Journal: CNS Neuroscience & Therapeutics

Article Title: Artesunate Ameliorates Experimental Autoimmune Encephalomyelitis by Inhibiting Leukocyte Migration to the Central Nervous System

doi: 10.1111/cns.12561

Figure Lengend Snippet: Artesunate reduces the migration of cells. Experimental autoimmune encephalomyelitis was induced in C57BL/6 mice (n = 10) through the immunization with 100 μg of MOG35–55 peptide and 200 ng/mice of pertussis toxin injection at 0 and 48 h from immunization. Mice were divided in two groups, where one group received artesunate (3 mg/kg) for five consecutive days starting the same day of immunization, and the other group received vehicle (PBS 0.02M pH7.4). (A) Ten days after immunization, mice were killed and the draining lymph nodes were collected. The organs were disrupted to prepare single‐cell suspensions, which were seeded (5 × 105 cells) on the upper chamber of transwell plates. The lower chamber was filled with complete medium alone (PBS) or with recombinant mouse CCL19 (20 ng/mL). Cells were incubated for 3 h at 37°C. At the end of culture time, cells in the lower chamber were counted in hemocytometer. Results are presented as mean ± SEM. *P < 0.05 in t‐test analysis. (B) Ten days after immunization, EAE‐bearing mice were killed and the spleen cells (5 × 105 cells/insert) were seeded in the upper chamber of a transwell plate. In the lower chamber, RPMI medium alone (PBS) supplemented with CCL19 (20 ng/mL) with or without artesunate (2.5 μM). The cultures were incubated for 3 h at 37°C. At the end of incubation time, cells in the lower chamber were counted in hemocytometer. Results are presented as mean ± SEM. *P < 0.05 in one‐way ANOVA multiple comparison analysis. (C) Spleen cells from untreated and Art‐treated EAE‐bearing mice at 10 dpi were adoptively transferred to RAG2−/− mice (5 × 106 cells/mice), and the clinical course of passive EAE was evaluated daily. Results are presented as mean ± SEM. (D) Spleen cells from untreated EAE‐bearing mice at 10 dpi were adoptively transferred to RAG2−/− mice (n = 12 mice). Group of mice also received Art (3 mg/kg) for five consecutive days starting day 3 from adoptive transfer. The clinical course of passive EAE was evaluated daily. Results are presented as mean ± SEM. *P < 0.05 in two‐way ANOVA multiple comparison analysis. Combined results from three independent experiments with similar observations.

Article Snippet: The lower chamber was filled with 500 μ L of medium containing recombinant mouse 10 ng of CCL19 chemokine (R&D Systems Inc., Minneapolis, MN, USA).

Techniques: Migration, Injection, Recombinant, Incubation, Comparison, Adoptive Transfer Assay