The study protocol was approved by the review committee of the Chinese PLA General Hospital. All experiments were performed in accordance 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 the Institutional Animal Care and Use Committee in China (grant no. SC2019-06-013).
Animal subjects
Thirty landrace pigs (aged approximately 3.5 months, weighing 55±5 kg), 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 indices were measured to ensure that these values were within the normal range. Animals were deprived of food and water for 12 h and 2 h before anesthesia, respectively.
Animal grouping
Envelope randomization was used 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 mean arterial pressure (MAP) decrease (MD) group, in which the MAP 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
The swine were induced with an injection of zoletil (10 mg/kg, intravenous [i.v.], French Virbac Company, batch number: 76bp) and pumped to maintain anesthesia (0.03 mg/kg/h, i.v.). After anesthetization, the pigs were cut into hair in the operation area. The experimental pigs were then intubated with a combined endotracheal tube without mechanical ventilation.
All operations followed the principle of sterility. Before surgery, all materials and instruments were sterilized, including rubber gloves, venipuncture kit, Picco puncture kit, 5 ml syringe, and 20 ml syringe, among others. While the pigs were placed in the supine position on the experimental console, a Picco catheter was placed in the left femoral artery and a double-lumen catheter was placed in the left internal jugular vein under ultrasound guidance (Fig 1A and B). Bladder puncture was performed in pigs under ultrasound guidance (Fig 1C). After catheterization, the swine were instrumented on 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 that the inner thigh faced the shooter. After 15 min, blood samples were collected and the indices of hemodynamics were recorded as baseline values.
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). Fifteen minutes after drawing blood, the shooter, who was employed by the No. 208 Research Institute of China Ordnance, fired a common 9-mm bullet along the ultrasonic probe longitudinal axis direction at 3 m apart. The pistols and bullets used were provided by this institute. All shots were performed by the same shooter, blinded to the experimental design and group assignment.
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 the active bleeding wound. Second, five pieces of sterile medical gauze were used to cover the packed wound for 3 min with well-distributed compression. Finally, an emergency bandage was used to dress the wound at an appropriate pressure. All processes were performed by the same person in accordance with 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 h or until they died. Upon reaching the end point, a lethal dose (30 ml) of 10% potassium chloride solution was administered intravenously as a veterinary euthanasia solution.
Data collection
We recorded weight, hemodynamic, coagulation, and arterial blood gas indices at baseline and at 10 min, 30 min, and 60 min post-injury. All dressing 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 death was recorded.
Blood analysis
A laboratory was prepared at the experimental site to test the blood specimens at a constant indoor temperature of 18–24 °C. Venous blood was obtained via internal jugular vein catheterization, placed in heparinized syringes, and centrifuged at 3000 r/min for 10 min. The activated clotting time of whole blood, prothrombin time (PT), international normalized ratio, thrombin time, activated partial thromboplastin time, and fibrinogen were measured using a semi-automatic coagulometer (Rayto, RAC-120) according to the manufacturer’s protocols. Arterial blood was obtained through femoral artery catheterization and placed into a syringe for blood gas analysis with an automatic blood gas analyzer (Rayto, ABL-80).
Anatomy
The length of the entrance and exit wounds were recorded (Fig 2A and B). A dissection of approximately 10 cm was performed in the groin area with removal of the adductor muscle overlying the femoral canal; the ruptures of the 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
SPSS software version 26.0 was used for statistical analysis. All measurement data met the normality of distribution assumption. The sex distribution of the animals was evaluated among the groups using the Pearson χ2 test. Single-factor ANOVA was used to compare all other baseline indices among the groups. Differences in free bleeding time, pre-/pro-tamponade blood loss, total blood loss, and the MAP decline were analyzed by Student's t-test. The overall change to each index over time was examined using repeated measures ANOVA. The Greenhouse-Geisser test was used to correct the data that did not coincide with the sphericity test. Pairwise comparisons were performed using the LSD post-hoc tests, with a significance threshold of P<0.0167. Survival analyses were performed using the Cox proportional hazards regression model. P-values of <0.05 were considered statistically significant.