Phase Contrast X-ray Imaging for rapid, non-invasive assessment of genetic therapies for Cystic Fibrosis
Common airspace diseases such as asthma, cystic fibrosis and chronic obstructive pulmonary disease are characterised by airway inflammation and abnormal mucous production or retention that compromises respiratory function and impairs the lung defences against infection. Although symptomatic and curative therapies are the subject of extensive research, there is no direct method of examining the airways non-invasively, at sufficiently high resolution, and in a timely fashion, to provide monitoring or rapid assessment of treatment efficacy.
We are actively investigating advances in Phase Contrast X-ray Imaging (PCXI) to develop new means of assessing airway health. PCXI has the ability to image soft tissues with startling clarity, and it is ideally suited to direct and rapid study of the physiological changes in airways due to disease.
When coupled to high flux synchrotron X-ray sources, extremely high resolution and very short exposures times are achievable, enabling the use of PCXI for dynamic imaging in vivo, an ability that has yet to be fully exploited. If successful, this novel imaging technique has significant potential for new and extremely high resolution diagnostic measures in humans. |
Figure 1: A conventional X-ray image (left, Faxitron) and a phase contrast X-ray image (right, SPring-8, 33keV) of a (different) mouse nose region. Note the bright fringes around the sinuses (arrows) |
Figure 2: PC-CT image of a mouse upper torso, with upper right quadrant of skull cut away to show nasal airways. Note the simultaneous bony and soft tissue contrast, for example, the high visibility of the trachea including cartilage rings. An animated PC-CT rendering can be seen here (7Mb). |
In cystic fibrosis (CF), the airway-cell ion-balance is dysfunctional, resulting in a reduction of depth/volume of the airway surface liquid (ASL), a thin film of fluid that overlies airway cells and is critical to normal lung defences. In this environment normal airway cleaning mechanisms cannot work properly, and unstoppable infection, tissue damage, and eventual lung failure occurs. Gene correction of the defect of CF should return ASL depth to, or towards, normal levels providing a rational basis for a lifelong cure.
Despite good progress with the use if invasive and/or terminal assessment procedures in CF animal models, the inability to non-invasively measure the functional effect and longevity of gene correction on airway epithelium in animals or humans is a major impediment to further advancement. Currently, no such outcome assessment exists, and researchers and clinicians must wait months or even years to know if treatments have had beneficial effects. Clinically, the primary outcome measure for any attemprts has been improvement in lung function, but changes are small and slow to appear. We believe that PCXI can pro vide an exciting new measurement approach that can assess the success of gene therapy by direct, in vivo examination of the ASL.
Phase contrast imaging utilizes changes in the phase of propagating X-rays to greatly enhance image contrast. The resulting image contrast can be several orders of magnitude larger than the contrast offered by conventional (absorption) X-ray imaging. Whilst absorption contrast effectively maps the density distribution of differing tissues, phase contrast imaging is particularly sensitive to the boundaries between different tissues. This manifests as a characteristic bright “fringe” that appears near tissue interfaces whether produced in normal light microscopes, or via PCXI (Fig. 1). This edge effect will be exploited for imaging of the airways, and in particular, for imaging the airway lumen/ASL/tissue boundary.
Phase contrast imaging can be coupled to computed tomography (CT) to provide three-dimensional (3D) data with greatly enhanced soft tissue contrast. Since phase contrast enhances edges or interfaces, the reconstructed phase contrast CT (PC-CT) volume conspicuously renders soft tissue interfaces highly visible, particularly at the airway interfaces (Fig. 2) |
Our own pilot studies in normal mice have indicated that PCXI is feasible to use in vivo in transgenic CF mice. Fig. 3(a) indicates the areas of interest for assessment of gene therapy in the mouse nose. The resolution allows even air bubbles and bubble interstices (Fig. 3(b)) to be detected filling the lumen to the airway surface edge (arrows). The ability to visualise bubbles provides a novel direct means assessing liquid bolus instillation as a means of treatment delivery, a method employed in gene therapy for CF, as well as other pulmonary treatments such as the partial liquid ventilation for respiratory distress syndrome or surfactant replacement delivery.
More recent pilot studies have directly quantified the nasal mucociliary clearance (MCC) rate of introduced 13um dia. glass beads, showing a rate of ~ 0.18 mm/min under Nembutal anaesthesia (Fig 4.). MCC has not been detected so directly before; this technique could provide alternate or complementary assessment of successful gene delivery, since MCC should also improve when the CF gene defect is corrected by successful insertion of the CFTR gene. |
Figure 3: (a) area of interest for CF gene therapy, and inset (b) airway surface edge (arrows) at edges of airway bubbles induced by fluid instillation; note clarity of bubble edges, visualization of bubble interstices. |
Figure 4: High resolution image sequence showing a glass bead (13um dia.) transiting the nasal airway. The airway edge is at the right. Arrows indicate bead, and positions in previous/subsequent frames (x). Frames were acquired at 10 second intervals, such that these images are 30 seconds apart (image frame numbers shown). |
Although we are developing new imaging methods specifically for cystic fibrosis, the assessment and therapy of almost any airspace disease pathology demands in vivo, non-invasive and quantitative measures of lung function at a spatial resolution sufficient to see the physiological effect of induced structural changes. By implementing the developed techniques in several animal models of widely-differing sizes, we aim to not only provide important physiological information regarding the effectiveness of treatment, but also to advance the techniques towards eventual pre-clinical trials of it's suitability for use in human subjects.
Aspects of this project are funded by a NH&MRC grant, “Correction and measurement of the basic defects in cystic fibrosis”.
Dr Karen Siu, School of Physics, Monash University
Dr David Parsons, Women's and Children's Hospital; and University of Adelaide, Dept of Paediatrics, Adelaide, South Australia |
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