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A Cannabinoid Receptor 2 Agonist Prevents Thrombin-Induced Blood-Brain Barrier Damage via the Inhibition of Microglial Activation and Matrix Metalloproteinase Expression in Rats


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Thrombin mediates the life-threatening cerebral edema and blood-brain barrier (BBB) damage that occurs after intracerebral hemorrhage (ICH). We previously found that the selective cannabinoid receptor 2 (CB2R) agonist JWH-133 reduced brain edema and neurological deficits following germinal matrix hemorrhage (GMH). We explored whether CB2R stimulation ameliorated thrombin-induced brain edema and BBB permeability as well as the possible molecular mechanism involved. A total of 144 Sprague-Dawley (S-D) rats received a thrombin (20 U) injection in the right basal ganglia. JWH-133 (1.5 mg/kg) or SR-144528 (3.0 mg/kg) and vehicle were intraperitoneally (i.p.) injected 1 h after surgery. Brain water content measurement, Evans blue (EB) extravasation, Western blot, and immunofluorescence were used to study the effects of a CB2R agonist 24 h after surgery. The results demonstrated that JWH-133 administration significantly decreased thrombin-induced brain edema and reduced the number of Iba-1-positive microglia. JWH-133 also decreased the number of P44/P42(+)/Iba-1(+) microglia, lowered Evans blue extravasation, and inhibited the elevated matrix metallopeptidase (MMP)-9 and matrix metallopeptidase (MMP)-12 activities. However, a selective CB2R antagonist (SR-144528) reversed these effects. We demonstrated that CB2R stimulation reduced thrombin-induced brain edema and alleviated BBB damage. We also found that matrix metalloproteinase suppression may be partially involved in these processes.
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A Cannabinoid Receptor 2 Agonist Prevents Thrombin-Induced
BloodBrain Barrier Damage via the Inhibition of Microglial
Activation and Matrix Metalloproteinase Expression in Rats
Lin Li
&Yihao Tao
&Jun Tang
&Qianwei Chen
&Ya n g Ya n g
&Zhou Feng
Yuji e C h e n
&Liming Yang
&Yunfeng Yang
&Gang Zhu
&Hua Feng
&Zhi Chen
Received: 21 July 2015 /Revised: 31 August 2015 /Accepted: 2 September 2015
#Springer Science+Business Media New York 2015
Abstract Thrombin mediates the life-threatening cerebral
edema and bloodbrain barrier (BBB) damage that occurs
after intracerebral hemorrhage (ICH). We previously found
that the selective cannabinoid receptor 2 (CB2R) agonist
JWH-133 reduced brain edema and neurological deficits fol-
lowing germinal matrix hemorrhage (GMH). We explored
whether CB2R stimulation ameliorated thrombin-induced
brain edema and BBB permeability as well as the possible
molecular mechanism involved. A total of 144 Sprague
Dawley (S-D) rats received a thrombin (20 U) injection in
the right basal ganglia. JWH-133 (1.5 mg/kg) or SR-144528
(3.0 mg/kg) and vehicle were intraperitoneally (i.p.) injected
1 h after surgery. Brain water content measurement, Evans
blue (EB) extravasation, Western blot, and immunofluores-
cence were used to study the effects of a CB2R agonist 24 h
after surgery. The results demonstrated that JWH-133 admin-
istration significantly decreased thrombin-induced brain ede-
ma and reduced the number of Iba-1-positive microglia. JWH-
133 also decreased the number of P44/P42(+)/Iba-1(+) mi-
croglia, lowered Evans blue extravasation, and inhibited the
elevated matrix metallopeptidase (MMP)-9 and matrix
metallopeptidase (MMP)-12 activities. However, a selective
CB2R antagonist (SR-144528) reversed these effects. We
demonstrated that CB2R stimulation reduced thrombin-
induced brain edema and alleviated BBB damage. We also
found that matrix metalloproteinase suppression may be par-
tially involved in these processes.
Keywords Cannabinoid receptor .Thrombin .Bloodbrain
barrier .p44/42 MAPK .Matrix metalloproteinase
Spontaneous intracerebral hemorrhage (ICH) is a devastating
disease. It constitutes 1015 % of all strokes in the USA,
Europe, and Australia and 2030 % of strokes in Asia.
Approximately 2 million cases of ICH are reported annu-
ally worldwide [1]. There is currently no effective treat-
ment for ICH, and it has a 1-month mortality rate of 30 to
50 %. Patients who survive typically have major neuro-
logical impairments [2].
Cerebral edema is primarily responsible for secondary in-
jury after ICH [3]. Edema increases mass effect and intracra-
nial pressure (ICP) following ICH, which may directly dam-
age brain tissue and ultimately result in herniation [3]. Edema
is also directly toxic to neurons and glia by changing osmotic
gradients and disrupting the bloodbrain barrier (BBB) [4,5].
Multiple pathways, including cytotoxic injury due to coagu-
lation factors and a robust inflammatory response, lead to
edema formation, and thrombin is the primary molecule that
mediates the development of acute cerebral edema after ICH
[68]. The inhibition of thrombin with agratroban or hirudin
also reduces edema after ICH in rats [8,9]. Thrombin infu-
sions into the caudate-putamen of the rat brain induces a rapid
increase in edema within several hours that peaks from the
first to the third day, and the edema declines gradually over
several weeks [10,11]. The trend of cerebral edema changes
Lin Li and Yihao Tao contributed equally to this work.
*Zhi Chen
Department of Neurosurgery, Southwest Hospital, Third Military
Medical University, No. 30, Gaotanyan Street, Chongqing 400038,
Peoples Republic of China
Department of Neurosurgery, Sichuan Provincial Corps Hospital,
Chinese Peoples Armed Police Forces, Leshan, PeoplesRepublicof
Transl. Stroke Res.
DOI 10.1007/s12975-015-0425-7
in parallel with changes in BBB permeability [11]. Thrombin
activates many intracellular signaling cascades in brain cells
[12]. P44/42 MAPK, also called extracellular signal-regulated
kinase (ERK), is one MAP kinase that is activated in the brain
after intracerebral infusions of thrombin [13].
The endocannabinoid system, including endogenous ligands,
cannabinoid receptors, and degrading enzymes, is an important
pharmacological target in many neurological diseases [14]. Can-
nabinoid receptor type 1 (CB1R) and cannabinoid receptor type
2 (CB2R) are the most studied cannabinoid receptors [15,16].
The psychoactive effects of cannabinoids are associated with
CB1R, which is predominantly expressed by neurons [17].
The psychoactive effects of CB1R agonists limit their therapeu-
tic potential, which leaves CB2R agonists as the practical option
[18]. CB2R is primarily expressed in immune cells, and it me-
diates anti-inflammatory actions, immune cell migration, cyto-
kine production, and antigen presentation [19]. The anti-
inflammatory and neuroprotective effects of cannabinoids in
the brain were studied in animal models of multiple sclerosis
(MS) and Alzheimersdisease(AD)[20]. These effects were
observed using pharmacological ligands that act on CB1R,
CB2R, or both receptors [18]. We previously demonstrated that
a specific CB2R agonist (JWH133) attenuated brain edema in
rat models of germinal matrix hemorrhage (GMH), but the un-
derlying mechanisms are not known [21].
Increased metalloproteinase (MMP) expression is a key
mechanism for increased BBB permeability after ICH [3].
MMPs disrupt BBB integrity and promote edema by
degrading tight junction proteins, type IV collagen, laminin,
and fibronectin [22,23].
The present study investigated the effects of a CB2R ago-
nist in a rat model of thrombin-induced BBB damage and the
role of MMPs in the neuroprotective process.
Materials and Methods
The selective CB2R agonist JWH133 (Tocris Bioscience) ex-
hibits a very high affinity for the CB2R (Ki=3.4 nmol/L) but
low affinity for the CB1R (Ki=677 nmol/L). SR144528 (Santa
Cruz) is a selective CB2R antagonist. JWH-133 and SR144528
were dissolved in DMSO/ethanol/0.9 % saline (1:1:18) and
injected intraperitoneally into each animal. Untreated animals
received an equal volume of vehicle (DMSO/ethanol/0.9 % sa-
line (1:1:18)). Thrombin (Sigma) was dissolved in saline at a
concentration of 4000 U/ml (163 mg/ml), and thrombin activity
was expressed in NIH units.
Animal Preparation and Groups
Adult male Sprague Dawley rats (250300 g) were housed
under specific pathogen-free conditions with free access to
food and water until use. Animal use procedures complied
with the guide for the care and use of laboratory animals,
and the animal care and use committee at the Third Military
Medical University approved all procedures. All experiments
were designed to minimize the number of animals used and
their suffering.
Animals were randomly assigned to the following groups:
sham-operated (sham group, n=36), thrombin+ vehicle (vehi-
cle group, n=36), thrombin+JWH133 (JWH group, n=36),
and thrombin+SR144528 + JWH133 (SR+JWH group, n=
36). All animals were sacrificed 24 h after surgery. All exper-
imental groups and analyses were performed in accordance
with RIGOR Guidelines for translational research [24,25].
Model induction was performed as previously reported [26].
Briefly, a feedback-controlled heating pad was used to main-
tain body temperature at 37.0 °C. Rats were anesthetized with
an intraperitoneal injection of chloral hydrate (5 %,
350 mg/kg) and placed in a stereotaxic frame. A cranial burr
hole (1 mm) was drilled 4.0 mm lateral to the bregma. Throm-
bin solution in a volume of 5 μl/rat was micro-infused using a
pump at a constant rate of 0.5 μl/min into the right basal
ganglia (coordinates: 0.2 mm anterior, 5.5 mm ventral, and
4.0 mm lateral to the bregma) through a 29-gauge needle.
The injection needle was left in place for at least 5 additional
min to prevent the backflow of drugs. The burr hole was
sealed with bone wax, and skin incisions were closed with
sutures after the needle was removed. A third group received
an intraperitoneal injection of JWH-133 (1.5 mg/kg, JWH
group) 1 h after surgery. A fourth group was treated with
SR144528 (3 mg/kg) with JWH-133 and 3 min later
(1.5 mg/kg) intraperitoneally (SR+JWH group). A second
group of animals was injected with an equal volume of vehicle
(vehicle group). The sham group only received a needle inser-
tion. The doses of JWH133 and SR144528 were selected
based on a previous publication [27].
Brain Water Content Measurement
Brain water content was examined in rats 24 h after surgery, as
previously described [26]. Animals (n= 12, per group) were
anesthetized with an intraperitoneal injection of chloral hy-
drate (5 %, 350 mg/kg). Brains were removed, and a coronal
tissue was sliced (4 mm thickness) around the injection needle
tract. Brain sections were divided into 4 parts: ipsilateral basal
ganglia (Ipsi-BG), ipsilateral cortex (Ipsi-CX), contralateral
basal ganglia (Cont-BG), and contralateral cortex (Cont-
CX). The cerebellum (Cerebel) was the internal control. Brain
sample weights were determined immediately after removal
Transl. Stroke Res.
and after drying for 24 h in a 100 °C oven using an electric
analytical balance. Brain water content (%) was calculated as
(wet weight-dry weight)/wet weight × 100 %.
Evans Blue Assay
Evans blue extravasation was performed 24 h post-surgery as
previously described [28]. Briefly, Evans blue dye from
Sigma-Aldrich (2 %, 4 mL/kg) was injected (>2 min) into
the left femoral vein and allowed to circulate for 60 min. Rats
under anesthesia (5 % chloral hydrate, 350 mg/kg) (n=6)were
euthanized by an intracardial perfusion with phosphate-
buffered solution (PBS), and brains were removed. The right
basal ganglia were harvested for homogenization. Samples
were weighed, homogenized in saline, and centrifuged at 15,
000gfor 30 min. An equal volume of trichloroacetic acid was
added to the resulting supernatant. Samples were incubated
overnight at 4 °C and centrifuged at 15,000gfor 30 min.
The resulting supernatants were spectrophotometrically quan-
tified at 615 nm for the detection of Evans blue dye
Brains for Evans blue fluorescence were removed and
fixed in 4 % paraformaldehyde at 4 °C for 24 h. Brains (n=
6) were prepared for coronal brain sectioning (30 μm), and red
auto-fluorescence of Evans blue was observed on the slides
using a confocal microscope (Zeiss, LSM780) equipped with
a 633 nm HeNe laser. A minimum of 4 images were captured
for each rat.
Immunofluorescence Staining
Immunofluorescence staining was performed as previ-
ously described [27]. The right basal ganglia were in-
fused for 24 h, and rats (n=6, each group) were re-
anesthetized (5 % chloral hydrate, 350 mg/kg) and per-
fused intracardially with PBS followed by 4 % parafor-
maldehyde. Brains were removed, post-fixed in 4 %
paraformaldehyde for 24 h, and dehydrated in a 30 %
sucrose solution for 35 days at 4 °C. Free-floating
coronal brain slices (30 μm thick) were cut using a
cryostat and stored at 20 °C until used. Sections were
rinsed with PBS and permeabilized with 0.3 % Triton
with 10 % goat serum for 1 h and incubated at 4 °C
overnight with a primary rabbit polyclonal anti-Iba1 an-
tibody (1:200; WAKO Pure Chemical Industries Ltd.)
followed by an Alexa 488-labeled goat anti-rabbit IgG
(H+L) (1:500, Beyotime, Wuhan, China) for 3 h at
37 °C. A sequential immunofluorescence protocol was
used for double immunofluorescence with anti-Iba1 and
anti-phospho-p44/42 MAPK antibodies with the
appropriate controls. Briefly, free-floating slices were
incubated with a primary mouse anti-phospho-p44/42
MAPK (1:200; CST) at 4 °C overnight followed by
an Alexa 555-labeled goat anti-mouse IgG (H+L)
(1:500; Beyotime, Wuhan, China) secondary antibody
(3 h, 37 °C). Sections were washed and blocked with
10 % normal goat serum for 1 h. Sections were incu-
bated overnight with the anti-Iba1 antibody followed by
Alexa 488-labeled goat anti-rabbit IgG (H +L) (1:500;
Beyotime, Wuhan, China) secondary antibody incuba-
tion (3 h, 37 °C). The same protocol was used to in-
vestigate the colocalization of ZO-1 and vWF. Sections
were permeabilized with 0.3 % Triton X-100 in PBS for
30 min, blocked with 10 % goat serum for 1 h, and
incubated at 4 °C overnight with primary antibodies:
goat antiZO-1 (1:200, Santa Cruz) and mouse anti-
vWF (1:200, Santa Cruz). Sections were incubated with
appropriate secondary antibodies for 3 h at 37 °C.
Colocalization was examined using a fluorescent micro-
scope (Zeiss, LSM780).
Western Blot Analysis
Western blot assays were performed as described previously
[29]. Protein extraction of the right basal ganglia tissue (n=6),
including the injection site, was performed 24 h after thrombin
injection by tissue homogenization in RIPA buffer (Santa
Cruz) supplemented with protease and phosphatase inhibitors
(Sigma). Homogenates were centrifuged at 14,000×gat 4 °C
for 20 min. Supernatants were whole cell protein extracts and
stored at 80 °C until usage. Tissue samples were taken from
6 rats in each group, and one sample was taken from each
brain. The protein concentration was determined using a
Bio-Rad Laboratories (Hercules, CA, USA) protein assay
kit. A total of 50 μg of protein from each sample was loaded
into each lane of SDS-PAGE gels. Gel electrophoresis was
performed, and proteins were transferred to a nitrocellulose
membrane. The membrane was blocked in Carnation® nonfat
milk and probed with primary and secondary antibodies. The
following primary antibodies were used: anti-phospho-p44/42
MAPK (T202/Y204) (1:1000, CST), anti-p44/42 MAPK
(1:1000, CST), antiβ-Tubulin (1:1000, Santa Cruz), anti-
MMP-9 (1:1000, CST), anti-MMP-12 (1:1000, Abcam),
anti-ZO-1 (1:500, Santa Cruz), and antiGAPDH (1:1000,
Santa Cruz). The membranes were incubated under gentle
agitation at 4 °C overnight, and the membranes were washed
in TBST. Membranes were incubated in the appropriate HRP-
conjugated secondary antibody (diluted 1:1000 in secondary
antibody dilution buffer) for 1 h at 37 °C. Protein bands were
visualized using a nickel-intensified DAB solution, and the
densitometric values were analyzed using ImageJ software.
Transl. Stroke Res.
The housekeeping protein β-tubulin and GAPDH were used
as internal controls.
Statistical Analysis
Data are reported as the means±standard derivation (SD).
SPSS 13.0 software package (SPSS, Inc., Chicago, IL,
USA) was used for statistical analyses. Data were analyzed
using one-way analysis of variance (ANOVA) tests followed
by Student-Newman-Keuls (SNK) tests. A nonparametric test
(Kruskal-Wallis H) was used if the data were not normally
distributed, followed by a Nemenyi test when a two-group
comparison was necessary. Differences were considered sig-
nificant at P<0.05.
Treatment with JWH-133 Decreased Brain Water
Content 24 h After Thrombin Infusion
Brain water content of rats in the vehicle group was signifi-
cantly greater than the sham group 24 h after thrombin infu-
sion, especially in the ipsilateral basal ganglia (Ipsi-BG: sham,
77.45± 0.21 % versus Vehicle, 80.24 ±0.40 %, p<0.05,
Fig. 1). Brain edema in the ipsilateral basal ganglia was sig-
nificantly reduced 24 h after JWH-133 administration (Ipsi-
BG: JWH, 79.32±0.46 % versus vehicle, 80.24± 0.40 %,
p<0.05; versus SR+JWH, 80.13±0.46 %, p< 0.05) compared
to the vehicle and SR+JWH groups. Brain edema in the ipsi-
lateral cortex (Ipsi-CX) was significantly increased at 24 h
(Ipsi-CX: sham, 78.47± 0.21 % versus vehicle, 79.35±
0.39 %, p<0.05), and JWH-133 treatment reduced edema
levels 24 h post-administration (Ipsi-CX: JWH 78.38±
0.40 % versus vehicle, 79.35±0.39 %, p< 0.05; versus SR+
JWH, 79.33±0.41 %, p<0.05) compared with the vehicle and
SR+ JWH groups (n=12).
JWH-133 Administration Suppressed Microglial
Activation Surrounding the Injury Boundary
Iba1 is an indicator of microglial activation. Iba1 immunore-
activity was revealed using fluorescence microscopy to inves-
tigate whether JWH-133 affected microglial activation after
surgery (Fig. 2). No obvious microglial activation was expect-
ed in the sham group. Many activated microglial cells were
widely observed in the vehicle group. The intraperitoneal ad-
ministration of JWH-133 post-surgery greatly reduced the
number of activated microglial cells (Fig. 2a). However, the
selective CB2R antagonist SR144528 reversed this treatment
effect. Similar results were obtained when the number of Iba-1
positive microglia was quantified (Fig. 2b). Cell number quan-
tification using ImageJ software was performed, as indicated
in Fig. 2c, in four pictures of the region surrounding the injury.
JWH-133 Protects Against Thrombin-Induced
BloodBrain Barrier Destruction
We used Evans blue extravasation to evaluate BBB integrity
after surgery. The results demonstrated increased Evans blue
dye leakage from vessels within the boundary of the injection
site 24 h after surgery, and JWH-133 treatment significantly
reduced Evans blue leakage (JWH, 1.70±0.32 versus vehicle,
3.04± 0.38, P<0.05; versus SR+ JWH, 2.65±0.34, P<0.05).
Simultaneous SR144528 administrated abolished this effect
(Fig. 3b,n=6). Evans blue immunofluorescence was per-
formed to confirm the extravasation results (Fig. 3a,n=6),
and the same results were obtained.
JWH-133 Reduces the Phosphorylation Level of p44/42
MAPK After Thrombin Infusion
We examined the phosphorylation level of p44/42 MAPK
using Western blot analysis to further clarify the role of the
p44/42 MAPK pathway. Western blots demonstrated that the
phosphorylation level of p44/42 MAPK markedly increased
in the ipsilateral basal ganglia 24 h after the intracerebral in-
fusion of thrombin compared to the sham group (Fig. 4c,
P<0.05, n=6). However, protein phosphorylation levels were
lower in the JWH-133-treated group compared to the vehicle
group (P<0.05). The combination treatment of JWH-133 with
the CB2R antagonist SR-144528 reversed protein phosphor-
ylation levels compared to the JWH-133 group (P<0.05).We
performed double immunofluorescence using a combination
of antibodies against phosphorylated p44/42 MAPK and cell
type-specific protein markers to identify the cell types
Fig. 1 CB2R agonist significantly reduced thrombin-induced brain ede-
ma 24 hr after injury. Brain sections (4 mm) were divided into 4 parts:
ipsilateral basal ganglia (Ipsi-BG), ipsilateral cortex (Ipsi-CX), contralat-
eral basal ganglia (Cont-BG), and contralateral cortex (Cont-CX). Cere-
bellum (Cerebel) was the internal control. Values are expressed as the
means±SD, n= 12. Vehicle vs. sham **P<0.01, vs. JWH
JWH vs. SR+JWH &P<0.05
Transl. Stroke Res.
exhibiting p44/42 MAPK phosphorylation 24 h after throm-
bin injection. Immunostaining revealed that most of the phos-
phorylated p44/42 MAPK-positive cells colocalized with the
microglial marker Iba1 (Fig. 4a,n=6). The number of p44/42
MAPK-positive microglial cells decreased after JWH-133 ad-
ministration 24 h after surgery. SR-144528 administration re-
versed this effect.
JWH-133 Prevents Thrombin-Induced ZO-1 Attenuation
We examined BBB integrity using immunofluorescence stain-
ing and Western blotting. The tight junction (TJ)-related pro-
tein ZO-1 was examined using immunofluorescence micros-
copy in conjunction with an endothelial marker, von
Willebrand factor (vWF), which is also a marker for the
BBB. ZO-1 and vWF signals aligned perfectly in the sham
group, but this alignment was interrupted while in the vehicle
group, which indicates damage to the BBB. However, in the
JWH-133 group, some rescue of the superimposed ZO-1 and
vWF lining was observed, which suggests an attenuation of
the BBB destruction after surgery. However, this effect was
abolished with simultaneous administration of SR-144528
(Fig. 5). Notably, Western blot analyses of lysates also dem-
onstrated a significant reduction in ZO-1 levels after surgery,
and JWH-133 upregulated the expression of the tight junction
protein ZO-1compared with the vehicle and SR+JWH groups
(P<0.05) (Fig. 6a, d). Immunofluorescence and Western blot
analyses demonstrated that JWH-133 treatment obviously
Fig. 2 Effect of JWH-133 on
microglial cell activation
surrounding the injection site.
Representative images of Iba-1(+)
cells at the injection site in sham,
vehicle, JWH and SR+JWH
groups (a). The number of Iba-
1(+) cells around the injection site
(b,n=6 in each group). Select
coronal sections of fields of view
for immunohistochemical
observation (c). Scale bars=
20 μm. Vehicle vs. sham
**P<0.01, vs. JWH
JWH vs. SR+JWH &P<0.05
Transl. Stroke Res.
protected against TJ protein reduction after injury, which was
indicated by the changes in fluorescence and immunoblotting
signal intensities in each experimental group. These results
further demonstrated that JWH-133 treatment effectively res-
cued BBB destruction after injury, possibly as a result of at-
tenuated neuroinflammation.
JWH-133 Downregulates MMP-9 and MMP-12
Expression After Infusion
MMPs degrade the TJ proteins of the BBB. Therefore, we
examined MMP expression in each experimental group. Our
results demonstrated that JWH-133 significantly reduced
Fig. 3 JWH-133 treatment
significantly reduced Evans blue
dye leakage around the lesion site.
Representative perivascular
Evans blue fluorescence around
the injection site (a). Scale bars=
20 μm. Assay of extravasation
demonstrated that thrombin
induced higher Evans blue dye
leakage, which can be reduced by
a CB2R agonist (JWH133).
Moreover, the effect of JWH133
was reversed by SR144528 (b,
P<0.05, n=6)
Transl. Stroke Res.
MMP-9 levels in brain tissues near the injury region 24 h post-
surgery compared to the vehicle and SR+JWH-133 groups
(Fig. 6a, b,P<0.05). JWH-133 significantly downregulated
MMP-12 expression compared to the vehicle and SR +JWH-
133 groups (Fig. 6a, c,P<0.05, n=6).
This study investigated the effects of CB2R activation using a
model of intracerebral infusion of 20 U thrombin in rats. Our
data demonstrated that the administration of the selective
CBR2 agonist JWH-133 after surgery reduced brain water
content and Evans blue extravasation.
Our results indicate that suppression of microglial activa-
tion and the downregulation of p44/42 MAPK phosphoryla-
tion mediated the neuroprotective effects of JWH-133,
improved BBB integrity, and restrained MMP-9/12 activity.
The CB2R selective antagonist SR-144528 reversed these
neuroprotective effects.
Thrombin is a serine protease that is produced immediately
after ICH and converts fibrin to fibrinogen to initiate clot
formation [3]. Low thrombin concentrations are neuroprotec-
tive in vitro and in vivo. A low dose of thrombin attenuated
brain edema induced by thrombin or intracerebral hemorrhage
and significantly reduced infarct size and brain edema in a rat
middle cerebral artery occlusion model via a phenomena
called thrombin preconditioning (TPC) [30,31]. In contrast,
high thrombin concentrations are deleterious to the brain after
intracerebral hemorrhage [30]. Thrombin is also primarily re-
sponsible for early brain edema formation following intrace-
rebral hemorrhage (ICH) [8].
Thrombin is activated through the coagulation cascade
once ICH occurs in humans or animal models, and it rapidly
Fig. 4 JWH-133 suppressed the
phosphorylation level of p44/42
MAPK, and phosphorylation of
p44/42 MAPK was primarily
visible in reactive microglia.
Confocal immunofluorescence
images of Iba1 (green), P-ERK
(red), and their merged image
24 h after thrombin injection (a).
Administration of JWH-133
reduced the phosphorylation of
p44/42 MAPK-positive
microglia. Phosphorylated p44/42
MAPK, total p44/42 MAPK and
β-tubulin proteins in right basal
ganglia 24 h after surgery (b).
Relative density analyses of
phosphorylation levels of p44/42
MAPK (c,p<0.05, n=6). Scale
bars=20 μm. Values are
expressed as the means±SD
Transl. Stroke Res.
diffuses into the brain parenchyma. Therefore, intracerebral
thrombin infusion provides a model for thrombin diffusion
into the brain after ICH [32]. One milliliter of whole blood
produces ~260 to 360 U of thrombin, and a 50-μLclot(used
experimentally in rats) produces up to ~15 U of thrombin [6].
Therefore, we injected 20 U of thrombin into the right basal
ganglia of the rat to achieve an approximate acute concentra-
tion of 35 U/ml of thrombin in the cerebrospinal fluid (CSF) in
this study [32] based on an estimated volume of CSF in a
300 g rat of ~580 μl[33]. Activation of the coagulation cas-
cade and production of thrombin disrupts the BBB approxi-
mately 24-h post-ICH, which promotes edema formation [6,
8]. Therefore, we chose 24 h after surgery as the time point for
our evaluations.
Microglia constitute up to 10 % of the total cell population
of the brain, and these cells act as resident macrophages and
immune cells of the brain [34,35]. Microglial activation may
contribute to the pathogenesis of brain injury in intracerebral
hemorrhage (ICH), and it is also associated with BBB dam-
age. Activated microglia undergo proliferation, chemotaxis,
and morphological alterations and generate immunomodula-
tory molecules [36]. We observed that JWH-133 decreased
microglial activation after surgery, as shown by the reduced
expression of Iba1 and the predominance of a resting mor-
phology in microglial cells located within the injuryboundary.
CB2R stimulation inhibits microglia/macrophage cell migra-
tion, which may participate in neuroprotection after intracere-
bral infusion of thrombin. Mitogen-activated protein kinases
Fig. 5 CB2R agonist reduced
thrombin-induced BBB damage
24 h after injury. Representative
immunohistochemistry staining
of ZO-1 (green) and von
Willebrand factor (vWF) (red)
24 h after surgery. Arrow
indicates the breakdown of
continuous endothelial cell layer.
Scale bars=20 μm
Transl. Stroke Res.
(MAPKs) are well-known cytoplasmatic signal transducers
that play an important role in thrombin-induced neurotoxicity
[37]. p44/42 MAPKs are activated in the brain after an intra-
cerebral infusion of thrombin. PD98059 is a specific p44/42
MAPK kinase inhibitor that abolished thrombin-induced acti-
vation of p44/42 MAPKs, and it also blocked thrombin-
induced brain neurotoxicity [37]. Thrombin treatment also
activated p44/42 MAPKs in vitro, and PD98059 completely
blocked the cytoprotective effect of thrombin pretreatment,
which indicates that the p44/42 MAPK system mediates the
thrombin-induced neuroprotective effect [38]. Our study dem-
onstrated that phosphorylation of p44/42 MAPK in reactive
microglia was also visible, which may mediate the detrimental
effects of thrombin.
ZO-1 anchors the transmembrane protein occludin to the
actin cytoskeleton, which confers the capacity of BBB to pre-
clude permeation of blood substances [39]. ICH increases BBB
permeability mediated via TJ disruptions with an involvement
of MMPs [40]. MMPs are classically known as matrix-
degrading enzymes that are involved in many physiological
processes, and MMP expression is a key mechanism underlying
increased BBB permeability after ICH [41]. Broad-spectrum
MMP inhibitors relieve brain injury [42]. An increase in plasma
MMP-9 following ICH in humans correlates with peri-
hematoma edema and early neurological deterioration. There-
fore, MMP-9 is closely associated with edema formation [43,
44]. MMP-12 is not expressed in the healthy brain [45]. MMP-
12 is a strong marker of brain injury in animal models. MMP-12
is also the most highly upregulated MMP of the MMPs that
were examined after ICH [46]. Microglial activation may re-
lease MMPs [47]. Maddahi et al. suggested that inhibition of
MEK/ERK signal transduction using a specific raf inhibitor
administeredupto6haftersubarachnoid hemorrhage in a rat
model normalized the expression of pro-inflammatory
mediators and MMP-9 [48]. Adhikary et al. demonstrated that
CB2R-selective agonists reduced MMP-9 expression in microg-
lia. Inhibition of MMP-9 is mediated through CB2R-induced
reduction in cAMP, inhibition of ERK1/2 AP-1 activation, and
the subsequent reduction in AP-1 binding to the MMP-9 pro-
moter [49]. Our Western blot results revealed that JWH-133
suppressed MMP expression after surgery. MMP reduction also
correlates with the de-phosphorylation levels of p44/42 MAPK,
which may explain why JWH-133 ultimately de-phosphorylates
the p44/42 MAPK pathway and suppresses MMPs.
In summary, our data demonstrated that the CB2R agonist
JWH-133 attenuated brain edema by preserving BBB integri-
ty following an intracerebral infusion of thrombin. We also
found that dephosphorylation of the p44/42 MAPK pathway
and the suppression of MMPs such as MMP-9 and MMP-12
were likely involved in the process. These data suggest that
C2R agonists are a promising treatment option for BBB pro-
tection after ICH.
Acknowledgments We would like to thank Dr. Ya Hua from the Uni-
versity of Michigan for her professional comments on this research. This
work was supported by grants 81571130 (Z.C) and 81070929 (Z.C) from
the National Natural Science Foundation of China and 2014CB541606
(H.F) from the National Key Basic Research Development Program (973
Program) of China.
Author Contributions ZC made substantial contributions to the con-
ception and design. LL and YHT performed the experiments and acquired
the data. JT and QWC measured the ventricular volume and cortical
length. YJC and YYF participated in tissue fixation and immunohisto-
chemistry. YY and LMY were responsible for supervising all experi-
ments, data analysis and drafting of the manuscript. HF and GZ read
Fig. 6 Changes in MMP-9,
MMP-12, and ZO-1 expression
after treatment 24 h post-
intracerebral infusion of
thrombin. Representative bands
(a) and relative density analyses
of MMP-9 (b), MMP-12 (c), and
ZO-1 (d) expression in the
ipsilateral right basal ganglia of
brain specimens 24 h after
surgery, n=6. Vehicle vs. sham
**P<0.01, vs. JWH
JWH vs. SR+JWH, &P<0.05
Transl. Stroke Res.
and revised some parts of the manuscript. All authors read and approved
the final manuscript.
Conflict of Interest Lin Li, Yihao Tao, Jun Tang, Qianwei Chen, Yang
Yang, Zhou Feng, Yujie Chen, Li Ming Yang, Yunfeng Yang, Hua Feng,
and Zhi Chen declare that they have no conflicts of interest.
Compliance with Ethics Requirements All institutional and national
guidelines for the care and use of laboratory animals were followed.
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... The immune microenvironment changes shift microglial polarization from M1 to M2. Intraventricular injection of IL-4 in mice increases the proportion of M2 microglia and accelerates the recovery of neurological function after ICH . Some other molecular targets have also been recently identified up-regulated on microglia during M2-polarization after ICH, including Dopamine D1 receptor (DRD1), Cannabinoid receptor-2 (CB2R), Melanocortin receptor 4 (MC4R), and especially sphingosine-1-phosphate receptor (S1PR; Xu et al., 2013;Li L. et al., 2015;Zhang et al., 2015). ...
Full-text available
Intracerebral hemorrhage (ICH) features extremely high rates of morbidity and mortality, with no specific and effective therapy. And local inflammation caused by the over-activated immune cells seriously damages the recovery of neurological function after ICH. Fortunately, immune intervention to microglia has provided new methods and ideas for ICH treatment. Microglia, as the resident immune cells in the brain, play vital roles in both tissue damage and repair processes after ICH. The perihematomal activated microglia not only arouse acute inflammatory responses, oxidative stress, excitotoxicity, and cytotoxicity to cause neuron death, but also show another phenotype that inhibit inflammation, clear hematoma and promote tissue regeneration. The proportion of microglia phenotypes determines the progression of brain tissue damage or repair after ICH. Therefore, microglia may be a promising and imperative therapeutic target for ICH. In this review, we discuss the dual functions of microglia in the brain after an ICH from immunological perspective, elaborate on the activation mechanism of perihematomal microglia, and summarize related therapeutic drugs researches.
... The brain water content was examined 72 h following ICH, as previously reported (Li et al., 2015). Briefly, the rats (n = 10/group) were euthanized and decapitated, the brains were quickly removed, and a 4-mm thick section of the coronal brain tissue surrounding the needle entry site was harvested. ...
Full-text available
Intracerebral hemorrhage (ICH) is a common disease in the elderly population. Inflammation following ICH plays a detrimental role in secondary brain injury, which is associated with a poor prognosis of patients with ICH, and no efficient pharmacological preventions are available. Here, we investigated the effects of glibenclamide (GLC) on neuroinflammation in an autoblood-induced aged rat (18 months old) model of ICH. Rats were randomized into the sham, vehicle, and GLC groups. First, we investigated the expression level of sulfonylurea receptor 1 (Sur1) surrounding the hematoma after ICH. Then, neurological scores were calculated, and water maze tests, brain water content analysis, western blotting, and immunofluorescence assays were implemented to detect the neuroprotective effect of GLC. The expression of the Sur1-Trpm4 channel was significantly increased in the perihematomal tissue following ICH in aged rats. The GLC administration effectively reduced brain edema and improved neurofunction deficits following ICH. In addition, GLC increased the expression of brain-derived neurotrophic factors and decreased the expression of proinflammatory factors [tumor necrosis factor (TNF)-α,interleukin (IL)-1, and IL-6]. Moreover, GLC markedly reduced Ikappa-B (IκB) kinase (IKK) expression in microglia and nuclear factor (NF)-κB-P65 levels in perihematomal tissue. GLC ameliorated ICH-induced neuroinflammation and improved neurological outcomes in aged rats. In part, GLC may exert these effects by regulating the NF-κB signaling pathway through the Sur1-Trpm4 channel.
... Second, thrombin activates matrix metalloproteinases (MMPs), which may degrade various extracellular matrix proteins (such as tight junction proteins), increasing blood-brain barrier permeability and aggravating posterior cerebral edema after SAH (45). Third, activation of microglia in the brain and peripheral blood immune cells, which enter the central nervous system through an impaired blood-brain barrier, may cause serious neuroinflammatory injury (46). Fourth, studies regarding ischemia-reperfusion injury in mouse models have reported that astrocytes originally activated by thrombin may aid in the production of MMP-2 and reduce myelin cells (47). ...
Full-text available
Aneurysmal subarachnoid hemorrhage (SAH) is one of the special stroke subtypes with high mortality and mobility. Although the mortality of SAH has decreased by 50% over the past two decades due to advances in neurosurgery and management of neurocritical care, more than 70% of survivors suffer from varying degrees of neurological deficits and cognitive impairments, leaving a heavy burden on individuals, families, and the society. Recent studies have shown that white matter is vulnerable to SAH, and white matter injuries may be one of the causes of long-term neurological deficits caused by SAH. Attention has recently focused on the pivotal role of white matter injury in the pathophysiological processes after SAH, mainly related to mechanical damage caused by increased intracerebral pressure and the metabolic damage induced by blood degradation and hypoxia. In the present review, we sought to summarize the pathophysiology processes and mechanisms of white matter injury after SAH, with a view to providing new strategies for the prevention and treatment of long-term cognitive dysfunction after SAH.
Background and purpose Traumatic brain injury (TBI) destroys white matter, and this destruction is aggravated by secondary neuroinflammatory reactions. Although white matter injury (WMI) is strongly correlated with poor neurological function, understanding of white matter integrity maintenance is limited, and no available therapies can effectively protect white matter. One candidate approach that may fulfill this goal is cannabinoid receptor 2 (CB2) agonist treatment. Here, we confirmed that a selective CB2 agonist, JWH133, protected white matter after TBI. Methods The motor evoked potentials (MEPs), open field test, and Morris water maze test were used to assess neurobehavioral outcomes. Brain tissue loss, WM damage, Endoplasmic reticulum stress (ER stress), microglia responses were evaluated after TBI. The functional integrity of WM was measured by diffusion tensor imaging (DTI) and transmission electron microscopy (TEM). Primary microglia and oligodendrocyte cocultures were used for additional mechanistic studies. Results JWH133 increased myelin basic protein (MBP) and neurofilament heavy chain (NF200) levels and anatomic preservation of myelinated axons revealed by DTI and TEM. JWH133 also increased the numbers of oligodendrocyte precursor cells and mature oligodendrocytes. Furthermore, JWH133 drove microglial polarization toward the protective M2 phenotype and modulated the redistribution of microglia in the striatum. Further investigation of the underlying mechanism revealed that JWH133 downregulated phosphorylation of the protein kinase R (PKR)-like endoplasmic reticulum (ER) kinase (PERK) signaling pathway and its downstream signals eukaryotic translation initiation factor 2 α (eIF2α), activating transcription factor 4 (ATF4) and Growth arrest and DNA damage-inducible protein (GADD34); this downregulation was followed by p-Protein kinase B(p-Akt) upregulation. In primary cocultures of microglia and oligodendrocytes, JWH133 decreased phosphorylated PERK expression in microglia stimulated with tunicamycin and facilitated oligodendrocyte survival. These data reveal that JWH133 ultimately alleviates WMI and improves neurological behavior following TBI. However, these effects were prevented by SR144528, a selective CB2 antagonist. Conclusions This work illustrates the PERK-mediated interaction between microglia and oligodendrocytes. In addition, the results are consistent with recent findings that microglial polarization switching accelerates WMI, highlighting a previously unexplored role for CB2 agonists. Thus, CB2 agonists are potential therapeutic agents for TBI and other neurological conditions involving white matter destruction.
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The COVID-19 pandemic, caused by SARS-CoV-2, is a deadly disease affecting millions due to the non-availability of drugs and vaccines. The majority of COVID-19 drugs have been repurposed based on antiviral, immunomodulatory, and antibiotic potential. The pathogenesis and advanced complications with infection involve the immune-inflammatory cascade. Therefore, a therapeutic strategy could reduce infectivity, inflammation, and immune modulation. In recent years, modulating the endocannabinoid system, particularly activation of the cannabinoid type 2 (CB2) receptor is a promising therapeutic target for modulation of immune-inflammatory responses. JWH133, a selective, full functional agonist of the CB2 receptor, has been extensively studied for its potent anti-inflammatory, antiviral, and immunomodulatory properties. JWH133 modulates numerous signaling pathways and inhibits inflammatory mediators, including cytokines, chemokines, adhesion molecules, prostanoids, and eicosanoids. In this study, we propose that JWH133 could be a promising candidate for targeting infection, immunity, and inflammation in COVID-19, due to its pharmacological and molecular mechanisms in numerous preclinical efficacy and safety studies, along with its immunomodulatory, anti-inflammatory, organoprotective, and antiviral properties. Thus, JWH133 should be investigated in preclinical and clinical studies for its potential as an agent or adjuvant with other agents for its effect on viremia, infectivity, immune modulation, resolution of inflammation, reduction in severity, and progression of complications in COVID-19. JWH133 is devoid of psychotropic effects due to CB2 receptor selectivity, has negligible toxicity, good bioavailability and druggable properties, including pharmacokinetic and physicochemical effects. We believe that JWH133 could be a promising drug and may inspire further studies for an evidence-based approach against COVID-19.
The pharmacological activation of cannabinoid type 2 receptors (CB2R) gained attention due to its ability to mitigate neuroinflammatory events without eliciting psychotropic activities, a limiting factor for the drugs targeting cannabinoid type 1 receptors (CB1R). Therefore, ligands activating CB2R are receiving enormous importance for therapeutic targeting in numerous diseases including neurodegenerative, neuropsychiatric and neurodevelopmental disorders as well as traumatic injuries and neuropathic pain where neuroinflammation is a common accompaniment. Since the characterization of CB2R, many CB2R selective synthetic ligands have been developed with high selectivity and functional activity. Among numerous ligands, JWH133 has been found one of the compounds with high selectivity for CB2R. JWH133 has been reported to exhibit numerous pharmacological activities including antioxidant, anti-inflammatory, anticancer, cardioprotective, hepatoprotective, gastroprotective, nephroprotective, and immunomodulatory. Recent studies have showed that JWH133 possesses potent neuroprotective properties in several neurological disorders, including neuropathic pain, anxiety, epilepsy depression, alcoholism, psychosis, stroke, and neurodegeneration. Additionally, JWH133 protects neurons from oxidative damage and inflammation, promotes neuronal survival and neurogenesis, and serves as an immunomodulatory agent. The present review comprehensively examined neuropharmacological activities of JWH133 in neurological disorders including neurodegenerative, neurodevelopmental and neuropsychiatric using synoptic tables and elucidated pharmacological mechanisms based on reported observations. Considering the available data, JWH133 appears to be a promising CB2R agonist molecule for further evaluation and it can be a prototype agent for a unique class of drugs in drug discovery and development for neurotherapeutics and neuroprotection. Further, regulatory toxicology and pharmacokinetic studies are required to determine safety and proceed for clinical evaluation.
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The mechanisms triggering brain damage after intracerebral hemorrhage (ICH) are pleiotropic and are in many respects distinct from those contributing to ischemic brain injury. The toxicity of extravasated blood toward all structural components of the neurovascular unit represents a unique feature of ICH-mediated brain damage. Inflammation and oxidative stress appear to play prominent roles in the pathobiology of ICH. The secondary injury after ICH develops over days suggesting the presence of a considerably wide window for therapeutic intervention. Approaches aimed at detoxification of blood-derived noxious components represent a promising target for the treatment of ICH. Pre-clinical animal models provide useful guidance on the pathogenesis of ICH. However, better models to assess re-bleeding (hematoma enlargement) are urgently needed.
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Thrombin is a Na+-activated allosteric serine protease of the chymotrypsin family involved in coagulation, inflammation, cell protection, and apoptosis. Increasingly, the role of thrombin in the brain has been explored. Low concentrations of thrombin are neuroprotective, while high concentrations exert pathological effects. However, greater attention regarding the involvement of thrombin in normal and pathological processes in the central nervous system is warranted. In this review, we explore the mechanisms of thrombin action, localization, and functions in the central nervous system and describe the involvement of thrombin in stroke and intracerebral hemorrhage, neurodegenerative diseases, epilepsy, traumatic brain injury, and primary central nervous system tumors. We aim to comprehensively characterize the role of thrombin in neurological disease and injury.
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Stroke continues to be a serious and significant health problem in the USA and worldwide. This article will emphasize the need for good laboratory practices, transparent scientific reporting, and the use of translational research models representative of the disease state to develop effective treatments. This will allow for the testing and development of new innovative strategies so that efficacious therapies can be developed to treat ischemic and hemorrhagic stroke. This article recommends guidelines for effective translational research, most importantly, the need for study blinding, study group randomization, power analysis, accurate statistical analysis, and a conflict of interest statement. Additional guidelines to ensure reproducibility of results and confirmation of efficacy in multiple species are discussed.
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The US National Institute of Neurological Disorders and Stroke convened major stakeholders in June 2012 to discuss how to improve the methodological reporting of animal studies in grant applications and publications. The main workshop recommendation is that at a minimum studies should report on sample-size estimation, whether and how animals were randomized, whether investigators were blind to the treatment, and the handling of data. We recognize that achieving a meaningful improvement in the quality of reporting will require a concerted effort by investigators, reviewers, funding agencies and journal editors. Requiring better reporting of animal studies will raise awareness of the importance of rigorous study design to accelerate scientific progress.
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Administration of cannabinoid receptor 2 (CB2R) agonists in inflammatory/autoimmune and CNS injury models results in significant attenuation of clinical disease, and reduction of inflammatory mediators. Previous studies reported that CB2R signaling also reduces leukocyte migration. Migration of dendritic cells (DC) to various sites is required for their activation and for the initiation of adaptive immune responses. In the present study we report for the first time that CB2R signaling affects DC migration in vitro and in vivo, primarily through the inhibition of metalloproteinase 9 (MMP-9) expression. Reduced MMP-9 production by DC results in decreased migration to draining lymph nodes in vivo and in vitro in the matrigel migration assay. The effect on MMP-9 expression is mediated through CB2R resulting in reduction in cAMP levels, subsequent decrease in ERK activation, and reduced binding of c-Fos and c-Jun to MMP-9 promoter AP-1 sites. We postulate that, by dampening production of MMP-9 and subsequent MMP-9-dependent DC migration, cannabinoids contribute to resolve acute inflammation and to reestablish homeostasis. Selective CB2R agonists might be valuable future therapeutic agents for the treatment of chronic inflammatory conditions by targeting activated immune cells including DC.
The authors previously found that pretreatment with a low dose of thrombin attenuates the brain edema induced by a large dose of thrombin or an intracerebral hemorrhage, and reduces infarct volume after focal cerebral ischemia (i.e., thrombin preconditioning). This study investigated whether thrombin preconditioning is caused by activation of the thrombin receptor, also called protease-activated receptor, In the in vivo studies, thrombin-induced brain tolerance was eliminated by RPPGF (Arg-Pro-Pro-Gly-Phe), a thrombin-receptor antagonist. Pretreatment with a thrombin-receptor agonist reduced the amount of edema induced by a large dose of thrombin infused into the ipsilateral basal ganglia 7 days later (81.3 +/- 0.7% vs. 82.6 +/- 0.8% in the control, P < 0.05). In the in vitro study, low doses of thrombin (1 or 2 U/mL) did not induce cell death. However, doses greater than 5 U/mL resulted in dose-dependent lactate dehydrogenase release (P < 0.01). Thrombin and thrombin receptor-activating peptide preconditioning reduced lactate dehydrogenase release induced by a high dose of thrombin (10 and 20 U/mL), whereas RPPGF blocked the effect of thrombin preconditioning in vitro. Western blots indicated that p44/42 mitogen-activated protein kinases were activated after thrombin preconditioning. Finally, inhibition of p44/42 mitogen-activated protein kinases activation by PD98059 abolished the thrombin-preconditioning effect. Results indicate that thrombin-induced brain tolerance is in part achieved through activation of the thrombin receptor. Activation of the thrombin receptor in the brain may be neuroprotective. The protective effect of thrombin preconditioning is achieved through the p44/42 mitogen-activated protein kinase signal-transduction pathway.
Norrin and its receptor Frizzled-4 have important roles in the blood-brain barrier development. This study is to investigate a potential role and mechanism of Norrin/Frizzled-4 on protecting blood-brain barrier integrity after subarachnoid hemorrhage (SAH). One hundred and seventy-eight male Sprague-Dawley rats were used. SAH model was induced by endovascular perforation. Frizzled-4 small interfering RNA was injected intracerebroventricularly 48 hours before SAH. Norrin was administrated intracerebroventricularly 3 hours after SAH. SAH grade, neurological scores, brain water content, Evans blue extravasation, western blots, and immunofluorescence were used to study the mechanisms of Norrin and its receptor regulation protein TSPAN12, as well as neurological outcome. Endogenous Norrin and TSPAN12 expression were increased after SAH, and Norrin was colocalized with astrocytes marker glial fibrillary acidic protein in cortex. Exogenous Norrin treatment significantly alleviated neurobehavioral dysfunction, reduced brain water content and Evans blue extravasation, promoted β-catenin nuclear translocation, and increased Occludin, VE-Cadherin, and ZO-1 expressions. These effects were abolished by Frizzled-4 small interfering RNA pretreated before SAH. Norrin protected blood-brain barrier integrity and improved neurological outcome after SAH, and the action of Norrin appeared mediated by Frizzled-4 receptor activation, which promoted β-catenin nuclear translocation, which then enhanced Occludin, VE-Cadherin, and ZO-1 expression. Norrin might have potential to protect blood-brain barrier after SAH. © 2014 American Heart Association, Inc.
Intracerebral hemorrhage (ICH) is a devastating type of stroke with no effective therapies. Clinical advances in ICH treatment are limited by an incomplete understanding of the molecular mechanisms responsible for secondary injury and poor outcome. Increasing evidence suggests that cerebral edema is a major contributor to secondary injury and poor outcome in ICH. ICH activates specific signaling pathways that promote edema and damage neuronal tissue. By increasing our understanding of these pathways, we may be able to target them pharmaceutically to reduce edema in ICH patients. In this review, we focus on three major signaling pathways that promote edema after ICH: (1) the coagulation cascade and thrombin, (2) the inflammatory response and matrix metalloproteinases, and (3) the complement cascade and hemoglobin toxicity. We will describe the experimental evidence that confirms these pathways promote edema in ICH, discuss potential targets for new therapies, and comment on important directions for future research.
Purpose: Hepatic resection is arguably the preferred treatment for huge hepatocellular carcinoma (H-HCC). Estimating the remnant liver volume is therefore essential. This study aimed to evaluate the feasibility of using computer-assisted volumetric analysis for this purpose. Methods: The study involved 40 patients with H-HCC. Laboratory examinations were conducted, and a contrast CT-scan revealed that 30 cases out of the participating 40 had single-lesion tumors. The remaining 10 had less than three satellite tumors. With the consensus of the team, two physicians conducted computer-assisted 3D segmentation of the liver, tumor, and vessels in each case. Volume was automatically computed from each segmented/labeled anatomical field. To estimate the resection volume, virtual lobectomy was applied to the main tumor. A margin greater than 1 cm was applied to the satellite tumors. Resectability was predicted by computing a ratio of functional liver resection (R) as (Vresected- Vtumor)/(Vtotal-Vtumor) x 100%, applying a threshold of 50% and 60% for cirrhotic and non-cirrhotic cases, respectively. This estimation was then compared with surgical findings. Results: Out of the 22 patients who had undergone hepatectomies, only one had an R that exceeded the threshold. Among the remaining 18 patients with non-resectable H-HCC, 12 had Rs that exceeded the specified ratio and the remaining 6 had Rs that were < 50%. Four of the patients who had Rs less than 50% underwent incomplete surgery due to operative findings of more extensive satellite tumors, vascular invasion, or metastasis. The other two cases did not undergo surgery because of the high risk involved in removing the tumor. Overall, the ratio of functional liver resection for estimating resectability correlated well with the other surgical findings. Conclusion: Efficient pre-operative resectability assessment of H-HCC using computer-assisted volumetric analysis is feasible.