Ovariectomy increases the incidence and diameter of abdominal aortic aneurysm in a hypoperfusion-induced abdominal aortic aneurysm animal model

Figures

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  Figure 1. Experimental schema. (A) Schema of the experiment 1 protocol. (B) Schema of the experiment 2 protocol. (C) Schema of the experiment 3 protocol.

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  Figure 2. Effects of OVX on abdominal aortic aneurysm (AAA) incident, rupture, and aortic diameter. The effect of OVX on AAA incidence rate (A) and AAA rupture rate (B). Data are presented as number of rats. P values of the Fisher’s exact test. AAA formation was defined as dilation ratio > 2.0. The dilation rate = maximal aneurysm diameter (sac) / non-dilated vascular diameter (neck). (C) Representative images of the abdominal aorta from AAA induction in the control ligation and the OVX ligation groups (scale bar = 5 mm). (D) Quantitative analysis of aortic diameter. Data are represented as mean ± S.E.M. (A) Control ligation group (n = 20), OVX ligation group (n = 14). (B) Control ligation group (n = 13), OVX ligation group (n = 9). (D) Control ligation group (n = 13), OVX ligation group (n = 14). Values with different letters are significantly different (P < 0.05).

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  Figure 3. Elastica van Gieson (EVG) and Picrosirius Red (PSR) staining of the AAA wall in experiment 1 (A–D) Representative images of EVG staining (scale bar = 100 µm). (E) Elastin degradation scores in the control ligation and OVX ligation groups. (F–K) Representative images of PSR staining (scale bar = 50 µm). (L) Quantification of the collagen-positive area of the vascular wall in the control ligation and OVX ligation groups. Data are represented as mean ± S.E.M. (E,L) Control ligation group (n = 13), OVX ligation group (n = 9). Values with different letters are significantly different (P < 0.05). The arrows show the direction of intima and adventitia. In the case of enlarged figure, all layers of arterial wall were not shown.

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  Figure 4. Immunohistochemical staining for matrix metalloproteinases (MMPs) and macrophage/monocyte of AAA wall in experiment 1. Abdominal aortic aneurysm (AAA) sac areas from the two experimental groups were divided into those without adipocytes (−) (B,E,H,K,N,Q) and those with adipocytes ( + ) (C,F,I,L,O,R). (A–F) Representative images of immunostaining for MMP-2 (scale bar = 30 µm). (G–L) Representative images of immunostaining for MMP-9 (scale bar = 30 µm). (M–R) Representative images of immunostaining for macrophage/monocyte (scale bar = 30 µm). (S) Quantification of MMP-2 positive areas of the vascular wall. (T) Quantification of MMP-9 positive areas of the vascular wall. (U) Quantification of macrophage/monocyte positive areas of the vascular wall. (S,T) Control ligation group (n = 12), OVX ligation group (n = 8). (T) Control ligation group (n = 11), OVX ligation group (n = 8). Values with different letters are significantly different (P < 0.05). The arrows show the direction of intima and adventitia. In the case of enlarged figure, all layers of arterial wall were not shown.

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  Figure 5. Elastica van Gieson (EVG), Picrosirius Red (PSR) and denatured collagen staining of vascular wall in experiment 2 (A,B,D,E) Representative images of EVG staining (scale bar = 50 µm). (G,H,J,K) Representative images of PSR staining (scale bar = 100 µm). (M,N,P,Q) Representative images of denatured collagen staining (scale bar = 100 µm). Elastin degradation scores in the control and OVX groups on 3 weeks after OVX (C) and on 7 weeks after OVX (F). Quantification of the collagen-positive area of the vascular wall in the control and OVX groups on 3 weeks after OVX (I) and on 7 weeks after OVX (L). Quantification of the denatured collagen positive area of the vascular wall in the control and OVX groups on 3 weeks after OVX (O) and on 7 weeks after OVX (R). (C,F,I) Control group (n = 8), OVX group (n = 8). (L,O) Control group (n = 7), OVX group (n = 9). (R) Control group (n = 7), OVX group (n = 8). *P < 0.05, ***P < 0.001. The arrows show the direction of intima and adventitia.

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  Figure 6. Immunohistochemical staining for matrix metalloproteinases (MMPs) and macrophage/monocyte of vascular wall in experiment 2. (A,B,D,E) Representative images of immunostaining for MMP-2 (scale bar = 30 µm). (G,H,J,K) Representative images of immunostaining for MMP-9 (scale bar = 30 µm). (M,N,P,Q) Representative images of immunostaining for macrophage/monocyte (scale bar = 30 µm). Quantification of MMP-2 positive areas of the vascular wall on 3 weeks after OVX (C) and on 7 weeks after OVX (F). Quantification of MMP-9 positive areas of the vascular wall on 3 weeks after OVX (I) and on 7 weeks after OVX (L). Quantification of macrophage/monocyte positive areas of the vascular wall on 3 weeks after OVX (O) and on 7 weeks after OVX (R). (E,K) Control group (n = 8), OVX group (n = 8). (F) Control group (n = 6), OVX group (n = 8). (L,Q) Control group (n = 7), OVX group (n = 9). (R) Control group (n = 7), OVX group (n = 8). *P < 0.05, ***P < 0,001. The arrows show the direction of intima and adventitia. In the case of enlarged figure, all layers of arterial wall were not shown.

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  Figure 7. Immunohistochemical staining for matrix metalloproteinases (MMPs) and macrophage/monocyte of vascular wall in experiment 3. (A,B) Representative images of immunostaining for MMP-2 (scale bar = 30 µm). (C,D) Representative images of immunostaining for MMP-9 (scale bar = 30 µm). (E,F) Representative images of immunostaining for macrophage/monocyte (scale bar = 30 µm). (G) Quantification of MMP-2 positive areas of the vascular wall. (H) Quantification of MMP-9 positive areas of the vascular wall. (I) Quantification of macrophage/monocyte positive areas of the vascular wall. (G,H) Control ligation group (n = 8), OVX ligation group (n = 8). (I) Control ligation group (n = 8), OVX ligation group (n = 9). The arrows show the direction of intima and adventitia. In the case of enlarged figure, all layers of arterial wall were not shown.