 27, compared with sham-operated animals.