Peroral Exposure to Cannabis Sativa Ethanol Extract Caused Neuronal Degeneration and Astrogliosis in Wistar Rats’ Prefrontal Cortex

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.

Introduction

Cannabis sativa is the most often consumed illegal drug. Its prevalence has grown with the passage of time, and its eventual global legalization is drawing nearer. ¹ In light of these tendencies, there has been an uptick in research into the possible therapeutic effects and possible adverse side effects of particular phytocannabinoids derived from cannabis for a 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 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. 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 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)

Figure 1. Experimental Protocol Summary.

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.

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 assessment^{18, 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:

$$\text{Spontaneous alternation \%}=\frac{\text{Number of spontaneous alternation}}{\text{Total number of arm entries}-\text{2}}\times 100$$

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 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 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 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).

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.

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).

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.

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 Y-maze spontaneous alterations when experimental groups were compared with control.

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.

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 intracellular aggregation of nuclear materials were observed (Figures 5 and 6b, c, d.)

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.

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.

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 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.

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.

Discussion

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 content of the psychoactive compound, THC. ²³ The PFC is one 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 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 Cheer ³⁰ 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. ³⁵ ACh 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 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 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.

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).

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 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.

Informed Consent

This article complies with ICMJE guidelines.