Repositório Institucional - Unifesp :: Navegando por Palavras-chave "pilocarpina"

Navegando por Palavras-chave "pilocarpina"

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Aplicação do método de interferência de rna na caracterização do papel do receptor p2x7 na epilepsia do lobo temporal
(Universidade Federal de São Paulo (UNIFESP), 2014-12-30) Amorim, Rebeca Padrao [UNIFESP]; Fernandes, Maria Jose da Silva Fernandes [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
Introduction: Epilepsy is a serious neurological disease, and temporal lobe epilepsy (TLE) is the most common type in adults. Hippocampal sclerosis is the major histopathological alteration in ELT. Hippocampal sclerosis can be reproduced in rats following pilocarpine-induced status epilepticus (SE). Pilocarpine, a cholinergic agonist, when injected in high doses, systemically, induces SE which is followed by neuronal death, synaptic reorganization and hyperexcitability, resulting in spontaneous and recurrent seizures. Cell signaling mediated by P2X7 receptors (P2X7R) has been suggested as an important epileptogenic mechanism. P2X7R activation has been associated with increased intracellular calcium, excitotoxicity, inflammation cascades activation and cell death. Aims: To standardize the RNA interference (RNAi) method to study the in vivo silencing of P2X7R and its role in epileptogenesis using the pilocarpine model. Methods: To standardize small interfering RNA (siRNA) was used a fluorescent oligonucleotide (BLOCK-ITTM), administered by three pathways: intranasal, tail intravenous and intra-hippocampal (ih). The BLOCK-ITTM was complexed to the transfection agent RVG-9dR. The P2X7R knockdown was studied measuring the protein level by western blot. Neuronal death was assayed by Fluoro-Jade B (FJ-B) in brain slices of rat after ih and intracerebroventricular (icv) P2X7R siRNA administration. The hippocampal formation and its subregions volume were studied 48 hours after icv RNAi. Behavioral alterations were performed within 60 days after SE induction in animals subjected to pilocarpine model or saline, that received icv RNAi or vehicle (Groups: Saline-Vehicle, Saline-RNA, Pilo-Vehicle and Pilo-RNA). Results: The ih application of BLOCK ITTM:RVG-9dR complex showed the intracellular fluorescence presence in the hippocampal formation. 48 hours after icv administration of siRNA there was lower P2X7R protein level in the hippocampal formation of Saline-RNA (-43%) and Pilo-RNA (-37%) groups than the respective vehicle groups. The P2X7R knockdown reduced the number of FJ-B-stained cells in the CA1 and CA3 regions, increased latency to the first spontaneous seizure, decreased the number of recurrent spontaneous seizures and normalized the volume of hilus, dentate gyrus suprapyramidal granule cells layer, CA1 and CA3. Conclusions: According to these results, we conclude that RNAi in vivo was effective in the P2X7R knockdown in the hippocampal formation and it protects against injury caused by SE, prevents edema, reduces cell death in the hippocampal formation and minimizes the epileptic seizures manifestations.
Acesso aberto (Open Access)
Avaliação de parâmetros cardíacos em animais com epilepsia: possível causa de morte súbita?
(Academia Brasileira de Neurologia - ABNEURO, 2005-12-01) Colugnati, Diego Basile [UNIFESP]; Gomes, Paulo Alberto Paes; Arida, Ricardo Mario [UNIFESP]; Albuquerque, Marly de [UNIFESP]; Cysneiros, Roberta Monterazzo [UNIFESP]; Cavalheiro, Esper Abrão [UNIFESP]; Scorza, Fulvio Alexandre [UNIFESP]; Universidade de Mogi das Cruzes Núcleo de Pesquisas Tecnológicas; Universidade Federal de São Paulo (UNIFESP); UMC NPT Laboratório de Neurociências; Centro Universitário São Camilo; UMC
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.
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Avaliação do tratamento com n-acetilcisteína nas manifestações comportamentais e histológicas causadas pela pilocarpina
(Universidade Federal de São Paulo (UNIFESP), 2015-06-30) Patsis, Vassiliki de Brito [UNIFESP]; Fernandes, Maria Jose da Silva Fernandes [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
Epilepsy is the most common neurological dysfunction, affecting more than 65 million people worldwide. Temporal lobe epilepsy (TLE) is the most prevalent form in adults, affecting 40% of all cases. Hippocampal sclerosis is frequently observed in patients with mesial TLE, characterized by cell loss in the hippocampal subregions (CA1, CA3 and hilus), gliosis, granular cell dispersion and mossy fiber sprouting. The increase in intracellular calcium has been associated with lesion resulting from seizures by production of reactive oxygen species, reactive nitrogen species and consequent oxidative stress. The control of oxidative stress by antioxidants may represent a challenge for epileptologists. N-acetylcysteine (NAC) is an antioxidant derivated of the L-cysteine aminoacid usually used in clinic as acetaminophen detoxified. NAC restores glutathione levels, the larger endogenous antioxidant, it modulates glutamatergic, neurotrophic and inflammatory pathways. However, little is known about the effect of NAC in epilepsy. Pilocarpine (Pilo) is a cholinergic agonist that mimics TLE in rats. Objectives: The present study verified the effects of the treatment with NAC considering their antioxidatives properties in the epileptogenic process caused by Pilo. Methods: Wistar rats, males, adults, 2 months old, weighing about 300g, submitted to Pilocarpine (360 mg/kg, intraperitoneal), treated with NAC ad libitum during 8 weeks. NAC was administered by gavage (24mg/day) for 7 days, and ad libitum in the water of the drinking fountains (600 mg/L) in the 7 weeks that followed. The following groups were studied: Saline-Water, Saline-NAC, Pilo-Water and Pilo-NAC. Behavioral analysis was done by video surveillance to check the amount and intensity of seizures during these 7 weeks. The Fluoro-Jade B (FJ-B) was used as marker of neuronal degeneration in the hippocampal subregions. The analysis of free radicals and antioxidants was taken respectively by measuring of NO, MDA, GSH and GSH total. Results: There was a reduction in seizure frequency in Pilo-NAC rats when compared to Pilo-Water. Histological analysis found neurodegeneration in CA1, CA3 and hilus in Pilo group (Pilo-Pilo-Water and NAC) and NAC did not change this pattern of cell loss. There was normalization of the levels of NO and MDA in treatment of NAC compared to Pilo-Water group. There was no statistical differences in the levels of GSH and GSH-total between experimental groups. Conclusions: According to our data, NAC normalized the levels of NO and MDA which were increased by seizures, but was not able to preventing the hippocampal neurodegeneration caused by Pilo. However, because of the improvement in behavior, the NAC can be considered as a possible adjunct therapy in the treatment of epilepsy.
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Avaliação dos efeitos da estimulação cerebral profunda no núcleo anterior do tálamo no modelo de epilepsia do lobo temporal induzida por pilocarpina
(Universidade Federal de São Paulo (UNIFESP), 2015-05-31) Amorim, Beatriz Oliveira [UNIFESP]; Covolan, Luciene Covolan [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
The epilepsies are considered one of the most common neurological disorders with the temporal lobe epilepsy (TLE) the most frequent among them. In clinical practice deep brain stimulation (DBS) has been used as alternative treatment for patients who are drug-resistent or refractory and are not good candidates for conventional surgery. As the anterior nucleus of the thalamus (ANT) occupies a central position in the limbic circuitry, it has been one of the targets commonly used for implantation of deep electrodes. Despite evidences that DBS reduces spontaneous seizures in clinical and experimental models, its cellular actions remain largely unknown. The purpose of this study was to investigate the mechanisms underlying the ANT DBS actions in the acute and chronic phases a model of TLE. To mimic the TLE in animals, here we have used systemic injection of pilocarpine in adult male rats. In the acute phase of this model DBS reduced the inflammation and apoptosis in the hippocampus with no change in the severity and duration of status epilepticus as measured by electrocorticography. In the chronic phase the continuous video-monitoring indicated that ANT DBS reduced spontaneous seizures. On the other hand, the EEG recordings indicated that there was no difference in seizure frequency between stimulatedand no stimulated-animals. In addition, no cellular changes (proliferation, expression of proteins present in inhibitory neurons and the expression of brain derived neurotrophic factor) could be associated with DBS treatment in chronically epileptic animals. The thalamic stimulation increased the activity of Na+K+ATPase ? subunit in the hippocampus, indicating that the extrassynaptic mechanism could be the responsible for the effects of high frequency stimulation in ANT.
Acesso aberto (Open Access)
Caracterização fisiopatológica do sistema renina-angiotensina durante o status epilepticus induzido pela pilocarpina em camundongos transgênicos que expressam tonina de rato
(Universidade Federal de São Paulo (UNIFESP), 2013-12-20) Iha, Higor Alves [UNIFESP]; Mazzacoratti, Maria da Graca Naffah Mazzacoratti [UNIFESP]; Gouveia, Telma Luciana Furtado Gouveia; http://lattes.cnpq.br/5872912520546019; http://lattes.cnpq.br/3202325307358102; http://lattes.cnpq.br/7202294193275511; Universidade Federal de São Paulo (UNIFESP)
Objectives: To evaluate the influence of pilocarpine induced long-term convulsive seizure in transgenic mice with overexpressed plasma and brain Ang II. Methods: Using a 320mg/kg pilocarpine dose to induce SE. Following the injection protocol of CAVALHEIRO (1995). We conducted an investigation of both strains (TGM (rTon) and WT C57Black/6) of the following groups: a) saline group; b) 3 hour in SE group; and c) Tonic-clonic seizure group. The last group was created upon the greater incidence of such crises in rTon when compared with WT. During the SE, we accessed several parameters such as latency for the first seizure, tonic-clonic seizure susceptibility and mortality. In their hippocampi was assessed by western-blot, real-time RT-PCR, respectively, protein and RNAm expression levels of AT1, AT2, B1 and B2 receptors as well as of the ACE ACE2, iNOS and eNOS enzymes. The same was made to AT1 receptor in heart. It was also assessed the enzymatic activity of hippocampal, cardiac and plasmatic ECA. Results: In an assessment of how both strains reacted to the pilocarpine induced SE we found no difference on first seizure latency time, but a significant higher frequency of death during the generalized tonic-clonic seizure in rTon mice, 66% in rTon and 34% in WT. In RAS, KKS and NO analyzed parameters we found in studied mice groups strain comparison that rTon showed: a) Saline group: in mRNA hippocampal expression, greater transcript level for iNOS and lower for B1 receptor, in protein quantifying, greater for hippocampal AT1 receptor and lower for cardiac AT1 receptor, thereafter, on ECA enzymatic activity evaluation, greater in hippocampi and heart a and on ACE activity, in heart on AT1 receptor protein levels and in plasma on ACE activity; b) in Tonic-Clonic group: mRNA levels were reduced transcripts of eNOS and iNOS, protein quantification showed raised in hippocampi raised ECA2 and lowered B1 receptor and in heart raised AT1 receptor, thereafter, in ECA enzymatic activity assessment, increased in hippocampal and in plasmatic forms. c) 3h of SE group: in hippocampi mRNA levels were upkeeped for ECA transcript, raised for ECA2 and eNOS transcript and reduced for iNOS transcript, protein analysis showed greater raised hippocampal and heart AT1 receptor and hippocampal lowered B2 receptor. Thereafter, on ECA enzymatic activity evaluation, showed raised activity on hippocampal and plasmatic forms. Conclusion: Transgenic mice showed in assessed tissues differences on RAS, KKS and iNOS expression. These differences resulted in a differentiated response to pilocarpine induced seizure in RAS, KKS, iNOS and eNOS which culminated in a greater propensity to tonic-clonic seizures and greater frequency of death throughout the SE, but didn´t affected the latency period for the first seizure.
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Composição celular absoluta do cérebro de animais com epilepsia em diferentes fases da vida adulta
(Universidade Federal de São Paulo (UNIFESP), 2014-06-30) Lopim, Glauber Menezes [UNIFESP]; Arida, Ricardo Mario Arida [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
Epilepsy is a chronic neurological disorder characterized by permanent brain predisposition to generate epileptic seizures, reaching approximately 50 million people around the world. Temporal lobe epilepsy is the most common form of human epilepsy, being reported extensive loss of pyramidal neurons from hippocampal region, hilar neurons from the dentate gyrus and gliosis. Many features of temporal lobe epilepsy are reproduced in animals by the pilocarpine model. This study investigated the frequency of epileptic seizures in animals throughout adulthood and subsequently quantified the number of cells in different brain regions (cortex, hippocampus, cerebellum and remaining regions) of these animals. Sixty Wistar rats were divided into control and epilepsy groups (9 animals per group). Seizures were video-monitored during 360 days, 24 hours a day. The number of cells was quantified by the technique of isotropic fractionation on the 30th, 180th and 360th day after the first spontaneous seizure. Our results showed a progressive increase in the frequency of spontaneous seizures in the early months, followed by a reduction from the seventh month, and later a stabilization from the tenth to the twelfth month. Statistical analysis (ANOVA for repeated measures) showed higher number of seizures in the fourth and sixth months compared to the first (p=0.001, p=0.006), second (p=0.006, p=0.005) and eleventh (p=0.012, p=0.002) months. The total number of brain cells was lower in rats with epilepsy compared to control animals (p<0.005). Animals with epilepsy had fewer cortical and cerebellar cells at 30, 180 and 360° (p<0.005, p<0.005). In the hippocampal for mation, this reduction occurred in 180° (p<0.001) and 360° days (p=0.01). In the re maining regions, the reduction occurred at 30° and 180° days (p<0.001). A correlat ion between the frequency of recurrent seizures and the number of cells in the hippocampal formation was noted. Animals presenting more seizures showed less cell number at the 30° (r=-0.828, p=0.006) and 360° days (r=-0.729 , p = 0.017). Our findings demonstrate that there is an relation between frequency of spontaneous seizures and number of brain cells throughout the life of animals with epilepsy, and this depends on investigated area and age of animals.
Acesso aberto (Open Access)
The contribution of the lateral posterior and anteroventral thalamic nuclei on spontaneous recurrent seizures in the pilocarpine model of epilepsy
(Academia Brasileira de Neurologia - ABNEURO, 2002-09-01) Scorza, Fulvio Alexandre [UNIFESP]; Arida, Ricardo Mario [UNIFESP]; Priel, Margareth [UNIFESP]; Calderazzo, Lineu [UNIFESP]; Cavalheiro, Esper Abrão [UNIFESP]; Universidade de Mogi das Cruzes; Universidade Federal de São Paulo (UNIFESP)
The pilocarpine model of epilepsy in rats is characterised by the occurrence of spontaneous seizures (SRSs) during the chronic period that recur 2-3 times per week during the whole animal life. In a previous study on brain metabolism during the chronic period of the pilocarpine model it was possible to observe that, among several brain structures, the lateral posterior thalamic nuclei (LP) showed a strikingly increased metabolism. Some evidences suggest that the LP can participate in an inhibitory control system involved in the propagation of the seizures. The aim of the present study was to verify the role of LP in the expression and frequency of spontaneous seizures observed in the pilocarpine model. Ten adult male rats presenting SRSs were monitored for behavioural events by video system one month before and one month after LP ibotenic acid lesion. Another group of chronic epileptic rats (n=10) had the anteroventral thalamic nuclei (AV) lesioned by ibotenic acid. After the surgical procedure, the animals were sacrified and the brains were processed for histological analysis by the Nissl method. The LP group seizure frequency was 3.1±1.9 before ibotenic acid injection and showed an increase (16.3±7.2 per week) after LP lesion. No changes in SRSs frequency were observed in the AV group after ibotenic lesion in these nuclei. These results seem to suggest that LP play a role in the seizure circuitry inhibiting the expression of spontaneous seizures in the pilocarpine model.
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Distribuição e expressão dos receptores de melatonina no hipocampo de ratos submetidos ao modelo de epilepsia induzida por pilocarpina
(Universidade Federal de São Paulo (UNIFESP), 2014-10-31) Rocha, Anna Karynna Alves de Alencar [UNIFESP]; Scerni, Debora Amado Scerni [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
Studies from our laboratory have shown a neuroprotective effect of melatonin on pilocarpine-induced epilepsy. The pinealectomy is able to facilitate the epileptogenesis while the melatonin exerted a neuroprotection in the epilepsy model induced by pilocarpine, indicating a major role of melatonin in this condition. However, no consistent data are published about the dynamic changes of melatonergic system in the natural course of temporal lobe epilepsy. Dysfunctions in the melatonin signaling pathway may be primarily based on changes in the density of their receptors. Moreover, altered receptor expression may be a consequence of neurodegenerative processes. Therefore, our study aimed to verify if status epilepticus-induced by pilocarpine cause alterations in the distribution and gene expression of melatonin receptors, MT1, MT2 and ROR? in rat hippocampus. For this, we compared the differences in mRNA expression of MT1, MT2 and ROR? receptors in acute, silent and chronic phases of the model compared to controls within a period of 24 hours by RT-PCR in real time as well as the difference in the location and intensity of immunostaining of these receptors in the same phase by immunohistochemistry.Therefore, our study found that the pilocarpine-induced status epilepticus caused short and long-term changes in the distribution and gene expression of melatonin receptors, MT1, MT2 and ROR? in rat hippocampus. There is an increase in the expression of MT1 and MT2 receptor in the initial hours of the status epilepticus. During the following phases of the model (silent and chronic) there is a reduction in the expression of these receptors in various hippocampal subregions. In contrast to membrane receptors, the expression of nuclear receptor ROR? presented a drastically reduction during the acute phase and the silent phase. In the chronic phase, the presence of these nuclear receptors returns to levels similar of controls. Therefore, we can suggest that the increase in the expression of MT1 and MT2 receptors may be a compensatory mechanism due to the loss of receptors during injury, as the attempt to ?capture? more melatonin. Also, we hypothesized that these receptors might be involved in mechanisms of neuroprotection. Moreover, taking into account the beneficial effect of melatonin via ROR? by modulating of the inflammation and excitotoxicity, we suggest that the reduction of these receptors in the early stages of the pilocarpine model of epilepsy may negatively influence the development of epilepsy, facilitating the epileptogenesis. The correct mechanism related to the decrease and later increase in the expression of this receptors are unknown and needs further investigations.
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Efeito de um programa de treinamento de força em animais com epilepsia do lobo temporal induzido pela pilocarpina
(Universidade Federal de São Paulo (UNIFESP), 2013-06-26) Pena, Luiz Fernando Peixinho [UNIFESP]; Arida, Ricardo Mario Arida [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
The beneficial effects of physical exercise on epilepsy, such as a decreased seizure frequency, have been observed following aerobic exercise programs in both clinical and experimental studies. However, it is not well clarified whether other types of exercise, including strength exercise, can provide similar benefits for epilepsy. Forty four animals with epilepsy were continuously monitored 24 h a day for 60 days and divided into two periods of 30 days. The first period was used to determine the number of seizures before beginning the physical exercise program, and the second period was utilized to determine the number of seizures during the strength training. The mean frequency of seizures in the control and SHAM groups increased significantly from period 1 to period 2. Although the frequency of seizures did not change significantly between the two periods of 30 days of observation in the strength exercise group, a significant reduction in the seizure frequency was observed compared with the control and SHAM groups in period 2. Our study demonstrated that a strength exercise program exerted a significant influence on the seizure frequency in animals with epilepsy and strengthens the observed beneficial effect of exercise on epilepsy that has been demonstrated in animal studies. The finding of this nonclinical study can open a new window to verify the beneficial contribution of strength exercise in epilepsy. Further experimental and clinical investigations are necessary to explore the extent to which strength exercise interferes with the epileptic condition.
Acesso aberto (Open Access)
Effect of aerobic physical exercise in pinealectomized animals submitted to the pilocarpine model of epilepsy
(Liga Brasileira de Epilepsia (LBE), 2006-06-01) Siliano, Marcelo Reina [UNIFESP]; Lima, Eliângela de [UNIFESP]; Valente, Sandra Gomes [UNIFESP]; Naffah-Mazzacoratti, Maria da Graca [UNIFESP]; Cavalheiro, Esper Abrão [UNIFESP]; Arida, Ricardo Mario [UNIFESP]; Amado, Débora [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Universidade de Mogi das Cruzes Núcleo de Pesquisas Tecnológicas Laboratório de Neurociências
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.
Acesso aberto (Open Access)
Effect of glycemic state in rats submitted to status epilepticus during development
(Academia Brasileira de Neurologia - ABNEURO, 2006-06-01) Santiago, Joselita Ferreira Carvalho [UNIFESP]; Carvalho, Fatima Ferreira [UNIFESP]; Perosa, Sandra Regina [UNIFESP]; Siliano, Marcelo Reina [UNIFESP]; Cruz, José Walber Miranda Costa; Fernandes, Maria Jose da Silva [UNIFESP]; Cavalheiro, Esper Abrão [UNIFESP]; Amado, Débora [UNIFESP]; Naffah-Mazzacoratti, Maria da Graca [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP); Instituto do Coração
The effect of glycemic state on status epilepticus (SE) development was studied in animals of different ages, submitted to pilocarpine model of epilepsy. Groups: I- Rats with 9-day-old (P9): IA. Submitted to 1SE; IB. Saline-treated; IC. Induced- hyperglycemia; ID. Induced- hyperglycemia+SE; II- Rats submitted to three consecutive episodes of SE at P7, P8 and P9; III- Rats submitted to 1SE at P17; IV- Rats submitted to 1SE at P21. Hippocampal cell death and the expression of glucose transporter GLUT3 were analyzed in group I. The results demonstrated normoglycemia in the groups IA, IB and II, hypoglycemia in group III and hyperglycemia in group IV, showing that the glycemia during SE is age dependent. Induced hyperglycemia during SE in P9 protected the hippocampal neurons from death and both groups IC and ID presented increased GLUT3 expression, showing high glucose consumption by the hippocampus.
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Estudo de mediadores de inflamação sistêmica durante epileptogênese
(Universidade Federal de São Paulo (UNIFESP), 2014-07-30) Sousa, Paula Viviane Vieira de [UNIFESP]; Mazzacoratti, Maria da Graca Naffah Mazzacoratti [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
Introduction: Several evidences show that inflammatory processes are involved in the pathophysiology of temporal lobe epilepsy (TLE ) . Infiltration of peptides such as cytokines, kinins , lymphocytes and other blood cells in the brain showed a close relationship between the central and peripheral immune system, showing the involvement of disruption of the blood-brain barrier during the process of epileptogenesis . Objective: The objective of this study was to measure blood levels of C-reactive protein ( CRP) and inflammatory cytokines ( IL - 1? , IL - 6 , IL - 10 and TNF - ? ) during acute , silent and chronic phases of rats submitted to the pilocarpine model of epilepsy . The effect of chronic treatment of mice was also analyzed with omega -3 levels of CRP and cytokines during generation of the epileptic focus . Methods : Male Wistar rats were analyzed in three periods of the pilocarpineinduced epilepsy (350mg/kg) into phases: acute ( 5h , 12h and 24h ) , silent ( 48 hours and 5 days ) and chronic (90 days after induction of SE) and the chronic period of the model we used 4 groups of animals: control vehicle ( CV ) , omega ( CO ) control , epilepsy vehicle ( EV ) , epilepsy omega ( EO) . The treatment with omega-3 (85 mg / kg) was initiated 3 hours after the induction of the model and was given daily for 90 days. Blood was collected and centrifuged 4000 rpm for 10 minutes at room temperature. Serum was separated and used for PCR quantification by ELISA . IL- 1? , IL-6 , IL-10 and TNF- ? were measured using the multiplex . Results: The results showed increased levels of CRP in the serum of mice during all phases of this model, which was reduced after treatment with omega - 3 . Even as the levels of IL - 6 . The levels of IL - 1? were higher in the acute and chronic phases of the model and there was no significant difference between groups treated with omega - 3 . The IL - 10 presented elevated only in the chronic phase of the model , showing its neuroprotective effect . However, the group treated with omega -3 had levels of IL - 10 less than the untreated group , assuming that due to the neuroprotective effect of omega - 3 , the body did not need to form IL - 10 as a neuroprotective agent . TNF - ? was not significantly different between groups . Conclusion : Since high levels of CRP in the blood have been linked to heart disease , sudden unexpected death ( SUDEP ) found in these animals may be related to these changes , suggesting that a disturbance in the central nervous system can be reflected in the peripheral system , especially markers of heart disease. Taken together, these data demonstrate for the first time , the accumulation of CRP in the blood of rats during epileptogenesis .
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Estudo do sistema renina-angiotensina em animais durante a epileptogênese
(Universidade Federal de São Paulo (UNIFESP), 2014-05-12) Brito, Joise Marques Vieira de [UNIFESP]; Mazzacoratti, Maria da Graca Naffah Mazzacoratti [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
Introduction: Recent data have associated the renin-angiotensin system (RAS) to some types of epilepsy. A previous study from our group showed an increased XVIII expression of angiotensin II (Ang II) receptors (AT1 and AT2 ) in the cortex and hippocampus of patients with Temporal Lobe Epilepsy (TLE).Objective: In order to verify the role of these components of RAS during epileptogenesis the present work studied the gene and protein expression of AT1, AT2 and Mas receptors, angiotensin converting enzyme (ACE) and the concentration of important peptides (Angl, Ang II and Ang 1-7), in the hippocampus of rats in pilocarpine-induced epilepsy. Methods: The animals were subjected to the experimental model with the injection of pilocarpine (350 mg / kg ip) or saline (control group). The animals were grouped according to the phase of the model: acute (status epilepticus), silent (seizure free), chronic (spontaneous recurrent seizures) and saline (control). HPLC techniques were performed to determine the concentration of Ang I, Ang II and Ang 1-7. RT-PCR, Western blotting and immunohistochemistry was used to evaluate the gene and protein expression of ACE and AT1, AT2 and Mas receptors levels and distribution. In the chronic phase the seizure frequency was assessed by video monitoring in the same group of rats, before and after treatment with perindopril (ACE-inhibitor). Changes in systolic blood pressure, neuronal death and mossy fiber sprouting after treatment with perindopril were also evaluated. Statistical analysis one-way ANOVA followed by Tukey-Kramer test was performed to verify the HPLC assays and RT-PCR. Unpaired Student t test was used to compare the results of Western blotting, quantification of the frequency of seizures and systolic blood pressure variation. p <0.05 was accepted. Results: Different concentration of Angl, AngII and Ang1-7 were founded on the 3 phases of this model, as well as AT1, AT2 and Mas receptor and enzyme ACE levels. Conclusion: Ang I appears to be converted to Ang II and Ang 1-7 at the hippocampus during the process of epileptogenesis. The increase in the concentration of Ang II and AT1 receptor in the chronic phase may be related to the generation of spontaneous seizures, since ACE-inhibitor was able to reduce the seizures frequency. High expression levels of Ang1-7, which would have antagonistic action of AngII, and the increased Mas receptor, in the silent phase, suggests that this peptide is involved in seizure blocking. Levels of Ang II and its receptors ( AT1 and AT2 ) was increased in the chronic phase , confirming the data reported by our group in another study , which showed increased expression of these receptors in hippocampal tissue from TLE patients already installed. The reduction of Ang II by perindopril induced a decrease in seizure frequency (chronic phase), reduced blood pressure and minimized the development of mossy fiber sprouting. However, perindopril was not able to modify the loss of hippocampal neurons. The results suggest that this antihypertensive can be beneficial in the treatment of epilepsy.
Acesso aberto (Open Access)
Estudo qualitativo da formação hipocampal de animais hipertensos com epilepsia
(Academia Brasileira de Neurologia - ABNEURO, 2005-06-01) Scorza, Fulvio Alexandre [UNIFESP]; Arida, Ricardo Mario [UNIFESP]; Cysneiros, Roberta Monterazzo [UNIFESP]; Scorza, Carla Alessandra [UNIFESP]; Albuquerque, Marly de [UNIFESP]; Cavalheiro, Esper Abrão [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Universidade de Mogi das Cruzes Universidade de Mogi das Cruzes Laboratório de Neurociências; Centro Universitário São Camilo
The aim of our study was to investigate the hippocampus and dentate gyrus neuropathological features of spontaneous hypertensive rats (SHR) with epilepsy. METHOD: 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.
Acesso aberto (Open Access)
Lovastatin reduces neuronal cell death in hippocampal CA1 subfield after pilocarpine-induced status epilepticus: preliminary results
(Academia Brasileira de Neurologia - ABNEURO, 2005-12-01) Rangel, Pauline; Cysneiros, Roberta Monterazzo [UNIFESP]; Arida, Ricardo Mario [UNIFESP]; Albuquerque, Marly de [UNIFESP]; Colugnati, Diego Basile [UNIFESP]; Scorza, Carla Alessandra [UNIFESP]; Cavalheiro, Esper Abrão [UNIFESP]; Scorza, Fulvio Alexandre [UNIFESP]; Universidade de Mogi das Cruzes Núcleo de Pesquisas Tecnológicas Laboratório de Neurociências; Universidade Federal de São Paulo (UNIFESP)
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 (350mg/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 20mg/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.
Acesso aberto (Open Access)
Nestin down-regulation of cortical radial glia is delayed in rats submitted to recurrent status epilepticus during early postnatal life
(Academia Brasileira de Neurologia - ABNEURO, 2009-09-01) Scorza, Carla Alessandra [UNIFESP]; Arida, Ricardo Mario [UNIFESP]; Scorza, Fulvio Alexandre [UNIFESP]; Cavalheiro, Esper Abrão [UNIFESP]; Naffah-Mazzacoratti, Maria da Graca [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
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.
Acesso aberto (Open Access)
Neurogenesis induced by seizures in the dentate gyrus is not related to mossy fiber sprouting but is age dependent in developing rats
(Academia Brasileira de Neurologia - ABNEURO, 2008-12-01) Sanabria, Yaima Del Carmen Garrido [UNIFESP]; Argañaraz, Gustavo Adolfo [UNIFESP]; Lima, Eliângela de [UNIFESP]; Priel, Margareth Rose [UNIFESP]; Trindade, Edvaldo da Silva [UNIFESP]; Loeb, Luana Mazzacoratti [UNIFESP]; Scorza, Fulvio Alexandre [UNIFESP]; Cavalheiro, Esper Abrão [UNIFESP]; Amado, Débora [UNIFESP]; Naffah-Mazzacoratti, Maria da Graca [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
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.
Acesso aberto (Open Access)
The pilocarpine model of epilepsy: what have we learned?
(Academia Brasileira de Ciências, 2009-09-01) Scorza, Fulvio Alexandre [UNIFESP]; Arida, Ricardo Mario [UNIFESP]; Naffah-Mazzacoratti, Maria da Graca [UNIFESP]; Amado, Débora [UNIFESP]; Calderazzo, Lineu [UNIFESP]; Cavalheiro, Esper Abrão [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
The systemic administration of a potent muscarinic agonist pilocarpine in rats promotes sequential behavioral and electrographic changes that can be divided into 3 distinct periods: (a) an acute period that built up progressively into a limbic status epilepticus and that lasts 24 h, (b) a silent period with a progressive normalization of EEG and behavior which varies from 4 to 44 days, and (c) a chronic period with spontaneous recurrent seizures (SRSs). The main features of the SRSs observed during the long-term period resemble those of human complex partial seizures and recurs 2-3 times per week per animal. Therefore, the pilocarpine model of epilepsy is a valuable tool not only to study the pathogenesis of temporal lobe epilepsy in human condition, but also to evaluate potential antiepileptogenic drugs. This review concentrates on data from pilocarpine model of epilepsy.
Somente Metadadados
Tratamento com ômega-3 abole a taquicardia de repouso de ratos com epilepsia induzida pelo modelo da pilocarpina
(Universidade Federal de São Paulo (UNIFESP), 2015-10-31) Lopes, Marcio Delascio [UNIFESP]; Scorza, Fulvio Alexandre Scorza [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
Since the 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 exerts 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 HR of rats with epilepsy. In agreement with our previous investigations, this study also showed that HR of animals with epilepsy is higher than that of control group. Quite interesting, 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 FAs supplementation against SUDEP.
Somente Metadadados
Uso de antagonista farmacológico no estudo do papel do receptor p2x7 nas alterações causadas por crises induzidas por pilocarpina
(Universidade Federal de São Paulo (UNIFESP), 2015-02-28) Araujo, Michelle Gasparetti Leao [UNIFESP]; Fernandes, Maria Jose da Silva Fernandes [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
Introduction: Temporal lobe epilepsy (TLE) is the most common type of focal epilepsy, most cases displaying seizures refractory to antiepileptic drugs, with hippocampal sclerosis (HS) the most common structural abnormality. The pilocarpine-induced epilepsy model reproduces the main features of TLE. Pilocarpine is a cholinergic agonist and when administered systemically in high doses, induces status epilepticus (SE), HS, synaptic reorganization and spontaneous recurrent seizures. SE induces changes in neurotransmitter systems, including the purinergic systems. Studies showed an increase in subtypes of the purinergic receptors P2X in the TLE model induced with pilocarpine, and the increase of P2X7 receptor (P2X7R) being considered as the mechanism in which promotes the excitotoxity and hyper-excitability. The increase in the purinergic signaling is, therefore, one of the alterations caused by seizures and needing elucidation. The use of antagonists has assisted in the study of this signaling in epilepsy. Aims: Elucidate the role of P2X7R, through the administration of the antagonist AZ-10606120 (AZ), in the cellular death process in the hippocampus and in the epileptogenesis in animals underwent the pilocarpine-induced model of TLE, and in calcium mobility in hipocampal slices. Methods: The AZ was administered intracerebroventricularly (icv) at doses of 1?g, 2?g and 3?g, 30 minutes following SE, in animals underwent to pilocarpine-induced mode and in control animals, and the hippocampal cellular death process analyzed by Fluoro Jade B (FJ-B) labelling. The dose that caused the lowest number of FJ-B positive cells was chosen for the study (3?g). Therefore, four groups were studied: Saline+Vehicle, Saline+AZ, Pilo+Vehicle, Pilo+AZ. The quantification of P2X7R was analyzed by Western Blot. For the intracellular calcium study, was used hippocampal slices of control animals, in which was incubated with Fluo-4AM calcium dye. Then, slices was incubated with AZ at concentrations of 2.5?M, 5?M and 10?M or saline, and stimulated with BzATP. The concentrations of AZ which have caused the lowest hate of intracellular calcium (2.5?M) was used in the groups studied: Salina+BzATP, Salina+AZ+BzATP, Pilo+BzATP e Pilo+AZ+BzATP. Behavioral assessment was performed over 30 days after SE in animals underwent the pilocarpine model or received saline, with or without AZ administration. Results: The dose of 3?g of AZ i.c.v. caused a reduction of the number of FJ-B labeled cell in the CA1, CA3 and the hilus of dentate gyrus in animals underwent the SE induced with pilocarpine. This dose also reduced the expression of P2X7R (-42%) in animals of Pilo+AZ group compared to Pilo+Vehicle group. The animals treated with AZ displayed a lower mortality rate 24 hours after SE, however there was an increase in the number of seizures, particularly for smaller stage (mainly Racine?s stage 2). The concentration of 2.5?M of AZ caused an inhibition on the intracellular calcium concentration in hippocampal slices of Saline rats after stimulation with BzATP. Nevertheless, this result did not occur in hippocampal slices Pilo+AZ+BzATP group, where we observed a significant increase in the concentration of calcium compared to saline group. Conclusion: AZ administration caused a significant reduction of the mortality rate, a reduction of P2X7R density, reduction of the cellular death in hippocampal formation and an increase of the intracellular calcium in the hippocampus in the acute period. In the chronic period, it cause an increase of the number of seizures, mainly the less severe ones, highlighting the role of P2X7R in epilepsy induced by pilocarpine. The heterogeneous responses induced by AZ indicate that new studies are necessary to elucidate mechanisms modulated by this antagonist.

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