Because the metal cannula used for tracheostomy was traumatic when used for orotracheal intubation, we tested other intubation catheters, and finally chose the cannula that offered the best compromise between a resistance close to that of the tracheostomy cannula and sufficient flexibility to enable orotracheal intubation. To facilitate intubation, the 18G catheter was narrowed by slightly heating the distal end of the tube. The resistance of the tube, measured during calibration of the FlexiVent system was 0.32 cmH2O.s/mL. This value was close to that of 18G metal cannula used for tracheostomy (0.27 cmH2O.s/mL). One of the 15 BALB/cJ mice (7%) and 5 of the 15 C57BL/6 J mice (33%) died during the intubation procedure. One of the 15 BALB/cJ mice (7%) was successfully intubated, but showed aberrant lung function data using intubation as well as tracheostomy. On average, 23 min and 26 min separated the measurements performed following intubation from those following tracheostomy in C57BL/6 J and BALB/cJ mice, respectively. No sign of respiratory drive was observed during the measurements. Overall, lung function was successfully measured in 13 of the 15 BALB/cJ mice (87%) and 10 of the 15 of the C57BL/6 J mice (67%) in response to NPFE maneuver, in 14 of the 15 BALB/cJ mice (93%) and 10 of the 15 of the C57BL/6 J mice (67%) using FOT single compartment model and PV loop maneuver, and in 12 of the 15 BALB/cJ mice (80%) and 9 of the 15 of the C57BL/6 J mice (60%) using FOT constant phase model. We then compared the lung function data obtained using intubation and subsequently tracheostomy in the same mice of each strains.
Regarding BALB/cJ mice, the expiratory flow-volume curve was downward shifted especially at the onset of expiration in intubated compared with tracheostomized mice (Fig. 1a). As a consequence, the peak expiratory flow (PEF) and the FEV0.1/FVC ratio were significantly decreased in intubated compared to tracheostomized BALB/cJ mice (Additional file 1: Figure S1A-B). Of note, the magnitude of FEV0.1/FVC ratio decrease was very small (around 2%), and does not probably reflect a physiologically significant change. Regarding C57BL/6 J mice, by contrast, the flow-volume curve obtained in intubated mice was similar to that measured in tracheostomized animals (Fig. 1b). As a consequence, we did not find any significant difference between intubated and tracheostomized C57BL/6 J mice for PEF and FEV0.1/FVC (Additional file 1: Figure S1A-B). For both strains, FVC and FEV0.1 were not different between intubated and tracheostomized animals (Fig. 1c-f). FVC of C57BL/6 J and BALB/cJ mice, and FEV0.1 of BALB/cJ mice measured under the 2 conditions were also positively and significantly correlated (Fig. 1g-j and Additional file 1: Table S1). The difference between the two measurements (“bias”), calculated by Bland-Altman analysis was close to 0 (Additional file 1: Table S2), even if, for FVC in BALB/cJ mice, one point is outside the confidence interval (Fig. 1k-n). Moreover, there was no correlation between the difference and the average in the two conditions for FVC and FEV0.1 (Additional file 1: Table S3). Thus, the measurement of both FVC and FEV0.1 using orotracheal intubation can be considered accurate.
To evaluate the possibility that lung function measured in tracheotomized C57BL/6 J mice could have been affected by previous intubation, we compared lung function obtained in animals that have been only intubated or tracheostomized. We observed a downward shift of the expiratory flow-volume curve for intubated C57BL/6 J animals compared with tracheostomized mice (Additional file 1: Figure S2A), with a subsequent decrease in peak expiratory flow (PEF), FEV0.1, FVC and FEV0.1/FVC ratio in intubated C57BL/6 J mice compared to tracheostomized mice (Additional file 1: Figure S2B-E).
Using single frequency FOT measurements fitted to the single compartment model, we found in both strains of mice a significant decrease of the coefficients of determination (COD) in intubated mice compared to tracheostomized animals (Additional file 1: Figure S3A). Excepted respiratory system resistance (Rrs) which was increased in intubated BALB/cJ mice, neither Rrs in C57BL/6 J mice nor respiratory system compliance (Crs) in both strains of mice, was different between intubated and tracheostomized animals (Fig. 2a-b). The presence of a significant positive association (Additional file 1: Table S1) together with the result of Bland-Altman analysis (Fig. 2c-d and Additional file 1: Table S2) and the absence of correlation between the difference and the average (Additional file 1: Table S3) indicated that the measurement of Crs using orotracheal intubation was accurate.
At first glance, the impedance curves obtained by multi-frequency FOT in intubated BALB/cJ and C57BL/6 J mice appeared almost similar to those obtained with tracheostomy (Fig. 3a-b). However, they revealed some subtle differences: in BALB/cJ mice, the real part of the recorded in tracheostomized BALB/cJ mice was slightly above the same curve obtained intubated BALB/cJ mice at low frequency (1 Hz), and slightly below at higher frequencies (1.5–20.5 Hz). In contrast, in C57BL/6 J mice, the real part of the curve obtained in tracheostomized mice was always slightly above the same the curve obtained in intubated mice. Finally, calculation of the threshold frequency that discriminates the central compartment alone (at high frequency) from the combination central and distal compartments (at low frequency) showed that it was different in both strains. In this connection, the newtonian resistance (Rn, Fig. 3c) derived from the real part was increased in intubated BALB/cJ mice as was tissue elastance ( Fig. 3h, i) in intubated C57BL/6 J mice, compared corresponding parameters in tracheostomized animals. With the exception of H in C57BL/6 J mice, there was no significant correlation between Rn, G, H assessed under the two conditions (Additional file 1: Table S1). Although the average number of measurements required to get 2 coefficients of determination for the constant phase model fit (CODcp) values above 0.95 in intubated mice (i.e, 4.6 for BALBc/J and 5.3 for C57BL/6) was higher than that required in tracheotomized mice (i.e, 3.2 for BALBc/J and 3.6 for C57BL/6), this only represented a significant change in BALBc/J mice. The analysis of CODcp did not show any difference in intubated mice compared to tracheostomized animals (Additional file 1: Figure S3B).
The PV curves recorded in intubated BALB/cJ and C57BL/6 J mice were very similar to those obtained with tracheostomy (Fig. 4a-b). The quality of the Salazar-Knowles model fit was unchanged in intubated mice as shown by the very similar coefficients of determination for the Salazar-Knowles model (CODsk) in intubated and tracheostomized animals (Additional file 1: Figure S3C). The compliance (C, Fig. 4c, e), the estimate of inspiratory capacity (A, Fig. 4d, f), the curvature of the upper portion of the deflation limb of the PV curve (K, Additional file 1: Figure S4A) and the area enclosed by the pressure volume loop (Area, Additional file 1: Figure S4B) were not significantly altered in intubated compared to tracheotomized mice. The result of the combination of correlation and Bland-Altman analysis thus indicated that C and A could be accurately measured in intubated mice (Fig. 4g-n, and Additional file 1: Tables S1, S2 and S3). Of note, C did not behave differently from Crs from a qualitative point of view: although the difference between C and Crs was always positive (close to 0.03 mL/cm H2O), C was strongly positively correlated with Crs in both strains, whatever the measurement method, orotracheal intubation or tracheostomy (Additional file 1: Figure S5B).
To distinguish the effect due to the cannula from that due to the procedure (i.e., tracheostomy vs intubation), we also performed another set of experiments using the same cannula. Because the metal cannula used for tracheostomy was traumatic when used for orotracheal intubation, we chose the intubation catheter for both procedures. Two of the 22 BALB/cJ mice (9%) died during the intubation procedure; 3 of the 22 BALB/cJ mice (14%) and 5 of the 22 C57BL/6 J mice (23%) could not be intubated. One of the 22 BALB/cJ mice (4%) was successfully intubated, but showed aberrant lung function data using intubation as well as tracheostomy. Overall, lung function was successfully measured in 16 of the 22 BALB/cJ mice (73%) and 16 of the 22 of the C57BL/6 J mice (73%) in response to NPFE maneuver, in 16 of the 22 BALB/cJ mice (73%) and 17 of the 22 of the C57BL/6 J mice (77%) using FOT single compartment model and PV loop maneuver, and in 8 of the 22 BALB/cJ mice (36%) and 15 of the 22 of the C57BL/6 J mice (68%) using FOT constant phase model.
In both strains of mice, the flow-volume curves obtained in intubated mice were similar to those measured in tracheostomized animals (Fig. 5a-b). Except for the FEV0.1/FVC ratio which was slightly and significantly increased in tracheostomized BALB/cJ mice (Additional file 1: Figure S6B), FVC, FEV0.1 (Fig. 5c-f) and PEF (Additional file 1: Figure S6A) were not different between intubated and tracheostomized animals. Agreement analyses confirmed that the measurements of FVC and FEV0.1 using orotracheal intubation were accurate (Fig. 5g-n and Additional file 1: Tables S4, S5 and S6). Using single frequency FOT measurements fitted to the single compartment model, we could also confirm our previous results obtained with different cannulas, i.e., the validation of Crs but not Rrs assessment in intubated BALB/cJ mice (Fig. 6 and Additional file 1: Tables S4, S5 and S6). By contrast, the existence of a positive significant correlation between the average and the difference of both Crs and C measurements using intubation and tracheostomy in C57BL/6 J mice suggests that these sole parameters should not be evaluated using intubation in this mouse strain (Fig. 6d, Fig. 8m and Additional file 1: Table S6).
In intubated C57BL/6 J mice, but not in BALB/cJ mice, Rn derived from the impedance curves was significantly increased compared to tracheostomized C57BL/6 J mice (Fig. 7c, e). Our evaluation of the agreement between the two different methods by means of intra-class correlation and Bland-Altman analysis for Rn, G and H did not allow to validate the measurements of those parameters using orotracheal intubation (Fig. 7 and Additional file 1: Tables S4, S5 and S6). The PV curves obtained in intubated BALB/cJ and C57BL/6 J mice appeared almost similar to those obtained with tracheostomy (Fig. 8a-b). C and A measured in both strains (Fig. 8c-f), K and the area enclosed by the pressure volume loop measured in BALB/cJ mice (Additional file 1: Figure S7A-B) were not significantly altered in intubated compared to tracheotomized mice. The result of the combination of correlation and Bland-Altman analysis thus indicated that C, for BALB/cJ mice, and A, for both strains, could be accurately measured in intubated mice (Fig. 8g-n, and Additional file 1: Tables S1, S2 and S3).
Altogether, with the exception of Crs and C for C57BL/6 J mice, the second set of experiments allowed us to identify exactly the same subsets of parameters that are accurately evaluated in intubated BALB/cJ animals: 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).
To test the possibility of repeated intubations, we performed sequential lung function measurements using intubation in C57BL/6 J mice with 19 days interval (Fig. 9a). Three of the 12 mice died during one of the procedures (survival rate of 75%), 2 mice (17%) could not be successfully intubated, and one mice (8%) was successfully intubated, but showed aberrant lung function data. The first orotracheal intubation is followed by weight loss (Fig. 9b). As a result, we tried without success to re-intubate mice days following the first intubation, but a second intubation was impossible to perform until the 5th day. Mice regain a normal weight around 12 days after the first intubation. In total, we could successfully measure lung function at day 0 and day 19 in 6 of the 12 mice (50%). Our results do not show any significant differences between the measurements at day 0 and 19 (Fig. 9c-k). Although no significant correlation could be evidenced for FEV0.1, the FEV0.1/FVC ratio, A, Area, Rn, G, H and Rrs measured by intubation at day 0 and day 19, probably due to the low number of mice, PEF, FVC, Crs and K measured by the first and second intubation were positively correlated (Additional file 1: Table S7). Altogether, this suggests that repetitive lung function measurements using intubation are feasible and seem to be reproducible.