ABC | Volume 112, Nº1, January 2019

Original Article Arq Bras Cardiol. 2019; 112(1):3-10 Tianshu-Chu et al Rapamycin combined with α -cyanoacrylate 1 ml of α -CA (taken by pipette) in a sterile EP tube. A magnetic stirrer was then used to mix them to α -CA-RPM of 8 mg/ml, stored in a refrigerator between 2-8°C. RPMwas hydrosolvent, prepared by the same method. 7 Models and groups Fifty SD rats (provided by Anhui Lab Animal Research Center and identified by the medical ethics committee of Anhui Medical University), male and female, aged 10-12 weeks, weighing 220-280 g, were randomized (completely randomized design) into 5 groups, each group containing 10 rats, and fed for 4 weeks after operation. Operating procedure and sample size were determined according to pilot experiments and previous studies, as subsequently described. Operating procedure: an intraperitoneal injection of 10% chloralic hydras was used to anaesthetize rats. Heparin (700 IU/Kg) was injected through the caudal vein to induce heparinization. A vertical incision of approximately 1 cm was made in the middle of the neck (deflected to the operation side), and veins were dissociated on one side. Epitheca of 1‑2 mm were taken from 20G red arterial puncture needle (BD Company), used as cannula. The carotid artery was isolated until the branches. Then two suture traction lines and hemoclips were placed at both ends of the artery to block blood flow. The middle of the artery was isolated and turned carefully to 1-1.2 mm above the cannula. A 6/0 silk suture was used to knot and fix in order to isolate the vein fromarteries; we were then able to open vascular clamps. The incision was sutured after we verified that the pulse of the grafted vein was normal and there was no bleeding. We checked rats’ vital status and incisions every day. We maintained the environment cool, changed their bedding regularly, and gave them sufficient fodder and water. Three days after the operation, 400,000 IU penicillin were delivered via intramuscular injection to every rat on a daily basis. Sham group: we merely simulated the operation process. Jugular veins were dissociated and collateral vessels were ligatured, without dividing or transplanting; Control group: jugular arteriovenous graft on the same side; α -CA group: jugular arteriovenous graft and application of α -CA glue to grafted veins; RPM group: jugular arteriovenous graft and application of RPM to grafted veins; α -CA-RPM group: jugular arteriovenous graft on the same side and application of α ‑CA‑RPM to grafted veins. Collection of samples Blood samples were taken preoperatively at 0 h and postoperatively at 12 h, 36 h, and 4 weeks after operation. Serum was collected by centrifugation and stored at -80°C until cytokine analysis. Four weeks later, we collected each group’s vein sample. Fully anaesthetized rats were fixed on the operating table, heparinized as previously described and operated in the same way through the same path. We observed grafted veins’ shapes and circulation and ligatured and isolated vessels at both ends of cannulas; we then removed intact and fresh veins and washed lumens fully with normal saline. Samples with HE staining and immunohistochemistry were placed in microtubes full of paraformaldehyde. Samples with RT-PCR were placed in microtubes full of RNA-EZ regents and then kept in the fridge at -80°C. Rats were euthanized by cervical dislocation method and handled properly. Enzyme-linked immunosorbent assay for ET-1 ET-1 was determined by ELISA Kits (R&D, USA) using 50 μl of serum for the assay. Three measurements were performed for each blood sample. The ELISA plate was read at 450 nm in a plate reader. Histological examination of graft tissue Immersed in formalin, grafted veins were cut into 4 mm sections. Hematoxylin-eosin (HE) staining was subsequently performed using a hematoxylin and eosin staining kit (Beyotime Biotechnology, Shang Hai, China). Olympus microscope image acquisition system was used to collect section images (×100 objective lens) and measure intima thickness. Two independent researchers performed the measurements and data analysis. Sections were selected randomly from grafted and non-grafted veins; we then measured 16 points’ thickness and calculated the mean. Three sections were selected and measured from every rat. We then calculated intima thickness. Determination of proliferation index Tissuesectionswereincubatedwiththeimmunohistochemistry analysis kit for proliferating cell nuclear antigen (PCNA) (Santa Cruz Biotechnology, Dallas, TX) at 4°C overnight. After washing with phosphate-buffered saline (PBS) (DAKO, Glostrup, Denmark) and incubating with the secondary antibody, color was developed using the DAB system. The tissue sections were dehydrated and installed on slides. All images (×200 objective lens) were captured by Olympus microscope image acquisition system and SPOT Digital Camera (Diagnostic Instruments, Sterling Heights, MI). PCNA-positive cells were counted in the intima. A total of 10 observation views were used to calculate the average percentage of PCNA-positive cells for each rat. RT-PCR Total RNA of the vessel tissues was isolated by the TRIzol Kit (Life Technology, USA). The RNA was reverse-transcripted to cDNA using the RNA reverse transcription kit (Promega, USA). 2 μg total RNA and 1 μl of random primer were denatured at 70°C for 10min and annealed at 4°C for 10min, and then 2 μl of 10× buffer, 2 μl of MgCl 2 (20.8 mol/l) and 1 μl of reverse transcriptase were added to the reaction system. Double distilled water (ddH 2 0) was added to bring the volume to 20 μl. The condition for cDNA synthesis was 37°C for 1 h and 4°C for 10min. The PCR also contained 10 μl 2×SYBRMixture (Takara, Japan), 7 μl ddH 2 0 and 1 μl forward and 1 μl reverse primers. The PCR conditions were 95°C for 5min, 95°C for 15 s, 60°C for 60 s, and 40 cycles. The sequences of the primers used for RT-PCR were as follows: Forward, 5'-CATCTCCGTGGTCCTGAAGT-3' and reverse, 5'-GGCAAGGGAAACGTCTAGTG-3' for von Willebrand factor; forward, 5'-CAGAGTCCAGCACAATACCAG-3' and reverse, 5'-GACCCAGATTATGTTTGAGACC for α -Smooth MuscleActin ; and forward, 5'-ACATGAATGACCTCGTCTCTGA-3' and reverse, 5'-CCTCTTCTTCTGCCTCCTCTCC-3' for GAPDH. The instrument for quantitative real-time PCR was purchased from ABI (USA). 4