Influence of Age and Sex on M1-Type Pro-Inflammatory Gene Expression Following Cerebral Ischemia and Reperfusion

Study objective: To examine the effects of age and sex on microglial/macrophage M1-type pro-inflammatory gene expression in mice subjected either to transient focal cerebral ischemia followed by reperfusion or to control surgery without occlusion. Hypothesis: We hypothesized that age and sex would differentially modulate baseline and post-ischemic expression of microglial/macrophage M1-type pro-inflammatory markers in mice. Methods: Young (3–4 months) and aged (17–19 months) male and female C57BL/6 mice (n = 6 per age/sex group) underwent 1-hour right middle cerebral artery occlusion (MCAO) or an identical control surgery without occlusion, followed by 24 hours of reperfusion. Ipsilateral brain tissue was collected for RNA isolation, cDNA synthesis, and real-time PCR analysis of mRNA expression for several M1-type pro-inflammatory markers (CD68, CD16, TLR2, TLR4, iNOS, MCP1, CXCL1, TNFα, IL-6, and IL-1β). Results: At baseline (non-ischemic), young females exhibited higher expression (approximately 2-6-fold) of all M1-type pro-inflammatory markers than young males, with significant increases in 9 of the 10 markers examined. Of these, five markers (CD16, TLR2, TLR4, iNOS, and TNFα) were selectively elevated in young females, whereas CD68, MCP1, IL-1ß, and IL-6 were elevated in females of both age groups, suggesting a stable core inflammatory profile in females. CXCL1 was significantly upregulated only in aged females compared with aged males. With aging, IL-6 was the only marker significantly elevated in both sexes. In addition, males showed significant upregulation of the M1-type markers CD16, TLR2, TLR4, and TNFα, whereas females showed significant upregulation of IL-1ß. Following MCAO, nearly all pro-inflammatory markers were significantly upregulated across age and sex groups; most markers increased by approximately 2–50-fold, whereas MCP1, CXCL1, IL-6, and IL-1β showed much larger inductions (up to ~1,560-fold for MCP1, 445-fold for CXCL1, 346-fold for IL-6, and 130-fold for IL-1β), highlighting their key roles in post-ischemic inflammation. Compared with aged females, young females showed stronger post-ischemic induction of several cytokines and chemokines, including CXCL1, IL-6, IL-1β, and TNFα, whereas aged females exhibited relatively greater induction of iNOS and MCP1. In aged mice, males showed much stronger induction of MCP1, CXCL1, IL-1β, and IL-6 than aged females, whereas aged females exhibited relatively higher induction of iNOS and CD16. Overall, young females showed higher baseline expression and marked post-ischemic induction of M1-type markers, whereas aging particularly enhanced post-ischemic M1-type responses in males. Conclusions: Age and sex exert pronounced effects on M1-type pro-inflammatory gene expression and differentially modulate post-ischemic pro-inflammatory responses. These effects likely contribute to variability in stroke severity and outcome and may underlie age- and sex-specific susceptibility to ischemic brain injury, with potential relevance to differences in stroke outcome in humans. Funding: This work was supported by NIH Grant Award (R01NS102573). This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.