Currently there is no complete cure for multiple sclerosis. Our protocol allows for the effective construction of experimental autoimmune encephalomyelitis model to help explore multiple sclerosis disease therapies. The main advantage of our protocol is a more comprehensive assessment of the treatment of experimental autoimmune encephalomyelitis from multiple perspectives.
First prepare myelin oligodendrocyte glycoprotein peptide emulsion by dissolving lyophilized peptide and sterile pre-cooled PBS without calcium and magnesium ions at pH 7.4. Add one sterilized five millimeter steel ball to a clean two milliliter micro centrifuge tube. Then add 500 microliters of complete Freuds's adjuvant containing five milligrams per milliliter of lyophilized microbacterium tuberculosis and 500 microliters of the myelin oligodendrocyte glycoprotein peptide emulsion to the tube.
Now oscillate the tube on a tissue lyser for 10 minutes. Then cool the tubes on ice for 10 minutes. Repeat the process four times until a white viscous solution is formed.
Next, dilute the pertussis toxin stalk solution 50 times in sterile PBS of pH 7.4 without calcium and magnesium ions to achieve a final concentration of 200 nanograms per 100 microliters. To precipitate all the initially prepared myelin oligodendrocyte glycoprotein peptide emulsion at the bottom of the tube, centrifuge the tube at four degree Celsius for two to three seconds, by pressing the pulse button on the equipment. Aspirate the myelin oligodendrocyte glycoprotein peptide emulsion with a one milliliter syringe equipped with a 22 gauge needle and transfer the emulsion to a new 1 milliliter syringe barrel.
Attach a 26 gauge needle to the barrel and secure the connection with a sealing film. Inject 100 microliters of the myelin oligodendrocyte glycoprotein peptide emulsion subcutaneously on each side of the dorsal spine of a mouse. After the injection, observe the automatic formation of bulbous masses under the skin and the dorsum.
Then intraperitoneally, inject 100 microliters of pertussis toxin into the same mouse. Prepare an open field reaction chamber and set up a video analysis system for recording mouse locomotion. To clean the reaction chamber before the experiment, spray 70%ethanol on the entire area and wipe with a clean paper towel.
Next place a mouse in the corner of a reaction chamber. To begin shooting, click on the start capture button in the menu bar of the video recording system and record the time. Keeping silence in the test room, let the mouse move freely for five minutes.
Then stop the recording and save the video. Put the mouse back in its cage. Then clean the test area with 70%ethanol and proceed to the next mouse.
Keep the euthanized mouse flat on a dissecting tray and fix the extremities. Hold the hind limb skin in the mouse with forceps and open the skin and muscles with scissors. Next, separate the femur from the tibia and the hip bone by carefully using the scissors.
Remove the muscles adhering to the femur with scissors. Then place the femur in 70%ethanol at room temperature. In this study, the ability of myelin oligodendrocyte glycoprotein derived peptide to induce EAE was assessed.
Mice injected with the peptide experienced progressive weight loss. After six to nine days of injection, they developed EAE symptoms that peaked after 14 to 16 days. Track plots from the open field test demonstrated that EAE alters the normal exploratory behavior of mice.
Compared to normal mice, the mice with EAE traveled significantly less distance in the early onset, peak, and remission phases. Mice with EAE also had significantly reduced activity duration in the peak and remission phases of the disease. Moreover, in all three phases the EAE mice had significantly less travel distance and time spent in the center.
Microcomputed tomography scans of femurs from EAE mice revealed a trabecular architecture different from normal mice. Femurs from EAE mice had significantly less bone mineral density and bone volume to tissue volume ratio than those from normal mice. Moreover, femurs from mice with EAE had less trabecular connection density, trabecular number, and trabecular thickness than those from normal mice.
Compared to normal mice, the trabecular spacing was enhanced and cortical bone thickness was reduced in EAE mice. We can also isolate the brain and spinal code of EAE mice at the peak of the disease and analyze the production of immune cells and cytokines by flu cytometry.
