Age and sex differences in primary microglia culture: A comparative study

Abstract Background Microglia play a central role in neuroinflammation in various CNS diseases.Neonatal microglial culture has been extensively used to in vitro study microglial activation; however, as many neuroinflammatory diseases occur in the elderly, the neonatal microglial culture may not fully replicate the aged microglial activity seen in these diseases. New method Primary microglia from both 18–24-month-old and P0-P4 C57BL/6 mice were cultured simultaneously. Morphology and activation profiles of the two age groups of microglia were examined following ischemic stimulation, by ELISA, RT-PCR, live microscopy, immunocytochemistry, and Western blotting. Results We showed that aged microglia had larger cell bodies, more cytoplasmic inclusions, and enhanced phagocytosis than neonatal microglia. Cytokine production in these cells exhibited heterogeneity either after or before ischemic stimulation. The baseline expression of microglial marker CD11b was significantly higher in aged vs. neonatal cells; ischemic stimulation increased the expression in neonatal vs. aged microglia only in males but not in females. Comparison with existing methods Previous primary microglia cultures have been limited to using neonatal/adult cells. This method is complementary to exiting methods and works for aged microglia, and does not suffer from potential limitations due to filtering artifacts. The protocol renders microglial culture no need for meningeal/hippocampal removal prior to brain tissue dissociation, and compares microglia between males vs. females, and between the aged vs. neonates. Conclusions We concluded that neonatal microglial culture is not appropriate for those in vitro studies that mimic the neuroinflammatory central nervous system disorders occurring in the elderly, in which case the aged microglial culture should be applied, and sex differences should be considered.