This method can help answer key questions in the field of pulmonary research, such as those related to the therapy of acute and chronic end-stage lung disease. The main advantage of this technique is that it facilitates the starting of molecular and pathological mechanisms of multi-organ damage during prolonged extracorporeal membrane oxygenation as a life-saving procedure. Under clean, non-sterile conditions, use a surgical blade and a dissecting microscope to introduce three fenestrations into the distal third of a two French polyurethane catheter under 16x magnification.
Next, place the outflow cannula into the priming solution, switch on the peristaltic pump to fill the extracorporeal membrane oxygenation, or ECMO machine, for priming of the circuit at a one millimeter per minute flow rate. After 30 minutes, add 0.5 liters per minute of 100%oxygen to the oxygenator and confirm the lack of response to toe pinch in an anesthetized adult mouse. After applying ointment to the animal's eyes, make a four millimeter lateral skin incision on the left side of the neck to visualize the jugular vein, and use micro-forceps and cotton swabs to further expose the vessel.
Use an 8-0 silk suture to ligate the distal end of the exposed vein, and place a slip knot at the proximal end. Incise the anterior wall of the vein with microscissors, and inject 2.5 international units per gram of heparin into the jugular vein via a 26-gauge branula, raising the head end of the surgical pad by 30 degrees to avoid excessive blood loss during the insertion. Carefully insert the fenestrated two French polyurethane cannula into the proximal end of the jugular vein, rotating the cannula slightly, while pushing the cannula to a four centimeter depth to reach the illiac bifurcation of the inferior vena cava.
It's important to push the cannula very gently, but continuously without the use of extra force. If resistance is encountered, pull the cannula back and insert it again. Secure the cannula with 8-0 silk knots, and expose the right jugular vein as just demonstrated.
Cannulate the right jugular vein with a one French polyurethane cannula, gently moving the cannula five millimeters toward the right atrium, and secure the cannula with 8-0 silk knots. Catheterize the left femoral artery with another one French polyurethane cannula for invasive pressure monitoring and blood gas analysis, and insert electrocardiogram needles connected to a data acquisition device subcutaneously into both forelimbs, and into the left thoracic wall. Then insert a rectal thermometer connected to a data acquisition device.
To initiate veno-venous extracorporeal membrane oxygenation, turn on the pump to an initial flow rate of 0.1 milliliter per minute. After two minutes, adjust the flow rate to three to five milliliters per minute. Under stable flow, monitor the vital parameters via the data acquisition device in the real-time mode, continuously checking the back flow from the venous drainage, and monitoring the level of blood in the air trapper reservoir.
Use a one milliliter syringe equipped with a 24-gauge branula to collect any blood leakage, and return the blood to the ECMO circuit via the air trapping reservoir. For blood gas analysis, 10 minutes after ECMO initiation, use a blood sampling cartridge to collect approximately 75 microliters of arterial blood from the inferior vena cava before the oxygenator, directly after the oxygenator, and from the femoral artery. 30 and 60 minutes after ECMO initiation, collect blood from the femoral artery only.
45 and 90 minutes after initiation, administer 0.1 milliliter of priming solution to compensate for intravasal liquid loss via the air trapper. Two hours after initiation, collect blood from the oxygenator, inferior vena cava, and femoral artery. After the last blood collection, reduce the flow rate on the pump over the course of five minutes, thereby stopping the ECMO, while continuing to record the vital parameters for another 10 minutes.
In an typical experiment, the animal's physiological parameters are recorded every 10 minutes. In this representative experiment, the hematological parameters demonstrated a relevant hemodilution during ECMO, although no blood transfusion was necessary to compensate for moderate anemia. The oxygenation parameters displayed a proper oxygenator performance at an oxygen air mixture at a fraction of inspired oxygen of 1.0.
Further, the metabolic changes during ECMO included respiratory alkalosis at the start and at the end of the experiment. No extra blood buffering was performed. It's important to use the proper cannulation technique to absorb blood flows through the ECMO circuit, to monitor the oxygenation parameters via blood gas analysis, and to replace any blood loss caused by blood sampling with extra priming solution.
This technique paves the way for researchers in the field of lung disease to significantly improve protocols for life-saving treatments through the use of over 80 available knock-in, knock-out mouse models.
