Specific pathogen free adult male Sprague Dawley rats (B&K Universal Ltd., U.K.) weighing 300 to 380 g were used in all experiments. Animals were housed for a minimum of 7 days prior to study, under controlled light/dark conditions where the light period was from 8:00 a.m. to 8:00 p.m. They were allowed access to ordinary rat chow and tap water ad libitum. Animals were maintained at constant ambient temperature of 21–22°C, while the humidity level was maintained between 45% and 55%.

The experimental model was based on that previously reported, with several modifications 89101112. Briefly, anesthesia was induced and maintained with Isoflurane (Abbott Laboratories, UK). After confirming depth of anesthesia by absence of response to paw compression, a tail vein was cannulated using a 24 gauge cannula (Becton Dickinson, NJ). Oleic acid (OA) (Sigma-Aldrich, United Kingdom), which was prepared as a 1:1 mixture with pure ethanol 17, was administered intravenously. Animals were then randomized to hypertonic saline (HTS) or control groups. The HTS group were then administered 4 ml.Kg^-1 of 7.5% hypertonic saline and the control animals administered an equal volume of 0.9% saline. The animals were then allowed to recover from anesthesia, and then placed in their cages.

Two preliminary series of experiments was carried out to determine the dose of OA and duration of time required to produce a significant evolving ALI, and to produce a model of resolving lung injury. A dose of 40 μl of OA was determined to produce a significant early lung injury over a 4 h period. In later studies, a dose of 9 μl per 100 g body weight was found to produce a lung injury of similar magnitude at 4 hours, and which was resolving at 6 hours following OA administration.

This series was carried out to determine the potential for HTS to attenuate evolving OA-induced injury. Following induction of anesthesia, 40 μl of OA was administered and each rat allowed to recover. Four hours following OA administration, the animals were re-anesthetized and the degree of ALI quantified.

This series was carried out to determine whether HTS could modulate the resolution of OA-induced injury. Following induction of anesthesia, 9 μl.100 g^-1 body weight of OA was administered and each rat was allowed to recover. Six hours following OA administration, the animals were re-anesthetized and the extent of lung injury quantified.

At four (Series I) or six (Series II) hours following OA administration, the animals were re-anesthetized with Isoflurane. After confirming depth of anesthesia by absence of response to paw compression, the dorsal penile vein and carotid artery were cannulated using 22G cannulae (Becton Dickinson, NJ). A tracheotomy was performed, and a tracheal tube was inserted to a depth of 2 cm and secured in place. Pancuronium (1 mg; Organon, The Netherlands) was administered intravenously and the lungs were then ventilated using a small animal ventilator (Model 683; Harvard Apparatus, United Kingdom) with a fractional inspired O₂ (FiO₂) of 0.21 (room air), respiratory rate 90 min.^-1, tidal volume 4.5 ml.kg^-1 and 2 cm H₂O positive end-expiratory pressure (PEEP).

Systemic arterial pressure, peak airway pressures and temperature were measured throughout the protocol. Arterial blood samples then were drawn for assessment of systemic oxygenation, ventilation and acid-base status (ABL 710, Radiometer, United Kingdom). Static inflation lung compliance was measured immediately prior to a recruitment manoeuvre, ensuring a standardized lung volume history. Incremental 1 ml volumes of room air were injected via the tracheal cannula, and the pressure attained 3 seconds after each injection was measured, until a total volume of 5 ml was injected. At the end of the protocol, the inspired gas was altered to FiO₂ of 1.0 for 5 minutes, and an arterial blood sample was then taken for calculation of alveolar-arterial oxygen gradient. The animals were then euthanized by exsanguination under anesthesia.

Immediately post mortem, the heart-lung block was dissected from the thorax and bronchoalveolar lavage (BAL) performed. BAL was carried out by intratracheal instillation of 3 aliquots (5 ml each) of isotonic saline and collection of the returned fluid by free drainage. Total cell numbers per ml in BAL fluid were counted and differential cell counts were performed following staining with Diff-Quik (BDH, United Kingdom). Samples of BAL fluid were centrifuged, snap frozen in liquid N₂ and stored at -80°C. The concentration of interleukin-6 (IL-6) in these BAL samples was determined using quantitative sandwich ELISA's (R&D Systems, United Kingdom). The BAL protein concentration was determined using a standard assay (Biorad Assay, Biorad, Hercules, CA).

The left lung was isolated and fixed with 4% paraformaldehyde 1819, and the extent of histologic lung damage determined using quantitative stereological techniques as previously described 20.

Results are expressed as mean ± (SEM) for normally distributed data, and as median (interquartile range, IQR) if non-normally distributed. Data were analyzed by one-way ANOVA followed by Student-Newman-Keuls, t-test or Kruskalis-Wallis followed by Mann-Whitney U test with the Bonferroni correction for multiple comparisons, as appropriate. P < 0.05 was considered statistically significant.

12 animals were entered into this study. All animals entered into the study survived the full duration of the protocol.

24 animals were entered into this study. All animals entered into the study survived the full duration of the protocol.

The neutrophil-endothelial cell interaction is central to the pathogenesis of ALI/ARDS. HTS is a potent inhibitor of neutrophil function, downregulating neutrophil oxidative burst activity 21, reducing neutrophil adhesion molecule expression 22 and suppressing neutrophil activation and the release of pro-inflammatory cytokines 523. HTS decreased lung neutrophil infiltration 2425 and ICAM-1 25 expression in the setting of hemorrhagic shock.

HTS has been demonstrated to reduce pulmonary edema, histologic injury, and lung neutrophil infiltration following systemic ischemia-reperfusion injury 1. HTS also reduced serum IL-6 and TNF-α cytokine levels in this study. HTS reduced the increase in lung permeability and lung neutrophil sequestration and activation following trauma-haemorrhagic shock 26. HTS resuscitation also increased survival, and reduced lung injury, in a two-hit injury model of hemorrhage followed by cecal ligation and puncture in mice 27. HTS transiently improved tissue oxygen delivery and increased oxygen consumption in a canine model of oleic acid-induced lung injury 28. However, the potential for HTS to modulate oleic acid-induced ALI has not been assessed

HTS did not reduce the severity of evolving oleic acid induced ALI. There was no difference between HTS and an equal volume of isotonic saline in terms of arterial oxygenation, pulmonary shunt or lung static or dynamic compliance. The degree of histologic injury on quantitative stereological analysis was similar with HTS versus isotonic saline, with no difference in the amount of tissue or airspace in the gas exchanging portion of the lung. In contrast to these findings, HTS did appear to reduce pulmonary inflammation, significantly reducing bronchoalveolar lavage neutrophil cell counts, in keeping with previous findings in hemorrhage-induced ALI models 222425. While the bronchoalveolar lavage protein and interleukin-6 levels showed a clear trend towards decrease in the HTS group, the results were not statistically significant. Therefore, while HTS reduced indices of pulmonary inflammation, it not attenuate the severity of oleic acid induced ALI.

A key feature of the oleic acid model of ALI is the fact that the injury is transient, permitting study of the effects of HTS on the resolution of the ALI. HTS did enhance the resolution of oleic acid induced ALI. Of importance, the degree of histologic injury on quantitative stereological analysis was clearly reduced by HTS. Specifically, there was a significantly reduced amount of tissue and a significantly greater amount of airspace in the gas exchanging portion of the lung. Furthermore, HTS reduced pulmonary inflammation, significantly reducing bronchoalveolar lavage neutrophil cell counts and interleukin-6 levels. Interestingly, HTS did not modulate physiologic indices of lung injury, in terms of arterial oxygenation, pulmonary shunt or lung static or dynamic compliance. This may reflect the fact that different parameters of lung injury and damage may resolve over differing time courses.

Our study has a number of limitations. In particular, it is not clear whether the effects seen are a function of the additional saline load, or the hypertonicity per se. We chose to use a control group of an equal volume of isotonic saline in order to compare the effects of equal infusion volumes. Additional studies, which compare the effects of HTS to an equi-osmolar load of isotonic saline, are required. The relatively short experimental time frames studied may have restricted the capacity to fully characterize the therapeutic potential of HTS, particularly during the recovery phase. However oleic acid injury is relatively transient and the time points utilized allowed characterisation of the effects of HTS during injury evolution and resolution.