Animal models of central cord syndrome could subsequently benefit pre-clinical research. The strategy in this study is advantageous because it's enabled the investigation of injury mechanisms by producing consistent results. The present protocol of simulating central cord syndrome in mice has improved repeatability, and minimized operational damage towards experimental animals.
Avoiding disruption of their anatomical structure. Begin by organizing the surgical instruments and the spinal cord injury coaxial platform. Once the mouse is properly anesthetized, identify the small protrusion along the midline behind the animal's neck, which is the spinous process of the second thoracic vertebra, or T2.Shave the hair surrounding the T2 spinous process behind the neck, and disinfect the skin with three alternating rounds of Iota 4 solution and 75%ethanol.
Then, place the mouse in a prone position on the operation table. Apply an eye ointment to protect the eyes. Position a 3 to 4 millimeter thick pad beneath the animal's chest to facilitate the arching of the cervical spine curve, which allows the exposure of the interlaminar space and also an unhindered air passage during surgery.
Next, while working under a microscope, use a scalpel to make a 1 to 1.5 centimeter longitudinal incision centered on the T2 spinous process, and expose the fascial layer. With the help of micro scissors, remove a portion of the adipose tissue above T2 to expose the T2 spinous process clearly. Using the micro scissors, separate the bilateral trapezius and rhomboid muscles from C5 to T2 along the midline.
Then, separate the muscles on the lamina of C5 to T2 vertebrae, and use micro retractors to retract the muscle layer to the sides. Cut the multifidus and cervical spinal muscles present on the surface of the vertebrae. Locate the T2 vertebra by identifying the highest point of the spinous processes.
Then, continue to probe the spinous processes successively from T2, moving towards the rostral end, until the sixth cervical vertebra or C6 is located. Using forceps, lift the C6 lamina to expose the spinal cord before cutting the lamina off. Clamp the C6 to C7 facet joints with the vertebral stabilizer, and lock it.
Align the weight tip with the exposed spinal cord, ensuring the bottom of the tip is parallel to the dorsal surface of the spinal cord. Adjust the sleeve to let the weight compress the spinal cord. Stop adjusting when the weight maintains a constant relative position with the spinal cord.
Remove the weight and the vertebral stabilizer after allowing a five-minute compression. Under the microscope, observe any color changes to the spinal cord resulting from the compression. Rinse the operation site using sterile phosphate buffered saline.
Following which, use suction to clean the area. Suture the muscles and skin in layers using a polypropylene non-absorbable 6-0 suture. After disinfecting the surgical area, place the mouse on a warm pad until it regains full consciousness before returning it to its cage.
The hematoxylin and eosin or H&E staining of the spinal cord sagittal section suggested that the area damaged in the gray matter was wider in the severe injury group than that in the mild injury group. The coronal HE sections showed that in both groups, the lesion mainly existed in gray matter. In the severe group, although the structure of the white matter surrounding the gray matter was more impacted, its outline was still maintained.
Immunofluorescent staining also revealed that though the white matter surrounding gray matter was affected in the severe group, it was still relatively intact. No red blood cells were found in sagittal HE sections at seven days post-injury in either the mild or the severe group. The Prussian blue staining revealed hemosiderosis only in the severe group.
Immunofluorescence revealed areas of elevated GFAP and Iba1 expression in both mild and severe injury, suggesting an inflammatory response and formation of a glial scar. Also, the severe group exhibited a larger lesion area than the mild group. Magnetic resonance imaging suggested that in both groups, there was a hypo intense signal change in the lesion with a high signal outline.
The severe group showed a significantly larger hypo intense signal area. Removing the soft tissues in front of and behind the lamina as much as possible facilitates lamina relaxation and proven damage to the span cut when parallelly cutting. The present protocol phase volume models central cord syndrome, and enables further understanding and exploration of the signal.
