Brain Injury

ISSN: 0269-9052 (Print) 1362-301X (Online) Journal homepage: http://www.tandfonline.com/loi/ibij20

Intra-cerebral cannabidiol infusion-induced

neuroprotection is partly associated with the
TNF-α/TNFR1/NF-кB pathway in transient focal
cerebral ischaemia

Sepideh Khaksar & Mohammad Reza Bigdeli

To cite this article: Sepideh Khaksar & Mohammad Reza Bigdeli (2017): Intra-cerebral cannabidiol
infusion-induced neuroprotection is partly associated with the TNF-α/TNFR1/NF-кB pathway in
transient focal cerebral ischaemia, Brain Injury, DOI: 10.1080/02699052.2017.1358397

To link to this article: http://dx.doi.org/10.1080/02699052.2017.1358397

Published online: 05 Sep 2017.

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 BRAIN INJURY
https://doi.org/10.1080/02699052.2017.1358397

Intra-cerebral cannabidiol infusion-induced neuroprotection is partly associated

with the TNF-α/TNFR1/NF-кB pathway in transient focal cerebral ischaemia
Sepideh Khaksar and Mohammad Reza Bigdeli
Department of Physiology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran

ABSTRACT ARTICLE HISTORY

Background: Stroke is a neurological disease, which, in addition to high mortality, imposes many Received 23 July 2016
financial and mental burdens on families and the society. The main objective of this study was to Revised 19 June 2017
investigate the effect of cannabidiol (CBD) on one of the major inflammatory pathways in cerebral Accepted 24 June 2017
ischaemia. Method: Using stereotaxic surgery, the cannula was implanted into the right lateral ventricle KEYWORDS
of rats. CBD (50, 100, and 200 ng/rat; i.c.v.) was administrated for five consecutive days. After pretreat- BBB breakdown; brain
ment, the rats were subjected to 60 min of right middle cerebral artery occlusion (MCAO). After 24 h,
Downloaded by [UC Santa Barbara Library] at 01:14 06 September 2017

oedema; cannabidiol;
neurological deficits score, infarct volume, brain oedema, and blood–brain barrier (BBB) permeability in cerebral ischaemia; nuclear
total, core, and penumbra areas were assessed. The expression of tumour necrosis factor alfa (TNF-α), factor-kappa B; tumour
tumour necrosis factor receptor 1 (TNFR1), and nuclear factor-kappa B (NF-кB) in the mentioned regions necrosis receptor 1
was also studied. Results: Administration of CBD (100 and 200 ng/rat) caused a significant reduction in
infarction, brain oedema, and BBB permeability compared with the vehicle-received group. Down-
regulation of TNF-α, TNFR1, and NF-кB expression was also observed by CBD. Conclusion: The results
achieved in this study support the idea that CBD has a cerebroprotective effect (partly through
suppression of TNF-α, TNFR1, and NF-кB) on ischaemic injury.
ABBREVIATIONS: CBD, cannabidiol; ANOVA, analysis of variance; PVDF, polyvinylidene difluoride;
SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; SEM, standard error of
mean.

Introduction prominent pathophysiologic processes consist of excitotox-

city (glutamate-mediated toxicity), increased intracellular
Cannabis sativa is a type of flowering plant that is indigen-
calcium(7), mitochondrial dysfunction, oxidative stress(8),
ous to central and south Asia(1). Eighty different terpeno-
inflammation(9), the blood–brain barrier (BBB) disruption,
phenol combinations have been extracted so far, collectively
cerebral oedema, and eventually cell death(5). Among the
known as phytocannabinoids or natural cannabinoids,
cannabinoid compounds of cannabis, there are outstanding
including cannabidiol (CBD) and Δ9-tetrahydrocannabinol
characteristics of CBD, such as the neuroprotective property,
(THC)(2). The harmful effects of cannabis were attributed to
which makes it a suitable candidate for the present study (10;
THC due to having psychoactive property, dependence, and
11). It was shown that CBD exerts protective effects in the
the development of tolerance(3), whereas CBD has non-
hypoxic–ischaemic model through the decrease of glutamate
psychoactive and protective properties, which reveal the
(12), suppression of calcium overload(13), inhibition of
positive aspects of cannabis plant(2). Therefore, the attention
apoptosis(12), and reduction of brain infarction(14). The
of researchers has been focused on CBD. CBD has, for many
antioxidant and anti-inflammatory effects of CBD have also
decades, been reported to possess a broad spectrum of ther-
been demonstrated (15; 16). A series of studies have identi-
apeutic potentials(4). On the other hand, cerebral ischaemia
fied that CBD exhibits its anti-inflammatory property
is followed by insufficient blood flow to the brain. This event
through the inhibition of interleukin1 (IL_1) and inducible
leads to ischaemic stroke(5). The incidence rate of ischaemic
nitric oxide synthase (iNOS)(15), the suppression of micro-
stroke is increasing rapidly in developing countries. Indeed,
glia activity, and the prevention of neutrophil migration
stroke is considered the second leading cause of death world-
(17). More relevantly, there are several reports about the
wide(6). Intriguingly, cerebral ischaemia happens in indivi-
anti-inflammatory effects of CBD on global and focal ischae-
duals with abnormalities including sickle cell anaemia,
mia. CBD induces a remarkable reduction of myeloperoxi-
compressed blood vessels, aneurysm, and patients with a
dase (MPO) activity, an index of neutrophil accumulation, in
history of heart attack. Accordingly, prevention of stroke
cerebral ischaemia (18). Furthermore, CBD exerts its neuro-
and induction of ischaemic tolerance are the major objects
protective role by the inhibition of IL_6, iNOS, and cycloox-
of stroke studies both in clinics and in sciences. The most
ygenase 2 (COX-2) in the in vitro model of newborn

CONTACT Mohammad Reza Bigdeli, Ph.D bigdelimohammadreza@yahoo.com Department of Physiology, Biological Sciences and technology Faculty,
Shahid Beheshti University, Daneshjoo BLV, Evin, Tehran, Iran.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/IBIJ.
© 2017 Taylor & Francis Group, LLC
 2 S. KHAKSAR AND M. R. BIGDELI
hypoxic–ischaemic brain damage (12). Consequently, CBD
can influence the most important events leading to brain
damages during ischaemia. However, the mechanisms
involved in CBD-induced neuroprotection in cerebral
ischaemia have not been fully revealed.
In this study, first we attempted to elucidate whether CBD
can ameliorate ischaemic damages such as neurological defi-
cits, infarction, brain oedema, and BBB permeability.
Subsequently, inflammation as the major consequence of
ischaemic injury was also considered. Hence, the role of
inflammatory factors such as tumour necrosis factor alfa
(TNF-α), tumour necrosis factor receptor 1 (TNFR1), and
nuclear factor kappa-light-chain-enhancer of activated B
cells (NF-кB) in the mentioned effects was studied.
Experimental procedures
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Animals and group assignment
Adult male Wistar rats 8–12 weeks of age (250–350 g) were
housed under conditions of controlled temperature (22 ± 2 °C)
and constant humidity, with a 12 h light/dark cycle for all
experiments. All experimental animals were managed in accor-
dance with National Institute of Health and Guide for the care
and use of laboratory animals (NIH Publications) revised in
2011. The study was approved by the ethics committee of the
Shahid Beheshti University of Iran (NO#920.667). We made
every effort to minimize the number of animals used for this
study. The rats were divided randomly into five main groups
(control, vehicle, 50, 100, and 200 ng/rat; i.c.v. of CBD) of 35
animals and a sham group with 29 animals. Each main group
was subdivided to ischaemia-operated (for evaluation of infarct
volume (n = 6), brain water content (BWC; total of hemisphere
n = 6; core and penumbra areas of hemisphere n = 6), and BBB
permeability (total of hemisphere n = 6; core and penumbra
n = 6)) and intact (for assessment of TNF-α, TNFR1 protein
expression as well as NF-кB in the core and penumbra (n = 5))
subgroups. In addition, five rats of the ischaemia-operated sub-
group of the control group were considered for evaluation of
TNF-α, TNFR1, and NF-кB expression in the core and penum-
bra (n = 5). The sham-operated group (n = 29) was assigned for
the assessment of BWC (n = 12), BBB permeability (n = 12), and
inflammatory factors (n = 5) separately (Table 1). Seven days
after stereotaxic surgery, treatment groups received CBD (50,
100, and 200 ng/rat) for five consecutive days through intracer-
ebroventricular (i.c.v.) injection. Ischaemia-operated subgroups
were subjected to 60 min of middle cerebral artery occlusion
(MCAO). Around 24 h later, measurement of neurological def-
icits and then infarct volume, oedema, and BBB integrity were
performed separately. It should be noted that the neurologic
Table 1. Evaluation of ischaemic tolerance in various experimental groups. CBD: cannabidiol; MCAO: middle cerebral artery occlusion; BBB: blood-brain barrier; TNF-α:
tumour necrosis factor alfa; TNFR1: tumour necrosis factor receptor 1; NF-кB: nuclear factor-kappa B.
Control Sham
Assessment MCAO Intact MCAO MCAO
Infarction + - - +
Brain Oedema + - + +
BBB + - + +
TNF-α, TNFR1, and NF-кB expression + + + -
deficits score in all animals was carried out. In the control
group, the whole methodological strategy was similar to the
stereotaxic/ ischaemia-operated group without receiving any
treatment. Sham-operated animals underwent the same surgery
procedure of MCAO, except that the filament was introduced.
For western blotting in intact subgroups, whole methodological
strategy was similar to the main groups without any surgery
procedure.
Drug administration
CBD (Tocris, UK) was dissolved in a mixture of dimethylsulf-
oxide (10% DMSO) and phosphate buffer solution (90%PBS)
(19). A volume of 2 μl of CBD and DMSO solution was
injected into the right lateral ventricle of treatment and vehi-
cle-received rats, respectively. Intracerebroventricular injec-
tion of CBD at the doses of 50, 100, and 200 ng/rat (19) was
given by lowering a 30-gauge injection. The injection cannula
was attached to a Hamilton syringe using a polyethylene tube.
According to a previous report, there is evidence about the
possible neuroprotective role of vehicle (DMSO) (20).
Therefore, in this study analysis between the control and
vehicle groups was performed.
Stereotaxic surgery
Anaesthetized rats were placed in a stereotaxic apparatus
(Stoelting Instruments, USA). A midline incision was made,
the skin was retracted, and a hole in the position of the right
lateral ventricle was created according to stereotaxic coordi-
nates: AP, −0.58 mm posterior to the bregma; L, -1.4 mm
from the midline; and V, −1.6 mm relative to the skull(21).
The cannula was anchored to the skull with dental cement.
Animals were then placed in the animal house for 7 days, after
stereotaxic operation. Injection of 2 μl of methylene blue
solution into the lateral ventricle was carried out to verify
the site of microinjection. After decapitation, the brain was
removed and fixed in 10% formalin solution for 48 h. Sections
were observed macroscopically to ascertain whether the can-
nula was correctly placed into the lateral cerebral ventricle.
Focal cerebral ischaemia
At the fifth day, rats received CBD 30 min prior to MCAO
surgery. After anaesthetizing rat, the middle cerebral artery
(MCA) was occluded by the intraluminal suture technique,
described by Longa et al. (22). Briefly, the right carotid region
was exposed through a midline incision and the external carotid
and common carotid arteries were ligated with a suture. A 3–0
CBD CBD CBD
Vehicle (50 ng/rat) (100 ng/rat) (200 ng/rat)
Intact MCAO Intact MCAO Intact MCAO Intact
- + - + - + -
- + - + - + -
- + - + - + -
+ - + - + - +

coated monofilament nylon suture (Nylon, Homemade), whose
tip had been rounded by heating and coated with poly-l-lysine
(Sigma, USA), was introduced from the carotid bifurcation into
the internal carotid artery until mild resistance was felt (18–
19 mm), thereby monofilament occluded the origin of the MCA
and blocked the blood flow to the MCA. Reperfusion was
established by the withdrawal of the suture after 60 min of
ischaemia. Rectal temperature was monitored (Citizen-513w,
CITIZEN, United Arab Emirates) and maintained at 37 ºC by
surface heating and cooling during surgery.
Neurobehavioural evaluation
Around 24 h after the MCAO surgery, the neurological
examination (sensorimotor responses) was performed. The
sum of partial scores was assigned as the total neurologic
score, with a maximum of 28 points and a minimum of 0
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points in normal rats. The neurological scoring system used
in this study evaluates changes in certain sensorimotor func-
tions, and describes the functional recovery. For instance,
the limb placing test examines sensorimotor integration,
which is the most common disability after a stroke, and
influences the quality of life in patients. The rats were
assessed neurologically by following a chart that describes
the sensorimotor abilities of rats (23).
Infarct volume
Rats were sacrificed 24 h after the induction of ischaemia and
the brains rapidly removed and cooled in saline at 4ºC for
15 min. The brain was cut into 2-mm-thick coronal sections
(Brain Matrix, Iran). The slices were immersed in 2% 2, 3, 5-
triphenyltetrazolium chloride solution (TTC) (Merck,
Germany), and kept at 37ºC in a water bath. The photos of
these slices were then taken by a digital camera (Nikon, D40x
digital), which was connected to a computer. Unstained area
was determined as an infarct area. Infarction volume was
measured using image analysis software (Image J, version
1.46r). The infarct volume of each slice was calculated by
multiplying the infarcted area of the slice by its thickness
(2 mm). The total infarct volume of each brain was defined
as the sum of the infarct volumes of the seven brain slices. The
involvement of the oedema in the infarct volume was corrected
by the following formula (24). Assessment of infarct volume in
the core and penumbra regions was also accomplished sepa-
rately as described previously (25). This classification was
determined in accordance with the cerebral arteries distribu-
tion, especially the territories of the brain that is supplied blood
by MCA (26). With regard to this classification, the major part
of the somatosensory cortex and its surrounding area were
considered as a core area of ischaemic damage and penumbra
following occlusion of MCA, respectively.
Corrected infarct volume = left hemisphere size – (right
hemisphere size – measured infarct size).
Cerebral oedema
After completion of the behavioural experiments, the rats
were sacrificed and the brains were removed. The cerebellum,
BRAIN INJURY 3
pons, and olfactory bulb were separated. Right and left wet
weights (WW) were measured separately. Subsequently, dry
weights (DW) were assessed after 24 h at 120 °C. BWC was
calculated as [(WW−DW)/WW] ×100 (27). In addition, BWC
was measured in the core and penumbra areas separately.
BBB integrity
Evans Blue dye (EBD, Sigma Chemicals, USA) extravasation was
used to evaluate the permeability of BBB. Briefly, 4 ml/kg of 2%
EBD solution was injected intravenously 30 min after MCAO.
Around 24 h after ischaemia, transcardial perfusion with 250 ml
saline was accomplished on anaesthetized rats to replace intravas-
cular EBD with saline. After decapitation, the brains were
removed and the hemispheres were separated and weighed. Two
hemispheres were separately homogenized in 2.5 ml phosphate-
buffered saline to extract the EBD. Then 2.5 ml of 60% trichlor-
oacetic acid was added to precipitate protein and mixed by vortex
for 3 min. The samples were placed at 4 °C for 30 min and
centrifuged for 30 min at 1000 rpm. Measuring of EBD absor-
bance in the supernatant was carried out by using a spectro-
photometer at 610 nm (Perkin-Elmer, Illinois, USA). EBD
concentration was expressed as μg/g of brain tissue through the
standard curve (28). BBB integrity was also evaluated in the core
and penumbra areas separately.
Western blot analysis
The expression levels of TNF-α, TNFR1, and NF-кB proteins were
assessed by the western blotting technique following sample
extraction and SDS-PAGE. Assessment of TNF-α, TNFR1, and
NF-кB expressions was carried out in the total, core, and penum-
bra areas of the rat brain hemisphere separately. Each tissue sample
was homogenized at 4 ºC for 1 min in lysis buffer. Tissue extract
was centrifuged at 12,000 rpm at 4 ºC for 20 min. The supernatant
was treated in the same way as the tissue extract. SDS sample buffer
was added to the aliquots of tissue extracts. Samples were heated at
100 ºC for 5 min. Proteins were separated by SDS-PAGE (10% gel).
Blotting was performed by semi-dry type blotting (BIORAD) and
PVDF membrane (Millipore). The blots were blocked with 2%
non-fat dry milk in Tris buffer saline in 0.1% Tween 20 at 4ºC for
75 min, and incubated for 18 h with rabbit anti-TNF-α polyclonal
(1:500 dilution; Abcam), goat anti-TNFR1 polyclonal (1:500 dilu-
tion; Santa Cruz), rabbit anti-NF-кB polyclonal (1:500 dilution;
Santa Cruz), and rabbit anti-β-actin (1:1000 dilution; Santa Cruz)
antibodies, followed by goat anti-rabbit and rabbit anti-goat sec-
ondary antibodies (1:500 dilution; Santa Cruz) for 90 min sepa-
rately. TNF-α, TNFR1, and NF-кB immune-reactive proteins
were detected with advanced chemiluminescence (Enhanced
Chemiluminescence, Amersham Biosciences) and film exposure.
The signal density of the blots was measured by an image analysis
system (Image J, version 1.46r).
Statistical analysis
Neurological deficits scores were compared using non-para-
metric Kruskal–Wallis analysis of variance on ranks fol-
lowed by the Dunn test (SPSS v22.0). Infarct volume,
BWC, EBD extravasations, and data from TNF-α, TNFR1,
 4 S. KHAKSAR AND M. R. BIGDELI
and NF-кB assays were analysed using two-way ANOVA
(SPSS v22.0 post hoc Bonferroni). Data were expressed as
mean±SEM. p < 0.05 was considered significant.
Results
The effect of CBD on neurological deficits
The total neurological deficits score of the vehicle group was
calculated 18.8 ± 1.9. CBD at the dose of 100 ng/rat (10.8 ± 1.8)
improved the neurological outcome significantly (p < 0.01). The
dose of 200 ng/rat CBD indicated its beneficial effects on the
reduction of neurological deficits (14.5 ± 2.1) in comparison
with the vehicle group (p = 0.02), whereas CBD at the dose of
50 ng/rat (19.5 ± 2.3) did not change the total neurological score
(Figure 1). Furthermore, the average score of sham-operated rats
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was very low, with significant difference from the MCA-
occluded animals in all signs (p = 0.004). The difference between
the vehicle and the control groups was not significant.
The effect of CBD-induced protection on infarct volume
The total infarct volume in the vehicle group was
208.13 ± 20.2 mm3. Administration of CBD at the doses of 100
(51.43 ± 12.1 mm3) and 200 ng/rat (91.44 ± 12.1 mm3) resulted
in a significant reduction of the total infarct volume (p < 0.001
and p < 0.01, respectively). Moreover, the dose of 50 ng/rat CBD
did not significantly change the infarct size (198.48 ± 15.1 mm3)
in comparison with the vehicle group. Two doses of CBD, 100
and 200 ng/rat (7.92 ± 10.7 mm3, p < 0.001 and 21.08 ± 6.9 mm3,
p = 0.03, respectively), induced decremental effects on the infarct
volume of the penumbra area. Moreover, the dose of 100 ng/rat
CBD decreased infarction in the core remarkably
(31.55 ± 10.5 mm3, p ˂ 0.01). The comparison between the
vehicle and control groups did not show any significant differ-
ence (Figures 2 and 3).
30
Total Neurological Deficits Score
25
20
15
10
0
Control
Figure 1. Effect of cannabidiol (CBD) at the doses of 50, 100, and 200 ng/rat; i.c.v. on total neurological deficits score at 24 h after cerebral ischaemia in rats.
Cannabidiol in a dose-dependent manner improved neurological deficits significantly. Scores in the sham-operated rats were observed with a significant difference
from the MCA-occluded animals. Values are expressed as the mean±SEM (n = 10). (*p < 0.05, **p < 0.01, ***p < 0.001) (Non-parametric Kruskal–Wallis analysis
followed by the Dunn test).
Dose-dependent effect of CBD on brain oedema
The effect of CBD on BWC as an indicator of brain oedema
was considered. Brain oedema is a major ischaemic injury,
which is associated with increasing BBB permeability. The
difference between ipsilateral and contralateral hemispheres
in control, vehicle-received, and 50 ng/rat of CBD groups was
significant (p < 0.001, p < 0.001, p = 0.008, respectively),
whereas this difference between the two hemispheres in 100
and 200 ng/rat of CBD groups was not significant as well as in
the sham-operated group. Focal cerebral ischaemia signifi-
cantly increased the brain oedema in total, the core, and
penumbra of the ipsilateral hemisphere in comparison with
the sham-operated group (p ˂0.05). CBD at the doses of 100
(78.06 ± 0.4%, p < 0.01) and 200 ng/rat (78.32 ± 0.2%,
p = 0.01) significantly decreased brain oedema of the infarcted
hemisphere compared with the vehicle group (79.42 ± 0.2%),
while the dose of 50 ng/rat of CBD was not statistically
effective (79.58 ± 0.3%). It should be noted that the anti-
oedematous effect of CBD at two doses of 100 and 200 ng/
rat was manifested in the penumbra (69.56 ± 0.5%, p = 0.001
and 70.2 ± 0.2%, p = 0.03, respectively). In addition, the
statistical analysis revealed that there was a significant differ-
ence in brain oedema of the core at the dose of 100 ng/rat of
CBD (70.01 ± 0.5%, p = 0.01) in comparison to the vehicle
group (71.34 ± 0.4%) (Figure 4).
Dose-dependent effect of CBD on BBB integrity
The effect of CBD on BBB permeability is shown in Figure 5.
MCAO surgery was able to significantly increase BBB permeability
in the ipsilateral hemisphere in comparison with the contralateral
hemisphere in the control, vehicle-received, 50, 100, and 200 ng/
rat of the CBD groups (p < 0.01, p < 0.01, p = 0.001, p = 0.03, and
p = 0.02, respectively). The breakdown of BBB was observed in
total, the core, and the penumbra of the ipsilateral hemisphere
compared with the sham-operated group (p˂0.05). In the vehicle
group, EBD concentration was measured as 10.55 ± 0.5 and
***
*
**
Sham Vehicle CBD (50 CBD (100 CBD (200
ng/rat) ng/rat) ng/rat)

a b
300
**
250 ***
Infarct Volume(mm3)
200
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150
100
50
0
Total
Figure 2. This image was used to classify the core and penumbra areas of the ipsilateral hemisphere (right) (a) (25). The classification was determined in accordance with
cerebral arteries distribution, especially territories of the brain supplied blood by MCA (b) (26). The graph shows the effect of various doses of cannabidiol (CBD) (50, 100, and
200 ng/rat; i.c.v.) on infarct volume in total, core, and penumbra areas (c). Total cerebral infarction was significantly reduced by the doses of 100 and 200 ng/rat of
cannabidiol. Values are expressed as the mean±SEM (n = 6). (*p < 0.05, **p < 0.01, ***p < 0.001) compared with the vehicle-treated group (two-way ANOVA test).
3.18 ± 0.4 µg/g cerebral tissue in ischaemic and non-ischaemic
hemisphere, respectively. Administration of CBD at the doses of
100 (right hemisphere = 7.57 ± 0.44, left hemisphere = 2.96 ± 0.44,
p = 0.001) and 200 ng/rat (right hemisphere = 8.13 ± 0.47, left
hemisphere = 3.16 ± 0.38, p = 0.01) significantly decreased BBB
disruption in comparison with the vehicle group. This effect was
not observed at the lower dose of CBD. Moreover, the dose of 100
ng/rat CBD attenuated Evans Blue extravasations both in the core
(right hemisphere = 2.22 ± 0.43, left hemisphere = 1.89 ± 0.25,
p < 0.01) and in the penumbra areas (right hemisphere = 2.04 ± 0.51,
left hemisphere = 1.99 ± 0.3, p = 0.01), while the dose of 200 ng/rat
CBD affected only the penumbra (right hemisphere = 2.56 ± 0.5,
left hemisphere = 1.89 ± 0.5, p = 0.03) (Figure 5).
Effects of CBD-induced neuroprotection on TNF-α
expression
Regarding the expression of TNF-α protein, induction of cerebral
ischaemia by MCAO surgery caused a significant increase in
TNF-α expression level in the core and penumbra (p = 0.03 and
p = 0.04, respectively). Analysis between the sham-operated and
control groups showed no significant difference (data not shown).
Besides, similar results were observed between the control and
vehicle-received groups. The present result expresses that the
TNF-α protein level was attenuated in the penumbra area at
doses of 100 and 200 ng/rat of the CBD-received groups in
BRAIN INJURY 5
Control
Vehicle
CBD (50 ng/rat)
CBD (100 ng/rat)
CBD (200 ng/rat)
**
***
*
Core Penumbra
c
comparison with the vehicle group (p < 0.01 and p = 0.03, respec-
tively). In addition, the TNF-α expression level in the core
decreased in the 100 ng/rat CBD group (p < 0.01) (Figure 6).
Effects of CBD-induced neuroprotection on TNFR1
expression
Western blotting technique indicates that TNFR1 protein is
expressed in the core and penumbra areas of the rat brain hemi-
sphere. The difference of TNFR1 expression between control,
MCAO-operated, and vehicle-received groups was not significant.
Furthermore, comparison between the control and sham-oper-
ated groups revealed no significant difference (data not shown).
Nevertheless, the expression of TNFR1 protein in the core was
reduced at the doses of 100 and 200 ng/rat of CBD compared with
the vehicle group (p < 0.01 and p = 0.01, respectively). In addition,
the doses of 100 and 200 ng/rat of CBD attenuated TNFR1 in the
penumbra (p = 0.01 and p = 0.03, respectively) (Figure 7).
Effects of CBD-induced neuroprotection on NF-кB
expression
The expression of NF-кB protein in the core and penumbra
was administrated by using western blotting. The analysis of
the result exhibits that MCAO surgery caused a significant
increase of NF-кB protein expression in the core (p = 0.03).
 6 S. KHAKSAR AND M. R. BIGDELI

rat of CBD (p = 0.01 and p = 0.03, respectively). Therefore,

our results demonstrate that CBD causes a considerable
reduction in the expression of NF-кB (Figure 8).

Discussion
It is necessary to assess the neurobehavioural responses in par-
allel to other studies when evaluating the efficacy of a candidate
neuroprotective substance. The results of this study show that
CBD at different doses of 100 and 200 ng/rat, i.c.v. improved
neurological impairment induced by MCA occlusion.
Consistent with these results, it has been confirmed that CBD
improves functional deficits such as neurological score and
motor coordination on the rota-rod test (29). Moreover, the
present work indicates that the intracerebroventricular admin-
istration of CBD (100 and 200 ng/rat) reduced the total infarct
volume significantly. The protective effect of CBD was also
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Figure 3. Sections of rat brain stained with TTC in the vehicle, 50, 100, and 200
ng/rat of cannabidiol (CBD) groups. The colourless regions (white) are related to
infarcted territory.
There was no significant difference between the control and
sham-operated groups (data not shown). Similar results were
observed between the control and vehicle-received groups.
Based on the present findings, the doses of 100 and 200 ng/
rat of CBD in the core decreased NF-кB expression when
compared with the vehicle group (p < 0.01 and p = 0.01,
respectively). Furthermore, the reduction of NF-кB expression
in the penumbra was induced by the doses of 100 and 200 ng/
observed in the core and penumbra zones. The core zone, the
centre of ischaemic damages, is caused by necrosis at the early
phase of cerebral ischaemia and the surrounding zone of the core
is known as the penumbra or the recordable tissue that the
apoptosis process develops gradually. In agreement with the
present study, CBD was shown to reduce infarction volume
(14; 29; 30). The i.c.v. route of CBD administration was carried
out for the first time in an animal model of cerebral ischaemia.
The previous investigations were mostly designed based on the
therapeutic approach, while the induction of ischaemic tolerance
is an innovation of this research. CBD prevents cerebral infarc-
tion via a cannabinoids receptor-independent mechanism.
According to the reported findings, the neuroprotective effect
of CBD in cerebral ischaemia might be, at least in part, related to
the activation of the serotonergic 5-HT1A, cannabinoid type 2,
and adenosine A2 receptors (12; 14). In addition, CBD can exert
protective effects via activation of the transient receptor potential
cation channel subfamily V member 1 (TRPV1) and the peroxi-
some proliferator-activated receptor γ (PPAR-γ) receptors
and the inhibition of the G protein-coupled receptor 55
(GPR55) receptor (31–33). As demonstrated, neuronal death
(infarction) in cerebral ischaemia is a consequence of several

* Sham
81
** Control
Vehicle
79
Brain Water Content (%)

CBD (50 ng/rat)

77 CBD (100 ng/rat)

CBD (200 ng/rat)

75 ***

73 * *

71

69
Total.R Core.R Penumbra.R Total.L Core.L Penumbra.L

Figure 4. Brain water content in various experimental groups including ischaemic hemisphere (Total. R), non-ischaemic hemisphere (Total. L), core (right and left),
and penumbra (right and left) areas of control, sham-operated, vehicle, 50, 100, and 200 ng/rat of cannabidiol (CBD). CBD (100 and 200 ng/rat) statistically decreased
brain oedema of infarcted hemisphere compared with the vehicle group. Values are expressed as the mean±SEM (n = 6). (*p < 0.05, **p < 0.01, ***p < 0.001)
compared with the vehicle-treated group (two-way ANOVA test).
 BRAIN INJURY 7

12 *** Sham
*
Control

Evans Blue (µg/g brain tissue)

10 Vehicle
CBD (50 ng/rat)
8 * CBD (100 ng/rat)
** CBD (200 ng/rat)
*
6

0
Total.R Core.R Penumbra.R Total.L Core.L Penumbra.L

Figure 5. EBD extravasations in various experimental groups including the ischaemic hemisphere (Total. R), the non-ischaemic hemisphere (Total. L), core (right and
left), and penumbra (right and left) of control, sham-operated, vehicle, 50, 100, and 200 ng/rat of cannabidiol (CBD). CBD (100 and 200 ng/rat) statistically decreased
the BBB permeability of the infarcted hemisphere compared with the vehicle group. Values are expressed as the mean±SEM (n = 6). (*p < 0.05, **p < 0.01,
Downloaded by [UC Santa Barbara Library] at 01:14 06 September 2017

***p < 0.001) compared with the vehicle-treated group (two-way ANOVA test).

Experimental Groups
Brain Areas Factors
CBD CBD CBD
Control MCAO Vehicle
(50 ng/rat) (100 ng/rat) (200 ng/rat)

TNF-α (16 KD)

Core
β-actin (42 KD)

TNF-α (16 KD)

Penumbra
β-actin (42 KD)

1.8 Control
*
1.6 MCAO
*
** **
Band Intensity of TNF-α

Vehicle
1.4 *
CBD (50 ng/rat)
1.2
CBD (100 ng/rat)
1
CBD (200 ng/rat)
0.8

0.6

0.4

0.2

0
Core Penumbra

Figure 6. Effect of different doses (50, 100, and 200 ng/rat; i.c.v.) of cannabidiol (CBD) on TNF-α expression in the core and penumbra areas of the hemisphere was
evaluated. Cannabidiol decreased the TNF-α protein level in the core and the penumbra. All the data were normalized on the basis of β-actin levels. Values are
expressed as the mean±SEM (n = 5). (*p < 0.05, **p < 0.01) (two-way ANOVA test).
 8 S. KHAKSAR AND M. R. BIGDELI

Experimental Groups
Brain Areas Factors
CBD CBD CBD
Control MCAO Vehicle
(50 ng/rat) (100 ng/rat) (200 ng/rat)

TNFR1 (55 KD)

Core
β-actin (42 KD)

TNFR1 (55 KD)

Penumbra
β-actin (42 KD)
Downloaded by [UC Santa Barbara Library] at 01:14 06 September 2017

1.4 ** * * Control
* MCAO
1.2
Vehicle
Band Intensity of TNFR1

1 CBD (50 ng/rat)

CBD (100 ng/rat)
0.8
CBD (200 ng/rat)
0.6

0.4

0.2

0
Core Penumbra

Figure 7. The expression of TNFR1 protein in the core and penumbra areas of the hemisphere in control, MCAO, vehicle, 50, 100, and 200 ng/rat cannabidiol (CBD)-
received groups. Analysis of TNFR1 protein bands after normalization with β-action as loading control was carried out. The decremental effect of cannabidiol was
revealed at the doses of 100 and 200 ng/rat in the core and penumbra. Values are expressed as the mean±SEM (n = 5). (*p < 0.05, **p < 0.01) (two-way ANOVA test).

pathophysiologic conditions, including glutamate-mediated progressive increase in impedance as an indicator of brain

toxicity, appearing shortly after ischaemia (34). It has also eluci-
dated that CBD attenuates brain damage in an in vitro model of
the immature brain ischaemia by decreasing the glutamate level
and apoptosis (12). Moreover, CBD indirectly suppresses apop-
tosis through scavenging the toxic free radicals (35). In fact, free
radicals participate in activating stress-induced apoptosis (36).
Therefore, protective agents such as CBD that can restore the
glutamate level, free radicals, and neuronal survival will amelio-
rate infarction considerably.
In line with these findings, CBD (100 and 200 ng/rat; i.c.v.)
suppressed brain oedema in a dose- dependent manner. After
the induction of ischaemia, the existence of brain oedema was
also confirmed in the ischaemic hemisphere in comparison
with the non-ischaemic hemisphere. Consistent with our find-
ings, EEG studies demonstrated an increase in the impedance
of piglets after hypoxia–ischaemia. Except Alvarez and collea-
gue’s study, no data regarding the anti-oedematous activity of
CBD during cerebral ischaemia exists (37). CBD blunted the
oedema (35). It is worth noting that the dose of 100 ng/rat of
CBD significantly decreased BWC in the core and penumbra
zones, while a high dose of CBD (200 ng/rat) exclusively
affected the penumbra. In this regard, this study approves
that CBD at a dose of 100 ng/rat showed the best protective
effect in experimentally induced cerebral ischaemia.
Disruption of ion homeostasis results in brain oedema.
Probably, CBD exerts its anti-oedematous effect via the reduc-
tion of massive calcium accumulation and pre-inflammatory
mediators such as interlukin_1 and NF-κB. Increasing intra-
cranial pressure and neurological deficits are the serious
sequels of brain oedema (38). Overall, our data suggest that
CBD-mediated anti-oedematous effect is likely due to the
reduction of infarct volume and neurological deficits.
Subsequently, the present data prove that CBD in a dose-
dependent manner (100 and 200 ng/rat) inhibited BBB perme-
ability significantly. This is the first study to evaluate the effect of
CBD in an in vivo BBB model. We hypothesize that the reduction
 BRAIN INJURY 9

Experimental Groups
Brain area Factor
CBD CBD CBD
Control MCAO Vehicle
(50 ng/rat) (100 ng/rat) (200 ng/rat)

NF-кB (65 KD)

Core
β-actin (42 KD)

NF-кB (65 KD)

Penumbra
β-actin (42 KD)
Downloaded by [UC Santa Barbara Library] at 01:14 06 September 2017

1.8 ** Control
* * MCAO
1.6
Vehicle
Band Intensity of NF-кB

1.4 ** *
CBD (50 ng/rat)
1.2
CBD (100 ng/rat)
1 CBD (200 ng/rat)
0.8

0.6

0.4

0.2

0
Core Penumbra

Figure 8. Effect of different doses (50, 100, and 200 ng/rat; i.c.v.) of cannabidiol (CBD) on NF-κB expression in the core and penumbra areas of the hemisphere was
measured. All the data were normalized on the basis of β-actin levels. Cannabidiol attenuated NF-κB expression at two doses (100 and 200 ng/rat) in the core and the
penumbra. Values are expressed as the mean±SEM (n = 5). (*p < 0.05, **p < 0.01) (two-way ANOVA test).

of brain oedema exerted by CBD might be related to an increase in
BBB integrity, because vosogenic oedema occurs due to the leak-
age of large molecules through the damaged BBB(39). These data
are partly supported by the recent study, suggesting that CBD
preserved BBB integrity in an in vitro BBB model through the
PPAR-γ receptor (40). Disruption of BBB is characterized by
releasing ROS and pro-inflammatory mediators (cytokines and
chemokines) from the injured tissues. These mediators elevate the
expression of the adhesion molecules such as the intercellular
adhesion molecule 1 (ICAM-1) and consequently promote the
adhesion of leukocytes to endothelia cells and migration out of the
vessels, eventually triggering BBB breakdown(41). It has been
reported that CBD reduces ICAM-1 expression in experimental
diabetes and, subsequently, inhibits the migration of leukocytes
(10; 17). The protective effect of CBD on BBB integrity may be
associated with its anti-inflammatory property. Several pieces of
evidence have indicated that ischaemia-induced increases in TNF-
α, iNOS, and IL-6 are abolished by CBD (12; 15). On the other
hand, the activation of microglia potentiates BBB permeability,
whereas the inhibition of microglial may protect the brain against
ischaemic damages by improving BBB viability (42). It has also
been reported that CBD attenuates microglial activation (17).
Therefore, CBD likely mediates the maintenance of BBB integrity
by regulating microglial activation, free radicals, and pro-inflam-
matory mediators. It should also be stated that CBD could reveal
further efficacy in the penumbra zone than in the core. Based on
these findings, the promising function of CBD in rescuing the
penumbra can be suggested. It has been recently asserted that
CBD would prevent the penumbra area from becoming necrotic,
and thus ameliorate brain injury (29).
Evaluation of TNF-α, TNFR1 (the major receptor responsible
for mediating TNFα-induced cell death), and NF-кB proteins
expression was carried out by western blotting. Based on the
data obtained in this study, CBD exerted its decremental effect
on TNFR1 and NF-кB proteins level at two doses (100 and 200 ng/
rat; i.c.v.) in the core and penumbra areas. The decrease in TNF-α
protein expression was observed in the core and penumbra areas
following the administration of a dose of 100 ng/rat. The dose of
 10 S. KHAKSAR AND M. R. BIGDELI
200 ng/rat of CBD also had a beneficial effect on the expression of
TNF-α in the penumbra. The TNF-α factor is particularly impor-
tant in triggering the inflammatory cascade. The involvement and
up-regulation of TNF-α and TNFR in many pathologic manifesta-
tions including cerebral ischaemia have been implicated (43; 44).
The detrimental effect of TNF-α in cerebral ischaemia is related to
an increase in the adhesion of leukocytes to blood vessels(45), the
stimulation of matrix metalloproteinases production(46), and the
decrease in BBB integrity(47). It has been reported that the inhibi-
tion of TNF-α signalling by pharmacologic agents decreases the
infarct volume (48). Therefore, we assume that the reduction of
infarct volume, oedema, and BBB permeability by CBD might be
mediated via the inhibition of TNF-α. TNF-α can play a pro-
inflammatory/pro-apoptotic role. Binding of TNF-α to TNFR1
will trigger a series of intracellular events that ultimately activates
NF-қB. NF-кB also is a mediator of the intracellular pathway that
regulates the transcription of genes involved in cell survival,
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inflammation, and apoptosis (49). Furthermore, the sustained
presence of TNF-α activates caspase 8, which leads to apoptosis
(50). Our results are in agreement with others who have asserted
that CBD ameliorates hypoxic–ischaemic damage by decreasing
TNF-α (12; 29; 51). In addition, a great many publications have
shown that NF-кB is activated in cerebral ischaemia and may
contribute to infarction in permanent MCAO-operated mice
(52; 53) as an increase in NF-кB protein expression in the core
area was found. Despite the well-known role of NF-кB as an anti-
apoptotic factor, it contributes to inflammatory responses and
neuronal death in cerebral ischaemia (54). Hypoxia, ROS, and
inflammatory mediators in cerebral ischaemia trigger cell death
pathways such as NF-кB and impair pro-survival signalling path-
ways (53). One possibility is that the hyperglycaemia-induced
augmentation of leukocyte adhesion to the endothelium (through
the up-regulation of adhesion molecules expression) and transen-
dothelial migration have been reported to be dependent on NF-кB
activation (55). Therefore, we hypothesize that CBD can reduce
NF-кB activity through the suppression of adhesion molecules
expression (10). Although there is no similar study, CBD attenu-
ated TNF-α release through the PPAR-γ receptor in an animal
model of Alzheimer’s. The decrease in NF-кB expression by the
PPAR-γ receptor further reinforces the importance of the cascade
PPAR-γ receptor activation, TNF-α, TNFR1, and NF-кB for
showing the anti-inflammatory effect of CBD (33). In another
study, it has been reported that CBD inhibits both nitrite produc-
tion and iNOS protein expression induced by Aβ. This effect of
CBD is mediated by the inhibition of p38-MAP kinase and NF-кB
(35). Then, the anti-inflammatory activity of CBD was highlighted
once again by attenuating the TNF-α, TNFR1, and NF-кB proteins
levels. Considering these data, our present study demonstrated
that CBD at the dose of 100 ng/rat showed the best protection
(neurological deficits, infarction volume, oedema, and BBB integ-
rity), as the lowest premature death observed at this dose confirms
this hypothesis.
Conclusion
Despite the large amount of data describing the significant
neuroprotective and anti-inflammatory properties of CBD
in neurodegenerative diseases, the mechanism of action of
CBD in cerebral ischaemia has remained unknown. In
order to further elucidate this vague mechanism, the pre-
sent study investigated the intracerebroventricular adminis-
tration of CBD on the main parameters of ischaemic injury.
Based on the data presented in this article, CBD-mediated
neuroprotection (amelioration of neurological deficits,
infarct volume, oedema, and BBB permeability) after
experimental brain ischaemia was partly associated with
the modulation of inflammation, especially the TNF-α,
TNFR1, and NF-кB pathways. However, more research is
required to extend these observations. It should be noted
that the considerable effect of CBD in the amelioration of
penumbra damage provides a new approach for rescuing
this area.
Acknowledgement
This study was supported by a grant (No: 1843) from cognitive Science
and technologies council. The authors would like to express theirs thanks
to Dr Homayoon Khazali at the Reproductive Physiology Lab for coop-
eration in this research by providing a stereotaxic apparatus and Dr Hiva
Alipanah at the Nerves and Heart Physiology Lab of the Shahid Beheshti
University.
Declaration of interest
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of the paper.
References
1. Clarke R, Watson DP. Marijuana and the cannabinoids. In:
ElSohly MA, editors. Forensic science and medicine. Totowa, N.
J.: Humana Press; 1995. 1–15.
2. ElSohly MA. Grotenhermen F, Russo E, editors. Cannabis and
cannabinoids: pharmacology, toxicology, and therapeutic poten-
tial. New York: Haworth Integrative Healing Press; 1995. 27–35.
3. Hayakawa K, Mishima K, Nozako M, Ogata A, Hazekawa M,
Liu AX, Fujioka M, Abe K, Hasebe N, Egashira N, et al.
Repeated treatment with cannabidiol but not Delta9-tetrahy-
drocannabinol has a neuroprotective effect without the devel-
opment of tolerance. Neuropharmacology. 2007;52(4):1079–87.
doi:10.1016/j.neuropharm.2006.11.005.
4. Scuderi C, Filippis DD, Iuvone T, Blasio A, Steardo A, Esposito G.
Cannabidiol in medicine: a review of its therapeutic potential in
CNS disorders. Phytother Res. 2009;23(5):597–602. doi:10.1002/
ptr.v23:5.
5. Dirnagl U, Iadecola C, Moskowitz MA. Pathobiology of ischaemic
stroke: an integrated view. Trends in Neurosciences. 1999;22
(9):391–97. doi:10.1016/S0166-2236(99)01401-0.
6. Donnan GA, Fisher M, Macleod M, Davis SM. Stroke. Lancet.
2008;371(9624):1612–23. doi:10.1016/S0140-6736(08)60694-7.
7. Obrenovitch TP, Richards DA. Extracellular neurotransmitter
changes in cerebral ischaemia. Cerebrovascular and Brain
Metabolism Reviews. 1995;7(1):1–54.
8. Moro MA, Almeida A, Bolanos JP, Lizasoain I. Mitochondrial respira-
tory chain and free radical generation in stroke. Free Radic Biol Med.
2005;39(10):1291–304. doi:10.1016/j.freeradbiomed.2005.07.010.
9. Lakhan SE, Kirchgessner A, Hofer M. Inflammatory mechanisms
in ischemic stroke: therapeutic approaches. J Transl Med.
2009;7:97. doi:10.1186/1479-5876-7-97.
10. El-Remessy AB, Al-Shabrawey M, Khalifa Y, Tsai NT, Caldwell
RB, Liou GI. Neuroprotective and blood-retinal barrier-preserving
effects of cannabidiol in experimental diabetes. Am J Pathol.
2006;168(1):235–44. doi:10.2353/ajpath.2006.050500.
11. Iuvone T, Esposito G, Esposito R, Santamaria R, Di Rosa M, Izzo
AA. Neuroprotective effect of cannabidiol, a non-psychoactive

component from Cannabis sativa, on beta-amyloid-induced toxi-
city in PC12 cells. J Neurochem. 2004;89(1):134–41. doi:10.1111/
jnc.2004.89.issue-1.
12. Castillo A, Tolon MR, Fernandez-Ruiz J, Romero J, Martinez-
Orgado J. The neuroprotective effect of cannabidiol in an in
vitro model of newborn hypoxic-ischemic brain damage in mice
is mediated by CB(2) and adenosine receptors. Neurobiol Dis.
2010;37(2):434–40. doi:10.1016/j.nbd.2009.10.023.
13. Ryan D, Drysdale AJ, Lafourcade C, Pertwee RG, Platt B. Cannabidiol
targets mitochondria to regulate intracellular Ca2+ levels. J Neurosci.
2009;29(7):2053–63. doi:10.1523/JNEUROSCI.4212-08.2009.
14. Mishima K, Hayakawa K, Abe K, Ikeda T, Egashira N, Iwasaki K,
Fujiwara M. Cannabidiol prevents cerebral infarction via a serotoner-
gic 5-hydroxytryptamine1A receptor-dependent mechanism. Stroke.
2005;36(5):1077–82. doi:10.1161/01.STR.0000163083.59201.34.
15. Esposito G, Scuderi C, Savani C, Steardo L Jr., De Filippis D,
Cottone P, Iuvone T, Cuomo V, Steardo L. Cannabidiol in vivo
blunts beta-amyloid induced neuroinflammation by suppressing
IL-1beta and iNOS expression. Br J Pharmacol. 2007;151(8):1272–
79. doi:10.1038/sj.bjp.0707337.
Downloaded by [UC Santa Barbara Library] at 01:14 06 September 2017
16. Hampson AJ, Grimaldi M, Axelrod J, Wink D. Cannabidiol and (-)
Delta9-tetrahydrocannabinol are neuroprotective antioxidants.
Proceedings of the National Academy of Sciences of the United
States of America. 1998;95(14):8268–73. doi:10.1073/pnas.95.14.8268.
17. McHugh D, Tanner C, Mechoulam R, Pertwee RG, Ross RA.
Inhibition of human neutrophil chemotaxis by endogenous can-
nabinoids and phytocannabinoids: evidence for a site distinct
from CB1 and CB2. Mol Pharmacol. 2008;73(2):441–50.
doi:10.1124/mol.107.041863.
18. Hayakawa K, Mishima K, Nozako M, Hazekawa M, Irie K,
Fujioka M, Orito K, Abe K, Hasebe N, Egashira N, et al.
Delayed treatment with cannabidiol has a cerebroprotective action
via a cannabinoid receptor-independent myeloperoxidase-inhibit-
ing mechanism. J Neurochem. 2007;102(5):1488–96. doi:10.1111/
j.1471-4159.2007.04565.x.
19. Shirazi-Zand Z, Ahmad-Molaei L, Motamedi F, Naderi N. The
role of potassium BK channels in anticonvulsant effect of canna-
bidiol in pentylenetetrazole and maximal electroshock models of
seizure in mice. Epilepsy & Behavior: E&B. 2013;28(1):1–7.
doi:10.1016/j.yebeh.2013.03.009.
20. Di Giorgio AM, Hou Y, Zhao X, Zhang B, Lyeth BG, Russell MJ.
Dimethyl sulfoxide provides neuroprotection in a traumatic brain
injury model. Restor Neurol Neurosci. 2008;26(6):501–07.
21. Paxinos G, Watson CR, Emson PC. AChE-stained horizontal
sections of the rat brain in stereotaxic coordinates. J Neurosci
Methods. 1980;3(2):129–49. doi:10.1016/0165-0270(80)90021-7.
22. Longa EZ, Weinstein PR, Carlson S, Cummins R. Reversible
middle cerebral artery occlusion without craniectomy in rats.
Stroke. 1989;20(1):84–91. doi:10.1161/01.STR.20.1.84.
23. Reglodi D, Tamas A, Lengvari I. Examination of sensorimotor per-
formance following middle cerebral artery occlusion in rats. Brain
Res Bull. 2003;59(6):459–66. doi:10.1016/S0361-9230(02)00962-0.
24. Swanson RA, Morton MT, Tsao-Wu G, Savalos RA, Davidson C,
Sharp FR. A semiautomated method for measuring brain infarct
volume. J Cereb Blood Flow Metab. 1990;10(2):290–93. doi:10.1038/
jcbfm.1990.47.
25. Hori M, Nakamachi T, Shibato J, Rakwal R, Tsuchida M, Shioda
S, Numazawa S. PACAP38 differentially effects genes and CRMP2
protein expression in ischemic core and penumbra regions of
permanent middle cerebral artery occlusion model mice brain.
Int J Mol Sci. 2014;15(9):17014–34. doi:10.3390/ijms150917014.
26. Blumenfeld H. Cerebral hemispheres and vascular supply. In:
Blumenfeld H. editor. Neuroanatomy through clinical cases. 1st
ed. Sunderland, Mass: Sinauer; 1995. 375 p.
27. Bigdeli MR, Hajizadeh S, Froozandeh M, Rasulian B,
Heidarianpour A, Khoshbaten A. Prolonged and intermittent
normobaric hyperoxia induce different degrees of ischemic toler-
ance in rat brain tissue. Brain Res. 2007;1152:228–33. doi:10.1016/
j.brainres.2007.03.068.
BRAIN INJURY 11
28. Mohagheghi F, Bigdeli MR, Rasoulian B, Hashemi P, Pour MR.
The neuroprotective effect of olive leaf extract is related to
improved blood-brain barrier permeability and brain edema in
rat with experimental focal cerebral ischemia. Phytomedicine.
2011;18(2–3):170–75. doi:10.1016/j.phymed.2010.06.007.
29. Pazos MR, Cinquina V, Gomez A, Layunta R, Santos M,
Fernandez-Ruiz J, Martinez-Orgado J. Cannabidiol adminis-
tration after hypoxia-ischemia to newborn rats reduces long-
term brain injury and restores neurobehavioral function.
Neuropharmacology. 2012;63(5):776–83. doi:10.1016/j.
neuropharm.2012.05.034.
30. Braida D, Pegorini S, Arcidiacono MV, Consalez GG, Croci L,
Sala M. Post-ischemic treatment with cannabidiol prevents elec-
troencephalographic flattening, hyperlocomotion and neuronal
injury in gerbils. Neurosci Lett. 2003;346(1–2):61–64.
doi:10.1016/S0304-3940(03)00569-X.
31. Hegde VL, Nagarkatti PS, Nagarkatti M. Role of myeloid-derived
suppressor cells in amelioration of experimental autoimmune
hepatitis following activation of TRPV1 receptors by cannabidiol.
PloS One. 2011;6(4):e18281. doi:10.1371/journal.pone.0018281.
32. Chiurchiu V, Lanuti M, De Bardi M, Battistini L, Maccarrone M.
The differential characterization of GPR55 receptor in human
peripheral blood reveals a distinctive expression in monocytes
and NK cells and a proinflammatory role in these innate cells.
Int Immunol. 2015;27(3):153–60. doi:10.1093/intimm/dxu097.
33. Esposito G, Scuderi C, Valenza M, Togna GI, Latina V, De
Filippis D, Cipriano M, Carratu MR, Iuvone T, Steardo L.
Cannabidiol reduces Abeta-induced neuroinflammation and pro-
motes hippocampal neurogenesis through PPARgamma involve-
ment. PLoS One. 2011;6(12):e28668. doi:10.1371/journal.
pone.0028668.
34. Fernandez-Lopez D, Martinez-Orgado J, Casanova I, Bonet B, Leza
JC, Lorenzo P, Moro MA, Lizasoain I. Immature rat brain slices
exposed to oxygen-glucose deprivation as an in vitro model of
neonatal hypoxic-ischemic encephalopathy. J Neurosci Methods.
2005;145(1–2):205–12. doi:10.1016/j.jneumeth.2005.01.005.
35. Esposito G, De Filippis D, Maiuri MC, De Stefano D, Carnuccio
R, Iuvone T. Cannabidiol inhibits inducible nitric oxide synthase
protein expression and nitric oxide production in beta-amyloid
stimulated PC12 neurons through p38 MAP kinase and NF-
kappaB involvement. Neurosci Lett. 2006;399(1–2):91–95.
doi:10.1016/j.neulet.2006.01.047.
36. Booz GW. Cannabidiol as an emergent therapeutic strategy for
lessening the impact of inflammation on oxidative stress. Free
Radic Biol Med. 2011;51(5):1054–61. doi:10.1016/j.
freeradbiomed.2011.01.007.
37. Alvarez FJ, Lafuente H, Rey-Santano MC, Mielgo VE, Gastiasoro
E, Rueda M, Pertwee RG, Castillo AI, Romero J, Martinez-Orgado
J. Neuroprotective effects of the nonpsychoactive cannabinoid
cannabidiol in hypoxic-ischemic newborn piglets. Pediatr Res.
2008;64(6):653–58. doi:10.1203/PDR.0b013e318186e5dd.
38. Lingwood BE, Dunster KR, Healy GN, Ward LC, Colditz PB.
Cerebral impedance and neurological outcome following a mild
or severe hypoxic/ischemic episode in neonatal piglets. Brain Res.
2003;969(1–2):160–67. doi:10.1016/S0006-8993(03)02295-9.
39. Simard JM, Kent TA, Chen M, Tarasov KV, Gerzanich V. Brain
oedema in focal ischaemia: molecular pathophysiology and theo-
retical implications. Lancet Neurol. 2007;6(3):258–68.
doi:10.1016/S1474-4422(07)70055-8.
40. Hind W, England T, O’Sullivan S, Eds. Cannabidiol protects an in
vitro model of the blood-brain barrier against increased perme-
ability following oxygen-glucose deprivation. Proceedings of the
6th European Workshop on Cannabinoid Research Trinity
College Dublin; 2013; Ireland. Year.
41. Yilmaz G, Granger DN. Cell adhesion molecules and ischemic
stroke. Neurological Research. 2008;30(8):783–93. doi:10.1179/
174313208X341085.
42. Yenari MA, Xu L, Tang XN, Qiao Y, Giffard RG. Microglia potenti-
ate damage to blood-brain barrier constituents: improvement by
 12 S. KHAKSAR AND M. R. BIGDELI
minocycline in vivo and in vitro. Stroke. 2006;37(4):1087–93.
doi:10.1161/01.STR.0000206281.77178.ac.
43. Botchkina GI, Meistrell ME 3rd, Botchkina IL, Tracey KJ.
Expression of TNF and TNF receptors (p55 and p75) in the rat
brain after focal cerebral ischemia. Mol Med. 1997;3(11):765–81.
44. Maddahi A, Kruse LS, Chen QW, Edvinsson L. The role of tumor
necrosis factor-alpha and TNF-alpha receptors in cerebral arteries
following cerebral ischemia in rat. J Neuroinflammation.
2011;8:107. doi:10.1186/1742-2094-8-107.
45. Robbins DS, Shirazi Y, Drysdale BE, Lieberman A, Shin HS, Shin
ML. Production of cytotoxic factor for oligodendrocytes by sti-
mulated astrocytes. Journal of Immunology. 1987;139(8):2593–97.
46. Rosenberg GA, Dencoff JE, Correa N Jr., Reiners M, Ford CC.
Effect of steroids on CSF matrix metalloproteinases in multiple
sclerosis: relation to blood-brain barrier injury. Neurology.
1996;46(6):1626–32. doi:10.1212/WNL.46.6.1626.
47. Megyeri P, Abraham CS, Temesvari P, Kovacs J, Vas T, Speer CP.
Recombinant human tumor necrosis factor alpha constricts pial
arterioles and increases blood-brain barrier permeability in new-
born piglets. Neurosci Lett. 1992;148(1–2):137–40. doi:10.1016/
Downloaded by [UC Santa Barbara Library] at 01:14 06 September 2017
0304-3940(92)90823-P.
48. Hosomi N, Ban CR, Naya T, Takahashi T, Guo P, Song XY,
Kohno M. Tumor necrosis factor-alpha neutralization reduced
cerebral edema through inhibition of matrix metalloproteinase
production after transient focal cerebral ischemia. J Cereb Blood
Flow Metab. 2005;25(8):959–67. doi:10.1038/sj.jcbfm.9600086.
49. Wajant H, Pfizenmaier K, Scheurich P. Tumor necrosis factor
signaling. Cell Death Differ. 2003;10(1):45–65. doi:10.1038/sj.
cdd.4401189.
50. Gaur U, Aggarwal BB. Regulation of proliferation, survival and apop-
tosis by members of the TNF superfamily. Biochem Pharmacol.
2003;66(8):1403–08. doi:10.1016/S0006-2952(03)00490-8.
51. Lafuente H, Alvarez FJ, Pazos MR, Alvarez A, Rey-Santano MC,
Mielgo V, Murgia-Esteve X, Hilario E, Martinez-Orgado J.
Cannabidiol reduces brain damage and improves functional recov-
ery after acute hypoxia-ischemia in newborn pigs. Pediatric
Research. 2011;70(3):272–77. doi:10.1203/PDR.0b013e3182276b11.
52. Nurmi A, Lindsberg PJ, Koistinaho M, Zhang W, Juettler E,
Karjalainen-Lindsberg ML, Weih F, Frank N, Schwaninger M,
Koistinaho J. Nuclear factor-kappaB contributes to infarction
after permanent focal ischemia. Stroke. 2004;35(4):987–91.
doi:10.1161/01.STR.0000120732.45951.26.
53. Ridder DA, Schwaninger M. NF-kappaB signaling in cerebral
ischemia. Neuroscience. 2009;158(3):995–1006. doi:10.1016/j.
neuroscience.2008.07.007.
54. Shih RH, Wang CY, Yang CM. NF-kappaB signaling pathways in
neurological inflammation: A mini review. Front Mol Neurosci.
2015;8:77. doi:10.3389/fnmol.2015.00077.
55. Hamuro M, Polan J, Natarajan M, Mohan S. High glucose
induced nuclear factor kappa B mediated inhibition of endothelial
cell migration. Atherosclerosis. 2002;162(2):277–87. doi:10.1016/
S0021-9150(01)00719-5.