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

Summary

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).

Abstract

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.

Introduction

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 outcome^2,3. 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.

Protocol

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

1. Withhold food for 6 h before anesthesia to reduce the risk of aspiration but allow free access to water to reduce stress.
2. 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.
3. Insert peripheral vein catheter (20 G) in an ear vein after local disinfection with alcohol.
4. Inject fentanyl (4 µg kg^-1), propofol (3 mg kg^-1) and atracurium (0.5 mg kg^-1) intravenously for the induction of anesthesia.
5. When the pig stops breathing, place it in supine position on the stretcher and immobilize it with bandages.
6. Start monitoring the peripheral oxygen saturation (SpO₂) by clipping the sensor on to one of the ears or the tail of the animal.
7. 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.
8. 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.
9. 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.
10. Person 1: Pull out the tongue with one hand and press the snout dorsally with the other.
11. Person 2: Insert laryngoscope and advance it as usual until the epiglottis comes into view.
12. Pull the laryngoscope ventrally to visualize the vocal cords.
    NOTE: Sometimes the epiglottis "sticks" to the soft palatine. In this case, mobilize it with the tip of the tube.
13. Insert the tube through the vocal cords and pull out the introducer.
14. Block the cuff of the tube with a syringe with 10 mL of air.
15. Connect the tube to the ventilator.
16. 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.
17. Start mechanical ventilation (tidal volume 6-8 mL/kg, positive PEEP 5 cm H₂0, FiO₂ to keep peripheral oxygen saturation (SpO₂) between 94 – 98%¹⁷, respiratory rate to keep end tidal pressure of carbon dioxide (etCO₂) between 35 – 45 mmHG).

2. Instrumentation

1. Retract the hindlegs with bandages to stretch the skin above the femoral area for catheterizing necessary vessels.
2. Prepare a 5 mL syringe, a 10 mL syringe, a Seldinger'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).
3. Generously disinfect the femoral area with skin disinfectant applying a wipe down technique.
4. Completely fill the catheters with saline.
5. Place the ultrasound-probe on the right inguinal ligament and scan for femoral vessels.
6. Turn the probe 90° to fully visualize the femoral artery in the long axis.
7. Cannulate right femoral artery under in-line ultrasound visualization with the Seldinger'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.
8. When pulsating bright blood flows out, introduce the guidance wire and retract the needle.
9. Visualize the femoral vein and cannulate the vein under in-line ultrasound visualization and continuous aspiration with the needle.
10. When venous blood is aspirable, disconnect the syringe and insert the guidance wire.
11. Retract the needle.
12. Check the position of the wires with ultrasound.
13. Insert the arterial introducer sheath (5 Fr) and central venous catheter using Seldinger's technique (for details on Seldinger's technique, refer to published method¹⁸).
14. Repeat the arterial and venous puncture on other side and insert the introducer sheaths using Seldinger´s technique as described above (artery 6 Fr, vein 8 Fr).
15. Connect the arterial introducer sheath and central venous catheter to a transducer system suitable to the monitoring equipment.
16. Calibrate the invasive monitoring against atmosphere (zero) by opening the three-way-stopcocks to the atmosphere and press Zero all on the monitor.
17. Turn the three-way-stopcocks back to measure hemodynamics.
18. Start monitoring hemodynamics.
19. Place all pressure transducers at the height of the right atrium.
20. 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.

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.

1. Open software NeoFox viewer and click Options.
2. Choose the Calibration tab and click the Open Calibration button.
3. Choose calibration file and click Open and Download.
4. Confirm the pop-up window by clicking Yes.
5. Open the Options dialogue.
6. Choose the Calibration tab and click Single point calibration.
7. Enter 21% in the field Oxygen and the temperature in the field Temperature.
8. Click Use current Tau and Download. Afterwards, confirm the pop-up window by clicking Yes.
9. Insert the probe for ultrafast measurements of pO₂ through the left arterial introducer sheath.

4. INSERTING PULMONARY ARTERY CATHETER

1. Check the balloon of pulmonary artery catheter for damage.
2. Connect to the transducer system suitable to the monitoring equipment.
3. 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.
4. Turn the three-way-stopcock back to measure pulmonary arterial pressure.
5. Start monitoring the pulmonary arterial pressure.
6. Insert the pulmonary artery catheter through the left venous introducer sheath (balloon deflated).
7. When the pulmonary artery catheter has passed through the introducer sheath, inflate the balloon with 1 mL of air.
8. 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.

5. Induction of Lung Injury

1. 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.
2. Take 2 mL of blood in another 20 mL syringe and add saline to a total volume of 20 mL in both syringes.
3. Connect the second syringe also to the 3-way-stopcock.
   CAUTION: Use gloves and eye protection when working with oleic acid.
4. Prepare norepinephrine (0.1 mg/mL) for continuous infusion and for bolus injection (10 µg/mL).
5. Connect the norepinephrine syringe pump to one of the ports of the central venous catheter without starting it.
6. Start the ultrafast pO₂-measurement.
7. Before the induction of lung injury, record the values (baseline) from all relevant parameters.
8. Set the F_iO₂ to 1.0 and conduct a lung recruitment maneuver (plateau pressure 40 cm H₂O for 10 s).
9. Connect the 3-way-stopcock to the proximal port of the pulmonary artery catheter.
10. 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.
11. 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.
12. Closely monitor the hemodynamics after the injection of oleic acid and keep the norepinephrine at hand. If necessary, give norepinephrine as bolus injection (10 – 100 µg) or continuous infusion to keep mean arterial pressure above 60 mmHg.
13. 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.
14. 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.
15. 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.
16. If PaO₂/FiO₂-ratio is between 100 and 200 mmHg, wait for 30 min and check again.
17. 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.
18. Set the ventilation according to the suggestions from the ARDS network²⁰.

6. End of Experiment and Euthanasia

1. 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.

Results

PaO₂/FiO₂-ratio decreases after fractionated application of oleic acid (Figure 1). In the presented study, 0.185 ± 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, f...

Discussion

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 emulsion^{23,24,25,26,27,28,29}. Our method offers a ...

Materials

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