This method can help answer questions related to the role of neural precursor cells in regenerative medicine. Specifically, we can ask how injury affects the behavior of neural precursor cells. The main advantage of this technique is that it provides an approach to study the effect of spinal cord injury on endogenous neural precursor cell kinetics.
Visual demonstration of these techniques are critical. As the surgical and dissection steps requires precision, and can be difficult to learn, although they can be perfected with practice. Before beginning the procedure, use hair clippers to remove the fur from the dorsum of an anesthetized mouse from the mid back to the neck and ears.
After securing the mouse in a stereotactic device, place four to five pieces of rolled gauze beneath the abdomen, while lightly pulling on the base of the tail to straighten out the body and spine. Push the thoracic gauze roll rostrally from the mouse abdomen toward the upper thorax to support the thoracic spine. And use laboratory tape to secure the tail and limbs in a star-like orientation.
After disinfecting the exposed skin with sequential 70%ethanol and povidone iodine swabs, use a number 10 scalpel blade to make a vertical incision parallel to the longitudinal axis of the animal, from the midpoint of both shoulder blades, to the curvature of the thoracic spine. Retract the skin to expose the soft tissue and the spinal column contour. And identify the lower border of the super scapular fat pad.
Then, use the scalpel to carefully but forcefully cut along both sides of the vertebral T5 to T8 and nine bones, to detach the back muscle tendons from the spinal column. Reposition retractors so that the teeth of the retractors are inserted into the incision site on either side of the spine. And expand each retractor to adjust the exposure, until the spine is sufficiently elevated without putting too much strain on the retracted muscle layers.
Under a surgical microscope, carefully clean the residual muscle and other soft tissue overlying the spine, to expose the vertebral bone. Clasping the spinous process of the vertebrae with toothed forceps, and moving the forceps slightly up and down to identify the vertebrae that will be removed. Next, insert one tip of a pair of curved blunted scissors into either side of the exposed intervertebral foramen, caudal to the vertebral lamina to be excised.
And cut the connecting intervertebral joints bilaterally. Then, lift the lamina upwards and cut off the upper attachment of the lamina to isolate and remove the bone. When cutting away and removing the vertebral lamina, take care to always angle the scissors upwards so not as to damage the underlying spinal cord.
With the dorsal midline veins serving as a landmark, insert the 45 degree bent shaft of a 30 gage needle tip, bevel side up, one millimeter deep into the dorsal lateral surface of the spinal cord. And approximately 0.5 millimeters lateral to either side of the midline. Move the needle about two millimeters from the caudal to rostral direction, so that the entire length of the bevel is inserted into the cord.
Then, retrace the path of the entry to remove the needle. To close the wound, remove the retractor and use a 6-0 absorbable suture to join the back muscles on either side of the injury along the midline. And a 4-0 silk suture to close the overlying skin.
To isolate the neurospheres, make a midline incision in the skin along the entire length of the back to expose the muscle and vertebral bone contour. Lift the medial aspect of each scapula and use scissors to cut away the soft tissue between the scapula and vertebrae. When the underlying vertebral column has been exposed, insert the scissors into the upper thoracic aperture, following the curvature of the spinal column, and cut away the attached ribs, muscle and internal organs to isolate the vertebral column.
Insert the scissors into the caudal vertebral foramen, and cut the intervertebral joints on either side of the laminae, taking care to not damage the intact cord. When the appropriate length of the cord is exposed, carefully cut any spinal nerves extending laterally from the cord, before transferring the spinal cord tissue into a Petri dish of regular, artificial cerebral spinal fluid on ice. Under a dissection microscope, cut the spinal cord tissue to include about two millimeters of tissue rostral and caudal to the injury site, and use two pairs of fine forceps to gently separate the cord into two longitudinal halves, along the dorsal and ventral fissures.
Use the micro scissors to cut out the injured dorsal lateral white matter. And place the injured tissue into a 15 milliliter conical tube. Holding one half of the spinal cord with the forceps, tease and remove the uninjured white matter with two pairs of forceps along the rostral caudal extent of the spinal cord, to isolate the appropriate periventricular region.
After isolating the injured white matter and associated periventricular regions from the other half of the spinal cord in the same manner, place the pieces of periventricular tissue into individual 15 milliliter conical tubes. And use the micro scissors to gently mince the tissue against the walls of the tubes. Once the tissue digestion procedure has been completed, and the single cell suspension has been obtained, set up a neurosphere culture by adding 10 milliliters of the appropriate medium into one 25 milliter tissue culture flask per cell condition.
And add the isolated neural stem cells up to a clonal density of 10 cells per microliter for their culture in a cell culture incubator. After 24 hours, gently swirl the flasks and decant the contents into individual 15 milliliter conical tubes for centrifugation. Resuspend the pellets in one to two milliliters of fresh neural basal medium for counting.
And plate the cells at clonal density in one 24 well tissue culture plate per condition, containing 500 microliters of the appropriate corresponding mitogen supplemented medium per well. Then, grow the neurospheres in the cell culture incubator for seven days. Five days following the minimal spinal cord injury, the absolute numbers of definitive neural stem cell derived neurospheres is greater than the number of primitive neural stem cell derived neurospheres.
Definitive neurospheres exhibit a larger diameter and a large dark center. While primitive neurospheres are smaller in size and more tightly packed. Minimal spinal cord injury induces a significant increase in neurospheres grown from the central canal at the level of injury, compared to a relatively modest increase at the central canal, rostral to the level of injury.
Definitive neurospheres can also be generated from the lesioned white matter at the level of the injury. A region that does not contain neurosphere forming cells in laminectomy alone animals. A similar increase in primitive neural stem cells is observed post injury, with the exception of primitive neurospheres at the white matter at the level of the lesion site.
Once mastered, the surgical technique can be performed in under 40 minutes. And the tissue dissection and isolation can be performed under 30 minutes, if performed properly. Following the surgical procedure, depending on the animal strain, additional methodologies such as immunohistochemistry and lineate shocking, can be used to answer additional questions such as proliferation kinetics, and self fate of the neural precursor cells after injury.
After watching this video, you should have a good understanding of how to utilize the neurosphere assay to assess neural stem cell activation, following spinal cord injury.
