Name: lipopolysaccharide infusion as a porcine endotoxemic shock model
Uploaded: 2023-12-08T13:40:00.000+00:00
Duration: 5 min 52 s
Description: Watch this Scientific Journal Video about Lipopolysaccharide Infusion as a Porcine Endotoxemic Shock Model at JoVE.com

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, TVA G21-1-080). The experiments were conducted in accordance with the ARRIVE guidelines. For this study, six healthy male German Landrace pigs aged 2-3 months and weighing 30-35 kg were used. The experimental timeline is summarized in Figure 1. The details related to all materials and instruments used in this protocol are listed in the Table of Materials.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66039/66039fig01.jpg" /> 
Figure 1: Experimental timeline. Baseline health measurements were taken after the preparation of the animal and a 30 min stabilization period. Endotoxemia was induced by LPS injection over 30 min and 0 h measurements were taken after another 30 min; after that, hourly measurements were continued for 4 h. Abbreviations: BLH = baseline healthy; LPS = lipopolysaccharide. <a href="https://www.jove.com/files/ftp_upload/66039/66039fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
1. Animal preparation
<ol>
	<li>Keep the animals in their usual environment for as long as possible to minimize stress. Withhold food for 6 h prior to the administration of anesthesia, while allowing free access to water.</li>
	<li>Sedate the animals with an intramuscular injection of azaperone (3 mg&#8729;kg-1) and midazolam (0.5 mg kg-1) while still in their normal environment.</li>
	<li>Once the sedation takes effect, which typically occurs within approximately 15-20 min after administration, transport the pigs to the laboratory. 
	NOTE: It is crucial to ensure that continuous sedation is maintained throughout the transfer; depending on the regional legislature, this may require the permanent supervision of a veterinarian.</li>
	<li>Pay close attention to maintaining the normal body temperature of the pigs (~38 &#176;C) during transport. For instance, consider covering the animal with a blanket to prevent hypothermia. 
	NOTE: It is important to limit the transport time to not exceed the duration of sedation, which usually ranges from 30 min to 60 min.</li>
	<li>Following disinfection, establish an intravenous access by inserting a 22 G catheter in the auricular vein. Before proceeding with any further movement of the pig or induction of anesthesia, ensure that the catheter is securely fixed to prevent dislocation by any sudden movements.</li>
	<li>Continuously monitor the peripheral oxygen saturation using a sensor clipped to the tail or ear.</li>
</ol>
2. Anesthesia and mechanical ventilation
<ol>
	<li>Administer intravenous fentanyl (4 &#181;g kg-1) and propofol (3 mg kg-1) to induce anesthesia.</li>
	<li>Place the pig in a supine position.</li>
	<li>Administer atracurium (0.5 mg kg-1) as a muscle relaxant and immediately initiate non-invasive ventilation using a dog ventilation mask. Place the mask over the snout and apply firm pressure with the thumbs while pulling the lower jaw forward using the middle/ring finger. Set the ventilator to the following parameters: inspiratory oxygen fraction (FiO2 ) = 100%, tidal volume = 6-8 mL kg-1, Positive End Expiratory Pressure (PEEP) = 5 mbar, peak inspiratory pressure &#8804; 20 mbar, respiratory rate = 18-20 min-1.</li>
	<li>Maintain anesthesia by initiating a continuous infusion of a balanced electrolyte solution (5 mL kg-1 h-1), fentanyl (10 &#181;g kg-1 h-1), and propofol (6 mg kg-1 h-1).</li>
	<li>Perform endotracheal intubation using a standard endotracheal tube (ID 6-7 mm), a guidewire, and a laryngoscope equipped with a Macintosh Blade (size 4).
	<ol>
		<li>Have an assistant open the mouth and hold the tongue to the left side.</li>
		<li>Insert the Macintosh Blade until the epiglottis is visible. Then, lift the laryngoscope upwards to move the epiglottis ventrally and visualize the vocal cords. Occasionally, the epiglottis may stick to the soft palate; in this case, mobilize it by gently swiping sideways with the tube or a bougie.</li>
		<li>Carefully insert the endotracheal tube through the vocal cords and remove the inducer. If encountering difficulty, try rotating the tube without applying excessive force. If necessary, use a smaller tube. Once the tube is in place, inflate the cuff with 10 mL of air.</li>
	</ol>
	</li>
	<li>Connect the endotracheal tube to the ventilator and initiate ventilation. Confirm proper tube positioning by detecting end-expiratory CO2 and performing bilateral auscultation. Use the following ventilation settings: FiO2 = 40%, tidal volume = 6-8 mL kg-1, PEEP = 5 mbar, inspiration to expiration ratio = 1:2, respiratory rate = adjusted to achieve an end-tidal CO2 level of &#60;45 mmHg, typically 30-40 min-1 . 
	NOTE: If the tube has been placed incorrectly in the esophagus, air will inflate the stomach, causing a visible bulge. In such cases, immediately remove the tube, administer non-invasive ventilation for 1-2 min, and reposition the tube correctly.</li>
	<li>Insert a gastric tube to prevent reflux or vomiting. If insertion proves challenging, use the laryngoscope to obtain a better view of the esophageal entrance.</li>
</ol>
3. Instrumentation
<ol>
	<li>Place an arterial and a central venous line in the femoral artery and vein, respectively, for hemodynamic monitoring and intravenous volume therapy.</li>
	<li>Use bandages to retract and secure the hind legs, providing better access to the femoral vessels.</li>
	<li>Prepare all necessary materials prior to instrumentation. Fill all catheters with saline solution and ensure easy access to wires and catheters to minimize the need for multiple catheterization attempts and unnecessary blood loss.</li>
	<li>Apply an alcoholic disinfectant to the inguinal area and wipe it with a sterile swab. Repeat this process twice. Apply the disinfectant again without wiping and wait for 3 min. Place a sterile fenestrated drape over the inguinal area.</li>
	<li>Use ultrasound to identify the femoral blood vessels. Use an in-plane ultrasound-guided Seldinger&#39;s technique for catheterization to minimize tissue damage and blood loss.</li>
	<li>Visualize the femoral artery longitudinally. Puncture the artery with a syringe attached to the needle for continuous aspiration. Bright red, pulsating blood confirms arterial puncture. Remove the syringe and insert the prepared wire. Remove the needle while leaving the wire in place.</li>
	<li>Repeat the same procedure for the femoral vein. Venous puncture is confirmed by slow-flowing, dark red blood.</li>
	<li>Confirm the correct positioning of both wires by visualizing both femoral vessels using ultrasound.</li>
	<li>Use Seldinger&#39;s technique to insert the arterial introducer sheath first, followed by the venous introducer sheath. Confirm proper positioning through blood gas analysis of blood samples drawn from the two lines.</li>
	<li>Ensure that blood can be aspirated from all lines. Flush all lines with saline solution to prevent clot formation.</li>
	<li>Securely fix the lines to the skin using surgical sutures to prevent dislocation.</li>
	<li>Connect the arterial and central venous lines to transducers for the measurement of hemodynamic parameters.</li>
	<li>Insert a pulse contour cardiac output (PiCCO) catheter into the arterial introducer sheath and connect it to the arterial pressure transducer and temperature interface cable of the PiCCO monitor.</li>
	<li>Connect a Swan-Ganz catheter to a transducer.
	<ol>
		<li>While continuously measuring pressure, insert the catheter into the central venous introducer sheath. Inflate the balloon after approximately 30 cm, when a central venous pressure curve becomes visible.</li>
		<li>Slowly advance the catheter while monitoring the pressure curve. As the catheter enters the right ventricle, look for a pulse curve with a high systolic and low diastolic value. Further advancement of the catheter will result in a consistent systolic value and an increased diastolic value, indicating placement in the pulmonary artery.</li>
		<li>Fix the catheter in this position (usually between 50 and 70 cm). Connect the injectate temperature sensor of the PiCCO system to the proximal lumen of the Swan-Ganz catheter.</li>
	</ol>
	</li>
	<li>Shave the pig&#39;s forehead and apply the adhesive sensor electrode to measure Cerebral Regional Oxygen Saturation.</li>
	<li>After anesthesia induction and instrumentation, allow the animal to stabilize for 30 min or until hemodynamic parameters have stabilized before conducting baseline measurements and inducing endotoxemic shock.</li>
</ol>
4. Shock induction
NOTE: When working with LPS, always wear gloves, protective goggles, a mask, and a lab coat. Avoid direct contact with LPS.
<ol>
	<li>Prepare an LPS solution with a concentration of 100 &#181;g mL-1 by dissolving 5 mg of LPS in 50 mL of 0.9% NaCl.</li>
	<li>Obtain baseline hemodynamic measurements immediately before initiating LPS infusion.</li>
	<li>Administer a 150 &#181;g kg-1 dose of LPS over 30 min (equivalent to a continuous infusion rate of 300 &#181;g kg-1&#8729;h-1 for 30 min).</li>
	<li>After 30 min, reduce the infusion rate to 15 &#181;g&#8729;kg-1&#8729;h-1 for the remainder of the experiment.</li>
	<li>Continuously monitor hemodynamic parameters, including arterial and pulmonary arterial blood pressure, heart rate, and ventilation parameters. Monitor the body temperature continuously to maintain normothermia.</li>
</ol>
5. Treatment of hemodynamic instability
<ol>
	<li>When the mean arterial blood pressure falls below 60 mmHg, use PiCCO to measure cardiac index (CI), Global End-Diastolic Volume Index (GEDI), and Extravascular Lung Water Index (ELWI). Treat the low blood pressure according to the recommendations in the flow chart in Figure 2.
	<ol>
		<li>On the PiCCO monitor, press the thermodilution button (TD).</li>
		<li>Press the button for central venous pressure (CVP) input and enter the current CVP value.</li>
		<li>Press the Start button.</li>
		<li>When instructed to do so, inject 10 mL of cold saline solution into the injectate temperature sensor connected to the Swan-Ganz catheter. 
		NOTE: Do not inject anything else directly before or during PiCCO measurement as this would compromise the measurement.</li>
	</ol>
	</li>
	<li>After obtaining measurements for CI, GEDI, and ELWI, treat hemodynamic instability according to the flow chart in Figure 2. If Volume loading is recommended, rapidly infuse 200 mL of balanced electrolyte solution. If catecholamine therapy is recommended, increase the rate of norepinephrine infusion by 1 &#181;g kg-1 h-1.</li>
	<li>Repeat this process whenever the mean arterial blood pressure falls below 60 mmHg. In case of severe hemodynamic instability, opt for rapid escalation of therapy.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66039/66039fig02.jpg" /> 
Figure 2: PiCCO-guided therapy of hemodynamic instability. After obtaining measurements for CI, GEDI, and ELWI, apply treatment according to the chart. This figure was adapted from the PiCCO user guidebook12. Abbreviations: PiCCO = pulse contour cardiac output; V+ = volume loading; cat = catecholamine therapy; V- = volume reduction; CI = cardiac index; GEDI = global end-diastolic volume index; ELWI = extravascular lung water index. <a href="https://www.jove.com/files/ftp_upload/66039/66039fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
6. End of experiment and euthanasia
<ol>
	<li>Inject 0.5 mg of fentanyl intravenously. Wait 5 min. Inject 200 mg of propofol.</li>
	<li>Euthanize the pig with a rapid injection of 40 mL of 1 M potassium chloride via the central venous line.</li>
</ol>

Dosage-Dependent Induction of Endotoxemia in Pigs via LPS Infusion

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The NIRS device was unconditionally provided by Medtronic PLC, USA, for experimental research purposes. Alexander Ziebart received a lecture fee from Medtronic PLC. None of the authors report any financial or other conflicts of interest. The manuscript was proofread and edited by ChatGPT&#174; (Python Software, Version: May 24, 2023).

We present a protocol for inducing experimental endotoxemia in pigs through LPS infusion, aiming to reliably induce changes commonly observed in sepsis and septic shock. Several critical steps need to be considered in this protocol. Adequate sedation of pigs prior to transport is crucial to prevent stress-induced elevation of catecholamine levels, which could potentially compromise the results. Intubation of pigs may pose challenges compared to humans due to the anatomical features of their elongated snouts. To address t...

Sepsis and septic shock rank among the leading causes of mortality in patients receiving intensive care treatment1,2,3. Sepsis arises when an infection triggers a dysregulated immune response resulting in multiorgan failure. It is characterized by life-threatening symptoms, including hemodynamic instability, respiratory distress, hepatic and renal failure, as well as cognitive impairment4,5. Septic shock represents a subset of sepsis with particularly severe symptoms that significantly increase mortality. These symptoms include persistent hypotension requiring vasopressor therapy and a serum lactate level exceeding 2 mmol&#8729;L-1&#160;4,5. Mortality rates in patients with septic shock have been estimated as high as 40%, even with hospital treatment1,3,5.
Gram-negative bacteria, such as Pseudomonas and Escherichia coli, often cause infections triggering this dysregulated immune response4. The underlying pathophysiological mechanisms are complex and not yet fully understood. One well-described aspect involves the activation of Toll-like receptors on immune cells by pathogen-associated molecular patterns (PAMPs), leading to the release of cytokines such as tumor necrosis factor-alpha (TNF&#945;) or Interleukin 1 (IL 1)4. One of these PAMPs is lipopolysaccharide (LPS), which constitutes a component of the cell membrane in gram-negative bacteria6. LPS has been employed in animal models to induce endotoxemia and endotoxemic shock7,8.
Animal models provide a controlled and standardized setting to develop and investigate novel treatment strategies. Due to its similar anatomy, immunological physiology, and comparable hemodynamic parameters, the pig model is particularly well-suited for studying the effects of endotoxemic shock9,10. Furthermore, standard medical equipment commonly used in human patients can be readily applied in pigs due to the similar size of their airways and blood vessels, facilitating instrumentation and hemodynamic monitoring.
With this protocol, we provide an experimental model for endotoxemic shock in pigs by intravenously infusing LPS derived from E. coli. To monitor the effects, we measured hemodynamic and pulmonary parameters, including arterial blood pressure, heart rate, peripheral oxygen saturation, pulmonary arterial pressure, and airway pressure. To evaluate the influence of endotoxemia on cerebral oxygen supply, we used near-infrared spectrometry (NIRS). With this method, the cerebral oxygen saturation can be evaluated via an adhesive electrode applied to the forehead11.

<table><tbody><tr><td>Atracurium Hikma 50 mg/5mL</td><td>Hikma Pharma GmbH, Martinsried</td><td></td><td></td></tr><tr><td>Azaperone (Stresnil) 40 mg/mL</td><td>Lilly Deutschland GmbH, Bad Homburg, Germany</td><td></td><td></td></tr><tr><td>BD Discardit II Spritze 2, 5, 10, 20 mL</td><td>Becton Dickinson S.A. Carretera, Mequinenza Fraga, Spain</td><td>syringe</td><td></td></tr><tr><td>BD Luer Connecta&amp;nbsp;</td><td>Becton Dickinson Infusion Therapy, AB Helsingborg, Schweden</td><td>3-way-stopcock</td><td></td></tr><tr><td>Curafix i.v. classics</td><td>Lohmann &amp;amp; Rauscher International GmbH &amp;amp; Co. KG, Rengsdorf, Germany</td><td>Cannula retention dressing</td><td></td></tr><tr><td>Datex Ohmeda S5</td><td>GE Healthcare Finland Oy, Helsinki, Finland</td><td>hemodynamic monitor</td><td></td></tr><tr><td>Engstr&amp;ouml;m Carestation</td><td>GE Heathcare, Madison USA</td><td>ventilator</td><td></td></tr><tr><td>Fentanyl-Janssen 0.05 mg/mL</td><td>Janssen-Cilag GmbH, Neuss</td><td>fentanyl</td><td></td></tr><tr><td>F&amp;uuml;hrungsstab, Durchmesser 4.3</td><td>R&amp;uuml;sch</td><td>endotracheal tube introducer</td><td></td></tr><tr><td>Incetomat-line 150 cm</td><td>Fresenius, Kabi Deutschland, GmbH</td><td>perfusor line</td><td></td></tr><tr><td>Intrafix Primeline</td><td>B. Braun Melsungen AG, Melsungen, Germany</td><td>Infusion line</td><td></td></tr><tr><td>Introducer sheath 5 Fr.</td><td>Terumo Healthcare</td><td>arterial introducer&amp;nbsp;</td><td></td></tr><tr><td>INVOS</td><td>Medtronic, Dublin, Ireland</td><td>near infrared spectrometry</td><td></td></tr><tr><td>JOZA Einmal Nitril Untersuchungshandschuhe&amp;nbsp;</td><td>JOZA, M&amp;uuml;nchen, Germany</td><td>disposable gloves</td><td></td></tr><tr><td>Laryngoscope, 45.48.50, KL 2000</td><td>Medicon</td><td>Laryngoscope handle</td><td></td></tr><tr><td>Littmann Classic III Stethoscope</td><td>3M Deutschland GmbH, Neuss, Germany</td><td>stethoscope</td><td></td></tr><tr><td>LPS (E. coli; Serotype O111:B4)</td><td>Sigma-Aldrich, Switzerland</td><td></td><td></td></tr><tr><td>MAC Two-Lumen Central venous access set</td><td>Arrow international inc. Reading, PA, USA</td><td>venous introducer</td><td></td></tr><tr><td>Maimed Vlieskompresse</td><td>Maimed GmbH, Neuenkirchen, Germany</td><td>Fleece compress to fix the tongue</td><td></td></tr><tr><td>Masimo LNCS Adtx SpO&lt;sub&gt;2 &lt;/sub&gt;sensor</td><td>Masimo Corporation Irvine, Ca 92618 USA</td><td>saturation clip for the tail</td><td></td></tr><tr><td>Masimo LNCS TC-I SpO2 ear clip sensor</td><td>Masimo Corporation Irvine, Ca 92618 USA</td><td>Saturation clip for the ear</td><td></td></tr><tr><td>Masimo Radical 7</td><td>Masimo Corporation Irvine, Ca 92618 USA</td><td>periphereal oxygen saturation&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Midazolam 15 mg/3 mL</td><td>B.Braun Melsungen AG, Germany</td><td></td><td></td></tr><tr><td>Midmark Canine Mask Small Plastic with Diaphragm FRSCM-0005</td><td>Midmark Corp., Dayton, Ohio, USA</td><td>dog ventilation mask</td><td></td></tr><tr><td>Monocryl surgical suture</td><td>Johnson &amp;amp; Johnson, Belgium</td><td></td><td></td></tr><tr><td></td><td>B.Braun Melsungen AG, Germany</td><td>saline solution</td><td></td></tr><tr><td>NaCl 0.9 %</td><td>Sanofi- Aventis, Seutschland GmbH</td><td></td><td></td></tr><tr><td>Octeniderm farblos</td><td>Sch&amp;uuml;lke &amp;amp; Mayr GmbH, Nordenstedt, Germany</td><td>Alcoholic disinfectant</td><td></td></tr><tr><td>Original Perfusor syringe 50 mL</td><td>B.Braun Melsungen AG, Germany</td><td>perfusor syringe</td><td></td></tr><tr><td>PA-Katheter Swan Ganz 7.5 Fr 110 cm</td><td>Edwards Lifesciences LLC, Irvine CA, USA</td><td>Swan-Ganz catheter</td><td></td></tr><tr><td>Perfusor FM Braun</td><td>B.Braun Melsungen AG, Germany</td><td>syringe pump</td><td></td></tr><tr><td>PiCCO catheter</td><td>PULSION Medical Systems SE, Feldkirchen, DE</td><td></td><td></td></tr><tr><td>Potassium chloride 1 M</td><td>Fresenius, Kabi Germany GmbH</td><td></td><td></td></tr><tr><td>Propofol 2% 20 mg/mL (50 mL flasks)</td><td>Fresenius, Kabi Deutschland, GmbH</td><td></td><td></td></tr><tr><td>Pulse-contour continous cardiac output System PiCCO2</td><td>PULSION Medical Systems SE, Feldkirchen, DE</td><td></td><td></td></tr><tr><td>R&amp;uuml;schelit Super Safety Clear &gt;ID 6/6.5 /7.0 mm</td><td>Teleflex Medical Sdn. Bhd, Malaysia</td><td>endotracheal tube</td><td></td></tr><tr><td>Sonosite Micromaxx Ultrasoundsystem</td><td>Sonosite Bothell, WA, USA&amp;nbsp;</td><td>ultrasound&amp;nbsp;</td><td></td></tr><tr><td>Stainless Macintosh Gr&amp;ouml;&amp;szlig;e 4</td><td>Welch Allyn69604</td><td>blade for laryngoscope</td><td></td></tr><tr><td>Sterofundin</td><td>B.Braun Melsungen AG, Melsungen, Germany</td><td>Balanced electrolyte solution</td><td></td></tr><tr><td>Vasco OP sensitive&amp;nbsp;</td><td>B.Braun Melsungen AG, Germany</td><td>sterile gloves</td><td></td></tr><tr><td>Vasofix Safety 22 G-16 G</td><td>B.Braun Melsungen AG, Germany</td><td>venous catheter</td><td></td></tr><tr><td>VBM Cuff Manometer</td><td>VBM Medizintechnik GmbH, Sulz a.N., Germany&amp;nbsp;</td><td>cuff pressure gauge</td><td></td></tr></tbody></table>

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.

For this study, six healthy male pigs aged 2-3 months and weighing 30-35 kg were anesthetized and received an infusion of lipopolysaccharide (LPS) to induce endotoxemia. To determine the appropriate dosage of LPS required to consistently induce symptoms of shock, the pigs were administered various induction doses of LPS ranging from 100 &#181;g kg-1 to 200 &#181;g kg-1 over a 30-min period, followed by a maintenance dose of 1/10 of the initial dose per hour for the remainder of the experiment. All a...

Watch this Scientific Journal Video about Lipopolysaccharide Infusion as a Porcine Endotoxemic Shock Model at JoVE.com

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.

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 ...

lipopolysaccharide-infusion-as-a-porcine-endotoxemic-shock-model

Research

JoVE Journal

Medicine

842 Views.  University Medical Center of the Johannes Gutenberg-University. We provide a protocol for an experimental endotoxemic shock model in pigs by infusion of lipopolysaccharide. 

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

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, ...

Oleic Acid-Injection in Pigs As a Model for Acute Respiratory Distress Syndrome

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit patients. Despite treatment advances over the last decades, ARDS patients still suffer mortality rates between 35 and 40%. There is still a need for further research to improve the outcome of patients suffering from ARDS. One problem is that no single animal model can mimic the complex pathomechanism of the acute respiratory distress syndrome, but several models exist to study different parts of it. Oleic acid injection (OAI)-induced lung injury is a well-established model for studying ventilation strategies, lung mechanics and ventilation/perfusion distribution in animals. OAI leads to severely impaired gas exchange, deterioration of lung mechanics and disruption of the alveolo-capillary barrier. The disadvantage of this model is the controversial mechanistic relevance of this model and the necessity for central venous access, which is challenging especially in smaller animal models. In summary, OAI-induced lung injury leads to reproducible results in small and large animals and hence represents a well-suited model for studying ARDS. Nevertheless, further research is necessary to find a model that mimics all parts of ARDS and lacks the problems associated with the different models existing today.

In this article, we present a protocol to induce acute lung injury in pigs by central-venous injection of oleic acid. This is an established animal model for studying the acute respiratory distress syndrome (ARDS).

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit ...

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.

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 ...

Halogenated Agent Delivery in Porcine Model of Acute Respiratory Distress Syndrome via an Intensive Care Unit Type Device

Acute respiratory distress syndrome (ARDS) is a common cause of hypoxemic respiratory failure and death in critically ill patients, and there is an urgent need to find effective therapies. Preclinical studies have shown that inhaled halogenated agents may have beneficial effects in animal models of ARDS. The development of new devices to administer halogenated agents using modern intensive care unit (ICU) ventilators has significantly simplified the dispensing of halogenated agents to ICU patients. Because previous experimental and clinical research suggested potential benefits of halogenated volatiles, such as sevoflurane or isoflurane, for lung alveolar epithelial injury and inflammation, two pathophysiologic landmarks of diffuse alveolar damage during ARDS, we designed an animal model to understand the mechanisms of the effects of halogenated agents on lung injury and repair. After general anesthesia, tracheal intubation, and the initiation of mechanical ventilation, ARDS was induced in piglets via the intratracheal instillation of hydrochloric acid. Then, the piglets were sedated with inhaled sevoflurane or isoflurane using an ICU-type device, and the animals were ventilated with lung-protective mechanical ventilation during a 4 h period. During the study period, blood and alveolar samples were collected to evaluate arterial oxygenation, the permeability of the alveolar-capillary membrane, alveolar fluid clearance, and lung inflammation. Mechanical ventilation parameters were also collected throughout the experiment. Although this model induced a marked decrease in arterial oxygenation with altered alveolar-capillary permeability, it is reproducible and is characterized by a rapid onset, good stability over time, and no fatal complications.
We have developed a piglet model of acid aspiration that reproduces most of the physiological, biological, and pathological features of clinical ARDS, and it will be helpful to further our understanding of the potential lung-protective effects of halogenated agents delivered through devices used for inhaled ICU sedation.

We describe a model of hydrochloric acid-induced acute respiratory distress syndrome (ARDS) in piglets receiving sedation with halogenated agents, isoflurane and sevoflurane, through a device used for inhaled intensive care sedation. This model can be used to investigate the biological mechanisms of halogenated agents on lung injury and repair.

Acute respiratory distress syndrome (ARDS) is a common cause of hypoxemic respiratory failure and death in critically ill patients, and there is an urgent ...

A Reproducible Intensive Care Unit-Oriented Endotoxin Model in Rats

Sepsis and septic shock remain the leading cause of death in intensive care units. Despite significant improvements in sepsis management, mortality still ranges between 20 and 30%. Novel treatment approaches in order to reduce sepsis-related multiorgan failure and death are urgently needed. Robust animal models allow for one or multiple treatment approaches as well as for testing their effect on physiological and molecular parameters. In this article, a simple animal model is presented.
First, general anesthesia is induced in animals either with the use of volatile or by intraperitoneal anesthesia. After placement of an intravenous catheter (tail vein), tracheostomy, and insertion of an intraarterial catheter (tail artery), mechanical ventilation is started. Baseline values of mean arterial blood pressure, arterial blood oxygen saturation, and heart rate are recorded.
The injection of lipopolysaccharides (1 milligram/kilogram body weight) dissolved in phosphate-buffered saline induces a strong and reproducible inflammatory response via the toll-like receptor 4. Fluid corrections as well as the application of norepinephrine are performed based on well-established protocols.
The animal model presented in this article is easy to learn and strongly oriented towards clinical sepsis treatment in an intensive care unit with sedation, mechanical ventilation, continuous blood pressure monitoring, and repetitive blood sampling. Also, the model is reliable, allowing for reproducible data with a limited number of animals in accordance with the 3R (reduce, replace, refine) principles of animal research. While animal experiments in sepsis research cannot easily replaced, repetitive measurements allow for a reduction of animals and keeping septic animals anesthetized diminishes suffering.

Here, we present a reproducible intensive care unit-oriented endotoxin model in rats.

Sepsis and septic shock remain the leading cause of death in intensive care units. Despite significant improvements in sepsis management, mortality still ...

Real-Time Assessment of Spinal Cord Microperfusion in a Porcine Model of Ischemia/Reperfusion

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its incidence is still considerably high and therefore, influences patient outcome. Microcirculation plays a key role in tissue perfusion and oxygen supply and is often dissociated from macrohemodynamics. Thus, direct evaluation of spinal cord microcirculation is essential for the development of microcirculation-targeted therapies and the evaluation of existing approaches in regard to spinal cord microcirculation. However, most of the methods do not provide real-time assessment of spinal cord microcirculation. The aim of this study is to describe a standardized protocol for real-time spinal cord microcirculatory evaluation using laser-Doppler needle probes directly inserted in the spinal cord. We used a porcine model of ischemia/reperfusion to induce deterioration of the spinal cord microcirculation. In addition, a fluorescent microsphere injection technique was used. Initially, animals were anesthetized and mechanically ventilated. Thereafter, laser-Doppler needle probe insertion was performed, followed by the placement of cerebrospinal fluid drainage. A median sternotomy was performed for exposure of the descending aorta to perform aortic cross-clamping. Ischemia/reperfusion was induced by supra-celiac aortic cross-clamping for a total of 48 min, followed by reperfusion and hemodynamic stabilization. Laser-Doppler Flux was performed in parallel with macrohemodynamic evaluation. In addition, automated cerebrospinal fluid drainage was used to maintain a stable cerebrospinal pressure. After completion of the protocol, animals were sacrificed, and the spinal cord was harvested for histopathological and microsphere analysis. The protocol reveals the feasibility of spinal cord microperfusion measurements using laser-Doppler probes and shows a marked decrease during ischemia as well as recovery after reperfusion. Results showed comparable behavior to fluorescent microsphere evaluation. In conclusion, this new protocol might provide a useful large animal model for future studies using real-time spinal cord microperfusion assessment in ischemia/reperfusion conditions.

Spinal cord microcirculation plays a pivotal role in spinal cord injury. Most methods do not allow real-time assessment of spinal cord microcirculation, which is essential for the development of microcirculation-targeted therapies. Here, we propose a protocol using Laser-Doppler-Flow Needle probes in a large animal model of ischemia/reperfusion.

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its ...

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 ...

Investigating Physiological Responses to Acute Volume Overload in Porcine Models

Adult and Pediatric Porcine Model of Acute Volume Overload

This protocol describes an acute volume overload porcine model for adult Yorkshire pigs and piglets. Both swine ages undergo general anesthesia, endotracheal intubation, and mechanical ventilation. A central venous catheter and an arterial catheter are placed via surgical cutdown in the external jugular vein and carotid artery, respectively. A pulmonary artery catheter is placed through an introducer sheath of the central venous catheter. PlasmaLyte crystalloid solution is then administered at a rate of 100 mL/min in adult pigs and at 20 mL/kg boluses over 10 min in piglets. Hypervolemia is achieved either at 15% decrease in cardiac output or at 5 L in adult pigs and at 500 mL in piglets. Hemodynamic data, such as heart rate, respiratory rate, end-tidal carbon dioxide, fraction of oxygen-saturated hemoglobin, arterial blood pressure, central venous pressure, pulmonary artery pressure, pulmonary capillary wedge pressure, partial arterial oxygen pressure, lactate, pH, base excess, and pulmonary artery fraction of oxygen-saturated hemoglobin, are monitored during experimentation. Preliminary data observed with this model has demonstrated statistically significant changes and strong linear regressions between central hemodynamic parameters and acute volume overload in adult pigs. Only pulmonary capillary wedge pressure demonstrated both a linear regression and a statistical significance to acute volume overload in piglets. These models can aid scientists in the discovery of age-appropriate therapeutic and monitoring strategies to understand and prevent acute volume overload.

Author Spotlight: Exploring Venous Waveforms in Porcine Models to Tackle Volume Overload in Medicine

The protocol here shows how continuous administration of crystalloids into the central veins of a euvolemic pig/piglet allows for the appropriate investigation of the physiological effects of acute volume overload.

This protocol describes an acute volume overload porcine model for adult Yorkshire pigs and piglets. Both swine ages undergo general anesthesia, ...

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 ...

A Porcine Model of Acute Autologous Pulmonary Embolism

Acute pulmonary embolism (PE) is a potentially life-threatening condition that causes abrupt obstruction of the pulmonary arteries, leading to acute right heart failure. Novel diagnostic methods and catheter-directed therapies are being developed rapidly, and there is an obvious need for a realistic PE animal model that can be used for pathophysiological evaluation and preclinical testing.
This protocol introduces a porcine model employing large autologous pulmonary emboli. Instrumentations are performed with minimally invasive techniques, creating a close-chest model that enables the investigation of various treatment options with high reproducibility. Three hours after drawing blood to create autologous emboli ex vivo, the induction of PE caused an immediate increase in the mean pulmonary arterial pressure (17&#160;&#177; 3 mmHg to 33&#160;&#177; 6 mmHg, p &#60; 0.0001) and heart rate (50&#160;&#177; 9 beats&#183;min-1 to 63&#160;&#177; 6 beats&#183;min-1,&#160;p &#60; 0.0003) accompanied by a decreased cardiac output (5.0&#160;&#177; 0.8 L/min to 4.5&#160;&#177; 0.9 L/min, p &#60; 0.037) compared to baseline. The CT pulmonary angiography revealed multiple emboli, and the pulmonary obstruction percentage was increased compared to baseline (0% [0-0] to 57.1% [38.8-63.3], p &#60; 0.0001). In the acute phase, the phenotype is comparable to intermediate-risk PE.
The model represents a realistic and well-characterized phenotype of intermediate-risk PE and creates an opportunity to test novel diagnostic methods, interventional and pharmaceutical treatments, and hands-on training for healthcare workers in interventional procedures.

This study presents a porcine model of pulmonary embolism (PE) using large autologous emboli that replicate acute intermediate-risk PE. The model is well-suited for the evaluation of both pathophysiology and treatment responses.

Acute pulmonary embolism (PE) is a potentially life-threatening condition that causes abrupt obstruction of the pulmonary arteries, leading to acute right ...