Adoptively-transferred EAE. It was performed as described previously by us [11 (
link), 12 (
link), 13 (
link), 14 (
link)]. Briefly, 4–5 weeks old female SJL/J mice were purchased from Harlan Sprague-Dawley (Indianapolis, IN). Donor mice were immunized s.c. with 400 µg bovine MBP and 60 µg
M. tuberculosis in IFA [11 (
link), 12 (
link), 13 (
link), 14 (
link)]. Animals were killed 10–12 days postimmunization, and the draining lymph nodes were harvested and single cell suspensions were cultured in RPMI 1640 supplemented with 10% FBS, 50 µg/mL MBP, 50 µM 2-ME, 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/ml streptomycin. On day 4, cells were harvested and resuspended in HBSS. A total of 2 × 10
7 viable cells in a volume of 200 µL were injected into the tail vein of naive mice. Pertussis toxin (150 ng/mouse; Sigma-Aldrich) was injected once via i.p. route on 0 day post-transfer (dpt) of cells. Animals were observed daily for clinical symptoms. Six mice were used in each group. Female mice (4–5 week old) were randomly selected for any group. Experimental animals were scored by a masked investigator, as follows: 0, no clinical disease; 0.5, piloerection; 1, tail weakness; 1.5, tail paralysis; 2, hind limb weakness; 3, hind limb paralysis; 3.5, forelimb weakness; 4, forelimb paralysis; 5, moribund or death.
A mouse was considered moribund when it showed any of the following criteria. Conditions for moribund were as follows: Prolonged inappetance; Evidence of muscle atrophy; Central nervous system disturbance (Head tilt, Seizures, Tremors, Circling, Spasticity, and Paresis); Chronic diarrhea or constipation; Rough coat and distended abdomen; Spreading area of alopecia caused by disease; Coughing, rales, wheezing and nasal discharge; Distinct jaundice and/or paleness (anemia); Markedly discolored urine, polyuria or anuria; Inability to remain upright; Frank bleeding from any orifice; Persistent self-induced trauma.
Relapsing EAE in 5B6 PLP-TCR Tg mice. Female Tg mice (4–5 weeks old) were immunized with 10 or 25 µg of PLP139–151 in
M. tuberculosis in IFA as described above. Mice also received pertussis toxin (150 ng/mouse) once on 0 day post-immunization (dpi). In the EAE group (
Fig. 1B), where female PLP-TCR transgenic mice were immunized with 25 μg PLP139–151, two mice died without humane intervention on 17 days post-immunization (dpi) and four moribund mice were decapitated after anesthesia. However, according to the disease scale, all six mice in this group received a score of 5.
Chronic EAE. C57BL/6 mice were immunized with 100 μg of MOG35–55 as described above. Mice also received two doses of pertussis toxin (150 ng/mouse) on 0 and 2 dpi.
Mondal S, & Pahan K. (2015). Cinnamon Ameliorates Experimental Allergic Encephalomyelitis in Mice via Regulatory T Cells: Implications for Multiple Sclerosis Therapy. PLoS ONE, 10(1), e0116566.