Assessment of endothelial-dependent vasodilation

Mice were euthanized by decapitations under isoflurane anesthesia. Mouse aortas were isolated and cleaned from the fat and connective tissue in a calcium and magnesium-free phosphate buffer solution (PBS, Lonza). A wire myograph from GlobalTown Microtech., Inc. (Sarasota, FL) was used to monitor the force generated by aortic arch rings. The isometric tension measurements were performed as described elsewhere [32–35]. Briefly, aortic arches were hung on the wires of the wire myograph and were placed into the 5-ml tissue baths filled with the standard Krebs buffer maintained at 37 °C and continuously oxygenated by bubbling a gas mixture of 95% O₂ and 5% CO₂ during all of the performed experiments. The preload in all experiments was set to 0.7–1 g. Increasing concentrations of phenylephrine and then acetylcholine was added directly into the tissue baths while the contraction force was measured. SNAP (S-Nitroso-N-acetyl-DL-penicillamine, a nitric oxide donor) was used to assess the maximal receptor-independent aortic ring dilations. The analog ring tension data were digitized with a frequency of 20 Hz and recorded on a computer’s hard drive.