Original Article
17β-Estradiol Attenuates Hematoma Expansion Through
Estrogen Receptor α/Silent Information Regulator 1/Nuclear
Factor-kappa B Pathway in Hyperglycemic Intracerebral
Hemorrhage Mice
Yun Zheng, MD, MS*; Qin Hu, MD, PhD*; Anatol Manaenko, PhD; Yang Zhang, MS;
Yan Peng, MD, PhD; Liang Xu, MD; Junjia Tang, MD; Jiping Tang, MD; John H. Zhang, MD, PhD
Downloaded from http://stroke.ahajournals.org/ by guest on June 14, 2017
Background and Purpose—17β-estradiol (E2) has been reported to reduce bleeding and brain injury in experimental
intracerebral hemorrhage (ICH) model. However, it is not clear if E2 can prevent early hematoma expansion (HE) induced
by hyperglycemia in acute ICH. The aim of this study is to evaluate the effects of E2 on HE and its potential mechanisms
in hyperglycemic ICH mice.
Methods—Two hundred, 8-week-old male CD1 mice were used. ICH was performed by collagenase injection. 50% dextrose
(8 mL/kg) was injected intraperitoneally 3 hours after ICH to induce acute HE (normal saline was used as control). The
time course of HE was measured 6, 24, and 72 hours after ICH. Two dosages (100 and 300 μg/kg) of E2 were administrated
1 hour after ICH intraperitoneally. Neurobehavioral deficits, hemorrhage volume, blood glucose level, and blood–brain
barrier disruption were measured. To study the mechanisms of E2, estrogen receptor α (ERα) inhibitor methyl-piperidinopyrazole, silent information regulator 1 (Sirt1) siRNA was administered, respectively. Protein expression of ERα, Sirt1,
and acetylated nuclear factor-kappa B, and activity of matrix metalloproteinases-9 were detected.
Results—Hyperglycemia enhanced HE and deteriorated neurological deficits after ICH from 6 hours after ICH. E2 treatment
prevented blood–brain barrier disruption and improved neurological deficits 24 and 72 hours after ICH. E2 reduced HE
by activating its receptor ERα, decreasing the expression of Sirt1, deacelylation of nuclear factor-kappa B, and inhibiting
the activity of matrix metalloproteinases-9. ERα inhibitor methyl-piperidino-pyrazole and Sirt1 siRNA removed these
effects of E2.
Conclusions—E2 treatment prevented hyperglycemia-enhanced HE and improved neurological deficits in ICH mice
mediated by ERα/Sirt1/nuclear factor-kappa B pathway. E2 may serve as an alternative treatment to decrease early HE
after ICH. (Stroke. 2015;46:00-00. DOI: 10.1161/STROKEAHA.114.006372.)
Key Words: 17β-Estradiol ◼ intracerebral hemorrhage ◼ MMP-9 ◼ Sirt1
S
the onset of ICH2 and is amenable to treatment.3,4 Because of
its strong relationship with poor prognosis and the potential
to prevent its development, HE is an appealing therapeutic
target after ICH.
Estrogens are steroid compounds that function as the primary sex hormone in females, and 17β-estradiol (E2) is the
most potent naturally occurring estrogen. In the last 2 decades,
estrogen is one of the most extensively studied neuroprotectants for the treatment of stroke.5–8 In experimental ICH model,
estrogens have been proven to reduce bleeding and brain
injury in rats,9–11 whereas the mechanisms underlying blood–
brain barrier (BBB) protection of E2 remain poorly explored,
pontaneous intracerebral hemorrhage (ICH) is a subtype of stroke featured by hematoma formation within
brain parenchyma, which accounts for ≈15% of all deaths
from stroke and with >75% of patients severely disabled
or deceased within the first year.1 The high mortality and
morbidity make ICH a major public health problem, and no
effective therapy has yet been established to counteract the
consequences of this detrimental subtype of stroke subtype.
After ICH, the initial hematoma forms and is untreatable.
However, ≈30% of ICH patients continue to bleed and demonstrate significant hematoma expansion (HE), which further
aggravates the outcome.2 Most HE occurs within 3 hours after
Received June 4, 2014; final revision received November 3, 2014; accepted November 14, 2014.
From the Departments of Physiology and Pharmacology (Y. Zheng, Q.H., A.M., Y. Zhang, Y.P., L.X., Junjia Tang, Jiping Tang, J.H.Z.), and Neurosurgery
(J.H.Z.), Loma Linda University School of Medicine, Loma Linda, CA; and Department of Physiology, Medical School of Yangtze University, Jingzhou,
Hubei, China (Y. Zheng).
*Drs Zheng and Hu contributed equally.
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.
114.006372/-/DC1.
Correspondence to John H. Zhang, MD, PhD, Department of Neurosurgery and Departments of Physiology and Pharmacology, Loma Linda University
School of Medicine, 11041 Campus St, Risley Hall, Room 219, Loma Linda, CA 92354. E-mail [email protected]
© 2014 American Heart Association, Inc.
Stroke is available at http://stroke.ahajournals.org
DOI: 10.1161/STROKEAHA.114.006372
1
2 Stroke February 2015
and there are no studies to investigate the effects of estrogens
on early HE after ICH. Matrix metalloproteinases (MMPs),
which are regulated by nuclear factor-kappa B ­(NF-κB), have
been proved to be the culprit for BBB disruption. Recently, E2
has been reported to upregulate silent information regulator 1
(Sirt1),12,13 which can inactivate NF-κB. In the present study,
we investigate whether E2 treatment will prevent the early
growth of HE induced by hyperglycemia in ICH mice and
explore the potential role of Sirt1/NF-κB in BBB protection.
Materials and Methods
All experiments were approved by the Institutional Animal Care and
Use Committee of Loma Linda University.
Animal Model and Experimental Protocol
Downloaded from http://stroke.ahajournals.org/ by guest on June 14, 2017
Two hundred, 8-week-old male CD1 mice (weight 25–35 g; Charles
River, Wilmington, MA) were used. ICH mouse model was performed
by collagenase injection as reported previously.14 50% dextrose (8 mL/
kg) was injected intraperitoneally 3 hours after ICH to induce acute HE
(normal saline was used as control). The time course of HE was measured 6, 24, and 72 hours after ICH. Two dosages (100 and 300 μg/kg)
of E2 (Sigma-Aldrich) were administrated 1 hour after ICH intraperitoneally. Neurobehavioral deficits, hemorrhage volume, and blood glucose were measured. To study the mechanisms of E2, estrogen receptor
α (ERα) inhibitor methyl-piperidino-pyrazole (MPP; Sigma-Aldrich;
100 μg/kg) and Sirt1 siRNA (OriGene Technologies) was administered,
respectively. Protein expression of ERα, Sirt1, deacetylated NF-κB, and
activity of MMP-9 were detected. The experimental design was included in Figure I in the online-only Data Supplement.
siRNA Injection
Two different formats of Sirt1 siRNA were applied 48 hours before ICH
to enhance the silencing effect. An intracerebroventricular injection was
then performed as previously described.14 The Sirt1 siRNA or scramble
siRNA mixed with the transfection reagent (OriGene Technologies; 100
pmol/2 μL) was delivered into the ipsilateral ventricle with a Hamilton
syringe and administered over 2 minutes. After the needle was removed,
the burr hole was sealed with bone wax. The incision was closed with
sutures, and the mice were allowed to recover.
Neurological Scores
Twenty-four or 72 hours after ICH, the Garcia test was performed by
a blinded investigator as previously described with modifications.15
The scores given to each of the mice at the completion of the evaluation was the summation of 7 individual test scores (spontaneous activity, symmetry in the movement of 4 limbs, forepaw outstretching,
climbing, body proprioception, response to vibrissae touch, and beam
walking). The neurological scoring ranged from 2 (most severe deficit) to 21 (maximum).
Hematoma Volume
Hemoglobin assay was performed as previously described.14 The ipsilateral hemisphere was homogenized for 60seconds in a tube with
distilled water (total volume 3 mL). After centrifuging (12 000g, 30
minutes), Drabkin’s reagent (400 μL; Sigma-Aldrich) was mixed in
with a supernatant (100 μL) and allowed to react for 15minutes. The
absorbance of the mixture was read with a spectrophotometer (540
nm), and the amount of blood in each brain was calculated using a
standard curve generated with known blood volumes.
Evan’s Blue Dye Extravasation
Disruption of the BBB was analyzed 24 hours after ICH using Evan’s
blue dye as reported previously.15 The amount of extravasated Evan’s
blue in the brain was determined by spectroflurophotometry with a
standard curve. Measurements were conducted at an excitation wavelength of 610 nm.
Immunohistochemistry
Immunofluorescent staining for brain tissue was performed on fixed
frozen ultrathin sections as previously described.15 Primary antibodies used were ERα (SAB4500810; Sigma-Aldrich), Sirt1 (S5447;
Sigma-Aldrich), glial fibrillary acidic protein (sc-6170; Santa Cruz
Biotechnology), and Von Willebrand factor (sc-8068; Santa Cruz
Biotechnology). Peri-hemorrhagic area of the brain coronal section
was imaged by Olympus-BX51.
Western Blot Analysis
Brain samples were collected 24 hours after the ICH. Western blotting was performed as described previously.15 Primary antibodies used were ERα (SAB4500810; Sigma-Aldrich), Sirt1 (S5447;
Sigma-Aldrich), acetylated NF-κB (SAB4502616; Sigma-Aldrich),
and β-actin (sc-1616; Santa Cruz Biotechnology).
Matrix Metalloproteinase Zymography
The ipsilateral brain cortex was used to analyze MMP-9.13 Briefly,
samples were homogenized, and the supernatant was collected.
Samples were loaded and separated by 10% Tris-tricine gel with
0.1% gelatin as a substrate. After separation by electrophoresis, the
gel was renatured and then incubated with development buffer at
37°C for 24 hours. The gel was stained with 0.5% coomassie blue
R-250 for 30 minutes and then destained appropriately. MMP-9 activity was quantified using ImageJ, version 1.32.
Statistical Analysis
The analysis of the data was performed using GraphPad Prism software.
Statistical differences between 2 groups were analyzed using the student’s
unpaired, 2-tailed t test. Multiple comparisons (without a rating scale)
were statistically analyzed with 1-way analysis of variance followed by
the Tukey method. The data are presented as means±SEM. In all statistical analysis, a value of P<0.05 represents statistical significance.
Results
Hyperglycemia Enhanced HE and Deteriorated
Neurobehavioral After ICH
To observe the effects of hyperglycemia on HE, we administrated saline or 50% dextrose (8 mL/kg) to the animals 3 hours
after ICH and measured the hemorrhagic volume and neurobehavioral at 6, 24, and 72 hours after ICH. There was distinct
hematoma formation at the basal ganglia from 6 hours after
collagenase injection. In ICH+saline group, the hemorrhagic
volume 6, 24, and 72 hours were 19.29±1.917 , 18.89±1.367,
and 13.40±1.056 μL, respectively. Hyperglycemia significantly
enhanced HE to 29.70±3.368, 31.87±1.972, and 27.40±2.100
μL, respectively (Figure 1A and 1B; P<0.05 versus ICH+saline)
and deteriorated neurological deficits (Figure 1C; P<0.05 versus
ICH+saline). Blood glucose peaked 1 hour after DX injection
and returned to baseline within 4 hours, and E2 had no effect on
blood glucose level (Figure 1D; P>0.05 versus ICH+DX).
E2 Suppressed HE and Improved Neurological
Deficits 24 and 72 Hours After ICH
A low dosage of E2 showed the tendency to attenuate the HE.
A high dosage of E2 significantly decreased hemorrhagic volume (Figure 2A and 2B; P<0.05 versus ICH+DX+vehicle).
This dosage of E2 (300 μg/kg) significantly improved the
Zheng et al 17β-Estradiol and Intracerebral Hemorrhage 3
Downloaded from http://stroke.ahajournals.org/ by guest on June 14, 2017
Figure 1. Effects of hyperglycemia on hematoma expansion and neurobehavioral 6, 24, and 72 h after intracerebral hemorrhage (ICH).
There was a distinct hematoma formation at the basal ganglia from 6 h after collagenase injection (A), hyperglycemia potently enhanced
hemorrhagic volume (B), and severely deteriorated the neurological deficits (C) 6, 24, and 72 h after ICH. *P<0.05 vs Sham; &P<0.05 vs
ICH+saline. n=6 for each group.
neurological scores compared with vehicle group 24 hours
after ICH (Figure 2C; P<0.05 versus ICH+DX+vehicle). The
beneficial effects of E2 on BBB disruption and neurological
deficits last ≤72 hours after ICH (Figure 3; P<0.05 versus
ICH+DX+vehicle).
ERα Inhibitor MPP and Sirt1 siRNA Abolished the
Effects of E2 24 Hours After ICH
To investigate the potential mechanisms of E2 in suppressing
HE, we administrated ERα inhibitor MPP 45 minutes after ICH
and Sirt1 siRNA 48 hours before ICH, respectively, with the
treatment of a high dosage of E2. MPP significantly increased
the hemorrhagic volume from 21.97±1.11 to 29.47±1.99 μL
(Figure 4A and 4B; P<0.05 versus ICH+DX+E2) and decreased
the neurological scores from 13.17±1.22 to 9.25±0.49 24
hours after ICH (Figure 4C; P<0.05 versus ICH+DX+E2).
Sirt1 siRNA also abolished the effects of E2 by increasing
the hemorrhagic volume to 28.25±0.68 μl (Figure 4A and 4B;
P<0.05 versus ICH+DX+E2) and decreased the neurological
scores from 13.17±1.22 to 9.75±0.62 (Figure 4C; P<0.05 versus ICH+DX+E2). These data suggested that E2 ameliorated
HE dependent on its receptor ERα and Sirt1.
Figure 2. E2 prevented hematoma expansion (A and
B) and improved Garcia neuroscores (C) 24 h after
intracerebral hemorrhage (ICH). *P<0.05 vs sham;
&P<0.05 vs ICH+DX+vehicle. n=6 for each group.
4 Stroke February 2015
Figure 3. E2 prevented hematoma expansion (A
and B), improved Garcia neuroscores (C), and preserved the integrity of blood–brain barrier (BBB; D)
72 h after intracerebral hemorrhage (ICH). *P<0.05
vs sham; & P<0.05 vs ICH+DX+vehicle. n=6 for
hematoma expansion and Garcia neuroscores in
each group; n=5 for Evan’s blue in each group.
Downloaded from http://stroke.ahajournals.org/ by guest on June 14, 2017
E2 Ameliorated HE by Inhibiting the Activity of
MMP-9 Through ERα/Sirt1/NF-κB Pathway
Double fluorescence immunostaining showed the expression
of ERα and Sirt1 were strong in astrocytes and endothelial cells
in sham animals and decreased after ICH (Figure 5). Western
blots showed that after ICH, there was a dramatic loss of ERα
in ipsilateral brain tissue (Figure 6A; P<0.05 versus Sham),
and administration of E2 significantly upregulated the expression of ER-α (Figure 6A; P<0.05 versus ICH+DX+vehicle),
whereas ERα inhibitor MPP did not affect the expression of
ERα significantly (Figure 6A; P>0.05 versus ICH+DX+E2).
The expression of Sirt1 decreased after ICH (Figure 6B;
P<0.05 versus Sham) and increased by E2 24 hours after ICH
(Figure 6B; P<0.05 versus ICH+DX+vehicle). Sirt1 siRNA
strongly knocked down the expression of Sirt1 in both Sham
and ICH mice compared with scramble siRNA (Figure II in
the online-only Data Supplement). MPP and Sirt1 siRNA
abrogated the results of E2 and decreased the expression of
Sirt1 (Figure 6B; P<0.05 versus ICH+DX+E2). ICH increased
the acetylation of NF-κB (Figure 6C; P<0.05 versus Sham),
which was decreased by E2 administration (Figure 6C; P<0.05
versus ICH+DX+vehicle). Administration of MPP or Sirt1
siRNA increased the acetylated NF-κB in E2-treated animals
(Figure 6C; P<0.05 versus ICH+DX+E2).
The activity of MMP-9 in ICH+DX+vehicle group was
intensely increased 24 hours after ICH (Figure 6D; P<0.05
versus Sham) and was significantly decreased by E2
(Figure 6D; P<0.05 versus ICH+DX+vehicle). ER-α inhibitor
Figure 4. Estrogen receptor α (ERα) inhibitor
methyl-piperidino-pyrazole (MPP) and Sirt1 siRNA
removed the effects of E2 on hematoma expansion (A, B) and Garcia neuroscores (C) 24 hours
after intracerebral hemorrhage (ICH). *P<0.05 vs.
sham, & P<0.05 vs. ICH+DX+vehicle, # P<0.05 vs.
ICH+DX+E2. n=6 for each group.
Zheng et al 17β-Estradiol and Intracerebral Hemorrhage 5
Figure 5. Representative immunostaining of estrogen receptor α (ERα) and estrogen receptor α (Sirt1) in astrocytes and endothelial cells
in Sham and intracerebral hemorrhage (ICH) animals 24 h after surgery. GFAP indicates glial fibrillary acidic protein; and vWF, Von Willebrand factor.
Downloaded from http://stroke.ahajournals.org/ by guest on June 14, 2017
MPP and Sirt1 siRNA abolished the effects of E2 on the activity of MMP-9 (Figure 6D; P<0.05 versus ICH+DX+E2). The
activity of MMP-2 did not show great changes in all groups.
The results suggested that E2 inhibited the activity of MMP-9
through ERα/Sirt1/NF-κB pathway in ICH mice.
Discussion
Hematoma volume is a major factor of both mortality and
poor outcome after ICH. Early HE aggravates initial outcomes and increase mortality. Early HE is, however, treatable,
and restriction of HE is a promising therapeutic approach. In
this study, our goals were to test, first, whether hyperglycemia enhanced early HE in ICH model, second, whether E2
ameliorated early HE and improved neurobehavioral performance, and third, investigated the mechanisms of E2 on BBB
protection. We found out that hyperglycemia can enlarge HE
and deteriorate neurological deficits as early as 3 hours after
dextrose injection in ICH mice. Administration of E2 significantly decreased the hemorrhagic volume and improved
the neurological deficits both 24 and 72 hours after ICH. E2
suppressed the activity of MMP-9 through ERα/Sirt1/NF-κB
signaling pathway, and inhibition of ERα/Sirt1 abolished the
Figure 6. E2 reduced hematoma expansion by
inhibiting the activity of matrix metalloproteinases
(MMP)-9 through estrogen receptor α (ERα)/silent
information regulator 1 (Sirt1)/nuclear factor-kappa
B (NF-κB) pathway. Western blots showed E2
treatment increased the level of ERα (A) and Sirt1
(B), decreased the deacetylation of NF-Κb (C)
and activity of MMP-9 (D). ERα inhibitor methylpiperidino-pyrazole (MPP) and Sirt1 siRNA reversed
these effects of E2. *P<0.05 vs sham; & P<0.05
vs ICH+DX+vehicle; #P<0.05 vs ICH+DX+E2.
n=6 for each group. ICH indicates intracerebral
hemorrhage.
6 Stroke February 2015
Downloaded from http://stroke.ahajournals.org/ by guest on June 14, 2017
effect of E2. These observations indicated that E2 might serve
as an alternative therapeutic strategy to prevent early HE in
ICH patients by protecting BBB.
In clinic, hyperglycemia is more common in patients with
preexisting diabetes mellitus, but is also present in a significant proportion of nondiabetic patients. About 60% ICH
patients might develop hyperglycemia even in the absence of
a previous history of diabetes mellitus,16 which is probably
a response to the stress and severity of ICH. Hyperglycemia
is strictly associated with greater HE and worse clinical
outcomes after ICH.16–20 The deleterious effects of hyperglycemia are attributed to its secondary effects of acidosis,
increased free radical formation, and release of inflammatory cytokines, which accelerated the degradation of BBB
components and impaired the integrity of adjacent vessels
surrounding the initial bleeding site.21–23 In streptozotocininduced diabetic rats and mice, hyperglycemia results in
greater HE after ICH.20,24 In the present study, we injected
dextrose 3 hours after ICH to mimic hyperglycemia on hospital admission. We found that a greater macroscopic bleeding area was observed around the injection site, and more
bleeding volume was confirmed in the hyperglycemic mice
from 3 hours after dextrose injection when compared with
saline controls. Our findings demonstrated for the first time
that hyperglycemia in the acute setting of ICH significantly
enhanced early HE and deteriorated neurological outcome.
Liu et al in their excellent study demonstrated that hyperosmolality caused by hyperglycemia let to a HE in autologous
blood model of ICH via activation of kallikrein/platelet signaling pathway.24 Our findings show that 50% mannitol did
not significantly affect the HE (Figure III in the online-only
Data Supplement). HE in Liu’s publication was specified as
hematoma area in the subarachnoid space. The hematoma
volume was not measured by the authors. We, on the contrary, investigated effect of dextrose on the hematoma volume, without examination of the hematoma location. We
think that, because of different scientific targets, there are no
contradictions in these 2 studies.
Even though the precise mechanism of early HE during
the acute phase of ICH is poorly understood, it is partly preventable. Inflammatory cascade activation and matrix metalloproteinases (MMPs) overexpression have been claimed to
be the major perpetrators in BBB disruption and HE formation after ICH.25 Recently, emerging evidence from basic
research suggests that estrogens showed potency and efficacy
on BBB protection,9,26–28 which might contribute to preventing
HE formation in ICH. After brain injury, estrogen exposure
ameliorated BBB disruption induced by transient focal cerebral ischemia through inhibition of MMP-2 and MMP-9 activation.29 In female rats, endogenous estrogen reduced brain
edema and improved neurological deficits after ICH when
compared with male rats. In collagenase-induced ICH rats,
estrogen treatment significantly reduced bleeding and lesion
volume.9 In agreement with this, we observed activation of
MMP-9 and HE in hyperglycemic ICH mice, and E2 dramatically suppressed the activity of MMP-9 and reduced early HE.
These results justified that early HE is a potential therapeutic
target in the acute phase of ICH, and E2 treatment may be an
accessible and effective strategy to restrict HE and improve
neurological functions in clinic.
Next we addressed the role of ERα in HE suppression of
E2 after ICH. There are two receptor isoforms of E2, ERα,
and ERβ; both of which are members of the nuclear receptor
transcription factor superfamily. Deficiency of ERα but not of
ERβ abolished the protective effect of E2 in ovariectomized
mice subjected to focal cerebral ischemia.30 Additional studies confirmed that in animals subjected to SAH, there was a
significant change in protein expression of ERα but not ERβ in
dentate gyrus, and E2 reversed SAH downregulated ERα and
phospho-Akt expression via an ERα-dependent mechanism.31
The results of these studies demonstrated that ERα, and not
ERβ, was the critical responsible for estrogen-mediated neuroprotection in the rodent cerebral cortex. Furthermore, Vegeto
et al revealed that ERα mediated anti-inflammatory activity
of E2 in brain through inhibiting the expression of MMP-9.32
In transient cerebral ischemia, E2 has been proved attenuated BBB disruption by suppressing the activity of MMP-2
and MMP-9.29 In our experiment, we observed a decreased
expression of ERα and activation of MMP-9 in hemorrhagic
hemisphere, which is in agreement with the previous studies.
Administration of E2 and ERα inhibitor MPP did not affect
the level of ERα, whereas MPP abolished the beneficial
effects of E2 on BBB broken. Our data demonstrated that the
protective role of E2 on BBB is partially mediated by inhibiting the activity of MMP-9 through ERα in ICH rats.
How does E2/ERα signaling regulate MMP-9? ERα is a
member of the nuclear receptors, and once ERα is activated,
ERα may interact with specific transcriptional coregulators to
modulate gene expression and mediate the effects of E2. The
role of sirtuins in the regulation of ER signaling has emerged
over the years. Sirtuins are a family of highly conserved protein deacetylases that have extensively implicated energy
metabolism, stress response, and cell/tissue survival. Sirt1
is the best characterized family member, resides mainly in
the nucleus and deacetylases numerous transcription factors,
including p53, NF-κB, forkhead box O, and so on. Exciting
researches have shown that in a rat model of postmenopausal
metabolic syndrome, E2 restored the protein expression of
Sirt1 and alleviated endothelial dysfunction.12 E2 treatment
also has been reported to result in an upregulation of Sirt1
in old mice13 and in Hela cells.33 The studies of Elangovan
showed that ERα bound to p53 promoter and suppressed its
expression and that the process is obligatorily dependent on
Sirt1 in breast cancer cells. In the present study, E2 treatment
promoted deacetylation of NF-κB and inactivated its transcription of MMP-9. Sirt1 knockdown abolished this effect,
indicating that Sirt1 is necessary for the ability of E2/ERα
signaling to suppress MMP-9 and attenuate HE in hyperglycemic ICH rats.
In conclusion, we demonstrated that hyperglycemia
enhanced early HE in acute setting of ICH, and administration
of E2 prevented the development of HE and improved neurological deficits in ICH mice. E2 preserved the integrity of BBB
by suppressing the activity of MMP-9, which is dependent
on ERα/Sirt1/NF-κB pathway. Our results suggested that E2
might be a promising approach to restrict early HE after ICH.
Zheng et al 17β-Estradiol and Intracerebral Hemorrhage 7
Sources of Funding
This study was supported partially by a grant from National Institute
of Health NS078755, NS081740, and NS082124 to J.H. Zhang.
Disclosures
None.
References
Downloaded from http://stroke.ahajournals.org/ by guest on June 14, 2017
1.Qureshi AI, Mendelow AD, Hanley DF. Intracerebral haemorrhage.
Lancet. 2009;373:1632–1644. doi: 10.1016/S0140-6736(09)60371-8.
2. Brott T, Broderick J, Kothari R, Barsan W, Tomsick T, Sauerbeck L, et
al. Early hemorrhage growth in patients with intracerebral hemorrhage.
Stroke. 1997;28:1–5.
3. Mayer SA, Brun NC, Begtrup K, Broderick J, Davis S, Diringer MN,
et al; FAST Trial Investigators. Efficacy and safety of recombinant
activated factor VII for acute intracerebral hemorrhage. N Engl J Med.
2008;358:2127–2137. doi: 10.1056/NEJMoa0707534.
4. Anderson CS, Huang Y, Wang JG, Arima H, Neal B, Peng B, et al;
INTERACT Investigators. Intensive blood pressure reduction in acute
cerebral haemorrhage trial (INTERACT): a randomised pilot trial.
Lancet Neurol. 2008;7:391–399. doi: 10.1016/S1474-4422(08)70069-3.
5. Carwile E, Wagner AK, Crago E, Alexander SA. Estrogen and stroke: a
review of the current literature. J Neurosci Nurs. 2009;41:18–25, quiz 26.
6. Dubal DB, Kashon ML, Pettigrew LC, Ren JM, Finklestein SP, Rau SW,
et al. Estradiol protects against ischemic injury. J Cereb Blood Flow
Metab. 1998;18:1253–1258. doi: 10.1097/00004647-199811000-00012.
7. Yang SH, He Z, Wu SS, He YJ, Cutright J, Millard WJ, et al. 17-Beta
estradiol can reduce secondary ischemic damage and mortality of subarachnoid hemorrhage. J Cereb Blood Flow Metab. 2001;21:174–181.
doi: 10.1097/00004647-200102000-00009.
8. Sudo S, Wen TC, Desaki J, Matsuda S, Tanaka J, Arai T, et al. Betaestradiol protects hippocampal CA1 neurons against transient forebrain
ischemia in gerbil. Neurosci Res. 1997;29:345–354.
9. Auriat A, Plahta WC, McGie SC, Yan R, Colbourne F. 17beta-Estradiol
pretreatment reduces bleeding and brain injury after intracerebral hemorrhagic stroke in male rats. J Cereb Blood Flow Metab. 2005;25:247–256.
doi: 10.1038/sj.jcbfm.9600026.
10. Nakamura T, Hua Y, Keep RF, Park JW, Xi G, Hoff JT. Estrogen therapy for experimental intracerebral hemorrhage in rats. J Neurosurg.
2005;103:97–103. doi: 10.3171/jns.2005.103.1.0097.
11. Nakamura T, Xi G, Keep RF, Wang M, Nagao S, Hoff JT, et al. Effects
of endogenous and exogenous estrogen on intracerebral hemorrhageinduced brain damage in rats. Acta Neurochir Suppl. 2006;96:218–221.
12. Bendale DS, Karpe PA, Chhabra R, Shete SP, Shah H, Tikoo K. 17-β
Oestradiol prevents cardiovascular dysfunction in post-menopausal
metabolic syndrome by affecting SIRT1/AMPK/H3 acetylation. Br J
Pharmacol. 2013;170:779–795. doi: 10.1111/bph.12290.
13.Elbaz A, Rivas D, Duque G. Effect of estrogens on bone marrow
adipogenesis and Sirt1 in aging C57BL/6J mice. Biogerontology.
2009;10:747–755. doi: 10.1007/s10522-009-9221-7.
14. Ma Q, Chen S, Hu Q, Feng H, Zhang JH, Tang J. NLRP3 inflammasome
contributes to inflammation after intracerebral hemorrhage. Ann Neurol.
2014;75:209–219. doi: 10.1002/ana.24070.
15. Hu Q, Chen C, Yan J, Yang X, Shi X, Zhao J, et al. Therapeutic application of gene silencing MMP-9 in a middle cerebral artery occlusioninduced focal ischemia rat model. Exp Neurol. 2009;216:35–46. doi:
10.1016/j.expneurol.2008.11.007.
16. Godoy DA, Piñero GR, Svampa S, Papa F, Di Napoli M. Hyperglycemia
and short-term outcome in patients with spontaneous intracerebral hemorrhage. Neurocrit Care. 2008;9:217–229. doi: 10.1007/
s12028-008-9063-1.
17. Lee SH, Kim BJ, Bae HJ, Lee JS, Lee J, Park BJ, et al. Effects of glucose
level on early and long-term mortality after intracerebral haemorrhage:
the Acute Brain Bleeding Analysis Study. Diabetologia. 2010;53:429–
434. doi: 10.1007/s00125-009-1617-z.
18. Kimura K, Iguchi Y, Inoue T, Shibazaki K, Matsumoto N, Kobayashi K,
et al. Hyperglycemia independently increases the risk of early death in
acute spontaneous intracerebral hemorrhage. J Neurol Sci. 2007;255:90–
94. doi: 10.1016/j.jns.2007.02.005.
19. Stead LG, Jain A, Bellolio MF, Odufuye A, Gilmore RM, Rabinstein
A, et al. Emergency Department hyperglycemia as a predictor of early
mortality and worse functional outcome after intracerebral hemorrhage.
Neurocrit Care. 2010;13:67–74. doi: 10.1007/s12028-010-9355-0.
20.Song EC, Chu K, Jeong SW, Jung KH, Kim SH, Kim M, et al.
Hyperglycemia exacerbates brain edema and perihematomal cell
death after intracerebral hemorrhage. Stroke. 2003;34:2215–2220. doi:
10.1161/01.STR.0000088060.83709.2C.
21.Allen CL, Bayraktutan U. Antioxidants attenuate hyperglycaemiamediated brain endothelial cell dysfunction and blood-brain barrier
hyperpermeability. Diabetes Obes Metab. 2009;11:480–490. doi:
10.1111/j.1463-1326.2008.00987.x.
22. Kamada H, Yu F, Nito C, Chan PH. Influence of hyperglycemia on oxidative stress and matrix metalloproteinase-9 activation after focal cerebral
ischemia/reperfusion in rats: relation to blood-brain barrier dysfunction.
Stroke. 2007;38:1044–1049. doi: 10.1161/01.STR.0000258041.75739.
cb.
23. Esposito K, Nappo F, Marfella R, Giugliano G, Giugliano F, Ciotola
M, et al. Inflammatory cytokine concentrations are acutely increased
by hyperglycemia in humans: role of oxidative stress. Circulation.
2002;106:2067–2072.
24. Liu J, Gao BB, Clermont AC, Blair P, Chilcote TJ, Sinha S, et al.
Hyperglycemia-induced cerebral hematoma expansion is mediated by
plasma kallikrein. Nat Med. 2011;17:206–210. doi: 10.1038/nm.2295.
25. Li Y, Ogle ME, Wallace GC IV, Lu ZY, Yu SP, Wei L. Erythropoietin
attenuates intracerebral hemorrhage by diminishing matrix metalloproteinases and maintaining blood-brain barrier integrity in mice. Acta
Neurochir Suppl. 2008;105:105–112.
26. Shin JA, Yang SJ, Jeong SI, Park HJ, Choi YH, Park EM. Activation
of estrogen receptor β reduces blood-brain barrier breakdown following ischemic injury. Neuroscience. 2013;235:165–173. doi: 10.1016/j.
neuroscience.2013.01.031.
27. Sandoval KE, Witt KA. Age and 17β-estradiol effects on blood-brain
barrier tight junction and estrogen receptor proteins in ovariectomized
rats. Microvasc Res. 2011;81:198–205. doi: 10.1016/j.mvr.2010.12.007.
28.Rutkowsky JM, Wallace BK, Wise PM, O’Donnell ME. Effects of
estradiol on ischemic factor-induced astrocyte swelling and AQP4 protein abundance. Am J Physiol Cell Physiol. 2011;301:C204–C212. doi:
10.1152/ajpcell.00399.2010.
29. Liu R, Wen Y, Perez E, Wang X, Day AL, Simpkins JW, et al. 17betaEstradiol attenuates blood-brain barrier disruption induced by cerebral
ischemia-reperfusion injury in female rats. Brain Res. 2005;1060:55–61.
doi: 10.1016/j.brainres.2005.08.048.
30. Dubal DB, Zhu H, Yu J, Rau SW, Shughrue PJ, Merchenthaler I, et al.
Estrogen receptor alpha, not beta, is a critical link in estradiol-mediated
protection against brain injury. Proc Natl Acad Sci U S A. 2001;98:1952–
1957. doi: 10.1073/pnas.041483198.
31.Kao CH, Chang CZ, Su YF, Tsai YJ, Chang KP, Lin TK, et al.
17β-Estradiol attenuates secondary injury through activation of Akt
signaling via estrogen receptor alpha in rat brain following subarachnoid hemorrhage. J Surg Res. 2013;183:e23–e30. doi: 10.1016/j.
jss.2013.01.033.
32. Vegeto E, Belcredito S, Etteri S, Ghisletti S, Brusadelli A, Meda C, et al.
Estrogen receptor-alpha mediates the brain antiinflammatory activity of
estradiol. Proc Natl Acad Sci U S A. 2003;100:9614–9619. doi: 10.1073/
pnas.1531957100.
33. Han L, Wang P, Zhao G, Wang H, Wang M, Chen J, et al. Upregulation
of SIRT1 by 17β-estradiol depends on ubiquitin-proteasome degradation
of PPAR-γ mediated by NEDD4-1. Protein Cell. 2013;4:310–321. doi:
10.1007/s13238-013-2124-z.
17β-Estradiol Attenuates Hematoma Expansion Through Estrogen Receptor α/Silent
Information Regulator 1/Nuclear Factor-kappa B Pathway in Hyperglycemic
Intracerebral Hemorrhage Mice
Yun Zheng, Qin Hu, Anatol Manaenko, Yang Zhang, Yan Peng, Liang Xu, Junjia Tang, Jiping
Tang and John H. Zhang
Downloaded from http://stroke.ahajournals.org/ by guest on June 14, 2017
Stroke. published online December 18, 2014;
Stroke is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2014 American Heart Association, Inc. All rights reserved.
Print ISSN: 0039-2499. Online ISSN: 1524-4628
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://stroke.ahajournals.org/content/early/2014/12/18/STROKEAHA.114.006372
Data Supplement (unedited) at:
http://stroke.ahajournals.org/content/suppl/2015/01/30/STROKEAHA.114.006372.DC1
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
in Stroke can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office.
Once the online version of the published article for which permission is being requested is located, click
Request Permissions in the middle column of the Web page under Services. Further information about this
process is available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Stroke is online at:
http://stroke.ahajournals.org//subscriptions/
ONLINE SUPPLEMENT
Supplementary Figures
A.)Experiment)1)
Blood)glucose)measure)
Collagenase)DX/Saline)
0h)
3h) 4h) 5h) 6h)
B.)Experiment)2)
Collagenase)
0h)
24h)
72h)
E2) DX)
Hemorrhagic)volume)
Garcia)scores)
(24h,)72h))
1h) 3h)
24h)
C.)Experiment)3)
Sirt1)
siRNA) Collagenase) MPP) E2) DX)
S48h)
Hemorrhagic)volume)
Garcia)scores)
(6h,)24h,)72h))
0h)
45min) 1h) 3h)
72h)
Hemorrhagic)volume)
Garcia)scores)
Western)blot:)ERα,)Sirt1,)NF)SκB))
Zymography:)MMPS9)
24h)
Supplementary figureⅠ. Experimental design. Experiment 1 is to investigate the effects and time course of hyperglycemia on HE (A). DX or saline was injected intraperitoneally 3 hours after ICH. Blood glucose was measured at 3h, 4h, 5h and 6h after ICH. Hemorrhagic volume and Garcia scores were detected at 6h, 24h, and 72h after ICH. Experiment 2 is to study the outcome of E2 on hyperglycemia enhanced HE and neurological deficits in ICH mice. E2 was administrated intraperitoneally at 1h after ICH, Hemorrhagic volume and Garcia scores were detected at 24h and 72h after ICH. C, Experiment 3 is to investigate the potential mechanisms of E2 on BBB protection (C). ERα inhibitor MPP or Sirt1 siRNA was administrated at 45min after or 48h before ICH respectively; hemorrhagic volume and Garcia scores were detected at 24h after ICH. Protein expression of ERα, Sirt1, acetylated NF-­‐κB, and activity of MMP-­‐9 were detected. 1""""""""""""""""2"""""""""""""""""3""""""""""""""4"
Sirt1&
β(ac+n&
Supplement figure Ⅱ. Representative western blots 48 hours after administration of Sirt1 siRNA in Sham and ICH animals. Sirt1 siRNA strongly knocked down the expression of Sirt1 in both Sham and ICH mice compared with scramble siRNA. *p<0.05 vs. Sham+Scramble siRNA; # p<0.05 vs. ICH+DX+Scramble siRNA. n=6 for each group. Supplement figure Ⅲ. Hemorrhagic volume and neurobehavioral scores with or without mannitol administration 24 hours after ICH. Mannitol had no effects on Hemorrhagic volume (A) and neurobehavioral scores (B) when injection at 3 hours after ICH compared with ICH+Vehicle group. *p<0.05 vs. Sham. Sham, n=6; ICH+Vehicle, n=5; ICH+Mannitol, n=5.