Sex differences in astrocyte and microglia responses immediately following middle cerebral artery occlusion in adult mice

Abstract

Epidemiological studies report that infarct size is decreased and stroke outcomes are improved in young females when compared to males. However, mechanistic insight is lacking. We posit that sex-specific differences in glial cell functions occurring immediately after ischemic stroke are a source of dichotomous outcomes. In this study we assessed astrocyte Ca2+ dynamics, aquaporin 4 (AQP4) polarity, S100β expression pattern, as well as, microglia morphology and phagocytic marker CD11b in male and female mice following 60 minutes of middle cerebral artery (MCA) occlusion. We reveal sex differences in the frequency of intracellular astrocyte Ca2+ elevations (F(1,86)=8.19, P=0.005) and microglia volume (F(1,40)=12.47, P=0.009) immediately following MCA occlusion in acute brain slices. Measured in fixed tissue, AQP4 polarity was disrupted (F(5,86)=3.30, P=0.009) and the area of non-S100β immunoreactivity increased in ipsilateral brain regions after 60 min of MCA occlusion (F(5,86)=4.72, P=0.007). However, astrocyte changes were robust in male mice when compared to females. Additional sex differences were discovered regarding microglia phagocytic receptor CD11b. In sham mice, constitutively high CD11b immunofluorescence was observed in females when compared to males (P=0.03). When compared to sham, only male mice exhibited an increase in CD11b immunoreactivity after MCA occlusion (P=0.006). We posit that a sex difference in the presence of constitutive CD11b has a role in determining male and female microglia phagocytic responses to ischemia. Taken together, these findings are critical to understanding potential sex differences in glial physiology as well as stroke pathobiology which are foundational for the development of future sex-specific stroke therapies.

Sex and regional differences in microglia morphology and complement receptor 3 are independent of constitutive neuroinflammatory protein concentrations in healthy mice

“…Each hemisphere was sectioned into 50µm coronal sections (Leica cryostat) and stored at -20°C in a cryoprotectant solution (50% 50mM PBS, 30% ethylene glycol, 20% glycerol) until IHC experiments. Our IHC protocol, similar to as previously published (Morrison and Filosa, 2013;Morrison and Filosa, 2016), results in double staining of microglia with anti-iba1 and CR3 (anti-CD18 and anti-CD11b). Free floating brain sections were first blocked in 10% horse serum solution (0.01M PBS, 0.05% Triton, and 0.04% NaN3) for 1h followed by a 72h incubation with primary antibodies as appropriate: rabbit antiiba1 at 1:1000 (Wako, 019-19741), rat anti-ITAM/CD11b at 1:100 (ThermoFisher, RM2800) and rat anti-Integrin beta-2/CD18 at 1:100 (Millipore, MABT42).…”

“…The extent of microglia ramified morphology was quantified using an objective and computeraided skeleton analysis method as previously published in detail (Morrison and Filosa, 2013;Morrison and Filosa, 2016). Briefly, a series of ImageJ plugins (adjust brightness, unsharp mask, and despeckle) were consistently applied in order to ensure adequate process visualization before the conversion to binary and skeletonized images.…”

“…Complement receptor 3 (CD18/CD11b) mean fluorescence intensity was determined from photomicrographs using Image J software and methods that have been previously published (Morrison and Filosa, 2013;Morrison and Filosa, 2016). Using consistent thresholding settings, positive staining for CD18 and CD11b were determined for all photomicrographs.…”

“…ischemic stroke) (Mozaffarian et al, 2016). Microglia are best known and vigorously studied in their roles as the brain immune cell and first responder to diseases and brain injury (Davalos et al, 2005;Morrison and Filosa, 2016;Nimmerjahn et al, 2005;Tremblay et al, 2011). Pre-clinical animal models have shown that sex hormones have a potent effect on microglia mediated neuroplasticity (Hansberg-Pastor et al, 2015) and neuroinflammation (Hanamsagar and Bilbo, 2016;Villa et al, 2016) during development, health and disease.…”

“…In order to capitalize on this potential, sensitive tools to precisely quantify microglia morphology (Karperien et al, 2013;Morrison and Filosa, 2013;Morrison and Filosa, 2016) have been employed here to distinguish the effects of sex, estrus cycle, and ovarian failure on microglia morphology in healthy brain regions. We examined the constitutive presence of complement receptor 3 (CR3) to gain insight into microglia phagocytic potential.…”