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Updated: Dec 30, 2025
Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation
Published on: February 16, 2018
Microglial depletion and repopulation in brain slice culture normalizes sensitized proinflammatory signaling
Leon G Coleman1,2, Jian Zou3, Fulton T Crews3,4,5
1Bowles Center for Alcohol Studies, The University of North Carolina at Chapel Hill, School of Medicine, CB#7178, 1021 Thurston-Bowles Building, Chapel Hill, NC, USA. leon_coleman@med.unc.edu.
Background:
Microglia are critical mediators of neuroimmune pathology across multiple neurologic disorders. Microglia can be persistently activated or "primed" by Toll-like receptor (TLR) activation, ethanol, stress, and other insults. Thus, strategies to prevent or reverse microglial priming may be beneficial for conditions that involve progressively increasing microglial activation. Microglial depletion with repopulation is emerging as a potential therapy to normalize chronic immune activation. Primary organotypic hippocampal slice culture (OHSC) allows for the study of neuroimmune activation as well as microglial depletion and repopulation without involvement of peripheral immune activation. OHSC undergoes functional maturation and retains cytoarchitecture similar to in vivo.
Methods:
OHSC underwent microglial depletion with the CSF1R antagonist PLX3397 with or without repopulation after removal of PLX3397. Immune, trophic, and synaptic gene changes in response to agonists of TLRs 2, 3, 4, 7, and 9 as well as ethanol were assessed in the settings of microglial depletion and repopulation. Gi-DREADD inhibition of microglia was used to confirm select findings seen with depletion. The ability of microglial repopulation to prevent progressive proinflammatory gene induction by chronic ethanol was also investigated.
Results:
Microglia were depleted (> 90%) by PLX3397 in OHSC. Microglial depletion blunted proinflammatory responses to several TLR agonists as well as ethanol, which was mimicked by Gi-DREADD inhibition of OHSC microglia. Removal of PLX3397 was followed by complete repopulation of microglia. OHSCs with repopulated microglia showed increased baseline expression of anti-inflammatory cytokines (e.g., IL-10), microglial inhibitory signals (e.g., CX3CL1), and growth factors (e.g., BDNF). This was associated with blunted induction (~ 50%) of TNFα and IL-1β in response to agonists to TLR4 and TLR7. Further, chronic cycled ethanol from 4 days in vitro (DIV) to 16DIV caused immediate 2-fold inductions of TNFα and IL-1β that grew to ~4-fold of age-matched control slices by 40DIV. This persistent inflammatory gene expression was completely reversed by microglial depletion and repopulation after chronic ethanol.
Conclusions:
Microglia in OHSCs mediate proinflammatory responses to TLR agonists and ethanol. Microglial repopulation promoted an anti-inflammatory, trophic neuroenvironment and normalized proinflammatory gene expression. This supports the possibility of microglial depletion with repopulation as a strategy to reverse chronic neuroimmune activation.
Insights
Microglial depletion and repopulation in brain slice cultures reversed chronic inflammation caused by ethanol. This approach normalized gene expression, suggesting a potential therapy for neuroimmune disorders.
Area of Science:
- Neuroscience
- Immunology
- Pharmacology
Background:
- Microglia are key players in neuroimmune responses and can become chronically activated (
Purpose of the Study:
- To investigate the therapeutic potential of microglial depletion and repopulation in reversing chronic neuroinflammation.
- To assess the impact of microglial repopulation on neuroimmune gene expression in response to inflammatory stimuli and ethanol.
Main Methods:
- Utilized organotypic hippocampal slice culture (OHSC) for studying neuroinflammation.
- Administered CSF1R antagonist PLX3397 for microglial depletion, followed by repopulation.
- Assessed gene expression changes in response to Toll-like receptor (TLR) agonists and ethanol.
Main Results:
- Microglial depletion significantly reduced proinflammatory responses to TLR agonists and ethanol.
- Repopulated microglia exhibited increased anti-inflammatory cytokine and growth factor expression.
- Microglial depletion and repopulation effectively reversed persistent inflammatory gene induction caused by chronic ethanol exposure.
Conclusions:
- Microglia in OHSCs drive neuroinflammation in response to TLR agonists and ethanol.
- Microglial repopulation establishes an anti-inflammatory and trophic environment.
- Microglial depletion-repopulation is a promising strategy for reversing chronic neuroimmune activation.
