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Cited by 149Neuroinflammation and Neurodegeneration MechanismsMultiple Sclerosis Research StudiesSingle-cell and spatial transcriptomics

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Analyzing microglial phenotypes across neuropathologies: a practical guide

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Schwabenland, Marius, Brück, Wolfgang, Priller, Josef, Stadelmann, Christine, Lassmann, Hans, Prinz, Marco·2021·Acta Neuropathologica
DOI 10.1007/s00401-021-02370-8PMC PMC8498770
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Abstract

As extremely sensitive immune cells, microglia act as versatile watchdogs of the central nervous system (CNS) that tightly control tissue homeostasis. Therefore, microglial activation is an early and easily detectable hallmark of virtually all neuropsychiatric, neuro-oncological, neurodevelopmental, neurodegenerative and neuroinflammatory diseases. The recent introduction of novel high-throughput technologies and several single-cell methodologies as well as advances in epigenetic analyses helped to identify new microglia expression profiles, enhancer-landscapes and local signaling cues that defined diverse previously unappreciated microglia states in the healthy and diseased CNS. Here, we give an overview on the recent developments in the field of microglia biology and provide a practical guide to analyze disease-associated microglia phenotypes in both the murine and human CNS, on several morphological and molecular levels. Finally, technical limitations, potential pitfalls and data misinterpretations are discussed as well.

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Figures

Fig. 1 The workflow of tissue processing for the analysis of microglial cells using conventional and novel state-of-the-art technologies is depicted. The technical spectrum for the characterization of microglia cells and the necessary tissue pretreatment are shown
Fig. 1 The workflow of tissue processing for the analysis of microglial cells using conventional and novel state-of-the-art technologies is depicted. The technical spectrum for the characterization of microglia cells and the necessary tissue pretreatment are shownCC BY · PubMed Central
Fig. 2 Visualization of microglia by using non-antibody-dependent staining techniques. a Different cell types including microglia can be identified in a Haematoxylin & Eosin section (H&E) of a healthy human cortex. Microglial nuclei (M) are small and dark, partially appear irregular and sometimes elongated with a “cigar-like” or “comma-like” shape. Perivascular macrophages (P) are located along blood vessels, in which endothelial cells (E) can be identified by their elongated nuclei. Arrows also point towards neurons (N), oligodendrocytes (O) and astrocytes (A). Scale bars: 50 µm and 20 µm (insert). b Lectins stain ramified microglia within the cortex of a mouse brain (asterisk). Cerebral vessels are stained as well (upper right). The staining with lectin (obtained from Lycopersicon Esculentum) is shown in green, DAPI in blue. Scale bar: 20 µm. c An electron microscopy image of a murine spinal cord is depicted. Microglia are typically small in size, partly have a “bean-shaped” nuclei (yellow asterisks) and only show little cytoplasm. Scale bars: 5 µm and 2 µm (insert)
Fig. 2 Visualization of microglia by using non-antibody-dependent staining techniques. a Different cell types including microglia can be identified in a Haematoxylin & Eosin section (H&E) of a healthy human cortex. Microglial nuclei (M) are small and dark, partially appear irregular and sometimes elongated with a “cigar-like” or “comma-like” shape. Perivascular macrophages (P) are located along blood vessels, in which endothelial cells (E) can be identified by their elongated nuclei. Arrows also point towards neurons (N), oligodendrocytes (O) and astrocytes (A). Scale bars: 50 µm and 20 µm (insert). b Lectins stain ramified microglia within the cortex of a mouse brain (asterisk). Cerebral vessels are stained as well (upper right). The staining with lectin (obtained from Lycopersicon Esculentum) is shown in green, DAPI in blue. Scale bar: 20 µm. c An electron microscopy image of a murine spinal cord is depicted. Microglia are typically small in size, partly have a “bean-shaped” nuclei (yellow asterisks) and only show little cytoplasm. Scale bars: 5 µm and 2 µm (insert)CC BY · PubMed Central
Fig. 3 Immunohistochemistry-based assessment of microglia phenotypes across neuropathologies. Using Iba1 immunohistochemistry (brown), the following six-step standardized sequence is recommended for the analysis of microglia cells. In short, microglia cells and CNS-associated macrophages first need to be identified by their spatial distribution. Secondly, the cell density should be evaluated. The cell shape is of particular importance when analyzing the microglial phenotype. An uneven, irregular distribution pattern and microglia cell territories that are potentially overlapping might point towards local pathological processes within a tissue section. Next, the examiner should look for distinct microglia phenotypes, e.g. dividing cells or cells that underwent foam cell transformation. Lastly, a potential interaction with other cell types should be explored. Combined immunohistochemical reactions with two different markers are indicated. Counterstaining with haematoxylin (blue)
Fig. 3 Immunohistochemistry-based assessment of microglia phenotypes across neuropathologies. Using Iba1 immunohistochemistry (brown), the following six-step standardized sequence is recommended for the analysis of microglia cells. In short, microglia cells and CNS-associated macrophages first need to be identified by their spatial distribution. Secondly, the cell density should be evaluated. The cell shape is of particular importance when analyzing the microglial phenotype. An uneven, irregular distribution pattern and microglia cell territories that are potentially overlapping might point towards local pathological processes within a tissue section. Next, the examiner should look for distinct microglia phenotypes, e.g. dividing cells or cells that underwent foam cell transformation. Lastly, a potential interaction with other cell types should be explored. Combined immunohistochemical reactions with two different markers are indicated. Counterstaining with haematoxylin (blue)CC BY · PubMed Central
Fig. 4 Spatial heterogeneity of the myeloid compartment within a single tissue section of a patient with encephalitis revealed by P2RY12 and TMEM119 immunohistochemistry. The immunohistochemical reactions for P2RY12 (brown, upper panel) and TMEM119 (brown, lower panel) are shown in two regions of the same tissue section of a patient with encephalitis. The microglia cells (as identified by P2RY12) positivity show a downregulation/loss of TMEM119 upon activation. Counterstaining with haematoxylin (blue). Scale bar: 100 µm
Fig. 4 Spatial heterogeneity of the myeloid compartment within a single tissue section of a patient with encephalitis revealed by P2RY12 and TMEM119 immunohistochemistry. The immunohistochemical reactions for P2RY12 (brown, upper panel) and TMEM119 (brown, lower panel) are shown in two regions of the same tissue section of a patient with encephalitis. The microglia cells (as identified by P2RY12) positivity show a downregulation/loss of TMEM119 upon activation. Counterstaining with haematoxylin (blue). Scale bar: 100 µmCC BY · PubMed Central
Fig. 5 Novel high-throughput technologies expand the diagnostic spectrum of research on microglia. Several emerging cutting-edge methods for studying the microglial phenotype are depicted in a graphical manner. The pictograms demonstrate a gene expression heatmap (Bulk RNA sequencing), a droplet-based analysis of single cells with subsequent t-SNE visualization (Single-cell RNA sequencing), the chromatin landscape using ATAC-seq (epigenetic changes and chromatin accessibility), an objective slide incubating with metal-coupled antibodies with subsequent single-cell analysis (CyTOF-based Imaging Mass Cytometry), barcoded antibodies in combination with droplet-based microfluidics system (combined transcriptomics and proteomics) and a tissue section analyzed by in-situ-sequencing as well as two microglia cells with different intracellular symbols representing different genes (transcriptomics with spatial resolution)
Fig. 5 Novel high-throughput technologies expand the diagnostic spectrum of research on microglia. Several emerging cutting-edge methods for studying the microglial phenotype are depicted in a graphical manner. The pictograms demonstrate a gene expression heatmap (Bulk RNA sequencing), a droplet-based analysis of single cells with subsequent t-SNE visualization (Single-cell RNA sequencing), the chromatin landscape using ATAC-seq (epigenetic changes and chromatin accessibility), an objective slide incubating with metal-coupled antibodies with subsequent single-cell analysis (CyTOF-based Imaging Mass Cytometry), barcoded antibodies in combination with droplet-based microfluidics system (combined transcriptomics and proteomics) and a tissue section analyzed by in-situ-sequencing as well as two microglia cells with different intracellular symbols representing different genes (transcriptomics with spatial resolution)CC BY · PubMed Central

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Metadata

Authors
Schwabenland, M., Brück, W., Priller, J., Stadelmann, C., Lassmann, H., & Prinz, M.
Affiliations
  • University of Freiburg (DE)
  • Universitätsmedizin Göttingen (DE)
  • German Center for Neurodegenerative Diseases (DE)
  • Medical University of Vienna (AT)
Corresponding author
  • Lassmann, Hans
  • Prinz, Marco
ORCID
Venue
Acta Neuropathologica
Publisher
Springer Science and Business Media LLC
Published
2021-10-08
Volume
142
Issue
6
Pages
923-936
ISSN
  • 0001-6322 (ISSN-L)
  • 1432-0533
DOI
10.1007/s00401-021-02370-8
PMC
PMC8498770
OpenAlex
W3201995754
Cited by
149
References
80
Citations by year
2026: 21 · 2025: 29 · 2024: 25 · 2023: 36 · 2022: 37 · 2021: 1
FWCI
2.89
Type
journal article
Language
en
Open access
Licence
Locations
  • Acta Neuropathologica (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Informationszentrum Lebenswissenschaften (ZB MED) (submittedVersion, cc-by)
  • mediaTUM – the media and publications repository of the Technical University Munich (Technical University Munich) (submittedVersion)
  • DZNE Pub (submittedVersion, cc-by)
  • GoeScholar The Publication Server of the Georg-August-Universität Göttingen (Georg-August-Universität Göttingen) (submittedVersion, cc-by)
Funders
Universitätsklinikum Freiburg
Topics
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Health Sciences › Medicine › Clinical Neurology › Multiple Sclerosis Research Studies
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
Keywords
microglia, microglial phenotype, microglial activation, neurodegenerative diseases, neuroinflammation, single-cell transcriptomics, enhancer landscape, epigenetics, microglial morphology, CNS homeostasis, mouse model
MeSH terms
Neuropathology, Animals, Central Nervous System Diseases, Humans, Phenotype, Microglia, Mice

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