Modeling Multiple Sclerosis in the Two Sexes: MOG_35-55-Induced Experimental Autoimmune Encephalomyelitis

Our research focuses on the experimental autoimmune encephalomyelitis, or, EIE, model, which can significantly enhance the understanding of peripheral immune-mediated mechanism and evaluate neuroinflammatory and partially demyelinating processes. This model has also been widely used to develop and test a wide range of therapeutic drugs. Due to the unknown etiology and complexity of multiple sclerosis, no animal model currently recapitulates all the clinical and radiological features displayed in humans.

Thus, it is quite challenging for the researchers to select the animal model most fitting for a specific experimental question. EAE is a widely used model of multiple sclerosis. However, due to several issue, only some studies are performed, and this model considered both sexes.

With our protocol, we want allow the possibility of including both sexes in the study conducted in this model. Specifically, our protocol proposed a clinical evaluation based of two methods, the clinical score assessment and the rotarod test, leading a more quantitative, less subjective, and more precise clinical assessment of the disease course. Even though this model does not entirely reproduce the sexual dimorphism of multiple sclerosis, it has potential advantages.

The combination of the induction with other possible risk factor, especially the environmental ones, can aid in understanding specific effects of such factors and identifying the specific role in some sexually dimorphic aspect of the disease. To begin, prepare the myelin oligodendrocyte glycoprotein peptide at 35 to 55, or, MOG 35-55 solution in a glass beaker. To do so, add the liquid component to the beaker, followed by adding the appropriate mycobacterium tuberculosis strain.

Place the glass beaker containing the solution in an ice bath. Using a glass syringe with an 18-gauge needle, initiate the emulsification process and continue emulsifying the solution for a minimum of 15 minutes. To assess the quality of the emulsion, dispense a drop of the emulsion into a transparent container filled with water.

Consider the emulsion ready if the drop maintains its structure and remains intact. Transfer the emulsion directly into the one-milliliter syringes designated for immunization purposes. Store the filled syringes at four degrees Celsius until they're ready for use.

Begin the immunization of the mouse by performing a PT intravenous injection into its lateral caudal vein. To do so, gently plug the tail of the properly anesthetized mouse with an ethanol solution, which acts as a vasodilator to make the veins visible. Focusing on one of the two lateral veins of the tail, use a 0.5 milliliter syringe fitted with a 30-gauge needle to inject 500 nanograms of PT.Next, proceed to administer subcutaneous injections of MOG 35-55 emulsion previously prepared in one-milliliter syringes.

Using the syringes with 26-gauge needles, perform two subcutaneous injections of 100 microliters each under the rostral part of the flanks. Then administer one injection of 100 microliters at the base of the tail. After inducing the experimental autoimmune encephalomyelitis in the mouse, have a blinded investigator perform daily assessment of the animal's clinical scores.

Score each animal on a scale from zero to five to evaluate the progression of the disease. To evaluate motor performance, initiate rotarod sessions for the mice daily, starting at one DPI until the study requires. Conduct each session for 300 seconds, during which increase the rotation speed of the rod linearly from four to 40 rotations per minute.

When the animal loses its balance and falls from the rotarod device, it lands on the ground, triggering a sensor that records the time. Record the animal's performance and score it as a latency to fall. A significant increase in the clinical score or, CS, with time was observed in both male and female mice, starting from 10 DPI until the endpoint.

Females displayed higher CS than males, but not significantly. A tendency for earlier disease onset was also observed in females compared to males. Furthermore, females showed a significantly higher cumulative CS compared to males.

A significant decrease in the rotarod performance with time was observed in both male and female mice. Particularly, males displayed the minimum performance at 16 DPI, while the females at 17 DPI. Males performed better than females, especially during the chronic phase of the disease, probably as a consequence of lower CS.