Fig 1: Characterization of TNF and TNFR in individuals with SCI. (a,b) Immunofluorescent double labeling of TNF (red) and Iba1 (green) expressing microglia/macrophages (arrows) in the spinal cord of an 33-year-old man with a 3-week-old C6–7 SCI. (c) Immunofluorescent double labeling of TNF (green) and CD68 (red) phagocytic cells in the spinal cord of an 65-year-old man with a 5-week-old C5 SCI. (d) Immunofluorescent double labeling of TNF+ (green) cells and GFAP+ astrocytes (red) in a 61-year-old man with a 2-week-old C1-2 SCI. (e) Immunofluorescent double labeling of TNFR1+ (red) and NF-L+ (green) neuronal fibers in an 67-year-old man with a 6-week-old C5-7 SCI. (f) Immunofluorescent double labeling of TNFR2 (red) and astroglial GFAP (green) in an 80-year-old woman with a 2-month-old C8-T1 SCI. (g) Immunofluorescent double labelling of TNFR2+ (red) and Iba1+ (green) microglia (arrow) in a 67-year-old man with a 6-week-old C5-7 SCI. (h) Immunofluorescent double labeling of TNFR2+ (red) cells and NF-L+ (green) neuronal fibers in an 33-year-old man with a 3-week-old C6-7 SCI. (i,j) Immunofluorescent double labeling of IL-1ß (red) and Iba1 (green) in a 65-year-old man with a 5-week-old C5 SCI. IL-1ß was found to co-localize to a subpopulation of Iba1+ microglia (arrow in h). (k,l) Immunofluorescent double labeling of CD68 (red) and Iba1 (green) in a 67-year-old man with a 6-week-old C5-7 SCI (k) and a 33-year-old man with a 3-week-old C6-7 SCI (l). Scale bars: (a,d,e,i,l) = 20 µm; (b,c,g,j) = 40 µm; (f,g,h) = 100 µm. (m,n) CSF TNF (m) and TNFR1 (n), Time: F3,30 = 10.33, p = 0.003) levels in individuals with SCI. Results are expressed as mean ± SEM, n = 5–12/group, * p < 0.05,** p < 0.01. CD, cluster of differentiation; GFAP, glial fibrillary acidic protein; Iba1, ionized calcium-binding adaptor molecule 1; IL, interleukin; NF-L, neurofilament light chain; TNF, tumor necrosis factor; TNFR, TNF receptor.
Fig 2: Spatiotemporal expression of TNF receptor 1 and 2 after SCI. (a,b) TNFR1 protein levels in the acute (a) and delayed (b) phases after SCI. Significant indications represent comparisons to sham mice in the acute phase (a, Interaction: F5,48 = 4.52, p = 0.002; Time: F5,48 = 45.74, p < 0.0001; SCI: F1,48 = 2.19, p = 0.15) and delayed phase (b, Interaction: F4,40 = 49.51, p < 0.0001; Time: F4,40 = 52.17, p < 0.0001; SCI: F1,40 = 406.5, p < 0.0001) after SCI. (c–g) Immunofluorescent double labelling for TNFR1 (red) and neuronal MAP2 (green) within the lesion site (c), peri-lesion area (d,f,g), as well as distant from the lesion site (e) at 21 (c–e) and 28 (f,g) days after SCI. (h) Immunofluorescent double labelling for TNFR1+ (red) and CD68+ (green) cells 21 days after SCI. (i,j) TNFR2 protein levels in the acute phase (i, Interaction: F5,47 = 0.42, p = 0.83; Time: F5,47 = 3.89, p < 0.005; SCI: F1,47 = 1.75, p = 0.19) and delayed phase (j, Interaction: F4,40 = 25.04, p < 0.0001; Time: F4,40 = 36.07, p < 0.0001; SCI: F1,40 = 2702.8, p < 0.0001) after SCI. (k–n) Immunofluorescent double labelling for TNFR2 (red: k,l and green: (m,n) and GFAP+ astrocytes (green: (k,l) and red: (m,n)) and CD11b+ microglia/macrophages (green: (l)) at 21 (k) and 28 (l–n) days after SCI. (o–q) Immunofluorescent double labeling of TNFR2+ cells (arrows in (o)) and CD68+ microglia/macrophages (arrow heads in (o)). (p) Only a few CD68+ cells co-expressed TNFR2. (q) represents a high magnification image of the area squared in (p), demonstrating TNFR2 + CD68+ cells located in the peri-lesion areas 21 days after SCI (arrow heads in (q)). DAPI was used as a nuclear marker. Scale bars: (k,m) = 100 µm, (c–f,h,n–p) = 40 µm, and (g,l,q) = 20 µm. Results are expressed as mean ± SEM, n = 5/group, **** p < 0.0001. GFAP, glial fibrillary acidic protein; MAP2, microtubule associated protein 2; TNFR, tumor necrosis factor receptor.
Fig 3: Cellular and temporal expression of Tnf mRNA in the thoracic spinal cord after SCI. (a–i) In situ hybridization was used to investigate the distribution of Tnf mRNA+ cells the first 24 h after SCI. Tnf mRNA expression was undetectable in naïve mice (a). Tnf mRNA+ cells in the white matter of the posterior funiculi (arrows in b) and in neuronal-like cells in the dorsal horn (arrowhead in c), at 1 h after SCI. Tnf mRNA+ cells in the white matter of the posterior funiculi (d) and in the grey matter of the ventral horn (e). At 3 h, most cells displayed macrophage- or glial-like morphology (arrows in f). By 6 h (g), 12 h (h), and 24 h (i), Tnf mRNA+ cells were mainly located in white matter areas of the damaged spinal cord (arrows). (j) Parallel spinal cord sections that were in situ hybridized for glyceraldehyde-3-phosphate dehydrogenase (Gapdh) mRNA showed a largely neuronal signal and confirmed the overall suitability of the tissue for in situ hybridization. (k,l) Parallel sections hybridized with buffer alone (k) or pretreated with RNAse A before the in situ hybridization (l) were devoid of signal. (m–o) RT-qPCR analysis of Tnf mRNA (m), Tnfrsf1a mRNA (n), and Tnfrsf1b mRNA (o) levels in naïve mice and in mice allowed 1, 3, 6, and 12 h and 1, 3, 7, 14, and 28-days survival after SCI. Tnf mRNA levels were significantly increased at 1, 3, and 6 h and 7 days after SCI, compared to naïve mice (Time: p < 0.0001, F9,39 = 58.14) (m). Tnfrsf1a mRNA levels significantly increased from 6 h to 28 days after SCI, compared to naïve mice (Time: p < 0.0001, F9,39 = 20.01) (n). Tnfrsf1b mRNA levels significantly increased at 3 h after SCI, compared to naïve mice (Time: p = 0.009, F9,39 = 2.965). Results are expressed as mean ± SEM, n = 5/group, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Scale bars: (a–c, f–i, k,l) = 40 µm, (d,e) = 100 µm, and (j) = 200 µm.
Supplier Page from Abcam for Human sTNF RI ELISA Kit