Journal of Cerebral Blood Flow & Metabolism (2013) 33, 19761982

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ORIGINAL ARTICLE
Inhibition of microRNA-181 reduces forebrain ischemia-induced
neuronal loss
Jeong-mi Moon1,2, Lijun Xu1 and Rona G Giffard1

MicroRNA (miRNA), miR-181a, is enriched in the brain, and inhibition of miR-181a reduced astrocyte death in vitro and infarct
volume after stroke in vivo. This study investigated the role of miR-181a in neuronal injury in vitro and hippocampal neuronal loss
in vivo after forebrain ischemia. miR-181a levels were altered by transfection with mimic or antagomir. N2a cells subjected to serum
deprivation and oxidative stress showed less cell death when miR-181a was reduced and increased death when miR-181a
increased; protection was associated with increased Bcl-2 protein. In contrast, transfected primary neurons did not show altered
levels of cell death when miR-181a levels changed. Naive male rats and rats stereotactically infused with miR-181a antagomir or
control were subjected to forebrain ischemia and cornus ammonis (CA)1 neuronal survival and protein levels were assessed.
Forebrain ischemia increased miR-181a expression and decreased Bcl-2 protein in the hippocampal CA1 region. miR-181a
antagomir reduced miR-181a levels, reduced CA1 neuronal loss, increased Bcl-2 protein, and signicantly prevented the decrease of
glutamate transporter 1. Thus, miR-181a antagomir reduced evidence of astrocyte dysfunction and increased CA1 neuronal survival.
miR-181a inhibition is thus a potential target in the setting of forebrain or global cerebral ischemia as well as focal ischemia.

Journal of Cerebral Blood Flow & Metabolism (2013) 33, 19761982; doi:10.1038/jcbfm.2013.157; published online 4 September 2013
Keywords: apoptosis; astrocytes; brain ischemia; global ischemia; glutamate; hippocampus

INTRODUCTION miR-181a, a miRNA that is highly conserved across most

Several clinical situations such as cardiac arrest and resuscitation
or severe hypotension can lead to transient global cerebral
ischemia. Global ischemia causes selective, delayed death
of pyramidal neurons in the hippocampal cornus ammonis
(CA)1 while sparing the neighboring dentate gyrus in rodents1
and humans, resulting in persistent severe memory decits.2,3
However, despite increasing knowledge of the pathologic
mechanisms of global ischemia, the only known clinically
effective treatment is hypothermia.4,5 Neuronal death in CA1
is not histologically detectable until 3 to 4 days after global/
forebrain ischemia, and this signicant delay between injury
and the onset of neuronal death holds promise for the
development of future therapies, including ones targeting
apoptosis.68
MicroRNAs (miRNAs) are small regulatory noncoding RNAs
B22 bp in length that modulate protein synthesis by post-
transcriptional silencing of gene expression via the recognition of
specic sequences in target messenger RNAs (mRNAs). Many
miRNAs are highly expressed in the adult nervous system in a
regulated manner, and pathologic conditions including focal and
global ischemia lead to altered regulation of brain miRNAs.911
These reports suggested that miRNAs could have a critical role in
molecular signaling associated with ischemic pathologic events.
Indeed, several laboratories have demonstrated that manipulation
of miRNAs can attenuate infarct volume in a focal ischemia
model.1214 However, in contrast to focal ischemia, it still remains
unknown whether modulation of miRNAs can protect against
global ischemia.
vertebrates, is enriched in the brain15,16 and increases during
maturation of hippocampal neurons.17 The overexpression of miR-
181a induces drug addiction-related synaptic changes in the
hippocampus17 and increases astrocyte death because of glucose
deprivation.18 miR-181a is upregulated in the infarct core and
downregulated in the penumbra after focal ischemia14,17 but it has
not yet been studied in forebrain ischemia. In the present study,
we test the effect of reducing miR-181a on forebrain ischemia,
and, because of the importance of apoptosis in this setting,
characterize the effects on levels of Bcl-2, a target of miR-181a. We
also examined the effect of miR-181a in N2a cells and primary
neurons subjected to stress.
MATERIALS AND METHODS
Cell Culture and Transfection
Mouse neuroblastoma (N2a) cells (a kind gift from Drs Kurt Lucin and Tony
Wyss-Coray at Stanford University) were grown in high-glucose Dulbeccos
modied Eagles medium (Invitrogen, Carlsbad, CA, USA) supplemented
with 8% fetal bovine serum (Hyclone, Logan, UT, USA) and 1% antibiotics
(50 U/mL penicillin and 50 mg/mL streptomycin; Invitrogen) in a humidied
atmosphere containing 5% CO2 at 37 1C. N2a cells were transfected with
control miRNA, miR-181a mimic, or miR-181a inhibitor (Thermo Scientic
Dharmacon, Chicago, IL, USA) using Lipofectamine 2000 (Invitrogen)
according to the manufacturers protocol.
Primary mouse neuronal cultures were prepared from 16-day-old
embryos as previously described19 in accordance with a protocol
approved by the Stanford University Animal Care and Use Committee
and after the NIH Guide for the Care and Use of Laboratory Animals. Isolated

1
Department of Anesthesia, Stanford University School of Medicine, Stanford, California, USA and 2Department of Emergency Medicine, Chonnam National University School of
Medicine, Gwangju, South Korea. Correspondence: Dr RG Giffard, Stanford University School of Medicine, 300 Pasteur Drive, Grant Building S272A, Stanford, CA 94305-5117, USA.
E-mail: rona.giffard@stanford.edu
This work was supported in part by NIH Grants NS053898 and GM49831 to RGG.
Received 18 March 2013; revised 31 July 2013; accepted 2 August 2013; published online 4 September 2013

cortical neurons were seeded on poly-D-lysine (Sigma, St Louis, MO, USA)
and laminin (Sigma)-coated 24- or 6-well plates in minimal essential
medium supplemented with 5% horse serum (Hyclone), 5% fetal bovine
serum (Hyclone), and 2 mM of L-glutamine (Sigma). On day 2, 60% of the
medium was replaced by glial-conditioned medium supplemented with
3% B-27 serum-free supplement (Invitrogen) and 3 mM cytosine
arabinoside. On day 4, primary neurons were transfected using Effectene
(Qiagen, Valencia, CA, USA) according to the manufacturers protocol and
on day 5, they were exposed to injury.
Injury Paradigms
Both N2a cells and primary neurons were subjected to injury 24 hours after
transfection.
N2a cells were exposed to 900 mM of H2O2 in Dulbeccos modied Eagles
medium supplemented with 1% antibiotics for 24 hours. Primary neurons
were deprived of serum for 48 hours by changing medium to a balanced
salt solution containing 5.5 mM glucose at pH 7.4, 116 mM NaCl, 1.8 mM
CaCl2, 0.8 mM MgSO4, 5.4 mM KCl, 1 mM NaH2PO4, 14.7 mM NaHCO3, 10 mM
N-(2-hydroxyethyl) piperazine-N-ethanesulfonic acid, and phenol red 10 mg.
Quantitation of Cell Death
The viability of N2a cells was quantied by measuring the concentration of
intracellular lactate dehydrogenase (with LDH kit from Sigma) released
from dead cells into the media.20 The results were expressed as the
percentage of lactate dehydrogenase release compared with lactate
dehydrogenase release after freeze thaw to disrupt all cells.
The viability of primary neurons was assessed by cell counting after
Hoechst 33342 (5 mmol/L; Sigma) and propidium iodide (5 mmol/L; Sigma)
staining. Hoechst stains all cells blue; propidium iodide stains only dead
cells red. Eight nonoverlapping uorescence images per well were
acquired at  20 magnication with a Zeiss Axiovert 200M and Axiovision
software (Carl Zeiss Microscopy, Thornwood, NY, USA). The percentage of
dead cells was calculated using NIH Image J (US National Institutes of
Health, Bethesda, MD, USA).
Imaging-Activated Caspases
N2a cells were stained with Magic Red (Immunochemistry, Bloomington,
MN, USA) to identify cells with activated caspases 3 and 7 and
counterstained with Hoechst 33342 (5 mmol/L; Sigma) after 4 hours of
exposure to injury. The uorescence images were taken at  20
magnication. Caspase activity was calculated by dividing the uorescence
intensity of Magic Red by total cell number in each eld using Adobe
Photoshop CS3, and then normalized to the uorescence per cell for
normal N2a cells without injury.
Animal Groups
Male SpragueDawley rats (Charles River, Wilmington, MA, USA) weighing
280 to 320 g were divided into the following groups: sham (n 8),
forebrain ischemia without treatment (n 6), ischemia with miR-181a
antagomir (n 29), ischemia with mismatch miR-181a antagomir (n 29),
ischemia with vehicle injection (n 3), injection of miR-181a antagomir,
and mismatch antagomir without ischemia (n 11, 11).
Transfection of Rat Hippocampus In Vivo
Rats were anesthetized with isourane and placed in a stereotaxic frame
with a rat head holder. Three picomoles per gram of miR-181a antagomir
or mismatch miR-81a antagomir (Thermo Scientic Dharmacon), mixed
with the cationic lipid DOTAP 1:3 (Roche Applied Science, San Francisco,
CA, USA), were infused stereotactically just outside the left hippocampus
(from bregma  3.8 mm, M-L 2.0 mm, deep 2.5 mm) at 1 mL/minute,
maximal total volume 16 mL via a burr hole.21 After injection the bone
wound was closed with bone wax.
Transient Forebrain Ischemia
Forebrain ischemia was induced 24 hours after transfection22 according to
an animal protocol approved by the Stanford University Animal Care and
Use Committee. Briey, hypotension (mean blood pressure o40 mm Hg)
was induced by removing blood into heparinized sterile tubing with
continuous femoral arterial blood pressure monitoring (PuritanBennett
Corporation, Wilmington, MA, USA). Both carotid arteries were clamped for
10 minutes, then unclamped and the shed blood reinfused. Rectal
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MiR-181 reduction protects from forebrain ischemia
J-m Moon et al
1977
temperature was 370.5 1C controlled by a homeothermic blanket
(Harvard Apparatus, Holliston, MA, USA). Respiratory rate, oxygen
saturation, and heart rate were monitored with a small animal oximeter
(STARR Life Sciences, Allison Park, PA, USA). After forebrain ischemia, rats
were killed at 5 or 24 hours after ischemia for protein and RNA isolation.
Control rats injected with antagomir or mismatch but not subjected to
ischemia were killed 1 and 3 days after injection to assess protein and
miRNA levels. For assessment of CA1 neuronal survival by cresyl violet
staining or assessment of protein by immunoblotting, rats were killed at 7
days after ischemia under deep anesthesia with isourane. Brains were
collected after perfusion with cold saline for protein or RNA, or saline
followed by ice-cold 4% phosphate-buffered paraformaldehyde for cresyl
violet staining. After 48 hours after xation in 4% paraformaldehyde, 50 mm
coronal sections were made with a vibratome (Leica VT1000S, Heidelberg,
Germany). The investigator making the injections was masked to the
treatment and the surgeon performing forebrain ischemia was masked to
the treatment group.
Assessment of CA1 Hippocampal Injury
Coronal sections were mounted on gelatin-coated slides and then
incubated in 1% cresyl violet acetate (Sigma) for 5 minutes followed by
dehydration. The density of cresyl violet staining was determined in a xed
area of CA1 on each micrograph using the histogram function of Adobe
Photoshop CS3 and normalized to sham by a researcher masked to
treatment, as previously described.21
Immunoblotting
Immunoblotting was performed as previously described.22 Homogenates
prepared from either cell cultures or subdissected CA1 and dentate gyrus
regions of the hippocampus from brain were studied. Briey, 20 to 40 mg of
protein were separated on a 4 to 12% polyacrylamide gel (Invitrogen) and
electrotransferred to an Immobilon polyvinylidene uoride membrane
(Millipore, Billerica, MA, USA). Membranes were blocked in phosphate-
buffered saline containing 0.1% tween 20 and 5% non-fat milk for 1 hour
at room temperature, and then incubated overnight with primary antibody
Figure 1. The effect of mimic and inhibitor on miR-181a level and
Bcl-2 protein expression in N2a cells. (A) miR-181a levels after
transfection with 10 pmol of miR-181a mimic (Mimic) and 20 pmol of
inhibitor (Inh) in N2a cells. n 6. (B) Effect of increased or decreased
miR-181a level on Bcl-2 protein expression, normalized to the
transfection control (Ctrl). n 8 to 11. Inset shows representative
western blots. *Po0.05, **Po0.01, ***Po0.001 compared with the
transfection control.
Journal of Cerebral Blood Flow & Metabolism (2013), 1976 1982
 MiR-181 reduction protects from forebrain ischemia
J-m Moon et al
1978

Figure 2. Decreasing miR-181a levels reduces serum deprivation and oxidative stress-induced N2a cell death and caspases 3 and 7 activation.
(A) Cells were deprived of serum and incubated in the presence of added H2O2 for 24 hours, then cell death was assessed by lactate
dehydrogenase release. The extent of cell death in all injury conditions was significantly different from wash control (Wash). n 12. (Mean cell
death valuess.e. were Wash 9.00.71, Trans 33.01.04, Mimic 47.51.14, Inh 28.50.70.) (B, C) Caspase 3 and 7 activity after exposure to
injury for 4 hours was determined by Magic Red staining and normalized to wash control. Cultures were stained for activated caspases 3 and 7
(red) and nuclei were counterstained with Hoechst dye (blue) after exposure to injury for 4 hours. n 18. SD, serum deprivation;
Mimic 10 pmol of miR-181a mimic; Inh 20 pmol of miR-181a inhibitor. *Po0.05, **Po0.01, ***Po0.001 compared with transfection control
injury. Bar is 50 mm.

against Bcl-2 (1:1000, Cell Signaling, Danvers, MA, USA), glutamate
transporter 1 (GLT-1; 1 : 500, Millipore, Temecula, CA, USA), and actin
(1 : 5000, Sigma). After washing, membranes were incubated with
secondary antibody for 1 hour. Immunoreactive bands were visualized
using the LI-COR Odyssey infrared imaging system (LI-COR Biosciences,
Lincoln, NE, USA). Densitometric analysis of bands was performed using
NIH image J and normalized to actin.
Real-Time Quantitative PCR
Total RNA was extracted with TRIzol reagent (Invitrogen), and the
concentration and purity of the RNA samples were quantied by
absorbance at 260 and 280 nm. Reverse transcription of miRNA was
performed using the TaqMan MicroRNA Reverse Transcription Kit (Applied
Biosystems, Carlsbad, CA, USA). Equal amounts of RNA (200 ng) were
reverse transcribed with 1.3 mM dNTPs (with dTTP), 50 U reverse
transcriptase, 10 U RNase inhibitor, and specic miRNA reverse trans-
criptase primers (Applied Biosystems) at 16 1C for 30 minutes, 42 1C for
30 minutes, and 851C for 5 minutes. PCR reactions were carried out using
the TaqMan MicroRNA Assay Kit (Applied Biosystems) at 95 1C for
10 minutes, followed by 40 cycles of 95 1C for 15 seconds and 60 1C for
1 minute. Predesigned primer/probes for miR-181a and mouse U6 were
also from Applied Biosystems. Relative change of miR-181a was calculated
using the 2  DDCT method.23 Normalization was rst to U6, which has
been shown to be stable under ischemic brain conditions11 and then to
control to give the relative fold change compared with the control
sample.
Statistical Analysis
All cell culture data represent at least three independent experiments. All
data reported are meanss.e.m. Statistical analysis was performed using
t-test if only two conditions, or one-way analysis of variance with the
Bonferroni post-test. Po0.05 was considered signicant.
RESULTS
Altering miR-181a Levels Alters Bcl-2 Levels and Apoptotic Cell
Death of N2a cells
To investigate the effect of altering miR-181a levels on N2a cells,
cells were transfected with miR-181a mimic, miR-181a inhibitor, or
control. Treatment with mimic increased miR-181a levels 4,700-
fold, whereas a decrease of 88% was seen with the inhibitor
(Figure 1A). As we previously found that Bcl-2 was a target of miR-
181a in astrocytes, we therefore determined whether changing
levels of miR-181a would affect Bcl-2 levels in N2a cells. miR-181a
inhibitor treatment signicantly increased Bcl-2 protein levels,
whereas mimic-treated cells had decreased levels relative to
control (Figure 1B).
N2a cells were next exposed to combined serum deprivation
and oxidative stress for 24 hours to induce apoptosis. Over-
expression of miR-181a signicantly increased cell death, whereas
inhibitor effectively reduced cell death (Figure 2A). There was no
signicant difference in cell death between injury control without
(data not shown) vs with treatment with transfection control
(Trans). To conrm that cell death was apoptotic, we stained the
N2a cells with Magic Red to detect active caspases 3 and 7.
Increasing miR-181a by treatment with mimic was associated with
many more cells showing activation of caspases 3 and 7, whereas
reduction of miR-181a by inhibitor treatment reduced the number
of cells activating these enzymes (Figures 2B and C).

Journal of Cerebral Blood Flow & Metabolism (2013), 1976 1982 & 2013 ISCBFM

Figure 3. Altering miR-181a levels in primary neurons does not alter
serum deprivation cell death. (A) miR-181a levels in primary neurons
after transfection with 10 or 50 pmol of miR-181a mimic, and 20 or
40 pmol of miR-181a inhibitor. n 9. (B) Serum deprivation induced
primary neuronal death assessed by cell counting. All injured groups
had significantly greater injury than wash control. There was no
significant difference in serum deprivation-induced cell death
between control transfected cultures (Trans) and not transfected
cultures (data not shown). n 10. *Po0.05, ***Po0.001 compared
with the transfection control.
Figure 4. Temporal change of miR-181a and Bcl-2 protein in the CA1
region of hippocampus after forebrain ischemia. (A) miR-181a levels
at 5 and 24 hours of reperfusion (Rep) after forebrain ischemia.
n 3. (B) Bcl-2 protein at 5 and 24 hours of reperfusion after
forebrain ischemia. n 3. *Po0.05 compared with sham, not
subjected to ischemia.
Effect of Altering miR-181a Levels in Primary Neurons
We compared the expression of miR-181a in primary neurons and
N2a cells by real-time quantitative PCR. There was no difference in
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MiR-181 reduction protects from forebrain ischemia
J-m Moon et al
1979
Table 1. Levels of miR-181a and Bcl-2 protein in the dentate gyrus
region of hippocampus after forebrain ischemia
Reperfusion
Sham 5 hours 24 hours
miR-181a levels 1.020.14 0.520.06* 0.750.20
Bcl-2 protein 0.970.03 1.270.05** 1.290.04**
Values are means.e.m. n 3 for miR-181a levels, n 3 for Bcl-2 protein
levels. *P 0.054 for miR-181a level at 5 hours reperfusion compared with
sham. **Po0.01 compared with sham.
threshold cycle for U6 between the two cell types, but the
threshold cycle for miR-181a was signicantly lower in primary
neurons (20.090.03 vs 25.920.28 for N2a cells; n 5),
indicating greater abundance of miR-181a in primary neurons.
Transfection of primary neuronal cultures with miR-181a mimic or
inhibitor led to a dose-dependent change in miR-181a levels
(Figure 3A). Despite this, when transfected neurons were exposed
to serum deprivation, no change in the extent of injury was
observed (Figure 3B). After transfection but without injury, levels
of Bcl-2 protein were unaltered despite changes in miR-181a levels
(data not shown), in contrast to the results with N2a cells.
Forebrain Ischemia Changes miR-181a and Bcl-2 Protein Levels in
CA1 and Dentate Gyrus in the Hippocampus
CA1 is the subregion of the hippocampus most susceptible to
ischemia. We assessed levels of miR-181a 5 and 24 hours after
10 minutes of forebrain ischemia. In CA1, miR-181a signicantly
increased 24 hours after ischemia (Figure 4A), whereas Bcl-2
protein decreased (Figure 4B). In contrast, in the dentate gyrus
miR-181a tended to decrease at 5 hours, whereas Bcl-2 protein
was increased at both 5 and 24 hours (Table 1).
Reducing Levels of miR-181a Reduces Forebrain Ischemic Injury
Real-time quantitative PCR was performed 1 and 3 days after
injection to determine the extent to which miR-181a levels changed
after transfection with antagomir by stereotactic injection. miR-181a
antagomir, but not mismatched antagomir, signicantly reduced
miR-181a expression 1 and 3 days after a single treatment
(Figure 5A). Bcl-2 protein levels also changed with a signicant
increase on day 1 and a trend to increase on day 3 (Figures 5B and
5C). Therefore, we performed forebrain ischemia on rats 1 day after
stereotactic infusion of antagomir or control. Cresyl violet staining
performed 7 days after forebrain ischemia revealed that when
brains were transfected with miR-181a antagomir CA1, neuronal cell
death was signicantly reduced compared with rats treated with
mismatch miR-181a antagomir (Figures 5D and 5E).
The Effect of miR-181a Antagomir on GLT-1 in CA1 After Forebrain
Ischemia.
As we previously found that astrocyte impairment was a critical
determinant of the extent of subsequent neuronal cell death in
forebrain ischemia,22 we hypothesized that miR-181 antagomir
might reduce astrocyte impairment. Earlier studies showed that
reducing miR-181a levels in primary astrocyte cultures could
reduce oxidative stress,18 and oxidative stress contributes to
astrocyte impairment and reduced GLT-1 levels after forebrain
ischemia. We therefore determined the level of GLT-1 in the CA1
subeld by immunoblot analysis. GLT-1 expression signicantly
decreased in CA1 24 hours after forebrain ischemia compared
with sham; however, treatment with miR-181a antagomir
Journal of Cerebral Blood Flow & Metabolism (2013), 1976 1982
 MiR-181 reduction protects from forebrain ischemia
J-m Moon et al
1980

Figure 5. Pretreatment with miR-181a antagomir increases CA1 neuronal survival after forebrain ischemia. (A) miR-181a levels 1 and 3
days after injecting miR-181a antagomir or mismatch miR-181a antagomir just outside the hippocampus relative to sham. n 5 to 6.
*Po0.05 and **Po0.01 compared with sham. (B) Representative blots of Bcl-2 protein expression after transfection with indicated antagomir
in vivo. (C) Levels of Bcl-2 protein 1 and 3 days after injecting miR-181a antagomir or mismatch miR-181a antagomir relative to sham. n 3 to
6. *Po0.05 compared with sham. (D) Cresyl violet staining of hippocampi. Sham, which was not subjected to ischemia, shows a normal
hippocampus. After forebrain ischemia in a rat pretreated with mismatch miR-181a antagomir, selective neuronal loss of CA1 hippocampal
neurons (between white arrows) and reduced neuronal loss (between white arrows) in a rat treated with miR-181a antagomir outside the left
hippocampus is shown. (E) Quantitation of neuronal survival in CA1 of rats treated with miR-181a antagomir (Ant) or mismatch miR-181a
antagomir (Mis) relative to sham. n 15. ***P o0.001, compared with rats treated with mismatch miR-181a antagomir.

signicantly inhibited this decrease of GLT-1 compared with

vehicle or mismatch control antagomir (Figure 6).

DISCUSSION
Pretreatment with miR-181a antagomir reduces loss of CA1
pyramidal neurons 7 days after forebrain ischemia. This adds
forebrain ischemia to the settings in which reducing miR-181
provides neuroprotection. Treatment with antagomir inhibited the
changes seen in untreated animals, reducing miR-181a, increasing
Bcl-2 protein levels, and preserving neurons in CA1. Although
pretreatment was tested in this study, future studies of the effect
of changing miRNA levels after the onset of ischemia will be an
essential step in further demonstrating the likely relevance for
clinical treatment.
Overexpression of Bcl-2, the prototype anti-apoptotic member
of the eponymous family of apoptosis regulatory proteins, was Figure 6. Pretreatment with miR-181a antagomir preserves GLT-1
previously shown to be protective against both focal and global expression after forebrain ischemia. GLT-1 expression 24 hours after
reperfusion in the CA1 subfield of rats treated with miR-181a
cerebral ischemia in vivo and brain cell injury in vitro.2427 antagomir, mismatch miR-181a antagomir, vehicle (Isch), and sham
However, gene therapy in the setting of cardiac arrest is less rats, which were not subjected to ischemia. Inset shows representa-
likely to translate to clinical application than manipulation of a tive western blot. n 3. *Po0.05 compared with both Isch and
miRNA in this setting. We previously demonstrated that Bcl-2 is a mismatch.

Journal of Cerebral Blood Flow & Metabolism (2013), 1976 1982 & 2013 ISCBFM

direct target of miR-181 by luciferase assay.18 Trials of mimic and
antagomir miRNAs are already underway for several diseases, and
as the chemistry is the same only the sequence will differ. Thus,
extending this approach to cerebral ischemia has strong
translational potential.
Although we demonstrate changes in one target of miR-181a,
a limitation of our study is that there are likely to be additional
targets of miR-181a that may have a role in the protection we
observe, because miRNAs target multiple mRNAs. Our prior work
showed that miR-181a can directly target Mcl-1 and Grp78/BIP in
some cells,14,18 and a recent report showed that X-linked inhibitor
of apoptosis is also a direct target, and that several antioxidant
enzymes are likely targets.28 These targets, which are all pro-
survival proteins and can reduce oxidative stress, could also have a
role here. We think this is relevant because our earlier studies
demonstrated a link between oxidative stress in astrocytes,
reduction in GLT-1, and CA1 neuronal cell death.21,22 miR-181
was also shown to sensitize glioblastoma cells to apoptosis by
reducing Bcl-2.29 Future studies are needed to explore additional
potential targets in this setting.
One interesting observation is the difference in effect of miR-
181a antagomir in different cell types. Reducing miR-181a
increased Bcl-2 in N2A cells and increased Bcl-2, Grp78, and Mcl-
1 in primary astrocytes while increasing survival of both these cell
types. It failed to signicantly change levels of Bcl-2 in primary
neurons, and it did not change outcome from serum deprivation-
induced neuronal cell death. This observation is consistent with an
increasing body of literature that suggests that individual miRNAs
may have modest effects on their target mRNAs, and that often
several miRNAs are required for larger, or in some cases,
detectable alteration of target protein levels. In prostate cancer
cells, miR-181a acts cooperatively with two other miRNAs to
regulate Grp78 protein levels,30 in contrast to the response of
astrocytes in which increasing miR-181a alone effectively
downregulates Grp78.14,30 Further, cell lines are in general
dependent on inducing anti-apoptotic pathways for survival;
hence, the N2A cells may be more sensitive to the ability of miR-
181 to reduce several anti-apoptotic proteins than are primary,
postmitotic neurons. Thus, the efciency of regulation of a target
protein depends on the cell type and likely the physiologic setting;
hence, the difference observed between N2A cells and primary
neurons may not be surprising.
Total hippocampal miR-181a was reported to be upregulated
30 minutes and 24 hours after 20 minutes of global ischemia,31
and hippocampal Bcl-2 protein levels were previously found
to be decreased 24 hours after 10 minutes of global ischemia.32
Although these studies evaluated changes in whole hippocampus,
we dissected out the selectively vulnerable CA1 region so as not to
average changes in the vulnerable region with changes in the
other hippocampal regions that survive. We found that miR-181a
increased in CA1 but tended to decrease in dentate gyrus
(Table 1), whereas Bcl-2 protein levels decreased in CA1 but
increased in dentate gyrus. These results are consistent with the
pattern of neuronal cell death in CA1 and survival in dentate gyrus.
The signicant protection observed here against forebrain
ischemia was associated with increased Bcl-2 protein levels and
preservation of the astrocyte glutamate transporter GLT-1. We
previously reported that inhibition of miR-181a in primary
astrocytes increased Bcl-2 protein and reduced oxidative stress
during glucose deprivation.18 A close association was seen
between oxidative stress, the early loss of astrocyte GLT-1, and
delayed CA1 neuronal death in forebrain ischemia.21,22 Thus, the
preservation of GLT-1 observed here is consistent with protection
of astrocyte function contributing to neuronal protection.
Astrocytic glutamate uptake prevents or limits excitotoxic
neuronal injury.21,22,3335 In global and forebrain ischemia,
reduced astrocytic glutamate uptake precedes neuronal loss.
Preventing this astrocyte dysfunction with metabotropic
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MiR-181 reduction protects from forebrain ischemia
J-m Moon et al
1981
glutamate receptor agonists35 or by overexpressing protective
proteins selectively in astrocytes21,22 reduces neuron cell
death. In this study, signicant decreases in GLT-1 protein were
demonstrated 24 hours after ischemia, consistent with other
studies in gerbils and rats,22,36 and antagomir treatment reduced
this loss.
Despite the observed changes in GLT-1, miR-181a does not
directly target GLT-1. Immunoblotting did not show an increase of
GLT-1 protein in rats pretreated with miR-181a antagomir (data
not shown), and no potential binding sites were identied in the
3-untranslated region of GLT-1 mRNA for miR-181a using the
miR-Target analysis database (http://www.targetscan.org, http://
www.microRNA.org). Thus, the protection is likely indirect
secondary to reduced oxidative stress in astrocytes. Reduced
oxidative stress may reect increased levels of Bcl-2, Grp78, and
Mcl-114,18 and possibly glutathione peroxidases 1 and 4, and
peroxiredoxin 2.28
Astrocytes support neurons and other surrounding cells
through multiple functions including regulation of blood ow,
provision of substrates, metabolic and ionic homeostasis, and
antioxidant defense.33,37,38 However, after ischemia or other
central nervous system insults, astrocytes become reactive and
may lose some supportive functions, thereby contributing to
neuronal death.39 Ischemic injury induces activation of astrocytes,
early loss of GLT-1 in the CA1 region, with recovery over days after
forebrain ischemia. Findings in forebrain ischemia are consistent
with reports using the four vessel occlusion model of global
ischemia.40,41 Thus, benecial effects of miR-181a on astrocytes
may lead to reduced or modulated activation and better
preserved protective function. Effects of miR-181a antagomir in
multiple cell types are probably important to the nal extent of
protection observed.
SUMMARY
Stereotactic delivery of miR-181a antagomir just outside the
hippocampus reduced neuronal cell death in rat forebrain
ischemia, associated with increased Bcl-2 and preserved GLT-1
levels, consistent with protection that includes preserving
astrocyte function.
DISCLOSURE/CONFLICT OF INTEREST
The authors declare no conict of interest.
ACKNOWLEDGEMENTS
The authors thank Robin White for assistance with microscopy, Yibing Ouyang for
help preparing gures, and William Magruder for assistance with preparing the
manuscript.
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