RESUMO
The pilocarpine-induced (PILO) model has helped elucidate the electrophysiological and molecular aspects related to mesial temporal lobe epilepsy. It has been suggested that the extensive cell death and edema observed in the brains of these animals could be induced by increased inflammatory responses, such as the rapid release of the inflammatory cytokine interleukin 1 beta (Il1b). In this study, we investigate the role of endogenous Il1b in the acute phase of the PILO model. Our aim is twofold. First, we want to determine whether it is feasible to silence Il1b in the central nervous system using a non-invasive procedure. Second, we aim to investigate the effect of silencing endogenous Il1b and its antagonist, Il1rn.We used RNA interference applied non-invasively to knockdown Il1b and its endogenous antagonist Il1rn. We found that knocking down Il1b prior to pilocarpine injection increased the mortality rate of treated animals. Furthermore, we observed that, when exposing the animals to more Il1b by silencing its endogenous antagonist Il1rn, there was a better response to status epilepticus with decreased animal mortality in the acute phase of the PILO model. Thus, we show the feasibility of using a novel, less invasive approach to study genes involved in the inflammatory response in the central nervous system. Furthermore, our results provide suggestive evidence that modulating endogenous Il1b improves animal survival in the acute phase of the PILO model and may have effects that extend into the chronic phase.
Assuntos
Epilepsia do Lobo Temporal , Epilepsia , Estado Epiléptico , Animais , Pilocarpina/efeitos adversos , Pilocarpina/metabolismo , Interleucina-1beta/metabolismo , Epilepsia/induzido quimicamente , Epilepsia/genética , Epilepsia do Lobo Temporal/induzido quimicamente , Epilepsia do Lobo Temporal/genética , Epilepsia do Lobo Temporal/metabolismo , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/genética , Estado Epiléptico/metabolismo , Modelos Animais de Doenças , Hipocampo/metabolismoRESUMO
Neonatal noxious stimulation has been proposed to model pain triggered by diagnostic/therapeutic invasive procedures in premature infants. Previous studies have shown that hippocampal neurogenesis rate and the behavioral repertoire of adult rats may be altered by neonatal noxious stimuli. The purpose of this study was to evaluate whether noxious stimulation during neonatal period alters the nociceptive response and dentate gyrus neurogenesis when compared to rats subjected to a single noxious stimulus in late infancy. Plasma corticosterone and hippocampal brain-derived neurotrophic factor (BDNF) levels were measured. Neurogenesis in the dentate gyrus was evaluated in adolescent rats (postnatal day 40; P40) exposed twice to intra-plantar injections of Complete Freund's adjuvant (CFA) on P1 and P21 (group P1P21) or P8 and P21 (P8P21) or exposed once on P21 (pubertal). On P21, one subset of animals received 5-bromo-2'-deoxyuridine (BrdU) and was euthanized on P40 for identification of proliferating cells in the dentate gyrus. Another subset was sampled for thermal response or plasma corticosterone measurement and hippocampal BDNF levels. Proliferative cell rate in dentate gyrus was the highest in all re-exposed groups (P < 0.001), except for P8 females (P8P21F), revealing also a sex difference, where P8P21 males showed higher rate than females (P < 0.001). Stimulated groups took longer than CTL animals to lick the paws (P < 0.001), regardless of the age when the noxious stimulus was applied. Re-exposed groups had lower corticosterone plasma level (P1P21 M and F, P8P21M) than controls. On the contrary, hippocampal BDNF was increased in males from both re-exposed groups. These results show that infant noxious stimulation in neonatally previously stimulated rats is related to high proliferation in the DG and this association seems to be modified by the animal's sex. The new generated dentate granule cells in the hippocampus may have a role in the long-term behavioral responses to neonatal nociceptive stimulation. Noxious stimulation in the neonatal period results in sex-dependent neurogenic response.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Giro Denteado/fisiologia , Hipocampo/fisiologia , Neurogênese/fisiologia , Neurônios/fisiologia , Nociceptividade/fisiologia , Animais , Animais Recém-Nascidos , Corticosterona/sangue , Giro Denteado/crescimento & desenvolvimento , Feminino , Adjuvante de Freund , Hipocampo/crescimento & desenvolvimento , Masculino , Distribuição Aleatória , Ratos , Ratos Wistar , Fatores SexuaisRESUMO
Here we describe a new non-human primate model of temporal lobe epilepsy (TLE) to better investigate the cause/effect relationships of human TLE. Status epilepticus (SE) was induced in adult marmosets by pilocarpine injection (250mg/kg; i.p.). The animals were divided in 2 groups: acute (8h post-SE) and chronic (3 and 5 months post-SE). To manage the severity of SE, animals received diazepam 5min after the SE onset (acute group: 2.5 or 1.25mg/kg; i.p.; chronic group/; 1.25mg/kg; i.p). All animals were monitored by video and electrocorticography to assess SE and subsequent spontaneous recurrent seizures (SRS). To evaluate brain injury produced by SE or SRS we used argyrophil III, Nissl and neo-Timm staining techniques. Magnetic resonance image was also performed in the chronic group. We observed that pilocarpine was able to induce SE followed by SRS after a variable period of time. Prolonged SE episodes were associated with brain damage, mostly confined to the hippocampus and limbic structures. Similar to human TLE, anatomical disruption of dentate gyrus was observed after SRS. Our data suggest that pilocarpine marmoset model of epilepsy has great resemblance to human TLE, and could provide new tools to further evaluate the subtle changes associated with human epilepsy.
Assuntos
Modelos Animais de Doenças , Epilepsia do Lobo Temporal/diagnóstico , Epilepsia do Lobo Temporal/fisiopatologia , Análise de Variância , Animais , Anticonvulsivantes/uso terapêutico , Benzoxazinas , Callithrix , Diazepam/uso terapêutico , Progressão da Doença , Diterpenos , Eletroencefalografia , Epilepsia do Lobo Temporal/induzido quimicamente , Epilepsia do Lobo Temporal/tratamento farmacológico , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , Sistema Límbico/efeitos dos fármacos , Sistema Límbico/metabolismo , Sistema Límbico/patologia , Imageamento por Ressonância Magnética , Masculino , Agonistas Muscarínicos/toxicidade , Oxazinas , Fosfopiruvato Hidratase/metabolismo , Pilocarpina/toxicidade , Gravação em VídeoRESUMO
INTRODUÇÃO: Existem contradições na literatura quanto aos efeitos dos genes il1β e il1rn nas epilepsias. Nosso objetivo foi avaliar os efeitos do silenciamento desses dois genes na fase aguda do modelo de epilepsia induzido pela pilocarpina. MÉTODOS: Para alterar a expressão dos genes il1β e il1rn utilizamos a técnica de interferência por RNA. RESULTADOS: Obtivemos taxas de silenciamento significativas para os dois genes no sistema nervoso central. Observamos efeitos fenotípicos significativos, incluindo a alteração na taxa de mortalidade dos animais 5 dias após a indução do modelo. CONCLUSÕES: A il1β parece exercer um papel protetor na fase aguda do modelo de epilepsia induzido pela pilocarpina.
INTRODUCTION: There is contradictory information regarding the of effects il1β and il1rn in epilepsy. We aimed to evaluate the effect of silencing both genes in the acute phase of the pilocarpine-induced epilepsy model. METHODS: We used RNA interference in order to achieve gene silencing. RESULTS: We obtained significant gene silencing in the central nervous system. In addition, we observed phenotypic effects including differences in mortality rates of animals 5 days after pilocarpine injections. CONCLUSION: Our results indicate that il1β seems to have a protective effect in the acute phase of the pilocarpine-induced epilepsy model.
Assuntos
Humanos , Modelos Animais , RNA Interferente Pequeno , Interleucina-1betaRESUMO
Epilepsy is known to influence hippocampal dentate granule cell (DGC) layer neurogenesis. In young adult rats, status epilepticus (SE) increases the number DGC newly borne cells and basal dendrites (BD), which persist at long-term. In contrast, little is known on whether these phenomena occur in elderly epileptic animals. In the present study, we compare DGC proliferation and the incidence of BD in young and aged pilocarpine-treated rats. Three epileptic groups were considered: Young animals given pilocarpine at 3 months of age. Aged animals treated with pilocarpine at 3 months of age that were sacrificed at 17-20 months. Aged animals that had pilocarpine and developed SE at 20 months, being sacrificed 2 months later. Nine days prior to sacrifice, animals underwent swimming sessions in the Morris water maze as a protocol for the development of hippocampal neurogenesis. We found a higher incidence of newly born DGC cells in young as compared to aged epileptic animals (P<0.001). This later group however, was not homogeneous. While a significant increase in DGC neurogenesis was observed when aged animals with long lasting epilepsy were compared to non-epileptic controls (P<0.01), this has not been recorded in aged animals that had epilepsy for only 2 months (P>0.05). When the number of DGC containing BD was considered, a significantly higher incidence was observed in young as compared to aged epileptic rats (P=0.001). Animals in this later group virtually lacked BD in newly formed dentate gyrus (DG) cells. Based on these results we conclude that plastic changes during epileptogenesis and the development of a pathological substrate in young animals is associated with DGC proliferation and the emergence of BD. As aging occurs, DGC neurogenesis can still be induced in rats with a long-term history of epilepsy but the emergence of BD is markedly reduced.
Assuntos
Dendritos/ultraestrutura , Giro Denteado/citologia , Neurônios/citologia , Estado Epiléptico/patologia , Fatores Etários , Animais , Doença Crônica , Giro Denteado/crescimento & desenvolvimento , Modelos Animais de Doenças , Masculino , Aprendizagem em Labirinto/fisiologia , Neurogênese/efeitos dos fármacos , Pilocarpina , Ratos , Ratos Wistar , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/fisiopatologiaRESUMO
Pilocarpine-induced (320 mg/kg, i.p.) status epilepticus (SE) in adult (2-3 months) male Wistar rats results in extensive neuronal damage in limbic structures. Here we investigated whether the induction of a second SE (N = 6) would generate damage and cell loss similar to that seen after a first SE (N = 9). Counts of silver-stained (indicative of cell damage) cells, using the Gallyas argyrophil III method, revealed a markedly lower neuronal injury in animals submitted to re-induction of SE compared to rats exposed to a single episode of pilocarpine-induced SE. This effect could be explained as follows: 1) the first SE removes the vulnerable cells, leaving behind resistant cells that are not affected by the second SE; 2) the first SE confers increased resistance to the remaining cells, analogous to the process of ischemic tolerance. Counting of Nissl-stained cells was performed to differentiate between these alternative mechanisms. Our data indicate that different neuronal populations react differently to SE induction. For some brain areas most, if not all, of the vulnerable cells are lost after an initial insult leaving only relatively resistant cells and little space for further damage or cell loss. For some other brain areas, in contrast, our data support the hypothesis that surviving cells might be modified by the initial insult which would confer a sort of excitotoxic tolerance. As a consequence of both mechanisms, subsequent insults after an initial insult result in very little damage regardless of their intensity.
Assuntos
Sistema Límbico/efeitos dos fármacos , Agonistas Muscarínicos/farmacologia , Neurônios/patologia , Pilocarpina/farmacologia , Estado Epiléptico/induzido quimicamente , Animais , Morte Celular/efeitos dos fármacos , Modelos Animais de Doenças , Sistema Límbico/patologia , Masculino , Ratos , Ratos Wistar , Coloração pela Prata , Estado Epiléptico/patologiaRESUMO
Pilocarpine-induced (320 mg/kg, ip) status epilepticus (SE) in adult (2-3 months) male Wistar rats results in extensive neuronal damage in limbic structures. Here we investigated whether the induction of a second SE (N = 6) would generate damage and cell loss similar to that seen after a first SE (N = 9). Counts of silver-stained (indicative of cell damage) cells, using the Gallyas argyrophil III method, revealed a markedly lower neuronal injury in animals submitted to re-induction of SE compared to rats exposed to a single episode of pilocarpine-induced SE. This effect could be explained as follows: 1) the first SE removes the vulnerable cells, leaving behind resistant cells that are not affected by the second SE; 2) the first SE confers increased resistance to the remaining cells, analogous to the process of ischemic tolerance. Counting of Nissl-stained cells was performed to differentiate between these alternative mechanisms. Our data indicate that different neuronal populations react differently to SE induction. For some brain areas most, if not all, of the vulnerable cells are lost after an initial insult leaving only relatively resistant cells and little space for further damage or cell loss. For some other brain areas, in contrast, our data support the hypothesis that surviving cells might be modified by the initial insult which would confer a sort of excitotoxic tolerance. As a consequence of both mechanisms, subsequent insults after an initial insult result in very little damage regardless of their intensity.
Assuntos
Animais , Masculino , Ratos , Sistema Límbico/patologia , Agonistas Muscarínicos/farmacologia , Neurônios/patologia , Pilocarpina/farmacologia , Estado Epiléptico/induzido quimicamente , Morte Celular/efeitos dos fármacos , Modelos Animais de Doenças , Ratos Wistar , Coloração pela Prata , Estado Epiléptico/patologiaRESUMO
Cell loss in the hippocampal formation is a common event in patients with temporal lobe epilepsy. The belief that dentate granule neurons are relatively resistant to excitotoxic injury has recently been challenged both, in epileptic patients and in animal models of temporal lobe epilepsy. The nature of dentate granule cell damage in epilepsy has been reported as either apoptotic, necrotic or both. The lack of a consensus on this topic stems from use of different animal models and different experimental techniques for characterizing the apoptotic/necrotic process. Using electron microscopy for defining the, nature of cell loss and one of the main animal models of status epilepticus (SE) we have focussed on the nature of the degenerative process in dentate granule cells. Ultrastructural morphological changes of these cells were evaluated 2.5-48 h after pilocarpine-induced status epilepticus. A variety of morphologies ranging from apoptosis to necrosis, could be seen at 2.5 h after SE onset and continued at least over the following 48 h. Some cells displayed coalescence of chromatin against nuclear membranes. In such cases however, chromatin did not have well-defined edges (as it should, if it were apoptosis). Condensation of cytoplasm. present in both processes was also frequently found. Neither obvious apoptotic budding-off of cytoplasm nor typical membrane-bound apoptotic bodies were found. Our results indicate that in the dentate granule cell layer pilocarpine-induced SE promotes a degenerative process in which apoptotic and necrotic features overlap.
Assuntos
Grânulos Citoplasmáticos/efeitos dos fármacos , Giro Denteado/patologia , Pilocarpina/toxicidade , Convulsões/patologia , Estado Epiléptico/patologia , Animais , Grânulos Citoplasmáticos/patologia , Grânulos Citoplasmáticos/ultraestrutura , Giro Denteado/efeitos dos fármacos , Giro Denteado/ultraestrutura , Modelos Animais de Doenças , Epilepsia do Lobo Temporal/patologia , Humanos , Masculino , Microscopia Eletrônica , Ratos , Ratos Wistar , Convulsões/induzido quimicamente , Estado Epiléptico/induzido quimicamente , Fatores de TempoRESUMO
Dentate granule cells are generally considered to be relatively resistant to excitotoxicity and have been associated with robust synaptogenesis after neuronal damage. Synaptic reorganization of dentate granule cell axons, the mossy fibers, has been suggested to be relevant for hyperexcitability in human temporal lobe epilepsy and animal models. A recent hypothesis suggested that mossy-fiber sprouting is dependent on newly formed dentate granule cells. However, we recently demonstrated that cycloheximide (CHX) can block the mossy-fiber sprouting that would otherwise be induced by different epileptogenic agents and does not interfere with epileptogenesis in those models. Here, we investigated cell damage and neurogenesis in the dentate gyrus of pilocarpine- or kainate-treated animals with or without coadministration of CHX. Dentate granule cells were highly vulnerable to pilocarpine induced-status epilepticus (SE), but were hardly damaged by kainate-induced SE. CHX pretreatment markedly reduced the number of injured neurons after pilocarpine-induced SE. Induction of SE dramatically increased the mitotic rate of KA- and KA + CHX-treated animals. Induction of SE in animals injected with pilocarpine alone led to 2-7-fold increases in the mitotic rate of dentate granule cells as compared to 5- and 30-fold increases for pilocarpine + CHX animals. We suggest that such increased mitotic rates might be associated with a protection of a vulnerable precursor cell population that would otherwise degenerate after pilocarpine-induced SE. We further suggest that mossy-fiber sprouting and neurogenesis of granule cells are not necessarily linked to one another.
Assuntos
Fibras Musgosas Hipocampais/patologia , Estado Epiléptico/patologia , Animais , Antimetabólitos/análise , Bromodesoxiuridina/análise , Cicloeximida/farmacologia , Agonistas de Aminoácidos Excitatórios , Ácido Caínico , Masculino , Mitose/efeitos dos fármacos , Fibras Musgosas Hipocampais/química , Agonistas Muscarínicos , Pilocarpina , Inibidores da Síntese de Proteínas/farmacologia , Ratos , Ratos Wistar , Coloração e Rotulagem , Estado Epiléptico/induzido quimicamenteRESUMO
Systemic administration of pilocarpine and kainic acid (KA) has been extensively used to model temporal lobe epilepsy in rats. Here the regional distribution of selectively vulnerable neurons and the temporal evolution of such neuronal injury after status epilepticus (SE) are compared in both models. Using the silver staining technique of Gallyas, argyrophilic neurons were measured on a 0-3 (least-most) scale in 53 different brain areas. Few neurons were silver-stained 2.5 h after kainate-induced SE, but many silver-stained cells could be seen in most neocortical, hippocampal, amygdaloid and hypothalamic structures for pilocarpine group. In general, 8 or 24 h intervals between SE onset and perfusion times yielded the most intense neuronal silver-impregnation. Pilocarpine-induced neuronal silver impregnation was more prominent than that induced by kainate treatment for many areas in cortex, hippocampus, endopiriform nucleus, amygdaloid complex and hypothalamus. On the other hand, in the thalamus, some cortical areas, claustrum, lateral septum and caudoputamen, kainate-induced neuronal silver staining was also prominent, but occurred later than in pilocarpine-treated animals. Neuronal injury was found in almost the same brain areas in both models of SE but with different intensity levels and time course profiles. It was suggested that such differences in the temporal profile of cell damage should be taken into account when searching for neuroprotective agents.