Curr Neurobiol 2020; 11(2): 37-47 ISSN 0975-9042

Garcinia kola Mitigates 3, 4-methylenedioxymethamphetamine- induced Derangement in Cellular and Molecular Characterization in the Cerebellum of Wistar rat Owolabi Joshua Oladele, Okoro Iheanyichukwu, Adelodun Stephen Taiye, Olatunji Sunday Yinka, Olanrewaju John Afeez, Fabiyi Oluseyi

Department of Anatomy, Ben Carson School of Medicine, Babcock University, Ilisan Remo, Ogun State Abstract Background: 3, 4-methylenedioxymethamphetamine (MDMA) causes neurological disorders. kola (GK) has neuroprotective property. We investigated the neuroprotective roles of GK on MDMA-induced cerebellar damage. Method: Sixty male Wistar rats (120 - 160g), 5 months old were grouped into six (n =10). Rats were fed with normal chow and water ad libitum. Group A was given distilled water, B and C received 100mg/kg and 200mg/kg GK respectively while D received 20mg/kg MDMA. Groups E and F received 100mg/kg and 200mg/kg GK respectively (pre-treatment), followed by intermittent 20mg/kg MDMA administration. All administrations were via oral route for 21days. Food, water intake, body temperature and weight were recorded. Rats were sacrificed and the brains were excised and used for assays. One-way ANOVA was used to analyze data followed by Student Neuman-Keauls test. Graph pad Prism 5 was used for analysis, p-values ≤ 0.05 were considered statistically significant. Results: Purkinje cells reduced in number and glial GFAP expression was increased. Serotonin, dopamine and glutamate levels showed no differences. MDMA treatment increased cytochrome c oxidase and glucose-6-phosphate dehydrogenase levels. Distance travelled was reduced while number of turns and foot slips were increased (p < 0.05). Weight loss and increase relative brain weight were observed (p < 0.05). It also increased the water intake and body temperature significantly. Conclusion: GK protected the cerebellum from MDMA toxicity in a dose-dependent manner through a conglomeration of evidences which include: Purkinje cells preservation, normalizing erratic gaits and acting as anti-diuretics. GK does not possess thermoregulatory function.

Keywords: cerebellar damage, garcinia kola, MDMA, neurotoxicity, stereotypic movement, wistar rats.

Introduction which is manifested as muscular tension, jaw clenching, tooth grinding (bruxism) and constant restless movement of the legs MDMA, otherwise known as Ecstasy, Adam, Lover’s speed, [8-10]. The increased muscle activity, together with a direct Superman etc. is one of the most commonly abused synthetic action of the drug on the thermoregulatory system in the brain drugs presently. The prevalence of its use in the recent time [11], leads to hyperthermia. Stiffness and pain in the lower-back among university students has been reported to be as high as and limb muscles are very common complaints during the first 13 to 39% in the U.S and United Kingdom [1-2]. Before now, 2-3 days after the use of MDMA. Users of MDMA from the mortalities and major adverse events due to recreational use of onset take the drug in pills or powder forms but there is now MDMA alone were uncommon and rarely reported [3]. However, a shift from those forms to the crystalline form due to its high the number of death and serious adverse effects of MDMA on purity. For example, the Ecstasy and Related Drugs Reporting users is on the increase in the recent time [4-5]. It has been System (EDRS) found that about 60 per cent of the users took postulated that life-threatening serotonin [5-hydroxytryptamine ecstasy in a high-purity crystal form. And they hypothesized (5-HT)] syndrome might contribute to mortalities and major those crystal MDMA users present riskier patterns of drug use adverse outcomes [6]. Ecstasy exposure has adverse effects on and lower health outcomes compared to non-crystal users. This new shift is the main reason for our choice of crystal forms of many physical functions even when taken in moderate doses for ecstasy for current this investigation [12]. the recreational purposes just as amphetamines do [7]. Because the basic action of the amphetamines involves increased arousal Cerebellum helps in the control of movement especially the and alertness, it is usually accompanied by an increase in tension, limbs. Although this structure is not necessary for the initiation 37 Curr Neurobiol 2020 Volume 11 Issue 2 Garcinia kola Mitigates 3, 4-methylenedioxymethamphetamine-induced Derangement in Cellular and Molecular Characterization in the Cerebellum of Wistar rat of motion, movements become erratic and distorted during known weight (250 g) of the powder was extracted in 1000 ml cerebellar damage due to toxicity insults on the movement of ethanol for 72 hours at room temperature. The extract was circuit [13]. Neurodegenerative diseases such as Alzheimer’s, filtered with Whatman No. 1 filter paper (Maidstone, UK) and Parkinson’s, Huntington’s, stroke and others are usually a the resulting filtrate concentrated in a Rotary Evaporator. The consequence of excess production of oxygen radicals from mixture was further transferred into steam bath where it was oxidative stress [14]. MDMA is known to induce neurotoxicity evaporated to give the required brownish-black residue. The in animal models, by the production of reactive species thus residue was stored in a desiccator until used. resulting in oxidative stress [15-16]. Chronic toxicity of MDMA Phytochemical screening: Basic phytochemical screening involves principally the brain, revealed through cerebral to detect the presence of alkaloids, saponins, tannins, flavonoids, imaging from previous studies [17-18] with consequences in steroids, anthraquinones, cardiac glycosides and carbohydrates memory loss especially short-term memory [19]. was carried out by adopting previous methods [27-31] (Table 1). Garcinia kola (GK) is a medium sized tree, ubiquitously Acute toxicity study (LD50) : The method described by found in West and Central African countries including Lorke [32] was adopted to determine the lethal dose (LD50). Nigeria, , and among others [20]. Almost every part of the contains phytochemicals Animal care and management that are reputed for their medicinal importance [21]. Among 5 months old sixty (60) male Wistar rats weighing between the phytochemicals found in GK seeds are tannins, saponins, 120 - 160 g were bred in the animal house of Babcock University alkaloids, cardiac glycosides and flavonoids (kolaflavone and Ilisan-Remo Ogun State, and used for this research. The rats 2 hydroxyflavonoids) [22]. Studies have shown that seeds of were grouped into six (A-F) n = 10. Rats were kept in a well GK possess anti-hepatotoxic, antioxidant [23], hypoglycaemic, ventilated standard housing conditions (temperature: 28-31°C; aphrodisiac, anti-cancer, anti-histamine, and antimicrobial photoperiod: 12 hours; humidity: 50-55%) in clean plastic cages properties among others [24]. Kolaviron (isolate of GK) has to acclimatise for one week. Ethical approval was obtained from specially been reported to elicit strong antioxidant activity, the Health Research Ethics Committee, Babcock University for in both in vivo and in vitro experimental models as seen in this research with a reference number (BUHREC-716/19). Rats its neuronal protection role against toxic substances [25]. A in all groups were fed with normal laboratory chow (Premier recent study has shown kolaviron to protect the neurons against Feed Mill Co. Ltd., Ibadan, Nigeria) and had access to water gamma radiation-induced oxidative stress in the brain of rats ad libitum. Group A served as control which was given distilled [26]. However, there is sparse report of the neuroprotective water. Groups B and C were given 100mg/kg GK and 200mg/ effect of GK on the cerebellum of Wistar rat against crystal kg GK respectively while group D received 20mg/kg of MDMA form of MDMA-induced neurotoxicity, hence, this study. This only. Groups E and F were also pre-treated with 100mg/kg GK research looked solely into the effect of crystal form of ecstasy and 200mg/kg GK respectively followed by 20mg/kg MDMA (at a dose of 20mg/kg given intermittently for the period of 21 administration every other day. All administrations were done days) on the cerebellum and the role of ethanolic extract of GK. through oral route for the period of 21days. The body weight, water-intake and food consumption of the rats were measured Materials and method throughout the period of treatment. Chemicals procurement MDMA stock preparation Crystal MDMA, ethanol (100%) as well as rabbit polyclonal 1g of crystalline MDMA was measured using sensitive anti-glial fibrillary acidic protein (GFAP) were purchased weighing balance (METTLER TOLEDO, China) in a cool room from Sigma Aldrich (St. Louis, MO, USA). Eosin 1% aqueous and dissolved in 100 ml of distilled water. This was kept inside was purchased from Biostain (Traralgon, Australia) while the fridge before use and the dosage for each rat was calculated Harris Hematoxylin was purchased from Harris Surgipath and administered using oral cannula. (Richmond, IL, USA) and Histofluid from Marien- feld (Lauda- Königshofen, Germany). Other chemicals were purchased from Table 1. Chemical compositions of GK. Preliminary ethanol screening of GK Sigma Aldrich excepted otherwise stated. reveals the following phytochemical constituents. Extraction of GK Phytochemical constituents Inference Alkaloids +++ GK seeds were procured from a local market at Ile-Ife, Flavonoids +++ Osun State, Nigeria (Latitude: 7º 27 ˈ 59.99 ̎ N and Longitude: Saponins ++ 4º 33 ˈ 59.99 ̎ E in the Rainforest zone). A sample was taken Tannins ++ Carbohydrates + to a taxonomist at the Department of Basic Sciences, Babcock Anthraquinones - University Ilisan-Remo for authentication. The seed were air- Steroids + dried, cut into smaller sizes and thereafter pulverized in a blender Cardiac glycosides - (PHILIPS, Model HR-1724, Brazil) to obtain smooth powder. A Key: - Absent; + Present; ++ Significantly present; +++ Abundantly present

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Measurement of core body temperature (ºC) Results The core body temperature of each rat was taken using a Body weight change and relative organ weight digital thermometer (METTLER TOLEDO, China) by inserting Body weight change (Figure 1): Changes in body weights of the instrument into the anal canal of the rats at about 10:00am of the animals following MDMA exposure and GK administration each day for the period of 20 days. were determined. From the graph, there is a significant reduction Neurobehavioral study in the body weight of GK-only (higher dose), MDMA-only and MDMA plus GK low dose groups when compared with Motor coordination and balance were assessed by the beam the control (p≤ 0.05). Worthy of note is the sudden shift from walking test using a beam apparatus. The goal of this test was this trend to an increase in the body weight of the MDMA plus for the rat to stay upright and walk across an elevated narrow GK higher dose. This indicates that both MDMA and GK have beam. This test took place over 3 consecutive days: 2 days of acute effect in reducing the body weights of the rat when taken training and 1 day of testing. Performance on the beam was individually. determined by measuring the number of foot slip, number of turns and distance travelled following previous method [33]. Relative brain weight (Figure 2): Result of the relative brain weight from the graph shows a significant increase in the Animal sacrifice MDMA-only, GK-only higher dose and MDMA plus GK lower Rats were sacrificed 24 hours after the last administration of dose when compared with the control (p≤ 0.05). GK and MDMA using chloroform diether as anesthesia. Two rats Food and water intake (Figures 3A & 3B) from each group were perfused with 10% formol saline and the Food and water consumption were measured following brain tissues were later fixed in 10% neutral buffered formalin exposure to MDMA. The result shows a significant decrease for the purpose of histological and immunohistochemical in food intake of the MDMA-only group when compared with investigations while the remaining were not perfused and used the control (*p < 0.05) and a significant increase in the groups for enzymatic and biochemical assays using ELISA kits. The of GK-only and the GK plus MDMA groups when compared brain weights were taken and recorded after excision. with the MDMA-only (^p < 0.05). On the other hand, there is a Preparation of tissue sample for biochemical analyses: significant increase in water intake in MDMA-only group (***p Cerebellar homogenate was prepared with cold 0.25 M < 0.001) and in MDMA plus GK (lower dose) group (**p < sucrose solution using an automated homogenizer at 4°C. The 0.01) when compared with the control. GK-only groups and GK (higher dose) plus MDMA group showed significant decrease tissue homogenate was centrifuged at 4000 revolution for 20 in water intake when compared with MDMA-only group (^^^p minutes at 4 °C using Potter AGV-8 Bunsen (Madrid, Spain). < 0.001). The supernatants obtained were thereafter aspirated into plain bottles and analyzed for the activities of 5-HT, dopamine (DA), glutamate, cytochrome C oxidase and glucose-6-phosphate dehydrogenase according to the manufacturer’s instruction on the ELISA kits (ALPCO Diagnostics, Salem, NH, USA). Glial Fibrillary Acidic Protein (GFAP) procedure The GFAP investigation was done using a modified previous method [34]. Photomicroscopy Slides were viewed using LEICA DM 750 microscope connected to a digital camera (LEICA ICC50) (Olympus, New Jersey, USA) and a desktop computer. Statistical analysis Results were presented as Mean ± SEM and analyzed using descriptive and inferential statistics. One-way ANOVA was used to analyze data, followed by Student Neuman-Keauls (SNK) test for multiple comparisons. GraphPad Prism 5 (GraphPad Software, CA, USA) was the statistical package for Figure 1. Showing the Effect of GK on the Body Weight Change of MDMA- induced Wistar rats data analysis and p-values ≤ 0.05 were considered statistically *= significantly lower when compared with control group (p≤ 0.05) significant. #= significantly higher when compared with group D (MDMA only) (p≤ 0.05)

39 Curr Neurobiol 2020 Volume 11 Issue 2 Garcinia kola Mitigates 3, 4-methylenedioxymethamphetamine-induced Derangement in Cellular and Molecular Characterization in the Cerebellum of Wistar rat

the numbers of turns and foot slip when compared with the control (p≤0.05). GK extract was able to correct these erratic movements, though not significant in distance travelled and number of foot slips (p≤0.05). Neurotransmitters Result from the assays of neurotransmitters shows that there is no significant difference in the levels of 5-HT, glutamate and DA in the MDMA and the GK treated groups when compared with the control. Enzymatic assays Cytochrome-C-Oxidase (Figure 5): The figure shows a significant increase in the activity of Cytochrome-C-Oxidase in MDMA-only and in GK (lower and higher doses) plus MDMA when compared with the control (p≤ 0.05). This indicates that ecstasy exposure increased the activity of the enzyme. G6PD (Figure 6): The figure shows a significantly higher activity of Glucose-6-Phosphate Dehydrogenase in the MDMA- only group indicting the effect of the drug on the enzyme Figure 2. Effect of GK on the Relative Brain Weight of MDMA-induced Wistar rats activities (p≤ 0.05). However, there is a significant reduction *= significantly higher when compared with the control (p≤ 0.05). in the levels of the activity of the enzyme in groups of GK-only and GK (lower and higher doses) plus MDMA when compared with MDMA-only. Histological and immunohistochemical Haematoxylin and eosin (Figure 7): The control group presents normal histoarchitecture while there is the evidence of Purkinje cells loss in MDMA-only group. Though the arrangement of these cells were also altered in other groups especially GK-only (lower dose), the numbers are still preserved anyway. Glial fibrillary acidic protein (Figure 8):This was carried Figure 3A & 3B. Effect of GK extract on food and water in-take in Wistar rat out to investigate the level of reactivity of astrocytes in the following exposure to MDMA cerebellum and in turn the expression of glial fibrillary acidic (**p < 0.01, and ***p < 0.001 when compared with control) protein as a marker for neurotoxicity. MDMA-only group shows (^^p < 0.01, and ^^^p < 0.001 when compared with MDMA) high reactivity of GFAP when compared with others. This is Body temperature (Table 2) evidence of extensive neuroinflammation. GK on the other hand caused very mild expression of this protein in groups of lower The core body temperature of each rat was taken to monitor and higher GK-only. the effect of MDMA as it is known to cause hyperthermia in the body of the users. The result shows an acute significant increase Discussion in the core body temperature of the animals of MDMA-only A number of factors including but not limited to increased group from day one of administration (p≤0.05). The increase wakefulness/alertness, endurance and sense of energy, sexual became consistent from the 3rd day and highest at day 20. arousal, and reduced fatigue and sleepiness have made the However, the groups that received the GK extract also revealed usage of MDMA more popular among the youths [35-36]. The significant rise in temperature from day one when compared aforementioned factors are its psychological effects, however, with the control (p≤0.05). The highest rise in temperature was the list of its undesired effects is endless. Majority of these interestingly recorded on day 16 in the group that received GK adverse effects are life-threatening and call for emergency (lower dose) plus MDMA (Figure 4). response. MDMA, like other amphetamines has adverse effects Neurobehavioral studies (Figure 5) on many physical functions in the body system, even when taken in moderate doses [8, 10], the crystal (purest) form of the The result revealed that MDMA caused reduction in drug however has more deleterious effect than its amorphous the distance travelled by the rat and a significant increase in form. Although the cerebellum only plays a synergistic role

Curr Neurobiol 2020 Volume 11 Issue 2 40 Owolabi JO, Okoro IO, Adelodun ST

Table 2. Effect of MDMA on the core body temperature (ºC) of the rats and the role of GK Day Control 100mg/kg GK 200mg/kg GK MDMA 100mg/kg GK+ MDMA 200mg/kg GK+ MDMA 1 34.06±0.31 34.52±0.29^^ 35.64±0.24* 36.26±0.33** 35.96±0.22** 36.58±0.38 2 36.06±0.30 35.90±0.29 36.26±0.11 35.48±0.14 35.80±0.24 36.95±0.41^^ 3 35.54±0.19 35.66±0.22^^ 35.94±0.23^^ 36.84±0.14** 37.04±0.23** 36.98±0.36** 4 35.08±0.17 35.52±0.22^^^ 35.90±0.19^^^ 37.78±0.51*** 36.68±0.10**^^ 36.63±0.23**^ 5 35.30±0.13 35.34±0.11^^^ 35.62±0.07^^^ 37.68±0.10*** 36.72±0.12**^^ 36.63±0.63**^ 6 35.10±0.36 35.30±0.28^^^ 35.24±0.24^^ 36.80±0.21** 37.20±0.29*** 37.03±0.32** 7 34.92±0.23 35.34±0.33^^ 34.92±0.23^^ 36.72±0.56** 37.12±0.16*** 37.60±0.29*** 8 34.70±0.20 35.20±0.12 34.70±0.20 36.06±0.12 36.54±0.62* 36.90±0.61** 9 34.84±0.14 35.50±0.03^^^ 34.84±0.14^^^ 37.36±0.25*** 37.60±0.31*** 37.15±0.33*** 10 35.46±0.16 35.38±0.10^ 35.50±0.16^^ 37.00±0.20* 37.00±0.72* 36.85±0.42* 11 35.32±0.18 35.46±0.05^^ 35.64±0.17^^ 37.24±0.67** 37.73±0.26*** 37.20±0.12** 12 34.78±0.21 35.26±0.23^^^ 35.56±0.41^^ 37.40±0.27*** 37.23±0.38*** 37.30±0.07*** 13 35.26±0.23 35.52±0.14^^^ 35.42±0.19^^^ 36.80±0.19*** 37.83±0.13***^^ 37.70±0.13**^^ 14 35.14±0.23 35.54±0.16^^^ 35.54±0.33^^^ 37.33±0.30*** 38.00±0.07*** 37.48±0.43*** 15 35.04±0.16 35.44±0.28^^^ 35.30±0.09^^^ 37.60±0.20*** 36.80±0.24***^ 37.85±0.29*** 16 35.48±0.32 35.75±0.22^^^ 35.60±0.21^^^ 37.43±0.31*** 38.75±0.17***^^ 37.90±0.17*** 17 35.88±0.32 35.90±0.31 35.90±0.31 36.35±0.23 37.65±0.43**^ 36.73±0.08 18 35.64±0.35 35.70±0.33^^ 35.70±0.31^^ 37.65±0.30** 37.65±0.37** 37.43±0.13** 19 35.34±0.15 35.50±0.03^^^ 35.44±0.11^^^ 37.38±0.09*** 36.50±0.41**^ 36.85±0.42** 20 35.26±0.12 35.52±0.04^^^ 35.58±0.06^^^ 38.25±0.31*** 37.60±0.14***^^ 37.15±0.06***^^^ Temperature was taken starting from the first day of administration of MDMA through the next 20 days of experiment. (*p < 0.05, **p < 0.01, and ***p < 0.001 when compared with control) (^p < 0.05, ^^p < 0.01, and ^^^p < 0.001 when compared with MDMA)

Figure 4A, 4B & 4C. Effect of MDMA on the distance travelled, number of foot slip and the number of turns made by the rats. This is to ascertain the alterations caused by MDMA in the role of cerebellum on skeletal movement and the role of GK administration *: Significantly lower or higher when compared with the control (P<0.05)

Figure 5. Effect of GK on the Level of Cytochrome C Oxidase Enzyme of MDMA-induced Wistar rats *= significantly higher when compared with control (p≤ 0.05). ^= significantly lower when compared with group D (p≤ 0.05). 41 Curr Neurobiol 2020 Volume 11 Issue 2 Garcinia kola Mitigates 3, 4-methylenedioxymethamphetamine-induced Derangement in Cellular and Molecular Characterization in the Cerebellum of Wistar rat

Figure 6. Effect of GK on the Level of Glucose-6-Phosphate Dehydrogenase Enzyme of MDMA-induced Wistar rats *= significantly higher when compared with control (p≤ 0.05). # = significantly lower when compared with the D (p≤ 0.05).

Figure 7. Photomicrograph of the Cerebellum showing the general histoarchitecture. Control group (A) shows normal arrangement of the molecular and granular layers as well as the Purkinje cells. This is also seen in groups B and C(GK-only groups). However, the administration of the MDMA caused the loss of certain Purkinje cells in the group that was only administered with the agent (D); these cells were relatively preserved when GK was administered as revealed in groups E and F (i.e 100mg/kg MDMA + GK and 200mg/kg MDMA + GK groups). ML = molecular layer; GL = granular layer; P = Purkinje cell. H & E.

Figure 8. Photomicrograph of the Cerebellum of Wistar rat showing the demonstration of glial fibrillary acidic protein (GFAP). The control group shows negative reactivity when compared with other groups. Groups B shows mild positive reactivity while C is more evident than B indicating that garcinia kola can also induce some toxicity at some concentration. MDMA group (D) shows high level of positive reactivity which administration of garcinia kola ameliorated to some extent as shown in groups E and F.

Curr Neurobiol 2020 Volume 11 Issue 2 42 Owolabi JO, Okoro IO, Adelodun ST

after motion initiation, there is distortion in movements when increase in the body temperature of the rats from the first it becomes damaged. The present study therefore looked at day of administration of ecstasy (Table 2). Many factors on the possible protective role of ethanolic extract of GK on the the other hand, can determine the hyperthermic response of cerebellum following MDMA in-take. laboratory animals. For instance, Broening and colleagues in their investigation reported that age is a big factor for animals Effect of MDMA on body weight change to either become hyperthermic or not when exposed to MDMA. The result from our study shows a significant reduction in Their young rats (post-natal day 10) exposed orally to MDMA body weight change of the rats following exposure to MDMA similar to ours (20 or 40 mg/kg) did not show any hyperthermic (Figure 1). The body weight loss may be ascribed to the reduced response, while rats at post-natal day 40 and post-natal day 70 feeding characteristic of the rats which was observed during the showed an hyperthermic response after being exposed to the same period of drug administration (Fig 3A). Bearing in mind that dose of the drug [46]. However, we also deduced that GK does not MDMA is primarily known to be an appetite suppressant in possess thermoregulatory function as it could not bring down the animals, right from its time of advent [37]. We noticed that the temperature of the animals that were pre-exposed to the drug. animals responded poorly to food but not water consumption Neurobehavioral after MDMA exposure. Previous studies have reported that headache, nausea, loss of appetite, blurred vision and many The reduction in the distance travelled (Figure 4A) reflects more are among the physical symptoms during the drug the adverse effect of MDMA on the functionality of cerebellum. experience and immediately afterwards [38]. Worthy of note is However, there is a noticeable but not significant improvement in the significant body weight gain in rats that were administered GK-only (higher dose) group, suggesting that the extract at this with GK (higher dose) plus MDMA. This is suggestive that the dose has great tendency in preventing or ameliorating impaired phytochemical components of GK probably have some anabolic locomotion and motor activity of rats. Result on the foot slip functions as a long term effect. also shows significant increase in foot slip of the MDMA and GK-only (low dose) groups (Figure 4B). This is pointing to Effect of MDMA on the relative brain weight uncoordinated movement (i.e ataxia) in the rats. It also proves One major symptom of MDMA toxicity on the user is that GK at low dose is unable to prevent this movement disorder hyperthermia and dehydration leading to the urge for water- caused by damage to the cerebellum through MDMA-induced intake. We noticed a significant increase in the relative brain neurotoxicity. On the other, higher dose of our agent was able weight of the rats following administration of ‘ecstasy’ (Figure to correct these abnormalities. This suggests that GK can 2). The factor responsible for the gain in brain weight could prevent and reduce ataxia in animal following MDMA-induced probably be as a result of fluid accumulation in the brain tissue. neurotoxicity. Our finding is in consonance with a previous report Excessive water-intake under this condition consequently led that kolaviron was able to cause a delay in the onset of stereotypic to hyponatremia which is a life-threatening effect of MDMA movement induced by methamphetamine in Wistar rat [47]. intake [39-41]. Our present study shows that MDMA caused Levels of 5-HT, DA and glutamate in rats treated with GK the rats to consume much water (Figure 3B) as expected based following MDMA intake on previous reports. Symptomatic hyponatremia which results from passive flow of water into cells is characterized with Long-term decrease in serotonergic markers which include cerebral edema and may result in brain stem herniation [42]. depletion of 5-HT and its main metabolite, 5-Hydroxyindoleacetic All these factors are probable pointers to the brain weight gain acid (5-HIAA), have been implicated in MDMA neurotoxicity observed in this study. However, it was observed that the brain [45, 48-51], consequently leading to ‘5-HT syndrome’ [52] which weight reduced down the groups that were treated with GK. occurs when there is high level of 5-HT in circulation. The high This suggests that GK might possess the potential to function level in circulation has been attributed to some pharmacological as an antagonist to the symptoms of inappropriate anti-diuretic mechanisms which include inhibition of the metabolism of 5-HT hormone secretion and abnormal water intake as the extract was by monoamine oxidase inhibitors, prevention of the reuptake able to reduce the excessive water intake by the animals. Our of 5-HT in nerve terminals, and up regulation of 5-HT release report seems to proffer solution to the challenge from a previous from the presynaptic membrane caused by MDMA [53]. Result report that “hyponatremia after MDMA use is thought to involve from our study shows that there is no significant difference in a combination syndrome of inappropriate antidiuretic hormone the levels of serotonin concentration in the brain tissue across (SIADH) secretion and increased hypotonic fluid intake” [43]. all the treated groups when compared with the control (Table 3). Though MDMA and other amphetamine derivatives have been Effect on the core body temperature postulated to act by increasing the net release of the monoamine A number of reports have shown that there is a consequent neurotransmitters (5-HT, noradrenaline and, to a smaller extent, hyperthermia following MDMA consumption leading to DA) from their respective axon terminals [54-55]. Almost all, if enhancement of MDMA neurotoxic actions in experimental not all the released 5-HT reuptake is being inhibited by one or all animals [44-45]. Our result from this present study also of the factors mentioned above hence, the low level of 5-HT in corroborates these existing claims, as there was a sharp the brain tissue in our study. However, it has been stated earlier

43 Curr Neurobiol 2020 Volume 11 Issue 2 Garcinia kola Mitigates 3, 4-methylenedioxymethamphetamine-induced Derangement in Cellular and Molecular Characterization in the Cerebellum of Wistar rat that serotonin syndrome is as a result of MDMA abuse among this present study when compared with the GK treated groups the users. 5-HT syndrome is usually being diagnosed clinically (Figure 5). This suggests that MDMA caused recruitment of and some of its symptoms include neuromuscular excitation like cytochrome c into the site of damage in the brain tissue due to tremor and muscle rigidity and changes in mental status, such induced oxidative stress and generated ROS. On the other hand, as disorientation, restlessness, and coma [56]. The behavioural GK extract through its phytochemical components (probably study shows some signs of disoriented limb movements on the kolaviron) was able to restore the level of the enzyme to normal beam as stated above, pointing to a damaged cerebellum. in an appreciable manner in groups of GK (low and high doses) plus MDMA, proving its anti-oxidant capability. As stated earlier, MDMA’s mechanism of action is also related to the net release of DA as applicable to 5-HT, but to a G6PDH is an enzyme that helps in the stability of catalase smaller extent. DA also faces the same fate of inhibited reuptake and in the regeneration of reduced glutathione. If any condition as 5-HT and that explains its insignificant levels across the initiates the release of reactive oxygen species (ROS) and treated groups when compared with the control in this present oxidative stress, the expression of G6PDH automatically study (Table 3). increases and this has been demonstrated in vitro and in vivo [65-66]. The result from our study shows an increased level of Several investigations have been done and are still being G6PDH in group MDMA-only, indicating tissue damage as a done on glutamate, not because it is used as a food enhancer but result of induced oxidative stress (Figure 6). This is in consonance because it is known to be the major excitatory neurotransmitter. with the early study which reported that an increased neuronal Glutamate exerts its neurotransmitter function from the G6PDH expression has been observed in the hippocampus extracellular fluid because its receptor proteins are expressed of Alzheimer’s disease patients. MDMA administration has on the surface of the cells in such a way that they can only be also been known to alter brain microenvironment leading activated from the outside. Therefore, level of glutamate can only to oxidative stress and ROS production [67]. However, our be controlled by releasing glutamate to the extracellular fluid result shows no significant difference in the levels of G6PDH and then removing glutamate from it [57-58]. It is detrimental in all the groups treated with GK (either only or plus MDMA to the brain when it is too high or too low in concentration. administration). This also suggests protective property of GK MDMA has been reported to produce a delayed and sustained against MDMA-induced oxidative stress in the brain tissue. increase in the extracellular concentration of glutamate in the Kolaviron, a biflavonoid complex of GK has been reputed for hippocampus [59]. Result from the present study shows an its strong antioxidant property, both in vivo and in vitro [60]. insignificant increase in the level of glutamate only in group This property has been elicited through its ability to protect MDMA-only group when compared with the control (Table the neurons of the brain from toxic damage. We can therefore 3). Others have no noticeable difference from the control. We say that, the reduced level of G6PD in the GK treated groups is suggest this outcome may be due to impairment in the reuptake probably elicited by kolaviron. of glutamate as applied to 5-HT. Our argument corroborates previous report as stated above that 5-HT released by MDMA Histological and immunohistochemical features activates 5-HT2A/C receptors, thereby promoting the release of Histological and Immunohistochemistry analyses are parts glutamate, presumably from astrocytes in the hippocampus. of the hypotheses upon which MDMA-induced axotomy relies Levels of Cytochrome-C-Oxidase and Glucose-6-Phosphate as semi-quantitative methods could produce misleading results Dehydrogenase (G6PDH) in the brain of rats treated with GK if not validated by other means. Purkinje cells and the granular following MDMA intake cells are the major targets in cerebellum for toxic substances. Purkinje cells which are among the largest neuron in the CNS are It has been hypothesized that any alterations in cytochrome very sensitive to ischemia, and other processes that could release c oxidase or complex IV may be a crucial factor that causes ROS in the brain. Result from the histological investigation several neurodegenerative diseases that are related to aging, in our present study shows reduction in the number of the such as Alzheimer’s disease [60]. Cytochrome c oxidase is a Purkinje cells in the MDMA-only group when compared with known antioxidant enzyme which has the ability to remove others (Figure 7). Eltony and his colleagues [68] reported that mitochondrially-produced ROS by reacting with superoxide and cerebellum exposure to toxic metals caused degeneration and hydrogen peroxide to produce O and H O, respectively [61- 2 2 reduction in the numbers of Purkinje cells, which corroborates 62]. Though MDMA-mediated toxicities are well established our report. However, GK was able to preserve the Purkinje and greatly researched on, there are still more yet to be known cells’ integrity (which are largely responsible for cerebellar and appreciated by the general public as regards the adverse output and integration of intra-cerebellar network) as observed effects associated with MDMA usage. MDMA has been shown on the photomicrograph. to be a factor that can suppress the mitochondrial function by directly inhibiting the activities of mitochondrial cytochrome A review has it that the general reaction to any damage c and its other complexes: I, II, and V [63] or interacting in the CNS is characterised by increase in size (hypertrophy) with various proteins in the mitochondria [64]. ‘Ecstasy’ of astrocytes, or “reactive gliosis” which is commonly administration increased the level of cytochrome c activities in accompanied by enhanced expression of glial-specific structural

Curr Neurobiol 2020 Volume 11 Issue 2 44 Owolabi JO, Okoro IO, Adelodun ST

proteins such as glial fibrillary acidic protein (GFAP). Because 6. Mueller PD, Korey WS. Death by “ecstasy”: The serotonin syndrome? GFAP has been investigated to be a good marker for neuronal Ann Emerg Med. 1998; 32: 377-380. toxicity, O’Callaghan and Miller [69] carried out extensive 7. Henry JA. Ecstasy and the dance of death. BMJ. 1996; 305: 5-6. studies to evaluate the effects of MDMA administration on 8. Shulgin AT. The background and chemistry of MDMA. J Psychoactive GFAP expression in rat brain. They discovered that rats that Drugs. 1986; 18: 291-304. were treated with 10–30mg/kg MDMA for 7 days did not show 9. Sherlock K, Wolff K, Hay AW, Conner M. Analysis of illicit ecstasy any increase in GFAP level until the doses were increased to tablets: implications for clinical management in the accident and 75–150 mg/kg then the level increased in various brain regions. emergency department. J Accid Emerg Med. 1999; 16: 194-197. Our immunohistochemical result shows increased expression of 10. Vollenweider FX, Gamma A, Liechti M, Huber T. Psychological and GFAP on MDMA-only group (Figure 8) which contradicts the cardiovascular effects and short-term sequelae of MDMA (“ecstasy”) in result of O’Callaghan and Miler as stated above. MDM Anaive healthy volunteers. Neuropsychopharmacol. 1998; 19: 241- 251. Conclusion 11. Olson KR, Benowitz NL. Environmental and drug-induced hyperthermia. We conclude that ethanolic extract of GK was able to protect Pathophysiology, recognition and management. Emerg Med Clin North Am. 1984; 2: 459-474. the cerebellum from damage incurred by MDMA exposure in a dose-dependent manner through a conglomeration of evidences 12. Peacock A, Gibbs D, Karlsson A, Uporova J, Sutherland R, et al. Ecstasy and Related Drugs Reporting System (EDRS) Interviews: Background and which include: preservation of the Purkinje cells integrity, Methods. Sydney, National Drug and Alcohol Research Centre, UNSW normalizing erratic gaits in the animals, normalizing abnormal Australia, 2018. enzymatic activities and acting as anti-diuretics. On the other 13. James CH, Lee EM. Cerebellum: Movement Regulation and Cognitive hand, GK does not possess thermoregulatory potential based Functions, Encyclopedia of Life Sciences /Nature Publishing Group / on our recent findings. Therefore, we recommend that further www.els.net 2001. studies be done to establish the optimal dose and also which 14. Butterfield DA, Hensley K, Cole P, Subramaniam R, Askenov M, et al. phytochemical (s) is (are) specifically responsible for the Oxidatively induced structural alteration of glutamine synthetase assessed neuroprotective function. by analysis of spin label incorporation kinetics: relevance to Alzheimer’s disease. J Neurochem 1997; 68: 2451-2457. Conflict of interest 15. Davidson C, Gow AJ, Lee TH, Ellinwood EH. Methamphetamine We declare no conflict of interest as regards this research. neurotoxicity: necrotic and apoptotic mechanisms and relevance to human abuse and treatment. Brain Research. Brain Res Rev 2001; 36: 1-22. Authors’ contributions 16. Escubedo E, Chipana C, Pérez-Sánchez M, Camarasa J, Pubill D. Methyllycaconitine prevents methamphetamine-induced effects in mouse OJO played role in formulating the research, arrangement striatum: involvement of alpha7 nicotinic receptors. J Pharmacol Exp of resources, and served as the project’s supervisor. OI played Ther. 2005; 315: 65867. role in research design, arranged funding, and reviewed the final 17. Obrocki J, Buchert R, Vaterlein O, Thomasius R, Beyer W, et al. Ecstasy manuscript. AST snapped the photomicrographs and interpreted – long term effects on the human central nervous system revealed by the Immunohistochemical and histological results with scale positron emission tomography. Br J Psychiatry. 1999; 175: 186-188. bar and wrote the manuscript. OSY conducted the experiments, 18. Kish SJ, Furukawa Y, Ang L, Vorce SP, Kalasinski KS. Striatal serotonin performed biochemical investigations and assisted in writing is depleted in brain of a human MDMA (Ecstasy) user. Neurology. 2000; of the manuscript. OJA performed statistical analysis and 55: 294-296. contributed to the interpretation of the results. FSO prepared 19. Wareing M, Fisk JE, Murphy PN. Working memory deficits in current and figures and helped in analyzing the neurobehavioral data. previous users of MDMA (“ecstasy”). Br J Psychol. 2010; 91: 181-188. 20. Adesuyi AO, Elumm IK, Adaramola FB, Nwokocha AGM. Nutritional References and phytochemical screening of Garcinia kola. Adv J Food Sci Technol. 1. Peroutka SJ. Incidence of recreational use of 2012; 4: 9-14. 3,4-ethylenedimethoxymethamphetamine (MDMA, ‘ecstasy’) on an 21. Adefule-Ositelu AO, Adefule AK, Oosa BO, Onyenefa PC. Antifungal undergraduate campus. N Engl J Med. 1987; 317: 1542-1543. activity of Garcinia extract as an ocular bacterial isolates in Lagos. 2. Webb E, Ashton CH, Kelly P, Kamali F. Alcohol and drug use in UK Nig Qt J Hosp Med. 2004; 14: 112-114. university students. Lancet. 1996; 348: 922-925. 22. Terashima K, Takaya Y, Niwa M. Powerful antioxidative agents based 3. Parrott AC, Lasky J. Ecstasy (MDMA) effects upon mood and cognition: on Garcinoic acid from Garcinia kola. Bioor Med Chem. 2002; 10: 1619- Before, during and after a Saturday night dance. Psychopharmacol. 1998; 1625. 139: 126-128 23. Olatunde FE, Hansen M, Rain-Haren P, Dragsted LO. Commonly 4. Centers for Disease Control and Prevention: Ecstasy overdoses at a new consumed and naturally occuring dietary substances affect biomarkers of year’s eve Rave—Los Angeles, California, MMWR Morb Mortal Wkly oxidative stress and DNA damage in healthy rats. Food Chem Toxicol. Rep. 2010; 59: 677-681. 2004; 42: 1315-1322. 5. Armenian P, Mamantov TM, Tsutaoka BT, Gerona RR, Silman EF, et al. 24. Ajibade AJ, Oyewopo AO, Fakunle PB, Ashamu EA. Effects of crude Multiple MDMA (Ecstasy) overdoses at a rave event: A Case Series. J ethanolic extract of Garcinia kola (Bitter Kola) on the histology of the Intensive Care Med. 2013; 28: 252-258. testis of male adult wistar rats. Wajar. 2011; 2: 24-32.

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Correspondence to: Adelodun Stephen Taiye Neuroscientist and Reproductive scientist Department of Anatomy Ben Carson School of Medicine Babcock University Ilisan Remo, Ogun State Nigeria E-mail: [email protected]

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