MOG peptide, sequence 35–55 (MEVGWYRSPFSRVVHLYRNGK; Auspep) was obtained from NeoMPS (San Diego, USA). Pertussis toxin and CFA were purchased from Sigma Chemical Co (St. Louis, MO, USA). Attenuated M. tuberculosis H37 RA was purchased from Difco Laboratories (Sparks, MD, USA). Each animal received 100 μL of the emulsion in the base of tail containing 100 μg of MOG35–55. Each animal received two i.p. doses of 300 ng of pertussis toxin in the day of the immunization and 48 h later. Animals were monitored daily and clinical score was evaluated using a standard scoring system. Briefly, the score is characterized as follows: 0 = no signs;

0.5 = tail weakness; 1 = tail paralysis; 2 = hind limb weakness; 3 = hind limb paralysis; 4 = hind limb paralysis and front limb weakness. Animals were also weighed daily. Spinal Selleck Veliparib cords were quickly removed after intravital microscopy and preserved in 10% buffered formalin. The sections (4 μm) were stained with hematoxylin and eosin (H&E) and analyzed for CNS inflammation in an Olympus BX51 microscope. The extent of macrophage sequestration was quantified indirectly by the measuring of N-acetyl-β-d-glucosaminidase (NAG) activity in brain supernatants,

as an index of monocyte influx ( Lacerda-Queiroz et al., 2010). In brief, the brains of control and immunized animals (on day 14 post immunization) were removed, weighed and the tissue was homogenized in extraction solution (100 mg of tissue per mL), containing 0.4M NaCl, 0.05%

Tween 20, 0.5% BSA, 0.1 mM phenyl methyl sulphonyl fluoride, Crenolanib 0.1 mM benzethonium chloride, 10 mM EDTA and 20 KIU aprotinin, using Ultra-Turrax. Brain homogenate was centrifuged at 3000×g for 10 min at 4 °C and the resultant pellet was resuspended in saline/Triton 0.1%. The NAG reaction was run at 37 °C for 10 min in a 96-well microplate following the addition of 100 μL p-nitrophenyl-N-acetyl-β-d-glucosaminide ALOX15 (Sigma-Aldrich, St. Louis, MO), dissolved in citrate/phosphate buffer (0.1 M citric acid, 0.1 M Na2HPO4, pH 4.5) at a final concentration of 2.24 mM. The reaction was terminated by the addition of 100 μL 0.2M glycine buffer (pH 10.6). NAG activity was assayed by measuring the change in absorbance (optical density [OD]) at 405nm in a spectrophotometer (Emax, Molecular Devices) and interpolated on a standard curve constructed with p-nitrophenol (0–500 nmol/ml) (Sigma-Aldrich). Results were expressed as change in O.D. per gram of tissue. Intravital microscopy of the mouse cerebromicrovasculature was performed at day 14 post immunization as previously described (Vilela et al., 2008). Briefly, mice were anesthetized by intraperitoneal injection of a mixture of 150 mg/kg ketamine and 10 mg/kg Xylazine and the tail vein was cannulated for administration of fluorescent dyes. A craniotomy was performed using a high-speed drill (Dremel, USA) and the dura mater was removed to expose the underlying pial vasculature.