Ethics
The protocol was approved by the review committee of Chinese PLA General Hospital. All experiments were accorded with the principles stated in the guide for the Care and use of Laboratory animals (national institutes of health publication, 1985) and were authorized by Institutional Animal Care and Use Committee in China (grant no. SC2019-06-013).
Animal subjects
Thirty landrace pigs (approximately 3.5 months old, weighing 55±5kg), purchased from Beijing Shichuangshiji Mini-pig Breeding Base, were utilized for this study. Before the study, all swine were fed a standard diet and were observed for 7 days to adapt to the experimental environment. Before the surgery date, blood samples were collected from pigs and coagulation, and arterial blood gas indexes were measured to ensure these values were in the normal range. Animals were deprived of food 12 hours before anesthesia and water 2 hours before.
Animal grouping
Envelope randomization was utilized to ensure that each group had the same sample capacity. A total of 30 pigs were randomly divided into the following three groups: (1) a sham-operated (SHAM) group that underwent the same anesthetic and surgical procedures but without the conduct of gunshot (n=10), (2) a MAP decrease (MD) group in which the mean arterial pressure of the pigs dropped by 30% of the basal value before the application of Combat Gauze (n=10), and (3) a bleeding time (BT) group, in which 30 seconds of free bleeding was allowed (n=10).
Procedure
Anesthesia preparation:Swine were induced with an injection of Zoletil (10mg/kg, intravenous [i.v.], French virbac company, batch number: 76bp) and pumped to maintain anesthesia (0.03mg/kg/h, i.v.). After anesthetized, pigs were cut the hair in the operation area. Then the experimental pigs were intubated with combined endotracheal tube without mechanical ventilation.
Instrument preparation: All operations followed the principle of sterility. Before operations, all materials and instruments had been sterilized, including rubber gloves, venipuncture kit, Picco puncture kit, 5ml syringe, 20ml syringe, etc. While the pigs were in a supine position on the experimental console, Picco catheter was placed in the left femoral artery and double lumen catheter was placed in the left internal jugular vein under ultrasound guidance (Fig.1A and B). A bladder puncture was performed in pigs under ultrasound guidance (Fig.1C). After catheterization, the swine were instrumented to a Picco monitor (PULSION, PC8500). The pigs were placed on the test bench in a lateral position, with the right hind limb fixed vertically to ensure the inner thigh facing the shooter. After 15 minutes stationary, the blood samples were collected and the indexes of hemodynamics were recorded as the baseline.
Gunshot procedures: In order to improve the accuracy of femoral artery injury, a portable ultrasonic diagnostic instrument (VINNO Q Series-7L) was used to locate the right femoral artery and the midpoint of the descending territory of the artery in the ultrasound images was marked as the shooting point (Fig.1D and E). After 15 min from blood draw, the shooter, who were employed by the No. 208 Research Institute of China Ordnance, fired a common 9 mm along the ultrasonic probe longitudinal axis direction at 3 meters apart. The pistols and bullets used were also provided by this institute. Shootings from the entire experiment were completed by the same shooter, who was blinded to the overall experimental design and grouping.
Hemostatic treatment: After the shooting, the pigs were considered ready for the experiment when spurting bleeding occurred in the wound (Fig.1F). Combat gauze was applied to the bleeding site in accordance with the Tactical Combat Casualty Care Guidelines. First, Combat Gauze was used to fill up the active bleeding wound. Second, five pieces of sterile medical gauze were used to cover the packed wound for 3 minutes with well-distributed compression. Finally, an emergency bandage was used to dress the wound with appropriate pressure. All processes were performed by the same person in accordance with the Tactical Combat Casualty Care, and no fluid resuscitation or drug intervention was administered throughout the experiment. The animals were monitored under general anesthesia for 3 hours or until they died. Upon reaching the end point, a lethal dose (30ml) of 10% potassium chloride solution was administered intravenously as veterinary euthanasia solution.
Data collection
We recorded weight, hemodynamic, coagulation, and arterial blood gas indexes at baseline and 10min, 30min, 60min postinjury. All dressings and gauze used during the operation were weighed before use and afterward to determine the volume of blood loss. The pigs were observed until they died, and the time of deaths was recorded.
Blood analysis
A laboratory was prepared in the experimental site to test the blood specimens at a constant indoor temperature of 18℃-24℃. Venous blood was obtained via internal jugular vein catheterization, placed in heparinized syringes, and centrifuged at 3000r/min for 10 minutes. Activated clotting time of whole blood (ACT), Prothrombin time (PT), International normalized ratio (INR), Thrombin time (TT), Activated partial thromboplastin time (APTT), Fibrinogen (FIB) were measured using a semi-automatic coagulometer (Rayto, RAC-120) according to the manufacturer’s protocols. The arterial blood was obtained through femoral artery catheterization and put into syringe for blood gas analysis with automatic blood gas analyzer (Rayto, ABL-80).
Anatomy
The length of the entrance and exit of the wound were recorded (Fig.2A and B). A approximately 10 cm dissection in the groin area was then conducted with removal of the adductor muscle overlying the femoral canal, and the ruptures of femoral artery and vein were found and recorded (Fig.2C). The femoral fracture site was also recorded if there was a fracture of the femur (Fig.2C and D).
Statistical analyses
The statistical analysis in this study was based on the SPSS 26.0 software. All measurement data were in accordance with or approximated normal distribution after normality test. Sex of animals was evaluated using Pearson’s χ2 test. Single factor ANOVA was used for other indexes of animal baseline characteristics. Differences of free bleeding time, pre-/pro-tamponade blood loss, total blood loss and MAP decline were analyzed by Student's t test. The overall effect of each index over time was compared using repeated measures ANOVA and Greenhouse-Geisser test was used to correct the data that did not coincide with the sphericity test. Pairwise comparisons were performed using LSD-t post hoc tests, with a significance threshold of P<0.0167. Survival analyses were made by using Cox proportional hazards regression model. Significance for results was established when p-values were less than 0.05.