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 ) models .  
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 level , and several sci studies have been performed using animal cervical injury models .  
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 ) impactor ( figure 2 ) , precision systems and instrumentation , llc infinite horizon ( ih ) device , ohio state university / electromagnetic spinal cord injury device , and the louisville injury system apparatus ( lisa ) , allowing for widespread use in sci research .
clean the surgical surface with 70% ethanol , pre - warmed with a heating pad . cover the surface with a sterile surgical drape before placing sterile gauze , cotton swabs , and
use a microbead sterilizer for inter - surgery sterilization of surgical tools .   anesthetize the rat with ketamine ( 87.7 mg / kg)/xylazine ( 12.3 mg / kg ) intraperitoneally ( ip ) .  the proper plane of anaesthesia is reached when the animal ceases to respond to a toe pinch stimulus . 
subcutaneously inject 0.01 - 0.05 mg / kg buprenorphine and 5 mg / kg carprofen prior to surgical procedure .  
buprenorphine should then be administered every 8 - 12 hr  and carprofen once daily , for the first 4 - 7 days following surgery . 
apply protective ointment to the eyes of the animal to prevent corneal drying during surgery .
shave the surgical area on the dorsal surface of the rat from the mid - thoracic region to the back of the head with clippers . 
apply betadine solution to the shaved area as a surgical scrub then clean the area with 70% isopropyl alcohol wipes .
use a scalpel blade to perform a 3 - 4 cm midline incision in the skin from the base of the head caudally to mid - thorax .
identify the midline of the fascia and subcutaneous muscles anterior to the hibernating gland at the lower neck ; cut through the trapezius and other muscles along the midline to reduce hemorrhage .  
find the midline of two regions of adipose tissue underlying the muscles ; cut the paraspinous muscles caudally strictly along the midline , and separate muscle layers using a small tissue retractor until the level of the thoracic t2 spinous process is reached .
identify and cut away the muscle connected to the t2 spinous process to utilize this structure as an anatomic landmark .   remove the cartilaginous tip of the t2 spinous process to improve visibility of the cervical vertebrae . 
separate the paraspinal muscles laterally from the spinous processes and laminae of c4-t1 ; however , spare the muscle covering on the c3 lamina to prevent bleeding .  
cut the muscles over the laminae from c4-t1 laterally towards the facets on both sides of the spinal column . 
after the spinal laminae are exposed , place the animal on its ventral surface in the u - shaped channel of the stabilizer . 
identify the c5 vertebra by counting the spinous processes rostrally from the t2 landmark to t1 , c7 , c6 , and finally c5 . position the two stainless steel arms of the stabilizer to suspend the animal by placing the serrated edges of the arms underneath the lateral facets of the c5 - 6 vertebrae ( figure 1c ) .   after securing the arms with vertebrae in place ( figure 2b ) ,
cut the ligaments between the spinal processes and laminae at c4 - 5 and c5 - 6 to identify the margin of the c5 lamina . using a micro - rongeur , clip away the half of the lamina on the right at c5 as intended for sci ( figure 5c - e ) . 
secure the animal together with stabilizer on a mount ( figure 3a - c ) to precisely align the plunger on the spinal cord target using a lateral micro - manipulator ( figure 3 ) . under high magnification , locate the c5 and c6 dorsal root entry zones ( drezs ) on the exposed dorsal spinal cord surface without durotomy .  aim the plunger at the middle of the two identified drezs and halfway between the midline and the lateral edge of the spinal cord ( figure 5b ) .
using an nyu / mascis impactor device with a 2.5 mm diameter tip ( figure 3a & b ) , produce a  c5 hemi - contusion  ( figure 5b & e ) by a 10 g rod x 12.5 mm height drop ( figure 2a ) . verify the injury visually by bruising on the spinal cord ( figure .
5e , arrow ) and check the injury parameters provided by the nyu software(figure 6 ) . suture muscles and soft tissue with sterile 4 - 0 vicryl suture , then close the skin incision with surgical staples ( ez clips ) .
place the animal in a heat - controlled environment ( recirculating hot water padcage on a heating pad )  ) with moist food provided on the bedding ( changed daily ) , and a water bottle with a long spout for easy access placed on the floor of the cage . 
as this is a unilateral cervical contusive injury , the animal will likely lose function of the ipsilateral forelimb , transiently , which begins to recover during the first few weeks after injury
.   however , contralateral function should remain intact , thus the animal should be able to eat and drink without impairment , and have only minor impairment in locomotion and grooming .
clean the surgical surface with 70% ethanol , pre - warmed with a heating pad . cover the surface with a sterile surgical drape before placing sterile gauze , cotton swabs , and
use a microbead sterilizer for inter - surgery sterilization of surgical tools .   anesthetize the rat with ketamine ( 87.7 mg / kg)/xylazine ( 12.3 mg / kg ) intraperitoneally ( ip ) .  the proper plane of anaesthesia is reached when the animal ceases to respond to a toe pinch stimulus . 
subcutaneously inject 0.01 - 0.05 mg / kg buprenorphine and 5 mg / kg carprofen prior to surgical procedure .  
buprenorphine should then be administered every 8 - 12 hr  and carprofen once daily , for the first 4 - 7 days following surgery . 
apply protective ointment to the eyes of the animal to prevent corneal drying during surgery .
shave the surgical area on the dorsal surface of the rat from the mid - thoracic region to the back of the head with clippers . 
apply betadine solution to the shaved area as a surgical scrub then clean the area with 70% isopropyl alcohol wipes .
use a scalpel blade to perform a 3 - 4 cm midline incision in the skin from the base of the head caudally to mid - thorax .
identify the midline of the fascia and subcutaneous muscles anterior to the hibernating gland at the lower neck ; cut through the trapezius and other muscles along the midline to reduce hemorrhage .  
find the midline of two regions of adipose tissue underlying the muscles ; cut the paraspinous muscles caudally strictly along the midline , and separate muscle layers using a small tissue retractor until the level of the thoracic t2 spinous process is reached .
identify and cut away the muscle connected to the t2 spinous process to utilize this structure as an anatomic landmark .   remove the cartilaginous tip of the t2 spinous process to improve visibility of the cervical vertebrae . 
separate the paraspinal muscles laterally from the spinous processes and laminae of c4-t1 ; however , spare the muscle covering on the c3 lamina to prevent bleeding .  
cut the muscles over the laminae from c4-t1 laterally towards the facets on both sides of the spinal column . 
after the spinal laminae are exposed , place the animal on its ventral surface in the u - shaped channel of the stabilizer . 
identify the c5 vertebra by counting the spinous processes rostrally from the t2 landmark to t1 , c7 , c6 , and finally c5 .
position the two stainless steel arms of the stabilizer to suspend the animal by placing the serrated edges of the arms underneath the lateral facets of the c5 - 6 vertebrae ( figure 1c ) .   after securing the arms with vertebrae in place ( figure 2b ) , adjust the stabilizing apparatus to ensure the vertebral column is level and centered . 
cut the ligaments between the spinal processes and laminae at c4 - 5 and c5 - 6 to identify the margin of the c5 lamina . using a micro - rongeur , clip away the half of the lamina on the right at c5 as intended for sci ( figure 5c - e ) . 
secure the animal together with stabilizer on a mount ( figure 3a - c ) to precisely align the plunger on the spinal cord target using a lateral micro - manipulator ( figure 3 ) . under high magnification , locate the c5 and c6 dorsal root entry zones ( drezs ) on the exposed dorsal spinal cord surface without durotomy .  aim the plunger at the middle of the two identified drezs and halfway between the midline and the lateral edge of the spinal cord ( figure 5b ) .
using an nyu / mascis impactor device with a 2.5 mm diameter tip ( figure 3a & b ) , produce a  c5 hemi - contusion  ( figure 5b & e ) by a 10 g rod x 12.5 mm height drop ( figure 2a ) . verify the injury visually by bruising on the spinal cord ( figure .
5e , arrow ) and check the injury parameters provided by the nyu software(figure 6 ) . suture muscles and soft tissue with sterile 4 - 0 vicryl suture , then close the skin incision with surgical staples ( ez clips ) . apply antibacterial ointment to the surgical site .
place the animal in a heat - controlled environment ( recirculating hot water padcage on a heating pad )  ) with moist food provided on the bedding ( changed daily ) , and a water bottle with a long spout for easy access placed on the floor of the cage . 
as this is a unilateral cervical contusive injury , the animal will likely lose function of the ipsilateral forelimb , transiently , which begins to recover during the first few weeks after injury .  
however , contralateral function should remain intact , thus the animal should be able to eat and drink without impairment , and have only minor impairment in locomotion and grooming .
upon following this protocol , consistent and reproducible cervical hemi - contusive sci is produced ( figure 5 & 6 ) .
the use of a vertebral stabilizer to stabilize the lateral processes of the same vertebra at the level intended for sci allows for such satisfactory results . using this method , not only the target c5 vertebra , but also adjacent c4 and c6 are rigidly fixed .
the nyu / mascis software provides a read - out with a graph set on an x and y - axis , and supports the use of our vertebral stabilization method , and equipment ( figure 6 ) .
this method of stabilization reduces injury variability that can result from the downward shifting of the target tissue and spinal column ( figure 1 ) . following injury , a clear unilateral bluish hematoma centered between the c5 and c6 drezs is visible ( figure 5e ) .
these injury parameters are consistent from animal to animal according to the readout provided by the nyu / mascis software ( figure 6 ) .
as the cervical hemi - contusion produces clear forelimb deficits , this model is ideal for assessing forelimb functional abilities such as reaching , grooming , and object manipulation .
as hindlimb motor deficits are less prominent , the basso , beattie and bresnahan ( bbb ) locomotor scoring scale is not appropriate for use in this model .
the functional outcome following injury is most noticeable in the ipsilateral forepaw extensor deficits , in which the rat exhibits a  clubbed 
all animals exposed to the same injury severity and level of the spinal cord should exhibit similar deficits to the ipsilateral forelimb illustrated in this protocol , upon correct injury .
histologically , this model produces extensive gray and white matter damage at the injury epicenter and rostral and caudal to the site of injury , leading to considerable lesion and cavity formation contained almost exclusively within the injured side of the spinal cord . a large ,
primarily astrocyte - based glial scar forms at the lesion borders with massive neuronal death .
yield  of the spine when spinous processes are clamped dorsally , allowing for improper impacts and inconsistent data .
the illustration shown in a displays much more flexibility upon impact ( red dashed line and large curved arrows ) compared to that shown in b ( smaller curved arrows ) , as the clamps are farther from the site of laminectomy and injury .
figure c shows lateral stabilization with our described device with the stabilizing arm securely tightened under the transverse process of the vertebra where the site of impact will be performed .
there is no flexibility of the spine during this procedure , as the vertebra of interest is completely stabilized .
figure a displays the parts and features of the nyu / mascis spinal cord injury device , with multiple rod height settings for injury severity ( inset ) .
figures b and c illustrate the u - shaped container that holds the rat , and the serrated stabilization arms that securely stabilize the vertebral column during surgery and injury ( designed and produced by y.p .
figure 3 : custom mounting system and lateral microadjuster on the nyu / mascis impactor .
figure a details the different components of the custom mounting system for the u - shaped rat stabilizer for spinal cord injury .
note the lateral microadjuster in figure a , crucial for precise alignment of the rat spinal cord for injury .
figures b and c provide further depiction of the stabilizer without ( b ) and with the u - shaped rat container ( c ) with respect to other important components of the injury device ( mounting system designed and produced by y.p .
figure 4 : measurements of the individual components of the surgical stabilization device and attachments .
each component of the custom stabilization system is highlighted to show the dimensions and scale ( a , c , and d ) .
thoracic stabilization arms ( b ) are shown to display the potential application of this device for use in different spinal surgical models .
figure 5 : surgical landmarks and preparation for cervical hemi - contusion spinal cord injury .
figures a and b portray the correct landmarks for proper impact alignment on the exposed rat spinal cord . the appropriate impact point is directly between the c5 and c6 dorsal nerve roots ( rostral - caudal ) and the midline and lateral edges of the spinal cord ( b ) .
figures c - e show , in higher magnification , the process of exposing the desired half of the cervical spinal cord for injury , through careful unilateral laminectomy .
also , figures d and e demonstrate the cord immediately before and after spinal cord contusion injury .
note the visible hemorrhage ( e ) caused by the impact ( black arrow ) .
examples of acceptable versus unacceptable data readouts following impact with the nyu / mascis impactor .
the top graph ( a ) and top data set ( c ) illustrate a readout of a very good impact , with data measurements of  % error  for impact rod velocity , initial height , and starting time , as indicated with the red arrow and underline .
conversely , the bottom panel demonstrates data produced by an improper impact caused by improper stabilization of the spinal column ( b ) and error during  zeroing  of the impactor rod and tip onto the spinal cord surface , prior to setting the height of the impactor rod ( c ) .
note the considerable error indicated for the initial height and start time of the impactor drop , as indicated by the red arrow and underline .
the software also provides a warning that error has been detected for these parameters ( bottom of panel c ) .
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 deficits .  
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 sci . 
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 journal .   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 .
 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 . 
Introduction
Protocol
1. Exposure of the Cervical Spinal Laminae
2. Stabilizing the Vertebrae and Performing the Impact Injury
Representative Results
Discussion
Disclosures