2.6. Vascular reactivity experiment

The thoracic aorta was immediately dissected and put in physiological salt solution (PSS) buffer (mmol/L): glucose 5.5, NaCl 119, NaHCO₃ 25, KCl 4.7, CaCl₂ 2.5, KH₂PO₄ 1.2 and MgSO₄ 1.2.31 Adhered tissues were removed, and aorta was chopped into 3‐mm rings and moved into the Multi Myograph System chambers (DMT). The rings were mounted on chambers filled with warmed (37℃), oxygenated (air mixture containing 95% O₂ and 5% CO₂) PSS. LabChart software (DMT620) was used to continuously record the vascular tension. The aortic rings were maintained at a basal tension of 2 g for 90 min and then stimulated twice with KCl (60 mmol/L)‐PSS in advance. Relaxation of norepinephrine (NE, 10⁻⁷ M) precontracted vessels to acetylcholine (ACh, 10⁻⁵ M) was used to determine endothelial integrity (vessels that relaxed by at least 80% were deemed endothelium intact). Following this, aortas were then precontracted with NE (10⁻⁷ M), until the aortic rings reached maximum contraction and the tension curve became stable, cumulative concentration‐response (10⁻⁹ M to 10⁻⁵ M) curves were constructed for ACh (endothelium‐dependent relaxation). Average concentration‐response curves were plotted.