An Investigation of Sex Difference in Microglia Morphology and Function

Abstract

Microglia are the resident immune cells of the brain. During development microglia progenitor cells migrate from the periphery into the central nervous system. During this critical developmental period, immature microglia differ greatly in their shape and function from those in the adult brain. Immune activation of microglia in the young, developing brain, via neonatal infection, can often lead to long‐term changes in microglial function with associated neuronal and cognitive dysfunction. Furthermore, microglial activation is associated with multiple neurodevelopmental disorders with known or suspected immune etiologies. Such neurological disorders include autism, ADHD, schizophrenia, and cerebral palsy – all exhibiting a strong sex bias in males (Schwarz et al. J. Neurochem., 2012; Bilbo et al. J. Neuroimmune Pharmacol., 2011). Given these rodent and well‐known human epidemiological data, we hypothesize that a hightened level of cytokine expression in developing male rat pups increases their vulnerability to an immune challenge. To test this hypothesis, we removed any variability in the number of microglia cells between males and females and isolated microglia cells from the remainder of the neural cell population. A primary culture of microglial cells was prepared from dissociated hippocampal tissue from P4 male and female rat brains. Microglial cells were selected for by immunoprecipitation (CD11b surface marker), using magnetic beads. Cells were counted and 30,000 cells were plated per well. An immune response was trigged by treating the cells with a 1,000ng/mL Lipopolysaccharide (LPS) dose or Saline (for control) prior to flash freezing and RNA extraction. PCR was used to determine the relative gene expression of pro‐inflammatory cytokines Interleukin‐1 Beta (IL‐1B) and Interleukin‐6 (IL‐6), Toll‐Like Receptor 4 (TLR4), Apoptosis Marker (Cideb1), and Hormone Receptors for estrogen (Esr‐1) and androgen (Ar). IL‐1B and IL‐6 expression for microglial populations had a significant main effect for treatment (F1,78 = 68.41; p < 0.001, η2 = 0.533) and (F1,80 = 91.28; p < 0.001, η2 = 0.467) respectively. TLR4 expression in microglial cells had a significant main effect for treatment (F1,81 = 11.36; p = 0.001, η2 = 0.123) and the other neural cells had a trending main effect for sex (F1,27 = 3.49; p = 0.073, η2 = 0.115). Cideb1 expression in the non‐microglial neural cell population had a significant sex by treatment interaction (F1,27 = 5.49; p = 0.027, η2 = 0.169). And there was no gene expression for either hormone receptor detected on microglial cells. We concluded that there is no sex by treatment differences at a 1000ng/mL LPS dose. However, the possible down‐regulation in TLR4 receptors could indicate that 1000ng/mL is too high of a dose for the isolated cells to tolerate and the extreme immune response washed out smaller differences between sexes. The significant increase in cell death of female non‐microglial cells despite the non‐significant difference in pro‐inflammatory cytokine expression between the sexes could indicate a possible sex‐based difference in the way neurons respond to immune challenges.Support or Funding InformationNIH grant R21 MH101883‐01 to Jaclyn M. Schwarz