Non-invasive measurements of exhaled NO and CO associated with methacholine responses in mice

Background

Nitric oxide (NO) and carbon monoxide (CO) in exhaled breath are considered obtainable biomarkers of physiologic mechanisms. Therefore, obtaining their measures simply, non-invasively, and repeatedly, is of interest, and was the purpose of the current study.

Methods

Expired NO (E_NO) and CO (E_CO) were measured non-invasively using a gas micro-analyzer on several strains of mice (C57Bl6, IL-10^-/-, A/J, MKK3^-/-, JNK1^-/-, NOS-2^-/- and NOS-3^-/-) with and without allergic airway inflammation (AI) induced by ovalbumin systemic sensitization and aerosol challenge, compared using independent-sample t-tests between groups, and repeated measures analysis of variance (ANOVA) within groups over time of inflammation induction. E_NO and E_CO were also measured in C57Bl6 and IL-10-/- mice, ages 8–58 weeks old, the relationship of which was determined by regression analysis. S-methionyl-L-thiocitrulline (SMTC), and tin protoporphyrin (SnPP) were used to inhibit neuronal/constitutive NOS-1 and heme-oxygenase, respectively, and alter NO and CO production, respectively, as assessed by paired t-tests. Methacholine-associated airway responses (AR) were measured by the enhanced pause method, with comparisons by repeated measures ANOVA and post-hoc testing.

Results

E_NO was significantly elevated in naïve IL-10^-/- (9–14 ppb) and NOS-2^-/- (16 ppb) mice as compared to others (average: 5–8 ppb), whereas E_CO was significantly higher in naïve A/J, NOS-3^-/- (3–4 ppm), and MKK3^-/- (4–5 ppm) mice, as compared to others (average: 2.5 ppm). As compared to C57Bl6 mice, AR of IL-10^-/-, JNK1^-/-, NOS-2^-/-, and NOS-3^-/- mice were decreased, whereas they were greater for A/J and MKK3^-/- mice. SMTC significantly decreased E_NO by ~30%, but did not change AR in NOS-2^-/- mice. SnPP reduced E_CO in C57Bl6 and IL-10^-/- mice, and increased AR in NOS-2^-/- mice. E_NO decreased as a function of age in IL-10^-/- mice, remaining unchanged in C57Bl6 mice.

Conclusion

These results are consistent with the ideas that: 1) E_NO is associated with mouse strain and knockout differences in NO production and AR, 2) alterations of E_NO and E_CO can be measured non-invasively with induction of allergic AI or inhibition of key gas-producing enzymes, and 3) alterations in AR may be dependent on the relative balance of NO and CO in the airway.