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Updated: Apr 28, 2026
Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates
Published on: January 30, 2014
Microglia change from a reactive to an age-like phenotype with the time in culture
Cláudia Caldeira1, Ana F Oliveira2, Carolina Cunha2
1Research Institute for Medicines - iMed.ULisboa, Faculdade de Farmácia, Universidade de Lisboa Lisboa, Portugal ; Centro de Investigação Interdisciplinar Egas Moniz, Egas Moniz - Cooperativa de Ensino Superior, CRL, Campus Universitário Monte de Caparica, Portugal.
Abstract:
Age-related neurodegenerative diseases have been associated with chronic neuroinflammation and microglia activation. However, cumulative evidence supports that inflammation only occurs at an early stage once microglia change the endogenous characteristics with aging and switch to irresponsive/senescent and dystrophic phenotypes with disease progression. Thus, it will be important to have the means to assess the role of reactive and aged microglia when studying advanced brain neurodegeneration processes and age-associated related disorders. Yet, most studies are done with microglia from neonates since there are no adequate means to isolate degenerating microglia for experimentation. Indeed, only a few studies report microglia isolation from aged animals, using either short-term cultures or high concentrations of mitogens in the medium, which trigger microglia reactivity. The purpose of this study was to develop an experimental process to naturally age microglia after isolation from neonatal mice and to characterize the cultured cells at 2 days in vitro (DIV), 10 DIV, and 16 DIV. We found that 2 DIV (young) microglia had predominant amoeboid morphology and markers of stressed/reactive phenotype. In contrast, 16 DIV (aged) microglia evidenced ramified morphology and increased matrix metalloproteinase (MMP)-2 activation, as well as reduced MMP-9, glutamate release and nuclear factor kappa-B activation, in parallel with decreased expression of Toll-like receptor (TLR)-2 and TLR-4, capacity to migrate and phagocytose. These findings together with the reduced expression of microRNA (miR)-124, and miR-155, decreased autophagy, enhanced senescence associated beta-galactosidase activity and elevated miR-146a expression, are suggestive that 16 DIV cells mainly correspond to irresponsive/senescent microglia. Data indicate that the model represent an opportunity to understand and control microglial aging, as well as to explore strategies to recover microglia surveillance function.
Insights
Researchers developed a method to age microglia in vitro, mimicking aged brain cells. This new model helps study neurodegeneration and potential treatments for age-related brain disorders.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Age-related neurodegenerative diseases are linked to chronic neuroinflammation and microglia activation.
- Microglia shift from reactive to senescent phenotypes with aging, complicating disease studies.
- Current methods for studying aged microglia are limited, often using neonatal cells or artificial stimulation.
Purpose of the Study:
- To develop an experimental process for naturally aging microglia in culture.
- To characterize microglia at different in vitro aging stages (2, 10, and 16 days in vitro).
- To provide a model for understanding microglial aging and developing therapeutic strategies.
Main Methods:
- Isolation of microglia from neonatal mice.
- Culturing microglia for extended periods (2, 10, and 16 days in vitro) to induce natural aging.
- Characterization of cellular morphology, marker expression, and functional assays (e.g., migration, phagocytosis).
Main Results:
- 2 DIV microglia showed amoeboid morphology and stressed/reactive markers.
- 16 DIV microglia exhibited ramified morphology, increased matrix metalloproteinase (MMP)-2, and decreased MMP-9.
- Aged microglia displayed reduced glutamate release, nuclear factor kappa-B activation, Toll-like receptor (TLR)-2/4 expression, migration, and phagocytosis.
- Senescence markers like beta-galactosidase activity increased, while specific microRNAs (miR-124, miR-155) decreased and miR-146a increased.
Conclusions:
- The in vitro model successfully replicates key features of aged, senescent microglia.
- This model offers a valuable tool for investigating the role of aged microglia in neurodegeneration.
- The findings pave the way for exploring interventions to restore microglial surveillance function.
