Aortic ring preparation

The contractile responses of the aortic smooth muscle of male SD rat to VAE were determined through an in vitro study. First, the SD rat was anesthetized by carbon dioxide inhalation before isolation of its aorta. The aorta was then transferred to a Petri dish filled with Krebs–Henseleit (Krebs) solution (118.0 mM NaCl, 4.7 mM KCl, 25.0 mM NaHCO₃, 1.8 mM CaCl₂, 1.2 mM NaH₂PO₄, 1.2 mM MgSO₄, and 11.0 mM glucose) while the connective tissues around the aorta were removed. During the process, the Krebs solution containing the aorta was continuously supplied with ‘carbogen’ (95% O₂ and 5% CO₂). After the aorta was cleaned, it was cut into 2–3 mm aortic ring segments and suspended horizontally in an organ bath that contains 10 mL of Krebs solution supplied constantly with carbogen gas at 37 °C. Special care was taken to avoid damage to the endothelium. When preparing endothelium-denuded aorta, the endothelium was removed with a cotton swab by gently rubbing the internal space of the aorta (Wang et al. 2014). The aortic rings were allowed to equilibrate at an optimal tension of 1 g for 30 min. Throughout this period, the Krebs solution was replaced every 10 min and the tension was readjusted to 1 g if needed. For confirmation of the condition of the aortic rings, they were pre-contracted with PE (1 µM) followed by relaxation with Ach (1 µM). After that, the rings were rinsed with Krebs solution and the tension was readjusted to 1 g, and 1 µM of PE was added to establish a stable contractile tone. When a plateau was achieved, a concentration–response curve was constructed by the cumulative addition of the VAE (0.01, 0.02, 0.04, 0.08, 0.16, 0.32, 0.64, and 1.28 mg/mL) at 20-min intervals on endothelium-denuded and endothelium-intact rings (Ameer et al. 2010; Loh et al. 2017; Tan, Ch’ng, et al. 2017; Tan, Loh, et al. 2017).

Changes of the contractile force on the aortic rings were measured with a force-electricity transducer (GRASS Force-Displacement Transducer FT03 C Isometric Force Measurements). Signals were amplified, generated by the LabChart 5 software, and the data were tabulated by using Microsoft Excel.