Pharmacological characterization of the cannabinoid receptor 2 agonist, β-caryophyllene on seizure models in mice

Open ArchivePublished:March 12, 2018DOI:https://doi.org/10.1016/j.seizure.2018.03.009

      Highlights

      • β-caryophyllene exerted a protection in the MES.
      • β-caryophyllene attenuated the KA-induced SE in rats.
      • β-caryophyllene decreased lipid peroxidation induced by SE.
      • β-caryophyllene dose-dependently improved spatial memory performance.

      Abstract

      Purpose

      Activation of CB1 receptors, produces anticonvulsant effect accompanied by memory disturbance both in animal seizure tests and in patients with epilepsy. Few reports considered the role of CB2 receptor on seizure susceptibility and cognitive functions. The aim of the present study was to explore the effect of a selective CB2 receptor agonist β-caryophyllene (BCP) in models of seizures and cognition in mice.

      Methods

      Dose-dependent effects of BCP was studied in maximal electroshock seizure (MES) test, subcutaneous pentylenetetrazole (scPTZ) test and Morris water maze test. Phenytoin and diazepam were used as reference drugs in seizure tests. The effect of sub-chronic treatment with BCP for 7 days (50 and 100 mg kg−1) was assessed on status epilepticus (SE) induced by kainic acid (KA) model and oxidative stress through measurement of malondialdehyde (MDA) level in the hippocampus. The acute neurotoxicity was determined by a rotarod test.

      Results

      The BCP exerted a protection in the MES test at the lowest dose of 30 mg kg−1 at the 4-h interval tested comparable to that of the referent drug phenytoin. The CB2 agonist was ineffective in the scPTZ test. The BCP displayed no neurotoxicity in the rotarod test. The BCP decreased the seizure scores in the KA-induced SE, which effect correlated with a diminished lipid peroxidation. The CB2 agonist exerted a dose-dependent decrease of latency to cross the target area during the three days of testing in the Morris water maze test.

      Conclusion

      Our results suggest that the CB2 receptor agonists might be clinically useful as an adjunct treatment against seizure spread and status epilepticus and concomitant oxidative stress, neurotoxicity and cognitive impairments.

      Keywords

      1. Introduction

      Epilepsy is a neurological disorder that is characterized by spontaneous seizures as well as learning and memory impairment as a comorbid risk factor. Treatment by antiepileptic drugs (AEDs) prevent seizures but in most cases their effectiveness against cognitive disturbance in patients with epilepsy is neglected and uncertain [
      • Agrawal N.
      • Govender S.
      Epilepsy and neuropsychiatric comorbidities.
      ]. Current research in this field is focused on design and development of alternative therapeutic approaches that prevent epileptogenesis after status epilepticus (SE) and its deleterious consequences, including oxidative stress, neuronal damages and resulting aggravation in cognitive functions. During the last decade, drugs with potential memory-enhancing effects are explored as adjuvant to anticonvulsant therapy for treatment of deletirious comorbid behavioral changes.
      Accumulated experimental and clinical data support the suggestion that endocannabinoid system (ECS) represent a potential therapeutic tool for treatment of pathological conditions in neurodegenerative diseases including epilepsy, Alzheimer's disease, multiple sclerosis, and Parkinson’s disease [
      • Rosenberg E.C.
      • Patra P.H.
      • Whalley B.
      Therapeutic effects of cannabinoids in animal models of seizures, epilepsy, epileptogenesis, and epilepsy-related neuroprotection.
      ,
      • Sharma C.
      • Sadek B.
      • Goyal N.S.
      • Sinha S.
      • Kamal A.M.
      • Ojha S.
      Small molecules from nature targeting G-protein coupled cannabinoid receptors: potential leads for drug discovery and development.
      ]. Until recently, numerous studies have been focused on the role of CB1 receptors in the CNS and in this line it has been suggested that ECS has a role in brain excitabiity mostly by activation of CB1 receptors [
      • Bambico R.F.
      • Gobbi G.
      The cannabinoid CB 1 receptor and the endocannabinoid anandamide: possible antidepressant targets.
      ,
      • Huizenga M.N.
      • Wicker E.
      • Beck V.C.
      • Forcelli P.A.
      Anticonvulsant effect of cannabinoid receptor agonists in models of seizures in developing rats. CB1 receptor agonists as novel antiepileptic drugs targeting epilepsy.
      ]. On the other hand, the role of CB2 receptors, which are mainly distributed in peripheral tissues and in particular in B lymphocytes, macrophages, mast cells, microglia and natural killer cells as well as spleen, tonsils, and the thymus [
      • Sharma C.
      • Sadek B.
      • Goyal N.S.
      • Sinha S.
      • Kamal A.M.
      • Ojha S.
      Small molecules from nature targeting G-protein coupled cannabinoid receptors: potential leads for drug discovery and development.
      ], is poorly explored in the central nervous system (CNS). Furthermore, ligands for CB2 receptors are concidered as promising drugs for treatment of peripheral pain with application in diabetic neuropathy [
      • Dhopeshwarkar A.
      • Mackie K.
      CB2 Cannabinoid receptors as a therapeutic target-what does the future hold.
      ]. Several studies, which reported encouraging data are in support of the presumption that drugs acting throught CB2 receptors could be exploited as novel pharmacological agents in the treatment of disturbances in the CNS [
      • Galdino P.M.
      • Nascimento M.M.
      • Florentino F.I.
      • Lino C.R.
      • Fajemiroye O.J.
      • Chaibub A.B.
      • de Paula R.J.
      • de Lima C.T.
      • Costa A.E.
      The anxiolytic-like effect of an essential oil derived from Spiranthera odoratissima A. St. Hil. leaves and its major component, β-caryophyllene, in male mice.
      ,
      • García-Gutiérrez M.S.
      • Pérez-Ortiz J.M.
      • Gutiérrez-Adán A.
      • Manzanares J.
      Depression-resistant endophenotype in mice overexpressing cannabinoid CB2 receptors.
      ,
      • García-Gutiérrez M.S.
      • García-Bueno B.
      • Zoppi S.
      • Leza C.J.
      • Manzanares J.
      Chronic blockade of cannabinoid CB2 receptors induces anxiolytic-like actions associated with alterations in GABAA receptors.
      ,
      • Bahi A.A.
      • Mansouri S.
      • Al Memari E.
      • Al Ameri M.
      • Nurulain S.M.
      • Ojha S.
      β-caryophyllene, a CB2 receptor agonist produces multiple behavioral changes relevant to anxiety and depression in mice.
      ]. Compounds, which effects are mediated by CB2 receptors deserve further exploration because of lack of side psychotropic activity of ligands of CB1 receptors. In the recent years, numerous novel synthetic and natural compounds showing high affinity for CB2 receptors are explored as good alternative of conventional drugs for disorders of CNS. Among them, a natural bicyclic sesquiterpene β-caryophyllene (BCP) is a common component of essential oils of spices (cinnamon, black pepper, oregano, clove, rosemary, thyme) and various plants, mainly Cannabis sativa and Copaifera spp. [
      • Gertsch J.
      • Leonti M.
      • Raduner S.
      • et al.
      Beta-caryophyllene is a dietary cannabinoid.
      ]. BCP posseses high affinity to CB2 receptors and is considered a promising dietary phytocannabinoid deserving further exploration. This CB2 receptor agonist has been found to exert a protective effect against alcohol addiction inflammation [
      • Gertsch J.
      • Leonti M.
      • Raduner S.
      • et al.
      Beta-caryophyllene is a dietary cannabinoid.
      ], [
      • Al Mansouri S.
      • Ojha S.
      • Al Maamari E.
      • Al Ameri M.
      • Nurulain S.M.
      • Bahi A.
      The cannabinoid receptor 2 agonist, β-caryophyllene, reduced voluntary alcohol intake and attenuated ethanol-induced place preference and sensitivity in mice.
      ], nociception [
      • Katsuyama S.
      • Mizoguchi H.
      • Kuwahata H.
      • Komatsu T.
      • Nagaoka K.
      • Nakamura H.
      • et al.
      Involvement of peripheral cannabinoid and opioid receptors in β-caryophyllene induced antinociception.
      ], depression [
      • Valenzano K.J.
      • Tafesse L.
      • Lee G.
      • Harrison J.E.
      • Boulet J.M.
      • Gottshall S.L.
      • et al.
      Pharmacological and pharmacokinetic characterization of the cannabinoid receptor 2 agonist, GW405833, utilizing rodent models of acute and chronic pain, anxiety, ataxia and catalepsy.
      ], cerebral ischemia [
      • Choi I.Y.
      • Ju C.
      • Anthony Jalin A.M.
      • Lee Da I.
      • Prather P.L.
      • Kim W.K.
      Activation of cannabinoid. CB2 receptor-mediated AMPK/CREB pathway reduces cerebral ischemic injury.
      ] and Alzheimer disease [
      • Cheng Y.
      • Dong Z.
      • Liu S.
      β-caryophyllene ameliorates the Alzheimer-like phenotype in APP/PS1 mice through CB2 receptor activation and the PPARγ pathway.
      ]. To date, a few studies have reported its potential activity against seizures in rodent tests [
      • Huizenga M.N.
      • Wicker E.
      • Beck V.C.
      • Forcelli P.A.
      Anticonvulsant effect of cannabinoid receptor agonists in models of seizures in developing rats. CB1 receptor agonists as novel antiepileptic drugs targeting epilepsy.
      ,
      • de Carvalho C.R.
      • Hoeller A.A.
      • Franco P.L.
      • Martini A.P.
      • Soares F.M.
      • Lin K.
      • Prediger R.D.
      • Whalley B.J.
      • Walz R.
      The cannabinoid CB2 receptor-specific agonist AM1241 increases pentylenetetrazole-induced seizure severity in Wistar rats.
      ,
      • de Oliveira C.C.
      • de Oliveira C.V.
      • Grigoletto J.
      • Rodrigo Ribeiro Leandro
      • Rafael Funck Vinícius
      • Beck Grauncke Ana
      • Lopes de Souza Thaíze
      • Schiefelbein Souto Naieli
      • Flávia Furian Ana
      • Rose Alencar Menezes Irwin
      • Schneider Oliveira Mauro
      Anticonvulsant activity of β-caryophyllene against pentylenetetrazol-induced seizures.
      ].
      Kainic acid (KA), an analog of the excitatory amino acid glutamate, has been widely used as a tool for SE induction. Previous studies revealed that some natural substances such as curcumin, vineatrol, resveratrol and capsaicin demonstrate promising anticonvulsant, neuroprotective and antioxidant activity in acute seizure tests and SE model in rodents [
      • Gupta Y.K.
      • Briyal S.
      • Chaudhary G.
      Protective effect of transresveratrol against kainic acid-induced seizures and oxidative stress in rats.
      ,
      • Gupta Y.K.
      • Briyal S.
      Protective effect of vineatrol against kainic acid induced seizures, oxidative stress and on the expression of heat shock proteins in rats.
      ,
      • Lee T.-H.
      • Lee J.-G.
      • Yon J.-M.
      • Oh K.-W.
      • Baek I.-J.
      • Nahm S.
      • et al.
      Capsaicin prevents kainic acid-induced epileptogenesis in mice.
      ,
      • Saudi M.A.
      Protective effects of curcumin against lithium–pilocarpine induced status epilepticus, cognitive dysfunction and oxidative stress in young rats.
      ]. The major rationale of this study was further to explore neurobiological activity of BCP on seizure models and spatial memory task in mice. For studying of anticonvulsant activity, we used maximal electroshock seizure (MES) and subcutanous pentylenetetrazole (scPTZ) test. Acute neurotoxicity of BCP was assesesed by test for motor coordination. Because of the observed protective effects of BCP described earlier, we hypothesized that CB2 receptor agonist might be effective against KA-induced SE by its antioxidant activity. Therefore, the effect of sub-chronically pretreatment with BCP on the KA-induced SE and concomitant lipid peroxidation in the hippocampus was investigated in mice. In addition, the effect of the CB2 receptor agonist on hippocampus-dependent cognitive functions was explored by Мîrris water maze test.

      2. Materials and methods

      2.1 Experimental animals

      Male albino ICR mice weighing 25–30 g were used as experimental animals. They were obtained from the animal facility of the Bulgarian Academy of Sciences and were maintained at an ambient temperature 22 ± 1 °C, in groups of six per cage under standard laboratory conditions (temperature: 26 ± 2 °C; humidity 50 ± 5%; 12 h dark/light cycle; food pellets and filtered water: ad libitum). All procedures were performed in agreement with the European Communities Council Directive 2010/63/EU. The experimental design was approved by the Institutional Ethics Committee at the Institute of Neurobiology.

      2.2 Maximal electroshock test (MES test)

      Initial screening in MES, scPTZ test and rota-rod test was performed at two time point intervals as follows: 0.5 h and 4 h, respectively. A drop of a local anesthetic was applied to the eyes of each animal and corneal electrodes were applied followed by an electric stimulus of 50 mA, 60 Hz delivered for 0.2 s (Constant Current Shock Generator). Abolition of the hind limb extensor component following drug treatment was taken as an end point of the test. The tonic component was considered abolished, if the hind limb extension did not exceed a 90° with the plane of the body. Abolition of hind limb tonic extensor component of the seizure in half or more of the animals was defined as protection.

      2.3 Subcutaneous pentylenetetrazole (scPTZ) seizure test

      For the scPTZ test, the compounds that raise seizure threshold were primarily identified. A dose of 85 mg kg−1 PTZ produced clonic seizures lasting for a period of at least five seconds in 97% (CD97) of control animals tested. The mice were further observed during a period of 30 min. The protection against clonic seizures in half or more of the animals was defined as an anticonvulsant activity.

      2.4 Rota-rod test

      The minimal motor impairment in mice was measured using the rota-rod test as described previously [
      • Angelova V.
      • Voynikov Y.
      • Andreeva-Gateva P.
      • Surcheva Sl Vassilev N.
      • Pencheva T.
      • Tchekalarova J.
      In vitro and in silico evaluation of chromene based aroyl hydrazones as anticonvulsant agents.
      ]. The mice are trained to stay on a rod of diameter 3.2 cm that rotates at a speed of 6 rpm and the animal can maintain their equilibrium for a long period of time. Neurotoxicity was indicated by the inability of the animal to maintain equilibration on the rod for at least one minute in each of the three trials. The dose at which 50% of the animals were unable to balance themselves and fell off the rotating rod was determined as toxic.

      2.5 Kainate induced status epilepticus

      On the 7th day of BCP/vehicle i.p. injection, mice received i.p. injection of KA (Merck, US) at a single dose of 30 mg kg−1 dissolved in sterile saline (0.9% NaCl) or saline in a volume of 10 ml kg−1 of body weight. Seizure intensity was scored according to the scale of Racine et al. [
      • Racine R.
      • Rose P.A.
      • Burnham W.M.
      Afterdischarge thresholds and kindling rates in dorsal and ventral hippocampus and dentate gyrus.
      ]: stage 1 (facial clonus), stage 2 (nodding), stage 3 (forelimb clonus), stage 4 (forelimb clonus with rearing), and stage 5 (rearing, jumping, and falling). The onset of SE was defined as the time when animals experienced continuous Stage 4 or 5 seizures. After a 3-h period of observation and detection of seizure intensity, the animals were decapitated with a guillotine after a mild CO2 anesthesia. Brains were quickly dissected on ice and the hippocampi were bilaterally removed. The tissue samples were frozen in liquid nitrogen, and stored at −20 °C before analysis.

      2.6 Drugs and dosage

      Control group (n = 8) (treated with vehicle, 0.05% Tween 80 dissolved in 0.9% saline, negative control) and experimental groups (n = 8) (treated with either referent drugs phenytoin sodium and diazepam (DZP) or BCP were injected intraperitoneally (i.p.) at different doses and were dissolved in Tween 80 at a volume of 10 ml kg−1. BCP (30, 100 and 300 mg kg−1) was administered 0.5 h and 4 h, respectively, before the MES, scPTZ test and rota-rod test. Phenytoin (30 mg kg−1) was used as a referent drug in the MES test. Diazepam (5 mg kg−1) was used as a referent drug in the scPTZ test. The selection of the doses was based on previous studies [
      • de Carvalho C.R.
      • Hoeller A.A.
      • Franco P.L.
      • Martini A.P.
      • Soares F.M.
      • Lin K.
      • Prediger R.D.
      • Whalley B.J.
      • Walz R.
      The cannabinoid CB2 receptor-specific agonist AM1241 increases pentylenetetrazole-induced seizure severity in Wistar rats.
      ,
      • de Oliveira C.C.
      • de Oliveira C.V.
      • Grigoletto J.
      • Rodrigo Ribeiro Leandro
      • Rafael Funck Vinícius
      • Beck Grauncke Ana
      • Lopes de Souza Thaíze
      • Schiefelbein Souto Naieli
      • Flávia Furian Ana
      • Rose Alencar Menezes Irwin
      • Schneider Oliveira Mauro
      Anticonvulsant activity of β-caryophyllene against pentylenetetrazol-induced seizures.
      ].
      Separate group was used for the Morris water maze test. Doses of 10, 25 and 50 mg kg−1, respectively, were applied 30 min before each “Probe test” on 2nd, 3rd and 4th day, respectively.
      The KA injection was performed 24 h after a sub-chronic treatment of BCP (50 and 100 mg kg−1) for 7 days.

      2.7 Morris water-maze test

      The maze, consisting of a circular pool (120 cm in diameter, 30 cm height) with warm water (23 °C), was located in a dimly lit (45 lx) and soundproof room with four extra-maze visual cues (A4-size sheets of white laminated paper with black geometric symbols). The pool was divided into four equal quadrants. An escape platform (9 cm in diameter, 21 cm height) at a fixed location was made of transparent Plexiglas and was immersed 1 cm under the surface of water in the center of the south-west quadrant (target quadrant). Four tests, from which the 1st one (the acquisition test) was compared to the following three tests (the probe test) that were performed for three consecutive days with one session a day. Each session consisted of four trials separated by 10 min interval. The animal was taken from the home cage and placed into the water maze at the semi-random start positions with its head facing the center of the water maze. When the mouse climbed onto the platform, the trial was stopped and the escape latency was recorded. The maximum trial length was 60 s. If a mouse failed to find a submerged platform within 60 s, it was placed on it for 15 s. The animal was kept on the platform before starting the next trial. Then it was placed in the pool again, but at a different location, and the next trial began upon its release. Thirty minutes after injection of BCP or vehicle, respectively, on the second, third and the fourth day, a ‘probe test’ was performed to estimate the rats' spatial learning capability of the location of the hidden platform. Navigation parameters were calculated by using a camera and a video tracking system (SMART PanLab software, Harvard Apparatus, USA). The calculated parameter was the latency in the first crossing of the platform location (target area). If a mouse was unable to find the platform within 60 s, it was given a latency score of 60 s.

      2.8 Lipid peroxidation assay (MDA)

      A 10% (w/v) tissue homogenates were prepared in 0.1 M phosphate buffer (pH 7.4). The homogenates were centrifuged at 10,000 × g for 15 min and aliquots of the supernatants were separated and used for biochemical estimation. Malondialdehyde (MDA) was measured by an ELISA reader at 540 (532 according to kit instructions) nm wavelength using Elisa test kit (Abcam, UK, ab118970), according the instructions of the manufacturer and was expressed as nmol gram tissue−1.

      2.9 Statistical analysis

      The results were expressed as mean ± standard error of mean (SEM). Significance of seizure stage was analyzed using ANOVA for non-parametric data (Kruskal-Wallis on ranks) followed by the Mann-Whitney U test. Other data were analyzed by one- or two- way ANOVA followed by Student–Newman–Keuls as post hoc test by GraphPad Prism (version 6.0; GraphPad San Diego, California, USA. copyright © 1994–1999). Differences were considered statistically significant when p < 0.05.

      3. Results

      3.1 Еffects of BCP on seizure susceptibility and acute neurotoxicity

      The anticonvulsant effect of BCP was assessed on MES and scPTZ tests, after i.p. injection of BCP at initial doses of 30, 100, and 300 mg/kg, respectively. In parallel, acute neurotoxicity was determined via the test for minimal motor impairment (rota-rod). Phenytoin and diazepam were used as reference drugs. Screening evaluation was executed at two time points, 0.5 h and 4 h. The results are shown in Table 1. In the MES test, BCP suppressed tonic-clonic seizures at the lowest dose of 30 mg kg−1 at 4.0-h, which provided 66.6% as compared to the 100% protection provided by phenytoin.
      Table 1Data representing anticonvulsant activity and neurotoxicity of Beta-caryophyllene (ICR mice, i.p.).
      DrugMES
      Maximal electroshock test (MES).
      scPTZ
      Subcutaneous pentylenetetrazole test (scPTZ).
      Rota-rod
      Test for neurotoxicity (rota-rod).
      0.5 h4.0 h0.5 h4.0 h0.5 h4.0 h
      Beta-caryophyllene30
      Phenytoin30303030
      Diazepam5555
      Data in the table indicate the minimum dose, whereby bioactivity or neurotoxicity was demonstrated in at least 50% of treated animals. A dash indicates the absence of activity or neurotoxicity at the maximum dose administrated (300 mg/kg).
      a Maximal electroshock test (MES).
      b Subcutaneous pentylenetetrazole test (scPTZ).
      c Test for neurotoxicity (rota-rod).
      In the scPTZ test, BCP was ineffective in the three doses used suggesting that the compound is unable to raise the seizure threshold. In the rota-rod test, no signs of neurotoxicity were observed in all applied doses compared to the referent drugs phenytoin and diazepam, respectively, at two time points (2 h and 4-h), respectively.
      Mice were pretreated with BCP (50 and 100 mg kg−1, i.p. for 7 days) to evaluate the protection of CB2 receptor agonist against the KA-induced SE. Matched group was treated with vehicle in the same manner before KA. Twenty four hours after the last injection of BCP the neurotoxin was administered at a dose of 30 mg kg−1. The seizure intensity was scored for a 3-h period of observation. The behavior observed after a systemic i.p. injection of a single excitotoxic dose of KA consisted of facial automatisms and head nodding which occured within 20 min after injection. Furthermore, this motor activity progressed to forelimb clonus and rearing in KA + Veh group (Fig. 1A). Decreased seizure scores were detected in the BCP groups, which reached a significant difference after 100th min (100 mg kg−1 BCP) and 120th min (50 mg kg−1), respectively.
      Fig. 1
      Fig. 1A The effect of β-caryophyllene (BCP) on behavioral seizure activity in kainic acid (KA)-treated mice. Mice were sub-chronically pretreated with BCP (50 mg kg−1 and 100 mg kg−1) for 7 days (n = 8) before KA (30 mg kg−1, i.p.). Behavioral seizure activity was evaluated according to the scales devised by Racine [
      • Racine R.
      • Rose P.A.
      • Burnham W.M.
      Afterdischarge thresholds and kindling rates in dorsal and ventral hippocampus and dentate gyrus.
      ]. Values are presented as mean ± S.E.M. (a) Significance: ap < 0.05 vs. KA + Veh, bp < 0.01 KA + BCP 50 vs. KA + BCP 100 (Kruskal-Wallis test). B. The effect of BCP on the levels of lipid peroxidation (MDA) in the hippocampus after the KA-induced status epilepticus in mice. Values are presented as mean ± S.E.M. (a) Significance: ap < 0.05 vs. Vehicle, bp < 0.05 vs. KA + Veh (One-way ANOVA). C. The effect of BCP on escape latency in the first crossing of the platform location (target area) in mice using Morris water maze test. Four groups were tested as follows: Vehicle, BCP 10 mg kg−1, BCP 25 mg kg−1, BCP 50 mg kg−1. Values are presented as the mean ± S.E. Significance: ap < 0.05 vs. Vehicle, bp < 0.01 vs. BCP 10 mg kg−1 vs. BCP 50 mg kg−1 (Two-way ANOVA).
      The effects of a sub-chronic i.p, injection of BCP on lipid peroxidation concomitant with the KA-induced SE are presented in Fig. 1B. The CB2 receptor agonist caused a significant increase in the level of lipid peroxidation in the hippocampus of KA + Veh group (p < 0.05 vs Vehicle group) (Fig. 1B). The activation of CB2 receptors significantly attenuated the KA-induced elevation of the lipid peroxidation in the hippocampus of mice sub-chronically treated with both 50 and 100 mg kg−1 BCP, respectively (p < 0.05 vs. KA + Veh group).

      3.2 Dose-dependent activity of BCP in Moris water maze test

      Single injection of BCP, 30 min before the each “probe test”, at three different doses of 10, 25 and 50 mg kg−1, respectively, given i.p., significantly improved spatial learning capacity. Two-way ANOVA showed a main effect of Treatment [F3,160 = 288; p < 0.0001] and Day [F3,160 = 472.9, p < 0.0001] as well as interaction between the two factors [F9,160 = 34.14, p < 0.0001] for the latency in the first crossing of the former platform location (target area). Post hoc comparison revealed a significant decrease in the latency to reach the former platform location in the three groups of mice given BCP from the 1st to the 3rd session (p < 0.05) (Fig. 1C). Moreover, a decrease in the latency to reach the platform target was significantly different in the group treated with the highest dose of 50 mg kg−1 BCP in comparison to the group treated with the lowest dose of 10 mg kg−1 (p < 0.05).

      4. Discussion

      Despite the broad range of seizure tests with different mechanisms of action, the two seizure test, MES and scPTZ are accepted as “gold standards” for initial screening tool for evaluation of newly designed substances. In the present work, we conducted a primary evaluation of a selective CB2 receptor agonist BCP following the protocol of the Epilepsy Therapy Screening Program [
      • Swinyard E.A.
      • Woodhead J.H.
      • White H.S.
      • Franklin M.R.
      Experimental selection, quantification and evaluation of anticonvulsants.
      ]. On the basis of this preliminary screening data in mice, we have been found that BCP is active in the MES test, suggesting a capability to inhibit seizure spread but not in the PTZ test, which is used to identify compounds that elevate the seizure threshold.
      Our results are in accordance with previous work revealing that i.c.v. microinfusion of the selective CB2 agonist AM1241 exerted a pro-convulsive effect against tonic-clonic seizures which was blocked by the selective CB receptor antagonist AM630 in Wistar rats [
      • de Carvalho C.R.
      • Hoeller A.A.
      • Franco P.L.
      • Martini A.P.
      • Soares F.M.
      • Lin K.
      • Prediger R.D.
      • Whalley B.J.
      • Walz R.
      The cannabinoid CB2 receptor-specific agonist AM1241 increases pentylenetetrazole-induced seizure severity in Wistar rats.
      ]. However, de Oliveira et al. [
      • de Oliveira C.C.
      • de Oliveira C.V.
      • Grigoletto J.
      • Rodrigo Ribeiro Leandro
      • Rafael Funck Vinícius
      • Beck Grauncke Ana
      • Lopes de Souza Thaíze
      • Schiefelbein Souto Naieli
      • Flávia Furian Ana
      • Rose Alencar Menezes Irwin
      • Schneider Oliveira Mauro
      Anticonvulsant activity of β-caryophyllene against pentylenetetrazol-induced seizures.
      ] reported that BCP exerted an anticonvulsant effect in the PTZ-induced myoclonic seizure test. However, the authors demonstrated that although the hight dose of 100 mg kg−1 of CB2 receptor agonist increased the latency for onset of myoclonic jerks, it was unable to prevent generalised seizures induced by a dose of 60 mg kg−1 PTZ in C57BL/6 mice. Huizenga et al. [
      • Huizenga M.N.
      • Wicker E.
      • Beck V.C.
      • Forcelli P.A.
      Anticonvulsant effect of cannabinoid receptor agonists in models of seizures in developing rats. CB1 receptor agonists as novel antiepileptic drugs targeting epilepsy.
      ] found that while CB1 and CB2 antagonism exacerbate seizures induced by the chemoconvulsant methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), only the mixed CB1/2 agonist and the selective CB1 receptor agonist possessed anticonvulsant activity but not the CB2 agonist HU-308 in Sprague-Dawley rat pups. Furthermore, the mixed CB1/2 agonist showed potency against PTZ seizures suggesting that activation of CB1 receptor underlie neuroprotective effect of the CB system in rat pups.
      In the literature there are data in support of the assumption that activation of CB1 receptor is an effective tool in the prevention of SE and its neurotoxic consequences but our results revealed for the first time that CB2 receptors also deserve exploration as a putative target against SE. Recently, Suleymanova [
      • Suleymanova E.M.
      • Shangaraeva V.A.1
      • van Rijn C.M.2
      • Vinogradova L.V.
      The cannabinoid receptor agonist WIN55. 212 reduces consequences of status epilepticus in rats.
      ] reported that acute injection of the CB1 aminoalkylindole agonist, WIN55, 212-2 after insult-induced by pilocarpine was able to prevent SE and exerted neuroprotection in the dentate hilus without preventing development of chronic epileptic state and spontaneous seizure activity in rats. It is well-known that the neurotoxicity of KA is due to its specific mechanism of action targeting ionotropic glutamate receptors in the hippocampus which contribute to neural damage mediated by oxidative stress. Furthermore, putative antioxidants and free radical scavengers are considered promising strategy to counteract the KA-induced neurotoxicity and neuronal damage. In the present study, we found that sub-chronic pretreatment with the CB2 receptor agonist BCP successfully attenuated seizure intensity in the KA test, which effect was accompanied by a suppression of concomitant with SE lipid peroxidation in the hippocampus. Recently, Stempel et al. [
      • Stempel A.V.
      • Stumpf A.
      • Zhang H.-Y.
      • Xi Z.-X.
      • Zimmer A.
      • Schmitz D.
      Cannabinoid type 2 receptors mediate a cell type-specific plasticity in the hippocampus.
      ], for the first time, demonstrated a detailed CB2 receptor expression and functionality in the hippocampus. Unlike the CB1 receptors, which were found to be preferably expressed presynaptically, the CB2 receptors were distributed on postsynaptic compartments of CA3/2 fields of the hippocampus indicative of divergence in CB receptor subtype functions. The authors concluded that the CB2 receptors in the CA3 and CA2 fields might exert a tonic inhibitory action. Unlike our findings, de Oliveira et al. [
      • de Oliveira C.C.
      • de Oliveira C.V.
      • Grigoletto J.
      • Rodrigo Ribeiro Leandro
      • Rafael Funck Vinícius
      • Beck Grauncke Ana
      • Lopes de Souza Thaíze
      • Schiefelbein Souto Naieli
      • Flávia Furian Ana
      • Rose Alencar Menezes Irwin
      • Schneider Oliveira Mauro
      Anticonvulsant activity of β-caryophyllene against pentylenetetrazol-induced seizures.
      ] reported that BCP was ineffective against oxidative stress (i.e., increase in the levels of thiobarbituric acid-reactive substances) produced by PTZ seizures in mice suggesting that BCP might exert a model-specific protection. However, we have confirmed the report of the above mentioned authors showing that CB2 agonist at a dose of 100 mg kg−1 improved recognition index in the object recognition test without affecting locomotion in the open-field, rota-rod, or forced swim tests in mice [
      • de Oliveira C.C.
      • de Oliveira C.V.
      • Grigoletto J.
      • Rodrigo Ribeiro Leandro
      • Rafael Funck Vinícius
      • Beck Grauncke Ana
      • Lopes de Souza Thaíze
      • Schiefelbein Souto Naieli
      • Flávia Furian Ana
      • Rose Alencar Menezes Irwin
      • Schneider Oliveira Mauro
      Anticonvulsant activity of β-caryophyllene against pentylenetetrazol-induced seizures.
      ]. We showed that BCP dose-dependently improved learning abilities in the hippocampus-dependent spatial memory test. Our preliminary data demonstrated that these low doses of BCP did not affect motor activity in the open field test suggesting that the effect of BCP on spatial memory was not due to the false positive effect.

      5. Conclusion

      The selective CB2 agonist BCP suppressed tonic-clonic seizures in the MES test and alleviated the KA-induced neurotoxicity and concomitant oxidative stress in mice suggesting that activation of CB2 receptors is protective against seizure spread and ROS activation resulting from SE. Based on our present results and few encouraging previous data that demonstrated anticonvulsant effect of CB2 receptor agonists in models of both generalized tonic–clonic and SE together with memory-improving capacity suggest that CB2 receptor agonists might be clinically useful as an adjunct treatment in SE to reduce oxidative stress, neurotoxicity and cognitive impairments.

      Conflict of interest

      The authors declare no conflict of interest.

      Acknowledgments

      This work was supported by the National Science Fund of Bulgaria (research grant # № DN 03/10 ) and the Medical University of Sofia (grant № D-74/2017 ). Keyla Machado was a visiting PhD student funded by the National Counsel of Technological and Scientific Development – MCTI/MEC/CNPq fellowship Programme [grant numbers CPF 028.823.723-45 ]. The authors would like to thank Tania Markovska for her technical assistance.

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