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Tanaka. Neuroimmunol Neuroinflammation 2020;7:73-91 I http://dx.doi.org/10.20517/2347-8659.2020.04 Page 75

have long been thought to be able to display almost completely spherical or amoeboid morphology
[5]
when they become fully activated in the core of severe brain insults such as stroke or trauma . Both cell
[26]
types share numerous immune cell markers . Therefore, distinguishing between them has been very
problematic. However, many methods have been now established for this purpose.

Morphological characteristics
Despite the historical view, even highly activated microglia do not display spherical morphology [9,27] . In a
rat stroke model prepared by transient occlusion of middle cerebral artery (tMCAO), activated microglia
in the peri-ischemic regions exhibit enlarged somata and shortened processes that are distinct from spikes.
The processes can be identified with immunoreactivity to microglia/macrophage markers such as Iba1 or
CD11b. Conversely, blood-borne macrophages and neutrophils, the latter of which infiltrate in abundance,
[27]
do not have processes even though they may have a polygonal shape and short spikes . Thus, microglia
[10]
can be distinguished from blood-borne cells via morphological observation . This may be the simplest
method for specific identification of microglia.

Specific markers to distinguish the two cell types in the pathologic brains
Iba1, CD11b, CD45, and CD68 have long been used to identify resident microglia; however, these markers
are more strongly expressed by infiltrating macrophages than by activated microglia . As CD11b is a
[9]
marker for myeloid cells and CD45 is a marker for all of the leukocytes, they are not suitable for identifying
microglia in the pathologic CNS. To examine specific roles of resident microglia in CNS pathology, they
must be distinguished from blood-borne cells that are macrophages, lymphocytes, and neutrophils. Thus,
considerable effort has been dedicated to finding microglia-specific markers that are not expressed by
[28]
macrophages and other blood-borne cells . Comprehensive gene analyses using RNAseq and/or microarray
analyses have identified genes that are expressed predominantly by microglia rather than by macrophages
such as Cx3cr1, Gpr34, P2ry12, P2ry13, Siglech, Tmem119, and Trem2 [29-32] . Of the specific marker candidates,
TMEM119 and Siglec-H may be the most promising for immunohistochemical discrimination of microglia
from macrophages.

[33]
Transmembrane protein 119, commonly known as TMEM119, was identified by Bennet et al. Specific
expression of TMEM119 by microglia has been demonstrated by immunohistochemical staining,
flow cytometry analyses, and in situ hybridization [33,34] . TMEM119 was identified originally as a type I
[35]
transmembrane protein expressed by murine osteoblasts and is responsible for their differentiation . It
is not expressed by microglia in immature murine brains, but its expression increases along with their
[33]
development or ramification . In aged human brains either with or without Alzheimer’s disease (AD),
[34]
microglia expressed TMEM119 . Activated microglia with enlarged somata in close proximity to amyloid
plaques are less immunoreactive to TMEM119 antibody than are resting (or homeostatic) microglia.
Moreover, TMEM119-expressing microglia in AD brains either do not or only weakly express the polarized
markers CD80, CD163, or CD206. In a TBI model, ramified or homeostatic microglia express TMEM119
[10]
at higher levels than do activated microglia . Therefore, TMEM119 is particularly suitable for identifying
homeostatic microglia with a ramified shape in both the normal and the injured mature brain.

Sialic-acid-binding immunoglobulin-like lectin-H (Siglec-H) is another promising marker for
[36]
immunohistochemical discrimination of microglia from macrophages . Siglec-H is a single-pass
transmembrane protein that was identified originally as a member of a CD33-related Siglec family. Siglec-H
is barely expressed by circulating monocytes and their derived macrophages. In contrast to TMEM119,
Siglec-H is expressed continually by activated microglia and by microglia in immature brains. Siglec-H
[37]
may mediate signals necessary for phagocytosis by microglia .

Bone marrow transplantation
Bone marrow transplantation (BMT) has long been a reliable method for identifying blood-borne cells
[28]
in the CNS with BBB breakdown . After ~10 Gy irradiation to cause near-total death of the host’s bone