Animals
Nineteen male C57Bl/6 mice were obtained from Charles River (Sulzfeld, Germany) and used for the experiments. Animals were randomly divided into sham-operated (n = 6) animals, 4 h vv-ECMO (n = 8) animals, and animals used in the design of the double lumen cannula (n = 5, see below). The weight of mice ranged between 25 and 35 g. This study was performed in compliance with the German Animal Protection Law (TierSchG) and was approved by the local animal welfare committee (Lower Saxony State Office for Consumer Protection and Food Safety, Protocol TSA 33.12-42502-04-16/2250).
Design and construction of the double lumen cannula
A 2F double lumen silicone-based catheter (Vygon GmbH & CO.KG Medizintechnik, Aachen, Germany) was used as the basis for the production of the double lumen cannula (Fig. 1). Using a sharp blade, outflow fenestrations were made in the catheter at the eventual site of the superior vena cava and inferior vena cava (Fig. 2). Similarly, inflow fenestrations were made at the height of the right atrium. To ensure optimal positioning of the fenestrations, multiple measurements were performed on five mouse cadavers of a similar age/size to the experimental mice. For optimal drainage and minimal shunting, venous outflow fenestrations were made 0.2 mm, 0.4 mm, and 20 mm from the distal end. Atrial inflow fenestrations were made 0.4 mm and 0.5 mm from the distal end. To prevent recirculation and shunting of the blood, the distal end of the inflow cannula lumen was sealed.
Surgical procedure and extracorporeal membrane oxygenation
Surgical preparation (Fig. 3) and ECMO setup were carried out as previously described [12]. In brief, all animals were anesthetized with isoflurane mask narcosis (Fig. 3 (a)) and spontaneous breathing was maintained. Subcutaneous carprofen (Zoetis, Parsippany, NJ, USA) injections were given as additional analgesia (5 mg/kg body weight). Perfusion solution consisted of a 1:1 solution of Tetraspan: Sterofundin (B Braun Medical, Melsungen, Hesse, Germany) that had been heparinized (30 IU/ml). Prior to cannulation, the ECMO circuit was primed with 500 μl of perfusion solution. Buffering of the solution was carried out using 2.5% v/v of an 8.4% solution of sodium bicarbonate. An arterial pressure line in the left femoral artery was used for blood sampling and blood pressure monitoring.
For cannulation, a lateral skin incision on the left side of the neck was made to expose the left jugular vein. An 8-0 silk suture was placed cranially to ligate the distal segment, and a slip knot was placed at the proximal end of the vein. After introducing the double lumen cannula into the left jugular vein (Fig. 3 (b)), it was moved 3.5 cm in the direction of the superior vena cava, and further into the inferior vena cava. The cannula was then secured using slip-knots. After confirmation of correct position of the cannula, extracorporeal circulation was started and continued for 4 h. In the sham group, the surgical procedure for cannulation was identical to the ECMO animals; however, no extracorporeal circulation was commenced. In both ECMO- and sham-treated animals, the procedure was performed for 4 h.
Blood gas analysis
Blood gas analysis (BGA) was performed from blood sampled from the femoral artery to evaluate the oxygenation and metabolic state of animals undergoing vv-ECMO.
Blood tests of kidney and liver function
Blood samples were collected via the femoral artery prior to the procedure and via exsanguination after termination of the experiment. Serum was stored at − 20 °C for later analysis. Clinical chemistry was done using an Olympus analyzer (AU 400) according to the manufacturer’s instructions to evaluate liver enzymes (glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT)) and kidney function (creatinine, urea) after vv-ECMO. All values were corrected for hemodilution as previously described [13]. Furthermore, to evaluate hemolysis during the experiment, lactate dehydrogenase (LDH) values were compared between the start of the experiment and after 4 hours.
Histology
For organ fixation, lungs were filled with 4% paraformaldehyde via injection into the trachea. Filled lungs were explanted and incubated in 4% formalin overnight at 4 °C. After dehydration and deparaffinization, lungs were stained with hematoxylin and eosin (H&E) and histologically assessed for pulmonary damage. The liver and kidney were also collected, fixed in 4% paraformaldehyde, and stored for 24 h at 4 °C. Two-micrometer paraffin sections were stained with periodic acid-Schiff (PAS) to evaluate kidney and liver morphology. Assessment of acute kidney injury (AKI) and liver damage was done using a method previously described [13].
Statistics
Statistical analyses were performed using GraphPad Prism version 5.0 software (GraphPad Software Inc., San Diego, CA, USA). The Kolmogorov-Smirnov test revealed the data was normally distributed. One-way ANOVA with post hoc Bonferroni tests were used for statistical analysis. Unless otherwise stated, data are presented as mean ± standard deviation (SD).