Animal Model of PAH
All protocols had been approved by the animal research committee of Kaohsiung Medical University (IACUC approval NO: 104226). Male Wistar rats weighing approximately 220 g were purchased from National Animal Center, Taiwan. after arriving at the Kaohsiung Medical University vivarium, the rats were acclimated for at least one week before being used in the experiment. They were housed in a room on a 12-hr light/dark cycle under controlled temperature of 22.1℃ and relative humidity of 55%, and they were provided with normal chow and water ad libitum. On the day of experiment, animals were anesthetized with a mixture of sodium pentobarbitone (20 mg/kg, i.p.) and ketamine (40 mg/kg, i.m.) under orotracheally ventilated rodent respirators (Harvard, South Natick, MA) and lateral thoracotomy in the third intercostal space was performed. Left lung of rats was resected and closed by clip ligation (Ligaclip multiple clip applier, medium size, Ethicon Endo-Surgery), and the animals were suture to prevent pneumothorax. The chest retractor was then removed after 15 minutes, and the thymus was moved back to its physiological position. The chest cavity and skin were closed. The entire procedure took less than 30 min for each rat. After operation, rats were extubated and received an additional injection of sterile saline (10 ml). On the following day, SU5416 (25 mg/kg) was injected subcutaneously in surviving rats. Sham-operated animals underwent identical operation without pneumonectomy (PNx) and received sterile saline (20 mg/kg) subcutaneously, instead. The day of SU5416 injection was designated as day 0. All animals were individually housed in a 12-h dark/light cycle-controlled room and fed a regular rat diet. The procedure was also shown in supplemental figure 1.