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Updated: Oct 19, 2025
Culturing Microglia from the Neonatal and Adult Central Nervous System
Published on: August 9, 2013
Age and sex differences in primary microglia culture: A comparative study
Conelius Ngwa1, Shaohua Qi1, Abdullah Al Mamun1
1Department of Neurology, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX 77030, USA.
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.
Insights
Neonatal microglial cultures do not accurately model aged microglia in neuroinflammation studies. Aged microglia exhibit distinct morphology and activation, necessitating their use in elderly-relevant research, considering sex differences.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Microglia are key players in central nervous system (CNS) neuroinflammation.
- Current in vitro studies often use neonatal microglial cultures.
- Neonatal cultures may not reflect aged microglial activity relevant to elderly neuroinflammatory diseases.
Purpose of the Study:
- To develop and validate a method for culturing aged microglia.
- To compare aged microglia with neonatal microglia in vitro.
- To investigate age- and sex-related differences in microglial activation.
Main Methods:
- Primary microglia were cultured from aged (18-24 months) and neonatal (P0-P4) C57BL/6 mice.
- Morphology and activation were assessed using ELISA, RT-PCR, live microscopy, immunocytochemistry, and Western blotting.
- Ischemic stimulation was applied to compare responses between age groups and sexes.
Main Results:
- Aged microglia displayed larger cell bodies, more cytoplasmic inclusions, and enhanced phagocytosis compared to neonatal microglia.
- Cytokine production showed heterogeneity in both age groups, both before and after stimulation.
- Baseline CD11b expression was higher in aged microglia; ischemic stimulation differentially affected CD11b expression in males versus females.
Conclusions:
- Neonatal microglial cultures are inadequate for studying elderly-associated neuroinflammatory CNS disorders.
- Aged microglial cultures are recommended for in vitro studies mimicking age-related neuroinflammation.
- Sex differences in microglial responses should be considered in research.
