EAE Induction and Assessment and Resveratrol Treatment Regimen

EAE was induced in 8–10-week-old female C57BL/6 mice as previously described (Rouse et al. 2013). Briefly,on day 0, mice were immunized via subcutaneous injection in each hind flank of 75 μg myelin oligodendrocyte peptide (MOG₃₅–₅₅) (for a total of 150 μg MOG₃₅–₅₅) emulsified in 50 μL complete Freund’s adjuvant (CFA) (Difco, Detroit, MI) containing 6 mg/Ml killed Mycobacterium tuberculosis H37Ra(Difco).Mice were given 200 ng and 400 ng pertussis toxin (List Biologicals, Campbell, CA) via intraperitoneal injection on day 0 and day 2, respectively. Following immunization, mice were monitored daily and assigned disease scores based on the severity of disease symptoms: 0 = no symptoms; 1 = partial loss of tail tonicity; 2 = complete tail atony,clumsygait;3 = hind limb weakness, partial paralysis; 4 = complete hind limb paralysis, fore limb weakness; and 5 = tetraplegia, moribund. Myelin oligodendrocyte glycol-protein (MOG_35–55) peptide and H-MEVGWYRSP FSRVVHLYRNGK-OH from PolyPeptide Laboratories San Diego (San Diego,CA). Additional measures were taken to ensure accessibility to fresh food and water for paralytic animals. Moribund animals were euthanized, as indicated in IACUC- and AALAS-approved protocols, by inhalant anesthetic isoflurane over dose. Death was not used as an index for clinical scores. Beginning on day two post-immunization, 100 mg/kg RES was administered as a suspension in 0 .2mL of water, and given daily for the duration of the experiement, as previously described. (Singh et al. 2007)