Abstract Computed tomography (CT) and spirometry are the mainstays of clinical pulmonary assessment. Spirometry is effort dependent and only provides a single global measure that is insensitive for regional disease and as such, poor for capturing the early onset of lung disease, especially patchy disease such as cystic fibrosis lung disease. CT sensitively measures change in structure associated with advanced lung disease. However, obstructions in the peripheral airways and early onset of lung stiffening are often difficult to detect. Furthermore, CT imaging poses a radiation risk, particularly for young children and dose reduction tends to result in reduced resolution. Here, we apply a series of lung tissue motion analyses, to achieve regional pulmonary function assessment in β-ENaC-overexpressing mice, a well-established model of lung disease. The expiratory time constants of regional airflows in the segmented airway tree were quantified as a measure of regional lung function. Our results showed marked heterogeneous lung function in β-ENaC-Tg mice compared to wild-type littermate controls; identified locations of airway obstruction and quantified regions of bimodal airway resistance demonstrating lung compensation. These results demonstrate the applicability of regional lung function derived from lung motion as an effective alternative respiratory diagnostic tool.
Bright synchrotron x-ray sources enable imaging with short exposure times, and hence in a high-speed image sequence. These x-ray movies can capture not only sample structure, but also how the sample changes with time, how it functions. The use of a synchrotron x-ray source also provides high spatial coherence, which facilitates the capture of not only a conventional attenuation-based x-ray image, but also phase-contrast and dark-field signals. These signals are strongest from air/tissue interfaces, which means that they are particularly useful for examining the respiratory system. We have performed a range of x-ray imaging studies that look at lung function, airway surface function, inhaled and instilled treatment delivery, and treatment effect in live small animal models [Morgan, 2019]. These have utilized a range of optical set-ups and phase-contrast imaging methods in order to be sensitive to the relevant sample features, and be compatible with high-speed imaging. For example, we have used a grating interferometer to measure how the airsacs in the lung inflate during inhalation, via changes in the dark-field signal [Gradl, 2018], a single-exposure, single-grid set-up to capture changes in the liquid lining of the airways [Morgan, 2015] and propagation-based phase contrast to image clearance of inhaled debris [Donnelley, 2019]. Studies have also utilized a range of analysis methods to extract how the sample features change within a time-sequence of two-dimensional projections or three-dimensional volumes. While these imaging studies began in large-scale synchrotron facilities, we have recently performed these kinds of studies at an inverse-Compton-based compact synchrotron, the Munich Compact Light Source (MuCLS) [Gradl, 2018b]. 1. Morgan, Kaye, et al., "Methods for dynamic synchrotron X-ray imaging of live animals.", under review 01/2019. 2. Gradl, R., et al. "Dynamic in vivo chest x-ray dark-field imaging in mice." IEEE Transactions on Medical Imaging (2018). 3. Morgan, Kaye S., et al. "In vivo X-ray imaging reveals improved airway surface hydration after a therapy designed for cystic fibrosis." American Journal of Respiratory and Critical Care Medicine 190.4 (2014): 469-472. 4. Donnelley, Martin, et al. "Live-pig-airway surface imaging and whole-pig CT at the Australian Synchrotron Imaging and Medical Beamline." Journal of Synchrotron Radiation 26.1 (2019). 5. Gradl, Regine, et al. "In vivo Dynamic Phase-Contrast X-ray Imaging using a Compact Light Source." Scientific Reports 8.1 (2018b): 6788.
Genetic therapies for cystic fibrosis (CF) must be assessed for safety and efficacy, so testing in a non-human primate (NHP) model is invaluable. In this pilot study we determined if the conducting airways of marmosets (n = 2) could be transduced using an airway pre-treatment followed by an intratracheal bolus dose of a VSV-G pseudotyped HIV-1 based lentiviral (LV) vector (LacZ reporter). LacZ gene expression (X-gal) was assessed after 7 days and found primarily in conducting airway epithelia as well as in alveolar regions. The LacZ gene was not detected in liver or spleen via qPCR. Vector p24 protein bio-distribution into blood was transient. Dosing was well tolerated. This preliminary study confirmed the transducibility of CF-relevant airway cell types. The marmoset is a promising NHP model for testing and translating genetic treatments for CF airway disease towards clinical trials.
The Australian Synchrotron Imaging and Medical Beamline (IMBL) was designed as the world’s widest synchrotron X-ray beam, enabling both clinical imaging and therapeutic applications for humans as well as the imaging of large animal models. Our group is developing methods for imaging the airways of newly developed CF animal models that display human-like lung disease, such as the CF pig, and we expect that the IMBL can be utilised to image airways in animals of this size. This study utilised samples of excised tracheal tissue to assess the feasibility, logistics and protocols required for airway imaging in large animal models such as pigs and sheep at the IMBL. We designed an image processing algorithm to automatically track and quantify the tracheal mucociliary transport (MCT) behaviour of 103 μm diameter high refractive index (HRI) glass bead marker particles deposited onto the surface of freshly-excised normal sheep and pig tracheae, and assessed the effects of airway rehydrating aerosols. We successfully accessed and used scavenged tracheal tissue, identified the minimum bead size that is visible using our chosen imaging setup, verified that MCT could be visualised, and that our automated tracking algorithm could quantify particle motion. The imaging sequences show particles propelled by cilia, against gravity, up the airway surface, within a well-defined range of clearance speeds and with examples of ‘clumping’ behaviour that is consistent with the in vivo capture and mucus-driven transport of particles. This study demonstrated that the wide beam at the IMBL is suitable for imaging MCT in ex vivo tissue samples. We are now transitioning to in vivo imaging of MCT in live pigs, utilising higher X-ray energies and shorter exposures to minimise motion blur.
To determine the efficacy of potential cystic fibrosis (CF) therapies we have developed a novel mucociliary transit (MCT) measurement that uses synchrotron phase contrast X-ray imaging (PCXI) to non-invasively measure the transit rate of individual micron-sized particles deposited into the airways of live mice. The aim of this study was to image changes in MCT produced by a rehydrating treatment based on hypertonic saline (HS), a current CF clinical treatment. Live mice received HS containing a long acting epithelial sodium channel blocker (P308); isotonic saline; or no treatment, using a nebuliser integrated within a small-animal ventilator circuit. Marker particle motion was tracked for 20 minutes using PCXI. There were statistically significant increases in MCT in the isotonic and HS-P308 groups. The ability to quantify in vivo changes in MCT may have utility in pre-clinical research studies designed to bring new genetic and pharmaceutical treatments for respiratory diseases into clinical trials.
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