Automated analysis and visualization of preclinical whole-body microCT data

In this thesis, several strategies are presented that aim to facilitate the analysis and visualization of whole-body in vivo data of small animals. Based on the particular challenges for image processing, when dealing with whole-body follow-up data, we addressed several aspects in this thesis. The developed methods are tailored to handle data of subjects with significantly varying posture and address the large tissue heterogeneity of entire animals. In addition, we aim to compensate for lacking tissue contrast by relying on approximation of organs based on an animal atlas. Beyond that, we provide a solution to automate the combination of multimodality, multidimensional data.* Advanced School for Computing and Imaging (ASCI), Delft, NL * Bontius Stichting inz Doelfonds Beeldverwerking, Leiden, NL * Caliper Life Sciences, Hopkinton, USA * Foundation Imago, Oegstgeest, NLUBL - phd migration 201

Doctor of Philosophy

dissertationImage-based biomechanics, particularly numerical modeling using subject-specific data obtained via imaging, has proven useful for elucidating several biomechanical processes, such as prediction of deformation due to external loads, applicable to both normal function and pathophysiology of various organs. As the field evolves towards applications that stretch the limits of imaging hardware and acquisition time, the information traditionally expected as input for numerical routines often becomes incomplete or ambiguous, and requires specific acquisition and processing strategies to ensure physical accuracy and compatibility with predictive mathematical modeling. These strategies, often derivatives or specializations of traditional mechanics, effectively extend the nominal capability of medical imaging hardware providing subject-specific information coupled with the option of using the results for predictive numerical simulations. This research deals with the development of tools for extracting mechanical measurements from a finite set of imaging data and finite element analysis in the context of constructing structural atlases of the heart, understanding the biomechanics of the venous vasculature, and right ventricular failure. The tools include: (1) application of Hyperelastic Warping image registration to displacement-encoded MRI for reconstructing absolute displacement fields, (2) combination of imaging and a material parameter identification approach to measure morphology, deformation, and mechanical properties of vascular tissue, and (3) extrapolation of diffusion tensor MRI acquired at a single time point for the prediction the structural changes across the cardiac cycle with mechanical simulations. Selected tools were then applied to evaluate structural changes in a reversible animal model for right ventricular failure due to pressure overload

ENHANCing the limb: from micro- to macro-evolution

Abstract Understanding the molecular basis of the diverse morphological forms found within and across species is a longstanding goal in evolutionary biology. One especially relevant class of cis-regulatory elements are enhancers. This is because mutations affecting enhancers tend to be tissue- or stage-specific, which allows adaptation to proceed with relatively less harmful side effects in other organs or tissues. In Chapter 2 I explore how enhancers help drive morphological selection response within-species. We scanned the genomes of the Longshanks mice, which are mice selectively bred over 20 generations, for a 13% increase in tibiae. Against a backdrop of polygenic response, we found the bone repressor Nkx3-2, and specifically its enhancers, to be among the strongest contributor towards increased tibia length. I used transgenics to compare the enhancer activity of the F0 and F17 alleles at 3 candidate enhancers (two near the Nkx3-2 gene; and one near the limb developmental regulator gene, Gli3). We found that both loss-of-function (Nkx3-2) and gain-of-function (Gli3) alleles contributed to the selection response. In Chapter 3, we explored an approach to study macro-evolutionary variations across species. One of the major barriers to such study is the inability to perform direct genetic crosses due to hybrid sterility. We tackle the species barrier problem by inducing mitotic recombination in vitro in hybrid embryonic stem cells (including cross-species hybrids between Mus musculus and Mus spretus). This was achieved via Blm inhibition by the small molecule ML216. We further show, that the resultant mitotic recombinant cells can be used for genetic mapping by connecting tioguanine drug resistance to variations at the Hprt locus. Furthermore, in vitro recombinant stem cells can be used for rederivation of animals through laser-assisted morula injection, thus allowing the acquisition of morphological data. Here, through a multidisciplinary approach, we show that enhancer modulation contributes to morphological diversity and selection response within-species and provide a new methodology for enhancer study across-species, thus enabling the study of evolutionary developmental variations in genetic backgrounds that would otherwise be challenging to obtain. Overall, these studies highlight the relevance of enhancers in morphological diversification and provide new tools for their study

Bone marrow derived cells as endothelial precursors and the role of multi-potent progenitor cells in repairing ischaemic tissues.

PhDIntroduction: Atherosclerosis and its complications are a major cause of death and disability and it remains a major challenge to develop new therapies for patients with irreversible end organ damage and ongoing ischaemia. The discovery of adult stem and progenitor cells with the ability to regenerate adult tissues holds great promise. Bone marrow is the source of both endothelial progenitor cells (EPCs) and multi-potent adult progenitor cells (MAPCs). MAPCs are rare pluripotent bone marrow derived cells with the theoretical potential to differentiate into tissues of all three germ cell layers, including endothelium. These cells may have the potential to facilitate cardiac repair. The aim of this thesis was to further characterise bone marrow derived endothelial progenitor cells including multi potent adult progenitor cells and assess their angiogenic potential and mechanisms of action in animal models of cardiovascular disease. Findings: EPCs were isolated from humans and mice and their phenotype, markers and function determined, including gene tracking experiments in mice utilising the Cre/Lox system. It was not possible to isolate cells with the same phenotype as MAPCs from rodent bone marrow. However, cells with pluri-potent properties, named rat multi-potent progenitor cells (rMPCs), were isolated from rat bone marrow. These cells had the ability to up regulate tissue specific antigens from all 3 germ cell lineages and in addition secreted multiple cytokines related to angiogenesis and inflammation. To investigate the in vivo properties of rMPCs a rat hind limb model of ischaemia was established and syngeneic rMPCs were transplanted into the ischaemic hind limbs. rMPCs engrafted selectively into the adventitia of arterioles of ischaemic muscles. However, engrafted cells did not differentiate into an endothelial or smooth muscle phenotype. Cytokine analysis of muscles 5 days after rMPC injection revealed raised levels of cytokines, including chemokines MCP1 and SDR. Limb perfusion, measured by microspheres, increased after rMPC injection. In addition a novel MRI based assessment of ischaemic muscles revealed a significant normalisation of MRI signal after rMPC transplantation. However, there was no improvement in limb function assessed by treadmill running distance 4 weeks after cell injection. These findings suggest that transplantation of rMPCs into ischaemic muscles may modulate local inflammatory and angiogenic responses through paracrine mechanisms. Conclusion: Despite the potential for stem and progenitor cells to be used for the treatment of chronic cardiac ischaemia the biology of stem cells is still relatively poorly understood, as is the mechanism of action of cells after transplantation. As set out in the aims, the work in this thesis adds further to our understanding of both EPCs and BM derived pluri-potent stem cells. In addition it provides insight into the hind limb ischaemia model and the mechanism of action of cell therapy after transplantation into ischaemic muscle

CHARACTERIZATION OF BRAIN TISSUE MICROSTRUCTURES WITH DIFFUSION MRI

Diffusion MRI is a useful medical imaging tool for noninvasive mapping of the neuroanatomy and brain connectivity. In this dissertation, we worked on developing diffusion MRI techniques to probe brain tissue microstructures from various perspectives. Spatial resolution of the diffusion MRI is the key to obtain accurate microstructural information. In Chapter 2 and 3, we focused on developing high-resolution in vivo diffusion MRI techniques, such as 3D fast imaging sequence and a localized imaging approach using selective excitation RF pulses. We demonstrated the power of the superior resolution in delineating complex microstructures in the live mouse brain. With the high resolution diffusion MRI data, we were able to map the intra-hippocampal connectivity in the mouse brain, which showed remarkable similarity with tracer studies (Chapter 4). Using the localized fast imaging technique, we were the first to achieve in utero diffusion MRI of embryonic mouse brain, which revealed the microstructures in the developing brains and the changes after inflammatory injury (Chapter 5). The second half of the dissertation explores the restricted water diffusion at varying diffusion times and microstructure scales, using the oscillating gradient spin-echo (OGSE) diffusion MRI. We showed in the live normal mouse brains that unique tissue contrasts can be obtained at different oscillating frequency. We demonstrated in a neonatal mouse model of hypoxia-ischemia, that in the edema brain tissues, diffusion MRI signal changed much faster with oscillating frequency compared to the normal tissue, indicating significant changes in cell size associated with cytotoxic edema (Chapter 6). In the mild injury mice, OGSE showed exquisite sensitivity in detecting subtle injury in the hippocampus, which may relate to microstructural changes in smaller scales, such as the subcellular organelles (Chapter 7). Finally, we addressed the technical issues of OGSE diffusion MRI, and proposed a new hybrid OGSE sequence with orthogonally placed pulsed and oscillating gradients to suppress the perfusion related pseudo-diffusion (Chapter 8). In conclusion, we developed in vivo high-resolution diffusion techniques, and time-dependent diffusion measurements to characterize brain tissue microstructures in the normal and diseased mouse brains. The knowledge gained from this dissertation study may advance our understanding on microstructural basis of diffusion MRI