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. 2014 Jan 21;11:12.
doi: 10.1186/1742-2094-11-12.

Morphometric Characterization of Microglial Phenotypes in Human Cerebral Cortex

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Free PMC article

Morphometric Characterization of Microglial Phenotypes in Human Cerebral Cortex

Susana G Torres-Platas et al. J Neuroinflammation. .
Free PMC article

Abstract

Background: Microglia can adopt different morphologies, ranging from a highly ramified to an amoeboid-like phenotype. Although morphological properties of microglia have been described in rodents, little is known about their fine features in humans. The aim of this study was to characterize the morphometric properties of human microglia in gray and white matter of dorsal anterior cingulate cortex (dACC), a region implicated in behavioral adaptation to neuroinflammation. These properties were compared to those of murine microglia in order to gain a better appreciation of the differences displayed by these cells across species.

Methods: Postmortem dACC samples were analyzed from 11 individuals having died suddenly without any history of neuroinflammatory, neurodegenerative, nor psychiatric illness. Tissues were sectioned and immunostained for the macrophage marker Ionized calcium binding adaptor molecule 1 (IBA1). Randomly selected IBA1-immunoreactive (IBA1-IR) cells displaying features corresponding to commonly accepted microglial phenotypes (ramified, primed, reactive, amoeboid) were reconstructed in 3D and all aspects of their morphologies quantified using the Neurolucida software. The relative abundance of each morphological phenotype was also assessed. Furthermore, adult mouse brains were similarly immunostained, and IBA1-IR cells in cingulate cortex were compared to those scrutinized in human dACC.

Results: In human cortical gray and white matter, all microglial phenotypes were observed in significant proportions. Compared to ramified, primed microglia presented an average 2.5 fold increase in cell body size, with almost no differences in branching patterns. When compared to the primed microglia, which projected an average of six primary processes, the reactive and amoeboid phenotypes displayed fewer processes and branching points, or no processes at all. In contrast, the majority of microglial cells in adult mouse cortex were highly ramified. This was also the case following a postmortem interval of 43 hours. Interestingly, the morphology of ramified microglia was strikingly similar between species.

Conclusions: This study provides fundamental information on the morphological features of microglia in the normal adult human cerebral cortex. These morphometric data will be useful for future studies of microglial morphology in various illnesses. Furthermore, this first direct comparison of human and mouse microglia reveals that these brain cells are morphologically similar across species, suggesting highly conserved functions.

Figures

Figure 1
Figure 1
Distribution of ionized calcium binding adaptor molecule 1 (IBA1)-immunoreactive (IR) cells in the gray and white matter human dorsal anterior cingulate cortex (dACC) counterstained with cresyl violet. Human microglial cells in the dACC appear evenly distributed across the cortical layer in the gray matter (a) and aligned to myelinated tracts in the white matter (b). Scale bars: 50 μm.
Figure 2
Figure 2
Four main phenotypes represent the population of resident IBA1-IR cells in human dACC gray matter. Ramified microglia (a) display a small circular cell body with highly ramified processes. Primed microglia (b) present a bigger and less round cell body with similar ramification patterns when compared to the ramified phenotype. Reactive microglia (c) display an amoeboid cell body but still present a few ramified processes compared to amoeboid microglia (d), which can present, at most, two unramified processes or be completely devoid of them. These cells are occasionally observed to be associated with blood vessels (asterisks). Scale bars: 10 μm.
Figure 3
Figure 3
Four main phenotypes represent the population of resident IBA1-IR cells in human dACC white matter. Ramified microglial cell body and highly ramified processes appear aligned to white matter tracts (a). Primed microglia display a wider cell body in the primed phenotype (b) compared to the ramified phenotype, but their processes and cell body retain a similar alignment. Reactive microglia present an amoeboid-shaped rounder cell body with a few ramified processes (c), whereas amoeboid microglia display a characteristic amoeboid-shaped cell body extending one or two unramified processes (top panel) or are completely devoid of processes (bottom panel) (d). Scale bars: 10 μm.
Figure 4
Figure 4
Microglial phenotypes in human dACC gray matter are characterized by significant changes in the cell body and processes. Primed microglia display a cell body of greater area (a) and of decreased roundness (b), as reflected by a significantly increased difference between the maximum and minimum feret (max-min feret) (c) compared to the ramified phenotype. The cell body morphology of reactive microglia appears statistically similar in area (a), roundness (b) and max-min feret (c) as primed microglia, but presents a decrease in roundness (b) when compared with amoeboid microglia. A reconstruction of a primed microglia shows the shortest (blue arrow; min) and longest chord (violet arrow, max) representing the maximum and minimum feret, respectively, and the ramification patterns represented by the ends and nodes of the processes (d). Ramified and primed microglia project similar numbers of primary (e) and higher-order processes (f-g). Reactive microglia display fewer first-order (and overall) branches (e-g), as well as shorter total process length (h) and volume (i). Amoeboid microglia have a significant decrease of primary and higher order branches (f-g), as well as a significant decrease in total process length (h) and volume (i) compared to reactive microglia, *P <0.01, **P <0.001, ***P <0.0001.
Figure 5
Figure 5
Microglial phenotypes display significant differences in cell body and ramification patterns. Compared to ramified, the primed phenotype displays a larger cell body area (a) as reflected by a significantly larger maximum (not shown) and minimum feret (b). Despite this significant increase in area, the max-min feret (c) and the roundness (d) of the cell body did not statistically differ between these or any of the other phenotypes. The cell body morphology of reactive, primed and amoeboid microglia appears comparable at all points (a-d). Ramified and primed microglia both extend similar numbers of primary processes (e), however, primed microglia hold fewer ends (f) and nodes, (g) as well as significantly shorter processes (h) with no significant changes in volume (i) compared to ramified microglia. Reactive microglia extend a significant decrease of primary (e) and higher-order branches (f-g), as well as shorter processes than primed microglia (h) and amoeboid microglia present almost an absence of overall processes (e-g) that were thus, of significantly reduced length (h) and volume (i) compared to reactive microglia; *P <0.01, **P <0.001, ***P <0.0001.
Figure 6
Figure 6
IBA1-IR cells in the cingulate cortex of a young adult mouse (1.5 months old) display cell bodies of heterogeneous shapes and sizes, and highly ramified processes with overlapping domains. Scale bars: 50 μm.
Figure 7
Figure 7
Representative reconstructions of gray matter (top row) and white matter (bottom row) ramified microglia in human dACC (left column) and mouse cingulate cortex (right column). Scale bar: 10 μm.
Figure 8
Figure 8
IBA1-IR cells in the non-perfused cingulate cortex of a of young adult mouse (3 months old) following a PMI of 43 h (11 h at room temperature and 32 h at 4˚C). The great majority of IBA1-IR cells observed remained ramified, and did not display noticeable signs of degradation. Sections were counterstained with cresyl violet. Scale bar: 25 μm.

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