Differential effects of aging and sex on stroke induced inflammation across the lifespan

Abstract

Aging and biological sex are critical determinants of stroke outcome. Post-ischemic inflammatory response strongly contributes to the extent of ischemic brain injury, but how this response changes with age and sex is unknown. We subjected young (5-6 months), middle aged (14-15 months) and aged (20-22 months), C57BL/6 male and female mice to transient middle cerebral artery occlusion (MCAO) and found that a significant age by sex interaction influenced histological stroke outcomes. Acute functional outcomes were worse with aging. Neutrophils, inflammatory macrophages, macrophages, dendritic cells (DCs) and microglia significantly increased in the brain post MCAO. Cycling females had higher Gr1(-) non-inflammatory macrophages and lower T cells in the brain after stroke and these correlated with serum estradiol levels. Estrogen loss in acyclic aged female mice exacerbated stroke induced splenic contraction. Advanced age increased T cells, DCs and microglia at the site of injury, which may be responsible for the exacerbated behavioral deficits in the aged. We conclude that aging and sex have differential effects on the post stroke inflammatory milieu. Putative immunomodulatory therapies need to account for this heterogeneity.

Keywords: Age; Estrogen; Flow cytometry; Immunity; Ischemia; Sex.

Fig. 1

A and B. Uterine weight (mg/g of body weight) and 17β estradiol levels (pg/ml) in females. Young and middle aged females had significantly higher uterine weights corresponding to higher estradiol levels versus aged females, p < 0.05. C. Representative cresyl violet stained sections of the brains (60 min MCAO, 24 h) in the young, middle aged (aging) and aged males and females. D. Total hemispheric infarct percentage at 24 hour endpoint after 60 min of MCAO. A significant age × sex interaction was seen, p < 0.02; young males had significantly higher infarct volumes vs. young females, p < 0.05; while middle aged females had significantly higher infarct volumes than middle aged males, p < 0.05. Aged males and females had no significant difference in their infarct volumes (n = 6–8/group).

Fig. 2

Representative plots showing gating strategy in the ipsilateral (lesion side- right) hemisphere of brain. A. Plot showing CD45 expression. The plot is gated on CD45 high (right) and CD45 intermediate (left) cell populations. B. CD3 gate is drawn on CD45 high population, T cells are the CD3⁺ cells (right). The gate on the left is drawn on CD45high/CD3⁻ cell populations. C. Plot on CD45high/CD3⁻/CD11b⁺ cell population gated on CD11c. DCs are the CD11c⁺ cells on the right. D. Plot on CD45high/CD3⁻/CD11b⁺/CD11c⁻ cells, gated on Gr1⁺ cells (red arrow, right) which are neutrophils and inflammatory monocytes/macrophages. Plot on CD45 high/CD3⁻/CD11b⁺/CD11c⁻ cells, gated on Gr1⁻ cells which are monocytes/macrophages. E. Plot on CD45 intermediate cells (green arrow down from 1a) gating on CD11b⁺ cells which are the microglia.

Fig. 3

A. T cells in the brains of stroke mice. A significant main effect of age, p < 0.001; sex, p < 0.05 and a significant age × sex interaction, p < 0.001 was seen. A significant negative correlation between estrogen in females and T cells, r = −0.54, higher T cell numbers in aged acyclic females than young and middle aged females. B. T cells in the spleen of control mice. A significant effect of age (p < 0.001) on the frequency of T cells in the spleen in control mice. Young vs. aged, p < 0.001*; T cells decrease significantly with age. C. T cells in the spleen of stroke mice. A significant effect of sex (p < 0.001) on the frequency of T cells in the spleen in MCAO mice. T cells are significantly lower in young males vs. females, p < 0.001*.

Fig. 4

A. DCs in the brains of stroke mice. A significant effect of sex (p < 0.001) on the % of DCs in the brain in MCAO mice. DCs are significantly lower in young males vs. female* and middle aged males vs. females**, respectively. B. Neutrophils and Gr1⁺ macrophages in the brains of stroke mice. No significant effect of age or sex on the percentage of neutrophils in the brain, p > 0.05. C. Neutrophils and Gr1⁺ macrophages in the spleen of stroke mice. A significant effect of age*, p < 0.04 on the frequency of neutrophils and Gr1⁺ macrophages in the spleen. D. Gr1⁻ macrophages in the brain of stroke mice. A significant effect of sex (p < 0.001)*, and age x sex interaction (p < 0.04) was seen. Aged females had significantly lower Gr1⁻ macrophages as compared to cycling females** (positive correlation with estrogen, r = 0.58, p < 0.009).

Fig. 5

A. Microglia in the brain of control mice. No significant effect of age or sex was seen. B. Microglia in the brain of stroke mice. There is no significant effect of age or sex. C. Iba1+ (microglia) cells in stroke and control mice quantified as average number of cells per field of view through IHC. An effect of stroke and age was seen, p < 0.05. Significantly higher number of microglia were seen with increasing age (*) and also with stroke (#). D. Cresyl violet stained coronal section of the brain showing the penumbral region where Iba1+ cells were counted using IHC. E. Iba1+ microglia in representative sections from young and aged male and female control mice showing increased number of Iba1+ cells in the aged (20×). F. Iba1+ microglia in representative sections from young and aged male and female MCAO mice showing increased number of Iba1+ cells in the aged (20×). Iba1 – green, DAPI – blue. Images were processed with a 1.6 gamma adjustment. Scale bars indicate 50 μm.

Fig. 6

Bar graphs showing the differential leukocyte percentage (expressed as a percentage of CD45 high) in the brains of males (6A), and females (6B), across the lifespan after MCAO.