Experimental protocol
We conducted a randomized, prospective study in sheep, using four different models of ARDS. The Animal Ethical Committee of the Maastricht University Medical Centre (MUMC+), the Netherlands, approved the study.
Pulmonary causes of ARDS were mimicked using lung lavage, intratracheal administrating of albumin 20%, or hydrochloric acid (10-14). Extra-pulmonary ARDS was induced by administrating intravenous lipopolysaccharide (LPS) from E.coli (10,12,14).
Twenty-seven adult Texel ewes, age 1 year plus minus 3 months, were intravenously anesthetized with thiopental, intubated, and mechanically ventilated with a pressure-controlled ventilation (PCV) (Servo 900 ventilator, Siemens, Germany) (18,19). The ventilator settings consisted of a peak pressure to maintain a tidal volume (Vt) of 6-8 mL/kg, a respiratory rate of 15 breaths per minute, an inspiratory time of 50%, a positive end expiratory pressure (PEEP) of 10 cm H2O, and a fraction of inspired oxygen (FiO2) of 1.0 (18,19). A central venous and arterial catheter were inserted. The sheep were sedated during the experiment with intravenously administered midazolam (0.2 mg/kg/h), ketamine (8 mg/kg/h), and paralyzed with repeated boluses of pancuronium (0.1 mg/kg) if needed (18,19). The sheep were randomly assigned to four different groups. The sheep were selected by the animal technician, who was not involved in the experiment and the insult was assigned according the date of the research. The groups varied according to the insult by which the ARDS was induced:
- Warm saline broncho-alveolar lavage (n = 6) (10,12,14). In this group, a broncho-alveolar lavage was performed using 500 mL per kilogram body weight of sterile 0.9% NaCl at 39°C. This procedure was repeated every 15 minutes until the arterial oxygen pressure (PaO2) had decreased to 15 kPa (112 mmHg) at PEEP of 10 cm H2O and FiO2 of 1.0.
- Intratracheal albumin 20% (n = 10) (15,16). In this group, 250 mg albumin 20% per kilogram body weight was administered intratracheally through a small tube that was temporarily placed in the endotracheal tube.
- Intratracheal hydrochloric acid (HCl) (n = 6) (10,12-14). This group was given HCl 0.5N 1.5 mL per kilogram body weight intratracheally through a small tube that was temporarily inserted in the endotracheal tube and advanced to the carina before injection.
- Intravenous lipopolysaccharide (LPS iv) (n = 5) (10,12,14). This group received 75 µg per kilogram body weight LPS of E. coli serotype 0.5B55 (Sigma Aldrich, Amsterdam The Netherlands) intravenously every hour over the course of the experiment, until the criteria of ARDS had been met.
ARDS was pre-defined as PaO2 < 15 kPa (112 mmHg) when ventilated with PEEP 10 cm H2O and FiO2 1.0 (15,16). The length of time that passed between the initiation of lung injury and meeting the ARDS criteria was recorded. In order to make a comparison between the groups possible, the time point at which the animals fulfilled the criteria of ARDS was taken as the starting point of the 4-hour study period (T= 0).
Arterial analyses for gas exchange were performed at baseline and every 30 minutes thereafter for the duration of the experiment. Cardio-respiratory settings were recorded every 30 min. Plasma from blood samples were collected every 30 min, spun down and stored at -80ºC. After four hours, the animals were euthanized by an intravenous injection of thiopental (18,19). Thoracotomy was performed and the lungs were removed. The left lung was lavaged with 0.9% NaCl and broncho-alveolar lavage fluid (BALF) was collected and analyzed for differential cell counts. The remaining volume was aliquoted and frozen for measurement of total protein, disaturated phospholipids (DSPL), and interleukin-6 (IL-6) and interleukin-8 (IL-8) at -80ºC. The inflated right lower lobe of the lung was fixed in buffered 4% formaldehyde and samples were taken for histology and lung injury score.
Oxygenation index (OI) and ventilation efficacy index (VEI)
As markers of gas exchange, OI and VEI were calculated from the cardio-respiratory parameters and blood gas analyses before and after lavage, and every 30 minutes during the 4-hour study period (18,19). OI was calculated by multiplying the oxygen fraction times mean airway pressure (cm H2O) and dividing this by PaO2 (mm Hg) ((FiO2 x MAP)/PaO2). The VEI was calculated by dividing 3800 by the pressure difference between the PEEP and peak pressure (in cm H2O) times the respiratory rate (breaths/min) times the PaCO2 (mm Hg) (3800/(PIP-PEEP) x RR x PaCO2).
Inflammatory cell count and differentiation in BALF
A cell count was performed after mixing the BALF with 0.4% trypan blue stain, (Gibco Invitrogen Corporation, California, USA) using the modified Neubauer Hemocytometer (Hirschmann EM Techcolor) (20). A May-Grünwald-Giemsa staining was performed on the BALF smears, in which all neutrophils, lymphocytes and monocytes were counted in 200 cells. For each cell type, the total number of cells was calculated per kilogram body weight (20). This was used as marker for inflammation.
Total protein in BALF
The protein concentration of the BALF was measured using the Micro BCA Protein Assay Reagent Kit (Thermo Fisher Scientific Inc., Illinois, USA), according to the manufacturer’s instructions. The microtiter plate was read at a wavelength of 562 nm. All photospectro-analyses were done using Multiskan Spectrum hardware and SkanIt RE for MSS 2.2 software (Thermo Electron Corporation, Massachusetts, USA). Translation of optical densities into concentrations was carried out using GraphPad Prism 5 software (GraphPad Software, California, USA) (21,22). Total protein in BALF was used as a marker for lung injury and interstitial edema.
Disaturated phospholipids in BALF
The DSPL in BALF was measured after 10 minutes of centrifugation of the BALF at 300xg and 4ºC, and 1 mL of the supernatant BALF was evaporated overnight at 60°C under continuous nitrogen gas flow. The dry BALF was dissolved in a mixture of carbon tetrachloride and osmium tetroxide and disaturated phospholipids were isolated according to Mason et al (23). The DSPL were dissolved in chloroform and quantified according to Stewart, with some minor modifications, as described by Been et al (24,25). The DSPL in BALF was taken as a marker for the surfactant pool size.
Interleukin (IL)-6 and IL-8 in BALF; IL-6 in plasma
In the BALF and in the plasma, respectively, the concentration of IL-6 and IL-8 were quantified with a sheep-specific sandwich ELISA (21,26) and used as markers of lung and systemic inflammation.
Histology and lung injury score
We evaluated lung injury using histology of the right lower lobe of the lung, by measuring the mean alveolar size (MAS), alveolar wall thickness (AWT) and the lung injury score system by Matute-Bello et al. (27). MAS was used as a marker of atelectasis and overinflation, while AWT was used as a marker for edema. The lung injury score system scored neutrophils in the alveolar space (A), neutrophils in the interstitial space (B), hyaline membranes (C), proteinaceous debris filling the airspace (D) and alveolar septal thickening (E). Every item was given a score between 0 to 2. The score was calculated by: ((20 x A) + (14 x B) + ( 7 x C ) + (7 x D) +(2 x E))/number of fields x 100, leading to a score between zero (no lung injury) and one (severe lung injury) (27). Samples of the lung were imbedded in paraffin, cut into 4 μm slices, deparaffinized in an ethanol series, stained with hematoxylin and eosin, dehydrated, and cover slipped. To measure MAS and AWT per location, 10 representative microscopic images were made with a 10-fold magnification using the Leica microscope (Axioskop 4.0; Zeiss) and Leica Qwin Pro version 3.4.0 software (Leica Microsystems, Mannheim, Germany). All images were uploaded and analyzed by Matlab 6.0 software (The Mathworks, Inc. Massachusetts, USA) to measure MAS and AWT in a blinded manner. To calculated the lung injury score 20 random high-power fields (400x total magnification) were scored per animal, by a blinded researcher, and the lung injury score was calculated per animal (27).
Statistical analysis
Statistical analysis was performed using one-way ANOVA with a Bonferroni post-hoc test in Graphpad Prism. Data are presented as mean ± SEM unless individual measurements are shown; p < 0.05 was considered to be statistically significant.