Early Sex Differences in the Immune-Inflammatory Responses to Neonatal Ischemic Stroke

Abstract

We recently reported that neonatal ischemia induces microglia/macrophage activation three days post-ischemia. We also found that female mice sustained smaller infarcts than males three months post-ischemia. The objective of our current study was to examine whether differential acute neuroinflammatory response and infiltrated immune cells occurs between male and females after three days post-ischemia. Permanent middle cerebral artery occlusion was induced in male and female postnatal 9-day-old (P9) mice, and mice were sacrificed three days after ischemia. Brains were analyzed for mRNA transcription after microglia magnetic cell sorting to evaluate M1 and M2 markers. FACS analysis was performed to assess myeloid infiltration and microglial expression of CX3 chemokine receptor 1 (CX3CR1). Inflammatory cytokine expression and microglia/macrophage activation were analyzed via in situ hybridization combined with immunofluorescence techniques. Lesion volume and cell death were measured. An increase in microglia/macrophages occurred in male versus female mice. The cells exhibited amoeboid morphology, and TNFα and ptgs2 (Cox-2) genes were more expressed in males. More myeloid cell infiltration was found in male versus female brains. However, we did not observe sex-dependent differences in the injured volume or cell death density. Our data show that sex differences in the acute microglial and immune responses to neonatal ischemia are likely both gene- and region-specific.

Keywords: lesion; macrophages; microgliosis; neonatal stroke; neuroinflammation; neuronal loss; sex differences.

Figure 1

No differences in lesion volume and cell death in male and female ischemic mice. (A) The lesion was delineated (yellow dots) in 4′,6-diamidino-2-phenylindole (DAPI)-stained sections of the ipsilateral hemisphere after three days post-ischemia (upper images, scale bar = 100 µm). The primary somatosensory area (S1) and the secondary somatosensory area (S2) are indicated in the images and correspond with the peri-infarct region in the cortex. Representative images for TUNEL⁺ nuclei (green) and DAPI (blue) show the core region in the ischemic cortex (lower images, scale bar = 50 µm). (B) Lesion volumes in M (blue) and F (pink) mice at three days post-ischemia represented as the percentage of the total ipsilateral hemisphere. (C) No sex differences in the number of TUNEL⁺ nuclei, n = 5/group.

Figure 2

Sex differences in microglial gene expression three days after neonatal ischemia. Brains were analyzed for mRNA transcription after microglia magnetic cell sorting. Gene expression of M1- and M2-like markers in CD11b⁺ microglia from ischemic M (blue) and F (pink) mice using RT-qPCR. Each datum in M and F is expressed as a ratio against control referenced gene expression in CD11b⁺ microglia isolated from naive M and F mice. Data are reported as the median and 25th–75th percentiles, n = 5–7/group. * p < 0.05 M versus F.

Figure 3

Sex-specific TNFα cytokine mRNA expression but not Il-1β after neonatal ischemia. (A,C) Representative images for TNFα- and Il-1β-positive cells (red), Iba-1 (microglia/macrophages, green), and DAPI (nuclei, blue) in the peri-infarct and core regions. Both TNFα- and Il-1β-positive cells were expressed in microglia/macrophage cells showing ramified or amoeboid morphology. Scale bars are 50 µm for the upper images and 20 µm for high-magnification images. (B) Increased mRNA Il-1β-positive cells in the core areas in M compared with F at three days post-injury. No significant differences in the peri-infarct region. (D) No significant differences in mRNA Il-1β density in M and F after injury, ** p < 0.01 (n = 5/group).

Figure 4

FACS analysis of myeloid infiltrate and microglial expression of CX3CR1. (A) Gating strategy for microglia (CD11b⁺/CD45 ^int/live) and myeloid infiltrate (CD11b⁺/CD45 ^high): cells and singlets were selected based on morphological parameters, then CD45 ^int and CD45 ^high cells were gated in CD11b⁺ cells, and dead cells were excluded in CD11b⁺/CD45 ^int cells. Forward scatter (FSC); (B) CX3CR1 expression was measured in microglia (CX3CR1 signal in red line; control isotype in black line). (C) Percentage of myeloid infiltrate in whole brain (% in side-scatter (SSC) singlets) in M (blue) and F (pink) control (circles) and ischemia (squares). Control (CTL); ischemia (Isch). (D) Mean fluorescence intensity (MFI) of CX3CR1 in microglia in M (blue) and F (pink) control (circles) and ischemia (squares). Data are reported as median and 25th–75th percentiles (n = 7 to 9/group). *** p < 0.001, non-parametric Mann–Whitney test vs. control, ^§ p < 0.05, non-parametric Mann–Whitney test vs. M.

Figure 5

Sex-dependent differences in microglia/macrophage density after neonatal ischemia. (A) Representative images of Iba-1 immunohistochemical staining (microglia/macrophages) show an increase in M ischemic brains compared to the F in the primary (S1) and secondary (S2) somatosensory area and in the cortical core. (B) Sex differences observed in the cortical S1 and S2 regions with higher Iba-1 staining in M compared to F at three days post-ischemia. No differences were observed in the corpus callosum (cc) region (C). Iba-1-positive cells classified according to amoeboid/hypertrophy microglia/macrophage morphology in the peri-infarct (S1 and S2 areas). M mice showed a higher increase in amoeboid cells than did F mice. Representative images of Iba-1 staining show an increase in cells with amoeboid morphology in M compared to F. *** p < 0.001, ** p < 0.01, * p < 0.05, n = 5/group.