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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.

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