Development of a Severe Blast Lung Injury Model in a Goat for Ultrasonographic Evaluation

Summary

This study successfully established a severe blast lung injury model in goats at a driving pressure of 4.5 MPa, causing significant injury and 41.67% mortality. Lung ultrasound scoring dynamically assessed damage, correlating with traditional indicators, demonstrating its diagnostic value. This reproducible model advances diagnostics and therapies for acute lung injury.

Abstract

Blast lung injury (BLI) causes severe lung damage and high morbidity, especially in military and industrial settings, leading to hemorrhage and potential respiratory failure, highlighting the need for improved diagnostics and treatments. This study aimed to develop a standardized severe BLI model in goats to evaluate the accuracy and feasibility of point-of-care ultrasound for dynamic assessment. Goats were subjected to controlled blast overpressure using the biologic shock tube (BST-Ⅰ) at driving pressures of 4.0 MPa (n = 4), 4.5 MPa (n = 12), and 5.0 MPa (n = 4), respectively. Key parameters, such as peak overpressure, were recorded. Vital signs, lung ultrasound scoring (LUS), oxygenation index (PaO₂/FiO₂), and extravascular lung water (EVLW) were monitored at baseline (0 h pre-injury) and 0.5 h, 3 h, 6 h, 9 h, 12 h post-injury. Thoracic computed tomography (CT) at 0 h and 12 h quantified lung injury ratio, and gross examination post-euthanasia assessed pulmonary hemorrhage and injury score. At 4.5 MPa, the peak overpressure was 396.92 kPa, with a 41.67% mortality rate post-injury, whereas at 4.0 MPa, the mortality rate was 0%. LUS increased over time, showing a negative correlation with PaO₂/FiO₂ and a positive correlation with EVLW at 3 h, 6 h, and 9 h, and a positive correlation with lung injury ratio at 12 h. Gross lung injury area ratio was 42.14% in the 4.5 MPa group, indicating severe injury, while 4.0 MPa showed moderate injury. The 4.5 MPa model was suitable for studying severe injury, unlike 4.0 MPa, which caused only moderate injury, and 5.0 MPa resulted in 100% mortality. A reproducible goat model of blast lung injury was established, effectively using LUS to non-invasively assess pulmonary damage over time, providing a basis for monitoring and exploring therapeutic strategies for acute lung injury.

Introduction

Blast exposures, whether in military or civilian settings, continue to represent a major contributor to morbidity and mortality, with lung injury being a primary determinant of outcome¹. Contemporary evidence confirms that pulmonary contusion is a critical risk factor for severe respiratory complications, such as pneumonia and acute respiratory distress syndrome (ARDS), which significantly impact the prognosis of affected individuals². Prompt and precise evaluation of the extent of lung injury is therefore essential to guide clinical management and improve survival.

Computed tomography (CT) is effective for evaluating the severity of blast lung injury (BLI)³. However, it entails exposure to ionizing radiation and may be impractical in scenarios involving mass casualties or limited medical resources. The clinical examination for tympanic membrane perforation has been investigated as a potential marker for pulmonary blast injury, but studies indicate that isolated tympanic membrane perforation is not a reliable indicator of concealed pulmonary blast injury or poor prognosis, and thus does not preclude the need for further investigation⁴. The clinical presentation of blast victims is highly variable; some patients present acutely, while others may develop respiratory failure 12–24 h later, underscoring the critical importance of frequent reassessment in the emergency department to detect missed injuries, particularly BLI⁵. These limitations highlight the urgent need for a rapid, bedside-compatible imaging technique to promptly evaluate lung contusion severity. In mass casualty events, tools capable of rapid triage and assessment are crucial for optimizing resource allocation and improving overall outcomes⁵.

Thoracic ultrasonography has emerged as an indispensable tool in critical care settings, owing to its real-time, non-invasive, and portable nature⁶. Its established role in diagnosing various pleural and parenchymal lung diseases makes it a highly promising modality for dynamic assessment of blast lung injury. Sonographic signs such as lung consolidation, bronchograms, pleural line abnormalities, and comet-tail artifacts can serve as indicators of the extent and severity of pulmonary contusion and edema⁷. Its lack of ionizing radiation makes it particularly suitable for patients requiring repeated assessments, and its accessibility in resource-limited environments is a significant advantage.

While the existing literature supports the use of point-of-care ultrasound (POCUS) for triage and monitoring, ultrasound is already routinely used in trauma assessment (e.g., extended focused assessment with sonography for trauma, eFAST) to detect pneumothorax and hemothorax⁸. Nevertheless, rigorous validation of its accuracy and feasibility for grading severe pulmonary contusion requires a translational and physiologically relevant animal model. This research seeks to establish a standardized severe blast lung injury model in goats, which will serve as a platform for systematically evaluating the utility of point-of-care ultrasound in the swift triage and ongoing monitoring of this life-threatening condition.

Protocol

All experimental methodologies detailed in this manuscript received approval from the Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University (SCXK (Yu) 2017-0002). Furthermore, all animal-related procedures were carried out in strict compliance with the animal use guidelines established by the approval committee.

1. Experimental animals

1. Prepare healthy goats, 3–6 months old, female, weighing around 15 kg.

2. Injury platform

1. Cause lung blast injuries by biologic shock tube (the large one) (BST-Ⅰ), which is 39.4 m long (Figure 1) and divided by two semi-rigid aluminum films into two sections--the driving section and the experimental section, using 4.0 MPa, 4.5 MPa, and 5.0 MPa driving pressure, respectively.

3. Animal preparation

1. Fast the animals for 24 h and deprive them of water for 8 h before the experiment.
2. Weigh and record.
3. Use midazolam to introduce anesthesia (intramuscular injection: 1 mg/kg).
4. Shave off the fur on the chest, neck, and groin of the goat, then apply the depilatory cream to completely remove the remaining fur.

4. Drawing partitions for ultrasonography detection

1. Anesthetize the goat with 3% pentobarbital sodium (10–20 mg/kg) through an indwelling needle in the saphenous vein. Keep it in a relaxed lateral position, with limbs perpendicular to the torso, and the head extended.
2. Move the ultrasonic probe parallel to the intercostal space from the bottom to the top, from the dorsal to the anterior to mark the edges of the lung.
3. Draw successive 2.5-centimeter-long rectangles along the intercostal space on the area of the lung as partitions for ultrasonography detection. There are a total of 66 partitions with 33 on the right and 33 on the left, and the number of partitions in each intercostal space is 1, 2, 2, 5, 5, 5, 5, 4, 3, 1, respectively, from the first intercostal space to the tenth (Figure 2).

5. Surgical operation

1. Make sure the goat is under anesthesia. If not, inject an additional 3% pentobarbital sodium of 10 mg/kg.
2. Cervical vein catheterization
   1. Position the goat in right lateral recumbency and properly restrain the goat.
   2. Make a longitudinal skin incision, approximately 4 cm in length, over the middle third of the jugular groove. Perform blunt dissection through the subcutaneous tissue and the cutaneous colli muscle to expose and isolate the external jugular vein.
   3. Insert a double-lumen central venous catheter (7Fr) under the guidance of an introducer needle. Advance the catheter approximately 15 to 20 cm until its tip is positioned within the goat’s precaval vein, approximately 2 cm from the right atrium. Ligate and securely fixate the catheter in place.
   4. Connect the distal lumen via a three-way stopcock to a temperature sensor and a pressure transducer for central venous pressure monitoring and ice-cold saline injection. Connect the proximal lumen via a three-way stopcock to an intravenous infusion line for fluid administration and supplemental anesthetic delivery.
3. Endotracheal Intubation
   1. Position the goat in dorsal recumbency and secure the neck in extension.
   2. Perform a 5–6 cm longitudinal skin incision along the midline. Use blunt dissection to separate the sternohyoid muscles and expose a 2–3 cm segment of the trachea.
   3. Place a length of non-absorbable suture beneath the exposed trachea to gently lift and stabilize the trachea. Make a horizontal "T" incision through 2–3 tracheal rings at the midpoint of the stabilized segment.
   4. Insert a size 6.0 cuffed endotracheal tube into the tracheal lumen and advance it distally. Inflate the cuff. Suture the tracheal incision loosely and securely suture the tube to the skin. Connect the endotracheal tube directly to the mechanical ventilator circuit.
      NOTE: Ventilator parameters: the mode of ventilation is intermittent positive pressure ventilation (IPPV); the tidal volume (VT) is set at 8 mL/kg; the respiratory rate (RR) is maintained at 15 breaths per min; the inspiratory to expiratory ratio (I: E) is configured as 1:1.5; the flow rate (V) is established at 7.2 L/min; the fraction of inspired oxygen (FIO₂) is adjusted to 21%; the positive end-expiratory pressure (PEEP) is set at 3.9 cm H₂O; and the inspiratory pressure (Pinsp) ranges from 9 to 12 cm H₂O.
4. Femoral artery catheterization
   1. Position the goat in right lateral recumbency and properly restrain the goat.
   2. Perform a 3–4 cm longitudinal skin incision along the medial aspect of the groin. Employ blunt dissection techniques to reveal the femoral neurovascular bundle, ensuring to meticulously separate the femoral artery from the neighboring vein and nerve through a combination of blunt and sharp dissection methods.
   3. Perform a small transverse arteriotomy on the isolated femoral artery segment and insert a Picco thermodilution catheter (4Fr) into the artery. Advance the catheter to 16 cm, ensuring the tip is in the common iliac artery. Securely suture the catheter to the surrounding skin or subcutaneous tissue.
      NOTE: Take care not to kink or force the catheter against the vessel wall, as this will compromise accurate pressure transduction and thermodilution measurements.
   4. Connect the catheter via a three-way stopcock to a pressure transducer for continuous hemodynamic monitoring, such as blood pressure and extravascular lung water. This line is also used for intermittent arterial blood sampling. Maintain a continuous heparinized saline flush (1–2 mL/h) to prevent catheter occlusion.

6. Monitoring and measurements

1. Vital signs
   1. Measure and record vital signs at the following time points: 0 h pre-injury, 0.5 h, 3 h, 6 h, 9 h, and 12 h post-injury.
   2. Connect all sensor lines to the PiCCO monitoring module. Position the pressure transducer at the level of the heart. Open the stopcock on the transducer to atmospheric air and perform zero calibration for both the arterial and venous pressure lines.
   3. Monitor and record the mean arterial pressure (MAP). Position the electrode pads for monitoring core temperature (T), respiratory rate (RR), and heart rate (HR) at the specified locations: on the left side of the chest, between the second and third ribs; on the left side of the chest, between the fifth and sixth ribs; and on the right side of the chest, between the second and third ribs.
2. Ultrasonography (B-Mode)
   1. Perform lung ultrasound examinations at the following time points: 0 h pre-injury, 0.5 h, 3 h, 6 h, 9 h, and 12 h post-injury. Maintain a consistent scanning sequence: examine the right lung first, then the left lung at each time point.
   2. Position the ultrasound probe perpendicular to the skin surface at the midpoint of each partition. Ensure the probe's marker dot is oriented toward the spine.
   3. Obtain video clips by scanning along the intercostal spaces from the 10th rib to the 1st rib. Save video recordings that include at least two full respiratory cycles.
   4. Apply the following standardized lung ultrasound score (LUS) to the saved images⁹: Assign two trained sonographers to perform the scoring process independently. Calculate the average of the two scores for the final assessment.
      NOTE: Score 0 indicates the presence of A-lines. Score 1 is characterized by multiple, distinctly separated B-lines, or a merging of B-lines that encompasses less than 50% of the pleural line. Score 2 indicates diffuse coalescent B-lines, commonly referred to as "white lung". Score 3 is assigned when there is evidence of consolidation, which may present as tissue-like echotexture or dynamic air bronchograms.
3. Arterial blood gas (ABG) analysis
   1. Collect arterial blood samples for analysis at the following time points: 0 h pre-injury, 0.5 h, 3 h, 6 h, 9 h, and 12 h post-injury.
   2. Using a syringe, first aspirate and discard the heparinized saline flush and approximately 5 mL of blood from the PiCCO arterial line. Subsequently, extract 0.3 mL of fresh arterial blood directly via the catheter.
   3. Immediately inject the blood sample into a CG4+ test cartridge. Insert the cartridge into the portable blood gas analyzer to perform the analysis.
   4. Record the measured values for pH, partial pressure of oxygen (PaO₂), and other relevant parameters. Calculate the oxygenation index (the ratio of partial pressure of oxygen to the fraction of inspired oxygen, PaO₂/FiO₂).
4. Extravascular lung water (EVLW) measurement
   1. Perform EVLW measurements via transpulmonary thermodilution at the following time points: 0 h pre-injury, 0.5 h, 3 h, 6 h, 9 h, and 12 h post-injury.
   2. Re-zero the arterial and venous pressure transducers as described in section 6.1.2. Ensure a stable arterial waveform is displayed on the monitor before proceeding.
   3. Inject exactly 10 mL of ice-cold (0 °C) saline as a bolus through the temperature sensor of the central venous catheter. Complete the injection within 7 s. Conduct three consecutive injections at each time point. Use the average of the three measurements for the final EVLW value.
   4. Record EVLW and other hemodynamic parameters provided by the PiCCO system.
5. Computed tomography (CT)
   1. Acquire thoracic CT scans at 0 h pre-injury and 12 h post-injury.
   2. Perform the scan using a 64-slice CT scanner with the following protocol: tube current: 200 mas; tube voltage: 100 kV; rotation time: 0.4 s.
   3. Have a qualified radiologist perform the scan and subsequent analysis without knowledge of the experimental conditions. Use the specialized workstation to perform three-dimensional volumetric analysis of the total lung volume and the injured lung volume with a defined hounsfield unit (HU) threshold to segment injured lung tissue. Correct the damage range manually.
   4. Calculate the percentage of injured lung volume (lung injury ratio) using the formula: (injured lung volume / total lung volume) * 100%.

7. Blast injury procedure of the goat

1. Randomly allocate the goats into one of three distinct blast overpressure categories utilizing the BST-Ⅰ bio-shock tube. Adjust the driving pressure to levels of 4.0 MPa (n = 4), 4.5 MPa (n = 12), and 5.0 MPa (n = 4), respectively, in order to produce the corresponding primary blast waves.
2. Complete all pre-injury measurements and monitoring as outlined in Section 6. Secure the goat with canvas straps in the standing position on the fixation frame within the shock tube. Position the goat 37.5 m from the blast source with its right side facing the epicenter of the blast (Figure 3).
3. Position the air pressure sensor at the animal location inside the BST-Ⅰ. Connect the signal conditioning module to the data acquisition system. Record injury parameters: the peak overpressure, rise time, positive phase duration, and positive phase impulse for every injury event.
   NOTE: Peak overpressure: The maximum value of the overpressure generated by the shock wave. Rise time: The time required for the pressure to rise from 0 to the peak value. Positive phase duration: The overpressure remains above ambient pressure after the shock front passes. Positive phase impulse: The total momentum per unit area imparted by the shock wave overpressure.
4. Immediately retrieve the goat from the shock tube after the blast. Perform a rapid assessment of the goat's overall condition. Use a suction device to promptly clear any bloody secretions from the endotracheal tube and airway. Initiate manual ventilation immediately if apnea is observed.
5. Complete all post-injury measurements and monitoring as outlined in Section 6.

8. Euthanasia and necropsy

1. Deeply anesthetize the goat with an intravenous injection of 3% sodium pentobarbital after completing the 12 h post-injury observation period. Perform exsanguination via the arterial catheter or another major vessel. Proceed with a sternotomy to carefully extract the lung.
2. Perform gross scoring using ImageJ software.
   1. Open the gross lung image in ImageJ and convert it to a 16-bit grayscale picture using the menu path: Image > Type > 16-bit. (Figure 4A–B)
   2. Set the grayscale value at the junction between the injured and normal tissue as the threshold. Designate all areas above this threshold in red to represent the injury zone (Figure 4C).
   3. Manually trace the outer edge of the lung tissue to define the boundary of the total lung area (Figure 4D).
   4. Calculate the pixel ratio (injury area / total lung area) for both the dorsal and ventral sides using the menu path： Analyze > Set Measurements > Ctrl + M. Ensure the ‘Area’, ‘Area Fraction’, and ‘Limit to Threshold’ options are selected. Average these two ratios to determine the final injury area ratio for the entire lung.
3. Grade the pulmonary contusion using a standardized scoring system adapted from Yelverton et al¹⁰, and our laboratory's historical criteria.
   NOTE: Gross lung injury scoring criteria: Grade 0 (Absence of Injury): No visible contusions or hemorrhages. Grade 1 (Mild): Scattered petechiae or contusions involving less than 10% of the overall lung surface area. Grade 2 (Moderate): Contusions or hepatization encompassing up to 30% of the entire lung surface area. Grade 3 (Severe): Contusions or hepatization impacting up to 50% of the total lung surface area. Grade 4 (Very Severe): Contusions or hepatization affecting more than 50% of the complete lung surface area.

9. Statistical analysis

1. Perform all statistical analyses using SPSS software (Version 22.0).
2. Express continuous measurement data as mean ± standard deviation (x ± s).
3. Test all datasets for normality.
4. Employ an independent‑samples t‑test to compare the means between two groups.
5. Use Pearson's correlation coefficient for correlation analyses.
6. Set the significance threshold at p < 0.05.
   NOTE: ‘#’ means p < 0.05, compared with pre-injury score; ‘##’ means p < 0.01, compared with pre-injury score. ‘*’ means p < 0.05, comparison between the 4.0 MPa group and the 4.5 MPa group; ‘**’ means p < 0.01, comparison between the 4.0 MPa group and the 4.5 MPa group.

Results

In the 4.5 MPa group, the measured injury parameters are as follows: peak overpressure 396.92 ± 23.11 kPa, rise time 9.41 ± 0.32 ms, positive phase duration 51.57 ± 1.39 ms, and positive phase impulse 5884.26 ± 231.44 kPa.ms. The pressure-time profile of the shock wave is shown in Figure 5. In the 4.0 MPa cohort, the mortality rate was 0% both immediately post-injury and at 12 h. In the 4.5 MPa cohort, the immediate post-injury mortality rate was 16.67% (2/12), which increased to 41.67% (5/1...

Discussion

Blast lung injury is a severe condition resulting from exposure to explosive blasts. It is characterized by pulmonary parenchymal damage, alveolar hemorrhage, inflammation, and coagulation disorders, and frequently progresses to ARDS with high mortality^11,12,13. Current diagnostic methods include CT for detailed morphological assessment^3,14 and techniques such as esophag...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
64-slice CT scanner	GE Lightspeed, USA	5133730-3
Babylog 8000	Dräger, Germany	https://www.draeger.com/en_in/Products/Babylog-8000-plus
Biologic shock Tube (the large one) (BST-I)	Daping Hosipital, Army Medical University	BST-I
CG4+ test cartridge	Abbott, USA	CG4+
Double-lumen central venous catheter	Braun, Germany	Duo V730
Electric Suction Apparatus	Jiangsu Yuyue Medical Equipment & Supply Co., Ltd.	7A-23D
Endotracheal tube	TAMPA, Hangzhou	ETT6014C
Heparin Sodium	Changzhou Qianhong Bio-pharma Co., Ltd.
I.V.Catheter for single use	Weihai Jierui Medical Products Co., Ltd.	22G × 25mm/Y-G
ImageJ software
IOTech  WaveView 7.15.6 software	IOtech, USA	https://iotechsoftware.com/
Midazolam	Nhwa Pharma Co. China	http://pharma-api.com/2-2-5-midazolam-injection/
PCB  sensor	PCB Piezotronics, Inc. (USA)	M102A02
Pentobarbital sodium	Shanghai Pharmaceutical	NA
Philips IntelliSpace Portal workstation		https://www.philips.ca/healthcare/product/HC881101/intellispace-portal-12
Picco Monitoring Kit	PULSION, Germany	PV8215
Portable blood gas analyzer	Abbott, USA	i-STAT 300G
Portable wireless color ultrasound device	Youkey Bio-Medical Electronics Co., China	D-236
Veet pure hair removal cream	Reckitt Benckiser	https://www.veet.co.in/en/products/hair-removal-creams/