The authors want to thank Dagmar Dirvonskis for her excellent technical support.

The experiments in this protocol were approved by the State and Institutional Animal Care Committee (Landesuntersuchungsamt Rheinland-Pfalz, Koblenz, Germany; Chairperson: Dr. Silvia Eisch-Wolf; reference number: 23 177-07/G 14-1-084; 02.02.2015). The experiments were conducted in accordance with the Animal Research&#160;Reporting of In Vivo Experiments (ARRIVE) guidelines. The study was planned and conducted between November 2015 and March 2016. After extended literature research, the pig model was chosen as a well-established model for hemorrhagic shock. Seven anesthetized male pigs (Sus scrofa domestica) with a mean weight of 28 &#177; 2 kg and an age of 2-3 months were included in the protocol. The animals were cared for by a local breeder that was recommended by the State and Institutional Animal Care Committee. The animals were kept in their known environment as long as possible to minimize stress. Food, but not water was denied 6 h before the experiment was scheduled, to reduce the risk of aspiration. The representative time course is displayed in Figure 1.
1. Anesthesia, Intubation, and Mechanical Ventilation
<ol>
	<li>Sedate pigs with a combined injection of ketamine (4 mg&#183;kg-1) and azaperone (8 mg&#183;kg-1) in the neck or the gluteal muscle with a needle for intramuscular injection (1.2 mm). Ensure that the animals remain stable until the sedation sets in. 
	CAUTION: Gloves are absolutely necessary when handling animals.</li>
	<li>Transport the sedated animals to the laboratory. 
	NOTE: The animals fall deeply asleep and do not awake during normal handling, like when they are lifted into the transport cage. In this setting, the transport time was about 20 min with a special van for animal transport.</li>
	<li>Monitor the peripheral oxygen saturation (SpO2) with a sensor clipped to the pig&#39;s tail or ear directly after arrival.</li>
	<li>Disinfect the skin with colorless disinfection tincture and wait for 3 min before inserting a peripheral vein catheter (1.2 mm) into an ear vein. Then, induce anesthesia by an intravenous injection of fentanyl (4 &#181;g&#183;kg-1) and propofol (3 mg&#183;kg-1).</li>
	<li>When all reflexes are absent and spontaneous breathing expires, place the pigs in supine position on a stretcher and fix them with bandages. 
	NOTE: Adequate levels of anesthesia have to be confirmed by an experienced researcher by the absence of an eyelid reflex and other reactions to external stimuli.</li>
	<li>Immediately start noninvasive ventilation with a dog ventilation mask (size 2). Use the following ventilation parameters: inspiratory oxygen fraction (FiO2) = 1.0; respiratory rate = 14-16 min-1; peak inspiratory pressure &#60;20 cm H2O, positive end-expiratory pressure (PEEP) = 5 cm H2O.</li>
	<li>Maintain anesthesia via a continuous infusion of fentanyl (0.1-0.2 &#181;g&#183;kg-1&#183;h-1) and propofol (8-12 mg&#183;kg-1&#183;h-1) and start an infusion of balanced electrolyte solution (5 mL&#183;kg-1&#183;h-1).</li>
	<li>Facilitate endotracheal intubation by the application of a muscle relaxant (atracurium 0.5 mg&#183;kg-1).</li>
	<li>Secure the airway via intubation with a common endotracheal tube (ID 6-7) and an introducer. Use a common laryngoscope with a Macintosh blade (size 4). Two persons facilitate the procedure.
	<ol>
		<li>Person 1: Fix the tongue outside with a piece of tissue and open the snout with the other hand.</li>
		<li>Person 2: Perform a laryngoscopy.</li>
		<li>Person 2: When the epiglottis comes into view, move the laryngoscope ventrally. Lift up the epiglottis and make sure the vocal cords are visible. 
		NOTE: If the epiglottis does not move dorsally, it sticks to the soft palatine and can be mobilized by the tip of the tube. Alternatively, a blade with another size (3 or 5) or type (Miller blade) can be used.</li>
	</ol>
	</li>
	<li>Move the tube carefully through the vocal cords. 
	NOTE: The narrowest point of the trachea is not on the level of the vocal cords but subglottic. If tube insertion is not possible, try to rotate the tube or use a smaller tube.</li>
	<li>Pull the introducer out of the tube, use a 10 mL syringe to block the cuff with 10 mL of air, and control the cuff pressure with a cuff manager (30 cm H2O).</li>
	<li>Start mechanical ventilation after the tube is connected to a ventilator (PEEP = 5 cm H2O; tidal volume = 8 mL&#183;kg-1; FiO2 = 0.4; inspiration-to-expiration ratio = 1:2; respiratory rate = variable to achieve an end-tidal CO2 of &#60;6 kPa). 
	NOTE: Avoid fluctuation of the CO2 to minimize any respiratory effects on the cerebral perfusion.</li>
	<li>Make sure that tube position is correct by regular and periodic exhalation of CO2 via capnography, and check the double-sided ventilation through auscultation. 
	NOTE: If the tube is placed incorrectly, air inflation into the stomach rapidly forms a visible bulge in the abdominal wall, even before the capnography is installed. In this case, replacement of the tube and the insertion of a gastric tube are absolutely necessary.</li>
	<li>With two persons, place a gastric tube into the stomach to avoid reflux and vomiting.
	<ol>
		<li>Person 1: Fix the tongue outside with a piece of tissue and open the snout with the other hand.</li>
		<li>Person 2: Perform a laryngoscopy of the porcine larynx.</li>
		<li>Person 2: Visualize the esophagus.</li>
		<li>Person 2: Push the gastric tube inside the esophagus with a pair of Magill forceps until gastric fluid is drained. 
		NOTE: Sometimes, visualization is not easy. In this case, move the laryngoscope dorsally to the tube and push it ventrally to open the esophagus. During the procedure, the animal body is covered with blankets to avoid hypothermia. If the animal&#39;s body temperature decreases, use a heating system to stabilize the temperature on a physiological level (see the Table of Materials). The body temperature is displayed on the screen of the PiCCO.</li>
	</ol>
	</li>
</ol>
2. Instrumentation
<ol>
	<li>Use bandages to pull back the hind legs to smoothen the folds in the femoral area for vessel catheterization.</li>
	<li>Prepare the following materials: one 5 mL syringe, one 10 mL syringe, one 50 mL syringe, one Seldinger needle, introducer sheaths (2 mm, 2.7 mm, 2.7 mm), guidewires for the sheaths, a central venous catheter with three ports (2.3 mm, 30 cm) with guidewire, and a PiCCO catheter (1.67 mm, 20 cm).</li>
	<li>Disinfect the inguinal area with colored disinfection, wait for 2 min, and wipe the disinfection off with a sterile tissue. Repeat this procedure 3x. After the third time, do not remove the disinfection.</li>
	<li>Fill all catheters with saline solutions.</li>
	<li>Apply ultrasound gel to the ultrasound probe. Cover the inguinal area with a sterile fenestrated drape and scan the right femoral vessels with ultrasound. Use the Doppler technique to distinguish between the artery and the vein18.</li>
	<li>Bright red pulsating blood confirms the aspired needle position. Disconnect the syringe and insert the guidewire into the right femoral artery.</li>
	<li>Visualize the longitudinal axis of the right femoral vein and insert the Seldinger needle under permanent aspiration with the 5 mL syringe.</li>
	<li>Aspirate dark red nonpulsating venous blood.</li>
	<li>Visualize the right femoral artery axially and switch to a longitudinal view of the artery by rotating the probe 90&#176;.</li>
	<li>Puncture the right femoral artery under ultrasound visualization with the Seldinger needle under permanent aspiration with the 5 mL syringe. 
	NOTE: The ultrasound-guided Seldinger technique is associated with significantly lower blood loss, tissue trauma, and time consumption than other methods of vascular access19,20.
	<ol>
		<li>If the correct position of the needle in the different vessels cannot be established for certain, take the blood probes and analyze the blood gas content with a blood gas analyzer (see the Table of Materials). A high oxygen&#160;level is a good sign of arterial blood, and a low oxygen level is a sign of venous blood.</li>
	</ol>
	</li>
	<li>Insert the guidewire for the central venous catheter into the right femoral vein after disconnecting the syringe and retracting the Seldinger needle.</li>
	<li>Visualize both right vessels with ultrasound to control the correct wire position.</li>
	<li>Push the arterial introducer sheath (2 mm) over the guidewire into the right artery and secure the position with blood aspiration.</li>
	<li>Use the Seldinger technique to position the central venous line into the right femoral vein. Aspirate all ports and flush them with saline solution.</li>
	<li>Perform the same procedure on the left inguinal side to insert the other introducer sheaths in the Seldinger technique into the left femoral artery (2.7 mm) and femoral vein (2.7 mm).</li>
	<li>Connect the right arterial introducer sheath and the central venous catheter with two transducer systems for the measurement of invasive hemodynamics, and position both transducers on the heart level to get appropriate values.</li>
	<li>Switch the three-way-stopcocks of both transducers open to the atmosphere to calibrate the systems to 0 as is prescribed in the operation instructions. 
	NOTE: It is absolutely necessary to avoid any air bubbles and bloodstains in the systems to generate plausible values.</li>
	<li>Switch all infusions for maintaining anesthesia from the peripheral vein to the central venous line.</li>
	<li>Take baseline values (hemodynamics, spirometry, NIRS (see section 4) and PiCCO&#160;(see section 3) after 15 min of recovery.</li>
	<li>Initiate hemorrhagic shock (see section 5).</li>
</ol>
3. PiCCO Measurement
NOTE: For the PiCCO equipment, see the Table of Materials.
<ol>
	<li>Insert the PiCCO catheter into the right arterial introducer sheath. 
	NOTE: In clinical medicine, PiCCO catheters are directly placed by the Seldinger technique. However, placement via an introducer sheath is feasible as well.</li>
	<li>Connect the catheter with the arterial wire of the PiCCO system and the arterial transducer directly with the PiCCO port. Then, recalibrate as described in step 2.17.</li>
	<li>Connect the venous measuring unit of the PiCCO&#160;system with the left venous introducer sheath. 
	NOTE: It is necessary to connect the venous and arterial probes at some distance from each other. Otherwise, the measurement will be disturbed, because the application of cold saline solution into the venous system will influence the arterial measurement. For more details on PiCCO, see&#160;Mayer and Suttner21.</li>
	<li>Turn on the PiCCO system and confirm that a new patient is measured.</li>
	<li>Enter the animal&#39;s size and weight and switch the category to adults.</li>
	<li>Enter the protocol name and ID and enter Exit.</li>
	<li>Set the injection volume to 10 mL. 
	NOTE: The volume of the chosen injection solution can be varied. A higher volume makes the measured values more valid. Chose a small volume to avoid any hemodilution effects through repetitive application.</li>
	<li>Enter the central venous pressure.</li>
	<li>Open the three-way stopcock to the atmosphere, click on Zero for system calibration, and click on Exit.</li>
	<li>Calibrate the continuous cardiac output measurement as described next and click on TD (Thermodilution). Prepare physiological saline solution with a temperature of 4 &#176;C in a 10 mL syringe and click on Start.</li>
	<li>Inject 10 mL of the cold saline solution quickly and steadily into the venous measuring unit and wait until the measurement is completed and the system requests a repetition.</li>
	<li>Repeat this procedure until three measurements are completed.</li>
	<li>Let the system calculate the mean of all parameters and click on Exit.</li>
	<li>After complete calibration, immediately start the measurement. To monitor shock induction, focus on the PiCCO-derived parameter cardiac index.</li>
</ol>
4. Cerebral Regional Oxygenation Saturation
NOTE: For the equipment to monitor cerebral regional oxygenation, see the Table of Materials.
<ol>
	<li>Shave the forehead of the pig with a disposable razor and water and stick two self-adherent sensors (see the Table of Materials) for NIRS to the forehead of the pig.</li>
	<li>Connect the preamplifier to the monitor and connect the sensor cable connectors color-coded to the preamplifier.</li>
	<li>Close the preamp locking mechanism and attach the sensors to the sensor cables. 
	NOTE: In order to record real-time data, a USB flash drive has to be connected to the NIRS monitor.</li>
	<li>Switch on the monitor, click on New Patient, enter the study name, and click on Done.</li>
	<li>Check the incoming signal. When the signal is stable, click on Baseline Menu and click on Set Baselines. If the baseline has already been entered, confirm the new baseline by clicking Yes and click on Event Mark.</li>
	<li>Chose the event with the arrow buttons on the keyboard and with Next Event; select the event 3 Induction and press Select Event. 
	NOTE: If further information is necessary, consult the operation manual of the NIRS system22.</li>
</ol>
5. Hemorrhagic Shock Induction
<ol>
	<li>Connect left introducer sheath with a tree-way stopcock. Connect one port of the three-way stopcock with a 50 mL syringe and one with an empty infusion bottle. 
	NOTE: Alternatively, the withdrawn blood may be collected in citrated bags for later autotransfusion. This is a major advantage of controlled blood withdrawal.</li>
	<li>Measure and document the exact hemodynamic parameters and calculate 40% of the cardiac index and mean arterial pressure as hemodynamic targets. Set the event 93 Blood Loss in the NIRS system as described in step 4.6. 
	NOTE: Hemorrhagic shock is achieved if the cardiac index and mean arterial pressure drops below 40% of the baseline value. A considerable cerebral regional oxygenation saturation (crSO2) decline of 20% is preferable to depict microcirculatory impairment. The average blood loss to achieve this lies within a range of 25-35 mL&#183;kg-1.</li>
	<li>Aspirate 50 mL of blood into the syringe and switch the three-way stopcock. Push the blood into the empty bottle.</li>
	<li>Note the removed blood volume.</li>
	<li>Monitor the arterial blood pressure, the cardiac index, and the crSO2 closely. Repeat the blood withdrawal until the target blood pressure and cardiac index are achieved (after 20-30 min).</li>
	<li>Set the event 97 Hypotension in the NIRS device as described in step 4.6. 
	NOTE: Do not withdraw the blood too quickly, because this bears the risk of immediate cardio-circulatory failure. After finishing the shock induction procedure, the animals can be used for various therapeutic interventions.</li>
</ol>
6. End of the Experiment and Euthanasia
<ol>
	<li>Inject 0.5 mg of fentanyl into the central venous line and wait for 5 min.</li>
	<li>Inject 200 mg of propofol into the central venous line and euthanize the animal with 40 mmol potassium chloride.</li>
</ol>

The NIRS device was provided unconditionally by Medtronic PLC, USA, for experimental research purposes. Alexander Ziebart, Andreas Garcia-Bardon, and Erik K. Hartmann received instructor honoraria for physician training courses from Medtronic PLC. None of the authors report financial or other conflicts of interest.

The protocol describes one method of inducing hemorrhagic shock via controlled arterial bleeding in pigs that is guided by systemic hemodynamics, as well as by cerebral microcirculatory impairment. Shock conditions were achieved by a calculated blood withdrawal of 25-35 mL kg-1 and confirmed by the mentioned composite of surrogate parameters indicating considerable cardio-circulatory failure. If untreated, this procedure was lethal within 2 h in 66% of the animals, which underlines the severity and reproducibi...

Massive blood loss is among the main causes of injury-related deaths1,2,3. The loss of circulatory fluid and oxygen carriers leads to hemodynamic failure and severe oxygen undersupply and can cause irreversible organ failure and death. The severity level of shock is influenced by additional factors like hypothermia, coagulopathy, and acidosis4. Particularly the brain, but also the kidneys lack compensation capacity due to high oxygen demand and the incapability of adequate anaerobic energy generation5,6. For therapeutic purposes, fast and immediate action is pivotal. In clinical practice, fluid resuscitation with a balanced electrolyte solution is the first option for treatment, followed by the administration of red blood cell concentrates and fresh frozen plasma. Thrombocyte concentrates, catecholamines, and the optimization of coagulation and the acid-base status support the therapy to regain normal physiological conditions after sustained trauma. This concept focuses on the restoration of hemodynamics and macrocirculation. Several studies, however, show that microcirculatory perfusion does not recover simultaneously with the macrocirculation. Especially, cerebral perfusion remains impaired and further oxygen undersupply may occur7,8.
The use of animal models allows scientists to establish novel or experimental strategies. The comparable anatomy, homology, and physiology of pigs and humans enable conclusions on specific pathological factors. Both species have a similar metabolic system and response to pharmacologic treatments. This is a great advantage in comparison to small animal&#160;models where differences in blood volume, hemodynamics, and overall physiology make it almost impossible to mimic a clinical scenario9. Furthermore, authorized medical equipment and consumables can be easily used in porcine models. Additionally, it is easily possible to obtain pigs from commercial suppliers, which allows a high diversity of genetics and phenotypes and is cost reducing10. The model of blood withdrawal via vessel cannulation is quite common11,12,13,14,15.
In this study, we extend the concept of hemorrhagic shock induction via arterial blood withdrawal with an exact titration of hemodynamic failure and cerebral oxygenation impairment. Hemorrhagic shock is achieved if the cardiac index and mean arterial pressure drops below 40% of the baseline value, which has been shown to cause considerable deterioration of the cerebral regional oxygenation saturation8. Pulse contour cardiac output (PiCCO) measurement is used for continuous hemodynamic monitoring. First, the system has to be calibrated by transpulmonary thermodilution, which enables the calculation of the cardiac index of the extravascular lung water content and the global end-diastolic volume. Subsequently, the continuous cardiac index is calculated by pulse contour analysis and also provides dynamic preload parameters like pulse pressure and stroke volume variation.
This technique is well established in clinical and experimental settings.Near-infrared spectroscopy (NIRS) is a clinically and experimentally established method to monitor changes in cerebral oxygen supply in real-time. Self-adherent sensors are attached to the left and right forehead and calculate the cerebral oxygenation non-invasively in the cerebral frontal cortex. Two wavelengths of infrared light (700 and 900 nm) are emitted and detected by the sensors after being reflected from the cortex tissue. To assess the cerebral oxygen content, contributions of arterial and venous blood are calculated in 1:3 relations and updated in 5 s intervals. The sensitivity in depth of 1-4 cm is exponential decreasing and influenced by the penetrated tissue (e.g., skin and bone), although the skull is translucent to infrared light. The technique facilitates quick therapeutic actions to prevent patients from adverse outcomes like delirium or hypoxic cerebral injury and serves as the target parameter in case of impaired cardiac output16,17. The combination of both techniques during experimental shock enables an exact titration of macrocirculation, as well as cerebral microcirculatory impairment, to study this life-threatening event.

<table border="0" cellpadding="0" cellspacing="0" style="width:824px;" width="825">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:166px;">3-way-stopcock blue</td>
			<td style="width:258px;">Becton Dickinson Infusion Therapy AB Helsingborg, Sweden</td>
			<td style="width:150px;">394602</td>
			<td style="width:252px;">Drug administration</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:166px;">3-way-stopcock red</td>
			<td style="width:258px;">Becton Dickinson Infusion Therapy AB Helsingborg, Sweden</td>
			<td style="width:150px;">394605</td>
			<td style="width:252px;">Drug administration/Shock induction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:166px;">Atracurium</td>
			<td style="width:258px;">Hikma Pharma GmbH , Martinsried</td>
			<td>AM03AC04*</td>
			<td style="width:252px;">Anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:22px;width:166px;">Canula 20 G</td>
			<td style="width:258px;">Becton Dickinson S.A. Carretera Mequinenza Fraga, Spain</td>
			<td style="width:150px;">301300</td>
			<td style="width:252px;">Vascular access</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:166px;">Datex Ohmeda S5</td>
			<td style="width:258px;">GE Healthcare Finland Oy, Helsinki, Finland</td>
			<td>-</td>
			<td style="width:252px;">Hemodynamic monitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:166px;">Desinfection&#160;</td>
			<td style="width:258px;">Sch&#252;lke &#38; Mayr GmbH, Germany</td>
			<td>104802</td>
			<td style="width:252px;">Desinfection&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:166px;">Heidelberger Verl&#228;ngerung 75CM</td>
			<td style="width:258px;">Fresenius Kabi Deutschland GmbH</td>
			<td>2873112 &#160;</td>
			<td style="width:252px;">Drug administration/Shock induction</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">INVOS 5100C Cerebral</td>
			<td style="width:258px;">Medtronic PLC, USA</td>
			<td>-</td>
			<td style="width:252px;">Monitore for cerebral regional oxygenation&#160;</td>
		</tr>
		<tr height="83">
			<td height="83" style="height:83px;width:166px;">INVOS Cerebral/Somatic Oximetry Adult Sensors</td>
			<td style="width:258px;">Medtronic PLC, USA</td>
			<td style="width:150px;">20884521211152</td>
			<td style="width:252px;">Monitoring of the cerebral regional oxygenation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:166px;">Endotracheal tube</td>
			<td style="width:258px;">Teleflex Medical Sdn. Bhd, Malaysia</td>
			<td style="width:150px;">112482</td>
			<td style="width:252px;">Intubation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:166px;">Endotracheal tube introducer</td>
			<td style="width:258px;">&#160; Wirutec GmbH, Sulzbach, Germany</td>
			<td>5033062</td>
			<td style="width:252px;">Intubation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:166px;">Engstr&#246;m Carestation</td>
			<td style="width:258px;">GE Heathcare, Madison USA</td>
			<td style="width:150px;">-</td>
			<td style="width:252px;">Ventilator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:166px;">Fentanyl</td>
			<td style="width:258px;">Janssen-Cilag GmbH, Neuss</td>
			<td style="width:150px;">AA0014*</td>
			<td style="width:252px;">Anesthesia</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:166px;">Gloves</td>
			<td style="width:258px;">Paul Hartmann, Heidenheim, Germany</td>
			<td>9422131</td>
			<td style="width:252px;">Self-protection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:166px;">Incetomat-line 150 cm</td>
			<td style="width:258px;">Fresenius, Kabi GmbH, Bad Homburg, Germany</td>
			<td style="width:150px;">9004112</td>
			<td style="width:252px;">Drug administration</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:166px;">Ketamine</td>
			<td style="width:258px;">Hameln Pharmaceuticals GmbH, Zofingen, Schweiz</td>
			<td style="width:150px;">AN01AX03*</td>
			<td style="width:252px;">Sedation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:166px;">Laryngoscope</td>
			<td style="width:258px;">Teleflex Medical Sdn. Bhd, Malaysia</td>
			<td style="width:150px;">671067-000020</td>
			<td style="width:252px;">Intubation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:166px;">Logical pressure monitoring system</td>
			<td style="width:258px;">Smith- Medical GmbH,&#160; Minneapolis, USA</td>
			<td>MX9606</td>
			<td style="width:252px;">Hemodynamic monitor</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:166px;">Logicath 7 Fr 3-lumen 30cm</td>
			<td style="width:258px;">Smith- Medical GmbH,&#160; Minneapolis, USA</td>
			<td>MXA233x30x70-E</td>
			<td style="width:252px;">Vascular access/Drug administration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:22px;width:166px;">Masimo Radical 7</td>
			<td style="width:258px;">Masimo Corporation, Irvine, USA</td>
			<td style="width:150px;">-</td>
			<td style="width:252px;">Hemodynamic monitor</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:166px;">Mask for ventilating dogs</td>
			<td style="width:258px;">Henry Schein, Melville, USA</td>
			<td style="width:150px;">730-246</td>
			<td style="width:252px;">Ventilation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:166px;">Original Perfusor syringe 50ml Luer Lock</td>
			<td style="width:258px;">B.Braun Melsungen AG, Melsungen, Germany</td>
			<td>8728810F</td>
			<td style="width:252px;">Drug administration</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:166px;">PICCO Thermodilution. F5/20CM EW&#160;</td>
			<td style="width:258px;">MAQUET Cardiovascular GmbH, Rastatt, Germany</td>
			<td>PV2015L20-A &#160;</td>
			<td style="width:252px;">Hemodynamic monitor</td>
		</tr>
		<tr height="104">
			<td height="104" style="height:104px;width:166px;">Percutaneous sheath introducer set 8,5 und 9 Fr, 10 cm with integral haemostasis valve/sideport</td>
			<td style="width:258px;">Arrow international inc., Reading, USA</td>
			<td>AK-07903</td>
			<td style="width:252px;">Vascular access/Shock induction</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:166px;">Perfusor FM Braun</td>
			<td style="width:258px;">B.Braun Melsungen AG, Melsungen, Germany</td>
			<td>8713820</td>
			<td style="width:252px;">Drug administration</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:166px;">Potassium chloride</td>
			<td style="width:258px;">Fresenius, Kabi GmbH, Bad Homburg, Germany</td>
			<td>6178549</td>
			<td style="width:252px;">Euthanasia</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:166px;">Propofol 2%</td>
			<td style="width:258px;">Fresenius, Kabi GmbH, Bad Homburg, Germany</td>
			<td style="width:150px;">&#160; AN01AX10*</td>
			<td style="width:252px;">Anesthesia</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:166px;">&#160;Pulse Contour Cardiac Output (PiCCO2)&#160;</td>
			<td style="width:258px;">Pulsion Medical Systems, Feldkirchen, Germany</td>
			<td>-</td>
			<td style="width:252px;">Hemodynamic monitor</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:166px;">Sonosite Micromaxx Ultrasoundsystem</td>
			<td style="width:258px;">Fujifilm, Sonosite Bothell, Bothell, USA&#160;</td>
			<td style="width:150px;">-</td>
			<td style="width:252px;">Vascular access</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:166px;">Stainless Macintosh Size 4</td>
			<td style="width:258px;">Teleflex Medical Sdn. Bhd, Perak,&#160; Malaysia</td>
			<td style="width:150px;">670000</td>
			<td style="width:252px;">Intubation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:166px;">Sterofundin</td>
			<td style="width:258px;">B.Braun Melsungen AG, Melsungen, Germany</td>
			<td style="width:150px;">AB05BB01*</td>
			<td style="width:252px;">balanced electrolyte infusion</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:166px;">Stresnil 40mg/ml&#160;&#160;</td>
			<td style="width:258px;">Lilly Germany GmbH, Wiesbaden, Germany</td>
			<td style="width:150px;">QN05AD90</td>
			<td style="width:252px;">Sedation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:166px;">Syringe 10 mL</td>
			<td style="width:258px;">Becton Dickinson S.A. Carretera Mequinenza Fraga, Spain</td>
			<td>309110</td>
			<td style="width:252px;">Drug administration</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:166px;">Syringe 2 mL</td>
			<td style="width:258px;">Becton Dickinson S.A. Carretera Mequinenza Fraga, Spain</td>
			<td>300928</td>
			<td style="width:252px;">Drug administration</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:166px;">Syringe 20 mL</td>
			<td style="width:258px;">Becton Dickinson S.A. Carretera Mequinenza Fraga, Spain</td>
			<td style="width:150px;">300296</td>
			<td style="width:252px;">Drug administration</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:166px;">Syringe 5 mL</td>
			<td style="width:258px;">Becton Dickinson S.A. Carretera Mequinenza Fraga, Spain</td>
			<td>309050</td>
			<td style="width:252px;">Drug administration</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:166px;">venous catheter 22G</td>
			<td style="width:258px;">B.Braun Melsungen AG, Melsungen, Germany</td>
			<td>4269110S-01</td>
			<td style="width:252px;">Vascular access</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:423px;">*ATC:&#160; Anatomical Therapeutic Chemical / Defined Daily Dose Classification&#160;</td>
			<td style="width:150px;"></td>
			<td style="width:252px;"></td>
		</tr>
	</tbody>
</table>

standardized hemorrhagic shock induction guided by cerebral oximetry and extended hemodynamic monitoring in pigs

Hemorrhagic shock ranks among the main reasons for severe injury-related death. The loss of circulatory volume and oxygen carriers can lead to an insufficient oxygen supply and irreversible organ failure. The brain exerts only limited compensation capacities and is particularly at high risk of severe hypoxic damage.This article demonstrates the reproducible induction of life-threatening hemorrhagic shock in a porcine model by means of calculated blood withdrawal. We titrate shock induction guided by near-infrared spectroscopy&#160;and extended hemodynamic monitoring to display systemic circulatory failure, as well as cerebral microcirculatory oxygen depletion. In comparison to similar models that primarily focus on predefined removal volumes for shock induction, this approach highlights a titration by means of the resulting failure of macro- and microcirculation.

After starting the shock induction, a short time of compensation can be registered. With ongoing blood removal, the aforementioned cardio-circulatory decompensation, as monitored by a significant decrease of crSO2, the cardiac index, the intrathoracic blood volume index, and the global end-diastolic volume index (Figure 2, Figure 3, and Figure 4), occurs. Furthermore, significant tachycard...

Watch this Scientific Journal Video about Standardized Hemorrhagic Shock Induction Guided by Cerebral Oximetry and Extended Hemodynamic Monitoring in Pigs at JoVE.com

Standardized Hemorrhagic Shock Induction Guided by Cerebral Oximetry and Extended Hemodynamic Monitoring in Pigs

Hemorrhagic shock is a severe complication in seriously injured patients, which leads to life-threatening oxygen undersupply. We present a standardized method to induce hemorrhagic shock via blood withdrawal in pigs that is guided by hemodynamics and microcirculatory cerebral oxygenation.

Hemorrhagic shock ranks among the main reasons for severe injury-related death. The loss of circulatory volume and oxygen carriers can lead to an ...

standardized-hemorrhagic-shock-induction-guided-cerebral-oximetry

Research

JoVE Journal

Medicine

7.3K Views.  University Medical Center of the Johannes Gutenberg-University. Hemorrhagic shock is a severe complication in seriously injured patients, which leads to life-threatening oxygen undersupply. We present a standardized method to induce hemorrhagic shock via blood withdrawal in pigs that is guided by hemodynamics and microcirculatory cerebral oxygenation. 

Video: Standardized Hemorrhagic Shock Induction Guided by Cerebral Oximetry and Extended Hemodynamic Monitoring in Pigs

Fixed Volume or Fixed Pressure: A Murine Model of Hemorrhagic Shock

It is common knowledge that severe blood loss and traumatic injury can lead to a cascade of detrimental signaling events often resulting in mortality. 1, 2, 3, 4, 5 These signaling events can also lead to sepsis and/or multiple organ dysfunction (MOD). 6, 7, 8, 9 It is critical then to investigate the causes of suppressed immune function and detrimental signaling cascades in order to develop more effective ways to help patients who suffer from traumatic injuries. 10 This fixed pressure Hemorrhagic Shock (HS) procedure, although technically challenging, is an excellent resource for investigation of these pathophysiologic conditions. 11, 12, 13 Advances in the assessment of biological systems, i.e. Systems Biology have enabled the scientific community to further understand complex physiologic networks and cellular communication patterns. 14 Hemorrhagic Shock has proven to be a vital tool for unveiling these cellular communication patterns as they relate to immune function. 15, 16, 17, 18 This procedure can be mastered! This procedure can also be used as either a fixed volume or fixed pressure approach. We adapted this technique in the murine model to enhance research in innate and adaptive immune function. 19, 20, 21 Due to their small size HS in mice presents unique challenges. However due to the many available mouse strains, this species represents an unparalleled resource for the study of the biologic responses. The HS model is an important model for studying cellular communication patterns and the responses of systems such as hormonal and inflammatory mediator systems, and danger signals, i.e. DAMP and PAMP upregulation as it elicits distinct responses that differ from other forms of shock. 22, 23, 24, 25 The development of transgenic murine strains and the induction of biologic agents to inhibit specific signaling have presented valuable opportunities to further elucidate our understanding of the up and down regulation of signal transduction after severe blood loss, i.e. HS and trauma 26, 27, 28, 29, 30. 
There are numerous resuscitation methods (R) in association with HS and trauma. 31, 32, 33, 34 A fixed volume resuscitation method of solely lactated ringer solution (LR), equal to three times the shed blood volume, is used in this model to study endogenous mechanisms such as remote organ injury and systemic inflammation. 35, 36, 38 This method of resuscitation is proven to be effective in evaluating the effects of HS and trauma 38, 39.

The Hemorrhagic Shock model has been a reliable and reproducible resource facilitating the identification and understanding of signaling cascades associated with inflammation and end-organ damage after trauma. This article provides a step-by-step description of surgical and mechanical aspects associated with the Hemorrhagic Shock experimental procedure in mice.

It is common knowledge that severe blood loss and traumatic injury can lead to a cascade of detrimental signaling events often resulting in mortality. 1, ...

Monitoring of Systemic and Hepatic Hemodynamic Parameters in Mice

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. However, due to the small size of the mouse, technical details of the intraoperative monitoring procedure suitable for the mouse were rarely described. Previously we have reported a monitoring procedure to obtain hemodynamic parameters for rats. Now, we adapted the procedure to acquire systemic and hepatic hemodynamic parameters in mice, a species ten-fold smaller than rats. This film demonstrates the instrumentation of the animals as well as the data acquisition process needed to assess systemic and hepatic hemodynamics in mice. Vital parameters, including body temperature, respiratory&#160;rate&#160;and heart&#160;rate were recorded throughout the whole procedure. Systemic hemodynamic parameters consist of carotid artery pressure (CAP) and central venous pressure (CVP). Hepatic perfusion parameters include portal vein pressure (PVP), portal flow rate as well as the flow rate of the common hepatic artery (table 1). Instrumentation and data acquisition to record the normal values was completed within 1.5 h. Systemic and hepatic hemodynamic parameters remained within normal ranges during this procedure.
This procedure is challenging but feasible. We have already applied this procedure to assess hepatic hemodynamics in normal mice as well as during 70% partial hepatectomy and in liver lobe clamping experiments. Mean PVP after resection (n= 20), was 11.41&#177;2.94 cmH2O which was significantly higher (P&#60;0.05) than before resection (6.87&#177;2.39 cmH2O). The results of liver lobe clamping experiment indicated that this monitoring procedure is sensitive and suitable for detecting small changes in portal pressure and portal flow rate. In conclusion, this procedure is reliable in the hands of an experienced micro-surgeon but should be limited to experiments where mice are absolutely needed.

This film demonstrates how to acquire systemic and hepatic hemodynamics in mice. The whole monitoring includes acquisition of vital parameters, systemic blood pressure, central venous pressure, common hepatic artery flow rate, and portal vein pressure as well as the portal flow rate in mice.

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. ...

Uncontrolled Hemorrhagic Shock Modeled via Liver Laceration in Mice with Real Time Hemodynamic Monitoring

Uncontrolled hemorrhage is an important cause of preventable deaths among trauma patients. We have developed a murine model of uncontrolled hemorrhage via a liver laceration that results in consistent blood loss, hemodynamic alterations, and survival.
Mice undergo a standardized resection of the left-middle lobe of the liver. They are allowed to bleed without mechanical intervention. Hemostatic agents can be administered as pre-treatment or rescue therapy depending on the interest of the investigator. During the time of hemorrhage, real-time hemodynamic monitoring via a left femoral arterial line is performed. Mice are then sacrificed, blood loss is quantified, blood is collected for further analysis, and organs are harvested for analysis of injury. Experimental design is described to allow for simultaneous testing of multiple animals.
Liver hemorrhage as a model of uncontrolled hemorrhage exists in the literature, primarily in rat and porcine models. Some of these models utilize hemodynamic monitoring or quantify blood loss but lack consistency. The present model incorporates quantification of blood loss, real-time hemodynamic monitoring in a murine model that offers the advantage of using transgenic lines and a high-throughput mechanism to further investigate the pathophysiologic mechanisms in uncontrolled hemorrhage.

Uncontrolled hemorrhage, an important cause of mortality among trauma patients, can be modeled using a standard liver laceration in a murine model. This model results in consistent blood loss, survival, and allows for testing hemostatic agents. This article provides the step-by-step process to perform this valuable model.

Uncontrolled hemorrhage is an important cause of preventable deaths among trauma patients. We have developed a murine model of uncontrolled hemorrhage via ...

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 ...

Complete and Partial Resuscitative Endovascular Balloon Occlusion of the Aorta for Hemorrhagic Shock

Resuscitative endovascular balloon occlusion of the aorta (REBOA) devices grew out of a military-civilian partnership to develop new capabilities for hemorrhage control. With the advent of purpose-built devices, REBOA has become increasingly common in civilian trauma and acute care settings. Currently available REBOA catheters were designed as complete aortic occlusion devices. However, the therapeutic window for complete aortic occlusion is time-limited due to ischemia-reperfusion injury. The partial procedure allows blood flow past the level of occlusion while maintaining targeted proximal pressure, which has been shown to reduce distal ischemia and adjunctive resuscitation requirements in preclinical studies with prolonged occlusion times as compared to traditional complete occlusion.
pREBOA-PRO is the first catheter designed to enable partial and complete aortic occlusion and is currently in limited market release at seven Level I trauma centers in North America. This paper will focus on procedural considerations for REBOA, including patient selection criteria and a comparison of complete and partial aortic occlusion in a simulator, along with highlighting critical steps to improve clinical outcomes. Additionally, this paper reviews a contrast-enhanced CT scan from a trauma patient that shows distal perfusion after 2 h of partial aortic occlusion using this newly designed catheter and discusses representative results from the limited market release to highlight the profound effect of technological innovation on outcomes in vascular emergencies.

A commercial catheter was designed to facilitate true partial resuscitative endovascular balloon occlusion of the aorta (REBOA) and address the complications associated with complete aortic occlusion. Initial clinical reports indicate that partial REBOA improves transition to reperfusion, reduction of distal ischemia, and extension of safe occlusion time compared to complete occlusion.

Resuscitative endovascular balloon occlusion of the aorta (REBOA) devices grew out of a military-civilian partnership to develop new capabilities for ...

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 ...

Dosage-Dependent Induction of Endotoxemia in Pigs via LPS Infusion

Lipopolysaccharide Infusion as a Porcine Endotoxemic Shock Model

Sepsis and septic shock are frequently encountered in patients treated in intensive care units (ICUs) and are among the leading causes of death in these patients. It is caused by a dysregulated immune response to an infection. Even with optimized treatment, mortality rates remain high, which makes further insights into the pathophysiology and new treatment options necessary. Lipopolysaccharide (LPS) is a component of the cell membrane of gram-negative bacteria, which are often responsible for infections causing sepsis and septic shock. 
The severity and high mortality of sepsis and septic shock make standardized experimental studies in humans impossible. Thus, an animal model is needed for further studies. The pig is especially well suited for this purpose as it closely resembles humans in anatomy, physiology, and size. 
This protocol provides an experimental model for endotoxemic shock in pigs by LPS infusion. We were able to reliably induce changes frequently observed in septic shock patients, including hemodynamic instability, respiratory failure, and acidosis. This will allow researchers to gain valuable insight into this highly relevant condition and evaluate new therapeutic approaches in an experimental setting.

Author Spotlight: Induction of Experimental Endotoxemic Shock in Pigs for Studying Hemodynamic and Respiratory Failure

We provide a protocol for an experimental endotoxemic shock model in pigs by infusion of lipopolysaccharide.

Sepsis and septic shock are frequently encountered in patients treated in intensive care units (ICUs) and are among the leading causes of death in these ...

Oleic Acid&#45;Induced Acute Respiratory Decompensation in Pigs

A Porcine Model of Acute Respiratory Failure with a Continuous Infusion of Oleic Acid

This protocol outlines an acute respiratory distress model utilizing centrally administered oleic acid infusion in Yorkshire pigs. Prior to experimentation, each pig underwent general anesthesia, endotracheal intubation, and mechanical ventilation, and was equipped with bilateral&#160;jugular vein central vascular access catheters. Oleic acid was administered through a dedicated pulmonary artery catheter at a rate of 0.2 mL/kg/h. The infusion lasted for 60-120 min, inducing respiratory distress. Throughout the experiment, various parameters including heart rate, respiratory rate, arterial blood pressure, central venous pressure, pulmonary artery pressure, pulmonary capillary wedge pressure, end-tidal carbon dioxide, peak airway pressures, and plateau pressures were monitored. Around the 60 min mark, decreases in partial arterial oxygen pressure (PaO2) and fraction of oxygen-saturated hemoglobin (SpO2) were observed. Periodic hemodynamic instability, accompanied by acute increases in pulmonary artery pressures, occurred during the infusion. Post-infusion, histological analysis of the lung parenchyma revealed changes indicative of parenchymal damage and acute disease processes, confirming the effectiveness of the model in simulating acute respiratory decompensation.

Author Spotlight: Exploring Venous Waveforms for Non&#45;Invasive Respiratory Monitoring in Pigs

Infusing oleic acid continuously into the pulmonary artery of an anesthetized adult pig induces acute respiratory failure, enabling controlled experimentation during acute respiratory decompensation.

This protocol outlines an acute respiratory distress model utilizing centrally administered oleic acid infusion in Yorkshire pigs. Prior to ...

Vascular Cannulation with Hemorrhagic Shock Induction in Rats

Developing a Clinically Relevant Hemorrhagic Shock Model in Rats

Over the recent decades, the development of animal models allowed us to better understand various pathologies and identify new treatments. Hemorrhagic shock, i.e., organ failure due to rapid loss of a large volume of blood, is associated with a highly complex pathophysiology involving several pathways. Numerous existing animal models of hemorrhagic shock strive to replicate what happens in humans, but these models have limits in terms of clinical relevance, reproducibility, or standardization. The aim of this study was to refine these models to develop a new model of hemorrhagic shock. Briefly, hemorrhagic shock was induced in male Wistar Han rats (11-13 weeks old) by a controlled exsanguination responsible for a drop in the mean arterial pressure. The next phase of 75 min was to maintain a low mean arterial blood pressure, between 32 mmHg and 38 mmHg, to trigger the pathophysiological pathways of hemorrhagic shock. The final phase of the protocol mimicked patient care with an administration of intravenous fluids, Ringer Lactate solution, to elevate the blood pressure. Lactate and behavioral scores were assessed 16 h after the protocol started, while hemodynamics parameters and plasmatic markers were evaluated 24 h after injury. Twenty-four hours post-hemorrhagic shock induction, the mean arterial and diastolic blood pressure were decreased in the hemorrhagic shock group (p&#160;&#60; 0.05). Heart rate and systolic blood pressure remained unchanged. All organ damage markers were increased with the hemorrhagic shock (p &#60; 0.05). The lactatemia and behavioral scores were increased compared to the sham group (p &#60; 0.05). In conclusion, we demonstrated that the protocol described here is a relevant model of hemorrhagic shock that can be used in subsequent studies, particularly to evaluate the therapeutic potential of new molecules.

Author Spotlight: Developing Innovative Therapeutic Strategies for Hemorrhagic Shock Research

Hemorrhagic shock kills 1.9 million people worldwide every year. Small animals are frequently used as hemorrhagic shock models but are associated with issues of standardization, reproducibility, and clinical significance, thus limiting their relevance. This article describes developing a new clinically relevant hemorrhagic shock model in rats.

Over the recent decades, the development of animal models allowed us to better understand various pathologies and identify new treatments. Hemorrhagic ...