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Updated: Apr 30, 2026
Identifying Microglia and Peripheral Infiltrating Macrophages in the Injured Spinal Cords Using Flow Cytometry
Published on: June 24, 2025
Differences in the phagocytic response of microglia and peripheral macrophages after spinal cord injury and its
Andrew D Greenhalgh1, Samuel David
1Centre for Research in Neuroscience, The Research Institute of the McGill University Health Center, Montreal, Quebec, Canada, H3G 1A4.
Abstract:
Macrophages in the injured spinal cord arise from resident microglia and infiltrating, peripherally derived monocytes. It is still not clear if macrophages derived from these two populations differ in their roles after CNS injury. The aims of this study are to investigate the phagocytic response and clearance of damaged axons and tissue debris by these distinct subsets of macrophages and assess their viability after spinal cord injury (SCI). The lysozyme M EGFP-knockin mouse tags hematogenous macrophages, but not microglia. Using a combination of immunofluorescence, flow cytometry, and neuronal tracing techniques, we show that microglia contact damaged axons early (24 h) after SCI and are the main type of macrophage to contain phagocytic material at 3 d. Thereafter, infiltrating macrophages become the predominant cell in contact with degenerating axons and contain more phagocytic material, which in contrast to microglia, persists for up to 42 d. Furthermore, after phagocytosis of myelin in vitro, bone marrow-derived macrophages are much more susceptible to apoptotic and necrotic cell death than CNS microglia, which is mirrored in vivo with apoptotic TUNEL-positive cells of infiltrating macrophage origin. This work suggests that microglia play a major role in the early response to SCI, by phagocytosing damaged and degenerating tissue, processing phagocytic material efficiently, and remaining viable. Later, macrophages of peripheral origin contribute predominantly to phagocytosis but are less efficient at processing CNS debris, and their death, in situ, may contribute to the secondary damage after CNS injury.
Insights
Resident microglia clear debris early after spinal cord injury (SCI), while infiltrating macrophages dominate later but are less efficient and more prone to death, potentially worsening secondary damage.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Macrophages in the injured spinal cord originate from resident microglia and infiltrating peripheral monocytes.
- The distinct roles and viability of these macrophage subsets after central nervous system (CNS) injury remain unclear.
Purpose of the Study:
- To investigate the phagocytic response and clearance of damaged axons and tissue debris by distinct macrophage subsets after spinal cord injury (SCI).
- To assess the viability of resident microglia versus infiltrating macrophages post-SCI.
Main Methods:
- Utilized lysozyme M EGFP-knockin mice to distinguish between microglia and hematogenous macrophages.
- Employed immunofluorescence, flow cytometry, and neuronal tracing techniques.
- Assessed phagocytic activity, material persistence, and cell death (TUNEL assay) in vivo and in vitro.
Main Results:
- Microglia initiate early axonal contact and phagocytosis (24h-3d post-SCI).
- Infiltrating macrophages become predominant later (up to 42d), containing more phagocytic material.
- Infiltrating macrophages exhibit higher susceptibility to apoptosis and necrosis compared to microglia post-myelin phagocytosis.
Conclusions:
- Microglia are crucial for the early, efficient clearance of debris and survival post-SCI.
- Peripheral macrophages contribute significantly to later phagocytosis but are less efficient and their death may exacerbate secondary injury.
