Riboflavin may ameliorate neurological motor disability but not spatial learning and memory impairments in murine model of multiple sclerosis Mahshid.

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Riboflavin may ameliorate neurological motor disability but not spatial learning and memory impairments in murine model of multiple sclerosis Mahshid Naghashpour, Reza Amani, Alireza Sarkaki, Ata A. Ghadiri, Alireza Samarbaf-Zadeh, Sima Jafarirad, Ahmad Rouhizadeh, Azadeh Saki Clinical Nutrition Experimental Volume 23, Pages 1-14 (February 2019) DOI: 10.1016/j.yclnex.2018.12.001 Copyright © 2018 The Authors Terms and Conditions

Fig. 1 Illustration of experimental design. MOG35-55 (Myelin Oligodendrocyte Glycoprotein-35-55); CFA (Complete Freund's adjuvant); PTX (Pertussis toxin); PBS (phosphate buffer saline); INF-β1a (Interferon beta -1a); MWM (Morris water maze). Clinical Nutrition Experimental 2019 23, 1-14DOI: (10.1016/j.yclnex.2018.12.001) Copyright © 2018 The Authors Terms and Conditions

Fig. 2 Gene expression and protein levels of BDNF in the brain and spinal cord of study groups. (a) Animals in the T3 group increased significantly BDNF expression in the whole brain compared to sham operated, sham treated, and treatment groups (**P < 0.01). (b) EAE mice in T3 group showed a significant increase in BDNF gene expression in the spinal cord compared to sham operated, sham treated, and treatment groups (***P < 0.001). (c) Animals in T3 group showed a significant increase in BDNF protein levels in the whole brain compared to ST2, T1, and T2 EAE mice (*P < 0.05). (d) Animals in SO1 and SO2 groups, and EAE animals in ST1, ST2, and T3 groups showed a significant decrease in BDNF expression in the spinal cord compared to EAE mice in T1 and T2 groups (*P < 0.05,**P < 0.01, ***P < 0.001). The data are given as mean ± SEM analyzed by one way ANOVA and LSD's and Bonferroni's post -Hoc tests. The results are shown as pg/mg tissue of BDNF. SO1 (sham operated 1, Healthy+PBS); SO2 (sham operated 2, healthy + PBS); ST1 (sham treatment 1, EAE+Water); ST2 (sham treatment 2, EAE+sodium acetate buffer); T1 (treatment 1, EAE + INFβ1A); T2 (treatment 2, EAE+riboflavin); T3 (Treatment 3, EAE + INFβ1A+riboflavin); BDNF (brain-derived neurotrophic factor). The experiments were repeated three times in duplicates. Clinical Nutrition Experimental 2019 23, 1-14DOI: (10.1016/j.yclnex.2018.12.001) Copyright © 2018 The Authors Terms and Conditions

Fig. 3 Clinical scores of EAE mice. Differences were observed between the EAE mice in different groups from 11 to 15 dpi (P < 0.05). ST1 (sham treatment 1, EAE+Water); ST2 (sham treatment 2, EAE+sodium acetate buffer); T1 (treatment 1, EAE + INFβ1A); T2 (treatment 2, EAE+riboflavin); T3 (Treatment 3, EAE + INFβ1A+riboflavin). Clinical Nutrition Experimental 2019 23, 1-14DOI: (10.1016/j.yclnex.2018.12.001) Copyright © 2018 The Authors Terms and Conditions

Fig. 4 Latency to find the hidden platform (s) in spatial learning in Morris water maze by mice treated with riboflavin and/or INFβ-1A. All data are shown as Mean ± SEM (N = 8). Repeated Measure test followed by Post-Hoc LSD tests demonstrated that during the 4 days of trials, the latency to the plate obtain showed no significant difference among the groups. SO1 (sham operated 1, PBS); SO2 (sham operated 2, PBS+riboflavin); ST1 (sham treatment 1, EAE+Water); ST2 (sham treatment 2, EAE+sodium acetate buffer); T1 (treatment 1, EAE + INF-β1a); T2 (treatment 2, EAE+riboflavin); T3 (Treatment 3, EAE + INF-β1a+riboflavin). Clinical Nutrition Experimental 2019 23, 1-14DOI: (10.1016/j.yclnex.2018.12.001) Copyright © 2018 The Authors Terms and Conditions

Fig. 5 Swimming speed to find the hidden platform (cm/s) in spatial learning in Morris water maze by mice treated with riboflavin and/or INFβ-1A. All data are shown as Mean ± SEM (N = 8). Repeated Measure test followed by Post-Hoc LSD tests showed that during the 4 days of trials, the speed to obtain the hidden plate indicated significant differences in all groups. a) There was no significant difference between SO1, ST1, and ST2 groups. b) There was no significant difference between SO2, ST1, and T2 groups. c) T2 mice treated with riboflavin swam significantly faster than ST2 and T1 mice. d) T3 mice treated with both riboflavin and INF-β1a swam significantly faster than ST1 mice on the 4th day of the trial. (†P = 0.045, δ P = 0.025, θ P = 0.024). SO1 (sham operated 1, PBS); SO2 (sham operated 2, PBS+ riboflavin); ST1 (sham treatment 1, EAE+Water); ST2 (sham treatment 2, EAE+sodium acetate buffer); T1 (treatment 1, EAE + INF-β1a); T2 (treatment 2, EAE+riboflavin); T3 (Treatment 3, EAE + INF-β1a+riboflavin). Clinical Nutrition Experimental 2019 23, 1-14DOI: (10.1016/j.yclnex.2018.12.001) Copyright © 2018 The Authors Terms and Conditions

Fig. 6 Performance in reference memory version of the Morris water maze by mice treated with riboflavin and/or INFβ1A. All data are shown as Mean ± SEM (N = 8). ANOVA test followed by Post-hoc LSD tests showed that no significant difference was found between the groups in terms of spatial memory variable (percentage of spent time in the goal quadrant). SO1 (sham operated 1, PBS); SO2 (sham operated 2, PBS+riboflavin); ST1 (sham treatment 1, EAE+Water); ST2 (sham treatment 2, EAE+sodium acetate buffer); T1 (treatment 1, EAE + INF-β1a); T2 (treatment 2, EAE+riboflavin); T3 (Treatment 3, EAE + INF-β1a+riboflavin). Clinical Nutrition Experimental 2019 23, 1-14DOI: (10.1016/j.yclnex.2018.12.001) Copyright © 2018 The Authors Terms and Conditions