Stroke sensitivity in the aged: sex chromosome complement vs. gonadal hormones

Abstract

Stroke is a sexually dimorphic disease. Elderly women not only have higher stroke incidence than age-matched men, but also have poorer recovery and higher morbidity and mortality after stroke. In older, post-menopausal women, gonadal hormone levels are similar to that of men. This suggests that tissue damage and functional outcomes are influenced by biologic sex (XX vs. XY) rather than the hormonal milieu at older ages. We employed the Four Core Genotype (FCG) mouse model to study the contribution of sex chromosome complement and gonadal hormones to stroke sensitivity in aged mice in which the testis determining gene (Sry) is removed from the Y chromosome, allowing for the generation of XX males and XY females. XXF, XXM, XYF, XYM and XYwt aged mice were subjected to middle cerebral artery occlusion (MCAO). XXF and XXM mice had significantly larger infarct volumes than XYF and XYM cohorts respectively. There was no significant difference in hormone levels among aged FCG mice. XXF/XXM mice also had more robust microglial activation and higher serum levels of pro-inflammatory cytokines than XYF/XYM cohort respectively. We concluded that the sex chromosome complement contributes to ischemic sensitivity in aged animals and leads to sex differences in innate immune responses.

Keywords: chromosome; hormone; immune response; ischemic stroke; microglia; sex difference.

Figure 1. Brief introduction of FCG mice

(A) Totally four genotypes of mice are produced by mating XXF to XYM mouse. XXF/XYF mice are gonadally females and XXM/XYM are males; among them, only XXF and XYM are fertile. (B) PCR results of FCG mice. MYO, autosomal gene (myosin) to confirm that PCR works (~250bp); YMT, Y gene to confirm presence of Y chromosome (~350bp); SRY, sex-determining region Y gene (~420bp).

Figure 2. Stroke outcomes of 72 hours after MCAO

(A) Representative CV staining pictures of stroked brain slices from FCG plus wide type male mice. (B-D) Quantitative data of infarct size in total hemisphere (B), cortex (C), and striatum (D). (E) Box-whisker figure of NDS. NDS was indicated as Median (interquartile range); white and gray boxes indicate the 25-50% and 50-75% interquartile range respectively; whiskers indicate the maximum and minimum NDS score. n=7~9 animals/group; *P < 0.05.

Figure 3. Serum levels of hormones and cytokines by Elisa

Estrodial (A) and testosterone levels (B) showed no differences between any two strains either in sham or stroke mice. (C) IL-1β level. (D) TNF-α level. n=6/sroke group; n=4/sham group; *P < 0.05.

Figure 4. Microglial responses after stroke

(A) Gating strategy indicating microglia as CD45^lowCD11b⁺, peripheral myeloid cells as CD45^highCD11b⁺, and lymphocytes as CD45^highCD11b⁻. (B) and (C) Representative histograms showing MHC II expression on microglia in stroke and sham groups respectively. (D) Mean fluorescence intensity (MFI) of MHC II in each groups. n=6/sroke group; n=3/sham group; *P < 0.05.

Figure 5. Infiltrating leukocytes in ischemic brains

(A) Representative flow plots from each mouse strain showing gate strategy for infiltrating peripheral leukocytes (CD45^high). (B) Total numbers of infiltrating peripheral leukocytes. (C) Representative plots indicating the percentage of inflammatory monocytes (Quardrant Q1) and neutrophils (Quardrant Q2) in infiltrating peripheral myeloid cells. Total numbers of each cell population were quantified in (D) monocytes, (E) neutrophils, and (F) lymphocytes. n=6/sroke group; n=3/sham group; *P < 0.05.