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Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry
Published on: June 22, 2017
Macrophages are comprised of resident brain microglia not infiltrating peripheral monocytes acutely after neonatal
Sheryl P Denker1, Shaoquan Ji, Andra Dingman
1Department of Neurology, University of California San Francisco, San Francisco, CA 94143-0663, USA.
Abstract:
Macrophages can be both beneficial and detrimental after CNS injury. We previously showed rapid accumulation of macrophages in injured immature brain acutely after ischemia-reperfusion. To determine whether these macrophages are microglia or invading monocytes, we subjected post-natal day 7 (P7) rats to transient 3 h middle cerebral artery (MCA) occlusion and used flow cytometry at 24 and 48 h post-reperfusion to distinguish invading monocytes (CD45high/CD11b+) from microglia (CD45low/medium/CD11b+). Inflammatory cytokines and chemokines were determined in plasma, injured and contralateral tissue 1-24 h post-reperfusion using ELISA-based cytokine multiplex assays. At 24 h, the number of CD45+/CD11b+ cells increased 3-fold in injured compared to uninjured brain tissue and CD45 expression shifted from low to medium with less than 10% of the population expressing CD45high. MCA occlusion induced rapid and transient asynchronous increases in the pro-inflammatory cytokine IL-beta and chemokines cytokine-induced neutrophil chemoattractant protein 1 (CINC-1) and monocyte-chemoattractant protein 1 (MCP-1), first in systemic circulation and then in injured brain. Double immunofluorescence with cell-type specific markers showed that multiple cell types in the injured brain produce MCP-1. Our findings show that despite profound increases in MCP-1 in injured regions, monocyte infiltration is low and the majority of macrophages in acutely injured regions are microglia.
Insights
In injured immature brains, most macrophages after ischemia-reperfusion are microglia, not invading monocytes. This finding is crucial for understanding brain injury responses and developing targeted therapies.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Macrophages play a dual role in central nervous system (CNS) injury.
- Previous studies indicated rapid macrophage accumulation in immature brain post-ischemia-reperfusion.
- The origin of these macrophages (microglia vs. infiltrating monocytes) remained unclear.
Purpose of the Study:
- To differentiate between microglia and infiltrating monocytes in the injured immature brain.
- To investigate the temporal dynamics of macrophage populations after stroke.
- To analyze the inflammatory cytokine and chemokine profiles during acute brain injury.
Main Methods:
- Transient middle cerebral artery (MCA) occlusion in post-natal day 7 (P7) rats.
- Flow cytometry (CD45/CD11b markers) to distinguish cell types at 24 and 48 hours post-reperfusion.
- ELISA-based multiplex assays for quantifying inflammatory cytokines and chemokines in plasma and brain tissue.
Main Results:
- A 3-fold increase in CD45+/CD11b+ cells in injured brain tissue at 24 hours.
- Predominantly CD45low/medium expression, indicating microglia, with <10% CD45high (monocytes).
- Rapid, transient increases in IL-1beta, CINC-1, and MCP-1, initially in circulation, then in injured brain.
Conclusions:
- Despite elevated MCP-1 in injured areas, monocyte infiltration into the immature brain is limited post-stroke.
- The majority of macrophages observed in the acute phase of CNS injury are resident microglia.
- This highlights the critical role of microglia in the early response to ischemic brain injury in young mammals.
