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 Table 1 – Mice deaths after surgery Needles (mm) for 18 g Needles (mm) for 22 g Needles (mm) for 26 g 0.35 0.40 0.45 0.35 0.40 0.45 0.35 0.40 0.45 0-24 h 10 4 1 10 7 1 10 8 3 24 h-3 d 0 0 0 0 2 0 0 1 2 3 d-5 d 0 0 1 0 1 2 0 1 1 There were no mice deaths in the AAC0.50-mm group or the sham group. Deaths were recorded during 3 time periods (0-24h, 24h-3d, and 3-5 d); 54 deaths occurred within 0-24h post-surgery. The total number of deaths was 65. Results Excessive AAC may lead to death We monitored mice deaths after surgery according to acute heart failure (AHF) criteria. Data (Table 1) showed that all deaths occurred within 5 days, and a high incidence of death occurred during the initial 24h post-surgery. AAC increases cardiac dimensions and reduces cardiac function Echocardiography was performed at the end of post- operative weeks 4 and 8. At week 4 post-surgery, data (Table 2) showed a trend of heart enlargement for mice with AAC, including thickening of the ventricular wall and an increase in chamber dilation; however, differences in EF and FS were not significant, indicating that changes in the heart structure did not have a pronounced effect on cardiac function at that time point. At week 8 post-surgery, the trend of heart enlargement continued; however, the EF and FS values for the AAC groups decreased significantly. This change in cardiac function from week 4 to week 8 was consistent with systolic function beginning to be markedly affected at week 4 after PO surgery. AAC increases HW, HW/BW, and HW/TL ratio Generally, the increased HW, HW/BW, and HW/TL ratio are the three main indicators of CH. In our study, as shown in Table 3, we found that AAC significantly increased HW and caused a significantly higher HW/BW ratio and HW/TL ratio compared to the sham groups for all weight levels. The HW, HW/BW, and HW/TL values for the AAC0.45 mm groups were significantly higher than those for the AAC0.50 mm groups. These HW-related indices for the 18 g/0.45 mm groups were even significantly higher than those for the 18 g/0.40 mm groups. AAC leads to cardiomyocyte hypertrophy and increases collagen depositions For mice undergoing AAC surgery, the hearts demonstrated different degrees of enlargement (Figure 1A), enlargement of the papillary muscles, and thickening of the ventricular walls (Figure 1B). Wall thickening increased significantly compared with that of the sham group (Table 4). The sham groups showed normal architecture of the cardiomyocytes compared with the AAC groups. Pathological changes including enlarged, disarrayed, and eosinophilic cardiomyocytes and cardiomyocytes rich in cytoplasm and trachychromatic and pantomorphic nuclei were observed in each of the AAC groups (Figure 1C). Scattered collagen depositions in the interstitial and perivascular spaces were observed in the sham groups. In comparison, in some AAC groups, a larger quantity and wider range of red deposits were observed in the interstitial space (Figure 1D), and thickened collagen was observed in the perivascular space, especially in the external vascular wall (Figure 1E). Statistical analysis indicated that the AAC group had a significantly greater collagen area than the sham group (Table 5). These results imply that AAC is capable of inducing PO-induced CH and fibrosis. AAC may restrict physical development Analysis showed that with AAC 0.45 mm, BW significantly increased in 18-g mice compared to 22-g and 26-g mice (Table 6), indicating that the 18-g groups had higher development potential. In the 18-g mice groups, data showed that the value of 18 g/0.40 mm was significantly lower than that of the 18 g/0.45 mm and 18 g/sham groups, and that there were no significant differences between the 18 g/0.45 mm and 18 g/sham groups (Table 7), indicating that the 18 g/0.45 mm group had nearly normal physical development. Development of the 18 g/0.40 mm group was limited. Discussion In this study, we performed AAC according to 4 different ligating intensities for mice of 3 different weight levels to evaluate the survival rates of mice and CH induced by PO under different conditions. This is the first study showing that CH diversities exist among groups under different ligations and BW. AAC is widely used in the modeling of CH induced by PO in mice. Needle ligation is usually used, and the efficiency of modeling is highly dependent on ligation intensity. Nevertheless, excessive constriction will lead to death, 1 and our research findings (Table 1) demonstrated this point. In this study, a 0.35-mm needle caused the death of all mice in the 3 weight levels, and the 0.40-mm needle caused the death of all mice in 22-g and 26-g groups. Contrarily, all mice with AAC that underwent surgery with a 0.50-mm needle or sham operation survived. Mice in the other groups had different mortality rates. Regarding the selection of needles for the BW ranges of this study, a needle smaller than 0.35mm in diameter caused stronger constriction and death. However, a needle larger than 0.50mm in diameter did not alternatively affect the survival rate, but it did reduce the efficiency of CH because of the reduced PO from weaker constriction. This is why we chose needles between 0.35 mm and 0.50 mm. 570