Quantification of muco-obstructive lung disease variability in mice via laboratory X-ray velocimetry.

To effectively diagnose, monitor and treat respiratory disease clinicians should be able to accurately assess the spatial distribution of airflow across the fine structure of lung. This capability would enable any decline or improvement in health to be located and measured, allowing improved treatment options to be designed. Current lung function assessment methods have many limitations, including the inability to accurately localise the origin of global changes within the lung. However, X-ray velocimetry (XV) has recently been demonstrated to be a sophisticated and non-invasive lung function measurement tool that is able to display the full dynamics of airflow throughout the lung over the natural breathing cycle. In this study we present two developments in XV analysis. Firstly, we show the ability of laboratory-based XV to detect the patchy nature of cystic fibrosis (CF)-like disease in ß-ENaC mice. Secondly, we present a technique for numerical quantification of CF-like disease in mice that can delineate between two major modes of disease symptoms. We propose this analytical model as a simple, easy-to-interpret approach, and one capable of being readily applied to large quantities of data generated in XV imaging. Together these advances show the power of XV for assessing local airflow changes. We propose that XV should be considered as a novel lung function measurement tool for lung therapeutics development in small animal models, for CF and for other muco-obstructive diseases.

Real-time in vivo imaging of regional lung function in a mouse model of cystic fibrosis on a laboratory X-ray source.

Most measures of lung health independently characterise either global lung function or regional lung structure. The ability to measure airflow and lung function regionally would provide a more specific and physiologically focused means by which to assess and track lung disease in both pre-clinical and clinical settings. One approach for achieving regional lung function measurement is via phase contrast X-ray imaging (PCXI), which has been shown to provide highly sensitive, high-resolution images of the lungs and airways in small animals. The detailed images provided by PCXI allow the application of four-dimensional X-ray velocimetry (4DxV) to track lung tissue motion and provide quantitative information on regional lung function. However, until recently synchrotron facilities were required to produce the highly coherent, high-flux X-rays that are required to achieve lung PCXI at a high enough frame rate to capture lung motion. This paper presents the first translation of 4DxV technology from a synchrotron facility into a laboratory setting by using a liquid-metal jet microfocus X-ray source. This source can provide the coherence required for PCXI and enough X-ray flux to image the dynamics of lung tissue motion during the respiratory cycle, which enables production of images compatible with 4DxV analysis. We demonstrate the measurements that can be captured in vivo in live mice using this technique, including regional airflow and tissue expansion. These measurements can inform physiological and biomedical research studies in small animals and assist in the development of new respiratory treatments.

Application of a novel in vivo imaging approach to measure pulmonary vascular responses in mice.

Noninvasive imaging of the murine pulmonary vasculature is challenging due to the small size of the animal, limits of resolution of the imaging technology, terminal nature of the procedure, or the need for intravenous contrast. We report the application of laboratory-based high-speed, high-resolution x-ray imaging, and image analysis to detect quantitative changes in the pulmonary vascular tree over time in the same animal without the need for intravenous contrast. Using this approach, we detected an increased number of vessels in the pulmonary vascular tree of animals after 30 min of recovery from a brief exposure to inspired gas with 10% oxygen plus 5% carbon dioxide (mean ± standard deviation: 2193 ± 382 at baseline vs. 6177 ± 1171 at 30 min of recovery; P < 0.0001). In a separate set of animals, we showed that the total pulmonary blood volume increased (P = 0.0412) while median vascular diameter decreased from 0.20 mm (IQR: 0.15-0.28 mm) to 0.18 mm (IQR: 0.14-0.26 mm; P = 0.0436) over the respiratory cycle from end-expiration to end-inspiration. These findings suggest that the noninvasive, nonintravenous contrast imaging approach reported here can detect dynamic responses of the murine pulmonary vasculature and may be a useful tool in studying these responses in models of disease.

Novel analysis of 4DCT imaging quantifies progressive increases in anatomic dead space during mechanical ventilation in mice.

Increased dead space is an important prognostic marker in early acute respiratory distress syndrome (ARDS) that correlates with mortality. The cause of increased dead space in ARDS has largely been attributed to increased alveolar dead space due to ventilation/perfusion mismatching and shunt. We sought to determine whether anatomic dead space also increases in response to mechanical ventilation. Mice received intratracheal lipopolysaccharide (LPS) or saline and mechanical ventilation (MV). Four-dimensional computed tomography (4DCT) scans were performed at onset of MV and after 5 h of MV. Detailed measurements of airway volumes and lung tidal volumes were performed using image analysis software. The forced oscillation technique was used to obtain measures of airway resistance, tissue damping, and tissue elastance. The ratio of airway volumes to total tidal volume increased significantly in response to 5 h of mechanical ventilation, regardless of LPS exposure, and airways demonstrated significant variation in volumes over the respiratory cycle. These findings were associated with an increase in tissue elastance (decreased lung compliance) but without changes in tidal volumes. Airway volumes increased over time with exposure to mechanical ventilation without a concomitant increase in tidal volumes. These findings suggest that anatomic dead space fraction increases progressively with exposure to positive pressure ventilation and may represent a pathological process.NEW & NOTEWORTHY We demonstrate that anatomic dead space ventilation increases significantly over time in mice in response to mechanical ventilation. The novel functional lung-imaging techniques applied here yield sensitive measures of airway volumes that may have wide applications.

X-ray specks: low dose in vivo imaging of lung structure and function.

Respiratory health is directly linked to the structural and mechanical properties of the airways of the lungs. For studying respiratory development and pathology, the ability to quantitatively measure airway dimensions and changes in their size during respiration is highly desirable. Real-time imaging of the terminal airways with sufficient contrast and resolution during respiration is currently not possible. Herein we reveal a simple method for measuring lung airway dimensions in small animals during respiration from a single propagation-based phase contrast x-ray image, thereby requiring minimal radiation. This modality renders the lungs visible as a speckled intensity pattern. In the near-field regime, the size of the speckles is directly correlated with that of the dominant length scale of the airways. We demonstrate that Fourier space quantification of the speckle texture can be used to statistically measure regional airway dimensions at the alveolar scale, with measurement precision finer than the spatial resolution of the imaging system. Using this technique we discovered striking differences in developmental maturity in the lungs of rabbit kittens at birth.

Tracking extended mucociliary transport activity of individual deposited particles: longitudinal synchrotron X-ray imaging in live mice.

To assess potential therapies for respiratory diseases in which mucociliary transit (MCT) is impaired, such as cystic fibrosis and primary ciliary dyskinesia, a novel and non-invasive MCT quantification method has been developed in which the transit rate and behaviour of individual micrometre-sized deposited particles are measured in live mice using synchrotron phase-contrast X-ray imaging. Particle clearance by MCT is known to be a two-phase process that occurs over a period of minutes to days. Previous studies have assessed MCT in the fast-clearance phase, ∼20 min after marker particle dosing. The aim of this study was to non-invasively image changes in particle presence and MCT during the slow-clearance phase, and simultaneously determine whether repeat synchrotron X-ray imaging of mice was feasible over periods of 3, 9 and 25 h. All mice tolerated the repeat imaging procedure with no adverse effects. Quantitative image analysis revealed that the particle MCT rate and the number of particles present in the airway both decreased with time. This study successfully demonstrated for the first time that longitudinal synchrotron X-ray imaging studies are possible in live small animals, provided appropriate animal handling techniques are used and care is taken to reduce the delivered radiation dose.