MOG administration

Upon arrival, the rats were randomly assigned to either the myelin oligodendrocyte glycoprotein (MOG) or saline group. For Experiments 1–8, the MOG assigned rats received a 4 μg injection of recombinant rat MOG_1–125 (VU University Medical Center, Netherlands, gifted by Dr. Anne-Marie Van Dam) in a vehicle consisting of sodium acetate (pH = 3) and incomplete Freund’s adjuvant (Sigma; St. Louis, MO; (Ledeboer et al., 2003). This dose of MOG is a lower dose than that which we have found induces typical EAE motor symptoms, including full hindlimb paralysis/partial upper limb paralysis (i.e. 16 µg, the dose employed for Experiment 9). This low-dose was specifically chosen for use in Experiments 1–8 so to avoid hind limb motor/sensory dysfunction that could confound behavioral assessment of mechanical allodynia by the von Frey test.

In experiments 1 (male rats) and 2 (female rats), rats were assessed for mechanical allodynia on the same days post-MOG administration. Due to higher than expected subject attrition in experiment 2 caused primarily by cerebellar deficits which required euthanasia, a second cohort of female rats was added to experiment 2 to achieve appropriate group sizes in a follow up experiment. In this second cohort of rats, the day 19 timepoint was omitted as there was no significant difference between this timepoint and the other timepoints that were nearest to it (i.e. days 17 and 22) in the first cohort of female rats. The data from these 2 cohorts of female rats were then pooled for experiment 2, thus the day 19 timepoint is omitted in this experiment for statistical analysis, however all other timepoints between Experiment 1 and 2 are the same. The timecourse of testing for Experiments 1 and 2 were chosen specifically to test every few days in an effort to fully characterize the effect of (+)-NTX on mechanical allodynia in both males and females. In the subsequent experiments in male rats (i.e. Experiments 3–6), as timepoints between days 14 and 28 were not statistically significant than one another in the vehicle-treated rats with EAE, the timepoints were chosen to answer the specific questions regarding those experiments. Experiment 3 tested later timepoints post MOG administration, Experiment 4 tested timepoints both after (+)-NTX dosing and before (+)-NTX, Experiment 5 tested XT-203, another TLR4 antagonist, for efficacy against EAE-induced mechanical allodynia, and lastly, Experiment 6 tested whether EAE-induced mechanical allodynia was reversed by intrathecal IL-1ra at day 15 and day 29 post MOG administration, to demonstrate that spinal IL-1 is necessary for low-dose EAE-induced mechanical allodynia. The higher dose (16 µg) was used in Experiment 9 as this one study focused on motor disturbances rather than allodynia. In all cases, the injection was given intradermally at the base of the tail and the syringe left in place for 3 minutes to avoid leakage from the injection site. The rats in the saline group received saline injections following the same procedure. Also in Experiment 9, female C57Bl6/J mice were induced with EAE by subcutaneous injection of 200 µg MOG_35–55 (Hooke Labs, Lawrence, MA) (100 µg injected on each flank in 100 µl complete Freund’s adjuvant vehicle), followed 1 and 24 h later by intraperitoneal administration of 400 ng pertussis toxin. Only female and not male C57BL6/J mice were used, as this model of EAE is commercially available, well-characterized, and easily reproducible in female mice for focus on motor disturbances (Hooke Labs, Lawrence, MA). All animals were between 10–12 weeks old upon receiving the injections.