All protocols were approved by the Lanzhou University Second Hospital (Lanzhou, Gansu, China) animal experimental ethics committee (approval number: D2021-020) and were in accordance with the animal care guidelines of the National Institutes of Health and Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. All efforts were made to minimize suffering and to reduce the number of rats used.
Experiments were performed in male Sprague-Dawley (SD) rats aged 12 weeks, weighing 350 ± 50g and purchased from Lanzhou Veterinary Research Institute, Chinese Academy of Sciences (Lanzhou, Gansu, China). All rats were housed in standard conditions, with a 12-h light/dark cycle, and ad-libitum access to food and water. Notably, all experiments were performed during the light cycle (from 9:00 am to 5:00 pm).
The experimental protocol
Animals were randomly assigned to two experimental groups (n=12 for each group): sevoflurane (Sevo) group and propofol (Pro) group. Rats were first anesthetized with 1% pentobarbital sodium 50mg/kg intraperitoneally, followed by fixing on the anatomical operating table in the supine position. Next, endotracheal intubation with a 16G needle was performed under direct vision and animals were connected to a rodent ventilator (Shanghai Alcott Biotech Co. Ltd., China). According to a previous study, the ventilator parameters were set as follows: tidal volume 6ml/kg, respiratory rate 70-75 breaths/min, positive end-expiratory pressure (PEEP) 2 cmH2O, and inhalation/exhalation ratio (I:E) 1:2. An electronic thermometer was placed in the anus to monitor the body temperature, and the temperature of the rats was maintained at 36±0.5℃ using a heating lamp. Notably, the right femoral artery and vein were exposed and free under local anesthesia with 1% lidocaine. A 24G intravenous indwelling needle was punctured into the femoral artery and a monitor was connected for continuous hemodynamic monitoring. ARDS was then induced by intravenous injection of 100mg/kg oleic acid (OA) (99%, Sigma-Aldrich, USA) as previously described. The arterial blood gas was measured 1 h after OA injection, and the PaO2/FiO2≤300 was used as the model standard of ARDS. The VV ECMO rat model was established as previously reported . After VV ECMO running, Sevo group rats inhaled 2% sevoflurane (Maruishi Pharmaceutical Co. Ltd., Japan), whereas Pro group rats were infused with 1% propofol 500μg·kg-1·min-1 (Corden Pharma S.P.A., USA). Notably, the whole experiment lasted for 3 h. Figure 1 shows the flow diagram of the experimental protocol.
Arterial blood gas analysis (BGA) and collection of bronchoalveolar lavage fluid (BALF)
Blood was collected from the femoral artery, and BGA was performed at the following three time points: T0 (baseline), T1 (the time to ARDS), and T2 (after weaning from ECMO for 1 h). At the end of the experiment, the left hilum was clipped with non-invasive blood vessels, and BALF was performed with 2.0 ml pre-cooled normal saline (0.9%). The lavage was repeated three times, and it was considered successful if the recovery rate was above 80%. Finally, the total protein content in the BALF was measured using Bicinchoninic acid (BCA) Protein Quantitation Kit (Solarbio, China) according to the manufacturer's protocol.
Lung wet/dry ratio (W/D)
Rats were sacrificed when the experimental period was complete. The left lung was isolated and weighed immediately (wet weight) after the lung surface liquid was absorbed using an absorbent paper. To obtain the dry weight, lungs were placed in a 60°C oven for 72 h until a constant weight was achieved. The lung wet/dry weight ratio was then calculated.
Hematoxylin-eosin (H&E) staining
The middle lobe of the left lung was fixed with 4% paraformaldehyde. After fixation and dehydration, the specimens were embedded in paraffin and cut into 5mm-thick sections. Next, sections were stained with hematoxylin and eosin in accordance with routine procedures of hematoxylin-eosin staining, followed by observation of the histopathological changes of lungs under a light microscope (Olympus, Japan). Two pathologists then observed and scored the extent of lung tissue damage using the double-blind method. The lung tissue was scored according to the following four items: congestion/edema in the alveolar cavity; lung tissue hemorrhage and thickening; inflammatory cell infiltration in the alveolar cavity and blood vessel cavity; and alveolar wall congestion, edema, thickening, and hyaline membrane form . The scores of the four aspects were added up to form a visual field lung injury score: 0 points for no pathological changes, 1 point for mild pathological changes, 2 points for moderate pathological changes, 3 points for severe pathological changes, and 4 points for very severe pathological changes. Finally, the 200 alveoli of lung tissue samples were examined at ×400 magnification to determine the lung injury score.
Sections were placed in the 60°C ovens to bake for 60 min. After dewaxing and rehydrating, antigen retrieval was performed with 10 mM citrate buffer (ZSGB-BIO, China) for 5 min. Next, sections were incubated with the rabbit anti-rat MPO (1:2000 dilution, Abcam, USA) primary antibody overnight at 4℃. On the next day, the sections were incubated with the biotinylated sheep anti-rabbit IgG secondary antibody at room temperature for 20 min, followed by staining with DAB from the SP-POD kit (ZSGB-BIO, China). Finally, sections were sealed with neutral resin and analyzed under a light microscope (Olympus, Japan). MPO-positive nuclei were counted in three randomly selected areas per section from each individual rat, and the percentage of MPO-positive nuclei was calculated for each area.
Enzyme-linked immunosorbent assay (ELISA)
BALF, serum, and lung specimens were used to evaluate the immunomodulatory function of the sedative. The cervical venous blood was collected and centrifuged at 3500rpm/min for 15 min at 4℃. The supernatant was then collected and stored at -80℃ until use. Next, lung tissues were homogenized and the supernatant was collected for analysis by ELISA. The level of TNF-α and IL-1b in different specimens was determined using the ELISA kit (Shanghai Enzyme-linked Biotechnology Co. Ltd., China) according to the manufacturer’s instructions.
All statistical analyses were performed using SPSS statistical software 22.0 (SPSS Inc, Chicago, IL, USA). Measurement data were expressed as mean ± standard deviation. Repeated measurement analysis of variance (ANOVA) was used to compare indexes at different time points in the same group, whereas one-way ANOVA was used to compare indexes at the same time points among different groups. P<0.05 was considered statistically significant.