Molecular Imaging

The present book gives an exceptional overview of molecular imaging. Practical approach represents the red thread through the whole book, covering at the same time detailed background information that goes very deep into molecular as well as cellular level. Ideas how molecular imaging will develop in the near future present a special delicacy. This should be of special interest as the contributors are members of leading research groups from all over the world

Detailed structure of the venous drainage of the brain: relevance to accidental and non-accidental traumatic head injuries

This project aimed to prove the existence of fine subdural veins hypothesised to be the source of intracranial bleeding seen in cases of accidental and non-accidental traumatic head injuries, and consequently illustrate their anatomical structure. This was important in contributing towards establishing the causal mechanism for traumatic intracranial bleeding, and was particularly applicable in unexplained traumatic head injuries in cases of possible child abuse. These issues are on-going, worldwide concerns that have been of public as well as scientific concern for many years. To illustrate the fine cerebral vessels, a unique modelling technique was recently developed involving polyurethane resin casting of the brain vasculature. Rat, marmoset, rhesus macaque and human brain tissue were all used. Tissue surrounding the resin perfused vessels were then either macerated to reveal the whole cast, or dissected to illustrate the cast as it would appear in situ. To allow analysis of these fine subdural vessels, various imaging techniques including fluorescence microscopy, light microscopy, confocal microscopy, scanning electron microscopy, transmission electron microscopy, magnetic resonance imaging, micro-computed tomography and 3D X-ray microscopy were used. The existence of subdural vessels was clearly illustrated via gross dissection of both primate and cadaveric material. Fluorescence imaging of resin-filled rat brain histological sections also showed orientation of fine vessels within the subdural space. Magnetic resonance imaging of the human head in vivo, as well as cadaveric material have shown signs of small calibre vessels that have never been previously documented, that are too fine to be bridging veins, yet seem to drain into the superior sagittal sinus. These results prove the existence of subdural vessels, present in a range of different species. Future work will further illustrate the exact morphological structure of these vessels, and biomechanical modelling will be applied to determine the exact forces required to cause them to rupture

Detailed structure of the venous drainage of the brain: relevance to accidental and non-accidental traumatic head injuries

This project aimed to prove the existence of fine subdural veins hypothesised to be the source of intracranial bleeding seen in cases of accidental and non-accidental traumatic head injuries, and consequently illustrate their anatomical structure. This was important in contributing towards establishing the causal mechanism for traumatic intracranial bleeding, and was particularly applicable in unexplained traumatic head injuries in cases of possible child abuse. These issues are on-going, worldwide concerns that have been of public as well as scientific concern for many years. To illustrate the fine cerebral vessels, a unique modelling technique was recently developed involving polyurethane resin casting of the brain vasculature. Rat, marmoset, rhesus macaque and human brain tissue were all used. Tissue surrounding the resin perfused vessels were then either macerated to reveal the whole cast, or dissected to illustrate the cast as it would appear in situ. To allow analysis of these fine subdural vessels, various imaging techniques including fluorescence microscopy, light microscopy, confocal microscopy, scanning electron microscopy, transmission electron microscopy, magnetic resonance imaging, micro-computed tomography and 3D X-ray microscopy were used. The existence of subdural vessels was clearly illustrated via gross dissection of both primate and cadaveric material. Fluorescence imaging of resin-filled rat brain histological sections also showed orientation of fine vessels within the subdural space. Magnetic resonance imaging of the human head in vivo, as well as cadaveric material have shown signs of small calibre vessels that have never been previously documented, that are too fine to be bridging veins, yet seem to drain into the superior sagittal sinus. These results prove the existence of subdural vessels, present in a range of different species. Future work will further illustrate the exact morphological structure of these vessels, and biomechanical modelling will be applied to determine the exact forces required to cause them to rupture

Biomechanical analysis of Ascending Thoracic Aortic Aneurysm (ATAA)

According to the reports of the World Health Organisation (WHO), cardiovascular diseases are the number one cause of death worldwide. Specifically, arterial disease and degeneration are the major reasons for cardiovascular death and disability. Because these diseases are dependent on the changes of the mechanical properties of the arterial wall, it is very important to know as much as possible about the structural composition of arteries. The human aorta is the biggest artery in the body and consists of three main parts, ascending aorta, aortic arch and descending aorta. The walls of the arteries consist of three layers, the intima, media and adventitia, where each of the layers has different physiological functions and therefore distinct mechanical properties. These were investigated using, i.e., uniaxial tensile, inflation or planar biaxial-testing. Purpose of this thesis was to apply the biomechanical approach by mean of numerical and experimental test referring to patient-specific aortic geometries with ascending thoracic aortic aneurysms. However, despite the ample literature and the related scientific and industrial activity in this field, many different phenomena are not yet consolidated. The PhD Thesis is then divided into two main sections: the first is composed by a brief introduction on ATAA, with some background about mechanical properties of soft tissues, the evolution of the constitutive model, some remarks of the continuum. The second section of the thesis is based on the different research activities developed during the PhD

Medical Robotics

The first generation of surgical robots are already being installed in a number of operating rooms around the world. Robotics is being introduced to medicine because it allows for unprecedented control and precision of surgical instruments in minimally invasive procedures. So far, robots have been used to position an endoscope, perform gallbladder surgery and correct gastroesophogeal reflux and heartburn. The ultimate goal of the robotic surgery field is to design a robot that can be used to perform closed-chest, beating-heart surgery. The use of robotics in surgery will expand over the next decades without any doubt. Minimally Invasive Surgery (MIS) is a revolutionary approach in surgery. In MIS, the operation is performed with instruments and viewing equipment inserted into the body through small incisions created by the surgeon, in contrast to open surgery with large incisions. This minimizes surgical trauma and damage to healthy tissue, resulting in shorter patient recovery time. The aim of this book is to provide an overview of the state-of-art, to present new ideas, original results and practical experiences in this expanding area. Nevertheless, many chapters in the book concern advanced research on this growing area. The book provides critical analysis of clinical trials, assessment of the benefits and risks of the application of these technologies. This book is certainly a small sample of the research activity on Medical Robotics going on around the globe as you read it, but it surely covers a good deal of what has been done in the field recently, and as such it works as a valuable source for researchers interested in the involved subjects, whether they are currently “medical roboticists” or not

Registration of histology and magnetic resonance imaging of the brain

Combining histology and non-invasive imaging has been attracting the attention of the medical imaging community for a long time, due to its potential to correlate macroscopic information with the underlying microscopic properties of tissues. Histology is an invasive procedure that disrupts the spatial arrangement of the tissue components but enables visualisation and characterisation at a cellular level. In contrast, macroscopic imaging allows non-invasive acquisition of volumetric information but does not provide any microscopic details. Through the establishment of spatial correspondences obtained via image registration, it is possible to compare micro- and macroscopic information and to recover the original histological arrangement in three dimensions. In this thesis, I present: (i) a survey of the literature relative to methods for histology reconstruction with and without the help of 3D medical imaging; (ii) a graph-theoretic method for histology volume reconstruction from sets of 2D sections, without external information; (iii) a method for multimodal 2D linear registration between histology and MRI based on partial matching of shape-informative boundaries

Capturing the first haematopoietic stem cell: the needle in the haystack

The most powerful cell in the blood differentiation hierarchy is the haematopoietic stem cell (HSC). It is the only cell capable of building an entire haematopoietic system from scratch, i.e. long-term (LT) repopulating a multilineage blood system within a lethally irradiated recipient. Though primitive blood is generated at early embryonic stages, it is only from embryonic day (E)10.5 of mouse development (4-5 weeks in human gestation) onwards that the definitive adult haematopoietic system is established with the formation of LT-HSCs. These first HSCs emerge in small ventral intra-aortic haematopoietic clusters (IAHCs) in the aorta-gonad-mesonephros (AGM) region. Like with many systems, a tight regulation of gene expression by master transcription factors (TF) is what ultimately drives cell fate change. Gata2 is one such TF known to play a crucial role in mouse definitive haematopoiesis. Through the work of de Pater et al. (2013) (Chapter 1) it became clear that Gata2 is both required for the generation of the very first HSCs and also, unlike other key TFs like Runx1, the survival of HSCs. The earliest HSCs are generated through an endothelial-to-haematopoietic transition (EHT), a process where flat arterial haemogenic endothelial cells (HECs) round up into IAHCs, containing HSCs. Solaimani Kartalaei et al. (2015) (Chapter 4) discovered that Gpr56 is one of the most highly upregulated genes during EHT (in mouse) and that it is essential for HSC generation (in zebrafish). Gpr56 is also a target of the key haematopoietic ‘heptad’ TFs in both mouse and human blood progenitors. To examine another signalling pathway required in the embryo for HSC generation, we used a BMP responsive element (BRE)-GFP transgenic mouse to show that all AGM HSCs are BMP-activated (Crisan et al., 2015) (Chapter 6). At slightly later stages, in the foetal liver and bone marrow, HSC heterogeneity starts to appear with the presence of genetically distinct BMP- and non-BMP activated HSCs. Given the importance of the ‘heptad’ TFs in establishing HSC identity, we next developed a novel Gata2-Venus (G2V) reporter mouse to isolate and examine the dynamics and function of live Gata2-expressing and non-expressing cells (Kaimakis et al., 2016) (Chapter 2). Gata2 is expressed in all HSCs, however haematopoietic progenitors can be generated either Gata2-dependently or -independently, the latter being less potent and genetically distinct from the first. With Eich et al. (2018) (Chapter 3) we went further and deciphered the role of Gata2 in haematopoietic fate establishment. By using time-lapse imaging of E10.5 live aortic sections of G2V embryos, we discovered rapid pulsatile level changes in Gata2 expression, specifically in those single cells undergoing EHT. This finding indicated the highly unstable genetic state of individual cells undergoing fate change. Furthermore, aortic cells haploinsufficient for Gata2 show many fewer pulsatile and EHT events, emphasizing the importance of Gata2 levels in this process. Having defined the specific (medium) levels of Gata2(Venus) protein in the population containing the first HSCs, we used our G2V model to isolate a pure population of HSCs as they are generated for the first time in development (Vink et al., 2020) (Chapter 5). Through iterations of index-sorting, single-cell transcriptomics and functional analyses we were able to greatly enrich for HSCs (~70x compared to Eich et al. (2018)). Based on refined CD31, cKit and CD27 expression and specific physical parameters we isolated the first cells with an HSC identity and identified their precise gene expression profile. Immunohistochemistry localized these HSCs to only the smallest IAHCs. The combined effort of my research and novel tools generated to facilitate the creation of this body of work/portfolio have now led us for the first time to study the one or two cells per embryo that harbour HSC identity at the developmental time when they are beginning to be made. Now that we have cracked the genetic code of the true HSC, the field is one step closer to translating these findings and applying them to producing human clinically relevant HSCs, which will ultimately improve transplantation therapies and benefit the fight against leukaemia

Characterisation and computational modelling of retinal stem cells in medaka (Oryzias latipes)

The central functional unit of the vertebrate eye is the retina, composed of neural retina (NR), retinal pigmented epithelium (RPE), and non-visual retina (NVR). In amphibians and fish, the retina grows throughout life via different pools of stem cells (SCs). In this work, I combined experimental and computational approaches to elucidate SC dynamics in the three retinal tissues of the teleost fish medaka (Oryzias latipes). I developed a cell centred agent based model to recapitulate post-embryonic growth of the NR and RPE. By accounting for 3D tissue geometry and continuous growth, the model reconciled conflicting hypotheses, demonstrating that competition between SCs is not mutually exclusive with lifelong coexistence of multiple SC lineages. To understand how NR and RPE regulate their proliferative output to coordinate growth rates, I developed quantitative methods to compare experiment and simulation. I tested the experimental data against simulations implementing two modes of feedback between cell proliferation and organ growth. Thus, I identified that the NR acts upstream to set the growth pace by sending an inductive growth signal, while the RPE responds downstream to this signal. Leveraging the model, I showed that NR SCs compete for niche space, but tissue geometry biases cells at certain positions to win this competition. Further, NR SCs modulate division axes and proliferation rate to change organ shape and retinal topology. Motivated by model predictions, I experimentally characterised the large SC population of the RPE, which consisted of both cycling and non-cycling quiescent cells. Putative sister cells exhibited similar temporal dynamics in local clusters, indicating that quiescence was the major mechanism for regulating proliferative output in the RPE. Finally, I experimentally showed that the NVR grows post-embryonically from a primordium, and shared all known markers for NR SCs in the same spatial distribution. Unlike NR and RPE, the NVR lacked a dedicated niche, instead proliferative cells were distributed throughout the tissue. Lineage tracing revealed a continuous relationship between RPE, NVR, and NR. Thus, the SCs of NR and RPE, and all cells of the NVR displayed plastic multipotency capable of generating all retinal tissues. By taking advantage of the positive feedback loop between experiment and simulation, this work shines a new light into a fundamental problem – growth coordination of different SC populations in a complex vertebrate organ