X-ray multiscale 3D neuroimaging to quantify cellular aging and neurodegeneration postmortem in a model of Alzheimer's disease.

PURPOSE: Modern neuroimaging lacks the tools necessary for whole-brain, anatomically dense neuronal damage screening. An ideal approach would include unbiased histopathologic identification of aging and neurodegenerative disease. METHODS: We report the postmortem application of multiscale X-ray phase-contrast computed tomography (X-PCI-CT) for the label-free and dissection-free organ-level to intracellular-level 3D visualization of distinct single neurons and glia. In deep neuronal populations in the brain of aged wild-type and of 3xTgAD mice (a triply-transgenic model of Alzheimer's disease), we quantified intracellular hyperdensity, a manifestation of aging or neurodegeneration. RESULTS: In 3xTgAD mice, the observed hyperdensity was identified as amyloid-ß and hyper-phosphorylated tau protein deposits with calcium and iron involvement, by correlating the X-PCI-CT data to immunohistochemistry, X-ray fluorescence microscopy, high-field MRI, and TEM. As a proof-of-concept, X-PCI-CT was used to analyze hippocampal and cortical brain regions of 3xTgAD mice treated with LY379268, selective agonist of group II metabotropic glutamate receptors (mGlu2/3 receptors). Chronic pharmacologic activation of mGlu2/3 receptors significantly reduced the hyperdensity particle load in the ventral cortical regions of 3xTgAD mice, suggesting a neuroprotective effect with locoregional efficacy. CONCLUSIONS: This multiscale micro-to-nano 3D imaging method based on X-PCI-CT enabled identification and quantification of cellular and sub-cellular aging and neurodegeneration in deep neuronal and glial cell populations in a transgenic model of Alzheimer's disease. This approach quantified the localized and intracellular neuroprotective effects of pharmacological activation of mGlu2/3 receptors.

A Multi-Scale and Multi-Technique Approach for the Characterization of the Effects of Spatially Fractionated X-ray Radiation Therapies in a Preclinical Model.

The purpose of this study is to use a multi-technique approach to detect the effects of spatially fractionated X-ray Microbeam (MRT) and Minibeam Radiation Therapy (MB) and to compare them to seamless Broad Beam (BB) irradiation. Healthy- and Glioblastoma (GBM)-bearing male Fischer rats were irradiated in-vivo on the right brain hemisphere with MRT, MB and BB delivering three different doses for each irradiation geometry. Brains were analyzed post mortem by multi-scale X-ray Phase Contrast Imaging-Computed Tomography (XPCI-CT), histology, immunohistochemistry, X-ray Fluorescence (XRF), Small- and Wide-Angle X-ray Scattering (SAXS/WAXS). XPCI-CT discriminates with high sensitivity the effects of MRT, MB and BB irradiations on both healthy and GBM-bearing brains producing a first-time 3D visualization and morphological analysis of the radio-induced lesions, MRT and MB induced tissue ablations, the presence of hyperdense deposits within specific areas of the brain and tumor evolution or regression with respect to the evaluation made few days post-irradiation with an in-vivo magnetic resonance imaging session. Histology, immunohistochemistry, SAXS/WAXS and XRF allowed identification and classification of these deposits as hydroxyapatite crystals with the coexistence of Ca, P and Fe mineralization, and the multi-technique approach enabled the realization, for the first time, of the map of the differential radiosensitivity of the different brain areas treated with MRT and MB. 3D XPCI-CT datasets enabled also the quantification of tumor volumes and Ca/Fe deposits and their full-organ visualization. The multi-scale and multi-technique approach enabled a detailed visualization and classification in 3D of the radio-induced effects on brain tissues bringing new essential information towards the clinical implementation of the MRT and MB radiation therapy techniques.

High-Spatial-Resolution Three-dimensional Imaging of Human Spinal Cord and Column Anatomy with Postmortem X-ray Phase-Contrast Micro-CT.

Background Modern high-spatial-resolution radiologic methods enable increasingly detailed volumetric postmortem investigations of human neuroanatomy for diagnostic, research, and educational purposes. Purpose To evaluate the viability of postmortem x-ray phase-contrast micro-CT to provide tissue-conserving, high-spatial-resolution, three-dimensional neuroimaging of the human spinal cord and column by comparing quality of x-ray phase-contrast micro-CT images of nondissected Thiel-embalmed human spines with images of extracted formalin-fixed human spinal cords. Specific focus was placed on assessing the detection of micrometric spinal cord soft-tissue structure and vasculature. Materials and Methods In this study from August 2015 to August 2019, three Thiel-embalmed human spinal column samples, unilaterally perfused with an iodinated vascular contrast agent, and three extracted formalin-fixed spinal cord samples were imaged postmortem at a synchrotron radiation facility. Propagation-based x-ray phase-contrast micro-CT was used with monochromatic 60-keV x-rays and a detector with either 46-µm or 8-µm pixel sizes. A single-distance phase-retrieval algorithm was applied to the acquired CT projection images in advance of filtered back projection CT reconstruction. The influence on image quality of Thiel versus formalin embalming was examined, and images were qualitatively evaluated in terms of the value of their anatomic representations. Results The x-ray phase-contrast micro-CT of Thiel-embalmed samples resulted in soft-tissue contrast within the vertebral canal, despite evident nervous tissue deterioration after Thiel embalming. Gross spinal cord anatomy, spinal meninges, contrast agent-enhanced spinal vasculature, and spinal nerves were all well rendered alongside surrounding vertebral bone structure. The x-ray phase-contrast micro-CT of formalin-fixed boneless cords led to much higher gray versus white matter contrast and to microscale visualization of deep medullary vasculature and neuronal perikarya. Conclusion This work demonstrated the use of x-ray phase-contrast micro-CT for detailed volumetric anatomic visualization of embalmed human spines. The method provided three-dimensional display of bone, nervous tissue, and vasculature at microscale resolutions without exogenous contrast agents. © RSNA, 2020 Online supplemental material is available for this article.

Multiscale pink-beam microCT imaging at the ESRF-ID17 biomedical beamline.

Recent trends in hard X-ray micro-computed tomography (microCT) aim at increasing both spatial and temporal resolutions. These challenges require intense photon beams. Filtered synchrotron radiation beams, also referred to as `pink beams', which are emitted by wigglers or bending magnets, meet this need, owing to their broad energy range. In this work, the new microCT station installed at the biomedical beamline ID17 of the European Synchrotron is described and an overview of the preliminary results obtained for different biomedical-imaging applications is given. This new instrument expands the capabilities of the beamline towards sub-micrometre voxel size scale and simultaneous multi-resolution imaging. The current setup allows the acquisition of tomographic datasets more than one order of magnitude faster than with a monochromatic beam configuration.

Establishing sample-preparation protocols for X-ray phase-contrast CT of rodent spinal cords: Aldehyde fixations and osmium impregnation.

BACKGROUND: Dense and unbiased cellular-resolution representations of extended volumetric central nervous system soft-tissue anatomy are difficult to obtain, even in experimental post-mortem settings. Interestingly, X-ray phase-contrast computed tomography (X-PCI-CT), an emerging soft-tissue-sensitive volumetric imaging technique, can provide multiscale organ- to cellular-level morphological visualizations of neuroanatomical structure. NEW METHOD: Here, we tested different nervous-tissue fixation procedures, conventionally used for transmission electron microscopy, to better establish X-PCI-CT-specific sample-preparation protocols. Extracted rat spinal medullas were alternatively fixed with a standard paraformaldehyde-only aldehyde-based protocol, or in combination with glutaraldehyde. Some specimens were additionally post-fixed with osmium tetroxide. Multiscale X-PCI-CT datasets were collected at several synchrotron radiation facilities, using state-of-the-art setups with effective image voxel sizes of 3.03 to 0.33 µm3, and compared to high-field magnetic resonance imaging, histology and vascular fluorescence microscopy data. RESULTS: Multiscale X-PCI-CT of aldehyde-fixed spinal cord specimens resulted in dense histology-like volumetric representations and quantifications of extended deep spinal micro-vascular networks and of intra-medullary cell populations. Osmium post-fixation increased intra-medullary contrast between white and gray-matter tissues, and enhanced delineation of intra-medullary cellular structure, e.g. axon fibers and motor neuron perikarya. COMPARISON WITH EXISTING METHODS: Volumetric X-PCI-CT provides complementary contrast and higher spatial resolution compared to 9.4 T MRI. X-PCI-CT's advantage over planar histology is the volumetric nature of the cellular-level data obtained, using samples much larger than those fit for volumetric vascular fluorescence microscopy. CONCLUSIONS: Deliberately choosing (post-)fixation protocols tailored for optimal nervous-tissue structural preservation is of paramount importance in achieving effective and targeted neuroimaging via the X-PCI-CT technique.

Micro-imaging of Brain Cancer Radiation Therapy Using Phase-contrast Computed Tomography.

PURPOSE: Experimental neuroimaging provides a wide range of methods for the visualization of brain anatomic morphology down to subcellular detail. Still, each technique-specific detection mechanism presents compromises among the achievable field-of-view size, spatial resolution, and nervous tissue sensitivity, leading to partial sample coverage, unresolved morphologic structures, or sparse labeling of neuronal populations and often also to obligatory sample dissection or other sample invasive manipulations. X-ray phase-contrast imaging computed tomography (PCI-CT) is an experimental imaging method that simultaneously provides micrometric spatial resolution, high soft-tissue sensitivity, and ex vivo full organ rodent brain coverage without any need for sample dissection, staining or labeling, or contrast agent injection. In the present study, we explored the benefits and limitations of PCI-CT use for in vitro imaging of normal and cancerous brain neuromorphology after in vivo treatment with synchrotron-generated x-ray microbeam radiation therapy (MRT), a spatially fractionated experimental high-dose radiosurgery. The goals were visualization of the MRT effects on nervous tissue and a qualitative comparison of the results to the histologic and high-field magnetic resonance imaging findings. METHODS AND MATERIALS: MRT was administered in vivo to the brain of both healthy and cancer-bearing rats. At 45 days after treatment, the brain was dissected out and imaged ex vivo using propagation-based PCI-CT. RESULTS: PCI-CT visualizes the brain anatomy and microvasculature in 3 dimensions and distinguishes cancerous tissue morphology, necrosis, and intratumor accumulation of iron and calcium deposits. Moreover, PCI-CT detects the effects of MRT throughout the treatment target areas (eg, the formation of micrometer-thick radiation-induced tissue ablation). The observed neurostructures were confirmed by histologic and immunohistochemistry examination and related to the micro-magnetic resonance imaging data. CONCLUSIONS: PCI-CT enabled a unique 3D neuroimaging approach for ex vivo studies on small animal models in that it concurrently delivers high-resolution insight of local brain tissue morphology in both normal and cancerous micro-milieu, localizes radiosurgical damage, and highlights the deep microvasculature. This method could assist experimental small animal neurology studies in the postmortem evaluation of neuropathology or treatment effects.