Rat models of acute lung injury: Exhaled nitric oxide as a sensitive, noninvasive real-time biomarker of prognosis and efficacy of intervention

• Exhaled NO (eNO) is an early, non-invasive biomarker for different types of acute lung injury.
• Its sensitivity is improved by normalizing eNO to exhaled CO2 and breathing frequency.
• eNO appears to be a particularly sensitive indicator for acute airway injury.
• Inhalation treatment with a nitric oxide synthase inhibitor is mirrored by decreased eNO.
• Real-time eNO is amenable to real-time dose targeting in drug-efficacy studies.

Exhaled nitric oxide (eNO) has received increased attention in clinical settings because this technique is easy to use with instant readout. However, despite the simplicity of eNO in humans, this endpoint has not frequently been used in experimental rat models of septic (endotoxemia) or irritant acute lung injury (ALI). The focus of this study is to adapt this method to rats for studying ALI-related lung disease and whether it can serve as instant, non-invasive biomarker of ALI to study lung toxicity and pharmacological efficacy. Measurements were made in a dynamic flow of sheath air containing the exhaled breath from spontaneously breathing, conscious rats placed into a head-out volume plethysmograph. The quantity of eNO in exhaled breath was adjusted (normalized) to the physiological variables (breathing frequency, concentration of exhaled carbon dioxide) mirroring pulmonary perfusion and ventilation. eNO was examined on the instillation/inhalation exposure day and first post-exposure day in Wistar rats intratracheally instilled with lipopolysaccharide (LPS) or single inhalation exposure to chlorine or phosgene gas. eNO was also examined in a Brown Norway rat asthma model using the asthmagen toluene diisocyanate (TDI). The diagnostic sensitivity of adjusted eNO was superior to the measurements not accounting for the normalization of physiological variables. In all bioassays – whether septic, airway or alveolar irritant or allergic, the adjusted eNO was significantly increased when compared to the concurrent control. The maximum increase of the adjusted eNO occurred following exposure to the airway irritant chlorine. The specificity of adjustment was experimentally verified by decreased eNO following inhalation dosing of the non-selective nitric oxide synthase inhibitor amoniguanidine. In summary, the diagnostic sensitivity of eNO can readily be applied to spontaneously breathing, conscious rats without any intervention or anesthesia. Measurements are definitely improved by accounting for the disease-related changes in exhaled CO2 and breathing frequency. Accordingly, adjusted eNO appears to be a promising methodological improvement for utilizing eNO in inhalation toxicology and pharmacological disease models with fewer animals.