Lack of intracranial atherosclerosis in various atherosclerotic mouse models

Figures

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  Figure 1. Three-dimensional reconstruction of the peripheral and cerebral arterial tree. (A) CT scan showing a slightly ventrolateral view of the skull and vessels. The dashed line indicates where the atherosclerotic plaques stop in most mice. The continuous line indicates the place where the internal carotid artery (ICA) enters the skull. Red indicates parts of the vessels without atherosclerosis. Black indicates the parts of the vessels with extensive atherosclerosis (based on 32 mice). Gray indicates atherosclerosis as observed in only one mouse. (B) Magnetic resonance angiogram showing the sagittal view of the mouse arterial tree from common carotid artery (CCA) to the circle of Willis (CoW) and its main branches. The numbered blue areas indicate the areas in which immunohistochemistry sections were taken.

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  Figure 2. Representative picture of the first transition point of the internal carotid artery (ICA) at the bifurcation (bif) with the pterygopalatine artery (Ptgpal) where the atherosclerotic lesion usually stopped in a 26-week-old ApoE−/−Fbn1C1039G+/− mouse on a WD. An atherosclerotic plaque containing a necrotic core with foamy macrophages is shown in the ICA on the rostral site of the bifurcation. There was no plaque at the cranial site of the bifurcation, the site of the vessel that is heading toward the brain. In the left panel (2.5×), an overview of the bifurcation of the ICA and Ptgpal is shown, while the right panel (10×) shows the first transition point in more detail.

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  Figure 3. Changes in vessel wall characteristics in 10-week-old ApoE−/− mice on normal chow measured in seven artery segments from the common carotid artery (CCA, 1) to the extracranial part of the internal carotid artery (ICA, 3) and the intracranial part of the ICA (7). (A) Hematoxylin and eosin and EvG staining and (B) the quantification of the vessel wall thickness and amount of elastin layers (n = 5–7). (C) Example pictures of changes in SMA and SM-MHC staining. (D) Quantification of the amount of positive SMA and SM-MHC/SMA staining (n = 6–7). Statistical significance was determined by the Friedman test and considered statistically significant if P < 0.05.

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  Figure 4. (A–E) Example pictures of changes in (A) ICAM-1, (B) VCAM-1, (C) ZO-1, (D) Claudin-5 and (E) Caveolin in 10-week-old ApoE−/− mice on normal chow stained in the common carotid artery (CCA, 1), the extracranial part of the internal carotid artery (ICA, 3) and the intracranial part of the ICA (7). (F) Quantification of the amount of positive staining overlapping with the positive CD31 signal (n = 6–7). Statistical significance was determined by the Friedman test and considered statistically significant if P < 0.05.

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  Figure 5. (A, B) Example pictures of changes in (A) HO-1 staining and (B) eNOS staining in 10-week-old ApoE−/− mice on normal chow in the common carotid artery (CCA, 1), the extracranial part of the internal carotid artery (ICA, 3) and the intracranial part of the ICA (7). (C) Quantification of the amount of positive HO-1 and eNOS staining overlapping with the positive CD31 signal (n = 6–7). Statistical significance was determined by the Friedman test and considered statistically significant if P < 0.05.

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  Figure 6. Examples of HO-1 expression in human formalin-fixed paraffin-embedded (FFPE) samples of the common carotid artery (CCA) and basilar artery (BA). (A) HO-1 was labeled with immunofluorescence staining (green), costained with endothelial marker CD31 (red) and counterstained with DAPI (blue) in the CCA and (B) BA (40× magnification).

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  Figure 7. oxLDL protects brain endothelial (hCMEC/D3) cells from oxidative stress. (A) HO-1 expression levels are increased after 6 and 24 h (n = 4). (B) Quantification of HO-1 protein expression as measured by western blot, corrected for actin (n = 3). (C) Western blot analysis of nuclear and cytoplasmic fractions of brain endothelial cells corrected for lamin B and β-actin, respectively, showed an increased nuclear presence of NRF2 (n = 4). Statistical significance was determined by two-tailed Student’s t-test. *P < 0.05, **P < 0.001, ***P < 0.0001.