Experimental autoimmune encephalomyelitis induction and monitoring

C57BL/6 mice (Jackson Labs; female, 10–12 weeks old; 20.3 ± 0.22 g, housed in standard housing conditions with 5 animals/cage in reusable plastic cages in accordance with institutional guidelines) were immunized with MOG_35-55 peptide (1 mg/mL) emulsified with complete Freud’s adjuvant (CFA) (Hooke laboratories, EK-0115/EK-2110, Lawrence, MA, USA) and injected with pertussis toxin (200 ng/mouse) at day 0 and day 2 according to the manufacturer’s instructions (n = 10 EAE mice) [34]. Controls (n = 10) received the same CFA and pertussis toxin administrations but not MOG peptide. EAE induction was performed under temporary isoflurane anesthetic with heat pad recovery in accordance with institutional guidelines. Cages (n = 5 animals/cage) were assigned to treatment groups using a random number generator (https://www.graphpad.com/ quickcalcs/randomN1.cfm). For the duration of each experiment, animals were fed a Bio-Serv NutraGel diet (BioServ #F4798-KIT) instead of conventional chow to prevent dehydration and aid wellbeing after onset of disease. EAE animals were assessed daily in late afternoon and scored for disease severity up to 24 days following EAE induction using an established 0–15 point scale [9, 34]. A score of 0 indicated no neurobehavioral signs; a score of 1–2 indicated tail paralysis (moderate or severe); a score of 3–8 indicated tail and hind leg paralysis (of varying degrees of severity, unilateral or bilateral); a score of 9–14 indicated tail, hindlimb, and forelimb paralysis (of varying degrees of severity, unilateral or bilateral); and a score of 15 indicated moribund. Three EAE animals met endpoint criteria for disease severity prior to the end of the experiment and were sacrificed. All remaining animals were sacrificed using cervical dislocation at the day 24 endpoint. Spinal cords were collected for tissue immunofluorescence.