Vertebral stabilization is necessary for minimizing variability, and for producing consistent experimental spinal cord injuries. 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.
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. 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). Here we introduce a new method to completely stabilize the cervical vertebra at the same level of the impact injury. This method effectively minimizes movement of the spinal column at the site of impact, which greatly improves the production of consistent SCIs. 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. 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. Improving spinal cord exposure and fixation through vertebral stabilization may be valuable for producing consistent and reliable injuries to the spinal cord. 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.
Consistent and replicable mechanical force on target spinal tissue is critical for minimizing functional and histological variability and for establishing successful contusive spinal cord injury (SCI) models1-7. The amount of force applied to a target region of the spinal cord depends on the methods utilized for spine stabilization. Positional shifting of the target spine during contact between the impact plunger and the spinal cord alters the resultant injury force. The cervical contusive SCI model is a more clinically relevant model than other forms of SCI, as approximately 50% of human SCI cases occur at this level8, and several SCI studies have been performed using animal cervical injury models9-14. Although contusive SCI models often utilize some form of stabilization by clamping the spinal processes anterior and posterior to the injury site, this preparation is difficult for producing cervical SCI. As shown in this demonstration, the stabilization method we developed is advantageous in its ability to increase both the quality and reproducibility of contusion injury. Particularly, this method of vertebral stabilization was established in an attempt to amend the shortcomings and challenges of other models: 1) variability in vertebral yielding under the impact force may occur by clamping adjacent dorsal spinous processes rostral and caudal to the laminectomy. The degree of vertebral shifting is dependent on the number of vertebral joints between the impact and the vertebrae being stabilized (Figure 1). Therefore, the more joints involved the less stable the spine becomes; 2) the dorsal spinous processes are fragile and cause clamp failure as a result of spinous process fracture or the clamp slipping off of the process; and 3) the spinous processes on these vertebrae are extremely short between the C3 to T1 vertebrae compared to those of the thoracic vertebrae, which makes it difficult using traditional clamps to grasp the spinous processes for stabilizing the cervical spine.
Here we describe a novel method of stabilizing the spine for producing C5 contusive SCI in adult female Sprague-Dawley rats. This method can be used for stabilization of other levels of the vertebral column and spinal cord, and conjugates well with other contusive SCI devices, including the New York University/Multicenter Animal Spinal Cord Injury Study (NYU/MASCIS) impactor15 (Figure 2), Precision Systems and Instrumentation, LLC Infinite Horizon (IH) device16, Ohio State University/Electromagnetic Spinal Cord Injury Device1, and the Louisville Injury System Apparatus (LISA)17, allowing for widespread use in SCI research.
Here we have demonstrated a cervical spine stabilization method for producing unilateral contusive SCI at C5. This stabilization method increases the precision of the trauma anatomically and produces consistent functional deficits13,18. In other models that rely on dorsal clamping of the spinous processes, the risk of spinous process damage or detachment of the clamps from the vertebra is quite high. These models may also allow considerable spine shifting and yielding from the contusion force and the flexible nature of the spine and vertebral columns (Figure 1A and B). The tissue yielding alters the plunger-tissue contact time and results in unpredictable injury force (Figure 1A-B & 6B). Our described vertebral stabilization also provides other benefits to the surgical preparation: 1) this method fully stabilizes the vertebrae centered at C5 under the surgical microscope which increases the accuracy of laminectomies (Figure 1C); 2) the animal mounted within the U-shaped stabilizer can be taken directly from the surgical location to the customized mounting attachment, which avoids the procedure of remounting the animal on SCI devices and saves time; and 3) stabilizing the vertebrae at the injury level and directly dorsal and caudal to the intended site of injury can greatly diminish the body movement caused by respiration, another measure to reduce variability.
The primary advantage of utilizing this stabilization method is the reduced amount of yielding, or ventral movement of the spinal cord and column upon impact. Based on simple physics of a contusion injury, the force and energy of the impact will transfer from the rod to the spinal cord, ideally with the cord absorbing this energy at the site of impact. However, if the spine yields beneath the cord, as is possible in the dorsal spinous clamping method (Figure 1A & B), the actual force applied to the cord is decreased and variable, depending upon the degree of yield.
Although this video illustrates the entire procedure of a cervical contusive SCI model, the essence of this article is the introduction of the spinal stabilization method we use in various applications in our lab, specifically for SCI studies. A modified version of this stabilization device and method has been used on mice SCI23. This simple method of spine stabilization is highly useful for SCI research, and we have previously used this method and equipment to perform thoracic contusion as well as laceration SCI models. Another lab has recently described a variation of this form of stabilization for cervical injury in this journal22. In summary, we introduce this novel vertebral stabilization method to several surgical procedures for generating reproducible experimental SCI ranging from laminectomy to injury production. The benefits of this stabilization device are not limited to cervical spinal cord contusion, as this stabilization method has been adapted for a wide variety of experiments such as intra-spinal injections, cellular transplantation, CSF collection from the cisterna magna, hemisection and transection injuries, thoracic contusion injuries, in vivo imaging employing two-photon microscopy, and spinal electrophysiological recording. Increasing the quality of the spinal surgical and injury procedures and reducing the experimental variability will help provide insight into true mechanisms of injury and recovery, and screen the effects of different therapies on the devastating disorder of SCI.
The authors have nothing to disclose.
This work was supported by the National Institutes of Health [NS36350, NS52290, and NS50243 to X–M.X.]; Mari Hulman George Endowment Fund; the State of Indiana; and a Ruth L. Kirschstein National Research Service Award (NRSA) 1F31NS071863 to C.L.W.
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Dukal Gauze Sponges | Fisher Scientific | 22-415-490 | |
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Antiobiotic Ointment | Webster Veterinary | 07-877-0876 | |
Cotton Tipped Applicators | Fisher Scientific | 1006015 | |
Rongeur | Fine Science Tools | 16000-14 | |
Surigical Scissors | Fine Science Tools | 15009-08 | |
Scissors (blunt dissection) | Fine Science Tools | 14040-10 | |
Surgical Retractor | Fine Science Tools | 17005-04 | |
Large Forceps | Fine Science Tools | 11024-18 | |
Fine Forceps | Fine Science Tools | 11223-20 | |
Hemostat | Fine Science Tools | 13004-14 | |
Scalpel | Fine Science Tools | 10003-12 | |
Scalpel Blade #15 | Fisher Scientific | 10015-00 | |
EZ Clips | Fisher Scientific | 59027 | |
Sterile sutures | Fine Science Tools | 12051-10 | |
Instrument Sterilizer | Fine Science Tools | 14040-10 | |
Surgical Stabilizer | Custom Manufactured | N/A | Contact Y. Ping Zhang for details. (yipingzhang50@gmail.com) |
Vertebral Stabilization Bars (clawed endfeet) | Custom Manufactured | N/A | Contact Y. Ping Zhang for details. (yipingzhang50@gmail.com) |
NYU/MASCIS Impactor Device | Custom Manufactured | W. M. Keck Center for Collaborative Neuroscience Rutgers, The State University of New Jersey e-mail: impactor@biology.rutgers.edu |