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 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.
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.
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
2. Instrumentation
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.
4. INSERTING PULMONARY ARTERY CATHETER
5. Induction of Lung Injury
6. End of Experiment and Euthanasia
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, 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'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).
Figure 1: 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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
Figure 3: Lungs after the injection of oleic acid. Photo of lungs 6 h after the injection of oleic acid. Hemorrhagic areas are visible. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
Animal 1 | Animal 2 | Animal 3 | Animal 4 | Animal 5 | Animal 6 | |
Body weight [kg] | 27 | 28 | 27 | 27 | 27 | 29 |
Right upper lobe wet [g] | 96 | 83 | 116 | 116 | 60 | 44 |
Right upper lobe dry [g] | 14 | 13 | 13 | 11 | 11 | 9 |
Wet-to-dry | 6,9 | 6,4 | 8,9 | 10,5 | 5,5 | 4,9 |
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' lungs.
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 ≥ 5 cm 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 – inflammatory lung injury with permeability changes, impairment in gas exchange and lung mechanics – 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 authors have nothing to disclose.
The authors want to thank Dagmar Dirvonskis for excellent technical support.
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 |