Experimental Autoimmune Encephalomyelitis (EAE)

EAE was induced in 10-12-week-old C57Bl6/J (H-2^b) male mice following a widely used EAE protocol (Fig 1A) [2], essentially as previously reported [24, 25]. Mice were subcutaneously immunized with 100 μg of 35–55 myelin oligodendrocyte glycoprotein (MOG_35–55, MEVGWYRSPFSRVVHLYRNGK, GeneCust), emulsified in Freund’s Adjuvant supplemented with 1mg of heat-inactivated Mycobacterium tuberculosis H37RA (Difco Laboratories) to the side flanks. In addition, mice received two intraperitoneal injections of 100 ng pertussis toxin at the time of immunization and 48h later. Mice were weighed and monitored for clinical signs of EAE throughout the experiment. EAE symptoms were scored as follows: 0, no clinical disease; 1, tail weakness; 2, paraparesis (incomplete paralysis of 1 or 2 hind limbs); 3, paraplegia (complete paralysis of 1 or 2 hind limbs); 4, paraplegia with forelimb weakness or paralysis; 5, dead or moribund animal. At the day of sacrifice, blood plasma and spinal cord tissue were harvested and stored.

(A) Schematic representation of the EAE protocol. Mice were (B) weighed and (C) monitored for clinical signs of EAE throughout the experiment. (D) Plasma ATX activity (nmol/min/ml) as measured with the TOOS assay; values are presented as mean (±SD). Total LPA (E) and LPC (F) levels in plasma; values were normalized to internal standards and presented as fold change (means ±SEM) to control samples. Statistical significance between experimental groups was assessed with one-way ANOVA complemented with Bonferroni or Dunn's multiple pair test accordingly; * denotes statistical significance (p<0.05).