Experimental autoimmune encephalomyelitis (EAE)

For the induction of EAE, five to seven-week old HBZ-Tg or control non-transgenic (non-Tg) C57BL/6J mice were immunized with an emulsion containing MOG peptide 35–55 (MEVGWYRSPFSRVVHLYRNGK). The emulsion was prepared by sonication, mixing 1 mL of MOG solution (1 mg/mL in PBS) with 1 mL of complete Freund’s adjuvant (Difco Laboratories, Detroit, MI, USA) containing desiccated Mycobacterium butyricum. The emulsion was injected subcutaneously in the area near the axillary lymph nodes and on both sides at the base of the tale of each mouse (50 μL/site, a total of 4 sites/mouse). On days 0 and 2 post-immunization, mice were injected intraperitoneally with 50 μL of a Pertussis toxin (Kaketsuken, Kumamoto, Japan) solution (4 μg/mL). Thereafter, mice were monitored daily for clinical signs of encephalomyelitis. A clinical score was assigned according to the following criteria: 0, no symptoms; 1, mild limp tail; 1.5, limp tail; 2, unilateral hind limb weakness or abnormal gait; 2.5, unilateral hind limb paralysis or bilateral hind limb weakness; 3, paraplegia; 3.5, unilateral fore limb weakness, with paraplegia; 4, unilateral fore limb paralysis or bilateral fore limb weakness; 4.5, bilateral fore limb paralysis; 5, moribund or dead.