RESUMEN
Focal cortical dysplasia (FCD) represents a group of diverse localized cortical lesions that are highly epileptogenic and occur due to abnormal brain development caused by genetic mutations, involving the mammalian target of rapamycin (mTOR). These somatic mutations lead to mosaicism in the affected brain, posing challenges to unravel the direct and indirect functional consequences of these mutations. To comprehensively characterize the impact of mTOR mutations on the brain, we employed here a multimodal approach in a preclinical mouse model of FCD type II (Rheb), focusing on spatial omics techniques to define the proteomic and lipidomic changes. Mass Spectrometry Imaging (MSI) combined with fluorescence imaging and label free proteomics, revealed insight into the brain's lipidome and proteome within the FCD type II affected region in the mouse model. MSI visualized disrupted neuronal migration and differential lipid distribution including a reduction in sulfatides in the FCD type II-affected region, which play a role in brain myelination. MSI-guided laser capture microdissection (LMD) was conducted on FCD type II and control regions, followed by label free proteomics, revealing changes in myelination pathways by oligodendrocytes. Surgical resections of FCD type IIb and postmortem human cortex were analyzed by bulk transcriptomics to unravel the interplay between genetic mutations and molecular changes in FCD type II. Our comparative analysis of protein pathways and enriched Gene Ontology pathways related to myelination in the FCD type II-affected mouse model and human FCD type IIb transcriptomics highlights the animal model's translational value. This dual approach, including mouse model proteomics and human transcriptomics strengthens our understanding of the functional consequences arising from somatic mutations in FCD type II, as well as the identification of pathways that may be used as therapeutic strategies in the future.
Asunto(s)
Epilepsia , Malformaciones del Desarrollo Cortical de Grupo I , Proteómica , Animales , Humanos , Malformaciones del Desarrollo Cortical de Grupo I/genética , Malformaciones del Desarrollo Cortical de Grupo I/metabolismo , Malformaciones del Desarrollo Cortical de Grupo I/patología , Ratones , Masculino , Serina-Treonina Quinasas TOR/metabolismo , Serina-Treonina Quinasas TOR/genética , Femenino , Modelos Animales de Enfermedad , Encéfalo/metabolismo , Encéfalo/patología , Proteoma/metabolismo , Displasia Cortical FocalRESUMEN
Tuberous Sclerosis Complex (TSC) is a multisystem genetic disorder characterized by the development of benign tumors in various organs, including the brain, and is often accompanied by epilepsy, neurodevelopmental comorbidities including intellectual disability and autism. A key hallmark of TSC is the hyperactivation of the mechanistic target of rapamycin (mTOR) signaling pathway, which induces alterations in cortical development and metabolic processes in astrocytes, among other cellular functions. These changes could modulate seizure susceptibility, contributing to the progression of epilepsy and its associated comorbidities. Epilepsy is characterized by dysregulation of calcium (Ca2+) channels and intracellular Ca2+ dynamics. These factors contribute to hyperexcitability, disrupted synaptogenesis, and altered synchronization of neuronal networks, all of which contribute to seizure activity. This study investigates the intricate interplay between altered Ca2+ dynamics, mTOR pathway dysregulation, and cellular metabolism in astrocytes. The transcriptional profile of TSC patients revealed significant alterations in pathways associated with cellular respiration, ER and mitochondria, and Ca2+ regulation. TSC astrocytes exhibited lack of responsiveness to various stimuli, compromised oxygen consumption rate and reserve respiratory capacity underscoring their reduced capacity to react to environmental changes or cellular stress. Furthermore, our study revealed significant reduction of store operated calcium entry (SOCE) along with strong decrease of basal mitochondrial Ca2+ concentration and Ca2+ influx in TSC astrocytes. In addition, we observed alteration in mitochondrial membrane potential, characterized by increased depolarization in TSC astrocytes. Lastly, we provide initial evidence of structural abnormalities in mitochondria within TSC patient-derived astrocytes, suggesting a potential link between disrupted Ca2+ signaling and mitochondrial dysfunction. Our findings underscore the complexity of the relationship between Ca2+ signaling, mitochondria dynamics, apoptosis, and mTOR hyperactivation. Further exploration is required to shed light on the pathophysiology of TSC and on TSC associated neuropsychiatric disorders offering further potential avenues for therapeutic development.
Asunto(s)
Epilepsia , Esclerosis Tuberosa , Humanos , Astrocitos/patología , Señalización del Calcio , Esclerosis Tuberosa/patología , Calcio/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Epilepsia/genética , Homeostasis , ConvulsionesRESUMEN
OBJECTIVE: Status epilepticus (SE) models in rodents are commonly used to research mesial temporal lobe epilepsy (mTLE) in translational epilepsy research. However, due to differences in susceptibility of mice strains to chemoconvulsants, developing this model in mice is challenging. Mice offer experimental advantages; in particular, the ability to use transgenic strains could provide novel insights about neurobiological mechanisms or ease of genetic modification to test potential therapeutic targets. This study aimed to characterise the neuroinflammation, epileptic seizures and behavioural comorbidities after self-sustained Electrical Status Epilepticus (SSSE) in C57BL/6J mice. METHODS: SSSE was induced in C57BL/6J mice via prolonged electrical stimulation through a bipolar electrode implanted in the ventral hippocampus. Video electroencephalography (vEEG) monitoring was then performed between 1st month (acute timepoint) and 4th month (chronic timepoint). Brain tissues were collected at two timepoints for gene expression and immunohistochemical analysis: 7-days and 16-weeks post-SE. Additionally, at the chronic timepoint, animals underwent a series of neurobehavioural tests. RESULTS: Sixty percent of animals that underwent SSSE developed spontaneous seizures within the first month, and an additional 25% developed seizures at the chronic timepoint. The number of seizures per week during the chronic period ranged from 0.2 to 15.7. Mortality rate was ~9% during or after SSSE. SSSE animals displayed significant spatial memory impairment and depression-like behaviour compared to sham animals. mRNA expression of inflammatory cytokines was upregulated at 7-days following SE, but equal to sham levels at 16-weeks. SIGNIFICANCE: This study provides evidence that SSSE in C57BL/6J mice induces epileptic seizures consistent with those seen in patients with mTLE, along with cognitive and behavioural comorbidities. This model therefore has the potential to be used experimentally to uncover mechanisms to target against epileptogenesis, or to test novel treatment approaches.
Asunto(s)
Epilepsia del Lóbulo Temporal , Epilepsia , Estado Epiléptico , Animales , Modelos Animales de Enfermedad , Electroencefalografía , Epilepsia/tratamiento farmacológico , Epilepsia del Lóbulo Temporal/metabolismo , Hipocampo/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Enfermedades Neuroinflamatorias , Convulsiones , Estado Epiléptico/metabolismoRESUMEN
AIMS: Focal cortical dysplasia (FCD) type 2 is an epileptogenic malformation of the neocortex associated with somatic mutations in the mammalian target of rapamycin (mTOR) pathway. Histopathologically, FCD 2 is subdivided into FCD 2a and FCD 2b, the only discriminator being the presence of balloon cells (BCs) in FCD 2b. While pro-epileptogenic immune system activation and inflammatory responses are commonly detected in both subtypes, it is unknown what contextual role BCs play. METHODS: The present study employed RNA sequencing of surgically resected brain tissue from FCD 2a (n = 11) and FCD 2b (n = 20) patients compared to autopsy control (n = 9) focusing on three immune system processes: adaptive immunity, innate immunity and cytokine production. This analysis was followed by immunohistochemistry on a clinically well-characterised FCD 2 cohort. RESULTS: Differential expression analysis revealed stronger expression of components of innate immunity, adaptive immunity and cytokine production in FCD 2b than in FCD 2a, particularly complement activation and antigen presentation. Immunohistochemical analysis confirmed these findings, with strong expression of leukocyte antigen I and II in FCD 2b as compared to FCD 2a. Moreover, T-lymphocyte tissue infiltration was elevated in FCD 2b. Expression of markers of immune system activation in FCD 2b was concentrated in subcortical white matter. Lastly, antigen presentation was strongly correlated with BC load in FCD 2b lesions. CONCLUSION: We conclude that, next to mutation-driven mTOR activation and seizure activity, BCs are crucial drivers of inflammation in FCD 2b. Our findings indicate that therapies targeting inflammation may be beneficial in FCD 2b.
Asunto(s)
Epilepsia/patología , Sistema Inmunológico/metabolismo , Malformaciones del Desarrollo Cortical de Grupo I/patología , Malformaciones del Desarrollo Cortical/patología , Serina-Treonina Quinasas TOR/metabolismo , Adolescente , Niño , Epilepsia/genética , Epilepsia/inmunología , Humanos , Masculino , Malformaciones del Desarrollo Cortical/genética , Malformaciones del Desarrollo Cortical/inmunología , Malformaciones del Desarrollo Cortical de Grupo I/genética , Malformaciones del Desarrollo Cortical de Grupo I/inmunología , Persona de Mediana Edad , Mutación/genética , Neocórtex/patología , Neuronas/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/inmunología , Sustancia Blanca/metabolismoRESUMEN
Neuronal dysfunction due to iron accumulation in conjunction with reactive oxygen species (ROS) could represent an important, yet underappreciated, component of the epileptogenic process. However, to date, alterations in iron metabolism in the epileptogenic brain have not been addressed in detail. Iron-related neuropathology and antioxidant metabolic processes were investigated in resected brain tissue from patients with temporal lobe epilepsy and hippocampal sclerosis (TLE-HS), post-mortem brain tissue from patients who died after status epilepticus (SE) as well as brain tissue from the electrically induced SE rat model of TLE. Magnetic susceptibility of the presumed seizure-onset zone from three patients with focal epilepsy was compared during and after seizure activity. Finally, the cellular effects of iron overload were studied in vitro using an acute mouse hippocampal slice preparation and cultured human fetal astrocytes. While iron-accumulating neurons had a pyknotic morphology, astrocytes appeared to acquire iron-sequestrating capacity as indicated by prominent ferritin expression and iron retention in the hippocampus of patients with SE or TLE. Interictal to postictal comparison revealed increased magnetic susceptibility in the seizure-onset zone of epilepsy patients. Post-SE rats had consistently higher hippocampal iron levels during the acute and chronic phase (when spontaneous recurrent seizures are evident). In vitro, in acute slices that were exposed to iron, neurons readily took up iron, which was exacerbated by induced epileptiform activity. Human astrocyte cultures challenged with iron and ROS increased their antioxidant and iron-binding capacity, but simultaneously developed a pro-inflammatory phenotype upon chronic exposure. These data suggest that seizure-mediated, chronic neuronal iron uptake might play a role in neuronal dysfunction/loss in TLE-HS. On the other hand, astrocytes sequester iron, specifically in chronic epilepsy. This function might transform astrocytes into a highly resistant, pro-inflammatory phenotype potentially contributing to pro-epileptogenic inflammatory processes.
Asunto(s)
Epilepsia del Lóbulo Temporal/complicaciones , Hipocampo/metabolismo , Trastornos del Metabolismo del Hierro/etiología , Hierro/metabolismo , Estado Epiléptico/complicaciones , Adulto , Anciano , Anciano de 80 o más Años , Animales , Astrocitos/metabolismo , Astrocitos/patología , Estudios de Casos y Controles , Técnicas de Cultivo de Célula , Modelos Animales de Enfermedad , Epilepsia del Lóbulo Temporal/metabolismo , Epilepsia del Lóbulo Temporal/patología , Femenino , Humanos , Trastornos del Metabolismo del Hierro/patología , Masculino , Persona de Mediana Edad , Estrés Oxidativo/fisiología , Ratas , Estado Epiléptico/metabolismo , Estado Epiléptico/patologíaRESUMEN
Epilepsy is one of the most common neurological disorders and affects both the young and adult populations. The question we asked for this review was how positron emission tomography (PET) imaging with translocator protein (TSPO) radioligands can help inform the epilepsy clinic and the development of future treatments targeting neuroinflammatory processes.Even though the first TSPO PET scans in epilepsy patients were performed over 20 years ago, this imaging modality has not seen wide adoption in the clinic. There is vast scientific evidence from preclinical studies in rodent models of temporal lobe epilepsy which have shown increased levels of TSPO corresponding to neuroinflammatory processes in the brain. These increases peaked sub-acutely (1-2 weeks) after the epileptogenic insult (e.g. status epilepticus) and remained chronically increased, albeit at lower levels. In addition, these studies have shown a correlation between TSPO levels and seizure outcome, pharmacoresistance and behavioural morbidities. Histological assessment points to a complex interplay between different cellular components such as microglial activation, astrogliosis and cell death changing dynamically over time.In epilepsy patients, a highly sensitive biomarker of neuroinflammation would provide value for the optimization of surgical assessment (particularly for extratemporal lobe epilepsy) and support the clinical development path of anti-inflammatory treatments. Clinical studies have shown a systematic increase in asymmetry indices of TSPO PET binding. However, region-based analysis typically does not yield statistical differences and changes are often not restricted to the epileptogenic zone, limiting the ability of this imaging modality to localise pathology for surgery. In this manuscript, we discuss the biological underpinnings of these findings and review for which applications in epilepsy TSPO PET could bring added value.
Asunto(s)
Epilepsia del Lóbulo Temporal , Epilepsia , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Epilepsia/diagnóstico por imagen , Humanos , Tomografía de Emisión de Positrones , Receptores de GABA/metabolismoRESUMEN
Different types of brain injury, such as status epilepticus (SE), trauma, or stroke may initiate the process of epileptogenesis and lead to the development of temporal lobe epilepsy. Epileptogenesis is characterized by an initial latent period during which impaired network communication and synaptic circuit alterations are occurring. Ultimately, these modifications result in the development of spontaneous recurrent seizures (SRS). Current knowledge on the functional connectivity network changes during epileptogenesis and how network alterations relate to seizure is very limited. To investigate these underlying network connectivity modifications, we imaged epileptic and control rats by means of resting-state functional MRI (rsfMRI) during epileptogenesis. A cohort of animals was video-electroencephalography (video-EEG) monitored continuously over 12â¯weeks to determine disease severity during the course of disease, with the first SRS appearing around 2â¯weeks post-SE for most of the animals. Epileptic animals displayed a significant wide-spread hyposynchrony at 2â¯weeks post-SE, followed by a significant increase in network synchronicity from 2 to 4â¯weeks post-SE. Interestingly, subjects with a delayed epilepsy onset demonstrated significantly lower synchronicity compared to controls and the epileptic group at 4â¯weeks post-SE. Finally, network connectivity at 4â¯weeks post-SE was found to correlate with seizure onset (râ¯=â¯0.858, pâ¯<â¯.0001) and disease severity measured over 12â¯weeks (e.g. cingulate cortex: râ¯=â¯0.863, pâ¯=â¯.002), suggesting a possible network strengthening upon seizure reoccurrence. Our findings indicate that epileptogenesis is characterized by an initial hyposynchrony of brain networks and the disease-associated progression reflects disease severity.
Asunto(s)
Encéfalo/fisiopatología , Epilepsia del Lóbulo Temporal/fisiopatología , Red Nerviosa/fisiopatología , Estado Epiléptico/fisiopatología , Animales , Encéfalo/diagnóstico por imagen , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Electroencefalografía , Epilepsia del Lóbulo Temporal/diagnóstico por imagen , Imagen por Resonancia Magnética , Masculino , Red Nerviosa/diagnóstico por imagen , Neuroimagen , Ratas , Ratas Wistar , Índice de Severidad de la Enfermedad , Estado Epiléptico/diagnóstico por imagenRESUMEN
Neuroimaging techniques applied to a variety of organisms-from zebrafish, to rodents to humans-can offer valuable insights into neuronal network properties and their dysfunction in epilepsy. A wide range of imaging methods used to monitor neuronal circuits and networks during evoked seizures in animal models and advances in functional magnetic resonance imaging (fMRI) applied to patients with epilepsy were discussed during the XIV Workshop on Neurobiology of Epilepsy (XIV WONOEP) organized in 2017 by the Neurobiology Commission of the International League Against Epilepsy (ILAE). We review the growing number of technological approaches developed, as well as the current state of knowledge gained from studies applying these advanced imaging approaches to epilepsy research.
Asunto(s)
Encéfalo/diagnóstico por imagen , Epilepsia/diagnóstico por imagen , Neuroimagen Funcional , Animales , Epilepsia Refractaria/diagnóstico por imagen , Educación , Epilepsia del Lóbulo Temporal/diagnóstico por imagen , Neuroimagen Funcional/métodos , HumanosRESUMEN
Brain inflammation is an important factor in the conversion of a healthy brain into an epileptic one, a phenomenon known as epileptogenesis, offering a new entry point for prognostic tools. The development of anti-epileptogenic therapies to treat before or at disease onset is hampered by our inability to predict the severity of the disease outcome. In a rat model of temporal lobe epilepsy we aimed to assess whether in vivo non-invasive imaging of brain inflammation at disease onset was predictive of spontaneous recurrent seizures (SRS) frequency and severity of depression-like and sensorimotor-related comorbidities. To this end, translocator protein, a biomarker of inflammation, was imaged by means of positron emission tomography (PET) 2 and 4weeks post-status epilepticus using [18F]-PBR111. Translocator protein was highly upregulated 2weeks post-status epilepticus in limbic structures (up to 2.1-fold increase compared to controls in temporal lobe, P<0.001), whereas 4weeks post-status epilepticus, upregulation decreased (up to 1.6-fold increase compared to controls in temporal lobe, P<0.01) and was only apparent in a subset of these regions. Animals were monitored with video-electroencephalography during all stages of disease (acute, latent - first seizures appearing around 2weeks post-status epilepticus - and chronic phases), for a total of 12weeks, in order to determine SRS frequency for each subject (range 0.00-0.83SRS/day). We found that regional PET uptake at 2 and 4weeks post-status epilepticus correlated with the severity of depression-like and sensorimotor-related comorbidities during chronic epilepsy (P<0.05 for each test). Regional PET imaging did not correlate with SRS frequency, however, by applying a multivariate data-driven modeling approach based on translocator protein PET imaging at 2weeks post-status epilepticus, we accurately predicted the frequency of SRS (R=0.92; R2=0.86; P<0.0001) at the onset of epilepsy. This study not only demonstrates non-invasive imaging of translocator protein as a prognostic biomarker to ascertain SRS frequency, but also shows its capability to reflect the severity of depression-like and sensorimotor-related comorbidities. Our results are an encouraging step towards the development of anti-epileptogenic treatments by providing early quantitative assessment of SRS frequency and severity of comorbidities with high clinical relevance.
Asunto(s)
Encéfalo/diagnóstico por imagen , Encefalitis/diagnóstico por imagen , Convulsiones/diagnóstico por imagen , Estado Epiléptico/diagnóstico por imagen , Animales , Progresión de la Enfermedad , Electroencefalografía , Masculino , Tomografía de Emisión de Positrones , Valor Predictivo de las Pruebas , Ratas , Ratas Wistar , RecurrenciaRESUMEN
OBJECTIVES: Neuroinflammation plays a critical role in the pathophysiology of mesial temporal lobe epilepsy. We aimed to evaluate whether intracerebral transplantation of interleukin 13-producing mesenchymal stem cells (IL-13 MSCs) induces an M2 microglia/macrophage activation phenotype in the hippocampus with an epileptogenic insult, thereby providing a neuroprotective environment with reduced epileptogenesis. METHODS: Genetically engineered syngeneic IL-13 MSCs or vehicle was injected within the hippocampus 1 week before the intrahippocampal kainic acid-induced status epilepticus (SE) in C57BL/6J mice. Neuroinflammation was evaluated at disease onset as well as during the chronic epilepsy period (9 weeks). In addition, continuous video-electroencephalography (EEG) (vEEG) monitoring was obtained during the chronic epilepsy period (between 6 and 9 weeks after SE). RESULTS: Evaluation of vEEG recordings suggested that IL-13 MSC grafts did not affect the severity and duration of SE or the seizure burden during the chronic epilepsy period, when compared to the vehicle treated SE mice. An M2-activation phenotype was induced in microglia/macrophages that infiltrated the -13 MSC graft site, as evidenced by the arginase1 expression at the graft site at both the 2-week and 9-week time-points. However, M2-activated immune cells were rarely observed outside the graft site and, accordingly, the neuroinflammatory response or cell loss related to SE induction was not altered by IL-13 MSC grafting. Moreover, an increase in the proportion of F4/80+ cells was observed in the IL-13 MSC group compared to the controls. SIGNIFICANCE: Our data suggest that MSC-based IL-13 delivery to induce M2 glial activation does not provide any neuroprotective or disease-modifying effects in a mouse model of epilepsy. Moreover, use of cell grafting to deliver bioactive compounds for modulating neuroinflammation may have confounding effects in disease pathology of epilepsy due to the additional immune response generated by the grafted cells.
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Modelos Animales de Enfermedad , Epilepsia del Lóbulo Temporal/fisiopatología , Hipocampo/efectos de los fármacos , Hipocampo/fisiopatología , Interleucina-13/farmacología , Activación de Macrófagos , Trasplante de Células Madre Mesenquimatosas , Microglía/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Animales , Sistemas de Liberación de Medicamentos , Electrodos Implantados , Electroencefalografía/efectos de los fármacos , Ingeniería Genética , Inyecciones , Interleucina-13/genética , Interleucina-13/metabolismo , Masculino , Ratones Endogámicos C57BLRESUMEN
Epilepsy can be a devastating disorder. In addition to debilitating seizures, epilepsy can cause cognitive and emotional problems with reduced quality of life. Therefore, the major aim is to prevent the disorder in the first place: identify, detect, and reverse the processes responsible for its onset, and monitor and treat its progression. Epilepsy often occurs following a latent period of months to years (epileptogenesis) as a consequence of a brain insult, such as head trauma, stroke, or status epilepticus. Although this latent period clearly represents a therapeutic window, we are not able to stratify patients at risk for long-term epilepsy, which is prerequisite for preventative clinical trials. Moreover, because of the length of the latent period, an early biomarker for treatment response would be of high value. Finally, mechanistic biomarkers of epileptogenesis may provide more profound insight in the process of disease development.
Asunto(s)
Biomarcadores/análisis , Epilepsia/inmunología , Inflamación Neurogénica/inmunología , Animales , Astrocitos/fisiología , Encéfalo/diagnóstico por imagen , Encéfalo/inmunología , Encéfalo/fisiopatología , Lesiones Encefálicas/complicaciones , Lesiones Encefálicas/diagnóstico por imagen , Lesiones Encefálicas/inmunología , Lesiones Encefálicas/fisiopatología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Electroencefalografía , Epilepsia/diagnóstico por imagen , Epilepsia/fisiopatología , Epilepsia/prevención & control , Humanos , Aumento de la Imagen , Imagen por Resonancia Magnética , Inflamación Neurogénica/diagnóstico por imagen , Inflamación Neurogénica/fisiopatología , Inflamación Neurogénica/prevención & control , Espectroscopía de Protones por Resonancia Magnética , Ratas , Factores de Riesgo , Convulsiones Febriles/diagnóstico por imagen , Convulsiones Febriles/fisiopatología , Estado Epiléptico/complicaciones , Estado Epiléptico/diagnóstico por imagen , Estado Epiléptico/inmunología , Estado Epiléptico/fisiopatología , Accidente Cerebrovascular/complicaciones , Accidente Cerebrovascular/diagnóstico por imagen , Accidente Cerebrovascular/inmunología , Accidente Cerebrovascular/fisiopatología , Molécula 1 de Adhesión Celular Vascular/análisis , Grabación en VideoRESUMEN
OBJECTIVE: Urokinase-type plasminogen activator (uPA) and kallikrein-related peptidase 8 (KLK8) are serine proteases that contribute to extracellular matrix (ECM) remodeling after brain injury. They can be labelled with the novel radiotracer [111 In]MICA-401. As the first step in exploring the applicability of [111 In]MICA-401 in tracing the mechanisms of postinjury ECM reorganization in vivo, we performed in vitro and ex vivo studies, assessing [111 In]MICA-401 distribution in the brain in two animal models: kainic acid-induced status epilepticus (KASE) and controlled cortical impact (CCI)-induced traumatic brain injury (TBI). METHODS: In the KASE model, in vitro autoradiography with [111 In]MICA-401 was performed at 7 days and 12 weeks post-SE. To assess seizure burden, rats were monitored using video-electroencephalography (EEG) for 1 month before the 12-week time point. In the CCI model, in vitro autoradiography was performed at 4 days and ex vivo autoradiography at 7 days post-TBI. RESULTS: At 7 days post-SE, in vitro autoradiography revealed significantly decreased [111 In]MICA-401 binding in hippocampal CA3 subfield and extrahippocampal temporal lobe (ETL). In the chronic phase, when animals had developed spontaneous seizures, specific binding was decreased in CA3 and CA1/CA2 subfields of hippocampus, dentate gyrus, ETL, and parietal cortex. Of interest, KASE rats with the highest frequency of seizures had the lowest hippocampal [111 In]MICA-401 binding (r = -0.76, p ≤ 0.05). Similarly, at 4 days post-TBI, in vitro [111 In]MICA-401 binding was significantly decreased in medial and lateral perilesional cortex and ipsilateral dentate gyrus. Ex vivo autoradiography at 7 days post-TBI, however, revealed increased tracer uptake in perilesional cortex and hippocampus, which was likely related to tracer leakage due to blood-brain barrier (BBB) disruption. SIGNIFICANCE: Strong association of reduced [111 In]MICA-401 binding with seizure burden in the KASE model suggests that analysis of reduced levels of active uPA/KLK8 represents a novel biomarker candidate to be explored as a biomarker for epilepsy severity. However, limited BBB permeability of [111 In]MICA-401 currently limits its application in vivo.
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Epilepsia del Lóbulo Temporal/metabolismo , Calicreínas/metabolismo , Convulsiones/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Animales , Modelos Animales de Enfermedad , Electroencefalografía , Masculino , Ratas , Ratas Sprague-Dawley , Estado Epiléptico/metabolismo , Grabación en VideoRESUMEN
Neuroimaging offers a wide range of opportunities to obtain information about neuronal activity, brain inflammation, blood-brain barrier alterations, and various molecular alterations during epileptogenesis or for the prediction of pharmacoresponsiveness as well as postoperative outcome. Imaging biomarkers were examined during the XIII Workshop on Neurobiology of Epilepsy (XIII WONOEP) organized in 2015 by the Neurobiology Commission of the International League Against Epilepsy (ILAE). Here we present an extended summary of the discussed issues and provide an overview of the current state of knowledge regarding the biomarker potential of different neuroimaging approaches for epilepsy.
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Biomarcadores , Epilepsia , Neuroimagen , Biomarcadores/metabolismo , Barrera Hematoencefálica/fisiopatología , Educación , Epilepsia/diagnóstico , Epilepsia/metabolismo , Epilepsia/terapia , Humanos , NeurobiologíaRESUMEN
MMP-9 is a zinc-dependent endopeptidase that is involved in the proteolytic degradation of the extracellular matrix and plays an important role in cancer migration, invasion, and metastasis. The aim of this study was to evaluate the potential of MMP-tracers [18 F]BR420 and [18 F]BR351 for MMP-9 imaging in a colorectal cancer xenograft model. [18 F]BR420 and [18 F]BR351 were synthesized using an automated synthesis module. For [18 F]BR420, a novel and improved radiosynthesis was developed. Plasma stability and MMP-9-targeting capacity of both radiotracers was compared in the Colo205 colorectal cancer model. MMP-9 and MMP-2 expression levels in the tumors were evaluated by immunohistochemistry and in situ zymography. µPET imaging as well as ex vivo biodistribution revealed a higher tumor uptake for [18 F]BR420 (3.15% ± 0.03% ID/g vs 0.94% ± 0.18% ID/g for [18 F]BR351 at 2 hours pi) but slower blood clearance compared with [18 F]BR351. [18 F]BR351 was quickly metabolized in plasma with 20.28% ± 5.41% of intact tracer remaining at 15 minutes postinjection (PI). By contrast, [18 F]BR420 displayed a higher metabolic stability with >86% intact tracer remaining at 2 hours PI. Immunohistochemistry revealed the presence of MMP-9 and MMP-2 in the tumor tissue, which was confirmed by in situ zymography. However, an autoradiography analysis of tracer distribution in the tumors did not correlate with MMP-9 expression. [18 F]BR420 displayed a higher tumor uptake and higher stability compared with [18 F]BR351 but a low tumor-to-blood ratio and discrepancy between tracer distribution and MMP-9 immunohistochemistry. Therefore, both tracers will not be usefulness for MMP-9 imaging in colorectal cancer.
Asunto(s)
Neoplasias Colorrectales/diagnóstico por imagen , Metaloproteinasa 9 de la Matriz/metabolismo , Pirimidinonas/síntesis química , Radiofármacos/farmacocinética , Sulfonamidas/síntesis química , Valina/análogos & derivados , Animales , Línea Celular Tumoral , Femenino , Radioisótopos de Flúor/química , Humanos , Tasa de Depuración Metabólica , Ratones , Ratones Desnudos , Tomografía Computarizada por Tomografía de Emisión de Positrones , Pirimidinonas/farmacocinética , Radiofármacos/efectos adversos , Radiofármacos/síntesis química , Sulfonamidas/farmacocinética , Distribución Tisular , Valina/síntesis química , Valina/farmacocinéticaRESUMEN
OBJECTIVE: The neurobiological factors underlying a predisposition towards developing epilepsy and its common behavioral comorbidities are poorly understood. FAST rats are a strain that has been selectively bred for enhanced vulnerability to kindling, while the SLOW strain has been bred to be resistant to kindling. FAST rats also exhibit behavioral traits reminiscent of those observed in neurodevelopmental disorders (autism spectrum disorder (ASD)/attention-deficit/hyperactivity disorder (ADHD)) commonly comorbid with epilepsy. In this study, we aimed to investigate neuroanatomical differences between these strains that may be associated with a differential vulnerability towards these interrelated disorders. METHODS: Ex vivo high-resolution magnetic resonance imaging on adult male FAST and SLOW rat brains was performed to identify morphological differences in regions of interest between the two strains. Behavioral examination using open-field, water consumption, and restraint tests was also conducted on a subgroup of these rats to document their differential ASD/ADHD-like behavior phenotype. Using optical stereological methods, the volume of cerebellar granule, white matter, and molecular layer and number of Purkinje cells were compared in a separate cohort of adult FAST and SLOW rats. RESULTS: Behavioral testing demonstrated hyperactivity, impulsivity, and polydipsia in FAST versus SLOW rats, consistent with an ASD/ADHD-like phenotype. Magnetic resonance imaging analysis identified brain structural differences in FAST compared with SLOW rats, including increased volume of the cerebrum, corpus callosum, third ventricle, and posterior inferior cerebellum, while decreased volume of the anterior cerebellar vermis. Stereological measurements on histological slices indicated significantly larger white matter layer volume, reduced number of Purkinje cells, and smaller molecular layer volume in the cerebellum in FAST versus SLOW rats. SIGNIFICANCE: These findings provide evidence of structural differences between the brains of FAST and SLOW rats that may be mechanistically related to their differential vulnerability to kindling and associated comorbid ASD/ADHD-like behaviors.
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Encéfalo/anatomía & histología , Encéfalo/fisiología , Ingestión de Líquidos/genética , Conducta Impulsiva/fisiología , Excitación Neurológica/patología , Animales , Trastorno por Déficit de Atención con Hiperactividad/diagnóstico por imagen , Trastorno por Déficit de Atención con Hiperactividad/genética , Trastorno por Déficit de Atención con Hiperactividad/psicología , Encéfalo/patología , Epilepsia/diagnóstico por imagen , Epilepsia/genética , Epilepsia/psicología , Masculino , Memoria/fisiología , Ratas , Ratas Wistar , Especificidad de la EspecieRESUMEN
AIMS: A hallmark in the neuropathology of temporal lobe epilepsy is brain inflammation which has been suggested as both a biomarker and a new mechanistic target for treatments. The translocator protein (TSPO), due to its high upregulation under neuroinflammatory conditions and the availability of selective PET tracers, is a candidate target. An important step to exploit this target is a thorough characterisation of the spatiotemporal profile of TSPO during epileptogenesis. METHODS: TSPO expression, microglial activation, astrocyte reactivity and cell loss in several brain regions were evaluated at five time points during epileptogenesis, including the chronic epilepsy phase in the kainic acid-induced status epilepticus (KASE) model (n = 52) and control Wistar Han rats (n = 33). Seizure burden was also determined in the chronic phase. Furthermore, ¹8F-PBR111 PET/MRI scans were acquired longitudinally in an additional four KASE animals. RESULTS: TSPO expression measured with in vitro and in vivo techniques was significantly increased at each time point and peaked two weeks post-SE in the limbic system. A prominent association between TSPO expression and activated microglia (p < 0.001; r = 0.7), as well as cell loss (p < 0.001; r = -0.8) could be demonstrated. There was a significant positive correlation between spontaneous seizures and TSPO upregulation in several brain regions with increased TSPO expression. CONCLUSIONS: TSPO expression was dynamically upregulated during epileptogenesis, persisted in the chronic phase and correlated with microglia activation rather than reactive astrocytes. TSPO expression was correlating with spontaneous seizures and its high expression during the latent phase might possibly suggest being an important switching point in disease ontogenesis which could be further investigated by PET imaging.
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Encéfalo/inmunología , Proteínas Portadoras/metabolismo , Encefalitis/metabolismo , Epilepsia/inmunología , Receptores de GABA-A/metabolismo , Animales , Autorradiografía , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Enfermedad Crónica , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Electrocorticografía , Encefalitis/diagnóstico por imagen , Encefalitis/patología , Epilepsia/diagnóstico por imagen , Epilepsia/patología , Estudios de Seguimiento , Inmunohistoquímica , Ácido Kaínico , Estudios Longitudinales , Imagen por Resonancia Magnética , Masculino , Degeneración Nerviosa/diagnóstico por imagen , Degeneración Nerviosa/inmunología , Degeneración Nerviosa/patología , Neuroglía/diagnóstico por imagen , Neuroglía/inmunología , Neuroglía/patología , Neuronas/diagnóstico por imagen , Neuronas/inmunología , Neuronas/patología , Tomografía de Emisión de Positrones , Ratas WistarRESUMEN
The standard uptake value (SUV), commonly used to quantify (18)F-FluoroDeoxyGlucose (FDG) uptake in small animal brain PET imaging, is affected by many factors. In this study the influence of fasting times, inter-scan duration and repetitive scanning on the variability of different SUV measures is investigated. Additionally it is demonstrated that these variables could adversely influence the outcome of a pharmacological challenge when not accounted for. Naive Sprague-Dawley rats (n=20) were randomly divided into five different fasting groups (no fasting up to 24h of fasting). SUV brain uptake values were reproducible in naive animals when a fasting period of at least 12h is used and for shorter fasting periods SUV values need to be corrected for the glucose level. Additionally, a separate animal group (n=6) was sufficiently fasted for 16h and in a longitudinal setting being scanned six times in three weeks. Especially with short inter-scan durations, increasing glucose levels were found over time which was attributed to increased stress due to repeated food deprivation, altered food intake or scan manipulations. As a result, even with controlled and sufficient fasting, blood glucose levels should be taken into account for data quantification. Strikingly, even the brain activation effects of an NMDA-antagonist challenge with memantine could not be detected in experiments with a short inter-scan duration if glucose levels were not taken into account. Correcting for glucose levels decreases the inter- and intra-animal variability for rat brain imaging. SUV corrected for glucose levels yields the lowest inter-animal variation. However, if the body weight changes significantly, as in a long experiment, quantification based on the glucose corrected percentage injected dose (and not SUV) is recommendable as this yields the lowest intra-animal variation.
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Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Ayuno/fisiología , Fluorodesoxiglucosa F18/farmacocinética , Memantina/farmacología , Tomografía de Emisión de Positrones/métodos , Animales , Encéfalo/efectos de los fármacos , Simulación por Computador , Aumento de la Imagen/métodos , Masculino , Tasa de Depuración Metabólica/efectos de los fármacos , Modelos Biológicos , Radiofármacos , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Modern functional neuroimaging provides opportunities to visualize activity of the entire brain, making it an indispensable diagnostic tool for epilepsy. Various forms of noninvasive functional neuroimaging are now also being performed as research tools in animal models of epilepsy and provide opportunities for parallel animal/human investigations into fundamental mechanisms of epilepsy and identification of epilepsy biomarkers. Recent animal studies of epilepsy using positron emission tomography, tractography, and functional magnetic resonance imaging were reviewed. Epilepsy is an abnormal emergent property of disturbances in neuronal networks which, even for epilepsies characterized by focal seizures, involve widely distributed systems, often in both hemispheres. Functional neuroimaging in animal models now provides opportunities to examine neuronal disturbances in the whole brain that underlie generalized and focal seizure generation as well as various types of epileptogenesis. Tremendous advances in understanding the contribution of specific properties of widely distributed neuronal networks to both normal and abnormal human behavior have been provided by current functional neuroimaging methodologies. Successful application of functional neuroimaging of the whole brain in the animal laboratory now permits investigations during epileptogenesis and correlation with deep brain electroencephalography (EEG) activity. With the continuing development of these techniques and analytical methods, the potential for future translational research on epilepsy is enormous. A PowerPoint slide summarizing this article is available for download in the Supporting Information section here.
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Encéfalo/patología , Epilepsia/patología , Neuroimagen/métodos , Animales , Imagen de Difusión por Resonancia Magnética , Modelos Animales de Enfermedad , Educación , Epilepsias Parciales/patología , Epilepsia Generalizada/patología , Fluorodesoxiglucosa F18 , Humanos , Imagen por Resonancia Magnética , Ratones , Tomografía de Emisión de Positrones/métodos , RatasRESUMEN
Structural epilepsies display complex immune activation signatures. However, it is unclear which neuroinflammatory pathways drive pathobiology. Transcriptome studies of brain resections from mesial temporal lobe epilepsy (mTLE) patients revealed a dysregulation of transforming growth factor ß, interferon α/ß, and nuclear factor erythroid 2-related factor 2 pathways. Since these pathways are regulated by ubiquitin-specific proteases (USP), in particular USP15, we hypothesized that USP15 blockade may provide therapeutic relief in treatment-resistant epilepsies. For validation, transgenic mice which either constitutively or inducibly lack Usp15 gene expression underwent intrahippocampal kainate injections to induce mTLE. We show that the severity of status epilepticus is unaltered in mice constitutively lacking Usp15 compared to wild types. Cell death, reactive gliosis, and changes in the inflammatory transcriptome were pronounced at 4 days after kainate injection. However, these brain inflammation signatures did not differ between genotypes. Likewise, induced deletion of Usp15 in chronic epilepsy did not affect seizure generation, cell death, gliosis, or the transcriptome. Concordantly, siRNA-mediated knockdown of Usp15 in a microglial cell line did not impact inflammatory responses in the form of cytokine release. Our data show that a lack of USP15 is insufficient to modulate the expression of relevant neuroinflammatory pathways in an mTLE mouse model and do not support targeting USP15 as a therapeutic approach for pharmacoresistant epilepsy.
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Epilepsia del Lóbulo Temporal , Animales , Humanos , Ratones , Regulación hacia Abajo , Gliosis , Hipocampo/metabolismo , Ácido Kaínico , Ratones Transgénicos , Proteasas Ubiquitina-Específicas/metabolismoRESUMEN
We have previously characterized the molecular mechanisms for variants in γ-aminobutyric acid transporter 1-encoding solute carrier family 6-member 1 (SLC6A1) in vitro and concluded that a partial or complete loss of γ-aminobutyric acid uptake due to impaired protein trafficking is the primary aetiology. Impairment of γ-aminobutyric acid transporter 1 function could cause compensatory changes in the expression of γ-aminobutyric acid receptors, which, in turn, modify disease pathophysiology and phenotype. Here we used different approaches including radioactive 3H γ-aminobutyric acid uptake in cells and synaptosomes, immunohistochemistry and confocal microscopy as well as brain slice surface protein biotinylation to characterize Slc6a1+/A288V and Slc6a1+/S295L mice, representative of a partial or a complete loss of function of SLC6A1 mutations, respectively. We employed the γ-aminobutyric acid transporter 1-specific inhibitor [3H]tiagabine binding and GABAA receptor subunit-specific radioligand binding to profile the γ-aminobutyric acid transporter 1 and GABAA receptor expression in major brain regions such as cortex, cerebellum, hippocampus and thalamus. We also determined the total and surface expression of γ-aminobutyric acid transporter 1, γ-aminobutyric acid transporter 3 and expression of GABAA receptor in the major brain regions in the knockin mice. We found that γ-aminobutyric acid transporter 1 protein was markedly reduced in cortex, hippocampus, thalamus and cerebellum in both mutant mouse lines. Consistent with the findings of reduced γ-aminobutyric acid uptake for both γ-aminobutyric acid transporter 1(A288V) and γ-aminobutyric acid transporter 1(S295L), both the total and the γ-aminobutyric acid transporter 1-mediated 3H γ-aminobutyric acid reuptake was reduced. We found that γ-aminobutyric acid transporter 3 is only abundantly expressed in the thalamus and there was no compensatory increase of γ-aminobutyric acid transporter 3 in either of the mutant mouse lines. γ-Aminobutyric acid transporter 1 was reduced in both somatic regions and nonsomatic regions in both mouse models, in which a ring-like structure was identified only in the Slc6a1+/A288V mouse, suggesting more γ-aminobutyric acid transporter 1 retention inside endoplasmic reticulum in the Slc6a1+/A288V mouse. The [3H]tiagabine binding was similar in both mouse models despite the difference in γ-aminobutyric acid uptake function and γ-aminobutyric acid transporter 1 protein expression for both mutations. There were no differences in GABAA receptor subtype expression, except for a small increase in the expression of α5 subunits of GABAA receptor in the hippocampus of Slc6a1S295L homozygous mice, suggesting a potential interaction between the expression of this GABAA receptor subtype and the mutant γ-aminobutyric acid transporter 1. The study provides the first comprehensive characterization of the SLC6A1 mutations in vivo in two representative mouse models. Because both γ-aminobutyric acid transporter 1 and GABAA receptors are targets for anti-seizure medications, the findings from this study can help guide tailored treatment options based on the expression and function of γ-aminobutyric acid transporter 1 and GABAA receptor in SLC6A1 mutation-mediated neurodevelopmental and epileptic encephalopathies.