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OPG/RANKL/RANK axis is a critical inflammatorysignaling system
in ischemic brain in miceMunehisa Shimamuraa, Hironori Nakagamia,
Mariana K. Osakoa,1, Hitomi Kurinamia, Hiroshi Koriyamaa,Pang
Zhengdab, Hideki Tomiokac, Akiko Tenmaa, Kouji Wakayamad, and
Ryuichi Morishitac,2
aDivision of Vascular Medicine and Epigenetics, Osaka University
United Graduate School of Child Development, Osaka 565-0871, Japan;
bDepartment ofGeriatric Medicine and Nephrology, Osaka University
Graduate School of Medicine, Japan; cDepartment of Clinical Gene
Therapy, Osaka University GraduateSchool of Medicine, Osaka
565-0871, Japan; and dDepartment of Advanced Clinical Science and
Therapeutics, Graduate School of Medicine, The University ofTokyo,
Tokyo 113-8655, Japan
Edited by Michael Karin, University of California, San Diego
School of Medicine, La Jolla, CA, and approved April 24, 2014
(received for reviewJanuary 13, 2014)
Osteoprotegerin (OPG) is a soluble secreted protein and a
decoyreceptor, which inhibits a receptor activator of nuclear
factor κB(NF-κB) ligand (RANKL)/the receptor activator of NF-κB
(RANK)signaling. Recent clinical studies have shown that a
high-serum-OPG level is associated with unfavorable outcome in
ischemicstroke, but it is unclear whether OPG is a culprit or an
innocentbystander. Here we demonstrate that enhanced RANKL/RANK
sig-naling inOPG−/− mice or recombinant RANKL-treated mice
contrib-uted to the reduction of infarct volume and brain edema
viareduced postischemic inflammation. On the contrary, infarct
vol-ume was increased by reduced RANKL/RANK signaling in OPG−/−
mice and WT mice treated with anti-RANKL neutralizing
antibody.OPG, RANKL, and RANK mRNA were increased in the acute
stageand were expressed in activated microglia and
macrophages.Although enhanced RANKL/RANK signaling had no effects
inglutamate, CoCl2, or H2O2-stimulated neuronal culture,
enhancedRANKL/RANK signaling showed neuroprotective effects with
re-duced expression in inflammatory cytokines in
LPS-stimulatedneuron-glia mixed culture, suggesting that RANKL/RANK
signalingcan attenuate inflammation through a Toll-like receptor
signalingpathway in microglia. Our findings propose that increased
OPGcould be a causal factor of reducing RANKL/RANK signaling and
in-creasing postischemic inflammation. Thus, the OPG/RANKL/RANK
axisplays critical roles in controlling inflammation in ischemic
brains.
cerebral ischemia | neuroprotection | immune cells
An elevated serum osteoprotegerin (OPG) level has beenreported
to be associated with the severity (1, 2), subtype (2),poor
functional outcome (1), and long-term mortality of ischemicstroke
(2, 3). However, it is still unclear why a high-serum-OPGlevel
could result in a poor prognosis in ischemic stroke.OPG is a
soluble secreted protein that lacks transmembrane
and cytoplasmic domains. It binds to a receptor activator of
nu-clear factor-кB ligand (RANKL) (4, 5), whose receptor is the
re-ceptor activator of NF-κB (RANK), and inhibits
RANKL/RANKsignaling. The OPG/RANKL/RANK system in bone (6)
andvasculature (7) is well known to work on bone metabolism (8)
andvascular calcification (7). In addition, immune cells (6, 9–12)
ex-press these molecules, and this system is believed to be
associatedwith the regulation of inflammatory and immune responses
(13,14). RANKL is expressed in CD4+T cells (6) andmacrophages
(9,10), whereas OPG is expressed in mature B cells (6) and
macro-phages (11, 12). RANK is expressed in macrophage and
dendriticcells (6). One of the functions of RANKL/RANK signaling in
theimmune system is to control the thymocyte-mediated
medullaformation and the formation of self-tolerance in T cells
(14), aswell as the number of regulatory T cells (Treg) (15).
Additionally,RANKLdirectly contributes to the regulation of
proinflammatorycytokine production in macrophages (13, 16).Despite
these characteristics of the OPG/RANKL/RANK
system, its action on inflammation in central nervous
systemdiseases has yet to be studied. Ischemic stroke is a typical
acute
inflammatory disease, and postischemic inflammation affects
theoutcome, i.e., the infarct volume (17). These inflammatory
cyto-kines are produced mainly from microglia in the early phase
andfrom mixed microglia/macrophage (M/M) in the delayed phase,i.e.,
12–24 h after ischemia, whereas neutrophilic granulocytes arenot
responsible for the production of those inflammatory cyto-kines
(17). Because OPG/RANKL/RANK is expressed in mac-rophages (6, 9–12)
and affects inflammatory responses (13), wehypothesized that one of
the mechanisms of poor outcome inhigh-level OPGmight reflect
themodulation of such postischemicinflammations by the
OPG/RANKL/RANK signaling system.Interestingly, OPG, RANKL, and RANK
have also been re-
ported to be expressed in normal brain in rodents, although
thedistribution of their expression is controversial. Early
reportsshowed that RANKL mRNA was expressed in neurons in
thecerebral cortex (18) and that RANK (19) and OPG (5) mRNAwas
expressed in normal brain. However, a recent report re-vealed that
the RANK protein was specifically expressed inneurons and
astrocytes in the preoptic area and the medial septalnucleus,
whereas RANKL mRNA was expressed in the lateralseptal nucleus (20).
In normal brain, RANKL/RANK signalingwas reported to be associated
with fever and body temperature
Significance
Although a high-serum osteoprotegerin (OPG) level is associ-ated
with an unfavorable outcome in ischemic stroke, it is
un-clearwhetherOPG is a culprit or an innocent bystander.
Hereweshow that the deletion of OPG and enhanced
RANKL/RANKsignaling contribute to the reduction of infarct volume
withlower brain edema, whereas infarct volume is increased by
re-duced RANKL/RANK signaling in OPG−/− mice and WT micetreated
with anti-RANKL neutralizing antibody. OPG, RANKL,and RANK mRNA
were increased in ischemic brain and wereexpressed in activated
microglia and macrophages. EnhancedRANKL/RANK signaling showed
neuroprotective effects withreduced expression in inflammatory
cytokines in LPS-stimulatedneuron-gliamixed culture.Our findings
propose anti-inflammatoryroles for RANKL/RANK signaling in ischemic
brains.
Author contributions: M.S. and R.M. designed research; M.S.,
M.K.O., and H. Kurinamiperformed research; H.N., H. Koriyama, P.Z.,
H.T., A.T., and K.W. analyzed data; and M.S.,H.N., and R.M. wrote
the paper.
Conflict of interest statement: The Department of Clinical Gene
Therapy is financiallysupported by AnGes MG, Novartis, Shionogi,
Boeringher, and Rohto. The Division ofVascular Medicine and
Epigenetics is financially supported by Bayer. R.M. is a founderand
stockholder of AnGes MG and a former board member.
This article is a PNAS Direct Submission.1Present address:
Department of Cell and Molecular Biology, Ribeirão Preto
MedicalSchool, University of São Paulo, Ribeirão Preto, 04044-010
São Paulo, Brazil.
2To whom correspondence should be addressed. E-mail:
[email protected].
This article contains supporting information online at
www.pnas.org/lookup/suppl/doi:10.1073/pnas.1400544111/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1400544111 PNAS | June 3, 2014
| vol. 111 | no. 22 | 8191–8196
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control (20). Because body temperature is associated with
theoutcome in ischemic stroke, we also hypothesized that
themodulation of body temperature by the OPG/RANKL/RANKsystem might
be another mechanism behind a poor outcome inischemic stroke. To
clarify these hypotheses, we examined theaction of the
OPG/RANKL/RANK signaling system in a transientmiddle cerebral
artery occlusion (MCAo) model using OPG−/−
mice and treated RANK fragment crystalizable (Fc) chimera
toinhibit the function of RANKL or recombinant RANKL.
ResultsEffect of OPG/RANKL/RANK Signaling on Infarct Volume.
Becausea high-serum-OPG level is associated with stroke severity
inclinical trials (1–3), we examined the effects of OPG in
ischemicbrain using OPG−/− mice (Fig. 1A). The OPG−/− mice showeda
significant reduction in infarct volume compared with WT mice(Fig.
1A), which was associated with a significant decrease incerebral
edema (Fig. 1B). Although the differences in thestructure of
cerebral vessels and microcirculations could affectstroke outcome,
no differences were observed in the pial arterialanastomoses in
middle cerebral arteries and anterior cerebralarteries and the
microcirculations in cerebral cortex betweennormal OPG−/− mice and
WT mice (Fig. S1). In addition, ce-rebral blood flow (CBF) was also
similar between OPG−/− miceand WT mice during surgery (Fig. S2A).
Although body tem-perature could be a critical factor for stroke
outcome, there wasno significant difference in the circadian rhythm
of rectal tem-perature between OPG−/− and WT mice (Fig. S2B) or
thechange in rectal temperature after MCAo (Fig. S2C). These
dataindicate that OPG might work as an exacerbation factor for
is-chemic insult, independent of body temperature and CBF.Because
OPG is a decoy receptor for RANKL and because
RANKL/RANK signaling was augmented in OPG−/− mice (21),we
hypothesized that an enhancement of RANKL/RANK sig-naling might be
responsible for the reduction of infarct volume
in OPG−/− mice. To elucidate the role of this system, we
ex-amined the inhibition of RANKL/RANK signaling using an
i.c.v.injection of a neutralizing anti-RANKL antibody, RANK
Fc/chimera, in OPG−/− mice. As expected, the OPG−/− mice
treatedwith RANK Fc/chimera exhibited a significant increase in
infarctvolume (Fig. 1C). To examine whether RANKL/RANK signal-ing
would be associated with the outcome even in WT mice,RANK
Fc/chimera was also injected i.c.v. in a mild ischemic model.The
infarct volume was also significantly exacerbated (Fig.
1D),although the extent was less than that observed in OPG−/−
mice (Fig. S3A). These results suggest that RANKL/RANKsignaling
might act as a protective signal in the ischemic brainin both
OPG−/− and WT mice. However, RANKL/RANK sig-naling might not be
fully stimulated in WT mice due to thepossibility that OPG partly
blocks the RANKL/RANK signalingin WT mice.To further examine the
action of RANKL, WT mice in a se-
vere ischemic model were treated with i.c.v. of RANKL startingat
4 h after MCAo (Fig. 1 E and F). The mice treated withRANKL showed
a tendency to have a low mortality rate (Fig.S3B), a significantly
lower infarct volume (Fig. 1E), and lessformation of cerebral edema
(Fig. 1F). Similar to OPG−/− mice,no differences in CBF were
observed during the surgery (Fig.S4A). Because a single i.c.v.
injection of RANKL was reported toaffect the core body temperature
(20), we examined the changeof core body temperature using data
loggers (Fig. S4 B–D). Thecore body temperature was not affected by
the i.c.v. injection ofRANKL in normal mice (Fig. S4B) and mice
exposed to MCAo(Fig. S4 C and D). These results indicated that the
stimulation ofRANKL/RANK signaling resulted in a reduction of
ischemicinjury without changes in body temperature or CBF.
Temporal Profile of RANK, RANKL, and OPG mRNA Expression.
Next,we examined the temporal expression of RANK, RANKL, andOPG
mRNA in the ischemic brain in WT mice (Fig. 2). RANKmRNA was
increased starting at 4 h after MCAo and peaked at12 h after MCAo.
RANKL and OPG mRNA showed a biphasicprofile. The peak of RANKL
expression occurred at 7 and 48 hafter MCAo, and OPG expression
peaked at 12–24 and 72 hafter MCAo. In OPG−/− mice, RANK and RANKL
mRNA werealso increased after MCAo (Fig. 2 A and B) although
the
Fig. 1. Infarct volume and brain edema in OPG−/− mice and mice
treatedwith anti-RANKL neutralizing antibody or RANKL. The infarct
volume at 72 hafter 45 min middle cerebral artery occlusion (MCAo)
(A–C), 35 min MCAo(D), and 70 min MCAo (E and F). (A and B) OPG−/−
mice showed a reducedinfarct volume and brain edema compared with
WT mice. **P < 0.01 vs. WTmice. n = 8 in WT mice and n = 7 in
OPG−/− mice. (C) Intracerebroventricularinjection of anti-RANKL
neutralizing antibody, which inhibited RANKL/RANKsignaling,
exacerbated the infarct volume in OPG−/− mice. *P < 0.05 vs.
BSA-injected mice. n = 6 in each group. (D) Treatment with
anti-RANKL neu-tralizing antibody in WT mice also showed an
increased infarct volume.**P < 0.01; *P < 0.05 vs. BSA
injected mice. n = 10 in BSA-treated mice; n = 11in RANKL-treated
mice. (E) WT mice treated with RANKL showed lower in-farct volume
when the mice survived to 72 h after MCAo (Fig. S3B) werecompared.
**P < 0.01; *P < 0.05 vs. BSA injected mice. (F) There was a
lowerformation of brain edema in RANKL-treated mice. n = 8 in
RANKL-treatedmice and n = 5 in BSA-treated mice.
Fig. 2. Temporal expression profile of RANK, RANKL, or OPG mRNA
in theischemic brain. The expression of RANK (A), RANKL (B), or OPG
(C) mRNAwas analyzed by real-time RT-PCR. RANK mRNA was increased
starting at 4 hafter MCAo and peaked at 12 h after MCAo. RANKL and
OPG mRNA showeda biphasic profile. The peak of RANKL expression was
at 7 and 48 h afterMCAo, and OPG expression peaked at 12–24 and 72
h after MCAo in WTmice. The expression of RANK and RANKL in OPG−/−
mice was also shown.(n = 3 in each time point.)
8192 | www.pnas.org/cgi/doi/10.1073/pnas.1400544111 Shimamura et
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expression level of RANK was lower than that in WT mice
(Fig.2A). Thus, OPG, RANKL, and RANK were up-regulated in theacute
phase of cerebral ischemia. Immunohistochemistry showedthat RANK,
RANKL, and OPG were expressed in F4/80-posi-tive or Iba1-expressing
M/M in the border between the infarctand intact areas, whereas the
nonischemic hemisphere showedno expression of RANK, RANKL, and OPG
(Fig. 3). OPG−/−
mice also showed expression of RANK and RANKL in M/M inthe
ischemic hemisphere (Fig. S5). These data indicate that OPG,RANKL,
and RANK work primarily in M/M in the acute stage ofischemic
brain.
Expression of Inflammatory Cytokines in the Ischemic Brain.
Becauseinflammatory cytokines produced from microglia have
modifyingeffects on the infarct volume in the early phase of
cerebral is-chemia (17), we hypothesized that RANKL/RANK signaling
inmicroglia modulated the inflammatory cytokine expression.
Asexpected, OPG−/− mice showed a significantly lower expressionof
interleukin-6 (IL-6) and inducible nitric oxide synthase (iNOS)and
a lower expression of tumor necrosis factor α (TNFα),
in-terleukin-1β (IL-1β), and monocyte chemotactic protein-1
(MCP-1), whereas the expression of arginase 1 (Arg1) was higher
(Fig.4A). Moreover, the number of F4/80 positive cells in
ischemicbrain was significantly lower in the OPG−/− mice (Fig. 5).
WhenRANK Fc/chimera was intracerebroventricularly injected inOPG−/−
mice, a higher expression of mRNA was observed inRANK
Fc/chimera-treated mice in IL-6, TNFα, IL-1β, MCP-1,and iNOS, but
not in Arg1 (Fig. S6). RANKL-treated mice in WTmice showed a
significant reduction of IL-1β and MCP-1 anda lower expression in
both IL-6 and TNFα (Fig. 4B). However,RANKL treatment did not
affect the expression of iNOS andArg1. These results indicate that
RANKL/RANK signaling hadanti-inflammatory properties in
postischemic injury.
RANKL Inhibited Toll-like receptor 4-Mediated Neuronal Death
inNeuron-Glia Mixed Cultures. In postischemic inflammation,
en-dogenous danger signals, such as high-mobility group box
1(HMGB-1) and peroxiredoxin family proteins, were reported tobe
responsible for exacerbating the ischemic insult (22).
BecauseToll-like receptor 4 (TLR-4) is one of the receptors for
thesemolecules (23), we examined the effect of RANKL on
neuronalcell death induced by a TLR-4 ligand and LPS using
neuron-gliamixed culture. In the neuron-glia culture, RANK
expression wasobserved in Iba1-positive microglia (Fig. S7). When
LPS wasadded to the mixed culture, the number of MAP-2 neurons
thatsurvived was decreased, whereas RANKL pretreatment for 24
hprevented neuronal death (Fig. 6 A and B). Because LPS in-directly
causes neuronal death by increasing the expression ofinflammatory
cytokines produced from activated microglia (24),we examined the
expression of IL-6 and TNFα in cultured me-dium (Fig. 6C). Although
RANKL itself did not affect the ex-pression of those cytokines,
RANKL pretreatment prevented theproduction of IL-6 and TNFα
stimulated by LPS (Fig. 6C). Toexclude the possibility of
neuroprotection of RANKL throughthe direct action on neurons, the
effect of RANKL was examinedin primary neuronal cultures (Fig. S8).
We examined the directneuroprotective effects of RANKL in a hypoxic
model inducedby CoCl2. However, no neuroprotective effects of RANKL
wereobserved (Fig. S8A). We further examined the effects of RANKLin
glutamate toxicity and H2O2-induced oxidative stress using
anincreased dose of RANKL (Fig. S8 B and C). Similarly, no
directneuroprotective effects of RANKL were observed. These
datasuggest that the neuroprotective effects of RANKL are not
throughits direct actions on neurons but instead through the
inhibition ofinflammatory cytokines from microglia.
Fig. 3. Expression of RANK, RANKL, or OPG in the ischemic brain
at 48 hafter MCAo in WT mice. Immunohistochemistry of RANK (A–E),
RANKL (F–J),or OPG (K–O) expression at 48 h after MCAo. Although
RANK, RANKL, andOPG were not expressed in intact regions in
ischemic cortex (A, F, and K) aswell as nonischemic cortex (E, J,
and O), these molecules were expressed inF4/80-positive (A–D) or
Iba-1–positive (F–I and K–N) activated M/M in theborder zone
between the intact and infarct region. Higher magnification ofthe
images in the rectangle area (A, B, F, G, K, and L) were shown in
C, D, H, I,M, and N. (Scale bar: 100 μm in A, B, E, F, G, J, K, L,
and O; 20 μm in C, D, H, I,M, and N.)
Fig. 4. Cytokine expressions in MCAo mice. Cytokine expression
was ex-amined using real-time RT-PCR at 48 h after MCAo in OPG−/−
mice (A) and at24 h after MCAo in RANKL-treated WT mice (B). *P
< 0.05 vs. WT micesubjected to MCAo (A), *P < 0.05 vs.
BSA-treated mice (B). n = 4 in eachgroup. BI, brain infarction.
Shimamura et al. PNAS | June 3, 2014 | vol. 111 | no. 22 |
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DiscussionIn the present study, we demonstrate that the
stimulation ofRANKL/RANK signaling through the deletion of OPG or
ex-ogenous RANKL addition prevented the further exacerbation
ofinfarct volume and cerebral edema by inhibiting the productionof
inflammatory cytokines. This stimulation was independent ofbody
temperature and CBF, whereas the blockade of RANKL/RANK signaling
resulted in the exacerbation of infarct volume.OPG, RANKL, and RANK
were up-regulated in M/M in theischemic border. Because microglia
are responsible for the pro-duction of inflammatory cytokines in
the early phase of ischemicbrain (17) and because RANK expression
starts to be up-regu-lated at 4 h after MCAo, RANKL could act on
microglia first.Thereafter, RANKL could work on M/M in the later
phase be-cause both macrophages and microglia produce
inflammatorycytokines 12–24 h after MCAo (17). Because LPS-induced
neu-ronal death in mixed neuronal glia was prevented by
RANKLthrough the inhibition of inflammatory cytokine production,
onepossiblemechanismmight involve an inhibitory effect of
RANKL/RANK signaling on TLR-4 signaling in activated microglia,
whichhas been reported to be responsible for the receptor of
intrinsicdanger signals in the ischemic brain (25). Similarly, in
the laterphase, inflammatory cytokines from macrophages might be
in-hibited by RANKL because a previous report demonstrated
theanti-inflammatory action of RANKL in LPS-stimulated
bone-marrow–derived macrophages and peritoneal macrophages
(13).Therefore, the association of high OPG with poor
functionaloutcome in clinics (1–3) could result from the attenuated
anti-inflammatory function of RANKL/RANK signaling due to thehigher
level of OPG, although the effects of the OPG/RANKL/RANK axis on
bone metabolism (8) and vascular calcification (7)should be
carefully considered.The molecular mechanisms that determine how
RANKL affects
TLR signaling inmicroglia have yet to be clarified, but one
possiblemechanism in the OPG−/− model could be associated with
drivingthe alternative activation of M/M (M2) because iNOS mRNA
wasdecreased and Arg1 mRNA was increased in the ischemic brainin
OPG−/− mice. Unexpectedly, the blockade of RANKL/RANKsignaling with
RANK Fc chimera did not decrease the expressionof Arg1 in OPG−/−
mice, but we speculate that this might be dueto the functional
compensation in the knockout mouse instead ofRANKL/RANK signaling.
Further studies are necessary to clarifythe mechanisms that
determine how OPG deletion influenceson phenotype of M/M. On the
contrary, the i.c.v. injection of
RANKL in WT mice did not affect the expression of iNOS andArg1
mRNA. This finding suggests that the temporal stimulationof
RANKLmight have anti-inflammatory effects without changesin M/M
polarization. Because the previous study showed thatmyeloid
differentiation primary response 88 (Myd88) expression wasdecreased
in bone-marrow–derived macrophages by recombinantRANKL treatment
(13), RANKL might work downstream ofTLR signaling in both microglia
and macrophages. Furtherstudies are necessary to clarify the
detailed mechanisms.In the present study, the expression level of
RANK mRNA was
lower in OPG−/− mice, probably due to the chronic stimulationof
RANKL in OPG−/− mice. Previous reports showed an in-consistent
expression level of RANK and RANKL in OPG−/−
mice compared with WT mice: increased mRNA levels of RANKand
RANKL in the aorta (21); the same expression level of RANKLmRNA
expression in osteoblasts (26, 27); and a decreased ex-pression
level of RANK mRNA in osteoclasts in 12-wk-old micefollowed by an
increased level at 20 and 28 wk (28). From thisviewpoint, the
expression level of RANK and RANKL in OPG−/−
might be dependent in cells or tissue.Compared with a previous
report showing the site-specific
expression of RANK and RANKL in the normal brain, i.e.,
thepreoptic area and medial septal nucleus or the lateral
septalnucleus (20), the present study showed that these
moleculesstarted to be diffusely expressed in the cerebral cortex
in theischemic border zone. These data indicate that the RANKL/RANK
axis might not work in neurons and astrocytes in thecortex but that
this axis might work in the activated microgliawhen the brain is
exposed to ischemia. This speculation is sup-ported by the data
indicating that RANKL exhibited neuro-protective effects in the
mixed glia-neuron culture but not incultured neurons from the
cerebral cortex. In addition, becauseOPG is expressed in activated
M/M with the expression ofRANK, the OPG/RANKL/RANK axis might work
through auto-crine and/or paracrine action in M/M. Thus, the
RANKL/RANK
WT OPG-/- WT OPG-/-0
500
1000
1500
2000
**
Ischemic Nonischemic
Tota
l num
ber
of F
4/80
posi
tive
cells
Intact
Infarct
A
C
Intact
Infarct
BWT OPG-/- WT OPG-/-
OPG-/- OPG-/-
Fig. 5. Activated macrophage/microglia in the peri-infarct
region in WT andOPG−/− mice. Immunohistochemistry for F4/80 was
shown in ischemic cortex(A) and nonischemic brain (B) in WT (Left)
or OPG−/− mice (Right). Thenumber of F4/80-positive cells was
counted at 0.7 mm from the bregma (C).n = 8 in WT mice and n = 7 in
OPG−/− mice. **P < 0.01 vs. WT mice. Ischemic,ischemic
hemisphere; Nonischemic, nonischemic hemisphere.
Fig. 6. Prevention of LPS-triggered neuronal death by RANKL
treatment.Typical immunohistochemical images for MAP2 in mixed
neuron-glia cul-tures at 5 d after LPS stimulation (A). The cells
were pretreated with 100 ng/mLRANKL for 24 h and exposed to 10
μg/mL LPS for 5 d. The LPS-treated cultureshowed a lower number of
MAP2-positive cells compared with RANKL-treatedculture (B, n = 4 in
each group). The expression of TNFα and IL-6 in medium at24 h after
exposure to LPS was less in the group treated with 100 ng/mL
RANKL(C). n = 3 in the cells without LPS; n = 10 in LPS-stimulated
cells. #P < 0.05 vs.cells without LPS and RANKL; *P < 0.05
vs. LPS-stimulated cells without RANKL.
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axis could be associated with other central nervous system
dis-eases such as multiple sclerosis and Alzheimer’s disease,
whereactivated microglia work as a deteriorative factor.Although
the role of the RANKL/RANK axis in the patho-
logical brain has not been reported, except in the present
study,its role in the control of body temperature and fever has
beenreported in normal rodent brain (20). In this previous
study,RANKL-injected mice (1, 10, or 100 ng single i.c.v.
injection)showed the induction of fever at 6 h after injection,
with theactivation of specific brain regions involved in
thermoregula-tion and induction via the COX2-PGE2/EP3R pathway
(20). Incontrast, the present study demonstrated that
RANKL-injectedWT mice and OPG−/− mice did not show any differences
incore body temperature in either normal mice or mice subjectedto
MCAo. Although the cause of discrepancy between the pre-vious
report (20) and the present study is still unclear, the
betteroutcome in OPG−/− mice and RANKL-treated WT mice couldnot be
explained by the differences in body temperature.Although we
focused on the OPG/RANKL/RANK axis in
M/M in the acute stage of ischemic brain, it is possible that
theOPG/RANKL/RANK axis might influence both the control
ofT-cell–mediated immune responses because RANKL regulatesthe
formation of self-tolerance in T cells (14) and any
immuno-suppressive effects by controlling the number of Treg in
allergiccontact hypersensitivity responses and the development of
sys-temic autoimmunity (15). Because T lymphocytes are
significantlyincreased at day 3 (29) and because Foxp3+ Treg cells
work ascerebroprotective immunomodulators in the more delayed
phase,i.e., 3–7 d after MCAo (30), the OPG/RANKL/RANK axis
couldalso be associated with the regulation of T-cell–mediated
immuneresponses in the delayed phase. Further studies are necessary
toclarify the actions of the OPG/RANKL/RANK axis on the
T-cell–mediated immune response.In conclusion, in the present study
we demonstrate that the
OPG/RANKL/RANK system might be one of the key signalsregulating
M/M-derived inflammatory responses in ischemicbrain. Because the
i.c.v. injection of RANKL a minimum of 4 hafter MCAo decreased the
infarct volume, RANKL/RANKsignaling might be a therapeutic target
to treat ischemic stroke.Although the development of a delivery
system and a modifi-cation of RANKL are needed to avoid
RANKL-induced osteo-porosis, further studies on the OPG/RANKL/RANK
system inthe brain might shed light on the molecular mechanism
involvedin postischemic inflammation and lead to the development
ofnew therapeutic options for ischemic stroke.
Materials and MethodsSurgical Procedure. All procedures were
approved by the Institutional AnimalCare and Use Committee of Osaka
University. OPG−/− mice on C57Bl6/J back-ground and C57Bl6/J mice
(wild-type) were obtained from CLEA Japan, Inc.
Transient Middle Cerebral Artery Occlusion. Mice were
anesthetized with iso-flurane (1.4%). Cerebral blood flow was
measured using a laser Dopplerflowmeter (Unique Acquisition
software; UniqueMedical). A 6.0monofilamentsurgical suture was
advanced into the internal carotid artery to obstruct theorigin of
themiddle cerebral artery. The filament was left in place for 35,
45, or70 min and then withdrawn. Because the effect of deleting OPG
was not clearin the first experiment, we chose 45 min of ischemia,
which caused moderateischemia and was suitable for observing both
deteriorating and improvingeffects. To examine the deteriorative
effect of neutralizing anti-RANKL anti-body both in WT mice and
OPG−/− mice, 45- and 35-min ischemic models wereapplied in OPG−/−
and WT mice because the average infarct volume betweenOPG−/− and WT
mice was almost the same in that ischemic duration. To assessthe
therapeutic effect of RANKL, 70 min of ischemia to cause a severe
infarctwas also applied. Only animals that exhibited a typical
reduction pattern andmore than 82% reduction in CBF during MCAo, in
which CBF recovered by 30–80% after 5 min of reperfusion, were
included in the study. In all mice, rectaltemperature was kept at
37.0 ± 0.5 °C during surgery and in the recoveryperiod until
animals regained consciousness. The overall mortality was 27% at72
h after ischemia.
Measurement of Infarction Volume. Ischemic damage was evaluated
usingsections stained with cresyl violet at 72 h after MCAo.
Coronal sections (12-μmthickness) were made at −1.4, −0.7, 0, 0.7,
and 1.4 mm from the bregma,mounted on the stereomicroscope, and
photographed. The hemisphericlesion area (HLA) in the coronal
sections was calculated. The corrected HLAwas calculated as HLA (%)
= [LT-(RT-RI)]/LT × 100, where LT is the area of theleft
hemisphere, RT is the area of the right hemisphere, and RI is the
infarctedarea (23). Brain edema was calculated as brain edema (%) =
[RT-LT]/LT × 100.
Measurement of Body Temperature. In OPG−/− mice, body
temperature wasmeasured by rectal probe thermometer (Unique
Medical) without anesthe-sia. Core body temperature was
continuously recorded using temperaturedata loggers (KN
Laboratories) implanted into the peritoneal cavity. Thedata loggers
were programmed to record body temperature every 3 minwith a
resolution of 0.1 °C. Following the implant, animals were allowed 4
dto recover.
Fluorescein Angiography. The structure of cerebral vessels,
capillary density,and blood–brain barrier leakage was assessed
using an angiographic tech-nique with FITC-conjugated albumin (31).
The depicted brain was fixed in4% (wt/vol) paraformaldehyde (PFA),
and the image was acquired usinga fluorescence stereoscope (SZX 12,
Olympus). The brains were frozen, cut into20-μm-long sections, and
examined using a Nikon A1 confocal scanning lasermicroscope. The
images were analyzed using NIS Elements software (Nikon).
Administration of Recombinant RANKL and RANK/Fc Chimera.
Recombinantmouse RANKL [5 ng in 2 μL artificial cerebrospinal fluid
(aCSF)] was purchasedfrom Peprotech EC. Recombinant mouse
RANK/TNFRSF11a Fc Chimera(neutralizing anti-RANKL antibody, 4 μg in
2 μL aCSF) was obtained fromR&D Systems. Intracerebroventricle
injection was performed using a pulledglass micropipette
(anteroposterior 1.0 mm, lateral 1.2 mm from bregma,depth 1.7 mm).
Recombinant RANKL was injected at 4, 24, and 48 h afterMCAo.
Recombinant mouse RANK/TNFRSF11a Fc Chimera was
intra-cerebroventricularly injected 2 h before MCAo. For the
controls, BSA (NacalaiTesque) was used at the same concentration
with each reagent. FITC- conju-gated albumin (10 μg/2 μl,
Sigma-Aldrich) was intracerebroventricularly injec-ted at 4 or 24 h
after MCAo to examine whether the injected proteins couldpenetrate
the cortex in ischemic brain. The concentration of
FITC-conjugatedalbumin was half that in the previous report
examining the fluorescein angi-ography (31). At 2 h after
injection, the fluorescence was observed around themicroglia in the
ischemic cortex (Fig. S9).
Real-Time RT-PCR. The ischemic hemisphere was collected at 48 h
after MCAoin OPG−/− mice. With the RANKL-treatment being 70-min
ischemia in WTmice, the sample was collected at 24 h because the
survival rate was low inmice at 48 h (Fig. S3B). The mRNAs were
isolated using QIAGEN RNeasy LipidTissue Mini Kit (Qiagen),
according to the manufacturer’s recommendations.The cDNA reaction
was performed using a High-Capacity cDNA Archive kit(Applied
Biosystems) according to the manufacturer’s instructions. The
oli-gonucleotide primers used exclusively in the in vitro
experiments werepurchased according to the identification RANK:
Mm00437135; RANKL:Mm01313944; OPG: Mm00435451; MCP1: Mm00441243;
IL-6: Mm00446190;Arg1: Mm00475988; iNOS: Mm00440502; IL-17a:
Mm004329618; IL-10:Mm01288386; IL-1β: Mm99999061; and GAPDH:
Mm99999915 (AppliedBiosystems). The 5′ nuclease assay PCRs were
performed in a MicroAmpOptical 384-well reaction plate using ABI
PRISM 7900 Sequence DetectionSystem. The levels of the target genes
were quantified by comparing thefluorescence generated by each
sample with that of the serially dilutedstandard, and the target
gene expressions were normalized by the level ofGAPDH expression in
each individual sample.
Cell Culture. Primary neuron-glial cultures were prepared from
postnatal days1–2 in C57BL/6J mice using the method of Araki with
some modifications(32). The entire cerebral cortex was dissected
and minced in Neurobasal-A(Invitrogen) with B-27 supplement
(Invitrogen, Neurobasal-A/B-27) afterremoving the meninx. Cells
were treated with papain (25 U/mL) and DNaseI(25 U/mL) for 30 min
at 30 °C. The cells were dissociated mechanically
inNeurobasal-A/B-27 with 0.25 mM Glutamax (Invitrogen) and 10%
horse serumby using a siliconized Pasteur pipette. Cells were
plated on polyethyleneimine-coated 24-well plates at 7 × 105/well.
Cultures were maintained at 37 °C in ahumidified atmosphere
containing 5% CO2. Half of the medium was replacedwith
Neurobasal-A/B-27 and 5% horse serum twice per week. After 9 d
ofplating, the cells were composed of 42 ± 5% neurons, 56 ± 5%
astrocytes, and2 ± 1% microglia. In the experiment on neural death,
cells were treated withRANKL (100 ng/mL) for 24 h, and the medium
was replaced with Neurobasal-A
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containing N2 supplement (Invitrogen, Neurobasa-A/N2), 1% horse
serum, LPS(10 μg/mL), and RANKL (100 ng/mL). Cells were
immunostained with MAP-2 todetermine the survival of the neurons at
5 d after the addition of LPS. Theimages were then digitized by
microscope (FSX-100, Olympus). The acquiredimages were converted to
grayscale with GIMP 2.8.6, and the stained area wascalculated using
ImageJ (National Institutes of Health). In the experiment
ex-amining the expression of TNFα or IL-6, the mediumwas collected
at 24 h afterLPS stimulation. The concentration of these cytokines
was checked usingcommercially available ELISA kits [TNFα:
Quantikine Mouse TNF-α ELISA Kit(R&D systems); IL-6: Quantikine
Mouse IL-6 ELISA Kit (R&D Systems)].
For the primary neuronal culture, mouse embryonic cerebral
cortexneurons were obtained from pregnant C57BL/6J mice on the 18th
day ofgestation and cultured (33). The cerebral cortex was
dissected, and individualcells were isolated by treatment with
papain and triturated in Leibovitz’sL-15 medium (Invitrogen). Cells
were plated on polyethyleneimine-coated24-well plastic culture
dishes with Neurobasal (Invitrogen)/B-27 (Invitrogen)with 0.25 nM
Glutamax (Invitrogen) at 37 °C in a humidified atmosphere of95%
air-5% CO2. The medium was changed on the fourth day. On day 9,
themedium was changed to Neurobasal/N2 (Invitrogen) containing BSA
orRANKL (10 or 100 ng/mL). Thereafter, CoCl2 (100 μM, 48 h),
glutamate (100μM, 10 min), or H2O2 (100 μM, 24 h) was added to the
medium. To check cellviability, cells were assessed using a
3-(4,5-dimethylthiazol-2-yl)-5-(3-car-boxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
(MTS) assay follow-ing the manufacturer’s protocol (CellTiter 96
AQueous One Solution CellProliferation Assay) at the following time
points after the stimulation:CoCl2—48 h; H2O2—24 h; and
glutamate—24 h.
Immunohistochemical Staining.Micewere perfusedwith 4%PFA, and
the brainwas cut into 12-μm-thick sections. For double
immunostaining, these sectionswere fixed and then blocked. The
sections were incubated with anti-F4/80(1:50, AbD Serotec),
anti-Iba1 (1:1,000, Wako), or anti-CD11b (1:100, AbDSerotec). Then,
the sections were incubated with anti-rat fluorescent antibody
(1:500 for F4/80 and CD11b, Alexa Flour 546, Invitrogen) or
anti-rabbit fluores-cent antibody (1:500 for Iba-1, Alexa Flour
546, Invitrogen). The sections wereblocked again and were incubated
in streptavidin/biotin blocking kit (VectorLab, Vector
Laboratories). Then they were incubated with anti-RANK
antibody(1:13, R&D systems) or RANKL (1:50, eBioscience). As a
negative control, thesame concentration of normal control IgG
(Santa Cruz) was applied. Then theywere incubated in biotinylated
anti-goat IgG antibody (1:200 for RANK; VectorLab) or anti-rat IgG
antibody (1:200 for RANKL; Vector Lab) followed by fluo-rescent
streptavidin conjugates (1:1,000, Alexa 488; Invitrogen). In the
immu-nohistochemistry for OPG, the sections were blocked with 20%
horse serum andthen incubated with streptavidin/biotin blocking kit
(Vector Lab), followed byincubation with biotin affinity-purified
polyclonal antibody for OPG (1:10, R&DSystems). Then the
sections were incubated with fluorescent streptavidinconjugates
(1:1,000, Alexa 488; Invitrogen).
Immunohistochemical staining was examined using Nikon A1
confocalscanning laser microscope, and the images were analyzed
using NIS Elementssoftware (Nikon). All parameters were set in a
similar manner when the signalintensity was compared.
Statistical Analysis. All values are expressed as the mean ±
SEM. Multiplecomparisons were evaluated by ANOVA followed by
Dunnett’s MultipleComparison Test. Two groups were compared using
an unpaired t test.Survival rates were evaluated using a log-rank
test. The core body temper-atures with data loggers were analyzed
by a two-way repeated-measuresANOVA with Bonferroni posttests.
Differences were considered significantat P < 0.05. The
statistical analysis were performed with the software Prism5.0
(GraphPad Software).
ACKNOWLEDGMENTS. This work was supported by the grants from
TheIchiro Kanehara Foundation (M.S.), the SENSHIN Medical Research
Founda-tion (M.S.), and Japan Society for the Promotion of Science
Grants-in-Aid forScientific Research (KAKENHI) Grant 25462214 (to
M.S.).
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