Figure 4

Induction, amelioration and histological analysis of EAE. Active EAE was induced in C57BL/6 female mice (8–12 weeks) by MOG_35–55. NTG-A-009 (2 mg/kg/day) or vehicle control was administered subcutaneously from the day 1 post immunization as described in materials and methods. (a) The disease severity was analyzed on the basis of sign and symptoms. Clinical score in the mice was recorded in daily basis. (b) Total no. of splenocytes and mononuclear cells (MNCs) obtained from EAE and NTG-A-009 treated mice. (c) Sections of spinal cord obtained from NTG-A-009 and EAE mice at day 21post immunization were analyzed for degree of inflammation by H&E (magnification 4X) which was quantified by analyzing the percentage of cell density in white matter. Luxol fast blue staining (magnification 4X) of spinal cord sections for analyzing the degree of demyelination and was quantified by the total cell density in white matter. (d) At day 21, total mononuclear cells were obtained from brain and spinal cord of EAE and NTG-A-009 treated mice. The percentage of CD4⁺ and CD8⁺ T cells was analyzed by flow cytometry. (e) The percentage CD11b⁺ and CD11c⁺ cells in MNCs derived from brain and spinal cord was analyzed by flow cytometry. Mean ± SEM of the triplicates are shown. Data represent three independent experiments. *P < 0.05. **P < 0.01; ***P < 0.001 was determined by student’s t- test or two-way ANOVA.