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Zheng et al. Neuroimmunol Neuroinflammation 2019;6:1 I http://dx.doi.org/10.20517/2347-8659.2018.52 Page 3 of 12

a functional means by which they release inflammatory factors that contribute to the neuroinflammation
responses in CNS disease.

MICROGLIAL POLARIZATION
Microglia generally polarize in two directions from a resting state. The classical activation is known as
M1, which is the mediator of pro-inflammatory responses. The alternative activation, known as M2, is
responsible for resolution and repair. The polarization of microglia has been clarified by measuring the
markers both in vitro and in vivo. In vitro treatment mouse microglia with lipopolysaccharide (LPS) and
[27]
IL-4 results in M1 and M2 phenotypes respectively . The M1 microglial polarization is characterized by
an increase in the expression of pro-inflammatory molecules. Alternatively, activated M2 microglia are
categorized into four subtypes, M2a, M2b, M2c, and M2d. The subtypes of the M2 phenotype are firstly
defined in the macrophage activation. Analogous to macrophage, we hypothesize the similar subtypes of M2
microglia [28-30] .

Recently, the mixture of M1 and M2-phenotyped response are reported in experimental studies, which raise
a controversy to the traditional concept of M1 vs. M2 microglial polarization. In addition, the classic M1
marker IL-6, which is induced by IL-4, functions as an anti-inflammatory mediator in a mouse model of
[31]
experimental autoimmune encephalomyelitis .

Although the classification of M1/M2 phenotypes is now recognized as an oversimplification, as the pure
M1 or M2 polarization is executively observed in in vitro studies, this conception remains useful for
understanding the functional role of microglia in CNS diseases. The recent studies on SAH address the
functional role of microglia by investigating the M1-like and M2-like markers, which are discussed below.

MICROGLIAL M1 POLARIZATION
Microglial polarization can be assessed by immunohistochemical analysis of the specific markers. Pro-
inflammatory cytokines are considered to be produced predominantly by classically activated M1 microglia,
and these pro-inflammatory factors are integral in the activation of downstream pathways. Therefore, the
changes in pro-inflammatory cytokine profiles and pathways can be indicators of microglial polarization
after SAH.

An intraparenchymal accumulation of KiM1P-positive microglia/macrophage cells is documented in the
SAH patients. The microglia accumulation is evident between day 5 and 15 . On experimental SAH, the
[32]
increase of Iba-1-positive microglial cells is observed around day 4 and 28 within the brain parenchyma.
The peak occurs on day 14 after SAH induction. The microglia accumulation presents a centrifugal pattern,
[32]
starting at the base to the cortex of both hemispheres and spreading globally to other regions of the brain .
[33]
[17]
[9]
Cell surface markers including CD11b , CD68 , and ED-1 are used to distinguish the activated from
resting microglia, both of which can be defined by immunostaining of iba1. In culture, microglia treated
[9]
with oxyhemoglobin leads to M1 polarization, indicated by CD16+/CD11b+ cells [Figure 1] . Other M1-
associated markers CD86 and inducible nitric oxide synthase (iNOS) increase immediately after SAH, peak
[11]
at 24-48 h, and remain highly expressed for 72 h .
TNF-α and IL-1β are known to be the most important pro-inflammatory cytokines in human SAH
pathology, as well as in other experimental models of SAH. M1-signature pro-inflammatory cytokines are
upregulated prominently after SAH. IL-1β, IL- 6, TNF-α increase rapidly within 24 h and last for 48 h on
experimental SAH model [9,17,33-35] . The increased pro-inflammatory cytokine expression is associated with
[36]
poor outcomes in SAH .