Non-invasive collection of exhaled breath condensate in rats: Evaluation of pH, H2O2 and NOx in lipopolysaccharide-induced acute lung injury
Introduction
There is an increasing amount of information on the investigation of human lung diseases following the collection and analysis of exhaled breath condensate (EBC) and the potential exists for its use in veterinary medicine. The central airways have been shown to be the origin of EBC (Bondesson et al., 2009) and collection is simple and non-invasive. Our general research theme is to evaluate markers of inflammation and oxidative stress caused by environmental and occupational pollution.
The development of inflammation can be followed in an animal model in response to an inflammatory stimulus or simulated occupational exposure. Animal studies on EBC have been performed using a number of different species (Wilhelm et al., 2003, Weissmann et al., 2004, Hatt et al., 2009, Reinhold and Knobloch, 2010) and two general applications, involving intubated (Wilhelm et al., 1999, Hatt et al., 2009) vs. non-intubated techniques of multiple different EBC collection systems have been described (Wilhelm et al., 2003, Reinhold et al., 2008, Duz et al., 2009, Hatt et al., 2009). Collection systems used in non-intubated animal studies have the advantage of avoiding interference with the inflammatory markers generated by intubation (Horváth et al., 2005), although none of these systems has unequivocally described the origin of the EBC collected.
Identification of mediators measurable in EBC in inflammatory airway disease might be relevant for differential diagnosis in respiratory medicine and EBC analysis of markers has been described in several inflammatory diseases. The pH of EBC has been used to study airway inflammation (Gessner et al., 2003, Prince et al., 2006), and hydrogen peroxide (H2O2) in EBC has been reported to be elevated in both human (Horváth et al., 1998) and animal inflammatory diseases (Deaton et al., 2003, Kirschvink et al., 2005) although other studies failed to show any association between pulmonary inflammation and increased concentrations of H2O2 in EBC (Duz et al., 2009, Deaton et al., 2005).
Administration of Lipopolysaccharide (LPS) has been used to induce sepsis in animals (Lee et al., 2002, Chen et al., 2006) and to induce lung inflammation (Jakubowski, 2009). Oxidant/anti-oxidant disorders associated with the inflammation/anti-inflammation response play an important role in the development of LPS-induced lung injury in animals (Bosma et al., 2005). Lung injury is characterised by an increase of oxygen free radicals, inflammatory cytokines and by an inflammatory cell sequestration. It has been hypothesised that mediators such as H2O2 and nitrogen oxides (NOx) may be detected in EBC within the first few hours following LPS challenge (Bosma et al., 2005).
The objective of the present study was to develop a non-invasive device for the collection of EBC from conscious rats, to examine the respiratory origin of the EBC collected. We then aimed to use the EBC collected to assess the acute inflammatory response in early and late phases of endotoxaemia in a model of LPS-induced acute lung injury.
Section snippets
Animals
Eighty-four adult male Sprague–Dawley rats (Harlan) weighing 320 ± 25 g were used in the study. The animal experiments were performed according to the principles advised by the European Council for the care of laboratory animals. The experimental protocol was approved by the local Animal Care and Use Committee (Number AF 01/2010).
EBC collection system in glass-chamber device
Rats were placed in a cylinder glass-chamber (DiSLab, MedicoLab) that had been designed in our laboratory. The device was aerated by continuous filtered air (Thermo
Experiment 1: EBC contamination during collection in the glass-chamber device (Table 1)
Urea was detected in EBC from both devices, but the concentrations of EBC were 100 times lower than the concentrations in urine and were not statistically different. No α-amylase was detected in any EBC samples. In addition, a comparison of EBC volume and concentrations of EBC markers (total proteins, NOx, H2O2) between nose-only and glass-chamber devices showed no significant differences.
Experiment 2: EBC fluorescence by Fluorescein isothiocyanate (FITC)
A higher level of FITC-fluorescence in EBC (expressed in arbitrary units – 600, 620 and 650) was found for
Discussion
To the best of our knowledge, this is the first study designed to describe a standardised method of collecting and analysing EBC markers in the conscious rat. Using a specifically designed device, we demonstrated that EBC originated mainly from the respiratory tract. The procedure of EBC collection described here is a repeatable method in conscious rats. Also, we found that analyses of EBC following acute lung injury induced by LPS, allowed early detection of increased levels of oxidative
Conclusions
A novel method was validated to collect EBC non-invasively and repeatedly without requiring anaesthesia. Induction of acute lung injury using LPS induced the release of H2O2 and NOx, which are useful non-specific markers for the investigation of lung diseases and were used to validate the device for EBC sampling.
Conflict of interest statement
None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper.
Acknowledgements
We thank Catherine Cimetta and Jessica Franczak for their technical assistance in this study. The work was supported by Environmental Health Research Programme, through convention no. 09 04 0055 (Conseil Régional Nord-Pas-de-Calais, France). We are grateful to Dr. Mike Howsam for reading and commenting on the manuscript, and to the anonymous reviewers for their help in significantly improving the text.
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