Fig. 2

Characterization of cellular IL-1 expression and serum IL-1 levels in ischemic stroke. a IL-1β immunohistochemical staining demonstrating IL-1β⁺ neurons (arrowheads) and IL-1β⁺ glial cells (insert) in a ≤ 2-day-old infarct. b IL-1β⁺ glial cell (arrow) located in a 3–7-day-old infarct. c IL-1β⁺ neurons (arrowheads) in a ≥ 8-day-old infarct. d Scoring of IL-1β staining intensity showing comparable IL-1β expression in neurons and glial cells in I/PI and NAT, whereas IL-1β was only expressed in macrophages located in I/PI. e IL-1Ra immunohistochemical staining demonstrating IL-1Ra⁺ glial cells (arrows) and IL-1Ra⁺ macrophages extravasating from capillaries (insert). f IL-1Ra⁺ macrophages (arrows) in I/PI in a 3–7-day-old infarct. g IL-1Ra⁺ glia (arrows) in a ≥ 8-day-old infarct. h Scoring of staining intensity showed that IL-1Ra expression was low in neurons in I/PI and NAT early after stroke and was absent in older infarcts. IL-1Ra was mainly expressed in glia located in NAT and I/PI, and in IL-1Ra⁺ macrophages located in I/PI. IL-1Ra⁺ macrophages were absent from NAT. i IL-1α immunohistochemical staining demonstrating IL-1α⁺ glial cell (arrow) and IL-1α⁺ macrophage (insert) in a ≤ 2-day-old infarct. j IL-1α⁺ macrophages in a 3–7-day-old infarct. k IL-1α⁺ macrophages in a ≥ 8-day-old infarct. l Scoring of IL-1α staining intensity showing comparable IL-1α expression in neurons and glia in I/PI and NAT, whereas IL-1α was primarily expressed in macrophages in I/PI and to a lesser extent in macrophages in NAT. m Immunofluorescent staining demonstrating co-localization of IL-1β (red) with Iba1⁺ (green) microglia (arrows) and Iba1⁺ (green) macrophages (insert). Note that not all IL-1β⁺ cells (arrowheads) co-localized to Iba1⁺ cells. n Immunofluorescent staining demonstrating co-localization of IL-1β (red) with NF⁺ (green) neurons (arrows). o Immunohistochemical double staining demonstrating co-localization of IL-1Ra⁺ (red) and CD45⁺ (brown) microglia. p Immunofluorescent staining demonstrating co-localization of IL-1α (red) with Iba1⁺ (green) microglia (arrows) and CD68⁺ (green) macrophages (insert). q-s V-Plex analysis demonstrating comparable plasma levels of IL-1β (q), IL-1Ra (r), and IL-1α (s) between controls (n = 7) and ischemic stroke patients at < 8 h (n = 34) and 72 h (n = 9) after symptom onset. For IL-1β, CV > 25% in one 72-h sample, for IL-1RA CV > 25% in one < 8-h sample and in one 72-h sample. IL-1α, interleukin-1alpha; IL-1β, interleukin-1beta; IL-1Ra, interleukin-1 receptor antagonist; I/PI, infarct/peri-infarct; NF, neurofilament; NAT, normal appearing tissue. 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 = 40 μm, m-n, p = 100 μm, and o = 10 μm