Richard Carnibella

Functional Lung Imaging during HFV in Preterm Rabbits

Although high frequency ventilation (HFV) is an effective mode of ventilation, there is limited information available in regard to lung dynamics during HFV. To improve the knowledge of lung function during HFV we have developed a novel lung imaging and analysis technique. The technique can determine complex lung motion information in vivo with a temporal resolution capable of observing HFV dynamics. Using high-speed synchrotron based phase contrast X-ray imaging and cross-correlation analysis, this method is capable of recording data in more than 60 independent regions across a preterm rabbit lung in excess of 300 frames per second (fps). This technique is utilised to determine regional intra-breath lung mechanics of preterm rabbit pups during HFV. Whilst ventilated at fixed pressures, each animal was ventilated at frequencies of 1, 3, 5 and 10 Hz. A 50% decrease in delivered tidal volume was measured at 10 Hz compared to 1 Hz, yet at the higher frequency a 500% increase in minute activity was measured. Additionally, HFV induced greater homogeneity of lung expansion activity suggesting this ventilation strategy potentially minimizes tissue damage and improves gas mixing. The development of this technique permits greater insight and further research into lung mechanics and may have implications for the improvement of ventilation strategies used to support severe pulmonary trauma and disease.

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.

Decoding the structure of granular and porous materials from speckled phase contrast X-ray images

Imaging techniques for studying the structure of opaque, granular and porous materials are limited by temporal resolution and radiation dose. We present a technique for characterising the structure of such materials by decoding three dimensional structural information from single, propagation based phase contrast X-ray images. We demonstrate the technique by measuring the distribution of diameters of glass microspheres in packed samples. We also present synthetic data, which shows that our inverse method is stable and that accuracy is improved by phase contrast X-ray imaging. Compared to computed tomography, our technique has superior temporal resolution and lower radiation dose.

Technical Note: Contrast free angiography of the pulmonary vasculature in live mice using a laboratory x-ray source

Purpose: In vivo imaging of the pulmonary vasculature in small animals is difficult yet highly desirable in order to allow study of the effects of a host of dynamic biological processes such as hypoxic pulmonary vasoconstriction. Here the authors present an approach for the quantification of changes in the vasculature. Methods: A contrast free angiography technique is validated in silico through the use of computer-generated images and in vivo through microcomputed tomography (μCT) of live mice conducted using a laboratory-based x-ray source. Subsequent image processing on μCT data allowed for the quantification of the caliber of pulmonary vasculature without the need for external contrast agents. These measures were validated by comparing with quantitative contrast microangiography in the same mice. Results: Quantification of arterial diameters from the method proposed in this study is validated against laboratory-based x-ray contrast microangiography. The authors find that there is a high degree of correlation (R = 0.91) between measures from microangiography and their contrast free method. Conclusions: A technique for quantification of murine pulmonary vasculature without the need for contrast is presented. As such, this technique could be applied for longitudinal studies of animals to study changes to vasculature without the risk of premature death in sensitive mouse models of disease. This approach may also be of value in the clinical setting.

Single-exposure dual-energy-subtraction X-ray imaging using a synchrotron source

Projection radiography of the chest has long been plagued by the presence of bony anatomy obscuring visibility of the lungs and heart. Dual-energy subtraction is a well known method for differentiating bone and soft tissue, but existing techniques are not ideally suited to dynamic imaging. Herein a new technique to address this problem is presented. The harmonic content of a monochromated X-ray beam is exploited, and two in-line detectors are used to perform single-exposure dual-energy imaging. Images of a phantom demonstrate the ability to both separate and quantitatively measure the thickness of constituent materials, whilst images of a mouse thorax demonstrate the ability to separate bone and soft tissue in a biological specimen. The technique is expected to improve the performance of dynamic lung imaging.

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.

Lung hypoplasia in newborn rabbits with a diaphragmatic hernia affects pulmonary ventilation but not perfusion

BackgroundA congenital diaphragmatic hernia (DH) can result in severe lung hypoplasia that increases the risk of morbidity and mortality after birth; however, little is known about the cardiorespiratory transition at birth.MethodsUsing phase-contrast X-ray imaging and angiography, we examined the cardiorespiratory transition at birth in rabbit kittens with DHs. Surgery was performed on pregnant New Zealand white rabbits (n=18) at 25 days’ gestation to induce a left-sided DH. Kittens were delivered at 30 days’ gestation, intubated, and ventilated to achieve a tidal volume (Vt) of 8 ml/kg in control and 4 ml/kg in DH kittens while they were imaged.ResultsFunctional residual capacity (FRC) recruitment and Vt in the hypoplastic left lung were markedly reduced, resulting in a disproportionate distribution of FRC into the right lung. Following lung aeration, relative pulmonary blood flow (PBF) increased equally in both lungs, and the increase in pulmonary venous return was similar in both control and DH kittens.ConclusionThese findings indicate that nonuniform lung hypoplasia caused by DH alters the distribution of ventilation away from hypoplastic and into normally grown lung regions. During transition, the increase in PBF and pulmonary venous return, which is vital for maintaining cardiac output, is not affected by lung hypoplasia.

Single-shot X-ray measurement of alveolar size distributions

Regional changes in lung microstructure are an important component of several common lung disorders and even in healthy lungs alveolar mechanics are poorly understood. Existing techniques capable of studying the lung microstructure have various limitations including poor temporal resolution. We present a technique, which can measure the distribution of alveolar diameters from a single, phase contrast chest X-ray. We present the results of analysis of synchrotron images of a rabbit pup’s lungs, which we compare with high-resolution computed tomography images. We demonstrate that measurements can be made with an exposure time of 40 ms, highlighting the unique potential for performing dynamic in vivo measurements. Applications include disease detection, assessment of therapeutics and physiological studies.

High resolution propagation-based imaging system for in vivo dynamic computed tomography of lungs in small animals

We have developed an x-ray imaging system for in vivo four-dimensional computed tomography (4DCT) of small animals for pre-clinical lung investigations. Our customized laboratory facility is capable of high resolution in vivo imaging at high frame rates. Characterization using phantoms demonstrate a spatial resolution of slightly below 50 μm at imaging rates of 30 Hz, and the ability to quantify material density differences of at least 3%. We benchmark our system against existing small animal pre-clinical CT scanners using a quality factor that combines spatial resolution, image noise, dose and scan time. In vivo 4DCT images obtained on our system demonstrate resolution of important features such as blood vessels and small airways, of which the smallest discernible were measured as 55-60 μm in cross section. Quantitative analysis of the images demonstrate regional differences in ventilation between injured and healthy lungs.