The authors would like to express their gratitude to Li Yang for designing the study&#39;s concept, Zhao Dongchu and Guo Yong for conducting the surgical procedures, Zhao Dongchu for data analysis and manuscript preparation, and Zhang Lianyang for supervising the research project. This work was supported by the Chongqing Municipal Health and Family Planning Commission and Science and Technology Commission Medical Scientific Research Project (No. 2024MSXM084), Construction of Key Clinical Specialties in the Military and Clinical Medical Research on Critical and Severe Diseases in Chongqing City.

All experimental procedures received approval from the Laboratory Animal Welfare and Ethics Committee of the Army Medical University (AMUWEC20201513), adhering to the 3Rs ethical guideline, which advocates for the reduction, refinement, and replacement of animal use. A total of 12 healthy 8-month-old (49-56 kg) male Yorkshire swine were selected as the animal model and randomized into three groups (n=4/group). The weight and anatomical structure of Yorkshire swine are similar to those of humans, making them suitable for biomedical research.
1. Surgical preparation
NOTE: A typical experiment spans 120 min, starting with 30 min dedicated to surgical preparation. This is followed by a sequence of events at 10 min intervals, specifically defined as T0, T10, T20, T30, T40, T50, T60, T70, T80, and T90, ensuring meticulous timing throughout the process. After surgical setup, the protocol includes 20 min for (VCH) and 40 min for applying external compression. The experiment concludes with 30 min allocated for follow-up monitoring to assess the outcomes of the interventions (Figure 1).
<ol>
	<li>Animal preparation
	<ol>
		<li>After arrival in the facility, house each swine in a separate room with a temperature of 24 &#176;C and a humidity of 60%. Dry fast (abstaining from food but only on water) the swine for 12 h before the surgery to prevent nausea, vomiting, and aspiration.</li>
	</ol>
	</li>
	<li>Preparation on the day of surgery
	<ol>
		<li>Premedicate the swine by intramuscular injection of ketamine (20 mg/kg) and atropine (0.01 mg/kg) in the lateral region of the neck. Assess the level of anesthesia by jaw tone, and both pedal and palpebral reflex responses. 
		NOTE: Based on our experience, the anesthetic doses administered are sufficient to sedate the animal, and the use of atropine can prevent excessive salivation. However, if the time taken to transfer the animal from the housing to the operating room is too long, additional doses of ketamine (20 mg/kg) can be injected safely. Sedate the animal in accordance with the approved IACUC protocol.</li>
		<li>Transfer the sedated animal to the operating room and secure its limb to an operating table in a supine position. Drape the operating table in a sterile fashion and equip it with a warming blanket.</li>
		<li>Puncture the ear vein with an intravenous indwelling needle. Attach a 60 inch extension line to the indwelling needle and connect the other end to a micro-pump. Induce general anesthesia and maintain by continuous infusion of fentanyl (4 &#181;g/kg/h), midazolam (0.05 mg/kg/h), and propofol (10 mg/kg/h).</li>
	</ol>
	</li>
	<li>Tracheostomy procedure
	<ol>
		<li>Disinfect the neck area by scrubbing with povidone-iodine scrub and alcohol for at least 3 alternating rounds.</li>
		<li>Prior to making the incision, assess the depth of anesthesia through jaw tone and pedal reflex to ensure adequate anesthesia. Using a scalpel, make an 8 cm longitudinal skin incision from the thyroid cartilage to the manubrium.</li>
		<li>Dissect along the medial surfaces of the two sternohyoid muscles using blunt-tip surgical scissors, deepening the dissection and making a 1 cm incision at the trachea.</li>
		<li>Insert an appropriately sized tracheostomy tube (7mm-10mm) based on the size of the pig and connect the other side to the ventilator. 
		NOTE: In this model, the animals are ultimately humanely euthanized. The intubation procedure for pigs is relatively complex and time-consuming. To save surgical time and reduce the pain experienced by the animals, we opted for a rapid tracheostomy after anesthesia rather than intubation before anesthesia induction.</li>
	</ol>
	</li>
	<li>Mechanical ventilation
	<ol>
		<li>Set the ventilator to intermittent positive-pressure ventilation mode, with a tidal volume of 8 mL/kg, respiratory rate of 20 breaths per minute, and FiO2 of 30%. Adjust ventilator to maintain PCO2 between 35-45 mmHg. Adjust ventilator settings to maintain vital parameters, ensuring ETCO2 and SPO2 are within the appropriate range.</li>
	</ol>
	</li>
	<li>Left carotid artery and jugular vein cannulation
	<ol>
		<li>For the cannulation of the left carotid artery, employ the cut-down technique, necessitating a minimum of two operators for the procedure.</li>
		<li>Ensure the availability of crucial instruments for cannulation, including micro-scissors, sutures with needles, an 18G needle, a tube, a 5 French catheter sheath with an introducer, and a Seldinger guide wire.</li>
		<li>Using the incision made during tracheostomy, dissect the tissue lateral to the sternohyoid muscle to isolate the left carotid artery from the surrounding fascia.</li>
		<li>Use non-absorbable silk sutures to loop around the isolated segment of the left carotid artery before puncture.</li>
		<li>Utilize silk sutures to elevate the artery with one hand while puncturing it with the needle in the other. To verify the accuracy of the needle placement, administer 5 mL of 0.9% NaCl solution through the needle and observe for an arterial wall expansion. Following confirmation of the needle&#39;s correct positioning, advance the guide wire through the tube while concurrently retracting the needle.</li>
		<li>Extract the tube, then proceed to insert the sheath, utilizing the introducer guided over the wire. Following insertion, ensure the removal of both the introducer and the wire. Secure the sheath to the skin by suturing the flanges attached with 3-0 silk.</li>
		<li>Use the same technique to expose and cannulate the left jugular vein.</li>
	</ol>
	</li>
	<li>Right carotid artery and jugular vein cannulation
	<ol>
		<li>For the isolation of the right carotid artery and jugular vein, perform a lateral dissection adjacent to the sternohyoid muscle on the contralateral side.</li>
		<li>Place a 7.0 Fr central venous catheter with dual ports into the right jugular vein, and promptly link a transducer system for central venous pressure measurement. 
		NOTE: Catheter size may vary based on pig size.</li>
		<li>Connect lactated Ringer&#39;s solution (7 mL/kg/h) to one port of the central line and administer a maintenance saline infusion (5 mL/kg/h) through the other port to mitigate the risk of catheter occlusion.</li>
		<li>Introduce a 4.0 Fr arterial catheter equipped with a thermistor into the right carotid artery, connecting it to the cardiac monitor while simultaneously connecting the monitor&#39;s venous measuring unit to the central venous transducer.</li>
		<li>Calibrate both the venous and arterial transducers to zero referencing the midaxillary line.</li>
		<li>Employ a thermal blanket to keep the animal&#39;s temperature within 37 &#176;C -39 &#176;C, with temperature monitoring facilitated by a thermistor at the arterial catheter&#39;s tip.</li>
		<li>Add mean arterial pressure to diastolic and systolic readings.</li>
	</ol>
	</li>
</ol>
2. Volume-controlled hemorrhage
<ol>
	<li>Following the outlined protocol, T0 marks the start of the VCH, immediately after the initial 30 min surgical preparation period. Use baseline data collected at T0 for the initiation of hemorrhagic shock by withdrawing blood from the left jugular vein at specific average rates for each group: 0.33 mL/kg/min for Group I, 0.67 mL/kg/min for Group II, and 1 mL/kg/min for Group III, over a duration of 20 min.</li>
	<li>T20 signifies the conclusion of the VCH phase. At this juncture, ensure that groups I, II, and III attain hemorrhage levels of 10%, 20%, and 30%, respectively, relative to the total blood volume (TBV).</li>
	<li>Determine the TBV of the swine, expressed in mL, through manual calculation, employing an estimated blood volume parameter of 70 mL/kg1,2,3. It is imperative to maintain the specific average rate during the blood withdrawal process.</li>
</ol>
3. Axillary artery injury
<ol>
	<li>Disinfect the axilla by scrubbing with povidone-iodine scrub and alcohol at least 3 alternating rounds.</li>
	<li>Employ ultrasonography to map the surface projection of the axillary artery, utilizing Doppler sonography for the distinction of arterial, venous, and neural structures within the axillary fossa. Align the probe perpendicular to the dermal layer above the pectoralis muscle, which allows for the precise anatomical localization of the axillary artery.</li>
	<li>Mark a 10 cm arc-shaped incision as the surgical entry based on the anatomical landmarks established through ultrasonographic imaging (Figure 2A). The axillary artery lumen is anechoic, while the wall appears as a round, hyperechoic structure, and is surrounded by the hyperechoic brachial plexus.</li>
	<li>Apply a force of approximately 20 N with the probe and see the vein occlude upon compression with the ultrasound probe. The axillary sheath manifests as a hyperechoic, linear structure encasing the neurovascular bundle (Figure 2B).</li>
	<li>Incise the 10 cm arc-shaped marked incision on the skin with a scalpel. Continue to incise the subcutaneous tissue along the incision to expose the underlying structures. Partially remove the superficial and deep pectoralis muscles to expose the axillary sheath (Figure 2C). 
	NOTE: The axillary artery of the swine originates from the subclavian artery that is covered by the subclavian muscle. After reaching the upper edge of the deep pectoral muscle, it passes between the superficial and deep pectoral muscles and continues as the brachial artery. A deep axillary sheath surrounds the axillary artery, vein, and brachial plexus.</li>
	<li>To expose the axillary artery, blunt dissect the axillary sheath with a micro-scissor and isolate a 6 cm segment of the artery from the surrounding vein and brachial plexus (Figure 2D).</li>
	<li>Upon isolation, loop two vascular blocking bands around the 6 cm segment to secure the vessel: place the red band at the proximal end and the green band at the distal end prior to the infliction of injury.</li>
	<li>Tie up the two vascular blocking bands to temporarily block the blood flow. If the vascular blocking bands are released, blood can be ejected from the vessel, simulating uncontrolled hemorrhage from axillary artery injury.</li>
	<li>Make a 2 mm transverse incision using a micro-scissor, which should be approximately 1/3 of the circumference of the vessel.</li>
	<li>Place 5 sheets of pre-weighted gauze (6 cm x 8 cm) over the incision on the axillary artery, with each sheet capable of absorbing approximately 30 mL of blood. Check the vascular blocking bands to ensure both are tied up and maintain tension to obstruct blood flow.</li>
</ol>
4. External compression
<ol>
	<li>Pre-set the external compression device
	<ol>
		<li>Attach the mechanical arm to the side of the operating table. Connect the inflatable hemostatic balloon (Figure 3) to the end of the arm. 
		NOTE: The external compression device consists of three parts: a mechanical arm, an inflatable hemostatic balloon, and a pressure measurement system.</li>
		<li>Immobilize the flexible film pressure sensor at the central point on the surface of the inflatable hemostatic balloon to measure the direct pressure. Connect the flexible film pressure sensor to an electrical wire, then link the wire to a data acquisition card.</li>
		<li>Attach the data acquisition card to a single-channel signal conditioning module and connect it to the data acquisition software. 
		NOTE: The pressure measurement system comprises a flexible film pressure sensor, an electrical wire, a data acquisition card, a single-channel signal conditioning conversion module (Figure 4), and data acquisition software.</li>
		<li>After installation, open the software. Click on the Connected Device, select the Single-Channel Option, and choose 1000 Data Points Per Second from the sampling rate menu. For the input channel, select Band Display; for the output channel, select Output Display.</li>
		<li>Before recording, ensure the pressure measurement system is calibrated to zero. Click the Circular Red Button to start recording.</li>
	</ol>
	</li>
	<li>To simulate external compression, target the mechanical arm on the axillary wound immediately after releasing the two vascular blocking bands. Manually inflate the hemostatic balloon to achieve local compression until the pressure reaches 210 mmHg. Record the pressure-time data and output in .CSV format in real time. 
	NOTE: Accurate pressure can be obtained through software processing after manually inflating the balloon. The pressure is measured in a range of 0-300 mmHg with a sampling rate of 1000 sampling points/second/channel.</li>
	<li>Allow 40 min for external compression to enable the formation of local blood clots. Observe the device during compression to ensure that there is no displacement. 
	NOTE: In this experiment, effective compression was defined as achieving a blood loss under compression of less than 10% of the animal&#39;s body weight.</li>
	<li>After the external compression, click the Red Square Button to stop data collection. Remove the device and weigh the gauze to calculate the blood loss under compression (Figure 5).</li>
</ol>
5. Follow-up and euthanasia
<ol>
	<li>Monitor the animal for 30 min after removal of the device.</li>
	<li>At the end of the experiment, increase the anesthetic infusion rates to a higher dose, such as fentanyl &#62;10 &#181;g/kg/h, propofol &#62;20 mg/kg/h, and midazolam &#62;0.1 mg/kg/h. Continue cardiac monitoring until the ECG shows no cardiac electrical activity and end-tidal CO2 tension reaches 0 mmHg.</li>
	<li>Confirm the absence of pulsatile flow on invasive arterial pressure monitoring and the absence of other vital signs. 
	NOTE: Follow the approved euthanasia procedure outlined in the IACUC protocol. A confirmatory method of euthanasia should also be performed, such as bilateral thoracotomy.</li>
</ol>

Swine Axillary Artery Injury Model for Testing Pre-Hospital Hemostatic Products

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The authors declared that no competing conflicts of interest exist.

Axillary artery injury is the main cause of massive hemorrhage at the upper extremity junctional site1. For active bleeding control, manual compression, gauze tamponade, and hemostatic agents are commonly used methods in combat or pre-hospital settings8. While compression alone can temporarily control junctional hemorrhage, it may not completely occlude the axillary artery due to the flexibility of the scapula. Additionally, most local hemostatic agents are in the form of gauze and membrane sheets that require manual compression to hold them in place at the bleeding site9. Currently, there are no relevant clinical studies that have confirmed a reliable compression pressure when combined with gauze tamponade or hemostatic agents. This study developed a new hybrid quantitative evaluation model for upper extremity junctional hemorrhage in swine that offers the advantage of further development of pre-hospital hemostatic products.
When constructing a hemorrhagic shock model, the primary challenge is to accurately replicate the clinical scenario while ensuring high reproducibility and standardization10. Therefore, we have developed a hybrid quantitative evaluation model that offers several advantages over the traditional hemorrhagic shock model:
This study introduces a swine axillary artery injury model for the first time. In recent publications, the majority of research has utilized the femoral artery injury model to evaluate the efficacy of new hemostatic agents11,12,13,14,15. However, limited attention has been given to describing the feasibility of the axillary artery injury model. This is attributed to the challenges associated with performing an axillary artery injury in swine, arising from the complex anatomical features of the swine axilla. To minimize unintentional injury or bleeding, an ultrasound-guided axillary approach is preferred to locate the axillary artery before dissection. This choice is made because the vessels are situated deep between the superficial and deep pectoral muscles and are surrounded by an axillary sheath. Meanwhile, another challenge is to achieve a consistent 2 mm transverse incision in all animals through visual estimation.
The vascular blocking bands provide traction force to temporarily occlude blood flow in the injured axillary artery (open artery). In the current landscape of research, one encounters critical challenges when developing novel hemostatic materials for junctional hemorrhages. These challenges stem from deep and narrow wounds in junctional regions that result in rapid and pressurized blood flow. These dynamics significantly hinder the ability of hemostatic materials to absorb and manage interfacial fluid. Under conditions of massive hemorrhage, this is especially detrimental, as the high-pressure, rapidly flowing blood can wash away hemostatic agents and disrupt any inadequately formed clots. The study introduces the use of two vascular blocking bands that temporarily control the pressurized blood flow. This strategy enables the evaluation of the efficacy of hemostatic materials without the confounding effects of active hemorrhage preceding the surgical intervention to manage bleeding.
The compression pressure in the experiment can be set to be a specific value according to the needs of the experiment and kept relatively stable throughout the experiment. The use of an external compression device to apply adjustable pressure to the tamponade gauze is a valuable development in the field of junctional hemorrhage research. The specific compression pressure of the upper extremity junctional hemorrhage has not yet been clarified. According to the case report of a gunshot wound, abdominal aortic and junctional tourniquet can be used in the axilla, but the required compression pressure was not clearly stated16. Johnson et al.5 verified the effectiveness and safety of Sam junctional tourniquet in the groin and axillary region, indicating occlusion of the axillary artery requires an average of 739 mmHg. In 2022, Avital et al.17 documented a case where a manual pressure points technique was utilized to control axillary hemorrhage during a 21 min flight transportation, following the repeated failure of a hemostatic bandage. This case report confirmed that the pressure points technique can successfully halt bleeding in the axillary junctional region and can be sustained over an extended period. However, the application of pressure was performed by an aeromedical evacuation medic, whose prolonged exertion led to fatigue, thereby compromising the ability to maintain consistent pressure throughout the procedure. In this model, the gauze tamponade and local compression were used simultaneously. We did not seek to block the axillary artery flow completely but used the method of gauze tamponade combined with external compression to achieve local hemostasis. The results indicate that only 210 mmHg of external pressure was required to effectively stop bleeding when combined with gauze tamponade. Additionally, the employment of a mechanical arm ensures a constant and unaltered pressure at the pressure point, thereby augmenting the effectiveness of this hemorrhage control strategy. For further research, the compression pressure of this device can be adjusted and customized based on the tamponade material. If various newly developed pressure-required hemostatic dressings need to be tested for their efficiency, the pressure can be adjusted to accommodate the experimental settings. For example, if the pressure is set to a specific value, it is possible to determine which hemostatic dressing is more effective by comparing the time to hemostasis and blood loss under compression.
Class I-III shock was induced by combining VCH with uncontrolled hemorrhage, accurately simulating the clinical scenario of axillary artery penetration injury while adjusting the total blood loss to the required range. The hemorrhagic shock model can be categorized into three types: volume-controlled, pressure-controlled, and uncontrolled. The volume-controlled hemorrhagic shock model simulates the process of hemorrhagic shock by releasing a certain proportion of blood based on the effective circulating volume over a set period. This model has advantages in evaluating hemodynamics (such as compensatory mechanisms), but it is difficult to control the degree of changes in blood pressure (such as hypotension). It has been found that rapid blood flow followed by slow exsanguination induces a more intense physiological response (heart rate, serum lactic acid, and amount of resuscitation fluid required) than constant exsanguination (traditional methods), and more realistically simulates the process of hemorrhagic shock. There are many ways to establish the model of uncontrolled hemorrhagic shock. The commonly used models of uncontrolled hemorrhagic shock include liver or spleen tear hemorrhage model, aortic and femoral artery injury hemorrhage model, etc.18,19,20,21,22. This hemorrhagic shock model is very close to the clinical situation of patients with trauma or severe bleeding and is widely used, especially in the study of fluid resuscitation of hemorrhagic shock. In this study, the blood drawing from the femoral artery of 10%, 20%, and 30% TBV combined with the fixed pressure compression of the mechanical arm induced Grade I-III hemorrhagic shock. Hemodynamic parameters were monitored every 10 min during the whole process to ensure the stability of the model.
This model has several significant limitations, including the aspect of uncontrolled hemorrhage. A major constraint is the precision required for the 2 mm transverse incision in the axillary artery. While our data show a narrow standard error for blood loss under compression, variability in incision size and, consequently, in uncontrolled hemorrhage may increase when different individuals perform the procedure. Another limitation is the anesthesia and pain management of the swine used in the experiment. Given the extensive surgical procedures involved in the surgical preparation phase of this model, managing analgesia and anesthesia depth is crucial. It is important to closely monitor for signs of pain and provide appropriate treatment during tracheostomy, cannulation, and axillary artery injury.
Conclusion 
In this study, we introduced a novel hybrid quantitative evaluation model for upper extremity junctional hemorrhage in swine, marking a significant advancement in the development of pre-hospital hemostatic products. The utilization of a swine axillary artery injury model, paired with the implementation of vascular blocking bands and an external compression device, addresses critical challenges in the control of junctional hemorrhages, such as achieving consistent and controlled pressure to enhance hemostatic efficacy. Our findings demonstrate that a combination of gauze tamponade with a precisely controlled external pressure of 210 mmHg, further supported by the constant force provided by a mechanical arm, effectively stops bleeding. This approach not only provides a practical solution for managing axillary artery injuries but also sets the stage for future research on adjustable pressure mechanisms for various hemostatic dressings. This enhances the clinical relevance and applicability of the findings. These results have significant potential to improve human trauma care, especially in pre-hospital settings.

The management of traumatic hemorrhage, especially in junctional regions where traditional tourniquets cannot be applied, presents a significant challenge in both military and civilian trauma care. Effective control of bleeding in areas such as the axillary and groin regions, characterized by complex vascular anatomy, is crucial for survival. Axillary artery injury is the main cause of massive hemorrhage at the upper extremity junctional site1. Dressings' packing and external compressing are the key steps in pre-hospital emergency hemorrhage control. The progress of new hemostatic dressings has greatly improved the hemostatic efficiency. However, external compression is essential for effective hemostasis in the early stage2,3. This study introduces a novel hybrid quantitative evaluation model using a swine model to simulate upper extremity junctional hemorrhage, aiming to advance pre-hospital hemostatic interventions.
Junctional injuries often require constant, direct manual pressure, which is challenging to achieve in prehospital settings due to limited personnel and unpredictable circumstances4. The rationale behind developing this technique lies in the limitations of existing methods for controlling junctional hemorrhage. The proposed hybrid quantitative evaluation model has a distinct advantage over alternative techniques. By using a swine model, which closely mimics human physiology, the study can provide more reliable and translatable results compared to a perfused cadaver model5.
Swine models are extensively utilized in vascular trauma research due to their physiological and anatomical similarities to humans, making them ideal for evaluating hemostatic agents and techniques6,7. The study's protocol meticulously combines volume-controlled hemorrhage (VCH) with external mechanical compression to accurately mimic the clinical scenario of axillary artery injury, thus addressing the urgent need for reliable hemostatic methods in junctional hemorrhage scenarios.
The integration of ultrasonography enhances the precision of hemorrhage control techniques and facilitates image interpretation, highlighting the interdisciplinary nature of modern trauma care. The development of a mechanical external compression device marks a significant advancement, offering a promising alternative to manual compression and traditional hemostatic methods. This research aims to determine the optimal pressure required for effective hemostasis in junctional areas, potentially filling a significant gap in trauma care literature.

<table><tbody><tr><td>Data Acquisition Software MCC DAQami</td><td>Measurement Computing Corporation</td><td>4.2.1f0</td><td>Advanced data acquisition and monitoring software designed to provide an intuitive and efficient user experience, developed by Measurement Computing Corporation based in Norton, MA, USA.</td></tr><tr><td>DF9-40 Flexible Film Pressure Sensor</td><td>Chengke Electronics</td><td>500g</td><td>A flexible film pressure sensor designed for applications requiring force measurement</td></tr><tr><td>DuPont Wire, Data Acquisition Card</td><td>J.X.</td><td>CT USB-1208LS</td><td>A data acquisition card equipped with DuPont connectors for interface and signal processing tasks</td></tr><tr><td>Single Channel Signal Conditioning Conversion Module</td><td>Risym</td><td>IMS-C04A</td><td>A module used for conditioning and converting signals in a single channel setup</td></tr></tbody></table>

a new hybrid quantitative evaluation model for axillary junctional hemorrhage in swine

In this study, we developed and validated a hybrid quantitative model for simulating upper extremity junctional hemorrhage in swine, aiming to advance the development of pre-hospital hemostatic products. Utilizing 12 healthy 8-month-old male Yorkshire swine, we demonstrated the feasibility of a swine axillary artery injury model for evaluating hemostatic efficacy. Animals were divided into three groups to undergo volume-controlled hemorrhage (VCH), mimicking Class I-III hemorrhagic shock by withdrawing blood at different rates. Subsequent external compression was applied using a novel device consisting of a mechanical arm and an inflatable hemostatic balloon, achieving controlled pressure to enhance clot formation. Hemodynamic parameters, including heart rate and blood pressure, were continuously monitored, highlighting the impact of controlled hemorrhage and external compression on physiological responses. The findings suggest that the combination of VCH with targeted external compression effectively simulates clinical scenarios of axillary artery injury, providing a valuable model for testing hemostatic interventions in a controlled, standardized manner. This study underscores the potential of the model in facilitating the development and evaluation of new hemostatic agents and devices for managing junctional hemorrhages.

All the 12 animals survived till the end of the experiment. All the statistical analyses were processed by statistical analysis software. The hybrid quantitative model produces quantitative volume-controlled blood loss and consistent blood loss under compression. In Figure 6A, after VCH, the mean blood loss in groups I, II, and III was 354.2 mL, 714.4 mL, and 1064.0 mL respectively, accounting for 10%, 20%, and 30% of the TBV. These results demonstrate that this model can be used in various experimental setups, allowing for quantitative blood loss to be achieved through VCH. Figure 6B shows that there was no statistically significant difference in blood loss under compression among the three groups (p &#62; 0.05). This consistent blood loss can be obtained by making an approximately 2 mm incision in the axillary artery using microscopic scissors, which is 1/3 of the circumference of the axillary artery. In Figure 6C, at the end of the experiment, the mean total blood loss for groups I, II, and III was 462.9 mL, 893.0 mL, and 1213.0 mL, respectively, achieving Class I, II, and III hemorrhagic shock, respectively.
The data depicted in Figure 7 elucidates the impact of controlled hemorrhage on hemodynamic parameters, indicating a marked increase in heart rate across all groups as a response to induced shock. The escalation in heart rate was most pronounced during the application of external compression and showed a decline upon its removal. The extent of heart rate elevation was directly proportional to the volume of blood withdrawn, with Group III exhibiting the most substantial increase, followed by Group II, and then Group I. This trend is quantitatively underscored by the maximal deviation in heart rate (&#916;HR = HR_peak - HR_baseline), where Group III recorded a significantly higher change (Group I: 11.28 &#177; 1.2 bpm, Group II: 30.08 &#177; 2.7 bpm, Group III: 106.80 &#177; 7.2 bpm, p &#60; 0.0001).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66974/66974fig01.jpg" /> 
Figure 1: The experimental setup and the timeline of the experiment. (A) Experimental setting with the swine. (B) The surgical preparation steps, along with the timeline of the experiment. The timeline of the procedure extends over 120 min, beginning with a 30 min surgical preparation phase, followed by designated time points every 10 min (T0 to T90), where specific actions are taken, such as volume-controlled hemorrhage, vascular blocking bands traction and release, external compression, and follow-up monitoring. <a href="https://www.jove.com/files/ftp_upload/66974/66974fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66974/66974fig02.jpg" /> 
Figure 2: Ultrasound-guided dissection of axillary anatomy in swine. (A) Probe placement for the ultrasonographic identification of axillary structures, with a 10 cm arc-shaped incision marked for surgical entry. (B) Sonographic image detailing the axillary space at 6 cm depth; (i) Sonoanatomy and (ii) revised ultrasound anatomy. The linear transducer is oriented in a parasagittal plane. The axillary artery (A) and vein (V) are indicated alongside the brachial plexus (arrows), deep pectoral muscle (DP), and superficial pectoral muscle (SP). (C) Initial incision along the projected path of the axillary artery. (D) Exposure of the axillary artery and neurovascular bundle following the incision, with the superficial and deep pectoral muscles partially excised. <a href="https://www.jove.com/files/ftp_upload/66974/66974fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66974/66974fig03.jpg" /> 
Figure 3: Components of the external compression device. The inflatable hemostatic balloon. <a href="https://www.jove.com/files/ftp_upload/66974/66974fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66974/66974fig04.jpg" /> 
Figure 4: Pressure measurement system. Hardware components of the pressure measurement system, including the flexible film pressure sensor connected via electrical wire to a single-channel signal conditioning conversion module. The module is then connected to a data acquisition card, which interfaces with a computer. <a href="https://www.jove.com/files/ftp_upload/66974/66974fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66974/66974fig05.jpg" /> 
Figure 5: Assessment of blood loss under compression via gauze saturation. (A) Gauze extracted from the surgical wound after external compression. (B) The blood-soaked gauze aligned for pre-weighing to quantify blood loss under compression. <a href="https://www.jove.com/files/ftp_upload/66974/66974fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66974/66974fig06.jpg" /> 
Figure 6: Assessment of blood loss and body weight. (A) Bar graph depicting volume-controlled blood loss as a percentage of total blood volume (TBV) for three different groups. (B) Bar graph showing blood loss under compression with no significant difference among the groups. (C) Bar graph representing total blood loss by the end of the experiment corresponding to three classes of hemorrhagic shock. The left Y-axis represents blood loss, while the right Y-axis represents the percentage of TBV. Each group has a sample size of N=6. All continuous data were expressed as mean &#177; standard deviation, and significance was set at 95% confidence intervals (C.I.s). Repeated Measures One-way analysis of variance (ANOVA) was employed to determine the effect of hemorrhage volume on hemodynamic parameters. <a href="https://www.jove.com/files/ftp_upload/66974/66974fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/66974/66974fig07.jpg" /> 
Figure 7: Hemodynamic responses during the experiment. (A-C) correspond to the three experimental groups subjected to different volumes of blood withdrawal. The graphs track the heart rate (red line), systolic blood pressure (blue line), and diastolic blood pressure (black line) over time, alongside the percentage of total blood volume (TBV) loss (shaded area). The data show the temporal progression of heart rate and blood pressure from the start of hemorrhage (T0) through to 90 min post-hemorrhage (T90). * indicates that the hemodynamic parameters at each time point have a statistically significant difference compared to T0 within each group (p&#60;0.05). All continuous data were expressed as mean &#177; standard deviation, and significance was set at 95% confidence intervals (C.I.s). Repeated Measures One-way analysis of variance (ANOVA) was employed to determine the effect of hemorrhage volume on hemodynamic parameters. <a href="https://www.jove.com/files/ftp_upload/66974/66974fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

A New Hybrid Quantitative Evaluation Model for Axillary Junctional Hemorrhage in Swine

This study presents a hybrid quantitative model for axillary junctional hemorrhage in swine, enhancing pre-hospital hemostatic intervention evaluation.

In this study, we developed and validated a hybrid quantitative model for simulating upper extremity junctional hemorrhage in swine, aiming to advance the ...

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182 Views.  Army Medical University. This study presents a hybrid quantitative model for axillary junctional hemorrhage in swine, enhancing pre-hospital hemostatic intervention evaluation.

Video: A New Hybrid Quantitative Evaluation Model for Axillary Junctional Hemorrhage in Swine

A Swine Model of Neonatal Asphyxia

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, perfusion deficit, hypoxic-ischemic encephalopathy, pulmonary hypertension, vasculopathic enterocolitis, renal failure and thrombo-embolic complications. Animal models are developed to help us understand the patho-physiology and pharmacology of neonatal asphyxia. In comparison to rodents and newborn lambs, the newborn piglet has been proven to be a valuable model. The newborn piglet has several advantages including similar development as that of 36-38 weeks human fetus with comparable body systems, large body size (&tilde;1.5-2 kg at birth) that allows the instrumentation and monitoring of the animal and controls the confounding variables of hypoxia and hemodynamic derangements.
We here describe an experimental protocol to simulate neonatal asphyxia and allow us to examine the systemic and regional hemodynamic changes during the asphyxiating and reoxygenation process as well as the respective effects of interventions. Further, the model has the advantage of studying multi-organ failure or dysfunction simultaneously and the interaction with various body systems. The experimental model is a non-survival procedure that involves the surgical instrumentation of newborn piglets (1-3 day-old and 1.5-2.5 kg weight, mixed breed) to allow the establishment of mechanical ventilation, vascular (arterial and central venous) access and the placement of catheters and flow probes (Transonic Inc.) for the continuously monitoring of intra-vascular pressure and blood flow across different arteries including main pulmonary, common carotid, superior mesenteric and left renal arteries. Using these surgically instrumented piglets, after stabilization for 30-60 minutes as defined by Z&lt;10% variation in hemodynamic parameters and normal blood gases, we commence an experimental protocol of severe hypoxemia which is induced via normocapnic alveolar hypoxia. The piglet is ventilated with 10-15% oxygen by increasing the inhaled concentration of nitrogen gas for 2h, aiming for arterial oxygen saturations of 30-40%. This degree of hypoxemia will produce clinical asphyxia with severe metabolic acidosis, systemic hypotension and cardiogenic shock with hypoperfusion to vital organs. The hypoxia is followed by reoxygenation with 100% oxygen for 0.5h and then 21% oxygen for 3.5h. Pharmacologic interventions can be introduced in due course and their effects investigated in a blinded, block-randomized fashion.

Large animal models have good translational values in the examination of physiology and pharmacology of neonatal asphyxia. Using newborn piglets, we develop an experimental protocol to simulate neonatal asphyxia which has advantages of studying the systemic and regional hemodynamics, oxygen transport with biochemical and pathologic pathways and correlations.

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

Complete and Partial Aortic Occlusion for the Treatment of Hemorrhagic Shock in Swine

Hemorrhage remains the leading cause of preventable deaths in trauma. Endovascular management of non-compressible torso hemorrhage has been at the forefront of trauma care in recent years. Since complete aortic occlusion presents serious concerns, the concept of partial aortic occlusion has gained a growing attention. Here, we present a large animal model of hemorrhagic shock to investigate the effects of a novel partial aortic balloon occlusion catheter and compare it with a catheter that works on the principles of complete aortic occlusion. Swine are anesthetized and instrumented in order to conduct controlled fixed-volume hemorrhage, and hemodynamic and physiological parameters are monitored. Following hemorrhage, aortic balloon occlusion catheters are inserted and inflated in the supraceliac aorta for 60 min, during which the animals receive whole-blood resuscitation as 20% of the total blood volume (TBV). Following balloon deflation, the animals are monitored in a critical care setting for 4 h, during which they receive fluid resuscitation and vasopressors as needed. The partial aortic balloon occlusion demonstrated improved distal mean arterial pressures (MAPs) during the balloon inflation, decreased markers of ischemia, and decreased fluid resuscitation and vasopressor use. As swine physiology and homeostatic responses following hemorrhage have been well-documented and are like those in humans, a swine hemorrhagic shock model can be used to test various treatment strategies. In addition to treating hemorrhage, aortic balloon occlusion catheters have become popular for their role in cardiac arrest, cardiac and vascular surgery, and other high-risk elective surgical procedures.

Here, we present a protocol demonstrating a hemorrhagic shock model in swine that uses aortic occlusion as a bridge to definitive care in trauma. This model has application in testing a wide range of surgical and pharmacological therapeutic strategies.

Hemorrhage remains the leading cause of preventable deaths in trauma. Endovascular management of non-compressible torso hemorrhage has been at the ...

Brachial Artery Catheterization in Swine

The video describes in detail the catheterization of the distal brachial artery in swine. This technique enables researchers to measure arterial blood pressure continuously and collect arterial blood samples to assess arterial blood gas measurements. Arterial blood pressures and arterial blood gases are important physiological parameters to monitor during experimental procedures. In swine, four common methods of arterial catheterization have been described, including catheterization of the carotid, femoral, auricular, and medial saphenous arteries. Each of these techniques have advantages, such as ease of access for the auricular artery, and disadvantages that include deep tissue dissection for carotid artery catheterization. The described alternative method of arterial catheterization in swine, the catheterization of the distal aspect of the brachial artery, is a rapid procedure that requires relatively minimal tissue dissection and provides information that is in line with data collected from other arterial catheterization sites. The procedure uses a medial approach along an oblique plane of the lower brachium, positioned between the olecranon and the flexor aspect of the elbow joint, and this approach allows researchers the major advantage of unimpeded freedom for procedures that involve the caudoventral, caudodorsal back, or hind limbs of the pig. Due to the location of the upper forelimb of the catheterized vessel and potential challenges of effective homeostasis following catheter removal from the artery, this technique may be limited to non-recovery procedures.

The video describes in detail the catheterization of the distal brachial artery in swine. This procedure accurately measures arterial blood pressure and is a simple and fast method to collect samples for arterial blood gas measurements.

The video describes in detail the catheterization of the distal brachial artery in swine. This technique enables researchers to measure arterial blood ...

Standardized Model of Ventricular Fibrillation and Advanced Cardiac Life Support in Swine

Cardiopulmonary resuscitation after cardiac arrest, independent of its origin, is a regularly encountered medical emergency in hospitals as well as preclinical settings. Prospective randomized trials in human subjects are difficult to design and ethically ambiguous, which results in a lack of evidence-based therapies. The model presented in this report represents one of the most common causes of cardiac arrests, ventricular fibrillation, in a standardized setting in a large animal model. This allows for reproducible observations and various therapeutic interventions under clinically accurate conditions, hence facilitating the generation of better evidence and eventually the potential for improved medical treatment.

Cardiopulmonary resuscitation and defibrillation are the only effective therapeutic options during cardiac arrest caused by ventricular fibrillation. This model presents a standardized regimen to induce, assess, and treat this physiological state in a porcine model, thus providing a clinical approach with various opportunities for data collection and analysis.

Cardiopulmonary resuscitation after cardiac arrest, independent of its origin, is a regularly encountered medical emergency in hospitals as well as ...

Measuring Cardiac Output in a Swine Model

Swine are frequently used in medical research given their similar cardiac physiology to that of humans. Measuring cardiac parameters such as stroke volume and cardiac output are essential in this type of research. Contrast ventriculography, thermodilution, and pressure-volume loop (PV-loop) catheters can be used to accurately obtain cardiac performance data depending on which resources and expertise are available. For this study,five Yorkshire swine were anesthetized and intubated. Central venous and arterial access was obtained to place the necessary measurement instruments.A temperature probe was placed in the aortic root. A cold saline bolus was delivered to the right atrium and temperature deflection curve was recorded. Integration of the area under the curve allowed for the calculation of the current cardiac output.A pigtail catheter was percutaneously placed in the left ventricle and 30 mL of iodinated contrast was power injected over 2 seconds. Digital subtraction angiography images were uploaded to volumetric analysis software to calculate the stroke volume and cardiac output. A pressure volume-loop catheter was placed into the left ventricle (LV) and provided continuous pressure and volume data of the LV, which allowed the calculation of both stroke volume and cardiac output.All three methods demonstrated good correlation with each other. The PV-loop catheter and thermodilution exhibited the best correlation with a 3% error and a Pearson coefficient of 0.99, with 95% CI=0.97 to 1.1, (p=0.002). The PV-loop catheter against ventriculography also showed good correlation with a 6% error and a Pearson coefficient of 0.95, 95% CI=0.96 to 1.1 (p=0.01). Finally, thermodilution against ventriculography had a 2% error with r=0.95, 95% CI=0.93 to 1.11, (p=0.01). In conclusion, we state that the PV-loop catheter, contrast ventriculography, and thermodilution each offer certain advantages depending on the researcher's requirements. Each method is reliable and accurate for measuring various cardiac parameters in swine such as the stroke volume and cardiac output.

Thermodilution, pressure-volume loop catheters, and contrast ventriculography are reliable and accurate methods for determining cardiac physiology such as stroke volume and cardiac output in a laboratory setting in swine.

Swine are frequently used in medical research given their similar cardiac physiology to that of humans. Measuring cardiac parameters such as stroke volume ...

Swine Models of Aneurysmal Diseases for Training and Research

Large animal models, specifically swine, are widely used to research cardiovascular diseases and therapies, as well as for training purposes. This paper describes two different aneurysmal swine models that may help researchers to study new therapies for aneurysmal diseases. These aneurysmal models are created by surgically adding a pouch of tissue to carotid arteries in swine. When the model is used for research, the pouch must be autologous; for training purposes, a synthetic pouch suffices.
First, the right external jugular vein (EJV) and right common carotid artery (CCA) must be surgically exposed. The EJV is ligated and a vein pouch fashioned from a short segment. This pouch is then sutured to an elliptical arteriotomy performed in the CCA. Animals must be kept heparinized during model creation, and local vasodilators may be used to decrease vasospasms. Once the suture is completed, correct blood flow should be inspected, checking for bleeding from the suture line and vessel patency. Finally, the surgical incision is closed by layers and an angiography performed to image the aneurysmal model.
A simplification of this aneurysmal carotid model that decreases invasiveness and surgical time is the use of a synthetic, rather than venous, pouch. For this purpose, a pouch is tailored in advance with a segment of a polytetrafluoroethylene (PTFE) prosthesis, one end of which is sutured close using polypropylene vascular suture and sterilized prior to surgery. This "sac" is then attached to an arteriotomy performed in the CCA as described. 
Although these models do not reproduce many of the physiopathological events related to aneurysm formation, they are hemodynamically similar to the situation found in the clinical setting. Therefore, they can be used for research or training purposes, allowing physicians to learn and practice different endovascular techniques in animal models that are close to the human system.

Description of two different aneurysmal swine models for neuroradiology training courses and research studies. This study provides evidence of the feasibility of these aneurysm porcine model creations and the reproducible methods that are close to the clinical setting.

Large animal models, specifically swine, are widely used to research cardiovascular diseases and therapies, as well as for training purposes. This paper ...

Hickman Catheter for Long-Term Vascular Access in Swine

Hickman Catheter Use for Long-Term Vascular Access in a Preclinical Swine Model

Central venous catheters (CVCs) are invaluable devices in large animal research as they facilitate a wide range of medical applications, including blood monitoring and reliable intravenous fluid and drug administration. Specifically, the tunneled multi-lumen Hickman catheter (HC) is commonly used in swine models due to its lower extrication and complication rates. Despite fewer complications relative to other CVCs, HC-related morbidity presents a significant challenge, as it can significantly delay or otherwise negatively impact ongoing studies. The proper insertion and maintenance of HCs is paramount in preventing these complications, but there is no consensus on best practices. The purpose of this protocol is to comprehensively describe an approach for the insertion and maintenance of a tunneled HC in swine that mitigates HC-related complications and morbidity. The use of these techniques in &gt;100 swine has resulted in complication-free patent lines up to 8 months and no catheter-related mortality or infection of the ventral surgical site. This protocol offers a method to optimize the lifespan of the HC and guidance for approaching issues during use.

Author Spotlight: Hickman Catheter Use for Long-Term Vascular Access in a Preclinical Swine Model  

A reliable and reproducible approach for the insertion and maintenance of a tunneled Hickman catheter for long-term vascular access in swine is described. Placement of a central venous catheter allows for convenient daily sampling of whole blood from awake animals and intravenous administration of medication and fluids.

Central venous catheters (CVCs) are invaluable devices in large animal research as they facilitate a wide range of medical applications, including blood ...

Ascending Aortic Banding for Studying Pressure-Overload Induced Heart Failure

A Minimally Invasive Model of Aortic Stenosis in Swine

Large animal models of heart failure play an essential role in the development of new therapeutic interventions due to their size and physiological similarities to humans. Efforts have been dedicated to creating a model of pressure-overload induced heart failure, and ascending aortic banding while still supra-coronary and not a perfect mimic of aortic stenosis in humans, closely resembling the human condition.
The purpose of this study is to demonstrate a minimally invasive approach to induce left ventricular pressure overload by placing an aortic band, precisely calibrated with percutaneously introduced high-fidelity pressure sensors. This method represents a refinement of the surgical procedure (3Rs), resulting in homogenous trans-stenotic gradients and reduced intragroup variability. Additionally, it enables swift and uneventful animal recovery, leading to minimal mortality rates. Throughout the study, animals were followed for up to 2 months after surgery, employing transthoracic echocardiography and pressure-volume loop analysis. However, longer follow-up periods can be achieved if desired. This large animal model proves valuable for testing new drugs, particularly those targeting hypertrophy and the structural and functional alterations associated with left ventricular pressure overload.

Author Spotlight: Development of a Minimally Invasive Large-Animal Model for Reliable and Reproducible Cardiovascular Research 

This protocol describes a minimally invasive surgical procedure for ascending aortic banding in swine.

Large animal models of heart failure play an essential role in the development of new therapeutic interventions due to their size and physiological ...

Description of a Swine Infant Model of Volume-Controlled Hemorrhagic Shock

Hemorrhagic shock is a leading cause of morbidity and mortality in pediatric patients. Interpretation of the clinical indicators validated in adults to guide resuscitation and comparison between different therapies is difficult in children due to the inherent heterogeneity of this population. As a result, compared to adults, appropriate management of pediatric hemorrhagic shock is still not well established. In addition, the scarcity of pediatric patients with hemorrhagic shock precludes the development of clinically relevant studies. For this reason, an experimental pediatric animal model is necessary to study the effects of hemorrhage in children as well as their response to different therapies. We present an infant animal model of volume-controlled hemorrhagic shock in anesthetized young pigs. Hemorrhage is induced by withdrawing a previously calculated blood volume, and the pig is subsequently monitored and resuscitated with different therapies. Here, we describe a precise and highly reproducible model of hemorrhagic shock in immature swine. The model yields hemodynamic data that characterizes compensatory mechanisms that are activated in response to severe hemorrhage.

This article aims to provide researchers with a detailed and accessible guide to set up an infant porcine model of hemorrhagic shock.

Hemorrhagic shock is a leading cause of morbidity and mortality in pediatric patients. Interpretation of the clinical indicators validated in adults to ...