RESUMEN
Febrile seizures (FSs) are the most common convulsion in infancy and childhood. Considering the limitations of current treatments, it is important to examine the mechanistic cause of FSs. Prompted by a genome-wide association study identifying TMEM16C (also known as ANO3) as a risk factor of FSs, we showed previously that loss of TMEM16C function causes hippocampal neuronal hyperexcitability [Feenstra et al., Nat. Genet. 46, 1274-1282 (2014)]. Our previous study further revealed a reduction in the number of warm-sensitive neurons that increase their action potential firing rate with rising temperature of the brain region harboring these hypothalamic neurons. Whereas central neuronal hyperexcitability has been implicated in FSs, it is unclear whether the maximal temperature reached during fever or the rate of body temperature rise affects FSs. Here we report that mutant rodent pups with TMEM16C eliminated from all or a subset of their central neurons serve as FS models with deficient thermoregulation. Tmem16c knockout (KO) rat pups at postnatal day 10 (P10) are more susceptible to hyperthermia-induced seizures. Moreover, they display a more rapid rise of body temperature upon heat exposure. In addition, conditional knockout (cKO) mouse pups (P11) with TMEM16C deletion from the brain display greater susceptibility of hyperthermia-induced seizures as well as deficiency in thermoregulation. We also found similar phenotypes in P11 cKO mouse pups with TMEM16C deletion from Ptgds-expressing cells, including temperature-sensitive neurons in the preoptic area (POA) of the anterior hypothalamus, the brain region that controls body temperature. These findings suggest that homeostatic thermoregulation plays an important role in FSs.
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Regulación de la Temperatura Corporal/genética , Canales de Cloruro/genética , Fiebre/genética , Hipertermia/genética , Área Preóptica/metabolismo , Convulsiones Febriles/genética , Potenciales de Acción/fisiología , Animales , Animales Recién Nacidos , Temperatura Corporal/efectos de los fármacos , Temperatura Corporal/fisiología , Canales de Cloruro/deficiencia , Femenino , Fiebre/inducido químicamente , Fiebre/metabolismo , Fiebre/fisiopatología , Expresión Génica , Hipocampo/metabolismo , Hipocampo/fisiopatología , Hipertermia/metabolismo , Hipertermia/fisiopatología , Ácido Kaínico/administración & dosificación , Masculino , Ratones , Ratones Noqueados , Neuronas/metabolismo , Neuronas/patología , Área Preóptica/fisiopatología , Isoformas de Proteínas/deficiencia , Isoformas de Proteínas/genética , Ratas , Convulsiones Febriles/inducido químicamente , Convulsiones Febriles/metabolismo , Convulsiones Febriles/fisiopatologíaRESUMEN
The structure of the brain is dramatically altered during the critical period. Physiological substances (neurotransmitters, hormones, etc.) in the body fluctuate significantly before and after sexual maturation. Therefore, the effect of chemical exposure on the central nervous system often differs depending on the developmental stage and sex. We aimed to compare the behavioural effects that emerged from the administration of chemicals to mice of different life stages (immature or mature) and different sex (male or female). We administered mice with domoic acid (DA), a marine poison, and ibotenic acid (IA), found in poisonous mushrooms. These excitatory amino acids act as agonists for glutamate and are potent neurotoxins. Interestingly, the behavioural effects of these chemicals were completely different. Following DA administration, we observed memory deficits only in groups of male mice treated at maturity. Following IA administration, we observed deviations in emotional behaviour in groups of male mice treated at both immaturity and maturity. In contrast, few characteristic changes were detected in all groups of females. Our results support the theory that the behavioural effects of chemical administration vary considerably with developmental stages and sex. In conclusion, our findings promote better understanding of individual differences in excitatory chemical-induced neurotoxicity and provide evidence for future risk strategies and treatments.
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Conducta Animal/efectos de los fármacos , Ácido Iboténico/toxicidad , Ácido Kaínico/análogos & derivados , Administración Oral , Animales , Conducta Animal/fisiología , Encéfalo/efectos de los fármacos , Encéfalo/crecimiento & desarrollo , Encéfalo/fisiología , Agonistas de Aminoácidos Excitadores/administración & dosificación , Agonistas de Aminoácidos Excitadores/toxicidad , Femenino , Ácido Iboténico/administración & dosificación , Ácido Kaínico/administración & dosificación , Ácido Kaínico/toxicidad , Masculino , Toxinas Marinas/administración & dosificación , Toxinas Marinas/toxicidad , Ratones , Ratones Endogámicos C57BL , Neurotoxinas/administración & dosificación , Neurotoxinas/toxicidad , Factores Sexuales , Maduración Sexual/fisiologíaRESUMEN
Treatment of epilepsy remains difficult because patients suffer from pharmacoresistant forms of the disease and drug side-effects. Thus, there is an urgent need to identify not only new antiepileptic drug candidates but also novel epileptic animal models. Here, we characterize seizures induced with kainic acid (KA) in the common marmoset (Callithrix jacchus). Adult marmosets received 0.1, 1, or 10 mg/kg of KA subcutaneously. All animals exhibited early convulsive behavior (seizure scores of I and II on the Racine scale). Seizure scores were low at lower KA doses, but the highest dose of KA tested triggered generalized seizures (scores IV and V on the Racine scale). We next performed preliminary evaluation of the efficacy of the antiepileptic drug diazepam. This drug at 1 mg/kg (delivered subcutaneously) prevented 10 mg/kg KA-induced stage V seizures. KA administration to marmosets reliably triggers generalized seizures; therefore, the marmoset is a useful animal model in which to analyze the seizures of a nonhuman primate brain and to develop new treatments for epilepsy.
Asunto(s)
Convulsiones/inducido químicamente , Convulsiones/patología , Animales , Conducta Animal/efectos de los fármacos , Callithrix , Diazepam/farmacología , Diazepam/uso terapéutico , Femenino , Ácido Kaínico/administración & dosificación , Masculino , Convulsiones/tratamiento farmacológicoRESUMEN
Immune changes occur in experimental and clinical epilepsy. Here, we tested the hypothesis that during epileptogenesis and spontaneous recurrent seizures (SRS) an impairment of the endogenous anti-inflammatory pathway glucocorticoid receptor (GR)-annexin A1 (ANXA1) occurs. By administrating exogenous ANXA1, we studied whether pharmacological potentiation of the anti-inflammatory response modifies seizure activity and pathophysiology. We used an in vivo model of temporal lobe epilepsy based on intrahippocampal kainic acid (KA) injection. Video-electroencephalography, molecular biology analyses on brain and peripheral blood samples, and pharmacological investigations were performed in this model. Human epileptic cortices presenting type II focal cortical dysplasia (IIa and b), hippocampi with or without hippocampal sclerosis (HS), and available controls were used to study ANXA1 expression. A decrease of phosphorylated (phospho-) GR and phospho-GR/tot-GR protein expression occurred in the hippocampus during epileptogenesis. Downstream to GR, the anti-inflammatory protein ANXA1 remained at baseline levels while inflammation installed and endured. In peripheral blood, ANXA1 and corticosterone levels showed no significant modifications during disease progression except for an early and transient increase poststatus epilepticus. These results indicate inadequate ANXA1 engagement over time and in these experimental conditions. By analyzing human brain specimens, we found that where significant inflammation exists, the pattern of ANXA1 immunoreactivity was abnormal because the typical perivascular ANXA1 immunoreactivity was reduced. We next asked whether potentiation of the endogenous anti-inflammatory mechanism by ANXA1 administration modifies the disease pathophysiology. Although with varying efficacy, administration of exogenous ANXA1 somewhat reduced the time spent in seizure activity as compared to saline. These results indicate that the anti-inflammatory GR-ANXA1 pathway is defective during experimental seizure progression. The prospect of pharmacologically restoring or potentiating this endogenous anti-inflammatory mechanism as an add-on therapeutic strategy for specific forms of epilepsy is proposed.-Zub, E., Canet, G., Garbelli, R., Blaquiere, M., Rossini, L., Pastori, C., Sheikh, M., Reutelingsperger, C., Klement, W., de Bock, F., Audinat, E., Givalois, L., Solito, E., Marchi, N. The GR-ANXA1 pathway is a pathological player and a candidate target in epilepsy.
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Anexina A1/metabolismo , Epilepsia , Receptores de Glucocorticoides/metabolismo , Animales , Anexina A1/genética , Recuento de Células Sanguíneas , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Corticosterona/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Hipocampo , Humanos , Inflamación/metabolismo , Inflamación/patología , Ácido Kaínico/administración & dosificación , Ácido Kaínico/farmacología , Ratones , Ratones Endogámicos C57BL , Receptores de Glucocorticoides/genéticaRESUMEN
OBJECTIVE: Intracranial (intrahippocampal or intra-amygdala) administration of kainate in rodents leads to spatially restricted brain injury and development of focal epilepsy with characteristics that resemble mesial temporal lobe epilepsy. Such rodent models are used both in the search for more effective antiseizure drugs (ASDs) and in the development of antiepileptogenic strategies. However, it is not clear which of the models is best suited for testing different types of epilepsy therapies. METHODS: In the present study, we performed a face-to-face comparison of the intra-amygdala kainate (IAK) and intrahippocampal kainate (IHK) mouse models using the same mouse inbred strain (C57BL/6). For comparison, some experiments were performed in mouse outbred strains. RESULTS: Intra-amygdala kainate injection led to more severe status epilepticus and higher mortality than intrahippocampal injection. In male C57BL/6 mice, the latent period to spontaneous recurrent seizures (SRSs) was short or absent in both models, whereas a significantly longer latent period was determined in NMRI and CD-1 outbred mice. When SRSs were recorded from the ipsilateral hippocampus, relatively frequent electroclinical seizures were determined in the IAK model, whereas only infrequent electroclinical seizures but extremely frequent focal electrographic seizures were determined in the IHK model. As a consequence of the differences in SRS frequency, prolonged video-electroencephalographic monitoring and drug administration were needed for testing efficacy of the benchmark ASD carbamazepine in the IAK model, whereas acute drug testing was possible in the IHK model. In both models, carbamazepine was only effective at high doses, indicating ASD resistance to this benchmark drug. SIGNIFICANCE: We found a variety of significant differences between the IAK and IHK models, which are important when deciding which of these models is best suited for studies on novel epilepsy therapies. The IAK model appears particularly interesting for studies on disease-modifying treatments, whereas the IHK model is well suited for studying the antiseizure activity of novel ASDs against difficult-to-treated focal seizures.
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Convulsivantes/administración & dosificación , Modelos Animales de Enfermedad , Epilepsia del Lóbulo Temporal/inducido químicamente , Ácido Kaínico/administración & dosificación , Amígdala del Cerebelo/efectos de los fármacos , Animales , Anticonvulsivantes/farmacología , Convulsivantes/toxicidad , Hipocampo/efectos de los fármacos , Ácido Kaínico/toxicidad , RatonesRESUMEN
Domoic acid (DA) is a marine neurotoxin that accumulates in filtering shellfish during harmful algal blooms. A health protection limit of 20 ppm DA in razor clams (RC) has been set based principally upon an episode of acute DA toxicity in humans that included Amnesic Shellfish Poisoning among survivors. The objective of this study was to determine the dose-response relationship between estimated DA exposure through RC consumption and memory loss in Washington state Native Americans from 2005 to 2015. Results from total learning recall (TLR) memory scores were compared before and after the highest DA exposures. A decrease in TLR was related to DA dose (p < 0.01) regardless whether the effect was assumed to be transient or lasting, and whether the dose was expressed as an average daily dose or an average dose per meal. Benchmark dose modeling identified BMDL10 values of 167 ng/kg-day and 2740 ng/kg-meal assuming a transient effect, and 196 ng/kg-day and 2980 ng/kg-meal assuming no recovery of function occurs. These DA dose thresholds for a measurable memory function reduction observed in this study of clam consumers are well below the safe acute dose underpinning the current regulatory DA limit of 20 ppm (ca. 60 µg/kg).
Asunto(s)
Indio Americano o Nativo de Alaska , Bivalvos , Ácido Kaínico/análogos & derivados , Trastornos de la Memoria/inducido químicamente , Trastornos de la Memoria/diagnóstico , Intoxicación por Mariscos/diagnóstico , Adolescente , Adulto , Anciano , Animales , Estudios de Cohortes , Bases de Datos Factuales , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Ácido Kaínico/administración & dosificación , Ácido Kaínico/toxicidad , Masculino , Trastornos de la Memoria/psicología , Persona de Mediana Edad , Fármacos Neuromusculares Despolarizantes/administración & dosificación , Fármacos Neuromusculares Despolarizantes/toxicidad , Intoxicación por Mariscos/psicología , Adulto JovenRESUMEN
Epilepsy is a neurological disorder characterized by recurrent epileptic seizures. Electrophysiological and neuroimaging studies in patients with epilepsy suggest that abnormal functional brain networks play a role in the development of epilepsy, i.e. epileptogenesis, resulting in the generation of spontaneous seizures and cognitive impairment. In this longitudinal study, we investigated changes in functional brain networks during epileptogenesis in the intraperitoneal kainic acid (IPKA) rat model of temporal lobe epilepsy (TLE) using resting state functional magnetic resonance imaging (rsfMRI) and graph theory. Additionally, we investigated whether these changes are related to the frequency of occurrence of spontaneous epileptic seizures in the chronic phase of epilepsy. Using a 7T MRI system, rsfMRI images were acquired under medetomidine anaesthesia before and 1, 3, 6, 10 and 16 weeks after status epilepticus (SE) induction in 20 IPKA animals and 7 healthy control animals. To obtain a functional network, correlation between fMRI time series of 38 regions of interest (ROIs) was calculated. Then, several graph theoretical network measures were calculated to describe and quantify the network changes. At least 17 weeks post-SE, IPKA animals were implanted with electrodes in the left and right dorsal hippocampus, EEG was measured for 7 consecutive days and spontaneous seizures were counted. Our results show that correlation coefficients of fMRI time series shift to lower values during epileptogenesis, indicating weaker whole brain network connections. Segregation and integration in the functional brain network also decrease, indicating a lower local interconnectivity and a lower overall communication efficiency. Secondly, this study demonstrates that the largest decrease in functional connectivity is observed for the retrosplenial cortex. Finally, post-SE changes in functional connectivity, segregation and integration are correlated with seizure frequency in the IPKA rat model.
Asunto(s)
Encéfalo/fisiopatología , Epilepsia del Lóbulo Temporal/fisiopatología , Convulsiones/fisiopatología , Animales , Encéfalo/efectos de los fármacos , Mapeo Encefálico , Modelos Animales de Enfermedad , Epilepsia del Lóbulo Temporal/inducido químicamente , Hipocampo/efectos de los fármacos , Hipocampo/patología , Procesamiento de Imagen Asistido por Computador , Ácido Kaínico/administración & dosificación , Estudios Longitudinales , Imagen por Resonancia Magnética , Masculino , Vías Nerviosas/efectos de los fármacos , Vías Nerviosas/fisiopatología , Ratas Sprague-Dawley , Convulsiones/inducido químicamenteRESUMEN
The secondary phase of spinal cord injury arising after the primary lesion largely extends the damage severity with delayed negative consequences for sensory-motor pathways. It is, therefore, important to find out if enhancing intrinsic mechanisms of neuroprotection can spare motoneurons that are very vulnerable cells. This issue was investigated with an in vitro model of rat spinal cord excitotoxicity monitored for up to 24 hr after the primary injury evoked by kainate. This study sought to pharmacologically boost the expression of heat shock proteins (HSP) to protect spinal motoneurons using celastrol to investigate if the rat spinal cord can upregulate HSP as neuroprotective mechanism. Despite its narrow range of drug safety in vitro, celastrol was not toxic to the rat spinal cord at 0.75 µM concentration and enhanced the expression of HSP70 by motoneurons. When celastrol was applied either before or after kainate, the number of dead motoneurons was significantly decreased and the nuclear localization of the cell death biomarker AIF strongly inhibited. Nevertheless, electrophysiological recording showed that protection of lumbar motor networks by celastrol was rather limited as reflex activity was impaired and fictive locomotion largely depressed, suggesting that functional deficit persisted, though the networks could express slow rhythmic oscillations. While our data do not exclude further recovery at later times beyond the experimental observations, the present results indicate that the upregulated expression of HSP in the aftermath of acute injury may be an interesting avenue for early protection of spinal motoneurons.
Asunto(s)
Proteínas HSP70 de Choque Térmico/metabolismo , Neuronas Motoras/metabolismo , Fármacos Neuroprotectores/administración & dosificación , Traumatismos de la Médula Espinal/metabolismo , Médula Espinal/metabolismo , Triterpenos/administración & dosificación , Animales , Animales Recién Nacidos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Femenino , Humanos , Ácido Kaínico/administración & dosificación , Locomoción/efectos de los fármacos , Masculino , Triterpenos Pentacíclicos , Ratas Wistar , Médula Espinal/efectos de los fármacos , Traumatismos de la Médula Espinal/inducido químicamenteRESUMEN
Antiepileptogenic agents that prevent the development of epilepsy following a brain insult remain the holy grail of epilepsy therapeutics. We have employed a label-free proteomic approach that allows quantification of large numbers of brain-expressed proteins in a single analysis in the mouse (male C57BL/6J) kainate (KA) model of epileptogenesis. In addition, we have incorporated two putative antiepileptogenic drugs, postsynaptic density protein-95 blocking peptide (PSD95BP or Tat-NR2B9c) and a highly selective inducible nitric oxide synthase inhibitor, 1400W, to give an insight into how such agents might ameliorate epileptogenesis. The test drugs were administered after the induction of status epilepticus (SE) and the animals were euthanized at 7 days, their hippocampi removed, and subjected to LC-MS/MS analysis. A total of 2,579 proteins were identified; their normalized abundance was compared between treatment groups using ANOVA, with correction for multiple testing by false discovery rate. Significantly altered proteins were subjected to gene ontology and KEGG pathway enrichment analyses. KA-induced SE was most robustly associated with an alteration in the abundance of proteins involved in neuroinflammation, including heat shock protein beta-1 (HSP27), glial fibrillary acidic protein, and CD44 antigen. Treatment with PSD95BP or 1400W moderated the abundance of several of these proteins plus that of secretogranin and Src substrate cortactin. Pathway analysis identified the glutamatergic synapse as a key target for both drugs. Our observations require validation in a larger-scale investigation, with candidate proteins explored in more detail. Nevertheless, this study has identified several mechanisms by which epilepsy might develop and several targets for novel drug development. OPEN PRACTICES: This article has been awarded Open Data. All materials and data are publicly accessible as supporting information. Learn more about the Open Practices badges from the Center for Open Science: https://osf.io/tvyxz/wiki.
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Amidinas/administración & dosificación , Anticonvulsivantes/administración & dosificación , Bencilaminas/administración & dosificación , Epilepsia/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Péptidos/administración & dosificación , Animales , Epilepsia/inducido químicamente , Ácido Kaínico/administración & dosificación , Masculino , Ratones Endogámicos C57BL , Proteómica , Estado Epiléptico/inducido químicamente , Estado Epiléptico/metabolismoRESUMEN
OBJECTIVE: More than one-third of patients with temporal lobe epilepsy (TLE) continue to have seizures despite treatment with antiepileptic drugs, and many experience severe drug-related side effects, illustrating the need for novel therapies. Selective expression of inhibitory Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) allows cell-type-specific reduction of neuronal excitability. In this study, we evaluated the effect of chemogenetic suppression of excitatory pyramidal and granule cell neurons of the sclerotic hippocampus in the intrahippocampal mouse model (IHKA) for temporal lobe epilepsy. METHODS: Intrahippocampal IHKA mice were injected with an adeno-associated viral vector carrying the genes for an inhibitory DREADD hM4Di in the sclerotic hippocampus or control vector. Next, animals were treated systemically with different single doses of clozapine-N-oxide (CNO) (1, 3, and 10 mg/kg) and clozapine (0.03 and 0.1 mg/kg) and the effect on spontaneous hippocampal seizures, hippocampal electroencephalography (EEG) power, fast ripples (FRs) and behavior in the open field test was evaluated. Finally, animals received prolonged treatment with clozapine for 3 days and the effect on seizures was monitored. RESULTS: Treatment with both CNO and clozapine resulted in a robust suppression of hippocampal seizures for at least 15 hours only in DREADD-expressing animals. Moreover, total EEG power and the number of FRs were significantly reduced. CNO and/or clozapine had no effects on interictal hippocampal EEG, seizures, or locomotion/anxiety in the open field test in non-DREADD epileptic IHKA mice. Repeated clozapine treatment every 8 hours for 3 days resulted in almost complete seizure suppression in DREADD animals. SIGNIFICANCE: This study shows the potency of chemogenetics to robustly and sustainably suppress spontaneous epileptic seizures and pave the way for an epilepsy therapy in which a systemically administered exogenous drug selectively modulates specific cell types in a seizure network, leading to a potent seizure suppression devoid of the typical drug-related side effects.
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Anticonvulsivantes/administración & dosificación , Epilepsia del Lóbulo Temporal/genética , Epilepsia del Lóbulo Temporal/prevención & control , Convulsiones/genética , Convulsiones/prevención & control , Animales , Clozapina/administración & dosificación , Clozapina/análogos & derivados , Electroencefalografía/efectos de los fármacos , Electroencefalografía/métodos , Epilepsia del Lóbulo Temporal/fisiopatología , Vectores Genéticos/administración & dosificación , Hipocampo/efectos de los fármacos , Hipocampo/fisiopatología , Ácido Kaínico/administración & dosificación , Ácido Kaínico/toxicidad , Masculino , Ratones , Ratones Endogámicos C57BL , Convulsiones/fisiopatologíaRESUMEN
Whisking and sniffing are predominant aspects of exploratory behaviour in rodents. Yet the neural mechanisms that generate and coordinate these and other orofacial motor patterns remain largely uncharacterized. Here we use anatomical, behavioural, electrophysiological and pharmacological tools to show that whisking and sniffing are coordinated by respiratory centres in the ventral medulla. We delineate a distinct region in the ventral medulla that provides rhythmic input to the facial motor neurons that drive protraction of the vibrissae. Neuronal output from this region is reset at each inspiration by direct input from the pre-Bötzinger complex, such that high-frequency sniffing has a one-to-one relationship with whisking, whereas basal respiration is accompanied by intervening whisks that occur between breaths. We conjecture that the respiratory nuclei, which project to other premotor regions for oral and facial control, function as a master clock for behaviours that coordinate with breathing.
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Movimientos de la Cabeza/fisiología , Respiración , Olfato/fisiología , Vibrisas/fisiología , Animales , Relojes Biológicos/fisiología , Cara/anatomía & histología , Cara/fisiología , Femenino , Ácido Kaínico/administración & dosificación , Ácido Kaínico/farmacología , Masculino , Bulbo Raquídeo/citología , Bulbo Raquídeo/fisiología , Músculo Esquelético/fisiología , Ratas , Ratas Long-Evans , Vibrisas/inervaciónRESUMEN
Epileptic seizures are associated with increased astrocytic Ca2+ signaling, but the fine spatiotemporal kinetics of the ictal astrocyte-neuron interplay remains elusive. By using 2-photon imaging of awake head-fixed mice with chronic hippocampal windows we demonstrate that astrocytic Ca2+ signals precede neuronal Ca2+ elevations during the initial bout of kainate-induced seizures. On average, astrocytic Ca2+ elevations preceded neuronal activity in CA1 by about 8 s. In subsequent bouts of epileptic seizures, astrocytes and neurons were activated simultaneously. The initial astrocytic Ca2+ elevation was abolished in mice lacking the type 2 inositol-1,4,5-trisphosphate-receptor (Itpr2-/-). Furthermore, we found that Itpr2-/- mice exhibited 60% less epileptiform activity compared with wild-type mice when assessed by telemetric EEG monitoring. In both genotypes we also demonstrate that spreading depression waves may play a part in seizure termination. Our findings imply a role for astrocytic Ca2+ signals in ictogenesis.
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Astrocitos/fisiología , Señalización del Calcio , Epilepsia/fisiopatología , Hipocampo/fisiopatología , Neuronas/fisiología , Convulsiones/fisiopatología , Animales , Epilepsia/inducido químicamente , Agonistas de Aminoácidos Excitadores/administración & dosificación , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/fisiología , Ácido Kaínico/administración & dosificación , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Convulsiones/inducido químicamenteRESUMEN
BACKGROUND This study aimed to discover the effect and mechanism of microRNA-27a-3p (miR-27a-3p) in epilepsy. MATERIAL AND METHODS To perform our investigation, in vivo and in vitro models of epilepsy were induced using kainic acid (KA). Expression of miR-27a-3p in the hippocampus of epileptic rats or normal rats or neuronal cells was detected using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Racine score was used to assess seizures in epileptic rats. Cell viability and cell apoptosis were analyzed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and flow cytometry. Enzyme-linked immunosorbent assay (ELISA) was performed to detect inflammatory factors expression. RESULTS Significantly higher expression of miR-27a-3p in the hippocampus of epileptic rats and in KA-induced neurons was observed. We found that miR-27a-3p inhibitor alleviated seizures in epileptic rats. miR-27a-3p inhibitor also inhibited apoptosis of hippocampal neurons in epileptic rats, promoted Bcl2 expression, and decreased Bax and Caspase3 expression. The results showed that miR-27a-3p inhibitor effectively reduced the expression levels of interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-alpha (TNF-alpha) in hippocampal tissues of epileptic rats. Dual luciferase reporter assay showed that mitogen-activated protein kinase 4 (MAP2K4) was a direct target of miR-27a-3p. miR-27a-3p inhibitor significantly promoted the cell viability of KA-induced neurons, inhibited cell apoptosis, promoted the expression of Bcl-2, and decreased Bax and Caspase3 expression, and all these changes were abolished by MAP2K4-siRNA co-transfection. CONCLUSIONS Our preliminary findings indicated that miR-27a-3p inhibitor protected against epilepsy-induced inflammatory response and hippocampal neuronal apoptosis by targeting MAP2K4.
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Epilepsia/metabolismo , Hipocampo/metabolismo , MAP Quinasa Quinasa 4/biosíntesis , MicroARNs/metabolismo , Neuronas/metabolismo , Animales , Apoptosis/fisiología , Supervivencia Celular/fisiología , Modelos Animales de Enfermedad , Regulación hacia Abajo , Epilepsia/genética , Epilepsia/patología , Regulación de la Expresión Génica , Células HEK293 , Hipocampo/patología , Humanos , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Ácido Kaínico/administración & dosificación , MAP Quinasa Quinasa 4/genética , MAP Quinasa Quinasa 4/metabolismo , Masculino , MicroARNs/antagonistas & inhibidores , MicroARNs/biosíntesis , MicroARNs/genética , Neuronas/patología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-Dawley , Activación Transcripcional , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Status epilepticus may decrease mitochondrial biogenesis, resulting in neuronal cell death occurring in the hippocampus. Sirtuin 1 (SIRT1) functionally interacts with peroxisome proliferator-activated receptors and γ coactivator 1α (PGC-1α), which play a crucial role in the regulation of mitochondrial biogenesis. In Sprague-Dawley rats, kainic acid was microinjected unilaterally into the hippocampal CA3 subfield to induce bilateral seizure activity. SIRT1, PGC-1α, and other key proteins involving mitochondrial biogenesis and the amount of mitochondrial DNA were investigated. SIRT1 antisense oligodeoxynucleotide was used to evaluate the relationship between SIRT1 and mitochondrial biogenesis, as well as the mitochondrial function, oxidative stress, and neuronal cell survival. Increased SIRT1, PGC-1α, and mitochondrial biogenesis machinery were found in the hippocampus following experimental status epilepticus. Downregulation of SIRT1 decreased PGC-1α expression and mitochondrial biogenesis machinery, increased Complex I dysfunction, augmented the level of oxidized proteins, raised activated caspase-3 expression, and promoted neuronal cell damage in the hippocampus. The results suggest that the SIRT1 signaling pathway may play a pivotal role in mitochondrial biogenesis, and could be considered an endogenous neuroprotective mechanism counteracting seizure-induced neuronal cell damage following status epilepticus.
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Región CA3 Hipocampal/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Neuronas/efectos de los fármacos , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Sirtuina 1/genética , Estado Epiléptico/genética , Animales , Región CA3 Hipocampal/metabolismo , Región CA3 Hipocampal/patología , Caspasa 3/genética , Caspasa 3/metabolismo , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Complejo I de Transporte de Electrón/genética , Complejo I de Transporte de Electrón/metabolismo , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Inyecciones Intraventriculares , Ácido Kaínico/administración & dosificación , Masculino , Mitocondrias/metabolismo , Mitocondrias/patología , Neuronas/metabolismo , Neuronas/patología , Biogénesis de Organelos , Estrés Oxidativo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Sirtuina 1/antagonistas & inhibidores , Sirtuina 1/metabolismo , Estado Epiléptico/inducido químicamente , Estado Epiléptico/metabolismo , Estado Epiléptico/patología , Técnicas EstereotáxicasRESUMEN
In this study, we report the development of a dual extraction protocol for RNA and lipids, including phospholipids, endocannabinoids, and arachidonic acid, at high spatial resolution, e.g., brain punches obtained from whole frozen brains corresponding to four brain subregions: dorsal hippocampus, ventral hippocampus, basolateral amygdala, and hypothalamus. This extraction method combined with LC/multiple reaction monitoring for lipid quantifi-cation and quantitative PCR for RNA investigation allows lipidomic and transcriptomic profiling from submilligram amounts of tissue, thus benefiting the time and animal costs for analysis and the data reliability due to prevention of biological variability between animal batches and/or tissue heterogeneity, as compared with profiling in distinct animal batches. Moreover, the method allows a higher extraction efficiency and integrity preservation for RNA, while allowing concurrently quantitative analysis of low and high abundant lipids. The method was applied for brain punches obtained 1 h after kainic acid-induced epileptic seizures in mice (n = 10) compared with controls (n = 10), and enabled the provision of valuable new insights into the subregional lipid and RNA changes with epilepsy, highlighting its potential as a new viable tool in quantitative neurobiology.
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Encéfalo/efectos de los fármacos , Modelos Animales de Enfermedad , Ácido Kaínico/toxicidad , Lípidos , Convulsiones/inducido químicamente , Enfermedad Aguda , Animales , Encéfalo/metabolismo , Perfilación de la Expresión Génica , Ácido Kaínico/administración & dosificación , Ratones , Convulsiones/genética , Convulsiones/metabolismoRESUMEN
Temporal lobe epilepsy (TLE), the most common pharmacoresistant focal epilepsy disorder, remains a major unmet medical need. Propofol is used as a short-acting medication for general anesthesia and refractory status epilepticus with issues of decreased consciousness and memory loss. Dipropofol, a derivative of propofol, has been reported to exert antioxidative and antibacterial activities. Here we report that dipropofol exerted anticonvulsant activity in a mouse model of kainic acid-induced seizures. Whole cell patch-clamp recordings of brain slices from the medial entorhinal cortex (mEC) revealed that dipropofol hyperpolarized the resting membrane potential and reduced the number of action potential firings, resulting in suppression of cortical neuronal excitability. Furthermore, dipropofol activated native tonic GABAA currents of mEC layer II stellate neurons in a dose-dependent manner with an EC50 value of 9.3 ± 1.6 µM (mean ± SE). Taken together, our findings show that dipropofol activated GABAA currents and exerted anticonvulsant activities in mice, thus possessing developmental potential for new anticonvulsant therapy. NEW & NOTEWORTHY The anticonvulsant effect of dipropofol was shown in a mouse model of kainic acid-induced seizures. Whole cell patch-clamp recordings of brain slices showed suppression of cortical neuronal excitability by dipropofol. Dipropofol activated the native tonic GABAA currents in a dose-dependent manner.
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Alcanos/administración & dosificación , Anticonvulsivantes/administración & dosificación , Corteza Entorrinal/efectos de los fármacos , Neuronas/efectos de los fármacos , Fenoles/administración & dosificación , Receptores de GABA-A/fisiología , Convulsiones/tratamiento farmacológico , Ácido gamma-Aminobutírico/fisiología , Animales , Relación Dosis-Respuesta a Droga , Corteza Entorrinal/fisiología , Ácido Kaínico/administración & dosificación , Masculino , Potenciales de la Membrana/efectos de los fármacos , Ratones Endogámicos C57BL , Neuronas/fisiología , Convulsiones/inducido químicamenteRESUMEN
Epilepsy is a chronic brain disease affecting millions of individuals. Kainate receptors, especially kainate-type of ionotropic glutamate receptor 2 (GluK2), play an important role in epileptogenesis. Recent data showed that GluK2 could undergo post-translational modifications in terms of S-nitrosylation (SNO), and affect the signaling pathway of cell death in cerebral ischemia-reperfusion. However, it is unclear whether S-nitrosylation of GluK2 (SNO-GluK2) contributes to cell death induced by epilepsy. Here, we report that kainic acid-induced SNO-GluK2 is mediated by GluK2 itself, regulated by neuronal nitric oxide synthase (nNOS) and the level of cytoplasmic calcium in vivo and in vitro hippocampus neurons. The whole-cell patch clamp recordings showed the influence of SNO-GluK2 on ion channel characterization of GluK2-Kainate receptors. Moreover, immunohistochemistry staining results showed that inhibition of SNO-GluK2 by blocking nNOS or GluK2 or by reducing the level of cytoplasmic calcium-protected hippocampal neurons from kainic acid-induced injury. Finally, immunoprecipitation and western blotting data revealed the involvement of assembly of a GluK2-PSD95-nNOS signaling complex in epilepsy. Taken together, our results showed that the SNO-GluK2 plays an important role in neuronal injury of epileptic rats by forming GluK2-PSD95-nNOS signaling module in a cytoplasmic calcium-dependent way, suggesting a potential therapeutic target site for epilepsy.
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Epilepsia/metabolismo , Hipocampo/metabolismo , Ácido Kaínico/administración & dosificación , Neuronas/metabolismo , Óxido Nítrico/metabolismo , Receptores de Ácido Kaínico/metabolismo , Animales , Calcio/metabolismo , Homólogo 4 de la Proteína Discs Large/metabolismo , Epilepsia/inducido químicamente , Hipocampo/efectos de los fármacos , Masculino , Neuronas/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo I/metabolismo , Cultivo Primario de Células , Ratas Sprague-Dawley , Transducción de Señal , Receptor de Ácido Kaínico GluK2RESUMEN
BACKGROUND: Ropivacaine, a long-acting amide local anesthetic agent, has been demonstrated to inhibit glutamatergic transmission. Glutamate neurotoxicity plays a pivotal role in the pathogenesis of brain disorders. The purpose of this study is to investigate the neuroprotective effect of ropivacaine against brain damage induced by kainic acid (KA), an analogue of glutamate. METHODS: Rats were injected with ropivacaine (0.4 or 2 mg/kg, intraperitoneal) 30 min before KA treatment (15 mg/kg, intraperitoneal). KA-induced memory impairment was evaluated using the Morris water maze test. Extracellular hippocampal glutamate levels were assessed using high-performance liquid chromatography. Neuronal death was verified using Fluoro-Jade B and neutral red staining, and apoptosis was determined through terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Western blotting was conducted to assay the levels of activated (cleaved) caspase-3 and the phosphorylation of different mitogen-activated protein kinases (MAPKs). -Results: Ropivacaine pretreatment effectively prevented KA-induced memory impairment. KA-induced elevations of -glutamate release in rat hippocampi were inhibited by pretreatment with ropivacaine. Histopathological and TUNEL staining analyzes showed that ropivacaine inhibited KA-induced neuronal death and apoptosis in the hippocampal CA3 region. KA-induced caspase-3 activation and MAPKs phosphorylation in the hippocampus were also reduced by ropivacaine pretreatment. CONCLUSIONS: This study -demonstrates that ropivacaine executes a protective action against KA-induced neuronal damage and apoptosis in vivo. Protective effects may be caused by glutamate level reduction, caspase-3 activation suppression, and MAPKs phosphorylation reduction. Our findings indicate that ropivacaine can benefit prevention or treatment of glutamate excitotoxicity-related neurodegenerative diseases.
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Hipocampo/efectos de los fármacos , Hipocampo/patología , Trastornos de la Memoria/prevención & control , Enfermedades Neurodegenerativas/prevención & control , Fármacos Neuroprotectores/farmacología , Ropivacaína/farmacología , Memoria Espacial/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Muerte Celular/efectos de los fármacos , Agonistas de Aminoácidos Excitadores/toxicidad , Ácido Glutámico/metabolismo , Hipocampo/metabolismo , Ácido Kaínico/administración & dosificación , Masculino , Trastornos de la Memoria/inducido químicamente , Trastornos de la Memoria/tratamiento farmacológico , Trastornos de la Memoria/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Enfermedades Neurodegenerativas/inducido químicamente , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Excitotoxicity is the pivotal mechanism of neuronal death. Prostaglandins (PGs) produced during excitotoxicity play important roles in neurodegenerative conditions. Previously, we demonstrated that initial burst productions of PGD2, PGE2, and PGF2α are produced by cyclooxygenase-2 (COX-2) in the hippocampus following a single systemic kainic acid (KA) administration. In addition, we showed that blocking of all PG productions ameliorated hippocampal delayed neuronal death at 30 days after KA administration. To investigate the role of individual PGs in the delayed neuronal death, we performed intracerebroventricular injection of PGD2, PGE2, or PGF2α in rats whose hippocampal PG productions were entirely blocked by pretreatment of NS398, a COX-2 selective inhibitor. Administration of PGD2 and PGF2α had a latent contribution to the delayed neuronal death, sustained over 30 days after a single KA treatment. Furthermore, PGD2 enhanced microglial activation, which may be involved in the delayed neuronal death in the hippocampus. These findings suggest that excitotoxic delayed neuronal death is mediated through microglia activated by PGD2.
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Ciclooxigenasa 2/metabolismo , Hipocampo/metabolismo , Ácido Kaínico/administración & dosificación , Prostaglandina D2/biosíntesis , Animales , Muerte Celular/efectos de los fármacos , Hipocampo/efectos de los fármacos , Microglía/efectos de los fármacos , Microglía/metabolismo , Microglía/patología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Nitrobencenos/administración & dosificación , Prostaglandina D2/metabolismo , Ratas , Sulfonamidas/administración & dosificaciónRESUMEN
Imaging markers for monitoring disease progression, recovery, and treatment efficacy are a major unmet need for many neurological diseases, including epilepsy. Recent evidence suggests that diffusion tensor imaging (DTI) provides high microstructural contrast even outside major white matter tracts. We hypothesized that in vivo DTI could detect progressive microstructural changes in the dentate gyrus and the hippocampal CA3bc in the rat brain after status epilepticus (SE). To test this hypothesis, we induced SE with systemic kainic acid or pilocarpine in adult male Wistar rats and subsequently scanned them using in vivo DTI at five time-points: prior to SE, and 10, 20, 34, and 79 days post SE. In order to tie the DTI findings to changes in the tissue microstructure, myelin- and glial fibrillary acidic protein (GFAP)-stained sections from the same animals underwent Fourier analysis. We compared the Fourier analysis parameters, anisotropy index and angle of myelinated axons or astrocyte processes, to corresponding DTI parameters, fractional anisotropy (FA) and the orientation angle of the principal eigenvector. We found progressive detectable changes in DTI parameters in both the dentate gyrus (FA, axial diffusivity [D||], linear anisotropy [CL] and spherical anisotropy [CS], p<0.001, linear mixed-effects model [LMEM]) and the CA3bc (FA, D||, CS, and angle, p<0.001, LMEM; CL and planar anisotropy [CP], p<0.01, LMEM) post SE. The Fourier analysis revealed that both myelinated axons and astrocyte processes played a role in the water diffusion anisotropy changes detected by DTI in individual portions of the dentate gyrus (suprapyramidal blade, mid-portion, and infrapyramidal blade). In the whole dentate gyrus, myelinated axons markedly contributed to the water diffusion changes. In CA3bc as well as in CA3b and CA3c, both myelinated axons and astrocyte processes contributed to water diffusion anisotropy and orientation. Our study revealed that DTI is a promising method for noninvasive detection of microstructural alterations in the hippocampus proper. These alterations may be potential imaging markers for epileptogenesis.