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.