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

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

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

Morphological and ultrastructural features of Iba1-immunolabeled microglial cells in the hippocampal dentate gyrus.

Microglia are found throughout the central nervous system, respond rapidly to pathology and are involved in several components of the neuroinflammatory response. Iba1 is a marker for microglial cells and previous immunocytochemical studies have utilized this and other microglial-specific antibodies to demonstrate the morphological features of microglial cells at the light microscopic level. However, there is a paucity of studies that have used microglial-specific antibodies to describe the ultrastructural features of microglial cells and their processes. The goal of the present study is to use Iba1 immuno-electron microscopy to elucidate the fine structural features of microglial cells and their processes in the hilar region of the dentate gyrus of adult Sprague-Dawley rats. Iba1-labeled cell bodies were observed adjacent to neurons and capillaries, as well as dispersed in the neuropil. The nuclei of these cells had dense heterochromatin next to the nuclear envelope and lighter chromatin in their center. Iba1-immunolabeling was found within the thin shell of perikaryal cytoplasm that contained the usual organelles, including mitochondria, cisternae of endoplasmic reticulum and Golgi complexes. Iba1-labeled cell bodies also commonly displayed an inclusion body. Iba1-labeled cell bodies gave rise to processes that often had a small side branch arise within 5 mum of the microglial cell body. These data showing "resting" Iba-1 labeled microglial cells in the normal adult rat dentate gyrus provide a basis for comparison with the morphology of microglial cells in disease and injury models where they are activated or phagocytotic

Astrocyte Alterations in the Hippocampus Following Pilocarpine-induced Seizures in Aged Rats

It is known that the incidence of epilepsy increases with age, but only a few studies have investigated the consequences and mechanisms of seizure and epilepsy in aged animals. Astrocytic changes are known to directly influence neuronal excitability and seizure susceptibility. However, information regarding alterations to astrocytes after seizures in aged animals is lacking in the literature. In the present study, the density and morphology of astrocytes expressing GFAP were investigated in the hippocampus of aged rats that experienced status epilepticus induced by pilocarpine. One month after seizures, astrocytes in aged rats have increased volume and present activated morphology. Despite these morphological changes, the density of astrocytes was not altered in the hippocampus of aged rats after seizures