H37ra

The encephalitogenic peptide MOG (35-55)(MEVGWYRSPFSRVVHLYRNGK) (GLBiochem) used to induce EAE had a purity of 95%. Female B7-H1 WT or transgenic mice 6-8 weeks of age were injected subcutaneously with 200μg MOG (35-55) and complete freund's adjuvant (CFA) containing 200μg of H37RA (BD Bioscience) in the posterior right and left flank. Pertussis toxin (Calbiochem) in PBS was injected intraperitoneally on day 0 and day 2. The mice were assigned a scores of EAE on a scale of 0-5 as follows [34 (link)]: 0. normal; 1. Paralyzed tail; 2. Moderate hind-limb weakness or mild ataxia;3. Severe hind-limb weakness;4.paraplegia with weakness or paralysis;5. Moribund state or death.
For analysis of CNS infiltrates, brain and spinal cord tissues were colletected from perfused mice and mononuclear cells were prepared by Percoll gradient centrifugation. For histological analysis, brain and spinal cord tissues were immediatedly fixed in 4% paraformaldehyde.

Four to five female B6J WT or Nqo1-deficient mice were injected subcutaneously in the back with 200 μg CFA-MOG mixture which premixed MOG (35–55) (BEX) with an equal volume of complete Freund’s adjuvant (DIFCO) containing of H37RA (BD Biosciences, San Jose, CA, USA). These mice were injected with 200 μg/ml Pertussis toxin i.p. (Merck Millipore, Burlington, MA, USA) in PBS on days 0 and 2. To examine infiltrating cells into the central nervous system, spinal cord and brain were collected from mice perfused with physiological saline containing heparin under anesthesia. For adoptive transfer EAE, CD4⁺ T cells were harvested from donor mice at peak of disease (days 21). These cells were cultured in the presence of MOG antigen under the Th17 (23) condition for 3 days. These cells were injected into recipient Rag2-deficient mice i.v. at 10 million cells /mice. Clinical score follows; 0: normal, 1: Partially limp tail, 2: Complete limp tail, 3: Partial hind limb paralysis, 4: Complete hind limb paralysis, 5: Moribund state (euthanasia), 6: death. Clinical score and animal health were monitored once every two days. Mice were euthanized at end of experiments (at 23–30 days), or at the humane endpoints defined by > 20% loss in maximal body weight, or appearance of general paralysis symptoms.

On day 0, the MOG_35-55 peptide (3038001, MD Bioproducts) was dissolved in PBS to 4 mg/ml. CFA is prepared by mixing H37Ra (231141, BD Biosciences) in IFA (263910, BD Biosciences) to a concentration of 6 mg/ml. The MOG-CFA emulsion was prepared by 1:1 mixed MOG and CFA, quality-checked by drop test then subcutaneously injected in a total volume of 100 μl to each mouse. After 2 h, 200 ng of Pertussis toxin (PTX) diluted in 200 μl of PBS were intraperitoneally injected into each mouse as a booster. On day 1, 1 mg of E4 antibody (n = 9) or equal volume of PBS (n = 10) was intravenously injected into the mice. Mice received another boost of PTX on day 2 and the same dose of E4 antibody on day 9. Mice were weighed and the disease was macroscopically scored in different time points, briefly, animals with EAE develop ascending flaccid paralysis that initially affects the tail (score 1–2), later involves hind limbs (score 3-6), forelimbs (score 7) and ultimately result in quadriplegia and death (score 8).

One week after insertion of the device, chronic EAE was induced by immunization of the mice with an emulsion (0.2 mL) containing 300 µg purified myelin oligodendrocyte glycoprotein (MOG) 35–55 peptide in phosphate-buffered saline (PBS) and an equal volume of complete Freund’s adjuvant containing 5 mg of H37Ra (Difco Laboratories, Detroit, MI, USA). In addition, Bordetella pertussis toxin (300 ng in 0.2 mL PBS) was injected intraperitoneally the same day and 48 h later. Animals with EAE were scored daily for neurological symptoms according to the EAE clinical severity scale: 0 = asymptomatic; 1 = partial loss of tail tonicity; 2 = tail paralysis; 3 = hind limb weakness; 4 = hind limb paralysis; 5 = 4-limb paralysis; 6 = death [48 (link)].