Experimental Demyelination and Remyelination of Murine Spinal Cord by Focal Injection of Lysolecithin

The overall goal of this procedure is to study aspects of demyelination and subsequent remyelination in the mouse spinal cord. This is accomplished by first injecting the detergent lyin from a pulled glass capillary into the ventral spinal cord white matter. Then the spinal cords are processed at different time points to study aspects of repair, including oligodendrocyte precursor cell recruitment differentiation, and remyelination.

This method is useful not only for studying the normal events involved in remyelination, but also as a preclinical tool for screening remyelination promoting therapeutics. Start by removing a 75 microliter aliquot of 1%detergent solution from minus 20 degrees Celsius, storage and thaw to room temperature on the bench if the detergent is undissolved. Sonicate the tube at 40 kilohertz for about 30 minutes to get a uniform solution.

Next, unscrew the nut of a 10 microliter injecting syringe and thread it onto the flat end of a PrepU glass capillary. Add two varials and to make sure their endign snug around the capillary. Now rinse the syringe with isopropanol and remove the plunger.

Once dry hand tighten the needle assembly onto the injecting syringe. Then attach a metal hub needle to the priming syringe. Pierce a rubber disc with the needle and slide it down to the base.

Now fill the priming syringe with the room temperature lyo than detergent. Gently depress the plunger to remove unwanted air until the solution is visible at the needle's tip for control surgeries. Load PBS.

Now insert the metal hub needle of the priming syringe into the injecting syringe, making sure that there is a tight seal with the rubber disc. Then slowly depress the priming syringe until the capillary fills to the tip with solution. With the capillary filled carefully pull out the priming syringe while continuing to fill the barrel of the injecting syringe.

Try to avoid introducing air bubbles. Now insert the plunger into the injecting syringe and make sure that solution will flow from the tip. If any air bubbles are visible in the capillary or injecting syringe, repeat the syringe preparation from the beginning.

Continuous fluid in the injecting apparatus is critical to ensure accurate injection volumes. To finish, attach the injecting syringe to the arm of a stereotactic micro manipulator. This setup can inject 15 to 20 animals before a new capillary is needed.

First, anesthetize the animal, in this case a young adult female, C 57 black six mouse. Use ketamine and xylazine and plan for an hour of anesthesia. Ensure adequate sedation based on a lack of response to a firm foot pinch.

Then shave a two to three centimeter square area on the dorsal side close to the ears. Wipe the skin down with 70%ethanol on a gauze pad, removing all the hair. Then disinfect the area with iodine.

Be sure to apply an atoma ointment and keep the animal in a heated recovery chamber. Ready to begin the procedure. 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 the tooth clamp and fasten the arms and tail with surgical tape. Stabilizing ear bars are not necessary. Now use a scalpel to make a three centimeter midline incision starting just below the ears and cutting in the cuddle 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 T two vertebra characteristic of the C 57 black six 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 T three and T four to confirm proper anatomical location. Now make shallow lateral cuts in the connective tissue between T three and T four. 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 cerebral spinal fluid leaks from the arachnoid, remove it with the sponge spear. Begin the injection by moving the capillary into position over the spinal cord.

The prominent coddle 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 micro manipulator, make one full rotation every five seconds for two minutes to inject a total volume of 0.5 microliters. Then leave the capillary in place for two minutes. After two 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 uninterrupted 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. Postoperative care and analysis are described in the text protocol focal injection of lyin into the ventral white matter.

Produced a discrete demyelinating lesion that spanned approximately three millimeters. Immunohistochemical staining of the lesion core was performed, labeling myelin with MVP and labeling axons with SM I three 12. The axons were stripped of myelin at seven days and by 14 days, MVP positive rings surrounded many axons suggesting remyelination was occurring.

Staining cells of the oligodendrocyte lineage using PDG FFR alpha LIC two and CC one showed a significant increase in both the total number of cells at 14 days compared to seven days, as well as a greater distribution of mature oligodendrocytes compared to OPCs. Consistent with this finding semi thin sections stained with toluidine, blue revealed a presence of thin myelin sheets at 14 days that were rarely detected at seven days. Indicating the presence of remyelinate inter nodes With proficiency.

The operation can be performed in 10 to 15 minutes per animal, particularly with the help of a second person tying sutures.