Fig. 3

Characterization of cellular localization and plasma levels of TNF, TNFR1, and TNFR2 in ischemic stroke. a TNF immunohistochemical staining showing a TNF⁺ neuron (arrowhead) in a ≤ 2-day-old infarct. b TNF⁺ glial- and macrophage-like cells (arrows) located in a 3–7-day-old infarct. c TNF⁺ cells (arrows) and TNF⁺ macrophages extravasating from capillaries (arrows in insert) in a ≥ 8-day-old infarct. d Scoring of TNF staining intensity showing significantly higher TNF expression in neurons located in I/PI in ≤2-day-old infarcts compared to ≥8-day-old infarcts and in neurons located in NAT in ≤2-day-old infarcts compared to both 3–7-day-old and ≥ 8-day-old infarcts (upper graph). TNF expression was comparable in glial cells located in I/PI and NAT in ≤2-day-old and 3–7-day-old infarcts, with significantly increased expression in I/PI in ≥8-day-old infarcts compared to ≤2-day- and 3–7-day-old infarcts and compared to NAT (middle graph). At all timepoints, TNF expression in macrophages was only observed in I/PI (lower graph). e TNFR1 immunohistochemical staining demonstrating a TNFR1⁺ glial cell (arrow) and TNFR1⁺ neuron (arrowhead) in a ≤ 2-day-old infarct. f TNFR1⁺ neurons (arrowheads) in a 3–7-day-old infarct. g TNFR1⁺ neuron (arrowhead) in a ≥ 8-day-old infarct. h Scoring of staining intensity showed that TNFR1 expression was comparable in neurons and glia in I/PI and NAT at all timepoints. TNFR1 was mainly expressed in macrophages located in I/PI and absent from NAT. i TNFR2 immunohistochemical staining demonstrating TNFR2⁺ glial end-feet encircling the blood vessels (arrow) and a TNFR2⁺ glia cell (insert) in ≤2-day-old infarcts. j TNFR2⁺ cell (arrow) in a 3–7-day-old infarct. k TNFR2⁺ glia in a ≥ 8-day-old infarct. l Scoring of TNFR2 staining intensity showing comparable TNFR2 expression in glia located in I/PI and NAT in ≤2-day-old and 3–7-day-old infarcts and with a significant increase in TNFR2 expression in glia located in I/PI compared to NAT in ≥8-day-old infarcts. TNFR2 expression was only found in macrophages located in I/PI. No neurons were found to express TNFR2. m Immunofluorescent staining demonstrating co-localization of TNF (red) with NeuN⁺ (green) neurons (arrows). n Immunofluorescent staining demonstrating co-localization of TNFR1 (red) with NF⁺ (green) neurons and their proximal dentrites (arrows). o Immunofluorescent staining demonstrating co-localization of TNFR2 (green) with GFAP⁺ astrocytes (red). p High magnification of (o). q V-Plex analysis demonstrating comparable plasma TNF levels between controls and ischemic stroke patients at < 8 and 72 h after symptom onset. r-s V-Plex analysis demonstrating significantly increased plasma TNFR1 (r) and TNFR2 (s) levels in ischemic stroke patients < 8 h after symptom onset compared to controls. For TNF, CV > 25% in three control samples and one < 8-h sample. For TNFR1, CV > 25% in four < 8-h samples, 12 control samples, and one 72-h sample. For TNFR2, CV > 25% in three < 8-h samples, 11 control samples, and three 72-h samples. GFAP, glial fibrillary acidic protein; I/PI, infarct/peri-infarct; NF, neurofilament; NAT, normal appearing tissue; TNF, tumor necrosis factor; TNFR1, tumor necrosis factor receptor 1; TNFR2, tumor necrosis factor receptor 2. Results are presented as mean ± SD (staining intensity) or mean with IQR (V-plex analysis). *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA followed by Sidak’s multiple comparisons test (scoring of staining intensity, n = 3–9/group) or Kruskal Wallis test followed by Dunn’s multiple comparison test (V-plex analysis). Scale bars: a-c, e-g, i-k, p, inserts = 40 μm, and m-o = 100 μm