However, the exact role played by astrocytes during the development of EAE is still debated. In the present study, we demonstrate that astrocytes are capable of inducing and suppressing lymphocyte functions during different phases of EAE. During the initial phases, astrocytes probably inhibit the activity of myelin oligodendrocyte glycoprotein (MOG)35–55-specific lymphocytes in part by secreting IL-27, which contributes to inhibition of proliferation

and lymphocyte secretion. During EAE progression, lymphocyte-derived IFN-γ might induce the up-regulation of major histocompatibility complex (MHC)-II on astrocytes, thereby promoting lymphocyte proliferation and activation and resulting in disease progression. These findings indicate that the changing physiological role of astrocytes is important to EAE development. The study contributes to a clearer understanding of EAE and adds new insights into the field of EAE research. Female C57BL/6 mice (6–8 weeks Fulvestrant solubility dmso of age) were purchased from the Beijing Vital River DMXAA Laboratory Animal Ltd (Beijing, China). All mice were bred and housed in a specific pathogen-free animal facility at the Harbin Medical University. Neonatal C57BL/6 mice aged 1–3 days were used for the isolation of astrocytes. All animal experiments were performed in compliance with the principles and procedures outlined in the Care and Use of Laboratory Animals guidelines, which is published by the China National

Institute of Health and approved by the Institutional Animal Care and Use Committee. C57BL/6 mice were immunized subcutaneously in the axillary

fossa with the MOG35–55 (MEVGWYRSPFSRVVHLYRNGK) peptide (200 μg) emulsified in complete Freund’s adjuvant (CFA) at a final volume of 100 μl. Mice were then injected intravenously (i.v.) with 200 ng pertussis toxin (PT) on days 0 and 2. The behavioural performance was assessed by a 0–5-point scale as follows: 0, no clinical signs; 1, floppy tail; 2, hind limb weakness; 3, full hind limb paralysis; 4, quadriplegia; and 5, death as described [34]. Astrocytes were isolated from newborn mice as described previously [35, 36]. Briefly, following removal of the meninges, Resminostat brains were minced with a Pasteur pipette and passed through a 150 μm nylon filter to remove debris. Cells were then seeded onto 10 μg/ml poly-D-lysine precoated flasks and cultures were incubated at 37°C in 5% CO2. After 72 h, non-adherent cells were removed by changing the media every 3–4 days. When cultures were 70–80% confluent, mixed glia were agitated rigorously for 2 h in an orbital incubator shaker at 0.23 g at 37°C to detach microglia. Cells were then shaken again at 0.23 g at 37°C overnight to ablate oligodendrocytes. Suspended cells were trypsinized [0·25% trypsin and 0·02% ethylenediamine tetraacetic acid (EDTA)] and replated onto flasks. Subcultured astrocytes were 92% positive for glial fibrillary acidic protein (GFAP) by immunofluorescence staining.