Age-Related Changes in Neuroinflammation and Epigenetic Regulation in Mouse Ischemic Stroke Model

Abstract

Background/Objectives: The incidence and prevalence of ischemic stroke, a leading cause of death and disability worldwide, are significantly higher in older adults than in younger individuals. Senescence induces a variety of biological changes that influence the pathogenesis of diseases such as ischemic stroke, thereby necessitating age-specific medical treatments. However, the molecular mechanisms underlying age-related differences in ischemic stroke progression remain poorly understood. Methods: We compared the histological and molecular features of ischemic stroke in a photothrombotic mouse model, focusing on 9-week-old (young) and 90-week-old (old) mice. Results: We found that microglial accumulation at the infarct region of the cerebral cortex was significantly lower in old mice than in young ones. This reduction in the microglial response was accompanied by a decrease in the morphological robustness of the astrocytes forming the glial scar. Furthermore, the mRNA expression of proinflammatory cytokines CXCL10, CCL2, and TNF-α, which were upregulated in the infarct region, was considerably higher in the old mice than in the young ones. Cytokine expression was well correlated with the mRNA levels of Toll-like receptor 4 (TLR4), a key regulator of neuroinflammation in old mice, but less correlated with them in young mice. Interestingly, Tlr4 mRNA expression in young mice was negatively correlated with the mRNA expression of the epigenetic regulator HDAC7, whereas this correlation was positive in old mice. Conclusions: These findings suggest that age-dependent changes in epigenetic regulation, such as the interaction between HDAC7 and TLR4, may contribute to the distinct pathological progression of ischemic stroke in older individuals.

Keywords: HDAC7; TLR4; astrocyte; cytokines; ischemic stroke; microglia.

Figure 1

Impaired formation of the glial scar by reactive astrocytes in old mice. (A,B) Cerebral cortex sections from the ipsilateral side of young and old mice, seven days after photothrombosis, were immunostained with the anti-GFAP antibody (n = 3). The yellow square regions in (A) and (B) are shown at higher magnification in (a) and (b), respectively. The white arrowheads indicate the boundary between the ischemic core and penumbra regions. Scale bars: 200 μm (A,B), 50 μm (a,b). (C) The intensity of the GFAP signal was quantified as described in the Section 2 of the manuscript. Values are expressed relative to the average in young mice. Statistical significance was determined using Student’s t-test.

Figure 2

Reduced microglial accumulation in the infarct region in old mice. Cerebral cortex sections from the ipsilateral side of young and old mice, three days after photothrombosis, were immunostained with an anti-IBA1/AIF1 antibody (n = 4). (A,B) Representative images. The yellow square regions in (A) are shown at higher magnification in (B). Scale bars: 200 μm (A), 50 μm (B). (C) Microglia in the penumbra and ischemic core regions within 200 μm from the boundary between these regions were counted and normalized to the area. Statistical significance was determined using Student’s t-test.

Figure 3

mRNA expression of inflammatory cytokines in the infarct region of young and old mice. The expression levels of inflammatory cytokines three days after photothrombosis were measured by RT-qPCR as described in the Materials and Methods section of the manuscript (young, n = 5; old, n = 4). Values are expressed relative to the average of the contralateral side in young mice. Statistical significance was determined using the Tukey–Kramer post hoc test. * p < 0.05, ** p < 0.01.

Figure 4

Relationship between the mRNA expression of inflammatory cytokines and Tlr4. (A) mRNA expression of Tlr4 in the contralateral and ipsilateral (infarct) regions of young (n = 5) and old (n = 4) mice three days after photothrombosis. Values are expressed relative to the average of the contralateral side in young mice. Statistical significance was determined using the Tukey–Kramer post hoc test. (B) The effects of ischemic stroke, aging, and their interaction on Tlr4 mRNA expression were analyzed using the two-way factorial ANOVA. * p < 0.05, *** p < 0.001. (C) Results of the correlation analysis between inflammatory cytokines and Tlr4. The correlation coefficients for each condition are displayed.

Figure 5

Relationship between the mRNA expression of Hdac7 and Tlr4. (A) mRNA expression of Hdac7 in the contralateral and ipsilateral (infarct) regions of young (n = 5) and old (n = 4) mice three days after photothrombosis. Values are expressed relative to the average of the contralateral side in young mice. Statistical significance was determined using the Tukey–Kramer post hoc test. (B) The effects of ischemic stroke, aging, and their interaction on Hdac7 mRNA expression were analyzed using the two-way factorial ANOVA. * p < 0.05. (C) Results of correlation analysis between Tlr4 and Hdac7. The correlation coefficients for each condition are displayed.

Figure 6

Schematic summary of this study. The gray and blue arrows represent the associations between Tlr4 mRNA expression and inflammatory cytokine expression.