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Rosmarinic acid is anticonvulsant against seizures induced by pentylenetetrazol and pilocarpine in mice

Open AccessPublished:July 19, 2016DOI:https://doi.org/10.1016/j.yebeh.2016.06.037

      Highlights

      • Rosmarinic acid delays the onset of PTZ-induced myoclonic and tonic–clonic seizures.
      • Rosmarinic acid increases the latency to pilocarpine-induced tonic–clonic seizures.
      • Rosmarinic acid does not modify the consequences of pilocarpine-induced SE.

      Abstract

      Epilepsy is a chronic neurological disease characterized by spontaneous recurrent seizures (SRS). Current anticonvulsant drugs are ineffective in nearly one-third of patients and may cause significant adverse effects. Rosmarinic acid is a naturally occurring substance which displays several biological effects including antioxidant and neuroprotective activity. Since oxidative stress and excitotoxicity play a role in the pathophysiology of seizures, we aimed the present study to test the hypothesis that rosmarinic acid displays anticonvulsant and disease-modifying effects. Female C57BL/6 mice received rosmarinic acid (0, 3, 10, or 30 mg/kg; p.o.) 60 min before the injection of pentylenetetrazol (PTZ, 60 mg/kg; i.p.) or pilocarpine (300 mg/kg, i.p.). Myoclonic and generalized tonic–clonic seizure latencies and generalized seizure duration were analyzed by behavioral and electroencephalographic (EEG) methods. The effect of acute administration of rosmarinic acid on mice behavior in the open-field, object recognition, rotarod, and forced swim tests was also evaluated. In an independent set of experiments, we evaluated the effect of rosmarinic acid (3 or 30 mg/kg, p.o. for 14 days) on the development of SRS and behavioral comorbidities in the pilocarpine post-status epilepticus (SE) model of epilepsy. Rosmarinic acid dose-dependently (peak effect at 30 mg/kg) increased the latency to myoclonic jerks and generalized seizures in the PTZ model and increased the latency to myoclonic jerks induced by pilocarpine. Rosmarinic acid (30 mg/kg) increased the number of crossings, the time at the center of the open field, and the immobility time in the forced swim test. In the chronic epilepsy model, treatment with rosmarinic acid did not prevent the appearance of SRS or behavioral comorbidities. In summary, rosmarinic acid displayed acute anticonvulsant-like activity against seizures induced by PTZ or pilocarpine in mice, but further studies are needed to determine its epilepsy-modifying potential.

      Graphical abstract

      Keywords

      1. Introduction

      Epilepsy is a chronic neurological disease defined by a pathologic and enduring tendency to have recurrent seizures [
      • Fisher R.S.
      • Acevedo C.
      • Arzimanoglou A.
      • Bogacz A.
      • Cross J.H.
      • Elger C.E.
      • et al.
      ILAE official report: a practical clinical definition of epilepsy.
      ]. Importantly, epilepsy is linked to numerous physical, neurological, mental health, and cognitive comorbidities, including heart disease, autism spectrum disorders, Alzheimer's disease, depression, anxiety, and learning and memory deficits [
      • England M.J.
      • Liverman C.T.
      • Schultz A.M.
      • Strawbridge L.M.
      Epilepsy across the spectrum: promoting health and understanding.
      ]. In this context, epilepsy has been considered a major worldwide public health problem [
      • Schmidt D.
      • Sillanpaa M.
      Evidence-based review on the natural history of the epilepsies.
      ]. Accordingly, the World Health Organization estimates that epilepsy affects around 70 million people worldwide; 80% of them are in developing countries. In these countries, although most cases can be treated, around 75% of people with epilepsy are not receiving appropriate treatment [
      • WHO
      World Health Organization.
      ].
      Despite the availability of a wide range of antiepileptic drugs (AEDs), about one-third of individuals with epilepsy still experience seizures that do not respond to medication [
      • Perucca E.
      • French J.
      • Bialer M.
      Development of new antiepileptic drugs: challenges, incentives, and recent advances.
      ]. Development of new therapeutic strategies for seizure activity is therefore important, and understanding the molecular basis of seizure onset and maintenance, as well as probing for new targets for antiepileptic drugs, is a fundamental part of this process. In this context, phytomedicines are important in the development of new antiepileptic drugs, since it has been shown that several plant extracts and products may be useful for the treatment of convulsions or seizures [
      • Sucher N.J.
      • Carles M.C.
      A pharmacological basis of herbal medicines for epilepsy..
      ].
      Rosmarinic acid is an ester of caffeic acids and 3,4-dihydroxyphenyl lactic acid commonly found in a broad range of plant species [
      • Petersen M.
      • Simmonds M.S.
      Rosmarinic acid.
      ]. A number of biological activities have been described for rosmarinic acid, including antioxidant, antiinflammatory, and neuroprotective activity [
      • Kim G.D.
      • Park Y.S.
      • Jin Y.H.
      • Park C.S.
      Production and applications of rosmarinic acid and structurally related compounds.
      ,
      • Bigford G.E.
      • Del Rossi G.
      Supplemental substances derived from foods as adjunctive therapeutic agents for treatment of neurodegenerative diseases and disorders.
      ]. Since oxidative stress [
      • Martinc B.
      • Grabnar I.
      • Vovk T.
      Antioxidants as a preventive treatment for epileptic process: a review of the current status.
      ], inflammatory processes in the brain [
      • Vezzani A.
      • French J.
      • Bartfai T.
      • Baram T.Z.
      The role of inflammation in epilepsy.
      ], and excitotoxicity [
      • Bazan N.G.
      • Marcheselli V.L.
      • Cole-Edwards K.
      Brain response to injury and neurodegeneration: endogenous neuroprotective signaling.
      ] may contribute to the pathophysiology of seizures, it is of particular interest to determine if antioxidant, anti-inflammatory and neuroprotective agents are also anticonvulsant. antiinflammatory, and neuroprotective agents are also anticonvulsant. In this context, a few studies have investigated the effect of rosmarinic acid on seizure activity. For instance, a seven-day pretreatment with rosmarinic acid reduced the severity of kainate-induced seizures in rats [
      • Khamse S.
      • Sadr S.S.
      • Roghani M.
      • Hasanzadeh G.
      • Mohammadian M.
      Rosmarinic acid exerts a neuroprotective effect in the kainate rat model of temporal lobe epilepsy: underlying mechanisms.
      ]. Conversely, in another study, administration of rosmarinic acid did not prevent kindling development induced by pentylenetetrazol (PTZ) in mice [
      • Coelho V.R.
      • Vieira C.G.
      • de Souza L.P.
      • Moyses F.
      • Basso C.
      • Papke D.K.
      • et al.
      Antiepileptogenic, antioxidant and genotoxic evaluation of rosmarinic acid and its metabolite caffeic acid in mice.
      ]. In light of these apparently conflicting results, and in order to further evaluate the anticonvulsant potential of rosmarinic acid, we aimed the present study to investigate the effect of this natural product on seizure activity in experimental models of epilepsy in which it has not been evaluated. In this context, we tested the effect of rosmarinic acid against the seizures induced by acute injection of PTZ or pilocarpine, two convulsants that have been widely used in the preclinical screening of new AEDs [
      • Pitkänen A.
      • Schwartzkroin P.A.
      • Moshé S.L.
      Models of seizures and epilepsy.
      ]. Furthermore, we evaluated the effect of rosmarinic acid on spontaneous recurrent seizures (SRS) and behavioral comorbidities in the pilocarpine-induced status epilepticus (SE) model of epilepsy.

      2. Materials and methods

      2.1 Animals and reagents

      Female C57BL/6 mice (20–30 g; 30–60 days old) were used. Animals were maintained under controlled light and environment (12:12 h light–dark cycle, 24 ± 1 °C, 55% relative humidity) with free access to water and food (Supra™, Santa Maria, RS, Brazil). All experimental protocols aimed to keep the number of animals used to a minimum, as well as their suffering. These were conducted in accordance with national and international legislation (guidelines of the Brazilian Council of Animal Experimentation — CONCEA and of the U.S. Public Health Service's Policy on Humane Care and Use of Laboratory Animals — PHS Policy) and with the approval of the Ethics Committee for Animal Research of the Federal University of Santa Maria.
      Pentylenetetrazol (PTZ) and pilocarpine were purchased from Sigma (Sigma-Aldrich, St. Louis, Missouri) and were dissolved in 0.9% NaCl to 6 mg/mL and 30 mg/mL, respectively. Rosmarinic acid was purchased from Sigma and dissolved in 0.9% NaCl containing 5% Tween 80. Doses and schedules for drug injections were selected based on the literature [
      • Khamse S.
      • Sadr S.S.
      • Roghani M.
      • Hasanzadeh G.
      • Mohammadian M.
      Rosmarinic acid exerts a neuroprotective effect in the kainate rat model of temporal lobe epilepsy: underlying mechanisms.
      ,
      • Coelho V.R.
      • Vieira C.G.
      • de Souza L.P.
      • Moyses F.
      • Basso C.
      • Papke D.K.
      • et al.
      Antiepileptogenic, antioxidant and genotoxic evaluation of rosmarinic acid and its metabolite caffeic acid in mice.
      ] and on pilot experiments.

      2.2 Acute seizure models

      Animals were individually placed in glass boxes and administered, by gavage, with increasing doses of rosmarinic acid (3, 10, or 30 mg/kg) or vehicle (0.9% NaCl containing 0.05% Tween 80). Sixty minutes thereafter, PTZ (60 mg/kg) or pilocarpine (300 mg/kg) was injected intraperitoneally. All solutions were administered at 10 mL per kg of body weight. After the injection of the convulsant, seizure behavior was followed for 15 min (PTZ) or 60 min (pilocarpine). The latency to myoclonic jerks, the latency to generalized seizures, and the duration of the first generalized seizure were recorded.

      2.3 Electroencephalography (EEG)

      Seizure activity and the effect of rosmarinic acid in acute seizure models induced by PTZ or pilocarpine were evaluated in a subset of animals (n = 3–4) by electroencephalographic (EEG) recordings. The procedures for recording electrode implantation and EEG recordings are described in detail elsewhere [
      • Oliveira C.C.
      • Oliveira C.V.
      • Grigoletto J.
      • Ribeiro L.R.
      • Funck V.R.
      • Grauncke A.C.
      • et al.
      Anticonvulsant activity of beta-caryophyllene against pentylenetetrazol-induced seizures.
      ]. Briefly, a 30-min baseline recording was obtained to establish an adequate control period. After the baseline recording, mice were injected with rosmarinic acid (30 mg/kg, p.o.) or vehicle (10 mL/kg, p.o.), 60 min before the injection of PTZ (60 mg/kg, i.p.) or pilocarpine (300 mg/kg, i.p.). Following convulsant injection, the behavior was monitored, and EEG was recorded for 15 min (PTZ) or 60 min (pilocarpine). The EEG signals were stored in a PC for offline analysis.

      2.4 Pilocarpine-induced SE and chronic model of epilepsy

      Status epilepticus was induced in C57BL/6 mice following an improved procedure in which repeated low doses of pilocarpine (100 mg/kg, i.p.) are injected until the onset of SE; this ramping protocol has been shown to reduce mortality after SE [
      • Groticke I.
      • Hoffmann K.
      • Loscher W.
      Behavioral alterations in the pilocarpine model of temporal lobe epilepsy in mice.
      ]. Briefly, 30 min before the injections of pilocarpine, methylscopolamine (a muscarinic antagonist) was administered intraperitoneally (1 mg/kg) to reduce adverse peripheral effects. Next, mice were intraperitoneally injected with repeated doses of pilocarpine hydrochloride every 20 min until onset of SE, defined by continuous limbic seizure activity. Status epilepticus was terminated after 60 min with diazepam (10 mg/kg, i.p.). Control animals received methylscopolamine and diazepam but received 0.9% NaCl instead of pilocarpine. All mice were hand-fed with moistened chow and fresh fruits (apples and bananas) and injected with 5% dextrose in lactated Ringer's solution for three days following the SE or control procedure for welfare purposes.
      In order to evaluate the effect of rosmarinic acid on SRS and behavioral comorbidities, control and post-SE mice received daily doses of vehicle or rosmarinic acid (3 or 30 mg/kg, p.o.) during 14 consecutive days. Treatment with rosmarinic acid started 3 h after diazepam, and all solutions were prepared fresh daily. Starting 24 h after the SE, all animals were monitored daily for the appearance of SRS. Monitoring was performed during the light phase of the circadian cycle (8–10 h per day) and was carried out until the end of the experimental period (14 days after SE), totaling approximately 100–120 h per animal. In addition, all SRS that occurred during handling (weighing, gavage, tagging, etc.) were noted.

      2.5 Behavioral tests

      The effect of rosmarinic acid on mice behavior or behavioral comorbidities of epilepsy was evaluated using two different protocols. The sequence of behavioral tests was organized from the least to the most aversive [
      • Oliveira C.C.
      • Oliveira C.V.
      • Grigoletto J.
      • Ribeiro L.R.
      • Funck V.R.
      • Grauncke A.C.
      • et al.
      Anticonvulsant activity of beta-caryophyllene against pentylenetetrazol-induced seizures.
      ].
      Protocol #1: In this set of experiments, an anticonvulsant dose of rosmarinic acid (30 mg/kg) was administrated in naïve mice 60 min before each test (open-field, object recognition, rotarod, and forced swim). Independent groups were used in each test, and each animal was used only once.
      Protocol #2: Starting one week after the induction of SE, control and SE mice were subjected to a behavioral test battery. All mice underwent all behavioral tests in the following sequence: open-field, object recognition, rotarod, sucrose preference, and forced swim.
      The procedures for open-field, object recognition, and forced swim tests are described in detail elsewhere [
      • Pitkänen A.
      • Schwartzkroin P.A.
      • Moshé S.L.
      Models of seizures and epilepsy.
      ]. The rotarod test for naïve animals was carried out as reported in [
      • Oliveira C.C.
      • Oliveira C.V.
      • Grigoletto J.
      • Ribeiro L.R.
      • Funck V.R.
      • Grauncke A.C.
      • et al.
      Anticonvulsant activity of beta-caryophyllene against pentylenetetrazol-induced seizures.
      ], but the paradigm was different for post-SE animals (cutoff latency was 300 s, and the procedure included an additional training session). For the sucrose preference, test mice were placed in individual cages which gave access to two bottles, one with water (100 mL) and the other with a 4% aqueous sucrose solution (100 mL). Water consumption and sucrose consumption over 48 h were measured, and sucrose preference was calculated as a percentage of the total fluid consumed.

      2.6 Statistical analyses

      Kolmogorov–Smirnov test was used to verify data normality, and Bartlett's test was used to verify homogeneity of variances. Nonparametric data (seizure latencies) were analyzed by Kruskal–Wallis test followed by post hoc analyses with Dunn's multiple comparisons test. Dose–response relationships were tested with Spearman's rank correlation test. Acute seizure duration, latency to the first SRS, and number of SRS were analyzed by one-way ANOVA. Data from behavioral testing were analyzed by two-tailed unpaired Student's t-test (protocol #1) or two-way ANOVA (protocol #2). Parametric post hoc analyses were carried out by Student–Newman–Keuls multiple comparison test. A probability of <0.05 was considered significant.

      3. Results

      The effect of increasing doses of rosmarinic acid on the seizures induced by PTZ is shown in Fig. 1. Statistical analysis revealed that the dose of 30 mg/kg increased the latency for the first myoclonic jerk [H(3) = 9.102; P < 0.05 — Fig. 1A] and the first generalized tonic–clonic seizure [H(3) = 10.55; P < 0.05 — Fig. 1B]. Spearman analysis revealed a significant positive correlation between the doses of rosmarinic acid and the latency to PTZ-induced myoclonic (rs = 0.5285; P < 0.05) and generalized (rs = 0.5548; P < 0.05) seizures. The duration of generalized tonic–clonic convulsions induced by PTZ was not altered by rosmarinic acid [F(3,30) = 0.451; P > 0.05 — Fig. 1C]. To confirm and extend these data, we also evaluated a subset of animals by electroencephalographic (EEG) recordings. Electroencephalographic experiments depicted in Fig. 1D and E confirmed that rosmarinic acid (30 mg/kg) delayed the appearance of PTZ-induced myoclonic jerks and tonic–clonic generalized seizures.
      Fig. 1
      Fig. 1Effect of oral administration of increasing doses of rosmarinic acid (0, 3, 10, or 30 mg/kg; p.o.) on seizures induced by pentylenetetrazol (PTZ, 60 mg/kg; i.p.). Latencies to (A) myoclonic jerks or (B) generalized seizure and the (C) duration of the first generalized seizure were measured. Rosmarinic acid or vehicle was administered 60 min before the injection of PTZ. Data are presented for n = 7–10 per group. In panels A and B, the box bounds the interquartile range divided by the median, and whiskers extend to minimum and maximum values beyond the box. Data in panel C are mean ± standard error of the mean. The asterisk indicates a statistically significant difference from the vehicle-treated group (P < 0.05). Also shown are representative electroencephalograms observed after administration of PTZ in animals treated with (D) vehicle (10 mL/kg, p.o.) or (E) rosmarinic acid (30 mg/kg, p.o.). The arrow indicates the administration of PTZ.
      We also evaluated the effect of rosmarinic acid on the seizures induced by pilocarpine. Statistical analysis revealed that the dose of 30 mg/kg increased the latency for the first myoclonic jerk [H(3) = 12.72; P < 0.05 — Fig. 2A ]. On the other hand, onset latency [H(3) = 1.123; P > 0.05 — Fig. 2B] and duration [F(3,35) = 0.2024; P > 0.05 — Fig. 2C] of the first pilocarpine-induced generalized tonic–clonic seizure did not change. Spearman analysis revealed a significant positive correlation between the doses of rosmarinic acid and the latency to myoclonic seizures induced by pilocarpine (rs = 0.5753; P < 0.05). Electroencephalographic experiments depicted in Fig. 2D and E confirmed that rosmarinic acid (30 mg/kg) treatment delayed the appearance of pilocarpine-induced myoclonic jerks.
      Fig. 2
      Fig. 2Effect of oral administration of increasing doses of rosmarinic acid (0, 3, 10, or 30 mg/kg; p.o.) on seizures induced by pilocarpine (300 mg/kg, i.p.). Latencies to (A) myoclonic jerks or (B) generalized seizure and the (C) duration of the first generalized seizure were measured. All solutions were administered 60 min before the injection of pilocarpine. Data are presented for n = 8–11 per group. In panels A and B, the box bounds the interquartile range divided by the median, and whiskers extend to minimum and maximum values beyond the box. Data in panel C are mean ± standard error of the mean. The asterisk indicates a statistically significant difference from the vehicle-treated group (P < 0.05). Also shown are representative electroencephalograms observed after administration of pilocarpine in animals treated with (D) vehicle (10 mL/kg, p.o.) or (E) rosmarinic acid (30 mg/kg, p.o.). The arrow indicates the administration of pilocarpine.
      In order to investigate whether effects on behavior and motor skills accompanied the anticonvulsant effect of rosmarinic acid, we tested independent groups of mice using the open-field, object recognition, rotarod, and forced swim tests (Fig. 3). Statistical analysis revealed that rosmarinic acid increased the number of crossings [t(10) = 3.790; P < 0.05 — Fig. 3A] and time spent exploring the center [t(10) = 2.327; P < 0.05 — Fig. 3B] in the open-field test. The number of rearing responses [t(10) = 1.503; P > 0.05 — Fig. 3C] or the latency to start the exploration [t(10) = 0.088; P > 0.05 — Fig. 3D] was not altered. In addition, no significant differences were found in the object recognition test regarding the time spent in object exploration during the habituation trial [t(8) = 0.6226; P > 0.05 — Fig. 3E] or in the recognition index 24 h after habituation [t(8) = 0.0815; P > 0.05 — Fig. 3F]. The latency to fall in the rotarod test was not altered by rosmarinic acid [t(22) = 0.0623; P > 0.05 — Fig. 3G]. On the other hand, rosmarinic acid increased the immobility time in the forced swim test [t(8) = 2.639; P < 0.05 — Fig. 3H].
      Fig. 3
      Fig. 3Effect of oral administration of rosmarinic acid (30 mg/kg, p.o.) on selected behavioral parameters. (A) Number of crossings, (B) time spent exploring the center, (C) number of rearing responses, and (D) latency to start exploration in the open-field test; (E) time spent in object exploration in the habituation session and (F) the recognition index in the object recognition test; (G) latency to fall off the rod in the rotarod test and (H) immobility time in the forced swim test. Independent groups of mice were used in each test. Data are presented as mean + standard error of the mean. The number of animals was 6 per group in the open-field test, 5 per group in the object recognition test, 12 per group in the rotarod test, and 5 per group in the forced swim test. The asterisk indicates a statistically significant difference from the vehicle-treated group (P < 0.05). Statistical analyses were performed with the two-tailed unpaired Student's t-test.
      In the post-SE model of epilepsy, we found no effect of rosmarinic acid on the latency to the first SRS [F(2,22) = 0.6065; P > 0.05 — Fig. 4A ] or on the total number of SRS during the 2-week monitoring period [F(2,22) = 0.5628; P > 0.05 — Fig. 4B]. In addition, no effect of rosmarinic acid was detected on the behavioral comorbidities of epilepsy. In the open-field test, the number of crossings was not altered by SE or rosmarinic acid [F(2,38) = 0.1561; P > 0.05 — Fig. 5A ], but post-SE mice spent less time exploring the center [F(1,38) = 20.14; P < 0.05 — Fig. 5B], exhibited fewer rearing responses [F(1,38) = 23.99; P < 0.05 — Fig. 5C], and showed increased latency to start exploration [F(1,38) = 5.499; P < 0.05 — Fig. 5D] compared with their control counterparts. In the object recognition test, post-SE mice spent less time exploring the objects in the habituation session [F(1,38) = 57.35; P < 0.05 — Fig. 5E]. The total exploration time was reduced to such an extent that made the analysis of the recognition index at 4 or 24 h unfeasible (data not shown). Considering the sucrose preference test, post-SE mice displayed reduced consumption of sucrose in comparison with their control counterparts [F(1,38) = 15.64; P < 0.05 — Fig. 5F]. In the rotarod test, the latency to fall was not altered by SE or rosmarinic acid [F(2,38) = 0.3215; P > 0.05 — Fig. 5G]. In the forced swim test, post-SE mice showed reduced immobility time when compared with controls [F(1,38) = 11.99; P < 0.05 — Fig. 5H]. Behavioral changes displayed by post-SE mice were not modified by rosmarinic acid.
      Fig. 4
      Fig. 4Effect of oral administration of increasing doses of rosmarinic acid (0, 3, or 30 mg/kg; p.o.) on the development of SRS after pilocarpine-induced SE. (A) Latency to the first SRS and (B) number of SRS. Data are presented as mean + standard error of the mean for n = 8–9 per group. Statistical analyses were carried out by one-way ANOVA.
      Fig. 5
      Fig. 5Effect of oral administration of increasing doses of rosmarinic acid (0, 3, or 30 mg/kg; p.o.) on behavioral comorbidities of epilepsy. (A) Number of crossings, (B) time spent exploring the center, (C) number of rearing responses, and (D) latency to start exploration in the open-field test; (E) time spent in object exploration in the habituation session of the object recognition test; (F) sucrose preference; (G) latency to fall off the rod in the rotarod test and (H) immobility time in the forced swim test. Independent groups of mice were used in each test. Data are presented as mean + standard error of the mean for n = 6–9 per group. The asterisk indicates a statistically significant difference from each respective control group (P < 0.05). Statistical analyses were carried out by two-way ANOVA followed by post hoc analyses with the Student–Newman–Keuls multiple comparisons test.

      4. Discussion

      In the present study, we showed that acute oral administration of rosmarinic acid (30 mg/kg) increased the latency to myoclonic jerks induced by PTZ or pilocarpine and the latency to PTZ-induced generalized seizures. Electroencephalographic experiments confirmed the results from behavioral seizure analysis, demonstrating that the rosmarinic-acid-induced delay of seizure onset also occurs at the electrophysiological level. Conversely, we found no effect of rosmarinic acid on the development of SRS or behavioral comorbidities in the chronic epilepsy model elicited by pilocarpine-induced SE. Altogether, data gathered in the present study confirm and extend literature data suggesting that rosmarinic acid displays anticonvulsant properties and limited, if any, antiepileptogenic or epilepsy-modifying activity.
      Several biological actions have been described for rosmarinic acid [
      • Kim G.D.
      • Park Y.S.
      • Jin Y.H.
      • Park C.S.
      Production and applications of rosmarinic acid and structurally related compounds.
      ], but only a few studies have investigated the effect of this natural product on seizure activity. For instance, in the study by Khamse et al. [
      • Khamse S.
      • Sadr S.S.
      • Roghani M.
      • Hasanzadeh G.
      • Mohammadian M.
      Rosmarinic acid exerts a neuroprotective effect in the kainate rat model of temporal lobe epilepsy: underlying mechanisms.
      ], rosmarinic acid was orally administered at the dose of 10 mg/kg/day for seven days, and 1 h after the last injection, a single dose of kainate was injected unilaterally into the right hippocampus of Wistar rats. Animals treated with rosmarinic acid exhibited only mild to moderate behavioral signs (lower seizure scores) when compared with the vehicle–kainate group [
      • Khamse S.
      • Sadr S.S.
      • Roghani M.
      • Hasanzadeh G.
      • Mohammadian M.
      Rosmarinic acid exerts a neuroprotective effect in the kainate rat model of temporal lobe epilepsy: underlying mechanisms.
      ]. In the study by Coelho et al. [
      • Coelho V.R.
      • Vieira C.G.
      • de Souza L.P.
      • Moyses F.
      • Basso C.
      • Papke D.K.
      • et al.
      Antiepileptogenic, antioxidant and genotoxic evaluation of rosmarinic acid and its metabolite caffeic acid in mice.
      ], authors investigated the effect of rosmarinic acid on PTZ-elicited kindling development, an experimental model of epileptogenesis in which repeated administrations of low doses of a convulsant drug led to a progressive decrease in the seizure threshold. Animals received intraperitoneal injections of rosmarinic acid (1, 2, or 4 mg/kg) 30 min before the subeffective dose of PTZ (50 mg/kg, s.c.), and seizure behavior was recorded for 30 min. Rosmarinic acid did not prevent the kindling development and had no effect on seizure latency, suggesting no antiepileptogenic effects [
      • Coelho V.R.
      • Vieira C.G.
      • de Souza L.P.
      • Moyses F.
      • Basso C.
      • Papke D.K.
      • et al.
      Antiepileptogenic, antioxidant and genotoxic evaluation of rosmarinic acid and its metabolite caffeic acid in mice.
      ].
      The mechanisms underlying the acute anticonvulsant effect of rosmarinic acid may include activation of the GABAergic system. For instance, rosmarinic acid is an in vitro inhibitor of the GABA-degrading enzyme, GABA transaminase, as identified in a bioassay-guided fractionation of Melissa officinalis L. methanolic extract [
      • Awad R.
      • Muhammad A.
      • Durst T.
      • Trudeau V.L.
      • Arnason J.T.
      Bioassay-guided fractionation of lemon balm (Melissa officinalis L.) using an in vitro measure of GABA transaminase activity.
      ]. Regarding this point, it is important to note that the clinically available anticonvulsant, vigabatrin, was rationally designed to inhibit GABA transaminase, which results in a widespread increase of GABA concentrations in the brain [
      • Ben-Menachem E.
      Mechanism of action of vigabatrin: correcting misperceptions.
      ]. On the other hand, the decrease of glutamatergic neurotransmission may also play a role in the anticonvulsant effect of rosmarinic acid, since it has been shown that this natural product produces a significant and dose-dependent (0.3–3 mg/kg) inhibition of the nociceptive response caused by intraplantar injection of glutamate [
      • Guginski G.
      • Luiz A.P.
      • Silva M.D.
      • Massaro M.
      • Martins D.F.
      • Chaves J.
      • et al.
      Mechanisms involved in the antinociception caused by ethanolic extract obtained from the leaves of Melissa officinalis (lemon balm) in mice.
      ].
      In the present study, we also found that the anticonvulsant dose of rosmarinic acid increased the immobility time in the forced swim test. Initially, this datum appears incongruent with previous studies showing antidepressant effects of rosmarinic acid. For instance, acute treatment with a low dose of rosmarinic acid (2 mg/kg), 30 min prior to the start of the test session of forced swimming, reduced the duration of immobility, suggesting an antidepressant-like effect [
      • Takeda H.
      • Tsuji M.
      • Inazu M.
      • Egashira T.
      • Matsumiya T.
      Rosmarinic acid and caffeic acid produce antidepressive-like effect in the forced swimming test in mice.
      ]. Of course, one important difference between the present study and that by Takeda et al. [
      • Takeda H.
      • Tsuji M.
      • Inazu M.
      • Egashira T.
      • Matsumiya T.
      Rosmarinic acid and caffeic acid produce antidepressive-like effect in the forced swimming test in mice.
      ] is the rosmarinic acid dosing, since the dose used here was 15 times larger (30 versus 2 mg/kg). Notwithstanding, the potential improvement of GABAergic transmission may also explain the increase in the immobility time after administration of rosmarinic acid. In fact, it has been shown that small doses of GABA or muscimol (a GABAA receptor agonist) decrease the forced-swimming-induced immobility period, while higher doses enhance the immobility period [
      • Aley K.O.
      • Kulkarni S.K.
      GABA-mediated modification of despair behavior in mice.
      ]. In addition, it is worth mentioning that prolonged forced-swim-test immobility was also observed with aminooxyacetic acid, a GABA-transaminase inhibitor [
      • Nagatani T.
      • TSaRK
      The effect of diazepam and of agents which change GABAergic functions in immobility in mice.
      ].
      Despite the increased immobility time in the forced swim test, it is important to note the presently reported anticonvulsant effect of rosmarinic acid accompanied by modest anxiolytic-like activity (as suggested by the increased number of crossings and time at the center of the open-field arena). In addition, rosmarinic acid did not cause adverse effects on spontaneous (open-field crossings) or forced (rotarod) motor performance. These results are in agreement with the recognized low toxicity of rosmarinic acid in mice, as shown by an LD50 of 561 mg/kg after intravenous administration [
      • Petersen M.
      • Simmonds M.S.
      Rosmarinic acid.
      ].
      Considering the lack of effect of rosmarinic acid on SRS and behavioral comorbidities of epilepsy in the chronic pilocarpine model, one possible explanation could be the well-known impairment of the GABAergic system that occurs during SE and in the course of epileptogenesis. For instance, increased internalization and consequent decreased surface expression of GABA receptor subunits occur during SE [
      • Goodkin H.P.
      • Yeh J.L.
      • Kapur J.
      Status epilepticus increases the intracellular accumulation of GABAA receptors.
      ]. In addition, GABAA receptor subunit composition is altered in epilepsy models, and these changes are associated with pharmacoresistance to GABAA receptor agonists [
      • Brooks-Kayal A.R.
      • Shumate M.D.
      • Jin H.
      • Rikhter T.Y.
      • Coulter D.A.
      Selective changes in single cell GABA(A) receptor subunit expression and function in temporal lobe epilepsy.
      ]. Alternatively, other schedules of rosmarinic acid administration may be tested in future studies, since it has been shown that the timing and schedule of drug administration critically affect the outcomes of SE in mice [
      • Jiang J.
      • Yang M.S.
      • Quan Y.
      • Gueorguieva P.
      • Ganesh T.
      • Dingledine R.
      Therapeutic window for cyclooxygenase-2 related anti-inflammatory therapy after status epilepticus.
      ]. In addition, it is also important to consider that most previous preclinical attempts to develop antiepileptogenic strategies based on monotherapies were unsuccessful [
      • Klee R.
      • Tollner K.
      • Rankovic V.
      • Romermann K.
      • Schidlitzki A.
      • Bankstahl M.
      • et al.
      Network pharmacology for antiepileptogenesis: tolerability of multitargeted drug combinations in nonepileptic vs. post-status epilepticus mice.
      ], and thus, combinatorial approaches (i.e., polytherapy) targeting multiple mechanisms relevant for epileptogenesis have been considered more promising options for future development of epilepsy-modifying drugs [
      • Loscher W.
      • Klitgaard H.
      • Twyman R.E.
      • Schmidt D.
      New avenues for anti-epileptic drug discovery and development.
      ]. In this context, given its acute anticonvulsant effects, rosmarinic acid associated with other drugs may be an interesting alternative to be tested in future studies.

      Conclusion

      Rosmarinic acid displays anticonvulsant activity against acute seizures induced by PTZ and pilocarpine in mice. No effects of rosmarinic acid on the development of SRS and behavioral comorbidities in the chronic pilocarpine model of epilepsy were found. Additional studies are needed to investigate the clinical implications of these findings as well as their underlying mechanisms.

      Acknowledgments

      The authors gratefully acknowledge the student fellowships from Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior ( CAPES ) (to J.G., C.V.O., N.S.S., and M.L.F.), CNPq / PIBIC (to A.C.B.G.), and FAPERGS / PROBIC (to T.L.S.). Authors A.F.F., A.R.S.S., and M.S.O. are grantees of CNPq research productivity fellowships. The authors thank Dr. Luiz Fernando Freire Royes and Michele Rechia Fighera for kindly providing EEG laboratory facilities.
      Conflict of interest statement
      The authors declare no conflict of interest.

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