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

Abstract

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.

Full text

ORIGINAL ARTICLE
A Cannabinoid Receptor 2 Agonist Prevents Thrombin-Induced
Blood–Brain Barrier Damage via the Inhibition of Microglial
Activation and Matrix Metalloproteinase Expression in Rats
Lin Li
1
&Yihao Tao
1
&Jun Tang
1
&Qianwei Chen
1
&Ya n g Ya n g
1
&Zhou Feng
1
&
Yuji e C h e n
1
&Liming Yang
1
&Yunfeng Yang
2
&Gang Zhu
1
&Hua Feng
1
&Zhi Chen
1
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 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 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 .Blood–brain
barrier .p44/42 MAPK .Matrix metalloproteinase
Introduction
Spontaneous intracerebral hemorrhage (ICH) is a devastating
disease. It constitutes 10–15 % of all strokes in the USA,
Europe, and Australia and 20–30 % 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 blood–brain 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
[6–8]. 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
zhichen8888@yeah.net
1
Department of Neurosurgery, Southwest Hospital, Third Military
Medical University, No. 30, Gaotanyan Street, Chongqing 400038,
People’s Republic of China
2
Department of Neurosurgery, Sichuan Provincial Corps Hospital,
Chinese People’s Armed Police Forces, Leshan, People’sRepublicof
China
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 Alzheimer’sdisease(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 (250–300 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].
Surgery
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
extravasation.
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 3–5 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
X-100inPBSfor30min.Specimenswereblocked
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 anti–ZO-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 anti–GAPDH (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.
Results
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
Blood–Brain 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
#
P<0.05,
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
#
P<0.05,
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).
Discussion
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
A
BC
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.
Conclusion
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
#
P<0.05,
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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