This study was supported by the National Key Research and Development Project of Stem Cell and Transformation Research (2019YFA0112100) and the State Key Program of National Natural Science of China (81930070).

Experiments were approved by the Laboratory Animal Ethical and Welfare Committee of Shandong University Cheeloo College of Medicine (approval number: 22021). They were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publications No. 85-23, revised 1996). All mice used in this study were 9-10 week old female C57BL/6J mice purchased from Jinan Pengyue Experimental Animal Company (Jinan, China). A total of 9 mice involved in this study were equally randomized to the control, mild, and severe groups. At 7, 28, and 70 days postinjury, one mouse from each group was sacrificed.
1. C6 laminectomy and spinal cord exposure
NOTE: The exposure was performed under a microscope. Bleeding can be avoided by paying attention to two aspects: (i) All blood vessels should be avoided. (ii) Muscles need to be separated at the origin and termination points of the muscle.
<ol>
	<li>Prepare surgical instruments and SCICP. 
	NOTE: The structure of SCICP has been reported in previous study11. The difference with respect to the previous study is that the present protocol accomplishes spinal cord injury by compression. Two different weights (10.4 g and 20.8 g) of this platform can produce compression of 5 g/mm2 and 10 g/mm2, respectively (Figure 1). The spinal cord exposure and compression steps are shown in Figure 2.</li>
	<li>Administer isoflurane to the mouse by inhalation using a nose cone (induction: 3%-5%, maintenance: 1.5%-2%).</li>
	<li>After the anesthesia takes effect, explore a small bulge at the midline behind the neck of mice, which is the spinous process of the second thoracic vertebra (T2).</li>
	<li>Shave the hair around&#160;this bulge. Disinfect the skin with three alternating applications of an iodophor solution followed by skin antiseptics 75% ethanol.</li>
	<li>Position the mouse prone on the operation table. Apply eye ointment to protect the eyes.</li>
	<li>Lay a 3-4 mm-thick pad under the chest to allow an arching cervical spine curve, facilitating exposure of the inter-lamina space and an unobstructed airway during operation. Inject buprenorphine as pre-operative analgesia (0.05-0.1&#160;mg/kg, SQ).</li>
	<li>Make a 1-1.5 cm longitudinal incision with a sterile scalpel centered on the 2nd thoracic vertebra spinous process to expose the fascial layer (Figure 2A).</li>
	<li>Remove a portion of the adipose tissue above T2 with sterile micro scissors to find the T2 spinous process.</li>
	<li>Separate the bilateral trapezius and rhomboid muscles from C5-T2 along the midline with&#160;micro scissors (Figure 2B).</li>
	<li>Separate the muscles on the lamina of C5-T2 vertebrae with micro scissors, and retract the muscle layer to the sides with sterile micro-retractors (Figure 2C).</li>
	<li>Cut the multifidus and cervical spinal muscles on the surface of the vertebrae.</li>
	<li>Locate T2 according to the highest point of the spinous processes. Probe the spinous processes successively toward the rostral end from T2 to locate C6 (Figure 3).</li>
	<li>Lift the C6 lamina with forceps, cut off the lamina, and the spinal cord is exposed (Figure 2D).</li>
</ol>
2. Cervical spinal cord compression injury
<ol>
	<li>Clamp the C6-7 facet joints with the vertebral stabilizator, and lock it (Figure 2E).</li>
	<li>Aim the sterile weight tip at the exposed spinal cord, and ensure the flat bottom of the tip is positioned parallel to the dorsal surface of the spinal cord (Figure 2F).</li>
	<li>Adjust the sleeve to make the weight compress the spinal cord. Stop adjusting when the weight keeps a constant relative position with the spinal cord (Figure 2G). 
	NOTE: Do not make this process too violent or quick in case the weight exerts contusion force on the spinal cord.</li>
	<li>Remove the weight and the vertebral stabilizator after a 5 min compression.</li>
	<li>Observe the color alterations of the spinal cord after compression under the microscope (Figure 2H).</li>
	<li>Rinse with sterile PBS and use suction to clean the operation site.</li>
	<li>Suture the muscles and skin in layers using polypropylene nonabsorbable suture (size: 6-0).</li>
	<li>Disinfect the surgical area, place the mouse on a warm pad until the mouse restores full consciousness, and then return the mouse to the mouse cage.</li>
	<li>Inject buprenorphine for analgesia (0.05-0.1&#160;mg/kg, SQ) every 8-12 h for 3 days.</li>
</ol>
3. Histological analysis
<ol>
	<li>Anesthetize the mouse by intraperitoneal injection of 1.25% tribromoethanol (0.02 mL/g body weight) on days 7, 28, or&#160;70 after injury. Transcardially infuse the mouse with 60 mL of phosphate-buffered saline (PBS) and 20 mL of 4% paraformaldehyde11.</li>
	<li>Transect the spinal cord at 0.5 cm from the lesion center from both sides with micro scissors, and preserve the 1 cm-long section.</li>
	<li>Immerse the preserved spinal cord section in 30% sucrose at 4 &#730;C for 48 h.</li>
	<li>Embed the tissues with OCT, slice the tissues into 6 &#181;m thick sections with a cryotome, and collect the sections on a glass slide.</li>
	<li>Hematoxylin and eosin staining
	<ol>
		<li>Rinse the 6 &#181;m sections with 1x PBS for 5 min 3 times to remove residual OCT.</li>
		<li>Immerse the sections in hematoxylin for 90 s. Wash the sections under running water for 3 min.</li>
		<li>Immerse the sections in eosin for 4 min. Soak in 95% alcohol for 30 s to remove excess eosin.</li>
		<li>Finally, dehydrate the slides with alcohol (95% alcohol and 100% alcohol twice, successively) for 30 s and put the slides in a xylene bath for clearing for 2 min. Then, seal the sections with a cover glass and resin gel.</li>
	</ol>
	</li>
	<li>Prussian blue staining
	<ol>
		<li>Submerge the slides for 20 min in an equal mixture of potassium ferrocyanide (10%) and hydrochloric acid (10%).</li>
		<li>Rinse 3 times with distilled water, and counterstain for 5 min with Nuclear Fast Red.</li>
		<li>Rinse three times with distilled water, followed by one rinse with 95% alcohol and two rinses with 100% alcohol for 5 min.</li>
		<li>Clear the sections in xylene twice for 3 min each and then seal with resin gel12.</li>
	</ol>
	</li>
	<li>Immunofluorescence staining
	<ol>
		<li>Incubate the slides with the following primary antibodies for 1 h at 37 &#176;C: rabbit anti-Ionized calcium-binding adapter molecule 1 (Iba-1) (1:500), which was up-regulated in microglia after nerve injury; mouse anti-glial fibrillary acidic protein (GFAP) (1:300), which is expressed in astrocytes in the central nervous system; rabbit anti-neurofilament-200 (NF-200) (1:2000), which is expressed in neurofilament.</li>
		<li>Incubate with secondary antibodies for 1 h at room temperature (RT): Alexa Fluor488 goat anti-mouse and Alexa Fluor594 goat anti-rabbit (1:1,000).</li>
		<li>Snap photographs and further analyze with a fluorescence microscope13.</li>
	</ol>
	</li>
</ol>
4. Magnetic resonance imaging
<ol>
	<li>Anesthetize the mouse at 7 days post-injury with Isoflurane anesthesia (1%-2% isoflurane, 20%-30% O2) administered through a mini mask.</li>
	<li>Scan the cervical spinal cord in sagittal orientation. Use the following settings for MRI imaging: Spin-echo (SE) sequence in multislice and interleaved fashion with TR/TE = 2500/12 ms, acquisition matrix =256 x 128 matrix over the field of view (FOV) = 12 x 8 mm2, slice thickness = 1 mm, and the number of excitations (NEX) = 2. 
	NOTE: Keep the mouse&#39;s respiration rate at 10-15/min during scanning to eliminate respiration-related image artifacts14.</li>
</ol>

<ol>
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The authors have nothing to disclose.

Of the numerous types of spinal cord injury, CCS is one of the most potentially treatable types of injury3,4. Due to the lack of laboratory research models, research on CCS from the 1950s focused on clinical studies and cadaveric dissection investigations3,16,17. The present study shows using compatible tools and minimally invasive procedures to establish mice&#39;s CCS model. From a technical perspective, this platform has strong operability and good reproducibility. Given that experiment results demonstrate the validity, our technique to establish the model closest to the standard previous studies have defined for CCS4.
Previous studies of compression injury have mainly employed aneurism clips, balloons, and calibrated forceps9,10,18. Moreover, most injuries occurred at the level of the thoracic spinal cord18. The spinal cord at the C6 level was chosen as the injured segment in this study to investigate the characteristics of CCS. It is worth the attention that the survival rate of the CCS model is also an essential factor in ensuring experimental consistency. The present study reports causing bilateral compression injury to the mouse cervical spinal cord, while high-level spinal cord traumatic injury, especially bilateral injury, could be fatal for experiment animals if too serious. According to El-Bohy, the C4/5 spinal cord is more likely to affect the descending bulbospinal tract and respiratory-related motoneurons, which leads experiment animals to respiratory depression and death18,19,20,21,22,23., In this study, mice with different degrees of compression on the C6 cervical spinal cord have significantly differentiated injury characteristics suggested by histological tests. Although there were significant behavioral and histological differences in the mouse cervical spinal cord clamping model reported by Forgione, disruption of the pedicles, articular processes, laminae, and even nerve roots were required in order to clamp the spinal cord with the modified clamps, which had a significant influence on the stability of the cervical structures24. Another study of cervical injuries reported using the transverse process as a fixation site5. Even though the articular processes were prevented from damage, over-muscular tissue breakdown could likewise lead to an impact on the stability of the spinal cord. In the present study, only the 6th cervical lamina was resected to maintain the stability of the cervical spinal cord, with the adjoining articular joints preserved and excessive muscular damage avoided. At the same time, compression from above the spinal cord prevents damage to the nerve roots.
The HE results suggest that the area of damage to the cervical spinal cord of the mice in each group was mainly in the gray matter near the central cord, which characterized CCS, with significant differences in the scope of injury between the different groups. Notably, the pathological sections we displayed may have alleviated injury manifestation because the specimens were collected at a few days post-injury. Immunofluorescence (NF-200) showed less damage to the neural tracts in the white matter region of the spinal cord, which also confirmed that the damage in CCS was mainly concentrated around the central cord. Immunofluorescence result was compounded by previous histological results of pathology. Previous studies have shown that CCS leads to edema near the central cord, leading to hematoma and, ultimately, dysfunction in the medial portion of the lateral corticospinal tract3. Hemorrhage has been reported as a typical component of CCS but is rarely seen in subsequent imaging and autopsy studies17. In this study, HE results at 7 days post-injury suggested signs of tissue edema in all groups; however, no residual red blood cells were found in the injury area. Therefore, Prussian blue was used to examine the injury area for hemorrhage, and the results corresponded with hemosiderosis observed in the injury area of the severe injury group at 7 days post-injury, whereas the mild group did not. MRI T2 images showed that both mild and severe injuries had low signal areas in the damaged area of the injury at 7 days post-injury, indicating the deposition of reticulocyte lysate here. These results provide circumstantial evidence that the discrepancy between the previously reported findings is probably due to the MRI test being potentially more sensitive than the histological test14, in addition to the severity of the injury, which may also influence the amount of hemorrhage in the area of injury. GFAP was also expressed extensively in the damaged area. At the same time, Iba-1 expression was also seen in intact areas, suggesting the persistence of an inflammatory response, consistent with the MRI results, where a ring of hyperintense signal around the hypointense signal area in the lesion suggests the presence of an inflammatory response. Ultimately, based on the results of the present study, the area of injury in the model was focused on the gray matter around the central cord, which is generally consistent with the descriptions previously reported13. Unfortunately, we did not perform MRI repetitively in every experiment animal to show how the injury site dynamically changes with time. Future researchers can include this in their work for better investigation into CCS. Also, Immunolabeling with neuronal markers like NeuN, which define the gray matter can be included in the study.
In conclusion, the characteristics of the findings on pathology and MRI scans have close similarities with those described for CCS in previous studies4. The present protocol feasibly modeling CCS enables further research into and understanding of CCS.

Recent years have witnessed a constantly rising incidence of spinal cord injury (SCI), with more injuries in older people from less violent tauma1. These injuries more frequently involve the cervical spine and more often lead to an incomplete neurologic dysfunction2.
In the twenty-first century, CCS is the most prevalent type of incomplete SCI, accounting for more than half of all SCI. Compared with conventional incomplete SCI, CCS is characterized by disproportionately more impairment of the upper than lower extremities3. It is characterized by predominantly upper extremity weakness with less significant sensory and bladder dysfunction. CCS is thought to be caused by post-traumatic central region hemorrhage and edema or, as recently proposed, by Wallerian degeneration from compression of the spinal cord in spinal canal stenosis. The management of CCS lacks high-level evidence to guide, which requires a comprehensive understanding of its pathophysiology4. However, models of CCS have not been reported. Suitable animal models are essential for the understanding of pathophysiology, which can provide a research base for clinical and preclinical studies5,6,7,8,9,10.
In this study, a CCS model in mice is established with a spinal cord injury coaxial platform (SCICP) and a minimally invasive operation plan, which allows for further research into and understanding of CCS. The model is proved valid in the course of the research process by histological, magnetic resonance imaging (MRI), and immunofluorescence analysis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4% fixative solution</td>
			<td>Solarbio</td>
			<td>P1110</td>
			<td>4%</td>
		</tr>
		<tr>
			<td>Anti-Neurofilament heavy polypeptide antibody</td>
			<td>Abcam</td>
			<td>ab8135</td>
			<td>Dilution ratio (1:2000)</td>
		</tr>
		<tr>
			<td>Eosin Staining Solution (water soluble)</td>
			<td>Biosharp</td>
			<td>BL727B</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fuyu Reagent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>KEYENCE</td>
			<td>BZ-X800</td>
			<td></td>
		</tr>
		<tr>
			<td>Frozen Slicer</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GFAP (GA5) Mouse mAb&#160;</td>
			<td>Cell Signaling TECHNOLOGY</td>
			<td>#3670</td>
			<td>Dilution ratio (1:600)</td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>A32723TR</td>
			<td>Dilution ratio (1:1000)</td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 594</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>A32740</td>
			<td>Dilution ratio (1:1000)</td>
		</tr>
		<tr>
			<td>Hematoxylin Staining Solution</td>
			<td>Biosharp</td>
			<td>BL702A</td>
			<td></td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>Jinan Pengyue Experimental AnimalCompany&#160;</td>
			<td>C57BL/6J&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsurgery apparatus&#160;</td>
			<td>Shandong ULT Biotechnology Co., Ltd</td>
			<td>All the surgey instruments are custom-made</td>
			<td>Ophthalmic scissors, micro mosquito forceps, microsurgery forceps, micro scissors</td>
		</tr>
		<tr>
			<td>Normal sheep serum for blocking (working solution)</td>
			<td>Zhong Shan Jin Qiao</td>
			<td>ZLI-9022</td>
			<td>working solution</td>
		</tr>
		<tr>
			<td>O.C.T. Compound</td>
			<td>SAKURA</td>
			<td>4583</td>
			<td></td>
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			<td>Solarbio</td>
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			<td>RWD Laboratory inhalation anesthetic station</td>
			<td>RWD Life Science Co., Ltd</td>
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			<td>Small animal in vivo microCT imaging system</td>
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			<td>Spinal cord injury coaxial platform</td>
			<td>Shandong ULT Biotechnology Co., Ltd</td>
			<td>Custom-made(Feng&#39;s standard)</td>
			<td>https://shop43957633.m.youzan.com/wscgoods/detail/367x5ovgn69q18g?banner_id=f.81386274~goods.7~ 
			1~b0yRFKOq&#38;alg_id= 
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			<td>Surgery microscope&#160;</td>
			<td>Zumax&#160;Medical Co., Ltd.</td>
			<td>zumax, OMS2355</td>
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			<td>Tris Buffered Saline+Tween (TBST)</td>
			<td>Solarbio</td>
			<td>T1082</td>
			<td>Dilution ratio (1:19)</td>
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			<td>Xylene</td>
			<td>Fuyu Reagent</td>
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establishment of central cord syndrome model in c57bl/6j mouse

Animal models of central cord syndrome (CCS) could substantially benefit preclinical research. Identifiable anatomical pathways can give minimally invasive exposure approaches and reduce extra injury to experimental animals during operation, enabling the maintenance of consistent and stable anatomical morphology during experiments to minimize behavioral and histological differences between individuals to improve the reproducibility of experiments. In this study, the C6 level spinal cord was exposed using a spinal cord injury coaxial platform (SCICP) and combination with a minimally invasive technique. With the assistance of a vertebral stabilizator, we fixed the vertebrae and compressed the spinal cords of C57BL/6J mice with 5 g/mm2 and 10 g/mm2 weights with SCICP to induce different degrees of C6 spinal cord injury. In line with the previous description of CCS, the results reveal that the lesion in this model is concentrated in the gray matter around the central cord, enabling further research into CCS. Finally, histological results are provided as a reference for the readers.

The sagittal HE section suggests that even though the damaged area in the gray matter was wider in the severe group, continuity over the white matter was present. In addition, the difference in damaged gray matter area between the severe and mild groups supports the reasonableness of the group setting in the protocol (Figure 4).
The coronal HE sections show that the lesion mainly exists in the gray matter in both groups. In the severe group, the structure of the white matter surrounding the gray matter was more likely to be impacted, but the outline of the white matter was still maintained (Figure 5). NF-200 immunofluorescence suggests that even though the white matter surrounding gray matter was affected in the severe group, the white matter was still relatively intact. These results are consistent with the characteristics described for CCS in the previous study4 (Figure 6).
No red blood cells were found in sagittal HE sections at 7 days post-injury in either the mild or the severe group. The Prussian blue staining revealed no hemosiderosis in the mild group but in the severe group. These results indicate the inducement of hemorrhage may require a relatively severe degree of damage (Figure 7).
Immunofluorescence revealed areas of elevated GFAP and Iba-1 expression in both mild and severe injury, suggesting an inflammatory response and the formation of a glial scar in the lesion. Also, the severe group exhibited a larger lesion area than the mild group (Figure 8).
MRI is a relatively minimally invasive method to observe the spinal cord. The results suggest that in both the mild and severe groups, there is a hypointense signal change in the lesion with a high signal outline. The severe group showed a significantly larger hypointense signal area (Figure 9). The hypointense signal suggests a precipitate from the reticulocyte lysate in this area, and the surrounding hyperintense signal suggests an inflammatory response. We did conduct several behavioral tests in our previous study. For instance, the grip strength test in the forelimbs reveals a significant difference15.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65028/65028fig01.jpg" /> 
Figure 1: The sleeve and weights of SCICP. The surface area of the tip was designed to be 1.3 mm x 1.6 mm based on the exposed area of the spinal cord measured after the C6 laminectomy. The weight is coated with PTFE, which effectively reduces friction between the sleeve&#39;s inner wall and the weight. <a href="https://www.jove.com/files/ftp_upload/65028/65028fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65028/65028fig02.jpg" /> 
Figure 2: Exposure and compression of the spinal cord. (A) Longitudinal incision of the skin; (B) Separate the muscles rostrally from T2 spinous process; (C) Separate the muscles above the laminae; (D) C6 laminectomy; (E) Fixing the vertebral body; (F) Determining the location of compression; (G) Compression of the spinal cord; (H) No significant damage to the white matter above the spinal cord after spinal cord compression. <a href="https://www.jove.com/files/ftp_upload/65028/65028fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65028/65028fig03.jpg" /> 
Figure 3: Mouse cervical skeleton anatomy. The site indicated by the arrow is the T2 spinous process. <a href="https://www.jove.com/files/ftp_upload/65028/65028fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65028/65028fig04.jpg" /> 
Figure 4: The sagittal HE stained sections. (A) Cervical spinal cord sagittal section. (B,C) The Severe group had more severe damage than the Mild group, but both focused on the gray matter around the central cord. The 7, 28, and 70 dpi images suggest no significant difference in the expression of injury in the same injury group at different periods and that the continuity of the white matter in the superior and inferior spinal cord is maintained. Scale bar: 1 mm. <a href="https://www.jove.com/files/ftp_upload/65028/65028fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65028/65028fig05.jpg" /> 
Figure 5: Cervical spinal cord injury coronal HE stained sections. (A-C) The injury primarily affects the gray matter surrounding the central cord, as seen in panels B and C. The severe injury group suffers from a more extensive range of damage than the mild injury group, which is more likely to affect the white matter. Scale bar: 400 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65028/65028fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65028/65028fig06.jpg" /> 
Figure 6: NF-200 coronal immunofluorescence after injury. NF-200 response with no significant difference at white matter outline. Scale bar: 400 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65028/65028fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65028/65028fig07.jpg" /> 
Figure 7: Prussian blue staining. (A-C) Hemosiderosis was observed in the severe group but not the mild group. Scale bar: 400 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65028/65028fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/65028/65028fig08.jpg" /> 
Figure 8: Sagittal GFAP and Iba-1 immunofluorescence after injury. (A-C) As the degree of injury increases, the area of GFAP and Iba-1 response increases. Scale bar: 1 mm. <a href="https://www.jove.com/files/ftp_upload/65028/65028fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/65028/65028fig09.jpg" /> 
Figure 9: Sagittal MRI after cervical spinal cord injury (T2-weighted images). The injury region was observed as a hypointense signal in the mild and severe injury groups, with a significantly wider area of hypointense signal in the severe injury group. <a href="https://www.jove.com/files/ftp_upload/65028/65028fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Establishment of Central Cord Syndrome Model in C57BL/6J Mouse at JoVE.com

Establishment of Central Cord Syndrome Model in C57BL/6J Mouse

The present protocol simulating central cord syndrome (CCS) in mice has improved repeatability and minimized operation damage to the experimental animals, avoiding disrupting the anatomical structure excessively. The strategy in this study is advantageous because it allows for research into injury mechanisms by producing consistent results.

Animal models of central cord syndrome (CCS) could substantially benefit preclinical research. Identifiable anatomical pathways can give minimally ...

establishment-of-central-cord-syndrome-model-in-c57bl6j-mouse

Research

JoVE Journal

Neuroscience

491 Views.  Qilu Hospital of Shandong University. The present protocol simulating central cord syndrome (CCS) in mice has improved repeatability and minimized operation damage to the experimental animals, avoiding disrupting the anatomical structure excessively. The strategy in this study is advantageous because it allows for research into injury mechanisms by producing consistent results.

Video: Establishment of Central Cord Syndrome Model in C57BL/6J Mouse

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.

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

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.
Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20.
We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

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.

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

A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied in vitro using the neurosphere assay. This colony-forming assay is a powerful tool to study the response of NSCs to exogenous factors in a dish; however, this can also be used to study the effect of in vivo manipulations with the proper understanding of the strengths and limitations of the assay. One manipulation of the clinical interest is the effect of injury on endogenous NSC activation. Current models of spinal cord injury provide a challenge to study this as the severity of common contusion, compression, and transection models cause the destruction of the NSC niche at the site of the injury where the stem cells reside. Here, we describe a minimal injury model that causes localized damage at the superficial dorsolateral surface of the lower thoracic level (T7/8) of the adult mouse spinal cord. This injury model spares the central canal at the level of injury and permits analysis of the NSCs that reside at the level of the lesion at various time points following injury. Here, we show how the neurosphere assay can be utilized to study the activation of the two distinct, lineally-related, populations of NSCs that reside in the spinal cord periventricular region - primitive and definitive NSCs (pNSCs and dNSCs, respectively). We demonstrate how to isolate and culture these NSCs from the periventricular region at the level of injury and the white matter injury site. Our post-surgical spinal cord dissections show increased numbers of pNSC and dNSC-derived neurospheres from the periventricular region of injured cords compared to controls, speaking to their activation via injury. Furthermore, following injury, dNSC-derived neurospheres can be isolated from the injury site &#8212; demonstrating the ability of NSCs to migrate from their periventricular niche to sites of injury.

Here, we demonstrate the performance of a minimal spinal cord injury model in an adult mouse that spares the central canal niche housing endogenous neural stem cells (NSCs). We show how the neurosphere assay can be used to quantify activation and migration of definitive and primitive NSCs following injury.

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

Portable Thermographic Screening for Detection of Acute Wallenberg's Syndrome

Wallenberg&#39;s syndrome (WS) is a type of brainstem infarction. WS patients often show Horner&#39;s syndrome, dissociated sensory disturbance, truncal ataxia, and hoarseness. However, they rarely show tactile sensory disturbance and paralysis of the extremities. Additionally, acute brainstem infarction is often not apparent in magnetic resonance images. These symptomatic and imaging characteristics sometimes lead to misdiagnosis of WS as a non-stroke disease, including auditory vertigo. Although careful neurological examination is necessary to prevent misdiagnosis of WS, this type of examination may be difficult for non-neurologists to whom affected patients initially present. Lateral differences in body surface temperature (BST) constitute a recognized and widespread symptom of WS. We previously reported that most acute WS patients exhibit lateral differences in BST at multiple locations and that these lateral differences in BST could easily be detected by thermographic measurement. Here, we present the method for use of portable thermography to detect acute WS, using a simple, rapid, noninvasive, and cost-effective approach. To assess lateral differences in BST among patients with suspected WS, BST was measured as soon as possible in the examination room or in the patient&#39;s bedroom. Measurements were performed bilaterally at four locations where images could easily be acquired (face, palm of the hand, abdomen, and dorsum of the foot) using a portable thermal camera. When lateral differences in BST are observed macroscopically, especially in multiple locations on the same side, a diagnosis of WS should be suspected. Macroscopic assessment of BST laterality can be made within 2 min of the acquisition of thermographic images. This method may be useful in preventing misdiagnosis of acute WS as a non-stroke disease, especially when such patients initially present to non-neurologists.

Acute Wallenberg&#39;s syndrome can be misdiagnosed as a non-stroke disease, such as auditory vertigo. Thus, careful neurological examination, which is sometimes difficult for non-neurologists, is necessary for precise diagnosis. Here, we present a simple, rapid, noninvasive, and cost-effective method for detection of acute Wallenberg&#39;s syndrome using portable thermography.

Wallenberg&#39;s syndrome (WS) is a type of brainstem infarction. WS patients often show Horner&#39;s syndrome, dissociated sensory disturbance, truncal ...

Thoracic Spinal Cord Hemisection Surgery and Open-Field Locomotor Assessment in the Rat

Spinal cord injury (SCI) causes disturbances in motor, sensory, and autonomic function below the level of the lesion. Experimental animal models are valuable tools to understand the neural mechanisms involved in locomotor recovery after SCI and to design therapies for clinical populations. There are several experimental SCI models including contusion, compression, and transection injuries that are used in a wide variety of species. A hemisection involves the unilateral transection of the spinal cord and disrupts all ascending and descending tracts on one side only. Spinal hemisection produces a highly selective and reproducible injury in comparison to contusion or compression techniques that is useful for investigating neural plasticity in spared and damaged pathways associated with functional recovery. We present a detailed step-by-step protocol for performing a thoracic hemisection at the T8 vertebral level in the rat that results in an initial paralysis of the hindlimb on the side of the lesion with graded spontaneous recovery of locomotor function over several weeks. We also provide a locomotor scoring protocol to assess functional recovery in the open-field. The locomotor assessment provides a linear recovery profile and can be performed both early and repeatedly after injury in order to accurately screen animals for appropriate time points in which to conduct more specialized behavioral testing. The hemisection technique presented can be readily adapted to other transection models and species, and the locomotor assessment can be used in a variety of SCI and other injury models to score locomotor function.

The rat thoracic spinal hemisection is a valuable and reproducible model of unilateral spinal cord injury to investigate the neural mechanisms of locomotor recovery and treatment efficacy. This article includes a detailed step-by-step guide to perform the hemisection procedure and to assess locomotor performance in an open-field arena.

Spinal cord injury (SCI) causes disturbances in motor, sensory, and autonomic function below the level of the lesion. Experimental animal models are ...

Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). Therefore, detection of the presence of A&#946; in AD brain tissue is a valuable tool for developing new treatments to prevent the progression of AD. Several classical amyloid binding dyes, fluorochrome, imaging probes, and A&#946;-specific antibodies have been used to detect A&#946; histochemically in AD brain tissue. Use of these compounds for A&#946; detection is costly and time consuming. However, because of its intense fluorescent activity, high-affinity, and specificity for A&#946;, as well as structural similarities with traditional amyloid binding dyes, curcumin (Cur) is a promising candidate for labeling and imaging of A&#946; plaques in postmortem brain tissue. It is a natural polyphenol from the herb Curcuma longa. In the present study, Cur was used to histochemically label A&#946; plaques from both a genetic mouse model of 5x familial Alzheimer&#39;s disease (5xFAD) and from human AD tissue within a minute. The labeling capability of Cur was compared to conventional amyloid binding dyes, such as thioflavin-S (Thio-S), Congo red (CR), and Fluoro-jade C (FJC), as well as A&#946;-specific antibodies (6E10 and A11). We observed that Cur is the most inexpensive and quickest way to label and image A&#946; plaques when compared to these conventional dyes and is comparable to A&#946;-specific antibodies. In addition, Cur binds with most A&#946; species, such as oligomers and fibrils. Therefore, Cur could be used as the most cost-effective, simple, and quick fluorochrome detection agent for A&#946; plaques.

Curcumin is an ideal fluorophore for labeling and imaging of amyloid beta protein plaques in brain tissue due to its preferential binding to amyloid beta protein as well as its structural similarities with other traditional amyloid binding dyes. It can be used to label and image amyloid beta protein plaques more efficiently and inexpensively than traditional methods.

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). ...