2.2. Active experimental autoimmune encephalomyelitis induction and control immunization

aEAE was induced in both male and female mice by injection of myelin oligodendrocyte glycoprotein peptide 35–55 (MOG_35‐55 peptide, 200 μg per animal, Genscript) and complete Freund's adjuvant (Incomplete Freund's Adjuvant, Santa Cruz Biotechnology; supplemented with Mycobacterium tubercolosis, Difco). An emulsion of MOG_35‐55 and CFA was injected subcutaneously in mouse flanks and at the tail base at day 0; in addition, 300 ng of pertussis toxin (List Biological Laboratories, Campbell, CA) was injected intraperitoneally at day 0 and day 2. As controls served mice treated as above, however with CFA emulsified in Dulbecco's phosphate‐ buffered saline (DPBS, Gibco, Paisley, UK) without MOG_35‐55. Immunized mice were weighted and disease development scored daily according to a previously established system (Tietz et al., ²⁰¹⁶). Four clinical time points were defined: weight loss (5 animals presenting a 3%–5% loss of weight shortly preceding clinical symptoms), clinical onset (9 animals showing a limb tail and partial hind leg paraparesis, average clinical score ± SD 1.03 ± 0.47), symptomatic peak of disease (10 animals presenting hind leg paraparesis and unsteady gait or hind leg paraplegia 4 days after EAE onset, average clinical score ± SD 1.6 ± 0.31), chronic EAE (7 animals showing a recovered, yet not complete strength in the hind limbs 10 days after disease onset, average clinical score ± SD 0.92 ± 0.31). At day 8 post treatment, 6 mice following CFA + PTx + MOG_35‐55 immunization and 5 mice following CFA + PTx control immunization were analyzed.