Assessment of vascular reactivity in isolated aortic rings.

The thoracic aorta was dissected and placed in cold physiological buffer: 119 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl₂ × 2H₂O, 1.17 mM MgSO₄ × 7H₂O, 20 mM NaHCO₃, 1.18 mM KH₂PO₄, 0.027 mM EDTA, 10.5 mM glucose; bubbled with a mixture of 95% O₂ and 5% CO₂, resulting in pH 7.4. Aortic rings (2–3 mm) were mounted onto a multichamber isometric myograph system (Model 620M, Danish Myo Technology) and equilibrated at 37°C. The resting tension was reached by the normalization process using the ADInstuments Normalization module, where the segments of aorta were normalized to their length and diameter. At the beginning of each experiment, the ability of the preparation to develop a contraction was assessed by exposing the aorta segments to a high KCl solution (124 mM). Endothelium-dependent relaxations were determined by administration of cumulatively increasing concentrations of acetylcholine (10^–9 to 10^–5 M, MilliporeSigma) to aortas precontracted with phenylephrine (10^–10 to 10^–6 M, MilliporeSigma). The aorta segments were then washed 3 times every 10 minutes for 30 minutes. The endothelium-independent relaxation was tested by sodium nitroprusside (SNP, 0.1 nM to 10 μM, Merck), following precontraction with phenylephrine (10 μM, MilliporeSigma). Data were recorded and analyzed with the LabChart Pro evaluation program (ADInstruments).