Determination of reliable lung function parameters in intubated mice

Abstract

Background: Animal models and, in particular, mice models, are important tools to investigate the pathogenesis of respiratory diseases and to test potential new therapeutic drugs. Lung function measurement is a key step in such investigation. In mice, it is usually performed using forced oscillation technique (FOT), negative pressure-driven forced expiratory (NPFE) and pressure-volume (PV) curve maneuvers. However, these techniques require a tracheostomy, which therefore only allows end-point measurements. Orotracheal intubation has been reported to be feasible and to give reproducible lung function measurements, but the agreement between intubation and tracheostomy generated-data remains to be tested.

Methods: Using the Flexivent system, we measured lung function parameters (in particular, forced vital capacity (FVC), forced expiratory volume in the first 0.1 s (FEV0.1), compliance (Crs) of the respiratory system, compliance (C) measured using PV loop and an estimate of inspiratory capacity (A)) in healthy intubated BALB/cJ mice and C57BL/6 J mice and compared the results with similar measurements performed in the same mice subsequently tracheostomized after intubation, by means of paired comparison method, correlation and Bland-Altman analysis. The feasibility of repetitive lung function measurements by intubation was also tested.

Results: We identified parameters that are accurately evaluated in intubated animals (i.e., FVC, FEV0.1, Crs, C and A in BALB/cJ and FVC, FEV0.1, and A in C57BL/6 J). Repetitive lung function measurements were obtained in C57BL/6 J mice.

Conclusion: This subset of lung function parameters in orotracheally intubated mice is reliable, thereby allowing relevant longitudinal studies.

Keywords: Asthma; COPD; Lung function; Mice model; Orotracheal intubation.

Fig. 1

Evaluation of lung function measurement assessed by the NPFE maneuver in mice using orotracheal intubation and tracheostomy. a, b, Average expiratory flow-volume curves of intubated (“OTI”, in blue) and tracheostomized (“TRA”, in red) mice. n = 13 BALB/cJ mice, n = 10 C57BL/6 J mice. Lower and upper error bars represent standard deviations for intubation and tracheostomy, respectively. c-f, Forced vital capacity (“FVC”, c and e), forced expired volume over 0.1 s (“FEV0.1”, d and f) in intubated and tracheostomized BALB/cJ mice (c, e) and C57BL/6 J mice (d, f). Data represent individual mice and are analyzed by the Wilcoxon signed-rank test or paired t tests. * P < 0.05. g, i, Relationships between FVC measured in intubated and tracheostomized BALB/cJ (g) and C57BL/6 J (i) mice. h, j, Relationships between FEV0.1 measured in intubated and tracheostomized BALB/cJ (h) and C57BL/6 J (j) mice. k-n, Bland-Altman plots to compare two measurements techniques for FVC in BALB/cJ (k) and C57BL/6 J (m), FEV0.1 in BALB/cJ (l) and C57BL/6 J (n). The upper and lower limits of agreement (95% confidence interval) are shown by a gray dotted line

Fig. 2

Evaluation of lung function measurement assessed by single compartment model obtained in mice using orotracheal intubation and tracheostomy. a-b, Comparaison of the variables compliance (“Crs”, a) and resistance (“Rrs”, b) of the respiratory system in intubated (“OTI”) and tracheostomized (“TRA”) mice. n = 14 BALB/cJ mice, n = 10 C57BL/6 J mice. Data represent individual mice and are analyzed by the Wilcoxon signed-rank test or paired t tests. ** P < 0.01. c, Relationships between Crs measured in intubated and tracheostomized BALB/cJ and C57BL/6 J mice. d, Bland-Altman plots to compare two measurements techniques for Crs. The upper and lower limits of agreement (95% confidence interval) are shown by a gray dotted line

Fig. 3

Evaluation of lung function measurement assessed by constant phase model obtained in mice using orotracheal intubation and tracheostomy. a, b, Average real (top) and imaginary (bottom) parts of respiratory system input impedance (Zrs) obtained in intubated (“OTI”, in blue) mice and tracheostomized (“TRA”, in red) mice. n = 12 BALB/cJ mice (a), n = 9 C57BL/6 J mice (b). Lower and upper error bars represent standard deviations respectively for respectively for intubation and tracheostomyintubation and tracheostomy. c-i, newtonian resistance (“Rn”, c and e), tissue damping (“G”, d, f), tissue elastance (“H”, g, i) in intubated and tracheostomized BALB/cJ mice (c, d, g) and C57BL/6 J mice (e, f, i). Data represent individual mice and are analyzed by the Wilcoxon signed-rank test or paired t tests. * P < 0.05, *** P < 0.001

Fig. 4

Evaluation of lung function measurement assessed by the Pressure-Volume (PV) curve maneuver in mice using orotracheal intubation and tracheostomy. a, b, Average PV curves of intubated (“OTI”, in blue) mice and tracheostomized (“TRA”, in red) mice. n = 14 BALB/cJ mice (a), n = 10 C57BL/6 J mice (b). Lower and upper error bars represent standard deviations for intubation and tracheostomy, respectively. c-f, compliance (“C”, c and e), estimate of inspiratory capacity (“A”, d and f) in intubated and tracheostomized BALB/cJ mice (c, e) and C57BL/6 J mice (d, f). Data represent individual mice and are analyzed by the Wilcoxon signed-rank test or paired t tests. g, i, Relationships between c measured in intubated and tracheostomized BALB/cJ (g) and C57BL/6 J (i) mice. h, j, Relationships between A measured in intubated and tracheostomized BALB/cJ (h) and C57BL/6 J (j) mice. k-n, Bland-Altman plots to compare two measurements techniques for C in BALB/cJ (k) and C57BL/6 J (m), A in BALB/cJ (l) and C57BL/6 J (n). The upper and lower limits of agreement (95% confidence interval) are shown by a gray dotted line

Fig. 5

Evaluation of lung function measurement assessed by the NPFE maneuver in mice using orotracheal intubation and tracheostomy, both using the intubation cannula. a, b, Average expiratory flow-volume curves of intubated (“OTI”, in blue) and tracheostomized (“TRA”, in red) mice. n = 16 BALB/cJ mice, n = 16 C57BL/6 J mice. Lower and upper error bars represent standard deviations for intubation and tracheostomy, respectively. c-f, Forced vital capacity (“FVC”, c and e), forced expired volume over 0.1 s (“FEV0.1”, d and f) in intubated and tracheostomized BALB/cJ mice (c, e) and C57BL/6 J mice (d, f). Data represent individual mice and are analyzed by the Wilcoxon signed-rank test or paired t tests. g, i, Relationships between FVC measured in intubated and tracheostomized BALB/cJ (g) and C57BL/6 J (i) mice. h, j, Relationships between FEV0.1 measured in intubated and tracheostomized BALB/cJ (h) and C57BL/6 J (j) mice. k-n, Bland-Altman plots to compare two measurements techniques for FVC in BALB/cJ (k) and C57BL/6 J (m), FEV0.1 in BALB/cJ (l) and C57BL/6 J (n). The upper and lower limits of agreement (95% confidence interval) are shown by a gray dotted line

Fig. 6

Evaluation of lung function measurement assessed by single compartment model obtained in mice using orotracheal intubation and tracheostomy, both using the intubation cannula. a-b, Comparaison of the variables compliance (“Crs”, a) and resistance (“Rrs”, b) of the respiratory system in intubated (“OTI”) and tracheostomized (“TRA”) mice. n = 16 BALB/cJ mice, n = 17 C57BL/6 J mice. Data represent individual mice and are analyzed by the Wilcoxon signed-rank test or paired t tests. * P < 0.05. C, Relationships between Crs measured in intubated and tracheostomized BALB/cJ and C57BL/6 J mice. d, Bland-Altman plots to compare two measurements techniques for Crs. The upper and lower limits of agreement (95% confidence interval) are shown by a gray dotted line

Fig. 7

Evaluation of lung function measurement assessed by constant phase model obtained in mice using orotracheal intubation and tracheostomy, both using the intubation cannula. a, b, Average real (top) and imaginary (bottom) parts of respiratory system input impedance (Zrs) obtained in intubated (“OTI”, in blue) mice and tracheostomized (“TRA”, in red) mice. n = 8 BALB/cJ mice (a), n = 15 C57BL/6 J mice (b). Lower and upper error bars represent standard deviations respectively for intubation and tracheostomy. c-i, newtonian resistance (“Rn”, c and e), tissue damping (“G”, d, f), tissue elastance (“H”, g, i) in intubated and tracheostomized BALB/cJ mice (c, d, g) and C57BL/6 J mice (e, f, i). Data represent individual mice and are analyzed by the Wilcoxon signed-rank test or paired t tests. * P < 0.05

Fig. 8

Evaluation of lung function measurement assessed by the Pressure-Volume (PV) curve maneuver in mice using orotracheal intubation and tracheostomy, both using the intubation cannula. a, b, Average PV curves of intubated (“OTI”, in blue) mice and tracheostomized (“TRA”, in red) mice. n = 16 BALB/cJ mice (a), n = 17 C57BL/6 J mice (b). Lower and upper error bars represent standard deviations for intubation and tracheostomy, respectively. c-f, compliance (“C”, c and e), estimate of inspiratory capacity (“A”, d and f) in intubated and tracheostomized BALB/cJ mice (c, e) and C57BL/6 J mice (d, f). Data represent individual mice and are analyzed by the Wilcoxon signed-rank test or paired t tests. g, i, Relationships between C measured in intubated and tracheostomized BALB/cJ (g) and C57BL/6 J (i) mice. h, j, Relationships between A measured in intubated and tracheostomized BALB/cJ (h) and C57BL/6 J (j) mice. k-n, Bland-Altman plots to compare two measurements techniques for C in BALB/cJ (k) and C57BL/6 J (m), A in BALB/cJ (l) and C57BL/6 J (n). The upper and lower limits of agreement (95% confidence interval) are shown by a gray dotted line

Fig. 9

Repeated lung function measurement in intubated C57BL/6 J mice. a, Study protocol. Lung function measurements were performed at day 0 and 19. n represents the number of surviving mice at a given time point. b, Average mice weight. c-k, Peak expiratory flow (“PEF”, c), Forced vital capacity (“FVC”, d), forced expired volume over 0.1 s (“FEV0.1”, e), FEV0.1/FVC ratio (f), newtonian resistance (“Rn”, g), tissue damping (“G”, h), tissue elastance (“H”, i), resistance (“Rrs”, j) and compliance (“Crs”, k) of the respiratory system in intubated mice at day 0 (“OTI 1”) and day 19 (“OTI 2”). Data represent individual mice and are analyzed by the Wilcoxon signed-rank test