Electrical Vagal Nerve Stimulation Ameliorates Pulmonary Vascular Remodeling and Improves Survival in Rats With Severe Pulmonary Arterial Hypertension

Figures

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  Figure 1. Photographic Images of Vagal Nerve Stimulation in Rats Electrodes are (A) attached to the right cervical vagal nerve and (B) connected to a neurostimulator (C) implanted subcutaneously in the back of a rat.

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  Figure 2. Protocols Protocol 1 assesses the therapeutic effects of 5-week chronic vagal nerve stimulation (VNS) delivered by an implantable neurostimulator in free-moving rats with pulmonary arterial hypertension (PAH). Protocol 2 assesses the effects of 90-min acute VNS delivered by an external stimulation device in anesthetized rats with advanced PAH. CTRL = control; O2 = oxygen; SS = sham stimulation.

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  Figure 3. Effects of Chronic VNS on Power Spectral Density of Heart Rate, Hemodynamic Variables, and Plasma BNP Values are mean ± SEM. Differences were tested by using 1-way analysis of variance, followed by post hoc Tukey-Kramer tests. ∗p < 0.05 and ∗∗p < 0.01 vs. CTRL. †p < 0.05 and ‡p < 0.01 vs. SS. (A) Representative traces of power spectral density of heart rate in CTRL, SS, and VNS in rats at 5 weeks after SU5416 injection. The frequency range of 0.04–0.73 Hz was defined as low frequency (LF) and 0.73–2.0 Hz as high frequency (HF). The HF of HRV that represents parasympathetic nerve activity is lower in SS than CTRL but is higher in VNS than in SS. (B to G) Mean pulmonary arterial pressure (mPAP), pulmonary vascular resistance (PVR), cardiac index (CI), right ventricular end-diastolic pressure (RVEDP), and maximum first derivation of right ventricular pressure normalized by RVEDP (Max +dP/dt/RVEDP) are shown. VNS significantly reduces mPAP and improves right ventricular function. CTRL, n = 5; SS, n = 6; and VNS, n = 6. BNP = brain natriuretic peptide; other abbreviations as in Figure 2.

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  Figure 4. Effects of Chronic VNS on Histology of PA Values are mean ± SEM. Differences were tested by using 1-way analysis of variance, followed by post hoc Tukey-Kramer test. ∗∗p < 0.01 vs. CTRL. ‡p < 0.01 vs. SS. (A) Representative photomicrographs of Verhoeff–van Gieson staining. Arrowheads indicate occlusive pulmonary artery (PA). (B) Pulmonary arterial occlusions are graded as grade 0 (no luminal occlusion; white), grade 1 (< 50% occlusion; light gray), and grade 2 (≧50% occlusion; dark gray). Percentage of occlusive PAs with outer diameter (OD) ≦50 μm (left) and 50 < OD <100 μm (right). Representative photomicrographs of (C) immunostained CD68-positive cells, (D) Ki67-positive cells, and (E) terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells are shown (n = 4 in each group). Arrowheads in (E) indicate TUNEL-positive cells. Other abbreviations as in Figure 2.

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  Figure 5. Effects of Chronic VNS on Histology of the Right Ventricle Values are mean ± SEM. Differences were tested by using 1-way analysis of variance, followed by post hoc Tukey-Kramer test. ∗p < 0.05 and ∗∗p < 0.01 vs. CTRL. †p < 0.05 and ‡p < 0.01 vs. SS. Representative photomicrographs of (A) Masson trichrome staining (MT), (B) immunostained CD34-positive cells, and (C) terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells are shown (n = 4 in each group). Arrowhead in (C) indicates TUNEL-positive cell. Other abbreviations as in Figure 2.

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  Figure 6. Effects of Chronic VNS on Inflammation of Lung Values are mean ± SEM. Differences were tested by using 1-way analysis of variance, followed by post hoc Tukey-Kramer test. ∗p < 0.05 vs. CTRL, †p < 0.05 and ‡p < 0.01 vs. SS. Quantitative analysis of cytokines and chemokine in CTRL (n = 5), SS (n = 6), and VNS (n = 6) rats at 5 weeks after SU5416 injection. Samples were obtained from the lung. The levels of protein expression of (A) interleukin (IL)-1β, (B) IL-6, (C) tumor necrosis factor (TNF)-α, (D) monocyte chemotactic protein (MCP)-1, and (E) IL-10 are shown. Other abbreviations as in Figure 2.

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  Figure 7. Effects of Chronic VNS on Survival Five-week VNS markedly improved survival both in the prevention (red, n = 14; p < 0.01) and treatment (purple, n = 14; p < 0.05) groups compared with the SS (blue, n = 25) group, with relative risk reductions of 53.0% and 34.2%, respectively. The survival assessment was started from 5 weeks after SU5416 injection. Prevention, 5-week VNS initiated from 0 weeks after SU5416 injection; treatment, 5-week VNS initiated from 5 weeks after SU5416 injection (0 weeks in Figure 7). Differences were tested by using the Kaplan-Meier method with log-rank testing among the SS-prevention, SS-treatment, and prevention-treatment groups. *p < 0.05 and **p < 0.01 vs. SS. n.s. = not significant; other abbreviations as in Figure 2.

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  Figure 8. Effects of Acute VNS on Hemodynamic Variables Values are mean ± SEM. Differences were tested by using a paired Student's t-test. ∗p < 0.05 vs. pre-stimulation. (A) Representative time series of hemodynamic variables under acute VNS in a rat at 5 weeks after SU5416 injection. Heart rate (HR) decreases soon after initiation of VNS, whereas no significant changes in pulmonary arterial pressure (PAP), cardiac output (CO), and left ventricular pressure (LVP) are observed. (B–F) Effects of acute VNS on hemodynamic variables. The white columns represent pre-stimulation, and the blue and red columns represent post SS (n = 5) and VNS (n = 4), respectively. CI = cardiac index; LVEDP = left ventricular end-diastolic pressure; other abbreviations as in Figures 2 and 3.

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  Figure 9. Effects of Acute VNS on NO Synthesis and Inflammation in the Lung Values are mean ± SEM. Differences were tested by using Student’s t-test. ∗p < 0.05 and ∗∗p < 0.01 vs. SS. Effects of acute VNS (n = 3) or SS (n = 3) on nitric oxide (NO) synthesis in the lung of rats 5 weeks after SU5416 injection. Representative Western blotting of (A) endothelial nitric oxide synthase (eNOS) and (B) phosphorylated eNOS (p-eNOS) are shown in the upper panels. Graphs indicate semi-quantitative analysis of Western blots of eNOS and p-eNOS normalized by β-actin. In addition, effects of acute VNS (n = 4) or SS (n = 5) on messenger ribonucleic acid expressions of (C)Il1b, (D)Il6, (E)Tnfa, (F)Mcp1, (G)Il10, and (H)Alpha7nachr are shown; 18s was used as the loading control. Each value was normalized by the averaged value in SS. Other abbreviations as in Figure 2.