This study investigates motor function in mice using a treadmill test and examines the effects of myelin-mimicking peptides on the nervous system. The methodology includes the induction of experimental autoimmune neuritis (EAN) to assess motor dysfunction.
Place a mouse on a treadmill to record its running motion, assessing its motor function, which represents the ability to control body movements.
After the test, inject Pertussis toxin into the abdominal cavity of the anesthetized mouse, to weaken its blood-nerve barrier.
Next, remove the hair between the mouse's shoulder blades, disinfect the area, and inject myelin-mimicking peptides with an adjuvant into the subcutaneous tissue.
Repeat both injections for optimal reactions.
The peptides stimulate antigen-presenting cells, which migrate to the lymph nodes and produce autoreactive T-cells.
These T-cells enter the bloodstream, cross the impaired blood-nerve barrier, and target peripheral nerves, leading to the release of cytokines.
This attracts macrophages and B-cells, which release effector molecules that degrade the myelin sheath.
The breakdown of myelin impairs nerve signal transmission and the onset of experimental autoimmune neuritis or EAN.
Finally, repeat the treadmill test. The mouse shows difficulty running and coordinating limb movements, indicating motor dysfunction.
Three days before the disease induction, turn on the motor function assessment apparatus and switch on the light button. Pick up the mouse and lower it onto a container of red food dye. Place the mouse into the walking compartment and set the treadmill speed to 15 centimeters per second. Turn on the treadmill, and click "record" to use the gait function imaging system to capture the running motion of the mouse for 36 seconds.
After 36 seconds, stop the recording and the treadmill, and save the practice run video file of the practice run in the designated folder.
One day before the first immunization, use a 0.5 milliliter syringe equipped with a 301/2 gauge needle to administer 1.6 micrograms per milliliter of pertussis toxin in 250 microliters of mouse isotonic PBS IP, to anesthetized six-to-eight-week-old male C57 black 6 mice.
The next day, combine equal volumes of freshly prepared solutions of peptide in 0.9% saline and 20 milligrams per milliliter of Mycobacterium tuberculosis in complete Freund's adjuvant in a bead beater, and mix the solutions at the maximum speed for 1 minute at room temperature.
Load a syringe equipped with a 23 gauge needle with the inoculum, and invert and shake the syringe before evacuating any air within the syringe shaft.
Before delivering the inoculum, remove the hair just lateral to the midline between the scapulae and disinfect the exposed skin with 70% ethanol.
Then, use a pair of Addison forceps to grasp the skin and deliver 50 microliters of the solution to the pertussis toxin-injected mouse via subcutaneous injection.
The next morning, deliver 1.2 micrograms per milliliter of pertussis toxin in 250 microliters of PBS IP, as just demonstrated, followed by another 1.2 micrograms per milliliter injection of pertussis toxin IP 48 hours later.
Three days later, after the third pertussis injection, administer another 50 microliters of freshly prepared inoculum at the same injection site as before, and monitor the clinical score and motor function of the animals twice a week until the end of the experiment.
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