Abstract
Exploring the three-dimensional (3D) morphology of neurons is essential to understanding spinal cord function and associated diseases comprehensively. However, 3D imaging of the neuronal network in the broad region of the spinal cord at cellular resolution remains a challenge in the field of neuroscience. In this study, to obtain high-resolution 3D imaging of a detailed neuronal network in the mass of the spinal cord, the combination of synchrotron radiation micro-computed tomography (SRμCT) and the Golgi-cox staining were used. We optimized the Golgi-Cox method (GCM) and developed a modified GCM (M-GCM), which improved background staining, reduced the number of artefacts, and diminished the impact of incomplete vasculature compared to the current GCM. Moreover, we achieved high-resolution 3D imaging of the detailed neuronal network in the spinal cord through the combination of SRμCT and M-GCM. Our results showed that the M-GCM increased the contrast between the neuronal structure and its surrounding extracellular matrix. Compared to the GCM, the M-GCM also diminished the impact of the artefacts and incomplete vasculature on the 3D image. Additionally, the 3D neuronal architecture was successfully quantified using a combination of SRμCT and M-GCM. The SRμCT was shown to be a valuable non-destructive tool for 3D visualization of the neuronal network in the broad 3D region of the spinal cord. Such a combinatorial method will, therefore, transform the presentation of Golgi staining from 2 to 3D, providing significant improvements in the 3D rendering of the neuronal network.
Highlights
The spinal cord, which transmits descending and ascending neural signals, is an essential component of the central nervous system
M‐Golgi-Cox method (GCM) achieved less background staining, fewer artefacts, and less stained incomplete vasculature compared to the GCM
In the GCM group, the residual blood, and Solution A/B left within the spinal cord tissue impacted Golgi staining, leading to high background staining, artefacts, and incomplete vasculature
Summary
The spinal cord, which transmits descending and ascending neural signals, is an essential component of the central nervous system. Histological staining techniques are widely used in research on neuronal morphology (Tsai et al 2003). These methods mainly rely on two-dimensional (2D) histological sections. To confirm that the M-GCM reduced the background staining, an area with the same size (30 μm × 45 μm) located at the unstained background from the 60 × images in each sample were randomly selected for the comparison of background staining. Artefact comparison: To confirm the M-GCM generated fewer artefacts (object does not have discernible dendrites or axons), we captured continuous 20 × images (n = 40) located at the gray matter from different sections. Artefacts in the 3D volume (740 μm × 900 μm × 80 μm) were manually counted
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