The spatiotemporal spread of cervical spinal cord contusion injury pathology revealed by 3D in-line phase contrast synchrotron X-ray microtomography.

Extensive structural changes occur within the spinal cord following traumatic injury. Acute tissue debris and necrotic tissue are broken down, proliferating local glia and infiltrating leukocytes remodel tissue biochemical and biophysical properties, and a chronic cavity surrounded by a scar forms at the injury epicentre. Serial-section 2D histology has traditionally assessed these features in experimental models of spinal cord injury (SCI) to measure the extent of tissue pathology and evaluate efficacy of novel therapies. However, this 2D snapshot approach overlooks slice intervening features, with accurate representation of tissue compromised by mechanical processing artefacts. 3D imaging avoids these caveats and allows full exploration of the injured tissue volume to characterise whole tissue pathology. Amongst 3D imaging modalities, Synchrotron Radiation X-ray microtomography (SRµCT) is advantageous for its speed, ability to cover large tissue volumes at high resolution, and need for minimal sample processing. Here we demonstrate how extended lengths of formalin-fixed, paraffin-embedded (FFPE) rat spinal cord can be completely imaged by SRµCT with micron resolution. Label-free contrast derived from X-ray phase interactions with low-density soft tissues, reveals spinal cord white matter, gray matter, tissue damage and vasculature, with tissue still viable for targeted 2D-histology after 3D imaging. We used SRµCT to quantify tissue pathology after a midline, cervical level (C6), 225 kDyne contusion injury over acute-to-chronic (24 h to 5 weeks) post injury time points. Quantification revealed acute tissue swelling prior to chronic atrophy across the whole imaged region (spanning 2 spinal segments above and below injury), along with rostro-caudal asymmetries in white and gray matter volume loss. 3D volumes revealed satellite damage in tissue far removed from the epicentre, and extensive rostro-caudal spread of damage through the base of the dorsal columns at 24 h post injury. This damage overlapped regions of vasogenic oedema, confirmed with subsequent histology. Tissue damage at later time points in border regions was most prominent in the dorsal columns, where it overlapped sites of damaged venous vasculature. Elaborating rostro-caudal and spatiotemporal asymmetries in reduced traumatic injury models centred on these regions may inform future treatments that seek to limit the spread of tissue pathology to these 'at-risk' regions.

Optimising complementary soft tissue synchrotron X-ray microtomography for reversibly-stained central nervous system samples.

Synchrotron radiation microtomography (SRµCT) is a nominally non-destructive 3D imaging technique which can visualise the internal structures of whole soft tissues. As a multi-stage technique, the cumulative benefits of optimising sample preparation, scanning parameters and signal processing can improve SRµCT imaging efficiency, image quality, accuracy and ultimately, data utility. By evaluating different sample preparations (embedding media, tissue stains), imaging (projection number, propagation distance) and reconstruction (artefact correction, phase retrieval) parameters, a novel methodology (combining reversible iodine stain, wax embedding and inline phase contrast) was optimised for fast (~12 minutes), high-resolution (3.2-4.8 µm diameter capillaries resolved) imaging of the full diameter of a 3.5 mm length of rat spinal cord. White-grey matter macro-features and micro-features such as motoneurons and capillary-level vasculature could then be completely segmented from the imaged volume for analysis through the shallow machine learning SuRVoS Workbench. Imaged spinal cord tissue was preserved for subsequent histology, establishing a complementary SRµCT methodology that can be applied to study spinal cord pathologies or other nervous system tissues such as ganglia, nerves and brain. Further, our 'single-scan iterative downsampling' approach and side-by-side comparisons of mounting options, sample stains and phase contrast parameters should inform efficient, effective future soft tissue SRµCT experiment design.