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

All animal experiments described here have been approved by the institutional and state animal care committee (Landesuntersuchungsamt Rheinland-Pfalz, Koblenz, Germany; approval number G14-1-077) and were conducted in accordance with the guidelines of the European and German Society of Laboratory Animal Sciences. The experiments were conducted in anesthetized male pigs (sus scrofa domestica) of 2-3 months age, weighing 27-29 kg.
1. Anesthesia, Intubation and Mechanical Ventilation
<ol>
	<li>Withhold food for 6 h before anesthesia to reduce the risk of aspiration but allow free access to water to reduce stress.</li>
	<li>For sedation, inject a combination of Ketamine (4 mg kg-1) and Azaperone (8 mg kg-1) in the neck or the gluteal muscle of the pig with a needle for intramuscular injection (20 G) while the animal is in the animal box. 
	Caution: Use gloves when working with the animal.</li>
	<li>Insert peripheral vein catheter (20 G) in an ear vein after local disinfection with alcohol.</li>
	<li>Inject fentanyl (4 &#181;g kg-1), propofol (3 mg kg-1) and atracurium (0.5 mg kg-1) intravenously for the induction of anesthesia.</li>
	<li>When the pig stops breathing, place it in supine position on the stretcher and immobilize it with bandages.</li>
	<li>Start monitoring the peripheral oxygen saturation (SpO2) by clipping the sensor on to one of the ears or the tail of the animal.</li>
	<li>Ventilate the pig with a mask for ventilating dogs, size 2, with a peak inspiratory pressure below 20 cm H2O, a positive end expiratory pressure (PEEP) of 5 cm H2O, a respiratory rate of 14-16 /min and an inspiratory oxygen fraction (FiO2) of 1.0.</li>
	<li>Start a continuous infusion with balanced electrolyte solution (5 mLkg-1 h-1), propofol (8-12 mg kg-1 h-1) and fentanyl (0.1-0.2 mg kg-1 h-1) to maintain anesthesia.</li>
	<li>For the intubation, prepare a common endotracheal tube suitable for the animal (e.g., weight of 25-30 kg, endotracheal tube internal diameter (ID) 6-7mm) armed with endotracheal tube introducer and a common laryngoscope with a Macintosh Blade 4. 
	NOTE: Two people are necessary for the intubation.</li>
	<li>Person 1: Pull out the tongue with one hand and press the snout dorsally with the other.</li>
	<li>Person 2: Insert laryngoscope and advance it as usual until the epiglottis comes into view.</li>
	<li>Pull the laryngoscope ventrally to visualize the vocal cords. 
	NOTE: Sometimes the epiglottis &#34;sticks&#34; to the soft palatine. In this case, mobilize it with the tip of the tube.</li>
	<li>Insert the tube through the vocal cords and pull out the introducer.</li>
	<li>Block the cuff of the tube with a syringe with 10 mL of air.</li>
	<li>Connect the tube to the ventilator.</li>
	<li>Check for the correct positioning of the tube by regular exhalation of carbon dioxide (CO2) with capnography and equal ventilation of both lungs with auscultation.</li>
	<li>Start mechanical ventilation (tidal volume 6-8 mL/kg, positive PEEP 5 cm H20, FiO2 to keep peripheral oxygen saturation (SpO2) between 94 &#8211; 98%17, respiratory rate to keep end tidal pressure of carbon dioxide (etCO2) between 35 &#8211; 45 mmHG).</li>
</ol>
2. Instrumentation
<ol>
	<li>Retract the hindlegs with bandages to stretch the skin above the femoral area for catheterizing necessary vessels.</li>
	<li>Prepare a 5 mL syringe, a 10 mL syringe, a Seldinger&#39;s needle, 3 introducer sheaths (5 Fr, 6 Fr, 8 Fr) with guidewires, a central venous catheter with 3 ports (7 Fr, 30 cm) with guidewire and a pulmonary artery catheter (7,5 Fr, 110 cm).</li>
	<li>Generously disinfect the femoral area with skin disinfectant applying a wipe down technique.</li>
	<li>Completely fill the catheters with saline.</li>
	<li>Place the ultrasound-probe on the right inguinal ligament and scan for femoral vessels.</li>
	<li>Turn the probe 90&#176; to fully visualize the femoral artery in the long axis.</li>
	<li>Cannulate right femoral artery under in-line ultrasound visualization with the Seldinger&#39;s needle. 
	NOTE: There are different ways to gain vascular access with or without ultrasound. Ultrasound-guided vascular cannulation is not necessary for this model.</li>
	<li>When pulsating bright blood flows out, introduce the guidance wire and retract the needle.</li>
	<li>Visualize the femoral vein and cannulate the vein under in-line ultrasound visualization and continuous aspiration with the needle.</li>
	<li>When venous blood is aspirable, disconnect the syringe and insert the guidance wire.</li>
	<li>Retract the needle.</li>
	<li>Check the position of the wires with ultrasound.</li>
	<li>Insert the arterial introducer sheath (5 Fr) and central venous catheter using Seldinger&#39;s technique (for details on Seldinger&#39;s technique, refer to published method18).</li>
	<li>Repeat the arterial and venous puncture on other side and insert the introducer sheaths using Seldinger&#180;s technique as described above (artery 6 Fr, vein 8 Fr).</li>
	<li>Connect the arterial introducer sheath and central venous catheter to a transducer system suitable to the monitoring equipment.</li>
	<li>Calibrate the invasive monitoring against atmosphere (zero) by opening the three-way-stopcocks to the atmosphere and press Zero all on the monitor.</li>
	<li>Turn the three-way-stopcocks back to measure hemodynamics.</li>
	<li>Start monitoring hemodynamics.</li>
	<li>Place all pressure transducers at the height of the right atrium.</li>
	<li>Switch the infusion of propofol (8-12 mg kg-1 h-1) and fentanyl (0.1-0.2 mg kg-1 h-1) to one of the ports of the central venous line to maintain anesthesia.</li>
</ol>
3. Ultrafast Measurement of Partial Oxygen Pressure (pO2)
NOTE: The measurement of pO2 with the probe for ultrafast pO2-measurement is not obligatory but helps visualizing the real-time changes in pO2.
<ol>
	<li>Open software NeoFox viewer and click Options.</li>
	<li>Choose the Calibration tab and click the Open Calibration button.</li>
	<li>Choose calibration file and click Open and Download.</li>
	<li>Confirm the pop-up window by clicking Yes.</li>
	<li>Open the Options dialogue.</li>
	<li>Choose the Calibration tab and click Single point calibration.</li>
	<li>Enter 21% in the field Oxygen and the temperature in the field Temperature.</li>
	<li>Click Use current Tau and Download. Afterwards, confirm the pop-up window by clicking Yes.</li>
	<li>Insert the probe for ultrafast measurements of pO2 through the left arterial introducer sheath.</li>
</ol>
4. INSERTING PULMONARY ARTERY CATHETER
<ol>
	<li>Check the balloon of pulmonary artery catheter for damage.</li>
	<li>Connect to the transducer system suitable to the monitoring equipment.</li>
	<li>Calibrate the pulmonary arterial pressure monitoring against the atmosphere (zero) by opening the three-way-stopcock to the atmosphere and press Zero on the monitor.</li>
	<li>Turn the three-way-stopcock back to measure pulmonary arterial pressure.</li>
	<li>Start monitoring the pulmonary arterial pressure.</li>
	<li>Insert the pulmonary artery catheter through the left venous introducer sheath (balloon deflated).</li>
	<li>When the pulmonary artery catheter has passed through the introducer sheath, inflate the balloon with 1 mL of air.</li>
	<li>Advance the pulmonary artery catheter and monitor the typical waveforms (venous vessels, right atrium, right ventricle, pulmonary arteria, and pulmonary capillary wedge pressure). Deflate the balloon and check, if it is possible to aspirate blood through all ports of the pulmonary artery catheter. 
	NOTE: For detailed instruction on how to insert pulmonary artery catheter, refer to previous publication19.</li>
</ol>
5. Induction of Lung Injury
<ol>
	<li>Prepare oleic-acid solution: 0.1 mL kg-1 of oleic acid in a 20 mL syringe and connect it to a 3-way-stopcock.</li>
	<li>Take 2 mL of blood in another 20 mL syringe and add saline to a total volume of 20 mL in both syringes.</li>
	<li>Connect the second syringe also to the 3-way-stopcock. 
	CAUTION: Use gloves and eye protection when working with oleic acid.</li>
	<li>Prepare norepinephrine (0.1 mg/mL) for continuous infusion and for bolus injection (10 &#181;g/mL).</li>
	<li>Connect the norepinephrine syringe pump to one of the ports of the central venous catheter without starting it.</li>
	<li>Start the ultrafast pO2-measurement.</li>
	<li>Before the induction of lung injury, record the values (baseline) from all relevant parameters.</li>
	<li>Set the FiO2 to 1.0 and conduct a lung recruitment maneuver (plateau pressure 40 cm H2O for 10 s).</li>
	<li>Connect the 3-way-stopcock to the proximal port of the pulmonary artery catheter.</li>
	<li>Mix the oleic acid and the blood/saline mixture thoroughly by injecting it repetitively from one syringe into the other via the 3-way-stopcock and keep mixing all the time.</li>
	<li>When it is a homogenous emulsion, inject 2 mL of the emulsion and continue mixing. 
	NOTE: If mixing is stopped, the emulsion may separate into a lipophilic and a hydrophilic part.</li>
	<li>Closely monitor the hemodynamics after the injection of oleic acid and keep the norepinephrine at hand. If necessary, give norepinephrine as bolus injection (10 &#8211; 100 &#181;g) or continuous infusion to keep mean arterial pressure above 60 mmHg.</li>
	<li>Repeat the injection of 2 mL of the solution every 3 min until the arterial partial pressure of oxygen (PaO2)/FiO2-ratio is below 200 mmHg.</li>
	<li>If the syringe is empty before the PaO2/FiO2-ratio is between 100 and 200 mmHg, prepare 2 more syringes as described in step 5.1.</li>
	<li>Wait 30 min and re-evaluate the PaO2/FiO2-ratio. If it is still over 200 mmHg, repeat steps 5.5-5.8 until PaO2/FiO2-ratio falls between 100 and 200 mmHg.</li>
	<li>If PaO2/FiO2-ratio is between 100 and 200 mmHg, wait for 30 min and check again.</li>
	<li>If it is persistent below 200 mmHg start experiment/treatment, otherwise prepare 2 more syringes as described in step 5.1 and repeat steps 5.5-5.9.</li>
	<li>Set the ventilation according to the suggestions from the ARDS network20.</li>
</ol>
6. End of Experiment and Euthanasia
<ol>
	<li>Inject 0.5 mg of fentanyl additionally to the continuous anesthesia and wait for 5 min. Inject 200 mg of propofol and 40 mmol of potassium chloride to euthanize the animal in deep anesthesia.</li>
</ol>

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All authors disclose no financial or any other conflict of interest.

This article describes one method of oleic acid-induced lung injury as a model for studying various aspects of severe ARDS. There are also other protocols with different emulsions, different injection sites, and different temperatures of the emulsion23,24,25,26,27,28,29. Our method offers a reproducible and stable deterioration in lung function. As the effect of oleic acid is dose dependent, it is necessary to define the individual threshold for the PaO2/FiO2-ratio, depending on the desired study, and find the necessary dose of oleic acid to achieve this ratio.
When using this method, there are some pitfalls. The first is the lipophilicity of the oleic acid. To keep it emulsified in the blood/saline mixture, it is necessary to continuously mix it. Another problem is the sudden change in hemodynamics after the injection of oleic acid. Directly after the injection of oleic acid, PAP values can increase abruptly to more than 60 mmHg, which can result in the sudden hemodynamic decompensation and the death of the animal. Therefore, it is necessary to keep sufficient rescue medication, e.g., norepinephrine, prepared and at hand. Nevertheless, the hemodynamic decompensation sometimes results in sudden death of the animal which cannot be prevented. The last pitfall is the after-effect of oleic acid. Similar to human ARDS, the time to symptom onset may vary and it is neither possible to predict exactly how much oleic acid is necessary in a given pig for the induction of lung injury, nor to predict the impact of a given dose on PaO2/FiO2-ratio. PaO2/FiO2-ratios can be nearly stagnating; but they might also improve or further decline. This is displayed in Figure 1. Once the PaO2/FiO2-ratio is between 100 and 200 mmHg at a PEEP &#8805; 5 cm&#160;H2O, we require oxygenation to remain impaired and below this threshold for more than 30 min. Usually, PaO2/FiO2 remains relatively constant during this time course, though it may drop further. Seldom, even an improvement is possible, reaching values above 200 mmHg. Under these circumstances, more oleic acid is needed.
The induction of lung injury by oleic acid does have certain limitations. The main disadvantage is the need for central venous access, which can be challenging particularly in small animals. Another is the question about the mechanistic relevance of this model. The oleic acid ARDS-model was developed in an attempt to reproduce ARDS due to lipid embolism as seen in trauma patients14. But trauma is only causative for about 10% of ARDS cases30 and whether or not other causes like sepsis or pneumonia share the same mechanism is still under discussion. The final disadvantage of this pig model for ARDS is the complex instrumentation and clinical experience needed to maintain anesthesia in hypoxic large animals with sudden hemodynamic changes. Therefore, only investigators with experience in large animal research and intensive care medicine should work with this model or at least closely supervise unexperienced researchers.
There are, however, distinct advantages to this model. It produces the basic pathologic changes of human ARDS &#8211; inflammatory lung injury with permeability changes, impairment in gas exchange and lung mechanics &#8211; very well and with good reproducibility7,31. This makes it superior to other models that usually lack one or more of the pathologic effects. Surfactant depletion by lavage induces only little alveolar epithelial changes7,19 and lipopolysaccharide administration, a sepsis model, usually induces only minimal changes of the alveolo-capillary barrier7. Oleic acid injection is feasible in large and small animals, so it can be used in various laboratories that use animal models8,9,10,12. Third, it not only mimics the early phase of ARDS, but also the later phases with deposition of fibrin on the alveolar surface16. Furthermore, when using large animals, it is possible to use extended clinical monitoring and instrumentation that is not fully available in small animals. This resembles the situation of a bedside setting which intensive care physicians are used to, thus allowing easier access for clinicians to this method and facilitating faster implementation in treatment algorithms.

The acute respiratory distress syndrome (ARDS) is an intensive care syndrome that has been extensively studied since its first description about 50 years ago1. This body of research led to a better understanding of the pathophysiology and causes the development of ARDS resulting in improved patient care and outcome2,3. Nevertheless, the mortality in patients suffering from ARDS remains very high with about 35-40%4,5,6. The fact that about 10% of ICU admissions and 23% of ICU patients who require mechanical ventilation is due to ARDS underscores the relevance for further research in this field.
Animal models are widely used in research to examine pathophysiologic changes and potential treatment modalities for different kinds of diseases. Due to the complexity of ARDS, there is no single animal model to mimic this disease, but different models representing different aspects7. One well-established model is oleic acid injection (OAI)-induced lung injury. This model has been used in a wide array of animals, including mice8, rats9, pigs10, dogs11, and sheep12. Oleic acid is an unsaturated fatty acid and the most common fatty acid in the body of healthy humans13. It is present in human plasma, cell membranes, and adipose tissue13. Physiologically, it is bound to albumin while it is carried through the bloodstream13. Increased levels of fatty acids in the blood stream are associated with different pathologies and the severity of some diseases correlates with serum fatty acid levels13. The oleic acid ARDS-model was developed in an attempt to reproduce ARDS caused by lipid embolism as seen in trauma patients14. Oleic acid has direct effects on innate immune receptors in the lungs13 and triggers neutrophil accumulation15, inflammatory mediator production16, and cell death13. Physiologically, oleic acid induces rapidly progressing hypoxemia, increase in pulmonary arterial pressure and accumulation of extravascular lung water. Furthermore, it induces arterial hypotension and myocardial depression7. The disadvantages of this model are the necessity for central venous access, the questionable mechanistic relevance and the potential lethal progress caused by rapid hypoxemia and cardiac depression. The advantage of this model compared to other models is the usability in small and large animals, the valid reproducibility of the pathophysiological mechanisms in ARDS, the acute onset of ARDS after injection of oleic acid, and the possibility to study isolated ARDS without systemic inflammation like in many other sepsis models7. In the following article, we give a detailed description of the oleic acid-induced lung injury in pigs and provide representative data to characterize the stability of the compromises in lung function. There are different protocols for OAI-induced lung injury. The protocol provided here is able to reliably induce acute lung injury.

<table><tbody><tr><td>3-way-stopcock blue</td><td>Becton Dickinson Infusion Therapy AB Helsingborg, Sweden</td><td>394602</td><td></td></tr><tr><td>3-way-stopcock red</td><td>Becton Dickinson Infusion Therapy AB Helsingborg, Sweden</td><td>394605</td><td></td></tr><tr><td>Atracurium</td><td>Hikma Pharma GmbH , Martinsried</td><td>4262659</td><td></td></tr><tr><td>Canula 20 G</td><td>Becton Dickinson S.A. Carretera Mequinenza Fraga, Spain</td><td>301300</td><td></td></tr><tr><td>Datex Ohmeda S5</td><td>GE Healthcare Finland Oy, Helsinki, Finland</td><td></td><td></td></tr><tr><td>Desinfection</td><td>Sch&amp;uuml;lke &amp;amp; Mayr GmbH, Germany</td><td>104802</td><td></td></tr><tr><td>Endotracheal tube</td><td>Teleflex Medical Sdn. Bhd, Malaysia</td><td>112482</td><td></td></tr><tr><td>Endotracheal tube introducer</td><td>R&amp;uuml;sch</td><td>5033062</td><td></td></tr><tr><td>Engstr&amp;ouml;m Carestation</td><td>GE Heathcare, Madison USA</td><td></td><td></td></tr><tr><td>Fentanyl</td><td>Janssen-Cilag GmbH, Neuss</td><td></td><td></td></tr><tr><td>Gloves</td><td>Paul Hartmann, Germany</td><td>9422131</td><td></td></tr><tr><td>Incetomat-line 150 cm</td><td>Fresenius, Kabi Germany GmbH</td><td>9004112</td><td></td></tr><tr><td>Ketamine</td><td>Hameln Pharmaceuticals GmbH</td><td></td><td></td></tr><tr><td>Laryngoscope</td><td>Teleflex Medical Sdn. Bhd, Malaysia</td><td>671067-000020</td><td></td></tr><tr><td>Logical pressure monitoring system</td><td>Smith- Medical Germany GmbH</td><td>MX9606</td><td></td></tr><tr><td>Logicath 7 Fr 3-lumen 30cm</td><td>Smith- Medical Germany GmbH</td><td>MXA233x30x70-E</td><td></td></tr><tr><td>Masimo Radical 7</td><td>Masimo Corporation Irvine, Ca 92618 USA</td><td></td><td></td></tr><tr><td>Mask for ventilating dogs</td><td>Henry Schein, Germany</td><td>730-246</td><td></td></tr><tr><td>Neofox Kit</td><td>Ocean optics Largo, FL USA</td><td>NEOFOX-KIT-PROBE</td><td></td></tr><tr><td>Norepinephrine</td><td>Sanofi- Aventis, Seutschland GmbH</td><td>73016</td><td></td></tr><tr><td>Oleic acid</td><td>Applichem GmbH Darmstadt, Germany</td><td>1,426,591,611</td><td></td></tr><tr><td>Original Perfusor syringe 50ml Luer Lock</td><td>B.Braun Melsungen AG, Germany</td><td>8728810F</td><td></td></tr><tr><td>PA-Katheter Swan Ganz 7,5 Fr 110cm</td><td>Edwards Lifesciences LLC, Irvine CA, USA</td><td>744F75</td><td></td></tr><tr><td>Percutaneous sheath introducer set 8,5 und 9 Fr, 10 cm with integral haemostasis valve/sideport</td><td>Arrow international inc. Reading, PA, USA</td><td>AK-07903</td><td></td></tr><tr><td>Perfusor FM Braun</td><td>B.Braun Melsungen AG, Germany</td><td>8713820</td><td></td></tr><tr><td>Potassium chloride</td><td>Fresenius, Kabi Germany GmbH</td><td>6178549</td><td></td></tr><tr><td>Propofol 2%</td><td>Fresenius, Kabi Germany GmbH</td><td></td><td></td></tr><tr><td>Saline</td><td>B.Braun Melsungen AG, Germany</td><td></td><td></td></tr><tr><td>Sonosite Micromaxx Ultrasoundsystem</td><td>Sonosite Bothell, WA, USA</td><td></td><td></td></tr><tr><td>Stainless Macintosh Size 4</td><td>Teleflex Medical Sdn. Bhd, Malaysia</td><td>670000</td><td></td></tr><tr><td>Sterofundin</td><td>B.Braun Melsungen AG, Germany</td><td></td><td></td></tr><tr><td>Stresnil 40mg/ml</td><td>Lilly Germany GmbH, Abteilung Elanco Animal Health</td><td></td><td></td></tr><tr><td>Syringe 10 mL</td><td>Becton Dickinson S.A. Carretera Mequinenza Fraga, Spain</td><td>309110</td><td></td></tr><tr><td>Syringe 2 mL</td><td>Becton Dickinson S.A. Carretera Mequinenza Fraga, Spain</td><td>300928</td><td></td></tr><tr><td>Syringe 20 mL</td><td>Becton Dickinson S.A. Carretera Mequinenza Fraga, Spain</td><td>300296</td><td></td></tr><tr><td>Syringe 5 mL</td><td>Becton Dickinson S.A. Carretera Mequinenza Fraga, Spain</td><td>309050</td><td></td></tr><tr><td>venous catheter 22G</td><td>B.Braun Melsungen AG, Germany</td><td>4269110S-01</td><td></td></tr></tbody></table>

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.

PaO2/FiO2-ratio decreases after fractionated application of oleic acid (Figure 1). In the presented study, 0.185 &#177; 0.01 ml kg-1 oleic acid was necessary for the induction of lung injury. All animals showed an impaired oxygenation after the induction of lung injury, with varieties in the further time course. In animal 1 and 3, it remained at one level with little fluctuations; in animal 2, we observe an initial increase, followed by a decrease at the end, while animal 4 shows a constant rise. Nevertheless, we find a marked impairment in oxygenation in all 4 animals after 6 h. Therefore, it is necessary to closely monitor PaO2/FiO2-ratio while inducing the lung injury. We use an ultrafast pO2-measurement probe to monitor the decrease in PaO2 in real time21. A different option is to take regular arterial blood gas samples from the time the SpO2 starts dropping. In vehicle-treated animals (5 and 6), there is no decrease in PaO2/FiO2-ratio.
The decrease in PaO2/FiO2-ratio is paralleled by an increase in pulmonary arterial pressure (PAP), which usually remains elevated for the rest of the experiment (Figure 2). Similar to PaO2/FiO2-ratio, it sometimes fluctuates a bit. In one animal (animal 3), MPAP stayed at this level afterwards; in two animals (animal 1 and 4), it fell a little; in one animal (animal 2), it initially fell to rise afterwards. In vehicle-treated animals (5 and 6), MPAP didn&#39;t change during the experiment.
Lung injury is also visually detectable in lungs taken out after the death of the animal. Figure 3 shows representative lungs of a pig with OAI-induced lung injury after the euthanasia. In histologic slices, processed according to earlier publications22, alveolar edema and hemorrhage are visible (Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57783/57783fig1.jpg" /> 
Figure 1:&#160;Development of PaO2/FiO2-Ratio during 6 h after the injection of oleic acid in 4 exemplary pigs and 2 pigs treated with vehicle. (A). Representative plots showing stable values with little fluctuations (animals 1 and 3), initial rise followed by a decrease (animal 2) or continuous rise (animal 4). Vehicle treated pigs (animals 5 and 6) show little variation over time. (B). Mean and standard deviation for all animals. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57783/57783fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57783/57783fig2.jpg" /> 
Figure 2: Development of mean pulmonary artery pressure (MPAP) during 6 h after injection of oleic acid in 4 exemplary pigs and 2 pigs treated with vehicle. (A). Representative plots showing an initial rise in all 4 animals. In one animal (animal 3), MPAP stayed at this level afterwards; in two animals (animal 1 and 4), it fell a little; in one animal (animal 2), it initially fell to rise afterwards. Vehicle treated pigs (animals 5 and 6) show little variation over time. (B). Mean and standard deviation for all animals. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57783/57783fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57783/57783fig3.jpg" /> 
Figure 3: Lungs after the injection of oleic acid. Photo of lungs 6 h after the injection of oleic acid. Hemorrhagic areas are visible. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57783/57783fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57783/57783fig4.jpg" /> 
Figure 4: Histologic images of lung injury after the oleic acid injection. The lungs were fixed in 10% formalin for paraffin sectioning and haematoxylin/eosin staining. Image magnification: 10X. (A). Alveolar edema. (B). Hemorrhage. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57783/57783fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td></td>
			<td>Animal 1</td>
			<td>Animal 2</td>
			<td>Animal 3</td>
			<td>Animal 4</td>
			<td>Animal 5</td>
			<td>Animal 6</td>
		</tr>
		<tr>
			<td>Body weight [kg]</td>
			<td>27</td>
			<td>28</td>
			<td>27</td>
			<td>27</td>
			<td>27</td>
			<td>29</td>
		</tr>
		<tr>
			<td>Right upper lobe wet [g]</td>
			<td>96</td>
			<td>83</td>
			<td>116</td>
			<td>116</td>
			<td>60</td>
			<td>44</td>
		</tr>
		<tr>
			<td>Right upper lobe dry [g]</td>
			<td>14</td>
			<td>13</td>
			<td>13</td>
			<td>11</td>
			<td>11</td>
			<td>9</td>
		</tr>
		<tr>
			<td>Wet-to-dry</td>
			<td>6,9</td>
			<td>6,4</td>
			<td>8,9</td>
			<td>10,5</td>
			<td>5,5</td>
			<td>4,9</td>
		</tr>
	</tbody>
</table>
Table 1: This table shows the weight of the animals, wet weight, dry weight and wet-to-dry-ratio of the right upper lobe of the animals&#39; lungs.

Watch this Scientific Journal Video about Oleic Acid-Injection in Pigs As a Model for Acute Respiratory Distress Syndrome at JoVE.com

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

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 ...

oleic-acid-injection-pigs-as-model-for-acute-respiratory-distress

Nanyang Technological University

Research

JoVE Journal

Medicine

9.8K Views.  University Medical Center of the Johannes Gutenberg-University. 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).

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

Bronchoalveolar Lavage (BAL) for Research; Obtaining Adequate Sample Yield

We describe a research technique for fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) using manual hand held suction in order to remove nonadherent cells and lung lining fluid from the mucosal surface. In research environments, BAL allows sampling of innate (lung macrophage), cellular (B- and T- cells), and humoral (immunoglobulin) responses within the lung.
BAL is internationally accepted for research purposes and since 1999 the technique has been performed in &#62; 1,000 subjects in the UK and Malawi by our group.
Our technique uses gentle hand-held suction of instilled fluid; this is designed to maximize BAL volume returned and apply minimum shear force on ciliated epithelia in order to preserve the structure and function of cells within the BAL fluid and to preserve viability to facilitate the growth of cells in ex vivo culture. The research technique therefore uses a larger volume instillate (typically in the order of 200 ml) and employs manual suction to reduce cell damage.
Patients are given local anesthetic, offered conscious sedation (midazolam), and tolerate the procedure well with minimal side effects. Verbal and written subject information improves tolerance and written informed consent is mandatory. Safety of the subject is paramount. Subjects are carefully selected using clear inclusion and exclusion criteria.
This protocol includes a description of the potential risks, and the steps taken to mitigate them, a list of contraindications, pre- and post-procedure checks, as well as precise bronchoscopy and laboratory techniques.

Bronchoalveolar Lavage BAL for Research; Obtaining Adequate Sample Yield

We describe a research technique for fiberoptic bronchoscopy and bronchoalveolar lavage using low pressure suction. The technique is used to harvest immune cells from the lung bronchoalveolar surfaces. Local anesthetic and mild conscious sedation (midazolam) is used. Subjects tolerate the procedure well and experience minimal side effects.

We describe a research technique for fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) using manual hand held suction in order to remove ...

A Research Method For Detecting Transient Myocardial Ischemia In Patients With Suspected Acute Coronary Syndrome Using Continuous ST-segment Analysis

Each year, an estimated 785,000 Americans will have a new coronary attack, or acute coronary syndrome (ACS). The pathophysiology of ACS involves rupture of an atherosclerotic plaque; hence, treatment is aimed at plaque stabilization in order to prevent cellular death. However, there is considerable debate among clinicians, about which treatment pathway is best: early invasive using percutaneous coronary intervention (PCI/stent) when indicated or a conservative approach (i.e., medication only with PCI/stent if recurrent symptoms occur).
There are three types of ACS: ST elevation myocardial infarction (STEMI), non-ST elevation MI (NSTEMI), and unstable angina (UA). Among the three types, NSTEMI/UA is nearly four times as common as STEMI. Treatment decisions for NSTEMI/UA are based largely on symptoms and resting or exercise electrocardiograms (ECG). However, because of the dynamic and unpredictable nature of the atherosclerotic plaque, these methods often under detect myocardial ischemia because symptoms are unreliable, and/or continuous ECG monitoring was not utilized.
Continuous 12-lead ECG monitoring, which is both inexpensive and non-invasive, can identify transient episodes of myocardial ischemia, a precursor to MI, even when asymptomatic. However, continuous 12-lead ECG monitoring is not usual hospital practice; rather, only two leads are typically monitored. Information obtained with 12-lead ECG monitoring might provide useful information for deciding the best ACS treatment.
Purpose. Therefore, using 12-lead ECG monitoring, the COMPARE Study (electroCardiographic evaluatiOn of ischeMia comParing invAsive to phaRmacological trEatment) was designed to assess the frequency and clinical consequences of transient myocardial ischemia, in patients with NSTEMI/UA treated with either early invasive PCI/stent or those managed conservatively (medications or PCI/stent following recurrent symptoms). The purpose of this manuscript is to describe the methodology used in the COMPARE Study.
Method. Permission to proceed with this study was obtained from the Institutional Review Board of the hospital and the university. Research nurses identify hospitalized patients from the emergency department and telemetry unit with suspected ACS. Once consented, a 12-lead ECG Holter monitor is applied, and remains in place during the patient's entire hospital stay. Patients are also maintained on the routine bedside ECG monitoring system per hospital protocol. Off-line ECG analysis is done using sophisticated software and careful human oversight.

Continuous 12-lead electrocardiographic (ECG) monitoring can identify transient myocardial ischemia, even when asymptomatic, among patients with suspected acute coronary syndrome (ACS). In this article we describe our method for initiating patient monitoring using a Holter device, downloading the ECG data for off-line analysis, and how to utilize the ECG software to identify transient ischemia.

Each year, an estimated 785,000 Americans will have a new coronary attack, or acute coronary syndrome (ACS). The pathophysiology of ACS involves rupture ...

Sublingual Immunotherapy as an Alternative to Induce Protection Against Acute Respiratory Infections

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been shown to effectively induce humoral and cellular responses at systemic and mucosal sites, namely the lungs and urogenital tract. Sublingual vaccination can promote protection against infections at the lower and upper respiratory tract; it can also promote tolerance to allergens and ameliorate asthma symptoms. Modulation of lung&#8217;s immune response by sublingual immunotherapy (SLIT) is safer than direct administration of formulations by intranasal route because it does not require delivery of potentially harmful molecules directly into the airways. In contrast to intranasal delivery, side effects involving brain toxicity or facial paralysis are not promoted by SLIT. The immune mechanisms underlying SLIT remain elusive and its use for the treatment of acute lung infections has not yet been explored. Thus, development of appropriate animal models of SLIT is needed to further explore its potential advantages. 
This work shows how to perform sublingual administration of therapeutic agents in mice to evaluate their ability to protect against acute pneumococcal pneumonia. Technical aspects of mouse handling during sublingual inoculation, precise identification of sublingual mucosa, draining lymph nodes and isolation of tissues, bronchoalveolar lavage and lungs are illustrated. Protocols for single cell suspension preparation for FACS analysis are described in detail. Other downstream applications for the analysis of the immune response are discussed. Technical aspects of the preparation of Streptococcus pneumoniae inoculum and intranasal challenge of mice are also explained.
SLIT is a simple technique that allows screening of candidate molecules to modulate lungs&#8217; immune response. Parameters affecting the success of SLIT are related to molecular size, susceptibility to degradation and stability of highly concentrated formulations.

The present work illustrates the convenience of using sublingual immunotherapy to boost the innate immune response in the lungs and confer protection against acute pneumococcal pneumonia in mouse.

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been ...

Lavage-induced Surfactant Depletion in Pigs As a Model of the Acute Respiratory Distress Syndrome (ARDS)

Various animal models of lung injury exist to study the complex pathomechanisms of human acute respiratory distress syndrome (ARDS) and evaluate future therapies. Severe lung injury with a reproducible deterioration of pulmonary gas exchange and hemodynamics can be induced in anesthetized pigs using repeated lung lavages with warmed 0.9% saline (50 ml/kg body weight). Including standard respiratory and hemodynamic monitoring with clinically applied devices in this model allows the evaluation of novel therapeutic strategies (drugs, modern ventilators, extracorporeal membrane oxygenators, ECMO), and bridges the gap between bench and bedside. Furthermore, induction of lung injury with lung lavages does not require the injection of pathogens/endotoxins that impact on measurements of pro- and anti-inflammatory cytokines. A disadvantage of the model is the high recruitability of atelectatic lung tissue. Standardization of the model helps to avoid pitfalls, to ensure comparability between experiments, and to reduce the number of animals needed.

Lavage-induced Surfactant Depletion in Pigs As a Model of the Acute Respiratory Distress Syndrome ARDS

Repeated pulmonary lavages in anesthetized pigs induce lung injury resembling major aspects of human acute respiratory distress syndrome (ARDS). For this purpose the lungs are repeatedly lavaged with 0.9% saline at 37 &#176;C. The goal of the protocol is a reproducible mitigation of gas exchange and hemodynamics for research in ARDS.

Various animal models of lung injury exist to study the complex pathomechanisms of human acute respiratory distress syndrome (ARDS) and evaluate future ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

A Component-resolved Diagnostic Approach for a Study on Grass Pollen Allergens in Chinese Southerners with Allergic Rhinitis and/or Asthma

Sensitization to grass pollen imposes a global risk for allergic airway diseases. Although prevention relies on local investigation of the pollen allergens, data on this topic are limited in southern China. Any available data were obtained by self-report questionnaires, skin prick tests, and total or specific IgE tests using crude extracts. For many reasons, these methods are unreliable. Serum sIgE reactivity to Bermuda grass, Timothy grass, and Humulus scandens allergens in a cohort of patients from Greater Guangzhou (southern China&#39;s largest city and its outskirts) with allergic rhinitis and/or asthma were examined using a fully-automated immunoassay analyzer as a component-resolved diagnostics (CRD) tool.
For the first time, a considerably high prevalence of Bermuda grass sIgE positivity was demonstrated in Chinese southerners with allergic rhinitis and/or asthma. In these patients, a subtle prevalence of sensitization to Timothy grass and Humulus scandens was also noted, which may arise from cross-reactivity, as the latter two are not common in the region. This was also supported by the detection of allergen components.
Fully-automated immunoassay analyzers may offer satisfactory consistency between regions, laboratories, and institutions and over time. The automaticity of the instrument may enable a standardized detection that would not have been readily revealed before the advent of CRD.
This is a study that uses a CRD approach to investigate sensitization to grass pollen allergens in southern China. It adds to current evidence in the literature. Future studies are needed to validate these findings. However, although CRD is a useful tool, the findings made with the fully-automated immunoassay analyzer should not substitute for other laboratory investigations, clinical evaluations, and physician expertise.

This work describes a protocol that uses a component-resolved approach to study sensitization to grass pollen allergens in a cohort of patients from southern China with allergic rhinitis and/or asthma.

Sensitization to grass pollen imposes a global risk for allergic airway diseases. Although prevention relies on local investigation of the pollen ...

Mouse Model of Oleic Acid-Induced Acute Respiratory Distress Syndrome

Acute respiratory distress syndrome (ARDS) is a significant threat to critically ill patients with a high fatality rate. Pollutant exposure, cigarette smoke, infectious agents, and fatty acids can induce ARDS. Animal models can mimic the complex pathomechanism of the ARDS. However, each of them has limitations. Notably, oleic acid (OA) is increased in critically ill patients with harmful effects on the lung. OA can induce lung injury by emboli, disrupting tissue, altering pH, and impairing edema clearance. OA-induced lung injury model resembles various features of ARDS with endothelial injury, increased alveolar permeability, inflammation, membrane hyaline formation, and cell death. Herein, induction of lung injury is described by injecting OA (in salt form) directly into the lung and intravenously in a mouse since it is the physiological form of OA at pH 7. Thus, the injection of OA in the salt form is a helpful animal model to study lung injury/ARDS without causing emboli or altering the pH, thereby getting close to what is happening in critically ill patients.

The present protocol describes a lung injury model in mice using oleic acid to mimic acute respiratory distress syndrome (ARDS). This model increases the inflammatory mediators on edema and decreases lung compliance. Oleic acid is used in the salt form (oleate) since this physiological form avoids the risk of embolism.

Acute respiratory distress syndrome (ARDS) is a significant threat to critically ill patients with a high fatality rate. Pollutant exposure, cigarette ...

Immunology and Infection

Bronchoalveolar Lavage and Oleic Acid-Injection in Pigs as a Double-Hit Model for Acute Respiratory Distress Syndrome (ARDS)

The treatment of ARDS continues to pose major challenges for intensive care physicians in the 21st century with mortality rates still reaching up to 50% in severe cases. Further research efforts are needed to better understand the complex pathophysiology of this disease. There are different well-established animal models to induce acute lung injury but none has been able to adequately mimic the complex pathomechanisms of ARDS. The most crucial factor for the development of this condition is the damage to the alveolar capillary unit. The combination of two well-established lung injury models allow us to mimic in more detail the underlying pathomechanism. Bronchoalveolar lavage (BAL) leads to surfactant depletion as well as alveolar collapse. The repeated instillation of fluid volumes causes subsequent hypoxemia. Surfactant depletion is a key factor of ARDS in humans. BAL is often combined with other lung injury approaches, but not with a second hit followed by oleic acid injection (OAI) yet. Oleic acid injection leads to severely impaired gas exchange, a deterioration of lung mechanics and disruption of the alveolo-capillary barrier. The OAI mimics most of the expected effects of ARDS consisting of extended inflammation of lung tissue with an increase of alveolar leakage and gas exchange impairment. A disadvantage of the combination of different models is the difficulty to determine the influence to the lung injury caused by BAL alone, OAI alone or both together. The model presented in this report represents the combination of BAL and OAI as a new double-hit lung injury model. This new model is easy to implement and an alternative to study different therapeutic approaches in ARDS in the future.

Bronchoalveolar Lavage and Oleic Acid-Injection in Pigs as a Double-Hit Model for Acute Respiratory Distress Syndrome ARDS

Different, complex animal models exist to study the pathophysiology of acute respiratory distress syndrome (ARDS). Bronchoalveolar lavage and oleic acid injection induced lung injury is suitable as a new double-hit animal model for studying the acute respiratory distress syndrome.

The treatment of ARDS continues to pose major challenges for intensive care physicians in the 21st century with mortality rates still reaching up to 50% ...

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 ...

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.