RESUMO
Epilepsies affecting the limbic regions are common and generate seizures often resistant to pharmacological treatment. Clinical evidence demonstrates that diverse regions of the mesial portion of the temporal lobe participate in limbic seizures; these include the hippocampus, the entorhinal, perirhinal and parahippocampal regions and the piriform cortex. The network mechanisms involved in the generation of olfactory-limbic epileptiform patterns will be here examined, with particular emphasis on acute interictal and ictal epileptiform discharges obtained by treatment with pro-convulsive drugs and by high-frequency stimulations on in vitro preparations, such as brain slices and the isolated guinea pig brain. The interactions within olfactory-limbic circuits can be summarized as follows: independent, region-specific seizure-like events (SLE) are generated in the olfactory and in the limbic cortex; SLEs generated in the hippocampal-parahippocampal regions tend to remain within these areas; the perirhinal region controls the neocortical propagation and the generalization of limbic seizures; interictal spiking in the olfactory regions prevents the invasion by SLEs generated in limbic regions. The potential relevance of these observations for human focal epilepsy is discussed.
Assuntos
Epilepsias Parciais , Epilepsia , Humanos , Animais , Cobaias , Convulsões , Hipocampo , Córtex CerebralRESUMO
We review here the neuronal mechanisms that cause seizures in focal epileptic disorders and, specifically, those involving limbic structures that are known to be implicated in human mesial temporal lobe epilepsy. In both epileptic patients and animal models, the initiation of focal seizures - which are most often characterized by a low-voltage fast onset EEG pattern - is presumably dependent on the synchronous firing of GABA-releasing interneurons that, by activating post-synaptic GABAA receptors, cause large increases in extracellular [K+] through the activation of the co-transporter KCC2. A similar mechanism may contribute to seizure maintenance; accordingly, inhibiting KCC2 activity transforms seizure activity into a continuous pattern of short-lasting epileptiform discharges. It has also been found that interactions between different areas of the limbic system modulate seizure occurrence by controlling extracellular [K+] homeostasis. In line with this view, low-frequency electrical or optogenetic activation of limbic networks restrain seizure generation, an effect that may also involve the activation of GABAB receptors and activity-dependent changes in epileptiform synchronization. Overall, these findings highlight the paradoxical role of GABAA signaling in both focal seizure generation and maintenance, emphasize the efficacy of low-frequency activation in abating seizures, and provide experimental evidence explaining the poor efficacy of antiepileptic drugs designed to augment GABAergic function in controlling seizures in focal epileptic disorders.
Assuntos
Epilepsias Parciais , Simportadores , Animais , Humanos , Ligantes , Convulsões , Receptores de GABA-A , Ácido gama-AminobutíricoRESUMO
OBJECTIVE: The influx of immune cells and serum proteins from the periphery into the brain due to a dysfunctional blood-brain barrier (BBB) has been proposed to contribute to the pathogenesis of seizures in various forms of epilepsy and encephalitis. We evaluated the pathophysiological impact of activated peripheral blood mononuclear cells (PBMCs) and serum albumin on neuronal excitability in an in vitro brain preparation. METHODS: A condition of mild endothelial activation induced by arterial perfusion of lipopolysaccharide (LPS) was induced in the whole brain preparation of guinea pigs maintained in vitro by arterial perfusion. We analyzed the effects of co-perfusion of human recombinant serum albumin with human PBMCs activated with concanavalin A on neuronal excitability, BBB permeability (measured by FITC-albumin extravasation), and microglial activation. RESULTS: Bioplex analysis in supernatants of concanavalin A-stimulated PBMCs revealed increased levels of several inflammatory mediators, in particular interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, interferon (INF)-γ, IL-6, IL-10, IL-17A, and MIP3α. LPS and human albumin arterially co-perfused with either concanavalin A-activated PBMCs or the cytokine-enriched supernatant of activated PBMCs (1) modulated calcium-calmodulin-dependent protein kinase II at excitatory synapses, (2) enhanced BBB permeability, (3) induced microglial activation, and (4) promoted seizure-like events. Separate perfusions of either nonactivated PBMCs or concanavalin A-activated PBMCs without LPS/human albumin (hALB) failed to induce inflammatory and excitability changes. SIGNIFICANCE: Activated peripheral immune cells, such as PBMCs, and the extravasation of serum proteins in a condition of BBB impairment contribute to seizure generation.
Assuntos
Leucócitos Mononucleares , Convulsões/sangue , Animais , Barreira Hematoencefálica/patologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Concanavalina A , Citocinas/sangue , Eletrodos Implantados , Endotélio Vascular/patologia , Cobaias , Humanos , Imunidade Celular , Mediadores da Inflamação/sangue , Ativação de Macrófagos , Microglia/imunologia , Microglia/patologia , Neurônios/efeitos dos fármacos , Fluxo Sanguíneo Regional , Convulsões/patologia , Albumina Sérica/farmacologia , Baço/irrigação sanguíneaRESUMO
Focal seizures are triggered by the pathological synchronization of a functionally altered group of neurons. In vivo and in vitro results in rodents and single unit studies in humans suggest that seizure can be initiated by increased activity in interneuronal networks. We review here the data derived from in vitro perparations to describe the function of GABAergic network in different phases of focal seizures. The data demonstrate that GABA-mediated synchronization of interneuronal activity has an active role in shaping focal seizure dynamics.
Assuntos
Interneurônios/metabolismo , Rede Nervosa/metabolismo , Receptores de GABA-A/metabolismo , Convulsões/metabolismo , Animais , Humanos , Rede Nervosa/fisiopatologia , Convulsões/fisiopatologiaRESUMO
The onset of focal seizures in humans and in different animal models of focal epilepsy correlates with reduction of neuronal firing and enhanced interneuronal network activity. Whether this phenomenon contributes to seizure generation is still unclear. We used the in vitro entorhinal cortex slices bathed in 4-aminopirydine (4-AP) as an experimental paradigm model to evaluate the correlation between interneuronal GABAergic network activity and seizure-like events. Epileptiform discharges were recorded in layer V-VI pyramidal neurons and fast-spiking interneurons in slices from male and female mice and in the isolated female guinea pig brain preparation during perfusion with 4-AP. We observed that 90% of seizure-like events recorded in principal cells were preceded by outward currents coupled with extracellular potassium shifts, abolished by pharmacological blockade of GABAA receptors. Potassium elevations associated to GABAA receptor-mediated population events were confirmed in the entorhinal cortex of the in vitro isolated whole guinea pig brain. Fast-rising and sustained extracellular potassium increases associated to interneuronal network activity consistently preceded the initiation of seizure-like events. We conclude that in the 4-AP seizure model, interneuronal network activity occurs before 4-AP-induced seizures and therefore supports a role of interneuron activity in focal seizure generation.SIGNIFICANCE STATEMENT The paper focuses on the mechanisms of ictogenesis, a topic that requires a step beyond the simplistic view that seizures, and epilepsy, are due to an increase of excitatory network activity. Focal temporal lobe seizures in humans and in several experimental epilepsies likely correlate with a prevalent activation of interneurons. The potassium channel blocker 4-aminopyridine reliably induces seizure-like events in temporal lobe structures. Herein, we show that a majority of seizures in the entorhinal cortex starts with interneuronal network activity accompanied by a fast and sustained increase in extracellular potassium. Our new findings reinforce and add a new piece of evidence to the proposal that limbic seizures can be supported by GABAergic hyperactivity.
Assuntos
Potenciais de Ação/fisiologia , Córtex Entorrinal/fisiologia , Interneurônios/fisiologia , Rede Nervosa/fisiologia , Convulsões/fisiopatologia , Animais , Feminino , Cobaias , Potenciais Pós-Sinápticos Inibidores/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Cultura de ÓrgãosRESUMO
OBJECTIVE: Systemic administration of kainic acid (KA) is a widely used procedure utilized to develop a model of temporal lobe epilepsy (TLE). Despite its ability to induce status epilepticus (SE) in vivo, KA applied to in vitro preparations induces only interictal-like activity and/or isolated ictal discharges. The possibility that extravasation of the serum protein albumin from the vascular compartment enhances KA-induced brain excitability is investigated here. METHODS: Epileptiform activity was induced by arterial perfusion of 6 µm KA in the in vitro isolated guinea pig brain preparation. Simultaneous field potential recordings were carried out bilaterally from limbic (CA1, dentate gyrus [DG], and entorhinal cortex) and extralimbic regions (piriform cortex and neocortex). Blood-brain barrier (BBB) breakdown associated with KA-induced epileptiform activity was assessed by parenchymal leakage of intravascular fluorescein-isothiocyanate albumin. Seizure-induced brain inflammation was evaluated by western blot analysis of interleukin (IL)-1ß expression in brain tissue. RESULTS: KA infusion caused synchronized activity at 15-30 Hz in limbic (but not extralimbic) cortical areas, associated with a brief, single seizure-like event. A second bolus of KA, 60 min after the induction of the first ictal event, did not further enhance excitability. Perfusion of serum albumin between the two administrations of KA enhanced epileptiform discharges and allowed a recurrent ictal event during the second KA infusion. SIGNIFICANCE: Our data show that arterial KA administration selectively alters the synchronization of limbic networks. However, KA is not sufficient to generate recurrent seizures unless serum albumin is co-perfused during KA administration. These findings suggest a role of serum albumin in facilitating acute seizure generation.
Assuntos
Albuminas/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Agonistas de Aminoácidos Excitatórios/efeitos adversos , Ácido Caínico/efeitos adversos , Sistema Límbico/fisiopatologia , Convulsões/induzido quimicamente , Animais , Permeabilidade Capilar/efeitos dos fármacos , Modelos Animais de Doenças , Eletroencefalografia , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Cobaias , Interleucina-1beta/metabolismo , Sistema Límbico/efeitos dos fármacos , Microscopia Confocal , Fosfopiruvato Hidratase/metabolismo , Albumina Sérica/farmacologia , Análise Espectral , Frações Subcelulares/metabolismoRESUMO
OBJECTIVE: Epilepsy is a common neurological disorder characterized by recurrent seizures often unresponsive to pharmacological treatment. Brain inflammation is considered a crucial etiopathogenetic mechanism of epilepsy that could be targeted to control seizures. Specific inflammatory mediators overexpressed in human epileptogenic foci are known to promote seizures in animal models. We investigated whether seizures induce brain inflammation independently on extracerebral factors. We also investigated whether brain-borne inflammation is required and sufficient to maintain seizure activity and whether it causes blood-brain barrier (BBB) impairment. We addressed these questions by studying the relation between seizures, inflammation, and BBB permeability in a brain preparation isolated from extracerebral compartments. METHODS: Epileptiform activity was induced by arterial perfusion of bicuculline in the in vitro isolated guinea pig brain. Seizure-induced brain inflammation was evaluated by quantitative immunohistochemical analysis of interleukin (IL)-1ß in parenchymal cells. BBB damage was assessed by extravasation of intravascular fluorescein isothiocyanate-albumin. The effects of arterially perfused anakinra, a human recombinant IL-1ß receptor antagonist, were investigated on epileptiform discharges, brain inflammation, and BBB damage. RESULTS: Seizure induction in the absence of extracerebral factors promoted the release of IL-1ß from brain resident cells and enhanced its biosynthesis in astrocytes. Anakinra rapidly terminated seizures, prevented their recurrence, and resolved seizure-associated BBB breakdown. INTERPRETATION: Seizures initiate brain inflammation in glia and promote BBB damage that is independent of either leukocytes or blood-borne inflammatory molecules. Brain inflammation contributes to the duration and recurrence of seizures. This study supports the use of specific anti-inflammatory drugs in clinical conditions that present with intractable recurrent seizures.
Assuntos
Barreira Hematoencefálica/patologia , Encefalite/patologia , Epilepsia/patologia , Convulsões/patologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Bicuculina , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Encefalite/tratamento farmacológico , Encefalite/metabolismo , Epilepsia/induzido quimicamente , Epilepsia/tratamento farmacológico , Epilepsia/metabolismo , Antagonistas de Receptores de GABA-A , Cobaias , Proteína Antagonista do Receptor de Interleucina 1/farmacologia , Proteína Antagonista do Receptor de Interleucina 1/uso terapêutico , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/metabolismo , Recidiva , Convulsões/induzido quimicamente , Convulsões/tratamento farmacológico , Convulsões/metabolismoRESUMO
PURPOSE: Early seizures are a frequent consequence of stroke. The main goal of the present study is to verify whether anoxic ischemia per se is able to induce early changes in excitability that may be a prelude to the generation of seizures and, ultimately, to epileptogenesis. Excitability changes in the very acute postischemic phase are here analyzed in a new model of ischemia developed in the isolated guinea pig brain preparation. METHODS: Permanent bilateral occlusion of the anterior cerebral arteries (ACAs) was performed in the isolated guinea pig brain maintained in vitro by arterial perfusion. Magnetic resonance imaging and immunohistochemistry were utilized to identify the penumbra and core regions induced by ACA occlusion (ACAo). Slow potentials and evoked responses recorded in olfactory cortices were utilized to evaluate excitability changes in the acute phase after ischemia. KEY FINDINGS: ACAo induces a core area located in the shell of the nucleus accumbens and a region of penumbra in the underlying olfactory cortices, where characteristic slow potential shifts, but no reduction of diffusion tensor magnetic resonance (MR) signal and microtubule associated protein 2 (MAP-2) immunostaining (typical of ischemic core) was observed. Recording of responses evoked by low- and high-frequency stimulations of the lateral olfactory tract showed no excitability changes in the early hours that follow ischemia in the olfactory cortical areas supplied by ACAs. SIGNIFICANCE: The absence of early hyperexcitability changes in an isolated whole brain model of ischemia, strongly suggests that brain anoxia per se does not contribute to the generation of early seizures. These findings support the view that blood-borne events (such as hemorrhage and inflammation) may play a major role in early postischemic seizures.
Assuntos
Infarto Encefálico/fisiopatologia , Isquemia Encefálica/fisiopatologia , Encéfalo/fisiopatologia , Epilepsia/fisiopatologia , Doença Aguda , Animais , Encéfalo/patologia , Infarto Encefálico/complicações , Infarto Encefálico/patologia , Isquemia Encefálica/complicações , Isquemia Encefálica/patologia , Modelos Animais de Doenças , Epilepsia/etiologia , Epilepsia/patologia , Cobaias , Técnicas de Cultura de ÓrgãosRESUMO
Under physiological conditions, neuronal network synchronization leads to different oscillatory EEG patterns that are associated with specific behavioral and cognitive functions. Excessive synchronization can, however, lead to focal or generalized epileptiform activities. It is indeed well established that in both epileptic patients and animal models, focal epileptiform EEG patterns are characterized by interictal and ictal (seizure) discharges. Over the last three decades, employing in vitro and in vivo recording techniques, several experimental studies have firmly identified a paradoxical role of GABAA signaling in generating interictal discharges, and in initiating-and perhaps sustaining-focal seizures. Here, we will review these experiments and we will extend our appraisal to evidence suggesting that GABAA signaling may also contribute to epileptogenesis, i.e., the development of plastic changes in brain excitability that leads to the chronic epileptic condition. Overall, we anticipate that this information should provide the rationale for developing new specific pharmacological treatments for patients presenting with focal epileptic disorders such as mesial temporal lobe epilepsy (MTLE).
Assuntos
Epilepsias Parciais , Epilepsia do Lobo Temporal , Epilepsia , Animais , Convulsões , Ácido gama-Aminobutírico , EletroencefalografiaRESUMO
The blood-brain barrier (BBB) represents a major obstacle to the delivery of drugs to the central nervous system. The combined use of low-intensity pulsed ultrasound waves and intravascular microbubbles (MB) represents a promising solution to this issue, allowing reversible disruption of the barrier. In this study, we evaluate the feasibility of BBB opening through a biocompatible, polyolefin-based plate in an in vitro whole brain model. Twelve in vitro guinea pig brains were employed; brains were insonated using a planar transducer with or without interposing the polyolefin plate during arterial infusion of MB. Circulating MBs were visualized with an ultrasonographic device with a linear probe. BBB permeabilization was assessed by quantifying at confocal microscopy the extravasation of FITC-albumin perfused after each treatment. US-treated brains displayed BBB permeabilization exclusively in the volume under the US beam; no significant differences were observed between brains insonated with or without the polyolefin plate. Control brains not perfused with MB did not show signs of FITC-albumin extravasation. Our preclinical study suggests that polyolefin cranial plate could be implanted as a skull replacement to maintain craniotomic windows and perform post-surgical repeated BBB opening with ultrasound guidance to deliver therapeutic agents to the central nervous system.
Assuntos
Barreira Hematoencefálica/metabolismo , Sistemas de Liberação de Medicamentos , Polienos , Ondas Ultrassônicas , Animais , Materiais Biocompatíveis , Estudos de Viabilidade , Cobaias , Técnicas In Vitro , Microbolhas , Modelos Anatômicos , Permeabilidade , Crânio , Sonicação/métodosRESUMO
Hypothermia is a promising therapeutic strategy for severe vasospasm and other types of non-thrombotic cerebral ischemia, but its clinical application is limited by significant systemic side effects. We aimed to develop an intraventricular device for the controlled cooling of the cerebrospinal fluid, to produce a targeted hypothermia in the affected cerebral hemisphere with a minimal effect on systemic temperature. An intraventricular cooling device (acronym: V-COOL) was developed by in silico modelling, in vitro testing, and in vivo proof-of-concept application in healthy Wistar rats (n = 42). Cerebral cortical temperature, rectal temperature, and intracranial pressure were monitored at increasing flow rate (0.2 to 0.8 mL/min) and duration of application (10 to 60 min). Survival, neurological outcome, and MRI volumetric analysis of the ventricular system were assessed during the first 24 h. The V-COOL prototyping was designed to minimize extra-cranial heat transfer and intra-cranial pressure load. In vivo application of the V-COOL device produced a flow rate-dependent decrease in cerebral cortical temperature, without affecting systemic temperature. The target degree of cerebral cooling (- 3.0 °C) was obtained in 4.48 min at the flow rate of 0.4 mL/min, without significant changes in intracranial pressure. Survival and neurological outcome at 24 h showed no significant difference compared to sham-treated rats. MRI study showed a transient dilation of the ventricular system (+ 38%) in a subset of animals. The V-COOL technology provides an effective, rapid, selective, and safe cerebral cooling to a clinically relevant degree of - 3.0 °C.
Assuntos
Hipotermia Induzida , Hipotermia , Animais , Ratos , Temperatura Corporal , Ratos Wistar , Bioengenharia , EncéfaloRESUMO
Introduction: It has been proposed that seizures induce IL-1ß biosynthesis in astrocytes and increase blood brain barrier (BBB) permeability, even without the presence of blood borne inflammatory molecules and leukocytes. In the present study we investigate if seizures induce morphological changes typically observed in activated glial cells. Moreover, we will test if serum albumin extravasation into the brain parenchyma exacerbates neuronal hyperexcitability by inducing astrocytic and microglial activation. Methods: Epileptiform seizure-like events (SLEs) were induced in limbic regions by arterial perfusion of bicuculline methiodide (BMI; 50 µM) in the in vitro isolated guinea pig brain preparation. Field potentials were recorded in both the hippocampal CA1 region and the medial entorhinal cortex. BBB permeability changes were assessed by analyzing extravasation of arterially perfused fluorescein isothiocyanate (FITC)-albumin. Morphological changes in astrocytes and microglia were evaluated with tridimensional reconstruction and Sholl analysis in the ventral CA1 area of the hippocampus following application of BMI with or without co-perfusion of human serum albumin. Results: BMI-induced SLE promoted morphological changes of both astrocytes and microglia cells into an activated phenotype, confirmed by the quantification of the number and length of their processes. Human-recombinant albumin extravasation, due to SLE-induced BBB impairment, worsened both SLE duration and the activated glia phenotype. Discussion: Our study provides the first direct evidence that SLE activity per se is able to promote the activation of astro- and microglial cells, as observed by their changes in phenotype, in brain regions involved in seizure generation; we also hypothesize that gliosis, significantly intensified by h-recombinant albumin extravasation from the bloodstream to the brain parenchyma due to SLE-induced BBB disruption, is responsible for seizure activity reinforcement.
RESUMO
Magnetic resonance imaging (MRI) during the acute phase of a stroke contributes to recognize ischemic regions and is potentially useful to predict clinical outcome. Yet, the functional significance of early MRI alterations during brain ischemia is not clearly understood. We achieved an experimental study to interpret MRI signals in a novel model of focal ischemia in the in vitro isolated guinea pig brain. By combining neurophysiological and morphological analysis with MR-imaging, we evaluated the suitability of MR to identify ischemic and peri-ischemic regions. Extracellular recordings demonstrated depolarizations in the ischemic core, but not in adjacent areas, where evoked activity was preserved and brief peri-infarct depolarizations occurred. Diffusion-weighted MRI and immunostaining performed after neurophysiological characterization showed changes restricted to the core region. Diffusion-weighted MR alterations did not include the penumbra region characterized by peri-infarct depolarizations. Therefore, by comparing neurophysiological, imaging and anatomical data, we can conclude that DW-MRI underestimates the extension of the tissue damage involved in brain ischemia.
Assuntos
Isquemia Encefálica/etiologia , Isquemia Encefálica/patologia , Encéfalo/patologia , Encéfalo/fisiopatologia , Infarto da Artéria Cerebral Média/complicações , Animais , Modelos Animais de Doenças , Eletrofisiologia , Potenciais Evocados/fisiologia , Cobaias , Técnicas In Vitro , Imageamento por Ressonância Magnética/métodos , Proteínas Associadas aos Microtúbulos/metabolismo , Oxigênio/sangueRESUMO
Astrocyte dysfunction, and in particular impaired extracellular potassium spatial buffering, has been postulated to have a potential role in seizure susceptibility and ictogenesis. Inwardly rectifying potassium (Kir) channels, and specifically KIR4.1, have a predominant role in K+ homeostasis and their involvement in neuronal excitability control have been hypothesized. To avoid the severe side effects observed in Kir4.1 cKO, we studied the effects of Kir4.1 down-regulation in cortical astrocytes by using Kir4.1 RNA interference (RNAi) technique combined with in utero electroporation (IUE) at E16 and a piggyBac transposon system. Kir4.1 down-regulation was confirmed by immunohistochemistry and field fraction analysis. To investigate if Kir4.1 silencing affects 4AP-induced seizure threshold and extracellular potassium homeostasis, simultaneous in vitro field potential and extracellular K+ recordings were performed on somatosensory cortex slices obtained from rats electroporated with a piggyBac-Kir4.1-shRNA (Kir4.1-) and scrambled shRNA (Kir4.1Sc). Electrophysiological data revealed no significant differences in terms of seizure onset and seizure-induced extracellular K+ changes between Kir4.1- and Kir4.1Sc rats. Intriguingly, immunohistochemical analysis performed on slices studied with electrophysiology revealed a reduced number of neurons generated from radial glial cells in Kir4.1- rats. We conclude that focal down-regulation of Kir4.1 channel in cortical astrocytes by Kir4.1 RNAi technique combined with IUE is not effective in altering potassium homeostasis and seizure susceptibility. This technique revealed a possible role of Kir4.1 during corticogenesis.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização , Animais , Astrócitos/metabolismo , Eletroporação , Potássio/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Interferência de RNA , RatosRESUMO
BACKGROUND AND PURPOSE: Increased mortality after stroke is associated with brain edema formation and high plasma levels of the acute phase reactant C-reactive protein (CRP). The aim of this study was to examine whether CRP directly affects blood-brain barrier stability and to analyze the underlying signaling pathways. METHODS: We used a cell coculture model of the blood-brain barrier and the guinea pig isolated whole brain preparation. RESULTS: We could show that CRP at clinically relevant concentrations (10 to 20 microg/mL) causes a disruption of the blood-brain barrier in both approaches. The results of our study further demonstrate CRP-induced activation of surface Fcgamma receptors CD16/32 followed by p38-mitogen-activated protein kinase-dependent reactive oxygen species formation by the NAD(P)H-oxidase. The resulting oxidative stress increased myosin light chain kinase activity leading to an activation of the contractile machinery. Blocking myosin light chain phosphorylation prevented the CRP-induced blood-brain barrier breakdown and the disruption of tight junctions. CONCLUSIONS: Our data identify a previously unrecognized mechanism linking CRP and brain edema formation and present a signaling pathway that offers new sites of therapeutic intervention.
Assuntos
Barreira Hematoencefálica/metabolismo , Edema Encefálico/metabolismo , Proteína C-Reativa/metabolismo , Transdução de Sinais/fisiologia , Acidente Vascular Cerebral/metabolismo , Junções Íntimas/metabolismo , Animais , Células Cultivadas , Técnicas de Cocultura , Endotélio Vascular/metabolismo , Cobaias , Cadeias Leves de Miosina/metabolismo , Fosforilação/fisiologia , Ratos , Espécies Reativas de Oxigênio/metabolismo , Receptores de IgG/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
OBJECTIVE: Network mechanisms responsible for focal seizure initiation are still largely unknown. One of the prevalent seizure patterns observed during diagnostic intracranial recordings performed in patients with mesial temporal lobe epilepsy is characterized by fast activity at 20 to 30 Hz. We reproduced 20 to 30 Hz oscillations at seizure onset in the temporal lobe of the in vitro isolated guinea pig brain to study cellular and network mechanisms involved in its generation. METHODS: Seizure-like activity was induced in the isolated brain by 3-minute arterial perfusion of 50 microM bicuculline. Intracellular, extracellular, and ion-selective electrophysiological recordings were performed simultaneously in the entorhinal cortex (EC) during interictal-ictal transition. RESULTS: Principal neurons in deep and superficial layers of the EC did not generate action potentials during fast activity at ictal onset, whereas sustained firing was observed in putative interneurons. Within 5 to 10 seconds from seizure initiation, principal neurons generated a prominent firing that correlated with the appearance of extracellular hypersynchronous bursting discharges. In superficial neurons, fast activity correlated with rhythmic IPSPs that progressively decreased in amplitude during the development of a slow depolarization associated with an increase in extracellular potassium. INTERPRETATION: We conclude that in an acute model of temporal lobe ictogenesis, sustained inhibition without firing of EC principal neurons correlates with the onset of a focal seizure. The progression of the ictal discharge is contributed by a potassium-dependent change in reversal potential of inhibitory postsynaptic potentials. These findings demonstrate a prominent role of inhibitory networks during the transition to seizure in the EC.
Assuntos
Córtex Entorrinal/fisiologia , Potenciais Pós-Sinápticos Inibidores/fisiologia , Rede Nervosa/fisiologia , Inibição Neural/fisiologia , Convulsões/fisiopatologia , Potenciais de Ação/fisiologia , Animais , Cobaias , Técnicas In VitroRESUMO
BACKGROUND: Multielectrodes are implanted in central and peripheral nervous systems for rehabilitation and diagnostic purposes. The physical resistance of intracranial devices to mechanical stress is critical and fractures or electrode displacement may occur. We describe here a new recording device with stretchable properties based on Supersonic Cluster Beam Implantation (SCBI) technology with high mechanical adaptability to displacement and movement. RESULTS: The capability of SCBI-based multichannel electrodes to record brain electrical activity was compared to glass/silicon microelectrodes in acute in vitro experiments on the isolated guinea pig brain preparation. Field potentials and power frequency analysis demonstrated equal recording features for SCBI and standard electrodes. Chronic in vivo epidural implantation of the SCBI electrodes confirmed excellent long-term recording properties in comparison to standard EEG metal electrodes. Tissue biocompatibility was demonstrated by neuropathological evaluation of the brain tissue 2 months after the implantation of the devices in the subarachnoid space. CONCLUSION: We confirm the biocompatibility of novel SCBI-based stretchable electrode devices and demonstrate their suitability for recording electrical brain activity in pre-clinical settings.
Assuntos
Encéfalo/fisiologia , Eletrodos Implantados , Fenômenos Eletrofisiológicos , Nanotecnologia/métodos , Polímeros/química , Potenciais de Ação , Animais , Cobaias , MicroeletrodosRESUMO
Potassium channels dysfunction and altered genes encoding for molecules involved in potassium homeostasis have been associated with human epilepsy. These observations are in agreement with a control role of extracellular potassium on neuronal excitability and seizure generation. Epileptiform activity, in turn, regulates potassium homeostasis through mechanisms that are still not well established. We review here how potassium-associated processes are regulated in the brain and examine the mechanisms that support the role of potassium in triggering epileptiform activities.
Assuntos
Epilepsia/metabolismo , Potássio/metabolismo , Convulsões/metabolismo , Animais , Encéfalo/metabolismo , Epilepsia/genética , Humanos , Canais de Potássio/genética , Canais de Potássio/metabolismo , Convulsões/genéticaRESUMO
The cerebral vasculature is a complex tridimensional network of arterial and venous vessels which are anatomically in proximity of and functionally coupled to neurons. Depending on the cellular composition of the vascular wall and size, cerebral vessels control regional blood flow, define interstitial homeostasis or cerebrospinal fluid circulation and influence immune cell patrolling. Pathological deviations from these functions promote or are a consequence of brain diseases, directly impacting neuronal firing. We propose that specific cerebrovascular segments are differentially implicated in the pathophysiology of epilepsy, including difference between white and grey matter. We offer plasticity of perivascular mural cells and endothelial-pericyte interactions as emerging players. We outline the potential for MRI vascular biomarkers tailored to the epileptic brain, specifically cerebral blood volume and flow, tissue oxygen saturation and microvessel permeability. Finally, we show the advantages of the guinea pig whole brain preparation to study the link between cerebrovascular permeability, expression of vascular adhesion molecules, inflammation and neuronal excitability.
Assuntos
Encéfalo/irrigação sanguínea , Circulação Cerebrovascular/fisiologia , Epilepsia/fisiopatologia , Microvasos/metabolismo , Neurônios/metabolismo , Animais , Encéfalo/fisiopatologia , Humanos , Fluxo Sanguíneo Regional/fisiologiaRESUMO
Diagnosis and treatment of brain disorders, such as epilepsy, neurodegenerative diseases and tumors, would benefit from innovative approaches to deliver therapeutic or diagnostic compounds into the brain parenchyma, with either a homogeneous or a targeted localized distribution pattern. To assess the mechanistic aspect of penetration of nanoparticles (NPs) into the brain parenchyma, a complex, yet controlled and facilitated environment was used: the isolated guinea pig brain maintained in vitro by arterial perfusion. In this unique preparation the blood-brain barrier and the interactions between vascular and neuronal compartments are morphologically and functionally preserved. In this study, superparamagnetic Au/Fe nanoparticles (MUS:OT Au/Fe NPs), recently studied as a promising magnetic resonance T2 contrast agent with high cellular penetration, were arterially perfused into the in vitro isolated brain and showed high and homogeneous penetration through transcytosis into the brain parenchyma. Ultramicroscopy investigation of the in vitro isolated brain sections by TEM analysis of the electron-dense core of the MUS:OT Au/Fe NPs was conducted to understand NPs' brain penetration through the BBB after in vitro arterial perfusion and their distribution in the parenchyma. Our data suggest that MUS:OT Au/Fe NPs enter the brain utilizing a physiological route and therefore can be exploited as brain penetrating nanomaterials with potential contrast agent and theranostics capabilities.