Original Article

Peroral Exposure to Cannabis Sativa Ethanol Annals of Neurosciences

30(2) 84­–95, 2023
Extract Caused Neuronal Degeneration and © The Author(s) 2022
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https://doi.org/10.1177/09727531221120988
DOI: 10.1177/09727531221120988
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Olatunji Sunday Yinka1,2, Ogunnaike Philip Olubunmi1, Abijo Ayodeji Zabdiel1 ,

Owolabi Joshua Oladele1,5, Adelodun Stephen Taiye1, Adeoye Ayodele3,
Fasesan Oluwatoyin Adetutu4 , Olanrewaju John Afees1 and Adegbite Ademola Kayode1
Abstract
Background: Despite widespread concerns about its possible side effects, notably on the prefrontal cortex (PFC), which
mediates cognitive processes, the use of Cannabis sativa as a medicinal and recreational drug is expanding exponentially. This
study evaluated possible behavioral alterations, neurotransmitter levels, histological, and immunohistochemical changes in
the PFC of Wistar rats exposed to Cannabis sativa.
Purpose: To evaluate the effect of graded doses of Cannabis sativa on the PFC using behavioural, histological, and
immunohistochemical approaches.
Methods: Twenty-eight juvenile male Wistar rats weighing between 70 g and 100 g were procured and assigned into groups
A-D (n = 7 each). Group A served as control which received distilled water only as a placebo; rats in groups B, C, and D
which were the treatment groups were orally exposed to graded doses of Cannabis sativa (10 mg/kg, 50 mg/kg, and 100 mg/
kg, respectively). Rats in all experimental groups were exposed to Cannabis sativa for 21 days, followed by behavioral tests
using the open field test for locomotor, anxiety, and exploratory activities, while the Y-maze test was for spatial memory
assessment. Rats for biochemical analysis were cervically dislocated and rats for tissue processing were intracardially perfused
following neurobehavioral tests. Sequel to sacrifice, brain tissues were excised and prefrontal cortices were obtained for the
neurotransmitter (glutamate, acetylcholine, and dopamine) and enzymatic assay (Cytochrome C oxidase (CcO) and Glucose
6- Phosphate Dehydrogenase-G-6-PDH). Brain tissues were fixed in 10% Neutral Buffered Formalin (NBF) for histological
demonstration of the PFC cytoarchitecture using H&E and glial fibrillary acidic protein (GFAP) for astrocyte evaluation.
Results: Glutamate and dopamine levels were significantly increased (F = 24.44, P = .0132) in groups D, and B, C, and D,
respectively, compared to control; likewise, the activities of CcO and G-6-PDH were also significantly elevated (F = 96.28, P =
.0001) (F = 167.5, P = .0001) in groups C and D compared to the control. Cannabis sativa impaired locomotor activity and spatial
memory in B and D and D, respectively. All Cannabis sativa exposed groups demonstrated evidence of neurodegeneration in the
exposed groups; GFAP immunoexpression was evident in all groups with a marked increase in group D.
Conclusion: Cannabis sativa altered neurotransmitter levels, energy metabolism, locomotor, and exploratory activity, and
spatial working memory, with neuronal degeneration as well as reactive astrogliosis in the PFC.
Keywords
Prefrontal cortex, cannabis sativa, neurodegeneration, astrocytes
Received 05 February 2022; accepted 25 July 2022

Introduction 1Department of Anatomy, School of Basic Medical Sciences, Benjamin Carson

(Snr.) College of Medical and Health Sciences, Ilishan-Remo, Ogun State Nigeria
Cannabis sativa is the most often consumed illegal drug. Its 2Anatomy Department, Adventist School of Medicine of East-Central

prevalence has grown with the passage of time, and its Africa, Adventist University of Central Africa, Kigali, Rwanda
3Department of Education, School of Education and Humanities, Babcock
eventual global legalization is drawing nearer.1 In light of University, Ilisan-Remo, Ogun State, Nigeria
these tendencies, there has been an uptick in research into the 4Department of Psychiatry, Ben Carson School of Medicine, Babcock

possible therapeutic effects and possible adverse side effects University, Ilisan-Remo, Ogun State, Nigeria
of particular phytocannabinoids derived from cannabis for a 5Anatomy Department, Division of Basic Medical Sciences, University of

wide range of psychiatric and nonpsychiatric health
conditions.2 Tetrahydrocannabinol (THC) and cannabidiol
(CBD) are the two most common bioactive phytocannabinoids
in cannabis.3 It is undeniable that chronic exposure to or
abuse of this substance can cause dependency. Addiction has
Global Health Equity, Kigali, Rwanda
Corresponding author:
Abijo Ayodeji Zabdiel, Neurobiology Unit, Department of Anatomy,
School of Basic Medical Sciences, Benjamin Carson (Snr.) College of
Medical and Health Sciences, Ilishan-Remo, Ogun State Nigeria.
E-mails: abijoayodeji@gmail.com, abijoa@babcock.edu.ng

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Yinka et al.
been linked to a wide range of factors, including those in the
individual's physical environment, social environment, and
emotional/psychological state. Cannabis is made from the
dried flower buds, leaves, stems, and seeds of the Cannabis
sativa Linn plant (Family Cannabidaceae). It has evolved
from a therapeutic practice to a leisure pursuit. There is
evidence that cannabinoids exert their effects via binding to
certain receptors. To date, the two most important subtypes of
cannabinoid receptors, CB1 and CB2 receptors, have been
discovered. The brain and spinal cord have more CB1
receptors than any other part of the body, while peripheral
tissues have more CB2 receptors.3,4
A plethora of studies have researched into how THC and
CBD are related, and how different neurotransmitter
pathways influence executive brain functions through their
interactions with the prefrontal cortex.2,5 One of the most
important roles of the prefrontal cortex is cognitive
flexibility, which allows behavior to change in response to
changes in the environment and demands. 6,7 THC can
impair memory in both humans and animals, but that the
effects are by affecting short and long term memories.8
According to research by Solowij and Battisti, chronic use
of Cannabis sativa is linked to a range of cognitive
impairments. These problems are identical to those seen
after only a brief exposure and manifest themselves most
noticeably in the areas of attention and working memory.9
Cannabinoid CB1 receptors are highly concentrated in the
striatum, amygdala, cerebellum, prefrontal cortex, and
hippocampus. THC has also been shown to cause structural
and functional changes in the brain that are proportional to
the amount consumed. 10,11,12
The prefrontal cortex has a role in executive function.
Knowing what is good and bad, better and worse, the same
and different, and what the future holds as a result of your
present activities is a key part of being able to think critically.
Group A (Control) Group B
n=7 n=7
Cannabis sativa
Distilled water
Low dose (10
(2 mL/kg, oral)
mg/kg,)
Treatments in groups A–D lasted for 21 days period and was orograstrically
Figure 1. Experimental Protocol Summary.
85
It also entails exerting effort toward an objective, making
plans based on past performance, and enforcing order in
social settings (the ability to suppress urges that, if not
suppressed, could lead to socially unacceptable outcomes).
The prefrontal cortex contributes to the learning of hard and
fast guidelines. Higher-level abstract rule learning is
supported by more front frontal regions along the rostrocaudal
axis of the brain.13 THC and CBD both have different
pharmacological effects, but they also have different
psychological effects. THC mostly acts as a partial agonist at
the cannabinoid receptor 1 (CB1) (CB1 R).14 However,
CBD's effects on receptors are more widespread.15
Delta-9-THC and CBD have diverse pharmacological and
state-dependent influences on cognitive and emotional
processing, as measured by neurobehavioral tests of factors
including working memory, anxiety, and social interaction.16
Despite this, there is no research that describes the effects
of subchronic Cannabis sativa exposure on PFC
cytoarchitecture, astrocyte morphology, neurotransmitter
level, enzyme activity, locomotor, and spatial working
memory. As a result, we hope to learn a great deal more about
this subject through this study.
Methods
Cannabis sativa Acquisition
Cannabis sativa L. plant leaves were acquired from National
Drug Law Enforcement Agency (NDLEA), Abeokuta, Ogun
State Command.
Rat Care and Management
Twenty-eight juvenile Wistar rats (Rattus norvegicus)
weighing between 70 g and 100 g were utilized for the
Group C Group D
n=7 n=7
Cannabis sativa Cannabis sativa
Medium dose High dose (100
(50 mg/kg) mg/kg)
86
study. The rats were randomly assigned into 4 groups
(A-D) (n = 7 each). Group A was the control and received
2 mL/kg distilled water while groups B, C, and D were
administered 10 mg/kg, 50 mg/kg, and 100 mg/kg Cannabis
sativa extract. The doses of Cannabis sativa were based on
the modification of the study carried out by Owolabi et al.,
and likewise the pilot study from our lab.17 Experimental
protocol of the study has been represented in Figure 1.
Rats in all groups were orally gavaged the extract
preparation and distilled water via the use of an oral
cannula. The rats were housed in Babcock University
animal holding in clean plastic cages, well-ventilated
environment, and room temperature. Rats in all groups
were fed with standard laboratory rat chow and allowed
free access to clean water. The rats were kept in the natural
photoperiodic condition of 2 hours of light and 12 hours of
darkness (12:12 hour dark/light cycle)
Cannabis sativa Preparation
Following the acquisition of Cannabis sativa from the
National Drug Law Enforcement Agency. Cannabis
sativa extract was prepared from the dried leaves of the plant.
The leaves were first identified by a taxonomist at the
Department of Botany. A voucher specimen was deposited at
the herbarium for reference purposes (BU-192030). The
leaves were air-dried until a constant weight was attained.
The dried leaves were ground to powder with a mechanical
grinder (Warring, Commercial and Torrington, CT). The
powdered leaves were then percolated in ethanol for 24 hours
while being agitated by a magnetic stirrer. A Whatmann No. 1
filter paper (Whatmann, Middlesex, UK) was used to filter
the extract. Next, a rotary vacuum evaporator was used to
concentrate the filtrate under vacuum. When not in use, the
extract was kept in a sealed, airtight container at room
temperature. The formula for determining the percentage
yield was W2/W1×100. The initial weight of the Cannabis
sativa leaves before the procedure was W1, and the weight of
the extracted material was W2 (final weight). As much as
10.15 g was obtained as the percentage yield through the
process of preparation.
Number of spontaneous alternation
Spontaneous alternation %  100
Total number of arm entries  2
Open Field Test
This was utilized in experimental Wistar rats to assess
locomotor and exploratory behaviors. Blanchard et al.’s work
was modified for this procedure.20 Seven rats from each group
were chosen and placed in the center of the device, a white
open box (72×72×36 cm) with black dividing lines, to assess
locomotor and exploratory activity. Ethanol (70%) was used to
clean both apparatuses in between tests to prevent olfactory
cues. While exploring, the animals were recorded with a
camcorder (DNE webcam, Porto Alegre, Brazil) placed at the
Annals of Neurosciences 30(2)
Preparation of Stock Solution
The stock solutions of the 10 mg/kg, 50 mg/kg, and 100 mg/
kg of Cannabis sativa leaf extract were prepared by dissolving
0.1 g, 0.5 g, and 1 g of the resultant yield each in 20 mL
distilled water. The solutions were freshly prepared each day
and then refrigerated. Volume administration was by
administration of 2 mL/kg/bw from the prepared Cannabis
sativa extract stock solutions.
Neurobehavior Assessment
The neurobehavioral assessment was carried out following
the last day of Cannabis sativa administration (day 22). All
neurobehavioral tests were recorded using a digital camcorder
and were scored later by two independent trained observers.
The following tests were carried out.
Y Maze Spontaneous Alternation Test
The procedure as carried out by Kraeuter et al. and modified
by Adelodun et al. was utilized for memory assessment18,19
This was a test designed to evaluate mice’ capacity for
learning and recalling spatial information. Three opaque
wooden arms formed a Y at a 120° angle from one another,
where the tests were conducted. The rats were introduced
at the center and allowed to move freely exploring the
three arms. The arms of the Y maze were labeled as A, B,
and C. The maze was cleaned with alcohol and allowed to
dry before subsequent use. The position was maintained
throughout the Y maze test of all the rats. Each rat was
taken and placed in one of the arms (same arm for all rats)
facing the center. After 5 minutes of quiet exploration,
each rat was released into the open arms of the Y maze.
The numbers of entries into each arm (A, B, and C) and the
number of spontaneous alternations were recorded. When
an experiment was over, the rats were returned to their
original groups.
The percentage of spontaneous alternation was calculated
using the following formula:
top and the video was analyzed by independent observers blind
to the procedure. The following parameters were scored: lines
crossed, center square entries, rearing, and freezing. After each
test, each rat was placed back in their respective groups.
Rat Sacrifice and Histological Analysis
Cervical dislocation and intracardiac perfusion were used to
euthanize the rats after their neurobehavioral performance
was evaluated. Exact weights were obtained using an AB204
Yinka et al.
Mettler Toledo weighing balance after the brains were
carefully removed with bone forceps, blotted dry, and then
weighed. Immersion in Neutral Buffered Formalin (NBF) at a
concentration of 10% was used to preserve the brains. The
prefrontal cortical regions of the brain were collected as 1
mm thick coronal slices and processed for standard paraffin
embedding. The general histoarchitectural organization of the
prefrontal cortex was demonstrated by staining sections with
H&E.
Immunohistochemistry
After transferring paraffin blocks of prefrontal cortex
sections to glass slides at a thickness of 5 μ, the slides were
heated on a hot plate set to 70°C for at least an hour. Following
a brief wash in water, sections were put through 2 xylene
changes, 3 alcohol changes (in progressively weaker
concentrations), and then another brief wash in water. Tissue
sections were heated in a citric acid solution (PH 6.0) for 25
minutes to retrieve their antigens. Tissue sections were
immersed in cold water to replace the heated citric acid
solution, a process that took at least 5 minutes. The sections
were pretreated for 15 minutes with a coating of 3% hydrogen
peroxide (H2O2) to inhibit peroxidase activity. After 15
minutes of protein blocking with Avidin, sections were rinsed
in Phosphate Buffered Saline (PBS).
After being rinsed in PBS, tissue sections were treated
with biotin to block the tissue’s own endogenous biotin for 15
minutes. Sections were washed in PBS and then treated with
a 1:100 dilution of the primary antibody (anti-GFAP). PBS
was used to remove any remaining antibody, and then a
secondary antibody (LINK) was applied to the section for 15
minutes. After 15 minutes, the sections were washed again
and treated with the labeling enzyme horseradish peroxidase.
Spectrophotometry for Enzyme Assay
Rat prefrontal cortex (PFC) tissues were analyzed by
spectrophotometry for cytochrome C oxidase (Cco) and G-6-
PDH activity determination. Each assay kit was purchased
from Cell Signaling Technologies, located in Danvers, USA.
Rat brains from all groups were dissected into 0.25 M sucrose
(Sigma) at 4°C, and then weighed before being pulverized in
an automatic homogenizer. In order to isolate organelle
fragments from PFC lysates, the lysates were centrifuged for
10 minutes at 12,000 rpm in a microfuge. The aspirated
supernatants were stored in a glass cuvette with a clear label
and ice. The assay of CcO and G-6-PDH activities was
performed in accordance with the assay kit manufacturer’s
instructions.
Neurotransmitter Assay
Brain sections were homogenized. Dopamine level
determination was by the modified method of Atack.21
87
Glutamate and acetylcholine (ACh) concentrations were
determined by the manufacturer’s instructions (Sigma,
Aldrich, 2016).22 The homogenates were then centrifuged
and the supernatant was decanted and levels of dopamine,
ACh, and glutamate were determined using these methods.
Statistics
The study’s findings were analyzed using the analysis of
variance and the Newman–Keuls posthoc test for multiple
comparisons in Graph Pad Prism® software (Version 6.1).
The threshold for significance was set at P < .05. The results
were depicted using bar charts with error bars displaying the
mean and standard error of the mean (SEM).
Results
Effect of Cannabis sativa on Neurotransmitters
Level
Following Cannabis administration for 21 days, levels of the
neurotransmitters glutamate, dopamine, and ACh were
assessed in the PFC. There were significantly higher
(F = 90.39, P = .0001) levels of glutamate in group D (100
mg/kg of Cannabis sativa) relative to control. Groups B (10
mg/kg of Cannabis sativa), C (50 mg/kg of Cannabis sativa),
and D showed significantly higher (F = 24.44, P = .0132)
levels of dopamine when compared to the control group,
while there was no significant difference in ACh level when
compared to the control group (Figures 2a, b, and c).
Effect of Cannabis sativa on Enzymatic Assays
Results obtained from the assessment of the activity of the
enzyme, CcO, in the PFC showed a marked increase (F =
96.28, P = .0001) in the activity of CcO in groups C (50 mg/
kg of Cannabis sativa) and D (100 mg/kg of Cannabis sativa)
(Figure 3a) when compared to the control group. Glucose 6-
Phosphate Dehydrogenase (G-6-PDH) activity (F = 167.5,
P = .0001) also followed similar fashion (Figure 3b).
Effect of Cannabis sativa on Neurobehavior
Following treatment with Cannabis for 21 days after which
locomotory activity, exploratory, and behavioral indices were
assessed on the 22nd day; results revealed a significant
reduction (F = 167.5, P = .0001) in the number of lines crossed
in groups B (10 mg/kg of Cannabis sativa) and D (100 mg/kg
of Cannabis sativa) when compared to the control and
significantly increased (F = 6.033, P = .0210) rearing frequency
in group C (50 mg/kg of Cannabis sativa) when compared with
the control, while there was no significant difference in freezing
and center square entries (Figures 4a, b, c, d). There was also
no significant difference in Y-maze total arm entries and
88 Annals of Neurosciences 30(2)

Figure 2. Bar chart represents glutamate, dopamine, and acetylcholine levels (A, B, C, respectively) in control (A) and experimental
groups exposed to Cannabis sativa (B—10 mg/kg, C—50 mg/kg, and D—100 mg/kg). Values are expressed as mean ±SEM.
Note: Statistically significant relative to control (P < .05). n = 7.

Figure 3. Bar chart represents cytochrome: c oxidase (CcO) and glucose 6-phosphate dehydrogenase (G-6-PDH) activity in control and
experimental groups exposed to Cannabis sativa (B—10 mg/kg, C—50 mg/kg, and D—100 mg/kg). Values are expressed as mean ±SEM.
Notes: Statistically significant relative to control (P < .05). n = 7.
Yinka et al. 89

Y-maze spontaneous alterations when experimental groups intracellular aggregation of nuclear materials were observed
were compared with control. (Figures 5 and 6b, c, d.)

Histological Assessment of the PFC

The assessment of the prefrontal cortices using the routine
Hematoxylin and Eosin stain showed a regular neuronal
population with normal cytoarchitecture, and well-
distinguished or delineated prefrontal cortical layers in the
control group (Figure 5A). However, in the groups
administered graded Cannabis sativa, several features of
neuronal degeneration are noticeable in the neuropil. Neurons
which are pyknotic and karyorrhectic in morphology with
Effect of Cannabis sativa on GFAP
Immunoexpression
Results revealed glial fibrillary acidic protein (GFAP)
immunoreactivity in all groups. The expression of GFAP was
in a dose-dependent manner from groups B to C and D. PFC
GFAP immunoreactivity was more in group D (100 mg/kg of
Cannabis sativa) administered the highest dose when
compared to the control group (Figures 7 and 8).

Figure 4. Bar chart represents number of lines crossed: (A) rearing frequency, (B) freezing time, (C) and center square entries, (D) in
control and experimental groups exposed to Cannabis sativa (B—10 mg/kg, C—50 mg/kg, and D—100 mg/kg). Values are expressed as
mean ±SEM.
Notes: Statistically significant relative to control (P < .05). n = 7.

Figure 5. Representative Photomicrographs of the histoarchitecture of the PFC shows regular neuronal population, normal
cytoarchitecture with regular neurons (A). Neurons with degenerative features scatters within neuropil of rats treated with graded
doses; 10 mg/kg (B), 50 mg/kg (C) and 100 mg/kg (D) of Cannabis sativa and are karyorrhectic in morphology. Scale bars—50 µm.
90 Annals of Neurosciences 30(2)

Figure 6. Representative Photomicrographs of the histoarchitecture of the PFC shows normal cytoarchitecture with intact neuronal
morphology (A). Neurons with degenerative features are evident by intensely stained eosinophilic cytoplasm, cytoplasmic fragmentation
and intracellular nuclear material aggregation in groups; 10 mg/kg (B), 50 mg/kg (C), and 100 mg/kg (D) treated with Cannabis sativa.
Scale bars—200 µm.

Figure 7. Immunohistochemical labelling of astrocytes (GFAP) in PFC sections of control and treated rats. Immunopositive cells within
prefrontal sections of control and rats treated with 10mg/kg (B), 50mg/kg (C) and 100mg/kg (D) Cannabis sativa are sparsely and evenly
expressed within the cortex, and shows identical morphological patterns. Evident in groups A, B, C are unreactive/resting astrocytes
while reactive astrocytes with modified processes appear in clusters in group D. Scale bars-50µm.
Yinka et al. 91

Figure 8. Immunohistochemical labelling of astrocytes (GFAP) in PFC sections of control and treated rats. Immunopositive cells within
prefrontal sections of control and rats treated with 10mg/kg (B), 50mg/kg (C) and 100mg/kg (D) Cannabis sativa are sparsely and evenly
expressed within the cortex, and shows identical morphological patterns in groups A, B and C. Group D showed more GFAP reactive
astrocytes with modified processes in clusters. Scale bars-200µm.

Figure 9. Bar chart represents Y-Maze total arm entries (A) and Y-Maze spontaneous alternation (B) in control and experimental
groups exposed to Cannabis sativa (B—10 mg/kg, C—50 mg/kg, and D—100 mg/kg). Values are expressed as mean ±SEM (n = 7).

Discussion content of the psychoactive compound, THC.23 The PFC is one

This study assessed the PFC cytoarchitecture, neurotransmitters,
and enzymes following exposure to Cannabis sativa. One of
the earliest plants with medicinal and recreational uses,
Cannabis sativa is a commonly abused plant due to its high
of the cortical regions to undergo phylogenetic and ontogenetic
development. It is rich in neurotransmitter systems such as
dopamine and cholinergic system. It has reciprocal and profuse
connections with various subcortical structures. It is highly
important in the control of various executive brain functions
92
through its various regions.24 Till date, there are several
conflicting reports on the role of Cannabis sativa.25 While
some have reported beneficial effects, the deleterious effects of
Cannabis sativa are also established; however, there is a need
to examine the state of the PFC, its associated neurotransmitters,
cognitive functions, learning, and memory following
subchronic exposure as this might help to unravel some
manifestations following exposure.
In the central nervous system (CNS) of vertebrates,
glutamate predominates as an excitatory neurotransmitter.26
The glutamate concentration in group D was significantly
greater than in the control group. Glutamate levels were
found to be higher in treated groups compared to controls,
correlating with findings by Owolabi et al.17 All excitatory
function in the vertebrate brain utilizes glutamate.
Glutamate is essential for synaptic plasticity and hence
plays a part in cognitive processes such as learning and
memory.27 When released in large amounts, glutamate is
excitotoxic and can damage neurons, accelerating their
degeneration. Dopamine is a neurotransmitter thought to
play a greater role in the regulation of arousal and drive
than in pleasure.28 Dopamine (DA) is a neurotransmitter
involved in a wide variety of cognitive and behavioral
processes, including but not limited to alertness,
vasodilation, executive functions, motor control,
motivation, working memory, arousal, reward, and lower-
level functions including sexual fulfillment.28 The ventral
tegmental area cell bodies provide the primary dopaminergic
input to the PFC via the mesocortical DA projection
(VTA).29 All three experimental groups (B, C, and D) had
significantly higher dopamine levels compared to the
control group, supporting the conclusion drawn by Oleson
and Cheer30 and Michel et al.31 that the cannabinoid system
is responsible for this effect.31,32 The involvement of
dopamine in working memory has a complex mechanism
although commonly accepted. Higher or lower levels of
dopaminergic activity impairing working memory have
been established.33 Cannabinoid-induced working memory
impairment might be a result of increased mesocortical
dopaminergic neuronal activity.34
To stimulate muscular contraction, the nervous system’s
motor neurons produce ACh from Acetyl-CoA and choline.
ACh is the parasympathetic nervous system’s final product
and an intrinsic transmitter for the sympathetic nervous
system.27 ACh is both a neurotransmitter and a neuromodulator
in the brain.35ACh is involved in the activation of all voluntary
skeletal muscular action, as well as in the regulation of
smooth and cardiac muscle, arousal, attention, and memory.36
Memory, both working and long-term, may be supported and
regulated by the neurotransmitter ACh.37 ACh is associated
with an increase in glutamatergic synaptic neurotransmission
and the functional support of synaptic plasticity. Although,
the level of ACh was not significantly different from the
control in the study, there was an observable increase in the
level of ACh suggesting some other possible mechanism of
Annals of Neurosciences 30(2)
Cannabis sativa involvement in increasing ACh level. In a
study by Tripathi et al., the effect of several cannabinoids was
determined on mouse brain ACh levels and on ACh turnover
within the cortex, hippocampus, striatum, midbrain, and
medulla-pons. Reportedly, Delta 9-THC (30 mg/kg) caused a
significant elevation of ACh in all5 brain areas.37 The reason
why Ach levels was not significantly increased or decreased
in the present study could be the route of exposure, period of
exposure, or possibly the dose administered. Alteration in the
neurotransmitter system may underlie some of the cognitive
deficits observed in this study.
Groups C and D, which received medium and high doses
of Cannabis sativa, respectively, showed a dose-dependent
increase in the activity of the enzyme G6PD in the PFC,
suggesting a potential role for this enzyme in cellular death.
The enzyme G6PD helps regulate reactive oxygen species
(ROS) production and inflammation. Maintaining an
appropriate G6PD concentration is critical for proper cellular
function. This enzyme’s levels are associated with the risk of
cellular damage from oxidative stress, and both high and low
levels have been implicated in cellular damages (Stanton,
2012).38 When it comes to important metabolic pathways
such as lipid, fatty acid, or cholesterol production, G6PD is a
significant NADPH-producing enzyme. Neurodegenerative
diseases, arthritis, muscular dystrophy, vascular damage, and
hormonal disarray are all caused by dysregulation in
this enzyme leading to uncontrolled inflammation and
metabolic stress.34 Since NAPDH is a reducing agent, it plays
a direct role in controlling oxidative molecules, which in turn
play a vital role in regulating metabolic stress and
inflammation.39 NADPH is necessary for the reduction of
oxidized-glutathione in the cellular antioxidant system;
without it, the generation of ROS is not inhibited, leading to
cellular oxidative damage.40
Results obtained from CcO activity in the PFC are in
tandem with G6PD showing a marked increase in groups
administered with medium and high doses and the ability of
Cannabis sativa to alter the levels of this complex which is a
basis for most neurodegenerative conditions. CcO is a
transmembrane protein complex that is large and located in
the mitochondria. It is the last enzyme in the respiratory
electron transport chain. The CNS is one of the systems with
high energy demand. CcO dysfunction predominantly
interferes with tissues with high energy demands (brain,
heart, muscle).41
Neurodegenerative disorders including Alzheimer’s and
Parkinson’s have been linked to CcO dysregulation.40 CcO is
the rate-limiting enzyme of the respiratory chain in many
tissues and cell types, highlighting its importance as a hub for
controlling energy metabolism and oxidative stress. CcO
isoform IV-2 is expressed at higher levels and swapped for
CcO IV-1 in the enzyme complex under hypoxic, toxic, and
degenerative circumstances. By switching to the CcO IV
isoform, the allosteric Adenosine Tri Phosphate (ATP)
feedback inhibition of CcO is removed, and so the ability to
Yinka et al.
sense energy levels is lost. There is a possibility that this
could raise CcO activity, which in turn would lead to higher
ATP levels in brain cells regardless of the energy state of the
cell. Moreover, there is an uptick in ROS generation, which
may contribute to cell death.41
The neurobehavioral assessments conducted following the
administration of Cannabis sativa showed that there was no
significant difference in freezing and center square entries,
while there was a significant difference in the number of lines
crossed when groups B and D were compared to the control.
Rearing frequency in group C was also relatively different
from the control. The changes observed in the decline in the
number of lines cross suggests the role of cannabis in
influencing locomotor activity and also the increase in rearing
frequency directly points to the role of cannabis in initiating
anxiety-like behaviors. Y-Maze (Figure 9) test was used to
test for cognition, spatial learning, and memory assessment in
rats exposed to Cannabis sativa. There was no significant
difference in the total arm entries and spontaneous alteration
across all groups, but there was an observable increase in
group C when compared with control.
Though cannabis has been used for medical purposes due
to its antioxidant, anticonvulsant, anti-inflammatory, and
neuroprotective properties, its adverse consequences should
not be underestimated.42
Despite reported claims of CBD, a nonpsychoactive
constituent of Cannabis sativa having some neuroprotective
ability, inhibiting neurodegeneration, and as a promising
agent in several neurodegenerative diseases,43,44 Results
from this study on the treatment of rats with cannabis
showed regular neuronal population, normal cytoarchitecture
with regular neuronal morphology in the control rats, while
there was extensive neuronal degeneration evidenced by
neurons with intensely stained eosinophilic cytoplasm,
karyorrhexis, and intracellular aggregation of nuclear
materials in treated groups. The exact mechanism by which
Cannabis sativa induces neuronal degeneration is not
completely understood but could be as a result of its
metabolite in causing the release of ROS or comprising
mitochondrial function leading to neuronal degeneration.
THC from Cannabis sativa has been identified to induce
brain mitochondrial respiratory chain dysfunction and
increases oxidative stress.45 This has been proposed as a
potential mechanism involved in cannabis-related stroke.
Another possible mechanism could be by reactive
astrogliosis. Astrocytes are in close proximity with neurons
and have a supportive role in the CNS aside from the other
numerous functions served. Assault to the CNS leads to
astrocytic reactivity or through release of excitotoxic
glutamate causing neuronal degeneration.46
The astrocytes’ primary intermediate filament (IF) protein
is GFAP. Increased GFAP expression is indicative of reactive
gliosis. Results obtained from this study on increased GFAP
immunoreactivity in all groups exposed orally to graded
93
doses of Cannabis sativa. Immunoreactivity of astrocytes to
GFAP was in a dose-dependent manner from the lowest to the
highest dose. GFAP immunoreactivity was more pronounced
in the group that received the highest dose of Cannabis sativa
when compared to the control group. There’s possibility that
the mechanism of neuronal degeneration may be by reactive
astrogliosis.
Conclusion
This study shows alteration in several neurotransmitters
mediating cognitive functions and spatial working memory,
and dysregulation in CcO and G6PDH involved in cellular
homeostasis which resulted in neuronal degeneration which
could also be secondary to astrogliosis. Despite the
documented neuroprotective efficacy of Cannabis sativa in a
variety of diseases, worthy of note is that it possesses the
potentiality to result in PFC neurodegeneration causing a
dysfunction in cognitive abilities.
Authors’ Contribution
OSY conducted the experiments, performed biochemical
investigations, and assisted in the writing of the manuscript.
OPO designed the study. He had a lead role in drafting the
article and reviewed the final manuscript. OJO played an
active role in the arrangement of resources and served as the
project’s supervisor. AST and AA performed statistical
analysis and contributed to the interpretation of the results.
FOA and OJA performed the immunohistochemical and
histological studies, interpreted the immunohistochemical
results with the scale bar, and also reviewed the manuscript.
AA and AAZ prepared figures and assisted in the writing of
the manuscript.
Statement of Ethics
Babcock University’s experimental animal holdings housed
the study rats in accordance with guidelines for the care and
use of animals in research and teaching put in place by the
institute of laboratory animal resources of the National
Research Council of the Department of Health and Human
Services (DHHS), publication number NIH86-23, 1885.
Babcock University Health Research Ethical Committee
(BUHREC) permission for the study was acquired; the study
was given the BUHREC number 084/19.
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with
respect to the research, authorship, and/or publication of this
article.
94
Funding
The authors received no financial support for the research,
authorship, and/or publication of this article.
Informed Consent
This article complies with ICMJE guidelines.
ORCID iDs
Abijo Ayodeji Zabdiel https://orcid.org/0000-0002-9187-
9908
Fasesan Oluwatoyin Adetutu https://orcid.org/0000-0002-
6138-0997
Olanrewaju John Afees https://orcid.org/0000-0002-9540-
4117
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