Aortic ring preparation and stress relaxation

At 13 weeks of age, mice were anesthetized by intraperitoneal injection of Avertin (350 mg/kg). Thoracic aortas were dissected, placed in ice-cold Hanks’ Balanced Salt Solution, and shipped overnight. As previously shown,^36–37 overnight storage does not affect the passive mechanical properties. Upon receipt, aortas were placed under a stereoscope and cleaned of excessive perivascular tissue before cutting them into 2 mm ring segments of equal length. Each ring segment was suspended in an organ chamber of a 610 M Multi Chamber Myograph System (Danish Myo Technology, Denmark) filled with 8 mL of oxygenated (95% O₂, 5% CO₂) physiological saline solution (118.31 mM NaCl, 4.69 mM KCl, 1.2 mM MgSO₄, 1.18 mM KH₂PO₄, 24.04 mM NaHCO₃, 0.02 mM EDTA, 2.5 mM CaCl₂, and 5.5 mM glucose) and allowed to equilibrate at 37°C for at least 30 min.

To assess the mechanical properties of the vessel, stress relaxation was assessed in thoracic aortic rings from Acta2^−/− and WT mice. Aortic rings were stretched in 4 mN increments from 0 mN until the calculated transmural pressure of 13.3 kPa (100 mmHg) was attained. Transmural pressure was calculated as p = 2π*T/L, where L is the internal circumference corresponding to wall tension T.³⁸ Length and tension were recorded immediately after each 4 mN increase in tension and again after 1 min.^39,40 Stress relaxation was calculated as the difference between the 4 mN increase in tension and the tension after 1 min, and was expressed as a percent decrease in tension. Passive tension curves were generated by plotting calculated transmural pressure (kPa) versus tension (mN). A repeated measures ANOVA followed by Bonferroni post-hoc analysis was used to assess strain differences for stress relaxation and passive tension curves. All values are presented as mean ± SD. Statistical significance was evaluated at P < 0.05.