Combined forced oscillation and forced expiration measurements in mice for the assessment of airway hyperresponsiveness

Abstract

Background: Pulmonary function has been reported in mice using negative pressure-driven forced expiratory manoeuvres (NPFE) and the forced oscillation technique (FOT). However, both techniques have always been studied using separate cohorts of animals or systems. The objective of this study was to obtain NPFE and FOT measurements at baseline and following bronchoconstriction from a single cohort of mice using a combined system in order to assess both techniques through a refined approach.

Methods: Groups of allergen- or sham-challenged ovalbumin-sensitized mice that were either vehicle (saline) or drug (dexamethasone 1 mg/kg ip)-treated were studied. Surgically prepared animals were connected to an extended flexiVent system (SCIREQ Inc., Montreal, Canada) permitting NPFE and FOT measurements. Lung function was assessed concomitantly by both techniques at baseline and following doubling concentrations of aerosolized methacholine (MCh; 31.25 - 250 mg/ml). The effect of the NPFE manoeuvre on respiratory mechanics was also studied.

Results: The expected exaggerated MCh airway response of allergic mice and its inhibition by dexamethasone were detected by both techniques. We observed significant changes in FOT parameters at either the highest (Ers, H) or the two highest (Rrs, RN, G) MCh concentrations. The flow-volume (F-V) curves obtained following NPFE manoeuvres demonstrated similar MCh concentration-dependent changes. A dexamethasone-sensitive decrease in the area under the flow-volume curve at the highest MCh concentration was observed in the allergic mice. Two of the four NPFE parameters calculated from the F-V curves, FEV0.1 and FEF50, also captured the expected changes but only at the highest MCh concentration. Normalization to baseline improved the sensitivity of NPFE parameters at detecting the exaggerated MCh airway response of allergic mice but had minimal impact on FOT responses. Finally, the combination with FOT allowed us to demonstrate that NPFE induced persistent airway closure that was reversible by deep lung inflation.

Conclusions: We conclude that FOT and NPFE can be concurrently assessed in the same cohort of animals to determine airway mechanics and expiratory flow limitation during methacholine responses, and that the combination of the two techniques offers a refined control and an improved reproducibility of the NPFE.

Figure 1

Block diagram of flexiVent system with extensions for negative pressure-driven forced expiration manoeuvres. During a negative pressure-driven forced expiration manoeuvre, the reservoir pressure (P_res) as well as the air flow into the plethysmograph () were recorded via precision differential pressure transducers attached respectively to the negative pressure reservoir and the pneumotachograph mounted on the plethysmograph chamber. These signals were collected in addition to the volume displaced by the piston (Vol), the pressure in the cylinder (P_cyl) and the pressure at airway opening (P_ao) typically recorded by the flexiVent. PEEP stands for positive end expiratory pressure.

Figure 2

Measurement protocol. Experimental trace in a sham control mouse illustrating the timing of a negative pressure-driven forced expiration (NPFE) manoeuvre following saline and methacholine (31.25 mg/ml) aerosol challenge, using closely-spaced (15s) single-frequency forced oscillation parameter Rrs to follow the time-course of the response. Rrs = respiratory system resistance; DI = deep lung inflation (30 cmH₂O); MCh = methacholine.

Figure 3

Impact of negative pressure-driven forced expiration manoeuvres on respiratory mechanics. Respiratory mechanics in naïve mice at baseline (BL), following the application of a negative pressure-driven forced expiration (NPFE) manoeuvre and following deep lung inflation (post-DI; 30 cmH₂O). Values are mean ± standard deviation from a group of 12 mice that were each studied once in the absence of methacholine challenge. (*p < 0.05; ANOVA).

Figure 4

Pressure-dependence of expiratory flow. Mean flow-volume curves from ovalbumin-sensitized and sham-challenged mice (A) and ovalbumin-sensitized and challenged mice (B) at varying negative pressures. Values are mean ± standard deviation from groups of 7-9 mice (1 determination per animal at each negative pressure).

Figure 5

Assessment of allergen-induced airway hyperresponsiveness by the forced oscillation technique. Forced oscillation parameters at peak Rrs response to each concentration of aerosolized methacholine in ovalbumin- and saline-challenged OVA-sensitized mice that were either vehicle- or dexamethasone-treated. Values are mean ± standard deviation from groups of 5-7 mice. (*p < 0.05 Veh/OVA vs Veh/Sal, ^#p < 0.05 Veh/OVA vs Dex/OVA; ANOVA).

Figure 6

Assessment of allergen-induced airway hyperresponsiveness by negative pressure-driven forced expiratory parameters. Forced expiration parameters at baseline (BL) and following aerosolized saline (Sal) or increasing methacholine concentrations in vehicle (Veh)- or dexamethasone (Dex)-treated, sham (Sal)- and ovalbumin (OVA)-challenged ovalbumin-sensitized mice. Values were obtained at peak response to each concentration of aerosolized methacholine and are expressed as mean ± standard deviation from groups of 4-6 mice where each animal was studied once. (*p < 0.05 Veh/OVA vs Veh/Sal, ^#p < 0.05 Veh/OVA vs Dex/OVA; ANOVA).

Figure 7

Flow-volume curves following increasing aerosolized methacholine concentrations. Mean flow-volume curves (mean ± standard deviation) from vehicle-treated saline- (A; Veh/Sal) and ovalbumin- (B; Veh/OVA) challenged ovalbumin-sensitized mice as well as from dexamethasone (1 mg/kg)-treated ovalbumin-sensitized and challenged mice (C; Dex/OVA) at baseline (BL) and following aerosolized saline (Sal) or increasing methacholine concentrations (MCh 31.25-250 mg/ml). Figure 7D represents the mean and standard deviation of the area under the flow-volume curves (AUC) under the varied experimental conditions. (*p < 0.05; ANOVA, n = 5-6 mice/group).

Figure 8

Normalized forced expiratory parameters. Forced expiration parameters normalized to baseline values at each concentration of aerosolized methacholine in ovalbumin-challenged (OVA) and sham-challenged (Sal) ovalbumin-sensitized mice that were either vehicle (Veh)- or dexamethasone (Dex)-treated. Values were normalized to individual baseline and expressed as mean ± standard deviation for each group (n = 4-6 mice/group, each mouse studied once). (*p < 0.05 Veh/OVA vs Veh/Sal; ^#p < 0.05 Veh/OVA vs Dex/OVA; ANOVA).

Figure 9

Assessment of drug effect via normalized parameters of the forced oscillation technique. Forced oscillation technique parameters normalized to baseline values at each concentration of aerosolized methacholine in vehicle treated- (Veh/OVA; closed circles) and dexamethasone treated- (Dex/OVA; closed squares) ovalbumin sensitized and challenged mice. Values were normalized to individual baseline and expressed as mean ± standard deviation. (*p < 0.05 Veh/OVA vs Veh/Sal; ^#p < 0.05 Veh/OVA vs Dex/OVA; ANOVA; n = 5-7 mice/group).