Fig 1: 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.
Fig 2: 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 3: 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 4: TNF receptor expression after SCI. (a) Temporal expression of Tnfrsf1a after SCI measured by real-time RT-qPCR analysis (Time: F6,53 = 24, p < 0.0001, Treatment: F1,53 = 1.00, p = 0.32, Interaction: F6,53 = 1.46, p = 0.21). (b,c) TNFR1 protein levels in the acute ((b), Time: F3,32 = 49.14, p < 0.0001, Treatment: F1,32 = 0.013, p = 0.72, Interaction: F3,32 = 0.25, p = 0.86) and delayed ((c), Time: F1,14 = 711.5, p < 0.0001, Treatment: F1,14 = 1.08, p = 0.32, Interaction: F1,14 = 1.08, p = 0.32) phases after SCI measured by chemiluminescence analysis. (d) Double immunofluorescent staining for TNFR1 (red) and neuronal MAP2 (green) 21, 28, and 35 days after SCI. (e) High magnification image of representative double immunofluorescent staining for TNFR1 (red) and MAP2 (green) in the peri-lesion area 28 days after SCI. (f) Temporal expression of Tnfrsf1b mRNA after SCI measured by real-time RT-qPCR analysis (Time: F6,55 = 23.09, p < 0.0001, Treatment: F1,55 = 0.04, p = 0.84, Interaction: F6,55 = 0.42, p = 0.87). (g,h) TNFR2 protein levels in the acute ((f), Time: F3,29 = 27.53, p < 0.0001, Treatment: F1,29 = 0.007, p = 0.93, Interaction: F3,29 = 0.61, p = 0.62) and delayed ((g), Time: F1,14 = 136.8, p < 0.0001, Treatment: F1,14 = 1.39, p = 0.26, Interaction: F1,14 = 1.39, p = 0.26) phases after SCI measured by chemiluminescence analysis. (i) Double immunofluorescent staining for TNFR2 (green) and astroglial GFAP (red) 21, 28, and 35 days after SCI. (j) High magnification image of representative double immunofluorescent staining for TNFR2 (red) and GFAP (green) in the peri-lesion area 21 days after SCI. 4',6-diamidino-2-phenylindole (DAPI) was used as a nuclear marker. Scale bars: (d,i) = 100 µm and (e,j) = 40 µm. Data are presented as mean ± SEM with n = 5 mice/treatment group/time point. * p < 0.05, **** p < 0.0001. GFAP, glial fibrillary acidic protein; MAP2, microtubule-associated protein 2; ns, not significant; SCI, spinal cord injury; TNFR, tumor necrosis factor receptor.
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