Beneficial Effects of Rosmarinic Acid In Vitro and In Vivo Models of Epileptiform Activity Induced by Pilocarpine.

Epilepsy is characterized by a predisposition to generate recurrent and spontaneous seizures; it affects millions of people worldwide. Status epilepticus (SE) is a severe type of seizure. In this context, screening potential treatments is very important. In the present study, we evaluated the beneficial effects of rosmarinic acid (RA) in pilocarpine-induced in vitro and in vivo models of epileptiform activity. Using an in vitro model in combined entorhinal cortex-hippocampal from Wistar rats we evaluated the effects of RA (10 µg/mL) on the lactate release and a glucose fluorescent analogue, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NDBG), after incubation in high potassium aCSF supplemented or not with pilocarpine. In the in vivo model, SE was induced in male C57BL/6 mice by pilocarpine. At 1, 24, and 48 h after the end of SE mice were treated with RA (30 mg/kg/v.o.). We evaluated the neuromotor impairment by neuroscore tests and protein carbonyl levels in the cerebral cortex. In both in vitro models, RA was able to decrease the stimulated lactate release, while no effect on 2-NBDG uptake was found. RA has beneficial effects in models of epileptiform activity in vivo and in vitro. We found that RA treatment attenuated SE-induced neuromotor impairment at the 48 h timepoint. Moreover, post-SE treatment with RA decreased levels of protein carbonyls in the cerebral cortex of mice when compared to their vehicle-treated counterparts. Importantly, RA was effective in a model of SE which is relevant for the human condition. The present data add to the literature on the biological effects of RA, which could be a good candidate for add-on therapy in epilepsy.

Beta-caryophyllene attenuates short-term recurrent seizure activity and blood-brain-barrier breakdown after pilocarpine-induced status epilepticus in rats.

BACKGROUND: Status epilepticus (SE) is a neurological life-threatening condition, resulting from the failure of the mechanisms responsible for seizure termination. SE is often pharmacoresistant and associated with significant morbidity and mortality. Hence, ceasing or attenuating SE and its consequences is of fundamental importance. Beta-caryophyllene is a functional CB2 receptor agonist and exhibit a good safety profile. Besides, it displays beneficial effects in several experimental conditions, including neuroprotective activity. In the present study we aimed to investigate the effects of beta-caryophyllene on pilocarpine-induced SE. METHODS: Wistar rats were submitted to pilocarpine-induced SE and monitored for 24 h by video and EEG for short-term recurrence of seizure activity (i.e. seizures occurring within 24 h after termination of SE). Rats received beta-caryophyllene (100 mg/kg, ip) at 1, 8- and 16-hours after SE. Twenty-four hours after SE we evaluated sensorimotor response, neuronal damage (fluoro jade C staining) and serum albumin infiltration into brain parenchyma. RESULTS: Beta-caryophyllene-treated animals presented fewer short-term recurrent seizures than vehicle-treated counterparts, suggesting an anticonvulsant effect after SE. Behavioral recovery from SE and the number of fluoro jade C positive cells in the hippocampus and thalamus were not modified by beta-caryophyllene. Treatment with beta-caryophyllene attenuated the SE-induced increase of albumin immunoreactivity in the hippocampus, indicating a protective effect against blood-brain-barrier breakdown. CONCLUSIONS: Given the inherent difficulties in the treatment of SE and its consequences, present results suggest that beta-caryophyllene deserve further investigation as an adjuvant therapeutic strategy for SE.

Anticonvulsant activity of ß-caryophyllene in association with pregabalin in a seizure model in rats.

Epilepsy is a common chronic neurological disease. The hallmark of epilepsy is recurrent, unprovoked seizures. Unfortunately, drug resistance is frequent in patients with epilepsy, and therefore improved therapeutic strategies are needed. In the present study, we tested the effect of pregabalin in association with beta-caryophyllene, an FDA-approved food additive and naturally occurring agonist of cannabinoid receptor subtype 2 against pentylenetetrazol (PTZ)-induced seizures in rats. In addition, selected neurochemical parameters were evaluated in the cerebral cortex. Adult male Wistar rats received beta-caryophyllene (100 mg/kg), pregabalin (40 mg/kg) or their combination before PTZ (60 mg/kg). Appropriated vehicle-treated control groups were included for each treatment. Animals were monitored by video-EEG and the latency to myoclonic seizures, latency to tonic-clonic seizures, tonic-clonic seizure duration and overall seizure score were measured. Glial fibrillary acidic protein (GFAP) release, erythroid-related factor 2 (Nrf2), c-fos and 3-nitrotyrosine (3-NT) levels were evaluated in the frontal cortex. We found that beta-caryophyllene plus pregabalin increased the latency to PTZ-induced myoclonic and tonic-clonic seizures and decreased the tonic-clonic seizure duration and overall seizure score. Interestingly, lower levels of GFAP, c-Fos and 3-NT were observed in animals receiving beta-caryophyllene and pregabalin treatments. Our results suggest a possible synergic effect of beta-caryophyllene plus pregabalin against PTZ induced-seizures.

TRPA1 involvement in depression- and anxiety-like behaviors in a progressive multiple sclerosis model in mice.

Progressive multiple sclerosis (PMS) is a neurological disease associated with the development of depression and anxiety, but treatments available are unsatisfactory. The transient receptor potential ankyrin 1 (TRPA1) is a cationic channel activated by reactive compounds, and the blockage of this receptor can reduce depression- and anxiety-like behaviors in naive mice. Thus, we investigated the role of TRPA1 in depression- and anxiety-like behaviors in a PMS model in mice. PMS model was induced in C57BL/6 female mice by the experimental autoimmune encephalomyelitis (EAE). Nine days after the PMS-EAE induction, behavioral tests (tail suspension and elevated plus maze tests) were performed to verify the effects of sertraline (positive control), selective TRPA1 antagonist (A-967,079), and antioxidants (α-lipoic acid and apocynin). The prefrontal cortex and hippocampus were collected to evaluate biochemical and inflammatory markers. PMS-EAE induction did not cause locomotor changes but triggered depression- and anxiety-like behaviors, which were reversed by sertraline, A-967,079, α-lipoic acid, or apocynin treatments. The neuroinflammatory markers (AIF1, GFAP, IL-1ß, IL-17, and TNF-α) were increased in mice's hippocampus. Moreover, this model did not alter TRPA1 RNA expression levels in the hippocampus but decrease TRPA1 levels in the prefrontal cortex. Moreover, PMS-EAE induced an increase in NADPH oxidase and superoxide dismutase activities and TRPA1 endogenous agonist levels (hydrogen peroxide and 4-hydroxynonenal). TRPA1 plays a fundamental role in depression- and anxiety-like behaviors in a PMS-EAE model; thus, it could be a possible pharmacological target for treating these symptoms in PMS.

Neuroprotective effects of thromboxane receptor antagonist SQ 29,548 after pilocarpine-induced status epilepticus in mice.

Thromboxane A2 (TXA2) is an important eicosanoid in the cardiovascular system, and increasing evidence suggests that TXA2 receptors (TPs) and their ligands may constitute valuable tools for the development of neuroprotective drugs. However, the role of TPs on seizure-induced damage has not been investigated. Therefore, we evaluated the effects of SQ 29,548, a potent and selective TP antagonist-on neuromotor performance, neurodegeneration, reactive astrocytosis, and c-Fos protein immunoreactivity after pilocarpine-induced status epilepticus (SE) in mice. Adult C57BL/6 mice received intracerebroventricular SQ 29,548 injections 90 min and 24 h after pilocarpine-induced SE. We found that SQ 29,548 prevented the impairment of neuromotor performance (Neuroscore test) 48 h after pilocarpine-induced SE. Data analysis suggested the existence of two subgroups of SQ 29,548-treated post-SE animals. Eight out of 12 SQ 29,548-treated animals displayed Neuroscore values identical to those of vehicle-treated controls, and were considered SQ 29,548 responders. However, 4 out of 12 SQ 29,548-treated animals did not show any improvement in Neuroscore values, and were considered SQ 29,548 non-responders. Treatment with SQ 29,548 attenuated SE-induced increase in the number of FJC- or GFAP-positive cells in the hippocampus of SQ 29,548 responders. In addition, SQ 29,548 prevented the SE-elicited increase of c-Fos immunoreactivity in the hippocampus. In summary, our results suggest that the TP antagonist (SQ 29,548) improves neurological outcome after pilocarpine-induced SE in mice. The existence of SQ 29,548 responders and non-responders was suggested by results from the Neuroscore test. Additional studies are needed to understand the mechanisms underlying these findings, as well as the potential uses of TP antagonists in the treatment of seizure-induced damage.

Anticonvulsant-like effect of thromboxane receptor agonist U-46619 against pentylenetetrazol-induced seizures.

Increasing evidence suggests that prostanoid receptors and their ligands may constitute valuable tools for development of new antiepileptic drugs. Thromboxane A2 (TXA2) is a major eicosanoid in cardiovascular homeostasis. TXA2 exerts its action through the specific G protein-coupled TXA2 receptor (TP). In addition to its crucial role in the cardiovascular system, TXA2 and TPs play a role in the brain. Nevertheless, previously identified roles have been limited to cell protection of neurotoxicity, and the role of TPs on seizure activity was not investigated. Here we evaluated the effect of potent and selective TP agonist U-46619 on seizures induced by pentylenetetrazol (PTZ). Adult C57BL/6 mice received increasing doses of U-46619 (0, 30, 100 or 300 µg/kg). After 30 min we measured the latencies to myoclonic and generalized seizures induced by PTZ (60 mg/kg). We found that U-46619 increased the latency to PTZ-induced myoclonic jerks and tonic-clonic seizures. Moreover, U-46619 increased the immunocontent of phosphorylated Ser657 at protein kinase C (PKC) alpha subunit, indicating PKC activation in the hippocampus and cerebral cortex. Levels of TPs were not altered by the agonist. Administration of a TP antagonist, SQ 29,548, did not alter seizures and did not blunt the anticonvulsant-like effect of the agonist. In summary, we showed that a potent and selective TP agonist, U-46619, increased seizure latency in mice. Activation of PKC signaling pathways may underlie the anticonvulsant-like effect. Further investigation is needed to understand the potential of TPs in seizure treatment.

Na+, K+-ATPase Activating Antibody Displays in vitro and in vivo Beneficial Effects in the Pilocarpine Model of Epilepsy.

Na+, K+-ATPase is an important regulator of brain excitability. Accordingly, compelling evidence indicates that impairment of Na+, K+-ATPase activity contributes to seizure activity in epileptic mice and human with epilepsy. In addition, this enzyme is crucial for plasma membrane transport of water, glucose and several chemical mediators, including glutamate, the major excitatory transmitter in the mammalian brain. Since glucose hypometabolism and increased glutamate levels occur in clinical and experimental epilepsy, we aimed the present study to investigate whether activation of Na+, K+-ATPase activity with specific antibody (DRRSAb) would improve glucose uptake and glutamate release in pilocarpine-treated mice. We found decreased uptake of the glucose fluorescent analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-il)amino]-2-desoxi-d-glucose (2-NBDG) in cerebral slices from pilocarpine-treated animals. Interestingly, decreased 2-NBDG uptake was not detected in DRRSAb-treated slices, suggesting a protective effect of the Na+, K+-ATPase activator. Moreover, DRRSAb prevented the increase in glutamate levels in the incubation media of slices from pilocarpine-treated mice. In addition, in vivo intrahippocampal injection of DRRSAb restored crossing activity of pilocarpine-treated mice in the open-field test. Overall, the present data further support the hypothesis that activation of the Na+, K+-ATPase is a promising therapeutic strategy for epilepsy.

Cerebral Malaria Causes Enduring Behavioral and Molecular Changes in Mice Brain Without Causing Gross Histopathological Damage.

Malaria, parasitic disease considered a major health public problem, is caused by Plasmodium protozoan genus and transmitted by the bite of infected female Anopheles mosquito genus. Cerebral malaria (CM) is the most severe presentation of malaria, caused by P. falciparum and responsible for high mortality and enduring development of cognitive deficits which may persist even after cure and cessation of therapy. In the present study we evaluated selected behavioral, neurochemical and neuropathologic parameters after rescue from experimental cerebral malaria caused by P. berghei ANKA in C57BL/6 mice. Behavioral tests showed impaired nest building activity as well as increased marble burying, indicating that natural behavior of mice remains altered even after cure of infection. Regarding the neurochemical data, we found decreased α2/α3 Na+,K+-ATPase activity and increased immunoreactivity of phosphorylated Na+,K+-ATPase at Ser943 in cerebral cortex after CM. In addition, [3H]-Flunitrazepam binding assays revealed a decrease of benzodiazepine/GABAA receptor binding sites in infected animals. Moreover, in hippocampus, dot blot analysis revealed increased levels of protein carbonyls, suggesting occurrence of oxidative damage to proteins. Interestingly, no changes in the neuropathological markers Fluoro-Jade C, Timm staining or IBA-1 were detected. Altogether, present data indicate that behavioral and neurochemical alterations persist even after parasitemia clearance and CM recovery, which agrees with available clinical findings. Some of the molecular mechanisms reported in the present study may underlie the behavioral changes and increased seizure susceptibility that persist after recovery from CM and may help in the future development of therapeutic strategies for CM sequelae.