This study was supported by Guangzhou Science and Technology Project of China (No. 201607010021) and the Nature Science Foundation of JiangXi (No. 20142BAB205065)

The study was approved by the local Medical Ethics Committee in Guangzhou First People&#8217;s Hospital in China. Signed informed consent forms were received from healthy volunteers and participants prior to participation. All of the studies were conducted in accordance with the World Medical Association Declaration of Helsinki.
1. Subject Preparation
<ol>
	<li>Ensure that each participant meets the following criteria for chronic spinal cord compression: a) a history of loss of significant neurological function, b) a positive myelopathy physical examination, and c) MRI evidence of cervical cord compression.&#160; 
	NOTE: The exclusion criteria are a) incapability of providing written consent and b) inability to obtain DTI parameters of artifacts. For controls, inclusion criteria are a) no history of significant back or neck injuries, neurological disorders, or spine surgeries; b) no MRI evidence of cervical cord compression.</li>
	<li>sk each participant to complete and sign a consent form that lists MRI safety guidelines and the imaging protocol. Specifically, patients with chronic spinal cord compression are examined by MRI preoperatively and 1 year postoperatively.</li>
	<li>rovide earplugs for each participant. Place them in a supine position with a head/neck coil enclosing the cervical region, and a landmark at the thyroid cartilage level. Ensure that each participant is in a comfortable position that effectively reduces movement.</li>
</ol>
2. Structural MRI Parameters&#160;
NOTE: Anatomical T1-weighted (T1 W) images, T2-weighted (T2 W) images, and DTI acquired on a 3 Tesla MRI scanner with a 16-channel head coil.
<ol>
	<li>Use fast perturbation gradient echo (FPGR) for localization scanning to obtain axial, sagittal, and coronal position maps.</li>
	<li>Position the sagittal positioning line with the coronal position maps to ensure that the positioning baseline is parallel to the spinal canal (spinal cord); first locate the sagittal plane T2 W, then copy and paste the sagittal T1 W positioning line to the T2 W positioning line.
	<ol>
		<li>Use the following imaging parameters for T1 W and T2 W sagittal imaging: field of view (FOV) = 240 mm x 240 mm, voxel size = 1.0 mm x 0.8 mm x 3.0 mm, slice gap = 0.3 mm, slice thickness = 3 mm, number of excitation (NEX) = 2, fold-over direction = feet/head (FH), and time of echo (TE)/time of repetition (TR) = 10/700 ms (T1 W) and 101/2,500 ms (T2 W). Obtain nine sagittal images covering the entire cervical spinal cord.&#160;</li>
	</ol>
	</li>
	<li>Position the axial positioning line on the sagittal T2 W image and cover the intervertebral disc from C2/3 to C6/7, centering on the anteroposterior diameter of the intervertebral space. Use the following imaging parameters: FOV = 180 mm x 180 mm, voxel size = 0.7 mm x 0.6 mm x 3.0 mm, slice thickness = 3 mm, fold-over direction = anterior/posterior (AP), NEX = 2, and TE/TR = 120/3,000 ms.</li>
	<li>Position the axial positioning line on the sagittal T2 W image, centering on the anteroposterior diameter of the intervertebral space, with 45 slices covering the cervical spinal cord from C1 to C7.
	<ol>
		<li>Obtain DTI via the following sequence: single-shot spin-echo echo-planar imaging (SE-EPI) with 20 orthogonal directions. Non-coplanar diffusion directions with b-value = 800 s/mm2.&#160;</li>
		<li>Use the following imaging parameters: FOV = 230 mm x 230 mm, acquisition matrix = 98 x 98, reconstructed resolution = 1.17 x 1.17, slice thickness = 3 mm, fold-over direction = AP, NEX = 2, EPI factor = 98, and TE/TR = 74/8,300 ms. Provide a time course summarizing the steps in the MRI protocol, as shown in Figure 1.&#160; 
		NOTE: The time course summarizing the MRI and DTI protocol is shown in Figure 1.</li>
	</ol>
	</li>
</ol>
3. Image Postprocessing and Data Measurement Indexes
<ol>
	<li>Automatically convey all scanning images to the Syngo MR B17. Load the T2 W sagittal and axial imaging of the intervertebral space in the filming interface and find the most compressed portion of the cervical spinal cord.</li>
	<li>In the 2:1 viewing interface, load the FA image and click the Position Display: Series tab. Count and record the level of highest compression from the top to the bottom of the location map.</li>
	<li>Click the File tab to select the tensor image, then use the applications toolbar at the top left of the screen to select Neuro 3D(MR) to automatically create ADC and FA colormaps.</li>
	<li>Turn to the level of the highest compression site, and create spherical regions of interest (ROIs) of identical volumes (with a size of 6 mm3) using the Start Evaluation Mode tab. The ROIs must be selected, including the inner spinal cord to exclude the partial volume effects of cerebrospinal fluid (CSF).</li>
	<li>Calculate and display the FA and ADC values at the bottom right of the screen automatically. Display the E1, E2, and E3 values by clicking the Diffusion toolbar and choosing them. 
	NOTE: All measurements were performed by two radiologists blinded to the patients&#8217; clinical details. The final results were determined as the average of the two.</li>
	<li>Perform image processing of the DTI datasets using a Syngo MR B17 Advantage Workstation, following the steps in Figure 2.</li>
</ol>

<ol>
	<li>Sun, G. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+progressive+compression+model+of+thoracic+spinal+cord+injury+in+mice:+function+assessment+and+pathological+changes+in+spinal+cord.">A progressive compression model of thoracic spinal cord injury in mice: function assessment and pathological changes in spinal cord.</a> Neural Regeneration Research. 12 (8), 1365-1374 (2017).</li><li>Watanabe, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurological+Recovery+after+Posterior+Spinal+Surgery+in+Patients+with+Metastatic+Epidural+Spinal+Cord+Compression.">Neurological Recovery after Posterior Spinal Surgery in Patients with Metastatic Epidural Spinal Cord Compression.</a> Acta Medica Okayama. 70 (6), 449 (2016).</li><li>Tatsui, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+Laser+Interstitial+Thermal+Therapy:+A+Novel+Alternative+to+Surgery+for+Metastatic+Epidural+Spinal+Cord+Compression.">Spinal Laser Interstitial Thermal Therapy: A Novel Alternative to Surgery for Metastatic Epidural Spinal Cord Compression.</a> Neurosurgery. 79 Suppl 1 (suppl_1), S73 (2016).</li><li>Zheng, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+Diffusion+Tensor+Imaging+Cutoff+Value+to+Evaluate+the+Severity+and+Postoperative+Neurologic+Recovery+of+Cervical+Spondylotic+Myelopathy.">Application of Diffusion Tensor Imaging Cutoff Value to Evaluate the Severity and Postoperative Neurologic Recovery of Cervical Spondylotic Myelopathy.</a> World Neurosurgery. 118, e849-e855 (2018).</li><li>Ellingson, B. M., Salamon, N., Holly, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+techniques+in+spinal+cord+injury.">Imaging techniques in spinal cord injury.</a> World Neurosurgery. 82 (6), 1351-1358 (2014).</li><li>Zhao, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+tensor+imaging+of+spinal+cord+parenchyma+lesion+in+rat+with+chronic+spinal+cord+injury.">Diffusion tensor imaging of spinal cord parenchyma lesion in rat with chronic spinal cord injury.</a> Magnetic Resonance Imaging. 47, 25-32 (2018).</li><li>Mohanty, C., Massicotte, E. M., Fehlings, M. G., Shamji, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Association+of+Preoperative+Cervical+Spine+Alignment+with+Spinal+Cord+Magnetic+Resonance+Imaging+Hyperintensity+and+Myelopathy+Severity:+Analysis+of+a+Series+of+124+Cases.">The Association of Preoperative Cervical Spine Alignment with Spinal Cord Magnetic Resonance Imaging Hyperintensity and Myelopathy Severity: Analysis of a Series of 124 Cases.</a> Spine. 40 (1), 11-16 (2015).</li><li>Tetreault, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+review+of+magnetic+resonance+imaging+characteristics+that+affect+treatment+decision+making+and+predict+clinical+outcome+in+patients+with+cervical+spondylotic+myelopathy.">Systematic review of magnetic resonance imaging characteristics that affect treatment decision making and predict clinical outcome in patients with cervical spondylotic myelopathy.</a> Spine. 38 (22 Suppl 1), S89 (2013).</li><li>Nouri, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Role of Magnetic Resonance Imaging in Predicting Surgical Outcome in Patients with Degenerative Cervical Myelopathy. , (2015).</li><li>Chen, C. J., Lyu, R. K., Lee, S. T., Wong, Y. C., Wang, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intramedullary+high+signal+intensity+on+T2-weighted+MR+images+in+cervical+spondylotic+myelopathy:+prediction+of+prognosis+with+type+of+intensity.">Intramedullary high signal intensity on T2-weighted MR images in cervical spondylotic myelopathy: prediction of prognosis with type of intensity.</a> Radiology. 221 (3), 789-794 (2001).</li><li>Vedantam, A., Jonathan, A., Rajshekhar, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+magnetic+resonance+imaging+signal+changes+and+outcome+prediction+after+surgery+for+cervical+spondylotic+myelopathy.">Association of magnetic resonance imaging signal changes and outcome prediction after surgery for cervical spondylotic myelopathy.</a> Journal of Neurosurgery Spine. 15 (6), 660 (2011).</li><li>Vedantam, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+tensor+imaging+of+the+spinal+cord:+insights+from+animal+and+human+studies.">Diffusion tensor imaging of the spinal cord: insights from animal and human studies.</a> Neurosurgery. 74 (1), 1-8 (2014).</li><li>Bazley, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DTI+for+assessing+axonal+integrity+after+contusive+spinal+cord+injury+and+transplantation+of+oligodendrocyte+progenitor+cells.">DTI for assessing axonal integrity after contusive spinal cord injury and transplantation of oligodendrocyte progenitor cells.</a> Conference Proceedings: Annual International Conference of the IEEE Engineering in Medicine and Biology Society. 2012 (4), 82-85 (2012).</li><li>Lewis, M., Yap, P. T., Mccullough, S., Olby, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+lesion+severity+characterized+by+diffusion+tensor+imaging+and+motor+function+in+chronic+canine+spinal+cord+injury.">The relationship between lesion severity characterized by diffusion tensor imaging and motor function in chronic canine spinal cord injury.</a> Journal of Neurotrauma. 35 (3), (2018).</li><li>Hagmann, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+diffusion+MR+imaging+techniques:+from+scalar+diffusion-weighted+imaging+to+diffusion+tensor+imaging+and+beyond.">Understanding diffusion MR imaging techniques: from scalar diffusion-weighted imaging to diffusion tensor imaging and beyond.</a> Radiographics. 26 Suppl 1 (suppl_1), S205 (2006).</li><li>Zheng, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+of+diffusion+tensor+imaging+metrics+in+the+chronic+spinal+cord+compression+rat+model.">Time course of diffusion tensor imaging metrics in the chronic spinal cord compression rat model.</a> Acta Radiologica. , 284185118795335 (2018).</li><li>Jones, J. G., Cen, S. Y., Lebel, R. M., Hsieh, P. C., Law, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+Tensor+Imaging+Correlates+with+the+Clinical+Assessment+of+Disease+Severity+in+Cervical+Spondylotic+Myelopathy+and+Predicts+Outcome+following+Surgery.">Diffusion Tensor Imaging Correlates with the Clinical Assessment of Disease Severity in Cervical Spondylotic Myelopathy and Predicts Outcome following Surgery.</a> American Journal of Neuroradiology. 34 (2), 471-478 (2013).</li><li>Kerkovsk&#253;, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+diffusion+tensor+imaging+in+patients+with+cervical+spondylotic+spinal+cord+compression:+correlations+between+clinical+and+electrophysiological+findings.">Magnetic resonance diffusion tensor imaging in patients with cervical spondylotic spinal cord compression: correlations between clinical and electrophysiological findings.</a> Spine. 37 (1), 48-56 (2012).</li><li>Zheng, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+Diffusion+Tensor+Imaging+Cutoff+Value+to+Evaluate+the+Severity+and+Postoperative+Neurologic+Recovery+of+Cervical+Spondylotic+Myelopathy.">Application of Diffusion Tensor Imaging Cutoff Value to Evaluate the Severity and Postoperative Neurologic Recovery of Cervical Spondylotic Myelopathy.</a> World Neurosurgery. 118, e849-e855 (2018).</li><li>Thurnher, M. M., Law, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion-weighted+imaging,+diffusion-tensor+imaging,+and+fiber+tractography+of+the+spinal+cord.">Diffusion-weighted imaging, diffusion-tensor imaging, and fiber tractography of the spinal cord.</a> Magnetic Resonance Imaging Clinics of North America. 17 (2), 225-244 (2009).</li><li>Cadotte, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+Cord+Segmentation+by+One+Dimensional+Normalized+Template+Matching:+A+Novel,+Quantitative+Technique+to+Analyze+Advanced+Magnetic+Resonance+Imaging+Data.">Spinal Cord Segmentation by One Dimensional Normalized Template Matching: A Novel, Quantitative Technique to Analyze Advanced Magnetic Resonance Imaging Data.</a> PLOS ONE. 10 (10), e0139323 (2015).</li></ol>

The authors have nothing to disclose.

Conventional MRI is usually utilized to assess the prognosis of patients with various spine conditions. However, this imaging modality provides macroscopic anatomic detail rather than microstructure evaluation14, which limits the prediction of neurological function. Furthermore, traditional MRI may underestimate the severity and extent of spinal cord damage. The emergence of DTI can help surgeons to evaluate spinal cord function more accurately by providing quantitative information on water molecu...

Chronic spinal cord compression is the most common cause of spinal cord impairment1. This condition can be due to posterior longitudinal ligament ossification, hematoma, cervical disc herniation, vertebral degeneration, or intraspinal tumors2,3. Chronic spinal cord compression can lead to various degrees of functional deficits; however, there are clinical cases with serious spinal cord compression without any neurological symptoms and signs, as well as patients with mild spinal cord compression but serious neurological deficits4. Under these circumstances, sensitive imaging is essential to evaluate compression severity and identify the range of damage.
Conventional MRI plays a significant role in elucidating spinal cord anatomy. This technique is usually utilized to evaluate the compression degree because of its sensitivity to soft tissues5. Many parameters can be measured from MRI, such as MR signal intensity, cord morphology, and spinal canal area. However, MRI has some limitations and only provides qualitative information rather than quantitative results6. Patients with chronic spinal cord compression often have abnormal signal changes of MRI intensity. However, discrepancies between clinical symptoms and MRI intensity changes make it hard to diagnose a functional condition based solely on MRI characteristics7. Previous studies highlight this controversy in terms of the prognostic value of MRI T2 hyperintensity in the spinal cord8. Two groups reported that T2 hyperintensity of the spinal cord is a poor prognostic parameter after surgery for chronic spinal cord compression8,9. In contrast, some authors found no significant association between T2 signal changes and prognosis8,9. Chen et al. and Vedantam et al. divided MRI T2 hyperintensities into two categories corresponding to different prognostic outcomes10,11. Type 1 showed faint, fuzzy, indistinct borders, and this category demonstrated reversible histologic changes. Type 2 images presented intense, well-defined borders, which corresponded to irreversible pathologic damage. Conventional T1/T2 MRI techniques do not provide adequate information to identify these two categories and evaluate patient prognosis. By contrast, DTI, a more sophisticated imaging technique, may help obtain more specific prognostic information by quantitatively detecting microstructural changes in tissues via water molecule diffusion.
In recent years, DTI has garnered increasing attention due to its ability to describe spinal cord microarchitecture. DTI can measure the direction and magnitude of water molecule diffusion in tissues. DTI parameters can quantitatively evaluate neural damage in patients with chronic spinal cord compression. FA and the ADC are the most commonly applied parameters during spinal cord evaluation. The FA value reveals the degree of anisotropy to orientate surrounding axonal fibers and describe anatomic boundaries12,13. The ADC value provides information on the characteristics of molecular motion in many directions in a three-dimensional space and reveals the mean of diffusivities along the three principal axes6,12. Changes in these parameters are associated with microstructural alterations that influence water molecule diffusion. Therefore, surgeons can utilize/measure DTI parameters to identify spinal cord pathology. The present study provides DTI methods and processes that provide more detailed prognostic information to treat patients with chronic spinal cord compression.

<table border="0" cellpadding="0" cellspacing="0" style="width:849px;" width="849">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">3-Tesla MRI scanner</td>
			<td style="width:123px;">Siemens</td>
			<td align="right" style="width:344px;">40708</td>
			<td style="width:167px;">Software: NUMARIS/4</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Syngo MR B17</td>
			<td>Siemens</td>
			<td align="right">40708</td>
			<td>Software: NUMARIS/4</td>
		</tr>
	</tbody>
</table>

diffusion tensor magnetic resonance imaging in chronic spinal cord compression

Chronic spinal cord compression is the most common cause of spinal cord impairment in patients with nontraumatic spinal cord damage. Conventional magnetic resonance imaging (MRI) plays an important role in both confirming the diagnosis and evaluating the degree of compression. However, the anatomical detail provided by conventional MRI is not sufficient to accurately estimate neuronal damage and/or assess the possibility of neuronal recovery in chronic spinal cord compression patients. In contrast, diffusion tensor imaging (DTI) can provide quantitative results according to the detection of water molecule diffusion in tissues. In the present study, we develop a methodological framework to illustrate the application of DTI in chronic spinal cord compression disease. DTI fractional anisotropy (FA), apparent diffusion coefficients (ADCs), and eigenvector values are useful for visualizing microstructural pathological changes in the spinal cord. Decreased FA and increases in ADCs and eigenvector values were observed in chronic spinal cord compression patients compared to healthy controls. DTI could help surgeons understand spinal cord injury severity and provide important information regarding prognosis and neural functional recovery. In conclusion, this protocol provides a sensitive, detailed, and noninvasive tool to evaluate spinal cord compression.

This is a summary of results obtained from healthy volunteers and patients with cervical spondylotic myelopathy. The protocol enabled the physician to view DTI maps. This technology could serve as an objective measure to measure functional status in myelopathic conditions. DTI maps of healthy volunteers are shown in Figure 3. The DTI parameters of healthy volunteers were as follows: FA = 0.661; ADC = 1.006 x 10-3 mm2/s; E1 = 1.893 x 10-3 mm2/s; E2 ...

Watch this Scientific Journal Video about Diffusion Tensor Magnetic Resonance Imaging in Chronic Spinal Cord Compression at JoVE.com

Diffusion Tensor Magnetic Resonance Imaging in Chronic Spinal Cord Compression

Diffusion tenseur imagerie par résonance magnétique dans la compression de la moelle épinière chronique

Here, we present a protocol for the application of diffusion tensor imaging parameters to evaluate spinal cord compression.

Chronic spinal cord compression is the most common cause of spinal cord impairment in patients with nontraumatic spinal cord damage. Conventional magnetic ...

diffusion-tensor-magnetic-resonance-imaging-chronic-spinal-cord

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JoVE Journal

Neuroscience

8.8K vues.  Guangzhou Medical University. Le détail anatomique de microstructure de moelle épinière fourni par MRI conventionnel n’estime pas exactement les dommages neuronaux réels ou le potentiel neuronal de rétablissement dans les patients chroniques de compression de moelle épinière. L’imagerie par tenseurs de diffusion, ou DTI, peut nous aider à obtenir des informations pronostiques plus spécifiques en détectant le changement de microstructure quantité dans les tissus par la diffusion de molécules d’eau. La com...

Vidéo: Diffusion Tensor Magnetic Resonance Imaging in Chronic Spinal Cord Compression

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Dissection et la culture de neurones de la moelle épinière commissurales embryonnaires

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

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Neurosciences

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

Multiple-Mouse Imaging neuroanatomiques par résonance magnétique

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

Dans l&#39;imagerie in vivo de la moelle épinière de souris utilisant microscopie à deux photons

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

Haute résolution magnétique fonctionnelle Méthodes d&#39;imagerie par résonance mésencéphale humain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

Spinal Cord Transection dans le poisson zèbre larvaire

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has been heavily studied. While many imaging studies treat the hippocampus as a single unitary neuroanatomical structure, it is, in fact, composed of several subfields that have a complex three-dimensional geometry. As such, it is known that these subfields perform specialized functions and are differentially affected through the course of different disease states. Magnetic resonance (MR) imaging can be used as a powerful tool to interrogate the morphology of the hippocampus and its subfields. Many groups use advanced imaging software and hardware (&#62;3T) to image the subfields; however this type of technology may not be readily available in most research and clinical imaging centers. To address this need, this manuscript provides a detailed step-by-step protocol for segmenting the full anterior-posterior length of the hippocampus and its subfields: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus (DG), strata radiatum/lacunosum/moleculare (SR/SL/SM), and subiculum. This protocol has been applied to five subjects (3F, 2M; age 29-57, avg. 37). Protocol reliability is assessed by resegmenting either the right or left hippocampus of each subject and computing the overlap using the Dice&#39;s kappa metric. Mean Dice&#39;s kappa (range) across the five subjects are: whole hippocampus, 0.91 (0.90-0.92); CA1, 0.78 (0.77-0.79); CA2/CA3, 0.64 (0.56-0.73); CA4/dentate gyrus, 0.83 (0.81-0.85); strata radiatum/lacunosum/moleculare, 0.71 (0.68-0.73); and subiculum 0.75 (0.72-0.78). The segmentation protocol presented here provides other laboratories with a reliable method to study the hippocampus and hippocampal subfields in vivo using commonly available MR tools.

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

Haute résolution<em&gt; In Vivo</em&gt; Protocole de segmentation manuelle pour les sous-zones de l&#39;hippocampe humain Utilisation 3T imagerie par résonance magnétique

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

Une procédure pour l&#39;implantation d&#39;une chambre pour la colonne vertébrale longitudinale<em&gt; In Vivo</em&gt; Imagerie du cordon spinal de souris

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Diffusion Imaging in the Rat Cervical Spinal Cord

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a diagnostic and prognostic tool in cases of disease or injury. Diffusion weighted imaging (DWI), is sensitive to the thermal motion of water molecules and allows for inferences of tissue microstructure. This report describes a protocol to acquire and analyze DWI of the rat cervical spinal cord on a small-bore animal system. It demonstrates an imaging setup for the live anesthetized animal and recommends a DWI acquisition protocol for high-quality imaging, which includes stabilization of the cord and control of respiratory motion. Measurements with diffusion weighting along different directions and magnitudes (b-values) are used. Finally, several mathematical models of the resulting signal are used to derive maps of the diffusion processes within the spinal cord tissue that provide insight into the normal cord and can be used to monitor injury or disease processes noninvasively.

The goal of this protocol is to obtain high-quality diffusion weighted magnetic resonance imaging (DWI) of the rat spinal cord for noninvasive characterization of tissue microstructure. This protocol describes optimizations of the MRI sequence, radiofrequency coil, and analysis methods to enable DWI images free from artifacts.

Diffusion d&#39;imagerie dans la moelle épinière cervicale Rat

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

Deux photons Imaging of Cellular Dynamics dans la moelle épinière de souris

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Experimental Strategies to Bridge Large Tissue Gaps in the Injured Spinal Cord after Acute and Chronic Lesion

After a spinal cord injury (SCI) a scar forms in the lesion core which hinders axonal regeneration. Bridging the site of injury after an insult to the spinal cord, tumor resections, or tissue defects resulting from traumatic accidents can aid in facilitating general tissue repair as well as regenerative growth of nerve fibers into and beyond the affected area. Two experimental treatment strategies are presented: (1) implantation of a novel microconnector device into an acutely and completely transected thoracic rat spinal cord to readapt severed spinal cord tissue stumps, and (2) polyethylene glycol filling of the SCI site in chronically lesioned rats after scar resection. The chronic spinal cord lesion in this model is a complete spinal cord transection which was inflicted 5 weeks before treatment. Both methods have recently achieved very promising outcomes and promoted axonal regrowth, beneficial cellular invasion and functional improvements in rodent models of spinal cord injury.
The mechanical microconnector system (mMS) is a multi-channel system composed of polymethylmethacrylate (PMMA) with an outlet tubing system to apply negative pressure to the mMS lumen thus pulling the spinal cord stumps into the honeycomb-structured holes. After its implantation into the 1 mm tissue gap the tissue is sucked into the device. Furthermore, the inner walls of the mMS are microstructured for better tissue adhesion.
In the case of the chronic spinal cord injury approach, spinal cord tissue - including the scar-filled lesion area - is resected over an area of 4 mm in length. After the microsurgical scar resection the resulting cavity is filled with polyethylene glycol (PEG 600) which was found to provide an excellent substratum for cellular invasion, revascularization, axonal regeneration and even compact remyelination in vivo.

Severe spinal cord injuries often result in tissue defects. Two possibilities are described to successfully bridge such gaps to promote tissue adaptation, regenerative responses and functional improvement in rats via implantation of a mechanical microconnector system after acute injury and five weeks after complete spinal cord transection.

Stratégies expérimentales pour combler les lacunes de tissus grandes dans la moelle épinière lésée après aiguë et chronique Lésion

After a spinal cord injury (SCI) a scar forms in the lesion core which hinders axonal regeneration. Bridging the site of injury after an insult to the ...