inducing a spinal cord contusion in a mouse model using a spinal cord injury coaxial platform

Inducing a Spinal Cord Contusion in a Mouse Model Using a Spinal Cord Injury Coaxial Platform

Once the T9 level spinal cord is exposed, and the vertebra is fixed, aim at the spinal cord by the tip, inside the sleeve, under the operating microscopic. After locating the interspinous space of the T12 to T13, lower the sleeve until the end of the impactor is consistent with the mark on the observation window, and the specified height of 22 millimeters is reached.Pull out the pull pin to release the weight. Remove the impactor when the contusion is done. And observe the degree of spinal cord injury under the operating microscope. In the mild group, the light red color alteration can be seen, while in the moderate group, the injury site exhibits dark red in 3 to 4 seconds, and possibly eminence can be observed.

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.&#160;1. Locating and laminectomy of the 9th thoracic vertebra (T9)NOTE: Female C57BL/6J mice 9-10 weeks old were purchased from Jinan Pengyue Experimental Animal Company (Jinan, China).<ol><li>Autoclave a suite of surgical instruments for the experiment and sterilize the operating table with 75% alcohol before surgery.</li><li>Inject buprenorphine for analgesia (0.05-2.0 mg/kg, subcutaneously) 30 minutes before anesthesia for injury operations. Then, anesthetize the mouse with isoflurane (induction: ~3-5%, maintenance: ~1.5-2%). Check if the animal is fully anesthetized by the reflexes of the tail or toe pinch. Once anesthesia is in effect, lay the mouse in a prone position in a designated part of the operating table and coat the cornea with ophthalmic ointment (apply ophthalmic ointment to the corneas to protect the eyes from drying during surgery).<ol><li>Shave the hair from the caudal to the rostral with an electric shaver over the thoracolumbar spine. Sterilize the skin several times in a circular motion with iodophor for 30 s, followed by 75% alcohol. Apply a sterile surgical drape and make a longitudinal incision of approximately 1.5 cm on the skin from approximately the 6th thoracic vertebra (T6) to the 13th thoracic vertebra (T13) with a scalpel and blade. &#160;NOTE: Palpate along the costal margin to the midline, where the 12th thoracic vertebra (T12) - T13 interspinous space is located. Make a 1.5 cm incision to the rostral, and the incision is approximately flush with T6-T13 vertebrae.</li></ol></li><li>Explore the 13th rib on one side from the bony portion under the operating microscope. Explore the spinous process in the midline by lightly touching the area of the costovertebral angle and then toward the rostral to locate the interspinous space of the T12-T13. Explore the interspinous space of the T9 - 10th thoracic vertebra (T10) from the space of the T12-T13 to the rostral side. (Figure 1A, 2A)</li><li>Dissect the paraspinal muscle along the spinous process of the T9 to the anterior and posterior facet joints of both sides with micro scissors (Figure 2B). Retract the paraspinal muscles with micro-retractors and clean the soft tissue on the lamina and in the interspinous space of the 8th thoracic vertebra (T8) - T9 and T9-T10 with micro scissors.</li><li>Perform T9 laminectomy, clamp the spinous process of T9 with microsurgery forceps, slightly lift it up, insert the micro scissors parallelly along the right dorsolateral side of the lamina, avoiding damage to the spinal cord, and cut off the lamina with micro scissors. Repeat on the left side, and the spinal cord can be exposed (Figure 1B, 2C).</li><li>Before fixing the vertebra, loosen the universal arm and slowly clamp the 9th to 10th facet joints on both sides of the vertebra with the micro mosquito forceps of the vertebral stabilizer. Tighten the screws on the micro mosquito forceps, and the vertebra is thus stabilized. Adjust the spinal cord to the horizontal plane, tighten the universal arm, and fix the vertebra (Figure 2D).&#160;</li></ol>2. T9 contusion injury<ol><li>Once the T9 level spinal cord is exposed and the vertebra is fixed, aim at the spinal cord by the tip inside the sleeve under the operating microscopic (Figure 2E).</li><li>Check if the surface of the tip is parallel to the spinal cord from the posterior and lateral aspects of the spinal cord since it is easy to observe the relationship between the spinal cord and the tip under the microscope, and the operating table can be turned easily.</li><li>Check if the surface of the tip is parallel to the bilateral borders of the spared lamina before the tip is in contact with the spinal cord after the laminectomy since it is a natural reference plane parallel to the spinal cord.</li><li>After locating the interspinous space of the T12-T13, lower the sleeve until the end of the impactor is consistent with the mark on the observation window and the specified height of 22 mm is reached. Pull out the pull pin to release the weight (1.3 g, 2.0 g, or 2.7 g according to the group, with each group including 3 mice and each group having one mouse for each time point). &#160;NOTE: The spinal cord should be parallel to the ground and perpendicular to the tip; move the operating table to ensure the microscopic visual field, since the table is very compact.</li><li>Remove the impactor when the contusion is done and observe the degree of spinal cord injury (SCI) under the operating microscope. In the mild group, a light red color alteration can be seen, while in the moderate group, the injury site exhibits dark red in 3-4 s, and, possibly, eminence can be observed. In the severe group, the dark red manifestations may appear immediately, and obvious eminence in the dura is manifested, but the dura is still in a consistent shape (Figure 2F).</li></ol>

<figure class='table' style='width:914pt;'><table class='ck-table-resized'><colgroup><col style='width:28.45%;'><col style='width:24.4%;'><col style='width:16.74%;'><col style='width:30.41%;'></colgroup><tbody><tr><td style='height:15.5pt;width:260pt;'>Ethanol</td><td style='width:223pt;'>Fuyu Reagent</td><td style='width:153pt;'>64-17-5</td><td style='width:278pt;'>&#160;</td></tr><tr><td style='height:15.5pt;width:260pt;'>Fluorescent microscope</td><td style='width:223pt;'>KEYENCE</td><td style='width:153pt;'>BZ-X800</td><td style='width:278pt;'>&#160;</td></tr><tr><td style='height:31.0pt;width:260pt;'>Mice</td><td style='width:223pt;'>Jinan Pengyue Experimental AnimalCompany</td><td style='width:153pt;'>C57BL/6J</td><td style='width:278pt;'>&#160;</td></tr><tr><td style='height:31.0pt;width:260pt;'>Microsurgery apparatus</td><td style='width:223pt;'>Shandong ULT Biotechnology Co., Ltd</td><td style='width:153pt;'>All the surgey instruments are custom-made</td><td style='width:278pt;'>Ophthalmic scissors, micro mosquito forceps, microsurgery forceps, micro scissors</td></tr><tr><td style='height:15.5pt;width:260pt;'>RWD Laboratory inhalation anesthetic station</td><td style='width:223pt;'>RWD Life Science Co., Ltd</td><td style='width:153pt;'>R550</td><td style='width:278pt;'>&#160;</td></tr><tr><td style='height:15.5pt;width:260pt;'>Small animal in vivo microCT imaging system</td><td style='width:223pt;'>PerkinElmer</td><td style='width:153pt;'>Quantum GX2</td><td style='width:278pt;'>&#160;</td></tr><tr><td style='width:260pt;'>Spinal cord injury coaxial platform</td><td style='width:223pt;'>Shandong ULT Biotechnology Co., Ltd</td><td style='width:153pt;'>Custom-made (Feng's standard)</td><td style='width:278pt;'>(https://shop43957633.m.youzan.com/wscgoods/detail/367x5ovgn69q18g?banner_id=f.81386274~goods.7~1~ b0yRFKOq&amp;alg_id=0&amp;slg=tagGood List-default%2COpBottom%2Cuuid %2CabTraceId&amp;components_style_ layout=1&amp;reft=1659409105184&amp;sp m=g.930111970_f.81386274&amp;alias =367x5ovgn69q18g&amp;from_uuid=136 2cc46-ffe0-6886-2c65-01903dbacbb a&amp;sf=qq_sm&amp;is_share=1&amp;shopAuto Enter=1&amp;share_cmpt=native_ wechat&amp;is_silence_auth=1)</td></tr></tbody></table></figure>

<a href='https://app.jove.com/v/64538/establishing-mouse-contusion-spinal-cord-injury-model-based-on'>Source: Elzat, E. Y. Y., et al.,&#160;Establishing a Mouse Contusion Spinal Cord Injury Model Based on a Minimally Invasive Technique. J. Vis. Exp.&#160;(2022).</a>This video demonstrates the method for inducing a contusion injury at the T9 level spinal cord in a mouse model, using a spinal cord injury coaxial platform. A weight is dropped on the T9 level spinal cord that creates a contusion that immediately causes tissue discoloration and swelling.

<img src='https://cloudfront.jove.com/files/ftp_upload/23331/Figure_2_editor_23331.jpg' alt='Figure_2.jpg' />Figure 1. An imaging graph of the T13 costovertebral vertebra locating method. (A) The 13th rib and T13 are relatively constant anatomical structures. The T13 costovertebral angle can be easily detected under the microscope, from which the operator can probe toward the spinous process and find the T12-T13 interspino...

Source: Elzat, E. Y. Y., et al.,&#160;Establishing a Mouse Contusion Spinal Cord Injury Model Based on a Minimally Invasive Technique. J. Vis. ...

Begin with an anesthetized mouse on the spinal cord injury coaxial platform, with the exposed 9th thoracic or T9 vertebrae level spinal cord.&#160;Using the platform&#8217;s stabilizer forceps, secure the vertebrae and prevent movement.Place the tubular sleeve containing the impactor tip with a weight secured with a pull pin over the exposed spinal cord.Adjust the tip position to accurately target the T9 spinal cord level.Lower the sleeve until the impactor tip reaches the predetermined height above the spinal cord.&#160;Release the pull pin to drop the weight onto the impactor tip, striking the spinal cord with controlled impact.&#160;The impact delivers a blunt force that causes tissue compression, leading to structural damage in axons and blood vessels.&#160;This results in a contusion or injury from tissue compression.&#160;Remove the impactor. Tissue discoloration and swelling indicate a successful spinal cord contusion.

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비디오: Inducing a Spinal Cord Contusion in a Mouse Model Using a Spinal Cord Injury Coaxial Platform - 실험

A Contusion Model of Severe Spinal Cord Injury in Rats

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is described to obtain a consistent model of severe SCI. Use of a stereotactic frame and computer controlled impactor allows for creation of reproducible injury. Hypothermia and urinary tract infection pose significant challenges in the post-operative period. Careful monitoring of animals with daily weight recording and bladder expression allows for early detection of post-operative complications. The functional results of this contusion model are equivalent to transection models. The contusion model can be utilized to evaluate the efficacy of both neuroprotective and neuroregenerative approaches.

A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model.

쥐에 심각한 척수 손상의 타박상 모델

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is ...

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A Novel Vertebral Stabilization Method for Producing  Contusive Spinal Cord Injury

Clinically-relevant animal cervical spinal cord injury (SCI) models are essential for developing and testing potential therapies; however, producing reliable cervical SCI is difficult due to lack of satisfactory methods of vertebral stabilization. The conventional method to stabilize the spine is to suspend the rostral and caudal cervical spine via clamps attached to cervical spinous processes. &#160;However, this method of stabilization fails to prevent tissue yielding during the contusion as the cervical spinal processes are too short to be effectively secured by the clamps (Figure 1). &#160;Here we introduce a new method to completely stabilize the cervical vertebra at the same level of the impact injury. &#160;This method effectively minimizes movement of the spinal column at the site of impact, which greatly improves the production of consistent SCIs. &#160;We provide visual description of the equipment (Figure 2-4), methods, and a step-by-step protocol for the stabilization of the cervical 5 vertebra (C5) of adult rats, to perform laminectomy (Figure 5) and produce a contusive SCI thereafter. &#160;Although we only demonstrate a cervical hemi-contusion using the NYU/MASCIS impactor device, this vertebral stabilization technique can be applied to other regions of the spinal cord, or be adapted to other SCI devices. &#160;Improving spinal cord exposure and fixation through vertebral stabilization may be valuable for producing consistent and reliable injuries to the spinal cord. &#160;This vertebral stabilization method can also be used for stereotactic injections of cells and tracers, and for imaging using two-photon microscopy in various neurobiological studies.

Vertebral stabilization is necessary for minimizing variability, and for producing consistent experimental spinal cord injuries.&#160; Using a customized stabilizing apparatus in conjunction with the NYU/MASCIS impactor device, we have demonstrated here the proper equipment and procedure for generating reproducible hemi-contusive cervical (C5) spinal cord injuries in adult rats.

Contusive 척수 손상의 제조 소설 척추 안정화 방법

Clinically-relevant animal cervical spinal cord injury (SCI) models are essential for developing and testing potential therapies; however, producing ...

Calibrated Forceps Model of Spinal Cord Compression Injury

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The calibrated forceps model of compression injury is a convenient, low cost, and very reproducible animal model for SCI. We used a pair of modified forceps in accordance with the method published by Plemel et al. (2008) to laterally compress the spinal cord to a distance of 0.35 mm. In this video, we will demonstrate a dorsal laminectomy to expose the spinal cord, followed by compression of the spinal cord with the modified forceps. In the video, we will also address issues related to the care of paraplegic laboratory animals. This injury model produces mice that exhibit impairment in sensation, as well as impaired hindlimb locomotor function. Furthermore, this method of injury produces consistent aberrations in the pathology of the SCI, as determined by immunohistochemical methods. After watching this video, viewers should be able to determine the necessary supplies and methods for producing SCI of various severities in the mouse for studies on SCI and/or treatments designed to mitigate impairment after injury.

Spinal cord injury models should be highly reproducible. We demonstrate that the calibrated forceps compression model of spinal cord injury is an easy to use surgical method for generating reproducible injuries to the murine spinal cord.

척수 압축 부상의 보정 집게 모델

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The ...

Inducing a Spinal Cord Contusion in a Mouse Model Using a Spinal Cord Injury Coaxial Platform

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Inducing a Spinal Cord Contusion in a Mouse Model Using a Spinal Cord Injury Coaxial Platform