Mouse aortic ring preparation for vascular function measurements

Preparation of the isolated mouse aorta was similar to that previously described⁶⁴. Briefly, the thoracic aorta was gently dissected from anaesthetized and heparinized adult, male Cygb^−/− mice or age-matched adult, male C57BL/6J mice (9–12 months of age), carefully cleaned of fat and connective tissues, and cut transversely into rings of 2–3 mm in length. The rings were mounted on a wire myograph (Multi Myograph System-610M, Danish Myo, Aarhus, Denmark) with care taken not to damage the endothelium, and then suspended in 5-ml organ baths containing modified KHB (containing (in mM) 118 NaCl, 24 NaHCO₃, 4.6 KCl, 1.2 NaH₂PO₄, 1.2 CaCl₂, 4.6 HEPES, and 18 glucose) and continuously purged with 95% O₂–5% CO₂ (37 °C, pH 7.4). Aortic rings were equilibrated for 90 min with an initial resting tension of 1 g, and the bathing solution was changed at 15-min intervals. Changes in isometric tension were recorded on a PowerLab/8sp multichannel data-acquisition system (AD Instruments, Colorado Springs, CO) using ADI Chart software (version 5.3) for digital processing and data analysis. After equilibration, the responsiveness and stability of each ring was checked by the successive administration of a maximally effective concentration of L-phenylephrine hydrochloride (phenylephrine; 1 μM). The integrity of the vascular endothelium was assessed pharmacologically by acetylcholine (Ach)-induced relaxation of phenylephrine-pre-contracted rings. Preparations were then washed three times with drug-free buffer and allowed to relax fully for 30 min before the experimental protocol began. To determine the vasodilatory response to ACh, the aortic rings were pre-contracted with phenylephrine, and dose–response curves for aortic relaxation were obtained by the cumulative addition of Ach or sodium nitroprusside to the organ bath. The concentration of agonist in the organ bath was increased in steps of 1-log units. ACh was added to yield the next higher concentration only when the response to the lower dose reached a steady state. One dose–response curve for ACh was constructed for each ring. The vasodilator (relaxant) responses were expressed as per cent decreases of phenylephrine-induced pre-contraction, where the contraction produced by 1 μM phenylephrine in each ring from its initial resting tension (1 g) was considered as 100%.