Edited by: Amanda Sierra, Achucarro Basque Center for Neuroscience, Spain
Reviewed by: Souvarish Sarkar, Brigham and Women's Hospital and Harvard Medical School, United States; Rishein Gupta, University of Texas at San Antonio, United States
This article was submitted to Multiple Sclerosis and Neuroimmunology, a section of the journal Frontiers in Immunology
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Microglia are specialized resident macrophages of the central nervous system (CNS) that have important functions during neurodevelopment, homeostasis and disease. This mini-review provides an overview of the current tools and approaches for studying microglia
The CNS (comprising the brain parenchyma, spinal cord, and neural retina) is populated with specialized resident macrophages called microglia. Microglia are derived from yolk sac progenitors (
The heterogeneous states of activated microglia exist on a continuum ranging from neuroprotective to neurotoxic/pathogenic (
Microglia are the subject of intense research efforts; however, there are several challenges associated with studying these cells.
In recent years, significant progress has been made to address these challenges by developing new cellular and molecular tools for microglia research. In this mini-review we discuss the current “microglia tool kit” for
Advantages, limitations, and applications of tools to study microglia
Antibodies widely available; most work well in fixed tissue sections and whole mounts. | Also expressed by BAMs and peripheral immune cells. | Phenotyping (e.g., analysis of MG density, distribution, morphology, marker co-expression, cell interactions). | |
Expression restricted to MG in healthy brain. |
Expression may be decreased by MG during disease. |
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GFP or Cre under control of |
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Higher specificity for MG compared to |
Non-specific recombination can occur in some Cre lines, resulting in subsets of BAMs and glia also being labeled. |
Phenotyping (fluorescent reporter lines); fate mapping in development, disease, and aging (Cre lines). | |
High resolution 3D datasets generated by collecting optical Z sections through tissue. |
Most confocal microscopes have limited imaging depth: requires specimen to be sectioned (brain) or microdissected (retina). |
Imaging fluorescently labeled microglia in fixed brain/spinal cord/retinal sections or whole mounts. | |
Can perform rapid 3D reconstructions of optically cleared tissues (deep imaging). |
Not all research facilities have access to light sheet microscopes and specialized objectives. |
Imaging fluorescently labeled microglia in fixed, optically cleared tissues (“global” tissue imaging). | |
Imaging MG in live animals. | Specialized instrumentation required; not available in all research facilities. |
Imaging dynamic MG behavior |
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Effective for short-term depletion studies. | MG depletion requires intracerebral or intravitreal injection (break “immune privilege” due to physical trauma). |
Depletion of MG to determine their functions in development, homeostasis or disease. |
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Cross the blood-brain/blood-retina barrier and can be administered orally. |
Not MG-specific | ||
Depletion requires injections of either tamoxifen, DT or ganciclovir. |
Immunohistochemistry and flow cytometry are common techniques in neuroimmunology. Traditionally, Iba-1 antibodies have been used to label/stain microglia using immunohistochemistry; however, Iba-1 is also expressed by BAMs and subsets of peripheral myeloid cells (
Bulk RNA-seq studies have identified several highly expressed genes that constitute the “microglia homeostatic signature” (
The development of microglia-specific antibodies is useful for labeling these cells during homeostatic conditions. However, these markers may not be reliable for the identification of microglia during development and disease. For example, embryonic microglia do not express Tmem119, and expression of this protein by all microglia does not occur until postnatal day 14 in mice (
The fractalkine receptor gene
Recently described reporter mice have taken advantage of microglia-specific signature genes.
The ability to label microglia using the above approaches enables researchers to visualize these cells
Tissue clearing techniques coupled with light sheet microscopy can be used to visualize microglia within intact transparent CNS tissues. Tissue clearing methods [reviewed in detail in (
Unlike the brain, the eye is naturally transparent. However, the retinal pigment epithelium, the interface between the retinal photoreceptors and the choroid, is highly melanized (darkly pigmented), which remains a challenge for standard clearing methods. Specialized clearing techniques including EyeCi (
Two-photon microscopy is a powerful technique that allows unparalleled imaging of microglia in live animals. Time-lapse
Specialized tools are available for
To attribute
CSF1R inhibitors are effective for microglia depletion, as adult microglia depend on CSF1R for survival (
Microglia depletion can also be achieved with genetic approaches. In
Genetic depletion systems targeting microglia-specific genes offer a more precise approach to microglia elimination without affecting BAMs and circulating myeloid cells. Using
Researchers have previously applied traditional immunological approaches to study microglia the CNS. Thanks to rapid advances in the neuroimmunology field, we now have a suite of tools and techniques for microglia research. The discovery of microglia-specific signature genes has elevated the choice of markers, antibodies and reporter mice for studying these cells
EE-S and SD conceived and researched data for the article and wrote the review. All authors contributed to the article and approved the submitted version.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The authors thank Dr. Antoine Corbin and Dr. Rejane Rua for insightful discussions and comments on early drafts of the manuscript, and the Master de Biologie of the École Normale Supérieure de Lyon for their support and guidance.