PAH model
SPF grade of Sprague-Dawley rats (all male, weighed 280-330g) were purchased from the Laboratory Animal Center, Chinese Academy of Science (Beijing, China). The rats were housed in a standard animal room at 21±1˚C temperature and 55±5% humidity. The animal room were under a 12-h light/dark cycle and the rats were free access to water and food. After feeding in the animal room for 7 days, he experiments were began. The animal studies performed were all approved by the Shandong University Institutional Animal Care and Use Committee and were conducted according to the standard protocols and guidelines. The rats were randomly divided into 4 groups: Sham group in which rats received water alone (n = 15); Sham + T group in which the rats were intraperitoneally injected with 14 mg/kg ticagrelor solution (AstraZeneca) every day (n = 15); PAH group in which PAH was induced by left pneumonectomy plus MCT injection [7] (n = 30); and PH + T group in which PAH rats were injected with ticagrelor solution (n = 20). Ticagrelor solution (a selective P2Y12R inhibitor) was made using a 360 mg tablet diluted with 25.5 ml saline water and injected from the time MCT injection to day 28 [8].
The animals were anaesthetized using 2% xylazine (4 mg/kg)/ketamine (100 mg/kg). The rats received an adjusted rate of 60 breaths/min. Respiratory support was given to the rats using a small animal ventilator (HX-300S; Chengdu TME Technology Co., Ltd.) at a tidal volume of 1.1-1.3 ml/100 g, followed by a left unilateral pneumonectomy [9]. One week following surgery, the rats were subcutaneously injected with 60 mg/kg MCT. All rats were under monitored every day until the PAH symptoms were developed, such as body weight loss and tachypnea.
Echocardiography and haemodynamic measurements
Cardiac function was evaluated using a 14 MHz linear transducer equipped with an echocardiographic machine (Visual Sonics, Toronto, Canada). According to Simpson's method, cardiac output (CO) and B-mode long axis was used to detect stroke volume, and pulmonary artery diameter and M-mode were used to measured RV wall thickness. The acceleration time of the pulmonary artery was obtained by applying ultrasonic Doppler to the pulmonary artery [10]. According to the tail-cuff method, a blood pressure recorder (BP-98A; Softron, Tokyo, Japan), was used to measure the blood pressure of the rats [11]. Pulmonary artery pressure transduction was conducted with correct jugular vein by a 1.4F Millar Mikro-Tip catheter transducer (Millar Instruments Inc., Houston, TX) directed to the main pulmonary artery after insertion into the right ventricular outflow duct, although RV systolic pressure (RVSP) was detected with a power laboratory monitoring device (Miller Instruments). Hemodynamic values were accurately computed by LabChart 7.0 physiological data acquisition system (AD Instruments, Sydney, Australia). The rats were anaesthetised during this process.
Tissue processing and histology
Following the test of echocardiography and haemodynamic measurements, the animals were sacrificed by inducing cardiac arrest by injection of 2 mmol KCl through the catheter. The lungs were isolated. The left one was weighed and the right one was inflated with 0.5% low melting agarose at a constant pressure of 25 cm H2O, and fixed in 10% formalin for 24 h. Subsequently, the heart was excised.
Western blot
The lysis buffer used for the extraction of proteins from tissues was a mixture of RIPA (Beyotime Institute of Biotechnology) and PMSF at a ratio of 100:1[11]. The extracted proteins were detected using a BCA protein assay reagent kit (Pierce). The proteins were then subjected to a 5–12% SDS-PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membrane. After blocking in the TBST for 1 h at 4˚C, the target proteins were probed by incubation with following antibodies: 1:2000 for P2Y12R (Abcam, USA) and 1:1500 for α-SMA (Abcam, USA). Primary antibodies were detected using horseradish peroxidase-conjugated antibodies: 1:5000 for anti-mouse (ZSJQ-BIO, Beijing, China) and 1:5000 for anti-rabbit (ZSJQ-BIO, Beijing, China), at room temperature for 2 h. The enhanced chemiluminescence (ECL) detection kit (Millipore) was used for blot development. The blots were visualized by the FluroChem E Imager (Protein-Simple, Santa Clara, CA, USA) and semi-quantified using ImageJ software (National Institutes of Health).
qRT-PCR
The RNAs in lung tissues were extracted using RNA-easyTM Isolation Reagent (cat. no. R701; Vazyme Biotech Co., Ltd.). The RNA was dissolved in DEPC-treated water and the concentration of the extracted RNA was determined using a superdifferential spectrophotometer (Thermo Fisher Scientific, Inc.). Subsequently, Mir-X™ miRNA First Strand Synthesis and TB Green™ (Cat. No. 638313; Takara Bio, Inc.) was used for reverse transcription and HiScript III RT SuperMix (cat. no. 323-01; Vazyme Biotech Co., Ltd.). The PCR procedure was conducted using an ABI 7500 fast Real Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) using ChamQ Universal SYBR qPCR Master Mix (cat. no. Q711-02; Vazyme Biotech Co., Ltd.). The thermocycling conditions were shown as following: 1 cycle of pre-denaturation at 95˚C for 30 sec, followed by 40 cycles of denaturation at 95˚C for 10 sec, annealing and extension at 60˚C for 30 sec, and step 3, 95˚C for 15 sec, 60˚C for 1 min and 95˚C for 15 sec is an additional cycle for melting curve. Target gene expression was calculated using the 2-ΔΔCT method [12] by normalizing to GAPDH. The primers used in this study were as following. GAPDH: forward, 5’-AGATCCACAACGGATACATT-3’, reverse, 5’-TCCCTCAAGATTGTCAGCAA-3’; α-SMA: forward, 5’-CCGACCGAATGCAGAAGGA-3’, reverse 5’-ACAGAGTATTTGCGCTCCGGA-3’; P2Y12R: forward 5’-CTTCGTTCCCTTCCACTTTG-3’, reverse 5’-AGGGTGCTCTCCTTCACGTA-3’.
Immunohistochemistry
The right lung tissues were formalin-fixed, paraffin-embedded and used for HE or regular immunohistochemistry staining [2]. The OCT-embedded tissue was placed into a freezing microtome (CM3050; Leica Microsystems GbmH) and tissue samples were cut into 5 μm sections[9]. In each lung section, 30 small PAs (50–100 μm in diameter) were analyzed at × 40 magnification in a blinded manner. The medial wall thickness was expressed as the summation of two points of medial thickness/ external diameter × 100 (%). Intraacinar (precapillary) PAs (20–30 μm in diameter, 25 vessels each) were assessed for occlusive lesions, defined as Grade 0 when there was no evidence of neointimal lesion, Grade 1 when there was less than 50% luminal occlusion, and Grade 2 when there was more than 50% luminal occlusion [13]. There was no evidence of neointimal lesion formation in any PAs from normal rats (all PAs were graded as 0). Anti-α-SMA (1:200; Abcam) antibodies were used as primary antibodies. After fixing the frozen sections with cold acetone at 25˚C for 5 min and blocking with QuickBlock™ Blocking Buffer for Immunol Staining (cat. no. P0260; Beyotime Institute of Biotechnology) for 10 min at 4˚C, they were treated overnight at 4˚C with anti-P2Y12R antibody (1:200; Novus) and α-SMA (1:200; Abcam). Following incubation with primary antibody, Alexa 546-conjugated donkey anti-rabbit (1:200; Invitrogen) and FITC-conjugated rabbit anti-mouse (1:200; Abcam) secondary antibodies were added, respectively, and the sections were incubated for 2 h at room temperature. The sections were counterstained with DAPI (Life Technologies) to identify nuclei. The sections were then washed and placed under a fluorescence microscope for observation and image capture. Nerve density was measured and evaluated using ImageJ software.
Statistics
Data are expressed as the mean ± SEM. The significant difference between two groups were analyzed by unpaired t-test. For three or more groups, analysis of variance (ANOVA) followed by a Newman-Keuls test was utilized. Statistical analyses were performed using SPSS 20.0 software (SPSS Inc. Chicago, IL, USA), and p-value < 0.05 was considered statistically significant.