Description

Introduction

An isolated perfused lung system for mice is a set of core products that facilitates ex vivo lung perfusion (EVLP) in fragile mouse lungs for ventilation and perfusion experiments. The system uses positive or negative pressure ventilation to perfuse the lung placed in the chamber. The procedure involves cannulation of the pulmonary artery and the left atrium. One lung is placed in the chamber under artificial thoracic pressure, whereas the other non-functional lung remains in the thoracic cavity. The purpose of this system is to keep the lung in physiological conditions for organ transplantation or other respiration-based experiments. An isolated perfused lung system for mice is used to measure respiratory mechanics and conduct drug and toxicology experiments on respiratory and vascular parameters.

Principle

The basic principle of ex vivo lung perfusion (EVLP) is to keep lungs in a viable state outside the animal’s body by perfusing them with a relevant perfusion buffer (perfusate). During the perfusion experiments, the animal’s pulmonary artery is cannulated. The perfusate is pumped through a roller pump at constant flow or pressure. As a result, the perfusion buffer passes through the heat exchanger to the pulmonary artery and then finally enters the lung vascular bed. The heart’s left atrium is cannulated to facilitate the effluent outflow. On the other hand, the non-functional lung is kept in the thoracic cavity, enabling easier cannula placement for the outflow of the perfusate.

Apart from perfusion, an isolated perfused lung system for mice can also be employed for ventilation experiments. The researcher can choose between positive and negative pressure ventilation. Positive pressure ventilation is used in experiments that imitate clinical EVLP systems. Due to substantial difference in vascular flow rates, sub-atmospheric pressure ventilation allow the researchers to establish ex vivo conditions quite close to the in vivo physiological conditions. In such experiments, the lung is placed in the chamber and can be ventilated using artificial thoracic pressure. This pressure can be positive or negative.

Apparatus and Equipment 

An isolated perfused lung system for mice comprises a moist lung chamber with an integrated heat exchanger and a bubble trap. The chamber is completely water-jacketed for accurate temperature monitoring and possesses a lid. Pressure balancing during ventilation experiments is achieved by a pressure-balancing vessel that eliminates transmural pressure difference. Additional options can be attached to the basic isolated lung perfusion system. For instance, the lung can be connected via a tracheal cannula to the pneumotachometer for measuring respiratory airflow. A ventilation control module (VCM) can be used to adjust respiration rate, and inspiratory, and end-expiratory pressure.

The basic unit of isolated perfused lung system offered by Conduct Science includes a negative pressure lung chamber, i.e., the “artificial thorax” with lid and Venturi jet, positive pressure ventilation head, connectors, pneumotachometer, air humidifier, pressure balancing vessel, and stainless steel tracheal, pulmonary, and atrial cannulas for the mouse. The internal diameter (ID) and outer diameter (OD) of these cannulas are listed below:

• Pulmonary Artery Cannula: ID 1mm and OD 1.3/1.6mm
• Tracheal Cannula: ID 1mm OD 1.3mm
• Atrial Cannula ID 1mm and OD 1.6mm (length 24mm)

Conduct Science also offers isolated perfused lung systems for other animals like rats, guinea pigs, and large rodents.

Protocol (Vanderpool and Chesler, 2011)

Ventilation 

1. Anesthetize the mouse by administering an intraperitoneal injection of 150mg/kg pentobarbital. Use the paw pinch reflex to ensure that the subject is deeply anesthetized. Pin its paws to the corkboard for stability.
2. Clean the subject’s skin using 95% alcohol spray and grab the skin using forceps. Make a 1cm long incision in the skin from the abdomen to the thorax using straight scissors.
3. Remove the white glandular tissue and superficial muscle and locate the trachea and esophagus. Isolate both structures from the tissue on both sides and posteriorly.
4. Place the bent forceps under the trachea and grasp a suture on the other side. Tie a loose surgeon’s knot. Be careful, do not tighten the knot.
5. Cut a small angled “v” in the trachea using small scissors and grasp the trachea under the “v” and the tracheal cannula using two blunt forceps. Place the mouse along with the corkboard into the heated isolated perfused lung system for mouse.
6.  Cannulate the trachea and tie the knot.
7. Start ventilation with room air at approximately 90 breaths/min, with deep inspiration.

Perfusion

1. Spray the animal’s chest again with alcohol and remove all the skin above the ribs using straight forceps and scissors. Cut along the sternum. Lift the skin on each side and cut the skin along the lower ribs.
2. Grab the xiphoid process at the bottom of the sternum using forceps, cut a hole in the diaphragm using straight scissors, and cut away the diaphragm.
3. Re-hold the xiphoid process with tweezers, and use balled-tip-angled scissors to cut the sternum through the ribs. But be careful! Do not cut the organ or the blood vessels. Hold the scissors against the sternum, so the lung and the heart are not cut.
4. Cut away as much of the ribs as possible and expose the heart. Now slowly inject 0.1ml heparin into the right ventricle while the heart is still beating.
5. Cut away the remaining ribs and place the microscope above the lungs. Remove glandular and fatty tissues from the heart.
6. Use tweezers to scoop the heart from left to right from the top such that you place the tip of the tweezers under the aorta and the pulmonary artery (PA). Following this, use a suture to tie a loose surgeon’s knot.
7. Pre-fill the cannula with 4ml perfusate (say RPMI in the given protocol) using a 10ml syringe. Make sure that there are no air bubbles, as lung perfusion with air bubbles can cause edema.
8. Insert the cannula into the pulmonary artery, so it is visible from the transparent wall of the PA. Use a suture to tie a knot to hold the cannula in place.
9. Cut a notch in the lower part of the left ventricle and cannulate the left atrium.
10. Infuse the perfusate at the rate of 0.3ml/min until the perfusate is visible in the tubing. If there is no flow in the tubing, re-position the LA cannula or check for leakage in the PA.
11. Attach a 60ml syringe to the pulmonary artery cannula through the isolated perfused lung perfusion system. Begin perfusing at 1ml/min until the lungs turn white. It indicates that RPMI has replaced the blood in the lungs. Perfuse at a slow flow for about 2 minutes.

Applications

The isolated perused lung system has manifold applications. Scientists use it to measure respiratory parameters like respiration rate, respiratory airflow, pulmonary artery pressure, intrapleural/tracheal pressure, and vascular resistance. The system is widely employed to assess the effect of drugs and toxins on respiration and vascular parameters. Out of several applications of isolated perfused lung systems for mice in perfusion and ventilation experiments, some are presented below.

IRI Diagnosis in Lung Transplantation 

Charles et al. (2020) studied how ischemia-reperfusion injury (IRI) is responsible for primary graft dysfunction. They used [99mTc]cFLFLF, a ligand of formyl peptide receptor 1 (FPR1), for the molecular targeting of FPR1 to perform SPECT imaging of IRI. The researchers used an isolated perfused long system for mouse to assess the combined function of right and left lungs after reperfusion. They anesthetized 9-12 weeks old C57BL/6 wild-type mice using ketamine-xylazine and then performed tracheotomy for tracheal cannulation. Following this, they ventilated the animal at a tidal volume of 7μL/g and a rate of 100 breaths per minute using 2 cmH2O positive end-expiratory pressure. They later exsanguinated via transection of the abdominal aorta and inferior vena cava. Later, the investigators cannulated the pulmonary artery and perfused the lungs using Krebs- Henseleit buffer warmed to 37oC and finally performed left ventriculotomy to facilitate the perfusate outflow. After 5 minutes pressure equilibration period, the data was collected using PULMODYN software. Then, the experimenters performed SPECT imaging and concluded that [99mTc]cFLFLF SPECT aids in non-invasive IRI diagnosis for timely intervention to improve lung transplantation outcomes.

Assessment of Airway Hyperresponsiveness

Tartaglione et al. (2018) the role of Nociceptin/orphanin Fq (N/OFQ) in inflammation and remodeling of small airways in murine models of airway hyperresponsiveness. The researchers took 6 weeks old female BALB/c mice and divided them into six groups including naïve, OVA alone (administered with allergen ovalbumin), N/OFQ alone, pre-OVA sensitized (i.p., administration of N/OFQ before OVA injection), post-OVA sensitized (OVA injection followed by N/OFQ administration). The experimenters used an isolated perfused lung system to determine airway hyperresponsiveness. They anesthetized the mice intraperitoneally and administered 40mg/kg ketamine HCl and 0.15mg/kg medetomidine hydrochloride. Following this, they incised the skin from the abdomen to the throat, thereby exposing the trachea, and cannulated it. Later, they incised the diaphragm to expose the heart and injected 50µl heparin. Then the investigators exsanguinated the mouse following renal vena incision and cut open the thorax. Two thoracic halves were fixed at the sides using two cannulas. They also cannulated PA and administered the drugs through the cannula into the pulmonary artery.

After this, they perfused the lungs in a non-circulating fashion via the PA at 1ml/min., resulting in a pressure of 1cmH2O. The perfusion buffer was RPMI 1640 medium without phenol red warmed to 37oC containing 4% low endotoxin grade albumin. The scientists ventilated the lungs at a negative pressure of -3 to -9 cmH2O with a tidal volume of 200μl and 90breaths/min. They used a pressure balancing vessel to measure pressure in the artificial thorax chamber and a pneumotachometer to measure airflow velocity. The lungs respired humidified air, and a pressure transducer was used to monitor arterial pressure constantly. All data was processed through PULMODYN software. The results suggested that N/OFQ protects against inflammation and remodeling in airway hyperresponsiveness.

Strengths and Limitations

An isolated perfused lung system for mice has several advantages. The foremost advantage of this system is that it allows researchers to study the effects of drugs and respiratory mechanics outside the body in near-physiological conditions without the influence of other body systems. The advantage of using small animal models like mice for EVLP is that they are budget friendly compared to isolated perfused lung systems for larger animals. The smaller initial cost means one can perform more perfusion experiments in less time and less money. Moreover, murine models have a better immune response as compared to rats. They have a greater number of species-specific antibodies and a greater number of gene probes available for experiments (Nelson et al., 2014).

The artificial thorax chamber in an isolated perfused lung system for mice provides near physiological conditions to study mouse lungs. The apparatus provides a quick switch between positive and negative pressure ventilation, thus increasing the experimental versatility. Its integrated surgery table lowers the damage during preparation. The system is designed to minimize perfusion artifacts and offers low flow resistance. A built-in humidifier prevents the lungs from drying. However, a potential disadvantage of using murine models in isolated lung perfusion experiments is that mouse organs are quite small and challenging to handle.

Summary 

• Isolated perfused lungs system for mice is a set of core products that facilitates ex vivo lung perfusion (EVLP) in fragile mouse lungs for ventilation and perfusion experiments.
• One lung is placed in the chamber under artificial thoracic pressure, whereas the other non-functional lung remains in the thoracic cavity.
• The purpose of this system is to keep the lung in physiological conditions for organ transplantation or other respiration-based experiments.
• The apparatus consists of an artificial thoracic chamber, ventilation head, pressure balancing vessel, and pulmonary, tracheal, and atrial cannulas.
• An isolated perfused lung system for mice is used to measure respiratory mechanics and conduct drug and toxicology experiments on respiratory and vascular parameters.
• A major advantage of this system is that it allows researchers to study the effects of drugs and respiratory mechanics outside the body in near-physiological conditions without the influence of other body systems.

References

1. Vanderpool, R. R., & Chesler, N. C. (2011). Characterization of the isolated, ventilated, and instrumented mouse lung perfused with pulsatile flow. JoVE (Journal of Visualized Experiments), (50), e2690.
2. Charles, E. J., Chordia, M. D., Zhao, Y., Zhang, Y., Mehaffey, J. H., Glover, D. K., … & Laubach, V. E. (2020). SPECT imaging of lung ischemia-reperfusion injury using [99mTc] cFLFLF for molecular targeting of formyl peptide receptor 1. American Journal of Physiology-Lung Cellular and Molecular Physiology, 318(2), L304-L313.
3. Tartaglione, G., Spaziano, G., Sgambato, M., Russo, T. P., Liparulo, A., Esposito, R., … & D’Agostino, B. (2018). Nociceptin/orphanin Fq in inflammation and remodeling of the small airways in experimental model of airway hyperresponsiveness. Physiological reports, 6(20), e13906.
4. Nelson, K., Bobba, C., Ghadiali, S., Hayes Jr, D., Black, S. M., & Whitson, B. A. (2014). Animal models of ex vivo lung perfusion as a platform for transplantation research. World journal of experimental medicine, 4(2), 7.