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Long-Term Cannabidiol Treatment Prevents the Development of Social Recognition Memory Deficits in Alzheimer's Disease Transgenic Mice

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Impairments in cognitive ability and widespread pathophysiological changes caused by neurotoxicity, neuroinflammation, oxidative damage, and altered cholesterol homeostasis are associated with Alzheimer's disease (AD). Cannabidiol (CBD) has been shown to reverse cognitive deficits of AD transgenic mice and to exert neuroprotective, anti-oxidative, and anti-inflammatory properties in vitro and in vivo. Here we evaluate the preventative properties of long-term CBD treatment in male AβPPSwe/PS1ΔE9 (AβPP × PS1) mice, a transgenic model of AD. Control and AD transgenic mice were treated orally from 2.5 months of age with CBD (20 mg/kg) daily for 8 months. Mice were then assessed in the social preference test, elevated plus maze, and fear conditioning paradigms, before cortical and hippocampal tissues were analyzed for amyloid load, oxidative damage, cholesterol, phytosterols, and inflammation. We found that AβPP × PS1 mice developed a social recognition deficit, which was prevented by CBD treatment. CBD had no impact on anxiety or associative learning. The prevention of the social recognition deficit was not associated with any changes in amyloid load or oxidative damage. However, the study revealed a subtle impact of CBD on neuroinflammation, cholesterol, and dietary phytosterol retention, which deserves further investigation. This study is the first to demonstrate CBD's ability to prevent the development of a social recognition deficit in AD transgenic mice. Our findings provide the first evidence that CBD may have potential as a preventative treatment for AD with a particular relevance for symptoms of social withdrawal and facial recognition.
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Journal of Alzheimer’s Disease 42 (2014) 1383–1396
DOI 10.3233/JAD-140921
IOS Press
Long-Term Cannabidiol Treatment Prevents
the Development of Social Recognition
Memory Deficits in Alzheimer’s Disease
Transgenic Mice
David Chenga,b, Adena S. Spiroc,d, Andrew M. Jennerc,d, Brett Garnerc,dand Tim Karla,b,e,
aNeuroscience Research Australia, Randwick, NSW, Australia
bSchool of Medical Sciences, University of New South Wales, NSW, Australia
cIllawarra Health and Medical Research Institute, University of Wollongong, NSW, Australia
dSchool of Biological Sciences, University of Wollongong, NSW, Australia
eSchizophrenia Research Institute, Darlinghurst, NSW, Australia
Handling Associate Editor: Gary Arendash
Accepted 14 May 2014
Abstract. Impairments in cognitive ability and widespread pathophysiological changes caused by neurotoxicity, neuroinflam-
mation, oxidative damage, and altered cholesterol homeostasis are associated with Alzheimer’s disease (AD). Cannabidiol
(CBD) has been shown to reverse cognitive deficits of AD transgenic mice and to exert neuroprotective, anti-oxidative, and
anti-inflammatory properties in vitro and in vivo. Here we evaluate the preventative properties of long-term CBD treatment in
male APPSwe/PS1E9 (APP ×PS1) mice, a transgenic model of AD. Control and AD transgenic mice were treated orally
from 2.5 months of age with CBD (20 mg/kg) daily for 8 months. Mice were then assessed in the social preference test, elevated
plus maze, and fear conditioning paradigms, before cortical and hippocampal tissues were analyzed for amyloid load, oxidative
damage, cholesterol, phytosterols, and inflammation. We found that APP ×PS1 mice developed a social recognition deficit,
which was prevented by CBD treatment. CBD had no impact on anxiety or associative learning. The prevention of the social
recognition deficit was not associated with any changes in amyloid load or oxidative damage. However, the study revealed a
subtle impact of CBD on neuroinflammation, cholesterol, and dietary phytosterol retention, which deserves further investigation.
This study is the first to demonstrate CBD’s ability to prevent the development of a social recognition deficit in AD transgenic
mice. Our findings provide the first evidence that CBD may have potential as a preventative treatment for AD with a particular
relevance for symptoms of social withdrawal and facial recognition.
Keywords: Alzheimer’s disease, amyloid load, behavior, cannabidiol, cholesterol, neuroinflammation, oxidative stress, phytos-
terol, social recognition memory, transgenic APPSwe/PS1E9 mice
Alzheimer’s disease (AD) is a neurodegenerative
disease, which is associated with progressive mem-
Correspondence to: Tim Karl, PhD, Neuroscience Research
Australia, Barker Street, Randwick, NSW 2031, Australia. Tel.: +61
2 9399 1838; Fax: +61 2 9399 1005; E-mail:
ory loss. Other behavioral and cognitive symptoms
include social withdrawal, poor facial recognition
ability, increased motor agitation, and likelihood of
wandering [1, 2]. AD is characterized by two main
postmortem pathological hallmarks; amyloid-(A)
protein aggregation forming plaque deposits and tau
protein hyperphosphorylation resulting in neurofib-
rillary tangles. Microglia, the resident immune cells
ISSN 1387-2877/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved
1384 D. Cheng et al. / Protective Effects of Cannabidiol
of the central nervous system, are activated for the
phagocytosis of A[3–5], but impaired clearance or
reuptake of Aresults in the release of inflamma-
tory cytokines such as interleukin-1(IL-1), tumor
necrosis factor-(TNF-), and chemokines, that cause
neuroinflammation. Brain tissue damage is further
exacerbated by the release of glutamate and reactive
oxygen and nitrogen species, resulting in neurotoxic-
ity and oxidative damage, respectively [6]. Increased
oxidative stress may be an early indication of AD risk
[7, 8]. Disturbances in brain cholesterol metabolism
are associated with the major pathological features
of AD (including Aand tau pathology). In partic-
ular, decreased cholesterol synthesis correlates with
the severity of neurodegeneration and dementia [9,
10], while late-stage AD patients also show decreased
cholesterol circulation [11, 12]. Interestingly, dietary
phytosterols (or plant sterols) found naturally in many
foods (such as vegetable oils, nuts, grains, and grain-
derived products) [13] can either interfere with critical
functional processes in AD or decrease amyloidogenic
processing [14]. Some phytosterols may even be rele-
vant additional biomarkers for AD [15].
Current treatments available to AD patients do
not slow the progression of the disease and only
offer limited benefits for the cognitive abilities of
patients (reviewed in [16]). Thus, it is important to
explore novel alternative treatment strategies. The phy-
tocannbinoid cannabidiol (CBD) may be a potential
new candidate for AD therapy (for review, see [17]).
CBD is derived from the cannabis sativa plant and is
devoid of psychoactive properties. It has neuroprotec-
tive, anti-inflammatory, and anti-oxidative properties
[18–22], thereby countering a number of AD-relevant
pathological symptoms. In vitro studies have found
that CBD prevents A-induced tau protein hyperphos-
phorylation [23], neurotoxicity [23, 24], attenuates cell
death, and promotes neurogenesis in mouse hippocam-
pal cells [25, 26]. These biological functions of CBD
promise therapeutic value for the neurodegenerative
and neurotoxic components of AD. Indeed, in vivo
studies reported that CBD reduced A-induced neu-
roinflammation in rats and mice [25, 27] and rescued
learning deficits in the Morris water maze in a phar-
macological mouse model of AD [28]. The memory
restoring properties of CBD were linked to a reduc-
tion in microglial activation and pro-inflammatory
cytokines (i.e., decreased IL-6) [28].
Current research suggests existing interventions
may be administered too late in the disease process
when the damage caused by AD pathology is already
too severe [17, 29, 30]. Thus, in the current study, we
evaluated for the very first time the effectiveness of
long-term oral CBD treatment to prevent the devel-
opment of cognitive deficits and AD-relevant brain
pathophysiology in an established transgenic mouse
model of familial AD [31]. The double transgenic
APPSwe/PS1E9 (APP ×PS1) mouse model co-
expresses mutant amyloid-protein precursor (APP)
and presenilin 1 (PS1) genes [31–34]. Amyloid plaques
are found as early as at 4 months of age in these AD
transgenic mice [35]. Our past research established
that male APP ×PS1 mice demonstrate social recog-
nition deficits, increased anxiety, and task-specific
hyperlocomotion whereas sensorimotor gating and
spatial memory were intact at 10–12 months of age
[36]. Importantly, we also demonstrated recently that
3-weeks of CBD treatment effectively reversed the
social and object recognition memory deficits of
APP ×PS1 males [37]. In the present study male
APP ×PS1 mice were treated with CBD (20 mg/kg)
or vehicle using a daily voluntary oral administration
scheme for 8 months beginning at 2.5 months of age
when AD-like pathophysiology is still sparse (i.e., no
Aburden reported for 4 months old APP ×PS1
mice: [35]). Following this, mice were assessed in
social recognition memory, associative memory (i.e.,
fear conditioning), and anxiety, before brain samples
were analyzed for amyloid load, oxidative damage
(i.e., markers of cerebral lipid oxidation), cholesterol
levels as well as dietary phytosterols, and neuroin-
flammation markers. We selected the cytokines TNF-
and IL-1as both have been most strongly implicated
in the promotion of AD pathology in humans and in
AD transgenic mouse models [38, 39]. Furthermore,
inflammation driven by these cytokines is attenuated
by CBD [27, 28].
Double transgenic mice expressing chimeric
mouse/human APP (Mo/HuAPP695swe/Swedish
mutations K595N/M596L) and mutant human
PS1 (PS1/E9) mice were obtained from Jackson
Laboratory [Bar Harbor, USA; strain name: B6C3-
Tg(APPSwe/PS1E9)85Dbo/Mmjax; stock no.
004462] and maintained as hemizygotes on the con-
genic C57BL/6JxC3H/HeJ background as described
previously [31–33, 40]. Male double transgenic mice
(APP ×PS1) and their non-transgenic littermates
(WT) were bred and group-housed in independently
ventilated cages (Airlaw, Smithfield, Australia) at
D. Cheng et al. / Protective Effects of Cannabidiol 1385
Animal BioResources (Moss Vale, Australia). Test
mice were transported to Neuroscience Research
Australia (NeuRA) at around 10 weeks of age,
where they were group-housed in Polysulfone cages
(1144B: Techniplast, Rydalmere, Australia) with corn
cob bedding (PuraCob Premium: Able Scientific,
Perth, Australia) and some tissues for nesting. Mice
were kept under a 12 : 12 h light:dark schedule
[light phase: white light (illumination: 210lx); lights
on 0700–1900 h]. Environmental temperature was
automatically regulated at 21 ±1C and relative
humidity was 40–60%. Food (Gordon’s Rat and
Mouse Maintenance Pellets: Gordon’s Specialty
Stockfeeds, Yanderra, Australia) and water were pro-
vided ad libitum, except where specified. Adult, male
A/J mice from Animal Resources Centre (Canning
Vale, Australia) were placed in the animal enclosures
as standard opponents for the social preference test.
Research and animal care procedures were approved
by the University of New South Wales Animal
Care and Ethics Committee in accordance with the
Australian Code of Practice for the Care and Use of
Animals for Scientific Purposes.
Drug treatment
Powdered cannabidiol (CAS: 13956-29-1, THC
Pharm GmbH, Frankfurt/Main, Germany) was used
at a dose of 20 mg/kg body weight, based on pre-
vious studies evaluating the behavioral properties of
different doses of CBD [41] and the effectiveness of
20 mg/kg CBD to reverse spatial memory deficits of a
pharmacological mouse model of AD [42]. Although
chronic administration of CBD appears to be well tol-
erated by transgenic mice [37, 43], the stress of chronic
injections may impact behavioral, cognitive, and/or
physiological results. Thus, the gel pellet preparation
and the oral administration regime were adapted from
Zhang and colleagues [44, 45]: CBD or vehicle were
dissolved in a highly palatable sweetened and choco-
late flavored gel pellet, and administered at a volume
of 8 ml/kg body weight. Due to the insolubility of CBD
in water, CBD was first dissolved in 100% ethanol and
an equal amount of Tween 80 (Sigma-Aldrich Co., St
Louis, USA), then vortexed vigorously. CBD was dis-
solved in gel pellets with a final composition of 2.0%
ethanol, 2.0% Tween 80, 15.2% Splenda (Splenda Low
Calorie Sweetener: Johnson & Johnson Pacific Pty,
Broadway, Australia), 8.7% gelatine (Davis Gelatine:
GELITA Australia Pty, Josephville, Australia), 20.1%
chocolate flavoring (Queen Flavouring Essence Imi-
tation Chocolate: Queen Fine Foods Pty, Alderley,
Australia), and 52.0% water for irrigation. Vehicle gel
pellets were identical but contained no CBD. Mice
were initially habituated to vehicle gel pellets in their
home cages for seven days. Following this, the mice
were isolated within their home cages for the treat-
ment by placing a plastic divider in the home cage.
Then animals were given either a vehicle or a CBD
gel pellet (treatments were quasi-randomized), which
they consumed within 2–5 min. The plastic divider was
removed once mice had consumed the gel pellets. Mice
were treated daily, late in the afternoon, to avoid poten-
tial acute effects of CBD confounding test outcomes
(Table 1).
Behavioral phenotyping
Starting at 10 months of age, mice were tested in a
number of behavioral tests (Table 1), with an inter-test
interval of at least 48 h as described earlier (n= 8–14
mice per genotype/treatment) [36, 46, 47]. All tests
were conducted during the first 5 h of the light phase
to minimize effects of circadian rhythm.
Social preference test (SPT)
The SPT was used to assess sociability and social
recognition memory [48] and performed as described
Table 1
Age of APPSwe/PS1E9 (APP ×PS1) mice and their WT counterparts (in days ±SEM) at the start of treatment, throughout behavioral
testing and at the end of treatment
Treatment Vehicle CBD
Genotype WT APP ×PS1 WT APP ×PS1
Age at start of treatment 91.5 ±11.5 97.1 ±18.3 89.0 ±8.2 95.9 ±12.5
Number of days treated prior to start of testing 228.1 ±38.5 234.8 ±31.3 226.1 ±33.0 237.8 ±35.5
Social Preference Test 319.6 ±34.5 331.9 ±41.5 315.1 ±30.8 333.8 ±38.6
Elevated plus maze 324.1 ±34.8 336.0 ±41.7 319.3 ±31.3 338.1 ±38.9
Y-Maze 326.9 ±34.5 338.4 ±41.5 321.8 ±31.2 340.6 ±38.6
Fear conditioning 329.9 ±34.5 341.4 ±41.5 324.8 ±31.2 343.6 ±38.6
Tissue collection 333.1 ±34.8 345.0 ±41.7 328.3 ±31.3 347.1 ±38.9
Total Days of treatment 241.6 ±38.9 247.9 ±31.6 239.3 ±33.4 251.2 ±35.8
1386 D. Cheng et al. / Protective Effects of Cannabidiol
previously [36, 37]. Test animals were isolated for an
hour prior to the start of testing. During the habit-
uation trial, mice were allowed to freely explore a
three-chamber apparatus, consisting of a center cham-
ber (length: 9 cm; width: 18 cm; depth: 20 cm) and
two outer chambers (16 cm ×18 cm ×20 cm), freely
for 5 min. For the sociability test, an unfamiliar stan-
dard opponent (male A/J mouse) was placed in one of
two animal enclosures (i.e., opponent chamber) in a
quasi-randomized fashion (mouse enclosures allowed
nose contact between mice but prevented fighting). The
test mouse was returned to the apparatus and allowed to
explore all three chambers and the animal enclosures
for 10 min. Finally, test animals were observed in a
10 min social recognition test. For this, a second, unfa-
miliar standard opponent was placed in the previously
empty chamber so that the test mouse had the choice
to explore either the familiar mouse (from the previ-
ous trial) or the novel, unfamiliar mouse. The inter-trial
interval (ITI) was 5 min. The chambers and enclosures
were cleaned with 30% ethanol in-between trials and
fresh corn cob bedding was added to the chambers
prior to each test trial. AnyMazeTM (Stoelting, Wood
Dale, USA) tracking software was used to determine
the time spent in the different chambers, number of
entries and distance travelled by the test mice in each
trial. Two mice (1 WT-VEH and 1 WT-CBD) were
excluded from the sociability test due to recording
Elevated plus maze (EPM)
The EPM assesses the natural conflict between
the tendency of mice to explore a novel environ-
ment and avoidance of a brightly lit, elevated, and
open area [49, 50] and was employed to determine
potential effects of chronic CBD treatment on anxiety
behavior. The ‘+’ apparatus consisted of two alter-
nate open arms (35 cm ×6cm; without side walls) and
two alternate enclosed arms (35 cm ×6 cm; height of
enclosing walls 28 cm) connected by a central platform
(6 cm ×6 cm), elevated 70 cm above the floor. Mice
were placed at the center of the ‘+’ of the grey PVC
plus maze (for further details of apparatus, see [51])
facing an enclosed arm and were allowed to explore
the maze for 5 min. The time spent on open arms,
the percentage of entries onto open arms over total
arm entries (open arm entries) and the distance trav-
elled on the open and enclosed arms were recorded
using AnyMazeTM tracking software. One mouse was
excluded (APP ×PS1-CBD group) for falling off the
Fear conditioning (FC)
FC assesses hippocampus- and amygdala-
dependent associative learning whereby a previously
neutral stimulus elicits a fear response after it has been
paired with an aversive stimulus. On conditioning
day, mice were placed into the test chamber (Model
H10-11R-TC, Coulbourn Instruments, USA) for
2 min. An 80 dB conditioned stimulus (CS) was
presented twice for 30 s with a co-terminating 0.4 mA
2-s foot shock (unconditioned stimulus; US) with an
inter-pairing interval of 2 min. The test concluded
2 min later. The next day (context test), mice were
returned to the apparatus for 7 min. On day 3 (cue
test), animals were placed in an altered context for
9 min. After 2 min (pre-CS/baseline), the CS was
presented continuously for 5 min. The test concluded
after another 2 min, absent the CS (for more details,
see [52, 53]). Time spent freezing was measured on all
three experimental days using Any-MazeTM software.
Biochemical analyses
Mice were anaesthetized and blood was collected
through cardiac puncture. Blood samples were cen-
trifuged (5000 rpm, 5 min, 4C) in a microcentrifuge
(Model No. 5415R, Eppendorf, Hamburg, Germany),
and the plasma fraction was collected and stored at
80C. Euthanized mice were perfused with phos-
phate buffered saline (PBS) transcardially as described
previously [54]. Brains were sagittally divided and the
right hemisphere was snap frozen in liquid nitrogen
before being stored at 80C. Cortex and hippocampal
samples were dissected and weighed on dry ice prior
to biochemical analyses [Sample numbers for ELISA
and GC-MS were: n= 8 for WT-vehicle, n= 10 for
APP ×PS1-vehicle, n= 10 for WT-CBD, and n=10
for APP ×PS1-CBD].
Aβenzyme-linked immunosorbent assay (ELISA)
for Aβpathology
Frozen cortex (20–30 mg) and hippocampal samples
(5 mg) were homogenized and prepared as TBS solu-
ble, and guanidine HCl (gHCl) soluble (TBS insoluble)
fractions and stored at 80C as described previously
[54]. Both TBS-soluble and gHCl-soluble fractions
were used in enzyme-linked immunosorbent assay
(ELISA) to investigate the effect of CBD on Alevels
in transgenic mice. Protein was quantified using the
bicinchoninic (BCA assay) method.
A40 and A42 protein in TBS-soluble and gHCl-
soluble fractions of brain homogenates were quantified
using Beta Amyloid x-40 and x-42 ELISA kits (Cat
D. Cheng et al. / Protective Effects of Cannabidiol 1387
No. SIG-38954 and SIG-38956 respectively, Covance,
Emeryville, USA) as described previously [54, 55].
Gas chromatography-mass spectroscopy (GC-MS)
for cholesterol, oxidative damage, and CBD
plasma levels
An Agilent 7000B triple quadrupole mass selective
detector interfaced with an Agilent 7890A GC sys-
tem gas chromatograph, equipped with an automatic
sampler and computer workstation (Agilent Technolo-
gies, Santa Clara, USA) was used to analyze markers
of oxidative damage in the cortical samples and CBD
presence in plasma samples. GC-MS triple quadrupole
provided very high analytical sensitivity for all ana-
lytes measured. Limits of detection (LOD: 0.05 ng/ml)
were significantly less (at least 10-fold) than the levels
of each analyte measured in plasma and brain. 150 l
of plasma were used for the analysis of CBD. The
concentration obtained from the GC-MS was there-
fore multiplied by a factor of 6.67 to give the total
amount of CBD per ml of plasma as shown in the
Results section. The injection port and GC-MS inter-
face were kept at 270C and separations were carried
out on a fused silica capillary column (20 m ×0.18 mm
i.d. ×0.18 m film thickness, Restek Rxi-5 ms). Helium
was the carrier gas with a flow rate of 0.8 ml/min (aver-
age velocity = 59 cm/s).
F2-isoprostanes, oxidised sterols (oxysterols), and
Frozen cortex samples (10–20 mg) were homoge-
nized and hydrolyzed overnight for GC-MS analysis as
described previously [56]. Samples were loaded into
solid phase extraction columns (UCT CUQAX223
3 ml; United Chemical Technologies, Bristol, USA).
Sterols and oxysterols, arachidonic acid, DHA, and
F2-isoprostanes were eluted from the SPE column
separately. The sterol/oxysterol fractions were deriva-
tized in 20 l acetonitrile and 20 l Selectra-SIL
BSTFA [N,O-bis(trimethylsilyl)trifluoroacetamide]
containing 1% TMCS (trimethylchlorosilane; United
Chemical Technologies, Bristol, USA) prior to GC-
MS analysis. Quantification of cholesterol oxidation
products (COP) was as previously described [57].
Cholesterol was quantified using lathosterol-d6heavy
isotope standard in a separate (0.6 l split ratio
25 : 1). Relative molar response factors of all analytes
were calculated from calibration curves constructed
from different concentrations in triplicate. The F2-
isoprostane and fatty acid fractions were prepared
and analyzed by GC-MS as described previously
[56]. Quantification of F2-isoprostanes and fatty
acids were calculated by comparison of specific SRM
transitions with their corresponding heavy isotope
internal standards.
Quantification of CBD in plasma
Concentration of cannabidiol in plasma was quan-
tified using the GC-MS as previously described with
slight modifications [58, 59]. Plasma samples (150 l)
were treated using sodium acetate buffer pH 4.0, with
MTBE and Hexane (1 : 1 v/v), rotated for 30 min and
centrifuged at 1500 rpm for 2 min at 4C, dried down,
derivatized in 20l BSTFA and 20 l 1% TMCS,
and incubated at 70C for 30 min. Derivatized sam-
ples were dried down, reconstituted in 40 l of toluene
and analyzed using the GC-MS (1 l splitless). MRM
was performed using EI mode similar to sterol anal-
ysis. Column temperature was held for 1 min and
increased 40C/min to 210C, then 20C/min to 300C
and held for 4 min. Quantification of CBD was calcu-
lated by comparison with specific MRM transitions
corresponding with its heavy isotope internal standard
(CBD-d3, Lipomed, Arlesheim, Switzerland).
Inflammatory markers (quantitative polymerase
chain reaction)
RNA extraction: Frozen cortex samples (10–20mg)
were homogenized in Tri-reagent (TRIzol Reagent,
cat no. 15596-018, Life Technologies, Mulgrave,
VIC, Australia) as described previously [57]. RNA
levels were quantified using a NanoDrop 2000 spec-
trophotometer (Thermo Fisher Scientific, Scoresby,
Australia), and diluted in RNAse-free water to obtain
a concentration of 0.5 g/l. cDNA was synthesized
using a Tetro cDNA Synthesis kit (Bioline, Alexandria,
Australia), according to manufacturer instructions.
The SensiFAST SYBR No-ROX kit (Bioline, Alexan-
dria, Australia) was used to determine levels of
inflammatory markers. Template concentration was
100 ng (1 : 10 dilution; 1l cDNA). Forward and
reverse primers for interleukin-1(IL-1; forward, 5-
necrosis factor-(TNF-; forward, 5-CATCTTCTC-
TAGACAAGGTACAACCC-3) were used as bio-
markers for the quantification of inflammation in trans-
genic mice, with -actin as a housekeeping gene.
Polymerase chain reaction (PCR) assays were reacted
(3-step cycling; IL-1: 45 cycles; TNF-: 50 cycles)
and analyzed using Roche LightCycler 480 (Roche
Diagnostics, Castle Hill, Australia). Three mice were
excluded (1×WT-CBD, 1×APP ×PS1-vehicle and
1388 D. Cheng et al. / Protective Effects of Cannabidiol
1×APP ×PS1-CBD) as outliers (2 standard devia-
tions away from mean).
Statistical analyses
One-way ANOVA was used to analyze effect
of ‘treatment’ on Alevels in APP ×PS1 mice.
Two-way analysis of variance (ANOVA) was used
to analyze behavioral parameters and biochemical
data obtained for oxidative damage, CBD levels and
quantification of inflammation for main effects of
‘genotype’ and ‘treatment’ in all tests. Repeated mea-
sures (RM) ANOVA was used to evaluate the effects of
‘chamber’ (SPT) and ‘1 min block’ (FC) as published
previously [36, 47]. Performance in the SPT was also
assessed using one sample t-tests to clarify whether
the percentage of time spent in the opponent/novel
chamber was greater than chance (50%). Differences
were regarded as significant if p< 0.05. Data are shown
as means ±standard error of means (SEM). F-values
and degrees of freedom are presented for ANOVAs
and significant ‘genotype’ and ‘treatment’ effects are
shown in figures and tables as ‘’(
p< 0.05, ∗∗p< 0.01)
and ‘#’ (#p<0.05) respectively whereas RM-ANOVA
results for social novelty preference are presented by
+p< 0.05, ++ p< 0.01, +++ p< 0.001). Analyses
were conducted using SPSS 20.0 for Windows.
Sociability and social recognition
RM ANOVA revealed an effect of ‘chamber’
[F(1,39) = 197.9, p< 0.001] (Fig. 1A). All mice spent
more time investigating the social opponent over the
empty chamber, indicating intact sociability for all
mice regardless of genotype and treatment. T-tests
for percentage of time spent with the novel mouse
confirmed that all mice demonstrated significant lev-
els of sociability above chance [WT-VEH: t(6) = 7.4,
p< 0.001; APP ×PS1-VEH: t(13) = 6.2, p< 0.001;
WT-CBD: t(8) = 12.3, p< 0.001; APP ×PS1-CBD:
t(12) = 8.6, p< 0.001] (data not shown).
In the SPT, RM ANOVA revealed a significant effect
of ‘chamber’ for time spent investigating the novel over
the familiar mouse [F(1,41) = 23.6, p< 0.001]. Impor-
tantly, a significant interaction between ‘genotype’
and ‘treatment’ was found [F(1,41) = 4.8, p< 0.05],
where only vehicle-treated AD transgenic mice did not
develop a preference for the novel mouse (Fig. 1B).
Two-way ANOVA also revealed a trend toward an
effect of CBD treatment [F(1,41) = 3.1, p= 0.09].
Indeed, ANOVA split by ‘genotype’ revealed that CBD
increased the time AD transgenic mice spent with the
novel mouse [F(1,25) = 5.0, p< 0.05], with no such
effect observed in WT mice [F(1,16) = 0.2, p= 0.7]
(Fig. 1B) showing that CBD had a beneficial effect
on social recognition memory. T-tests confirmed that
all animals, except vehicle-treated APP ×PS1 mice,
spent a significantly greater percentage of time with
the novel mouse than the familiar mouse [WT-VEH:
t(7) = 2.5, p< 0.05; APP ×PS1-VEH: t(13) = 0.3,
p= 0.8; WT-CBD: t(9) = 3.3, p< 0.01; APP ×PS1-
CBD: t(12) = 3.7, p< 0.01] (data not shown).
APP ×PS1 transgenic mice demonstrated WT-like
locomotion and anxiety (p> 0.05 for total distance
travelled, time spent on open arms and open arm
entries). Chronic treatment with CBD had no effect
on EPM behaviors (all p> 0.05; Table 2).
Associative learning
All mice responded to the electric foot shocks during
conditioning (i.e., vocalization detected in all mice).
Two-way ANOVA found transgenic mice demon-
strated increased amounts of freezing at baseline (i.e.,
first 2 min pre-conditioning) regardless of treatment
[F(1,41) = 4.5, p< 0.05]. However, freezing duration
during the first 2 min of the context and cue trials
was similar for all mice across test conditions (all
p> 0.05; Table 3) and all mice exhibited intact context
memory regardless of treatment [p> 0.05; Fig. 2A].
Furthermore, memory of the cue was intact as all ani-
mals showed increased freezing post cue presentation
[RM ANOVA: F(1,41) = 52.9, p< 0.001], regardless of
‘genotype’ or ‘treatment’ (Fig. 2B and Table 3).
Brain pathophysiology
Amyloid load
One-way ANOVA revealed that CBD had no
effect on soluble and insoluble A40 or A42 in
the cortex of APP ×PS1 mice, although insolu-
ble A42 was slightly higher after CBD treatment
[‘treatment’: Soluble A40: F(1,18) = 0.3, p= 0.6;
Insoluble A40: F(1,18) = 2.4, p= 0.1; Soluble A42:
F(1,18) = 0.1, p= 0.7; Insoluble A42 : F(1,18) = 3.5,
p= 0.08] (Table 4). Similarly, Alevels remained
unchanged after CBD treatment in the hippocam-
pus [‘treatment’: Soluble A40: F(1,17) = 0.4, p= 0.6;
Insoluble A40: F(1,18) = 1.1, p= 0.3; Soluble A42:
D. Cheng et al. / Protective Effects of Cannabidiol 1389
Fig. 1. Sociability and social recognition were measured using the social preference test. Graphs show total time spent [s] in test chambers by
the test mice containing A) either an unfamiliar mouse (i.e., opponent) or an empty mouse enclosure (i.e., empty); or B) either a familiar or an
unfamiliar (i.e., novel) mouse. Data for non-transgenic wild type-like control (WT) and double transgenic APPSwe/PS1E9 (APP ×PS1)
male mice after vehicle or cannabidiol (CBD) treatment are shown as means + SEM. Significant ‘treatment’ effects are indicated with ‘#’
(#p<0.01). RM ANOVA for novelty preference are presented by ‘+’(
+p< 0.05, ++ p< 0.01, +++ p< 0.001).
Table 2
Anxiety-related behaviors (i.e., time spent on and entries onto open arms) and locomotion (total distance travelled) in the elevated plus
maze (EPM). Parameters for wild type-like control mice (WT) and double transgenic APPSwe/PS1E9 (APP ×PS1) mice after vehicle
or cannabidiol (CBD) treatment are shown as mean ±SEM
Vehicle CBD
Time spent on open arms [s] 7.2 ±2.7 6.0 ±2.3 6.7 ±1.6 7.3 ±2.7
Entries onto open arms [%] 13.3±4.0 7.8 ±3.1 11.6 ±2.1 11.2 ±2.7
Total distance travelled [m] 7.1 ±0.9 8.0 ±1.4 7.2 ±0.9 7.9 ±1.0
Fig. 2. Fear-associated learning was assessed in the fear conditioning test. Time spent freezing during A) the context test and B) the cue test for
each is shown per ‘1 min bin’. Data for non-transgenic control wild type-like (WT) and double transgenic APPSwe/PS1E9 (APP ×PS1)
mice after vehicle or cannabidiol (CBD) treatment are shown as means ±SEM.
F(1,15) = 0.3, p= 0.6; Insoluble A42 : F(1,18) = 0.1,
p= 0.7] (Table 4).
Oxidative damage
Total F2-isoprostanes (free and esterified corrected
for arachidonic acid; AA) were not significantly
altered in APP ×PS1 mice when compared to
their WT littermates, regardless of ‘treatment’
(all p> 0.05) (Table 5). We also measured the
levels of oxysterols in the cortex. For enzymati-
cally oxidized sterols, APP ×PS1 mice demon-
strated significantly decreased overall levels of
24-hydroxycholesterol compared to WT littermates
[‘genotype’: F(1,34) = 4.9, p< 0.05], whereas ‘treat-
ment’ had no effect on sterols [F(1,34) = 0.07, p= 0.8]
and no ‘genotype’ by ‘treatment’ interactions were
found. No differences were found across all four
groups for 27-hydroxycholesterol, and the reactive
species oxidized sterols, 7-hydroxycholesterol and
7-ketocholesterol (all p> 0.05) (Table 5).
1390 D. Cheng et al. / Protective Effects of Cannabidiol
Table 3
Fear-associated memory in the fear conditioning paradigm. Freezing (i.e., time spent freezing [s]) at baseline, and during context test and cue test
for non-transgenic wild type-like control (WT) and double transgenic APPSwe/PS1E9 (APP ×PS1) male mice after vehicle or cannabidiol
(CBD) treatment are presented as mean ±SEM
Vehicle CBD
Baseline (first 2 min)
Conditioning freezing [s] 2.3 ±1.1 5.8 ±1.7 4.7 ±1.5 8.5 ±1.9
Context freezing [s] 32.0 ±7.8 47.1 ±7.4 37.0 ±6.0 36.7 ±7.0
Total time spent freezing [s] 132.2 ±29.6 152.3 ±28.1 131.6 ±15.4 150.7 ±25.8
Time spent freezing 2min prior to cue onset [s] 18.0 ±5.9 29.1 ±5.7 25.5 ±5.2 28.6±4.4
Time spent freezing 2min post cue onset [s] 40.4 ±7.3+++ 37.4 ±5.8+38.7 ±6.7+40.4 ±5.2+
Significant effects of cue presentation on freezing response are indicated by ‘+’(
+p< 0.05 and +++ p< 0.001)
Fig. 3. Neuroinflammation markers in cortical tissue. Quantitative PCR was used to measure the concentration of A) interleukin-1and B)
TNF-derived from the cortex of control (WT) and double transgenic APPSwe/PS1E9 (APP ×PS1) male mice after vehicle or cannabidiol
(CBD) treatment. Concentrations [ng/ l] are presented as mean±SEM.
Table 4
Amyloid-Soluble and insoluble amyloid load in double trans-
genic APPSwe/PS1E9 (APP ×PS1) male mice after vehicle or
cannabidiol (CBD) treatment are shown as means ±SEM
APP ×PS1 Vehicle CBD
Soluble A40 [pg/mg] 1033.2 ±211.2 904.6 ±118.8
Soluble A42 [pg/mg] 654.3 ±102.6 613.4 ±65.8
Insoluble A40 [pg/mg] 8184.6 ±701.0 9758.3 ±751.6
Insoluble A42 [pg/mg] 25601.4 ±2138.6 30897.8 ±1847.6
Soluble A40 [pg/mg] 464.4 ±99.3 390.8 ±69.6
Soluble A42 [pg/mg] 155.7 ±196.8 196.8 ±54.1
Insoluble A40 [pg/mg] 9854.5 ±2217.7 12776.1 ±1738.7
Insoluble A42 [pg/mg] 22295.1 ±7937.3 26370.0 ±8467.1
Cholesterol was increased in cortical tissues
of APP ×PS1 mice compared to WT animals
[F(1,34) = 12.1, p< 0.01] and CBD increased choles-
terol levels [F(1,34) = 11.0, p= 0.01]. Further one-way
ANOVA revealed that cholesterol was signifi-
cantly higher in vehicle-treated AD transgenic mice
[F(1,16) = 7.7, p< 0.05] compared to control mice,
while CBD increased the cholesterol levels in WT mice
[F(1,16) = 25.1, p< 0.001] but not APP ×PS1 mice
[F(1,18) = 1.3, p= 0.3] (Table 5).
Two-way ANOVA revealed a significant ‘genotype’
by ‘treatment’ interaction for the cortical levels of
the dietary phytosterol, brassicasterol [F(1,34) = 6.1,
p< 0.05], which was caused by CBD increasing bras-
sicasterol levels in APP ×PS1 mice only [WT:
F(1,16) = 0.5, p= 0.5 APP ×PS1: F(1,18) = 6.9,
p< 0.05; Table 5]. Furthermore, the analysis detected
a ‘genotype’ effect in CBD-treated mice [vehicle:
F(1,16) = 0.2, p= 0.7, CBD: F(1,18) = 9.9, p< 0.01;
Table 5]. The dietary phytosterol, campesterol, was
also increased in APP ×PS1 mice [F(1,34) = 4.4,
p< 0.05]. More specifically, cortical campesterol was
elevated in CBD-treated APP ×PS1 mice [vehicle:
F(1,16) = 0.2, p= 0.6, CBD: F(1,18) = 9.0, p< 0.01].
D. Cheng et al. / Protective Effects of Cannabidiol 1391
Table 5
Oxysterols, F2-isoprostanes, cholesterol, and phytosterol levels. Oxidative damage and total cholesterol in the cortex of non-transgenic wild
type-like control (WT) and double transgenic APPSwe/PS1E9 (APP ×PS1) male mice after vehicle or cannabidiol (CBD) treatment.
Concentrations (in pg) are presented as mean ±SEM
Vehicle CBD
Oxidized sterols
Reactive species oxidized
7-hydroxycholesterol [ng/mg] 0.29 ±0.02 0.31 ±0.05 0.30 ±0.02 0.32 ±0.02
7-ketocholesterol [ng/mg] 0.53 ±0.04 0.50 ±0.04 0.47 ±0.03 0.57 ±0.05
Enzymatically oxidized
24-hydroxycholesterol [ng/mg] 38.5 ±2.8 34.2 ±3.0 39.4 ±2.7 31.9 ±1.8*
27-hydroxycholesterol [pg/mg] 48.3 ±5.3 38.9 ±5.7 46.1 ±2.5 43.4 ±4.6
F2-isoprostanes (normalized for arachidonic acid)
Total [pg/ng] 10.0 ±1.0 8.1 ±0.5 9.1 ±0.4 8.3 ±0.5
Total cholesterol [ng/mg] 20.5 ±1.0 29.6 ±2.8 29.3 ±1.4### 33.1 ±1.4
Dietary phytosterol
Brassicasterol [pg/mg] 51.2 ±3.8 48.3 ±6.1 46.7 ±4.9 70.4 ±5.8∗∗#
Campesterol [ng/mg] 15.1 ±1.8 16.5 ±2.3 15.0 ±1.7 21.2 ±1.1
Significant effects of ‘genotype’ are indicated by ‘’(
p< 0.05 and ∗∗ p< 0.01) and effects of ‘treatment’ by ‘#’ (#p< 0.05 and ### p< 0.001)
There was also a trend for CBD to increase the corti-
cal levels of this phytosterol in transgenic mice [WT:
F(1,16) = 0.0, p= 1.0, APP ×PS1: F(1,18) = 3.4,
p= 0.08] (Table 5).
Inflammatory markers
Two-way ANOVA revealed no significant differ-
ences in the levels of mRNA for two inflammatory
cytokine markers across test conditions [‘genotype’:
IL-1: F(1,39) = 1.0, p= 0.3 – ‘TNF-’: F(1,39) = 1.1,
p= 0.3] (Fig. 3A-B). There was no significant effect of
‘treatment’ on these cytokines [IL-1: F(1,39) = 1.3,
p= 0.3 – TNF-: F(1,39) = 2.5, p= 0.1] either,
although cytokine levels of CBD-treated APP ×PS1
mice appeared closer to corresponding WT levels
than levels of vehicle-treated AD transgenic mice
(Fig. 3A-B).
CBD plasma levels
Two-way ANOVA revealed that all mice treated
with CBD demonstrated significantly increased levels
of plasma CBD (ng/ml) [WT-CBD: 2.1 ±0.6;
APP ×PS1-CBD: 1.9 ±0.4 – ‘treatment’:
F(1,30) = 21.3, p< 0.001]. No significant ‘geno-
type’ differences or interactions were found (all
p> 0.05). CBD could not be detected in mice that
were treated with vehicle (values < 0.05 ng/ml).
Our study demonstrates for the first time the effects
of long-term oral CBD treatment on the social recog-
nition memory and pathophysiology of a double trans-
genic APP ×PS1 mouse model for AD. We provide
first evidence of a possible impact of CBD on dietary
phytosterols, which can exert beneficial effects on cog-
nition. We also suggest that the therapeutic effect of
CBD may be linked to neuroinflammatory processes
or changes in cholesterol but further research using
additional CBD doses will be necessary to clarify this.
The SPT determined that vehicle-treated
APP ×PS1 mice exhibit a social recognition
memory deficit, confirming our earlier findings [36,
37]. Importantly, long-term CBD treatment pre-
vented this social recognition deficit from occurring
in APP ×PS1 mice. We previously found that
intraperitoneal administration of CBD for three weeks
reversed this cognitive deficit in the same AD mouse
model [37]. Other recent studies also report social
recognition deficits in AD transgenic mouse models,
providing evidence for the increasing relevance of
social recognition memory testing for AD research
[60, 61]. Anxiety can confound the performance
of mice in cognitive tests [62] and acute CBD has
been found to modify anxiety-related behaviors [41,
63–67]. However, the APP ×PS1 transgene did not
influence anxiety parameters nor did CBD treatment.
The beneficial effect of CBD on social recognition
memory was not associated with a direct effect on A
levels. Insoluble and soluble levels of A40 and A42
were no different between vehicle and CBD-treated
APP ×PS1 mice in cortex and hippocampus. Simi-
larly, another study described improvements in spatial
memory in APPSwe/PS1E9 mice on a C57BL/6J
background, which was not accompanied by changes
1392 D. Cheng et al. / Protective Effects of Cannabidiol
in Alevels [68]. The same study also found that lev-
els of insoluble A40 and A42 in the parietal cortex
did not correlate with cognitive deficits [68]. Nonethe-
less, various in vitro studies show CBD can attenuate
A-induced processes [23–26], reverse A-induced
memory impairments in rodents [28] and reduce A
formation [69].
The in vivo formation of isoprostanes is a marker for
cerebral lipid oxidation and directly correlated with
an increase in oxidative stress [7, 70–72]. Patients
with AD are also known to have increased concentra-
tions of F2-isoprostanes in CSF even prior to disease
diagnosis [7, 8, 71]. Levels of oxidation were not sig-
nificantly altered in APP ×PS1 mice in comparison
to their age-matched WT littermates, nor did we detect
changes in the level of lipid oxidation in the cortex
of CBD-treated animals, despite its known antioxidant
properties [73, 74]. These findings may be due to age
as nucleic acid oxidation is significantly higher in 3
and 5 months old APP ×PS1 mice compared to age-
matched control mice. Importantly, this phenomenon
is not evident in 10 and 15 month old mice, which is
the age when brain tissue was collected for the current
study [75].
Cholesterol was increased in APP ×PS1 mice
compared to WT mice, while CBD treatment increased
cholesterol levels in WT mice. Our finding of increased
cholesterol in APP ×PS1 mice could indicate either
an impaired reuptake process, or a compensatory
mechanism for protection against neurodegeneration
in AD mice. Maintenance of sufficient cholesterol is
important to help combat synapse loss and neurode-
generation [76] and such a response is consistent with
the reduced levels of 24-OH cholesterol detected in
the APP ×PS1 mice compared to WT. Formation of
24-OH cholesterol is the major pathway of cholesterol
removal from the brain [77]. Insufficient amounts of
cholesterol may interrupt essential processes such as
myelin formation, synaptic transmission and cognitive
ability in mice [78, 79], while a reduction in oxys-
terols has been shown to correlate with the severity
of dementia and brain atrophy [9, 10, 80]. Interest-
ingly, 8-month old APPSwe/PS1E9 mice on a pure
C57BL/6J background did not demonstrate signifi-
cantly different levels of cholesterol, while 15-month
old transgenic mice had significantly lower cholesterol
levels compared to control mice [81]. It is noteworthy
that decreased levels of cholesterol in cerebral spinal
fluid and plasma have been found in patients with AD
[11, 12].
Phytosterols are present naturally in a variety of dif-
ferent foods, including grains (e.g., sorghum and bran)
found in mouse food pellets. CBD increased the lev-
els of brassicasterol and campesterol in APP ×PS1
mice. The accumulation and long-term consump-
tion of dietary phytosterols do not interfere with
memory [82, 83]. On the other hand, dietary sup-
plementation of a fish oil-rich diet with phytosterols
reduced insoluble A42 in APPSwe/PS1E9 mice on
a C57BL/6J background [68], while the phytosterol
stigmasterol attenuated scopolamine-induced spatial
memory deficits of mice [84]. These findings suggest
a potentially beneficial effect of increased phytosterol
levels for cognitive symptoms in the AD brain. It is
possible that CBD interacted with AD pathophysiol-
ogy by increasing the retention of specific phytosterols.
Further research needs to be conducted in order to
understand the effect of increased phytosterol levels
in AD brains and how CBD might be involved in these
Daily long-term administration of 20 mg/kg CBD
did not result in a statistically significant effect.
However, APP ×PS1 mice have previously been
shown to exhibit elevated levels of neuroinflam-
mation (increased nitric oxide species and TNF-)
in the hippocampus [85] and inflammatory changes
have been linked to impaired spatial memory of
APP ×PS1 mice [86]. Furthermore, Martin-Moreno
and colleagues have demonstrated that A-induced
neuroinflammation was decreased after CBD treatment
[28]. Thus, we suggest that CBD might be able to
combat increased inflammation in APP ×PS1 mice
thereby impacting on the cognitive performance of
these mice. Future research should consider additional
CBD doses to determine the effects of long-term CBD
treatment on neuroinflammation in AD mouse models.
Furthermore, an escalating CBD dosage regime could
be used as the dosage of AD-approved treatments is
often increased over time as the condition of patients
deteriorates [87].
In conclusion, our study is the first to demonstrate
that long-term CBD treatment can prevent the devel-
opment of a social recognition deficit in APP ×PS1
mice. The findings suggest the mechanism involved
in this prevention may be linked to CBD-induced
retention of dietary phytosterols or neuroinflammatory
processes in the brain of AD mice. We provide the first
evidence that CBD has potential to be used as a long-
term preventative treatment option for AD and may
be especially relevant for symptoms of social with-
drawal and facial recognition. The behavioral inertness
of CBD and the fact that CBD is well tolerated in
humans [88, 89] suggests that preclinical research find-
ings could easily be followed up in clinical trials.
D. Cheng et al. / Protective Effects of Cannabidiol 1393
Future studies using cytokine arrays or an ‘omics’
approach may reveal which biochemical/genetic path-
ways contribute to the beneficial effects of CBD.
It will also be important to clarify what receptors
mediate the therapeutic-like effects of CBD: the per-
oxisome proliferator-activated receptor-[25, 69],
N-methyl-D-aspartate receptors [90, 91], and recep-
tors of the endocannabinoid system [92] are promising
TK is supported by the Schizophrenia Research
Institute utilizing infrastructure funding from NSW
Ministry of Health and a career development fellow-
ship (1045643) from the National Health and Medical
Research Council (NHMRC). BG is supported by an
NHMRC Senior Research Fellowship (630445). DC
received an Australian Postgraduate Award scholar-
ship from the University of New South Wales and a
supplementary scholarship provided by Neuroscience
Research Australia. We thank Jac Kee Low and Warren
Logge (Neuroscience Research Australia, Randwick,
Australia) for assisting in treatment of our test mice,
Kalani Ruberu and Hongyun (Henry) Li (University
of Wollongong, Wollongong, Australia) for provid-
ing extensive ELISA and qPCR training, Lei Zhang
(Garvan Institute of Medical Research, Darlinghurst,
Australia) for providing assistance in the gel pel-
let setup, Jerry Tanda for critical comments on the
manuscript, and the staff of the Australian BioRe-
sources and Adam Bryan at Neuroscience Research
Australia for taking care of our test mice.
Authors’ disclosures available online (http://www.j-
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... In addition to the remedial effects (i.e., CBD administered when behavioural impairment is present), CBD has been found to prevent the development of AD-relevant behavioural impairments. When CBD is administered orally for 8 months from 2.5 months of age, CBD prevents the development of social recognition impairment in male APPxPS1 mice (Cheng et al., 2014c). In this study, there were also subtle effects of CBD on neuroinflammation and cholesterol in the cortex and dietary phytosterol retention in the cortex and hippocampus (Cheng et al., 2014c). ...
... When CBD is administered orally for 8 months from 2.5 months of age, CBD prevents the development of social recognition impairment in male APPxPS1 mice (Cheng et al., 2014c). In this study, there were also subtle effects of CBD on neuroinflammation and cholesterol in the cortex and dietary phytosterol retention in the cortex and hippocampus (Cheng et al., 2014c). This suggests CBD has potential preventive and pro-cognitive effects on AD in male animals. ...
... Thus, the present study was designed to complement earlier behavioural research in our laboratory (Cheng et al., 2014c), to determine if 20 mg/kg CBD treatment given orally via gel pellets for 8 months prevents the development of the AD-relevant behavioural phenotype in APPxPS1 female mice. ...
Full-text available
Cannabidiol is a promising potential therapeutic for neurodegenerative diseases, including Alzheimer’s disease (AD). Our laboratory has shown that oral CBD treatment prevents cognitive impairment in a male genetic mouse model of AD, the amyloid precursor protein 1 x presenilin 1 hemizygous (APPxPS1) mouse. However, as sex differences are evident in clinical populations and in AD mouse models, we tested the preventive potential of CBD therapy in female APPxPS1 mice. In this study, 2.5-month-old female wildtype-like (WT) and APPxPS1 mice were fed 20 mg/kg CBD or a vehicle via gel pellets daily for 8 months and tested at 10.5 months in behavioural paradigms relevant to cognition (fear conditioning, FC; cheeseboard, CB; and novel object recognition test, NORT) and anxiety-like behaviours (elevated plus maze, EPM). In the CB, CBD reduced latencies to find a food reward in APPxPS1 mice, compared to vehicle-treated APPxPS1 controls, and this treatment effect was not evident in WT mice. In addition, CBD also increased speed early in the acquisition of the CB task in APPxPS1 mice. In the EPM, CBD increased locomotion in APPxPS1 mice but not in WT mice, with no effects of CBD on anxiety-like behaviour. CBD had limited effects on the expression of fear memory. These results indicate preventive CBD treatment can have a moderate spatial learning-enhancing effect in a female amyloid-β-based AD mouse model. This suggests CBD may have some preventive therapeutic potential in female familial AD patients.
... It has been reported that CB1/CNR1 binding may contribute to an increase in HDL-cholesterol in treated adults [28]. CBD consumption of 20 mg/kg/day for 8 months in rats has also been reported to have an impact on neuroinflammation and cholesterol concentration [29]. These studies support the conclusions of this study, highlighting the significance of an increase in HDL-C following cannabis use. ...
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Background: The effect of cannabis on cholesterol and lipid balance has been reported for decades. However, there are conflicting reports on the reduction of low-density lipoprotein (LDL-C) and total cholesterol. The purpose of this study was to determine the immediate changes of Cannabis spp. consumption by pyrolytic route in heavy users. Methods: A cross-sectional study on 20 Peruvian heavy cannabis users (mean age: 31 ± 9.5 years). The inclusion criteria were males with an average weight of 50–70 kg, normal BMI, and having used cannabis, without association with other drugs, for at least one year with a high frequency per week (use: 4–7 days/week). High-density lipoprotein (HDL-C), LDL-C, and total cholesterol were evaluated 30 and 120 min after the administration of Cannabis spp. (~0.2 g by inhalation). Results: Of the total 12 (60%), 10 (50%), and 11 (55%) had desirable total cholesterol, fairly good HDL-C (40–60 mg/dL) and fairly good LDL-C (100–129 mg/dL) values, respectively. The mean basal concentration of total cholesterol, HDL-cholesterol, and LDL-cholesterol was 193.37 ± 20.18 mg/dL, 60.05 ± 6.36 mg/dL, and 129.65 ± 14.50 mg/dL, respectively. HDL-cholesterol showed progressive increases in participants with desirable HDL-C > 60 mg/dL at 30 min (10 vs. 14 participants, p < 0.001) and at 120 min (10 vs. 16 participants, p < 0.001), while LDL-C peaked in participants with concentrations < 100 mg/dL at 30 min (desirable cholesterol: 0 vs. 2, p = 0.001). HDL-C concentration showed differences after cannabis consumption, showing increases at 30 (63.25 ± 7.68 mg/dL) and 120 min (69.15 ± 18.67 mg/dL) and total cholesterol concentration changed to 180.95 ± 19.3 mg/dL (95%CI 172.5 to 189.4) at 120 min (p = 0.007). Conclusions: HDL-C cholesterol increased 30 and 120 min after Cannabis spp. ingestion, while LDL-C and total cholesterol showed partial reductions in heavy-users from Cusco, Peru.
... The Lepidium sativum seed oil overall phytosterol content (0.825 mg/g) was lower than those cited by Zarrouk et al. (2019) in olive oil (1726 ± 45 mg/kg to 2936 ± 59 mg/kg), Milk thistle seed oil (5088 ± 71 mg/kg to 5891 ± 118 mg/kg), and argan oil (1881 ± 25 mg/kg to 2330 ± 79 mg/kg). Along with the effect of the intake of phytosterols on the development of neurodegenerative diseases, these bioactive nutrients have been recently reported to possess anti-neurodegenerative effects (Cheng et al., 2014;Dierckx et al., 2019). This has been confirmed by treating neuroinflammation which is a common pathological feature of many neurodegenerative diseases, namely Alzheimer and Parkinson (Ransohoff, 2016;Russo and McGavern, 2016;Subhramanyam et al., 2019). ...
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Lepidium sativum is cultivated mainly for the edible oil from its seeds, and considered as an unutilized and neglected crop despite its important properties. Its oil fraction is used to produce soap and stabilize linseed oil when it is mixed with wild mustard seed oil. Once converted into fatty acid methyl esters, it represents a good substitute for imported petroleum diesel after alkaline transesterification reaction. In the current study, Lepidium sativum seeds cultivated in Tunisia and the physicochemical properties and nutrient profile of its cold pressed seed oil were investigated. The antioxidant, antibacterial, and anti-inflammatory activities of the above oil were also assessed. Lepidium sativum seed oil was abundant in both linolenic (35.59 ± 1.9 %) and oleic (21.14 ± 0.63 %) acids, and high amounts of β-sitosterol (42.57 ± 2.96 mg/100g), campesterol (20.04 ± 1.4 mg/100g) and Δ 5,24 stigmastadienol (11.82 ± 0.45 mg/100g) were detected. The total tocopherol content of Lepidium sativum seed oil reached 136.83 ± 7.6 mg/100g with a predominance of γ-tocopherol (86.23 %). Its seed oil exhibited an IC50 of 10.33 ± 0.05 mg/mL and a radical scavenging activity of 415.6 ± 40 Trolox Equivalent Antioxidant Capacity (TEAC) for the DPPH and the ABTS assays, respectively. While the thermal analysis proved a high thermal stability of Lepidium sativum seed oil, that of eight bacteria and one fungal strain showed no noticeable bacterial or antifungal effects. It was also revealed that Lepidium sativum seed oil held a remarkable anti-inflammatory activity. Hence, the obtained results evidenced remarkable chemical, antioxidant and anti-inflammatory properties of Lepidium sativum seed oil, which might potentially be promising for enhancing human health and preventing age-related diseases.
Cannabis sativa L. was used for medicinal purposes for millennia, being useful for the treatment of several conditions such as pain, convulsions, insomnia, and lack of appetite, among others. However, due to its psychotropic side effects and the impossibility to prepare standardized formulations it was banned in the late 1930s and removed from therapeutics, being considered as a drug that was solely used for recreational purposes. In the last two decades, this situation has changed, and the therapeutic use of cannabis has resurged again, demonstrating that standardized cannabis preparations can be safe and useful for the treatment of a broad range of pathologies. In fact, several cannabis-based formulations have been approved with medicinal purposes. This chapter analyzes the medicinal use of cannabis, describing their benefits, risks, and current and future perspectives.
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Alzheimer’s disease (AD) is a debilitating neurodegenerative disease characterized by declining cognition and behavioral impairment, and hallmarked by extracellular amyloid-β plaques, intracellular neurofibrillary tangles (NFT), oxidative stress, neuroinflammation, and neurodegeneration. There is currently no cure for AD and approved treatments do not halt or slow disease progression, highlighting the need for novel therapeutic strategies. Importantly, the endocannabinoid system (ECS) is affected in AD. Phytocannabinoids, including cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC), interact with the ECS, have anti-inflammatory, antioxidant, and neuroprotective properties, can ameliorate amyloid-β and NFT-related pathologies, and promote neurogenesis. Thus, in recent years, purified CBD and THC have been evaluated for their therapeutic potential. CBD reversed and prevented the development of cognitive deficits in AD rodent models, and low-dose THC improved cognition in aging mice. Importantly, CBD, THC, and other phytochemicals present in Cannabis sativa interact with each other in a synergistic fashion (the “entourage effect”) and have greater therapeutic potential when administered together, rather than individually. Thus, treatment of AD using a multi-cannabinoid strategy (such as whole plant cannabis extracts or particular CBD:THC combinations) may be more efficacious compared to cannabinoid isolate treatment strategies. Here, we review the current evidence for the validity of using multi-cannabinoid formulations for AD therapy. We discuss that such treatment strategies appear valid for AD therapy but further investigations, particularly clinical studies, are required to determine optimal dose and ratio of cannabinoids for superior effectiveness and limiting potential side effects. Furthermore, it is pertinent that future in vivo and clinical investigations consider sex effects.
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Normal aging and many age-related diseases such as Alzheimer disease cause deficits in olfaction, however it is currently unknown how natural and pathological aging impact the detection of social odors which might contribute to the impoverishment of social behavior at old age further worsening overall health. Here, we investigated the effect of aging in the recognition of social cues and the display of social behavior. Our findings indicate that aging distinctively disrupts the processing of social olfactory cues decreasing social odor exploration, discrimination and habituation in both wild type senescent (2-year-old) mice and in 1-year-old double mutant model of Alzheimer disease (APP/PS1). Furthermore, social novelty was diminished in 1-year-old APP/PS1 mice, indicating that alterations in the processing of social cues are accelerated during pathological aging. Analysis of the vomeronasal organ, the main gateway to pheromone-encoded information, indicated that natural and pathological aging distinctively reduce the neurogenic ability of the vomeronasal sensory epithelium. Cell proliferation remained majorly preserved in 1-year model of Alzheimer disease (APP/PS1), whereas naturally aged animals exhibited significant deficiencies in the number of mature, proliferative and progenitor cells. This study reveals fundamental differences in the cellular processes by which natural and pathological aging disrupt the exploration of social cues and social behavior.
Legalization of cannabis in the US and other countries highlight the need to understand the health consequences of this substance use. Research indicates that some cannabis ingredients may play beneficial role in treating various medical conditions while other ingredients may pose health risks. This review is focused on the brain and mental health effects of cannabis use. The rationale for examining cannabis use in behavioral and neural conditions is that these conditions are highly widespread in the US and account for high level of medical healthcare and associated cost. The purpose of this review is to provide an overview of the known medicinal benefits of selected cannabis cannabinoids in conditions like pediatric epilepsy, attention deficit hyperactivity disorder, autism spectrum disorder, and the known side effects or contraindications in conditions such as addiction, cognition, and psychosis. Several recommendations are made as to studies that will help further understanding the increasing role of cannabis in neuropsychiatric health and disease.
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The present study provides the fatty acid, tocopherol, phytosterol, and polyphenol profiles of some Mediterranean oils extracted from pumpkin, melon, and black cumin seed oils and those of dietary argan seed oil. Gas chromatography analysis revealed that oleic and linoleic acids were the most abundant fatty acids. Argan and melon seed oils exhibited the highest levels of oleic acid (47.32±0.02%) and linoleic acid (58.35±0.26%), respectively. In terms of tocopherols, melon seed oil showed the highest amount (652.1±3.26 mg/kg) with a predominance of γ-tocopherol (633.1±18.81 mg/kg). The phytosterol content varied between 2237.00±37.55 µg/g for argan oil to 6995.55±224.01 µg/g for melon seed oil. High Performance Liquid Chromatography analysis also revealed the presence of several polyphenols: vanillin (0.59 mg equivalents Quercetin/100 g) for melon seed oil, and p-hydroxycinnamic acid (0.04 mg equivalents Quercetin/100 g), coumarine (0.05 mg equivalents Quercetin/100 g), and thymoquinone (1.2 mg equivalents Quercetin/100 g) for black cumin seed oil. The "Kit Radicaux Libres" (KRL) assay used to evaluate the scavenging properties of the oils showed that black cumin seed oil was the most efficient. On the light of the richness of all Mediterranean oil samples in bioactive compounds, the seed oils studied can be considered as important sources of nutrients endowed with cytoprotective properties which benefits in preventing age-related diseases which are characterized by an enhanced oxidative stress.
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Supplementation with cannabidiol (CBD) may expedite recovery when consumed after exercise. The purpose of this study was to determine if supplementation with CBD reduces inflammation and enhances performance following strenuous eccentric exercise in collegiate athletes. Twenty-four well-trained females (age = 21.2 ± 1.8 years, height = 166.4 ± 8 cm, weight = 64.9 ± 9.1 kg) completed 100 repetitions of unilateral eccentric leg extension to induce muscle damage. In this crossover design, participants were randomized to receive 5 mg/kg of CBD in pill form or a placebo 2 h prior to, immediately following, and 10 h following muscle damage. Blood was collected, and performance and fatigue were measured prior to, and 4 h, 24 h, and 48 h following the muscle damage. Approximately 28 days separated treatment administration to control for the menstrual cycle. No significant differences were observed between the treatments for inflammation, muscle damage, or subjective fatigue. Peak torque at 60°/s (p = 0.001) and peak isometric torque (p = 0.02) were significantly lower 24 h following muscle damage, but no difference in performance was observed between treatments at any timepoint. Cannabidiol supplementation was unable to reduce fatigue, limit inflammation, or restore performance in well-trained female athletes.
Cannabis has been well known for centuries due to its medicinal properties. In recent decades, the inclination of researchers towards its important phytoconstituents as a potential therapeutic alternative has been propounded due to the discovery of its major active constituent, i.e., Δ9-tetrahydrocannabinol (Δ9-THC). Besides this, the presence of other phytoproducts, including cannabidiol (CBD), cannabigerol (CBG), cannabichromene (CBC), etc., also contribute towards its medicinal importance. Interestingly, due to the effectiveness of cannabis against various pathological conditions, its use for medicinal purposes has been revolutionized worldwide. Despite these facts, it has become obligatory to explore synergistic interactions and mode of action of its phytoconstituents involving various biological pathways. Current advancements have allowed medical practitioners to better understand cannabis-derived products as a pharmacological choice in several conditions, including pain treatment, stress, anxiety, neurodegenerative disorders, and cancer. However, there exists a lacuna in the literature regarding its beneficial doses. Since medicinal exploration and the legalization of cannabis depend upon various factors, the present review deals with the important phytocannabinoids, their biogenesis, types of drugs obtained, mode of action, therapeutic implications, and new approaches for supporting this plant as a critical therapeutic agent for pharmaceutical drugs. Overall, this may provide an insight into the role of cannabis as a potent candidate for future drug discovery and generate efficient products for human welfare.
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Patients suffering from Alzheimer's disease (AD) exhibit a decline in cognitive abilities including an inability to recognise familiar faces. Hallmark pathological changes in AD include the aggregation of amyloid-β (Aβ), tau protein hyperphosphorylation as well as pronounced neurodegeneration, neuroinflammation, neurotoxicity and oxidative damage. The non-psychoactive phytocannabinoid cannabidiol (CBD) exerts neuroprotective, anti-oxidant and anti-inflammatory effects and promotes neurogenesis. CBD also reverses Aβ-induced spatial memory deficits in rodents. Thus we determined the therapeutic-like effects of chronic CBD treatment (20 mg/kg, daily intraperitoneal injections for 3 weeks) on the APPswe/PS1∆E9 (APPxPS1) transgenic mouse model for AD in a number of cognitive tests, including the social preference test, the novel object recognition task and the fear conditioning paradigm. We also analysed the impact of CBD on anxiety behaviours in the elevated plus maze. Vehicle-treated APPxPS1 mice demonstrated impairments in social recognition and novel object recognition compared to wild type-like mice. Chronic CBD treatment reversed these cognitive deficits in APPxPS1 mice without affecting anxiety-related behaviours. This is the first study to investigate the effect of chronic CBD treatment on cognition in an AD transgenic mouse model. Our findings suggest that CBD may have therapeutic potential for specific cognitive impairments associated with AD.
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The amyloidogenic cascade is regarded as a key factor at the basis of Alzheimer's disease (AD) pathogenesis. The aberrant cleavage of amyloid precursor protein (APP) induces an increased production and a subsequent aggregation of beta amyloid (Aβ) peptide in limbic and association cortices. As a result, altered neuronal homeostasis and oxidative injury provoke tangle formation with consequent neuronal loss. Cannabidiol (CBD), a Cannabis derivative devoid of psychotropic effects, has attracted much attention because it may beneficially interfere with several Aβ-triggered neurodegenerative pathways, even though the mechanism responsible for such actions remains unknown. In the present research, the role of CBD was investigated as a possible modulating compound of APP processing in SHSY5Y(APP+) neurons. In addition, the putative involvement of peroxisome proliferator-activated receptor-γ (PPARγ) was explored as a candidate molecular site responsible for CBD actions. Results indicated the CBD capability to induce the ubiquitination of APP protein which led to a substantial decrease in APP full length protein levels in SHSY5Y(APP+) with the consequent decrease in Aβ production. Moreover, CBD promoted an increased survival of SHSY5Y(APP+) neurons, by reducing their long-term apoptotic rate. Obtained results also showed that all, here observed, CBD effects were dependent on the selective activation of PPARγ. Copyright © 2013 John Wiley & Sons, Ltd.
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Amyloid-β (Aβ), major constituent of senile plaques in Alzheimer's disease (AD), is generated by proteolytic processing of the amyloid precursor protein (APP) by β- and γ-secretase. Several lipids, especially cholesterol, are associated with AD. Phytosterols are naturally occurring cholesterol plant equivalents, recently been shown to cross the blood-brain-barrier accumulating in brain. Here, we investigated the effect of the most nutritional prevalent phytosterols and cholesterol on APP processing. In general, phytosterols are less amyloidogenic than cholesterol. However, only one phytosterol, stigmasterol, reduced Aβ generation by (1) directly decreasing β-secretase activity, (2) reducing expression of all γ-secretase components, (3) reducing cholesterol and presenilin distribution in lipid rafts implicated in amyloidogenic APP cleavage, and by (4) decreasing BACE1 internalization to endosomal compartments, involved in APP β-secretase cleavage. Mice fed with stigmasterol-enriched diets confirmed protective effects in vivo, suggesting that dietary intake of phytosterol blends mainly containing stigmasterol might be beneficial in preventing AD.
Dietary fish oil, providing n3 polyunsaturated fatty acids like eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), associates with reduced dementia risk in epidemiological studies and reduced amyloid accumulation in Alzheimer mouse models. We now studied whether additional nutrients can improve the efficacy of fish oil in alleviating cognitive deficits and amyloid pathology in APPswe/PS1dE9 transgenic and wild-type mice. We compared four isocaloric (5% fat) diets. The fish oil diet differed from the control diet only by substituted fish oil. Besides fish oil, the plant sterol diet was supplemented with phytosterols, while the Fortasyn diet contained as supplements precursors and cofactors for membrane synthesis, viz. uridine-monophosphate; DHA and EPA; choline; folate; vitamins B6, B12, C and E; phospholipids and selenium. Mice began the special diets at 5 months and were sacrificed at 14 months after behavioral testing. Transgenic mice, fed with control chow, showed poor spatial learning, hyperactivity in exploring a novel cage and reduced preference to explore novel odors. All fish-oil-containing diets increased exploration of a novel odor over a familiar one. Only the Fortasyn diet alleviated the spatial learning deficit. None of the diets influenced hyperactivity in a new environment. Fish-oil-containing diets strongly inhibited β- and γ-secretase activity, and the plant sterol diet additionally reduced amyloid-β 1-42 levels. These data indicate that beneficial effects of fish oil on cognition in Alzheimer model mice can be enhanced by adding other specific nutrients, but this effect is not necessarily mediated via reduction of amyloid accumulation.
Alzheimer's disease (AD) is a chronic neurodegenerative disease leading to cognitive decline, dementia, and ultimately death. Despite extensive R&D efforts, there are no diseases modifying treatments for AD available. The stage in which patients receive a clinical diagnosis of probable AD may be too late for disease modifying pharmacotherapy. Prevention strategies may be required to successfully tackle AD. Preclinical AD applies to over half of all healthy elderly subjects and manifests by signs of amyloid deposition and/or neuronal injury in the brain, preceding the stage in which symptoms of dementia, cognitive and functional impairment become observable. Prevention trials in preclinical AD require longer and larger clinical trials using biomarkers and cognitive endpoints, which requires collaboration across academia, government and industry.
Giving rats opportunity to explore an enclosed or an elevated alleyway permitted study of exploratory drive in situations arousing different intensities of fear. It was concluded that such novel situations may induce approach avoidance conflict behavior. It was further found that strength of fear drive to novel situations declines as a function of time of direct exposure; after a period of non-exposure, spontaneous recovery of the fear reaction may be observed, "the magnitude of which depends upon the amount of fear extinguished to that stimulation on previous occasions."