Hormonal and biochemical changes in female Proechimys guyannensis, an animal model of resistance to pilocarpine-induced status epilepticus.

The Amazon rodent Proechimys guyannensis is widely studied for hosting various pathogens, though rarely getting sick. Previous studies on male Proechimys have revealed an endogenous resistance to epilepsy. Here, we assess in female Proechimys, whether sex hormones and biochemical aspects can interfere with the induction of status epilepticus (SE). The lithium-pilocarpine ramp-up protocol was used to induce SE, and blood sera were collected at 30 and 90 min after SE, alongside brains, for biochemical, western blot and immunohistochemical analyses. Results from non-ovariectomised (NOVX) Proechimys were compared to ovariectomised (OVX) animals. Data from female Wistars were used as a positive control of SE inductions. SE latency was similar in NOVX, OVX, and female Wistars groups. However, the pilocarpine dose required to induce SE in Proechimys was higher (25- to 50-folds more). Despite a higher dose, Proechimys did not show strong SE like Wistars; they only reached stage 2 of the Racine scale. These data suggest that female Proechimys are resistant to SE induction. Glucose and progesterone levels increased at 30 min and returned to normal at 90 min after SE. A relevant fact because in humans and rodents, SE leads to hypoglycaemia after 30 min of SE and does not return to normal levels in a short time, a typical adverse effect of SE. In OVX animals, a decrease in GABAergic receptors within 90 min of SE may suggest that ovariectomy produces changes in the hippocampus, including a certain vulnerability to seizures. We speculate that progesterone and glucose increases form part of the compensatory mechanisms that provide resistance in Proechimys against SE induction.

Characterization of the estrous cycle in the Amazon spiny rat (Proechimys guyannensis).

Males of Proechimys guyannensis, a rodent living in the Amazon rainforest are studied in biomedical research because of their antiepileptogenic mechanism. Females are usually taken from experimental designs, because of limited data of this sex. This study aimed to characterize the estrous cycle to include females together with males in research in a more balanced approach. The estrous cycle of P. guyannensis based through exfoliative cytology, determination of the vaginal occlusion membrane state, and hormonal analysis. In this study, cytological analyses of vaginal smears were performed for three months, three times a day. The observed length of the estrous cycle was 247 ± 81 h (mean ± SD) with a reproductive phase of 27.08 ± 17.39 h (estrus stage). We observed a frequent presence of both the open and closed states of the vaginal membrane in the estrus stage (fertile period) although only the open stage is a prerequisite for successful copulation. High levels of progesterone and estradiol were detected in proestrus. Levels of follicle-stimulating hormone peaked at the estrus stage. These data will establish the parameters and subsidies to set the grounds for future research either for investigating the biology of this species or to use P. guyannensis in research that previously excluded females. Information regarding female Proechimys is relevant to not only describe the species but also explain the interaction between sex hormones and physiological responses. Moreover, the present results will enhance rigor and reproducibility in preclinical studies. In conclusion, P. guyannensis reproductive cycles can occur spontaneously and cyclically independent of mating stimulation and the high levels of FSH in the estrus stage, suggest that ovulation occurs in the late phase of the estrus.

Altered MT1 and MT2 melatonin receptors expression in the hippocampus of pilocarpine-induced epileptic rats.

Clinical and experimental findings show that melatonin may be used as an adjuvant to the treatment of epilepsy-related complications by alleviates sleep disturbances, circadian alterations and attenuates seizures alone or in combination with AEDs. In addition, it has been observed that there is a circadian component on seizures, which cause changes in circadian system and in melatonin production. Nevertheless, the dynamic changes of the melatoninergic system, especially with regard to its membrane receptors (MT1 and MT2) in the natural course of TLE remain largely unknown. The aim of this study was to evaluate the 24-hour profile of MT1 and MT2 mRNA and protein expression in the hippocampus of rats submitted to the pilocarpine-induced epilepsy model analyzing the influence of the circadian rhythm in the expression pattern during the acute, silent, and chronic phases. Melatonin receptor MT1 and MT2 mRNA expression levels were increased in the hippocampus of rats few hours after SE, with MT1 returning to normal levels and MT2 reducing during the silent phase. During the chronic phase, mRNA expression levels of both receptors return to levels close to control, however, presenting a different daily profile, showing that there is a circadian change during the chronic phase. Also, during the acute and silent phase it was possible to verify MT1 label only in CA2 hippocampal region with an increased expression only in the dark period of the acute phase. The MT2 receptor was present in all hippocampal regions, however, it was reduced in the acute phase and it was found in astrocytes. In chronic animals, there is a reduction in the presence of both receptors especially in regions where there is a typical damage derived from epilepsy. Therefore, we conclude that SE induced by pilocarpine is able to change melatonin receptor MT1 and MT2 protein and mRNA expression levels in the hippocampus of rats few hours after SE as well as in silent and chronic phases.

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.

Effects of different physical exercise programs on susceptibility to pilocarpine-induced seizures in female rats.

In epilepsy, the most common serious neurological disorder worldwide, several investigations in both humans and animals have shown the effectiveness of physical exercise programs as a complementary therapy. Among the benefits demonstrated, regular exercise can decrease the number of seizures as well as improve cardiovascular and psychological health in people with epilepsy. While many studies in animals have been performed to show the beneficial effects of exercise, they exclusively used male animals. However, females are also worthy of investigation because of their cyclical hormonal fluctuations and possible pregnancy. Considering the few animal studies concerning seizure susceptibility and exercise programs in females, this study aimed to verify whether exercise programs can interfere with seizure susceptibility induced by pilocarpine in adult female Wistar rats. Animals were randomly divided into three groups: control, forced, and voluntary (animals kept in a cage with a wheel). After the final exercise session, animals received a pilocarpine hydrochloride (350 mg/kg i.p.; Sigma) injection to induce seizures. To measure the intensity of pilocarpine-induced motor signs, we used a scale similar to that developed by Racine (1972) in the kindling model. During a 4-h period of observation, we recorded latency for first motor signs, latency for reaching SE, number of animals that developed SE, and intensity of pilocarpine-induced motor signs. No difference was observed among groups in latency for first motor signs and in the number of animals that developed SE. Although the voluntary group presented more intense motor signs, an increased latency for developing SE was observed compared with that in forced and control groups. Our behavioral results are not enough to explain physiological and molecular pathways, but there are mechanisms described in literature which may allow us to propose possible explanations. Voluntary exercise increased latency to SE development. Further investigation is necessary to elucidate the pathways involved in these results, while more studies should be performed regarding gender specific differences.

Maternal seizures can affect the brain developing of offspring.

To elucidate the impact of maternal seizures in the developing rat brain, pregnant Wistar rats were subjected to the pilocarpine-induced seizures and pups from different litters were studied at different ages. In the first 24 h of life, blood glucose and blood gases were analyzed. (14)C-leucine [(14)C-Leu] incorporation was used to analyze protein synthesis at PN1, and Western Blot method was used to analyze protein levels of Bax, Bcl-2 and Poly(ADP-ribose) polymerase-1 (PARP-1) in the hippocampus (PN3-PN21). During the first 22 days of postnatal life, body weight gain, length, skull measures, tooth eruption, eye opening and righting reflex have been assessed. Pups from naive mothers were used as controls. Experimental pups showed a compensated metabolic acidosis and hyperglycemia. At PN1, the [(14)C-Leu] incorporation into different studied areas of experimental pups was lower than in the control pups. During development, the protein levels of Bax, Bcl-2 and PARP-1 in the hippocampus of experimental pups were altered when compared with control pups. A decreased level of pro- and anti-apoptotic proteins was verified in the early postnatal age (PN3), and an increased level of pro-apoptotic proteins concomitant with a reduced level of anti-apoptotic protein was observed at the later stages of the development (PN21). Experimental pups had a delay in postnatal growth and development beyond disturb in protein synthesis and some protein expression during development. These changes can be result from hormonal alterations linked to stress and/or hypoxic events caused by maternal epileptic seizures during pregnancy.

Pilocarpine-induced epilepsy alters the expression and daily variation of the nuclear receptor RORα in the hippocampus of rats.

It is widely known that there is an increase in the inflammatory responses and oxidative stress in temporal lobe epilepsy (TLE). Further, the seizures follow a circadian rhythmicity. Retinoic acid receptor-related orphan receptor alpha (RORα) is related to anti-inflammatory and antioxidant enzyme expression and is part of the machinery of the biological clock and circadian rhythms. However, the participation of RORα in this neurological disorder has not been studied. The aim of this study was to evaluate the RORα mRNA and protein content profiles in the hippocampus of rats submitted to a pilocarpine-induced epilepsy model at different time points throughout the 24-h light-dark cycle analyzing the influence of the circadian rhythm in the expression pattern during the acute, silent, and chronic phases of the experimental model. Real-time PCR and immunohistochemistry results showed that RORα mRNA and protein expressions were globally reduced in both acute and silent phases of the pilocarpine model. However, 60days after the pilocarpine-induced status epilepticus (chronic phase), the mRNA expression was similar to the control except for the time point 3h after the lights were turned off, and no differences were found in immunohistochemistry. Our results indicate that the status epilepticus induced by pilocarpine is able to change the expression and daily variation of RORα in the rat hippocampal area during the acute and silent phases. These findings enhance our understanding of the circadian pattern present in seizures as well as facilitate strategies for the treatment of seizures.

Influence of melatonin treatment on the survival and seizures frequency in pilocarpine-induced epilepsy in rats / Influência do tratamento com melatonina na sobrevivência e frequência das crises epiléticas em ratos induzidas por pilocarpina

Objetivo: verificar se o tratamento com melatonina no período crônico pode alterar a frequência de crises e sobrevida de ratos Wistar submetidos ao modelo de epilepsia induzido por pilocarpina. Métodos: os animais foram divididos em dois grupos: Epi+MEL (n=8), animais tratados com melatonina (10 mg/kg) no período crônico; Epi+VEI (n=5), animais tratados com solução veículo no período crônico. Para analisar a duração e a frequência de crises os animais foram vídeo-monitorados antes do tratamento no 5 o e 7o mês de vida e após o início do tratamento no 9°, 11° e 16° mês de vida. Resultados: os animais tratados com melatonina não apresentaram alterações na duração e frequência de crises. Embora tenhamos observado uma taxa de sobrevida de 87,5% nos animais tratados com melatonina e 40% nos animais tratados com veículo, não observamos diferença estatística. Conclusão: o tratamento com melatonina não foi eficaz no controle da frequência e duração das crises, bem como não alterou a sobrevida dos animais. Contudo, acreditamos que a melatonina tenha forte potencial no aumento da expectativa de vida, porém mais estudos são necessários para uma melhor compreensão da sua ação neuroprotetora, bem como seu papel na expectativa de vida...

Objective: to verify if treatment with melatonin in the chronic period can modify the seizures frequency and survival in Wistar rats submitted to pilocarpine-induced model of epilepsy. Methods: animals were divided in two groups: Epi+MEL (n=8) animals treated with melatonin (10 mg / Kg) in the chronic period; EPI+VEH (n=5) animals treated with vehicle solution in the chronic period. To analyze duration and frequency of seizure, all animals were video-monitored during the 5th and 7th month of life and during the treatments in the 9th, 11th and 16th month of life. Results: the animals treated with melatonin in the chronic phase not presented changes in the duration and frequency of seizures. Although, the animals treated with melatonin have shown a survival rate of 87.5% and the animals treated with vehicle 40%, this finding was not statistically significant. Conclusion: Chronic treatment with melatonin was not effective in the control of frequency and duration of seizures, as well did not modify the survival of the animals. Nevertheless, we believe that melatonin has strong potential to increase life expectancy, however, more studies are needed for a better understanding of its neuroprotective action, as well astheir role in life expectancy...

Changes in aminoacidergic and monoaminergic neurotransmission in the hippocampus and amygdala of rats after ayahuasca ingestion.

AIM: To evaluate changes in neurotransmission induced by a psychoactive beverage ayahuasca in the hippocampus and amygdala of naive rats. METHODS: The level of monoamines, their main metabolites and amino acid neurotransmitters concentrations were quantified using high performance liquid chromatography (HPLC). Four groups of rats were employed: saline-treated and rats receiving 250, 500 and 800 mg/kg of ayahuasca infusion (gavage). Animals were killed 40 min after drug ingestion and the structures stored at -80 °C until HPLC assay. The data from all groups were compared using Analysis of variance and Scheffé as post test and P < 0.05 was accepted as significant. RESULTS: The results showed decreased concentrations of glycine (GLY) (0.13 ± 0.03 vs 0.29 ± 0.07, P < 0.001) and γ-aminobutyric acid (GABA) (1.07 ± 0.14 vs 1.73 ± 0.25, P < 0.001) in the amygdala of rats that received 500 of ayahuasca. Animals that ingested 800 mg/kg of ayahuasca also showed a reduction of GLY level (0.11 ± 0.01 vs 0.29 ± 0.07, P < 0.001) and GABA (0.98 ± 0.06 vs 1.73 ± 0.25, P < 0.001). In the hippocampus, increased GABA levels were found in rats that received all ayahuasca doses: 250 mg/kg (1.29 ± 0.19 vs 0.84 ± 0.21, P < 0.05); 500 mg/kg (2.23 ± 038 vs 084 ± 0.21, P < 0.05) and 800 mg/kg (1.98 ± 0.92 vs 0.84 ± 0.21, P < 0.05). In addition, an increased utilization rate of all monoamines was found in the amygdala after ayahuasca administration in doses: 250 mg/kg (noradrenaline: 0.16 ± 0.02 vs 0.36 ± 0.06, P < 0.01; dopamine: 0.39 ± 0.012 vs 2.39 ± 0.84, P < 0.001; serotonin: 1.02 ± 0.22 vs 4.04 ± 0.91, P < 0.001), 500 mg/kg (noradrenaline: 0.08 ± 0.02 vs 0.36 ± 0.06, P < 0.001; dopamine: 0.33 ± 0.19 vs 2.39 ± 0.84, P < 0.001; serotonin: 0.59 ± 0.08 vs 4.04 ± 0.91, P < 0.001) and 800 mg/kg (noradrenaline: 0.16 ± 0.04 vs 0.36 ± 0.06, P < 0.001; dopamine: 0.84 ± 0.65 vs 2.39 ± 0.84, P < 0.05; serotonin: 0.36 ± 0.02 vs 4.04 ± 0.91, P < 0.001). CONCLUSION: Our data suggest increased release of inhibitory amino acids by the hippocampus and an increased utilization rate of monoamines by the amygdala after different doses of ayahuasca ingestion.

Sexual response in female rats with status epilepticus.

PURPOSE: Female sexual function is complex and may be disrupted by disease, in particular epilepsy. Chronic seizures in women can have adverse effects on reproductive function, but it has been difficult to dissociate the effects of epilepsy from those related to anticonvulsant medications. The purpose of this study was to evaluate sexual behavior in female rats submitted to pilocarpine-induced status epilepticus (SE). METHODS: Adult female Wistar rats were given saline or pilocarpine (350 mg/kg, i.p.) to induce SE. The groups were distributed according to the treatment or response to pilocarpine: CTRL (control rats maintained in the home-cage after saline administration); NSE (non-status epilepticus, rats that did not display convulsive and intermittent seizures after pilocarpine injection) and SE (status epilepticus, rats that displayed convulsive and intermittent seizures after pilocarpine injection). After 50 days, sexual receptivity in the female rats was artificially induced via administration of a combination of estradiol and progesterone. Sexual behavior was evaluated during three sessions in the presence of a sexually experienced male rat. Receptivity and proceptivity behaviors, as well as hormones concentrations, were monitored. KEY FINDINGS: Significant decreases in proceptivity and receptivity behaviors during the three tests were observed in SE female rats. The rejection response was significantly increased in SE rats compared with CTRL or NSE groups. Progesterone, testosterone, and corticosterone were unchanged between the groups. SIGNIFICANCE: The SE female rats showed lower sexual motivation and performance regardless of their steroid hormones levels.

The levels of renin-angiotensin related components are modified in the hippocampus of rats submitted to pilocarpine model of epilepsy.

We previously showed that patients with temporal lobe epilepsy (TLE) present an increased expression of angiotensin II (AngII) AT1 and AT2 receptors in the hippocampus, supporting the idea of an upregulation of renin-angiotensin system (RAS) in this disease. This study aimed to verify the relationship between the RAS and TLE during epileptogenesis. Levels of the peptides angiotensin I (AngI), angiotensin II (AngII) and angiotensin 1-7 (Ang 1-7), were detected by HPLC assay. Angiotensin AT1 and AT2 receptors, Mas mRNA receptors and angiotensin converting enzyme (ACE), tonin and neutral endopeptidase (NEP) mRNA were also quantified at the hippocampus of Wistar rats by real time PCR, during acute (n=10), silent (n=10) and chronic (n=10) phases of pilocarpine-induced epilepsy. We observed an increased peptide level of Ang1-7 into acute and silent phases, decreasing importantly (p≤0.05) in the chronic phase, suggesting that AngI may be converted into Ang 1-7 by NEP, which is present in high levels in these periods. Our results also showed increased peptide level of AngII in the chronic phase of this model. In contraposition, the ACE expression is reduced in all periods. These data suggest that angiotensinogen or AngI may be cleaved to AngII by tonin, which showed increased expression in all phases. We found changes in AT1, AT2 and Mas mRNA receptors levels suggesting that Ang1-7 could act at Mas receptor during the silent period. Herein, we demonstrated for the first time, changes in angiotensin-related peptides, their receptors as well as the releasing enzymes in the hippocampus of rats during pilocarpine-induced 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.

Neuroglobin is up-regulated in the cerebellum of pups exposed to maternal epileptic seizures.

To evaluate a potential insult in the cerebellum of pups exposed to maternal epileptic seizures during intrauterine life, female rats were subjected to pilocarpine-induced epilepsy. Pups from different litters were sacrificed at 1, 3, 7 and 14 post-natal days (PN) and neuroglobin (Ngb) and gliosis were analyzed in the cerebellum by Western blotting (WB) and RT-PCR. (14)C-l-leucine-[(14)C-Leu] incorporation was used to analyze protein synthesis at PN1. Nitric Oxide (NO) and thiobarbituric acid-reactive substances (TBARS) levels were also measured. Pups from naive mothers were used as controls. The mRNA level of Ngb was increased in experimental animals at PN1 ((**)p ≤ 0.001) and PN3 ((**)p ≤ 0.001), at PN7 ((***)p ≤ 0.0001) and at PN14 ((**)p ≤ 0.001) compared to the respective controls. The protein level of Ngb increased significantly in the experimental pups at PN1 ((*)p ≤ 0.05) and at PN3 ((**)p ≤ 0.001), when compared to the control pups at PN1 and PN3. At PN7 and PN14 no difference was found. The mRNA level of GFAP increased significantly about two times at PN3 ((*)p ≤ 0.05) and PN7 ((*)p ≤ 0.05) in the experimental pups when compared to the respective controls, but was unchanged in the other studied ages. Data showed that experimental pups at PN1 exhibited reduced (about 2 times, (*)p ≤ 0.05) total protein synthesis in the cerebellum when compared to control. No differences were found in the NO and TBARS levels. Our data support the hypothesis that an up-regulation of Ngb could be a compensatory mechanism in response to the hypoxic-ischemic insults caused by seizures in pups during intrauterine life.

Melatonin administration after pilocarpine-induced status epilepticus: a new way to prevent or attenuate postlesion epilepsy?

OBJECTIVE: The goal of this study was to verify the effects of treatment with melatonin and N-acetylserotonin on the pilocarpine-induced epilepsy model. METHODS: The animals were divided into four groups: (1) animals treated with saline (Saline); (2) animals that received pilocarpine and exhibited SE (SE); (3) animals that exhibited SE and were treated with N-acetylserotonin (30 minutes and 1, 2, 4, 6, 12, 24, 36, and 48 hours) after SE onset (SE+NAS); (4) animals that exhibited SE and were treated with melatonin at the same time the SE+NAS group (SE+MEL). Behavioral (latency to first seizure, frequency of seizures, and mortality) and histological (Nissl and neo-Timm) parameters were analyzed. RESULTS: The animals treated with melatonin (SE+MEL) had a decreased number of spontaneous seizures during the chronic period (P<0.05), a reduction in mossy fiber sprouting, and less cell damage than the SE group. Animals treated with N-acetylserotonin did not exhibit any kind of significant change. CONCLUSION: Melatonin exerts an important neuroprotective effect by attenuating SE-induced postlesion and promoting a decrease in the number of seizures in epileptic rats. This suggests, for the first time, that melatonin could be used co-therapeutically in treatment of patients exhibiting SE to minimize associated injuries in these situations.

Relationship between fluid-attenuated inversion-recovery (FLAIR) signal intensity and inflammatory mediator's levels in the hippocampus of patients with temporal lobe epilepsy and mesial temporal sclerosis.

We investigated a relationship between the FLAIR signal found in mesial temporal sclerosis (MTS) and inflammation. Twenty nine patients were selected through clinical and MRI analysis and submitted to cortico-amygdalo-hippocampectomy to seizure control. Glutamate, TNFα, IL1, nitric oxide (NO) levels and immunostaining against IL1ß and CD45 was performed. Control tissues (n=10) were obtained after autopsy of patients without neurological disorders. The glutamate was decreased in the temporal lobe epilepsy (TLE) -MTS group (p<0.001), suggesting increased release of this neurotransmitter. The IL1ß and TNFα were increased in the hippocampus (p<0.05) demonstrating an active inflammatory process. A positive linear correlation between FLAIR signal and NO and IL1ß levels and a negative linear correlation between FLAIR signal and glutamate concentration was found. Lymphocytes infiltrates were present in hippocampi of TLE patients. These data showed an association between hippocampal signal alteration and increased inflammatory markers in TLE-MTS.

Relationship between fluid-attenuated inversion-recovery (FLAIR) signal intensity and inflammatory mediator's levels in the hippocampus of patients with temporal lobe epilepsy and mesial temporal sclerosis / Correlação da intensidade do sinal em FLAIR e os níveis de mediadores inflamatórios no hipocampo de pacientes com epilepsia do lobo temporal e esclerose mesial temporal

We investigated a relationship between the FLAIR signal found in mesial temporal sclerosis (MTS) and inflammation. Twenty nine patients were selected through clinical and MRI analysis and submitted to cortico-amygdalo-hippocampectomy to seizure control. Glutamate, TNFα, IL1, nitric oxide (NO) levels and immunostaining against IL1β and CD45 was performed. Control tissues (n=10) were obtained after autopsy of patients without neurological disorders. The glutamate was decreased in the temporal lobe epilepsy (TLE) -MTS group (p<0.001), suggesting increased release of this neurotransmitter. The IL1β and TNFα were increased in the hippocampus (p<0.05) demonstrating an active inflammatory process. A positive linear correlation between FLAIR signal and NO and IL1β levels and a negative linear correlation between FLAIR signal and glutamate concentration was found. Lymphocytes infiltrates were present in hippocampi of TLE patients. These data showed an association between hippocampal signal alteration and increased inflammatory markers in TLE-MTS.

Este estudo foi delineado para investigar a presença de relação entre a intensidade de sinal em FLAIR e níveis de citocinas, óxido nítrico (NO) e glutamato no hipocampo de pacientes com epilepsia do lobo temporal refratária, associada com esclerose mesial (TLE-MTS). Vinte e nove pacientes foram selecionados através de análise clínica e de ressonância magnética (RM) que foram submetidos a cortico-amigdalo-hipocampectomia para o controle das crises. Os níveis de glutamato foram avaliados por HPLC, as citocinas TNFα e IL1β por ELISA e os níveis de NO via NO system. Avaliamos também por imuno-histoquímica a expressão de IL1β e CD45 em tecidos controles e com esclerose. Tecido controle foi obtido após autópsia de indivíduos mortos sem disfunções inflamatórias e neurológicas (n=10). A concentração de glutamato se mostrou reduzida no tecido TLE-MTS (p<0,001) sugerindo aumento na liberação desse neurotransmissor. TNFα e IL1β também apresentaram níveis elevados no hipocampo dos pacientes (p<0,05), demonstrando um processo inflamatório crônico. Houve uma correlação linear positiva entre a intensidade do sinal em FLAIR e os níveis de NO e IL1β. Em contraste, uma correlação linear negativa foi encontrada entre a intensidade do sinal em FLAIR e níveis de glutamato no hipocampo com esclerose. Infiltrado linfocitário hipocampal também foi visualizado pela imuno-marcação com CD45 em pacientes com TLE-MTS. Esses dados mostraram uma associação entre alteração de sinal na RM e marcadores inflamatórios em pacientes com TLE-MTS.

Kallikrein 1 is overexpressed by astrocytes in the hippocampus of patients with refractory temporal lobe epilepsy, associated with hippocampal sclerosis.

Kallikrein 1 (hK1) is a tissue enzyme responsible for kinin release in inflammatory cascade. This study was delineated to study the distribution and the co-localization of hK1 and kinin B1 and B2 receptors with glial and/or neuronal proteins markers, in the hippocampus of patients with refractory temporal lobe epilepsy, associated with hippocampal sclerosis (TLE-HS), comparing with control tissues. Hippocampal levels of KLK1 mRNA were also measured. hK1, kinin B1 and B2 receptors, NeuN and GFAP were analyzed using immunohistochemistry and confocal microscopy and KLK1 mRNA was quantified with real time PCR. Increased expression of hK1 by astrocytes co-localized with GFAP was found, contrasting with kinin B1 and B2 receptors, which were co-localized with NeuN in the sclerotic hippocampus. In addition, KLK1 mRNA was also up-regulated in same tissues. These data suggest an overexpression of kallikrein-kinin system and a neuron-glia interaction in the inflammatory process present in refractory TLE-HS.

Neuroprotective effect of pyruvate and oxaloacetate during pilocarpine induced status epilepticus in rats.

Recent research data have shown that systemic administration of pyruvate and oxaloacetate causes an increased brain-to-blood glutamate efflux. Since increased release of glutamate during epileptic seizures can lead to excitotoxicity and neuronal cell death, we tested the hypothesis that glutamate scavenging mediated by pyruvate and oxaloacetate systemic administration could have a neuroprotective effect in rats subjected to status epilepticus (SE). SE was induced by a single dose of pilocarpine (350mg/kgi.p.). Thirty minutes after SE onset, a single dose of pyruvate (250mg/kgi.p.), oxaloacetate (1.4mg/kgi.p.), or both substances was administrated. Acute neuronal loss in hippocampal regions CA1 and hilus was quantitatively determined five hours after SE onset, using the optical fractionator method for stereological cell counting. Apoptotic cascade in the hippocampus was also investigated seven days after SE using caspase-1 and -3 activity assays. SE-induced neuronal loss in CA1 was completely prevented in rats treated with pyruvate plus oxaloacetate. The SE-induced caspase-1 activation was significantly reduced when rats were treated with oxaloacetate or pyruvate plus oxaloacetate. The treatment with pyruvate and oxaloacetate caused a neuroprotective effect in rats subjected to pilocarpine-induced SE.

Lovastatin decreases the synthesis of inflammatory mediators in the hippocampus and blocks the hyperthermia of rats submitted to long-lasting status epilepticus.

Statins may act on inflammatory responses, decreasing oxidative stress and also reducing temperature after a brain ischemic insult. Previous data have indicated that statins protect neurons from death during long-lasting status epilepticus (SE) and attenuate seizure behaviors in animals treated with kainic acid. In this context, the study described here aimed to investigate the effect of lovastatin on body temperature and on mRNA expression levels of hippocampal cytokines such as interleukin-1ß, interleukin-6, tumor necrosis factor α, and kinin B1 and B2 receptors of rats submitted to pilocarpine-induced SE. Quantitative real-time polymerase chain reaction showed a significant decrease in mRNA expression of interleukin-1ß, interleukin-6, tumor necrosis factor α, and kinin B1 receptor in animals with SE treated with lovastatin, compared with untreated animals with SE (P<0.001). Lovastatin also reduced SE-induced hyperthermia, indicating that mechanisms related to brain protection are triggered by this drug under conditions associated with acute excitotoxicity or long-lasting SE.