Early TBI-Induced Cytokine Alterations are Similarly Detected by Two Distinct Methods of Multiplex Assay

Annually, more than a million persons experience traumatic brain injury (TBI) in the US and a substantial proportion of this population develop debilitating neurological disorders, such as, paralysis, cognitive deficits, and epilepsy. Despite the long-standing knowledge of the risks associated with TBI, no effective biomarkers or interventions exist. Recent evidence suggests a role for inflammatory modulators in TBI-induced neurological impairments. Current technological advances allow for the simultaneous analysis of the precise spatial and temporal expression patterns of numerous proteins in single samples which ultimately can lead to the development of novel treatments. Thus, the present study examined 23 different cytokines, including chemokines, in the ipsi and contralateral cerebral cortex of rats at 24 h after a fluid percussion injury (FPI). Furthermore, the estimation of cytokines were performed in a newly developed multiplex assay instrument, MAGPIX (Luminex Corp), and compared with an established instrument, Bio-Plex (Bio-Rad), in order to validate the newly developed instrument. The results show numerous inflammatory changes in the ipsi and contralateral side after FPI that were consistently reported by both technologies

Increased CCL2, CCL3, CCL5, and IL-1β cytokine concentration in piriform cortex, hippocampus, and neocortex after pilocarpine-induced seizures

BACKGROUND: Cytokines and chemokines play an important role in the neuroinflammatory response to an initial precipitating injury such as status epilepticus (SE). These signaling molecules participate in recruitment of immune cells, including brain macrophages (microglia), as well as neuroplastic changes, deterioration of damaged tissue, and epileptogenesis. This study describes the temporal and brain region pattern expression of numerous cytokines, including chemokines, after pilocarpine-induced seizures and discusses them in the larger context of their potential involvement in the changes that precede the development of epilepsy. FINDINGS: Adult rats received pilocarpine to induce SE and 90 min after seizure onset were treated with diazepam to mitigate seizures. Rats were subsequently deeply anesthetized and brain regions (hippocampus, piriform cortex, neocortex, and cerebellum) were freshly dissected at 2, 6, and 24 h or 5 days after seizures. Using methodology identical to our previous studies, simultaneous assay of multiple cytokines (CCL2, CCL3, CCL5, interleukin IL-1β, tumor necrosis factor (TNF-α)), and vascular endothelial growth factor (VEGF) was performed and compared to control rats. These proteins were selected based on existing evidence implicating them in the epileptogenic progression. A robust increase in CCL2 and CCL3 concentrations in the hippocampus, piriform cortex, and neocortex was observed at all time-points. The concentrations peaked with a ~200-fold increase 24 h after seizures and were two orders of magnitude greater than the significant increases observed for CCL5 and IL-1β in the same brain structures. TNF-α levels were altered in the piriform cortex and neocortex (24 h) and in the hippocampus (5 days) after SE. CONCLUSIONS: Pilocarpine-induced status epilepticus causes a rapid increase of multiple cytokines in limbic and neocortical regions. Understanding the precise spatial and temporal pattern of cytokines and chemokine changes could provide more viable therapeutic targets to reduce, reverse, or prevent the development of epilepsy following a precipitating injury

Transparent brain with CLARITY histology

A microscopia e as técnicas histológicas avançaram muito nossa compreensão da estrutura celular do tecido nervoso. No entanto, a complexa organização tridimensional dos neurônios, células gliais e da vasculatura é perdida no fatiamento do tecido nervoso para sua observação ao microscópio. Métodos elaborados e demorados são utilizados para reconstruir a organização tridimensional dessas células. A técnica histológica CLARITY preserva a estrutura das proteínas celulares “in situ” e torna o tecido nervoso transparente à luz, removendo a grande quantidade de lipídios presente nesse tecido, permitindo estudos envolvendo grandes volumes de diferentes regiões do cérebro. Esta histologia é feita por perfusão de monômeros que, após a polimerização criam um hidrogel híbrido da proteína com o monômero, permitindo a remoção eletroforética dos lipídios, tornando o tecido transparente à luz. Isso permite a visualização de grandes volumes do tecido em microscópios de fluorescência após marcação das proteínas de interesse por imunohistoquímica.Microscopy and histological techniques had greatly advanced our understanding of the fine cellular structure of nervous tissue. Nonetheless, the complex tridimensional organization of neurons, glial cells and vasculature is lost in sectioning the brain in order to analyze it under the microscope. Laborious and time consuming methods are employed to reconstruct the 3D organization of cells. Advanced histological techniques like CLARITY, that preserves the cellular protein structure in situ and renders the nervous tissue transparent to light by removing the cellular lipid layers, will allow studies encompassing large volumes of different brain regions. This histology is performed by perfusion of hydrogel monomers that after polymerization creates a protein-monomer hybrid that allows electrophoretic removal of the lipids. The transparent hydrogel can be scanned in a fluorescent microscope after protein immunolabeling

Chemokine CCL2 and its receptor CCR2 are increased in the hippocampus following pilocarpine-induced status epilepticus

<p>Abstract</p> <p>Background</p> <p>Neuroinflammation occurs after seizures and is implicated in epileptogenesis. CCR2 is a chemokine receptor for CCL2 and their interaction mediates monocyte infiltration in the neuroinflammatory cascade triggered in different brain pathologies. In this work CCR2 and CCL2 expression were examined following status epilepticus (SE) induced by pilocarpine injection.</p> <p>Methods</p> <p>SE was induced by pilocarpine injection. Control rats were injected with saline instead of pilocarpine. Five days after SE, CCR2 staining in neurons and glial cells was examined using imunohistochemical analyses. The number of CCR2 positive cells was determined using stereology probes in the hippocampus. CCL2 expression in the hippocampus was examined by molecular assay.</p> <p>Results</p> <p>Increased CCR2 was observed in the hippocampus after SE. Seizures also resulted in alterations to the cell types expressing CCR2. Increased numbers of neurons that expressed CCR2 was observed following SE. Microglial cells were more closely apposed to the CCR2-labeled cells in SE rats. In addition, rats that experienced SE exhibited CCR2-labeling in populations of hypertrophied astrocytes, especially in CA1 and dentate gyrus. These CCR2+ astroctytes were not observed in control rats. Examination of CCL2 expression showed that it was elevated in the hippocampus following SE.</p> <p>Conclusion</p> <p>The data show that CCR2 and CCL2 are up-regulated in the hippocampus after pilocarpine-induced SE. Seizures also result in changes to CCR2 receptor expression in neurons and astrocytes. These changes might be involved in detrimental neuroplasticity and neuroinflammatory changes that occur following seizures.</p

Morphological Alterations in Newly Born Dentate Gyrus Granule Cells That Emerge after Status Epilepticus Contribute to Make Them Less Excitable

Computer simulations of external current stimulations of dentate gyrus granule cells of rats with Status Epilepticus induced by pilocarpine and control rats were used to evaluate whether morphological differences alone between these cells have an impact on their electrophysiological behavior. The cell models were constructed using morphological information from tridimensional reconstructions with Neurolucida software. To evaluate the effect of morphology differences alone, ion channel conductances, densities and distributions over the dendritic trees of dentate gyrus granule cells were the same for all models. External simulated currents were injected in randomly chosen dendrites belonging to one of three different areas of dentate gyrus granule cell molecular layer: inner molecular layer, medial molecular layer and outer molecular layer. Somatic membrane potentials were recorded to determine firing frequencies and inter-spike intervals. The results show that morphologically altered granule cells from pilocarpine-induced epileptic rats are less excitable than control cells, especially when they are stimulated in the inner molecular layer, which is the target area for mossy fibers that sprout after pilocarpine-induced cell degeneration. This suggests that morphological alterations may act as a protective mechanism to allow dentate gyrus granule cells to cope with the increase of stimulation caused by mossy fiber sprouting.Conselho Nacional de Desenvolvimento Cientifico e Tecnologico National Counsel of Technological and Scientific Development (CNPq) BrazilConselho Nacional de Desenvolvimento Cientifico e Tecnologico - "National Counsel of Technological and Scientific Development" (CNPq) -Brazil [156597/2101-1]CNPq FellowshipCNPq FellowshipCNPqCNPqFundacao de Amparo a Pesquisa do Estado de Sao Paulo Foundation for Research Support of the State of Sao Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo - "Foundation for Research Support of the State of Sao Paulo" (FAPESP)Cooperacao Interinstitucional de Apoio a Pesquisas sobre o Cerebro da FAPESP Interinstitutional Cooperation in Support of Brain Research Program of FAPESP (FAPESPCinapce)Cooperacao Interinstitucional de Apoio a Pesquisas sobre o Cerebro da FAPESP "Inter-institutional Cooperation in Support of Brain Research Program of FAPESP" (FAPESP-Cinapce)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior Federal Agency of Support and Evaluation of Postgraduate Education (CAPES)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior - "Federal Agency of Support and Evaluation of Postgraduate Education" (CAPES)CAPES program of Academic Excellence (CAPES-PROEX), BrazilCAPES program of Academic Excellence (CAPESPROEX), Brazi

Increased CCL2, CCL3, CCL5, and IL-1β cytokine concentration in piriform cortex, hippocampus, and neocortex after pilocarpine-induced seizures

BACKGROUND: Cytokines and chemokines play an important role in the neuroinflammatory response to an initial precipitating injury such as status epilepticus (SE). These signaling molecules participate in recruitment of immune cells, including brain macrophages (microglia), as well as neuroplastic changes, deterioration of damaged tissue, and epileptogenesis. This study describes the temporal and brain region pattern expression of numerous cytokines, including chemokines, after pilocarpine-induced seizures and discusses them in the larger context of their potential involvement in the changes that precede the development of epilepsy. FINDINGS: Adult rats received pilocarpine to induce SE and 90 min after seizure onset were treated with diazepam to mitigate seizures. Rats were subsequently deeply anesthetized and brain regions (hippocampus, piriform cortex, neocortex, and cerebellum) were freshly dissected at 2, 6, and 24 h or 5 days after seizures. Using methodology identical to our previous studies, simultaneous assay of multiple cytokines (CCL2, CCL3, CCL5, interleukin IL-1β, tumor necrosis factor (TNF-α)), and vascular endothelial growth factor (VEGF) was performed and compared to control rats. These proteins were selected based on existing evidence implicating them in the epileptogenic progression. A robust increase in CCL2 and CCL3 concentrations in the hippocampus, piriform cortex, and neocortex was observed at all time-points. The concentrations peaked with a ~200-fold increase 24 h after seizures and were two orders of magnitude greater than the significant increases observed for CCL5 and IL-1β in the same brain structures. TNF-α levels were altered in the piriform cortex and neocortex (24 h) and in the hippocampus (5 days) after SE. CONCLUSIONS: Pilocarpine-induced status epilepticus causes a rapid increase of multiple cytokines in limbic and neocortical regions. Understanding the precise spatial and temporal pattern of cytokines and chemokine changes could provide more viable therapeutic targets to reduce, reverse, or prevent the development of epilepsy following a precipitating injury

Nervous and immune systems signals and connections: Cytokines in hippocampus physiology and pathology

Signaling through secretion of small molecules is a hallmark of both nervous and immune systems. the scope and influence of the intense message exchange between these two complex systems are only now becoming objects of scientific inquiry. Both neurotransmitters and cytokines affect their target cells through surface receptors and also by other molecular mechanisms. Cytokine receptors are present in neurons and glial cell populations in discrete brain regions. This review firstly focuses on the role of cytokines in hippocampal physiological processes, such as memory and learning, and secondly on the pathological involvement of cytokines in diseases like depression and epilepsy. Interleukin-1 beta is necessary for long-term potentiation (LTP) maintenance in the hippocampus. On the other hand, interleukin-6 has a negative regulatory role in long-term memory acquisition. Astrocyte-secreted tumor necrosis factor plays a role in synaptic strength by increasing surface translocation of glutamate AMPA receptors, and the chemokine CXCL12 can silence the tonic activity of Cajal-Retzius neurons in the hippocampus. Manifold increased concentrations of interleukin-10, interferon-gamma, ICAM1, CCL2, and CCL4 are observed in the hippocampi of patients with temporal lobe epilepsy. A contemporary view of the role of cytokines as neuromodulators is emerging from studies in humans and manipulations of experimental animals. Despite the accumulating evidence of the role of cytokines on nervous system physiology and pathology, it is important not to exaggerate its relevance. (C) 2014 Elsevier Inc. All rights reserved.Universidade Federal de São Paulo, Neurobiol Lab, Dept Physiol, Escola Paulista Med, São Paulo, BrazilUniversidade Federal de São Paulo, Neurobiol Lab, Dept Physiol, Escola Paulista Med, São Paulo, BrazilWeb of Scienc

The synaptic vesicle cycle: a molecular overview

A presente revisão aborda um ponto específico dentro da sinapse, provavelmente o mais crucial: as interações moleculares entre proteínas da membrana das vesículas sinápticas e da membrana plasmática pré-sináptica. Uma linguagem molecular muito precisa permite a fusão entre as membranas da vesícula sináptica e a plasmática, fusão que libera o neurotransmissor contido na vesícula para a fenda sináptica. A vesícula sináptica foi alvo, nos últimos anos, de uma verdadeira dissecção molecular. É a organela celular com a mais completa descrição estrutural e cinética de seus componentes protéicos. A descoberta de famílias de proteínas homólogas, presentes em todos os tipos celulares eucariotos, como a Rab e a SNARE (SNAP receptors), demonstrou que o ciclo da vesícula sináptica é uma interação entre sistemas protéicos, universais e específicos, de regulação do tráfego vesicular e de fusão de membranas lipídicas. O endereçamento e o controle do fluxo das estruturas precursoras das vesículas sinápticas até o terminal sináptico são realizados pela família Rab de pequenas GTPases. As proteínas da superfamília das kinesinas são as responsáveis pela ação mecânica no transporte anterógrado das estruturas precursoras, ao longo dos microtúbulos do citoesqueleto axonal. As proteínas SNARE realizam a fusão das vesículas com a membrana do terminal pré-sináptico. A proteína sinaptotagmina controla a formação do complexo SNARE em um modo dependente de cálcio. Embora já se tenha conhecimento da maior parte das proteínas envolvidas no ciclo da vesícula sináptica, tem-se ainda que elucidar muitas das funções e interrelações entre elas.The present review approaches a specific point in the synapse, probably the main one: the molecular interactions between the synaptic vesicle proteins and the presynaptic membrane proteins. A very precise language is talked between the vesicular and presynaptic membranes that allow the neurotransmitter release to the synaptic cleft. In recent years, the synaptic vesicle has been molecularly dissected, and it is probably the cell organelle with the most complete structural and kinetical description of its protein components. The discovery of families of homologue proteins in all eucariotic cells such as Rabs and SNAREs (SNAP receptors), demonstrated that the synaptic vesicle cycle depends on a refined interaction between a specific and universally distributed system of proteins that regulates the vesicle motion and fusion. The kinesin family is in charge of the mechanics for the anterograde transport of the raw material from the neuronal soma along microtubules in the axonal cytoskeleton. Sorting and flux control of the synaptic vesicle structural precursors is done by the Rab family of small GTPases. SNARE proteins make the fusion of the vesicles with the presynaptic terminal. Synaptotagmin regulates the formation of the SNARE complex in a calcium dependent mode. Although many of these proteins and their functions are well known, there is a large segment of knowledge on structure and interactions yet to be known