Diffusion MR microscopy of cortical development in the mouse embryo

Cortical development in the mouse embryo involves complex changes in the microstructure of the telencephalic wall, which are challenging to examine using three-dimensional (3D) imaging techniques. In this study, high-resolution 3D diffusion magnetic resonance (dMR) microscopy of the embryonic mouse cortex is presented. Using diffusion-weighted gradient- and spin-echo based acquisition, dMR microimaging data were acquired from fixed mouse embryos at 7 developmental stages from embryonic day (E)12.5 to E18.5. The dMR imaging (dMRI) contrasts revealed microscopic structural detail in the mouse telencephalic wall, allowing delineation of transient zones in the developing cortex based on their unique diffusion signatures. With the high-resolution 3D data of the mouse embryo, we were able to visualize the complex microstructure of embryonic cerebral tissue and to resolve its regional and temporal evolution during cortical formation. Furthermore, averaged dMRI contrasts generated via deformable registration revealed distinct spatial and temporal gradients of anisotropy variation across the developing embryonic cortical plate and the ventricular zone. The findings of this study demonstrate the potential of 3D dMRI to resolve the complex microstructure of the embryonic mouse cortex, and will be important for investigations of corticogenesis and its disruption in embryonic mouse models

Histological Correlates of Diffusion-Weighted Magnetic Resonance Microscopy in a Mouse Model of Mesial Temporal Lobe Epilepsy

Mesial temporal lobe epilepsy (MTLE) is the most common type of focal epilepsy. It is frequently associated with abnormal MRI findings, which are caused by underlying cellular, structural, and chemical changes at the micro-scale. In the current study, it is investigated to which extent these alterations correspond to imaging features detected by high resolution magnetic resonance imaging in the intrahippocampal kainate mouse model of MTLE. Fixed hippocampal and whole-brain sections of mouse brain tissue from nine animals under physiological and chronically epileptic conditions were examined using structural and diffusion-weighted MRI. Microstructural details were investigated based on a direct comparison with immunohistochemical analyses of the same specimen. Within the hippocampal formation, diffusion streamlines could be visualized corresponding to dendrites of CA1 pyramidal cells and granule cells, as well as mossy fibers and Schaffer collaterals. Statistically significant changes in diffusivities, fractional anisotropy, and diffusion orientations could be detected in tissue samples from chronically epileptic animals compared to healthy controls, corresponding to microstructural alterations (degeneration of pyramidal cells, dispersion of the granule cell layer, and sprouting of mossy fibers). The diffusion parameters were significantly correlated with histologically determined cell densities. These findings demonstrate that high-resolution diffusion-weighted MRI can resolve subtle microstructural changes in epileptic hippocampal tissue corresponding to histopathological features in MTLE

Recommendations and guidelines from the ISMRM Diffusion Study Group for preclinical diffusion MRI: Part 1 -- In vivo small-animal imaging

The value of in vivo preclinical diffusion MRI (dMRI) is substantial. Small-animal dMRI has been used for methodological development and validation, characterizing the biological basis of diffusion phenomena, and comparative anatomy. Many of the influential works in this field were first performed in small animals or ex vivo samples. The steps from animal setup and monitoring, to acquisition, analysis, and interpretation are complex, with many decisions that may ultimately affect what questions can be answered using the data. This work aims to serve as a reference, presenting selected recommendations and guidelines from the diffusion community, on best practices for preclinical dMRI of in vivo animals. In each section, we also highlight areas for which no guidelines exist (and why), and where future work should focus. We first describe the value that small animal imaging adds to the field of dMRI, followed by general considerations and foundational knowledge that must be considered when designing experiments. We briefly describe differences in animal species and disease models and discuss how they are appropriate for different studies. We then give guidelines for in vivo acquisition protocols, including decisions on hardware, animal preparation, imaging sequences and data processing, including pre-processing, model-fitting, and tractography. Finally, we provide an online resource which lists publicly available preclinical dMRI datasets and software packages, to promote responsible and reproducible research. An overarching goal herein is to enhance the rigor and reproducibility of small animal dMRI acquisitions and analyses, and thereby advance biomedical knowledge.Comment: 69 pages, 6 figures, 1 tabl

SHEEP AS ANIMAL MODEL IN MINIMALLY INVASIVE NEUROSURGERY IN EDEN2020

Glioblastomas (GBMs) is a malignant type of central nervous system tumours and its presentation is almost 80% of all malignant primary brain neoplasia. This kind of tumour is highly invasive infiltrating the white matter area and is confined to the central nervous with a very poor patient outcome survival around 10 months. Of the existing treatment approaches, Convection Enhanced drug Delivery (CED) offers several advantages for the patient but still suffers from significant shortcomings. Enhanced Delivery Ecosystem for Neurosurgery in 2020 (EDEN2020) is a European project supported with a new catheter development as the key project point in an integrated technology platform for minimally invasive neurosurgery. Due to the particular anatomy and size, sheep (Ovis aries) have been selected as experimental large animal model and a new Head Frame system MRI/CT compatible has been made and validated ad hoc for the project. In order to understand experimentally the best target point for the catheter introduction a sheep brain DTI atlas has been created. Corticospinal tract (CST), corpus callosum (CC), fornix (FX), visual pathway (VP) and occipitofrontal fascicle (OF), have been identified bilaterally for all the animals. Three of these white matter tracts, the corpus callosum, the fornix and the corona radiata, have been selected to understand the drugs diffusion properties and create a computational model of diffusivity inside the white matter substance. The analysis have been conducted via Focused Ion Beam using scanning Electron Microscopy combined with focused ion beam milling and a 2D analysis and 3D reconstruction made. The results showed homogeneous myelination via detection of ~40% content of lipids in all the different fibre tracts and the fibrous organisation of the tissue described as composite material presenting elliptical tubular fibres with an average cross-sectional area of circa 0.52\u3bcm2 and an estimated mean diameter of 1.15\u3bcm. Finally, as the project is currently ongoing, we provided an overview on the future experimental steps focalised on the brain tissue damage after the rigid catheter introduction

Examining the development of brain structure in utero with fetal MRI, acquired as part of the Developing Human Connectome Project

The human brain is an incredibly complex organ, and the study of it traverses many scales across space and time. The development of the brain is a protracted process that begins embryonically but continues into adulthood. Although neural circuits have the capacity to adapt and are modulated throughout life, the major structural foundations are laid in utero during the fetal period, through a series of rapid but precisely timed, dynamic processes. These include neuronal proliferation, migration, differentiation, axonal pathfinding, and myelination, to name a few. The fetal origins of disease hypothesis proposed that a variety of non-communicable diseases emerging in childhood and adulthood could be traced back to a series of risk factors effecting neurodevelopment in utero (Barker 1995). Since this publication, many studies have shown that the structural scaffolding of the brain is vulnerable to external environmental influences and the perinatal developmental window is a crucial determinant of neurological health later in life. However, there remain many fundamental gaps in our understanding of it. The study of human brain development is riddled with biophysical, ethical, and technical challenges. The Developing Human Connectome Project (dHCP) was designed to tackle these specific challenges and produce high quality open-access perinatal MRI data, to enable researchers to investigate normal and abnormal neurodevelopment (Edwards et al., 2022). This thesis will focus on investigating the diffusion-weighted and anatomical (T2) imaging data acquired in the fetal period, between the second to third trimester (22 – 37 gestational weeks). The limitations of fetal MR data are ill-defined due to a lack of literature and therefore this thesis aims to explore the data through a series of critical and strategic analysis approaches that are mindful of the biophysical challenges associated with fetal imaging. A variety of analysis approaches are optimised to quantify structural brain development in utero, exploring avenues to relate the changes in MR signal to possible neurobiological correlates. In doing so, the work in this thesis aims to improve mechanistic understanding about how the human brain develops in utero, providing the clinical and medical imaging community with a normative reference point. To this aim, this thesis investigates fetal neurodevelopment with advanced in utero MRI methods, with a particular emphasis on diffusion MRI. Initially, the first chapter outlines a descriptive, average trajectory of diffusion metrics in different white matter fiber bundles across the second to third trimester. This work identified unique polynomial trajectories in diffusion metrics that characterise white matter development (Wilson et al., 2021). Guided by previous literature on the sensitivity of DWI to cellular processes, I formulated a hypothesis about the biophysical correlates of diffusion signal components that might underpin this trend in transitioning microstructure. This hypothesis accounted for the high sensitivity of the diffusion signal to a multitude of simultaneously occurring processes, such as the dissipating radial glial scaffold, commencement of pre-myelination and arborization of dendritic trees. In the next chapter, the methods were adapted to address this hypothesis by introducing another dimension, and charting changes in diffusion properties along developing fiber pathways. With this approach it was possible to identify compartment-specific microstructural maturation, refining the spatial and temporal specificity (Wilson et al., 2023). The results reveal that the dynamic fluctuations in the components of the diffusion signal correlate with observations from previous histological work. Overall, this work allowed me to consolidate my interpretation of the changing diffusion signal from the first chapter. It also serves to improve understanding about how diffusion signal properties are affected by processes in transient compartments of the fetal brain. The third chapter of this thesis addresses the hypothesis that cortical gyrification is influenced by both underlying fiber connectivity and cytoarchitecture. Using the same fetal imaging dataset, I analyse the tissue microstructural change underlying the formation of cortical folds. I investigate correlations between macrostructural surface features (curvature, sulcal depth) and tissue microstructural measures (diffusion tensor metrics, and multi-shell multi-tissue decomposition) in the subplate and cortical plate across gestational age, exploring this relationship both at the population level and within subjects. This study provides empirical evidence to support the hypotheses that microstructural properties in the subplate and cortical plate are altered with the development of sulci. The final chapter explores the data without anatomical priors, using a data-driven method to extract components that represent coordinated structural maturation. This analysis aims to examine if brain regions with coherent patterns of growth over the fetal period converge on neonatal functional networks. I extract spatially independent features from the anatomical imaging data and quantify the spatial overlap with pre-defined neonatal resting state networks. I hypothesised that coherent spatial patterns of anatomical development over the fetal period would map onto the functional networks observed in the neonatal period. Overall, this thesis provides new insight about the developmental contrast over the second to third trimester of human development, and the biophysical correlates affecting T2 and diffusion MR signal. The results highlight the utility of fetal MRI to research critical mechanisms of structural brain maturation in utero, including white matter development and cortical gyrification, bridging scales from neurobiological processes to whole brain macrostructure. their gendered constructions relating to women

Quantitative analysis of fiber tractography in cervical spondylotic myelopathy

Background context: Diffusion tensor fiber tractography is an emerging tool for the visualization of spinal cord microstructure. However, there are few quantitative analyses of the damage in the nerve fiber tracts of the myelopathic spinal cord. Purpose: The aim of this study was to develop a quantitative approach for fiber tractography analysis in cervical spondylotic myelopathy (CSM). Study design/setting: Prospective study on a series of patients. Materials and methods: A total of 22 volunteers were recruited with informed consent, including 15 healthy subjects and 7 CSM patients. The clinical severity of CSM was evaluated using modified Japanese Orthopedic Association (JOA) score. The microstructure of myelopathic cervical cord was analyzed using diffusion tensor imaging. Diffusion tensor imaging was performed with a 3.0-T magnetic resonance imaging scanner using pulsed gradient, spin-echo, echo-planar imaging sequence. Fiber tractography was generated via TrackVis with fractional anisotropy threshold set at 0.2 and angle threshold at 40. Region of interest (ROI) was defined to cover C4 level only or the whole-length cervical spinal cord from C1 to C7 for analysis. The length and density of tracked nerve bundles were measured for comparison between healthy subjects and CSM patients. Results: The length of tracked nerve bundles significantly shortened in CSM patients compared with healthy subjects (healthy: 6.85-77.90 mm, CSM: 0.68-62.53 mm). The density of the tracked nerve bundles was also lower in CSM patients (healthy: 086±0.03, CSM: 0.80±0.06, p<.05). Although the definition of ROI covering C4 only or whole cervical cord appeared not to affect the trend of the disparity between healthy and myelopathic cervical cords, the density of the tracked nerve bundle through whole myelopathic cords was in an association with the modified JOA score in CSM cases (r=0.949, p=.015), yet not found with ROI at C4 only (r=0.316, p=.684). Conclusions: The quantitative analysis of fiber tractography is a reliable approach to detect cervical spondylotic myelopathic lesions compared with healthy spinal cords. It could be employed to delineate the severity of CSM. © 2013 Published by Elsevier Inc. All rights reserved.postprin

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

Magnetic resonance imaging, in situ hybridization, and immunohistochemistry-based analyses of early prenatal ethanol exposure-induced central nervous system abnormalities

Fetal alcohol spectrum disorders (FASD), the collection of defects resulting from prenatal alcohol (ethanol) exposure, has been the subject of basic and clinical investigation for four decades, but remains a major public health problem. At the severe end of the spectrum is fetal alcohol syndrome (FAS), which is characterized by the presence of growth retardation, craniofacial anomalies, and brain deficits. The research described herein was designed to advance our knowledge regarding ethanol's insult to the developing brain, with much of it directed toward testing the hypothesis that the application of magnetic resonance-based imaging to the examination of brain morphology, regional volumes and fiber tracts in ethanol-exposed fetal mice would facilitate new discoveries. As with other teratogens, it is well known that the type and severity of abnormality induced by ethanol is dependent upon the dose, timing, and pattern of maternal exposure. For this study, the CNS dysmorphology resulting from acute gestational day (GD) 7 maternal ethanol administration was examined in fetal mice utilizing state of the art imaging techniques. This time in mouse development is consistent with that in the third week of human gestation. Magnetic resonance microscopy (MRM) allowed for linear, volumetric and 3-dimensional morphologic analyses of ethanol-induced alterations in the fetal CNS and diffusion tensor imaging (DTI) provided for assessment of fiber tract abnormalities. In addition, routine histological techniques were utilized for detailed examination of the ventromedian forebrain in ethanol-exposed embryos and fetuses. Major new findings from these studies include the following regarding the consequences of acute GD7 ethanol exposure in mice 1) cerebral cortical heterotopias are induced; a discovery that was facilitated by MRM-based analyses, 2) fiber tract abnormalities involving the corpus callosum, anterior commissure, and fornix/fimbria occur, as evidenced by DTI, 3) fiber tract abnormalities, as identified in fetal mice, persist into periadolescent stages, 4) ventral forebrain insult preferentially involving the preoptic area and medial ganglionic eminences reduces Olig2 and GABA expression and alters the morphology of somatostatin-expressing cells. Overall, the results of this work promise to aid in clinical recognition, diagnosis, and prevention of FASD