Neuroprotector effect of melatonin and N-acetilserotonin in the epileptogenesis and in the control of seizures in animals submitted to the pilocarpine model

PURPOSE: The aim of this research was to study the effects of treatment with melatonin and N-acetilserotonin in the development of pilocarpina model of epilepsy in adult male rats. METHODS: Part I - The animals were divided in 4 groups: SALINE - animals that received only saline; SE - animals submitted to status epilepticus (SE); NAS + SE - animals that received pre-treatment with N-acetylserotonin and were submitted to SE and MEL + SE - animals that received pre-treatment with melatonin and were submitted to SE. Part II - The animals were divided in 6 groups: SALINE - animals that received only saline; SE - animals submitted to status epilepticus (SE); PX + SE - animals submitted to pinealectomy and to SE 7 days later; SH + SE - animals submitted to sham-surgery and to SE 7 days later; SE + NAS - animals submitted to SE and treated with N-acetylserotonin (2,5 mg/kg), 30 min, 1 h, 2 h, 4 h, 6 h, 12 h, 24 h, 36 h and 48 h after the SE and SE + MEL - animals submitted to SE and treated with melatonin (2,5 mg/kg), 30 min, 1 h, 2 h, 4 h, 6 h, 12 h, 24 h, 36 h and 48 h after the SE. Following the treatment the animals were continuously video-recorded for 60 days. The behavioral parameters were observed: latency for the SE in minutes, latency for the first spontaneous seizures (ie, duration of the silent period), number of spontaneous seizures during the chronic period and mortality. Five animals per group were perfused for neo-Timm assay. RESULTS: Part I - The animals treated with melatonin and N-acetylserotonin presented an increased of latency for the status epilepticus and decreased number of spontaneous seizures during the chronic period when compared to SE group. The mortality was reduced 100% in animals treated with melatonin and theses animals presented a minor mossy fibers sprouting. Part II - The latency for the first spontaneous seizures and mortality were similar in all groups. The animals treated with melatonin presented a decreased number of spontaneous seizures during the chronic period when compared to PX + SE group and a minor mossy fibers sprouting when compared to SE, SH + SE and PX + SE groups. CONCLUSION: Our data show that the melatonin and N-acetylserotonin have an important neuroprotector effect in the epileptogenesis and in the control of seizures during the chronic period of the pilocarpina model of epilepsy induced by pilocarpina.Universidade Federal de São Paulo (UNIFESP) Escola Paulista de MedicinaUNIFESP, EPMSciEL

Atraso no desaparecimento da nestina na glia radial cortical de ratos submetidos a recorrentes status epilepticus durante o desenvolvimento pós-natal precoce

OBJECTIVE: Nestin is temporarily expressed in several tissues during development and it is replaced by other protein types during cell differentiation process. This unique property allows distinguishing between undifferentiated and differentiated cells. This study was delineated to analyze the temporal pattern of nestin expression in cortical radial glial cells of rats during normal development and of rats submitted to recurrent status epilepticus (SE) in early postnatal life (P). METHOD: Experimental rats were submitted to pilocarpine-induced SE on P7-9. The cortical temporal profile of nestin was studied by immunohistochemistry at multiple time points (P9, P10, P12, P16, P30 and P90). RESULTS: We observed delayed nestin down-regulation in experimental rats of P9, P10, P12 and P16 groups. In addition, few radial glial cells were still present only in P21 experimental rats. CONCLUSION: Our results suggested that SE during early postnatal life alters normal maturation during a critical period of brain development.OBJETIVO: A nestina, temporariamente expressa em diversos tecidos durante o desenvolvimento, é substituída no processo de diferenciação celular, o que permite a distinção entre células diferenciadas e indiferenciadas. O objetivo deste estudo foi verificar o padrão temporal da expressão da nestina nas células da glia radial cortical de ratos durante o desenvolvimento normal e nos ratos submetidos a sucessivos status epilepticus (SE) no periodo pós-natal precoce (P). MÉTODO: Os animais foram submetidos ao SE induzido pela pilocarpina em P7-9. O perfil temporal da nestina foi estudado por imuno-histoquímica em P9, P10, P12, P16, P30 e P90. RESULTADOS: Nos ratos experimentais, observamos atraso no desaparecimento da nestina nos grupos P9, P10, P12 e P16. Ainda, encontramos algumas glias radiais corticais apenas em P21 experimental. CONCLUSÃO: Nossos resultados sugerem que o SE durante o desenvolvimento pós-natal precoce altera o processo de maturação durante um periodo crítico do desenvolvimento encefálico.Universidade Federal de São Paulo (UNIFESP) Laboratório de Neurologia ExperimentalUniversidade Federal de São Paulo (UNIFESP) Departamento de FisiologiaUniversidade Federal de São Paulo (UNIFESP) Departamento de BioquímicaUNIFESP, Laboratório de Neurologia ExperimentalUNIFESP, Depto. de FisiologiaUNIFESP, Depto. de BioquímicaSciEL

Indomethacin can downregulate the levels of inflammatory mediators in the hippocampus of rats submitted to pilocarpine-induced status epilepticus

OBJECTIVE: Refractory status epilepticus is one of the most life-threatening neurological emergencies and is characterized by high morbidity and mortality. Additionally, the use of anti-inflammatory drugs during this period is very controversial. Thus, this study has been designed to analyze the effect of a low dose of indomethacin (a COX inhibitor) on the expression of inflammatory molecules.METHOD: The hippocampus of rats submitted to pilocarpine-induced long-lasting status epilepticus was analyzed to determine the expression of inflammatory molecules with RT-PCR and immunohistochemistry.RESULTS: Compared with controls, reduced levels of the kinin B2 receptors IL1β and TNFα were found in the hippocampus of rats submitted to long-lasting status epilepticus and treated with indomethacin.CONCLUSIONS: These data show that low doses of indomethacin could be employed to minimize inflammation during long-lasting status epilepticus.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPQ)Institutos Nacionais de Ciencia e Tecnologia (INCT)Programa de Apoio a Nucleos de Excelencia (PRONEX)Universidade Federal de São Paulo (UNIFESP) Departamento de Neurologia/NeurocirurgiaSociedade Beneficente Israelita Brasileira Hospital Albert Einstein Neurofisiologia ClínicaUniversidade de Brasília Faculdade de Ciências da Saúde Laboratório de Patologia MolecularUniversidade Nove de Julho Departamento de Educação Física Programa de Pós-Graduação em Ciências da ReabilitaçãoUniversidade Federal de São Paulo (UNIFESP) Departamento de BioquímicaUNIFESP, Depto. de Neurologia/NeurocirurgiaUNIFESP, Depto. de BioquímicaSciEL

Efeito do exercício físico aeróbio em animais pinealectomizados submetidos ao modelo de epilepsia induzida por pilocarpina

OBJECTIVE: To better clarify the positive effects of physical exercise in the epilepsy, we analyzed the effect of the pinealectomy in animals with temporal lobe epilepsy (TLE) induced by pilocarpine submitted to an aerobic physical program. MATERIAL AND METHODS: Forty adults Wistar rats were used: 1) PX + CHRONIC - Pinealectomized animals (PX) with TLE (CHRONIC) without exercise (n = 9); 2) PX + CHRONIC + EXERCISE - submitted to an aerobic physical exercise program (n = 5); 3) CHRONIC - without exercise (n = 8); 4) CHRONIC + EXERCISE (n = 8); 5) CTRL - control without exercise (n = 5); 6) CTRL + EXERCISE (n = 5). The physical exercise program consisted of 1 hour of treadmill, 5 days/week, during 30 days, at 60% VO2max. The Nissl and neo-Timm methods were used. RESULTS: The pinealectomy increased the frequency of seizures in animals with epilepsy. It was observed a reduction of the neuronal death and mossy fiber sprouting in the animals with epilepsy submitted to an aerobic physical exercise program. However, the physical exercise program did not modify the frequency of the seizures in the pinealectomized animals.OBJETIVO: Buscando elucidar os efeitos positivos do exercício físico aeróbio na epilepsia, analisamos a influên-cia da pinealectomia em animais com epilepsia do lobo temporal (ELT) induzida por pilocarpina e submetidos a um programa de exercício físico. MATERIAL E MÉTODOS: Quarenta ratos Wistar adultos foram usados: 1) PX + CRÔNICO - pinealectomizados (PX) com ELT (CRÔNICO) sem exercício (n = 9); 2) PX + CRÔNICO + EXERCÍCIO - submetidos a um programa de exercício físico aeróbio (n = 5); 3) CRÔNICO - sem exercício (n = 8); 4) CRÔNICO + EXERCÍCIO (n = 8); 5) CTRL - controle sem epilepsia, sem exercício (n = 5); 6) CTRL + EXERCÍCIO (n = 5). O programa de exercício físico consistiu de corrida em esteira, 5 dias/semana (30 dias) a 60% VO2max. Os métodos de Nissl e neo-Timm foram utilizados. RESULTADOS: A pinealectomia aumentou a freqüência de crises em animais com epilepsia. Foi observada uma diminuição da morte neuronal e do brotamento de fibras musgosas em animais com epilepsia, submetidos ao programa de exercício físico. Entretanto, este programa não alterou a freqüência de crises em animais pinealectomizados.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo (UNIFESP)Universidade de Mogi das Cruzes Núcleo de Pesquisas Tecnológicas Laboratório de NeurociênciasUNIFESPSciEL

Exercise-induced hippocampal anti-inflammatory response in aged rats

Aging is often accompanied by cognitive decline, memory impairment and an increased susceptibility to neurodegenerative disorders. Most of these age-related alterations have been associated with deleterious processes such as changes in the expression of inflammatory cytokines. Indeed, higher levels of pro-inflammatory cytokines and lower levels of anti-inflammatory cytokines are found in the aged brain. This perturbation in pro-and anti-inflammatory balance can represent one of the mechanisms that contribute to age-associated neuronal dysfunction and brain vulnerability. We conducted an experimental study to investigate whether an aerobic exercise program could promote changes in inflammatory response in the brains of aged rats. To do so, we evaluated the levels of tumor necrosis factor alpha (TNF alpha), interleukin 1 beta (IL1 beta), interleukin 6 (IL6) and interleukin 10 (IL10) in the hippocampal formation of 18 month old rats that underwent treadmill training over 10 consecutive days. Quantitative immunoassay analyses showed that the physical exercise increased anti-inflammatory cytokine levels IL10 in the hippocampal formation of aged rats, when compared to the control group. the hippocampal levels of pro-inflammatory cytokines IL1 beta, IL6 and TNF alpha were not statistically different between the groups. However, a significant reduction in IL1 beta/IL10, IL6/IL10 and TNF alpha/IL10 ratio was observed in the exercised group in relation to the control group. These findings indicate a favorable effect of physical exercise in the balance between hippocampal pro-and anti-inflammatory during aging, as well as reinforce the potential therapeutic of exercise in reducing the risk of neuroinflammation-linked disorders.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Instituto Nacional de Neurociencia Translacional (INNT) (Brazil)Universidade Federal de São Paulo, Dept Fisiol, BR-04023900 São Paulo, BrazilFac Clube Naut Mogiano, BR-08773000 Mogi Das Cruzes, BrazilUniversidade Federal de São Paulo, Dept Neurol & Neurosurg, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Fisiol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Neurol & Neurosurg, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, BR-04023900 São Paulo, BrazilWeb of Scienc

A neurogênese induzida por crises no giro denteado não está relacionada ao brotamento de fibras musgosas, mas é dependente da idade, em ratos durante o desenvolvimento

Neurogenesis in the dentate gyrus (DG) has attracted attention since abnormal supragranular mossy fiber sprouting occurs in the same region, in temporal lobe epilepsy. Thus, we submitted developing rats to pilocarpine-induced status epilepticus (SE) to study the relationship between neurogenesis and mossy fiber sprouting. Groups were submitted to SE at: I-P9, II-P7, P8 and P9, III-P17 e IV-P21. Neurogenesis was quantified using BrdU protocol and confirmed through double staining, using neuronal pentraxin. Other animals were monitored by video system until P120 and their brain was studied (Timm and Nissl staining). The neurogenesis at P17 (p=0.007) and P21 (p=0.006) were increased. However, only P21 group showed recurrent seizures and the mossy fiber sprouting in the same region, during adult life, while P17 did not. Thus, our results suggest that neurogenesis is not related to mossy fiber sprouting neither to recurrent spontaneous seizures in pilocarpine model.A neurogênese no giro dentado tem atraído atenção já que ela ocorre na mesma região do hipocampo que o brotamento das fibras musgosas, na epilepsia do lobo temporal. Assim, submetemos ratos em desenvolvimento ao status epilepticus induzido (SE) por pilocarpine. Grupos foram submetidos em I-P9, II-P7, P8, P9; III-P17 e IV-P21. A neurogênese foi observada usando o protocolo do BrdU e confirmada por dupla marcação com pentraxina neuronal. Outros animais foram monitorados até P120 e seus cérebros analisados (Nissl e Timm). A neurogênese nos grupos P17 (p=0,007) e P21 (p=0,006) aumentaram. Entretanto, o P21 apresentou crises espontâneas e brotamento de fibras musgosas, na mesma região onde ocorreu a neurogênese, enquanto o grupo P17 apresentou somente aumento na neurogênese. Assim, nossos resultados sugerem que o fenômeno da neurogênese não está relacionado com o brotamento de fibras musgosas nem com o aparecimento de crises espontâneas e recorrentes no modelo da pilocarpina.Universidade Federal de São Paulo (UNIFESP) Escola Paulista de MedicinaUNIFESP, EPMSciEL

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 (350 mg/kg i.p.). Thirty minutes after SE onset, a single dose of pyruvate (250 mg/kg i.p.), oxaloacetate (1.4 mg/kg i.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. (C) 2010 Elsevier B.V. All rights reserved.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)UNIFESP São Paulo, Dept Neurol & Neurosurg, São Paulo, BrazilUNIFESP Santos, Dept Biosci, Santos, SP, BrazilUNIFESP São Paulo, Dept Neurol & Neurosurg, São Paulo, BrazilUNIFESP Santos, Dept Biosci, Santos, SP, BrazilWeb of Scienc