5 mm thickness,

NA = 8, experimental time = 16 min. Three measures were used to estimate the morphological change of the brain, the first one Line 1 going from the Pituitary gland to Sylvius aqueduct, the second one Median line crossing the medial cerebellar nucleus and the third one Medium line stemming from the cerebellar obex. Measurements of vascular cerebral blood flow was performed by MRA using a Fast Low Angle Shot sequence, with the following parameters: FOV = 18 × 18 mm2, matrix = 256 × 256, TR/TE = 16/5 ms, 55 axial slices 0.2 mm thickness, NA = 4, experimental time = 11 min. Angiograms were produced by generating maximum intensity projections after interpolating raw data to obtain an isotropic resolution (72 μm3). Image analysis and processing were performed with the public domain software Image J (NIH, click here http://rsb.info.nih.gov/ij) Total RNA was isolated from homogenized mouse brain using TRI-Reagent (Sigma), purified by RNeasy Mini Kit (Qiagen, Valencia, CA), and quantified by NanoDrop (Nd-1000). Reverse transcription was performed in with SuperScript®III Kit according to manufacturers’ instructions (Invitrogen). cDNA was subjected selleck compound to quantitative real-time PCR using primers for CD3, CD8α, Granzyme B, IFN-γ, IL-12Rβ2, CXCL9, CXCL10, CXCL11, and CXCR3 (Qiagen) and GoTaq® qPCR-Master Mix (Promega). GAPDH and 18S expression was used for normalization. Raw data were

analyzed using the Relative Expression Software Tool (REST, http://www.rest.de.com/). Mice were euthanized and Pyruvate dehydrogenase perfused with intracardiac PBS/2 mM-EDTA. Brain leukocytes were isolated as described [41]. Briefly, brains were gently homogenized in RPMI 1640 medium containing 2% FCS. The mononuclear cells were then separated over a 35% Percoll gradient (Amersham Biosciences AB, Uppsala, Sweden) and analyzed by flow cytometry with hamster antibodies anti-mouse CD3ε-PerCP (BD Pharmingen, clone 145-2C11), anti-mouse CD69-PE-Cy-7 (BD Pharmingen, clone H1.2F3), anti-mouse CXCR3/CD183-FITC (eBioscience, clone CXCR3-173), and with rat antibodies anti-mouse CD8α-allophycocyanin (BD Pharmingen, clone 53-6.7),

and anti-mouse CD4-V450 (BD Pharmingen, clone RM4-5). Data were analyzed using a BD CANTO II flow cytometer and FlowJO software. Statistical significance was determined with GraphPad Prism (GraphPad Software, La Jolla, CA). Differences were analyzed by mean of nonparametric tests (Kruskal–Wallis followed by Dunn’s multiple comparison) or Logrank test for survival. p-values < 0.05 were considered statistically significant. The authors are grateful to Prof. U. Kalinke (Paul-Ehrlich Institut, Langen, Germany) for the kind gift of IFNAR1-deficient mice and to Prof. F. Erard for helpful discussions. The authors acknowledge the support from University of Orleans and CNRS through International Associated Laboratory TB IMMUNITY (LIA N°236) between CNRS INEM and UCT IIDMM.