Selective deletion of interleukin-1 alpha in microglia does not modify acute outcome but may regulate neurorepair processes after experimental ischemic stroke

Abstract

Inflammation is a key contributor to stroke pathogenesis and exacerbates brain damage leading to poor outcome. Interleukin-1 (IL-1) is an important regulator of post-stroke inflammation, and blocking its actions is beneficial in pre-clinical stroke models and safe in the clinical setting. However, the distinct roles of the two major IL-1 receptor type 1 agonists, IL-1α and IL-1β, and the specific role of IL-1α in ischemic stroke remain largely unknown. Here we show that IL-1α and IL-1β have different spatio-temporal expression profiles in the brain after experimental stroke, with early microglial IL-1α expression (4 h) and delayed IL-1β expression in infiltrated neutrophils and a small microglial subset (24-72 h). We examined for the first time the specific role of microglial-derived IL-1α in experimental permanent and transient ischemic stroke through microglial-specific tamoxifen-inducible Cre-loxP-mediated recombination. Microglial IL-1α deletion did not influence acute outcome after ischemic stroke. However, microglial IL-1α knock out (KO) mice showed reduced peri-infarct vessel density and reactive astrogliosis at 14 days post-stroke, alongside long-term impaired functional recovery. Our study identifies for the first time a critical role for microglial IL-1α on post-stroke neurorepair and recovery, highlighting the importance of targeting specific IL-1 mechanisms in brain injury to develop effective therapies.

Keywords: Conditional gene knockout; interleukin-1 alpha; microglia; neurorepair; stroke.

Figure 1.

IL-1 brain expression during the early phase of distal pMCAo. (a) Schematic representation of the experimental design. (b) RT-qPCR analysis showing different spatio-temporal expression of IL1A (left) and IL1B (right) in the contralateral, peri-infarct and infarct areas at 4, 24 and 72 h after stroke (mRNA levels normalized to contralateral at 4 h); n = 6/group, ns = non-significant, ^#p < 0.100, *p < 0.05, **p < 0.01 vs. contralateral, Friedman test followed by Dunn’s post hoc test. (c) Representative immunostaining of IL-1α (red), microglia (Iba1, white), neutrophils (Ly6G, green), IL-1β (red), and DAPI (blue) in the infarct at 4, 24 and 72 h after stroke. Scale bar is 50 µm (large image) and 20 µm (insert) and (d) Percentage of IL-1α+ microglia in the infarct at 4, 24 and 72 h after stroke. (left; n = 3/group, ***p < 0.001, one-way ANOVA followed by Dunnet’s post hoc test). Percentage of IL-1β+ microglia and IL-1β+ neutrophils in the contralateral, peri-infarct and infarct areas at 4, 24 and 72 h after stroke. (right; n = 3/group, one-way ANOVA and Kruskal-Wallis followed by Dunnet’s or Dunn’s post hoc test, respectively). All data are shown as median (IQR), except graphs in (d) showing the percentage of IL-1α+ and IL-1β+ microglia, where data are shown as mean ± SD.

Figure 2.

Microglial IL-1α deletion does not influence acute brain damage after pMCAo nor tMCAo. (a) Schematic representation of the experimental design to study acute outcome after pMCAo. (b) Representative LSCI (left) and T2-WI (right) and (c) CBF quantification (left) and lesion volume quantification (right) at 24 h after pMCAo in IL-1α^fl/fl:Cx3cr1^CreERT2 mice. (d) Schematic representation of the experimental design to study acute outcome after tMCAo and (e) representative cresyl violet-stained brains (lesion delineated by dashed lines) and lesion volume quantification (n = 5–9/group, unpaired t-test). All data are shown as mean ± SD, except the graph in (c) showing the lesion volume at 24 h post-pMCAo, where data are shown as median (IQR).

Figure 3.

Microglial IL-1α deletion does not influence brain lesion progression up to 8 days after pMCAo. (a) Schematic representation of the experimental design. (b) Representative T2-WI at 4 h, 24 h and 8 days after pMCAo and (c) Lesion volume quantification showing no differences between IL-1α^fl/fl and IL-1α^fl/fl:Cx3cr1^CreERT2 mice (n = 4/group, mixed effects model (REML) followed by Sidak’s post hoc test). Data are shown as mean ± SD.

Figure 4.

Effect of microglial IL-1α deletion on long-term stroke outcome. (a) Schematic representation of the experimental design. (b) 28-point neurological score showing significant functional deficit IL-1α^fl/fl:Cx3cr1^CreERT2 compared to in IL-1α^fl/fl mice, 14 and 28 days after tMCAo (n = 15–16, *p < 0.05 mixed effects model (REML) followed by Sidak’s post hoc test). (c) Representative immunostaining of vascular density (lectin-FITC+) in the ipsilateral peri-infarct and corresponding contralateral subcortical brain regions in IL-1α^fl/fl and IL-1α^fl/fl:Cx3cr1^CreERT2 mice, and quantification (Scale bar: 50 µm, n = 4/group, *p < 0.05, **p < 0.01, ***p < 0.001, two-way ANOVA followed by Sidak’s post hoc test) and (d) Representative immunostaining of the glial scar (GFAP+) in the ipsilateral peri-infarct subcortical brain region in IL-1α^fl/fl and IL-1α^fl/fl:Cx3cr1^CreERT2 mice, and quantification of the GFAP+ density (left) and glial scar (GS) thickness (right) (Scale bar: 50 µm, n = 4–5/group, unpaired t-test, **p < 0.01). Data are shown as mean ± SD.