Animal study design
The animal care and use committee of the Academic Medical Center, University of Amsterdam, Netherlands approved this study. Animal procedures were carried out in compliance with Institutional Standards for Use of Animal Laboratory Animals.
Induction of lung injury, anesthesia, instrumentation, and mechanical ventilation
Male Sprague–Dawley rats (Harlan, The Hague, The Netherlands), weighing 350 to 400 g, were randomized into four experimental groups. Two groups were anesthetized using a transoral miniature nebulizer under light anesthesia (97% oxygen with 3% isoflurane) and intratracheally instilled with 1 mg/kg of Escherichia coli lipopolysaccharide (LPS) (L4131, 7.5 mg/kg, Sigma Aldrich, Steinheim, Germany). Control groups received no instillation. Two hours after LPS instillation, the animals were anesthetized by intraperitoneal injection of 90 mg/kg ketamine (Nimatek®; Eurovet Animal Health BV, Bladel, the Netherlands), 0.125 mg/kg dexmedetomidine (Dexdomitor®; Orion Pharma, Espoo, Finland), and 0.05 mg/kg atropine (atropine sulfate; Centrafarm BV, Etten-Leur, the Netherlands). Via a tail vein Venflon cannula, anesthesia was maintained by infusion of 10 mg/ml ketamine at 2.7 ml/h. A solution of saline and 4.2 mg/ml bicarbonate (Fresenius Kabi Nederland BV, Hertogenbosch, the Netherlands) was administered at 2.5 ml/h.
A tracheotomy was performed and a metal cannula was inserted into the trachea. Two sutures were placed around the exposed part of the trachea into which the cannula was tied down thoroughly. The cannula was then connected to a ventilator (Servo 900C, Siemens, Upplands Vasby, Sweden). The ventilators were calibrated for the heliox gas mixture according to the instruction of the manufacturer using a pressure reduction valve to allow the high-pressure of the heliox tank to be reduced to safe and usable pressures for ventilation (Linde Gas Therapeutics, Eindhoven, the Netherlands).
Hemodynamic parameters were monitored by inserting a polyethylene heparinized-saline (1:1,000)-filled catheter into the right carotid artery (Braun, Melsungen, Germany) that was connected to a monitor (Siemens SC900, Danvers, MA, USA). Temperature was monitored rectally (Ama-digit ad 15th, Amarell, Kreuzwertheim, Germany) and maintained at 37°C by a thermo mattress.
The rats were ventilated in a pressure-controlled mode for 4 h, with either heliox (technical gas 50% oxygen; 50% helium; blended by Linde Gas Therapeutics) or 50% oxygen-in-air gas mixture. In total, 32 animals were studied of which 16 received heliox (8 LPS, 8 healthy controls) and 16 received 50% oxygen-in-air gas mixture (8 LPS, 8 healthy controls).
Lung protective (LP) ventilation was maintained, according to a fixed protocol, by applying 6 ml/kg and 5 cm H2O positive end-expiratory pressure (PEEP). FiO2 was set at 50% with an inspiration to expiration ratio of 1:2 and adjustment of respiratory rate to maintain arterial PaCO2 within 4.5 to 6.0 kPa, according to hourly drawn arterial blood gases (RAPIDlab 865 blood gas analyzer, Bayern, Mijdrecht, the Netherlands).
Tidal volumes were strictly maintained using a pneumotachometer (Hugo Sachs Elektronik, Harvard apparatus, March-Hugstetten, Germany) specific for rats. The pneumotachometer is a transducer for airflow measurement, placed between the metal cannula and the ventilator. For both heliox and oxygen ventilation, the pneumotachometer was calibrated using a 1-ml syringe according to the manufacturer's instruction. Tidal volumes were recorded using respiration software (HSE-BDAS basic data acquisition, Harvard apparatus, March-Hugstetten, Germany) and displayed on a computer screen throughout the whole experiment. We set a pressure controlled ventilation mode and started with an inspiratory pressure of 15 cm H2O. The tidal volume was targeted by adjusting the inspiratory pressure [17, 19–21].
The inspiratory pressures were recorded every hour. The driving pressure was calculated by inspiratory pressure minus PEEP. Compliance was calculated by dividing the tidal volume per kilogram by the driving pressure. Minute volume was calculated in milliliter per minute by multiplying respiratory rate with the measured tidal volume.
Inflammation measurements
After 4 h of mechanical ventilation, the rats were bled and plasma was centrifuged at 1,800 × g for 10 min at 4°C. The lungs were removed en bloc and the right lung was ligated. A bronchoalveolar lavage was done by flushing the left lung three times with 2.0 ml NaCl, yielding approximately 5.5 to 6.0 ml of bronchoalveolar lavage fluid (BALF).
In BALF, the cells were counted using a hematocytometer (Z2 Coulter Particle Counter, Beckman Coulter Corporation; Hialeah, Florida, USA). After centrifugation of BALF (300 × g for 10 min at 4°C), protein levels were measured (Oz Biosciences, Marseille, France) and levels of interleukin (IL)-1β, IL-6, cytokine-induced neutrophil chemoattractant 3 (CINC-3), and tumor necrosis factor alpha (TNF-α) were determined by ELISA in BALF and blood, according to instructions of the manufacturer (R&D Systems, Abingdon, UK).
The upper lobe of the right lung was fixed in 1% buffered formaldehyde and subsequently embedded in paraffin and afterwards cut into 5-μm-thick sections. The lung sections were fixed on glass slides and stained with hematoxylin and eosin and were analyzed by a pathologist, who was blinded to group identity, with the use of total histology score. This score consists of several parameters, including interstitial inflammation, endothelialitis, edema, bronchitis, thrombus, and pleuritis. All parameters were scored on a scale of 0 to 4: 0 for normal lungs, 1 for <25% lung involvement, 2 for 25% to 50% involvement, 3 for 50% to 75% involvement, and 4 for >75% lung involvement. The total histology score was calculated as the sum score of these parameters, with a maximum of 24. Furthermore, the pathologist was asked to choose one representative illustration per group.
Statistical analysis
To compare time points (T = 0 vs. T = 4) within the same subject, a paired t test with Bonferroni correction was used if data were normally distributed, or Wilcoxon signed rank test in case of non-normal distribution. The effect of heliox versus oxygen-in-air at specific time points was compared using a one-way ANOVA or Kruskal-Wallis test, with either a Bonferroni's or Dunn's multiple comparison test, depending on the distribution of the data. Statistical significance was considered to be at P < 0.05 or at P < 0.0125 after Bonferroni correction. Data are expressed as mean ± SD.