Broadband low-frequency forced oscillations (BBFO) provide an exhaustive assessment of the respiratory system over an extended range of frequencies (1–20.5 Hz) and that in a single perturbation (e.g., Quick Prime-3). As an outcome, this analysis first generates impedance spectra, which are then physiologically interpreted using the constant phase model [17]. This more advanced mathematical model distinguishes between central airway and peripheral tissue mechanics, thus allowing a detailed phenotype characterization of lung diseases in mice. The parameters associated with this analysis include: (1) Newtonian resistance (R_N): this parameter represents the resistance of the central or conducting airways. (2) Tissue damping (G): this parameter closely relates to tissue resistance and reflects the energy dissipation in the lung tissue. (3) Tissue elastance (H): this parameter closely relates to tissue elastance and reflects the energy conservation in the lung tissue. These parameters can provide valuable detailed information and could prove helpful to dissect underlying mechanisms. BBFO can be incorporated in an automated measurement sequence that can include other perturbations for a more comprehensive functional assessment of the respiratory system.

In the example shown in Fig. 5.2, mice were given increasing concentrations of Mch administered as an aerosol through an in-line nebulizer to generate a concentration response curve. This figure illustrates the response over time acquired from multiple mice using this approach. The raw R_N, G, and H values are calculated directly from the software and can later be exported for analysis. Investigators have reported changes of the R_N, G, and H parameters of the constant phase model in studies of lung disease models [23, 24, 26, 36] including allergen-induced pulmonary physiology changes [6, 19, 34]. Despite that, our group, which has been using the flexiVent system for studies of allergic lung inflammation for many years, has been focusing on the SSFO described above to assess AHR in these models primarily because the protocol used in our laboratory (i.e., increasing doses of Ach administered intravenously) induces a quick response and high levels of bronchoconstriction. Since the constant phase model is best suited for moderate levels of bronchoconstriction, this often leads in our experimental conditions to a poor fit of the constant phase model to impedance spectra, especially at higher doses of Ach. As a consequence, we are often faced with the impossibility to use parameters, such as R_N, G, or H to detect AHR because of rejected datasets. However, the impedance spectra remain valid and can always be used to assess AHR at a specific frequency [10] or with other mathematical models describing the mechanical properties of the respiratory system. Alternatively, the situation can be addressed by modifying the dose-range of the bronchoconstrictor agent or by assessing AHR using aerosol deliveries of increasing Mch concentrations, where the exposure would be less. In this latter approach, care must be taken to avoid that condensation accumulates inside the tracheal catheter and obstructs the delivery of the test signal, which would have an important negative effect on the measurement. Carefully cleaning the aerosol delivery line between each subject is, in our view, very helpful in preventing that technical issue.

The flexiVent system can be used in studies that link tissue destruction with altered pulmonary function. Pressure-volume (PV) maneuvers can reliably measure the static compliance of the lung, which can increase significantly in models of emphysema and decrease after pneumonectomy and in models of acute lung injury or pulmonary fibrosis. PV loops capture the quasi-static mechanical properties of the respiratory system. The Salazar-Knowles equation can be fit to the expiratory branch of the PV loop, and quasi-static elastance and compliance values can be calculated. The commonly used outcomes in this analysis include: (1) A: this parameter represents the asymptote of the exponential function described by the Salazar-Knowles equation and estimates the subject’s inspiratory capacity. (2) K: this parameter reflects the curvature of the upper portion of the deflation PV curve. (3) C_st: this parameter is the quasi-static compliance which reflects the static elastic recoil pressure of the lungs at a given lung volume. (4) Area: this parameter describes the area between the inflation and deflation limb of the PV curve. (5) PV curve: The comparative visual representation of PV curves can be very informative.

As with other perturbations, the animals need to be anesthetized, tracheotomized or orally intubated and then connected to the computer-controlled ventilator for mechanical ventilation. This analysis can often be performed in combination with other perturbations in an automated manner using a script. Shown in Fig. 5.3 is a PV curve acquired from a single control mouse in a stepwise manner. The outcomes derived from this analysis provide valuable information on the elastic properties of lung at baseline or in disease studies. Voltz et al. [37] reported that male mice had significantly higher basal quasi-static lung compliance than female mice and a more pronounced decline in static compliance after bleomycin administration. Since the data suggest that male C57BL/6 mice are more susceptible than female mice to bleomycin-induced lung function decline, a special attention to animals’ sex, and genetic background would be advisable when designing experiments.

For the investigators who are interested in evaluating the inspiratory capacity while using the pneumonectomy model of lung regeneration, the deep lung inflation maneuver should be used instead of parameter A from the Salazar-Knowles equation to evaluate changes related to that specific lung volume. The reported values will more reliably reflect the corresponding pathologic changes because, at the difference of parameter A which offers an estimate of the inspiratory capacity from the Salazar-Knowles model, the deep lung inflation maneuver provides a direct and reliable measurement of that specific lung capacity.

While the in vivo invasive analyses described above have been most widely applied in models of allergic airway disease [14, 20], they are also very useful in studies of models of COPD [25], pulmonary fibrosis [5, 33] and acute lung injury [1, 15]. The analyses mentioned above have been applied to studies of acute lung injury, where the pulmonary edema associated with that condition causes alveolar filling and dramatically affects the mechanical properties of the respiratory system. This is reflected by significant changes in C_st, R_rs, C_rs, or H. In mice challenged with LPS, Håkansson et al. [15] reported a 70 % decrease in C_rs and a 45 % increase in H at 48 h post-challenge relative to saline controls. By 96 h, all respiratory mechanics parameters had returned to baseline levels, with no significant difference compared with the time-matched saline controls.