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International audience; Synchrotron-generated microplanar beams (microbeams) provide the most stereo-selective irradiation modality known today. This novel irradiation modality has been shown to control seizures originating from eloquent cortex causing no neurological deficit in experimental animals. To test the hypothesis that application of microbeams in the hippocampus, the most common source of refractory seizures, is safe and does not induce severe side effects, we used microbeams to induce transections to the hippocampus of healthy rats. An array of parallel microbeams carrying an incident dose of 600 Gy was delivered to the rat hippocampus. Immunohistochemistry of phosphorylated gamma-H2AX showed cell death along the microbeam irradiation paths in rats 48 hours after irradiation. No evident behavioral or neurological deficits were observed during the 3-month period of observation. MR imaging showed no signs of radio-induced edema or radionecrosis 3 months after irradiation. Histological analysis showed a very well preserved hippocampal cytoarchitecture and confirmed the presence of clear-cut microscopic transections across the hippocampus. These data support the use of synchrotron-generated microbeams as a novel tool to slice the hippocampus of living rats in a minimally invasive way, providing (i) a novel experimental model to study hippocampal function and (ii) a new treatment tool for patients affected by refractory epilepsy induced by mesial temporal sclerosis.

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-β 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.

Simple Summary This study aims at using a multi-technique approach to detect and analyze the effects of high dose rate spatially fractionated radiation therapies and to compare them to seamless broad beam irradiation targeting healthy and glioblastoma-bearing rat brains and delivering three different doses per each irradiation geometry. Brains were analyzed post mortem by multi-scale X-ray phase contrast imaging–computed tomography, histology, immunohistochemistry, X-ray fluorescence, and small- and wide-angle X-ray scattering to achieve detailed visualization, characterization and classification in 3D of the radio-induced effects on brain tissues. The original results bring new insights to the understanding of the response of cerebral tissue and tumors treated with high dose rate spatially fractioned radiotherapies and put the basis for channeling studies of in-vivo applications for monitoring RT effects. Abstract 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.

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 exvivo 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 invitro imaging of normal and cancerous brain neuromorphology after invivo 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 invivo to the brain of both healthy and cancer-bearing rats. At 45days after treatment, the brain was dissected out and imaged exvivo 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 exvivo 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. &nbsp