A combinatorial method to visualize the neuronal network in the mouse spinal cord: combination of a modified Golgi-Cox method and synchrotron radiation micro-computed tomography

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.

Bone Marrow Mesenchymal Stem Cell-Derived Exosome-Educated Macrophages Promote Functional Healing After Spinal Cord Injury

“…Since spinal cord neurovasculature has unique 3D morphological features ( Li P. et al, 2020 ; Li R. et al, 2020 ; Jiang et al, 2021a ), we performed a comparative analysis of the vasculature and neuronal network between control and EEM-treated mice and examined changes in the 3D morphological parameters of the spinal cord neurovascular structure after SCI using SRμCT. SRμCT is now routinely used to perform investigations on a wide range of biospecimen.…”

“…For neural network visualization, mice were anesthetized and perfused transcranially with artificial cerebrospinal fluid to remove the blood from the vessels. The spinal cord at the injured site was isolated and processed by staining with Golgi-Cox solutions in a FD Rapid GolgiStain TM (KitNeuro Technologies, Inc.) as previously described ( Jiang et al, 2021a ). Briefly, after Golgi-Cox staining, the spinal cord was prepared for SRμCT analysis.…”

In focus in HCB

“…To this day, further modifications to this neuronal staining method to achieve optimal results under different experimental conditions and protocols continue to be reported (Narayan et al 2020). In this light, Jiang et al (2021) now provide an improved Golgi-Cox staining method optimized for 3D visualization of neurons in the mouse spinal cord using synchrotron radiation micro-computed tomography (SRμCT). Their main modifications of the traditional Golgi-Cox Fig.…”

“…3 The neuronal network in the ventral horn of the mouse spinal cord as visualized by a modified Golgi-Cox method (left) and its 3D-rendering by synchrotron radiation micro-computed tomography (right). From Jiang et al (2021) staining protocol included (1) perfusion of animals with artificial cerebrospinal fluid prior to removal of the spinal cord to remove blood cells from the vasculature; (2) increased rinsing cycles of tissue with distilled water to provide a clearer background; and (3) clearing of sections with methyl salicylate prior to application of a coverslip and imaging (the authors provide a very detailed protocol comparison in a supplementary figure). Their protocol modifications resulted in stained spinal cord sections with clearer background, a reduction in both staining artefacts and incomplete vasculature structures (Fig.…”

3D visualization and morphometric analysis of spinal motion segments and vascular networks: A synchrotron radiation‐based micro‐CT study in mice

“…This technique has been used to visualize detailed images of the architecture of bone and its blood supply system (Langer et al, 2009). The three‐dimensional neuronal and vascular networks of the spinal cord could also be simultaneously vividly detected using SRμCT (Fratini et al, 2015; Jiang et al, 2021a, 2021b). In our previous study, the 3D morphologic changes of the disc and end plate during aging were vividly depicted (Cao et al, 2017b).…”