Measurement of mPAP and pulmonary arteriole morphology

Measurement of mPAP was performed as described previously (14). Briefly, after 8 weeks, the rats were anesthetized by an intraperitoneal injection of pentobarbital sodium (40 mg/kg; Shanghai Haling Biotechnology Co., Ltd.). A longitudinal skin incision was made on the right side of the neck, and blunt layer-by-layer separation of the tissues was performed until the right external jugular vein was exposed. A polyethylene catheter was gradually inserted into the pulmonary artery through an incision in the right external jugular vein, and the mPAP was recorded using a pressure transducer, which was interfaced to a BL-420S Bio Lab System (Chengdu TME Technology Co., Ltd., Chengdu, China). Following measurement of mPAP, blood samples (2.0 ml per rat) were drawn from the pulmonary artery of the rats. These were centrifuged for 10 min at 3,000 × g and the supernatant was collected and stored at −80°C until use. The rats were then sacrificed by exsanguination and the lungs were isolated and washed with physiological saline repeatedly. Three lobes of the right lung were surgically removed, immediately snap frozen in liquid nitrogen and stored at −80°C until use. The upper lobe of the left lung was removed and fixed in a 10% formalin solution overnight, which was followed by paraffin embedding. Subsequently, lung sections (4-µm) were prepared and stained with hematoxylin and eosin. Sections were examined under a light microscope (Eclipse 55i; Nikon Corporation, Tokyo, Japan) for pulmonary arteriolar morphological analysis.