Martin Donnelley

High-resolution visualization of airspace structures in intact mice via synchrotron phase-contrast X-ray imaging (PCXI)

Anatomical visualization of airspace‐containing organs in intact small animals has been limited by the resolution and contrast available from current imaging methods such as X‐ray, micro‐computed tomography and magnetic resonance imaging. Determining structural relationships and detailed anatomy has therefore relied on suitable fixation, sectioning and histological processing. More complex and informative analyses such as orthogonal views of an organ and three‐dimensional structure visualizations have required different animals and image sets, laboriously processed to gather this complementary structural information. Precise three‐dimensional anatomical views have always been difficult to achieve in small animals. Here we report the ability of phase‐contrast synchrotron X‐ray imaging to provide detailed two‐ and three‐dimensional visualization of airspace organ structures in intact animals. Using sub‐micrometre square pixel charge‐coupled device array detectors, the structure and anatomy of hard and soft tissues, and of airspaces, is readily available using phase‐contrast synchrotron X‐ray imaging. Moreover, software‐controlled volume‐reconstructions of tomographic images not only provide unsurpassed image clarity and detail, but also selectable anatomical views that cannot be obtained with established histological techniques. The morphology and structure of nasal and lung airways and the middle ear are illustrated in intact mice, using two‐ and three‐dimensional representations. The utility of phase‐contrast synchrotron X‐ray imaging for non‐invasively localizing objects implanted within airspaces, and the detection of gas bubbles transiting live airways, are other novel features of this visualization methodology. The coupling of phase‐contrast synchrotron X‐ray imaging technology with software‐based reconstruction techniques holds promise for novel and high‐resolution non‐invasive examination of airspace anatomy in small animal models.

Measuring Airway Surface Liquid Depth in Ex Vivo Mouse Airways by X-Ray Imaging for the Assessment of Cystic Fibrosis Airway Therapies

In the airways of those with cystic fibrosis (CF), the leading pathophysiological hypothesis is that an ion channel defect results in a relative decrease in airway surface liquid (ASL) volume, producing thick and sticky mucus that facilitates the establishment and progression of early fatal lung disease. This hypothesis predicts that any successful CF airway treatment for this fundamental channel defect should increase the ASL volume, but up until now there has been no method of measuring this volume that would be compatible with in vivo monitoring. In order to accurately monitor the volume of the ASL, we have developed a new x-ray phase contrast imaging method that utilizes a highly attenuating reference grid. In this study we used this imaging method to examine the effect of a current clinical CF treatment, aerosolized hypertonic saline, on ASL depth in ex vivo normal mouse tracheas, as the first step towards non-invasive in vivo ASL imaging. The ex vivo tracheas were treated with hypertonic saline, isotonic saline or no treatment using a nebuliser integrated within a small animal ventilator circuit. Those tracheas exposed to hypertonic saline showed a transient increase in the ASL depth, which continued for nine minutes post-treatment, before returning to baseline by twelve minutes. These findings are consistent with existing measurements on epithelial cell cultures, and therefore suggest promise for the future development of in vivo testing of treatments. Our grid-based imaging technique measures the ASL depth with micron resolution, and can directly observe the effect of treatments expected to increase ASL depth, prior to any changes in overall lung health. The ability to non-invasively observe micron changes in the airway surface, particularly if achieved in an in vivo setting, may have potential in pre-clinical research designed to bring new treatments for CF and other airway diseases to clinical trials.

In Vivo X-Ray Imaging Reveals Improved Airway Surface Hydration after a Therapy Designed for Cystic Fibrosis

Kaye S. Morgan, Martin Donnelley, Nigel Farrow, Andreas Fouras, Naoto Yagi, Yoshio Suzuki, Akihisa Takeuchi, Kentaro Uesugi, Richard C. Boucher, Karen K. W. Siu, David W. Parsons

A CAD System for Long-Bone Segmentation and Fracture Detection

Medical imaging has advanced at a tremendous rate since x-rays were discovered in 1895. Today, x-ray machines produce extremely high-quality images for radiologists to interpret. However, the methods of interpretation have only recently begun to be augmented by advances in computer technology. Computer aided diagnosis (CAD) systems that guide healthcare professionals in making the correct diagnosis are slowly becoming more prevalent throughout the medical field. Detection of long-bone fractures is an important orthopaedic and radiologic problem, and it is proposed that a novel CAD system could help reduce the number of fractures missed during x-ray diagnosis. A number of image processing software algorithms useful for assisting the fracture detection process are described, and their accuracy evaluated on a database of fracture images from trauma patients. Incorporating these methods will further expand the capabilities of todays CAD systems, and result in more accurate diagnosis of fractures and a reduction of the fracture miss rate.

Real-time non-invasive detection of inhalable particulates delivered into live mouse airways.

Fine non-biological particles small enough to be suspended in the air are continually inhaled as we breathe. These particles deposit on airway surfaces where they are either cleared by airway defences or can remain and affect lung health. Pollutant particles from vehicles, building processes and mineral and industrial dusts have the potential to cause both immediate and delayed health problems. Because of their small size, it has not been possible to non-invasively examine how individual particles deposit on live airways, or to consider how they behave on the airway surface after deposition. In this study, synchrotron phase-contrast X-ray imaging (PCXI) has been utilized to detect and monitor individual particle deposition. The in vitro detectability of a range of potentially respirable particulates was first determined. Of the particulates tested, only asbestos, quarry dust, fibreglass and galena (lead sulfate) were visible in vitro. These particulates were then examined after delivery into the nasal airway of live anaesthetized mice; all were detectable in vivo but each exhibited different surface appearances and behaviour along the airway surface. The two fibrous particulates appeared as agglomerations enveloped by fluid, while the non-fibrous particulates were present as individual particles. Synchrotron PCXI provides the unique ability to non-invasively detect and track deposition of individual particulates in live mouse airways. With further refinement of particulate sizing and delivery techniques, PCXI should provide a novel approach for live animal monitoring of airway particulates relevant to lung health.

Live small-animal X-ray lung velocimetry and lung micro-tomography at the Australian Synchrotron Imaging and Medical Beamline.

The high flux and coherence produced at long synchrotron beamlines makes them well suited to performing phase-contrast X-ray imaging of the airways and lungs of live small animals. Here, findings of the first live-animal imaging on the Imaging and Medical Beamline (IMBL) at the Australian Synchrotron are reported, demonstrating the feasibility of performing dynamic lung motion measurement and high-resolution micro-tomography. Live anaesthetized mice were imaged using 30 keV monochromatic X-rays at a range of sample-to-detector propagation distances. A frame rate of 100 frames s(-1) allowed lung motion to be determined using X-ray velocimetry. A separate group of humanely killed mice and rats were imaged by computed tomography at high resolution. Images were reconstructed and rendered to demonstrate the capacity for detailed, user-directed display of relevant respiratory anatomy. The ability to perform X-ray velocimetry on live mice at the IMBL was successfully demonstrated. High-quality renderings of the head and lungs visualized both large structures and fine details of the nasal and respiratory anatomy. The effect of sample-to-detector propagation distance on contrast and resolution was also investigated, demonstrating that soft tissue contrast increases, and resolution decreases, with increasing propagation distance. This new capability to perform live-animal imaging and high-resolution micro-tomography at the IMBL enhances the capability for investigation of respiratory diseases and the acceleration of treatment development in Australia.

Long-term therapeutic and reporter gene expression in lentiviral vector treated cystic fibrosis mice.

Persistent reporter gene and cystic fibrosis transmembrane conductance regulator (CFTR) nasal airway gene expression can be achieved with a single lentiviral (LV) gene vector dosing when coupled with a preparatory lysophosphatidylcholine (LPC) airway pre‐treatment. In the present study, we characterised the duration of gene expression in individual cystic fibrosis (CF) knockout mice (cftrtm1unc) over their lifetimes.

A new technique to examine individual pollutant particle and fibre deposition and transit behaviour in live mouse trachea

During respiration, particles suspended in the air are inhaled and unless cleared by airway defences they can remain and affect lung health. Their size precludes the use of standard imaging modalities so we have developed synchrotron phase-contrast X-ray imaging (PCXI) methods to non-invasively monitor the behaviour of individual particles in live mouse airways. In this study we used these techniques to examine post-deposition particle behaviour in the trachea. PCXI was used to monitor the deposition and subsequent behaviour of particles of quarry dust and lead ore; fibres of asbestos and fibreglass; and hollow glass micro-spheres. Visibility was examined in vitro and ex vivo to avoid the complicating effects of surrounding tissue and respiratory or cardiac motion. Particle behaviour was then examined after deposition onto the tracheal airway surfaces of live mice. Each particle and fibre looked and behaved differently on the airway surface. Particles lodged on the airway shortly after deposition, and the rate at which this occurred was dependent on the particle type and size. After the live-imaging experiments, excised airway samples were examined using light and electron microscopy. Evidence of particle capture into the airway surface fluids and the epithelial cell layer was found. PCXI is a valuable tool for examining post-deposition particulate behaviour in the tracheal airway. These first indications that the interaction between airways and individual particles may depend on the particle type and size should provide a novel approach to studying the early effects of respired particles on airway health.

Challenges of up-scaling lentivirus production and processing

Lentiviruses are becoming an increasingly popular choice of gene transfer vehicle for use in the treatment of a variety of genetic and acquired human diseases. As research progresses from basic studies into pre-clinical and clinical phases, there is a growing demand for large volumes of high purity, concentrated vector, and accordingly, the means to produce such quantities. Unlike other viral vectors, lentiviruses are difficult to produce using stable cell lines, therefore transient transfection of adherent cell lines is conventionally used, and this method has proven challenging to up-scale. Furthermore, with the required increases in the volume of vector needed for larger animal and human use, comes the need for more efficient and sophisticated supernatant purification and concentration techniques. This review presents the challenges of up-scaling lentivirus production and processing approaches, novel systems for overcoming these issues, and the quality assessments recommended for producing a clinical grade lentiviral gene therapy product.

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.