Non-Status Epilepticus female rats show seizure-like behaviors in the chronic phase of Pilocarpine experimental model / Ratas sem Status Epilepticus apresentam comportamento do tipo crise epiléptica na fase crônica do modelo experimental da Pilocarpina

Abstract Only few studies have focus on animals that received Pilocarpine (Pilo) and did not develop behavioral status epilepticus (SE) and, whether they may become epileptic in the model's chronic phase. Previews works observed mossy fiber sprouting in the hippocampus of Non-SE (NSE) rats, while others observed spontaneous and recurrent seizures (SRS) 6 - 8 months after animals received Pilo. It is known that neuronal excitability is influenced by female hormones, as well as, the occurrence of SE in castrated and non-castrated female rats. However, it is not known whether females that received Pilo and did not show SE, may have SRS. The aim of this work was to investigate whether castrated and non-castrated female rats that did not show behavioral SE after Pilo, will develop SRS in the following one-year. For that, animals received 360 mg/kg of Pilo and were video monitored for 12 months. SE females from castrated and non-castrated groups became epileptic since the first month after drug injection. Epileptic behaviors were identified watching video monitoring recordings in the fast speed. Castrated and Non-castrated NSE animals showed behaviors resembling seizures described by Racine Scale stages 1 - 3. Motor alterations showed by NSE groups could be observed only when recordings were analyzed in slow speed. In addition, behavioral manifestations as, rhythmic head movements, sudden head movements, whole body movements and immobility were also observed in both, SE and NSE groups. We concluded that NSE female rats may have become epileptic. Adding to it, slow speed analysis of motor alterations was essential for the observation of NSE findings, which suggests that possibly many motor alterations have been underestimated in epilepsy experimental research.

Resumo Poucos são os estudos com foco em animais que receberam Pilocarpina (Pilo) e não desenvolveram status epilepticus (SE) comportamental e, se os mesmos se tornarão epilépticos na fase crônica do modelo. Autores observaram o brotamento das fibras musgosas no hipocampo de ratos Não-SE (NSE), enquanto outros observaram crises espontâneas e recorrentes (CER) 6 - 8 meses após receberam a droga. A excitabilidade neuronal é influenciada pelos hormônios femininos e, da mesma forma, a ocorrência de SE em ratas castradas e não-castradas. Entretanto, não é sabido se as fêmeas que não apresentam SE terão CER. O objetivo deste trabalho foi investigar se fêmeas castradas e não castradas que não tiveram SE comportamental após a injeção de Pilo desenvolverão CER dentro de um ano. Para isto, os animais receberam 360 mg/kg de Pilo e foram videomonitorados por 12 meses. As fêmeas SE castradas e não-castradas se tornaram epilépticas desde o primeiro mês pós Pilo. O comportamento epiléptico foi identificado assistindo as gravações na velocidade rápida. As fêmeas NSE castradas e não-castradas apresentaram comportamentos similares aos estágios 1 - 3 da Escala de Racine. As alterações motoras nestes grupos (NSE) foram observadas apenas quando as videomonitoração foi analisada na velocidade lenta. Além destas, manifestações comportamentais como movimentos rítmicos da cabeça, movimentos súbitos da cabeça, movimentos de todo o corpo e imobilidade também foram observadas em ambos grupos, SE e NSE. Concluímos que as fêmeas NE podem ter se tornado epilépticas. Adicionado a isto, a análise das alterações motoras na velocidade lenta foi essencial para a observação dos achados das fêmeas NSE, o que sugere que possivelmente muitas alterações motoras têm sido subestimados na pesquisa em epilepsia experimental.

Non-Status Epilepticus female rats show seizure-like behaviors in the chronic phase of Pilocarpine experimental model.

Only few studies have focus on animals that received Pilocarpine (Pilo) and did not develop behavioral status epilepticus (SE) and, whether they may become epileptic in the model's chronic phase. Previews works observed mossy fiber sprouting in the hippocampus of Non-SE (NSE) rats, while others observed spontaneous and recurrent seizures (SRS) 6 - 8 months after animals received Pilo. It is known that neuronal excitability is influenced by female hormones, as well as, the occurrence of SE in castrated and non-castrated female rats. However, it is not known whether females that received Pilo and did not show SE, may have SRS. The aim of this work was to investigate whether castrated and non-castrated female rats that did not show behavioral SE after Pilo, will develop SRS in the following one-year. For that, animals received 360 mg/kg of Pilo and were video monitored for 12 months. SE females from castrated and non-castrated groups became epileptic since the first month after drug injection. Epileptic behaviors were identified watching video monitoring recordings in the fast speed. Castrated and Non-castrated NSE animals showed behaviors resembling seizures described by Racine Scale stages 1 - 3. Motor alterations showed by NSE groups could be observed only when recordings were analyzed in slow speed. In addition, behavioral manifestations as, rhythmic head movements, sudden head movements, whole body movements and immobility were also observed in both, SE and NSE groups. We concluded that NSE female rats may have become epileptic. Adding to it, slow speed analysis of motor alterations was essential for the observation of NSE findings, which suggests that possibly many motor alterations have been underestimated in epilepsy experimental research.

Pilocarpine-induced status epilepticus reduces chemosensory control of breathing.

One of the possible causes of death in epilepsy is breathing disorders, especially apneas, which lead to an increase in CO2 levels (hypercapnia) and/or a decrease in O2 levels in arterial blood (hypoxemia). The respiratory neurons located in the ventral brainstem respiratory column are the main groups responsible for controlling breathing. Recent data from our group demonstrated respiratory changes in two experimental models of epilepsy, i.e. audiogenic epilepsy, and amygdala rapid kindling. Here, we aimed to evaluate respiratory changes in the classic model of temporal lobe epilepsy induced by intra-hippocampal injection of pilocarpine. Adult Wistar rats with stainless-steel cannulas implanted in the hippocampus region were used. The animals were submitted to pilocarpine injection (2.4 mg/µL, N = 12-15) or saline (N = 9) into the hippocampus. The respiratory parameters analyzed by whole-body plethysmography were respiratory rate (fR), tidal volume (VT) and ventilation (VE). Respiratory mechanics such as Newtonian airway resistance (Rn), viscance of the pulmonary parenchyma (G) and the elastance of the pulmonary parenchyma (H) were also investigated. No changes in baseline breathing were detected 15 or 30 days after pilocarpine-induced status epilepticus (SE). However, 30 days after pilocarpine-induced SE, a significant reduction in VE was observed during hypercapnic (7% CO2) stimulation, without affecting the hypoxia (8% O2) ventilatory response. We also did not observe changes in respiratory mechanics. The present results suggest that the impairment of the hypercapnia ventilatory response in pilocarpine-induced SE could be related to a presumable degeneration of brainstem respiratory neurons but not to peripheral mechanisms.

Annexin A1-derived peptide Ac2-26 in a pilocarpine-induced status epilepticus model: anti-inflammatory and neuroprotective effects.

BACKGROUND: The inflammatory process has been described as a crucial mechanism in the pathophysiology of temporal lobe epilepsy. The anti-inflammatory protein annexin A1 (ANXA1) represents an interesting target in the regulation of neuroinflammation through the inhibition of leukocyte transmigration and the release of proinflammatory mediators. In this study, the role of the ANXA1-derived peptide Ac2-26 in an experimental model of status epilepticus (SE) was evaluated. METHODS: Male Wistar rats were divided into Naive, Sham, SE and SE+Ac2-26 groups, and SE was induced by intrahippocampal injection of pilocarpine. In Sham animals, saline was applied into the hippocampus, and Naive rats were only handled. Three doses of Ac2-26 (1 mg/kg) were administered intraperitoneally (i.p.) after 2, 8 and 14 h of SE induction. Finally, 24 h after the experiment-onset, rats were euthanized for analyses of neuronal lesion and inflammation. RESULTS: Pilocarpine induced generalised SE in all animals, causing neuronal damage, and systemic treatment with Ac2-26 decreased neuronal degeneration and albumin levels in the hippocampus. Also, both SE groups showed an intense influx of microglia, which was corroborated by high levels of ionised calcium binding adaptor molecule 1(Iba-1) and monocyte chemoattractant protein-1 (MCP-1) in the hippocampus. Ac2-26 reduced the astrocyte marker (glial fibrillary acidic protein; GFAP) levels, as well as interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and growth-regulated alpha protein (GRO/KC). These effects of the peptide were associated with the modulation of the levels of formyl peptide receptor 2, a G-protein-coupled receptor that binds to Ac2-26, and the phosphorylated extracellular signal-regulated kinase (ERK) in the hippocampal neurons. CONCLUSIONS: The data suggest a neuroprotective effect of Ac2-26 in the epileptogenic processes through downregulation of inflammatory mediators and neuronal loss.

Induction of Type 2 Iodothyronine Deiodinase After Status Epilepticus Modifies Hippocampal Gene Expression in Male Mice.

Status epilepticus (SE) is an abnormally prolonged seizure that results from either a failure of mechanisms that terminate seizures or from initiating mechanisms that inherently lead to prolonged seizures. Here we report that mice experiencing a 3 hours of SE caused by pilocarpine exhibit a rapid increase in expression of type 2 iodothyronine deiodinase gene (Dio2) and a decrease in the expression of type 3 iodothyronine deiodinase gene in hippocampus, amygdala and prefrontal cortex. Type 3 iodothyronine deiodinase in hippocampal sections was seen concentrated in the neuronal nuclei, typical of ischemic injury of the brain. An unbiased analysis of the hippocampal transcriptome of mice undergoing 3 hours of SE revealed a number of genes, including those involved with response to oxidative stress, cellular homeostasis, cell signaling, and mitochondrial structure. In contrast, in mice with targeted disruption of Dio2 in astrocytes (Astro D2KO mouse), the highly induced genes in the hippocampus were related to inflammation, apoptosis, and cell death. We propose that Dio2 induction caused by SE accelerates production of T3 in different areas of the central nervous system and modifies the hippocampal gene expression profile, affecting the balance between adaptive and maladaptive mechanisms.

Long-Term Effects of Anterior Thalamic Nucleus Deep Brain Stimulation on Spatial Learning in the Pilocarpine Model of Temporal Lobe Epilepsy.

INTRODUCTION AND OBJECTIVES: Cognitive impairment is a significant comorbidity of temporal lobe epilepsy that is associated with extensive hippocampal cell loss. Deep brain stimulation (DBS) of the anterior thalamic nucleus (ANT) has been used for the treatment of refractory partial seizures. In the pilocarpine model of epilepsy, ANT DBS applied during status epilepticus (SE) reduces hippocampal inflammation and apoptosis. When given to chronic epileptic animals it reduces hippocampal excitability and seizure frequency. Here, we tested whether ANT DBS delivered during SE and the silent phase of the pilocarpine model would reduce cognitive impairment when animals became chronically epileptic. MATERIALS AND METHODS: SE was induced by a systemic pilocarpine injection (320 mg/kg). Immediately after SE onset, rats were assigned to receive DBS during the first six hours of SE (n = 8; DBSa group) or during SE + the silent period (i.e., 6 h/day until the animals developed the first spontaneous recurrent seizure; n = 10; DBSs group). Four months following SE, animals underwent water maze testing and histological evaluation. Nonstimulated chronic epileptic animals (n = 13; PCTL group) and age-matched naïve rats (n = 11, CTL group) were used as controls. Results were analyzed by repeated-measures analyses of variance (RM_ANOVA) and one-way ANOVAs, followed by Newman-Keuls post hoc tests. RESULTS: Although all groups learned the spatial task, epileptic animals with or without DBS spent significantly less time in the platform quadrant, denoting a spatial memory deficit (p < 0.02). Despite these negative behavioral results, we found that animals given DBS had a significantly higher number of cells in the CA1 region and dentate gyrus. Mossy fiber sprouting was similar among all epileptic groups. CONCLUSIONS: Despite lesser hippocampal neuronal loss, ANT DBS delivered either during SE or during SE and the silent phase of the pilocarpine model did not mitigate memory deficits in chronic epileptic rats.

Modification of the natural progression of epileptogenesis by means of biperiden in the pilocarpine model of epilepsy.

Brain injuries are often associated with the later development of epilepsy. Evidence suggests that morphological and functional changes occur in the remaining neural tissue during a silent (or latent) period in which no seizures are expressed. It is believed that this silent (reorganization) period may provide a therapeutic window for modifying the natural history of disease progression. Here we provide evidence that biperiden, a muscarinic anticholinergic agent, is able to alter disease progression in an animal model of epilepsy. We observed that biperiden was capable of slowing the manifestation of the first spontaneous epileptic seizure and effectively reduced the severity and number of recurrent, spontaneous epileptic seizures during the animals' lifespan. Biomolecular (microdialysis) and electrophysiological (extracellular field recordings) studies determined that biperiden was capable of elevating the threshold of hippocampal excitability, thereby making the hippocampal glutamatergic pathways less responsive to stimuli when high concentrations of potassium were used in vivo or in vitro. Notably, there was no hindrance of long-term memory or learning (a potential problem given the amnestic nature of biperiden). We conclude that biperiden has antiepileptogenic potential and may represent an opportunity for the prevention of post-traumatic epilepsy.

Propylparaben applied after pilocarpine-induced status epilepticus modifies hippocampal excitability and glutamate release in rats.

Propylparaben (PPB) induces cardioprotection after ischemia-reperfusion injury by inhibiting voltage-dependent Na+ channels. The present study focuses on investigating whether the i.p. application of 178mg/kg PPB after pilocarpine-induced status epilepticus (SE) reduces the acute and long-term consequences of seizure activity. Initially, we investigated the effects of a single administration of PPB after SE. Our results revealed that compared to rats receiving diazepam (DZP) plus vehicle after 2h of SE, animals receiving a single dose of PPB 1h after DZP injection presented 126% (p<0.001) lower extracellular levels of glutamate in the hippocampus. This effect was associated with an increased potency of low-frequency oscillations (0.1-13Hz bands, p<0.001), a reduced potency of 30-250Hz bands (p<0.001) and less neuronal damage in the hippocampus. The second experiment examined whether the subchronic administration of PPB during the post-SE period is able to prevent the long-term consequences of seizure activity. In comparison to animals that were treated subchronically with vehicle after SE, rats administered with PPB for 5 days presented lower hippocampal excitability and interictal glutamate release, astrogliosis, and neuroprotection in the dentate gyrus. Our data indicate that PPB, when applied after SE, can be used as a therapeutic strategy to reduce the consequences of seizure activity.

Epilepsy and exercise: An experimental study in female rats.

OBJECTIVE: Epilepsy is the most common neurological chronic condition worldwide, affecting about 2% of world population. Temporal lobe epilepsy (TLE) reaches 40% of all cases of this condition, and it is highly refractory to pharmacological treatment. Physical activity has been suggested as complementary therapy for epilepsy. However, there is no consistent information whether all these effects are plenty applicable to females, since clinical and experimental studies concerning physical exercise and epilepsy are largely performed in males. Females are worthy of special attention due to gender specific particularities such as hormonal cyclical rhythm and possible pregnancy. Therefore, this study aimed to investigate the impact of two types of exercise programs (Forced and Voluntary) in female Wistar rats submitted to temporal lobe epilepsy induced by pilocarpine. METHODS: Animals were divided into four groups: Control (healthy), Epilepsy, Epilepsy/Forced (exercise in a treadmill) and Epilepsy/Voluntary (free access to wheel). Behavioral and histological analyses were evaluated among groups. RESULTS: Voluntary exercise was able to reduce seizure frequency and anovulatory estrous cycle occurrence. Yet, both types of exercise attenuated the mossy fiber sprouting in dentate gyrus. CONCLUSION: Our results indicate that voluntary exercise exerts a positive effect on epilepsy in female gender. Further investigations are necessary to better elucidate mechanisms involved in these responses, since these effects do not act in the same manner in male and female rats.

Medial Ganglionic Eminence Cells Freshly Obtained or Expanded as Neurospheres Show Distinct Cellular and Molecular Properties in Reducing Epileptic Seizures.

AIMS: Medial ganglionic eminence (MGE) progenitors give rise to inhibitory interneurons and may serve as an alternative cell source for large-scale cell transplantation for epilepsy after in vitro expansion. We investigated whether modifications in the culture medium of MGE neurospheres affect neuronal differentiation and expression of MGE-specific genes. In vivo, we compared anticonvulsant effects and cell differentiation pattern among neurospheres grown in different culture media and compared them with freshly harvested MGE cells. METHODS: We used four variations of cell culture: standard, containing growth factors (EGF/FGF-2) (GF); addition of retinoic acid (GF-RA); withdrawal of EGF/FGF-2 (WD); and addition of retinoic acid and withdrawal of EGF/FGF-2 (WD-RA). Based on in vitro results neurosphere-grown (WD-RA or GF conditions) or fresh MGE cells were transplanted into the hippocampus. RESULTS: In vitro WD-RA showed increased neuronal population and higher expression of Dlx1, Nkx2.1, and Lhx6 genes in comparison with GF culture condition. After transplantation, fresh MGE cells and neurospheres (GF) showed anticonvulsant effects. However, fresh MGE cells differentiated preferentially into inhibitory neurons, while GF gave rise to glial cells. CONCLUSION: We conclude that freshly isolated and neurosphere-grown MGE cells reduced seizures by different mechanisms (inhibitory interneurons vs. astrocytes). Fresh MGE cells appear more appropriate for cell therapies targeting inhibitory interneurons for conferring anticonvulsant outcomes.

Characterization of Intracranial Pressure Behavior in Chronic Epileptic Animals: A Preliminary Study.

Intracranial pressure (ICP) is a major neurological parameter in animals and humans. ICP is a function of the relationship between the contents of the cranium (brain parenchyma, cerebrospinal fluid, and blood) and the volume of the skull. Increased ICP can cause serious physiological effects or even death in patients who do not quickly receive proper care, which includes ICP monitoring. Epilepsies are a set of central nervous system disorders resulting from abnormal and excessive neuronal discharges, usually associated with hypersynchronism and/or hyperexcitability. Temporal lobe epilepsy (TLE) is one of the most common forms of epilepsy and is also refractory to medication. ICP characteristics of subjects with epilepsy have not been elucidated because there are few studies associating these two important neurological factors. In this work, an invasive (ICPi) and the new minimally invasive (ICPmi) methods were used to evaluate ICP features in rats with chronic epilepsy, induced by the experimental model of pilocarpine, capable of generating the main features of human TLE in these animals.

PI3Kγ deficiency enhances seizures severity and associated outcomes in a mouse model of convulsions induced by intrahippocampal injection of pilocarpine.

Phosphatidylinositol 3-kinase (PI3K) is an enzyme involved in different pathophysiological processes, including neurological disorders. However, its role in seizures and postictal outcomes is still not fully understood. We investigated the role of PI3Kγ on seizures, production of neurotrophic and inflammatory mediators, expression of a marker for microglia, neuronal death and hippocampal neurogenesis in mice (WT and PI3Kγ(-/-)) subjected to intrahippocampal microinjection of pilocarpine. PI3Kγ(-/-) mice presented a more severe status epilepticus (SE) than WT mice. In hippocampal synaptosomes, genetic or pharmacological blockade of PI3Kγ enhanced the release of glutamate and the cytosolic calcium concentration induced by KCl. There was an enhanced neuronal death and a decrease in the doublecortin positive cells in the dentate gyrus of PI3Kγ(-/-) animals after the induction of SE. Levels of BDNF were significantly increased in the hippocampus of WT and PI3Kγ(-/-) mice, although in the prefrontal cortex, only PI3Kγ(-/-) animals showed significant increase in the levels of this neurotrophic factor. Pilocarpine increased hippocampal microglial immunolabeling in both groups, albeit in the prelimbic, medial and motor regions of the prefrontal cortex this increase was observed only in PI3Kγ(-/-) mice. Regarding the levels of inflammatory mediators, pilocarpine injection increased interleukin (IL) 6 in the hippocampus of WT and PI3Kγ(-/-) animals and in the prefrontal cortex of PI3Kγ(-/-) animals 24h after the stimulus. Levels of TNFα were enhanced in the hippocampus and prefrontal cortex of only PI3Kγ(-/-) mice at this time point. On the other hand, PI3Kγ deletion impaired the increase in IL-10 in the hippocampus induced by pilocarpine. In conclusion, the lack of PI3Kγ revealed a deleterious effect in an animal model of convulsions induced by pilocarpine, suggesting that this enzyme may play a protective role in seizures and pathological outcomes associated with this condition.

Evaluation of potential gender-related differences in behavioral and cognitive alterations following pilocarpine-induced status epilepticus in C57BL/6 mice.

Together with pharmacoresistant seizures, the quality of life of temporal lobe epilepsy (TLE) patients is negatively impacted by behavioral comorbidities including but not limited to depression, anxiety and cognitive deficits. The pilocarpine model of TLE has been widely used to study characteristics of human TLE, including behavioral comorbidities. Since the outcomes of pilocarpine-induced TLE might vary depending on several experimental factors, we sought to investigate potential gender-related differences regarding selected behavioral alterations in C57BL6 mice. We found that epileptic mice, independent of gender, displayed increased anxiety-like behavior in the open-field test. In the object recognition test, epileptic mice, regardless of gender, showed a decreased recognition index at 24 (but not at 4) hours after training. On the other hand, no significant differences were found regarding mice learning and memory performance in the Barnes maze paradigm. Motor coordination and balance as assessed by the beam walk and rotarod tests were not impaired in epileptic mice of both genders. However, female mice, independent of epilepsy, performed the beam walk and rotarod tasks better than their male counterparts. We also found that only male epileptic mice displayed disturbed behavior in the forced swim test, but the mice of both genders displayed anhedonia-like behavior in the taste preference test. Lastly, we found that the extent of hilar cell loss is similar in both genders. In summary, both genders can be successfully employed to study behavioral comorbidities of TLE; however, taking the potential gender differences into account may help choose the more appropriated gender for a given task, which may be of value for the minimization of the number of animals used during the experiments.

Temporally unstructured electrical stimulation to the amygdala suppresses behavioral chronic seizures of the pilocarpine animal model.

Electrical stimulation applied to the basolateral amygdala in the pentylenetetrazole animal model of seizures may result in either a proconvulsant or an anticonvulsant effect depending on the interpulse intervals used: periodic or nonperiodic, respectively. We tested the effect of this electrical stimulation temporal coding on the spontaneous and recurrent behavioral seizures produced in the chronic phase of the pilocarpine animal model of temporal lobe epilepsy, an experimental protocol that better mimics the human condition. After 45 days of the pilocarpine-induced status epilepticus, male Wistar rats were submitted to a surgical procedure for the implantation of a bipolar electrical stimulation electrode in the right basolateral amygdala and were allowed to recover for seven days. The animals were then placed in a glass box, and their behaviors were recorded daily on DVD for 6h for 4 consecutive days (control period). Spontaneous recurrent behavioral seizures when showed in animals were further recorded for an extra 4-day period (treatment period), under periodic or nonperiodic electrical stimulation. The number, duration, and severity of seizures (according to the modified Racine's scale) during treatment were compared with those during the control period. The nonperiodically stimulated group displayed a significantly reduced total number and duration of seizures. There was no difference between control and treatment periods for the periodically stimulated group. Results corroborate previous findings from our group showing that nonperiodic electrical stimulation has a robust anticonvulsant property. In addition, results from the pilocarpine animal model further strengthen nonperiodic electrical stimulation as a valid therapeutic approach in current medical practice. Our working hypothesis is that temporally unstructured electrical stimulation may wield its effect by desynchronizing neural networks involved in the ictogenic process.

Lovastatin decreases the synthesis of inflammatory mediators during epileptogenesis in the hippocampus of rats submitted to pilocarpine-induced epilepsy.

Statins may act on inflammatory responses, decreasing oxidative stress and also reducing brain inflammation in several brain disorders. Epileptogenesis is a process in which a healthy brain becomes abnormal and predisposed to generating spontaneous seizures. We previously reported that lovastatin could prevent neuroinflammation in pilocarpine-induced status epilepticus (SE). In this context, this study investigated the long-lasting effects of lovastatin on mRNA expression of proinflammatory cytokines (interleukin-1ß, tumor necrosis factor α, interleukin-6) and the antiinflammatory cytokine IL-10 in the hippocampus during epileptogenesis by immunohistochemistry and real time polymerase chain reaction (RT-PCR) during the latent and chronic phases in the epilepsy model induced by pilocarpine in rats. For these purposes, four groups of rats were employed: saline (CONTROL), lovastatin (LOVA), pilocarpine (PILO), and pilocarpine plus lovastatin (PILO+LOVA). After pilocarpine injection (350mg/kg, i.p.), the rats were treated with 20mg/kg of lovastatin via an esophagic probe 2h after SE onset. All surviving rats were continuously treated during 15days, twice/day. The pilocarpine plus lovastatin group showed a significant decrease in the levels of IL-1ß, TNF-α, and IL-6 during the latent phase and a decreased expression of IL-1ß and TNF-α in the chronic phase when compared with the PILO group. Moreover, lovastatin treatment also induced an increased expression of the antiinflammatory cytokine, IL-10, in the PILO+LOVA group when compared with the PILO group in the chronic phase. Thus, our data suggest that lovastin may reduce excitotoxicity during epileptogenesis induced by pilocarpine by increasing the synthesis of IL-10 and decreasing proinflammatory cytokines in the hippocampus.

NK1 receptors antagonism of dorsal hippocampus counteract the anxiogenic-like effects induced by pilocarpine in non-convulsive Wistar rats.

Recent evidence supports a role for the substance P (SP) in the control of anxiety and epilepsy disorders. Aversive stimuli alter SP levels and SP immunoreactivity in limbic regions, suggesting that changes in SP-NK1 receptor signaling may modulate the neuronal excitability involved in seizures and anxiogenesis. The involvement of NK1 receptors of the dorsal hippocampus and lateral septum in the anxiogenic-like effects induced by a single injection of pilocarpine (PILO) was examined in non-convulsive rats evaluated in the elevated plus-maze (EPM). Male Wistar rats were systemically injected with methyl-scopolamine (1mg/kg) followed 30 min later by saline or PILO (350 mg/kg) and only rats that did not present status epilepticus were used. One month later, vehicle or FK888 (100 pmol) - an NK1 receptor antagonist - were infused in the dorsal hippocampus or the lateral septum of the rats and then behaviorally evaluated in the EPM. Previous treatment with PILO decreased the time spent in and the frequency of entries in the open arms of the EPM, besides altering risk-assessment behaviors such as the number of unprotected head-dipping, protected stretch-attend postures and the frequency of open-arms end activity, showing thus a long-lasting anxiogenic-like profile. FK888 did not show any effect per se but inhibited the anxiogenic responses induced by PILO when injected into the dorsal hippocampus, but not into the lateral septum. Our data suggest that SP-NK1 receptor signaling of the dorsal hippocampus is involved in the anxiogenic-like profile induced by PILO in rats evaluated in the EPM test.

The effects of sleep deprivation on microRNA expression in rats submitted to pilocarpine-induced status epilepticus.

Epilepsy is a neurological disorder with significant prevalence and the individuals affected by this disease have a great probability of occurrence of a lethal phenomenon known as Sudden Unexpected Death in Epilepsy (SUDEP). SUDEP occurs mainly during the night and probably during sleep. The pathophysiological mechanisms involved in this lethal phenomenon are still obscure and new evidences that could corroborate in this area are warranted. Thus, the aim of the present study was to evaluate the effect of sleep deprivation in the expression of microRNA (miRNA) in the frontal cortex and heart tissues of adult male rats after 50days of saline (SAL) or pilocarpine-induced status epilepticus (PILO). Initially 389 miRNA expressions were evaluated between SAL and PILO groups by microarray. Subsequently, 3 differentially expressed miRNAs of each tissue were investigated after total sleep deprivation (TSD 6h) and paradoxical sleep deprivation (PSD 24h). Still, it was analyzed that the effects of sleep rebound with equivalent duration of PSD and TSD. There was a significant increase of miR-146a expression, an important inflammatory modulator in the frontal cortex of PILO rats when compared to SAL animals. Animals treated with pilocarpine were affected by TSD (through overexpression of miRNAs related to inflammatory process) and these changes were maintained even after a sleep window of 6h. In contrast, miRNAs associated with heart diseases were down-regulated in PSD rebound, suggesting a possible restoration of homeostasis in cardiovascular system of SAL and PILO groups.

Activation and involvement of the lateral-posterior nucleus of the thalamus after a single generalized tonic-clonic seizure.

The lateral-posterior thalamic nuclei (LP) have been shown to play an important role in controlling epileptic activity. In addition, thalamic atrophy and neuronal loss have been observed in epilepsy. The objective of this study was to investigate whether lateral-posterior neuronal activation may be observed shortly after a single generalized seizure in rats submitted to the pilocarpine model of epilepsy. The results showed an increased lateral-posterior activation as soon as the seizure occurred, suggesting that neuronal loss in the thalamus is not only the consequence of chronic epilepsy.

Changes in gene expression in the frontal cortex of rats with pilocarpine-induced status epilepticus after sleep deprivation.

Sleep and epilepsy present a bidirectional interaction. Sleep complaints are common in epilepsy, and sleep deprivation may provoke seizures. However, the mechanisms underlying this relationship are unknown. Thus, this study investigated the effects of paradoxical sleep deprivation (PSD24h) and total sleep deprivation (TSD6h) in the expression of genes related to reactive oxygen species and nitric oxide production in the frontal cortex of a rodent model of temporal lobe epilepsy (PILO). The data show that PILO rats had increased NOX-2 expression and decreased SOD expression, independent of sleep. Higher NOX-2 expression was observed only in PILO rats subjected to the control condition and TSD6h. Also, eNOS and DDAH1 were increased in the PILO group submitted to TSD6h. Moreover, CAT expression in the frontal cortex of PILO rats submitted to PSD24h was reduced compared to that of PILO rats that were not sleep-deprived. The molecular changes found in the frontal cortex of PILO rats following sleep deprivation suggest a mechanism via oxidative stress.

Omega-3 fatty acid supplementation reduces resting heart rate of rats with epilepsy.

Since cardiovascular dysfunction may contribute to sudden unexpected death in epilepsy (SUDEP), the consumption of omega-3 fatty acids (omega-3 FAs) might be beneficial as an adjunctive therapy for SUDEP prevention. It is well recognized that omega-3 FAs exert positive effects on the cardiovascular system including heart rate (HR) reduction, a major risk factor to sudden death. Thus, we evaluated the effects of chronic supplementation of omega-3 FAs on the HR of rats with epilepsy. In agreement with our previous investigations, this study also showed that the HR of animals with epilepsy is higher than that of the control group. Quite interestingly, chronic supplementation with omega-3 FAs restored the HR of rats with epilepsy toward control values. In conclusion, although further investigations are still required, our preliminary results showed a potential preventive effect of omega-3 FA supplementation against SUDEP.