Methods
Experimental animals: The healthy male Balb/c mice, purchased from Wu’s Animal Center, Fuzhou City, Fujian Province, were free to drink water in separate cages and fed with granular feed. Weigh the mice regularly. The ambient temperature was 24℃~26 ℃, and the humidity was 60%~80%. The animals were randomly divided into two groups, blank control group and 5-fluorouracil + bevacizumab group. In the 5- fluorouracil + bevacizumab group, 5mg/kg bevacizumab and 30mg/kg fluorouracil were injected intraperitoneally once every two day for 14 days. All animal procedures were approved by the Animal Ethics Committee of Fujian Medical University.
Echocardiography: Echocardiography was performed before injection and on the 4th, 7th and 14th day after injection. The mice were anesthetized with isoflurane inhalation at an induced concentration of 3%. Then, the isoflurane at a maintained concentration of 1%~1.5% was administered to maintain anesthesia, and the target heart rate was maintained at 400~500 times per minute. After anesthesia, the mice were lying on the operating platform with a temperature of 40℃, and the heart rate was recorded by sticking all four limbs on the electrode piece of the operating platform.
The function of heart was analyzed by echocardiography with Visual Sonic Vevo 2100, and B mode and M mode echocardiography of long axis and short axis of left ventricle were detected by MS550D high frequency ultrasonic probe. And then measure IVS (interventricular septum thickness), LVPW (left ventricular posterior wall thickness), lVID;d (left ventricular end diastolic dimension), lVID;s (left ventricular end systolic dimension), LVEF (left ventricular ejection fraction) and LVFS (left ventricular fraction shortening). Then the apical four-chamber view was used the color doppler mode and spectrum doppler mode to record MVE (early diastolic mitral valve flow velocity), MVA (early diastolic mitral valve flow velocity) and e’, measured by tissue doppler. Using Vevo strain software analyzed and generated the left ventricular wall strain time curve, and records LS (longitudinal strain), RS (radial strain), CS (circumferential strain), LSR (longitudinal strain rate), RSR (radial strain rate) and CSR (circumferential strain rate).
Electrocardiograms: ECG (electrocardiograms) signals were collected with a multi-channel signal acquisition and processing system (RM6240BD, Chengdu, China) before injection and on the 7th and 14th day after injection. The ECG of mice in each group were collected by subcutaneously connecting the corresponding electrodes in the right upper limb, the right lower limb and the left upper limb. The software automatically analyzed the Q-wave amplitude, S-wave amplitude, T-wave amplitude, ST offset, PR interval, ST interval and average heart rate.
PET-CT : PET-CT was performed on the 7th and 14th day after injection. PET imaging in vivo was obtained by Siemens Inveon PET scanner. The mice were anesthetized with isoflurane, the induction concentration of inhaled isoflurane was 3%, the maintenance concentration was 2%, and 18F-FDG (120μCi/ mouse) was injected into tail vein. The mice were fixed on the scanning bed and kept still. The whole body of the mice was displayed in the field of vision. The data were collected in three-dimensional mode 20 minutes after the injection of 18F-FDG, manual location of mouse heart.
B-type natriuretic peptide: Before injection and the 7th and 14th day after injection, some mice were sacrificed and blood was taken. And then blood centrifugation, serum separation, storage backup. The BNP (B-type Natriuretic Peptide) in serum was calculated by mouse BNP ELISA (enzyme-linked immunosorbent assay) kit. The results were expressed in pg/ml.
Histomorphometry: On the 7th and 14th day after injection, the hearts of mice were sacrificed and the large blood vessels were removed and weighed. The cardiac tissues in different groups were prepared into frozen sections for histomorphometry analysis. Cardiac histology was examined by HE staining of thin myocardial sections. The pathological and morphological changes in the myocardial tissue were observed under an optical microscope.
Statistical analysis: The results of the experiment were analyzed by GraphPad Prism 5 software. All the data were expressed by Mean ± SEM. T test was used for comparison between groups. WhenP < 0.05, the difference was statistically significant.