Experiments were performed with healthy male C57Bl/6 (n = 66) and BALB/c mice (n = 66) (Charles River, Someren, the Netherlands), aged 8 to 10 weeks, with weights ranging from 19 to 25 g. Two groups of control animals served either as non-ventilated controls for blood gas analysis at baseline (n = 6 for each strain) or as non-ventilated controls after five hours (n = 12 for each strain). The other animals were all mechanically ventilated with two different MV-strategies and two different fluid support strategies. Thus, five groups of animals of each mice strain were compared.

Throughout the experiments, rectal temperature was maintained between 36.5 and 37.5°C using a warming path. Anaesthesia was achieved with intraperitoneal injection of a mix of 100 mg/ml ketamine (Eurovet Animal Health B.V., Bladel, the Netherlands), 1 mg/ml medetomidine (Pfizer Animal Health B.V., Capelle a/d IJssel, the Netherlands) and 0.5 mg/ml atropine (Pharmachemie, Haarlem, the Netherlands; KMA). Induction of anaesthesia was performed by injecting 7.5 μl/g of induction KMA mix (consisting of 1.26 ml ketamine, 0.2 ml medetomidine and 1 ml atropine). To maintain anaesthesia, 10 μl/g of maintenance KMA mix (consisting of 0.72 ml ketamine, 0.08 ml medetomidine and 0.3 ml atropine) was given, via an intraperitoneally placed catheter every hour.

A Y-tube connector, 1.0 mm outer diameter and 0.6 mm inner diameter (VBM Medizintechnik GmbH, Sulz am Neckar, Germany) was surgically inserted into the trachea under general anaesthesia. Mice were placed in a supine position and connected to a ventilator (Servo 900 C, Siemens, Sweden). Simultaneously, six mice were pressure-controlled ventilated with either an inspiratory pressure of 10 cmH₂O (resulting in V_T of about 7.5 ml/kg; low V_T (LV_T)) or an inspiratory pressure of 18 cmH₂O (resulting in V_T of about 15 ml/kg; high V_T (HV_T)).

In C57Bl/6 mice, respiratory rate was set at 120 breaths/minute and 70 breaths/minute with LV_T and HV_T, respectively; in BALB/c mice, respiratory rate was set at 100 breaths/minute and 70 breaths/minute with LV_T and HV_T, respectively. Preliminary studies showed these respiratory settings resulted in normal partial pressure of arterial carbon dioxide (PaCO₂) values after five hours of MV in the different mice strains. Positive end-expiratory pressure (PEEP) was set at 2 cmH₂O with both MV strategies. The fraction of inspired oxygen was kept at 0.5 throughout the experiment. The inspiration to expiration ratio was kept at 1:1 throughout the experiment.

Mice received an intraperitoneal bolus of 1 ml normal saline one hour before the start of MV, followed by 0.2 ml normal saline (sodium chloride (NaCl) 0.9%) or 0.2 ml sodium bicarbonate (containing 200 mM sodium and bicarbonate) administered via the intraperitoneal catheter every 30 minutes. Preliminary studies showed this fluid strategy to adequately compensate for insensible and observed fluid loss, and to keep the animals haemodynamically stable.

Systolic blood pressure and heart rate were non-invasively monitored using a murine tail-cuff system (ADInstruments, Spenbach, Germany). Blood pressure and pulse were measured directly after the start of MV, after 2.5 hours and 5 hours of MV. The data were recorded on a data acquisition system (PowerLab/4SP, ADInstruments, Spenbach, Germany). An average systolic blood pressure and heart rate were taken from three consecutive measurements.

V_T was checked hourly with a specially designed Fleisch-tube connected to the body-plethysmograph. The flow signal was integrated from a differential pressure transducer and data were recorded and digitised online using a 16-channel data acquisition program (ATCODAS, Dataq Instruments Inc, Akron, OH) and stored on a computer for post acquisition off-line analysis. A minimum of five consecutive breaths were selected for analysis of the digitised V_T signals.

Non-ventilated control mice were selected for blood gas analysis at baseline (for both strains n = 6): animals were handled one week before the experiment to decrease stress activation. After induction of anaesthesia with isoflurane arterial blood was taken from the left ventricle by heart puncture within 30 seconds.

LV_T mice receiving either normal saline (n = 12) or sodium bicarbonate (n = 12) and HV_T mice receiving either saline (n = 12) or sodium bicarbonate (n = 12) were mechanically ventilated for five hours and then euthanased. Non-ventilated control mice (n = 12) received half the dose of induction anaesthesia, were spontaneously breathing and then euthanased after five hours.

The first series of mice (n = 6) were euthanased and blood was drawn from the vena cava inferior into a sterile syringe, transferred to EDTA-coated tubes and immediately placed on ice. Blood samples of two mice were pooled together. Bronchoalveolar lavage fluid (BALF) was obtained from the right lung; the left lung was used to measure the wet-to-dry ratio. In a second series of mice (n = 6), blood was sampled from the carotid artery for blood gas analysis. The lungs of these mice were used for homogenate (right lung) and histopathology (left lung).

For wet-to-dry ratios the lung was weighed and subsequently dried for three days in an oven at 65°C. The right lung was removed and snap frozen in liquid nitrogen. These frozen specimens were suspended in four volumes of sterile isotonic saline and subsequently lysed in one volume of lysis buffer (150 mM NaCl, 15 mM Tris (tris(hydroxymethyl)aminomethane), 1 mM MgCl.H₂O, 1 mM CaCl₂, 1% Triton X-100, 100 μg/mL pepstatin A, leupeptin and aprotinin, pH 7.4) and incubated at 4°C for 30 minutes. Homogenates were spun at 3400 rpm at 4°C for 15 minutes after which the supernatants were stored at -20°C until assayed.

BALF was obtained by instilling three times 0.5 ml aliquots of saline by a 22-gauge Abbocath–T catheter (Abbott, Sligo, Ireland) into the trachea. About 1.0 ml of BALF was retrieved per mouse and cell counts were determined using a haemacytometer (Beckman Coulter, Fullerton, CA). Subsequently, differential counts were performed on citospin preparations stained with a modified Giemsa stain, Diff-Quick (Dade Behring AG, Düdingen, Switzerland). Supernatant was stored at -80°C for meausrement of total protein level, thrombin-antithrombin complexes (TATc) and plasminogen activator inhibitor (PAI)-1.

For histopathology lungs were fixed in 4% formalin and embedded in paraffin. Sections 4 μm in diameter were stained with H&E and analysed by a pathologist who was blinded for group identity. To score lung injury we used a modified VILI histopathology scoring system as previously described 2. VILI was scored according to the following four items: alveolar congestion; haemorrhage; infiltration or aggregation of neutrophils in airspace or vessel wall; and thickness of the alveolar wall/hyaline membrane formation. A score of 0 represented normal lungs; 1 represented mild, less than 25% lung involvement; 2 represented moderate, 25 to 50% lung involvement; 3 represented severe, 50 to 75% lung involvement; and 4 represented very severe, more than 75% lung involvement. An overall score of VILI was obtained based on the summation of all the scores from normal or ventilated lungs (n = 12 per group).

Total protein levels in BALF were determined using a Bradford Protein Assay Kit (OZ Biosciences, Marseille, France) according to the manufacturers' instructions with BSA as standard. Cytokine levels in blood lung homogenates were measured by ELISA according to the manufacturer's instructions. Tumour necrosis factor (TNF) α, interleukin (IL) 6, macrophage inflammatory protein (MIP) 2 and keratinocyte-derived chemokine (KC) assays were all obtained from R&D Systems (Abingdon, UK). TATc levels in BALF were measured with a mouse specific ELISA as previously described 16. Levels of PAI-1 were measured by means of a commercially available ELISA (Kordia, Leiden, the Netherlands).

All data in the results are expressed as mean ± standard deviation or median ± interquartile range (IQR), where appropriate. To detect differences between groups the Dunnett method or Mann Whitney U test, in conjunction with two-way analysis of variance was performed. Haemodynamics were measured in 12 animals, all other measurements were performed in six animals. A p value of less than 0.05 was considered significantly. All statistical analyses were carried out using SPSS 12.0.2 (SPSS, Chicago, IL).

All animals survived five hours of MV after which they were euthanased; control animals survived anaesthesia and were also euthanased after five hours. The systolic blood pressure and heart rate remained stable in all animals for the entire duration of the experiment, with no differences noted between mice strains, MV strategies and fluid strategies. Although blood gas analysis from LV_T mice and HV_T mice using normal saline revealed metabolic acidosis after five hours of MV (in C57Bl/6 mice pH with LV_T = 7.17 ± 0.07 and pH with HV_T = 7.23 ± 0.06, and in BALB/c mice pH with LV_T = 7.22 ± 0.04 and pH with HV_T = 7.11 ± 0.07, Tables 1 and 2) with the use of sodium bicarbonate metabolic acidosis was prevented (in C57Bl/6 mice pH with LV_T = 7.41 ± 0.07 and pH with HV_T = 7.49 ± 0.02, and in BALB/c mice pH with LV_T = 7.42 ± 0.05 and pH with HV_T = 7.37 ± 0.08). Arterial oxygenation in C57Bl/6 mice was significantly higher in HV_T-mice as compared with LV_T-mice (205 ± 33 vs. 141 ± 22 mmHg, p < 0.001). No differences regarding oxygenation were found between MV-groups in BALB/c mice (partial pressure of arterial oxygen (PaO₂) for HV_T = 167 ± 50 and PaO₂ for LV_T = 181 ± 42 mmHg).

The histopathological changes were minor (Figure 1 and Table 3). For both mice strains the lung histopathology score was higher in HV_T mice as compared with controls. However, no differences were noted between mice strains, MV strategies and fluid strategies.

In C57Bl/6 mice lung wet-to-dry ratios were significantly higher with both MV strategies compared with controls (LV_T mice = 4.8 ± 0.3 and HV_T mice = 5.3 ± 0.5, as compared with control mice = 4.2 ± 0.2; p < 0.01). Wet-to-dry ratios in HV_T mice were also significantly higher as compared with LV_T mice (p = .009). For BALB/c mice higher lung wet to dry ratios were found in HV_T mice (5.6 ± 0.6 as compared with 4.6 ± 0.4 in LV_T mice (p < 0.001) and 4.5 ± 0.2 in control mice (p < 0.001), respectively). No significant differences were found between LV_T mice and control mice.

Total BALF protein levels in C57Bl/6 were significantly higher in HV_T mice as compared with LV_T mice (p = .012) and control mice (p = .008; Figure 2). No significant difference was found between LV_T mice and control mice. In BALB/c mice, total BALF protein levels were significantly higher in HV_T mice as compared with LV_T mice and control mice (p < .001). No significant difference was found between LV_T mice and control mice.

The numbers of neutrophils in BALF were significantly higher in HV_T mice as compared with control mice in both mice strains (Figure 1 and Table 3). Neutrophil counts in BALF from HV_T mice did not differ from LV_Tmice.

In the HV_T group of both mice strains, higher pulmonary levels of TNF-α were found as compared with the LV_T group (p < 0.05) and control group (p≤ 0.001; Figure 3). In BALBc mice only, pulmonary levels of TNF-α in LV_T mice were higher as compared with control mice (p = 0.018). Pulmonary levels of IL-6 in the HV_T group of both mice strain were higher as compared with the LV_T group and control group. Only for BALBc mice a significant difference between LV_T mice and control mice were found. For pulmonary levels of MIP-2 in C57Bl/6 mice higher levels were found in HV_T mice and LV_T mice as compared with control (p = 0.001). No difference was found between LV_T mice and HV_T mice in this mice strain. In BALBc mice, higher pulmonary levels of MIP-2 in the HV_T group were found as compared with the LV_T group and control group, with also a significant difference between HV_T mice and LV_T mice. In both mice strain higher pulmonary levels of KC were found in the HV_T group as compared with the LV_T group and control group (p = 0.001). Only in BALBc mice, there was also a significant difference between LV_T mice and control mice.

Plasma levels of IL-6 and KC were elevated in the both ventilation groups, with higher levels in the HV_T group (Figure 4). Plasma levels of TNF-α and MIP-2 were below the detection limit of the assay (data not shown).

TATc levels in BALF were significantly higher in HV_T mice in both mice strain as compared with LV_T mice and control (p < 0.001; Figure 5). No significant difference was found between LV_T mice and control mice in both mice strain. Levels of PAI-1 were not significantly different in C57Bl/6 mice. BALB/c mice did show increased PAI-1 levels in the HV_Tgroup as compared with the LV_T group and control group (p < 0.001). No differences were found between LV_T mice and control mice.

The different fluid support strategies showed no difference in endpoint of VILI, except for pulmonary MIP-2 and IL-6 levels in C57Bl/6 mice. MIP-2 levels were significantly higher in HV_T mice and LV_T mice that received sodium bicarbonate as compared with mice that received normal saline (p < 0.01; Figure 3). Pulmonary IL-6 levels were significantly higher in HV_T mice receiving sodium bicarbonate as compared with mice receiving normal saline (p = 0.026).