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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 ...

a-new-hybrid-quantitative-evaluation-model-for-axillary-junctional

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JoVE Journal

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177 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 ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks performed by the RPE are phagocytosis and processing of outer photoreceptor segments through lysosome-derived organelles. These degradation products, stored and referred to as lipofuscin granules, are composed partially of bisretinoids, which have broad fluorescence absorption and emission spectra that can be detected clinically as fundus autofluorescence with confocal scanning laser ophthalmoscopy (cSLO). Lipofuscin accumulation is associated with increasing age, but is also found in various patterns in both acquired and inherited degenerative diseases of the retina. Thus, studying its pattern of accumulation and correlating such patterns with changes in the overlying sensory retina are essential to understanding the pathophysiology and progression of retinal disease. Here, we describe a technique employed by our lab and others that uses cSLO in order to quantify the level of RPE lipofuscin in both healthy and diseased eyes.

The retinal pigment epithelium (RPE) supports the sensory retina through recycling visual cycle byproducts, which accumulate as lipofuscin. These products are autofluorescent and can be qualitatively imaged in vivo. Here, we describe a method to quantitatively image RPE lipofuscin using confocal scanning laser ophthalmoscopy.

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks ...

Integrated Compensatory Responses in a Human Model of Hemorrhage

Hemorrhage is the leading cause of trauma-related deaths, partly because early diagnosis of the severity of blood loss is difficult. Assessment of hemorrhaging patients is difficult because current clinical tools provide measures of vital signs that remain stable during the early stages of bleeding due to compensatory mechanisms. Consequently, there is a need to understand and measure the total integration of mechanisms that compensate for reduced circulating blood volume and how they change during ongoing progressive hemorrhage. The body&#39;s reserve to compensate for reduced circulating blood volume is called the &#39;compensatory reserve&#39;. The compensatory reserve can be accurately evaluated with real-time measurements of changes in the features of the arterial waveform measured with the use of a high-powered computer. Lower Body Negative Pressure (LBNP) has been shown to simulate many of the physiological responses in humans associated with hemorrhage, and is used to study the compensatory response to hemorrhage. The purpose of this study is to demonstrate how compensatory reserve is assessed during progressive reductions in central blood volume with LBNP as a simulation of hemorrhage.

The purpose of this protocol is to demonstrate the techniques for measuring compensatory responses to reduced central blood volume using lower body negative pressure as a noninvasive experimental model of human hemorrhage which can be used to quantify the total integration of compensatory mechanisms to blood volume deficit in humans.

Hemorrhage is the leading cause of trauma-related deaths, partly because early diagnosis of the severity of blood loss is difficult. Assessment of ...

A Chronic Cardiac Ischemia Model in Swine Using an Ameroid Constrictor

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but regardless of the approach, an in vivo model is needed to test each treatment. The pig is one of the most used large animal models for cardiovascular disease. Its heart is very similar in anatomy and function to that of a human. The ameroid placement technique creates an ischemic area of the heart, which has many useful applications in studying myocardial infarction. This model has been used for surgical research, pharmaceutical studies, imaging techniques, and cell therapies.
There are several ways of inducing an ischemic area in the heart. Each has its advantages and disadvantages, but the placement of an ameroid constrictor remains the most widely used technique. The main advantages to using the ameroid are its prevalence in existing research, its availability in various sizes to accommodate the anatomy and size of the vessel to be constricted, the surgery is a relatively simple procedure, and the post-operative monitoring is minimal, since there are no external devices to maintain. This paper provides a detailed overview of the proper technique for the placement of the ameroid constrictor.

The purpose of this protocol is to demonstrate the placement of a delayed constricting device (an ameroid constrictor) around a coronary artery in a swine model. This device creates an ischemic area of the heart that is useful for studying new diagnostic imaging techniques and new methods of treatment.

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but ...

Tachycardia-Induced Cardiomyopathy As a Chronic Heart Failure Model in Swine

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment methods. Here, we present such a model by tachycardia-induced cardiomyopathy, which can be produced by rapid cardiac pacing in swine.
A single pacing lead is introduced transvenously into fully anaesthetized healthy swine, to the apex of the right ventricle, and fixated. Its other end is then tunneled dorsally to the paravertebral region. There, it is connected to an in-house modified heart pacemaker unit that is then implanted in a subcutaneous pocket. 
After 4 - 8 weeks of rapid ventricular pacing at rates of 200 - 240 beats/min, physical examination revealed signs of severe heart failure - tachypnea, spontaneous sinus tachycardia, and fatigue. Echocardiography and X-ray showed dilation of all heart chambers, effusions, and severe systolic dysfunction. These findings correspond well to decompensated dilated cardiomyopathy and are also preserved after the cessation of pacing.
This model of tachycardia-induced cardiomyopathy can be used for studying the pathophysiology of progressive chronic heart failure, especially hemodynamic changes caused by new treatment modalities like mechanical circulatory supports. This methodology is easy to perform and the results are robust and reproducible.

Here, we present a protocol to produce tachycardia-induced cardiomyopathy in swine. This model represents a potent way to study the hemodynamics of progressive chronic heart failure and the effects of applied treatment.

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment ...

Severe Burn Injury in a Swine Model for Clinical Dressing Assessment

Wound healing is a dynamic repair process and is the most complex biological process in human life. In response to burn injury, alterations in biological pathways impair the inflammation response, resulting in delayed wound healing. Impaired wound healing frequently occurs in patients with diabetes leading to unfavorable outcomes such as amputation. Hence, dressings having beneficial effect in promoting burn wound repair are needed. However, studies on burn wound treatment are limited due to lack of proper animal models. Our previous study demonstrated wound-healing performance in rat and swine models using a minimally invasive surgical technique. This study aimed to demonstrate a swine model of severe burn injury that eliminates wound contraction and more closely approximates the human processes of re-epithelialization and new tissue formation. This protocol provides a detailed procedure for creating consistent burn wounds and examining the wound-healing performance under the treatment of an experimental dressing in a swine model. Six burn wounds were created symmetrically on the dorsum, which were covered with a clinical dressing composed of four layers: an inner contact layer of experimental materials, an inner intermediate layer of waterproof film, an outer intermediate layer of gauze, and an outer layer of adhesive plaster. Upon the completion of experiments, wound closure, wound area, and Vancouver Scar Scale score were examined. The samples of skin resected from each animal post-sacrifice were histologically prepared and stained using hematoxylin and eosin staining. Antibacterial activity of each dressing in the context of wound healing was also examined. The application of the clinical dressing to the wounds in swine model mimics the biological processes of human wound healing with respect to the processes of epithelialization, cellular proliferation, and angiogenesis. Therefore, this swine model provides an easy-to-learn, cost-effective, and robust method to assess the effect of clinical dressings in severe burn injury.

To closely mimic the mode of burn injuries requires the interplay between clinical observation and studies in animal models. In this study, a swine model of severe burn injury was established to assess an experimental dressing in physiological and pathophysiological settings.

Wound healing is a dynamic repair process and is the most complex biological process in human life. In response to burn injury, alterations in biological ...

Retinal Pathophysiological Evaluation in a Rat Model

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal detachment, breakdown of the blood-retinal barrier (BRB), and retinal angiogenesis. An in vivo rat model is a valuable experimental tool to examine the changes in the structure and function of the retina. We propose three different experimental techniques in the rat model to identify morphological changes of retinal cells, retinal vasculature, and compromised BRB. Retinal histology is used to study the morphology of various retinal cells. Also, quantitative measurement is performed by retinal cell count and thickness measurement of different retinal layers. A BRB breakdown assay is used to determine the leakage of extraocular proteins from the plasma to vitreous tissue due to the breakdown of BRB. Fluorescence angiography is used to study angiogenesis and leakage of blood vessels by visualizing retinal vasculature using FITC-dextran dye.

Diabetic retinopathy is one of the leading causes of blindness. Histology, blood-retinal barrier breakdown assay, and fluorescence angiography are valuable techniques to understand the pathophysiology of the retina, which could further enhance the efficient drug screening against diabetic retinopathy.

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal ...

Myocardial Infarction by Percutaneous Embolization Coil Deployment in a Swine Model

Myocardial infarction (MI) is the leading cause of mortality worldwide. Despite the use of evidence-based treatments, including coronary revascularization and cardiovascular drugs, a significant proportion of patients develop pathological left-ventricular remodeling and progressive heart failure following MI. Therefore, new therapeutic options, such as cellular and gene therapies, among others, have been developed to repair and regenerate injured myocardium. In this context, animal models of MI are crucial in exploring the safety and efficacy of these experimental therapies before clinical translation. Large animal models such as swine are preferred over smaller ones due to the high similarity of swine and human hearts in terms of coronary artery anatomy, cardiac kinetics, and the post-MI healing process. Here, we aimed to describe an MI model in pig by permanent coil deployment. Briefly, it comprises a percutaneous selective coronary artery cannulation through retrograde femoral access. Following coronary angiography, the coil is deployed at the target branch under fluoroscopic guidance. Finally, complete occlusion is confirmed by repeated coronary angiography. This approach is feasible, highly reproducible, and emulates the pathogenesis of human non-revascularized MI, avoiding the traditional open-chest surgery and the subsequent postoperative inflammation. Depending on the time of follow-up, the technique is suitable for acute, sub-acute, or chronic MI models.

Myocardial infarction (MI) animal models that emulate the natural process of the disease in humans are crucial to understanding pathophysiological mechanisms and testing the safety and efficacy of new emergent therapies. Here, we describe an MI swine model created by deploying a percutaneous embolization coil.

Myocardial infarction (MI) is the leading cause of mortality worldwide. Despite the use of evidence-based treatments, including coronary revascularization ...