Journal of Neurology and Epidemiology, 2017, 5, 25-34
25
Acute Effects of Cannabis sativa on Ischaemia/Reperfusion Injury
in the Rat Brain
Omar M.E. Abdel-Salam1,*, Gehad Abdel Jaleel2 and Fatma A Morsy3
1
Department of Toxicology and Narcotics, National Research Centre, Cairo, Egypt
2
Department of Pharmacology, National Research Centre, Cairo, Egypt
3
Department of Pathology, National Research Centre, Cairo, Egypt
Abstract: We investigated the effect of Cannabis sativa extract on brain damage, oxidative stress and inflammation in
rats with transient global cerebral ischaemia. Rats were subjected to bilateral common carotid artery (CCA) occlusion for
9
45 minutes followed by 4 h of reperfusion. Rats were treated with cannabis at a dose of 20 mg/kg (expressed as Δ THC) intraperitoneally (i.p) either before CCA, at time of reperfusion or after reperfusion. Alternatively, cannabis was
given i.p. daily for 2 days before surgery. Markers of oxidative stress (malondialdehyde, reduced glutathione, nitric oxide)
and the proinflammatory cytokine tumour necrosis factor-alpha (TNF-α) were determined in brain tissue.
Histopathological evaluation was also done. Compared with the sham-treatment group, CCA occlusion resulted in
increased brain malondialdehyde (54.5 ± 2.0 vs. 26.0 ± 1.45 nmol/g. tissue; p<0.05) and nitric oxide (75.2 ± 3.2 vs. 31.3
± 3.0 µmol/g. tissue; p<0.05) contents along with decreased brain reduced glutathione (6.6 ± 0.14 vs. 8.28 ± 0.31µmol/g.
tissue; p<0.05). There was also a pronounced rise in brain TNF-α concentrations (2248 ± 105 vs. 51.42 ± 3.21 pg/g.
tissue; p<0.05). Cannabis sativa significantly increased reduced glutathione (by 30.3%-60.6%; p<0.05) and alleviated the
increase in nitric oxide levels (by 51.5%- 58.5%; p<0.05) in the ischaemic brain tissue. Cannabis given before or at time
of CCA occlusion significantly reduced brain TNF-α by 24.4% and 26.7%, respectively (1699 ± 80 and 1647 ± 54 vs.
2248 ± 105 pg/g. tissue; p<0.05). Histopathological examination of the cerebral cortex from rats subjected to CCA
occlusion revealed gliosis, vacuolation and widespread neuronal degeneration. Cannabis given as a single dose 1h prior
to CCA ligation or as 2 days pretreatment conferred protection against the ischaemic neuronal injury. It is concluded that
in cerebral ischaemia the prior administration of cannabis exerted neuroprotective effects which could be accounted for
by a decrease in nitric oxide and in the inflammatory response.
Keywords: Cannabis, brain ischemia and reperfusion, oxidative stress, neuroinflammation.
INTRODUCTION
Cannabis sativa L. (Family Cannabinaceae) has
long been known for its psychotropic effects and is
considered the most widely abused substance
worldwide [1]. Cannabis preparations mostly abused
are marijuana which is the dried leaves and flowering
tops or hashish, the compressed resin of the female
plant [2]. Cannabis exerts a wide spectrum of acute
central nervous system effects including euphoria or
“high”, short-term memory impairment, distorted time
perception, increased sensory awareness, tachycardia,
increased appetite, psychomotor and locomotor
impairments [3]. Chronic and heavy users of cannabis
exhibit cognitive decline, impaired memory processing
and attention [4], structural brain changes [5] and are
liable to develop psychosis [6]. These central effects of
cannabis are mediated through interaction with CB1
cannabinoid receptor, G-protein coupled receptors,
expressed in neurons in brain and spinal cord [7]. The
main active psychotropic ingredient in herbal cannabis
9
9
is Δ -tetrahydrocannabinol (Δ -THC) [8] but other
phytocannabinoids
eg.,
cannabigerol
(CBG),
*Address correspondence to this author at the Department of Toxicology
and Narcotics, National Research Centre, Dokki 112311, Cairo, Egypt;
Tel: +20 233 371 362; Fax: +20 233 370 931; E-mail: omasalam@hotmail.com
E-ISSN: 2309-6179/17
cannabichromene
(CBC),
cannabidiol
(CBD),
9
cannabidivarin (CBDV), Δ -tetrahydrocannabivarin
(THCV) are found and these might enhance or
9
antagonize some of the Δ -THC effects [9].
Ischaemic stroke has been reported in heavy users
of marijuana or hashish [10-14]. In these case reports a
temporal association has been found between
cannabis use and the development of stroke in that
cerebral or cerebellar infarcts occurred during or shortly
after consuming cannabis [10-12]. Moreover, recurrent
stroke developed upon re-exposure to cannabis [12].
Users of cannabis who developed ischaemic stroke
were more frequently young men and consumed
tobacco and alcohol compared with non-users [13, 14].
Cannabis might cause ischaemic stroke by inducing
cerebral vasospasm, cerebral hypoperfusion secondary
to systemic hypotension or via cardiac embolization
[14].
In cerebral ischaemia, strong evidence indicates an
important role for increased oxidative/nitrosative stress
and neuroinflammation that act to exacerbate the
inflammatory milieu and the initial ischemic injury
through the release of reactive oxygen metabolites and
inflammatory mediators [151]. Reactive oxygen
metabolites are produced by the cell’s metabolic
© 2017 Savvy Science Publisher
26
Journal of Neurology and Epidemiology, 2017, Vol. 5
machinery. The mitochondrial complexes I and II leak
electrons to molecular oxygen forming superoxide
anion which in turn could result in the formation of
hydrogen peroxide (H2O2), hydroxyl radical (·OH) or
react with nitric oxide forming peroxynitrite. The brain is
susceptible to free radical-mediated oxidative damage
due to high rate of oxygen consumption, its rich content
of polyunsaturated fatty acids, the presence of redoxactive transition metals, namely iron and copper
combined with paucity of antioxidant mechanisms
compared to other tissues [16]. Significant increase in
reactive oxygen species occurs early in ischaemia
while the increase in cerebral blood flow during reperfusion results in robust reactive oxygen species
generation with damage to the cell macromolecules
causing protein and DNA oxidation, lipid peroxidation
and membrane damage [17]. Cerebral ischaemia also
results in the expression of inflammatory genes and the
release of several inflammatory mediators eg.,
interleukin-6 (IL-6), IL-1β, tumour necrosis factor-alpha
(TNF- α) and inducible nitric oxide synthase (iNOS)
and the influx of neutrophils into the injured tissue. It
was suggested that this post-ischaemic local
inflammatory response might contribute to the
secondary brain damage [18, 19].
In this study, we aimed to investigate the effect of
acute
cannabis
treatment
on
cerebral
ischemia/reperfusion (I/R) injury induced in the rat by
bilateral CCA occlusion, a well-recognized model of
transient global cerebral ischaemia [20, 21].
MATERIALS AND METHODS
Animals
Male Sprague-Dawley rats weighing 230-250 g
were obtained from the Animal House of the National
Research Centre, Cairo. Rats were kept under
temperature- and light-controlled conditions and given
standard laboratory rodent chow and water ad libitum.
The
experimental
procedures
followed
the
recommendations of the institutional Ethics Committee
of the National Research Centre and that of the
National Institutes of Health Guide for Care and Use of
Laboratory Animals (Publication No. 85-23, revised
1985).
Abdel-Salam et al.
spec”. The dry extract was prepared according to the
method of Turner and Mahlberg [22] with modification.
In brief, 10 g of hashish was divided into small pieces,
well grounded and heated in a glass baker in boiling
water at 100 ºC for two hours for decarboxylation of the
acidic cannabinoids [22]. Cannabis was then placed in
chloroform overnight, extracted three times with
chloroform and fractions were combined, filtered over
filter paper and finally collected in a 100 mL volumetric
flask. The filtrate was then evaporated under a gentle
stream of nitrogen. The dry extract was protected from
light and stored at 4ºC. For use in experiments, the
extract was suspended in 2 mL of 96 % ethanol and
the total volume in the volumetric flask increased to
100 mL by distilled water. The extract was injected at
9
the dose of 20 mg/kg (expressed as Δ -THC). The
injection volume was 0.3 ml/rat.
Induction of Transient Global Cerebral Ischaemia
Cerebral ischaemia was induced by ligation of both
carotid arteries for 45 min followed by 4 h reperfusion.
Briefly, animals were fasted 12 hours before surgery
and then anesthetized with thiopental (20 mg/kg; i.p.).
A longitudinal cervical incision (2 cm) was made lateral
to the midline and the common carotid artery (CCA)
was carefully dissected. Ischemia was induced by
placing non traumatic micro-vascular clip on either
CCA just prior to its bifurcation. During ischemia, body
temperature was maintained at 36.5 ± 0.5˚C using
heating pad and respiration pattern monitored. The
vascular occlusion was maintained for 45 minutes, and
then the clips were removed to resume blood flow to
the ischemic region. Finally, the incisions were sutured,
the animal was allowed to recover from anesthesia,
and returned to a warm cage during the 4 h reperfusion
period.
Experimental Design
The effect of Cannabis sativa extract on the rat
cerebral ischaemia/reperfusion injury was studied.
Cannabis was given at the dose of 20 mg/kg
9
(expressed as Δ -THC).
Rats were randomly allocated into 7 groups (8 rats
each):
Plant Material and Extraction
Group I: Non-operated rats.
Cannabis sativa L. resin (Hashish) was a gift from
the Ministry of Justice- Egypt. Extract of Cannabis
sativa L. was obtained by chloroform treatment and
9
contained ∼ 20% Δ -THC as determined by “GC mass
Group II: Sham-operated rats.
Group III: Untreated rats received i.p. saline and
underwent cerebral ischemia- reperfusion injury (I/R).
Acute Effects of Cannabis sativa on Ischaemia/Reperfusion Injury
Group IV: Rats treated with a single dose of cannabis
extract (20 mg/kg; i.p.) 1h before CCA ligation (i.e.
before ischemia).
Group V: Rats treated with a single dose of cannabis
extract (20 mg/kg; i.p.) at time of CCA occlusion.
Journal of Neurology and Epidemiology, 2017, Vol. 5
27
IV. Tumour Necrosis Factor Alpha
TNF-α was measured in serum using a doubleantibody sandwich enzyme-linked immunosorbent
assay (ELISA) Kit purchased from Glory Science Co.,
Ltd. (Del Rio, TX, USA) according to the manufacture
instructions.
Group VI: Rats treated with a single dose of cannabis
extract (20 mg/kg; i.p.) 1 h after reperfusion.
Histopathological Examination
Group VII: Rats treated for two days with cannabis
extract (20 mg/kg/day; i.p.) followed by cerebral I/R
injury.
The brain of different groups was removed
immediately and fixed in 10% formol saline. Paraffin
sections (5µm thick) were stained with haematoxlin and
eosin and investigated by light microscope.
Brain Homogenate Preparation
After 4 h of reperfusion, rats were decapitated under
light diethyl ether anesthesia and their brain were
carefully isolated and dissected through the midline into
two hemispheres. 0.5 g of the affected hemisphere was
homogenized (using MPW–120 homogenizer, Med
instruments, Poland); the homogenate was then
centrifuged
using
a
cooling
centrifuge
(Laborezentrifugen, 2k15, Sigma, Germany) at 3000
r.p.m for 10 min and the supernatant was used for the
determination of brain level of MDA, nitric oxide
metabolites, and reduced glutathione.
Measurements
I. Lipid Peroxidation Products
Lipid peroxidation was assessed by measuring the
level of malondialdehyde (MDA) according to the
method of Ruiz-Larrea et al. [23]. The thiobarbituric
acid reactive substances react with thiobarbituric acid
to produce a red colored complex that exhibits a peak
absorbance at 532 nm.
II. Reduced Glutathione
Brain reduced glutathione (GSH) was determined
spectrophotometrically by the Ellman's method [24].
The procedure is based on the reduction of the
Ellman's reagent by –SH groups of GSH to form 2nitro-5-mercaptobenzoic acid, which is determined at
412 nm.
III. Nitric Oxide
Nitrite, the stable end product of nitric oxide, is
mostly used as an indicator for the production of nitric
oxide. Nitric oxide measured as nitrite was determined
in brain homogenates using the Griess reagent,
according to the method described by Moshage et al.
[25].
RESULTS
Biochemical Results
Data are presented in Table 1.
I. Carotid Artery Ligation Only
Rats subjected to bilateral CCA ligation for 45 min
exhibited a significant increase in brain lipid
peroxidation as indicated by malondialdehyde level
which rose to 109.6% of its sham control value (54.5 ±
2.0 vs. 26.0 ± 1.45 nmol/g. tissue; p<0.05). Meanwhile,
there was marked and significant increase in brain
nitric oxide by 140.2% (75.2 ± 3.2 vs. 31.3 ± 3.0
µmol/g. tissue; p<0.05) and a decrease in reduced
glutathione by 20.3% (6.6 ± 0.14 vs. 8.28 ± 0.31µmol/g.
tissue; p<0.05) of their corresponding sham values. On
the other hand, the level of the proinflammatory
cytokine TNF-α showed pronounced increase by
4271.8% compared with that in the sham control group
(2248 ± 105 vs. 51.42 ± 3.21 pg/g. tissue; p<0.05).
II. Cannabis Given Before CCA Occlusion
There was no significant difference in brain
malondialdehyde levels between the ischaemic control
group and that treated with cannabis prior to CCA
occlusion (54.5 ± 3.0 vs. 54.5 ± 2.0 nmol/g. tissue;
p>0.05). Nitric oxide, however, decreased by 41.5 %
(44.0 ±1.8 vs. 75.2 ± 3.2 µmol/g. tissue; p<0.05) and
reduced glutathione increased by 48.5 % (9.8 ± 0.34
vs. 6.6 ± 0.14 µmol/g. tissue; p<0.05). Cannabis
treatment also resulted in significant decrease in brain
TNF-α by -24.4 % (1699 ± 80 vs. 2248 ± 105 pg/g.
tissue; p<0.05) compared with the ischaemic untreated
group.
III. Cannabis Given at Time of CCA Occlusion
No significant difference in brain malondialdehyde
was observed between rats treated with cannabis at
28
Journal of Neurology and Epidemiology, 2017, Vol. 5
Abdel-Salam et al.
Table 1: The effect of cannabis on reduced glutathione (GSH), malondialdehyde (MDA), nitric oxide and tumour
necrosis factor-alpha (TNF-α) in the brain of rats subjected to iscahemia/reperfusion (I/R) injury
Groups
GSH (µmole/g.
tissue)
MDA (nmol/g.
tissue)
Nitric oxide (µmole/g.
tissue)
TNF-α (Pg/g.
tissue)
Control
8.43 ±0.26
23.4 ± 0.95
30.32 ± 1.92
44.6 ±1.80
Sham
8.28 ± 0.31
26.0 ± 1.45
31.30 ± 3.0
51.42 ± 3.21
Cerebral I/R control
6.6 ± 0.14
Single dose cannabis before
occlusion
Single dose cannabis at occlusion
*
54.5 ± 2.0
9.8 ± 0.34
+
54.5 ± 3.0
(48.5 %)
(0.0%)
+
*
75.2 ± 3.2
*
*
44.0 ±1.8
*+
(-41.5 %)
*
*+
*
2248 ± 105
*+
1699 ± 80
(-24.4 %)
*+
9.6 ± 0.38
48.5 ± 0.75
31.2 ± 1.7
1647 ± 54
(45.5 %)
(-11.0%)
(-58.5 %)
(-26.7 %)
+
*
*+
*
Single dose cannabis after
reperfusion
8.6 ± 0.10
55.4 ± 1.7
42.4 ± 1.3
2370 ± 146
(30.3 %)
(1.6%)
(-43.6 %)
(5.4%)
Cannabis pretreatment for 2 days
before I/R
10.6 ± 0.74
59.0 ± 4.2
32.2 ± 1.3
1970 ±102
(60.6 %)
(8.3%)
(-57.2%)
(-12.4 %)
*+
*
*+
*
Results are mean ± SEM. Abbreviations: IR, ischemia/reperfusion.
*p<0.05 vs. Sham group. +p<0.05 vs. I/R control. One-way ANOVA and Duncan multiple comparison test. The percent change from the cerebral I/R control group is
shown in parenthesis.
time of vessel occlusion and the control ischaemic
group (48.5 ± 0.75 vs. 54.5 ± 2.0 nmol/g. tissue;
p>0.05). Cannabis treatment, however, was associated
with a decrease in nitric oxide by 58.5% (31.2 ± 1.7 vs.
75.2 ± 3.2 µmol/g. tissue; p<0.05) and an increase in
reduced glutathione by 45.5 % (9.6 ± 0.38 vs. 6.6 ±
0.14 µmol/g. tissue; p<0.05). A significant decrease in
brain TNF-α by 26.7% was also noted compared with
the ischaemic control value (1647 ± 54 vs. 2248 ± 105
pg/g. tissue; p<0.05).
IV. Cannabis Given After Reperfusion
In this group, brain malondialdehyde was not
significantly altered (55.4 ± 1.7 vs. 54.5 ± 2.0 nmol/g.
tissue; p>0.05) but nitric oxide decreased by 43.6%
(42.4 ± 1.3 vs. 75.2 ± 3.2 µmol/g. tissue; p<0.05) and
reduced glutathione increased by 30.3% (8.6 ± 0.10 vs.
6.6 ± 0.14 µmol/g. tissue; p<0.05) compared with the
ischaemic untreated group. On the other hand,
cannabis administered following reperfusion did not
significantly alter brain TNF-α (2370 ± 146 vs. 2248 ±
105 pg/g. tissue; p>0.05).
V. Cannabis Pretreatment for 2 Days Before CCA
Occlusion
Pretreatment with cannabis for 2 days prior to brain
ischaemia has no significant effect on the level of
malondialdehyde (59.0 ± 4.2 vs. 54.5 ± 2.0 nmol/g.
tissue; p>0.05). Cannabis treatment was effective in
increasing brain reduced glutathione (60.6% increment:
10.6 ± 0.74 vs. 6.6 ± 0.14 µmol/g. tissue; p<0.05).
There was also a significant and marked decrease in
brain nitric oxide by 57.2% (32.2 ± 1.3 vs. 75.2 ± 3.2
µmol/g. tissue; p<0.05). Meanwhile, there was a nonsignificant decrease in TNF-α concentrations by 12.4%
as compared to the ischaemic untreated group (1970
±102 vs. 2248 ± 105 pg/g. tissue; p>0.05).
Histopathological Results
I. Carotid Artery Ligation Only
Microscopic examination of brain tissue from unoperated rats showed the normal structure of the
cerebral cortex with its innermost granular layer,
Purkinje cell layer, and the outermost molecular layer
(Figure 1A). No apparent morphological changes were
observed in the sham group, although some congested
blood vessels were seen (Figure 1B). Sections of the
cerebral cortex from rats subjected to I/R showed
congestion of cerebral blood vessels, hemorrhage in
meninges above the surface and thrombotic vessels.
Sings of degeneration in some neurons in the form of
pyknotic nuclei (anoxic neurons) with eosinophilic
cytoplasm were present. Foci of empty neural cells with
identifiable gliosis and vacuolation were also observed
(Figures 1C & D).
II. Cannabis Given Before CCA Occlusion
In rats given cannabis 1h before CCA occlusion,
most of neuronal cells appeared normal and a few
appeared degenerated in the form of karyorrhexis and
cytoplasmic vacuolation. Some neurons were seen
shrunken with esinophilic cytoplasm and pyknotic
nuclei (anoxic neurons) (Figures 2A & B).
Acute Effects of Cannabis sativa on Ischaemia/Reperfusion Injury
Journal of Neurology and Epidemiology, 2017, Vol. 5
A
B
C
D
29
Figure 1: H & E stained brain sections. (A): Control non-operated rat showing normal histological structure of brain tissue (HX&E
x200). (B) Sham-operated rat showing normal appearance (Hx&E x400). (C): Brain tissue after I/R showing gliosis (red arrow)
and vacuolation (black curved arrow) (Hx & E x400). (D): Another filed of the ischemic brain showing hemorrhage in meninges
above the surface and thrombotic vessels show a vessel with membrane bound vacuoles (yellow curved arrow). Sings of
degeneration in some neurons and eosinophilic in others with pyknotic nuclei (anoxic neurons) (red arrow) are seen (Hx & E
x400).
A
B
30
Journal of Neurology and Epidemiology, 2017, Vol. 5
Abdel-Salam et al.
(Figure 2). Continued.
C
D
Figure 2: H & E stained brain sections. (A): Cannabis given 1h before CCA occlusion: most of neuronal cells appeared normal
and few degenerated in the form of karyorrhexis (yellow arrow) and cytoplasmic vacuolation (red arrow) (Hx &E x200). (B):
Another filed showing easinophilic neuron (black arrow) (Hx &E x400). (C): Cannabis given at time of CCA occlusion: more
pronounced pathological changes are seen in the form of widespread severe spongiform change with accompanying neuronal
loss and some neuron appeared degenerated (yellow arrow) and gliosis (black arrow) (Hx & E x400). (D) Another filed in brain
after cannabis being given after CCA occlusion showing multiple numbers of focal homogenous deeply eosinophilic plaques of
various sizes and shapes (star). Within the plaque structure, nuclei of microglia cells could be seen and cytoplasmic
vacuolations. Congestion of cerebral blood vessel (red arrow) and oedema are seen (Hx &E x400).
A
B
C
D
Figure 3: H & E stained brain sections. (A): Cannabis given after reperfusion showing congestion of cerebral blood vessel
(yellow arrow) and degenerated neuron (red arrow) (Hx&E x400). (B) Another filed showing neuronal loss, eosinophilic neurons
and perineuronal vacuolation develops (yellow arrow) and others with shrunken eosinophilic cytoplasm and pyknotic nuclei
(anoxic neurons) (black arrow) with gliosis (red arrow) (Hx&E x400). (C) Cannabis given for two consecutive days prior to I/R
showing some improvement in pathological changes as compared to previous group in the form of no pyknotic neurons; most
neurons appeared normal but some degenerated neurons are still present (red arrow). Hemorrhage in meninges above the
surface (red arrow) is seen (Hx&E x200). (D) Another filed showing congestion of cerebral blood vessel (black arrow) and few
neurons with coagulation necrosis (red arrow) (Hx&E x400).
Acute Effects of Cannabis sativa on Ischaemia/Reperfusion Injury
III. Cannabis Given at Time of CCA Occlusion
Sections of the cerebral cortex of ischemic brain of
rats given cannabis after CCA occlusion showed more
pronounced pathological changes in the form of
multiple numbers of focal homogenous deeply
eosinophilic plaques of various sizes and shapes,
within the plaque structure, nuclei of microglia cells
could be seen and cytoplasmic vacuolations.
Congestion of cerebral blood vessels and edema were
present. Widespread severe spongiform change with
accompanying neuronal loss was observed (Figures
2C & D).
IV. Cannabis Given After Reperfusion
Sections of the cerebral cortex of ischemic brain of
rats given cannabis at time of reperfusion revealed
degeneration of neurons and foci of empty neural cells.
Few of neurons appeared shrunken with eosinophilic
cytoplasm, pyknotic nuclei (anoxic neurons) and
perineuronal vacuolation. Congestion of cerebral blood
vessel and identifiable gliosis are seen (Figures 3A &
B).
V. Cannabis Pretreatment for 2 Days Before CCA
Occlusion
In rats treated with cannabis for two consecutive
days prior to I/R there was some improvement in the
pathological changes as compared to previous group in
the form of no pyknotic neurons. Most neurons
appeared normal but some degenerated neurons were
still present. Hemorrhage in meninges above the
surface and congestion of cerebral blood vessels were
also seen (Figures 3C & D).
DISCUSSION
In this study, bilateral CCA occlusion for 4h resulted
in brain oxidative stress as evidenced by the rise in the
lipid peroxidation end product malondialdehyde, the
increase in nitric oxide and by the depletion of the
antioxidant molecule reduced glutathione. These
observations are in accordance with other studies
following transient or global cerebral ischaemia in
rodents [20, 21]. In brain ischemia there is robust
increase in reactive oxygen metabolites especially
during the reperfusion period owing to the increase in
O2 delivery to brain tissue with a resultant oxidative
damage to cell membrane fatty acids, proteins and
DNA [17]. Our results shows that reduced glutathione,
an important intracellular antioxidant and free radical
scavenger [26] decreased significantly in brain tissue of
rats with I/R. Glutathione (γ-glutamylcysteinylglycine) is
the most abundant intracellular non-protein thiol and by
Journal of Neurology and Epidemiology, 2017, Vol. 5
31
switching between its reduced (GSH) and oxidized
(glutathione disulfide: GSSG) forms helps to maintain
the redox equilibrium of the cell [26]. The decrease in
reduced glutathione following cerebral ischaemia and
reperfusion is likely to reflect consumption of reduced
glutathione by reactive oxygen metabolites. Thus in
brain ischaemia, the antioxidant defenses are
overwhelmed by the high level of reactive free radicals
generated by the mitochondrial electron transport chain
with consequent neuronal damage [27]. Studies
indicated an important role of oxidative stress in brain
ischaemic injury [28, 29]. It has been shown that mice
with 50% decrease in the activity of the antioxidant
enzyme superoxide dismutase exhibited higher rate of
neuronal death and increased mortality after brain
ischaemia [28]. Conversely, overexpression of the
enzyme protected the brain in transient global cerebral
ischemia and reperfusion [29].
Our results also indicated marked increase in nitric
acid content in the brain of rats subjected to
ischaemia/reperfusion (I/R). Increased nitric oxide level
has been observed in the rat brain following middle
cerebral artery ligation and bilateral CCA occlusion [20,
30]. Nitric oxide is a short-lived signaling molecule
produced by nitric oxide synthase enzyme in a reaction
that converts arginine to nitric oxide and citrulline. In
physiologic concentrations, nitric oxide derived from the
constitutive nitric oxide synthases in neuronal cells and
endothelium plays an important role in maintaining
vascular tone, neurotransmission and neuroplasticity.
Excessive generation of nitric oxide for prolonged time,
however, can be damaging to neurons [31]. Nitric oxide
is an important contributor to the development of brain
ischaemic injury. The latter induces the expression of
the neuronal and endothelial nitric oxide synthase
(NOS) isoforms [32]. The increase in brain nitric oxide
during ischaemia also stems from the inducible isoform
of NOS (iNOS) in neutrophils infiltrating the ischaemic
brain and from the resident brain immune cells
microglia [33]. This increase in nitric oxide can cause
neuronal damage by inactivation of mitochondrial
electron transport complexes and cellular energy
depletion. Nitric oxide can also be cytotoxic via the
formation of more reactive nitrogen species like
peroxynitrite (ONOO-) generated by the reaction with
superoxide anion radical. Peroxynitrite can also result
in the formation of hydroxyl free radicals and nitrogen
dioxide and result in nitrosylation of protein tyrosine
residues [31]. Studies indicated a decrease in brain
infarct volume after iNOS inhibition in rats subjected to
middle cerebral artery occlusion [34]. Inhibition of nitric
oxide would therefore be of benefit in reducing
neuronal loss in acute cerebral ischaemia.
32
Journal of Neurology and Epidemiology, 2017, Vol. 5
In this study, a pronounced increase in brain TNF-α
concentration
after
I/R
was
observed.
The
neuroinflammatory response that accompany acute
cerebral ischaemia is mediated by cytokines eg., such
as TNF-α and interleukin-1β released by ischaemic
neurons and glia and results in the increased
endothelial adhesion molecules, breakdown of the
blood brain barrier and recruitment of immune cells into
the
damaged
tissue,
thereby,
exacerbating
inflammation and brain damage [18, 19]. The
pleotrophic cytokine TNF-α is synthesized by
macrophages and monocytes and TNF-α receptors are
expressed on neurons and glia cells [35]. When
released in low concentrations, TNF-α could be
neuroprotective via TNF-α receptor 2 and this has been
shown
in
hippocampal
slice
culture
during
excitotoxicity.
Higher
concentration,
however,
increased neuronal damage [36].
Cannabis
has
been
shown
to
cause
neurodegenerative changes in the rat brain in the form
of dark and small-sized neurons, cellular infiltration,
and
gliosis
[37].
Cannabis
also
intensified
neuroinflammation due to systemic lipopolysaccharide
injection in mice with the development of neuronal
atrophy and shrinkage, pyknosis and cell necrosis.
Cannabis increased the expression of the apoptotic
marker caspase-3 in rodent brain [37, 38]. In vitro,
cannabis caused oxidative stress and impaired brain
mitochondrial respiratory chain function [39, 40].
Hippocampal neuronal cell body and nuclear shrinkage
also occurred after cannabis application [41] while THC
given to rats impaired hippocampal neurogenesis [42].
Yet, other studies have shown neuroprotective effects
9
for Δ -THC against excitotoxic brain injury, most likely
via cannabinoid receptor-independent antioxidant
mechanism [43, 44]. Chen et al. [43] showed that Nmethyl-D-aspartate (NMDA) neuronal toxicity in vitro
9
was reduced by Δ -THC. Higher concentrations,
however, were toxic, via CB1 receptor stimulation.
Glutamate-mediated death of rat hippocampal neurons
9
in culture was also reduced by Δ -THC [44].
The findings of this study indicates that the
administration of an extract of Cannabis sativa rich in
9
Δ -THC shortly before the development of brain
ischaemia is associated with neuroprotection in a rat
model of transient global cerebral I/R injury. In contrast,
no protection or even increased neuronal damage was
observed upon later administration of the herbal
cannabis. These observations are intriguing in view of
the reports associating cannabis intake with the
development of ischaemic brain injury in man [10-14].
Abdel-Salam et al.
In this study, cannabis was found to result in recovery
of brain reduced glutathione and nitric oxide levels.
Moreover,
cannabis
given
before
ischaemia
significantly reduced the brain concentrations of the
proinflammatory cytokine TNF-α. These findings are in
accordance with our previous observations in which
cannabis decreased brain oxidative stress, nitric oxide
and TNF-α [38, 45, 46]. Other studies have also shown
9
a modulatory effect for cannabinoids Δ -THC on the
release of inflammatory cytokines (IL-6, IL-1β, IL-12
and IL-23 and TNF-α) by macrophages and brain
microglia in culture [47-49]. The mechanisms by which
acute cannabis given prior to brain ischaemia prevents
neuronal damage might thus involve an antioxidant
action and decreased release of TNF-α.
CONFLICTS OF INTEREST
The authors declare that there are no conflicts of
interest
ACKNOWLEDGEMENTS
This works is was not supported by research grants
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Received on 15-06-2017
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Accepted on 15-08-2017
Published on 11-10-2017
DOI: https://doi.org/10.12974/2309-6179.2017.05.06
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