Simultaneous submicrometric 3D imaging of the micro-vascular network and the neuronal system in a mouse spinal cord

Abstract Faults in vascular (VN) and neuronal networks of spinal cord are responsible for serious neurodegenerative pathologies. Because of inadequate investigation tools, the lacking knowledge of the complete fine structure of VN and neuronal system represents a crucial problem. Conventional 2D imaging yields incomplete spatial coverage leading to possible data misinterpretation, whereas standard 3D computed tomography imaging achieves insufficient resolution and contrast. We show that X-ray high-resolution phase-contrast tomography allows the simultaneous visualization of three-dimensional VN and neuronal systems of ex-vivo mouse spinal cord at scales spanning from millimeters to hundreds of nanometers, with nor contrast agent nor sectioning and neither destructive sample-preparation. We image both the 3D distribution of micro-capillary network and the micrometric nerve fibers, axon-bundles and neuron soma. Our approach is very suitable for pre-clinical investigation of neurodegenerative pathologies and spinal-cord-injuries, in particular to resolve the entangled relationship between VN and neuronal system.

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Nondestructive imaging of the internal microstructure of vessels and nerve fibers in rat spinal cord using phase-contrast synchrotron radiation microtomography

Journal of Synchrotron Radiation ◽

10.1107/s1600577517000121 ◽

2017 ◽

Vol 24 (2) ◽

pp. 482-489 ◽

Cited By ~ 9

Author(s):

Jianzhong Hu ◽

Ping Li ◽

Xianzhen Yin ◽

Tianding Wu ◽

Yong Cao ◽

...

Keyword(s):

Spinal Cord ◽

Synchrotron Radiation ◽

Phase Contrast ◽

Three Dimensional ◽

Nerve Fibers ◽

The Body ◽

Rat Spinal Cord ◽

Nondestructive Imaging ◽

Synchrotron Radiation Microtomography ◽

The Brain

The spinal cord is the primary neurological link between the brain and other parts of the body, but unlike those of the brain, advances in spinal cord imaging have been challenged by the more complicated and inhomogeneous anatomy of the spine. Fortunately with the advancement of high technology, phase-contrast synchrotron radiation microtomography has become widespread in scientific research because of its ability to generate high-quality and high-resolution images. In this study, this method has been employed for nondestructive imaging of the internal microstructure of rat spinal cord. Furthermore, digital virtual slices based on phase-contrast synchrotron radiation were compared with conventional histological sections. The three-dimensional internal microstructure of the intramedullary arteries and nerve fibers was vividly detected within the same spinal cord specimen without the application of a stain or contrast agent or sectioning. With the aid of image post-processing, an optimization of vessel and nerve fiber images was obtained. The findings indicated that phase-contrast synchrotron radiation microtomography is unique in the field of three-dimensional imaging and sets novel standards for pathophysiological investigations in various neurovascular diseases.

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White matter regeneration induced by aligned fibrin nanofiber hydrogel contributes to motor functional recovery in canine T12 spinal cord injury

Regenerative Biomaterials ◽

10.1093/rb/rbab069 ◽

2021 ◽

Author(s):

Zheng Cao ◽

Weitao Man ◽

Yuhui Xiong ◽

Yi Guo ◽

Shuhui Yang ◽

...

Keyword(s):

Spinal Cord ◽

Spinal Cord Injury ◽

White Matter ◽

Nerve Regeneration ◽

Spinal Cord Injuries ◽

Diffusion Tensor ◽

Nerve Fibers ◽

Functional Restoration ◽

Large Animal ◽

Cord Injury

Abstract A hierarchically aligned fibrin hydrogel (AFG) that possesses soft stiffness and aligned nanofiber structure has been successfully proven to facilitate neuroregeneration in vitro and in vivo. However, its potential in promoting nerve regeneration in large animal models that is critical for clinical translation has not been sufficiently specified. Here, the effects of AFG on directing neuroregeneration in canine hemisected T12 spinal cord injuries were explored. Histologically obvious white matter regeneration consisting of a large area of consecutive, compact, and aligned nerve fibers is induced by AFG, leading to a significant motor functional restoration. The canines with AFG implantation start to stand well with their defective legs from 3 to 4 weeks postoperatively and even effortlessly climb the steps from 7 to 8 weeks. Moreover, high-resolution multi-shot diffusion tensor imaging illustrates the spatiotemporal dynamics of nerve regeneration rapidly crossing the lesion within 4 weeks in the AFG group. Our findings indicate that AFG could be a potential therapeutic vehicle for spinal cord injury by inducing rapid white matter regeneration and restoring locomotion, pointing out its promising prospect in clinic practice.

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Multifunctionalized hydrogels foster hNSC maturation in 3D cultures and neural regeneration in spinal cord injuries

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1818392116 ◽

2019 ◽

Vol 116 (15) ◽

pp. 7483-7492 ◽

Cited By ~ 21

Author(s):

Amanda Marchini ◽

Andrea Raspa ◽

Raffaele Pugliese ◽

Marina Abd El Malek ◽

Valentina Pastori ◽

...

Keyword(s):

Spinal Cord ◽

Spinal Cord Injuries ◽

Culture Media ◽

Three Dimensional ◽

Cholinergic Neurons ◽

Good Manufacturing Practice ◽

Neural Regeneration ◽

Neuronal Markers

Three-dimensional cell cultures are leading the way to the fabrication of tissue-like constructs useful to developmental biology and pharmaceutical screenings. However, their reproducibility and translational potential have been limited by biomaterial and culture media compositions, as well as cellular sources. We developed a construct comprising synthetic multifunctionalized hydrogels, serum-free media, and densely seeded good manufacturing practice protocol-grade human neural stem cells (hNSC). We tracked hNSC proliferation, differentiation, and maturation into GABAergic, glutamatergic, and cholinergic neurons, showing entangled electrically active neural networks. The neuroregenerative potential of the “engineered tissue” was assessed in spinal cord injuries, where hNSC-derived progenitors and predifferentiated hNSC progeny, embedded in multifunctionalized hydrogels, were implanted. All implants decreased astrogliosis and lowered the immune response, but scaffolds with predifferentiated hNSCs showed higher percentages of neuronal markers, better hNSC engraftment, and improved behavioral recovery. Our hNSC-construct enables the formation of 3D functional neuronal networks in vitro, allowing novel strategies for hNSC therapies in vivo.

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Visualization of mouse spinal cord intramedullary arteries using phase- and attenuation-contrast tomographic imaging

Journal of Synchrotron Radiation ◽

10.1107/s1600577516006482 ◽

2016 ◽

Vol 23 (4) ◽

pp. 966-974 ◽

Cited By ~ 5

Author(s):

Yong Cao ◽

Xianzhen Yin ◽

Jiwen Zhang ◽

Tianding Wu ◽

Dongzhe Li ◽

...

Keyword(s):

Spinal Cord ◽

High Resolution ◽

Synchrotron Radiation ◽

Three Dimensional ◽

Small Vessel ◽

Imaging Method ◽

Central Sulcus ◽

Contrast Imaging ◽

Mouse Spinal Cord ◽

Small Vessels

Many spinal cord circulatory disorders present the substantial involvement of small vessel lesions. The central sulcus arteries supply nutrition to a large part of the spinal cord, and, if not detected early, lesions in the spinal cord will cause irreversible damage to the function of this organ. Thus, early detection of these small vessel lesions could potentially facilitate the effective diagnosis and treatment of these diseases. However, the detection of such small vessels is beyond the capability of current imaging techniques. In this study, an imaging method is proposed and the potential of phase-contrast imaging (PCI)- and attenuation-contrast imaging (ACI)-based synchrotron radiation for high-resolution tomography of intramedullary arteries in mouse spinal cord is validated. The three-dimensional vessel morphology, particularly that of the central sulcus arteries (CSA), detected with these two imaging models was quantitatively analyzed and compared. It was determined that both PCI- and ACI-based synchrotron radiation can be used to visualize the physiological arrangement of the entire intramedullary artery network in the mouse spinal cord in both two dimensions and three dimensions at a high-resolution scale. Additionally, the two-dimensional and three-dimensional vessel morphometric parameter measurements obtained with PCI are similar to the ACI data. Furthermore, PCI allows efficient and direct discrimination of the same branch level of the CSA without contrast agent injection and is expected to provide reliable biological information regarding the intramedullary artery. Compared with ACI, PCI might be a novel imaging method that offers a powerful imaging platform for evaluating pathological changes in small vessels and may also allow better clarification of their role in neurovascular disorders.

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Monoaminergic Establishment of Rostrocaudal Gradients of Rhythmicity in the Neonatal Mouse Spinal Cord

Journal of Neurophysiology ◽

10.1152/jn.00299.2005 ◽

2005 ◽

Vol 94 (2) ◽

pp. 1554-1564 ◽

Cited By ~ 26

Author(s):

Kimberly J. Christie ◽

Patrick J. Whelan

Keyword(s):

Spinal Cord ◽

Locomotor Activity ◽

Spinal Cord Injuries ◽

Neonatal Mouse ◽

Network Activity ◽

Neonatal Rats ◽

Mouse Spinal Cord ◽

Bath Application ◽

Rats And Mice ◽

Calcium Green

Bath application of monoamines is a potent method for evoking locomotor activity in neonatal rats and mice. Monoamines also promote functional recovery in adult animals with spinal cord injuries by activating spinal cord networks. However, the mechanisms of their actions on spinal networks are largely unknown. In this study, we tested the hypothesis that monoamines establish rostrocaudal gradients of rhythmicity in the thoracolumbar spinal cord. Isolated neonatal mouse spinal cord preparations (P0–P2) were used. To assay excitability of networks by monoamines, we evoked a disinhibited rhythm by bath application of picrotoxin and strychnine and recorded neurograms from several thoracolumbar ventral roots. We first established that rostral and caudal segments of the thoracolumbar spinal cord had equal excitability by completely transecting preparations at the L3 segmental level and recording the frequency of the disinhibited rhythm from both segments. Next we established that a majority of ventral interneurons retrogradely labeled by calcium green dextran were active during network activity. We then bath applied combinations of monoaminergic agonists [5-HT and dopamine (DA)] known to elicit locomotor activity. Our results show that monoamines establish rostrocaudal gradients of rhythmicity in the thoracolumbar spinal cord. This may be one mechanism by which combinations of monoaminergic compounds normally stably activate locomotor networks.

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Interpreting the biochemical specificity of mouse spinal cord by confocal raman microspectral imaging

Journal of Innovative Optical Health Sciences ◽

10.1142/s1793545817430076 ◽

2017 ◽

Vol 10 (05) ◽

pp. 1743007 ◽

Cited By ~ 3

Author(s):

Yuze Gong ◽

Zhuowen Liang ◽

Yaning Yin ◽

Jiwei Song ◽

Xueyu Hu ◽

...

Keyword(s):

Spinal Cord ◽

Ex Vivo ◽

Distribution Patterns ◽

Chemical Components ◽

Spinal Cord Tissue ◽

Essential Requirement ◽

Mouse Spinal Cord ◽

Molecular Investigation ◽

Specific Substance ◽

Biochemical Mechanisms

Interpreting the biochemical specificity of spinal cord tissue is the essential requirement for understanding the biochemical mechanisms during spinal-cord-related pathological course. In this work, a longitudinal study was implemented to reveal a precise linkage between the spectral features and the molecular composition in ex vivo mouse spinal cord tissue by microspectral Raman imaging. It was testified that lipid-rich white matter could be distinguished from gray matter not only by the lipid Raman peaks at 1064, 1300, 1445 and 1660[Formula: see text]cm[Formula: see text], but also by protein (1250 and 1328[Formula: see text]cm[Formula: see text] and saccharides (913 and 1137[Formula: see text]cm[Formula: see text] distributions. [Formula: see text]-means cluster analysis was further applied to visualize the morphological basis of spinal cord tissue by chemical components and their distribution patterns. Two-dimensional chemical images were then generated to visualize the contrast between two different tissue types by integrating the intensities of the featured Raman bands. All the obtained results illustrated the biochemical characteristics of spinal cord tissue, as well as some specific substance variances between different tissue types, which formed a solid basis for the molecular investigation of spinal cord pathological alterations.

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