X-Ray based Lung Function measurement-a sensitive technique to quantify lung function in allergic airway inflammation mouse models

Abstract

In mice, along with the assessment of eosinophils, lung function measurements, most commonly carried out by plethysmography, are essential to monitor the course of allergic airway inflammation, to examine therapy efficacy and to correlate animal with patient data. To date, plethysmography techniques either use intubation and/or restraining of the mice and are thus invasive, or are limited in their sensitivity. We present a novel unrestrained lung function method based on low-dose planar cinematic x-ray imaging (X-Ray Lung Function, XLF) and demonstrate its performance in monitoring OVA induced experimental allergic airway inflammation in mice and an improved assessment of the efficacy of the common treatment dexamethasone. We further show that XLF is more sensitive than unrestrained whole body plethysmography (UWBP) and that conventional broncho-alveolar lavage and histology provide only limited information of the efficacy of a treatment when compared to XLF. Our results highlight the fact that a multi-parametric imaging approach as delivered by XLF is needed to address the combined cellular, anatomical and functional effects that occur during the course of asthma and in response to therapy.

Figure 1. Breathing modulates the x-ray transmission at the chest region over time.

(a) shows an exemplified radiograph: the average x-ray transmission function over time (XTF) is measured in a region of interest of the lung (ROI) and normalized by the background intensity (ROI_Bgk); and (b) shows exemplary two breathing cycles of a healthy control animal CN (blue curve); a mouse from the severe acute airway inflammation model (SAA) two days after the last challenge (red curve) and a mouse from the same model that had been treated with dexamethasone before each challenging step (SAA-DEX, green curve). The SAA mouse demonstrates a strong reduction in the area under the curve due to the lower air content within the lung at peak inspiration and an asymmetric shape of the breathing event with a shorter time to peak. The treated mouse (SAA-DEX) shows XTF properties comparable to those obtained in the healthy control mouse CN.

Figure 2. Comparison of XLF parameters with UWBP for healthy mice (CN), mice with severe acute airway inflammation (SAA) and dexamethasone treated mice of the same model (SAA-DEX).

XLF shows significant differences between CN and SAA for b₁, b₃, b₅ und t_in. The treated mice (SAA-DEX) show intermediate values suggesting an inefficient treatment in terms of lung function. UWBP failed to display significant differences in all parameters between CN, SAA and SAA-DEX mice, indicating that our XLF approach - as the only other in-vivo LF method used in this study - has a higher sensitivity and specificity than in-vivo UWBP. Statistical significance difference of the results is indicated by (*p < 0.1, **p < 0.05).

Figure 3. Airway responsiveness to methacholine as measured by XLF.

Relative change of XLF parameters to baseline values are shown for SAA and CN in dependence of increasing concentrations of methacholine (0, 3, 10, and 50 mg/ml). In SAA animals b₁, b₂ and b₄ are significantly affected by a lower concentration of methacholine (10 mg/ml) than CN (50 mg/ml) relative to 0 mg/ml (**p < 0.05).

Figure 4. Correlation of XLF and UWBP to ex-vivo techniques BAL, pSRμCT, and histology for healthy mice (CN), mice with severe acute airway inflammation (SAA) and dexamethasone treated mice of the same model (SAA-DEX).

The analysis of BAL shows a significant increase in total cell number and in the relative amount of eosinophils (EOS[%]) in SAA mice when compared to CN mice, verifying the presence of asthma. SAA-DEX still reveals an increased number of eosinophils suggesting inefficient treatment. pSRμCT suggests a strongly increased soft-tissue vol.ratio within the lungs of SAA and intermediate values for SAA-DEX mice. SAA and SAA-DEX lungs show no difference in the rel. δ-value. Histology shows an increased amount of infiltrating cells in SAA (HE-score) and an increased mucus production (PAS-score), SAA-DEX mice demonstrate intermediate results, suggesting only partially successful treatment. Statistical significance difference of the results is indicated by (*p < 0.1, **p < 0.05).