Animals
Fifteen Sprague-Dawley (SD) rats (350 ± 50 g) were used for all the experiments and randomly divided into 2 groups: OA group (n = 3) and VV-ECMO group (n = 12). All animals received humane care in compliance with the ‘Principles of laboratory animal care’ formulated by the National Society for Medical Research and the ‘Guide for the care and use of laboratory animal resources’ published by the US National Institute of Health (NIH publication No. 85-23, revised 1996). The following experimental protocol was approved by the local Ethical Committee of Lanzhou Second Hospital, Lanzhou University, Gansu.
Induction of lung injury
ARDS and impaired gas exchange were modeled in rats by intravenous administration of 99% pure OA (Sigma-Aldrich) as previously described [4, 5]. OA and 9mL physiologic saline solution (negative control) were given at a final concentration of 25mg/mL. 100mg/kg of OA was administered via the femoral vein at an infusion rate of 20mg/kg/min over 5 mins to avoid sudden death due to massive pulmonary embolism [6].
Extracorporeal membrane oxygenation circuit
The ECMO circuit consisted of a peristaltic pump (PreFluid Co. Ltd.) (Figure 1A), membrane oxygenator and tubing (Xijing Medical Co. Ltd.) (Figure 1B), 24G catheter (Figure 1C), and a 5.5 F specially designed three-cavity catheter (Figure 1D). In contrast to cardiopulmonary bypass circuits, this circuit lacked a reservoir unit, minimizing blood-air contact [7-9]. The membrane oxygenator (oxygenation area 0.02 m2) used during this procedure was specifically designed for this study. The entire circuit priming volume was 6 mL, with the membrane oxygenator constituting 3 mL. Priming was done using 6% HES130/0.4 (Fresenius Co. Ltd.) (3 mL) and Ringer’s solution (3 mL), without blood.
Surgical procedure
Rats were anesthetized using 2% sevoflurane to achieve stable anesthesia during the entire operation. Initial ventilatory parameters included 70-75 breaths/min, a tidal volume of 6 mL/kg, a 1:2 I:E ratio, and a 3 cmH2O positive end-expiratory pressure (PEEP). A 24-gauge catheter was inserted in the right femoral artery for continuous arterial pressure monitoring. Heparin was administered at 300 U/kg. The right jugular vein was exposed and cannulated with a 5.5 F specially designed three-cavity catheter. Central temperature was monitored using a rectal probe and maintained at 36 ± 0.5℃ using a heating lamp. Venous return was exclusively drained using a roller pump. ECMO was established at a flow rate of 80-90 mL/kg/min. Upon reaching optimal flow rate, it was adjusted to a level that could maintain desired arterial pressure. Gas flow (90% O2) was initiated at around 80-100mL/min and adjusted in terms of sweep rate and FiO2 to maintain blood gasses within the physiological range. 1mL of sodium bicarbonate is usually added to the circuit during ECMO to maintain acid-base balance. All volume substituting or buffering solutions were given via the femoral artery. After 3 h, the animals were weaned off ECMO and the remaining priming volume re-infused (Additional file video 1).
Blood gas analysis, protein concentration in bronchoalveolar lavage, and histology
To assess the oxygenation and metabolic states of the animals during VV-ECMO (EG7+, iStar, Abbott Co. Ltd.), blood gas analysis (BGA) was done on blood samples from the femoral artery. Femoral arterial blood gas measurements were done at 0 (T0, baseline), 1 h (T1, after OA modeling), and 3.5 h (T2, after VV-ECMO support). At the end of the experiment, bronchoalveolar lavage fluid (BALF) was harvested by lavaging the lungs with 2.5mL PBS 1X and BALF protein concentration determined using BCA analysis. The lung was collected and fixed in 4% paraformaldehyde for 24 h at 4 °C. Four micrometer paraffin sections were stained with hematoxylin and eosin (H&E), and organ damaged evaluated histologically. Lung injury score was evaluated using a modified, previously described scoring system [6]. Various degrees of lung injury score were designated degree 0, 1, 2, and 3 for mild, moderate, and severe edema, respectively. Similar scoring was used for inflammatory cell infiltration, with degree 0, 1, 2, and 3, indicating none, mild, moderate, and severe cellular infiltration, respectively. Histopathological assessment was done by several blinded laboratory assistants with each giving scores for edema and cell infiltration. The individual edema and cell filtration scores were then summed to obtain a final score ranging from 0-6.