Vascular reactivity testing

Rats in all four groups were euthanised by exsanguination via abdominal aorta after anaesthetising by 2.5-3.0% isoflurane inhalation on pregnancy day 20. Following euthanasia, the thoracic aorta was collected immediately for vascular reactivity testing. During sample collection, care was taken to avoid damage to the vascular endothelium. After collection, the aorta was immediately placed in a petri dish containing Krebs-Henseleit (KH) solution (cat. no. PB180348; Procell Life Science & Technology Co., Ltd.) at 37˚C. A mixture of 95% oxygen + 5% carbon dioxide gas was continuously injected into the KH solution. The fatty tissue on the surface of the blood vessels was removed and blood vessels were cut into rings (length, 4 mm). Two L-shaped stainless-steel hooks were passed through the lumen, with one end fixed and the other end connected to the tension transducer of a physiological recorder (PL3508 PowerLab 8/35; ADInstruments, Ltd.). Blood vessels were immersed in the KH solution and the physiological recorder was adjusted to apply a baseline tension of 1 g to the blood vessels to simulate physiological tension. The tension was equilibrated for 60 min, during which the KH solution was replaced every 15 min. After stabilisation of the tension, 10^-5 mol/l phenylephrine was added to the container, and 10^-9-10^-6 mol/l acetylcholine was added after vasoconstriction was equilibrated. The time interval between additions of different concentrations of acetylcholine was 2 min. The rings were continuously aerated while phenylephrine and acetylcholine were added. The relaxation of the thoracic aorta vascular ring of each group of rats was recorded.