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Page 76 Tanaka. Neuroimmunol Neuroinflammation 2020;7:73-91 I http://dx.doi.org/10.20517/2347-8659.2020.04

marrow, the bone marrow from transgenic animals that ubiquitously express fluorescent proteins such as
[12]
enhanced green fluorescent protein is transplanted . In the transplanted brain, blood-borne macrophages
[27]
bear fluorescence but not resident microglia . However, BMT usually leads to partial chimera, which can
make it difficult to analyze the results. With reconstruction of the bone marrow, it is a long time before
the anemia disappears. Radiation may cause degeneration of neural cells, such as NG2 glia and neurons,
which may affect the results . As radiation disrupts the BBB while increasing monocyte infiltration, BMT
[38]
[28]
produces donor-derived microglia or microglia-like ramified cells in the brain parenchyma . Thus, the
BMT may provide firm evidence showing the presence of blood-borne cells in the CNS, but it should be
noted that BMT itself will significantly change the brain functions due to the toxic effects on the neural
cells.

Flow cytometry
Microglial cells belong to a group of myeloid leukocytes and express a macrophage colony-stimulating
factor receptor known as CSF-1R. Therefore, microglia express a myeloid cell marker CD11b and a pan-
leukocyte marker CD45. Based on this finding, microglia have been analyzed by flow cytometry using
antibodies to CD11b and CD45 [10,39] . Because of faint expression of CD45 by homeostatic microglia,
immunohistochemical detection of this expression tends to be difficult. Conversely, macrophages and
neutrophils express CD45 rather strongly . However, weak CD45 expression can be detected easily by
[10]
+
flow cytometry and is an advantage in flow cytometry analysis; microglia can be defined as CD11b /
hi
CD45 and macrophages as CD11b /CD45 . Even activated microglia express at lower levels of CD45 than
lo
+
do macrophages. Moreover, activated and homeostatic microglia can be distinguished by flow cytometry
based on forward and side scatter value; activated microglia have larger somata (larger forward scatter
[10]
values) and more intracellular organelles (larger side scatter values) than do homeostatic microglia . Flow
cytometry analyses can be used to isolate the cells by cell sorting [10,39] . If the cells are treated appropriately
to prevent degradation of proteins, RNA, or DNA, the sorted cells can be used for either Western blotting
or PCR.

Flow cytometry analyses of either cultured microglia or circulating monocytes are simple [40,41] .
Nevertheless, analyses of microglia and macrophages in brain tissues have been difficult because of
difficulties in dissociating the tissues into single cells. However, dissociation kits and apparatus are now
available for preparing neural cell suspensions and are designed appropriately for dissociation of rodent
and human brains [10,39] . The sorted cells can also be used for culturing and/or functional analyses.

RESPONSE OF MICROGLIA IN BRAIN PATHOLOGY IN THE ABSENCE OF BBB BREAKDOWN
Microglia become activated in response to even minor pathological events that do not involve BBB
disruption. This section discusses microglia in Parkinson’s disease (PD), peripheral nerve injury, and
Carbon monoxide (CO) intoxication. Minute activation of homeostatic microglia accompanying circadian
changes can be observed in the normal mature brain; microglia exhibit weakly activated phenotypes
[39]
around the time of onset of sleep .
PD
PD is the second most frequent neurodegenerative disorder after AD. As BBB breakdown is not apparent
in PD, infiltration of leukocytes, including monocytes, is not often seen. In PD pathophysiology,
dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc) in the mesencephalon primarily
undergo degeneration, leading to microglial activation in the vicinity of the degenerating neurons [42-44] .
The activated microglia release potentially neurotoxic substances, such as either reactive oxygen/nitrogen
species or glutamate [45,46] . The microglia-derived proinflammatory cytokines and chemokines may also
contribute to aggravation. Injection of 6-hydorxydopamine (6-OHDA) into the striatum or medial
forebrain bundle is used to prepare the PD rat model [47,48] . In the model, DA neurons in the SNc primarily