Figure 8.
TNF-α expression is induced after a demyelinating injury and increases the number of premyelinating oligodendrocytes. (A and B) Confocal maximum intensity z-projections of RNAscope in situ hybridization targeting TNF-α in mouse spinal cord lesions at 4 dpi. TNF-α was determined by the total amount of signal in the lesion. n = 4 or 5 lesions. (C) TNF-α mRNA in spinal cord lesions of zebrafish larvae was determined by quantitative RT-PCR of lesions at 6 hpi. n = 3 independent experiments. (D–G) After 5 or 6 DIV, OCSCs were treated with 50 or 100 ng/ml recombinant mouse TNF-α and EdU for 48 h. (D and E) Newly formed myelinating oligodendrocytes were labeled by EdU (green) and expressed BCAS1 (magenta; examples of colocalization, white arrowheads) in the cell body. n = 5–11 slices in two experiments. (F and G) Newly generated oligodendrocytes that expressed OLIG2 (magenta) and incorporated EdU (green; examples of colocalization, white arrowheads). n = 6–13 slices in two experiments. (H) Schematic representation of cortical injection of TNF-α or vehicle in WT mice. (I) IHC of BCAS1 on day 5 after stereotactic injection of vehicle or TNF-α in WT mice. (J) Quantification of BCAS1+ cells with a premyelinating morphology in the subpial cortex day 5 after stereotactic injection of vehicle or TNF-α. (K and L) 2 h after myelin debris treatment, TNF-α (50 ng/ml) was added to cultured microglia for 6 h. The amount of PLP per cell at 6 h was normalized to the initial amount of PLP per cell (0 h). Myelin treatment control versus TNF-α: P = 3.125 × 10−6; effect size (Cohen’s d) = 0.224; 703 cells in the control group and 1,252 cells in the TNF-α treatment group were sampled randomly. n = 3 independent experiments. (M and N) After 7 DIV, OCSCs were treated with 0.5 mg/ml LPC for 16 h and subsequently with TNF-α (100 ng/ml) for 48 h, and BCAS1 immunoreactivity was determined in the slices. n = 9–17 slices in four to six independent experiments. All data are mean ± SEM (error bars). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by unpaired t test with Welch's correction in B, J, and L; one-way ANOVA test with Tukey’s multiple comparisons test in C, E, and G; and two-way ANOVA test with Tukey’s and Sidak’s multiple comparisons tests in N. Scale bars: 20 µm in A; 100 µm in D, F, and I; 60 µm in K; and 500 µm in M.

TNF-α expression is induced after a demyelinating injury and increases the number of premyelinating oligodendrocytes. (A and B) Confocal maximum intensity z-projections of RNAscope in situ hybridization targeting TNF-α in mouse spinal cord lesions at 4 dpi. TNF-α was determined by the total amount of signal in the lesion. n = 4 or 5 lesions. (C) TNF-α mRNA in spinal cord lesions of zebrafish larvae was determined by quantitative RT-PCR of lesions at 6 hpi. n = 3 independent experiments. (D–G) After 5 or 6 DIV, OCSCs were treated with 50 or 100 ng/ml recombinant mouse TNF-α and EdU for 48 h. (D and E) Newly formed myelinating oligodendrocytes were labeled by EdU (green) and expressed BCAS1 (magenta; examples of colocalization, white arrowheads) in the cell body. n = 5–11 slices in two experiments. (F and G) Newly generated oligodendrocytes that expressed OLIG2 (magenta) and incorporated EdU (green; examples of colocalization, white arrowheads). n = 6–13 slices in two experiments. (H) Schematic representation of cortical injection of TNF-α or vehicle in WT mice. (I) IHC of BCAS1 on day 5 after stereotactic injection of vehicle or TNF-α in WT mice. (J) Quantification of BCAS1+ cells with a premyelinating morphology in the subpial cortex day 5 after stereotactic injection of vehicle or TNF-α. (K and L) 2 h after myelin debris treatment, TNF-α (50 ng/ml) was added to cultured microglia for 6 h. The amount of PLP per cell at 6 h was normalized to the initial amount of PLP per cell (0 h). Myelin treatment control versus TNF-α: P = 3.125 × 10−6; effect size (Cohen’s d) = 0.224; 703 cells in the control group and 1,252 cells in the TNF-α treatment group were sampled randomly. n = 3 independent experiments. (M and N) After 7 DIV, OCSCs were treated with 0.5 mg/ml LPC for 16 h and subsequently with TNF-α (100 ng/ml) for 48 h, and BCAS1 immunoreactivity was determined in the slices. n = 9–17 slices in four to six independent experiments. All data are mean ± SEM (error bars). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by unpaired t test with Welch's correction in B, J, and L; one-way ANOVA test with Tukey’s multiple comparisons test in C, E, and G; and two-way ANOVA test with Tukey’s and Sidak’s multiple comparisons tests in N. Scale bars: 20 µm in A; 100 µm in D, F, and I; 60 µm in K; and 500 µm in M.

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