Effect of long-term antihypertensive treatment on cerebrovascular structure and function in hypertensive rats

Figures

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  Figure 1. Blood pressure and heart rate before and during treatment with antihypertensive drugs, and heart-to-body weight ratio. (A,B) During treatment, both systolic and diastolic blood pressure were significantly lower in SHR treated with amlodipine and atenolol when compared to the untreated hypertensive control group. (C) Heart rate was significantly lower in atenolol treated SHR and was comparable to the heart rate of normotensive control rats. (D) Heart weights were normalized to the body weight. Untreated SHR showed higher heart-to-body weights as compared to all other groups, which was partially normalized by treatment with amlodipine and atenolol. n = 12 for WKY Ctrl and SHR Amlo, and n = 11 for SHR Ctrl and SHR Aten. ***p ≤ 0.001, *p ≤ 0.05 (A–C mixed-effects model with Bonferroni correction, D one-way ANOVA with Bonferroni correction).

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  Figure 2. Hemodynamic and vascular parameters as measured using ultrasound imaging. (A) Common carotid artery diameter was significantly larger in untreated and amlodipine treated SHR when compared to normotensive WKY rats. (B) Control SHR showed a reduced diameter pulsatility, while treatment with atenolol increased diameter pulsatility to a similar level as in WKY. (C) Maximal flow, as measured during hypercapnia, was not different among the four groups. (D) Minimal vascular resistance was significantly increased in control SHR under hypercapnic conditions. This was partially corrected in amlodipine, but not in atenolol treated SHR. n = 12 for WKY Ctrl and SHR Amlo, and n = 11 for SHR Ctrl and SHR Aten. ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05 (A,C,D one-way ANOVA with Bonferroni correction, and B Kruskal–Wallis test with Dunn’s correction).

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  Figure 3. Smooth muscle cell and endothelial function of the basilar artery. (A,B) To determine smooth muscle cell function, basilar artery segments were constricted with U46619 and KPSS. The contractile force generated in response to these compounds was lowest in untreated SHR. Both treatment with amlodipine and atenolol significantly increased the contractile force, and nearly restored this to the level of WKY. (C) The effective pressure equivalent to the contraction with KPSS was significantly lower in untreated SHR when compared to WKY and treated SHR. (D,E) Endothelial function was determined by assessing the concentration-dependent relaxation to bradykinin and methacholine. (D) Control SHR showed an impaired relaxation to bradykinin, while WKY, amlodipine and atenolol treated SHR demonstrate a comparable relaxation. (E) In response to methacholine, treated animals showed a reduced relaxation when compared to WKY and untreated SHR. n = 12 for WKY Ctrl and SHR Amlo, and n = 11 for SHR Ctrl and SHR Aten. ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05 (A–C one-way RM ANOVA with Bonferroni correction, D–E two-way RM ANOVA with Bonferroni correction).

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  Figure 4. Remodelling of the superior cerebellar artery. (A) Diameter of the superior cerebellar artery was determined by measurement of the intraluminal diameter over a pressure range of 1–150 mmHg. Arteries from untreated SHR showed a smaller diameter when compared to control WKY. Amlodipine and atenolol treatment increased the diameter to the same extent, but did not normalize to normotensive levels. (B) No differences were observed in the wall cross-sectional area between the four groups. (C) Untreated, control SHR showed an increased wall-to-lumen ratio, which was partially normalized by treatment with amlodipine and atenolol. n = 12 for WKY Ctrl and SHR Amlo, and n = 11 for SHR Ctrl and SHR Aten. ***p ≤ 0.001, **p ≤ 0.01 (A two-way RM ANOVA with Bonferroni correction, B,C one-way ANOVA with Bonferroni correction).

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  Figure 5. Extracellular matrix and smooth muscle cell quantification in superior cerebellar arteries. (A,B,D,E) Representative cross-sectional images of the superior cerebellar artery wall of normotensive WKY, untreated SHR, amlodipine and atenolol treated SHR, respectively, in which the lumen of the artery is shown at the top. The black arrow indicates the extracellular matrix (ECM), whereas the white arrow indicates a smooth muscle cell (SMC). (C) The ECM/vessel wall ratio was not different between the four groups. (F) SMC/vessel wall ratios did also not differ between any of the groups. n = 8 for WKY Ctrl, SHR Ctrl, SHR Amlo, and SHR Aten. (C,F one-way RM ANOVA with Bonferroni correction). Scale bar represents 5 µm.

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  Figure 6. Volume measurements of the brain and specific anatomical structures. (A–D) Representative single slices of the brains of normotensive WKY, untreated SHR, amlodipine and atenolol treated SHR, respectively. (E) Total intracranial volumes were significantly smaller in all SHR groups when compared to control WKY. (F) Lateral ventricles were substantially enlarged in both untreated and treated rats. (G) No differences were noted in the third ventricle volume between the four groups. (H) The brain parenchyma volume was significantly decreased in untreated, control SHR, as well as amlodipine and atenolol treated SHR. (I,J) The corpus callosum volume was smaller in control and amlodipine treated SHR, while the hippocampal volume was significantly decreased in untreated hypertensive rats only. n = 12 for WKY Ctrl and SHR Amlo, and n = 11 for SHR Ctrl and SHR Aten. ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05 (E–J one-way ANOVA with Bonferroni correction). IC intracranial, LV lateral ventricle, 3V third ventricle, BP brain parenchyma, cc corpus callosum, Hc hippocampus.

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  Figure 7. Amyloid-β1–40, amyloid-β1–42, and neurofilament light expression levels in the brain and CSF. (A,B) Amyloid-β1–40 (Aβ1–40) and amyloid-β1–42 (Aβ1–42) concentrations in brain homogenates were not different between the four groups. (C) Additionally, the Aβ1–40/Aβ1–42 ratios were similar in all groups. (D) Neurofilament light (NfL) levels were significantly elevated in the cerebrospinal fluid (CSF) of untreated and treated SHR as compared to WKY. n = 11–12 for WKY Ctrl, n = 10–11 for SHR Ctrl and SHR Aten, n = 12 for SHR Amlo. **p ≤ 0.01, *p ≤ 0.05 (A–C one-way ANOVA with Bonferroni correction, D Kruskal–Wallis test with Dunn’s correction).

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  Figure 8. Number of cells and neurons in the cortex and hippocampus. (A) Representative images of NeuN (red) immunostaining in the cortex and hippocampus. Cell nuclei are visualised using DAPI staining (blue). (B,C) The number of cells did not differ between the four groups in the cortex and hippocampus. (D,E) Additionally, the number of neurons was not different between groups. n = 12 for WKY Ctrl and SHR Amlo, n = 10 for SHR Ctrl, n = 11 for SHR Aten. (C Kruskal–Wallis test with Dunn’s correction, D–F one-way ANOVA with Bonferroni correction). Cx cerebral cortex, Hc hippocampus. Scale bar represents 100 µm.