Fig. 1: Increased inflammatory responses in aged mouse brains.

a Immunohistochemical staining for Iba-1 in the cerebral cortex of young (2–3 months old) and old (21–22 months old) mice (arrow). Scale bars = 200 µm (A, C) and 50 µm (B, D). b The relative intensity of Iba-1-immunopositive cells in the brains of young and old mice was quantified (n = 6, means ± SDs, *p-value < 0.05). c Cytokine array results for the cerebral cortex of young (3 months old) and old (15 months old) mice. The relative expression of the protein spots detected by the array is presented in a graph based on a comparison of the signal intensities between young and old mice. d Immunoblots showing the levels of IκBα and p65 in young and old mice (n = 6). Tubulin was used as the loading control. The graph presents the intensities of IκBα normalized to those of tubulin (means ± SDs, **p-value < 0.01). e–i Bulk RNA sequencing of the cerebral cortex of young (3 month-old, n = 6 (4 females, 2 males)) and old (15 month-old, n = 7 (3 females, 4 males)) mice. e Principal component analysis (PCA) plot of the RNA-seq data obtained from the brains of young and old mice. f Volcano plot showing −log10 (adjusted p-value) and log2 (fold change) values for all genes, with those that were significantly upregulated in old mice (Benjamini‒Hochberg (BH)-adjusted p-value < 0.05 and log2 fold change > 2) indicated by red highlighting, and those that are significantly downregulated in old mice (BH-adjusted p-value < 0.05 and log2 fold change < −2) indicated by blue highlighting. g Gene Ontology (GO) terms enriched among significantly upregulated differentially expressed genes (DEGs) in old mice. The dot color and size represent the p-value and gene ratio (gene count in a specific term divided by the total number of genes), respectively. The top 10 GO terms, according to the gene ratio with BH-adjusted p-values < 0.05, are listed. h Heatmap showing the results of gene set variation analysis (GSVA). The color represents the Z scored and normalized enrichment score. i Gene set enrichment analysis (GSEA) of GO biological processes for significant DEGs. Significant GO terms (BH-adjusted p-value < 0.05) involved in the immune response are presented.