Effect of SAM junctional tourniquet on respiration when applied in the axilla: A swine model

Abstract

Purpose

SAM junctional tourniquet (SJT) has been applied to control junctional hemorrhage. However, there is limited information about its safety and efficacy when applied in the axilla. This study aims to investigate the effect of SJT on respiration when used in the axilla in a swine model.

Methods

Eighteen male Yorkshire swines, aged 6-month-old and weighing 55 – 72 kg, were randomized into 3 groups, with 6 in each. An axillary hemorrhage model was established by cutting a 2 mm transverse incision in the axillary artery. Hemorrhagic shock was induced by exsanguinating through the left carotid artery to achieve a controlled volume reduction of 30% of total blood volume. Vascular blocking bands were used to temporarily control axillary hemorrhage before SJT was applied. In Group I, the swine spontaneously breathed, while SJT was applied for 2 h with a pressure of 210 mmHg. In Group II, the swine were mechanically ventilated, and SJT was applied for the same duration and pressure as Group I. In Group III, the swine spontaneously breathed, but the axillary hemorrhage was controlled using vascular blocking bands without SJT compression. The amount of free blood loss was calculated in the axillary wound during the 2 h of hemostasis by SJT application or vascular blocking bands. After then, a temporary vascular shunt was performed in the 3 groups to achieve resuscitation. Pathophysiologic state of each swine was monitored for 1 h with an infusion of 400 mL of autologous whole blood and 500 mL of lactated ringer solution. T_b and T₀ represent the time points before and immediate after the 30% volume-controlled hemorrhagic shock, respectively. T₃₀, T₆₀, T₉₀ and T₁₂₀, denote 30, 60, 90, and 120 min after T₀ (hemostasis period), while T₁₅₀, and T₁₈₀ denote 150 and 180 min after T₀ (resuscitation period). The mean arterial pressure and heart rate were monitored through the right carotid artery catheter. Blood samples were collected at each time point for the analysis of blood gas, complete cell count, serum chemistry, standard coagulation tests, etc., and thromboelastography was conducted subsequently. Movement of the left hemidiaphragm was measured by ultrasonography at T_b and T₀ to assess respiration. Data were presented as mean ± standard deviation and analyzed using repeated measures of two-way analysis of variance with pairwise comparisons adjusted using the Bonferroni method. All statistical analyses were processed using GraphPad Prism software.

Results

Compared to T_b, a statistically significant increase in the left hemidiaphragm movement at T₀ was observed in Groups I and II (both p < 0.001). In Group III, the left hemidiaphragm movement remained unchanged (p = 0.660). Compared to Group I, mechanical ventilation in Group II significantly alleviated the effect of SJT application on the left hemidiaphragm movement (p < 0.001). Blood pressure and heart rate rapidly increased at T₀ in all three groups. Respiratory arrest suddenly occurred in Group I after T₁₂₀, which required immediate manual respiratory assistance. PaO₂ in Group I decreased significantly at T₁₂₀, accompanied by an increase in PaCO₂ (both p < 0.001 vs. Groups II and III). Other biochemical metabolic changes were similar among groups. However, in all 3 groups, lactate and potassium increased immediately after 1 min of resuscitation concurrent with a drop in pH. The swine in Group I exhibited the most severe hyperkalemia and metabolic acidosis. The coagulation function test did not show statistically significant differences among three groups at any time point. However, D-dimer levels showed a more than 16-fold increase from T₁₂₀ to T₁₈₀ in all groups.

Conclusion

In the swine model, SJT is effective in controlling axillary hemorrhage during both spontaneous breathing and mechanical ventilation. Mechanical ventilation is found to alleviate the restrictive effect of SJT on thoracic movement without affecting hemostatic efficiency. Therefore, mechanical ventilation could be necessary before SJT removal.

Introduction

Massive bleeding is the leading cause of death on the battlefield. Up to 90% of preventable deaths on the battlefield are caused by bleeding, and more than 1/3 of those cases are due to junctional injuries.^1,2 As it is routinely outside the range of body armor, the axilla is also known as the bare protective area, where large blood vessels pass through. However, due to the unique anatomical location, a conventional limb tourniquet cannot control bleeding, which is the most difficult hemostatic problem today. Axilla bleeding is one of the most challenging types of hemorrhage, with an extremely high mortality rate due to poor access to the isolated hemorrhage in a prehospital setting. The subclavian vessels and their shoulder branches, including the subclavian, axillary, and thoracoacromial artery, as well as brachial arteriovenous, are the primary sources of bleeding in the axilla. Patients with traumatic upper arm amputations may have a stump that is too short to apply a tourniquet, and as such, this type of bleeding should also be treated as an axillary hemorrhage. In a prehospital setting, the difficulty in effectively controlling hemorrhage at the axilla leads to a high mortality rate.

Currently, prehospital treatment for axillary hemorrhage is to seat the wounded and apply hemostatic and compression dressings at the bleeding site. There are 4 devices for external hemorrhage control that can be applied to the junctional site: abdominal aortic and junctional tourniquet, combat ready clamp, junctional emergency treatment tool, and SAM junctional tourniquet (SJT).³ Furthermore, only 3 of them (abdominal aortic and junctional tourniquet, combat ready clamp, and SJT) were approved by the Food and Drug Administration for controlling axillary hemorrhage.

However, current junctional hemostatic devices can restrict thoracic movement and affect breathing when used in the axilla, which is also a concern in clinical use.⁴ Therefore, this study is designed to experimentally verify the effect of SJT on respiration in a swine model of axillary hemorrhage with different respiratory states.

Methods

SJT instructions for the axilla in a swine model

Considering the different anatomical structures between swine and humans, the axilla was dissected to explore the optimal binding strategy in the swine model. As shown in Fig. 1A–D, we developed an SJT instruction for the axilla in the swine model. SJT was applied to the left axilla as shown in Fig. 1E & F.

Fig. 1.

SAM junctional tourniquet (SJT) instructions for the axilla in the swine model. (A) Place SJT loosely around the upper thorax of the swine and place a 25 mm-diameter pressure sensor between the target compression device (TCD) surface and the swine to measure the pressure exerted by SJT. Then, position the TCD over the area to be compressed; (B) Connect the belt using the buckle after the TCD is held in place, and secure the buckle to hear the first “click”; (C) Press the extra belt on the Velcro to hear the second “click”; (D) Manually inflate SJT via using the hand pump to control hemorrhage; (E) Swine applicated with SJT pressure sensor and TCD; and (F) Swine before SJT application.

Experiment animals

The study was approved by the Ethics Committee of the Army Medical University (animal experiment number: AMUWEC20201513) and strictly followed the international and national animal testing regulations. The Animal Care and Use Committee, Experimental Animal Center of Daping Hospital, Army Medical University provided all swine. Eighteen male Yorkshire swines, aged 6 months old and weighing 55 – 72 kg were divided into 3 groups using the random number method (n = 6 for each group). All swines were housed in a facility with a temperature of 24 °C and a humidity of 60%. Before the experiment, the swines were fasted for 8 h and had free access to water.

Experimental design

SJT was tested in Groups I and II, while Group III underwent the same surgical procedures but no SJT application. Anesthesia was induced by intramuscular injection of 20 mg/kg ketamine (Gutian Pharmaceutical Co., Ltd., Fujian, China) in the 3 groups.⁵ Groups I and III were allowed to breathe spontaneously and anesthetized by continuous intravenous administration of 4 mg/kg/h of propofol (propofol medium and long chain fat emulsion injection, Guorui Pharmaceutical Co., Ltd., Chengdu, China) and 4 μg/kg/h of remifentanil (Ruijie, Renfu Pharmaceutical Co. Ltd., Wuhan, China).⁵ Tracheostomy was performed in Group II, and a 7.0 mm tube (Shiley, COVIDIEN, Minneapolis, USA) was inserted for mechanical ventilation. The ventilator was set to the intermittent positive-pressure ventilation mode, with a tidal volume of 5 – 10 mL/kg adjusted to maintain PaCO₂ between 35 – 40 mmHg. General anesthesia was maintained with a mixture of isoflurane and 30% oxygen in Group II.

The swine was kept in a supine position during the experiment. Intravenous access was established via catheterization. The right carotid artery was catheterized with a 4.0 Fr arterial catheter (Braun, Hessen, Germany) to monitor the mean arterial pressure (MAP) and heart rate (HR). The left jugular vein was cannulated to collect venous blood samples for analysis of biochemical metabolism (complete cell count, serum metabolites and chemistry, standard coagulation tests, and thromboelastogram). The left carotid artery was catheterized to collect arterial blood samples for analysis of arterial blood gas. Baseline parameters were recorded before isolating a 6 cm segment of the left axillary artery, where two vascular blocking bands were placed at the distal and proximal ends. Due to the traction of the blocking bands, the blood flow was temporarily blocked. A 2 mm incision, approximately 1/3 of the vessel's circumference, was made using a scissor. A 30% volume-controlled hemorrhagic shock was induced by drawing blood through the left carotid artery at a speed of 1 mL/kg⁻¹/min⁻¹. The process lasted for 20 min, followed by a 10 min stabilization period. SJT was then loosely placed around the upper thorax of the swine, and target compression device (TCD) were attached to the pneumatic balloon. The axilla wound was packed with 5 sheets of gauze (6 cm × 8 cm). After releasing the vascular blocking bands, the blood spurted out and was quickly controlled by SJT inflation. Furthermore, the inflated SJT was placed on the left axilla for 2 h and observed in the following experiment to prevent rebleeding. Before SJT deflation, 2 vascular bands were pulled again to control bleeding. After SJT removal, a temporary vascular shunt was performed to achieve resuscitation. Each swine was monitored for 1 h with an infusion of 400 mL of autologous whole blood and 500 mL of lactated ringer solution. T_b and T₀ represent the time points before and after a 30% volume-controlled hemorrhagic shock, respectively. T₃₀, T₆₀, T₉₀, T₁₂₀, T₁₅₀, and T₁₈₀ denote 30, 60, 90, 120, 150, and 180 min after T₀. Blood samples were collected at each time point. Movement of the left hemidiaphragm was measured by ultrasonography at T_b and T₀ to assess respiration. After the experiment, all swines were euthanized. The experimental protocol is shown in Fig. 2.

Fig. 2.

Experimental protocol. (A) The algorithm of the experiment; and (B) Experimental flow chart.

SJT: SAM junctional tourniquet; TVS: Temporary vascular shunt.

Measurement of diaphragm movement

Ultrasonography was adopted to measure the movement of the left hemidiaphragm at T_b and T₀. The subcostal approach was used for ultrasonic visualization of the diaphragm. The left hemidiaphragm dome position changes could be followed by a selected sound path with a beam angle ≥ 70° in the M-mode. The excursion amplitude of the left hemidiaphragm during respiration was measured 4 times, and the highest waveform was chosen (Fig. 3).

Fig. 3.

Movement of the left hemidiaphragm was measured by M-mode ultrasonography.

Statistical analysis

Data are expressed as mean ± standard deviation. Blood test results were analyzed using repeated measures of two-way analysis of variance with pairwise comparisons adjusted using the Bonferroni method. All statistical analyses were processed using GraphPad Prism software (version 6.01, GraphPad Software, Inc., USA). A p < 0.05 was considered statistically significant.

Results

General results

No complications related to anesthesia or surgery occurred during the instrumentation to create a hemorrhagic shock model or SJT application. All swines survived after T₀. The compression pressure of 210 mmHg was effective in hemostasis with less than 55 mL of local free blood loss during the 2 h of SJT application in Group I and II.

Diaphragm movement

The movement of the left hemidiaphragm at T_b showed no statistically significant difference among 3 groups (p > 0.05). After the 30% volume-controlled hemorrhagic shock was successfully established at T_0, the left hemidiaphragm movement showed a significant increase in Groups I and II (both p < 0.001, but remained unchanged in Group III (p = 0.660).

Compared to Group I, mechanical ventilation in Group II significantly alleviated the effect of SJT application on the left hemidiaphragm movement (p < 0.001). Detailed data are shown in Table 1.

Table 1.

Diaphragm movement of the left hemidiaphragm in swine after 30% volume-controlled hemorrhagic shock in Groups Ⅰ - Ⅲ (n = 6 for each group)

Group	Diaphragm movement at Tb (cm)	Diaphragm movement at T0 (cm)	p value
Ⅰ	1.18 ± 0.13	1.98 ± 0.13b	< 0.001
Ⅱ	1.42 ± 0.01	1.72 ± 0.02a,b	< 0.001
Ⅲ	1.15 ± 0.08	1.17 ± 0.11	0.660

Note: Group Ⅰ: Spontaneous breathing swine with SAM junctional tourniquet application; Group Ⅱ: Mechanical ventilation swine with SAM junctional tourniquet application; Group Ⅲ: Spontaneous breathing swine with vascular blocking bands controlling the axillary hemorrhage. The diaphragm movement was measured by M-mode ultrasound at the end-expiration and end-inspiration. T_b and T₀ represent the time points before and after the 30% volume-controlled hemorrhagic shock, respectively.

^ap < 0.001 compared to Group Ⅰ;^bp < 0.001 compared to Group Ⅲ.

Hemodynamic parameters

MAP in Groups I, II, and III decreased by 28%, 30%, and 26%, respectively, from T_b to T₀. In contrast, HR in the 3 groups increased by 38%, 35%, and 41%, respectively. The MAP in all three groups sharply increased and then gradually returned to baseline levels from T₀ to T₁₂₀, concurrently with slow growth in HR. After T₁₂₀, MAP in these 3 groups sharply decreased (Fig. 4A), stimulating a more significant increase in HR (Fig. 4B). The tachycardia persisted until the end of the experiment. There were no significant differences in respiratory rate between the 3 groups, but the respiratory rate changed noticeably in Groups I and III from T₀ to T₁₂₀. Respiratory arrest suddenly occurred at T₁₂₀ in Group I, which required 10 min of immediate manual temporary respiratory assistance. One out of 6 swines in Group I died from respiratory arrest 40 min after T₁₂₀.

Fig. 4.

Hemodynamic changes in Groups Ⅰ, Ⅱ and Ⅲ. (A) Mean arterial pressure (MAP) and (B) heart rate (HR); (C) The arterial partial pressure of PaO₂ and (D) PaCO₂ gases. MAP in Groups Ⅰ, Ⅱ and Ⅲ from T_b to T₀ was accompanied by an increase in HR in the 3 groups. From T₀ to T₁₂₀, MAP sharply increased and then gradually returned to baseline levels, concurrently with slow growth in HR. However, after T₁₂₀, MAP sharp decreased, while HR significant increased. In addition, the blood gases changed significantly at T₁₂₀, particularly in Group Ⅰ (p < 0.001 vs. Groups II and III), and then returned to baseline levels from T₁₂₀ to T₁₈₀.

The changes of PaO₂ and PaCO₂ are shown in Fig. 4C & D. PaO₂ and PaCO₂ remained unchanged from T_b to T₁₂₀, despite the different respiratory modes of the 3 groups. However, PaO₂ in Group I decreased significantly at T₁₂₀, accompanied by an increase in PaCO₂ (both p < 0.001 vs. other groups). PaO₂ and PaCO₂ gradually returned to baseline levels from T₁₂₀ to T₁₈₀.

Biochemical metabolic parameters

As shown in Fig. 5A–C, potassium, lactate concentrations increased sharply at T₁₂₀ in all 3 groups, accompanied by a sharp drop in pH. These changes were most significant in Group I (both p < 0.001 vs. other groups) and gradually returned to baseline levels in all surviving swines. As shown in the Fig. 5D, blood urea nitrogen gradually increased in the 3 groups from T_b to T₁₈₀, which was statistically significant (all p < 0.001). The kidney function measurements showed a slight increase in serum creatinine concentrations at T₁₈₀ compared to T₁₂₀ (Fig. 5E), but were not statistically significant (all p < 0.05). Furthermore, hematocrit increased significantly in Groups I, II, and III from T₁₂₀ to T₁₈₀ (all p < 0.001), respectively. (Fig. 5F). The increase in hematocrit co-occurred with a decrease in platelet counts from T₁₂₀ to T₁₈₀ (all p < 0.001) and was statistically significant in the 3 groups (Fig. 5G). As shown in the Fig. 5H & I, creatine kinase (all p < 0.001) and aspartate aminotransferase (all p < 0.001) were significantly elevated from T₁₂₀ to T₁₈₀ in the 3 groups. No statistically significant differences existed in standard clotting tests throughout the experiments (data not shown). However, D-dimer measurements showed a more than 16-fold increase from T₁₂₀ to T₁₈₀ in all 3 groups (all p < 0.001), which was statistically significant (Fig. 5J).

Fig. 5.

Metabolic changes in blood samples collected at different time points (T_b, T₀, T₃₀, T₆₀, T₉₀, T₁₂₀, T₁₅₀ and T₁₈₀) during the experiments. (A) Potassium; (B) Lactate concentrations; (C) pH; (D) Blood urea nitrogen; (E) Serum creatinine concentration; (F) Percentage of hematocrit; (G) Platelet count; (H) Creatine kinase; (I) Aspartate aminotransferase enzymatic activities; and (J) D-dimer.

∗p < 0.001 comparison between T_b and T₁₈₀; #p < 0.001 comparison between T₁₂₀ and T₁₈₀.

Of the measured thromboelastogram parameters, the clot initiation, the clot polymerization and clotting angle did not vary significantly over time in the 3 groups (Fig. 6A–C). However, clot strength increased significantly in Group I (p < 0.001), Group II (p = 0.003), and Group III (p < 0.001) from T₀ to T₆₀. The changes were concomitant with a significant decrease in the clotting index (all p < 0.001), as shown in Fig. 6D & E.

Fig. 6.

Thromboelastogram changes in blood samples collected at different time points (A) Clot initiation; (B) Clot polymerization; (C) Clotting angle; (D) Clot strength; and (E) Clotting index.

#p < 0.001 comparison between T₀ and T₆₀.

Discussion

This research explored the effect of applying SJT on respiration when used in the axilla in a swine hemorrhagic shock model in 2 respiratory modes: spontaneous breathing and mechanical ventilation. The former mode mimics the prehospital setting, while the latter is often used in patients with severe hemorrhagic shock. It remains unclear whether mechanical ventilation affects the hemostatic effect of SJT and its efficacy in patients with both respiratory states. The sequence of mechanical ventilation and SJT deflation in patients with axillary hemorrhage combined with severe hemorrhagic shock is also unclear. Therefore, this research established an axillary injury model for axillary hemorrhage (Class III, 30% volume-controlled hemorrhagic shock). The primary hemodynamic response to SJT application was a sharp increase in blood pressure compared to the baseline levels. This hypertensive response was expected due to volume-controlled blood loss and increased peripheral vascular resistance in compressed tissues. In Group I, SJT application had an external compression effect on the thoracic side, leading to increased intrathoracic pressure, which was compensated by increased left-side diaphragm movement on the compressed side. In contrast, this effect was alleviated by mechanical ventilation in Group II. We assumed this was because mechanical ventilation extended the thoracic wall against the external compression pressure. SJT application was tolerated in spontaneous breathing swine, despite the external compression pressure on the thorax.

However, when SJT was deflated, swines in Group I suffered sudden respiratory arrest and cyanosis. Previous research showed that this emergency required manual positive-pressure ventilation assistance until the swine resumed spontaneous respiration.^6,7 One out of 6 swines died, while the rest survived until the end of the experiment. We assume that the cause of respiratory and cardiac arrest was sudden hyperkalemia. High plasma potassium may have been due to the noticeably changed respiratory rate of swine in Group I during SJT application. A sudden increase in respiratory rate can lead to acute hyperventilation and a net potassium efflux from cells. Other possible causes include hemolysis of vascular epithelial cells induced by axillary artery incision and traumatic vascular cannulation during surgical preparation. Potassium can also be released due to cellular damage caused by tourniquet compression. There were other abnormal biochemical metabolic changes during resuscitation in Groups I, II and III. The sharp increase in hematocrit during resuscitation suggested increased vascular permeability and plasma leakage into interstitial space. These changes could potentially compromise blood circulation in the upper extremities. The 16-fold increase in D-dimer and decrease in platelet counts indicated fibrin deposition and intravascular thrombosis.

The present experiment had several limitations. Firstly, we only measured differences in diaphragm movement. Further research on the effect of SJT on thoracic wall movement is required. Secondly, we did not perform a kidney autopsy to confirm a presumed kidney injury. A previous study showed that tourniquet-induced injury/reperfusion of the upper extremities affected the localized skeletal muscle and distant organs such as the kidneys.⁸ The mechanisms of this remote response to injury/reperfusion are related to microvascular dysfunction.9, 10, 11 In our research, slight kidney damage was highly suspected due to increased blood urea nitrogen and serum creatinine concentration. Lastly, we did not use the SJT shoulder straps at the axilla in the swine model. According to the SJT instruction manual in the human axilla, the TCD is attached to the extender, which is placed on the strap of the brown Velcro.^1,12 The strap is then connected to the D-ring of the SJT using the large clip. However, this method does not apply to swine. We dissected the axilla of 2 swine in the preliminary experiment and found that the collarbone was absent. Due to the absence of a bony structure around the axilla, shoulder straps cannot be used. Instead, the oblique binding method was adopted in this experiment.

SJT controlled the axillary hemorrhage in the swine model under both spontaneous breathing and mechanical ventilation conditions. Mechanical ventilation could alleviate the restricted effect of SJT on thoracic movement without affecting hemostatic efficacy. Mechanical ventilation could be necessary before the removal of SJT. However, the resulting ischemia-induced metabolic derangements or ischemia reperfusion injury were life-threatening for spontaneously breathing subjects. Upon SJT deflation, immediate cardiopulmonary resuscitation and mechanical ventilation appear to be needed to avoid possible respiratory arrest.

Funding

1. Clinical Technology Innovation and Cultivation Project, Army Medical University, number (CX2019JS109). 2.Innovative Project of Daping Hospital for Clinical Medicine, Daping Hospital (number 2019CXLCA002). 3.Innovation Developing Program of Army Medical University, Daping Hospital (number2021XJS27).

Ethical statement

All procedures were approved by the Ethics Committee of the Army Medical University, and all animals were provided by Animal Care and Use Committee, Experimental Animal Center of Daping Hospital, Army Medical University. Ethical approval for animal experimentation number: AMUWEC20201513.

Declaration of competing interest

The authors declare that they have no conflicts of interest.

Author contributions

Dong-Chu Zhao collected and analyzed the data and drafted the original manuscript. Yang Li designed the experiment protocol, reviewed and edited the manuscript. Yong Guo and Hua-Yu Zhang contributed to the implementation of the current experiment and the interpretation of the results. Lian-Yang Zhang supervised the whole experimental process. All authors read and approved the final manuscript.