Non-invasive collection of exhaled breath condensate in rats: Evaluation of pH, H₂O₂ and NOx in lipopolysaccharide-induced acute lung injury

Abstract

The analysis of exhaled breath condensate (EBC) offers the potential for identifying lung disease markers in humans and animals, but methodological issues and standardised procedures need to be addressed before the technique can be considered for use in applications to help understand the role of environmental pollution in respiratory diseases. The purpose of this study was to develop and implement a new device using a glass-chamber for collecting EBC non-invasively from rats in order to analyse EBC markers in lipopolysaccharide (LPS)-induced acute lung injury. Eighty-four adult rats were used in five different series of experiments to determine the source of EBC formation, intra-day and inter-day variability, and the influence of environmental parameters on EBC markers. The hypothesis that inflammation induces an oxidative stress was assessed by measuring pH, nitrogen oxides (NOx) and hydrogen peroxide (H₂O₂) in EBC.

The results confirmed that EBC fluid was generated at the level of the respiratory tract. The repeatability studies of disease markers indicated higher concentrations of NOx and H₂O₂ at midday compared to the morning, but there were no significant difference between measurements on consecutive days. EBC volume was influenced by both ambient temperature and humidity. Moreover, 3 h after LPS challenge, significantly increased concentrations of both NOx and H₂O₂ were observed in EBC of the LPS group compared with controls (P = 0.005 and P = 0.027, respectively). These results suggested that EBC collection may be a valuable tool to monitor the presence of markers, such as NOx and H₂O₂, in an animal model of LPS-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.