Veno-Venous Extracorporeal Membrane Oxygenation in a Mouse

Summary

Here we present a protocol describing the technique of veno-venous extracorporeal membrane oxygenation (ECMO) in a non-intubated, spontaneously breathing mouse. This murine model of ECMO can be effectively implemented in experimental studies of acute and end-stage lung diseases.

Abstract

The use of extracorporeal membrane oxygenation (ECMO) has increased substantially in recent years. ECMO has become a reliable and effective therapy for acute as well as end-stage lung diseases. With the increase in clinical demand and prolonged use of ECMO, procedural optimization and prevention of multi-organ damage are of critical importance. The aim of this protocol is to present a detailed technique of veno-venous ECMO in a non-intubated, spontaneously breathing mouse. This protocol demonstrates the technical design of the ECMO and surgical steps. This murine ECMO model will facilitate the study of pathophysiology related to ECMO (e.g., inflammation,bleeding and thromboembolic events). Due to the abundance of genetically modified mice, the molecular mechanisms involved in ECMO-related complications can also be dissected.

Introduction

Extracorporeal membrane oxygenation (ECMO) is a temporary life support system that takes over functions of the lungs and heart to allow adequate gas exchange and perfusion. Hill et al¹ described the first use of ECMO in patients in 1972; however, it only became widely used after its successful application during the H1N1 influenza pandemic in 2009². Today, ECMO is routinely used as a lifesaving procedure in end-stage heart and lung diseases³. Veno-venous ECMO is increasingly employed as an alternative to invasive mechanical ventilation in awake, non-intubated, spontaneously breathing patients with refractory respiratory failure⁴.

Despite its widespread adoption, diverse complications have been reported for ECMO^5,6,7. Complications that can be experienced by patients on ECMO include bleeding, thrombosis, sepsis, thrombocytopenia, device-related malfunctions, and air embolism. Moreover, a systemic inflammatory response syndrome (SIRS) resulting in multi-organ damage is well-described both clinically and in experimental studies^8,9. Neurological complications such as brain infarction are also frequently reported in patients undergoing long-term ECMO therapy. To confuse matters, it is often difficult to distinguish whether complications are caused by ECMO itself or arise from the underlying disorders accompanying acute and end-stage diseases.

To specifically study the effects of ECMO on a healthy organism, a reliable experimental animal model must be established. There are very few reports on performance of ECMO on small animals and are all limited to rats. To date, no mouse model of ECMO has been described in the literature. Due to the availability of a large number of genetically modified mouse strains, establishment of a mouse ECMO model would allow further investigation of the molecular mechanisms involved in ECMO-related complications^10,11.

Based on our previously described murine model of cardiopulmonary bypass (CPB)¹², we have developed a stable method of veno-venous ECMO in non-intubated, spontaneously breathing mice. The ECMO circuit (Figure 1), containing outflow and inflow cannulas, a peristaltic pump, oxygenator, and air-trapping reservoir, is similar to our previously described model of murine CPB¹² with the exception of having a smaller priming volume (0.5 mL). This protocol demonstrates the detailed techniques, physiological monitoring, and blood gas analysis involved in a successful ECMO procedure.

Protocol

Experiments were performed on male C57BL/6 mice, aged 12 weeks. This study was conducted in compliance with guidelines of the German Animal Law under Protocol TSA 16/2250.

1. Materials Preparation

NOTE: All steps are performed under clean, non-sterile conditions. Sterile conditions would be required if animal is to be survived postoperatively.

1. Introduce 3 fenestrations into a 2-Fr polyurethane tube using a surgical blade under a microscope with 16X magnification.
   NOTE: All fenestrations must be located in the distal third of the cannula to ensure optimal blood drainage.
2. Prepare the priming solution (Materials Table). Include 30 IU/mL heparin and 2.5% v/v of an 8.4% solution of NaHCO₃. Refrigerate this solution at 4 °C until it is ready to use. Prime the circuit with 500 uL of priming solution.
3. Place the outflow cannula into the priming solution and fill the ECMO machine by switching on the peristaltic pump. Continue to circulate the priming solution through the machine for the next 30 min at a flow rate of 1 mL/min.
4. Give 0.5 L/min of 100% oxygen to the oxygenator.

2. Anesthesia

1. Place the animal in an induction chamber filled with a 2.5% v/v isoflurane/oxygen mixture. Provide 0.5 L/min of 100% oxygen to the vaporizer. Before surgery, check that full anesthesia is achieved by testing pedal withdrawal and pain reflexes. Apply eye gel to prevent drying damage.
2. Use a warming pad to maintain the body temperature at 37 °C.
3. Perform inhalation mask anesthesia using an isoflurane vaporizer and inject 5 mg/kg carprofen subcutaneously.
4. Regularly observe spontaneous breathing and adjust the concentration of isoflurane so that it is between 1.3 and 2.5%.

3. Surgery

1. Expose the left jugular vein by using a lateral skin incision of 4 mm with the help of fine scissors on the left side of the neck. Together with sharp and blunt preparation using micro-forceps and cotton swabs, use bipolar coagulation of the small vessels.
2. Once the left jugular vein is exposed, ligate the distal part using an 8-0 silk suture with the help of micro-forceps.
3. Place a slip knot at the proximal end of the vein. Incise the anterior wall of the vein using micro-scissors.
4. To achieve full heparinization, inject 2.5 IU/g heparin into the jugular vein via a 26 G braunula.
5. Raise the head side of the animal pad by 30° to avoid excessive blood loss from the vein during insertion of the cannula.
6. Insert a 2-Fr polyurethane (PU) cannula into the proximal part of the jugular vein, rotating it slightly while pushing it to a depth of 4 cm; while doing so, the iliac bifurcation of inferior vena cava (IVC) will be reached.
7. Secure the cannula with 8-0 silk knots using microforceps.
8. Expose the right jugular vein using the steps described in 3.1, 3.2, and 3.3.
9. Cannulate the right jugular vein with a 1-Fr PU cannula and gently move it 5 mm towards the direction of right atrium.
10. Repeat step 3.7.
11. Catheterize the left femoral artery with another 1-Fr PU cannula and use it for invasive pressure monitoring as well as blood sampling for blood gas analysis (BGA).
12. Insert electrocardiogram (ECG) needles connected to a data acquisition device subcutaneously into both forelimbs and into the left thoracic wall.
13. Insert a rectal thermometer connected to a data acquisition device.

4. Veno-Venous Extracorporeal Membrane Oxygenation and Blood Gas Analysis

NOTE: For a schematic of the complete ECMO circuit, see Figure 1.

1. Initiate ECMO on the animal by turning on the pump with an initial flow rate of 0.1 mL/min. Adjust the flow rate of the pump within the next 2 min to 3-5 mL/min.
2. In case of air suction in the outflow cannula via the cannulation site, reduce the flow and add 0.1 mL of priming solution to the circuit via an air-trapping reservoir.
3. Under stable flow, continue to monitor in real-time mode all vital parameters via the data acquisition device.
4. Constantly observe backflow from the venous drainage and monitor the level of the blood in the air-trapper reservoir.
5. Collect any blood leaking from wounds into a 1 cc syringe with the tip of a 24 G branula andreturn it to the ECMO circuit via the air-trapping reservoir.
6. For BGA, use a blood sampling cartridge to collect approximately 75 µL of arterial blood at the following time points and from the following locations:
   1. 10 min after the initiation of ECMO, collect blood from the IVC via an extra tube built in before the oxygenator, via similar extra tube after oxygenator (control), and directly from the femoral artery.
   2. 30 min after the initiation of ECMO, collect blood from the femoral artery.
7. Give an extra 0.1 mL of priming solution to compensate for intravasal liquid loss every 45 min via the air-trapper or femoral artery catheter or by sucking the air bubbles through the blood draining cannula.
8. For BGA, use a blood sampling cartridge to collect approximately 75 µL of arterial blood:
   1. 1 h after the initiation of ECMO from the femoral artery.
   2. 2 h after the initiation of ECMO, collect blood from the IVC via an extra tube built in before the oxygenator, via similar extra tube after oxygenator (control), and directly from the femoral artery.
9. After 2 h, reduce the flow rate on the pump gradually (over the course of 5 min), thereby stopping ECMO.
10. Continue to record vital parameters for another 10 min.
11. Finish the experiment by exsanguinating the animal and harvesting the blood and organs.

Results

This protocol describes the method of veno-venous ECMO in a mouse. This model is reliable and reproducible, and compared to our previously described model of CPB with respiratory and circulatory arrest^12,13, it is less technically demanding to establish.

ECMO flow in the venous system was maintained between 1.5 and 5 mL/min. The mean arterial pressure was kept between 70...

Discussion

Previously, we described a successful model of CPB in a mouse^12,13. To implement such a model for acute or end-stage lung disorders we developed an easy-to-use veno-venous ECMO circuit for mice. Different to the CPB model, veno-venous ECMO does not require complicated surgical procedures such as sternotomy and clamping of the aorta, thus reducing the risk of wound bleeding in a fully heparinized animal. To avoid embolization of the oxygenator with blood clots, 2....

Materials

Name	Company	Catalog Number	Comments
Sterofundin	B.Braun Petzold GmbH	PZN:8609189	in 1:1 with Tetraspan
Tetraspan 6% Solution	B. Braun Melsungen AG	PZN: 05565416	in 1:1 with Sterofundin
Heparin Natrium 25.000	Ratiopharm GmbH	PZN: 3029843	2,5 IU per ml of priming
NaHCO3 8,4% Solution	B. Braun Melsungen AG	PZN: 1579775	3% in priming solution
Carprofen	Zoetis Inc., USA	PZN:00289615	5mg/kg/BW
1 Fr PU Catheter	Instechlabs INC., USA	C10PU-MCA1301	carotide artery
2 Fr PU Catheter	Instechlabs INC., USA	C20PU-MJV1302	jugular vein
8-0 Silk suture braided	Ashaway Line & Twine Co., USA	75290	ligature
Isoflurane	Piramal Critical Care GmbH	PZN:9714675	narcosis
Spring Scissors - 6mm Blades	Fine Science Tools GmbH	15020-15	instruments
Spring Scissors - 2mm Blades	Fine Science Tools GmbH	15000-03	instruments
Halsted-Mosquito Hemostat	Fine Science Tools GmbH	13009-12	instruments
Dumont #55 Forceps	Fine Science Tools GmbH	11295-51	instruments
Castroviejo Micro Needle Holder - 9cm	Fine Science Tools GmbH	12060-02	instruments
Micro Serrefines	Fine Science Tools GmbH	18555-01	instruments
Bulldog Serrefine	Fine Science Tools GmbH	18050-28	instruments
Isoflurane Vaporizer Drager 19.1	Drägerwerk AG & Co. KGaA		anesthesia 1,3 -2,5%
Multichannel Data Aquisition Device with ISOHEART Software	Hugo Sachs Elektronik GmbH, Germany		invasive pressure, ECG, t °C
i-STAT portable device	Abbott Laboratories, Lake Bluff, Illinois, USA		blood gas analysis
i-STAT CG4+ and CG8+ cartridges	Abbott Laboratories, Lake Bluff, Illinois, USA		blood gas analysis
C57Bl/6 mice, male, 30 g, 14 weeks old	Charles River Laboratories		housed 1 week before