The isolated rabbit lung preparation is a gold standard tool in pulmonary research. This publication aims to describe the technique as developed for the study of physiological and pathological mechanisms involved in airway reactivity, lung preservation, and preclinical research in lung transplantation and pulmonary edema.
The isolated lung perfusion system has been widely used in pulmonary research, contributing to elucidate the lungs' inner workings, both micro and macroscopically. This technique is useful in the characterization of pulmonary physiology and pathology by measuring metabolic activities and respiratory functions, including interactions between circulatory substances and the effects of inhaled or perfused substances, as in drug testing. While in vitro methods involve the slicing and culturing of tissues, the isolated ex vivo lung perfusion system allows to work with a complete functional organ making possible the study of a continuous physiological function while recreating ventilation and perfusion. However, it should be noted that the effects of the absence of central innervation and lymphatic drainage still have to be fully assessed. This protocol aims to describe the assembly of the isolated lung apparatus, followed by the surgical extraction and cannulation of lungs and heart from experimental lab animals, as well as to display the perfusion technique and signal processing of data. The average viability of the isolated lung ranges between 5-8 h; during this period, the pulmonary capillary permeability increases, causing edema and lung injury. The functionality of preserved pulmonary tissue is measured by the capillary filtration coefficient (Kfc), used to determine the extent of pulmonary edema through time.
Brodie and Dixon first described the ex-vivo lung perfusion system in 1903 1. Since then, it has become a gold standard tool for studying the physiology, pharmacology, toxicology, and biochemistry of the lungs2,3. The technique offers a consistent and reproducible way to evaluate the viability of lung transplants, and to determine the effect of inflammatory mediators such as histamine, arachidonic acid metabolites, and substance P, among others, as well as their interactions during pulmonary phenomena such as bronchoconstriction, atelectasis, and pulmonary edema. The isolated lung system has been a key technique in unveiling the important role of the lungs in the elimination of biogenic amines from general circulation4,5. Additionally, the system has been used to evaluate the biochemistry of pulmonary surfactant6. Over the last few decades, the ex-vivo lung perfusion system has become an ideal platform for lung transplantation research7. In 2001 a team lead by Stig Steen described the first clinical application of the ex-vivo lung perfusion system by using it to recondition the lungs of a 19-year-old donor, who was initially rejected by transplantation centers due to its injuries. The left lung was harvested and perfused for 65 min; afterward, it was successfully transplanted into a 70-year-old man with COPD8. Further research into lung reconditioning using the ex-vivo perfusion led to developing the Toronto technique for extended lung perfusion to assess and treat injured donor lungs9,10. Clinically, the ex-vivo lung perfusion system has shown to be a safe strategy to increase donor pools by treating and reconditioning sub-standard donor lungs, presenting no significant difference in risks or outcomes against standard criteria donors10.
The main advantage of the isolated lung perfusion system is that the experimental parameters can be evaluated in a complete functional organ that preserves its physiological function under an artificial laboratory setup. Furthermore, it allows the measurement and manipulation of pulmonary mechanical ventilation to analyze the components of pulmonary physiology such as airway resistance, total vascular resistance, gas exchange, and edema formation, which to date cannot be measured precisely in vivo on lab animals2. Notably, the composition of the solution with which the lung is perfused can be fully controlled, enabling the addition of substances to evaluate their effects in real-time and sample collection from perfusion for further study11. Researchers working with the isolated lung system should bear in mind that mechanical ventilation causes decay of the pulmonary tissue shortening its useful time. This progressive fall in mechanical parameters can be significantly delayed by hyperinflating the lungs occasionally during the time of the experiment4. Still, the preparation cannot usually last more than eight hours. Another consideration for the ex-vivo lung perfusion system is the absence of central nervous regulation and lymphatic drainage. The effects of their absence are not yet fully understood and could potentially be a source of bias in certain experiments.
The isolated lung perfusion system technique can be performed in the rabbit model with a high degree of consistency and reproducibility. This work describes the technical and surgical procedures for the implementation of the ex-vivo isolated lung perfusion technique as developed for the rabbit model at Instituto Nacional de Enfermedades Respiratorias in Mexico City, intending to share the insights and provide a clear guide on key steps in the application of this experimental model.
The isolated perfusion system in the rabbit model has been widely used in the Bronchial Hyperresponsiveness Laboratory at the Instituto Nacional de Enfermedades Respiratorias. The protocol includes New Zealand rabbits with an approximate weight of 2.5-3 kg. All animals were kept in standard vivarium conditions and ad libitum feeding in compliance with the official Mexican guidelines for laboratory animals (NOM 062-ZOO-1999) and under the Guide for the Care and Use of Laboratory Animals (8th edition, 2011). All the animal procedures and animal care methods presented in this protocol were previously approved by the Ethics Committee of the Instituto Nacional de Enfermedades Respiratorias.
NOTE: The preparation of the isolated lung perfusion system involves the deliberate death of an animal under anesthesia and via euthanasia.
1. Equipment and preparation of apparatus.
2. Surgical extraction of the cardiopulmonary block.
3. Perfusion technique.
The isolated lung perfusion system allows organ manipulation for biopsy, sample collection from perfusion, and real-time data collection of physiological parameters. The isolated system can be used to test many hypotheses involving different functions and lung phenomena, from metabolic and enzymatic activity to edema formation and preservation periods for lung transplants.
Figure 1 displays a diagram of the fully assembled isolated lung perfusion system along with the ventilation system and the computed data acquisition. The perfusion component of the system ensures that the perfusate is constantly flowing through the isolated lungs. The pulmonary artery is cannulated to provide inflow perfusion, while perfusate outflow is provided by cannulating the left atrium of the heart. The perfusate is passed using the roller pump so that perfusate passes through the heat exchanger, then through the bubble trap into the pulmonary artery, and finally into the lung vascular bed. The ventilation component of the system allows the ventilation medium to flow constantly past the distal end of the pneumotachometer directly via the tracheal cannula into the lungs.
Figure 2 shows the concentration of MAO (Figure 2A) and 5-HT (Figure 2B) in an isolated lung preserved at 4 °C through 24 h. Serotonin and monoamine oxidase levels were determined from intravascular fluid samples obtained at different times and analyzed by ELISA. 5-HT concentration peaked after 15 min of preservation and then decreased during the next 6 h. Afterward, perfusion levels showed a non-statistically significant increase up to the 24th hour. MAO levels showed a similar behavior, peaking after 15 minutes of preservation, then decreasing during the next six hours up to the 24th hour12. Figure 3 shows 5-HT and MAO release rates, expressed as a percentage of the initial value, measured through 24 h in an isolated lung preparation at 4 °C. During the first hour of preservation, 5-HT levels rose higher than MAO and decreased within 6 h after being recaptured by endothelial cells and platelets as well as MAO mediated catabolism12.
Figure 4 shows NEP (optic densities/mg protein/min), and ACE enzymatic activity (optic densities/mg protein/min) through time in an isolated lung preparation. NEP activity (Figure 4A) was determined by spectrophotometric analysis using N-Dansyl-D-Ala-Gly-pnitro-Phe-Gly as NEP substrate followed by enalapril addition to inhibit ACE. ACE activity (Figure 4B) was determined by spectrophotometric analysis using enalapril as ACE substrate, followed by phosphoramidon addition to inhibit NEP. Since both solutions contained enalapril, ACE activity was calculated as the difference in fluorescence between samples with and without enalapril13.
Figure 5 shows the effect of pulmonary preservation in capillary permeability (mKfc) through a period of 24 h in the isolated lung perfusion system in the rabbit model. A control group (n = 6), assessed immediately after harvesting, had an mKfc of 2.8 ± 0.8 (mL/min/cmH2O/g) standard error, in contrast, the perfused lung suffered a progressive increase on mKfc scoring 7.5 ± 1.4 (n = 6) at 6 h, 10.8 ± 2.3 (n = 6) at 12 h and reached 16.3 ± 2.5 (n = 6) after 24 h of preservation13.
Figure 6 shows the effect of different additives in the capillary permeability of the isolated lung perfusion system under diverse conditions. A sudden pressure increment of 10 cmH2O is generated by a partial obstruction of the venous outflow to measure the permeability of the capillary bed through the capillary filtration coefficient (Kfc). To measure the Kfc, the outflow tubing that goes out of the left ventricle to the Krebs reservoir was partially clamped. Then, the partial clamp was maintained for 3 min making sure that the pressure increment reached 10 cmH2O. The clamping was released, and the normal flow continued. This maneuver was registered as an increment of the arterial pressure and a lung weight augmentation. This last parameter is considered the Kfc.
Figure 1: Diagram for the isolated lung perfusion system. This figure has been modified from Hugo Sachs Elektronik (HSE), Harvard Apparatus14. Please click here to view a larger version of this figure.
Figure 2: Concentration of serotonin (5-HT) and monoamine oxidase (MAO) involved in lung metabolism and vascular permeability. The concentration of (A) MAO and (B) 5-HT in an isolated lung preserved at 4°C through 24 h. Please click here to view a larger version of this figure.
Figure 3: Release rates of serotonin (5-HT) and monoamine oxidase (MAO). The release rates of 5-HT and MAO, expressed as a percentage of the initial value, measured through 24 h in an isolated lung preparation at 4 °C. Please click here to view a larger version of this figure.
Figure 4: Enzymatic activity of Neutral endopeptidase (NEP) and Angiotensin-converting enzyme (ACE). Enzymatic activity of (A) NEP and (B) ACE through time in an isolated lung preserved at 4 °C through 24 h. Please click here to view a larger version of this figure.
Figure 5: Effect of pulmonary preservation in capillary permeability (mKfc). The data shows the effect of pulmonary preservation in capillary permeability (mKfc) through a period of 24 h in the isolated lung perfusion system in the rabbit model. Please click here to view a larger version of this figure.
Figure 6: Effect of different additives in the capillary permeability. The effect of different additives in the capillary permeability of the isolated lung perfusion system under diverse conditions. Please click here to view a larger version of this figure.
This work displays a general view of the isolated lung perfusion system, an essential technique in pulmonary physiology research. The isolated lung perfusion system offers a great degree of versatility in its uses and allows the evaluation of several parameters relevant in the testing of a wide range of hypotheses15. An isolated lung system is a tool with worldwide presence that, in the last decade, has further established its relevance for organ-specific evaluations and also expanded its usefulness as an extension of state-of-the-art technologies and novel therapies involving mesenchymal stem cells16 and CRISPR/Cas9 genome engineering17, among others. Current ex vivo lung perfusion research areas broadly cover anti-inflammatory strategies, ventilation injury management and prevention, anti-rejection treatment, and anti-pulmonary edema performance15.
A proper assembly of the apparatus is required to guarantee correct data recollection. As shown in Figure 1 the whole system consists of a negative pressure wet chamber attached to a ventilation system and a perfusion system that mimics the respiratory and circulatory functions of the lungs, respectively. Both systems are connected to a data acquisition system that allows the addition of measurement devices that can be tailored for the needs of any protocol. The surgical process of harvesting the cardio-pulmonary block should be performed quickly, preferably by experienced personnel, to avoid additional tissue injury to maintain the lung as intact as possible so the physiological function can continue without further interference during the experiment. The system also allows for real-time perfusion sample collection that can be used to determine the effect of certain molecules in different pulmonary functions (for instance, heparin effect on pulmonary preservation).
In order to achieve a proper distribution of the perfusion flow among pulmonary vessels, namely capillaries, zone 3 conditions should be procured. Zone 1 conditions are defined as the region where the arterial pressure drops below the alveolar pressure, typically approaching atmospheric pressure. When this happens, the capillaries flatten, making blood or perfusion flow impossible. Under normal circumstances zone 1 cannot exist since arterial pressure is enough to guarantee flow distribution. However, zone 1 conditions can appear if arterial pressure drops, or alveolar pressure increases (as it does during positive pressure ventilation). Zone 1 conditions lead to an unperfused ventilated lung that is incapable of performing a gas exchange. In zone 2 conditions, arterial pressure is higher than alveolar pressure. However, the venous pressure remains below the alveolar pressure resulting in a perfusion flow determined by the difference between arterial and alveolar pressures. This behavior can be modeled using a Starling resistor. Zone 3 conditions are determined by the difference between arterial and venous pressures. The increase in perfusion flow in zone 3 occurs because the capillaries distend, conditioning the opening of a maximum number of pulmonary vessels.
The system's unit consists of seven modules: two analog transducer amplifier modules (TAM-A) equipped with an analog LED bar graph signal to monitor dynamic signals (blood pressure, respiratory airflow, contraction force, etc.), one digital transducer amplifier module (TAM-D) with a digital numeric display designed to monitor slow-changing pulsatile signals; a servo controller for perfusion module (SCP) that works together with TAM-A and TAM-D amplifiers for perfusion control of isolated organ perfusions using the peristaltic pump, the pump speed can be set in constant pressure mode or manually controlled through the SCP; an edema balance module (EBM) that measures lung weight; a ventilation control module (VCM) to control positive and negative pressure ventilation, and a timer counter module (TCM) that can be set to trigger the VCM to perform deep inspiration cycles.
The high global prevalence of pulmonary and respiratory affections and the limitations of current therapeutic options are forcing a greater demand for lung transplants, as it remains the gold standard treatment for patients with terminal lung disease18. The ex-vivo lung perfusion system represents an excellent platform to test targeted therapies in both basic and clinical research. On a clinical level, the ex-vivo perfusion system can be used to evaluate graft tissue outside the body, allowing to test the isolated organ before transplant, helping to gather clinical data for a more precise prognostic on the effectiveness of the transplant. Rational use of the isolated lung perfusion system could to help optimize lung transplant surgery, making them a safer and more elective procedure. The isolated lung model is also useful in the basic research of advanced diagnosis and therapy techniques such as instillation of mesenchymal stem cells and other immune-mediated therapies; many reports have shown the potential of the ex-vivo perfusion technique as a platform to make further research on pulmonary preservation in the development of techniques to avoid ischemia-reperfusion injury and pulmonary edema, prolonging organ viability15. Some troubleshooting steps and limitations associated to the isolated lung model are mainly the short-available time of this technique for possible edema generation induced by lymphatic drain limitation as well as the systemic effect of the technique. The capillary filtration coefficient (Kfc) determination is a reliable criterion to measure the functionality of preserved pulmonary tissue and establish the extent of edema through time. No difference has been found between the manual and automatic determinations of Kfc19.
As the use of the isolated lung perfusion system popularizes and new therapies change the clinical landscape, the ex-vivo perfusion technique is becoming an elective choice to improve patient outcomes in different pulmonary pathologies, as well as to increase the pool of potential lung donors without compromising recipient safety, promising a new era in pulmonary preservation and lung transplant. The emergence of the Covid-19 pandemic and the increase of COPD's prevalence18,20 in the global population highlights the need for further basic research into pulmonary physiology, pulmonary preservation, and lung transplant, as well as preclinical research of novel therapies with views towards translational medicine. Furthermore, the ex-vivo rabbit model is an accessible and practical model to train residents and students in the area of pulmonology, particularly those involved with thoracic surgery and ECMO. Any laboratory involved in respiratory or thoracopulmonary research protocols is encouraged to consider the isolated lung perfusion system as part of their daily tools for their experiments.
The authors have nothing to disclose.
The authors would like to thank Ph.D. Bettina Sommer Cervantes for her support in the writing of this manuscript, and Kitzia Elena Lara Safont for her support with the illustrations.
2-Stop Tygon E-Lab Tubing, 3.17 mm ID, 12/pack, Black/White | Hugo Sachs Elektronik (HSE) | 73-1864 | |
Adapter for Positive Pressure Ventilation on IPL-4 | Hugo Sachs Elektronik (HSE) | 73-4312 | |
Adapter for Positive Pressure Ventilation on IPL-4 | Hugo Sachs Elektronik (HSE) | 73-4312 | |
Alternative Pressure-Free Gas Supply for IPL-4: To supply the trachea with gas mixture different from room air during negative ventilation | Hugo Sachs Elektronik (HSE) | 73-4309 | |
Base Unit for the Rabbit to Fetal Pig Isolated Perfused Lung | Hugo Sachs Elektronik (HSE) | 73-4138 | |
Bovine serum A2:D41albumin lyophilized powder | sigma | 3912 | 500 g |
Calcium chloride, CaCl2·2H2O. | JT Baker | 10035-04-8 | |
Cryogenic vials | Corning | 430659 | 2 mL |
D-glucosa, C6H12O6. | sigma | G5767 | |
Differential Low Pressure Transducer DLP2.5, Range +- 2.5 cmH2O, HSE Connector | Hugo Sachs Elektronik (HSE) | 73-3882 | |
Differential Pressure Transducer MPX, Range +- 100 cmH2O, HSE Connector | Hugo Sachs Elektronik (HSE) | 73-0064 | |
Eppendorf tubes | |||
Ethanol absolute HPLC grade | Caledon | ||
Falcon tubes | 14 mL | ||
Harvard Peristaltic Pump P-230 (Complete with Control Box and P-230 Motor Drive) | Hugo Sachs Elektronik (HSE) | 70-7001 | |
Heated Linear Pneumotachometer 0 to 10 L/min flow range | Hugo Sachs Elektronik (HSE) | 59-9349 | |
Heater Controller for Single Pneumotachometer 230 VAC, 50 Hz | Hugo Sachs Elektronik (HSE) | 59-9703 | |
Heparin | PISA | 5000 UI | |
HPLC Column (C18 100A 5U) | Alltech | 98121213 | 150 mm x 4.6 mm |
Hydrophilic Syringe Filter | Millex | SLLGR04NL | 4 mm |
IPL-4 Core System for Isolated Rabbit to Fetal Pig Lung, 230 | Hugo Sachs Elektronik (HSE) | 73-4296 | |
IPL-4 Core System for Isolated Rabbit to Fetal Pig Lung, 230 V | Hugo Sachs Elektronik (HSE) | 73-4296 | |
Jacketed Glass Reservoir for Buffer Solution, with Frit and Tubing, 6.0 L | Hugo Sachs Elektronik (HSE) | 73-0322 | |
Lauda Thermostatic Circulator, Type E-103, 230 V/50 Hz, 3 L Bath Volume, Temperature Range 20 to 150°C | Hugo Sachs Elektronik (HSE) | 73-0125 | |
Left Atrium Cannula for Rabbit with Basket, OD 5.9 mm | Hugo Sachs Elektronik (HSE) | 73-4162 | |
Low Range Blood Pressure Transducer P75 for PLUGSYS Module | Hugo Sachs Elektronik (HSE) | 73-0020 | |
Magnesium sulfate heptahydrate, MgSO4·7H2O | JT Baker | 10034-99-8 | |
Microcentrifuge Tube | Corning | 430909 | |
Negative Pressure Ventilation Control Option with Pressure Regulator for IPL-4 | Hugo Sachs Elektronik (HSE) | 73-4298 | |
New Zeland rabbits | |||
PISABENTAL (Pentobarbital sodium) | PISA | Q-7833-215 | |
PLUGSYS Case, Type 603* 7 | Hugo Sachs Elektronik (HSE) | 73-0045 | |
PLUGSYS TCM Time Counter Module | Hugo Sachs Elektronik (HSE) | 73-1750 | |
PLUGSYS Transducer Amplifier Module (TAM-A) | Hugo Sachs Elektronik (HSE) | 73-0065 | |
PLUGSYS Transducer Amplifier Module (TAM-D) | Hugo Sachs Elektronik (HSE) | 73-1793 | |
PLUGSYS VCM-4R Ventilation Control Module with Pressure Regulator | Hugo Sachs Elektronik (HSE) | 73-1755 | |
Potassium chloride, KCl. | JT Baker | 3040-01 | |
Potassium dihydrogen phosphate, KH2PO4 | JT Baker | 7778-77-0 | |
PROCIN (Xylacine clorhydrate) | PISA | Q-7833-099 | |
Pulmonary Artery Cannula for Rabbit with Basket, OD 4.6 mm | Hugo Sachs Elektronik (HSE) | 73-4161 | |
Scalpel knife | |||
Serotonin 5-HT | |||
Servo Controller for Perfusion (SCP | Hugo Sachs Elektronik (HSE) | 73-2806 | |
Snap Cap Microcentrifuge Tube | Costar | 3620 | 1.7 mL |
Sodium bicarbonate, NaHCO3 | sigma | S6014 | |
Sodium chloride, NaCl. | sigma | S9888 | |
Surgical gloves No. 7 1/2 | |||
Surgical gloves No. 8 | |||
Taygon tubes | Masterflex | ||
Tracheal Cannula for Rabbit, OD 5.0 mm | Hugo Sachs Elektronik (HSE) | 73-4163 |