Demyelination and Remyelination of Mouse Spinal Cord Neurons

Begin with an anesthetized mouse positioned on a stereotactic frame, with an incision exposing a region of the spinal cord.

Take the syringe containing lysolecithin, a detergent. Position it over the exposed area and touch one side of the midline.

Inject lysolecithin into the spinal cord's white matter, targeting the neurons with myelinated axons, which facilitate faster signal transmission.

Keep the syringe in place briefly to prevent backflow, then remove it.

Suture the incision, apply an antiseptic to prevent infection, and allow the mouse to recover.

Injected lysolecithin disrupts the myelin sheath around the neurons, impairing axonal signal transmission.

This process also triggers inflammation, recruiting immune cells to clear the myelin debris, causing localized demyelination of axons, a neuropathological condition.

Over time, oligodendrocyte precursor cells migrate to this site and differentiate into oligodendrocytes.

Later, these oligodendrocytes regenerate the myelin sheath around the demyelinated neurons and gradually restore the signal transmission.

Move the animal to a stereotactic frame, dorsal side up, elevated at the midsection with folded paper towels, to exaggerate the curvature of the spine. Then, secure the head with a tooth clamp, and fasten the arms and tail with surgical tape. Stabilizing ear bars are not necessary.

Now, use a scalpel to make a 3 centimeter midline incision, starting just below the ears and cutting in the caudal direction. Then, locate the divide between the two large adipose structures and use fine forceps in each hand to pull these apart. Spread the retractors to open the surgical field.

Under a surgical microscope, locate the prominent bony outgrowth of the T2 vertebra, characteristic of the C57 black 6 mouse strain. To view the vertebra, make a blunt dissection of the overlaying musculature with closed spring scissors. Then, feel for the hard surfaces of T3 and T4 to confirm proper anatomical location.

Now, make shallow lateral cuts in the connective tissue between T3 and T4. Be mindful that too deep a cut will pierce and damage the cord. Some bleeding is common and can be cleared with a sponge spear. The spinal cord is covered with a thick layer of dura, and will be visible if this meningeal layer was not yet cut.

If the dura remains intact, carefully remove it using gentle lateral scrapes with a 32-gauge metal needle. Avoid piercing the remaining underlying meninges, which are not as thick and are harder to see. If any cerebrospinal fluid leaks from the arachnoid, remove it with a sponge spear. Begin the injection by moving the capillary into position over the spinal cord.

The prominent caudal rostral blood vessel should not be used as a midline landmark. Instead, use the gray white matter boundaries flanking the dorsal column to estimate the midline. Now, slowly lower the capillary until the tip just barely touches the spinal cord, immediately lateral of either side of the midline. Lock the arm in place at this position.

Then, make note of the baseline position on the graded measurements of the z direction on the stereotactic arm. From this reading, subtract 1.3 millimeters, and use a quick and shallow downward motion to pierce the tissue, carefully lowering the capillary to the desired depth.

Now, using the micromanipulator, make one full rotation every 5 seconds for 2 minutes to inject a total volume of 0.5 microliters. Then, leave the capillary in place for 2 minutes. After 2 minutes, carefully retract the capillary from the tissue. This allows the solution to seep in. It could otherwise backflow.

Move the injecting apparatus away from the animal and remove the retractors. Close up the animal, by tying a single suture in the muscle adipose tissue overlaying the spinal column. Then, use a non-interrupted suture to close the skin, and apply more iodine to the incision site. Finish by placing the animal in a heated recovery chamber until it recovers. Then, return it to its cage. Post-operative care and analysis are described in the text protocol.