Fig 1: The functional state of NTPDase1-positive microglia/macrophages. (A,B) Representative micrographs showing triple immunofluorescence labeling directed to NTPDase1 (red fluorescence), Iba1 (blue fluorescence) and iNOS or Arg1 (green fluorescence) at cross-sections of lumbar spinal cord white matter in control and during EAE. Integrated density corresponding to Iba1 (C), iNOS (D), and Arg1 (E) immunofluorescence in control sections, and during EAE. Bars represent mean fluorescence ± SEM, from n ≥ 4 images per section, n ≥ 6 sections per animal, from 3 animals per experimental group, from two separate experiments. Significance inside the graph: ∗∗∗p < 0.0001 in comparison control. (F) MCC values obtained from multi-image colocalization analyses of micrographs presented in (A), showing level of co-occurrence of pair of signals, as indicated inside the graph. ∗∗p < 0.01, ∗∗∗p < 0.0001 in comparison to control. (G) MCC values calculated for pairs of signals, as indicated inside the graph. Bars at mean MCC value ± SEM from n ≥ 4 images per section, from n ≥ 6 sections per animal, from 3 animals per experimental group, from two separate experiments. Significance inside the graph: ∗p < 0.05, ∗∗p < 0.001, ∗∗∗p < 0.0001 in comparison to control. (C–G) Kruskal–Wallis with Dunn’s post hoc test.
Fig 2: Effect of Agm on two main enzymes of arginine metabolism: iNOS and ARG1 and markers of a microglial activation state. BV-2 cells were pretreated with Agm (100 µm) for 30 min and then stimulated with Lps (1 µg/mL) for an additional 4 or 6 h (qPCR analysis) or 24 h (Western blot analysis and fluorescent microscopy). Data represent gene expressions, protein levels, and immunofluorescence of iNOS (a), ARG1 (b), COX-2 (c), and CD206 (d). Levels of target genes (Nos2, Arg1, Ptgs2, and Mrc1) are expressed relative to the expression of the Gapdh gene. The ratios of iNOS, ARG1, COX2, and CD206 to β-actin are expressed relative to the control group (fold change). Data are presented as mean ± SEM, from three separate determinations. The results were analyzed by Kruskal–Wallis test followed by Dunn’s multiple comparisons tests (qPCR) or by two-way ANOVA followed by Bonferroni’s post hoc tests (Western blot). iNOS protein level was analyzed by t test for comparing the mean values between two groups (Lps vs. LpsAgm) because Ctrl and Agm groups were not detectable. * p < 0.05 compared with the control group; # compared with Lps group. Photomicrographs on the right represent immunofluorescence labeling against target protein (red) counterstained with Hoechst (blue). If not otherwise specified, in this and following figures experimental groups are: Ctrl—untreated control cells; Agm—agmatine sulfate (100 µm) treated cells; Lps—cells stimulated with 1 µg/mL Lps; LpsAgm—cells pretreated with 100 µm Agm and then stimulated with Lps. Plotted scale bar applies to all micrographs.
Fig 3: Functional state of NTPDase1 positive phagocytically active microglia/macrophages. (A,B) Representative micrographs showing triple immunofluorescence labeling directed to NTPDase1 (red fluorescence), CD68 (blue fluorescence), and iNOS or Arg1 (green fluorescence) at spinal cord cross-sections obtained from control animals and during EAE. Scale bar applicable to all micrographs = 20 μm. (C,D) MCC values obtained by multi-image colocalization analysis of micrographs represented in (A,B), showing fractional overlap between pairs of signals, as indicated inside the graph. Bars represent mean value ± SEM, from n ≥ 4 images per spinal cord region, from n ≥ 6 sections per animal, from 3 animals per experimental group, from two separate experiments. Significance inside the graph: ∗p < 0.05; ∗∗∗p < 0.0001 in respect to control, Kruskal–Wallis with Dunn’s post hoc test.
Fig 4: Assessment of the functional state of reactive microglia after TMT exposure. Ramified morphology of Iba1+cells corresponds to control microglia but also to ramified Iba1+ cells in the hippocampal areas distant from the site of neurodegeneration at both 7 and 21 dpi. Double immunofluorescent staining of Iba1 and iNOS, Arg1, CD68, P2Y12 receptor (R), and eN/CD73, and triple immunofluorescent staining of Iba1, GFAP and P2X7R in the injured area 7 and 21 dpi, reveal Iba1-ir morphotypes that expressed Arg1-, CD68-, P2Y12-, P2X7- as well as eN-ir. Scale bar = 50 μm.
Fig 5: Proinflammatory status of the rat hippocampal region after TMT exposure The abundance of transcripts coding IL-1β, TNF-α, IL-6, IL-10, C3, S100a10, iNOS, and Arg1. Bars represent mean mRNA expression of target gene relative to CycA ± SD. Significance shown inside the graphs: *p < .05 or less compared to age-match Ctrl.
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