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 °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 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.
The mortality of human acute respiratory distress syndrome (ARDS) remains high with values between 40 and 50% 1 despite more than 4 decades of intense research. Animal models of lung injury play a major role in investigating the complex pathomechanisms or novel therapeutical approaches to reduce mortality and limit long term disabilities.
Various models have been established to induce lung injury that simulates aspects of human ARDS in either large (e.g. pigs) or small animals (e.g. rodents). Methods differ strongly, including pulmonary arterial infusion of oleic acid, intravenous (i.v.) infusion of bacteria, and endotoxins or cecal ligation and puncture (CLP) models causing sepsis-induced ARDS. In addition, direct lung injuries due to large tidal volumes and high peak inspiratory pressures (ventilator-induced lung injury; VILI), smoke/burn injuries or lung ischemia/reperfusion (I/R) models are often used 2. One major disadvantage of CLP models, as well as models working with endotoxins, is the underlying inflammation which hinders the analysis of biotrauma caused by lung injury alone. Furthermore, it may take hours to days to result in lung injury, as is the case for VILI in large animals.
The induction of lung injury by surfactant washout with repeated lung lavages, as it was first described by Lachmann et al. in guinea pigs 3, is a time efficient method to induce lung injury with reproducible functional and mechanical compromises, as well as changes in pulmonary vascular resistance. The adaption of this model to mechanically ventilated pigs of about 30-60 kg body weight supports basic research with clinically used mechanical ventilators, catheters and monitors, while the compromises in gas exchange and hemodynamics are highly reproducible at the same time 4. In addition, induction of lung injury by lavages does not require specific equipment that is not commonly available in respiratory laboratories designed for experiments in large animals. The model presented in this article is suitable for research demanding equipment (e.g. ventilators) that is designed for use in humans, and furthermore ensures a high reproducibility in the occurring deteriorations in lung function. Standardization of this model helps to ensure comparability between experiments and reduce the number of animals needed. The potential recruitability of atelectatic lung regions with deliberate or unknown recruitment maneuvers is a severe limitation of this specific model. In the following article we give a detailed description of the lavage model for the induction of lung injury and provide representative data to characterize the stability of the compromises in lung function.
The experiments were conducted at the Department of Experimental Medicine, Charité – Universitätsmedizin, Berlin, Germany (certified according to the EN DIN ISO 9001:2000), and were approved by the federal authorities for animal research in Berlin, Germany prior to the experiments. The principles of laboratory animal care, which were used in all experiments, were in accordance with the guidelines of the European and German Society of Laboratory Animal Sciences.
1. Animal Welfare and Laboratory Animals
2. Anesthesia, Intubation, and Mechanical Ventilation
3. Instrumentation Techniques
4. Introduction of the Pulmonary Artery Catheter
5. Pulmonary Artery Thermodilution Technique and Hemodynamic Measurements
6. Lung Lavages to Induce Lung Injury
7. End of Experiment and Euthanasia
PaO2/FIO2-ratio decreases during lung lavages, but the exact impact of a single lavage is difficult to predict. We start to take arterial blood gas samples from the third lavage onwards to detect a decrease in PaO2/FIO2-ratio below 100 mmHg. Once a decrease in PaO2/FIO2-ratio below 100 mmHg is achieved, we require this ratio to remain below 100 mmHg for one hour at a PEEP ≥ 5 cm H2O. This ensures the induction of lung injury, which will formally meet the Berlin definition of ARDS. The concomitant changes in blood gases and hemodynamics will remain 'stable' for hr, deteriorate further, or even improve depending on the ventilator settings (Figure 5). In the case that the PaO2/FIO2-ratio does increase above 100 mmHg during the one hour baseline period, further lavages are performed as described above to prevent spontaneous recovery of the animal during the time course of the experiment (Figure 5). PAP increases with each lavage due to increasing atelectatic regions of the lungs, hypercapnia and hypoxemia (Figure 5). PAP values usually increase two- to triple-fold, but can increase above 60-70 mmHg during a single lavage. This may result in sudden hemodynamic decompensation and death of the animal. Overall death rate of this model averages 10-15%.
Figure 1: Instrument Table for Introducing a Central Venous Catheter and an Introducer Sheath by Seldinger Technique after a Cut Down Procedure. Note, do not use a vasodilator for direct cannulization of a blood vessel. PAC means pulmonary artery catheter. Please click here to view a larger version of this figure.
Figure 2: Pulmonary Artery Catheter. Please click here to view a larger version of this figure.
Figure 3: Pulmonary Artery Catheter with Inflated Balloon. Please click here to view a larger version of this figure.
Figure 4: Schematic Sketch of the Waveforms Visible while Advancing a Pulmonary Artery Catheter. The sketch depicts which waveform can be usually seen at which insertion depth of the catheter in pigs of about 40 kg body weight. PCWP means pulmonary capillary wedge pressure. Please click here to view a larger version of this figure.
Figure 5: Individual Measured Values for PaO2/FIO2 Ratio and Mean Pulmonary Arterial Pressure (MPAP) of Three Pigs. PaO2 means partial arterial pressure of oxygen, FIO2 means fraction of inspired oxygen. The data was recorded during workshops at our institution. Note, that the PaO2/FIO2 ratio increases after lung lavages in one animal, whereas it remains below 100 mmHg in the other two. Thus, this animal should have received further lavages as described in the article. Please click here to view a larger version of this figure.
This article describes a step by step instruction to induce severe lung injury in pigs due to surfactant washout by repeated lung lavages. This specific method enables a reproducible and comparable deterioration in lung function and pulmonary vascular resistance. It is imperative to lavage the pigs until the PaO2/FIO2 ratio decreased below 100 mmHg and stays below 100 mmHg for one hr. Once, this is achieved the animals usually do not recover from the lung injury for at least 4 to 8 hr as long as no recruitment maneuvers are conducted 4,8. Adherence to this protocol helps to increase the comparability between the findings from different experiments using the same animal model.
The induction of lung injury with lavages has several limitations. First, repeated lavages result in some of the histopathological properties of human ARDS including the formation of major atelectasis, perivascular edema formation and an increase of the alveolar-capillary membrane thickness. Yet, some important features like severe epithelial damage or the formation of hyaline membranes are not found in this model 2,9.
Second, the recruitment effect of high inspiratory pressures and increased PEEP seem to be higher in lavage-induced lung injury in dogs than in lung injury induced by the infusion of oleic acid or intratracheal installation of E. coli (pneumonia model) 10. Thus, lavage models may be a quick, suitable method to test e.g. the effect of different ventilation regimes, but the investigator has to be careful to avoid any alveolar recruitment whenever it is not desired. In our experience, the compromises in lung function and pulmonary vascular resistance remain stable for hours, as long as no accidental recruitment maneuvers are performed. But, the animal can deteriorate or even improve depending on the ventilator settings.
Third, the inflammatory response to lung injury greatly differs between models and furthermore between species. The role of e.g. inflammatory mediators like TNFα in pig lavage models are still controversial 9.
Fourth, this model requires complex instrumentation and monitoring procedures typically used in critical care medicine. In addition, the maintenance of anesthesia in hypoxic large animals exposed to sudden hemodynamic changes is necessary. Thus, only experienced investigators trained in large animal research and intensive care medicine should work with this model.
Finally, the induction of lung injury with lung lavages may result in a sudden hemodynamic decompensation and ultimately death of the animal. Up to 10-15 % of the animals may die during the induction period. In our experience this is usually the case, when the MAP decreases below 50 mmHg or the SpO2 falls below 70% resulting in sudden ischemic heart failure. Monitoring mean pulmonary arterial pressure (MPAP) during the lavage is also possible to reduce mortality because a rise of MPAP above 50-60 mmHg will result in right ventricular failure and death of the animal. In our experience right and left ventricular failure may occur simultaneously during lavages and monitoring hemodynamics during the procedure is essential to reduce mortality. We stop an ongoing lavage, drain the lavage fluid, and ventilate the animal whenever we record a decrease in MAP below 50 mmHg. Nevertheless, the lavages should be performed in a quick succession to washout a significant amount of surfactant. When the PaO2/FIO2 ratio decreases below 100 mmHg it should not increase above this threshold for at least one hour. This practical approach enables a time efficient induction of lung injury.
The advantage of this model is reproducibility with respect to lung function and pulmonary vascular resistance while allowing their precise quantification in the evaluation of therapeutic strategies. Furthermore, the size of the animals supports the use of clinically used catheters, endotracheal tubes, ventilators and monitors that are not fully available in smaller mammals (e.g. rodents). In addition, the acquired data format (e.g. cardiac output measurements with the thermodilution technique) is comparable to the bedside situation known to intensive care physicians.
The authors have nothing to disclose.
All authors disclose no financial or any other conflicts of interests.
GRANTS:
This study was supported by a grant from the Deutsche Forschungsgemeinschaft to P. Pickerodt and W. Boemke (Pi795/2-2).
Evita Infinity V500 | Dräger | intensive care ventilator | |
Vigilance I | Edwards | monitor | |
Vasofix Braunüle 20G | B Braun | 4268113B | peripheral vein catheter |
Mallinckrodt Tracheal Tube Cuffed | Covidien | 107-80 | 8.0 mm ID |
MultiCath3 | Vygon | 157,300 | 3 lumen central venous catheter, 20 cm length |
Leader Cath Set | Vygon | 115,805 | arterial catheter |
Percutaneus Sheath Introducer Set | Arrow | SI-09600 | introducer sheath for pulmonary artery catheter of 4-6 Fr., 10 cm length |
Swan-Ganz True Size Thermodilution Catheter | Edwards | 132F5 | pulmonary artery catheter, 75 cm length |
Flow through chamber thermistor | Baxter | 93-505 | for measuring cardiac output |
urinary catheter | no specific model requiered |