doi:10.

1093/brain/awm306 Brain (2008), 131, 616 ^ 629

Anti-inflammatory mechanism of intravascular neural

stem cell transplantation in haemorrhagic stroke
Soon-Tae Lee,1,2,3,* Kon Chu,1,2,* Keun-Hwa Jung,1,2,4 Se-Jeong Kim,1 Dong-Hyun Kim,1 Kyung-Mook Kang,1
Nan Hyung Hong,1 Jin-Hee Kim,1,2 Jae-Joon Ban,1 Hee-Kwon Park,1,2 Seung U. Kim,5,6 Chung-Gyu Park,7
Sang Kun Lee,1,2 Manho Kim1,2 and Jae-Kyu Roh1,2
1
Stroke & Stem Cell Laboratory, Clinical Research Institute, Stem Cell Research Center, Department of Neurology, Seoul
National University Hospital, 2Program in Neuroscience, Neuroscience Research Institute of SNUMRC, Seoul National
University, 3Program in Public Health Service, Seoul National Hospital, 4Department of Epidemic Intelligence Service, Korea
Center for Disease Control and Prevention, Seoul, 5Institute for Regenerative Medicine, Gachon Medical University, Inchon,
South Korea, 6Division of Neurology, Department of Medicine, UBC Hospital, University of British Columbia, Vancouver,
Canada and 7Department of Microbiology and Immunology, Xenotransplantation Research Center, Transplantation Research
Institute, Tumor Immunity Medical Research Center, Seoul National University, Seoul, South Korea

*These authors contributed equally to this work.

Correspondence to: Jae-Kyu Roh, MD, PhD, Department of Neurology, Seoul National University Hospital, 28, Yongon-Dong,
Chongro-Gu, Seoul, 110 -744, Republic of Korea

Downloaded from by guest on November 6, 2014

E-mail: rohjk@snu.ac.kr

Neural stem cell (NSC) transplantation has been investigated as a means to reconstitute the damaged brain
after stroke. In this study, however, we investigated the effect on acute cerebral and peripheral inflammation
after intracerebral haemorrhage (ICH). NSCs (H1 clone) from fetal human brain were injected intravenously
(NSCs-iv, 5 million cells) or intracerebrally (NSCs-ic, 1 million cells) at 2 or 24 h after collagenase-induced ICH
in a rat model. Only NSCs-iv-2 h resulted in fewer initial neurologic deteriorations and reduced brain oedema
formation, inflammatory infiltrations (OX- 42, myeloperoxidase) and apoptosis (activated caspase-3, TUNEL)
compared to the vehicle-injected control animals. Rat neurosphere-iv-2 h, but not human fibroblast-iv-2 h, also
reduced the brain oedema and the initial neurologic deficits. Human NSCs-iv-2 h also attenuated both cerebral
and splenic activations of tumour necrosis factor-alpha (TNF-a), interleukin- 6 (IL- 6), and nuclear factor-kappa B
(NF-kB). However, we observed only a few stem cells in brain sections of the NSCs-iv-2 h group; in the main,
they were detected in marginal zone of spleens. To investigate whether NSCs interact with spleen to reduce
cerebral inflammation, we performed a splenectomy prior to ICH induction, which eliminated the effect of
NSCs-iv-2 h transplantation on brain water content and inflammatory infiltrations. NSCs also inhibited in vitro
macrophage activations after lipopolysaccharide stimulation in a cell-to-cell contact dependent manner. In sum-
mary, early intravenous NSC injection displayed anti-inflammatory functionality that promoted neuroprotec-
tion, mainly by interrupting splenic inflammatory responses after ICH.

Keywords: neural stem cell; spleen; cerebral inflammation; intracerebral haemorrhage; macrophage

Abbreviations: bFGF = basic fibroblast growth factor; ChAT = choline acetyltransferase; DMEM = Dulbecco’s Modified
Eagle’s Medium; EAE = experimental autoimmune encephalitis; EGF = epidermal growth factor; GFAP = glial fibrillary acidic
protein; HPF = high power field; ICH = intracerebral haemorrhage; IL = interleukin; LPS = lipopolysaccharide;
MPO = myeloperoxidase; MLPT = modified limb placing test; NF-kB = nuclear factor-kappaB; NSCs = neural stem cells;
SDF-1a = stromal cell derived factor-1alpha; TGF = tumour growth factor; TNF-a = tumour necrosis factor-alpha;
TUNEL = terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling
Received June 25, 2007. Revised November 15, 2007. Accepted November 26, 2007. Advance Access publication December 21, 2007

ß The Author (2007). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
Neural stem cells and spleen
Introduction
Neural stem cell (NSC) transplantation has been studied as
a promising tool for inducing regeneration of damaged
brain tissue in many neurological disorders (Miller, 2006).
We have shown that the intravenous transplantation of
human NSCs promotes neurological recovery with cell
replacement in intracerebral haemorrhage (ICH) (Jeong
et al., 2003). However, recent evidence indicates that NSC
transplantation may protect the CNS from inflammatory
damage via a ‘bystander’ mechanism rather than by direct
cell replacement (Martino and Pluchino, 2006). NSCs
appear to rescue degenerating neurons by modulating
the host environment by adopting a chaperone-like role
(Ourednik et al., 2002; Hagan et al., 2003). In experimental
autoimmune encephalomyelitis (EAE) models, NSCs exert
immune-like functions and induce the apoptosis of blood-
borne encephalitogenic T cells (Pluchino et al., 2003, 2005),
as well as decrease CNS inflammation via peripheral
suppression of the adaptive immune response (Einstein
et al., 2007). This indicates the feasibility of using NSCs to
reduce cerebral inflammation and secondary brain damage
after stroke. However, no previous study has been per-
formed on the possible anti-inflammatory potential of
NSCs on innate inflammatory response after acute stroke.
Intracerebral haemorrhage (ICH) represents at least
15–20% of all strokes in the Caucasian population
(Qureshi et al., 2001), and occurs with increasing frequency
as a complication of the thrombolytic treatment of
ischaemic stroke (Lapchak, 2002). ICH induces neurological
damage due to local tissue deformation and the subsequent
developments of excitotoxicity, apoptosis, and inflamma-
tion (Aronowski and Hall, 2005). Of these, ICH-induced
inflammation appears to be a key factor of secondary brain
damage, which suggests that anti-inflammatory approaches
may lessen the outcome of haemorrhagic stroke (Jung et al.,
2004; Chu et al., 2004a; Aronowski and Hall, 2005; Wang
and Dore, 2007; Sinn et al., 2007).
Both innate and adaptive inflammatory mechanisms
have been clearly shown to participate in the progression
of cellular injury after stroke (Hallenbeck et al., 2005).
A prominent inflammatory response, which includes the
activation of resident brain microglia and inflammatory
infiltration into the brain, is initiated by neutrophils
followed by macrophages (Del Bigio et al., 1996). This
process is accompanied by the massive, rapid activation of
the peripheral immune system and the dynamic and wide-
spread activation of inflammatory cytokines and chemo-
kines in spleen (Offner et al., 2006). In particular, the
spleen is the key organ that activates tumour necrosis
factor-alpha (TNF-a) and nuclear factor-kappa B (NF-kB)
in systemic inflammatory response (Huston et al., 2006;
Tracey, 2007). TNF-a is one of the major inflammatory
mediators in stroke (Zheng and Yenari, 2004), and is largely
produced by infiltrating macrophages (Gregersen et al.,
2000). Thus, the issue as to whether NSCs can modulate
Brain (2008), 131, 616 ^ 629 617
cerebral and peripheral inflammatory response in acute
stroke has important therapeutic relevance that should be
clarified before developing NSC transplantation strategies.
In this study, we examined the influence of systemic NSC
transplantation on brain and spleen inflammatory reactions
during the acute period of ICH, and we observed that NSCs
have an important ‘bystander’ anti-inflammatory effect on
the spleen-macrophage system.
Material and Methods
Human neural stem cell culture
All experimental procedures were approved by the Care of
Experimental Animals Committee at Seoul National University
Hospital and human cell use was approved by our institutional
review board. Primary dissociated neural stem cells were prepared
from the ventricular zone (VZ) of fetal human brain (15 weeks
gestation) and maintained. Detailed characteristics of this human
NSC line (H1 clone) have been described elsewhere (Ourednik
et al., 2002; Jeong et al., 2003; Chu et al., 2004b,c,d; Imitola et al.,
2004; Chu et al., 2005; Lee et al., 2005; Parker et al., 2005; Lee
et al., 2006a). In brief, suspensions of dissociated NSCs
(5
105 cells per ml), isolated from the telencephalic VZ of a
15-week human fetal cadaver, were cultured in 98% DMEM
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(Dulbecco’s Modified Eagle’s Medium)/F12 (GIBCO, Invitrogen,
Carlsbad, CA, USA), 1% N2 supplement (GIBCO), 1% penicillin/
streptomycin (GIBCO), 8 mg/ml heparin (Sigma), 10 ng/ml
leukemia inhibiting factor (Chemicon-Millipore), 20 ng/ml basic
fibroblast growth factor (bFGF, Calbiochem) in uncoated 25 cm2
flasks (FalconTM, BD Bioscience) at 37 C in 5% CO2. NSCs were
cultured in poly-L-lysine-coated culture dishes or flasks as single
cells or large clusters, which can be subcultured and passaged
weekly over a period of 6 months. This cell line can give rise to
multiple neural cell types throughout the neural axis during
development and has the ability to reconstitute these regions when
perturbed (Snyder et al., 1997; Ourednik et al., 2001; Parker et al.,
2005). In addition, NSC clones generated using the same method
expresses all commonly accepted NSC markers (nestin, musashi,
vimentin, etc.) and can form neurospheres (Kitchens et al., 1994;
Parker et al., 2005). When injected into rats for histologic analysis,
NSCs were pre-labelled with VybrantÕ DiO cell-labelling solution
(Invitrogen) before transplantation, according to the manufac-
turer’s instructions.
Rat neurosphere and human fibroblast culture
We cultured neurospheres using the published method (Singec
et al., 2006), on postnatal days 5–7 from Sprague-Dawley rats
(Orient Bio, South Korea). We used microdissected sections
containing the subventricular zone (SVZ) for the culture, and
used DMEM/F12 medium (GIBCO) supplemented with B27
(Invitrogen), 20 ng/ml bFGF (PeproTech, Rocky Hill, NJ, USA),
and 20 ng/ml epidermal growth factor (EGF, PeproTech) for the
culture medium. For passaging, we collected neurospheres with
37 C warmed 0.05% trypsin, centrifuged them, and mechanically
triturated them, and we plated single cells again after counting
cells. For the transplantation, neurospheres (at three passages)
were dissociated into single cells, and suspensions containing
5
106 cells in 0.5 ml PBS were used. Primary human fibroblasts
were cultured from a normal skin biopsy specimen with the
patient’s consent using the published method (Lee et al., 2006c).
618 Brain (2008), 131, 616 ^ 629
Cell suspensions containing 5
106 fibroblasts in 0.5 ml PBS were
used for transplantation.
Induction of ICH and human NSC
transplantation
One hundred and seventy nine male Sprague-Dawley rats (Orient
Bio), each weighing 210–240 g, were utilized. Experimental ICH
was induced via the stereotaxic intrastriatal administration of
bacterial collagenase type VII (0.23 IU dissolved in 1 ml saline,
Sigma), as described previously (Jeong et al., 2003; Chu et al.,
2004a; Jung et al., 2004; Lee et al., 2006b). Sham ICH was induced
with a stereotaxic needle insertion and an injection of equal
volume (1 ml) of saline instead of collagenase. Two or 24 h after
ICH induction, human NSCs were administered intravenously
(via tail vein, 5 million cells in 0.5 ml PBS/rat) or intracerebrally
[1 million cells in 2 ml PBS/rat, divided at three co-ordinates
(anterior–posterior, medial–lateral, dorsal–ventral): (+1 mm,
+1.4 mm, 3.0 mm), (+0.2 mm, +2.0 mm, 3.0 mm), (0.4 mm,
+2.2 mm, 3.0 mm), respectively], as previously described (Jeong
et al., 2003; Kelly et al., 2004), namely intravenous transplantation
at 2 h (the NSCs-iv-2 h group), intravenous transplantation at
24 h (the NSCs-iv-24 h group), intracerebral transplantation at 2 h
(the NSCs-ic-2 h group), or intracerebral transplantation at 24 h
(the NSCs-ic-24 h group). Vehicle was administered to the ICH-
vehicle group by injecting 0.5 ml of PBS via tail vein at 2 h after
the ICH induction.
Splenectomy
Rats were anesthetized with ketamine and xylazine (Sigma).
Spleens were identified by midline laparotomy and removed after
appropriate blood vessel ligation.
Behavioural testing
Behavioural testing (n = 10 for each group) was conducted weekly
for 5 weeks after ICH using modified limb placing tests (MLPT), as
previously described (Jeong et al., 2003; Chu et al., 2004a; Lee et al.,
2006b). This test consists of two limb-placing tasks, which assess the
sensorimotor integration of the forelimb and the hindlimb, by
monitoring the subject’s responses to tactile and proprioceptive
stimulation. First, the rat is suspended 10 cm above a table, and the
stretch of the forelimbs towards the table is observed and evaluated:
a normal stretch is scored as 0 points; abnormal flexion is scored
as 1 point. Next, the rat is positioned along the edge of the table,
with its forelimbs suspended over the edge, and is then allowed to
move freely. Each forelimb (forelimb-second task, hindlimb-third
task) is gently pulled down, and retrieval and placement is evaluated.
Finally, the rat is placed near the table edge, in order to assess the
lateral placement of the forelimb. The three tasks are scored in the
following manner: normal performance is scored as 0 points; delayed
(at least 2 s) and/or incomplete performance is scored as 1 point; no
performance is scored as 2 points. A total score 7 points indicates
maximal neurological deficit, and a score of 0 points denotes normal
performance.
Spectrophotometric assay of haemorrhage
volume
Haemorrhage volumes were quantified at 3 days post-ICH using a
spectrophotometric assay, as described previously (n = 6 for each
S.-T. Lee et al.
group) (Choudhri et al., 1997; Asahi et al., 2000; Lee et al., 2006b).
In brief, hemispheric brain tissue was acquired from normal rats
who had been subjected to complete transcardial perfusion for the
removal of intravascular blood. Incremental volumes of homol-
ogous blood (0–200 ml) were added to each hemispheric sample,
along with PBS, to a total volume of 3 ml, followed by 30 s of
homogenization, 1 min of sonication on ice, and 30 min of
centrifugation at 12 000 r.p.m. Drabkin’s reagent (1.6 ml; Sigma)
was then added to 0.4 ml aliquots, and allowed to stand for 15 min
at room temperature. Optical density (OD) was then measured
and recorded at 540 nm with a spectrophotometer (Molecular
Devices). These procedures showed a linear relationship between
the haemoglobin concentrations in the perfused rat brain and the
volume of added blood. Measurements from the perfused ICH
brains were then compared with this standard curve, allowing us
to obtain data regarding haemorrhage volume (in ml).
Measurements of brain water content
Water contents were measured at 3 days after ICH (n = 6 for each
group), as previously described (Jeong et al., 2003; Chu et al.,
2004a; Jung et al., 2004; Lee et al., 2006b). In brief, the rats were
anesthetized and sacrificed via decapitation. The rats’ brains were
removed immediately, and divided into two hemispheres along the
midline, after which the cerebellum was removed from each brain.
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The brain samples were then immediately weighed on an
electronic analytical balance to obtain wet weights, then dried in
a gravity oven at 100 C for 24 h, in order to obtain dry weights.
Water contents were expressed as a percentage of wet weight: the
formula used to calculate the water contents was as follows: (wet
weight  dry weight)/(wet weight).
Tissue preparation and immunohistochemistry
At 3 days or 35 days after ICH, rats (n = 4 in each of the groups)
were re-anesthetized and perfused through the heart with 50 ml of
cold saline and 50 ml of 4% paraformaldehyde, in 0.1 mol/l PBS.
Sections (30 mm thickness) of brains were immunostained
with antibodies against myeloperoxidase (MPO; DAKO), Ox-42
(Chemicon), or activated caspase-3 (BD biosciences), and sections
of spleens were stained with antibodies against ED1 (Serotec,
Oxford, UK), CD11b (Chemicon), choline acetyltransferase
(ChAT, Serotec), and human nuclear antigen (HuN, Chemicon),
as previously described (Jeong et al., 2003; Chu et al., 2004a; Jung
et al., 2004; Lee et al., 2006b). The terminal deoxynucleotidyl
transferase biotin-dUTP nick end labelling (TUNEL) was used to
detect in situ DNA fragmentation using a commercial kit (R&D
systems). For fluorescent double staining of TUNEL and cell
markers, we used anti-NeuN (Chemicon), anti-glial fibrillary
acidic protein (GFAP, Chemicon) antibodies, and a fluorescent
TUNEL assay kit (Chemicon). Fluoro-Jade C staining was
performed according to the published protocol to detect neurons
undergoing degeneration (Schmued et al., 2005). To confirm any
cell-to-cell contact or co-labelled cell markers, sections were
analyzed with a laser-scanning confocal microscopic (LSM 510,
Carl Zeiss microimaging, Thornwood, NY, USA) imaging system.
One coronal brain section taken through centers of needle tracts
and two adjacent sections (1 mm width) were analysed by
counting marker-specific cells throughout entire sections (three
sections per antibody staining). Total counts were converted to
cell densities in order to facilitate quantification. We investigated
NSC distributions in brain, spleen, lungs, livers and mesenteric
Neural stem cells and spleen
lymph nodes of the NSCs-iv-2 h groups with DAPI staining at 3
or 35 days after ICH. ED1+-activated macrophages in the spleen
were counted in three marginal zone areas immediately adjacent
to the white pulp in at least three sections per animal using a
magnification of 400
(high power field, HPF) over a microscopic
field. For determining hemispheric atrophy, three sections through
the needle entry site were Nissl stained at 35 days after ICH
(n = 6 per group). The total hemispheric areas of each section were
traced and measured using image analyzer (Image Pro-Plus,
Media Cybernetics, Bethesda, MD, USA) as described previously
(Matsushita et al. 2000; Chu et al. 2004a; Jung et al. 2004). The
hemispheric atrophy was expressed as a percentage of contralateral
hemispheric area.
Western blotting and RT-PCR
Twenty four hours after ICH induction, rats were sacrificed by
decapitation, and brains, spleens and mesenteric lymph nodes
were immediately extracted (n = 4 in each group). Homogenates
of ipsilateral (haemorrhagic) hemispheres and spleens were
serially processed for western blotting, as described previously
(Jung et al., 2004; Lee et al., 2006d), using anti-NF-kB (1 : 200;
BD Biosciences) and anti-b-actin antibody (Santa Cruz Biotech-
nology). Relative optical densities were calculated versus measured
values of b-actin. We also conducted RT-PCR for TNF-a,
interleukin (IL)-1b, IL-4, IL-6, tumour growth factor (TGF)-b1,
Fas and Fas ligand (FasL) at 24 h after ICH induction (n = 3 in
each group), as previously described (Lee et al., 2006d,). In brief,
total RNA was isolated from homogenates (50 mg) of haemor-
rhagic hemispheres, spleens, or mesenteric lymph nodes using TRI
reagent (Sigma). RT-PCRs were conducted using First strand
cDNA Synthesis kits (Roche, Basel, Switzerland) over 25 cycles of
95 C, 58 C and 72 C for 40 s each using the following primer sets:
TNF-a: 50 -TAC TGA ACT TCG GGG TGA TTG GTC C-30 (sense)
and 50 -CAG CCT TGT CCC TTG AAG AGA ACC-30 (antisense);
IL-1b: 50 -ATG GCA ACT GTC CCT GAA CT (sense) and 50 -GTC
ATC ATC CCA CGA GTC AC (antisense); IL-4: 50 -CTG CTT
TCT CAT ATG TAC CGGG (sense) and 50 -TTT CAG TGT TGT
GAG CGT GG (antisense); IL-6: 50 -CTT GGG ACT GAT GTT
GTT GAC-30 (sense) and 50 -CTC TGA ATG ACT CTG GCT TTG-
30 (antisense); TGF-b1: 50 -CTA ATG GTG GAC CGC AAC AAC-
30 (sense) and 50 -CGG TTC ATG TCA TGG ATG GTG-30
(antisense); Fas: 50 -CAA GGG ACT GAT AGC ATC TTT GAG
G-30 (sense) and 50 -TCC AGA TTC AGG GTC ACA GGT TG-30
(antisense); Fas-L: 50 -CAG CCC CTG AAT TAC CCA TGT C-30
(sense) and 50 -CAC TCC AGA GAT CAA AGC AGT TC-30
(antisense). Transcript levels were quantified by analyzing the
scanned photographs of gels using appropriate imaging software
(Molecular AnalystÕ , Bio-Rad). Relative optical densities were
determined by comparing measured values with GAPDH mean
values.
FACS analysis
At 3 days after the ICH induction, single cell suspensions (5
105
cells) were prepared from spleens in phosphate buffered saline
(PBS) supplemented with 2% fetal calf serum (FCS) and 0.05%
sodium azide. To remove contaminating erythrocytes, the cell
suspensions were subjected to osmotic lysis using a hypotonic
ammonium chloride solution. With using FITC anti-rat CD11b
(2 mg/ml, BD Biosciences) and Phycoerythrin (PE) anti-rat TNF-a
(2 mg/ml, eBioscience, San Diego, CA, USA) antibodies, 5
104
Brain (2008), 131, 616 ^ 629 619
cells for each sample were analyzed on a FACS II flow cytometer
(BD Bioscience).
Co-culture of rat macrophages with NSCs
Peritoneal macrophages were isolated from Sprague-Dawley rats,
as previously described (Falcone and Ferenc, 1998). Briefly, rats
were injected i.p. with 3 ml of 4% thioglycollate. Four days later
cells were harvested by lavage with cold PBS. Peritoneal cells were
recovered by centrifugation and resuspended in 24-well plates in
500 ml of RPMI-10% FBS containing penicillin (200 U/ml) and
streptomycin (0.2 mg/ml). Cells were allowed to adhere for 2 h and
then washed free of non-adherent cells. Adherent cells were then
re-seeded (106 cells per well) in 500 ml of RPMI. Two hours before
or after lipopolysaccharide (LPS) stimulation, NSCs (106 cells per
well) were added to these macrophages as mixed culture. For the
double chamber system, equal numbers of NSCs were plated into
transwell membrane inserts (Costar) with a 0.4 mm pore size to
avoid direct cell-to-cell interactions and to allow the diffusion of
soluble factors between two cell populations (Shen et al., 2004).
LPS was added to the cultures at a final concentration of
100 ng/ml. Supernatants were collected 4 or 24 h after adding LPS
and prepared for TNF-a and IL-6 ELISA using ELISA kits (R&D
Systems).
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Statistical analysis
All data in this study are presented as means  SD. Error bars in
the figures mean SD except Fig 1, in which the error bars are SE.
The Mann–Whitney U-test was used for inter-group comparisons
and we did not specify the test in these cases. For a nonparametric
comparison among three and more unpaired groups, we used
Kruskall-Wallis analysis of variances (ANOVA) and specified
the test. When P-values from Kruskall–Wallis was 50.05, Mann–
Whitney U-test was further used for post-hoc inter-group
comparisons. A two-tailed probability value of 50.05 was
considered to be significant.
Results
NSCs-iv-2 h reduced initial neurologic
deterioration
We previously reported favourable neurologic recovery in
the intravenous NSCs transplantation at 24 h after the ICH
induction (NSCs-iv-24 h group) compared to the vehicle-
treated ICH group (ICH-vehicle) (Jeong et al., 2003). Thus,
we first investigated whether an earlier NSCs transplanta-
tion at 2 h after ICH (NSCs-iv-2 h) can induce more
favourable results. Although the ICH-vehicle group scored
over six points on the MLPT at 1 day after ICH
(6.85  0.38), the NSCs-iv-2 h group exhibited less pro-
found initial deficits (4.66  0.98, P50.01, Fig. 1). The
NSCs-iv-2 h group then continued to recover, and the
difference between the two groups was statistically sig-
nificant until at least 5 weeks after ICH (all P50.01).
Although the MLPT score of NSCs-iv-24 h group started to
differ from that of the ICH-vehicle group at 4 weeks, the
NSCs-iv-2 h group showed less neurologic deficits than the
NSCs-iv-24 h group from day 1 to day 35 (all P50.01). We
also investigated the neurologic deficits of the NSCs-ic-2 h
620 Brain (2008), 131, 616 ^ 629 S.-T. Lee et al.

and NSCs-ic-24 h groups for comparison. Although the
MLPT scores of these two groups started to differ from that
of the ICH-vehicle group at 21 or 28 days after the ICH,
the neurologic deficits during the early periods (day 1–14)
were not different to those of the ICH-vehicle group
(Fig. 1). In summary, only the NSCs-iv-2 h group showed
less initial neurologic deficits compared to the other groups.
NSCs-iv-2 h reduced brain water content
Because the lower levels of neurologic deficits observed in
the NSCs-iv-2 h group at 1 day after ICH could not be
explained by cell replacement or regeneration, we hypothe-
sized that some form of initial neuroprotection or anti-
inflammation had been initiated in the NSCs-iv-2 h group.
Thus, we measured brain water contents at 3 days after
ICH, because water content represents brain oedema which
is one of the most important surrogate markers of
perihematomal inflammation and tissue damage and
peaks at 3–4 days after ICH (Xi et al., 2006). ANOVA
suggested an intergroup difference among the ipsilateral
brain water contents of the five groups (p50.05, Kruskall–
Wallis ANOVA, Fig. 2A). Mean brain water content in
ipsilateral (haemorrhagic) hemispheres was 81.63  0.55%
in the ICH-vehicle group, and 80.10  0.27% in the
NSCs-iv-2 h group (P50.01), whereas in contralateral
(non-haemorrhagic) hemispheres these were 79.63  0.36%

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Fig. 1 Modified limb placing test (MLPT). At 24 h after ICH, the
NSCs-iv-2 h group exhibited less profound deficits than the ICH-
vehicle group, and then progressively recovered. The MLPT score
of NSCs-iv-24 h group started to differ from that of the ICH-
vehicle group at 4 weeks after ICH. NSCs-iv-2 h group showed less
neurologic deficits than NSCs-iv-24 h group from day 1 to day 35.
The MLPT scores of the NSCs-ic-2 h and the NSCs-ic-24 h groups
started to differ from that of the ICH-vehicle group at 21 or 28
days respectively, and the neurologic deficits during the early
periods (day 1^14) were not different to those of the ICH-vehicle
group. P50.05, P50.01 versus ICH-vehicle. yP50.01 versus
NSCs-iv-24 h. ôP50.05, ‰P50.01 versus NSCs-ic-2 h.
in the ICH-vehicle group and 79.30  0.25% in the NSCs-
iv-2 h group (P = 0.08), demonstrating that NSCs-iv-2 h
significantly reduced brain oedema in haemorrhagic hemi-
spheres. However, the brain water contents in the other
groups, i.e. the NSCs-iv-24 h group [81.18  0.45% (ipsi-
lateral); 79.58  0.26% (contralateral)], the NSCs-ic-2 h
group [80.95  0.35% (ipsilateral); 79.82  0.31% (contra-
lateral)], and the NSCs-ic-24 h group [81.25  0.45%
(ipsilateral); 79.93  0.36% (contralateral)], were not dif-
ferent from those of the ICH-vehicle group. These results

Fig. 2 Brain water content and haemorrhage volume. Brain water contents were significantly lower in the ipsilateral (haemorrhagic)
hemispheres of the NSCs-iv-2 h group than in all other groups at 3 days after ICH (A). Water contents of the other groups were not
different from those of the ICH-vehicle group. NSC transplantations either intravenously or intracerebrally at 2 or 24 h did not influence
haemorrhagic volume (B). Intravenous injection of rat neurosphere cells at 2 h (ICH-rat neurosphere-iv-2 h group) also reduced the mean
brain water content in the ipsilateral hemisphere, while the human fibroblast injection (ICH-human fibroblast-iv-2 h group) showed no
significant effect on the brain water content compared to the vehicle injection (C). P50.01 versus all other groups.
Neural stem cells and spleen Brain (2008), 131, 616 ^ 629 621

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Fig. 3 Histological analysis of inflammation and apoptosis. As representative photographs show (A), the NSC-iv-2 h group showed lower
numbers of OX 42+ and MPO+ inflammatory cells and lower levels of activated caspase-3+ cells and TUNEL+ apoptotic cells in perihe-
matomal regions at 3 days after ICH than the other four groups, as representative photographs show (B). The NSCs-iv-24 h group showed
reductions of MPO+ and caspase-3+ cells versus the ICH-vehicle group. The # signs indicate hematoma areas. Bar = 100 mm. P50.05
versus all other four groups. yP50.01 versus the ICH-vehicle group.

indicate that intravenous transplantation at 24 h, or
intracerebral. transplantation at 2 h or 24 h failed to
reduce brain oedema. Only early intravenous transplanta-
tion effectively reduced the brain oedema.
According to the results of spectrophotometric assays of
haemorrhage volumes at 3 days after ICH, NSC transplan-
tation by either method (intravenous, intracerebral, 2 h and
24 h) exerted no influence on haemorrhage volumes
(P40.05 in Kruskall–Wallis ANOVA; all P-values 40.05
versus ICH-vehicle, Fig. 2B), suggesting that equal burdens
of haemorrhage were initiated.
To investigate whether allogenic neural progenitor cells
can also reduce the brain oedema formation, we measured
brain water contents in two more experimental groups made
by intravenous injections of rat neurosphere cells (ICH-rat
neurosphere-iv-2 h) or human fibroblasts (ICH-human
fibroblast-iv-2 h) at 2 h after the ICH. The ICH-rat neuros-
phere-iv-2 h group showed the reduced brain water content
in the ipsilateral hemisphere (80.05  0.36%, P50.01 versus
ICH-vehicle). However, the ipsilateral brain water contents
of the ICH-human fibroblast-iv-2 h group (81.01  0.36%)
was not significantly different to that of the ICH-vehicle
group (P = 0.078). In addition, the ICH-rat neurosphere-
iv-2 h group, but not ICH-human fibroblast-iv-2 h group,
showed the reduced initial neurologic deficits (MLPT score)
compared to the ICH-vehicle group (ICH-rat neurosphere-
iv-2 h: 4.00  0.82, P50.01 versus ICH-vehicle; ICH-human
fibroblast-iv-2 h: 6.2  0.84, P = 0.104 versus ICH-vehicle).
Accordingly, NSCs attenuated the brain oedema formation
and initial neurologic deficits regardless of their allogenic or
xenogenic transplantation conditions.
NSCs-iv-2 h reduced brain inflammatory
infiltration and apoptotic cell numbers
Histologic quantifications of inflammatory infiltrations
and apoptotic cells were undertaken in perihematomal
areas to confirm the anti-inflammatory effects of NSCs-
iv-2 h at 3 days after ICH (Fig. 3A). Initially, we observed
intergroup differences among the five treatment groups in
the inflammatory/apoptotic cell counts including OX42,
MPO, Caspase-3 and TUNEL (P50.05, Kruskall–Wallis
ANOVA), and further analyzed the values by intergroup
comparison. It was found that the NSCs-iv-2 h group
possessed significantly lower numbers of OX42+ microglia
and macrophages than the ICH-vehicle group (P50.05),
whereas the other groups showed no difference versus the
ICH-vehicle group (ICH-vehicle: 105.0  20.8 cells/mm2,
NSCs-iv-2 h: 24.0  6.32 cells/mm2, NSCs-iv-24 h:
79.8  13.8 cells/mm2, NSCs-ic-2 h: 103.8  14.4 cells/mm2,
NSCs-ic-24 h: 113.5  12.6 cells/mm2) (Fig. 3B).
MPO+ neutrophil counts in perihematomal areas
revealed the most significant reduction in the NSCs-iv-2 h
group (ICH-vehicle: 212.5  29.9 cells/mm2, NSCs-iv-2 h:
44.3  9.2 cells/mm2, NSCs-iv-24 h: 111.3  19.7 cells/mm2,
NSCs-ic-2 h: 190.3  20.3 cells/mm2, NSCs-ic-24 h:
148.9  66.6 cells/mm2, P50.05 between NSCs-iv-2 h and
622 Brain (2008), 131, 616 ^ 629
ICH-vehicle) (Fig. 3). The NSCs-iv-24 h group also showed
a reduction in MPO+ count versus the ICH-vehicle group
(P50.05), but this was less pronounced than of the NSCs-
iv-2 h group (P50.05 versus NSCs-iv-24 h). However, the
NSCs-ic-2 h and NSCs-ic-24 h groups showed no reduction
in MPO+ neutrophil infiltration.
An analysis of active caspase-3+ apoptotic cell numbers
showed that the NSCs-iv-2 h and NSCs-iv-24 h groups had
lower counts than the ICH-vehicle group (all P50.05), and
the NSCs-iv-2 h group had lowest counts than NSCs-iv-24 h
(P50.05) (ICH-vehicle: 72.0  10.6 cells/mm2, NSCs-iv-2 h:
17.8  3.1 cells/mm2, NSCs-iv-24 h: 38.0  6.7 cells/mm2,
NSCs-ic-2 h: 76.5  9.4 cells/mm2, NSCs-ic-24 h:
63.5  11.0 cells/mm2) (Fig. 3).
TUNEL staining revealed a high density of positively-
stained cells within and at the peripheries of haemorrhagic
lesions (Fig. 3A). By quantitative analysis, the NSCs-iv-2 h
group exhibited a significantly lower number of TUNEL+
cells (82.5  43.7 cells/mm2) than the ICH-vehicle group
(314.5  50.8 cells/mm2; P50.05, Fig. 3B). However,
the NSCs-iv-24 h (254  44.6 cells/mm2), NSCs-ic-2 h
(349.5  63.6 cells/mm2), and NSCs-ic-24 h
(325.5  32.7 cells/mm2) groups were similar to the ICH-
vehicle group. The perihematomal TUNEL+ cells were
mostly positive for the neuronal marker (NeuN)
(Supplementary Fig. 1A). Nevertheless, overall immunor-
eactivity against NeuN was diminished in the perihemato-
mal area because of the loss of neuronal nuclear and/or
cytoskeletal structure (Matsushita et al., 2000), and it
seemed to be inaccurate to quantify TUNEL+NeuN+ cells.
Thus, we used Fluoro-Jade C staining to detect
degenerating neurons in the perihematomal area (Wang
and Tsirka, 2005). The ICH-NSCs-iv-2 h group had a lower
number of Fluoro-Jade C stained neurons than the ICH-
vehicle group at 3 days after ICH (ICH-vehicle:
176  33 cells/mm2, ICH-NSCs-iv-2 h: 49  18 cells/mm2,
P50.05, Supplementary Fig. 1B). To confirm the sustained
tissue protection, we also measured the hemisphere
atrophies of the ICH-vehicle and the NSCs-iv-2 h groups
at 35 days after ICH. The NSCs-iv-2 h group exhibited
lesser degree of hemispheric atrophies than did the ICH-
vehicle group (ICH-vehicle: 24.5  3.5%, NSCs-iv-2 h:
16.0  8.2%, P50.05). Accordingly, the NSCs-iv-2 h group
demonstrated the most prominent and reproducible anti-
inflammatory and neuroprotective effects.
NSCs-iv-2 h showed attenuated levels of
cerebral and splenic inflammatory mediators
We then analysed changes in the levels of inflammatory
mediators induced by NSCs-iv-2 h in brains, spleens and
lymph nodes at 1 day after ICH. We have previously shown
that ICH increases the expressions of TNF-a, IL-6, Fas and
FasL in the brain samples at 1 day after ICH (Sinn et al.,
2007). In the present study, we could confirm the
upregulations of TNF-a, IL-1b, IL-4 and IL-6 in the
S.-T. Lee et al.
haemorrhagic brains and spleens compared to the normal
samples, with using RT-PCR supported by optical density
analysis (all P50.05, n = 4 per group) (Fig. 4A and B). In
lymph nodes, only IL-4 was upregulated (P50.05) and
TNF-a, IL-1b, and IL-6 were unchanged after ICH. The
levels of TGF-b were not affected by the ICH induction in
all the three kinds of tissue.
In brain samples, the NSCs-iv-2 h group showed
decreases of 79% in TNF-a, 62% in IL-4, 47% in IL-6
mRNA levels versus the ICH-vehicle group (all P50.05,
Fig. 4A and B). The NSCs-iv-2 h group also showed
decreases in Fas and FasL mRNA levels compared to the
ICH-vehicle group (by 35% and 66%, respectively, P50.05,
Supplementary fig. 2A). In spleens, the NSCs-iv-2 h group
showed decreases of 85% in TNF-a and 58% in IL-6
mRNA levels versus the ICH-vehicle group (all P50.05,
Fig. 4A and B). In the lymph nodes, the NSCs-iv-2 h
injection attenuated IL-4 levels by 69% (P50.05,
Supplementary Fig. 2B). In addition, western blotting for
NF-kB expressions in the brain and spleen samples showed
that levels were significantly attenuated in the NSCs-iv-2 h
group compared to the ICH-vehicle group (36% decrease in
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the brains and 81% in the spleens, all P50.05 versus ICH-
vehicle, Fig. 4C).
To investigate whether NSCs-iv-2 h changed TNF-a
expressions in the spleen macrophages, we performed
flow cytometric analysis of splenocytes using anti-CD11b
and anti-TNF-a antibodies. In the ICH-vehicle spleen,
24.2% of CD11b+ macrophages expressed TNF-a, while
17.6% of CD11b+ macrophages expressed TNF-a in the
normal spleen (Fig. 4D). In the NSCs-iv-2 h group,
however, only 19.1% of CD11b+ macrophages expressed
TNF-a. In addition, the spleens from the NSCs-iv-2 h
group showed reduced numbers of ED1+ activated macro-
phages in the marginal zone compared to those from the
ICH-vehicle group (ICH-vehicle: 63  9 cells/HPF, NSCs-iv-
2 h: 30  8 cells/HPF, p50.05, Supplementary Fig. 2C). In
summary, these findings demonstrated that NSCs-iv-2 h
reduced the levels of the important inflammatory mediators
in both the brain and the spleen.
Distributions of injected NSCs
We previously observed that a number of NSCs migrated
to perihematomal areas and differentiated into NeuN+,
Neurofilament+, or GFAP+ cells in NSCs-iv-24 h group
when we analyzed brains at 6 weeks after the transplanta-
tion (Jeong et al., 2003). However, at 1 day after the
transplantation, only occasional NSCs were observed in a
cerebral ischaemia model (Jin et al., 2005), although robust
in situ proliferation was observed from 7 days after
transplantation (Chu et al., 2004b). In the present study,
we analyzed the distribution of injected NSCs in the NSCs-
iv-2 h group at 3 days after ICH when their anti-
inflammatory effects were apparent. However, only small
numbers were observed in brain [41 cell per high-power
Neural stem cells and spleen Brain (2008), 131, 616 ^ 629 623

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Fig. 4 Changes in the expressions of cerebral and splenic inflammatory mediators. RT-PCR (A) and the optical density measure
(B) revealed the upregulations of TNF-a, IL-1b, IL- 4 and IL- 6 in the haemorrhagic brains and spleens compared to the normal samples. In
lymph nodes, only IL- 4 was upregulated. The NSCs-iv-2 h group showed decreases of TNF-a, IL- 4 and IL- 6 mRNA levels in brain samples,
as well as decreases of TNF-a and IL- 6 mRNA levels in spleen samples (B). In the lymph nodes, the NSCs-iv-2 h injection attenuated the
IL- 4 upregulation. Western blotting for NF-kB expressions in the brain and spleen samples showed that the levels were significantly atte-
nuated in the NSCs-iv-2 h group compared to the ICH-vehicle group (C). In the flow cytometric analysis of splenocytes, 24.2% of CD11b+
macrophages expressed TNF-a in the ICH-vehicle group, while 17.6% of CD11b+ macrophages expressed TNF-a in the normal spleen
(D, from each single representative sample). In the NSCs-iv-2 h group, however, only 19.1% of CD11b+ macrophages expressed TNF-a.

P50.05 versus normal. yP50.05 versus ICH-vehicle.

field (HPF)], lung (2–3 cells per HPF), and liver (about 1
cell per HPF) (Fig. 5). This distribution is in accord with
previous observations in an EAE model, where more NSCs
were found to be distributed to the systemic organs rather
than to the CNS just after transplantation (Pluchino et al.,
2005). However, we observe few NSCs in mesenteric lymph
nodes. In an EAE model, many transplanted NSCs were
found in lymph nodes during the early stage, although they
disappeared over several days (Einstein et al., 2007).
However, in the present study, large numbers of NSCs
were detected in spleen, especially in the marginal zone area
(20–30 cells per HPF). Because this splenic zone contains
many macrophages (Mebius and Kraal, 2005), we immu-
nostained the spleen macrophages with anti-CD11b Ab. The
results obtained confirmed that NSCs were located in the
vicinity of marginal zone macrophages (Fig. 5F). In addition,
we found that a number of NSCs were in cell-to-cell contact
with CD11b+ spleen macrophages under the confocal
microscopic views (Fig. 5G).
At 35 days after the transplantation, those NSCs-iv-2 h
cells were still detected in lungs, livers and spleens
(Supplementary Fig. 3A–E). However, we could observe
few NSCs in the brains. The DiO-labelled cells were positive
for human nuclear antigen, confirming that DiO -labelled
cells were transplanted human cells (Supplementary
Fig. 3F). About 80% of the NSCs observed in the spleen
were positive for the cholinergic neuronal marker (ChAT)
(Supplementary Fig. 3G and H). When we analyzed the
distribution of NSCs in the NSCs-ic-2 h and NSCs-ic-24 h
groups at 3 days after ICH, we observed abundant NSCs in
the perihematomal area as well as transplantation foci of
the brain, but not in the systemic organs (Supplementary
Fig. 3I).
Splenectomy eliminated the effect of
NSCs-iv-2 h transplantation
Because of the splenic bias shown by NSCs and the
observed attenuations of inflammatory mediators in spleen,
we hypothesized that this organ may facilitate inflammatory
response modulation in our stroke model. To prove this,
we performed splenectomy 3 days prior to the ICH
induction, and then measured brain water contents at 3
days after ICH (n = 6 per each group). Mean brain water
content was found to be 81.63  0.55% in the ICH-vehicle
624 Brain (2008), 131, 616 ^ 629 S.-T. Lee et al.

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Fig. 5 Distributions of NSCs in vivo. NSCs were pre-labelled with DiO fluorescent dye before transplantation. In the NSCs-iv-2 h group,
only a few NSCs were detected in brain (A), lung (B) and liver (C) at 3 days after the transplantation (arrows). No NSCs were observed in
mesenteric lymph nodes (D). However, a number of NSCs are detected in spleen, especially in the marginal zone (MZ) (E). Immunostaining
for CD11b demonstrated that NSCs in the spleen are in the vicinity of the marginal zone macrophages (F). We could observe that a number
of NSCs were in cell-to-cell contact with CD11b+ spleen macrophages under the confocal microscopy (G, arrow heads, 1 mm step-size
optical sections along the z-axis). WP = white pulp. Dotted line indicates the WP border. Bar = 100 mm in A^F, and 10 mm in G.

group, and 80.10  0.27% in the NSCs-iv-2 h group
(P50.01, Fig. 6). Interestingly, the mean brain water
content of splenectomized ICH rats (splenectomy-
ICH-vehicle) was 80.8  0.26% and was much lower than
that of the ICH-vehicle group (P50.01), which suggests
that the spleen is involved in cerebral oedema formation
after ICH. Moreover, NSCs-iv-2 h transplantation in
splenectomized ICH rats (splenectomy-ICH-NSCs-iv-2 h)
resulted in a mean brain water content of 80.82  0.25%,
which was similar to that of the splenectomized ICH rats
(splenectomy-ICH-vehicle) (P = 0.986). The mean brain
water content of non-ICH normal control rats was
78.86  0.30, and that of splenectomized non-ICH rats
was 78.91  0.13 (P = 0.714; no difference).
In histologic quantifications of perihematomal inflam-
matory cells, the splenectomy-ICH-vehicle group showed
less OX-42+ or MPO+ cells compared to the ICH-vehicle
group (all P50.05, Fig. 6B). However, NSCs-iv-2 h trans-
plantation in the splenectomy-ICH rats (splenectomy-ICH-
NSCs-iv-2 h) failed to further reduce the OX-42+ or MPO+
inflammatory cell numbers (all P40.05). In addition,
there was also no difference in the initial neurologic scores
(MLPT) at 1 day between the two groups (splenectomy-
ICH-vehicle: 3.62  0.52, splenectomy-ICH-NSCs-iv-2 h:
3.33  0.52, P = 0.269), while the splenectomy-ICH-vehicle
group showed less deficits compared to the ICH-vehicle
group (P50.01). In summary, NSCs-iv-2 h transplantation
failed to reduce brain oedema, perihematomal inflammatory
cells and initial neurologic deficits in splenectomized rats,
which indicated that the spleen plays a central role in the
attenuation of ICH-associated brain injuries by intravenously
administered NSCs.
NSCs inhibited in vitro macrophage
activations via contact-dependency
TNF-a levels are one of the most important predictors of
cerebral inflammation and oedema formation after ICH
(Castillo et al., 2002). Because transplanted NSCs were
found to localize in macrophage-rich splenic areas, we
further focused on the interaction between NSCs and
macrophages. Thioglycollate-elicited macrophages stimu-
lated with lipopolysaccharide (LPS) (namely macrophage +
LPS) robustly secreted TNF-a in vitro (referred to as
Neural stem cells and spleen Brain (2008), 131, 616 ^ 629 625

Fig. 6 Splenectomy eliminated the effects of early systemic NSCs transplantation. Splenectomy before ICH (Third bar, splenectomy-
ICH-vehicle) reduced ipsilateral hemisphere water contents as compared with the ICH-vehicle group (First bar) (A). NSCs-iv-2 h
transplantation in splenectomized ICH rats (Fourth bar, splenectomy-ICH-NSCs-iv-2 h) had similar levels of brain water content as
splenectomy-ICH-vehicle rats (Third bar), which suggested that splenectomy eliminated the effects of NSCs. In histologic counts of
perihematomal inflammatory infiltrations (B), the splenectomy-ICH-vehicle group showed less OX- 42+ or MPO+ cells compared to the
ICH-vehicle group, and NSCs-iv-2 h transplantation in the splenectomy-ICH rats (splenectomy-ICH-NSCs-iv-2 h) failed to further reduce
the inflammatory cell numbers compared to the splenectomy-ICH-vehicle group. P50.05, P50.01. ns = not significant.

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Fig. 7 NSCs inhibited in vitro macrophage activation via cell-to-cell contact. Macrophages stimulated with LPS robustly secreted TNF-a
(second bar), and macrophage/NSC mixed-cultures showed lower TNF-a levels in culture medium (third bar) at 4 h after the LPS stimula-
tion (A). However, this effect was eliminated when NSCs were cultured in transwell membrane inserts which prevented cell-to-cell
contact and permitted only the diffusion of soluble factors (fourth bar). NSCs alone showed no response to LPS stimulation (fifth bar).
When we added NSCs to the cultured macrophages at 2 h after the LPS stimulation and measured cytokine levels at 24 h after the LPS
stimulation, NSCs still attenuated the TNF-a secretion of macrophages (B). IL- 6 secretion were not inhibited by NSCs. P50.05, P50.01.
ns = not significant.

100  1.5%, at 4 h after the LPS stimulation, Fig. 7).
However, when macrophages are co-cultured with NSCs
before LPS stimulation, TNF-a secretion induced by LPS
stimulation significantly reduced to 70.9  6.9% (P50.01
versus macrophage + LPS, n = 6 wells per group). However,
when NSCs were cultured in transwell membrane inserts
that prevented their contacting macrophages and allowed
the diffusion of soluble factors, mean TNF-a secretion was
97.9  1.8%, i.e., no different to the macrophage + LPS
situation (P = 0.149). These results suggest that NSCs can
inhibit in vitro macrophage activations via a contact-
dependent mechanism.
As an adjuvant experiment to simulate the in vivo
condition of NSCs-iv-2 h transplantation after ICH,
we added NSCs to the cultured macrophages at 2 h after
the LPS stimulation and measured cytokine levels at 24 h
after the LPS stimulation. In this experiment, NSCs again
attenuated the TNF-a secretion of macrophages by 56%
(P50.05, n = 3 wells per group, Fig. 7B). However,
the levels of IL-6 secretions were not changed by NSCs
co-culture (P = 0.369).
Discussion
In this study, we undertook to characterize the anti-
inflammatory effects of NSCs, and the mechanisms
involved. Intravenous NSC transplantation during the
hyperacute stage (at 2 h after ICH) was found to reduce
626 Brain (2008), 131, 616 ^ 629
initial neurologic deterioration, and to exhibit anti-
inflammatory and anti-apoptotic properties with attenuat-
ing brain oedema and the expressions of inflammatory
mediators in brain and spleen. Unexpectedly, we also
observed that the spleen participates in cerebral inflamma-
tion as splenectomy reduced cerebral oedema and inflam-
matory cell counts, and that NSCs modulate the splenic
inflammatory pathway to reduce the cerebral inflammation.
In addition, NSCs inhibited in vitro macrophage activation
via a contact-derived mechanism.
Our results suggest that earlier intravenous NSC admin-
istration can attenuate systemic inflammatory response after
haemorrhagic stroke and protect the brain via a bystander
mechanism rather than via any direct cell replacement. To
date, NSCs have been transplanted to regenerate damaged
brain tissue, and previously, we showed that human NSCs
administered intravenously at 24 h after ICH promoted
neurological recovery and replaced lost neuronal cells
(Jeong et al., 2003). Moreover, suppressions of T-lymphocyte
activity by mesenchymal stem cells or neural precursors have
been demonstrated in EAE models (Pluchino et al., 2003,
2005; Zappia et al., 2005; Einstein et al., 2007; Gerdoni et al.,
2007), and it has been suggested that the immunomodulatory
properties are a characteristics of stem cells rather than
neural cells (Miller and Bai, 2007). However, the present
study is the first to report that intravenous NSCs adminis-
tered during the hyperacute stage in stroke can modulate
innate cerebral inflammatory responses with interacting with
peripheral inflammatory systems.
As demonstrated by this study, the spleen is important
both as an inducer of cerebral inflammation and as a target
for NSC-based intervention. The link between brain and
spleen inflammation can be referred to as ‘brain-spleen
inflammatory coupling’. Humoral pathways facilitate brain/
systemic immunity communication. Cytokines like TNF-a,
IL-1b and IL-6 secreted by cells in different tissues and organs
link their inflammatory responses (Chamorro et al., 2007).
In addition, neural and hormonal mechanisms like the
cholinergic anti-inflammatory pathway, the adrenergic path-
way and the hypothalamic pituitary axis (HPA) link the brain
and peripheral immune organs (Prass et al., 2003; Meisel
et al., 2005; Tracey, 2007). Thus, changes in the peripheral
immune system can occur within hours of brain injury, and
may later cause stroke-induced immune depression syn-
drome (SIDS) or increase the risk of infection (Dirnagl et al.,
2007). In the present study, reductions in splenic inflamma-
tion by NSCs without significant migration to the brain were
coupled with a reduction in brain inflammation and a
favourable neurologic outcome. In addition, splenectomy
prior to ICH reduced the cerebral oedema formation and the
number of perihematomal inflammatory cells per se.
Accordingly, modulations of spleen-mediated inflammation
may provide new solutions for cerebral anti-inflammatory
treatment and brain protection.
The splenic marginal zone is an important transit area
for cells that are leaving the bloodstream and entering the
S.-T. Lee et al.
white pulp. Moreover, based on spleen structure, most
blood flows through the marginal zone and directly along
the white pulp, which leads to efficient monitoring of the
blood by the peripheral immune system (Mebius and Kraal,
2005). Macrophages represent a major component of the
marginal zone and express pattern-recognition receptors
(e.g. toll-like receptors) that are utilized for pathogen and
antigen clearance (Mebius and Kraal, 2005). Accordingly,
early splenic inflammatory activation after stroke (Offner
et al., 2006) and the localization of systemically injected
NSCs in the splenic marginal zone well correspond.
TNF-a activation can be detected a few hours after
stroke; even before significant neuronal death has occurred
(Allan and Rothwell, 2001), and the spleen secretes robust
amounts of TNF-a into circulating blood at this early stage
(Offner et al., 2006). TNF-a promotes early-stage inflam-
mation by increasing the expressions of chemotactic factors
and adhesion molecules by vascular endothelium, which
leads to the early infiltration of monocytes/macrophages,
neutrophils, and T cells into sites of injury (Barone and
Feuerstein, 1999; Stevens et al., 2002). Early modulation of
inflammatory cell response by administering an antibody
blocking TNF-a at 2 h after stroke reduced brain damage
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and improved neurologic outcomes in an experimental
model (Hosomi et al., 2005). Accordingly, interventions
targeting splenic TNF-a activation require a prompt
approach. Our result that NSC transplantation at 24 h
after ICH had no attenuating effect on brain oedema/
inflammation confirm this idea, because TNF-a has
probably been activated systemically and centrally by this
time, as shown by the present study and others (Offner
et al., 2006; Sinn et al., 2007). Thus, the observed
differences between NSC-iv-2 h and NSC-iv-24 h probably
depend on the pathogenic time course.
The homing of NSCs to damaged brain is dependent on
stromal cell derived factor-1a (SDF-1a)-CXC chemokine
receptor 4 signalling, or on adhesion molecule-receptor
signalling (Imitola et al., 2004; Mueller et al., 2006).
Although the expressions of SDF-1a and other adhesion
molecules in brain might be significant at later times, brain
homing signals might not be significant at 2 h after ICH
when NSCs-iv-2 h cells were injected in the present study.
In addition, blood-brain barrier might be less penetrable for
NSCs at this time window. This might answer why NSCs-
iv-2 h cells failed to migrate to the brain. However,
migration of NSCs to spleen might not be dependent on
inducible factors. The H1 cell line used in this study highly
expresses CD44+ (Chu et al., 2004b), which is also
expressed on inflammatory cells and mediates their
infiltration via CD44/hyaluronan interaction (Pure and
Cuff, 2001). In an EAE model, intravenously administered
neurosphere cells were predominantly detected in lymph
nodes at 1 day after transplantation, and were not detected
in brain, spinal cord, or even in spleen (Einstein et al.,
2007). These two different models, i.e. the haemorrhagic
stroke and the EAE models, with predominantly innate and
Neural stem cells and spleen
adaptive immune responses, respectively, may affect the
biodistribution patterns and immunologic effects of neural
stem cells. Although the NSCs-iv-2 h treatment also reduced
IL-4 levels in ICH brains and lymph nodes in the present
study, IL-4 is an anti-inflammatory molecule, mainly
secreted by a negative feedback mechanism responsive to
the other inflammatory cytokines (Tedgui and Mallat,
2001), and is not associated with the clinical outcome of
stroke (Vila et al., 2003). Accordingly, further studies are
warranted to reveal how NSCs migrate to immune organs
and how they interact with inflammatory cells, although the
present study demonstrates initial evidences that the
mechanism of macrophage inactivation by NSCs involves
a contact-dependent interaction.
The systemic transplantation of NSCs is probably the
least invasive method of cell administration. Intracerebral
direct neural transplantation requires invasive surgery and
leaves needle tracks and mass effects. In human, the local
stereotaxic infusion of NSCs may be severely limited
because infused cells must migrate substantial distances
and the time taken to embrace entire disease sites may be
unacceptable. Thus, to overcome these obstacles, NSCs
must be administered at numerous sites. In contrast, an
intravenous injection can disperse NSCs according to
natural cues and allow them to migrate along the host’s
‘chemotactic gradients’ (Pluchino et al., 2003; Chu et al.,
2004b; Martino and Pluchino, 2006). A similar systemic
approach has been studied using hematopoietic stem cells
in stroke models (Taguchi et al., 2004; Nomura et al., 2005;
Honma et al., 2006). Moreover, the transplantation of
NSCs into circulating blood may be easier and more
effective in multifocal and large-lesioned CNS disorders
(Jeong et al., 2003; Pluchino et al., 2003; Chu et al., 2004d;
Fujiwara et al., 2004; Pluchino et al., 2005). In addition,
based on the present study, the migration of NSCs to the
spleen can be therapeutically beneficial and be another
major contribution in stroke models. Nevertheless, the
immune responses in the brain and other systemic organs
are complex, and cannot be ignored when it comes to
repair strategies involving cellular transplants (Barker and
Widner, 2004). Accordingly, further studies are warranted
in this issue.
Collectively, our results suggest that systemic injections
of human NSCs during hyper-acute stage ICH have
potential therapeutic relevance, because they were found
to display strong anti-inflammatory functions that promote
secondary neuroprotection by interrupting splenic inflam-
matory response. The present study demonstrates that
the spleen is centrally involved in innate immune response
after ICH, and thus, represents an important target for the
therapeutic development of stem cell therapies in acute
stroke.
Supplementary material
Supplementary material is available at Brain online.
Brain (2008), 131, 616 ^ 629 627
Acknowledgements
This research was supported by a grant (SC3060) from
Stem Cell Research Center of the 21st Century Frontier
Research Program funded by the Ministry of Science and
Technology, Republic of Korea.
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