Preparation of Aortic Rings for Tension Measurements

The ex vivo experiment was conducted on thoracic aortas of rats according to the protocol described previously (Chen et al., 2019). Namely, the rats were anesthetized by intraperitoneal injection of chloral hydrate [400 mg/kg body weight (BW)] and were then sacrificed by cervical dislocation. After opening the chest, the rats’ thoracic aortas were obtained and were immediately placed in ice-cold Krebs–Henseleit (KH) solution of the following composition (in mmol/L): NaCl 120, KCL 4.8, MgSO₄•7H₂O 1.2, KH₂PO₄ 1.2, CaCl₂ 2.5, NaHCO₃ 25, and glucose 11 (pH 7.4). The fat tissue adhering to arteries was carefully removed to avoid endothelial cell damage, and the blood vessel was cut into approximately 3-mm-long rings. In the endothelium-denuded experiments, the endothelium was mechanically removed by gently rubbing the luminal surface of the aortic ring back and forth several times with plastic tubing. Aortic rings were suspended in organ baths containing 5 ml KH solution at 37°C gassed with 95% O₂ + 5% CO₂, which was maintained constantly throughout the experiments. After equilibration under no tension for 20 min, the aortic rings were allowed to equilibrate for 90 min at a resting tension of 1.0 g. During the equilibration period, the KH solution was changed every 20 min. Changes in tension were recorded by force transducers (FT-102, Chengdu Techman Software Co., Ltd. China) connected to a data acquisition system (BL-420F, Chengdu Techman Software Co., Ltd. China) and were stored in a computer. Moreover, in order to ensure the accuracy and repeatability of the study, 3.0–4.5 g was selected as the inclusion criterion of precontractile force according to preliminary experiments.