Blast injury simulation in pigs using an air-driven shock wave generator: effects of shock waves of lateral shock wave exposure

Abstract

Purpose

We have hypothesized that respiratory arrest caused by temporary brainstem dysfunction is the primary cause of death during the hyperacute phase of blast injury and have demonstrated that this is highly likely. To date, we have examined the effects of shock waves on pigs by exposing them to shock waves from both the dorsal and ventral sides. Therefore, in this study, we investigated the effects of lateral shock wave exposure in pigs and identified priority regions for protection against blast injuries.

Methods

Eight male hybrid pigs (age, 10–12 weeks; mean weight, 36.6 kg) were anesthetized and fitted with a femoral arterial line and subcutaneous electrocardiography leads for continuous monitoring of vital signs and respiration. Pigs were fixed prone with the right lateral thorax aligned to the aperture. A compressed air-driven shock tube generated a shock wave (driving pressure: 3.0 MPa). Arterial blood gases were obtained before and 0, 3, 5, and 10 min post-exposure. Whole-body computed tomography was performed pre- and post-exposure. Survivors were observed for 3 h, then underwent necropsy. Regarding lung injury, since “primary blast lung injury” is defined as “radiological and clinical evidence of acute lung injury occurring within 12 h of exposure and not due to secondary or tertiary injury”, this study focuses on organ injury and mortality in the hyperacute and acute phases.

Results

All pigs developed immediate apnea after blast exposure. Two pigs died: one from persistent apnea leading to progressive hypoxia and cardiac arrest and one from delayed cardiac arrest approximately 1 h later. Necropsy in the delayed death revealed thrombus extending from the right subclavian vein into the superior vena cava, suggesting embolism. Marked bradycardia was absent, and hypotension only occurred in one pig. Computed tomography depicted no air embolism and suggested increased right lung injury. Gross anatomy revealed pulmonary contusion and splenic injury in all the pigs; pulmonary bullae were rare.

Conclusion

Lateral shock wave exposure can induce profound apnea and death, likely owing to transient brainstem respiratory dysfunction rather than a vagal reflex mechanism. To reduce mortality, protective strategies should prioritize shielding the brainstem region, specifically the lateral and posterior sides of the occipital region and neck.

Background

Blast-related casualties occur daily worldwide due to combat and terrorism; therefore, adequate preparation for blast trauma is critical. While blast lung is a well-known cause of death in blast trauma patients, severe conditions can arise from the rapid physiological changes occurring in the hyperacute phase, such as apnea, bradycardia, and hypotension [1, 2]. Most importantly, physical protection aimed at preserving life is essential; however, current protective gear prioritizes counter-bullet capacity and does not sufficiently account for blast-wave protection [3–5].

To simulate blast injury, we installed a shock wave generator at our facility, and conducted experiments using pigs. The air-driven shock wave generator releases compressed air in a single burst, forming a shock wave [6]. In our past experiments in the early stage, since all pigs that died experienced respiratory arrest, we concluded that respiratory arrest was likely to be associated with death during the hyperacute phase of blast injury. Subsequently, based on experiments using mice to expose the posterior neck and lungs to shock waves [7], it was considered highly likely that the primary cause of death during the hyperacute phase of blast injury was respiratory arrest due to probable temporary brainstem dysfunction. Based on this hypothesis, we conducted shock wave experiments on pigs and have demonstrated that this is indeed highly likely. A previous study investigated the effects of shock waves applied to the dorsal side of pigs and demonstrated the protective efficacy of bulletproof vests and steel body armor fully covering the neck and chest [6, 8]. Subsequent studies have investigated the effects of shock waves applied from the ventral side and indicated that while they did not cause death in the acute phase, they resulted in multiple injuries (bullae) on the lung surface [9].

Considering blast waves can originate from any direction in actual battlefields or terrorist incidents, investigating the effects of shock waves from various orientations is essential. Nevertheless, most studies to date have been confined to numerical simulations, with few attempts to validate findings through animal experiments [10–14].

To date, we have applied shock waves to pigs from both the dorsal and ventral sides to investigate their effects. Therefore, the current study investigated the effects of shock waves on the lateral aspects of pigs. The results of this study provide insights into which body regions should receive greater protective emphasis when developing new body armor against blast shock waves.

Methods

Pig preparation

Eight male hybrid pigs (10–12 weeks old, mean weight 36.6 kg) were used in the study. The pigs were housed in individual cages in a room maintained at 24 °C with a 12-h light–dark cycle. Water was freely accessible, and a standard experimental diet was provided.

Anesthesia

Midazolam (0.3 mg/kg), butorphanol tartrate (0.4 mg/kg), and medetomidine chloride (0.04 mg/kg) were administered intramuscularly to all pigs. An intravenous line was secured via the auricular vein, and normal saline was administered at 30 mL/h. After achieving adequate anesthesia with an intravenous injection of alphaxalone (2.0 mg/kg), atropine (0.5 mg) was administered intravenously to suppress salivation, and tracheal intubation was performed. During the experiment, alphaxalone was continuously administered at 1–2 mg/kg/h for analgesia. If a pig exhibited signs of distress, such as body movement, hypertension, or tachycardia, additional doses of alphaxalone were administered as appropriate.

Surgical procedure

An arterial line was surgically inserted into the femoral artery for continuous monitoring of vital signs. All electrocardiography monitoring electrodes were implanted subcutaneously, and the lead wires were secured to the skin.

Shock tube setup

The shock wave generator (8 × 12 m) was located indoors. The shock tube (8 m long) features a high-pressure section at one end to store compressed air and a 40-cm diameter opening at the opposite end facing the measurement room. Driving pressure was set at 3.0 MPa, consistent with previous methodologies [6, 8, 9].

Figure 1 shows the pressure-time curve measured in the measurement area. The peak pressure, the duration, and the impulse were 232 kPa, 5.0 ms, and 494 kPa·ms, respectively.

Fig. 1.

Pressure - Time curve in the measurement area

Measurements and outcomes

After the surgical procedures, whole-body computed tomography (CT) was performed. Next, the pigs were transported to the shock wave generator measurement area, placed on a table in the prone position, and secured to the table using two belts and a net, with the right side of the chest facing the aperture.

After ensuring both the pig and measurement area were secured, compressed air was accumulated up to the target pressure. When the pressure reached 3.0 MPa, the compressed air was released, generating a shockwave that traveled through the shock tube and directly impacted the pig. Changes in vital signs after administration of the shock wave were observed continuously. Respiration was confirmed by monitoring end-tidal carbon dioxide. Arterial blood gases were measured immediately before, immediately after, and 3, 5, and 10 min after shock wave application to monitor changes.

A second whole-body CT scan was conducted, and then all pigs were returned to the experimental room for observation. No fluid resuscitation was performed after injury, and normal saline was administered intravenously (60 mL/h). Any pigs that remained alive after 3 h of observation were euthanized. At the time of euthanasia, in addition to alphaxalone, propofol (10 mg/kg), rocuronium (1 mg/kg), and KCl (40 mmol) were administered, followed by necropsy to investigate the extent of gross organ damage.

Regarding lung injury, since “primary blast lung injury” is defined as “radiological and clinical evidence of acute lung injury occurring within 12 h of exposure and not due to secondary or tertiary injury”, this study focuses on organ injury and mortality in the hyperacute and acute phases.

Quantitative lung volumetry

Additional lung volumetry was performed, in which areas exhibiting consolidation within the lungs were specifically extracted and their volumes measured. While pulmonary contusion is generally visualized on CT as ground-glass opacities (GGOs) to consolidation, attenuation increases due to insufficient inspiration or motion artifacts can obscure accurate delineation. Therefore, to evaluate pulmonary contusion with high reproducibility, volumetric extraction was intentionally limited to regions presenting with consolidation. All image processing was conducted using a dedicated 3D workstation (Ziostation; Ziosoft Inc., Tokyo, Japan).

The total lung volume and consolidation volume were measured separately for the right and left lungs. The extent of consolidation was expressed as the percentage of consolidation volume relative to the total volume of the corresponding lung (consolidation percentage = consolidation volume / total ipsilateral lung volume × 100). The consolidation percentage was calculated before and after shock-wave exposure for each lung. The change in consolidation percentage from baseline (Δ consolidation percentage) was calculated by subtracting the pre-exposure value from the post-exposure value.

Statistical analysis

Continuous variables are presented as mean ± standard deviation or median (interquartile range), as appropriate. Normality was assessed using the Shapiro–Wilk test. To evaluate whether shock-wave-induced lung injury differed between the right and left lungs, the changes in consolidation percentage from before to after shock-wave exposure (Δ consolidation percentage) were compared between the right and left lungs using a paired t-test when the paired differences were normally distributed. When the assumption of normality was not met, the Wilcoxon signed-rank test was used. The corresponding 95% confidence interval for the mean paired difference was calculated when applicable. A two-sided P value < 0.05 was considered statistically significant.

Ethics approval

This study was conducted in accordance with the ARRIVE guidelines and was approved by the Animal Ethics Committee of the National Defense Medical College (approval number: 23022-3).

Results

Vital sign changes after shock wave exposure are shown in Table 1. “Missing data” refers to unattainable values due to damaged monitoring cables or other equipment during the experiment. Apnea occurred in all eight pigs, and two pigs died. One of the two pigs that died exhibited respiratory arrest immediately after exposure, and spontaneous heartbeat was maintained. Respiratory recovery was not achieved, leading to cardiac arrest due to hypoxia; a typical sequela seen in our previous studies. The other pig that died resumed spontaneous breathing after a brief period of respiratory arrest, and survival was expected. However, approximately 1 h later, the peripheral oxygen saturation (SpO₂) and blood pressure suddenly dropped, leading to cardiac arrest. Necropsy revealed a thrombus in the right subclavian vein extending into the superior vena cava. Necropsy revealed a thrombus in the right subclavian vein extending into the superior vena cava, which meant venous thrombosis with highly suspected embolism.

Table 1.

Results of vital sign changes in pigs exposed to shock waves from the right lateral direction

Case	Apnea	Hypotension	Bradycardia	Survival
1	Yes	Missing	No	Survived
2	Yes	Yes	No	Survived
3	Yes	No	No	Died
4	Yes	No	No	Survived
5	Yes	No	No	Died (delayed)
6	Yes	No	No	Survived
7	Yes	No	No	Survived
8	Yes	No	No	Survived

In one surviving pig, respiratory arrest persisted, SpO₂ dropped to an unmeasurable level, and death was anticipated; however, spontaneous respiration resumed from agonal breathing, leading to survival. Similarly, in another surviving pig, SpO₂ dropped to 21%, the heart rate decreased to 36 beats/min, and cardiac arrest was expected; nonetheless, respiration resumed, resulting in survival (Fig. 2).

Fig. 2.

Transition of arterial blood gas during spontaneous breathing at room air and heart rate after shock wave exposure

Hypotension was only observed in one pig, and data for another pig were unattainable due to arterial line damage. Bradycardia was not observed in any pig; thus, no pig exhibited all three characteristic neurogenic reflexes—apnea, bradycardia, and hypotension—simultaneously in response to the shock wave. Arterial blood gas values temporarily decreased due to respiratory arrest, but subsequently improved (Fig. 1).

Quantitative evaluation of the CT images concerning lung injury was performed. Before shock-wave exposure, the consolidation percentage did not significantly differ between the right and left lungs (6.80 ± 5.60% vs. 8.48 ± 5.74%, respectively; P = 0.277). After shock-wave exposure, the consolidation percentage was significantly greater in the right lung than in the left lung (11.91 ± 5.11% vs. 7.97 ± 3.83%, respectively; P = 0.035). The change in consolidation percentage from baseline was greater in the right lung than in the left lung (+ 5.11 ± 4.96 vs. −0.51 ± 5.35% points, respectively), although the difference did not reach statistical significance (P = 0.058). No obvious air embolism was observed (Fig. 3).

Fig. 3.

CT scan images of the lungs at the level just above the diaphragm of the Case 3 pig before and after shock wave exposure. The right lung contusion (black arrows) was more severe than the left lung’s

Regarding organ damage, splenic injury and pulmonary contusion were observed in all cases (Table 2). While bullae on the lung surface were frequently observed in a previous study in which a shock wave was applied to the ventral side [9], only one instance of such bulla was observed in the present study. Macroscopically, the right lung—the exposed side—exhibited more severe damage. Spleen injury was observed in all the pigs, likely due to their anatomical characteristics—compared to humans, the spleen is much larger and more mobile in pigs.

Table 2.

Macroscopic findings of organ injuries following shock wave exposure

Case	Remarks on organ injury
1	Bilateral LC; single bulla in the right middle lobe; slight IAH due to SI
2	Bilateral LC; mild IAH due to splenic rupture; small serous pericardial effusion
3	Bilateral LC (right > left); moderate IAH due to SI
4	Bilateral LC (right > left); mild IAH due to SI
5	Bilateral LC (right > left); splenic rupture with slight IAH; slight hemothorax; mediastinal hematoma extending from the heart to the aorta; thrombus extending from the right subclavian vein to the superior vena cava; left renal injury with retroperitoneal hematoma; small bowel injury
6	Bilateral LC; splenic rupture with slight IAH; colonic mesenteric hematoma
7	Right LC; moderate IAH due to SI; small serosanguinous pericardial effusion; myocardial contusion; right renal injury with retroperitoneal hematoma
8	Bilateral LC (right > left); splenic rupture with mild IAH; right renal and adrenal injuries with retroperitoneal hematoma

LC, lung contusion; IAH, intra-abdominal hemorrhage; SI, splenic injury

IAH severity was classified as follows: slight, < 100 mL; mild, 100–300 mL; moderate, 300–600 mL; severe, > 600 mL

Discussion

There has recently been an increase in the use of pigs in blast injury experiments [15–17]. In the current study, two of the eight pigs died. Only one death occurred during the hyperacute phase. Apnea occurred immediately after a shock wave was applied; despite maintenance of the heart rate and blood pressure, respiratory recovery was not achieved, leading to hypoxia-induced cardiac arrest—a typical sequela observed in our previous studies [6, 8]. At least two other pigs were considered to have fallen into a nearly fatal state due to respiratory arrest. This suggests that shock wave appliance from the lateral side resulted in more severe damage and that respiratory arrest played a significant role in mortality. In our previous and current studies, mortality was highest when shock waves were applied dorsally (8 of 18 animals died) [6, 8], lower when applied laterally, and lowest when applied ventrally (no deaths) [9]. These collective findings indicate that the mortality risk may differ depending on the direction of shock wave exposure. We have insisted the hypothesis that the direct effect of a shock wave on the brainstem causes respiratory arrest, leading to death, and this difference can be considered directly proportional to the distance from the body surface to the brainstem, relative to the shock wave. Regarding shock wave exposure from the ventral side, the attenuation effects of the mandible and the skull base are likely included.

Previous numerical simulation studies have demonstrated that the biomechanical effects of blast exposure vary according to the direction of blast-wave propagation [10–14]. Most of these studies focused primarily on brain biomechanics [10–12, 14], while Mathis and Clutter investigated the effects of blast orientation in bomb-suit wearers [13]. Therefore, their specific outcomes cannot be directly compared with the cardiopulmonary injuries and mortality observed in the present porcine study. Nevertheless, these studies consistently indicate that blast-induced biological responses are orientation-dependent. Our experimental observations, together with our previous studies using the same shock-wave generator, similarly suggest direction-dependent differences in injury patterns and mortality, with mortality being highest following dorsal exposure, followed by lateral exposure, and lowest following ventral exposure. Thus, the present findings are broadly consistent with the concept of orientation-dependent blast effects suggested by previous simulation studies.

In goats, Yang et al. [18] reported increased mortality with proximity to the blast epicenter and noted higher injury severity on the right side, which they attributed to the anatomy of goats. Bouitllier et al. reported that during blast exposure, lung injury occurs in the lung closest to the blast wave [19]. Similarly, our experiments showed a tendency toward higher injury severity in the right lung, closer to the shockwave aperture. It is understandable that proximity to the blast epicenter is associated with higher mortality and that organs closer to the epicenter are more susceptible to injury.

Because the porcine spleen is anatomically different from the human spleen and is relatively large and mobile, it may be particularly susceptible to inertial forces. Therefore, the near-universal occurrence of splenic injury in the present porcine model may, at least in part, reflect species-specific anatomical characteristics. Therefore, this finding should not be directly extrapolated to human blast trauma.

Acute deaths from blast injury have been thought to result from air embolism associated with severe lung injury [20, 21]. In the current study, one death was suspected to be caused by embolism, although no findings suggestive of clear air embolism were observed on CT. There is a report indicating that nearly 1 in 10 patients with severe wartime injuries show signs of pulmonary thrombus on CT imaging [22]. While PBLI is thought to lead to a hypercoagulable state [23], there are also reports stating that isolated primary blast injury is not responsible for acute coagulopathy of trauma in victims of explosions but seems to lead to an early hypercoagulable state [24].

Neutrophil extracellular traps (NETs) are thought to play an important role as one of the causes of the hypercoagulable state [25]. NETs are the key factors causing inflammation and coagulation disorders of PBLI. They can promote the coagulation cascade, platelet activation, and platelet thrombosis formation by acting as ligands of platelet toll-like receptors. Furthermore, NETs can also promote coagulation by eliminating main blood clotting inhibitors, such as antithrombin III and tissue factor pathway inhibitors. It is possible that the massive thrombosis of the dead pig observed in this study also occurred through such a mechanism.

Based on rodent experiments, three extensively studied physiological sequelae have been associated with blast injury: apnea, bradycardia, and hypotension [2, 26]. The absence of marked bradycardia following shock wave exposure in the present study should be interpreted with caution because the anesthetic protocol may have modified autonomic cardiovascular responses. Atropine (0.5 mg) was administered before endotracheal intubation, approximately 2.5–3 h before shock wave exposure. Because atropine blocks cardiac muscarinic receptors, a residual antimuscarinic effect may have attenuated or masked vagally mediated bradycardia at the time of exposure. Indeed, a previous experimental study of primary thoracic blast demonstrated that atropine attenuated blast-induced bradycardia, whereas it did not affect blast-induced apnea. Nevertheless, blast-induced bradycardia has been observed in rats maintained under continuous alphaxalone/alphadolone anesthesia [27], indicating that vagally mediated cardiovascular responses can still occur under this anesthetic regimen. Continuous alfaxalone infusion may also have influenced autonomic tone and cardiovascular reflex responsiveness, although its specific effect on blast-induced vagal responses remains uncertain. Therefore, the absence of prominent bradycardia in the present study cannot be interpreted as evidence against the involvement of a vagally mediated reflex. The effects of these pharmacological interventions represent an important confounding factor when interpreting the cardiovascular response to shock wave exposure.

These sequelae are thought to result from vagal nerve (pulmonary nociceptive C-fiber) reflexes triggered by distension stimuli to the lung parenchyma. When shock waves were applied to the thorax, bradycardia and decreased blood pressure were observed, followed by apnea lasting approximately 15 s, then shallow breathing [28]. These three signs of blast lung injury were thought to play a significant role in mortality [23]. In a study exposing rats to shock waves, Mishra et al. [29] concluded that the cause of death was not related to lung injury. Similarly, Garman et al. [30] reported that rats exposed to shock waves only to the head died due to respiratory arrest.

Considering the aforementioned prior findings, the mechanism underlying the prolonged respiratory arrest observed in the present study remains uncertain. A vagally mediated pulmonary reflex may have contributed to the initial apnea, and the absence of marked bradycardia cannot exclude this mechanism because of the potential effects of atropine and alfaxalone. However, respiratory arrest caused by pulmonary vagal reflexes in previous experimental studies has generally been transient, whereas apnea persisted for several minutes in some animals in the present study. This prolonged respiratory arrest, together with previous observations of fatal respiratory arrest following shock wave exposure confined to the head, raises the possibility that transient dysfunction of the brainstem respiratory center may contribute to prolonged or irreversible apnea [30]. Further studies designed to distinguish pulmonary vagal reflexes from direct effects on the central respiratory network are required to clarify this mechanism.

During armed conflict, gunshot wounds and blast injuries are significant causes of cardiopulmonary arrest, yet their treatment is likely futile [31]. The paramount priority is preventing cardiopulmonary arrest. To achieve this, avoiding a fatal respiratory arrest state—that is, protecting the brainstem—is considered critically important.

The current study had some limitations. Owing to financial constraints, the sample size was small, thus limiting the generalizability of the findings. Second, the pigs were observed for only 3 h after shock wave exposure rather than for 12 h as per the definition of PBLI [23]; thus, the assessment of other symptoms of blast lung may have remained incomplete. Third, quantitative CT assessment has several technical limitations in this experimental setting. Because breath-holding could not be performed during CT acquisition, insufficient inspiration and respiratory motion may have affected the attenuation and delineation of pulmonary lesions. To minimize the influence of these factors and improve the reproducibility of volumetric assessment, the analysis was intentionally restricted to areas of consolidation rather than including GGOs. Furthermore, precise separation of the right and left lungs was challenging in some regions because their boundaries were not always clearly delineated on CT images. Although the results of the image analysis were visually reviewed for appropriateness, some degree of segmentation and measurement variability may have remained. Therefore, these technical limitations should be considered when interpreting the quantitative differences between the right and left lungs. Lastly, despite using straps and a net to tightly secure the pigs to the table within the experimental area, their bodies became airborne during shock-wave exposure. This in itself may have resulted in injuries that could not be isolated from those caused by the shock wave. The whole pig body was wrapped in netting and secured to the steel plate downstream of the experimental table (which, in this experiment, was adjacent to the pig’s left side). Furthermore, two sturdy industrial straps were used to wrap around the pig’s torso and secure it in the space beneath the experimental table so that there was no slack. Nevertheless, despite these efforts, it was difficult to prevent the pig’s body from floating against the shock wave exposure. Quantifying impact forces and other factors requires a new experimental setup and is therefore difficult. We are considering placing thin cushion covers on itself and the steel plate downstream of the experimental table in the future. It is possible that a vagal reflex was triggered by blunt trauma equivalent to a tertiary blast injury. However, respiratory arrest caused by a typical vagal reflex often resolves within a short period (ranging from a few seconds to several tens of seconds); therefore, the respiratory arrest lasting several minutes observed in this experiment suggests that mechanisms other than, or in addition to, a transient vagal reflex may have contributed. To mitigate this confounding factor, it is necessary to place the pig in an appropriate position within the shock tube, where the pig is secured in a suspended position at the point where the dynamic pressure is lowest. However, placing the pig inside a 40 cm-diameter shock tube was not practically feasible due to the nature of our shock tube setup.

Conclusion

After exposure to lateral shock waves, the blast injury can induce respiratory arrest and ultimately death. When developing specific body armor designed to counter blast waves, the foremost priority should be to protect the brainstem—specifically the lateral and posterior sides of the occipital region and neck—to prevent acute fatalities, namely death from respiratory arrest. In response to these findings, it is necessary to conduct verification tests using body armor that provides enhanced protection for the neck, particularly the lateral and posterior sides, to assess its effectiveness against shock waves.

Author contributions

N.K. contributed to conceptualization, data curation, formal analysis, investigation, methodology, project administration, visualization, and writing the original draft. Y.S., R.S., K.Y., M.A., and T.N. contributed to investigation. K.N. and D.S. contributed to supervision. T.K. and S.T. contributed to writing—review and editing. All authors read and approved the final manuscript.

Funding

This study was supported by the Japan Ministry of Defense under the Research Promotion Program for Defense Medicine.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethical approval

This study was approved by the Animal Ethics Committee of the National Defense Medical College (approval number: 23022-3).

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.