All neuronal circuits of the human brain make up the so-called connectome. Within, three spatial dimensions have been identified, ranging from the microscopic to the mesoscopic to the macroscopic scale. Most studies on connectivity focus on the macroscopic scale by performing magnetic resonance imaging (MRI) in and ex vivo. The bottleneck of this method is the limited resolution accompanied by limited accuracy and validity of 3D MRI data. Thereby, the challenge arises to provide anatomical ground-truth information and create microstructure-informed MRI measurements. One way to overcome these resolution differences between macroscopic and microscopic imaging methods is to generate (sub)cellularly resolved (< 100 nm), mesoscopic tissue blocks (mm-sized). This can be reached, by pairing novel tissue clearing techniques with high-end, large-scale microscopic imaging and is the first goal of the presented thesis. In combining different processing and imaging techniques, that are sensitive to different spatial scales and microstructural properties, a comprehensive understanding of the wiring of the human brain can be generated. Transparency of biological tissue is reached by matching heterogeneous refractive indices (RI) of intra- and extracellular compartments, divided by lipid membranes, to each other and to the RI of their surrounding medium. In doing so, scattering and absorption of light traveling through the sample is reduced. Tissue clearing methods use different approaches to homogenize RI. While hydrophilic and hydrogel-based methods wash out lipids by using strong detergents (e.g., sodium dodecyl sulfate or urea), hydrophobic methods use organic solvents (dibenzylether or ethylcinnamate) to dissolve tissue lipids. The clearing of human brain tissue is particularly challenging as it is densely packed, highly myelinated, and usually aged (i.e., by donors of high age). Initially seven tissue clearing techniques were tested to determine the most efficient one. Efficiency was defined in a high degree of tissue transparency, preserved ability for immunohistochemical staining before or after clearing to obtain highly resolved microscopic imaging results, and short experimental processes. All methods were tested on mm-sized, fixed post mortem brain tissue samples and included the CLARITY, CUBIC, iDISCO, MASH, ECi, Visikol and Ce3D protocol. The CLARITY, CUBIC, iDISCO, and MASH techniques were able to clear aged human brain tissue, whereas the ECi, Visikol and Ce3D techniques were not. Generally, water-based CLARITY and CUBIC methods are gentler than solvent-based iDISCO and MASH techniques. The expansion of CLARITY-treated samples appears to be advantageous as well. However, all technical aspects (i.e., hydrogel pre-treatment, electrophoretic chamber acquisition) of the CLARITY method are time and cost consuming. Here, the iDISCO, MASH, and ECi protocols are more efficient. Their material costs are lower and, if successful, the processing duration is short compared to the CLARITY technique. Although, the Visikol (commercial clearing kit) and Ce3D technique claim to be quickly applied and to clear tissue fast, they are less affordable and more complex to perform as well. After attaining transparency, optical properties of the tissue samples are altered. Hence, the application of immunohistochemistry remains crucial. Microscopic imaging of cleared, immunohistochemically labelled human brain tissue samples revealed that the CLARITY and iDISCO techniques are most suitable. Here, immunohistochemical reagents as well as light penetrated sufficiently deep into the tissue. In addition to the evaluation of different clearing techniques, three microscopic setups, specifically built or equipped for imaging large-scale specimen, were tested to identify suitability, benefits, and downfalls. The results showed that the most suitable and efficient approaches are the combination of i) the CLARITY method coupled with imaging at the Zeiss LSM 880 Airyscan and ii) the iDISCO method coupled with the Miltenyi Biotec Ultramicroscope ll. Both combinations enable three-dimensional histology of aged human brain tissue. There is growing demand for visualizing and examining human cyto- and myeloarchitecture in 3D. Successfully clearing aged human brain tissue whilst preserving its microstructure and ability for immunohistochemical staining will bridge standard histological thin-sectioning (2D) and non-invasive imaging techniques, such as MRI. Once mastered in post mortem human brain blocks in 3D, tools such as fiber tracking and co-localisation are going to provide a powerful tool for multi-modal validation of in vivo microstructure-informed MRI, unraveling the human connectome. Next to unraveling the human connectome based on myelinated axonal fiber pathways, the second goal of this thesis is to investigate the distribution patterns of astrocytes. As they support oligodendrocytes during myelination processes, astrocytes are proposed to be co-localised with myelinated fibers. Revealing their spatial organization offers another great approach to study the wiring of the human brain. Astrocytes are the most common subtype of glia cells in the central nervous system and can serve many functions depending on their interaction partner and surroundings. Those include but are not limited to the regulation of synaptic plasticity, ion and pH homeostasis of neurons and the vasculature. Moreover, astrocytes can adapt morphologically, physiologically, and molecularly during their response to neurodegeneration or demyelination. This remodelling is facilitated, amongst others, by the glial fibrillary acidic protein (GFAP), a cytoskeletal protein and the primary intermediate filament of mature astrocytes. Hence, antibodies against GFAP are commonly and validly used. Furthermore, great heterogeneity occurs among astrocytes with distinguishable morphologies in white matter (fibrous astrocytes) and grey matter areas (protoplasmic astrocytes). Accordingly, GFAP is expressed differently depending on the brain region. Focusing on the co-localisation of myelinated fibers and astrocytes, immunohistochemical analyses were performed to identify myelin- and GFAP-positive structures in aged human cerebral and brainstem areas. Up to three different regions of interest of four different human brains were examined to analyse myelinated fiber and astrocyte content in white and grey matter areas. Subsequently, two image processing approaches were deployed to extract fluorescence intensity values. The first, semi-manual approach, performed with the Zeiss ZEN blue software, generated distribution patterns within a wider range. Hereby, each channel underwent linear unmixing to co-localize the detected fluorescent signals to each other. The second, semi-automated approach, performed with the Segmensation application software, generated distribution patterns within a narrower range. As GFAP dominates in fibrillary astrocytes of white matter regions consistent results were obtained with both image analysis tools. As GFAP expression in grey matter astrocytes is limited, less consistent results were obtained. Therefore, immunohistochemical analyses of different white and grey matter regions of the human brain align with regional distribution patterns of GFAP-positive astrocytes. Remarkably, as proposed in white matter regions, astrocytes and myelinated axons share a similar spatial organization across all investigated regions of interest. In 1992, Suzuki and Raisman first observed this intertwined organization of axons and glial processes in the rat’s brain, coining the term glial framework. The presented thesis confirms this spatial organization of myelinated fibers and astrocytic branches in the human brain. Although this phenomenon was known as well, it was unclear whether this is a global feature. Moreover, the scarcity of studies on the human glial framework, especially in brainstem areas, needs to be acknowledged. This thesis starts to fill the gap, providing evidence for the presence of the glial framework, created by GFAP-positive astrocytes, within white matter regions of cerebral and brainstem areas of the aged human brain. Additionally, the application of immunohistochemistry in cleared, mesoscopic-sized brain tissue samples adds another promising perspective to successfully unraveling the human glial framework globally. As there is growing demand for sensitizing non-invasive imaging techniques to the heterogeneity of astrocytes, studying the spatial organization of myelinated fibers in correlation with astrocytes will provide deeper insights into the impact astrocytes have on neurological integrity and degeneration. Both points of focus of the presented thesis aim to untangle and give a clearer understanding of the human connectome. Myelin, as the primary component of structural and functional integrity of all neuronal fiber pathways, needs to remain the centre of investigations. However, expanding these investigations by studying the glial framework is going to propel our understanding of the human myeloarchitecture forward. Using the coherent approach as is tissue clearing, is a great way to examine structural and functional correlations in 3D and needs to remain a central element of future studies.

No description available.

The female reproductive organs are among the few adult tissues in which periodic angiogenesis normally occurs. Pathological angiogenesis can occur in various conditions, such as solid tumors. Vascular endothelial growth factor (VEGF) signaling often represents a critical rate-limiting step in physiological and pathological angiogenesis. This study utilizes development of utero-ovarian vasculature during pregnancy in goats as a model of physiological angiogenesis. Non-pregnant does and does at 4, 7, 10, 13, 16, and 18 weeks of gestation were used. Arteries of the reproductive tract were injected <i>in situ</i> with Microfil®. The tracts were fixed, dehydrated, and rendered transparent to reveal the paths of arteries. The ovarian artery was tortuous and lay in close apposition to the uterine tributary of the ovarian vein in all specimens studied. In non-pregnant does, this arrangement may serve as a local utero-ovarian pathway for the corpus luteum (CL) luteolysis at the end of non-fertile estrous cycle. During pregnancy, this arterio-venous arrangement may transfer luteotropic substances from uterus to ovary, which may serve in maternal recognition of pregnancy and fit the fact that the goat is CL dependent throughout gestation. In some cases of triplets, the size of the uterine branch of the ovarian artery was equal to or even larger than that of its parent artery and/or the ipsilateral uterine artery; and the vaginal artery contributed a connecting branch to the uterine artery. These physiological adaptations of the ovarian and/or vaginal arteries correlate well with the increasing nutrient demands of the growing multiple fetuses. In a second experiment, the vasculature of the uterus and ovaries was injected <i>in situ</i> with a mixture of Batson's No.17® and methyl methacrylate and then processed for observation by SEM. The microvasculature differed between non-pregnant and pregnant does, and with advancing gestation. We concluded that goats possess a <i>multivillous</i> type placenta. Capillary sinusoids and crypts on the fetal surface of the caruncle may compensate for the negative effect of the increased interhemal distance. Intussusceptive angiogenesis should be considered as equally possible and important mechanism as sprouting angiogenesis during placental development. Capillary diameters increased significantly during pregnancy especially after 4 weeks. Capillary density index was 66.8, 68.7, 55.5, 63.5, 70.1, 70.4, 64.5 percent in non-pregnant, 4, 7, 10, 13, 16, and 18 weeks of pregnancy, respectively. In the ovary, coiling of the ovarian branch of the ovarian artery around the ovarian tributary of the ovarian vein was observed. This may represent a local channel required for product transport from ovarian vein to ovarian artery and might have a role in regulating blood pressure to various ovarian structures. Immunolocalization of VEGF was performed as a third experiment. Immunostaining was observed in cyto- trophoblasts, maternal epithelial tissues, and vascular endothelium and smooth muscle, but not in binucleate giant cells or connective tissue. No apparent differences were observed in intensity and pattern of VEGF staining associated with advancing gestation. Luteal and follicular cells, and endothelium and smooth muscles of the ovarian vasculature positively stained. Patterns and intensity of staining of VEGF suggest that the fetus is directing its own survival by producing growth factors affecting fetal and maternal tissues. VEGF may have a role in growth and differentiation of cytotrophoblasts, as well as, development and maintenance of CL. In the fourth experiment, the sequential expression of VEGF and its receptors (fms-like tyrosine kinase, Flt-1 and kinase-insert domain-containing receptor, KDR) was measured using real-time quantitative PCR. Targets were detected in all studied tissues; however, levels of expression differed according to the stage of pregnancy. Expression of VEGF and its receptor mRNAs increased with advancing pregnancy, which correlates with the expansion of vasculature during pregnancy. Differences in the time-courses of the expression of Flt-1 and KDR mRNAs during pregnancy suggest that each receptor plays a different role in the angiogenic process. As an application of our model of angiogenesis, we tested the effect of swainsonine (active compound of locoweed and a potential anti-cancer drug) on the process. Does treated with swainsonine were euthanized at 7 and 18 weeks. No significant differences were found in sinusoidal diameters in treated does at 7 weeks, but a decrease in capillary density index was noted. In the ovary, focal avascular areas were observed in the corpus luteum of swainsonine-treated does at 7 weeks of pregnancy. Swainsonine caused great distortion in the uterine and ovarian vasculature at 18 weeks. A decrease in intensity of the immunoreactivity to VEGF antibody was observed in tissues from swainsonine-treated does at 7 and 18 weeks. There was no substantial effect of swainsonine on the expression of VEGF and its receptors' mRNAs in any of the studied tissues (except in the left ovary, where it had an inhibitory effect) at 7 weeks of pregnancy, but it had an inhibitory effect at 18 weeks. Demonstration of swainsonine's potential to negatively affect vascular development and suppress genes likely involved in angiogenesis at critical stages of blood vessel proliferation lends credibility to its potential as anti-cancer drug. / Ph. D.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24505号 / 医博第4947号 / 新制||医||1064(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邉 大, 教授 林 康紀, 教授 井上 治久 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The 15q13.3 microdeletion is a common genetic disorder associated with multiple neurodevelopmental disorders including autism spectrum disorder, epilepsy, and schizophrenia. Patients have diverse clinical presentations, often prompting genetic assays that identify the CNV in the clinic. This late-stage screening leaves a considerable gap in our understanding of the prenatal and prediagnostic developmental impairments in these individuals, providing a barrier to understanding the disease pathobiology. We provide the first investigation into embryonic brain development of individuals with the 15q13.3 microdeletion by generating multiple 3D neural organoid models from the largest clinical cohort in reported literature. We incorporated unguided and guided forebrain organoid models into our multi-transcriptomic phenotyping pipeline to uncover changes in cell type emergence and disruptions to circuit development, all of which had underlying changes to cell adhesion pathways. Specifically, we identified accelerated growth trajectories in 15q13.3del unguided neural organoids and used single cell RNA sequencing to identify changes in radial glia dynamics that affect neurogenesis. We measured changes in the pseudotemporal trajectory of matured unguided neural organoids, and later identified disruptions in synaptic signaling modules amongst the primary constituents to neural circuitry, excitatory and inhibitory neurons. We leveraged dorsal and ventral forebrain organoid models to better assess circuit dynamics, as they faithfully produce the excitatory and inhibitory neurons in the pallium and subpallium, respectively. We then used the entire 15q13.3del cohort and performed bulk RNA sequencing on each tissue type at two timepoints and discovered convergence on transcriptional dysregulation and disruptions to human-specific zinc finger proteins localized to chromosome 19. We also identified cell type-specific vulnerabilities to DNA damage and cell migration amongst the dorsal and ventral organoids, respectively, which was consistent with the excitatory and inhibitory neural subpopulations amongst the unguided neural organoids scRNA Seq, respectively. We then examined neuron migration in a 3D assembloid model by sparsely labeling dorsal-ventral forebrain organoids from multiple genotype-lineage combinations. Light sheet microscopy identified deficits in inhibitory neuron migration and morphology, but not migration distance, suggesting a complex disruption to cortical circuitry. This novel combination of cell type characterization, pathway identification, and circuitry phenotyping provides a novel perspective of how the 15q13.3 deletions impair prenatal development and can be applied to other NDD models to leverage understanding of early disease pathogenesis. / Dissertation / Doctor of Science (PhD) / The development of the human brain is a highly complex and tightly regulated process that requires the participation of multiple cell types throughout development. Disturbances to the emergence, differentiation, or placement of these cell types can cause disruptions and local miswiring of neural circuits, which is often associated with neurodevelopmental disorders (NDDs). The 15q13.3 microdeletion syndrome is a highly complex condition associated with multiple NDDs and has seldom been studied in a human context. To address this, we used stem cells derived from a 15q13.3 microdeletion syndrome cohort and their typically developing familial controls to generate unguided (“whole brain”) and region-specific organoids to investigate early fetal development across time. We used the largest 15q13.3 microdeletion cohort in reported literature to identify shared disruptions in early developmental milestones such as neurogenesis, neural migration, and neural patterning. We identified expansion of specific cell populations, including progenitors that later give rise to mature neurons. Abnormalities persisted in more mature cell populations, including the inhibitory neurons responsible for establishing critical microcircuitry in the human cortex. By generating guided organoids that enrich for excitatory and inhibitory neural populations, we were able to merge the models to form assembloids, where we captured early migratory and morphological deficits in inhibitory neuron populations, which is supported by the multi-transcriptomics experiments performed in both organoid models. This study provides a framework for examining fetal development in a neurodevelopmental disorder context. By using the 15q13.3 microdeletion background, we found novel disruptions in cell type emergence and circuit formation previously unreported in mouse or 2D neuron models, highlighting the utility of the phenotyping platform for disease modeling.

Solid tumors are complex structures comprising besides the cancer cells vasculature, extracellular matrix (ECM), soluble molecules and a plethora of various other cell types. These components form a so-called tumour microenvironment. From the numerous cell types that are part of tumor microenvironment, tumor infiltrating lymphocytes (TILs) play a major role in patient prognosis. Their presence is also of major importance with regard to new biological therapies based on immune checkpoint inhibitors. Crucial role of TILs is also reflected by the new approaches in cancer diagnostics namely by Immunoscore method (currently used in clinical settings). Immunoscore is based on localization and quantification of CD3+ and CD8+ TILs in thin histological sections of tumor tissue. The question remains to which extent the information obtained from 2D slices reflects the situation in tumor microenvironment considering its spatial heterogeneity. The development of new methodological approaches allowing evaluation of histological information in 3D is the key to answer this question. The theoretical part of this work first describes the heterogeneity of the tumor microenvironment and the role of immune cells within it. Then, the role of spatial heterogeneity and its possible influence on the histopathological...