Measurements of vascular reactivity.

Isometric tension studies of mesenteric arterioles were performed using 2-mm segments of second-order mouse mesenteric arterioles dissected free of perivascular fat. Studies were performed in a small vessel horizontal wire myograph (Danish Myo Technology, models 610M and 620M) containing a physiological salt solution composed of 130 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 25 mM NaHCO₃, 5 mM glucose, and 1.6 mM CaCl₂. The isometric tone was recorded for each vessel using LabChart Pro v7.3.7 (AD Instruments). The vessels were equilibrated over a 20-minute period at 37°C. A passive circumference-tension curve was generated for each vessel to determine optimum passive tension to simulate an in vivo transmural pressure of 100 mmHg according to the manufacturer’s protocol, as previously described with modifications (37). After normalization, vessels were contracted with 60 mM KCl to assess the integrity of the vessel, and then endothelium-dependent and -independent vascular relaxations were tested using increasing concentrations of acetylcholine or sodium nitroprusside, respectively, after preconstriction with norepinephrine (10 μM).