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Pharmaceutical Sciences, 2022, 28(2), 239-250
doi:10.34172/PS.2021.59
https://ps.tbzmed.ac.ir/
Research Article
Broad-Spectrum Cannabis Oil Alleviates Behavioral Symptoms
Associated with Stress-Related Anxiety and Depression in Mice
*Corresponding Author: Rafael Cypriano Dutra, E-mail: rafaelcdutra@gmail.com & Nádia Rezende Barbosa Raposo, E-mail: nadia.barbosa@uf.br
©2021 e Author(s). is is an open access article and applies the Creative Commons Attribution Non-Commercial License (http://creativecommons.
org/licenses/by-nc/4.0/). Non-commercial uses of the work are permitted, provided the original work is properly cited.
Pollyana Mendonça de Assis1, Eduarda Gomes Ferrarini2,3, Gabriela Mantovani Baldasso2, Rodrigo Sebben Paes2,
Murilo Chaves Gouvêa4, Carlos Espínola Neto Segundo4, Francesca Borrelli5, Marcos Antônio Fernandes Brandão1,
Raaele Capasso6,7, Rafael Cypriano Dutra2,3* , Nádia Rezende Barbosa Raposo1*
1Center of Research and Innovation in Health Sciences (NUPICS), School of Pharmacy, Universidade Federal de Juiz de Fora, Juiz de Fora 36036-330, Brazil.
2Laboratory of Autoimmunity and Immunopharmacology (LAIF), Department of Health Sciences, Campus Araranguá, Universidade Federal de Santa
Catarina, Araranguá 88906-072, Brazil.
3Post-Graduate Program of Neuroscience, Center of Biological Sciences, Universidade Federal de Santa Catarina, Florianópolis 88040-900, Brazil.
4Associação Brasileira de Apoio Cannabis e Esperança – ABRACE, Parque Sólon de Lucena, 697, João Pessoa 58028-470, Brazil.
5Department of Pharmacy, University of Naples Federico II, 80131 Naples, Italy.
6Department of Agricultural Sciences, University of Naples Federico II, 80055 Portici, Italy.
7Endocannabinoid Research Group, Naples, Italy.
Abstract
Background: Posttraumatic stress disorder (PTSD) is a psychiatric condition that manifests
through a broad range of symptoms and shares several phenotypes with anxiety and depression.
Refractory PTSD aects 10–30% of the patients and highlights the need for alternative
pharmacotherapy. e suggested involvement of the endocannabinoid system (ECS) with the
emotional processes has enlightened the use of Cannabis sp. en, this study aimed to evaluate
the therapeutic eects of a broad-spectrum Cannabis oil on anxiety- and depressive-like
behaviors triggered by stressors from combined nature. In addition, this study investigated the
eect of the oil on central cannabinoid receptor 1 and serum levels of cytokines, chemokines,
and growth factors.
Methods: Mice were randomized into ve groups (vehicle; Cannabis oil; uoxetine; single
oral dose) and submitted to acute restraint and chronic unpredictable stress. en, they were
behaviorally assessed in the elevated plus-maze test (EPMT), forced swimming test (FST),
splash test (ST), and open eld test (OFT). e tetrad cannabinoid assay evaluated the central
eect of the oil. Serum biomarkers levels were measured by a multiplex bead-based assay.
Results: Cannabis oil (0.1 mg/kg, p.o.) signicantly reduced the anxiety-like behavior in EPMT
in the acute restraint stress model (p < 0.05) as compared to vehicle. Moreover, compared to the
vehicle, Cannabis oil signicantly reverted the despair and anhedonic-like behaviors in FST (p
< 0.05) and ST (p < 0.05), respectively, in chronically stressed mice. Yet, compared to vehicle,
therapy with Cannabis oil did not induce cannabinoid-tetrad (p < 0.0001); downregulated
granulocyte-macrophage colony-stimulating factor (GM-CSF; p < 0.01) and advanced glycation
end-products (RAGE; p < 0.0001); and upregulated vascular endothelial growth factor (VEGF;
p < 0.01) serum levels.
Conclusion: Altogether, our data suggest the potential of the broad-spectrum Cannabis oil to
improve symptoms related to anxiety and depression caused by traumatic events.
Article Info
Article History:
Received: 11 June 2021
Accepted: 2 September 2021
ePublished: 10 September 2021
Keywords:
-Anxiety
-Broad-spectrum Cannabis oil
-Cannabis sp
-Depression
-Posttraumatic stress disorder
Introduction
Globally, the knowledge of the real prevalence of
posttraumatic stress disorder (PTSD) is unclear.1 It has
been estimated that 61% to 80% of individuals are going
to experience some type of traumatic event during their
lifetime.2 From those individuals, approximately 5% to
10% will develop PTSD.3 is psychiatric ailment aects
the self and social functions of individuals by a broad range
of symptoms, including the involvement of cognition,
emotion, and mood.4 In these people, PTSD is frequently
comorbid with depression and anxiety. According to the
National Epidemiologic Survey on Alcohol and Related
Conditions, 59% and 35.2% of those who met the criteria
for PTSD also met the criteria for anxiety and depression,
respectively.5
Although there are signicant proportions of people
Mendonça de Assis, et al.
240 | Pharmaceutical Sciences, 2022, 28(2), 239-250
suering from PTSD and its comorbidities, such as
depression and anxiety, the molecular mechanisms
underlying the pathophysiology are still poorly
understood. e current knowledge suggests alterations
in neurogenesis, neurohormonal, and neurotransmitter
functioning.6 Another aspect that has attracted attention
is the probable involvement of the immune system and
its dysregulation in PTSD7-9 and PTSD-related disorders
– anxiety and depression.10 en, a better understanding
of the molecular basis of PTSD is worth for future
development of diagnosis, prognosis, and therapeutic
strategies.
inking about new pharmacotherapy options is
particularly important if considering the challenges faced
by patients with the available drugs. To date, the US Food
and Drug Administration (FDA) has only approved
two selective serotonin reuptake inhibitors for the
treatment of PTSD: sertraline and paroxetine.11 O-label
pharmacotherapy includes uoxetine and venlafaxine.12
However, 10 – 30% of the patients are still refractory
to the conventional prescriptions,13 highlighting the
demand for alternative strategies that are safe and have
good tolerability. In this scenario, the endocannabinoid
system (ECS) has emerged as a promising pharmaceutical
target for the modulation of the synaptic transmission
involved with cognition, stress responses, and emotional
stability,14 besides its connection with the immune system
and neurogenesis.15 Consequently, the therapeutic role of
Cannabis sp. has attracted more attention.
Among the more than 400 compounds identied in
Cannabis sp. so far, delta-9-tetrahydrocannabinol (Δ9-
THC) and cannabidiol (CBD) are the most studied
phytocannabinoids. e psychomimetic eects of Cannabis
sp. have been attributed mainly to Δ9-THC, which is an
agonist for central cannabinoid receptor type 1 (CB1R).
CBD, instead, does not elicit euphoria.16 Indeed, CBD is
known for its pharmacological properties, which include
analgesic and anti-inammatory actions.17 Additionally,
this phytocannabinoid has proved to have neuroprotective,
anxiolytic, antipsychotic, antiemetic, and antioxidant
properties through a multi-target mechanism.18-21 Despite
all the pieces of evidence suggesting the eectiveness of
Cannabis sp. on psychiatric disorders, the FDA has only
approved this compound for children who suer from
Lennox-Gastaut Syndrome and Dravet syndromes.22,23 It
is worth mentioning that the approved drugs consist of
derivatives of isolated phytocannabinoids, which are way
dierent from Cannabis oil. According to the extraction
techniques, broad-spectrum Cannabis oil can be almost
free of Δ9-THC but contains all the phytochemicals found
in the plant, including terpenes, avonoids, and other
phytocannabinoids, such as CBD.24 is composition is
said to contribute to the synergistic eects of Cannabis
sp. and might be an alternative treatment for complex
psychiatric diseases.
Animal models are widely used to research new
treatments for PTSD. It helps to comprehend the
molecular basis of the disease and, consequently, identify
potential targets for new drugs or even drug repositioning.
Using animals in pre-clinical research is also a strategy for
screening new potential drugs to treat PTSD.25 Recently,
Deslauriers et al.26 have reviewed >600 articles to examine
the ability of current rodent models to probe biological
and behavioral phenotypes of PTSD. e authors have
evaluated several paradigms, including the restraint
stress and the chronic unpredictable stress, for their
ecacy in stimulating PTSD-like constructs (learned fear
and extinction, avoidance, reduced motivation/reward,
arousal, and cognitive decits) in addition to biological
and physiological phenotypes associated with PTSD.
All the reviewed paradigms produced lasting eects on
general depression- and/or anxiety measures.26 at said,
behavioral tests are methodological tools that represent
the best approach to measure anxiety- and depressive-like
phenotypes in a PTSD model.27
us, considering the high prevalence of PTSD; the
patients’ refractory to medical treatment; and the suggested
therapeutic potential of Cannabis sp. on psychiatric illness,
this study aimed to evaluate in mice the eects of a broad-
spectrum Cannabis oil on anxiety- and depressive-like
behaviors triggered by stressors from combined nature.
Further, it was investigated the central eects of the oil
on CB1R, as well as its inuence on the serum levels of
cytokines, chemokines, and growth factors.
Materials and Methods
Broad-spectrum Cannabis oil
e broad-spectrum Cannabis oil was produced and
analyzed by the Brazilian Association ABRACE (Associação
Brasileira de Apoio Cannabis Esperança, Paraíba - Brazil)
that is enrolled with the National Register of Legal
Entities (CNPJ) under the number 23.877.015/0001-38.
e chromatographic analysis reported a CBD:Δ9-THC
proportion of 11:1 and total cannabinoids of 40.2% (Figure
1). Regarding the microbiological assessment, the oil was
under the current quality parameters.
Animals
A total of 120 male Swiss mice (30-50 g, 50–90 days of age)
were provided by the breeding unit of the Universidade
Federal de Santa Catarina (UFSC). e animals (maximum
of 12 mice group-housed in clear-transparent plastic cages
with dust-free sawdust bedding) were housed under
regulated conditions that included a 12-h-light/-dark cycle
(articial light at 07:00 a.m. to 07:00 p.m.), controlled
temperature (22 ± 2 °C), and standard food and water
ad libitum. All of the tests were conducted between 8:00
a.m. and 5:00 p.m., and the animals were acclimatized to
the laboratory settings for at least 1 h before testing. Each
animal was used only once throughout the experiments.
ey were randomly assigned before treatment or
behavioral evaluation. All eorts were made to minimize
their suering and reduce the number of animals required
for the experiments. e experiments herein described were
Cannabis Oil for Mood Disorders
Pharmaceutical Sciences, 2022, 28(2), 239-250 | 241
Figure 1. High-performance liquid chromatography (HPLC) analysis of broad-spectrum Cannabis oil. THC (retention time = 18.2 min);
CBD (retention time = 10.6 min). THC: tetrahydrocannabinol; CBD: cannabidiol.
reported in compliance with the ARRIVE guidelines28-30
and approved by the Animal Ethics Committee of the
UFSC (CEUA-UFSC) under protocol 7176240920. All the
experimental procedures were conducted according to the
guidelines of CONCEA and CEUA/UFSC, based on the
3R’s principles: replacement, reduction, and renement. A
blind operator performed both the nociception assessments
and the statistical analysis.
Experimental design
Broad-spectrum Cannabis oil (0.1, 1, 3 and 10 mg/kg) and
uoxetine (10 mg/kg), a selective inhibitor of serotonin
used as the positive control, were dissolved in medium-
chain triglycerides (MCT) (Vitafor®, Araçoiaba da Serra,
São Paulo, Brazil) and saline, respectively, and administered
by oral gavage (p.o.). Fluoxetine has been used and proved
to be eective on pre-clinical models of PTSD27,31,32 and
clinical studies.33,34
e chronic protocol was also featured with an additional
group treated continuously with Cannabis oil at 0.1 mg/
kg (p.o.; 1x/administration; 5-day treatment). is extra
group aimed to verify if repeated treatment could result
in a long-lasting anxiolytic-/antidepressant-like eect. All
drugs were prepared right before the treatment, and the
volume of the administration was 10 ml/kg, 1 h before
the constraint stress or immediately aer the last stressful
stimulus (14-day protocol). e choice of the doses used
was based on pilot experiments (Supplemental Data) or on
previous data described in the literature.35-37
Acute restraint stress-induced paradigm
Five groups of randomized mice were tested through
this protocol: vehicle, uoxetine (10 mg/kg, p.o., 1x/
administration), or broad-spectrum Cannabis oil (0.1, 3,
and 10 mg/kg, p.o., 1x/administration), as shown in Figure
2A. For the acute restraint stress-induced protocol, mice
were rst maintained inside their home cages, with free
access to water and food, during the drug administration
and the period before the restriction (1 h). en, each animal
was removed from the group-house and introduced into a
fenestrated punctured plastic tube (18 cm × 4 cm) placed
in a horizontal, so the normal orientation of the animal’s
body was kept. e animal remained in that position for 7
h, with all physical movements restrained but without any
pain. No food or water was oered to the animal during
the 7-hour period. Aer release, the animals waited for 40
min before being behaviorally assessed.38-41 e researcher
was blind for the treatments, and the behavioral tests were
manually scored.
Chronic unpredictable stress (CUS) paradigm
Five groups of randomized mice were evaluated: vehicle,
uoxetine (10 mg/kg, p.o., 1x/administration), or broad-
spectrum Cannabis oil (0.1, 1 and 3 mg/kg, p.o., 1x/
administration), as shown in Figure 2B. is protocol
was also featured with an additional group that was
continuously treated with Cannabis oil at 0.1 mg/kg (p.o.) –
administered in the last 5 days of the protocol. e stressful
stimuli were designed to maximize the unpredictable
nature of the stressor. erefore, several stressors varying in
duration and time were randomly applied for 14 days, and
food and water were oered ad libitum. As soon as mice
were exposed to the stressor, they returned to their home
cage and were kept under laboratorial standard conditions.
Mice received the aforementioned treatments on the 14th
and last day of the protocol and were behaviorally assessed
24 h aer the treatment.42,43
Briey, the CUS paradigm consisted of exposure, once
daily, to one of the following aversive stressors: restraint –
mice were placed into a plastic tube (18 cm × 4 cm) sealed
at the extremities and properly perforated to promote
air circulation. is stimulus occurred on the 1st and 7th
Mendonça de Assis, et al.
242 | Pharmaceutical Sciences, 2022, 28(2), 239-250
days of the protocol; forced swimming – mice were into
a cylindrical recipient (10 cm × 25 cm) containing 19 cm
of water on the 6th day of the protocol; cold bath – mice
were placed inside a group-house with 2 cm of water on the
2nd and 9th days of the protocol; wet wood shaving – wood
shavings were wet with 400 ml of water, and the group-
house was tilted at 45° angle. is stimulus occurred on the
3rd and 12th days of the protocol; footshock – test took place
at the passive avoidance apparatus (Insight® – Ribeirão
Preto, São Paulo, Brazil). Mice were placed on the bars and
received paw shocks (0.7 mA; 0.5 s/min) every 30 sec for
3 min on the 8th, 10th, and 14th days of the protocol; tail
compression – on the 4th and 13th days, the tail compression
stimulus was conducted by positioning a clothespin 1 cm
from the base of the animal’s tail. Figure 2B summarizes all
the steps aforementioned.
Behavioral tests
Forced swimming test
e FST assessed antidepressant-like behavior and followed
the method described by Porsolt et al.44 Each one of the
animals was placed into a transparent cylindrical tank (30
cm × 20 cm) with 15 cm of water at a temperature of 22-25
ºC. e test was conducted for 6 min, with a habituation
period of 2 min. e antidepressant-like eects of the
treatments were assessed in the function of the latency to
immobility.45,46 e FST was only executed in the chronic
protocol.
Splash test
e splash test (ST) was used to measure the anhedonia-
like state. It consisted of squirting a sucrose solution (200
µl, 20%) on the dorsal coat of the animals. Because of the
high viscosity of sucrose in this concentration, the animals
initiate the self-cleaning behavior, a typical symptom of
anhedonia used to a depression diagnosis. Aer squirting,
the latency to grooming was recorded for 5 min as an
indicator of self-care and motivational behavior.27,47 e ST
was only executed in the chronic protocol.
Elevated plus-maze test
e elevated plus-maze test (EPMT) assessed anxiety-like
behavior in mice. e apparatus comprises two open arms
(36 cm × 5 cm) and two closed arms (36 cm × 5 cm × 18
cm) that are connected to a central platform (5 cm × 5 cm)
and elevated 50 cm from the ground. For the test, an animal
per time was placed at the center of the platform, facing
toward the closed arm, and allowed to move through the
arms. e time spent in the open arms and the number
of entries made into the open arms were recorded for 5
min. Subsequently, the percentage of time spent in open
arms was calculated from the total spent there divided by
the total time spent in both open and closed arms. e
percentage of entries made into the open arms was given
from the total entries made into these arms divided by the
total entries made in both open and closed arms.48
Open eld test
e open-eld test (OFT) evaluated whether the animals
Figure 2. Experimental design. (A) The acute restraint stress-induced protocol was conducted in four different stages as illustrated above
(I-IV), which included the drug administration 1 h before the restraint, the containment in tubes for 7 h, the ambiance of 40 min, and the
behavioral tests followed by euthanasia. Behavioral tasks were evaluated by the elevated plus-maze and open eld tests. (B) The chronic
unpredictable stress (CUS) protocol, applied for 15 days, was divided into periods of stressful stimuli and behavioral tests/euthanasia. The
stressful stimuli consisted of containment, forced swimming, cold bath, wet wool shavings, shock, and tail compression in alternated days
and hours, always unpredictably. In this protocol, the drug was administered on the last day (14) or daily for the group under continuous
treatment. Behavioral tasks were assessed by the forced swimming (FST), open eld (OFT), elevated plus-maze (EPMT), and splash
(ST) tests.
Cannabis Oil for Mood Disorders
Pharmaceutical Sciences, 2022, 28(2), 239-250 | 243
had any locomotor impairment during the experimental
protocols and treatments. Each one of the mice was placed
in the center of an acrylic box (30 cm × 30 cm) that possess
nine square areas equally divided (Insight® – Ribeirão
Preto, São Paulo, Brazil). e crossing number (scored by
the number of segments crossed with the four paws) was
used to assess locomotor activity. e test lasted 5 min per
animal, and the apparatus was cleaned with a solution of
ethanol 10% aer each test to avoid clues and smells from
the predecessor animal.49
Cannabinoid-induced tetrad
e classical cannabinoid-induced tetrad is a preclinical
model that evaluates the “safety-pharmacology” of new
cannabinoid-related molecules.50 e main purpose
of the test was to monitor the central eects of broad-
spectrum Cannabis oil on cannabinoids by measuring the
following parameters: spontaneous locomotor activity,
rectal temperature, catalepsy, and antinociception.51 Four
groups were assessed for each parameter aforementioned:
vehicle (MCT); broad-spectrum Cannabis oil (3 or 30
mg/kg, p.o.); or WIN 55,212 (1.5 mg/kg, i.p. – a potent
cannabinoid receptor agonist). Tests were conducted every
1 h, during the period of 6 h following the drug or vehicle
administration.
Spontaneous locomotor activity was evaluated using
the rotarod apparatus (Ugo Basile, Italy). It was xed at a
rotational speed of 4 revolutions per minute (rpm). Before
the experiment, mice had been trained for 60 sec on two
consecutive days. Latencies were determined in the case
the animals had fallen o the apparatus.
Core temperatures were measured using a clinic digital
thermometer (BD Basics, New Jersey, USA) that was
lubricated with intimate gel before inserting it into the
rectum to a constant depth of 3 cm.
To measure catalepsy mice were hung by their frontal
paws from a plastic stem (12 cm of diameter) xed
horizontally at a height of 2-3 cm, allowing them to stay
standing. is test measured the time that the animal spent
moving and touching the bottom of the box. e test cut-
o point was 180 sec.
To evaluate antinociception, the tail-ick apparatus with
a xed temperature of 45 °C. e test had a cut-o point
of 15 sec.
Serum biomarkers
Blood was collected from each animal on the 15th and
last day of CUS protocol, and serum was separated by
centrifugation (5500 × g for two 15-min cycles) and stored
until further analysis. Briey, 50 µl of the sample was
processed with a multiplex bead-based assay (R&D Systems,
Minneapolis, USA) according to the manufacturer’s
instructions. e assay determined the serum levels of
dierent cytokines, chemokines, and growth factors [for
instance, monocyte chemoattractant protein-1 (MCP-
1), eotaxin (EOTX), granulocyte-macrophage colony-
stimulating factor (GM-CSF), interleukin- (IL-) 1β, IL-4,
IL-17A, IL-33, IL-2, IL-6, IL-17E, vascular endothelial
growth factor (VEGF), macrophage inammatory protein
(MIP)-1α, keratinocytes-derived chemokine (KC),
intercellular adhesion molecule (ICAM), and receptor for
advanced glycation end products (RAGE)]. Measurements
and analysis were performed by the Luminex platform
(Luminex® 100/200™ System, Texas, EUA). Each multiplex
immunoassay was performed in quintuplicate, and results
were expressed as pg/ml serum.
Statistical analysis
All data are expressed as the mean ± standard error of
the mean (SEM) of 4 – 10 animals/group. A statistical
comparison of the data was performed by one- or two-way
ANOVA followed by Bonferroni to multiple comparisons
post hoc test. P < 0.05, 0.01, and 0.001 were considered
signicant. Statistical analyses were performing using
GraphPad Prism 8.2.1 soware (GraphPad Soware Inc.,
San Diego, CA, USA).
Results
Eects of single administration doses of broad-spectrum
Cannabis oil on acute restraint stress-induced behaviors
in the EPMT and OFT
e EPMT assessed the anxiolytic eect of the treatment
with a single dose of broad-spectrum Cannabis oil (0.1, 3,
and 10 mg/kg, p.o.) during acute restraint stress-induced
behaviors. As shown in Figure 3A, the oil signicantly
increased the time spent in open arms at 0.1 mg/kg as
compared to vehicle (p < 0.05). Figure 3B shows that the
more entries made into the open arms by the animals from
the groups treated with Cannabis oil were not signicantly
dierent from the vehicle (p > 0.05). Finally, no changes
were observed in the locomotor activity (p > 0.05) in the
OFT (Figure 3C), conrming that the anxiolytic eect of
Cannabis oil occurs independently of any motor changes.
Compared to the positive group, no signicant changes
were observed.
Eects of the treatment with broad-spectrum Cannabis oil
on CUS-induced behaviors
As shown in Figure 4A, the treatment with broad-spectrum
Cannabis oil at 0.1 mg/kg signicantly diminished the
latency to immobility in the FST as compared to the
vehicle group (p < 0.05), similarly to the eect seen in the
uoxetine group (p < 0.05). Moreover, Figure 4B showed
that mice treated with Cannabis oil (0.1, 1, and 3 mg/kg)
started grooming signicantly faster than animals treated
with vehicle (p < 0.05) in the ST. Relevantly, the OFT
(Figure 4C) showed no change in the locomotor behavior
of the animals (p > 0.05). Regarding the EPMT (Figure
4D, E) no signicant dierences were observed in the time
spent in the open arms (p > 0.05), although mice treated
with Cannabis oil (0.1 mg/kg) signicantly demonstrated
more entries into the open arms (p < 0.01). Finally, mice
under CUS protocol were continuously treated with broad-
spectrum Cannabis oil (0.1 mg/kg) for 5 days and then
Mendonça de Assis, et al.
244 | Pharmaceutical Sciences, 2022, 28(2), 239-250
Figure 3. Effect of the treatment with broad-spectrum Cannabis oil (0.1, 3, and 10 mg/kg, p.o.) or uoxetine (10 mg/kg, p.o.) on mice
induced to acute restraint stress and submitted to EPMT (A and B) and OFT (C). Values are expressed as mean ± SEM of 7-10 animals
per column. ***p < 0.001 versus vehicle (one-way ANOVA followed by Bonferroni post-hoc-test). EPMT: elevated plus-maze test; OFT:
open eld test.
behaviorally compared to vehicle and uoxetine groups,
although no signicant changes were observed (data not
shown).
Broad-spectrum Cannabis oil did not induce cannabinoid-
like eects on tetrad assay
e eects of treatment with broad-spectrum Cannabis
oil (3 and 30 mg/kg, p.o.) on locomotion, nociception,
catalepsy, and body temperature are shown in Figure 5. As
expected, WIN 52,212-2 (1.5 mg/kg, i.p.), a potent CB1R
agonist, induced cannabinoid-like eects during tetrad
assay. is agonist reduced signicantly the locomotor
activity (p < 0.0001; Figure 5A), increased the threshold
sensitivity (p < 0.0001, Figure 5B), induced catalepsy (<
0.0001; Figure 5C), and decreased the body temperature (p
< 0.0001; Figure 5D) as compared to the vehicle. Otherwise,
no signicant behavior changes were observed following
the oral administration of Cannabis oil (p > 0.05).
Serum biomarkers
Table 1 shows the cytokines, chemokines, and growth
factors levels following a single dose or continuous
Figure 4. Effects of the treatment with broad-spectrum Cannabis oil (0.1, 1 and 3 mg/kg; p.o.) or uoxetine (10 mg/kg; p.o.) on mice
chronically stressed by unpredictable stressors and submitted to the FST (A), ST (B), OFT (C), and EPMT (D and E). Treatments were
administered 24 h before behavioral assessments. Values are expressed as mean ± SEM of 7-10 animals per column. *p < 0.05 and **p
< 0.01 versus vehicle (one-way ANOVA followed by Bonferroni post-hoc-test). FST: forced swimming test; ST: splash test; OFT: open eld
test; EPMT: elevated plus-maze test.
Cannabis Oil for Mood Disorders
Pharmaceutical Sciences, 2022, 28(2), 239-250 | 245
Figure 5. Evaluation of the treatment with broad-spectrum Cannabis oil on cannabinoid tetrad assay. The tests included locomotor ac-
tivity (A), threshold hyperalgesia (B), catalepsy-like behavior (C), and thermal body measurement (D). WIN 52,212-2 (1.5 mg/kg, i.p.) – a
potent cannabinoid receptor agonist – was used as the positive control. Data are presented as mean ± SEM of 4-5 mice per group. **p
< 0.001 versus naïve (two-way ANOVA followed by Bonferroni post-hoc-test). B: baseline withdrawal threshold refers to the evaluation
performed before treatment.
Table 1. Cytokines, chemokines, and growth factors serum levels following single and continuous treatment with broad-spectrum Cannabis
oil (0.1 mg/kg, p.o.).
Measurement Vehicle Fluoxetine Single Dose Continuous Treatment P-value
MCP-1 281.5 (12.42) 294.8 (14.27) 340.8 (28.86) 294.9 (24.54) 0.2354
EOTX 255.7 (35.97) 249.9 (16.86) 282.1 (11.32) 256.7 (16.64) 0.7447
GM-CSF 4.525 (0.10) 4.289 (0.10) 4.018 (0.09)** 4.304 (0.08) 0.0081
IL-1β 49.76 (6.89) 51.49 (8.62) 45.40 (9.03) 44.98 (7.39) 0.9244
IL-4 ND ND ND ND -
IL-17A 7.769 (1.55) 6.971 (0.95) 10.55 (2.43) 10.37 (3.43) 0.6031
IL-33 54.86 (1.57) 47.57 (2.10) 56.12 (2.88) 52.04 (2.75) 0.0775
VEGF 12.88 (0.37) 12.77 (0.65) 13.66 (0.54) 22.26 (4.06)** 0.0075
MIP-1α 0.2138 (0.04) 0.2583 (0.08) 0.3688 (0.08) 0.3550 (0.03) 0.1943
KC 65.79 (3.41) 54.06 (2.27) 60.59 (1.82) 68.68 (6.25) 0.0590
ICAM 25450 (2138) 24210 (900.7) 20710 (1517) 22360 (1420) 0.1734
IL-2 3.630 (0.13) 3.423 (0.27) 3.718 (0.22) 3.208 (0.15) 0.3012
IL-6 3.370 4.430 (1.12) 5.810 (1.22) 7.030 0.1596
IL-17E 5.296 (5.296) 5.296 (5.296) 8.406 (5.369) 15.84 (8.925) 0.6196
RAGE 36.40 (1.97) 18.13 (2.12)* 36.62 (1.18) 20.19 (2.98)*** <0.0001
Measurements were in ng/ml. Values are reported as mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 versus vehicle (one-way ANOVA
followed by Bonferroni post-hoc test). ND: not detected.
treatment with broad-spectrum Cannabis oil (0.1 mg/kg,
p.o.) during CUS protocol. Serum levels of GM-CSF were
signicantly reduced (p < 0.01) when compared to vehicle,
aer single-dose administration. Under continuous
treatment and compared to the vehicle group, serum
levels of VEGF were signicantly upregulated (p < 0.01),
and RAGE levels were signicantly downregulated (p <
0.0001). No statistically signicant dierences were found
for the other measurements. e level of IL-4 could not be
detected in the assay.
Discussion
e present results showed that broad-spectrum Cannabis
oil reduced the anxiety-like behavior triggered by the
acute restraint paradigm. e oil also reverted despair and
anhedonic-like behaviors triggered by unpredictable stress.
Mendonça de Assis, et al.
246 | Pharmaceutical Sciences, 2022, 28(2), 239-250
Importantly, cannabinoid-like eects were not observed at
the doses of 3 and 30 mg/kg of Cannabis oil during tetrad
assay. Lastly, oral administration of the oil downregulated
the serum levels of GM-CSF and RAGE, whereas VEGF
serum levels were found to be upregulated.
Cannabis sativa, a botanical plant with a millenary
history of medicinal use, and its phytocannabinoids have
been under investigation by their potential eects on a
wide range of conditions, including psychiatric illnesses.52
Stressful episodes inuence homeostasis by changing the
physiological and neurobehavioral proles throughout
adaptive processes. Stress is one of the external causes
of anxiety and depression, the two of the most common
psychiatric illnesses, and is known for playing critical roles
in the pathophysiology of these conditions.48,53 Interestingly,
the involvement of the ECS with emotional processing has
gained attention in the last few years, suggesting the use of
Cannabis sp. as a therapeutic alternative for the treatment
of symptoms related to PTSD.52
In the rst set of experiments, mice were treated with
broad-spectrum Cannabis oil and then submitted to the
acute restraint stress protocol. Substantial ndings have
supported this model as able to evoke PTSD-like constructs,
including depressive- and anxiety-like symptoms.26,38,53-56
Moreover, this protocol has been employed to screen the
therapeutic potential of drugs to manage mood symptoms
related to PTSD since behavioral changes in mice can be
monitored. Herein, we found that Cannabis oil (0.1 mg/
kg; p.o.), rich in CBD, showed an anxiolytic eect while
it did not change depressive-like symptoms. Previously,
Resstel et al.57 also demonstrated that a single dose of CBD
(10 mg/kg, i.p.) increased the percentage of open arm
entries in the EPMT of rats that have had their movements
restrained. is anxiolytic action was attributed to the
activation of 5-HT1A receptors. Yet, a recent study showed
that mice exposed to traumatic brain injury had the
anxiety- and depressive-like behaviors reestablished by a
commercially available 10% CBD oil.58 Contradicting these
data and the study of Sales et al.59, which demonstrated the
antidepressant-like eect of CBD (10 mg/kg, i.p.) on mice
in the FST, we found that Cannabis oil was ineective to
revert the depressive-like symptom in the FST. It is worth
mentioning that those studies evaluated depressant-like
symptoms triggered by other protocols as compared to
ours. Yet, we are comparing the results of isolated CBD
with a broad-spectrum oil, even though the pieces of
evidence suggest that there is a positive contribution from
the combination of phytocannabinoid and other molecules,
such as terpenes, also called as “entourage eect”.60
Otherwise, neuropsychiatric alterations related to
anxiety- and depressive-like behaviors are also described
as consequences of exposure to chronic unpredictable
stress.43,61,62 In the second set of experiments, dierent
types of stressors applied to mice for 14 days signicantly
altered the parameters related to latency to immobility
in the FST and grooming behavior in the ST. In rodents,
a reduced persistence of swimming and a sucrose
indierence are commonly associated with depressive-
like symptoms.39 Since we observed an improvement of
the anhedonia and depressive patterns, we could interpret
these data as a consequence of the treatment with broad-
spectrum Cannabis oil. Previously, isolated CBD (10 mg/
kg; i.p.) had exerted pro-hedonic eects on rats subjected
to chronic unpredictable mild stress by increasing their
sucrose preference.39 Still, other reports demonstrated that
CBD isolated (30 and 200 mg/kg) signicantly diminished
depressive-like behavior in mice during FST.63,64 Taken
together, one may conclude that Cannabis and Cannabis
derivatives reduce depressant-like behaviors in rodents in
a large range of dosages.
When evaluating the potential psychoactive eects of
cannabinoids, the tetrad assay is very useful to characterize
their biological activity. Mainly, the cannabinoid tetrad
reveals cannabimimetic eects related to those elicited by
CB1R agonist ∆9-THC.65 Our ndings showed no eects
of broad-spectrum Cannabis oil at 3 and 30 mg/kg in
the tetrad assay, conrming the analytical parameters of
quality attested by the supplier (CBD:Δ9-THC proportion
of 11:1 and total cannabinoids of 40.2%). CBD is well
known for its low anity by CB1R and as expected, it does
not activate the cannabinoid tetrad.66 Our ndings are in
accordance with these previous data since the Cannabis oil
we tested is rich in CBD, but it is not the only compound.
us, the psychoactive eects on mood-related symptoms
appear to be independent of CB1R activation, although
further experiments are needed to conrm this hypothesis.
Earlier experiments have suggested the psychoactive
action of phytocannabinoids, mainly CBD, throughout
the 5-HT1A signaling pathway.57,67 Besides, the pieces of
evidence suggesting the anti-inammatory properties
of Cannabis68,69 contributed to the increasing interest
in its therapeutic potential in mood disorders. Earlier
experiments have demonstrated that GM-CSF levels are
downregulated with phytocannabinoids treatment.70,71
We also found a signicant decrease of GM-CSF serum
levels following a single dose administration of broad-
spectrum Cannabis oil (0.1 mg/kg, p.o.). is chemokine
plays a critical role in regulating leukocyte counts,72 of
which are elevated in PTSD patients.73,74 Another nding
of our study was the signicant downregulation of RAGE
following continuous treatment with broad-spectrum
Cannabis oil (0.1 mg/kg, p.o.). To the light of our current
knowledge, there are no previous reports regarding the
inuence of Cannabis, or any isolated phytocannabinoid,
upon RAGE expression. is receptor is known for its
ability to recognize danger-associated molecular patterns
(DAMPs) that can be released on a larger scale because of
psychological and physical stress.75 A recent review showed
that several DAMPs, including the high mobility group
box-1 (HMGB-1), may trigger depressive-like behaviors in
the stress-induced depression model.76 HMGB-1 is a well-
known ligand of RAGE and, interestingly, a prospective
study had associated the high plasma levels of HMGB-1
with the more likely to develop PTSD.77 Taken together,
Cannabis Oil for Mood Disorders
Pharmaceutical Sciences, 2022, 28(2), 239-250 | 247
the accumulating data suggest the role of the HMGB-
1/RAGE signaling in mood processes, and the eects
observed in our study suggest that Cannabis may mediate
immunomodulatory eects through this cellular pathway.
Last, but not least, the continuous treatment with broad-
spectrum Cannabis oil (0.1 mg/kg, p.o.) signicantly
upregulated the serum levels of VEGF. In dierent cells
and tissues, VEGF plays key roles in physiologic vascular
homeostasis but is also associated with the molecular
pathogenesis of tumor growth and metastasis.78 Previously,
Wheal et al.79 showed the increase of the circulating levels
of VEGF in ZDF diabetic rats treated with CBD, while
Leishman et al.80 showed a signicant lowering of the
mRNA encoding for VEGF in mice following acute Δ9-
THC administration (3 mg/kg; i.p.). We are not aware of
any studies that have investigated the eects of Cannabis
oil, and not only isolated phytocannabinoids, on VEGF
levels in stress-induced mice, thus justifying additional
investigation.
Conclusion
In summary, our data showed the potential of broad-
spectrum Cannabis oil to change behavioral patterns related
to stress-induced anxiety- and depressive-like symptoms.
Importantly, the oil did not trigger psychomimetic eects
related to CB1R activation, as shown by the tetrad assay.
Moreover, our data suggest the potential of the oil to
regulate biomarkers involved with inammation and
angiogenesis, although further investigations are required
to conrm these hypotheses.
Ethical Issues
e experiments were approved by the Animal Ethics
Committee of the UFSC (CEUA-UFSC) under protocol
7176240920.
Acknowledgments
Grants from the Conselho Nacional de Desenvolvimento
Cientíco e Tecnológico (CNPq), Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES),
Fundação de Amparo à Pesquisa e Inovação do Estado de
Santa Catarina (FAPESC), Programa INCT-INOVAMED
(Grant no. 465430/2014-7) and Programa de Pós-
Graduação em Neurociências (PPG NEURO), all from
Brazil, supported this work. P.M.A. and E.G.F. are PhD
student on the Health and Neuroscience Program,
respectively, receiving grants from the CAPES. G.M.B. and
R.S.P. are graduate student receiving grant from the CNPq.
R.C.D. is recipient of a research productivity fellowship
from the CNPq. e authors are grateful to the ABRACE
association in Paraíba – Brazil for providing the broad-
spectrum CBD oil obtained from Cannabis sp. used in this
study.
Author Contributions
RCD, NRBR, MAFB, and RC contributed to the
conception and design of the experiments. MCG,
CENS, and FB contributed to the acquisition and
analysis of data. EGF, GMB, and RSP contributed to
the acquisition, analysis, and interpretation of data.
PMA contributed to the analysis and interpretation
of data; writing the manuscript with input from all
authors. RC and NRBR supervised the project and
revised it critically for important intellectual content.
Conict of Interest
e authors report no conicts of interest.
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