Microglia are specialized glial cells that serve as the primary immune sentinels of the central nervous system (CNS), residing throughout the brain and spinal cord. Originating from yolk sac-derived myeloid cells during embryonic development, microglia are uniquely adapted to monitor and maintain the health of the CNS. Their ability to rapidly alter gene expression and morphology in response to environmental cues underpins their remarkable functional versatility.

A defining feature of microglia is their capacity for phenotype plasticity. Depending on the context, microglia can adopt a spectrum of activation states, commonly categorized as M1 and M2 phenotypes. M1 microglia are characterized by a pro-inflammatory gene expression profile, amplifying microglia activation and contributing to the defense against pathogens. In contrast, M2 microglia are associated with anti-inflammatory responses, tissue repair, and the resolution of inflammation. The dynamic balance between these microglia phenotypes is essential for CNS homeostasis, and disruptions in this balance can lead to the emergence of disease-associated microglia, which are implicated in various neurodegenerative and psychiatric conditions.

Microglia play a central role in the clearance of apoptotic cells and cell debris, a process vital for preventing chronic inflammation and supporting neuronal function. They are adept at recognizing and engulfing apoptotic neurons, utilizing a range of surface receptors, including receptor tyrosine kinases such as Axl and Mertk, to mediate phagocytosis. Chronic myelin phagocytosis, for example, can induce shifts in microglia phenotype, sometimes resulting in a disease-associated state that may exacerbate CNS pathology.

The activation of microglia is a tightly regulated process, influenced by signals from the CNS microenvironment. Upon encountering injury, infection, or prolonged exposure to phagocytosis substrates, microglia can transition from a resting to an activated state, altering their gene expression and functional properties. This microglia activation is a double-edged sword: while it is essential for defense and repair, excessive or prolonged activation can contribute to neuroinflammation and neuronal damage, as seen in disorders such as Alzheimer’s disease and multiple sclerosis.

Research utilizing primary murine glial cultures has been instrumental in dissecting the mechanisms underlying microglia polarization and function. In vitro assays, including the phagocytosis assay kit, enable scientists to determine microglia phagocytosis capacity and to compare the responses of distinct microglia subtypes under controlled conditions. These studies have revealed that microglia subtypes differ in their ability to respond to apoptotic cells, pathogens, and myelin debris, further emphasizing the importance of microglia phenotype in health and disease.

As our understanding of microglia plasticity and activation deepens, it becomes increasingly clear that these cells are not only guardians of the CNS but also key players in the pathogenesis of neurological disorders. Ongoing research aims to unravel the complex gene expression networks and signaling pathways that govern microglia responses, with the goal of developing targeted therapies to modulate microglia activation and restore CNS health.

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.

Amoeboid microglia are considered phagocytosis-competent CNS cells due to their high phagocytic activity during development.

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.

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 cells, characterized by their highly branched, ramified morphology, are often referred to as 'resting microglia' due to their continuous surveillance role in the healthy CNS.

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.

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 against nuclear antigens such as Ki67, proliferating cell nuclear antigen (PCNA), or 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. Microglia-triggered secretion of cytokines and chemokines is a hallmark of their response to injury and infection, influencing their phenotype and function.

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.

CD45 - microglia

A paucity of ​specific microglial-only antigens has hindered microglial identification. Markers used to detect microglia are also present in macrophages since both cell types exhibit the same lineage.

Microglia are often identified using flow cytometry, which enables quantification of differences in antigen expression levels. CD45 expression levels are commonly used to identify and distinguish microglia from other CNS macrophages. Ramified parenchymal microglia have been demonstrated to possess the phenotype CD11b+, CD45low, whilst other CNS macrophages and peripheral macrophages exhibit the phenotype CD11b⁺, CD45^high.

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, and then analyzed by flow cytometry using anti-CD45. The black shaded populations represent labeled cells, 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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CD45 - macrophage

Microglia can also be detected immunologically using antibodies raised against macrophage-specific antigens; however, such antibodies fail to distinguish microglia from macrophages.

CD11b

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, and 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).​

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, and 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).

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.

Silver carbonate staining technique

Identification of microglia can also be achieved using Río Hortega’s original silver carbonate staining technique; Río Hortega was the pioneering scientist who developed this method and first described microglia subtypes, laying the foundation for understanding their morphology and function.

Lectin staining

Lectins are a diverse group of carbohydrate-binding proteins that play a crucial role in identifying and labeling microglial cells, particularly in studies exploring microglial heterogeneity. These proteins recognize specific glycan structures on the surface of glycoproteins, with a notable affinity for terminal alpha-D-galactose residues. This selective binding enables researchers to visualize and differentiate microglia based on their glycosylation patterns, varying across distinct subtypes and activation states. In microglial subtypes, lectin-based labeling provides a valuable tool for distinguishing between homeostatic microglia and those involved in neuroinflammatory or neurodegenerative processes. For example, changes in glycoprotein expression and glycan composition during microglial activation can be detected using lectins, offering insights into subtype-specific functions and responses. Thus, lectins not only facilitate the identification of microglia in situ but also contribute to characterizing their phenotypic diversity, enhancing our understanding of their roles in central nervous system health and disease.

Vimentin

Vimentin is a type III intermediate filament protein that plays a pivotal role in neural development's structural and functional dynamics. Its expression is notably upregulated during the epithelial-to-mesenchymal transition (EMT), a critical phase in which neuroepithelial (NE) cells differentiate into radial glia. This transition marks the beginning of gliogenesis and is essential for the emergence of various glial subtypes, including astrocytes and microglia. In the context of microglial subtypes, vimentin serves as a marker of cellular plasticity and activation. Although traditionally associated with astrocytic lineage, vimentin expression has also been observed in reactive microglia, particularly under pathological conditions such as neuroinflammation or injury. This suggests that vimentin may contribute to the cytoskeletal remodeling and migratory capacity of specific microglial subtypes. Its persistent expression through astrocyte development and in activated microglia highlights its relevance in distinguishing glial populations and understanding the dynamic interplay between glial subtypes during central nervous system maturation and disease.

Figure. Immunocytochemistry/ Immunofluorescence - Anti-Vimentin antibody [EPR3776] - Cytoskeleton Marker (ab92547).

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Low-density lipoprotein

Microglial cells can be effectively identified using acetylated low-density lipoprotein (acLDL) conjugated to a fluorescent tag, a method that exploits the presence of LDL receptors on the microglial surface. These receptors are involved in lipid metabolism and are particularly active in microglia due to their role in clearing cellular debris and maintaining homeostasis in the central nervous system. In the context of microglial subtypes, acLDL labeling provides a functional marker that can help distinguish between different activation states and phenotypes. For instance, pro-inflammatory microglia often exhibit increased LDL receptor expression, reflecting their heightened phagocytic activity during neuroinflammation. Conversely, homeostatic microglia may show lower uptake, indicating a more surveillance-oriented role. By tagging acLDL with fluorescent dyes, researchers can visualize and quantify microglial populations in situ, enabling subtype-specific analysis in both healthy and diseased brain tissue. This technique thus contributes to the growing toolkit for characterizing microglial diversity and function.

References

1. Dalmau, I.  et al.  Development of microglia in the prenatal rat hippocampus.  J. Comp. Neurol.  377, 70–84 (1997).
2. Innocenti, G. M., Clarke, S. & Koppell, H. Transitory macrophages in the white matter of the developing visual cortex. II. Development and relations with axonal pathways.  Dev. Brain Res.  11, 55–66 (1983).
3. Kaur, C. & Ling, E. A. Study of the transformation of amoeboid microglial cells into microglia labelled with the isolectin  Griffonia simplicifolia  in postnatal rats.  Acta Anat. (Basel)  142, 118–125 (1991).
4. Ling, E. A. Transformation of monocytes into amoeboid microglia and into microglia in the corpus callosum of postnatal rats, as shown by labelling monocytes by carbon particles.  J. Anat.  128, 847–858 (1979).
5. Ling, E. A., Penney, D. & Lebond, C. P. Use of carbon labelling to demonstrate the role of blood monocytes as precursors of the ‘amoeboid cells’ present in the corpus callosum of postnatal rats.  J. Comp. Neurol.  193, 631–657 (1980).
6. Marin-Teva, J. L.  et al.  Microglia promote the death of developing Purkinje cells.  Neuron  41, 535–547 (2004).
7. Polazzi, E. & Contestabile, A. Reciprocal interactions between microglia and neurons: from survival to neuropathology.  Rev. Neurosci.  13, 221–242 (2004).
8. Banati, R. Neuropathological imaging: in vivo detection of glial activation as a measure of disease and adaptive change in the brain.  Br. Med. Bull.  65, 121–131 (2003).
9. Booth, P. L. & Thomas, W. E. Evidence for motility and pinocytosis in ramified microglia in tissue culture.  Brain Res.  548, 163–171 (1991).
10. Fetler, L. & Amigorena, S. Brain under surveillance: the microglia patrol.  Science  309, 392–393 (2005).
11. Lawson, L. J., Perry, V. H. & Gordon, S. Turnover of resident microglia in the normal adult mouse brain.  Neuroscience  48, 405–415 (1992).
12. Thomas, W. E. Brain macrophages: evaluation of microglia and their functions.  Brain Res. Rev.  17, 61–74 (1992).
13. Vaughan, D. W. & Peters, A. Neuroglial cells in the cerebral cortex of rats from young adult to old age: an electron microscopy study.  J. Neurocytol.  3, 405–429 (1974).
14. Yokoyama, A., Yang, L.  et al.  Microglia, a potent source of neurons, astrocytes and oligodendrocytes.  Glia  45, 96–104 (2004).
15. Kreutzberg, G. W. Microglia: a sensor for pathological events in the CNS.  Trends Neurosci.  19, 312–318 (1996).
16. Stence, N., Waite, M. & Dailey, E. Dynamics of microglial activation: a confocal time-lapse analysis in hippocampal slices.  Glia  33, 256–266 (2001).
17. Schluter, C., Duchrow, M.  et al.  The cell proliferation-associated antigen of antibody Ki-67: a very large, ubiquitous nuclear protein with numerous repeated elements, representing a new kind of cell cycle maintaining proteins.  J. Cell Biol.  123, 513–522 (1993).
18. Bellver-Landete, V., Bretheau, F.  et al.  Microglia are an essential component of the neuroprotective scar that forms after spinal cord injury.  Nat. Commun.  10, 518 (2019).
19. Tam, W. & Ma, C. Bipolar/rod-shaped microglia are proliferating microglia with distinct M1/M2 phenotypes.  Sci. Rep.  4, 7279 (2015).
20. Askew, K., Kaizhen, L.  et al.  Coupled proliferation and apoptosis maintain the rapid turnover of microglia in the adult brain.  Cell Rep.  4, 391–405 (2017).
21. Kawabori, M. & Yenari, M. A. The role of the microglia in acute CNS injury.  Metab. Brain Dis.  30, 381–392 (2015).
22. Donat, C. K., Scott, G.  et al.  Microglial activation in traumatic brain injury.  Front. Aging Neurosci.  9, (2017).
23. Aloisi, F., Ria, F.  et al.  Microglia are more efficient than astrocytes in antigen processing and in Th1 but not Th2 cell activation.  J. Immunol.  160, 4671–4680 (1998).
24. Benveniste, E. N., Nguyen, V. T. & O'Keefe, G. M. Immunological aspects of microglia: relevance to Alzheimer’s disease.  Neurochem. Int.  39, 381–391 (2001).
25. Navarro, V., Sanchez-Mejias, E.  et al.  Microglia in Alzheimer's Disease: Activated, Dysfunctional or Degenerative.  Front. Aging Neurosci.  10, (2018).
26. Regen, F., Hellmann-Regen, J.  et al.  Neuroinflammation and Alzheimer's disease: implications for microglial activation.  Curr. Alzheimer Res.  14, 1140–1148 (2017).
27. Luo, C., Chongdong, J.  et al.  The role of microglia in multiple sclerosis.  Neuropsychiatr. Dis. Treat.  13, 1661–1667 (2017).
28. Calabrese, M., Magliozzi, R.  et al.  Exploring the origins of grey matter damage in multiple sclerosis.  Nat. Rev. Neurosci.  16, 147–158 (2015).
29. Tikka, T. M. & Koistinaho, J. E. Minocycline provides neuroprotection against N-methyl-D-aspartate neurotoxicity by inhibiting microglia.  J. Immunol.  166, 7527–7533 (2001).
30. Lees, G. J. The possible contribution of microglia and macrophages to delayed neuronal death after ischaemia.  J. Neurol. Sci.  114, 119–122 (1993).
31. Ford, A. L., Goodsall, A. L.  et al.  Normal ramified microglia separated from other central nervous system macrophages by flow cytometric sorting.  J. Immunol.  154, 4309–4321 (1995).
32. Becher, B. & Antel, J. P. Comparison of phenotypic and functional properties of immediately ex vivo and cultured human adult microglia.  Glia  18, 1–10 (1996).
33. Kingham, P. J., Cuzner, M. L. & Pocock, J. M. Apoptotic pathways mobilized in microglia and neurones as a consequence of chromogranin A-induced microglial activation.  J. Neurochem.  73, 538–547 (1999).
34. Hooper, C., Taylor, D. L. & Pocock, J. M. Pure albumin is a potent trigger of calcium signalling and proliferation in microglia but not macrophages or astrocytes.  J. Neurochem.  92, 1363–1376 (2005).
35. Graeber, M. B., Banati, R. B.  et al.  Immunophenotypic characterisation of rat brain macrophages in culture.  Neurosci. Lett.  103, 241–246 (1989).
36. Perry, V. H., Hume, D. A. & Gordon, S. Immunohistochemical localization of macrophages and microglia in the adult and developing mouse brain.  Neuroscience  15, 313–326 (1985).
37. Graeber, M. B., Streit, W. J.  et al.  New expression of myelomonocytic antigens by microglia and perivascular cells following lethal motor neurone injury.  J. Neuroimmunol.  27, 121–131 (1990).
38. Slepko, N. & Levi, G. Progressive activation of adult microglial cells in vitro.  Glia  16, 241–246 (1996).
39. Wang, Y. & Wei, W. Microglia-like or microglia: results of the weak silver carbonate staining method of del Rio-Hortega.  Biotech. Histochem.  87, 346–349 (2012).
40. Taylor, D. L., Diemel, L. T.  et al.  Activation of group III metabotropic glutamate receptors underlies microglial reactivity and neurotoxicity following stimulation with chromogranin A, a peptide up-regulated in Alzheimer’s disease.  J. Neurochem.  82, 1179–1191 (2002).
41. Streit, W. J. & Kreutzberg, G. W. Lectin binding by resting and reactive microglia.  J. Neurocytol.  16, 249–260 (1987).
42. Bargagna-Mohan, P., Hamza, A., Kim, Y. E.  et al.  The tumor inhibitor and antiangiogenic agent withaferin A targets the intermediate filament protein vimentin.  Chem. Biol.  14, 623–634 (2007).
43. Lamouille, S., Xu, J. & Derynck, R. Molecular mechanisms of epithelial–mesenchymal transition.  Nat. Rev. Mol. Cell Biol.  15, 178–196 (2014).
44. Graeber, M. B., Streit, W. J. & Kreutzberg, G. W. The microglial cytoskeleton: vimentin is localised within activated cells in situ.  J. Neurocytol.  17, 573–580 (1988).
45. Wohl, S. G., Schmeer, C. W.  et al.  In situ dividing and phagocytosing retinal microglia express nestin, vimentin, and NG2 in vivo.  PLoS ONE  6, e21059 (2011).
46. Giulian, D. & Baker, T. J. Characterisation of amoeboid microglia isolated from developing mammalian brain.  J. Neurosci.  6, 2163–2178 (1986).
47. Paresce, D. M., Chung, H. & Maxfield, R. Slow degradation of aggregates of the Alzheimer’s disease amyloid β protein by microglial cells.  J. Biol. Chem.  272, 29390–29397 (1997).