Objectives In this study we investigated the potentials of dual-modal CT-MRI macrophage tracking, by a intratracheal instillation of a mixture of either gadolinium nanoparticles or barium sulphate loaded alveolar macrophages into mice of an allergic airway inflammation (asthma) model and their respective healthy control, imaged with Synchrotron X-rays microtomography (SR μCT) and Micro Magnetic Resonance Imaging (μMRI).

Materials and Methods The mice were scanned ex vivo using SRμCT at 22 keV and with a 9.4 Tesla μMRI scanner. The CT and MRI data sets were registered and fused together, followed by quantitative and statistical analysis.

Results The asthmatic sample injected with contrast agent loaded macrophages showed high absorbing spots inside the soft-tissue regions of the lung for the CT data set, as well as higher contrast for the soft-tissue in the MRI data set. Furthermore, the correlation analysis showed a perfect negative correlation between the soft tissue mean grey value in CT and the soft tissue mean grey value in MRI.

Conclusion The dual-modal CT-MRI cell tracking of intratracheally administered macrophages (loaded with contrast agent) in an asthmatic mouse helps to extract synergistic information about the migration  behaviour of macrophages, where clusters of cells were detected in CT, while as a general increase of the soft-tissue contrast could be observed in MRI, due to a homogeneous cell distribution.

¨This thesis focuses on evaluating the dual-modal Computed Tomography (CT) and Magnetic Resonance Imaging (MRI) capabilities of contrast agents. For such purposes a gadolinium based contrast agent is of high interest, due to its paramagnetic properties, which while present inside a magnetic field will hence interact with the protons spins of water (in tissue and fat) and shorten their the T1 relaxation time, thereby creating a positive image contrast in MRI. Furthermore, the X-ray Mass Attenuation Coefficient (MAC) of gadolinium is relatively high, thus suggesting its potential use, also as a CT contrast agent.

Gadolinium nanoparticles (GdNPs) can be loaded into cells, such as macrophages, which offers the possibility to track cells inside entire organisms. In the first step the uptake of GdNPs inside cells was investigated, together with a test for toxicity. To show the potential of using GdNP loaded macrophages for functional imaging of inflammation, an acute allergic airway inflammation mouse model (mimicking asthma in humans) was used and analyzed by in-situ synchrotron phase contrast CT. In the first step this approach was evaluated using macrophages loaded with a clinical contrast agent containing barium sulphate (BaSO4), since this agent is known to provide high contrast in CT. In the ultimate step a combination of both BaSO4 and GdNP loaded macrophages was used in the same asthmatic mouse model and analyzed by dual modal Synchrotron phase contrast CT and Micro Magnetic Resonance Imaging (μ-MRI).

Complementary results in terms of the biodistribution of injected macrophages could only be obtained by the combination of both synchrotron phase contrast CT and μ-MRI, where the first modality allows a detailed localization of clustered BaSO4 loaded macrophages, but fails to detect single macrophages, which could instead be indirectly observed by μ-MRI as an increase of the T1-contrast, coming from the soft tissue of mice injected with GdNP loaded macrophages.

Functionalized computed tomography (CT) in combination with labelled cells is virtually non-existent due to the limited sensitivity of X-ray-absorption-based imaging, but would be highly desirable to realise cell tracking studies in entire organisms. In this study we applied in-line free propagation X-ray phase-contrast CT (XPCT) in an allergic asthma mouse model to assess structural changes as well as the biodistribution of barium-labelled macrophages in lung tissue. Alveolar macrophages that were barium-sulfate-loaded and fluorescent-labelled were instilled intratracheally into asthmatic and control mice. Mice were sacrificed after 24 h, lungs were kept in situ, inflated with air and scanned utilizing XPCT at the SYRMEP beamline (Elettra Synchrotron Light Source, Italy). Single-distance phase retrieval was used to generate data sets with ten times greater contrast-to-noise ratio than absorption-based CT (in our setup), thus allowing to depict and quantify structural hallmarks of asthmatic lungs such as reduced air volume, obstruction of airways and increased soft-tissue content. Furthermore, we found a higher concentration as well as a specific accumulation of the barium-labelled macrophages in asthmatic lung tissue. It is believe that XPCT will be beneficial in preclinical asthma research for both the assessment of therapeutic response as well as the analysis of the role of the recruitment of macrophages to inflammatory sites.

We present novel cell uptake methodologies related to the usage of MRI/CT contrast agents for the purpose of performing dual-modal cell tracking with macrophages in both MRI and CT. Two different techniques, namely Synchrotron X-rays microtomography and Micro Magnetic Resonance Imaging were used to investigate the contrast  enhancement, as an effect of the MRI/CT contrast agent cell uptake of mouse alveolar macrophages. Macrophages loaded with the  commercial contrast agent Micropaque® CT, containing barium sulphate (BaSO4) immersed in Sorbitol, showed a much higher contrast enhancement in CT, than an MRI/CT contrast agent based on Gadolinium nanoparticles (GdNPs). The CT contrast of GdNPs (at 5 mM of Gd) could be increased, by immersing the GdNPs in Sorbitol, while still maintaining a positive T1-contrast in MRI. The idea of co-loading macrophages with both BaSO4 and GdNP inside the same cells  presented a valid "trade off" between the optimal contrast in CT vs. MRI etc. It was concluded that while optimizing the cell uptake of contrast agent for cell tracking in MRI/CT, it is important to make a "trade off" between the following 3 parameters, 1) optimal contrast in CT, 2) optimal contrast in MRI and 3) metabolic cell activity, depending on the given application. These cell optimization ideas may be of importance to every field aiming to image an inflammatory disease, based on the utilization of contrast agent loaded macrophages.

Lung imaging in mouse disease models is crucial for the assessment of the severity of airway disease but remains challenging due to the small size and the high porosity of the organ. Synchrotron inline free-propagation phase-contrast computed tomography (CT) with its intrinsic high soft-tissue contrast provides the necessary sensitivity and spatial resolution to analyse the mouse lung structure in great detail. Here, this technique has been applied in combination with single-distance phase retrieval to quantify alterations of the lung structure in experimental asthma mouse models of different severity. In order to mimic an in vivo situation as close as possible, the lungs were inflated with air at a constant physiological pressure. Entire mice were embedded in agarose gel and imaged using inline free-propagation phase-contrast CT at the SYRMEP beamline (Synchrotron Light Source, Elettra, Trieste, Italy). The quantification of the obtained phase-contrast CT data sets revealed an increasing lung soft-tissue content in mice correlating with the degree of the severity of experimental allergic airways disease. In this way, it was possible to successfully discriminate between healthy controls and mice with either mild or severe allergic airway disease. It is believed that this approach may have the potential to evaluate the efficacy of novel therapeutic strategies that target airway remodelling processes in asthma.

In this paper we have established a general investigative methodology for quantitative computed tomography (CT) lung image analysis in the sagittal, coronal and transversal orientation of lungs with various lung diseases. Mean values were recorded for the two parameters percentage volume and structural thickness based on stripe shaped volumes of interest (VOIs) from the XY (transversal), YZ (sagittal) and ZX (coronal) orientation, placed out in the left and right lung side. A one-way ANOVA with Tukey–Kramer 90% simultaneous confidence intervals for pair wise comparison of means was performed on each considered parameter, in order to detect any statistically significant differences in between the samples. This methodology was first tested on high resolution synchrotron micro-computed tomography images of a preclinical asthma mouse model, injected with barium sulfate filled alveolar macrophages, with the purpose of marking out asthmatic inflammation sites. Preclinical mouse models are today commonly used as artificial models for studying various human diseases, e.g. asthma. Therefore, in order to translate our methodology protocol also to clinical applications the proposed methodology was also tested on lung data sets of patients, with various lung diseases. The presented general methodology was proven to be successful for the quantification of lung structural differences in an asthma mouse model, as well as being applicable also on patient lungs with various lung diseases. The outlined analysis protocol was tested on images obtained only by means of CT, but could also potentially be applied on images of the lung obtained by other 3D-imaging techniques.

When attempting to establish accurate models for the human diploe, micro-scale morphological differences in the four main areas of the calvaria could also be considered. In this study, X-ray computed microtomography (μ-CT) images were analyzed in order to quantitatively characterize the micro-architecture of the human calvarium diploe. A bone specimen from each area of the skull (temporal, frontal, parietal and occipital) was extracted from a set of 5 human donors and each specimen was characterized in terms of density, specific surface area, trabecular thickness, trabecular spacing. The obtained results revealed that subject-individual structural differences could be related with the diploic as well as the total cranial thickness of the human skull bones. Some tendencies of dependency could also be made with respect to the age of the subject. A consideration of these individual variations can improve traditional models that assume equal conditions throughout the skull. 

Propagation-based X-ray phase-contrast computed tomography (PBI) has already proven its potential in a great variety of soft-tissue-related applications including lung imaging. However, the strong edge enhancement, caused by the phase effects, often hampers image segmentation and therefore the quantitative analysis of data sets. Here, the benefits of applying single-distance phase retrieval prior to the three-dimensional reconstruction (PhR) are discussed and quantified compared with three-dimensional reconstructions of conventional PBI data sets in terms of contrast-to-noise ratio (CNR) and preservation of image features. The PhR data sets show more than a tenfold higher CNR and only minor blurring of the edges when compared with PBI in a predominately absorption-based set-up. Accordingly, phase retrieval increases the sensitivity and provides more functionality in computed tomography imaging.