Engineering, 2013, 5, 491-495
http://dx.doi.org/10.4236/eng.2013.510B101 Published Online October 2013 (http://www.scirp.org/journal/eng)
The Phenotypic Conversion of Macrophage Detected by
FACS in EAE Mice Treated with or without Fasudil*
Chunyun Liu1, Yong Xie1, Yanhua Li1, Jiezhong Yu1, Ling Feng1, Shaowei Hou1,
Haifei Zhang1, Cungen Ma1#, Baoguo Xiao2#
1Institute of Brain Science, Department of Neurology, Medical School, Shanxi Datong University, Datong, China
2Institute of Neurology, Huashan Hospital, Fudan University, Shanghai, China
Received 2013
ABSTRACT
We studied the changes of macrophage populations in splenic mononuclear cells of experimental autoimmune ence-
phalomyelitis (EAE) mice treated with or without Fasudil. Phenotypic analysis using flow cytometry showed that the
levels of TLR4, CD11c and CD40 which represent the type 1 macrophage, were depressed in Fasudil-treated mice. In
contrast, it was observed the expressions of CD200 and CD14 which typify the type 2 macrophage were elevated in
Fasudil-treated mice as compared to EAE mice. And we also found that Fasudil at dose of 40 mg/kg alleviated the se-
verity of symptom in EAE mice. Based on the evidence that M1 macrophages are neurotoxic and M2 macrophages
promote a regenerative growth, indicating that polarization and shifting of macrophages into M2 cells may also play
key roles in treatment of EAE.
Keywords: Experiment Autoimmune Excephomeylitis; Fasudil; Flow Cytometry; Macrophage
1. Introduction
Multiple sclerosis (MS) is a chronic inflammatory de-
myelinating disease of th e central nervous system (CNS)
that afflicts approximately 1.4 million people worldwide
[1]. Experimental autoimmune encephalomyelitis (EAE)
is an animal model of MS, which exhibits some of the
symptomatology and pathology observed in MS patients.
The immunopathogenesis of EAE involves the disruption
of the BBB and the integrated attack of T cells, macro-
phages, and dendritic cells [2].
Fasudil (1-(5-isoquinolinesulfonyl)-homo-piperazine),
a selective Rho-kinase (ROCK) inhibitor, has been used
to treat cerebral vasospasm and stroke. In EAE model,
the clinical severity of EAE is tigh tly related to the infil -
tration of in f l amma to r y macrophages and T cells in the
CNS [3,4]. In the present study, we observed therapeutic
potential of fasudil and explored action mechanisms in
EAE.
2. Materials and Methods
2.1. Animals
Female C57BL/6 mice, 8 - 10 weeks old, weight 20 - 22
g, were purchased from Vital River Laboratory Animal
Technology Co. Ltd. (Beijing, China). All experiments
were conducted in accordance with the guidelines of the
International Council for Laboratory Animal Science.
The study was approved by the Ethics Committee of
Shanxi Datong University, Datong, China. All mice were
housed under pathogen-free conditions, received food
and water ad libitum, and maintained in a reversed 12:12
hours (h) light /da rk c ycle in a temper at ure-controlle d room
(25˚C ± 2˚C) for one week prior to experimental ma-
nipulation.
2.2. Induction and Clinical Evaluation of EAE in
C57BL/6 Mice
Mouse myelin oligodendrocyte glycoprotein peptide35-55
(MOG35-55) was produced in an automatic synthesizer
(CL. Bio-Scientific. Company, Xian, China), aminophenol
sequence for MOG35-55 is MEVGWYRSPFSRVVH-
LYRNGK. The purity of the peptide was >95% as de-
termined by HPLC.The Chronic EAE was induced by
subcutaneous immunization on the upper dorsal flanks
with 300 μg of MOG35-55 in complete Freund’s adjuvant
(Sigma, USA) supplemented with 3 mg/ml of M. Tuber-
culosis H37Ra (BD Difco, USA) (400 μg/mice). Mice
were injected with 400 ng of pertussis toxin (Enzo Life
Sciences, USA) via abdominal cavity at the same time of
*
This work was supported by National Natural Science Foundation of
China (No. 81070957).
#Corresponding author.

C. Y. LIU ET AL.
492
immunization and again 48 hours later. Animals were
weighed and evaluated for clinical score every other day
in a blinded fashion by at least two investigators. Clinical
scores of EAE were graded according to the following
criteria: 0. healthy; 1. limp tail; 2. ataxia and/o r pare sis of
hindlimbs; 3. paralysis of hindlimbs and/or paresis of
forelimbs; 4. tetraparalysis; 5. moribund or death. All ex-
periments were repeated 3 - 4 times.
2.3. Treatment of Fasudil
Fasudil (TIANJIN CHASE SUN PHARMACEUTICAL
CO., LTD) dissolved in normal saline (NS) was injected
intraperitoneally (i.p.) at 40 mg/kg/d on day 3 post-im-
munization (p.i.) (fasudil early treatment) or at onset of
EAE (fasudil late treatment) till d ay 28 p.i. The injection
of NS was set up as control (EAE group) in similar
manner.
2.4. Preparation of Mononuclear Cells
At day 28 p.i., mice were sacrificed and spleens were
removed under aseptic conditions. MNCs from spleens
were prepared by grinding the organ through a 40 mm
nylon mesh in medium. Erythrocytes in the suspensions
were osmotically lysed. Cells were then washed 3 times
and re-suspended in medium. Cells were adjusted to 3 ×
106 ml.
2.5. Flow Cytometry (FACS)
For phenotypic analysis, MNCs were stained for 20 min
at room temperature in 1% BSA-PBS buffer with the
following antibodies: PE-TLR4, PE-CD40, Percp-cy5-
CD11c, Percp-cy5-CD200, Percp-cy5-CD14, PE-CD23.
(BD Biosciences, San Diego, CA). Cells were gated us-
ing forward and sideward scatter characteristics and at
least 10,000 gated events were collected using FACS
Calibur flow cytometer (BD Biosciences, USA). Data
were analyzed using CellQuest software.
2.6. Statistical Analysis
Data were presented as mean ± S.E.M. Body weight,
clinical scores and incidence were performed using the
two-tailed Student’s t test and were analyzed by one-way
ANOVA using GraphPad Prism 4 (GraphPad Software,
Inc.). The multiple comparisons were performed by the
two-way ANOVA using GraphPad Prism 4. A statisti-
cally significant difference was assumed at P < 0.05.
3. Results
3.1. Fasudil Alleviated the Severity of Symptom
in EAE Mice
As shown in Table 1, the incidence of EAE model was
Table 1. The clinical symptoms of mice in each group.
Group n Incidence
(%) Mean onset
date Mean score of maximal sympt om
EAE control 14 100 12.57 ± 1.55 2.50 ± 1.13
Fasudil late 14 78.6 13.45 ± 2.07 1.36 ± 1.06b
Fasudil early 14 28.6a 16.50 ± 4.20a 0.32 ± 0.70a
ap < 0.05 vs. EAE control; bp < 0.05 vs. EAE control.
100%, while the incidence of Fasudil early treatment and
late treatment was 78.6% and 28.6% (P < 0.05), respec-
tively. Mean onset was 12.37 ± 1.55 p.i. in EAE control,
13.45 ± 2.07 p.i. in fasudil early treatment and 16.50 ±
4.20 p.i. in Fasudil late treatment (P < 0.05). In early
treatment, Fasudil delayed the onset of sign and dramati-
cally reduced clinical symptoms. In late treatment, Fasu-
dil also ameliorated clinical scores in EAE mice, as
compared with EAE controls (Figure 1(A), P < 0.05), In
addition, the improvement of EAE clinical scores were
accompanied by a decrease in body weight from days 10
to 28 p.i. (Figure 1(B)), providing further evidence that
Fasudil exhibits a ther apeutic potential for future clin ical
application.
3.2. Fasudil Shifts M1 to M2
To characterize the changes of macrophage populations
in spleen on EAE, we assessed the expression of TLR4,
CD40, CD11c, CD200, CD23 and CD14 using FACS.
Phenotypic analysis of macrophage showed that the le-
vels of TLR4, CD11c and CD40 which represent the type
1 macrophage (M1), were significantly depressed in Fa-
sudil late treatment mice compared with EAE control
(Figure 2(A), p < 0.001 for TLR4, p < 0.05 for CD11c
and p < 0.001 for CD40). The expre ssion of CD11c and
CD40 were significantly reduced in Fasudil early treat-
ment mice compared with EAE control (Figure 2(A), p <
0.001 for CD11c and p < 0.05 for CD40). As expected,
we found that the augmented tendency of CD200, CD23
and CD14 which labeled the type 2 macrophage (M2)
was observed in Fasudil treatment mice，especially in
Fasudil early treatment mice (Figure 2(B)). Mice in fa-
sudil early treatment exhibited increased expression of
CD200 and CD14 in comparison to EAE control mice (P
< 0.01 and P < 0.001, respectively), whereas the elev a-
tion of CD200 expression in Fasudil late treatment mice
was not obvious (P > 0.05). Our results indicate that Fa-
sudil treatment can shift macrophages from M1 to M2
phenotype.
4. Discussion
FACS was developed in 1971 by Leonard Herzenberg’s
team at Stanford University, which was widely used in
the world for clinical and experiment research. Under

C. Y. LIU ET AL.
493
Figure 1. Fasudil treatment suppresses MOG-induced EAE. (A) Fasudil could prevent the severity of clinical symptoms to
EAE, and there were significant differences of clinical symptoms score between EAE control and fasudil late treatment group
on days 24 - 28 p.i. (*P < 0.017) and between EAE control and fasudil early treatment group on days 14 - 28 p.i. (*P < 0.017).
(B) EAE control group had more marked loss of body weights as compared with that of fasudil late treatment group on days
18 - 28 p.i. (*P < 0.05) and that of fasudil early treatment group on days 12 - 28 p .i . (*P < 0.05). Data are presented as mean ±
S.E.M.
continuous development, this technology has been enable
to perform single cell multiparametric analysis and stem
cells sorting, which will open a new way to FACS uses
in the future [5,6]. By using FACS assay, we further ex-
plore the therapeutic mechanisms of Fasudil in treatment
of EAE.
In the present study, Fasudil treatment delayed onset
of EAE, and ameliorated clinical scores. The known
beneficial effects of Fasudil on EAE could partly be ex-
plained by inhibition of the GTPase Rho, which resulted
in a Th2 shift acting on T cells [7]. In addition, T-cell
proliferation specific to MOG (35-55) was markedly re-
duced, together with a significant down-regulation of
interleukin (IL)-17, IL-6, and MCP-1. In contrast, secre-
tion of IL-4 was increased, and IL-10 was slightly ele-
vated [4]. Our results demonstrate that Fasudil inhibited
TLR4, CD11c, CD40 expression and increased CD200,
CD23, CD14 expression on MNCs.
Macrophages play an important role in innate and
adaptive immunity and are a heterogeneous cell popula-
tion of the myeloid linage derived from monocytes.
These cells show two different polarization states, M1
and M2 macrophages in response to different immune
responses . It is postulated that the development of au-
toimmune diseases may be mediated by activating ma-
crophages to a cytolytic M1 phenotype and by suppress-
ing the activation of proregenerative macrophages to an
M2 phenotype. Different phenotype and function of ma-
crophages can be distinguished using M1 markers such
as TLR4, CD11c and CD40 as well as M2 markers such
as CD14, CD23 and CD200. To our knowledge, CD40
positive microglia were observed in the CNS of marmo-
set monkeys with acute EAE, a newly described non-
human primate model for MS, which is important for the
infiltration and retention of inflammatory leuko cytes into
the CNS [8]. TLR4 and CD11c could provoke antigen
presenting cells to activate autoreactive immune cells and
induce Ag-loaded dendritic cells to produce a marked
increase in inflammatory cytokine secretion [9,10]. These
molecules play the critical roles in driving the immune
response necessary to develop EAE, and therefore, the
down-regulation of TLR4, CD11c and CD40 which re-
veals the inhibition of proinflammatory M1 macrophages
could have a direct consequence on EAE progression.
CD200 is a non-signaling glycoprotein that belongs to
the Ig superfamily and has been shown to inhibit immune
stimulatory responses [11]. Our findings indicate that
Fasudil ameliorates the d evelopment of EAE at least par-
tially through sifting inflammatory M1 to anti-inflam-
matory M2 macroph a ges.

C. Y. LIU ET AL.
494
Figure 2. Effects of fasudil on splenic macrophages. The level of TLR4, CD40, CD11c, CD200, CD23 and CD14 were shown
by blue histograms. It is noteworthy that Fasudil could downregulate the expression of TLR4, CD11c, CD40 and upregulat e
the expression of CD200, CD14. Representative histograms of three groups are shown. p-Values are i ndicated by as terisks as
follows: *P < 0.05, **P < 0.01 and ***P < 0.001.

C. Y. LIU ET AL.
495
In conclusion, the therapeutic effect of F a sudil in EAE
may be related to shifting of macrophages from M1 to
M2 phenotype. By using FACS assay, the treatment of
Fasudil inhibits M1 macrophages, and elevates M2 ma-
crophages, providing novel mechanism of Fasudil in the
treatment of EAE.
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