Microglia are the immune effector cells of the central nervous system (CNS) existing in three distinct forms which serve different functional roles. Find out more about these cell types and how to use markers to identify them.
Amoeboid microglia
Amoeboid microglia are associated with the developing CNS. In rats, amoeboid microglia have been shown to appear late in gestation and disappear soon after birth
These cells exhibit a round cell body, possess pseudopodia and thin filopodia-like processes and contain numerous lysosomes; all traits indicative of a motile phagocytic phenotype.
During the post-natal period, amoeboid microglia are believed to play a role in tissue histogenesis through the removal of inappropriate and superfluous axons and through the promotion of axonal migration and growth.
Ultimately, amoeboid microglia grow long crenulated processes and transform into ramified microglia found in the adult CNS.
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Ramified microglia
Ramified microglia are present in abundance in the brain parenchyma and constitute approximately 10–20% of the total population of glial cells in the adult.
These small round cells comprise numerous branching processes and possess little cytoplasm. In the adult brain, the resident population of ramified microglia is maintained through local cell division and through the recruitment of circulating peripheral blood monocytes.
Under physiological conditions, microglia exhibit pinocytotic activity and localized motility. It has been suggested that ramified microglia contribute to metabolite removal and to the clearance of toxic factors released from injured neurons.
A study has demonstrated that microglia have the propensity to transform into neurons, astrocytes or oligodendrocytes. Hence, ramified microglia may represent a unique population of multipotent stem cells in the adult CNS, which strongly implicates microglia in CNS repair.
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Reactive microglia
In response to injury or pathogen invasion, quiescent ramified microglia proliferate and transform into active 'brain macrophages' otherwise known as reactive microglia.
Microglial proliferation can be studied using antibodies raised against nuclear antigens such as Ki67 or proliferating cell nuclear antigen (PCNA) or by bromodeoxyuridine (BrdU) staining.
Reactive microglia are rod-like, devoid of branching processes, and contain numerous lysosomes and phagosomes. The reactive cell form represents a population of macrophages, which are associated with brain injury and neuroinflammation.
Following a damaging event, reactive microglia accumulate at the site of injury where they play a neuroprotective role phagocytosing damaged cells and debris.
In acute lesions the peak of microglial activation occurs 2-3 days post insult, but if the pathological stimulus persists microglial activation continues.
Reactive microglia express MHC class II antigens and other surface molecules necessary for antigen presentation including CD40, B7, and ICAM-1. Consequently, microglia are considered to be the most potent antigen-presenting cells in the CNS. Like macrophages, reactive microglia secrete inflammatory mediators, which orchestrate the cerebral immune response.
Chronic microglial activation is associated with neurological disorders including Alzheimer's disease, multiple sclerosis, and delayed neuronal death occurring after ischaemia.
In these instances, the persistent activation of microglia accompanied by the sustained secretion of inflammatory mediators is thought to have a deleterious effect on neuronal function and survival, thereby exacerbating disease processes.
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CD45 - microglia
A paucity of specific microglial-only antigens has hindered microglial identification. Markers used for the detection of microglia are also present in macrophage since both cell types exhibit the same lineage.
Microglia are often identified using flow cytometry as it enables to quantify the differences in antigen expression levels. Ramified parenchymal microglia have been demonstrated to possess the phenotype CD11b+, CD45low, whilst other CNS macrophages and peripheral macrophages exhibit the phenotype CD11b+, CD45high.
CD45 expression by microglia (mic) extracted from 5-day old rat neonates. Microglia were isolated and left in culture for 24 hours. The cells were subsequently harvested, fixed, then analyzed by flow cytometry using anti-CD45. Labeled cells are represented by the black shaded populations, whereas the unlabeled cells are depicted by the grey line (%: % of cells in M1 or M2 region, MFI: mean fluorescence intensity).
Microglia can also be detected immunologically using antibodies raised against macrophage-specific antigens; however, such antibodies fail to distinguish microglia from macrophages.
CD45 expression by microglia (mic) extracted from 5-day old rat neonates. Microglia were isolated and left in culture for 24 hours. The cells were subsequently harvested, fixed, then analyzed by flow cytometry using anti-CD45. Labeled cells are represented by the black shaded populations, whereas the unlabeled cells are depicted by the grey line.
The OX-42 antibody recognizes the CR3 complement receptor (CD11b/CD18) expressed by rat or mouse microglia. Clone F4/80 binds a 60 kDa glycoprotein on murine ramified microglia.
CD11b expression by microglia (mic) extracted from 5-day old rat neonates. Microglia were isolated and left in culture for 24 hours. The cells were subsequently harvested, fixed, then analyzed by flow cytometry using the OX-42 antibody. Labeled cells are represented by the black shaded populations, whereas unlabeled cells are depicted by the grey line (%: % of cells in M1 or M2 region, MFI: mean fluorescence intensity).
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CD68 - microglia
CD68 is a lysosomal protein and can be used to stain microglia.
CD68 expression by microglia (mic). Cells were extracted from 5-day old rat neonates. Microglia were isolated and left in culture for 24 hours. The cells were subsequently harvested, fixed then analyzed by flow cytometry using anti-CD68 (ED-1) antibodies. Labeled cells are represented by the black shaded populations, whereas the unlabeled cells are depicted by the grey line (%: % of cells in M1 or M2 region, MFI: mean fluorescence intensity).
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CD68 - macrophage
High levels of CD68 expression are associated with macrophages and activated microglia, while low levels of expression are associated with quiescent ramified microglia.
Expression of CD68 by peritoneal macrophages.
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Silver carbonate staining technique
Identification of microglia can also be achieved using Rio Hortega's original silver carbonate staining technique.
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Lectin staining
Lectins are carbohydrate-binding proteins that label microglia through the recognition of glycoproteins containing terminal alpha-D-galactose residues.
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Vimentin
Gene name VIM
Vimentin is an intermediate filament protein, whose expression is upregulated during the epithelial-to-mesenchymal transition of NE cells to radial glia and persists until astrocyte development.
Rhesus monkey brain tissue sections stained with anti-vimentin (ab92547).
Microglia cells can be identified using acetylated low-density lipoprotein (LDL) conjugated to a fluorescent tag, which enables the labeling of LDL receptors.
