ABC | Volume 110, Nº6, June 2018

Original Article Zhen et al AAC leads to obvious CH using 0.45 mm needle Arq Bras Cardiol. 2018; 110(6):568-576 Methods Animal groups and handling One-hundred fifty male C57BL/6 wild-type mice were obtained from the Shanghai SLAC Laboratory Animal Co. Ltd (Shanghai, China). All animals were treated and cared for in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Washington, DC, 1996). Experimental protocols were approved by our Institutional Animal Care and Use Committee of Zhejiang University (Hangzhou, China). Mice were selected according to weights of approximately 18 g (range, 17.3-18.7 g), 22 g (range, 20.8‑23.0 g), and 26 g (range, 25.1-27.0 g), and they were divided into the following 3 weight levels: 18 g (18.0 ± 0.3 g; n = 50), 22 g (22.0 ± 0.6 g; n = 50), and 26 g (26.1 ± 0.5 g; n = 50). All weight levels were divided using sortition randomization method to create a sham group (n = 10) and 4 AAC groups according to ligating intensities (0.35, 0.40, 0.45, and 0.50mm; n = 10 per group). Regarding BW, no significant differences were found among the 5 groups for each weight level (Table S1), and the preoperative BWs of mice that died and those that survived were not significant (Table S2). Mice were anesthetized with 4% chloralhydrate (0.1ml/1g BW, intraperitoneal injection). When the mice did not respond when their toe was pinched, the limbs were fixed on the operating board in the supine position and the skin was prepared by shaving and disinfection with alcohol. Sterile gauze was placed on the right side of the abdomen and a ventrimesal incision approximately 1.5 cm was created starting from the xiphoid. The skin was fixed with a spreader and the viscera was pulled out gently with a swab and placed on the gauze. Then, the abdominal aorta was isolated using a blunt dissection technique with curved microforceps under a microscope. A 6–0 silk suture was snared and pulled back around the aorta 1mm above the superior mesenteric artery. A 2-mm blunt acupuncture needle (external diameters: 0.35 mm, 0.40 mm, 0.45 mm, and 0.50 mm; Huatuo; Suzhou Medical Appliance Factory, Suzhou, China; criterion number GB2024-1994) was then placed next to the aorta. The suture was tied snugly around the needle and the aorta. The needle was removed immediately after ligation, the viscera were replaced, the peritoneum and skin were sutured, and the mice were allowed to recover. Aortic ligation was omitted only for the sham group. After surgery, the ears were cut to differentiate the mice. Then, mice were placed in an incubator at 30°C until they woke, and they were returned to their cages. Survival status was recorded daily. To observe the physical development of mice under different conditions, BW differences before surgery and at week 8 post-surgery were calculated as the change in BW. Echocardiography imaging After post-surgery weeks 4 and 8, mice were weighed and anesthetized with 4% chloralhydrate and placed on a warming pad after skin preparation. Transthoracic 2-dimensional (2D) echocardiography was performed using the GE Vivid E9 Ultrasound echocardiographic system (General Electric Company, Fairfield, CT, USA) with the GE 9L probe (8-MHz linear array transducer; General Electric Company). M-mode parasternal long-axis scans of the left ventricle at the mitral chordae level were used to quantify the interventricular septum thickness at end-diastole (IVSd), interventricular septum thickness at end-systole (IVSs), left ventricular internal dimension at end-diastole (LVIDd), left ventricular internal dimension at end-systole (LVIDs), left ventricular posterior wall thickness at end-diastole (LVPWd), left ventricular posterior wall thickness at end-systole (LVPWs), ejection fraction (EF), and fractional shortening (FS). All mice were tested using the same parameters. Heart weight, heart weight/body weight, and heart weight/ tibial length After echocardiographic analysis at 8 weeks post-surgery, mice were sacrificed by cervical dislocation and the hearts were dissected. Then, atrial and vascular tissues were snipped carefully, leaving the ventricles. The hearts were rinsed with phosphate-buffered saline (PBS), drained by gently squeezing on absorbent paper, weighed, photographed under natural light, and fixed in 4% paraformaldehyde. The tibial lengths (TLs; mean value of the bilateral tibia) were recorded. Heart weight (HW), BW, and TL were measured, and the HW/BW ratio and HW/TL ratio were calculated to evaluate the hypertrophic response to PO. Histological examination of the heart Extracted hearts were fixed in 4% paraformaldehyde for 24h and dehydrated. After routine histologic procedures, the hearts were embedded in paraffin and cut into 4-μm sections. Sections were stained with hematoxylin and eosin (HE) and picrosirius red (PSR). Cardiac cross-sections were captured at 20 × microscopic views from HE sections, and 5 thicknesses of the left ventricle in each view were selected in systematic sampling, and measured using Image-Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). Then, the mean values were calculated. Cardiomyocyte morphological changes were captured at 400×microscopic views from HE sections. Interstitial and/or perivascular collagen depositions were captured at 200×microscopic views under standard lights. Collagen was stained red using PSR, thereby indicating fibrosis. At least 6 views were selected in a blinded manner, and each photograph was analyzed to reveal the ratio of red collagen to the entire tissue area using Image-Pro Plus 6.0. Then, the mean values were calculated. Statistical Analysis SPSS 17.0 statistical software (SPSS Inc., Chicago, IL, USA) was used for all statistical analyses. The Kolmogorov-Smirnov (K-S) test was used to verify the normality of the quantitative variables as appropriate. Data are presented as mean ± standard deviation (SD). One-way ANOVA and post-hoc Tukey tests were used to evaluate differences between groups. p < 0.05 was considered statistically significant. 569