Mog35 55

All experimental protocols were approved by the UCLA Animal Protection Committee and carried out in compliance with the ARRIVE guidelines and all relevant guidelines and regulations were followed for the experiment. Nine weeks old female C57BL/6 or SJL mice were obtained from the Jackson Laboratory and housed in a specific pathogen-free facility with free access to food and water. C57BL/6 mice were immunized subcutaneously with MOG_35-55 (200 µg, > 95% purity, GenScript)) in 50% IFA containing Mycobacterium tuberculosis H37R (5 mg/ml, Difco) in multiple sites near the base of their tail on day 1 and injected intraperitoneally with pertussis toxin (200 ng/mouse) on day 0 and 2. Individual SJL mice were immunized with PLP_139-151 (100 µg/mouse, > 95% purity, GenScript) using a similar protocol to that described for C57BL/6 mice. The mice were monitored for EAE onset daily: 0, no disease; 1, limp tail; 2, hind limb weakness; 3, complete hind limb paralysis; 4, quadriplegia; and 5, death. Mice that were in between the clear-cut gradations were scored intermediate in increments of 0.5. When the mice developed EAE with a score of 1 at 10–13 days post-immunization, they were randomized to receive plain water or water containing homotaurine (0.25 mg/ml) or GABA (6 mg/ml).

EAE was induced as in prior studies28 (link). Briefly, at 8 weeks of age, mice were anesthetized with isoflurane and were injected subcutaneously at two sites on the back with a total of 200 μg of myelin oligodendrocyte glycoprotein (MOG) peptide (MOG_35–55; Genscript, Piscataway, NJ, USA) emulsified in Complete Freund’s Adjuvant (Difco, Detroit, MI, USA) containing 2.5 mg/ml mycobacterium tuberculosis (Difco). Control mice were injected with an equal volume of phosphate buffered saline (PBS) and Complete Freund’s Adjuvant. In addition, each animal received 200 ng pertussis toxin (List Biological, Campbell, CA, USA) in 0.1 ml PBS by i.p. injection at 0 h and 48 h post-immunization. Severity of EAE was scored using a previously published28 (link)29 (link)30 31 (link)32 (link) 5-point scale: no disease = 0; partial tail paralysis = 0.5; tail paralysis or waddling gait = 1.0; partial tail paralysis and waddling gait = 1.5; tail paralysis and waddling gait = 2.0; partial limb paralysis = 2.5; paralysis of one limb = 3.0; paralysis of one limb and partial paralysis of another = 3.5; paralysis of two limbs = 4.0; moribund state = 4.5; death = 5.0.