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>

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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><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&amp;nbsp;</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&amp;nbsp;</td><td>C57BL/6J&amp;nbsp;</td><td></td></tr><tr><td>Microsurgery apparatus&amp;nbsp;</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></tr><tr><td>Phosphate buffered solution (PBS)&amp;nbsp;</td><td>Solarbio</td><td>P1020</td><td>pH 7.2&amp;ndash;7.4</td></tr><tr><td>Prussian Blue Iron Stain Kit (With Eosin)</td><td>Solarbio</td><td>G1424</td><td></td></tr><tr><td>RWD Laboratory inhalation anesthetic station</td><td>RWD Life Science Co., Ltd</td><td>R550</td><td></td></tr><tr><td>Small animal in vivo microCT imaging system</td><td>PerkinElmer&amp;nbsp;</td><td>Quantum GX2</td><td></td></tr><tr><td>Spinal cord injury coaxial platform</td><td>Shandong ULT Biotechnology Co., Ltd</td><td>Custom-made(Feng&#039;s standard)</td><td>https://shop43957633.m.youzan.com/wscgoods/detail/367x5ovgn69q18g?banner_id=f.81386274~goods.7~&lt;br/&gt; 1~b0yRFKOq&amp;amp;alg_id=&lt;br/&gt; 0&amp;amp;slg=tagGoodList-default%2COpBottom%2Cuuid%&lt;br/&gt; 2CabTraceId&amp;amp;components_&lt;br/&gt; style_layout&lt;br/&gt; =1&amp;amp;reft=1659409105184&amp;amp;spm=&lt;br/&gt; g.930111970_f.81386274&amp;amp;alias=&lt;br/&gt; 367x5ovgn69q18g&amp;amp;from_uuid=&lt;br/&gt; 1362cc46-ffe0-6886-2c65-01903&lt;br/&gt; dbacbba&amp;amp;sf=qq_sm&amp;amp;is_share=&lt;br/&gt; 1&amp;amp;shopAutoEnter=1&amp;amp;share_cmpt&lt;br/&gt; =native_wechat&amp;amp;is_silence_auth=1</td></tr><tr><td>Surgery microscope&amp;nbsp;</td><td>Zumax&amp;nbsp;Medical Co., Ltd.</td><td>zumax, OMS2355</td><td></td></tr><tr><td>Tris Buffered Saline+Tween (TBST)</td><td>Solarbio</td><td>T1082</td><td>Dilution ratio (1:19)</td></tr><tr><td>Xylene</td><td>Fuyu Reagent</td><td></td><td></td></tr></tbody></table>

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="Static equilibrium concept; steel sleeve, rods of 10 g/mm², 5 g/mm²; mechanics experiment setup." class="xfigimg" src="/files/ftp_upload/65028/65028fig01.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
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'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="Surgical procedure sequence on animal subject, showing incision and instrument use for implant insertion." class="xfigimg" src="/files/ftp_upload/65028/65028fig02.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
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="Skeleton diagram depicting vertebral anatomy: Sixth cervical vertebra, T2 spinous process." class="xfigimg" src="/files/ftp_upload/65028/65028fig03.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
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="Histological sections showing tissue at 0, 7, 28, 70 dpi comparing mild to severe conditions." class="xfigimg" src="/files/ftp_upload/65028/65028fig04.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
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="Histology comparison of spinal cord sections; control, mild, severe; microscopy image." class="xfigimg" src="/files/ftp_upload/65028/65028fig05.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5"> 
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 µm. <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="Neurofilament NF-200 staining, spinal cord sections, varying damage: control, mild, severe." class="xfigimg" src="/files/ftp_upload/65028/65028fig06.jpg" data-altupdatedat="1747911167000" data-alt="Figure 6"> 
Figure 6: NF-200 coronal immunofluorescence after injury. NF-200 response with no significant difference at white matter outline. Scale bar: 400 µm. <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="Histopathology sections showing tissue damage severity; control, mild, severe; labeled A-C." class="xfigimg" src="/files/ftp_upload/65028/65028fig07.jpg" data-altupdatedat="1747911167000" data-alt="Figure 7"> 
Figure 7: Prussian blue staining. (A-C) Hemosiderosis was observed in the severe group but not the mild group. Scale bar: 400 µm. <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="Fluorescence microscopy images of spinal cord sections; Iba-1, GFAP staining; neuroinflammation analysis." class="xfigimg" src="/files/ftp_upload/65028/65028fig08.jpg" data-altupdatedat="1747911167000" data-alt="Figure 8"> 
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="Spinal MRI comparison showing control, mild, severe injury at 7 dpi; analysis of injury severity." class="xfigimg" src="/files/ftp_upload/65028/65028fig09.jpg" data-altupdatedat="1747911167000" data-alt="Figure 9"> 
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

556 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

Chronic Constriction of the Sciatic Nerve and Pain Hypersensitivity Testing in Rats

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of the population1,2. Symptoms include spontaneous (tingling, burning, electric-shock like) pain, dysaesthesia, paraesthesia, allodynia (pain resulting from normally non-painful stimuli) and hyperalgesia (an increased response to painful stimuli). The sensory symptoms are co-morbid with behavioural disabilities, such as insomnia and depression. To study chronic neuropathic pain several animal models mimicking peripheral nerve injury have been developed, one of the most widely used is Bennett and Xie's (1988) unilateral sciatic nerve chronic constriction injury (CCI)3 (Figure 1). Here we present a method for performing CCI and testing pain hypersensitivity.
CCI is performed under anaesthesia, with the sciatic nerve on one side exposed by making a skin incision, and cutting through the connective tissue between the gluteus superficialis and biceps femoris muscles. Four chromic gut ligatures are tied loosely around the sciatic nerve at 1 mm intervals, to just occlude but not arrest epineural blood flow. The wound is closed with sutures in the muscle and staples in the skin. The animal is then allowed to recover from surgery for 24 hrs before pain hypersensitivity testing begins.
For behavioural testing, rats are placed into the testing apparatus and are allowed to habituate to the testing procedure. The area tested is the mid-plantar surface of the hindpaw (Figure 2), which falls within the sciatic nerve distribution. Mechanical withdrawal threshold is assessed by mechanically stimulating both injured and uninjured hindpaws using an electronic dynamic plantar von Frey aesthesiometer or manual von Frey hairs4. The mechanical withdrawal threshold is the maximum pressure exerted (in grams) that triggers paw withdrawal. For measurement of thermal withdrawal latency, first described by Hargreaves et al (1988), the hindpaw is exposed to a beam of radiant heat through a transparent glass surface using a plantar analgesia meter5,6. The withdrawal latency to the heat stimulus is recorded as the time for paw withdrawal in both injured and uninjured hindpaws. Following CCI, mechanical withdrawal threshold, as well as thermal withdrawal latency in the injured paw are both significantly reduced, compared to baseline measurements and the uninjured paw (Figure 3). The CCI model of peripheral nerve injury combined with pain hypersensitivity testing provides a model system to investigate the effectiveness of potential therapeutic agents to modify chronic neuropathic pain. In our laboratory, we utilise CCI alongside thermal and mechanical sensitivity of the hindpaws to investigate the role of neuro-immune interactions in the pathogenesis and treatment of neuropathic pain.

Due to the simplicity of surgery and the robust behavioural outcome, chronic constriction of the sciatic nerve is one of the pre-eminent animal models of neuropathic pain. Within 24 hrs following surgery, pain hypersensitivity is established and can be quantitatively measured using a von Frey aesthesiometer (mechanical test) and plantar analgesia meter (thermal test).

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of ...

Medicine

Controlled Cervical Laceration Injury in Mice

Use of genetically modified mice enhances our understanding of molecular mechanisms underlying several neurological disorders such as a spinal cord injury (SCI). Freehand manual control used to produce a laceration model of SCI creates inconsistent injuries often associated with a crush or contusion component and, therefore, a novel technique was developed. Our model of cervical laceration SCI has resolved inherent difficulties with the freehand method by incorporating 1) cervical vertebral stabilization by vertebral facet fixation, 2) enhanced spinal cord exposure, and 3) creation of a reproducible laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm in depth without associated contusion. Compared to the standard methods of creating a SCI laceration such as freehand use of a scalpel or scissors, our method has produced a consistent lesion. This method is useful for studies on axonal regeneration of corticospinal, rubrospinal, and dorsal ascending tracts.

A novel technique to create a reproducible in vivo model of cervical spinal cord laceration injury in the mouse is described. This technique is based on spine stabilization by fixation of the cervical facets and laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm.

Use of genetically modified mice enhances our  understanding of molecular mechanisms underlying several neurological disorders  such as a spinal cord ...

Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) below the site of injury. The secondary ischemia that develops following the initial mechanical insult is a serious complication of the SCI and severely impairs the function and viability of surviving neuronal and non-neuronal cells in the SC. In addition, ischemia is also responsible for the growth of lesion during chronic phase of injury and interferes with the cellular repair and healing processes. Thus there is a need to develop a spinal cord ischemia model for studying the mechanisms of ischemia-induced pathology. Focal ischemia induced by photothrombosis (PT) is a minimally invasive and very well established procedure used to investigate the pathology of ischemia-induced cell death in the brain. Here, we describe the use of PT to induce an ischemic lesion in the spinal cord of mice. Following retro-orbital sinus injection of Rose Bengal, the posterior spinal vein and other capillaries on the dorsal surface of SC were irradiated with a green light resulting in the formation of a thrombus and thus ischemia in the affected region. Results from histology and immunochemistry studies show that PT-induced ischemia caused spinal cord infarction, loss of neurons and reactive gliosis. Using this technique a highly reproducible and relatively easy model of SCI in mice can be achieved that would serve the purpose of scientific investigations into the mechanisms of ischemia induced cell death as well as the efficacy of neuroprotective drugs. This model will also allow exploration of the pathological changes that occur following SCI in live mice like axonal degeneration and regeneration, neuronal and astrocytic Ca2+ signaling using two-photon microscopy.

Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice.

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

A Tissue Displacement-based Contusive Spinal Cord Injury Model in Mice

Producing a consistent and reproducible contusive spinal cord injury (SCI) is critical to minimizing behavioral and histological variabilities between experimental animals. Several contusive SCI models have been developed to produce injuries using different mechanisms. The severity of the SCI is based on the height that a given weight is dropped, the injury force, or the spinal cord displacement. In the current study, we introduce a novel mouse contusive SCI device, the Louisville Injury System Apparatus (LISA) impactor, which can create a displacement-based SCI with high injury velocity and accuracy. This system utilizes laser distance sensors combined with advanced software to produce graded and highly-reproducible injuries. We performed a contusive SCI at the 10th thoracic vertebral (T10) level in mice to demonstrate the step-by-step procedure. The model can also be applied to the cervical and lumbar spinal levels.

We introduce a tissue displacement-based contusive spinal cord injury model that can produce a consistent contusive spinal cord injury in adult mice.

Producing a consistent and reproducible contusive spinal cord injury (SCI) is critical to minimizing behavioral and histological variabilities between ...

Induction of Complete Transection-Type Spinal Cord Injury in Mice

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, such as cell transplantation methods, are being researched to overcome the debilitating disabilities arising from SCI. Most pre-clinical animal trials are conducted in rodent models of SCI. While rat models of SCI have been widely used, mouse models have received less attention, even though mouse models can have significant advantages over rat models. The small size of mice equates to lower animal maintenance costs than for rats, and the availability of numerous transgenic mouse models is advantageous for many types of studies. Inducing repeatable and precise injury in the animals is the primary challenge for SCI research, which in small rodents requires high-precision surgery. The transection-type injury model has been a commonly used injury model over the last decade for transplantation-based therapeutic research, however a standardized method for inducing a complete transection-type injury in mice does not exist. We have developed a surgical protocol for inducing a complete transection type injury in C57BL/6 mice at thoracic vertebral level 10 (T10). The procedure uses a small tip drill instead of rongeurs to precisely remove the lamina, after which a thin blade with rounded cutting edge is used to induce the spinal cord transection. This method leads to reproducible transection-type injury in small rodents with minimal collateral muscle and bone damage and therefore minimizes confounding factors, specifically where behavioral functional outcomes are analyzed.

This protocol describes how to create a precise laminectomy for induction of stable transection-type spinal cord injury in the mouse model, with minimal collateral damage for spinal cord injury research.

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, ...

Establishing a Mouse Contusion Spinal Cord Injury Model Based on a Minimally Invasive Technique

Using minimally invasive methods to model spinal cord injury (SCI) can minimize behavioral and histological differences between experimental animals, thereby improving the reproducibility of the experiments.
These methods need two requirements to be fulfilled: clarity of the surgical anatomical pathway and simplicity and convenience of the laboratory device. Crucially for the operator, a clear anatomical pathway provides minimally invasive exposure, which avoids additional damage to the experimental animal during the surgical procedures and allows the animal to maintain a consistent and stable anatomical morphology during the experiment.
In this study, the use of a novel integrated platform called the SCI coaxial platform for spinal cord injury in small animals to expose the T9 level spinal cord in a minimally invasive way and stabilize and immobilize the vertebra of mice using a vertebral stabilizer is researched, and, finally, a coaxial gravity impactor is used to contuse the spinal cord of mice to approach different degrees of T9 spinal cord injury. Finally, histological results are provided as a reference for the readers.

Minimally invasive techniques and a simple laboratory device improve the reproducibility of the spinal cord injury model by reducing operative damage to the experimental animals and allowing anatomical morphology maintenance. The method is worthwhile because the reliable results and reproducible procedure facilitate investigations of the mechanisms of disease reparation.

Using minimally invasive methods to model spinal cord injury (SCI) can minimize behavioral and histological differences between experimental animals, ...

Whole Central and Peripheral Nervous System Mice Dissection

Animal models represent the workhorse of the neuroscience field. Despite this, today, there is still no step-by-step protocol to dissect a complete rodent nervous system, nor is there a complete schematic representing it that is freely available. Only methods to harvest the brain, the spinal cord, a specific dorsal root ganglion, and the sciatic nerve (separately) are available. Here, we provide detailed pictures and a schematic of the central and peripheral murine nervous system. More importantly, we outline a robust procedure to perform its dissection. The 30 min pre-dissection step allows isolating the intact nervous system within the vertebra with muscles free of viscera and skin. A 2-4 h dissection follows it under a micro-dissection microscope to expose the spinal cord and the thoracic nerves, and finally peel the whole central and peripheral nervous system off the carcass. This protocol represents a significant step forward in studying the anatomy and pathophysiology of the nervous system globally. For example, the dissected dorsal root ganglions from a neurofibromatosis type I mice model can be further processed for histology to unravel changes in tumor progression.

Here, we present a protocol to complete the dissection of central and peripheral nervous systems of mice. The dissected tissues are further analyzed in downstream applications, such as taking gross pictures or histology.

Animal models represent the workhorse of the neuroscience field. Despite this, today, there is still no step-by-step protocol to dissect a complete rodent ...

Establishing a Transcranial Middle Cerebral Artery Occlusion Mice Model

A Modified Transcranial Middle Cerebral Artery Occlusion Model to Study Stroke Outcomes in Aged Mice

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. The filament MCAO model typically exhibits a massive cerebral infarction in C57Bl/6 mice that sometimes includes brain tissue in the territory supplied by the posterior cerebral artery, which is largely due to a high incidence of posterior communicating artery atresia. This phenomenon is considered a major contributor to the high mortality rate observed in C57Bl/6 mice during long-term stroke recovery after filament MCAO. Thus, many chronic stroke studies exploit distal MCAO models. However, these models usually produce infarction only in the cortex area, and consequently, the assessment of post-stroke neurologic deficits could be a challenge. This study has established a modified transcranial MCAO model in which the MCA at the trunk is partially occluded either permanently or transiently via a small cranial window. Since the occlusion location is relatively proximal to the origin of the MCA, this model generates brain damage in both the cortex and striatum. Extensive characterization of this model has demonstrated an excellent long-term survival rate, even in aged mice, as well as readily detectable neurologic deficits. Therefore, the MCAO mouse model described here represents a valuable tool for experimental stroke research.

Author Spotlight: Innovative Techniques and Future Directions in Stroke Research 

This protocol demonstrates a unique mouse stroke model with a medium-sized infarct and an excellent survival rate. This model allows preclinical stroke researchers to extend the ischemia duration, use aged mice, and assess long-term functional outcomes.

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. ...

A Spinal Cord Injury Contusion Device for Accurate Mouse Models

Automated Impactor for Contusive Spinal Cord Injury Model in Mice

Spinal cord injury (SCI) due to traumatic injuries such as car accidents and falls is associated with permanent spinal cord dysfunction. Creation of contusion models of spinal cord injury by impacting the spinal cord results in similar pathologies to most spinal cord injuries in clinical practice. Accurate, reproducible, and convenient animal models of spinal cord injury are essential for studying spinal cord injury. We present a novel automated spinal cord injury contusion device for mice, the Guangzhou Jinan University smart spinal cord injury system, that can produce spinal cord injury contusion models with accuracy, reproducibility, and convenience. The system accurately produces models of varying degrees of spinal cord injury via laser distance sensors combined with an automated mobile platform and advanced software. We used this system to create three levels of spinal cord injury mice models, determined their Basso mouse scale (BMS) scores, and performed behavioral as well as staining assays to demonstrate its accuracy and reproducibility. We show each step of the development of the injury models using this device, forming a standardized procedure. This method produces reproducible spinal cord injury contusion mice models and reduces human manipulation factors via convenient handling procedures. The developed animal model is reliable for studying spinal cord injury mechanisms and associated treatment approaches.

Author Spotlight: Insight Into Innovations in Spinal Cord Injury Research

Presented here is a novel automated spinal cord injury contusion device for mice, which can accurately produce spinal cord injury contusion models with varying degrees.

Spinal cord injury (SCI) due to traumatic injuries such as car accidents and falls is associated with permanent spinal cord dysfunction. Creation of ...