Long-term monotherapy treatment with vitamin E reduces oxidative stress, but not seizure frequency in rats submitted to the pilocarpine model of epilepsy.

The imbalance between antioxidant system and reactive oxygen species (ROS) generation is related to epileptogenesis, neuronal death, and seizure frequency. Treatment with vitamin E has been associated with neuroprotection and control of seizures. In most experimental studies, vitamin E treatment has short duration. Therefore, the aim of this study was to verify the role of long-term treatment with vitamin E in rats submitted to the pilocarpine model of epilepsy. Rats were divided into two main groups: control (Ctr) and pilocarpine (Pilo). Each one was subdivided according to treatment: vehicle (Ctr V and Pilo V) or vitamin E at dosages of 6 IU/kg/day (Ctr E6 and Pilo E6) or 60 IU/kg/day (Ctr E60 and Pilo E60). Treatment lasted 120 days from status epilepticus (SE). There were no statistical differences concerning treatment in the Ctr group for all variables, so the data were grouped. Carbonyl content in the hippocampus of Pilo V and Pilo E6 was higher compared with that of the Ctr group (8 ±â€¯1.5, 7.1 ±â€¯1, and 3.1 ±â€¯0.3 nmol carbonyl/mg protein, respectively for Pilo V, Pilo E6, and Ctr; p < 0.05). Carbonyl content was restored to control values in Pilo E60 rats (4.2 ±â€¯1.1 and 3.1 ±â€¯0.3 nmol carbonyl/mg protein, respectively for Pilo E60 and Ctr; p > 0.05). The volume of the hippocampal formation (6.5 ±â€¯0.3, 6.6 ±â€¯0.4, 6.3 ±â€¯0.3, and 7.4 ±â€¯0.2, respectively for Pilo V, Pilo E6, Pilo E60, and Ctr) and subfields CA1 (1.6 ±â€¯0.1, 1.4 ±â€¯0.2, 1.5 ±â€¯0.1, and 2 ±â€¯0.05, respectively for Pilo V, Pilo E6, Pilo E60, and Ctr) and CA3 (1.7 ±â€¯0.1, 1.5 ±â€¯0.2, 1.4 ±â€¯0.1, and 2 ±â€¯0.1, respectively for Pilo V, Pilo E6, Pilo E60, and Ctr) was reduced in the Pilo group regardless of treatment. Parvalbumin immunostaining was increased in the hilus of the Pilo E60 group compared with that in the Ctr group (26 ±â€¯2 and 39.6 ±â€¯8.3 neurons, respectively for Ctr and Pilo E60). No difference was found in seizure frequency and Neo-Timm staining. Therefore, long-term treatment with 60 IU/kg/day of vitamin E prevented oxidative damage in the hippocampus and increased hilar parvalbumin expression in rats with epilepsy without a reduction in seizure frequency.

Early life seizures in female rats lead to anxiety-related behavior and abnormal social behavior characterized by reduced motivation to novelty and deficit in social discrimination.

Previously, we demonstrated that male Wistar rats submitted to neonatal status epilepticus showed abnormal social behavior characterized by deficit in social discrimination and enhanced emotionality. Taking into account that early insult can produce different biological manifestations in a gender-dependent manner, we aimed to investigate the social behavior and anxiety-like behavior in female Wistar rats following early life seizures. Neonate female Wistar rats at 9 days postnatal were subject to pilocarpine-induced status epilepticus and the control received saline. Behavioral tests started from 60 days postnatal and were carried out only during the diestrus phase of the reproductive cycle. In sociability test experimental animals exhibited reduced motivation for social encounter and deficit in social discrimination. In open field and the elevated plus maze, experimental animals showed enhanced emotionality with no changes in basal locomotor activity. The results showed that female rats submitted to neonatal status epipepticus showed impaired social behavior, characterized by reduced motivation to novelty and deficit in social discrimination in addition to enhanced emotionality.

High serum levels of proinflammatory markers during epileptogenesis. Can omega-3 fatty acid administration reduce this process?

During the epileptogenic process, several events may occur, such as an important activation of the immune system in the central nervous system. The response to seizure activity results in an inflammation in the brain as well as in the periphery. Moreover, CRP and cytokines may be able to interact with numerous ligands in response to cardiac injury caused by sympathetic stimulation in ictal and postictal states. Based on this, we measured the serum levels of C-reactive protein (CRP) and cytokines during acute, silent, and chronic phases of rats submitted to the pilocarpine model of epilepsy. We have also analyzed the effect of a chronic treatment of these rats with omega-3 fatty acid in CRP and cytokine levels, during an epileptic focus generation. C-reactive protein and cytokines such as IL-1ß, IL-6, and TNF-α presented high concentration in the blood of rats, even well after the occurrence of SE. We found reduced levels of CRP and all proinflammatory cytokines in the blood of animals with chronic seizures, treated with omega-3, when compared with those treated with vehicle solution. Taken together, our results strongly suggest that the omega-3 is an effective treatment to prevent SUDEP occurrence due to its capability to act as an anti-inflammatory compound, reducing the systemic inflammatory parameters altered by seizures.

Fish oil provides protection against the oxidative stress in pilocarpine model of epilepsy.

Temporal lobe epilepsy (TLE), the most common form of epilepsy is often resistant to pharmacological treatment. Neuronal loss observed in epileptic brain may be result of an overproduction of free radicals (oxidative stress). Oxidative stress is characterized by an imbalance between antioxidant defenses and oxidizing agents (free radicals), which can lead to tissue injury. The n-3 PUFAs are important for the development and maintenance of central nervous system functions. Research by our group has shown that chronic treatment with fish oil, immediately after status epilepticus (SE), exhibits both neuroprotective effects and effects on neuroplasticity. The main purpose of this research was to evaluate if fish oil exhibits a protective effect against oxidative stress. Animals were subjected to TLE model by pilocarpine administration. After 3 h of SE they were randomly divided into the following groups: control animals treated daily with vehicle or with 85 mg/kg of fish oil and animals with epilepsy treated daily with vehicle or with 85 mg/kg of fish oil. After 90 days, superoxide anion production, enzymatic activity of superoxide dismutase (SOD) and catalase (CAT) and protein expression of NAD(P)H oxidase subunits (p47(PHOX) and gp91(PHOX)) were analyzed. Our results showed evidences that reactive oxygen species are increased in animals with epilepsy and that fish oil supplementation could counteract it. Fish oil supplementation promoted protection against oxidative stress by multiple ways, which involved the reduction of activity and expression of NAD(P)H oxidase subunits and increased the activity and expression of antioxidants enzymes, contributing to well-known neuroprotective effect in epilepsy.

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.

Social behavior impairment in offspring exposed to maternal seizures in utero.

Human and animal models have demonstrated that maternal seizures in utero could be deleterious to the development of the offspring. This study focused on the social behavior of offspring exposed to seizures in utero. A pilocarpine model of temporal lobe epilepsy was induced in female Wistar rats that were mated after the first spontaneous seizure. Early after birth, pups from an epileptic mother were reared by a control mother. To evaluate the influence of the adoption process, two other groups were added: rat pups from control mothers cross-fostered with other control mothers, and rat pups reared by their birth mother. Animals exposed to seizures in utero showed impaired social behavior with no signs of anxiety-like behavior. This study demonstrated that epileptic seizures during pregnancy could be harmful to brain development and may increase the risk of developing neurodevelopmental disorders. The mechanisms underlying the abnormalities of social behavior are not well understood, and further studies in this field are warranted.

Effects of fish oil supplementation on spatial memory in rats with pilocarpine-induced epilepsy assessed using the Morris Water Maze test.

OBJECTIVE: Patients with temporal lobe epilepsy (TLE) are at high risk of experiencing cognitive impairment. Such dysfunction is also observed in an animal model of TLE, the rat model of pilocarpine-induced epilepsy. METHODS: We investigated the effects of fish oil supplementation on spatial memory in rats with pilocarpine-induced epilepsy using the Morris Water Maze (MWM) test. RESULTS: Although rats with pilocarpine-induced epilepsy treated with fish oil learned the platform location significantly faster by Day 7 of the acquisition phase, spatial memory performance of these rats was unaffected by fish oil supplementation during probe trials. SIGNIFICANCE: Our study provides insights into the importance of considering nutraceutical strategies for enhancing cognitive abilities in patients with TLE.

Social play impairment following status epilepticus during early development.

Neonatal status epilepticus (SE) disrupts prefrontal cortex and thalamus, brain regions related to social play. Juvenile play was evaluated using the "intruder-resident" paradigm following SE in 9-day-old Wistar pups of both genders. Quite interestingly, we demonstrated for the first time that neonatal SE produces social impairment in male rats, reduces locomotor activity in both genders and enhances self-grooming in female. Additional studies are necessary to clarify if these effects can impair social behavior across the life span.

Increased Endocannabinoid Signaling Reduces Social Motivation in Intact Rats and Does Not Affect Animals Submitted to Early-Life Seizures.

The early life status epilepticus (SE) causes high anxiety and chronic socialization abnormalities, revealed by a low preference for social novelty and deficit in social discrimination. This study investigated the involvement of the endocannabinoid system on the sociability in this model, due to its role in social motivation regulation. Male Wistar rats at postnatal day 9 were subjected to pilocarpine-induced neonatal SE and controls received saline. From P60 the groups received vehicle or JZL195 2 h before each behavioral test to increase endocannabinoids availability. In the sociability test, animals subjected to neonatal SE exhibited impaired sociability, characterized by social discrimination deficit, which was unaffected by the JZL195 treatment. In contrast, JZL195-treated control rats showed low sociability and impaired social discrimination. The negative impact of JZL195 over the sociability in control rats and the lack of effect in animals subjected to neonatal SE was confirmed in the social memory paradigm. In this paradigm, as expected for vehicle-treated control rats, the investigation toward the same social stimulus decreased with the sequential exposition and increased toward a novel stimulus. In animals subjected to neonatal SE, regardless of the treatment, as well as in JZL195-treated control rats, the investigation toward the same social stimulus was significantly reduced with no improvement toward a novel stimulus. Concerning the locomotion, the JZL195 increased it only in control rats. After behavioral tests, brain tissues of untreated animals were used for CB1 receptor quantification by Elisa and for gene expression by RT-PCR: no difference between control and experimental animals was noticed. The results reinforce the evidence that the early SE causes chronic socialization abnormalities, revealed by the low social interest for novelty and impaired social discrimination. The dual FAAH/MAGL inhibitor (JZL195) administration before the social encounter impaired the social interaction in intact rats with no effect in animals subjected to early-life seizures.

Neurogenesis in the amygdala: a new etiologic hypothesis of autism?

Neurogenesis studies had an increased development after BrdU (5-bromo-3'-deoxyuridine), a marker of cell proliferation. Today, several studies have showed the relevance of neurogenesis in the hippocampal formation. Notwithstanding, other brains areas have been described presenting neurogenesis, including the amygdala. This key structure is a complex cerebral region which has been associated with social behaviors and the emotional significance of the daily experiences. Several studies have associated the amygdala to the autism, a severe neurodevelopmental disorder. In this paper, we discuss the hypothesis of neurogenesis in the amygdala as a contributing cause of autism. The social skills require competent new neuronal connections, including efficient plasticity synaptic rearranging. Interestingly, emotional context cannot be imprinting in mature neurons in the presence of GABA, a neurotransmitter release during new environments experiences. However, it is known that new neurons are not well responsive to GABA stimulation, allowing the long-term potentiation necessary for the learning process. Based on these evidence it is tantalizing to hypothesize that the sociability impairment seen in some individuals with autism may partly be assigned to impaired regulation of the GABAergic system and to the impact of this impairment on the adequate functioning of the amygdala and on its capacity to store new experiences and to modulate the plasticity of the corticostriatal connections.

Fish Oil Supplementation Reduces Heart Levels of Interleukin-6 in Rats with Chronic Inflammation due to Epilepsy.

Sudden unexpected death in epilepsy (SUDEP) is a major cause of premature death related to epilepsy. The causes of SUDEP remain unknown, but cardiac arrhythmias and asphyxia have been suggested as a major mechanism of this event. Inflammation has been implicated in the pathogenesis of both epilepsy and ventricular arrhythmia, with interleukin-6 (IL-6) being recognized as a crucial orchestrator of inflammatory states. Our group previously reported that levels of IL-6 were increased in the hearts of epileptic rats. In this scenario, anti-inflammatory actions are among the beneficial effects of fish oil dietary supplementation. This investigation revealed that elevated levels of IL-6 in the heart were markedly reduced in epileptic rats that were treated in the long-term with fish oil, suggesting protective anti-inflammatory actions against dangerously high levels of IL-6. Based on these findings, our results suggest beneficial effects of long-term intake of fish oil in reducing the inflammation associated with chronic epilepsy.

Impaired executive functions in experimental model of temporal lobe epilepsy.

OBJECTIVE: The present study aimed to investigate cognitive and behavioural changes consistent with attention deficit hyperactivity disorder (ADHD -like behavior in male Wistar rats with temporal lobe epilepsy (TLE). METHOD: Male Wistar rats at 25 day of age were submitted to animal model of TLE by pilocarpine injection (350 mg/kg, ip) and a control group received saline 0.9%. The animals were continuously video monitored up to the end of experiments. The behavioural tests (open field, elevated plus maze and operant conditioning box) started from 60 days postnatal. RESULTS: Animals with TLE exhibited elevated locomotor activity, reduced level of anxiety-related behavior, impulsivity and impaired visuospatial working memory. CONCLUSION: Taken as a whole, we concluded that animals with TLE exhibited some cognitive and behavioural changes consistent with ADHD-like behavior.

Lovastatin reduces neuronal cell death in hippocampal CA1 subfield after pilocarpine-induced status epilepticus: preliminary results.

OBJECTIVE: To further characterize the capacity of lovastatin to prevent hippocampal neuronal loss after pilocarpine-induced status epilepticus (SE) METHOD: Adult male Wistar rats were divided into four groups: (A) control rats, received neither pilocarpine nor lovastatin (n=5); (B) control rats, received just lovastatin (n=5); (C) rats that received just pilocarpine (n=5); (D) rats that received pilocarpine and lovastatin (n=5). After pilocarpine injection (350 mg/kg, i.p.), only rats that displayed continuous, convulsive seizure activity were included in our study. Seizure activity was monitored behaviorally and terminated with an injection of diazepam (10 mg/kg, i.p.) after 4 h of convulsive SE. The rats treated with lovastatin received two doses of 20 mg/kg via an oesophagic probe immediately and 24 hours after SE induction. Seven days after pilocarpine-induced SE, all the animals were perfused and their brains were processed for histological analysis through Nissl method. RESULTS: The cell counts in the Nissl-stained sections performed within the hippocampal formation showed a significant cell loss in rats that received pilocarpine and presented SE (CA1=26.8 +/- 13.67; CA3=38.1 +/- 7.2; hilus=43.8 +/- 3.95) when compared with control group animals (Group A: CA1=53.2 +/- 9.63; CA3=63.5 +/- 13.35; hilus=59.08 +/- 10.24; Group B: CA1=74.3 +/- 8.16; CA3=70.1 +/- 3.83; hilus=70.6 +/- 5.10). The average neuronal cell number of CA1 subfield of rats that present SE and received lovastatin (44.4 +/- 17.88) was statically significant increased when compared with animals that just presented SE. CONCLUSION: Lovastatin exert a neuroprotective role in the attenuation of brain damage after SE.

[Analysis of cardiac parameters in animals with epilepsy: possible cause of sudden death?]. / Avaliação de parâmetros cardíacos em animais com epilepsia: possível causa de morte súbita?

Among the causes for sudden death in epilepsy, cardiac dysfunction has been an area of interest. Based on this, the aim of our study was to evaluate the heart rate (in vivo and in vitro) and ventricular pressure in vitro of rats with epilepsy induced by pilocarpine. Adult male Wistar rats (n=6) were given pilocarpine hydrochloride to induce status epilepticus. Control rats (n=6) received saline solution instead pilocarpine. Our results showed significant differences in the mean of heart rate in vivo between the groups. In contrast, we did not find differences during in vitro experiments. Our results suggest a central nervous system modulation on the heart, which could explain the sudden unexpected death in epilepsy.

[Qualitative study of hippocampal formation in hypertensive rats with epilepsy]. / Estudo qualitativo da formação hipocampal de animais hipertensos com epilepsia.

UNLABELLED: The aim of our study was to investigate the hippocampus and dentate gyrus neuropathological features of spontaneous hypertensive rats (SHR) with epilepsy. METHODS: Animals were randomly divided into 4 groups: control Wistar, Wistar with epilepsy, control SHR and SHR with epilepsy. The pilocarpine model of epilepsy was used in this experiement. After spontaneous recurrent seizures, all animals were perfused and their brains processed for histological analysis through Nissl and neo-Timm methods. RESULTS: In the Wistar rats with epilepsy we observed cell loss in hippocampal subfields CA1, CA3 and hilus of the dentate gyrus when compared with control animals. In the SHR with epilepsy we observed hippocampal formation atrophy with ventricular dilatation. No morphological alterations were observed in SHR and Wistar control rats. The neo-Timm staining of hippocampal formation has shown supragranular sprouting in Wistar and SHR with epilepsy. CONCLUSION We found neuropathological alterations in hippocampal formation in Wistar with epilepsy and SHR with epilepsy, suggesting that epilepsy per se or associated to hypertention are able to cause neuronal damage.

Seizure occurrence in patients with chronic renal insufficiency in regular hemodialysis program.

Hemodialysis-associated seizure is a complication of hemodialysis. This report describes the occurrence of seizures in patients with end stage renal disease on dialysis therapy at the Nephrology Institute of Mogi das Cruzes, São Paulo State, Brazil. A retrospective medical history of 189 patients was reviewed to investigate the occurrence of convulsive seizures during dialytic program. Seven patients with history of seizures were selected but five of them were included in our study. Three patients presented generalized tonic-clonic seizures, one had partial seizure with secondary generalization, and one presented unclassified seizure. Three patients presented seizure just during the dialysis (unique seizure) and one of them presented convulsive status epilepticus. The two other patients had already presented seizures prior the beginning of dialysis. We conclude that seizures in renal failure could be considered as occasional events that do not usually become chronic.

The effects of alcohol intake and withdrawal on the seizures frequency and hippocampal morphology in rats with epilepsy.

The aim of our study, using the pilocarpine model of epilepsy, was to investigate the effects of alcohol administration and withdrawal on the spontaneous recurrent seizures (SRSs). Four groups of adult, male Wistar rats were studied: (A). control rats (n=10), received neither pilocarpine nor alcohol, (B). alcohol-treated rats (n=10), received a daily dose of 3.0 g x kg(-1) of a 30% alcohol solution via an oesophagic probe for 30 days, (C). rats with epilepsy (n=10), (D). rats with epilepsy with alcohol intake (n=10). SRSs were induced by a single dose of pilocarpine (i.p.) and the basal frequency of SRSs was video monitored (24h per day) for 30 days. Following this period, the animals of group D received a daily dose of alcohol solution as described above and at the end of this period, alcohol administration was stopped and the seizure frequency was assessed for more 30 days. The basal seizure frequency observed in groups C and D during the first 30 days was 2.2+/-1.8 seizures per week per animal. In group D, it was observed an increase to 12.2+/-5.8 during the first 2 weeks of alcohol administration. During the last 2 weeks of alcohol administration, the number of SRSs returned to the previous basal level. During alcohol withdrawal the seizure frequency increased to 14.3+/-7.4 seizures per week per animal for the first 2 weeks, and returned to the basal level in the remaining period of observation. The Neo-Timm and Nissl staining of hippocampal formation and of the dentate gyrus in rats with epilepsy showed a cell loss in the hippocampal subfield CA1 and in the hillus of dentate gyrus. In rats with epilepsy with alcohol intake, we observed a cell loss in hippocampal subfields CA3 and hillus of the dentate gyrus, with significant neuronal death in subfield CA1, when compared with control animals. The alcohol withdrawal syndrome is a crucial event for the development of functional and neuropathological alterations associated with epilepsy.

Altered anxiety-related and abnormal social behaviors in rats exposed to early life seizures.

Neonatal seizures are the most common manifestation of neurological dysfunction in the neonate. The prognosis of neonatal seizures is highly variable, and the controversy remains whether the severity, duration, or frequency of seizures may contribute to brain damage independently of its etiology. Animal data indicates that seizures during development are associated with a high probability of long-term adverse effects such as learning and memory impairment, behavioral changes and even epilepsy, which is strongly age dependent, as well as the severity, duration, and frequency of seizures. In preliminary studies, we demonstrated that adolescent male rats exposed to one-single neonatal status epilepticus (SE) episode showed social behavior impairment, and we proposed the model as relevant for studies of developmental disorders. Based on these facts, the goal of this study was to verify the existence of a persistent deficit and if the anxiety-related behavior could be associated with that impairment. To do so, male Wistar rats at 9 days postnatal were submitted to a single episode of SE by pilocarpine injection (380 mg/kg, i.p.) and control animals received saline (0.9%, 0.1 mL/10 g). It was possible to demonstrate that in adulthood, animals exposed to neonatal SE displayed low preference for social novelty, anxiety-related behavior, and increased stereotyped behavior in anxiogenic environment with no locomotor activity changes. On the balance, these data suggests that neonatal SE in rodents leads to altered anxiety-related and abnormal social behaviors.