The overall goal of this procedure is to investigate dorsal root regeneration in order to discover a means to induce significant regeneration after spinal root injuries. This is accomplished by first creating a spinal hemi laminectomy that exposes the right L five dorsal root and allows visualization of YFP labeled positive axons in the spinal cord and dorsal root. Next, the L five dorsal root is crushed carefully to ensure minimal bleeding.
The crush site is then imaged after crushing to verify the position of the injured dorsal root in the lesion. Over the course of several time points, the injury site and the dorsal root entry zone are imaged to determine the ability of the damaged axons to regenerate into the spinal cord. Ultimately, results can be obtained that show regenerating axons are rapidly immobilized and remain stationary as they enter the dorsal root entry zone or DREZ.
The main advantage of this technique over existing techniques such as imaging in a a dead animal, is that we can image this system in a living animal repeatedly over a period of weeks or even months. First place a thermostatically controlled heating pad under a fluorescent stereo microscope and adjust the output to 32.5 degrees Celsius on a heating pad, adjusted to 32.5 degrees Celsius prewarm sterile ringer solution to 32.5 degrees Celsius for irrigation of the spinal cord During surgery, anesthetize the animal with an intraperitoneal injection of a xylazine and ketamine cocktail. An initial dose of the analgesic buprenorphine is also given subcutaneously.
A suitable level of anesthesia is achieved when the palpable reflex is absent and the animal does not respond to a toe pinch. Once anesthesia has been confirmed, shave the upper back with small animal clippers and spread one small drop of hair removal lotion over the shaved area with cotton tipped swabs. Minutes later, remove the applied lotion and any remaining fur with soapy water.
Finally, using 70%ethanol soaked gauze sponges to completely clean the surgical field. Place the animal on the heating pad and disinfect the skin with 70%ethanol soaked swabs. A sterile surgical drape should be applied though for demonstration purposes.
It is not shown in this video. Under brightfield illumination on the stereo microscope, make a two to three centimeter midline incision in the skin of the back. If necessary, use cotton tipped swabs to stop bleeding.
Reflect the spinal musculature with fine forceps and bone jaws. To expose the underlying lumbar vertebrae. Expose the L three to S one spinal segments by right-sided hemi laminectomy using small r jurors and then perfuse the cavity with warm sterile ringer solution.
Position the animal on a support cushion made of rolled cotton gauze. To flatten the vertebral column. Use retraction hooks to widen the exposed area.
At this point, approximately 30 minutes after the first injection of anesthetic, a supplement should be injected in order to keep the animal fully anesthetized. Switch to fluorescent excitation to visualize why FP labeled axons. Using the tip of a 27 and a half gauge needle, perform a small incision in the dura overlying the L five dorsal root perfuse repeatedly with ringer solution and clean gently with cotton tipped swabs.
Identify the site to be crushed and capture images of the whole exposed undamaged area. This will aid in identifying the injury site in subsequent imaging sessions. Then insert one side of a pair of fine forceps.
Duly close the forceps gently but firmly holding the medial portion of the L five root for 10 seconds, and then gently release the forceps. Wash repeatedly with physiological solution and clean gently with cotton tipped swabs. Obtain multiple images of the whole exposed area, including the crush site and the DREZ before and right after the crush at both low and high magnification.
After imaging tightly, apply a piece of thin synthetic matrix membrane followed by artificial dura on top of the exposed spinal cord, such that they fit precisely in the exposed spinal cord window. Close the musculature with sterile five oh sutures and close the midline incision with wound clips. Inject ringer solutions subcutaneously and administer buprenorphine as postoperative analgesia via intramuscular injection every 12 hours for two days.
Anesthetize the animal with xylazine and ketamine as previously described, and remove the wound clips and sutures. Remove the artificial dura and thin synthetic matrix membrane patches and keep them in a sterile tube containing ringer solution for later reuse. Next, gently remove any accumulated connective scar tissue with the bent tip of a 26 gauge needle and fine forceps perfusing frequently with warm ringer solution.
Re-expose the operative field, including crush site and dorsal root entry zone, and relocate the YFP labeled axons imaged in previous sessions for continued imaging over regeneration. Here four superficial YFP labeled axons indicated by colored arrows are shown immediately after the crush. Three days after the crush, all four axons extend a single neurite that grows through the crush site as highlighted in a two five days after the crush.
Neurites remain stable and there is no additional growth from these or other proximal axons. At day four, regenerating neurites are thinner and dimmer than spared axons. Repeated imaging of these axons in their tips every two or three days for two more weeks reveal that they did not grow forward or retract, but remained immobile with swelling of the tips and shafts of some axons.
After watching this video, you should have a good understanding of how to expose, crush, and observe the L five do root in order to better understand the mechanisms that prohibit or promote axonal regeneration.
