Following spinal cord injury (SCI) the degree of functional (motor, autonomous, or sensory) loss correlates with the severity of nervous tissue damage. An imaging technique able to capture non-invasively and simultaneously the complex mechanisms of neuronal loss, vascular damage, and peri-lesional tissue reorganization is currently lacking in experimental SCI studies. Synchrotron X-ray phase-contrast tomography (SXPCT) has emerged as a non-destructive three-dimensional (3D) neuroimaging technique with high contrast and spatial resolution. In this framework, we developed a multi-modal approach combining SXPCT, histology and correlative methods to study neurovascular architecture in normal and spinal level C4-contused mouse spinal cords (C57BL/6J mice, age 2–3 months). The evolution of SCI lesion was imaged at the cell resolution level during the acute (30 min) and subacute (7 day) phases. Spared motor neurons (MNs) were segmented and quantified in different volumes localized at and away from the epicenter. SXPCT was able to capture neuronal loss and blood–brain barrier breakdown following SCI. Three-dimensional quantification based on SXPCT acquisitions showed no additional MN loss between 30 min and 7 days post-SCI. In addition, the analysis of hemorrhagic (at 30 min) and lesion (at 7 days) volumes revealed a high similarity in size, suggesting no extension of tissue degeneration between early and later time-points. Moreover, glial scar borders were unevenly distributed, with rostral edges being the most extended. In conclusion, SXPCT capability to image at high resolution cellular changes in 3D enables the understanding of the relationship between hemorrhagic events and nervous structure damage in SCI.
Get full access to this article
View all access options for this article.
References
1.
OyinboCA. Secondary injury mechanisms in traumatic spinal cord injury: a nugget of this multiply cascade. Acta Neurobiol Exp (Wars), 2011; 71(2):281–299.
2.
NorenbergMD, SmithJ, MarcilloA. The pathology of human spinal cord injury: defining the problems. J Neurotrauma, 2004; 21(4):429–440. doi: 10.1089/089771504323004575
3.
ChooAM, LiuJ, DvorakM, et al.Secondary pathology following contusion, dislocation, and distraction spinal cord injuries. Exp Neurol, 2008; 212(2):490–506; doi: https://doi.org/10.1016/j.expneurol.2008.04.038
4.
GrossmanS, RosenbergL, WrathallJ. Temporal–spatial pattern of acute neuronal and glial loss after spinal cord contusion. Exp Neurol, 2001; 168(2):273–282; doi: 10.1006/exnr.2001.7628
5.
LiuXZ, XuXM, HuR, et al.Neuronal and glial apoptosis after traumatic spinal cord injury. J Neurosci, 1997; 17(14):5395–5406; doi: 10.1523/JNEUROSCI.17-14-05395.1997
6.
LouJ, LenkeL, LudwigF, et al.Apoptosis as a mechanism of neuronal cell death following acute experimental spinal cord injury. Spinal Cord, 1998; 36(10):683–690; doi: https://doi.org/10.1038/sj.sc.3100632
7.
LingX, LiuD. Temporal and spatial profiles of cell loss after spinal cord injury: reduction by a metalloporphyrin. J Neurosci Res, 2007; 85(10):2175–2185; doi: 10.1002/jnr.21362
8.
FehlingsMG, VaccaroA, BoakyeM, et al. (eds). Essentials of Spinal Cord Injury: Basic Research to Clinical Practice. Thieme Medical Publishers; 2013.
9.
WilsonJR, HashimotoRE, DettoriJR, et al.Spinal cord injury and quality of life: a systematic review of outcome measures. Evid Based Spine Care J, 2011; 2(1):37–44; doi: 10.1055/s-0030-1267085
10.
CalabreseE, AdilSM, CoferG, et al.Postmortem diffusion MRI of the entire human spinal cord at microscopic resolution. Neuroimage Clin, 2018; 18(963–971); doi: 10.1016/j.nicl.2018.03.029
11.
NijeholtGJ, BergersE, KamphorstW, et al.Post-mortem high-resolution MRI of the spinal cord in multiple sclerosis: a correlative study with conventional MRI, histopathology and clinical phenotype. Brain, 2001; 124(Pt 1):154–166.
12.
ArthursOJ, ThayyilS, Pauliah, et al. Diagnostic accuracy and limitations of post-mortem mri for neurological abnormalities in fetuses and children. Clini Radiol, 2015; 70(8):872–880.
13.
SchmiererK, McDowellA, PetrovaN, et al.Quantifying multiple sclerosis pathology in post mortem spinal cord using MRI. NeuroImage, 2018; 182(251–258); doi: 10.1016/j.neuroimage.2018.01.052
14.
BarboneGE, BravinA, MittoneA, et al.High-spatial-resolution three-dimensional imaging of human spinal cord and column anatomy with postmortem x-ray phase-contrast micro-CT. Radiology, 2021; 298(1):135–146; doi: 10.1148/radiol.2020201622
15.
BravinA, CoanP, SuorttiP. X-ray phase-contrast imaging: from pre-clinical applications towards clinics. Phys Medicine Biol, 2013; 58(1):R1–R35; doi: https://doi.org/10.1088/0031-9155/58/1/R1
16.
BukreevaI, MittoneA, BravinA, et al.Virtual unrolling and deciphering of Herculaneum papyri by X-ray phase-contrast tomography. Sci Rep, 2016; 6(1):1–7; doi: https://doi.org/10.1038/srep27227
17.
FratiniM, BukreevaI, CampiG, et al.Simultaneous submicrometric 3D imaging of the micro-vascular network and the neuronal system in a mouse spinal cord. Sci Rep, 2015; 5(8514); doi: https://doi.org/10.1038/srep08514
18.
MassimiL, FratiniM, BukreevaI, et al.Characterization of mouse spinal cord vascular network by means of synchrotron radiation X-ray phase contrast tomography. Physica Medica, 2016; 32(12):1779–1784; doi: https://doi.org/10.1016/j.ejmp.2016.09.015
19.
StrottonMC, BodeyAJ, WanelikK, et al.The spatiotemporal spread of cervical spinal cord contusion injury pathology revealed by 3D in-line phase contrast synchrotron X-ray microtomography. Exp Neurol, 2021; 336(113529); doi: https://doi.org/10.1016/j.expneurol.2020.113529
20.
CaoF, JiangY, WuY, et al.Apolipoprotein E-mimetic COG1410 reduces acute vasogenic edema following traumatic brain injury. J Neurotrauma, 2016; 33(2):175–182.
21.
CaoY, WuT-D, WuH, et al.Synchrotron radiation micro-CT as a novel tool to evaluate the effect of agomir-210 in a rat spinal cord injury model. Brain Res, 2017; 1655(55–65).
22.
HuJ, NiS, CaoY, et al.The angiogenic effect of microRNA-21 targeting TIMP3 through the regulation of MMP2 and MMP9. PLoS One, 2016; 11(2):e0149537.
23.
HuJ-Z, WangX-K, CaoY, et al.Tetramethylpyrazine facilitates functional recovery after spinal cord injury by inhibiting MMP2, MMP9, and vascular endothelial cell apoptosis. Curr Neurovasc Res, 2017; 14(2):110–116.
24.
LiaoS, NiS, CaoY, et al.The 3D characteristics of post-traumatic syringomyelia in a rat model: a propagation-based synchrotron radiation microtomography study. J Synchrotron Radiat, 2017; 24(6):1218–1225; doi: doi:10.1107/S1600577517011201
25.
NiS, CaoY, JiangL, et al.Synchrotron radiation imaging reveals the role of estrogen in promoting angiogenesis after acute spinal cord injury in rats. Spine, 2018; 43(18):1241–1249.
26.
WuT, CaoY, NiS, et al.Morphometric analysis of rat spinal cord angioarchitecture by phase contrast radiography: from 2D to 3D visualization. Spine, 2018; 43(9):E504–E511.
27.
NicaiseC, PutatundaR, HalaTJ, et al.Degeneration of phrenic motor neurons induces long-term diaphragm deficits following mid-cervical spinal contusion in mice. J Neurotrauma, 2012; 29(18):2748–2760; doi: 10.1089/neu.2012.2467
28.
SprimontL, JanssenP, De SwertK, et al.Cystine–glutamate antiporter deletion accelerates motor recovery and improves histological outcomes following spinal cord injury in mice. Sci Rep, 2021; 11(1):12227; doi: 10.1038/s41598-021-91698-y
29.
MittoneA, ManakovI, BrocheL, et al.Characterization of a sCMOS-based high-resolution imaging system. J Synchrotron Radiat, 2017; 24(6):1226–1236; doi: 10.1107/S160057751701222X
30.
BrunF, MassimiL, FratiniM, et al.SYRMEP Tomo Project: a graphical user interface for customizing CT reconstruction workflows. Adv Struct Chemi Imaging, 2017; 3(1):1–9; doi: https://doi.org/10.1186/s40679-016-0036-8
31.
BukreevaI, CampiG, FratiniM, et al.Quantitative 3D investigation of neuronal network in mouse spinal cord model. Sci Rep, 2017; 7(41054); doi: https://doi.org/10.1038/srep41054
32.
FerrucciM, LazzeriG, FlaibaniM, et al.In search for a gold-standard procedure to count motor neurons in the spinal cord. Histol Histopathol, 2018; 33(1021–1046); doi: 10.14670/HH-11-983.
33.
BolteS, CordelièresFP. A guided tour into subcellular colocalization analysis in light microscopy. J Microscopy, 2006; 224(3):213–232; doi: https://doi.org/10.1111/j.1365-2818.2006.01706.x
34.
NicaiseC, FrankDM, HalaTJ, et al.Early phrenic motor neuron loss and transient respiratory abnormalities after unilateral cervical spinal cord contusion. J Neurotrauma, 2013; 30(12):1092–1099; doi: 10.1089/neu.2012.2728
35.
NicaiseC, HalaTJ, FrankDM, et al.Phrenic motor neuron degeneration compromises phrenic axonal circuitry and diaphragm activity in a unilateral cervical contusion model of spinal cord injury. Exp Neurology, 2012; 235(2):539–552; doi: 10.1016/j.expneurol.2012.03.007
36.
BilgenM, DancauseN, Al-HafezB, et al.Manganese-enhanced MRI of rat spinal cord injury. Magn Reson Imaging, 2005; 23(7):829–832; doi: https://doi.org/10.1016/j.mri.2005.06.004
37.
FratiniM, AbdollahzadehA, DiNuzzoM, et al.Multiscale imaging approach for studying the central nervous system: methodology and perspective. Front Neurosci, 2020; 14(72–72); doi: 10.3389/fnins.2020.00072
38.
HuberE, DavidG, ThompsonAJ, et al.Dorsal and ventral horn atrophy is associated with clinical outcome after spinal cord injury. Neurology, 2018; 90(17):e1510–e1522; doi: 10.1212/WNL.0000000000005361
39.
MartinA, De LeenerB, Cohen-AdadJ, et al.Clinically feasible microstructural MRI to quantify cervical spinal cord tissue injury using DTI, MT, and T2*-weighted imaging: assessment of normative data and reliability. Am J Neuroradiol, 2017; 38(6):1257–1265; doi: 10.3174/ajnr.A5163
40.
ScholtesF, TheunissenE, Phan-BaR, et al.Post-mortem assessment of rat spinal cord injury and white matter sparing using inversion recovery-supported proton density magnetic resonance imaging. Spinal Cord, 2011; 49(3):345–351; doi: https://doi.org/10.1038/sc.2010.129
41.
KellerD, EröC, MarkramH. Cell densities in the mouse brain: a systematic review. Front Neuroanat, 2018; 12(83); doi: https://doi.org/10.3389/fnana.2018.00083
42.
RitzelRM, HooverJE. An unbiased stereological estimate of the number of motor neurons in the cervical enlargement of the rat spinal cord. J Penn Acad Sci, 2010; 26–30; doi: https://www.jstor.org/stable/44149711
43.
MoutonPR.Quantitative Anatomy Using Design-Based Stereology. In: Approximate Analytical Methods for Solving Ordinary Differential Equations. CRC Press; 2014.
44.
WestMJ. Stereological methods for estimating the total number of neurons and synapses: issues of precision and bias. Trends Neurosci, 1999; 22(2):51–61.
45.
TatorCH, KoyanagiI. Vascular mechanisms in the pathophysiology of human spinal cord injury. J Neurosurg, 1997; 86(3):483–492; doi: https://doi.org/10.3171/jns.1997.86.3.0483
46.
AdamsKL, GalloV. The diversity and disparity of the glial scar. Nat Neurosci, 2018; 21(1):9–15; doi: https://doi.org/10.1038/s41593-017-0033-9
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
