Background: Closed intra-abdominal hemorrhage remains the increasing leading cause of death both on the battlefield of modern warfare and in the civilian environment.
Objective: Developing a temporary hemostatic device to control of closed intra-abdominal hemorrhage, and to determine the hemostatic effect in a swine model.
Methods: A swine model of closed, lethal liver injury was established. The animals were randomly divided into group A (blank control), group B (gauze pack), group C (intra-abdominal compression by the device), group D (drug injection by the device), and group E (drug injection and intra-abdominal compression by the device) with 4 swine per group. Survival time, blood loss, and vital signs were monitored for all groups, and histologic examination after death was performed. One swine in group E was given liver CT-enhanced-scan at 4-time points to observe the device application.
Results: Group E had a significantly longer survival time and less blood loss than groups A, C, and D, while there were no statistical differences between groups B and E, both of which had more stable mean arterial pressure and cardiac output curves after liver injury. A histologic examination after death revealed no obvious secondary injuries in other visceral organs. Liver CT-enhanced-scan showed that intrahepatic hemorrhage was controlled to a certain extent.
Conclusion: This device prolongs survival time and controls blood loss in a swine model of closed lethal liver injury, which is equivalent to the gauze packing under this model with no obvious thrombosis or embolus formation in other organs.
Despite advances in medical treatment and protective equipment, uncontrolled bleeding remains the leading cause of death in potential survivors (PS) on the battlefield in modern warfare [1, 2], as well as one of the leading causes of death in the civilian environment [3, 4]. In recent years, with improvements in first-aid technology and evacuation strategies, and the extensive use of tourniquets and hemostatic agents, the mortality caused by massive hemorrhage involving the extremities and body junctions have decreased, while the mortality caused by closed intra-abdominal hemorrhage has significantly increased [5-7]. US military war injury data has shown that closed intra-abdominal hemorrhage accounts for > 50% of PS, and most of the casualties die before reaching a Level II military medical treatment facility [2, 8]. An epidemiologic analysis of civilian trauma also showed that the mortality caused by closed intra-abdominal hemorrhage was approximately 44.5%, and the delay in definitive surgery was positively correlated with mortality [7, 9].
Hemostatic agents include zeolite, chitosan, and kaolin, which are used as part of a temporary and rapid hemostasis strategy. Hemostatic agents are widely used in hemostasis caused by war injuries involving the extremities and body junctions, and the efficacy of hemostatic agents has been confirmed by many reports [10, 11]. Celox with chitosan as the main ingredient is an ideal hemostatic agent and can be used in cardiothoracic surgery without any apparent side effects . Closed intra-abdominal hemorrhage is mostly caused by blunt trauma that leads to abdominal organ injury. It is not possible to achieve hemostasis by tamponade or compression during the evacuation, as in other open injuries. Truly effective treatment requires quick transfer to a treatment facility for surgical intervention to control bleeding. A considerable number of casualties have shock due to the inability to control abdominal bleeding [5, 9]. However, direct application of hemostatic agents to closed intra-abdominal hemorrhage may not result in good hemostasis due to insufficient contact with the bleeding site. Based on the above, we have developed a temporary hemostasis device that combines Celox particles and high-expansion, compressed sponge tablets containing Celox ingredients that together with direct intra-abdominal compression will control closed intra-abdominal hemorrhage during evacuation and buy time for casualties to undergo definitive surgery in the hospital. In this paper, the hemostatic effect of the device was verified in a swine model of closed, lethal liver injury.
Introduction of the hemostatic device
In response to the characteristics of closed intra-abdominal hemorrhage, we developed an injectable hemostasis device. The device is equipped with a double-cavity structure (Figures 1-1 and 1-2) and an injection cavity prefilled with procoagulant hemostatic particles (Celox, 20 g; Med trade Biopolymers, Crewe, United Kingdom). Celox is distributed in the United States by SAM Medical Products (Portland, OR) and uses chitosan produced by the deacetylation of chitin, which is derived from the exoskeleton of shrimp. The mechanism of action for Celox is the formation of an adhesive complex upon exposure of positively charged Celox to negatively charged red blood cells . Celox is an entirely biodegradable and biocompatible substance that has been shown to function independently of coagulation factors, based on proprietary research . The other injection cavity is prefilled with a high-expansion, compressed sponge tablet containing Celox ingredients (20 pieces, 10 × 10 × 1 mm, each containing about 0.4 g of Celox; Guangzhou Master Meditech CO., LTD., Guangzhou, China), which can expand 25 times after absorbing blood, is tough after expansion, and binds to red blood cells as well as oppresses bleeding. The device is equipped with a blade for opening the abdomen, and an inflatable balloon (maximum, 15 × 15 cm) for pressing the injury directly after injection of the drug (Figures 1-3 and 1-4).
Establishment of a swine model of closed, lethal liver injury
Swine were observed for 3 days to ensure a good state of health, and were fasted for 12 h and had no access to water for 4 h before surgery. After anesthesia with ketamine (30 mg/kg) and atropine (0.04 mg/kg), the swine were placed in the supine position, the head and limbs were fixed, an intravenous indwelling needle was placed in the ear vein, and anesthesia was maintained by the intravenous injection of 3% sodium pentobarbital (6 mg/h/ kg). Hair was scraped and skin was disinfected on the abdomen, neck, and groin bilaterally. The skin and subcutaneous tissues were dissected in the groin, and the right femoral artery and vein were separated with single-lumen vascular catheters. The femoral artery catheter was connected to the sensor and a Philips IntelliVue MP60 monitoring system (Boeblingen, Germany) by a tee catheter for real-time monitoring of mean arterial pressure (MAP), systolic blood pressure, diastolic blood pressure, heart rate, and cardiac output (CO). The femoral vein catheter was used as an injection channel for CT-enhanced contrast agent, a blood collection channel, and an infusion channel during resuscitation. The body temperature was measured through the anus.
According to the liver injury model reported by Duggan et al. , a closed lethal liver injury swine model was made with two cutting lines (Figure 2). The abdomen was opened through a mid-abdominal incision. One line was perforated within 1 cm of the lateral margin of the left and right medial lobes at approximately 10 cm from the distal tip of the organ, such that pulling the line would result in a full transaction of the left and right medial liver lobes. Another line was positioned approximately 10 cm from the distal tip running anteroposteriorly along the midline between the right and left medial lobes and perforating the intrahepatic portal vein. Finally, two lines were attached to the distal tip of the medial liver lobes to allow retraction of the transected lobes after the initial injury. All lines were externalized through the laparotomy incision. Routine abdominal fascial closure was performed using no.1 nylon suture (Ethicon, Somerville, NJ, USA). According to the definition of the American Association of the Surgery of Trauma (AAST), pulling the cutting lines resulted in a standardized grade IV liver injury in our research .
All animal procedures were performed in accordance with protocols approved by the Third Military Medical University Institutional Animal Care and Use Committee. Twenty-three healthy local adult male swine, weighing 50.8 ± 2.0 kg, were used. Three swine that died within 10 min after anesthesia were excluded from the study. Twenty swine were randomly divided into 5 groups (4 per group) and the corresponding treatment was performed 5 min after the injury. The hemostasis interventions were completed by the same person. Group A was given extra- abdominal pressure for 10 min. The abdomen was opened again in group B through the mid-abdominal incision, and the liver was packed with medical absorbent gauze (length [8 cm] × width [6 cm] × number of layers , with 4 pieces on the top and bottom and 2 pieces on the left and right). The pressure was applied to the liver 10 min before closing the abdomen. In groups C, D, and E, the blade of the device was used to open the abdomen under the rib of the right clavicle. The incision was suitable for the hemostatic device to be placed in the abdominal cavity. Group C received intra-abdominal compression alone for 10 min with the inflated balloon of the device. Celox particles (20 g) and high-expansion, compressed sponge tablets containing Celox ingredients (20 pieces) alone were distributed as evenly as possible around the liver in group D. On the basis of group D, group E also received intra-abdominal compression for 10 min with the inflated balloon of the device. The incisions in groups C, D, and E were sutured after the operation. The compression pressure in the four groups (A, B, C, and E) was applied by the same person. According to Christen et al. , extra-abdominal pressure (50 mmHg) is equivalent to the intra-abdominal pressure (11-13 mmHg) during laparoscopic surgery. Therefore, we chose to apply 50 mmHg (1 mmHg = 0.133 kPa) of pressure in our experiments.
Death was defined as apnea and asystole for 5 continuous min. Five hundred milliliters of 6% hetastarch was infused 5 min after the liver injury. From 10 min before liver injury to animal death, the heart rate, systolic blood pressure, diastolic blood pressure, respiratory rate, pulse, rectal temperature, MAP, and CO were recorded every 10 min. After each animal died, the amount of blood loss was estimated by collecting the intra-abdominal blood, including blood in the abdominal cavity, free blood clots, and blood clots in the wound (volume = weight / 1.05, pig blood density was 1.05 kg/m³), then the liver, spleen, pancreas, lungs, heart, kidneys, and other tissues of the animal were extirpated for pathologic examination. Select one animal in group E to have a liver CT-enhanced-scan at 10 minutes before modeling, 5 minutes after modeling, 30 minutes after device application, and 60 minutes after device application, respectively.
The data were analyzed using SPSS 22.0 statistical software. The measurement data were expressed as the mean ±SD. Multiple sample means were compared using one-way ANOVA, and the LSD test was used as a post hoc test for pairwise comparisons of group means following ANOVA. Differences were statistically significant at a P < 0.05.
Survival time and blood loss
As shown in (Table 1), there was no significant difference in body weight among all groups (F = 022, P = 0.883). A comparison of the five groups revealed significant differences in mean survival time (F = 7.32, P = 0.001). Furthermore, comparisons of pairs between groups showed that the mean survival time of groups B and E were longer than those of groups A and D (A vs. B, P = 0.001; A vs. E, P = 0.004; B vs. D, P = 0.001; D vs. E, P = 0.003), while group B was also better than group C (B vs. C, P = 0.039). There was no significant difference between groups B and E. A comparison of the five groups showed significant differences in mean blood loss (F = 5.49, P = 0.006). Comparisons of pairs between groups of all groups showed that the mean blood loss of groups B and E was less than those of groups A and D (A vs. B, P = 0.003; A vs. E, P = 0.005; B vs. D, P = 0.004; D vs. E, P = 0.009). There was no significant difference between groups B and E.
The vital parameters were disordered from 10 minutes before liver injury to animal death in all groups. We mainly observed changes in the MAP and CO in each animal. As shown in Figs. 3 and 4, the changes in MAP and CO were more stable in groups B and E than groups A, C, and D. The MAP was maintained at approximately 60 mmHg and the CO was maintained at approximately 2.5 L/min 50 min after liver injury in groups B and E.
A pathologic examination of the main organs and tissues was performed for each animal. no thrombus or emboli was present in the spleen, pancreas, lungs, heart, and kidneys. Examination of blood clots showed that compared with common blood clots, the Celox blood clot in group E had increased red thrombus, a small gap, and tight connection between the clot and Celox (Figure 5). Examination of liver stump showed that compared with group A, group E had hepatic lobule destruction, hepatic congestion, central vein destruction, hepatic sinus congestion, and leukocyte infiltration (Figure 6).
As shown in (Figure 7), the same liver cross-sectional observations were taken at 4 time points, from 10 minutes before modeling, to 5 minutes after modeling, and to 30 minutes after device application, the maximum diameter of hepatic subcapsular hemorrhage gradually increased (0 mm vs 6.63 mm vs 18.69 mm); and as well as the infiltration of intrahepatic hemorrhage was also increasing. Comparison from 30 minutes to 60 minutes after device application, the maximum diameter of hepatic subcapsular hemorrhage did not change significantly (18.69 mm VS 18.84 mm), and the infiltration of intrahepatic hemorrhage was not increasing significantly, and there is an obvious leakage of CT contrast agent in the hepatic subcapsular at 60 minutes after device application.
Tactical combat casualty care of the US military requires that tourniquets be applied and hemostatic agents be effectively used to quickly control major bleeding involving the extremities and body junctions as first-aid maneuvers on the battlefield [14, 18]. Rapid and massive loss of blood not only leads to immediate danger, but can also lead to hypotension, coagulopathy, acidosis, infection, and multiple organ failure, and these complications result in an increase in mortality, even after successful resuscitation . Therefore, it is especially important to control blood loss caused by major bleeding. In the case of closed intra-abdominal hemorrhage on the battlefield or in the pre-hos-pital treatment environment, it is impossible to carry out definitive surgery to stop bleeding. How to maintain the life of the closed intra-abdominal hemorrhage casualty during evacuation has become one of the foci of trauma treatment in recent years. Based on current research, the main treatments are resuscitative endovascular balloon occlusion of the aorta (Reboa) and self-expanding poly(urea) urethane foam (Sepuf).
Reboa directly occludes the aorta in the blood vessel through an inflated balloon to stop bleeding, and there are risks of vascular injury, embolism, ischemia, and even multiple organ failure . Although research has shown that the use of Reboa for intra-abdominal hemorrhage can benefit 19% of the casualties, 27% of such casualties who did not use Reboa died during evacuation, and 12% died after arriving at the treatment facility [21, 22], but further clinical evidence and related operational practices and guidelines need to be established [20, 23].
Sepuf is polymerized into a solid by injecting two liquid substances into the closed abdominal cavity to provide intra-abdominal compression and stop bleeding. Currently, the effectiveness, dose selection, and long-term safety of Sepuf in an animal model have been confirmed, and the only side effect is bowel injury [24-26]. Similar to research conducted in humans, the recommended dose and effectiveness is preliminary [27, 28]; however, Sepuf has not been approved by the Food and Drug Administration (FDA), and safety for human use remains to be determined.
Although hemostatic agents are only temporary hemostasis measures, hemostatic agents cannot completely replace the role of surgical repair, but rapid control of bleeding can enable evacuation and subsequent treatment of the casualty; a number of products have received FDA approval. Of the soldiers who died from hemorrhage in Vietnam, 40% had bleeding that could have been controlled with a hemostatic agent . Compared with hemostatic agents, such as kaolin, zeolites and montmorillonite, Celox with chitosan as the main component has been evaluated as an ideal hemostatic agent (presented by Pusateri et al.  for civilian and military use. In a number of reports, Celox has the same or better hemostatic effect than other types of hemostatic agents, and Celox has a non-thermal reaction with an antibacterial effect, and thus can function under conditions of low temperature and coagulation disorders, is easy to remove, and is low in cost [10, 11, 30]
The temporary hemostasis device described in this paper is to achieve hemostasis of closed intra-abdominal hemorrhage by two means: (1) Celox particles and high-expansion, compressed sponge tablets containing Celox ingredients; and (2) direct intra-abdominal compression. We established the experimental group: group C (intra-abdominal compression by the device), group D (drug injection by the device), and group E (drug injection and intra-abdominal compression by the device). Based on the survival time and blood loss results, there was no significant difference between groups D and A, which may be because Celox mainly occludes the blood vessel through the clot, but did not come in close contact with the bleeding site in the abdomen. Although group C was superior to groups A and D with respect to mean survival time and mean blood loss, the corresponding MAP and CO curves of group C were more gradual and not statistically different. This finding may be because the tissue in the abdomen is relatively soft and cannot stop bleeding with pressure. While group E combined with the two means performed better with respect to survival time and blood loss. Although the animals did not completely recover, the MAP and CO remained in a relatively normal state 50 min after the injury in group E. The hemostatic effect was basically equivalent to the effect of gauze packing. Based on the pathologic examination, the short-term use of the device did not increase the risk of thrombosis and emboli formation in other organs. Meanwhile, liver CT-enhanced-scan at 4 time points in one animal showed that intrahepatic hemorrhage was controlled to a certain extent through the device application.
The research had the following limitations: (1) Animal studies are not universally accepted to reflect the effect of hemostatic agents on wounds in humans, and the limitation of sample size also led to a lack of a deeper analysis for effectiveness and safety (2). Otrocka-Domaga?a et al.  reported that the long-term (24 h) use of Celox in a model of porcine femoral artery injury may result in residue entering the systemic circulation and increasing the risk of emboli formation. We should also assess the long-term effects to clarify the safe time range for the device (3). The current device is a combination of Celox particles and high-expansion, compressed sponge tablets containing Celox ingredients. In order to meet the needs for multiple complex injuries of closed intra-abdominal massive hemorrhage, we should choose more hemostatic drug combinations as alternatives to achieve the desired hemostatic effect (4). The value and time of pressure performed by compression of the device in the abdomen requires further research to find a safe and appropriate range, and the design of the device should be improved to achieve automatic compression and time monitoring.
The temporary hemostasis device designed in this paper was filled with Celox particles and high-expansion, compressed sponge tablets containing Celox ingredients and direct intra-abdominal compression was performed to control the closed intra-abdominal hemorrhage. The efficacy of the device was verified by experiments in a swine model of closed, lethal liver injury; which can prolong survival time and control blood loss and is basically equivalent to the hemostatic effect of gauze packing under this model, with no thrombosis or embolus formation in other visceral organs.
This work was supported by the “Twelfth Five-Year Plan” Special Project in Military Logistics Scientific Program [AWS14L012], and the Special Project of Social Undertakings & People’s Livelihood Protection of Chongqing [cstc2016shmszx130018].
Hao Qin contributed to the device design, the experimental design and performance, and writing of the manuscript. Zhaowen Zong contributed to the study conception and design, and supervision. Lei Yang, Sixu Chen, Mingrui Lu, Quanwei Bao and Huayu Liu contributed to conducting experiments and acquiring data. Daocheng Liu, Qiang Chen, and Xinan Lai contributed to the data analysis and interpretation. Zhaowen Zong takes responsibility for the integrity of the data analysis.
“AB” indicates that group A is significantly different from group B; “AE” indicates that group A is significantly different from group E; “BC” indicates that group B is significantly different from group C; “BD” indicates that group B is significantly different from group D; “DE” indicates that group D is significantly different from group E.
Image: 1-1 “CELOX particles” and “high expansion compressed sponge tablets containing CELOX ingredients”.
Table 1: Comparison of weight, survival time, and blood loss among groups.
Figure 1: Details of the hemostatic device.
Figure 2: Establishment of a model of closed fatal liver injury in swine.
Figure 3: Changes in MAP from 10 minutes before the liver injury to death in each swine.
Figure 4: Changes in CO from 10 minutes before liver injury to death in each swine.
Figure 5: Pathologic examination of a blood clot. The results of group E; the Celox granules are shown by the white arrow, and the clot is shown by the black arrow. The two were tightly combined, and the Celox blood clot had more red thrombi than the common blood clot.
5-1 Common blood clot.
5-2 CELOX blood clot.
6-1 Group A
6-2 Group E
Figure 6: Pathologic examination of the liver stump. The hepatic lobules are shown by the blue arrow, the hepatic sinus congestion is shown by the black arrow, and the central vein destruction is shown by the white arrow.
7-1 10 minutes before modeling
7-2 5 minutes after the modeling?maximum diameter of hemorrhage under the liver capsule is 6.63 mm.
7-3 30 minutes after device application, the maximum diameter of hemorrhage under the liver capsule is 18.69 mm.
7-4 60 minutes after device application, the maximum diameter of hemorrhage under the liver capsule is 18.84 mm. The black arrow shows the exudation of the contrast agent.
Figure 7: Liver CT scan.
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