Additional file 4 of Carnosine quenches the reactive carbonyl acrolein in the central nervous system and attenuates autoimmune neuroinflammation

Additional file 4: Figure S1. Graphical summary of experimental designs. Graphical representation of the timing of carnosine treatment, EAE induction and sacrifice (tissue collection) for (a) analyses of control vs. EAE mice (data shown in Figure 2, Figure 3, Figure S2, Figure S3, Figure S4); (b) analysis of tissue carnosine loading in EAE (data shown in Figure 4); and (c) analyses of dose-dependent carnosine treatment effects (data shown in Figure 5, Figure 6, Figure S5, Figure S6). Figure S2. Spinal cord inflammatory activity during EAE. (a) Spinal cord mRNA expression in different stages of EAE compared to healthy controls (dashed line). n=6 animals/group. (b) Immunohistochemistry-based quantification of F4/80+ microglia/macrophage numbers in the spinal cord in different stages of EAE compared to healthy controls (dashed line). n=6 animals/group. Gene expression (a) and microglia/macrophage numbers (b) were increased in EAE vs. healthy controls (statistics not shown). Data are mean ± SD. One-way ANOVA or Kruskal-Wallis, post hoc testing *p<0.05, **p<0.01 between the indicated groups. Figure S3. HNE-protein adducts are not affected by EAE. Detection of HNE-protein adducts by western blot in different stages of EAE compared to healthy controls. n=5-7 animals/group. Data are mean ± SD. One-way ANOVA indicated no significant between-group differences. Figure S4. Spinal cord GSH levels in EAE. UPLC-ESI-MS/MS-based quantification of spinal cord GSH in different stages of EAE compared to healthy controls. n=11-22 animals/group. GSH, reduced glutathione. Data are mean ± SD. One-way ANOVA, post hoc testing *p<0.05, **p<0.01 between the indicated groups. Figure S5. Effects of carnosine treatment on homocarnosine, anserine and GSH levels in chronic EAE. UPLC-ESI-MS/MS-based quantification of spinal cord and plasma (a, d) homocarnosine, (b, e) anserine and (c, f) GSH in healthy controls, untreated EAE animals (0%) and following treatment with different doses of carnosine (0.3%, 1.5%, 3%) in EAE. n=7-11 animals/group. Data are mean ± SD. One-way ANOVA or Kruskal-Wallis, post hoc testing *p<0.05, **p<0.01 between the indicated groups. Figure S6. No effect of EAE and carnosine treatment on HNE-protein adduct levels. Detection of HNE-protein adducts by western blot in healthy controls, untreated EAE animals (0%) and following treatment with different doses of carnosine (0.3%, 1.5%, 3% in drinking water) in EAE. n=7-15 animals/group. Data are mean ± SD. One-way ANOVA indicated no significant between-group differences. Figure S7. Effect of carnosine on acrolein quenching, anserine levels and cell viability in primary mouse microglia cultures. UPLC-ESI-MS/MS-based quantification of (a) carnosine and carnosine-propanal and (b) anserine in primary mouse microglia exposed to carnosine or vehicle control (PBS) for 24 h, followed by LPS, H2O2 or vehicle control (PBS) for 24 h. n=4-5 biological replicates/condition. Carnosine-propanal values (~0.004 to 0.012 nmol/1e6 cells) were transformed (×103) to facilitate visualisation on the Y axis. Anserine was undetectable in all experimental conditions lacking carnosine pre-treatment. (c) Cell viability of primary microglia assessed by flow cytometry (Fixable Viability Dye eFluor 506). Cells were exposed to carnosine or vehicle control (PBS) for 24 h, followed by LPS, H2O2 or vehicle control (PBS) for 24 h. Data from n=7 different experiments, normalized to the respective control condition in each experiment and expressed as % change. Car, carnosine. H2O2, hydrogen peroxide; LPS, lipopolysaccharide; PBS, phosphate-buffered saline. Data are mean ± SD. Mann-Whitney U test with Bonferroni-adjusted p values for multiple testing *p<0.05 (b). One-way ANOVA indicated no significant between-group differences (c).