2.1 Pre-experiment and experimental grouping
2.1.1. Preparation of experimental animals
Male Babl/c mice, aged 6 weeks, weighing 22.1g±0.7g, were purchased from Beijing Vital River Laboratory Animal Technology (Beijing, China), and housed in a laboratory environment for 7 days to acclimate to the laboratory environment. All protocols for animal experiments were based on guidelines of the National Institutes of Health and preapproved by the Animal Use and Care Committee of Tianjin Medical University General Hospital (Tianjin, China).
2.1.2. Experimental animals were screened for hypobaric hypoxia pre-experiment
All experimental animals were placed in the hypobaric hypoxia animal model chamber, and the parameters in the chamber were set as follows: simulated altitude 3500 meters; The velocity of elevation change was 0.45m/s. The pressure was 65.7KPa. Oxygen concentration 14.60%; Temperature range 16-28℃; Humidity range 40-70RH%; The noise is controlled at 59 decibels at 3500 m altitude for 3 hours, the state of the experimental animals was observed and recorded. Animals with limbs extended and no activity during the experiment were defined as poor mobility and weak constitution. The experimental animals with poor activity were excluded, and the animal experiments of acute plateau hypobaric hypoxia myocardial injury were carried out. Information about the hypobaric hypoxia culture chamber: company name: Tianjin XianRenzhang Medical Technology Co., Ltd. Trademark: EXPBOX. (Note: The English name “EXPBOX” is assumed to be the corresponding trademark of “XIANRENZHANG (Tianjin) Medical Technology Co., Ltd.”)
2.1.3. Experimental grouping
After the screening experiment, the experimental animals were divided into control group and four experimental groups. The experimental groups were divided into 72h group (72h), 144h group (144h), 216h group (216h), and 216h continuous group (216ch). In the 216h continuous group, 18F-FDG myocardial metabolism imaging and echocardiography were performed before the experiment, 96h after the experiment, and 216h after the experiment. According to the random number principle, 8 experimental animals were included in each group.
2.2. Experimental condition setting
2.2.1. Feeding conditions of experimental animals.
The experimental animals in the experimental group were kept in the hypobaric hypoxia animal model chamber, and the altitude was set to be 8500 meters, the pressure was 33.1KPa, and the oxygen content was 8.25% during the high altitude stage of the experiment. The altitude of the experimental animals was 4500 m, the pressure was 57.7KPa, and the oxygen content was 12.1%. The altitude change rate of the hypobaric hypoxia animal model chamber was set as 0.45m/s.
2.2.2. The experiment was divided into three stages.
A. In the elevation stage, the pumping speed of the vacuum pump assembly was greater than that of the air supply assembly, which reduced the air pressure and oxygen content in the model chamber, and the simulated elevation was from the simulated sea level to 8500 meters.
B. In the stage of maintaining high altitude, the two vacuum pumps of the vacuum pump assembly were separated by 12 hours alternately to maintain the same amount of oxygen in the model chamber as that at the simulated altitude of 8500 meters, and to maintain a long time operation at the altitude of 8500 meters to meet the experimental requirements. The total time of the experiment reached 216 hours.
C. In the descending altitude stage, the pumping speed of the vacuum pump assembly was less than that of the filling air assembly, so that the total amount of oxygen in the model chamber remained unchanged and the air pressure increased, and the simulated descending altitude was dropped from 8500 meters to the simulated sea level. The rate of elevation change was 0.45±0.04m/s.
2.3. Validation of an animal model of acute plateau hypobaric hypoxia myocardial injury.
2.3.1. 18F-FDG myocardial metabolic imaging
A. 18F-FDG working solution was prepared and diluted to 0.5-1.2μCi/μL;
B. The animal model was anesthetized and 18F-FDG solution was injected 75-250μCi through the tail vein.
C. PET/CT imaging was performed 30-120 minutes after injection of 18F-FDG working solution, the region of interest of the heart was delineated, and the SUVmean value of the heart was calculated.
D. Interpretation of experimental results. The results were reviewed by two experienced nuclear medicine physicians in a double-blind manner and analyzed by statistical methods.
2.3.2. Small animal cardiac ultrasound experiment
2.3.3. FITC-BSA vascular leakage assay
A. 0.5-2.0ug/μL FITC-BSA solution was prepared and 50-200μL was injected through the tail vein.
B. After 2-4 hours, the heart tissue of the animal model was dissected and frozen section was performed.
C. The fluorescence level of FITC-BSA in frozen sections of heart tissue was observed under fluorescence microscope
D. ImageJ software was used to analyze the mean fluorescence intensity of myocardial vascular leakage.
2.3.4. A variety of pathological special staining experiments were performed on the myocardial tissue of the animal model.
HE staining, Masson staining, immunohistochemical CD163 staining and immunohistochemical CD34 staining were used to verify the myocardial cell injury, progressive fibrosis, macrophage infiltration and new capillary proliferation in the animal model, respectively.
2.4. Statistical methods
SPSS 22.0 software was used for statistical calculation of all data results. First, normal distribution was used to detect the result data. For the data conforming to normal distribution, two groups of data were tested by two independent samples t-test, and multiple groups of data were tested by one-way ANOVA. The non-parametric test method was used for the data that did not conform to the normal distribution. Statistical results were shown by calculating the mean±standard deviation, and a p-value less than 0.05 represented a statistically significant difference between the groups.