Brain Research 1722 (2019) 146361

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Research report

Kaempferol attenuates neuroinflammation and blood brain barrier T

dysfunction to improve neurological deficits in cerebral ischemia/
reperfusion rats
⁎
Wei-Han Lia,b, Xiao Chenga,b, Ying-Lin Yanga,b, Man Liub, Shan-Shan Zhangb, Yue-Hua Wanga,b, ,
⁎
Guan-Hua Dua,b,
a
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union
Medical College, Beijing 100050, China
b
Beijing Key Laboratory of Drug Target Identification and New Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical
College, Beijing 100050, China

H I GH L IG H T S

• Kaempferol attenuates inflammation after cerebral ischemia reperfusion.

• Kaempferol protects blood brain barrier integrity after cerebral ischemia reperfusion.
• The anti-inflammatory mechanism of kaempferol might be mediated by NF-κB pathway.

A R T I C LE I N FO A B S T R A C T

Keywords: Kaempferol has been reported to act as an anti-inflammatory agent in LPS-induced neuroinflammation in vitro
Cerebral ischemia/reperfusion (I/R) and in vivo, but its role in the inflammation after cerebral ischemia/reperfusion (I/R) is unclear. The present
Inflammation study was to investigate the effect of kaempferol on inflammation in ischemic brain tissue and explore its me-
Cytokines chanisms in cerebral I/R rats. Cerebral I/R rat model was established by middle cerebral artery occlusion for
Microglial activation
60 min and following reperfusion. Kaempferol at doses of 25, 50 and 100 mg/kg was administered for 7 days
NF-κB pathway
after cerebral I/R. Kaempferol treatment significantly reduced cerebral infarct volume, attenuated inflammation
and blood-brain barrier (BBB) disruption after cerebral I/R, thus improved neurological outcomes at the day 7
after cerebral I/R. Furthermore, the results also showed kaempferol treatment decreased the phosphorylation
and nuclear transposition of transcription factor NF-κB p65, thus inhibited expression of various pro-in-
flammatory proteins. In conclusion, kaempferol attenuates neuroinflammation and blood brain barrier dys-
function to improve neurological deficits in cerebral I/R rats, its mechanism is related to NF-κB pathway.

1. Introduction
Ischemic stroke is a neurologic disorder characterized by blocking
cerebral artery, reducing oxygen and glucose supply to brain and
causing damage to brain cells (Dawson and Dawson, 2017). Restoring
blood supply is critical in the treatment of ischemic stroke, one standard
therapeutic approach is to recanalize cerebral vascular and restore
blood flow using thrombolytic drug such as tissue plasminogen acti-
vator (Lekoubou et al., 2017). However, the thrombolytic drug needs to
be administered at a very limited time (e.g., 4.5 h) after the onset of
symptom (Manning et al., 2014; Thomalla et al., 2018). This poses a
challenge for the patients to obtain an effective therapy. Also, even
though the therapy is initiated by restoring blood flow, the damage can
still continue in neurons and glial cells due to reperfusion injury (Leech
et al., 2019). Therefore, controlling the pathological processes after
reperfusion is important to limit neurological damage (Wicha et al.,
2017; Zhang et al., 2018a,b).
Many evidences suggest the damage of reperfusion in neurons and
glial cells is mainly caused by inflammatory injury (Hoogland et al.,
2015; Mu et al., 2016). Many cellular events have been proposed to

⁎
Corresponding authors at: Beijing Key Laboratory of Drug Target Identification and New Drug Screening, Institute of Materia Medica, Chinese Academy of
Medical Sciences & Peking Union Medical College, Beijing 100050, China.
E-mail addresses: wangyuehua@pku.org.cn (Y.-H. Wang), dugh@imm.ac.cn (G.-H. Du).

https://doi.org/10.1016/j.brainres.2019.146361
Received 19 June 2019; Received in revised form 29 July 2019; Accepted 31 July 2019
Available online 01 August 2019
0006-8993/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
W.-H. Li, et al.
contribute to inflammatory process after cerebral ischemia/reperfusion
(I/R), such as activation of microglia. However, the roles of microglia in
the inflammatory mechanisms following cerebral I/R are not fully un-
derstood (Jiang et al., 2014; Jin et al., 2010; Lin et al., 2016; Zhan et al.,
2016). In addition, inflammation causes mitochondrial dysfunction,
reducing superoxide dismutase (SOD) activities, disturbing balance
between generating and detoxifying of reactive oxygen species (ROS)
(Dawson and Dawson, 2017; Goyal et al., 2017; Li et al., 2017). Such
radicals attack cell membrane lipids, proteins, and glycosaminoglycans,
causing further damage (Sies et al., 2017). Based on this information,
understanding and interfering with the inflammatory process following
cerebral I/R are promising to find new therapeutic strategies for is-
chemic stroke.
Kaempferol (3, 4, 5, 7, -tetrahydroxyflavone, KAE), a natural fla-
vonoid in variety of plants and some of traditional medicines, has been
increasingly investigated on its anti-inflammation and anti-oxidative
effects (Rajendran et al., 2014; Yan et al., 2014). Its anti-inflammatory
effect has been reported in cultured microglia and animal inflammatory
models induced by LPS (Cheng et al., 2018; Yang et al., 2019b; Park
et al., 2011). Although the anti-inflammatory effect of kaempferol is
clear, its therapeutic potential and molecular mechanisms on in-
flammation caused by stroke remain unclear. In present study, we in-
vestigated kaempferol therapeutic effect in cerebral I/R rats and ex-
plored its potential mechanisms.
2. Results
2.1. Kaempferol reduced infarct volume and improved neurological deficits
in cerebral I/R rats
The TTC staining and neurological deficits score were evaluated to
investigate the protective effects of kaempferol on the ischemic brain
tissues after cerebral I/R. The data showed that the infarct size of brain
tissues was decreased in a dose-dependent manner by kaempferol
treatment of 25-100 mg/kg. (Fig. 1A, 1B). Additionally, the brain tis-
sues in kaempferol treatment groups showed a larger proportion of is-
chemic penumbra, especially at the high dose of 100 mg/kg. Neurolo-
gical deficits score was calculated to evaluate the protection of
kaempferol on neurological function after I/R. We chose rats exhibiting
neurological deficits with the score of 1-3 at the 24 h after reperfusion
for the following experiments. On the day 1, neurological deficits
showed no difference among ischemic groups. On the day 7, kaempferol
treatment at the dose of 100 mg/kg significantly decreased neurological
deficits score (Fig. 1C).
2.2. Kaempferol attenuated microglia activation in cerebral I/R rats
Microglia is the major immune cell in the central nervous system, its
activation is one of the hallmarks of neuroinflammation. We performed
immunofluorescence staining to assess the effect of kaempferol on ac-
tivation of microglia. Iba-1 is a marker of activated microglia and often
used to characterize microglia cells, the activation of microglia cells
includes the increasing in number, size and morphological change of
microglia cells.
As shown in the results (Fig. 2A), the number and size of microglia
were significantly increased in cerebral I/R group as compared to Sham
group. Also, the number and size of activated microglia was decreased
in a dose-dependent manner by kaempferol treatment of 25–100 mg/kg
(Fig. 2B). These data suggested that kaempferol treatment attenuated
inflammation of ischemic brain tissues by inhibiting the microglia ac-
tivation after cerebral I/R.
2.3. Effect of kaempferol on inflammatory cytokines in cerebral I/R rats
To further evaluate the effect of kaempferol treatment on in-
flammatory cytokines secretion, we conducted V-PLEX assays using
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Brain Research 1722 (2019) 146361
Meso-scale Multiplex Discovery plate to measure total nine in-
flammatory cytokines in brain tissues. As shown in Fig. 3, there was a
significant increase in the production of KC/GRO (Fig. 3A), TNFα
(Fig. 3B), IL-1β (Fig. 3C), IL-5 (Fig. 3D) and IL-6 (Fig. 3E) in cerebral I/
R rats as compared to sham group (All p < 0.01). However, there were
no significant changes on IFN-γ (Fig. 3F), IL-4 (Fig. 3G), IL-13 (Fig. 3H),
IL-10 (Fig. 3I) in I/R group as compared to sham group. Following
kaempferol treatment, the elevated production of KC/GRO (Fig. 3A)
and IL-5 (Fig. 3D) was significantly decreased at doses of 25, 50 and
100 mg/kg. While TNF-α (Fig. 3B), IL-1β (Fig. 3C) and IL-6 (Fig. 3E)
was decreased significantly at higher dose of 100 mg/kg (Fig. 3B).
2.4. Effects of kaempferol on the transcription and expression of iNOS and
COX-2 in cerebral I/R rats
To determine whether kaempferol inhibits other pro-inflammatory
proteins in addition to inflammatory cytokines, the mRNA and protein
expression of iNOS and COX-2 were examined by RT-PCR and Western
blotting. Compared with the Sham group, the mRNA and protein ex-
pression of iNOS and COX-2 were both significantly increased in cere-
bral I/R group (Fig. 4A and Fig. 4B). Following kaempferol treatment,
the expression of iNOS and COX-2 were dramatically attenuated
(Fig. 4C and Fig. 4D). These results indicated that the inhibition of iNOS
and COX-2 transcription and protein expression might be one of me-
chanisms of kaempferol to attenuate the inflammatory response after
cerebral I/R.
2.5. Cytokine profile in ischemic cerebral tissue after in cerebral I/R rats
To clarify the roles of cytokines playing in cerebral I/R, we set out a
cytokine profile using the Rat Cytokine Array Q67 (RayBiotech). The
above results have shown kaempferol at dose of 100 mg/kg exerts
strong anti-inflammatory effect. Therefore, 100 mg/kg kaempferol
treatment was selected for further study. We measured the 67 cytokines
expression in ischemic cerebral tissue from Sham, cerebral I/R, cerebral
I/R + KAE-100 groups. In comparison with Sham group, 21 cytokines
were found to be differently expressed in response to I/R (Fig. 5A). Out
of the 21 cytokines, 13 cytokines marked in red (Fig. 5A) are upregu-
lated and other 9 cytokines marked in blue (Fig. 5A) are down-
regulated. From KEGG enrichment (Fig. 5B), we found the regulated
genes mainly enriched in Th cell differentiation and TNF, JAK-STAT
signaling pathway. The results also showed kaempferol treatment suc-
cessfully regulated proteins expression of 12 cytokines inculding TIMP-
1, MCP-1, ICAM-1, Notch-1, Notch-2, CNTF, P-Cadherin, Decorin,
Neuropilin-1, Galectin-1, Gas 1, and PDGF-AA after cerebral I/R
(Fig. 5C).
2.6. Effect of kaempferol on phosphorylation and nuclear translocation of
NF-κB p65 in cerebral I/R rats
Microglia activation results in the activation of many signaling
pathways during the inflammatory response. The above results showed
that kaempferol attenuated inflammatory response in cerebral I/R rats.
However, the detailed signaling pathways and its regulation mechan-
isms are still unclear. We performed Western blotting to measure the
phosphorylation of NF-κB p65 and IκB on day 7 in Sham, cerebral I/R
and cerebral I/R + 100 mg/kg kaempferol treatment groups. The re-
sults showed that kaempferol treatment at dose of 100 mg/kg sig-
nificantly decreased phosphorylation of NF-κB p65 (Fig. 6A). In com-
parison, IκB was moderately phosphorylated in I/R group and
kaempferol had less effect on IκB phosphorylation (Fig. 6B). These re-
sults suggested kaempferol inhibited NF-κB activation by decreasing
phosphorylation of NF-κB p65.
The phosphorylated NF-κB p65 can function as a transcriptional
activator to increase expression of pro-inflammatory cytokines and
exacerbate inflammation. To investigate translocation of NF-κB p65, we
W.-H. Li, et al.
Fig. 1. Effect of kaempferol on neurological function and cerebral infarction in cerebral I/R rats. (A) Representative images of TTC staining. (B) Statistical analysis of
infarct volume ratio (n = 4–5 in each group). (C) Statistical analysis of neurological scores at the day 7 (n = 6 in each group). Data were presented as mean ± SEM.
##
p < 0.01 vs. sham group; *p < 0.05 vs. I/R group.
extracted nuclear protein and cytoplasm protein using nuclear-cytosol
extraction kit and performed Western blotting to detect the expression
of NF-κB p65 in each sample. There were only small amounts of NF-κB
p65 detected in nuclear fraction in Sham group but significantly higher
in I/R group (p < 0.01, Fig. 6C). The results indicated I/R activated
NF-κB signal pathway and triggered NF-κB p65 nuclear translocation
from cytoplasm to nucleus. When treated with kaempferol at the dose of
100 mg/kg, the NF-κB p65 nuclear transposition was significantly
blocked (p < 0.05, Fig. 6C). In comparison, the amounts of NF-κB p65
had no significant difference in the cytoplasm fraction of Sham, cere-
bral I/R cerebral I/R and cerebral I/R + kaempferol treatment groups
(Fig. 6D).
2.7. Kaempferol inhibited MMP-3 expression in ischemic cerebral tissue and
decreased BBB permeability in cerebral I/R rats
Studies suggested inflammatory cytokines directly affected the
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Brain Research 1722 (2019) 146361
permeability of BBB and exaggerated BBB disruption. In our study, we
tested BBB permeability by measuring EB leakage. The amounts of EB
leakage in ischemic hemisphere significantly increased in I/R group at
the day 7 after surgery. Kaempferol treatment markedly attenuated the
leakage of EB in ischemic hemisphere (Fig. 7A). The finding demon-
strated kaempferol treatment protected BBB integrity, decreasing the
BBB permeability in cerebral I/R rats.
Matrix metalloproteinase 3 (MMP-3) is capable of breaking down
many matrix compounds and activating other MMPs. In our study, we
found the expression of this matrix metalloproteinase was upregulated
significantly after I/R and kaempferol treatment decreased its expres-
sion (Fig. 7B). Kaempferol attenuated MMP-3 expression in cerebral I/R
rats might be its one of the mechanisms to protect BBB integrity.
3. Discussion
The roles played by inflammation are changing in different stages of
W.-H. Li, et al.
Fig. 2. Effect of kaempferol on microglia activation in ischemic cortices of
cerebral I/R rats. (A) Representative images of microglia cells in cortices of
ischemic hemispheres with positive immunochemical staining to Iba-1, scale
bar = 50 μm. (B) Statistical analysis of integrated fluorescence intensity of Iba-
1. Data were presented as mean ± SEM (n = 3 in each group). ##p < 0.01 vs.
sham group; *p < 0.05, **p < 0.01 vs. I/R group.
stroke (Dawson and Dawson, 2017). During the acute phase, ischemic
tissue secretes pro-inflammatory cytokines rapidly, causing activation
of microglia and inflammatory response. Increasing MCP-1 and ICAM-1
promote leukocytes across the BBB into brain parenchyma, releasing
more pro-inflammatory cytokines, which further amplify the in-
flammation and cause death of neurons (Jin et al., 2013). However, in
chronic phase, inflammation is regarded as a beneficial factor to tissue
repair and functional recovery. Cytokines orchestrate the pathological
processes in different stages after stroke (Lambertsen et al., 2012).
Accordingly, understanding mechanisms after stroke is important to
find new therapeutic strategies to stroke.
In this study, we evaluated the protective effect of kaempferol to
cerebral I/R injury and explored its mechanism. We found kaempferol
treatment at dose of 100 mg/kg reduced infarct volume and neurolo-
gical deficits significantly after 7 days treatment. Then we set out a
cytokine profile using protein array to explore the regulatory roles of
different cytokines. From KEGG enrichment, we found the significantly
regulated genes after ischemic stroke mainly enriched in immune cells
differentiation and inflammatory signal pathways. This means in-
flammatory response still plays a key role at the day 7 after cerebral I/
R.
Kaempferol was reported as an anti-inflammatory agent eliciting
protection to LPS-induced inflammation and microglia activation
(Cheng et al., 2018; Park et al., 2011). It also attenuated the expression
of adhesion molecules, reduced oxidative stress. However, its protective
effect on cerebral I/R rats have not been documented. In this study, we
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Brain Research 1722 (2019) 146361
firstly assessed effect of kaempferol on activation of microglia after
cerebral I/R. If microglia increased in number, size and changed in
morphology, they were judged as activated (Hoogland et al., 2015). In
the current study, we found that kaempferol inhibited the activation of
microglia after 7 days treatment in cerebral I/R rats.
Pro-inflammatory cytokines are biomarkers to evaluate extent of
inflammation (Doll et al., 2014). However, inflammatory cytokines are
complicated regulatory systems and strokes include a lot of subtypes in
clinical. Therefore, the prognostic value of inflammatory markers is
controversial in stroke patients. Active microglia secrete lots of in-
flammatory cytokines such as TNF-α, IL-6, IL-1β, which orchestrate
various molecular mechanisms involved in inflammation (Shabab et al.,
2017). Initial activation of microglia is beneficial to protect neurons,
but excessive inflammatory cytokines led to over-activation of micro-
glia which caused a cascade of inflammatory cytokines and un-
controlled inflammation. In our results, TNF-α, IL-6, IL-1β, IL-5, KC/
GRO were increased after cerebral I/R and attenuated by kaempferol
treatment. Interestingly, we found IL-2 and its receptor were down-
regulated in cerebral I/R group. This might be related to the decreasing
number of regulatory T cells (Tregs). Tregs are known to protect against
ischemic stroke, prevent autoimmunity and modulate inflammation
after cerebral I/R (Zhang et al., 2018a,b). However, kaempferol treat-
ment has no significant effect to the IL-2 level in cerebral I/R rats.
Besides, protein array results showed the increasing MCP-1 and
ICAM-1 could be significantly attenuated by kaempferol treatment.
MCP-1 is a chemokine that regulates migration and infiltration of
monocytes. ICAM-1 is an adhesion molecule promoting leukocyte trans-
endothelial migration (Jin et al., 2013). Both of two proteins promote
immune cell across the brain blood barrier into ischemic tissue, re-
leasing more pro-inflammation cytokines and exacerbating in-
flammatory response. Another important inflammatory biomakers,
iNOS and COX-2 were also decreased by kaempferol. They were vastly
secreted by microglia after being activated by reperfusion damage and
caused series of inflammatory response (Balami et al., 2013;
Lambertsen et al., 2012). In conclusion, kaempferol inhibited over-ac-
tivation of microglia and expression of pro-inflammatory protein in
cerebral I/R rats.
BBB integrity is important to function of nervous system (Zuo et al.,
2019). However, many neurologic diseases such as stroke caused neu-
roinflammation, oxidative stress and apoptosis, which break the BBB
integrity (Li et al., 2019). In this study, we performed EB leakage ex-
periment to test BBB integrity. The results showed EB leakage increased
significantly in cerebral I/R rats and kaempferol decreased EB leakage
into brain. From the result of the cytokine profile, we found the TIMP-1
expression increasing in I/R group and downregulated by kaempferol
treatment. TIMP-1 is natural inhibitors of MMPs and the balance be-
tween TIMP-1 and MMPs plays an important role in inflammation
(Eisner et al., 2017). It has been reported that kaempferol could sup-
press expression of MMPs (especially for MMP-3) in LPS-induced mi-
croglia activation (Park et al., 2011). Therefore, we measured the MMP-
3 expression in cerebral I/R rats, data showed MMP-3 expression in-
creased after I/R and was suppressed by kaempferol treatment. These
results indicated kaempferol protected BBB integrity, which might be
mediated by inhibiting MMP-3 expression.
NF-κB p65 is an essential transcription factor involved in many
types of cellular processes, phosphorylation of NF-κB p65 is required for
NF-κB activation and promoting transcription factor activity (Diel et al.,
2011; Oeckinghaus et al., 2011; Yang et al., 2019a). NF-κB p65 was
activated in ischemic hemisphere, as determined by increased expres-
sion of an NF-κB driven reporter transgene (Schneider et al., 1999).
Inhibiting NF-κB prior to MCAO protected ischemic cerebral tissue and
reduced infarct size (Anttila et al., 2017). Based on the information,
pharmacological inhibition of NF-κB is a rational therapeutic target to
diseases involved with NF-κB. Kaempferol was reported to inhibit NF-
κB activation in isoproterenol-induced heart failure in diabetic rats
(Zhang et al., 2019). However, the effect of kaempferol on NF-κB
W.-H. Li, et al. Brain Research 1722 (2019) 146361

Fig. 3. Effect of kaempferol on inflammatory cytokines in ischemic cortices of cerebral I/R rats. The concentrations of each inflammatory cytokines were shown as
pg/mg protein. Data were presented as mean ± SEM (n = 6 in each group). ##p < 0.01 vs. sham group; *p < 0.05, **p < 0.01 vs. I/R group.

activation after ischemic stroke has not been reported. In this study, we
investigated the mechanisms to attenuate neuroinflammation following
kaempferol treatment and determined if NF-κB signal pathway was
involved in the pathological processes after cerebral I/R. Since phos-
phorylation is a critical post-translational modification that regulates
the activities of key components in NF-κB signaling pathway, we per-
formed Western blotting to verify the phosphorylation changes of NF-
κB p65 and its inhibitory protein IκB on day 7 after surgery. Phos-
phorylation of NF-κB p65 was significantly reduced at treatment of
100 mg/kg kaempferol, while kaempferol treatment has less effect on
the phosphorylation of IκB. This demonstrated that kaempferol in-
hibited NF-κB pathway mainly from inhibiting phosphorylation of NF-
κB p65. In additon, the effect of kaempferol on nuclear translocation of
NF-κB p65 was also investigated in this study. The data showed
kaempferol treatment significantly inhibited NF-κB p65 nuclear trans-
location after cerebral I/R. NF-κB p65 functions as a direct transcrip-
tional activator in cell nuclear, it could also affect promoter accessi-
bility to transcription factors and thereby indirectly regulate their gene
expression. In conclusion, the results suggested that the inhibition of
phosphorylation and nuclear translocation of NF-κB p65 is the one of
the mechanisms of kaempferol to attenuate inflammation after cerebral
I/R. This data is consistent with the studies that has shown transcription
factor NF-κB p65 is involved in the regulation of inflammation response
after stroke (Schneider et al., 1999).
In our study, kaempferol was proved to be a potential therapeutic
strategy to attenuate inflammation, reducing cerebral infarct volume
and improving neurological outcome in experimental rat stroke model.
Inhibiting NF-κB pathway might be key factor among the mechanisms
of kaempferol protecting from brain tissue damage after I/R injury.
However, the pathological processes are complicated during cerebral I/
R, further studies are needed to explore the other signal pathways and
investigate their interaction with NF-κB. In addition, the inflammatory
response is very complicated and lasts months after stroke. Further
studies are needed to evaluate kaempferol in a long term. The results
obtaining from the current study provided useful evidences in devel-
oping kaempferol as a promising therapeutic drug to treat stroke pa-
tients, while the clinical effects have to be proved in the future.
4. Material and methods
4.1. Animals and experiment design
110 male Sprague-Dawley rats (240–260 g) were purchased from
Vital River Laboratory Animal Technology Co. (SCXK (Jing) 2016-
0006, Beijing, China). Animals were housed under standard tempera-
ture and humidity, diurnal lighting conditions and allowed food and tap
water ad libitum. All animal care and experimental procedures were
approved by the ethic committees of Institute of Materia Medical,
Chinese Academy of Medical Sciences & Peking Union Medical College.
The focal cerebral I/R was established by transient MCAO for 60 min
and following reperfusion. Briefly, after acclimatized for 3 days, rats
were anesthetized by the intraperitoneal injection of sodium

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W.-H. Li, et al.
pentobarbital (30 mg/kg). Heating pad was used to maintain a rectal
temperature of 37.0 ± 0.5 °C during ischemia period. The right
common carotid artery (CCA), external carotid artery (ECA) and in-
ternal carotid artery (ICA) were isolated. Then, the standardized nylon
suture (0.36 ± 0.02 mm, 2636A2, Beijing Cinontech, China) was in-
serted from the ECA into the ICA until the maker on suture shown on
the intersection of ECA and ICA, which indicated that the suture suc-
cessfully blocked the MCA. Regional cerebral blood flow (rCBF) was
monitored using Laser Doppler Flowmetry (LDF) (PeriFlux 5000,
PERIMED, Sweden), LDF showed that MCAO reduced blood flow to
20% of the baseline during MCAO. After 60 min MCAO, nylon suture
was removed to allow reperfusion, LDF showed blood flow returned
comparably in all animals. Rats were randomly divided into five
groups: Sham group (Sham), cerebral I/R group (I/R), cerebral I/R with
different doses of kaempferol groups: I/R + KAE 25 mg/kg, I/R + KAE
50 mg/kg and I/R + KAE 100 mg/kg. Rats in the sham operation group
underwent the same procedure without occlusion of the MCA. Rats that
died or were not successfully established after MCAO surgery were
excluded from the experiments. Mortality in ischemic groups was
13.8% (12/87) because of brain damage but not observed in Sham
group. According to a five-point scale used in the study (Yang, et al.,
2003), the rats exhibiting no neurological deficits (score 0) after 24 h
reperfusion were considered not successfully established stroke models
(2/75) and excluded from the experiments. The experiment design and
using of each animal was shown in Fig. 8.
Kaempferol, purchased from Nanjing Zelang Biotechnology Co.,
Ltd., was prepared in saline containing 0.5% sodium carbox-
ymethylcellulose (CMC-Na) and delivered by intragastric at doses of 25,
50 and 100 mg/kg body weight per day for 7 days, respectively. The
rats in the sham group and I/R group were treated with the vehicle
(0.5% CMC-Na in saline) in the same manner. Drug and vehicle were
administered in a blinded fashion.
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Brain Research 1722 (2019) 146361
Fig. 4. Effect of kaempferol on transcription
and expression of iNOS and COX-2 in is-
chemic cortice of cerebral I/R rats. (A)
Protein expression of COX-2 (n = 4 in each
group). (B) mRNA expression of COX-2
(n = 6 in each group). (C) Protein expres-
sion of iNOS (n = 4 in each group). (D)
mRNA expression of iNOS (n = 6 in each
group). Data were presented as
mean ± SEM. #p < 0.05, ##p < 0.01 vs.
sham group; *p < 0.05 vs. I/R group.
4.2. Neurological assessments and infarct volume evaluation
To evaluate the degree of stroke in each animal following the sur-
gical procedure, neurological deficits assessments and brain infarct
volume were evaluated. Neurological deficits were assessed after
kaempferol treatment using neurobehavioral test, which is graded on a
five point scale as described in previous report (Yang, et al., 2003): 0,
no neurological deficits; 1, cannot extend left forelimb fully; 2, cannot
walk straightly; 3, circle to the left; 4, inability to walk spontaneously or
losing consciousness.
After neurological assessments, the rats were decapitated infarct
volume analysis immediately. The brain tissues were dissected cor-
onally into 2-mm slices using a scalpel. The sections were stained by 1%
2,3,5-tripenyltetrazolium chloride (TTC, Sigma, St. Louis, MO, USA) at
37 °C for 30 min, and then fixed in 4% paraformaldehyde at room
temperature overnight prior to imaging. In the stained sections, the red
region represents the viable tissues and the white area indicates the
infarct tissue. The infarct area in each section was evaluated using the
Image J/Fiji processing system (Schindelin, et al., 2012). Infarct volume
ratio = (The sum of infract area/ the sum of sections area) * 100%.
4.3. Immunofluorescence detection of Iba-1
After kaempferol treatment, rats were anesthetized and perfused
intracardially with PBS and then with 4% paraformaldehyde (PFA).
Subsequently, the brain sections were fixed in 4% PFA overnight at 4 °C.
The brain tissue sessions (20 μm) were obtained using a rotary micro-
tome (RM2016, Leica, Germany) by paraffin section method, then im-
munofluorescence was performed on paraffin sections. Firstly, dewax
paraffin section to water and repair antigen in citric acid antigen repair
buffer. Then blocked by 2% BSA at RT for 30 min. Spun the BSA slightly
and incubated the samples with ionized calcium binding adaptor mo-
lecule 1 (Iba-1) primary antibody (Wako: 019-19741, 1:200) at 4 °C
overnight. Primary antibodies were detected by using goat anti-rabbit
secondary antibodies (Wuhan Servicebio technology, China) for 50 min
W.-H. Li, et al. Brain Research 1722 (2019) 146361

Fig. 5. Cytokine profile assayed by Protein Array in ischemic cortices of cerebral I/R rats. (A) Interaction network of significantly regulated genes after cerebral I/R
(upregulation marked by red and downregulation marked by blue). (B) KEGG enrichment of significantly regulated genes after cerebral I/R. (C) Cytokine changes
regulated by kaempferol after cerebral I/R, the concentrations of regulated cytokines were shown as pg/mg protein in Fig. 6. Data were presented as mean ± SEM
(n = 4 in each group). #p < 0.05, ##p < 0.01 vs. sham group; *p < 0.05, **p < 0.01 vs. I/R group.

at room temperature. After washing with PBS for three times, spun PBS
slightly and incubated with CY3 (Servicebio technology) for 10 min and
wash it with TBST for 3 times. Spun slightly and added fluorescent
quencher to the slice, incubated for 5 min. After washing with flowing
water for 10 min, cell nuclei was stained with DAPI (Servicebio
technology) for 10 min. All images were acquired using fluorescence
microscope (Eclipse C1, Nikon, Japan). Fluorescence intensity was
analyzed using the image processing package Image J/Fiji (Schindelin,
et al., 2012). Cortices of ischemic hemispheres were defined to areas to
be analyzed and representative images were presented (3 rats in each

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Fig. 6. Effect of kaempferol on phosphorylation in NF-κB pathway in ischemic cortice of cerebral I/R rats. (A) Phosphorylation of NF-κB p65. (B) Phosphorylation of
IκB. (C) NF-κB p65 in nuclear fraction. (D) NF-κB p65 in cytoplasmic fraction. Data were presented as mean ± SEM (n = 4 in each group). ##p < 0.01 vs. sham
group; *p < 0.05 vs. I/R group.
group and 3 slides per rat).
4.4. Pro-inflammatory cytokine measurement by multiplex enzyme-linked
immunosorbent assay
At the necropsy, the brain tissues were quickly removed and the
cortices of ischemic hemispheres were dissected for frozen in liquid
nitrogen. When conducting the experiment, the tissue samples were
homogenized in PBS buffer and then centrifuged at 13,000 rpm at 4 °C
for 15 min. The supernatant was collected and the total amounts of
protein were determined by BCA protein assay kit (Thermo, Rockford,
USA) according to the manufacturer’s instructions. The levels of IL-1β,
IL-6, TNF-α, IFN-γ, IL-2, IL-4, IL-5, IL-10, and KC/GRO were measured
by immunoassay using V-PLex Pro-inflammmatory Panel 1 (rats) kit
(Meso scale Discovery, Gaithersburg, USA) on the Sector Imager 2400
according to the manufacturer’s instructions. The concentration of each
cytokine was normalized by the total amount of protein as measured
from BCA protein assay.
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Brain Research 1722 (2019) 146361
4.5. Analysis of cytokine profile by protein array assay
The supernatant of ischemic cortices was handled with the same
procedure as Western blot, the amounts of protein were determined by
BCA protein assay kit (Thermo, Rockford, USA). Then we conduct
Protein Array assay (Cat#: QAR-CAA-67, RayBiotech, Norcross, GA)
according to the manufacturer’s instructions. Firstly, block each chip
with blocking buffer for 30 min. Decant the buffer and add 50 μg
samples to each well, incubating chips overnight at 4 °C. Decant the
sample and wash 5 times with washing buffer. After that, incubate each
chip with biotinylated antibody for 2 h, then incubate with Cy3
Equivalent Dye Streptavidin for 1 h, wash 5 times after each incubation.
Finally, detect fluorescence at wave length of 532 nm with InnoScan
300 Microarray Scanner (Innopsys, France). Gene interaction network
was generated by Cytoscape (Shannon et al., 2003) and KEGG enrich-
ment was performed by clusterProfiler database (R/Bioconductor).
4.6. Evans blue (EB) leakage
At the day 7 after surgery, 4% Evans blue dye (Solarbio, Beijing,
China) dissolved in 0.9% saline was injected (0.25 ml/100 g) into the
W.-H. Li, et al. Brain Research 1722 (2019) 146361

Fig. 7. Effect of kaempferol on BBB permeability and

MMP-3 expression in ischemic cortice of cerebral I/R
rats. (A) Representative images of Evans blue
staining and quantitative analysis of Evans blue
content in ischemic hemisphere (n = 6 in each
group). (B) MMP-3 expression. (n = 4 in each group).
Data were presented as mean ± SEM. #p < 0.05 vs.
sham group; *p < 0.05 vs. I/R group.

tail vein of rats in each group. Two hours after injection, the rats were phospho-IκBα was normalized with total amount of NF-κB p65, IκBα,
anesthetized and transcardially perfused with saline until the outflow respectively.
fluid from the right atrium was clear. The brains were removed, dis-
sected coronally into 2-mm and imaged. The ischemic hemispheres
were collected immediately and homogenized in 50% trichloroacetic 4.8. Quantitative Real-Time PCR
acid (100 mg/300 μl) and centrifuged at 13,000 rpm at 4 °C for 5 min.
The supernatant was collected and determined their optical density Total RNA was extracted from the cortices of ischemic brain tissue
(OD) at 620 nm (Cheng et al., 2018). using Trizol reagent (Ambion, Carlsbad, CA, USA) according to the
manufacturer’s instructions. The concentration of RNA was measured
by ultraviolet spectrophotometry and determined using a formula A
4.7. Isolation of proteins and Western blot analysis
260 * dilution * 40 = μg RNA/mL. The cDNA was synthesized using the
PrimeScript™ RT reagent Kit (Takara Bio, Otsu, Japan). The primer
At the end of study, the cortices of ischemic hemisphere were col-
sequences used to measure mRNA expression were listed as followings:
lected to examine the expression of iNOS and COX-2 in each group,
5′-GCTGTACAAGCAGTGGCAAA-3′ (forward) and 5′- GTCTGGAGT
phosphorylation of NF-κB p65 and IκB in Sham, I/R and I/R + KAE-100
GGGAGGCACT-3′ (reverse) for COX-2;
treatment groups and also expression of MMP-3 in the three groups.
5′-TCATTGACCTCAACTACATGGT-3′ (forward) and 5′- CTAAGCA
Besides, the nuclear fraction and cytoplasmic fraction were extracted in
GTTGGTGGTGCAG-3′ (reverse) for GAPDH;
Sham, I/R and I/R + KAE-100 groups with nuclear-cytosol extraction
5′-GCATCCCAAGTACGAGTGGT-3′ (forward) and 5′- CCATGATGG
kit (Applygen Technologies Inc, Beijing, China) according to the man-
TCACATTCTGC-3′ (reverse) for iNOS.
ufacturer’s instructions. Following the extraction, nuclear translocation
The iNOS and COX-2 mRNA expression was measured using quan-
of NF-κB p65 was measured in three groups. The cortices of ischemic
titative Real-Time Polymerase Chain Reaction (RT-PCR) analysis. The
brain tissue were mixed with RIPA buffer including cocktail protease
samples were mixed in a final volume of 25 μL including 2 μL cDNA
inhibitor (100 mg/mL). Then the tissue was homogenized and cen-
synthesized before using SYBR® Premix Ex Taq II (TakaraBio, Otsu,
trifuged at 13000 rpm for 15 min. The supernatant was collected and
Japan). PCR was performed using the following protocol: 30 s at 95 °C,
mixed with loading buffer (Applygen Technologies Inc, Beijing, China)
followed by 40 cycles of 5 s at 95 °C, 30 s at 60 °C, and 15 s at 95 °C, 30 s
and heated at 100 ℃ for 10 min. Proteins were separated by sodium
for 60 °C and 15 s for 95 °C. With the relative quantification of 2−⊿⊿Ct
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
method, the transcription expression of target genes of iNOS, and COX-
transferred to PVDF membrane. The membrane was blocked with 5%
2 was determined using GAPDH as internal reference. GAPDH was
BSA in Tris-buffered saline with Tween-20 (TBS-T) for 1 h and then
wildly reported as a reference gene and validated no change on the day
incubated with anti-COX-2 antibody (ab15191, 1:1000), anti-iNOS
7 after I/R in our study.
antibody (ab204017, 1:500), anti-MMP-3 antibody (ab52915, 1:1000),
anti-phospho NF-κB p65 antibody (CST#3033, 1:1000), anti-NF-κB p65
antibody (CST#8242, 1:1000), anti-phospho IκBα antibody
4.9. Statistical analysis
(CST#2859, 1:1000), anti-IκBα antibody (CST#4814, 1:1000), anti-β-
actin antibody (C1313, 1:1000) and anti-histone H3 antibody
GraphPad Prism software version 7.04 (GraphPad Software, 7825
(orb136531, 1:1000) overnight at 4 °C, respectively. After washed with
Fay Avenue, Suite 230 La Jolla, CA 92037 USA) was performed to
TBS-T for 3 times, the membranes were incubated with secondary an-
compare the differences between each group. Data were presented as
tibody for 2 h at room temperature and then detected using enhanced
mean ± SEM. One-way ANOVA was used to calculate differences be-
ECL system. Optical density values were analyzed using the ImageJ/Fiji
tween the various groups. Tukey’s multiple comparison post hoc test
processing system. The signal density of iNOS, COX-2 and NF-κB p65 in
was performed to determine significance levels. Statistical significance
cytoplasm were normalized by total amount of internal β-actin protein.
was considered at p < 0.05.
The signal density of NF-κB p65 in nuclear was normalized by total
amount of histone H3. The signal density of phospho-NF-κB p65 and

9
W.-H. Li, et al. Brain Research 1722 (2019) 146361

Fig. 8. Experiment design. The rats in Experiment 1 were evaluated for infract volume. The rats in Experiment 2 were evaluated for Immunofluorescence detection of
Iba-1. In experiment 3 and 4, Western blot was performed to detect activation of NF-κB pathway and protein expression of MMP-3, iNOS and COX-2, PCR was
performed to detect mRNA expression, MSD was performed to measure inflammatory cytokine levels and Protein Array was also performed to make the Cytokine
Profile. The rats in Experiment 5 were evaluated for Evens blue leakage.

5. Authors’ contributions Declaration of Competing Interest

Yue-Hua Wang and Guan-Hua Du designed the experiments; Wei-
Han Li, Xiao Cheng, Ying-Lin Yang, Man Liu, and Shan-Shan Zhang
performed the animal experiments; Wei-Han Li and Yue-Hua Wang
analyzed the data and wrote the manuscript; and all authors read and
approved the final manuscript.
All authors declare that they have no competing interests.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.brainres.2019.146361.

Funding
References

This project was supported by the National Key Research &

Development Plan of China (2018YFC0311005); the National Natural
Science Foundation of China (81473383), the Significant New-Drugs
Creation of Science and Technology Major Projects of China
(2018ZX09711001-003-019), the Medical and Health Innovation
Project of Chinese Academy of Medical Sciences in China (2016-I2M-3-
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