GoKawiil(a) Flow cytometry analysis of Igf1 expression in microglia from Igf1-Egfp transgenic mice on day 21 after stroke onset. ASO-control or ASO-Zfp384 was administered on day 8 after stroke onset. The gate indicates Igf1-EGFPhi cells (n = 5 for each group). (b) Representative immunohistochemistry images and quantification of IGF1-positive IBA1+ cells in the surviving peri-infarct brain tissue on day 28 after stroke onset. ASOs were administered on days 8 and 22 after stroke onset (n = 3 per group) (bar: 20 μm). (c) A violin plot shows the cumulative expression levels of genes associated with inflammatory response (gene list is described in the Method section) in the microglia on day 28 after stroke onset. A dot plot shows the expression levels of inflammatory mediators in microglia. ASOs were administered on days 8 and 22 after stroke onset. (d) UMAP plots of total brain cells obtained from the post-ischaemic day 14 brain by scRNA-seq analysis. The heatmap shows the expression level of Igf1, Spp1, and Bdnf. (e) Violin plots show the Igf1, Spp1, and Bdnf expression levels in each brain cell population. (f) Violin plots of Igf1, Spp1, and Bdnf expression levels in microglia or astrocytes on day 28 after stroke onset. ASOs were administered on days 8 and 22 after stroke onset. (g) Neurological deficits after stroke onset in ASO-Zfp384-administered mice with the administration of control IgG, anti-IGF1, or anti-SPP1 neutralizing antibody. ASO-Zfp384 was administered on days 8 and 22. The neutralizing antibody was administered intracerebroventricularly on days 8, 13, 18, and 23 after stroke onset (n = 7 for control IgG, n = 11 for anti-IGF1, and n = 11 for anti-SPP1). (h) Neurological deficits after stroke onset in ASO-Zfp384-administered Igf1flox/flox and Cx3cr1-CreER; Igf1flox/flox mice (n = 9 for Igf1flox/flox and n = 7 for Cx3cr1-CreER; Igf1flox/flox mice). (i) Relative mRNA expression levels of the indicated genes in microglia isolated from the ipsilateral hemisphere on post-stroke day 6, compared to Igf1flox/flox mice (n = 4). (j) The measurement of ER-TR7+ area (fibrotic scar) and GFAP+ area (glial scar) in the ipsilateral hemisphere on day 28 after stroke onset (n = 4 for Igf1flox/flox and n = 5 for Cx3cr1-CreER; Igf1flox/flox mice). *p < 0.05, **p < 0.01, ***p < 0.001 vs. ASO-control (a,b), control IgG (g), or Igf1flox/flox (h,i,j). (two-sided Student’s t-test [a,b,i,j]; two-way ANOVA with Dunnett’s test [g]; two-way ANOVA [h]). Error bars represent the mean ± standard error of the mean (SEM).
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