Oligodendrogenesis from neural stem cells: Perspectives for remyelinating strategies

Mobilization of remyelinating cells spontaneously occurs in the adult brain. These cellular resources are specially active after demyelinating episodes in early phases of multiple sclerosis (MS). Indeed, oligodendrocyte precursor cells (OPCs) actively proliferate, migrate to and repopulate the lesioned areas. Ultimately, efficient remyelination is accomplished when new oligodendrocytes reinvest nude neuronal axons, restoring the normal properties of impulse conduction. As the disease progresses this fundamental process fails. Multiple causes seem to contribute to such transient decline, including the failure of OPCs to differentiate and enwrap the vulnerable neuronal axons. Regenerative medicine for MS has been mainly centered on the recruitment of endogenous self-repair mechanisms, or on transplantation approaches. The latter commonly involves grafting of neural precursor cells (NPCs) or neural stem cells (NSCs), with myelinogenic potential, in the injured areas. Both strategies require further understanding of the biology of oligodendrocyte differentiation and remyelination. Indeed, the success of transplantation largely depends on the pre-commitment of transplanted NPCs or NSCs into oligodendroglial cell type, while the endogenous differentiation of OPCs needs to be boosted in chronic stages of the disease. Thus, much effort has been focused on finding molecular targets that drive oligodendrocytes commitment and development. The present review explores several aspects of remyelination that must be considered in the design of a cell-based therapy for MS, and explores more deeply the challenge of fostering oligodendrogenesis. In this regard, we discuss herein a tool developed in our research group useful to search novel oligodendrogenic factors and to study oligodendrocyte differentiation in a time- and cost-saving manner

Argonaute-2 protects the neurovascular unit from damage caused by systemic inflammation

Funding PPBI—Portuguese Platform of BioImaging, POCI-01-0145-FEDER-022122; FCT—Fundação para a Ciência e Tecnologia, UID/Multi/00709/2013, SFRH/ BD/137440/2018 (MMP), IF/00178/2015/CP1300/CT0001 (RF); FCT—L’Oréal— UNESCO Portugal for Women in Science (RF).Background: The brain vasculature plays a pivotal role in the inflammatory process by modulating the interaction between blood cells and the neurovascular unit. Argonaute-2 (Ago2) has been suggested as essential for endothelial survival but its role in the brain vasculature or in the endothelial–glial crosstalk has not been addressed. Thus, our aim was to clarify the significance of Ago2 in the inflammatory responses elicited by these cell types. Methods: Mouse primary cultures of brain endothelial cells, astrocytes and microglia were used to evaluate cellular responses to the modulation of Ago2. Exposure of microglia to endothelial cell-conditioned media was used to assess the potential for in vivo studies. Adult mice were injected intraperitoneally with lipopolysaccharide (LPS) (2 mg/kg) followed by three daily intraperitoneal injections of Ago2 (0.4 nM) to assess markers of endothelial disruption, glial reactivity and neuronal function. Results: Herein, we demonstrated that LPS activation disturbed the integrity of adherens junctions and downregulated Ago2 in primary brain endothelial cells. Exogenous treatment recovered intracellular Ago2 above control levels and recuperated vascular endothelial-cadherin expression, while downregulating LPS-induced nitric oxide release. Primary astrocytes did not show a significant change in Ago2 levels or response to the modulation of the Ago2 system, although endogenous Ago2 was shown to be critical in the maintenance of tumor necrosis factor-α basal levels. LPS-activated primary microglia overexpressed Ago2, and Ago2 silencing contained the inflammatory response to some extent, preventing interleukin-6 and nitric oxide release. Moreover, the secretome of Ago2-modulated brain endothelial cells had a protective effect over microglia. The intraperitoneal injection of LPS impaired blood–brain barrier and neuronal function, while triggering inflammation, and the subsequent systemic administration of Ago2 reduced or normalized endothelial, glial and neuronal markers of LPS damage. This outcome likely resulted from the direct action of Ago2 over the brain endothelium, which reestablished glial and neuronal function. Conclusions: Ago2 could be regarded as a putative therapeutic agent, or target, in the recuperation of the neurovascular unit in inflammatory conditions.publishersversionpublishe

Tumor necrosis factor-alpha modulates survival, proliferation, and neuronal differentiation in neonatal subventricular zone cell cultures

Tumor necrosis factor (TNF)- has been reported to modulate brain injury, but remarkably, little is known about its effects on neurogenesis. We report that TNF- strongly influences survival, proliferation, and neuronal differentiation in cultured subventricular zone (SVZ) neural stem/progenitor cells derived from the neonatal P1-3 C57BL/6 mice. By using single-cell calcium imaging, we developed a method, based on cellular response to KCl and/or histamine, that allows the functional evaluation of neuronal differentiation. Exposure of SVZ cultures to 1 and 10 ng/ml mouse or 1 ng/ml human recombinant TNF- resulted in increased differentiation of cells displaying a neuronal-like profile of [Ca2+]i responses, compared with the predominant profile of immature cells observed in control, nontreated cultures. Moreover, by using neutralizing antibodies for each TNF- receptor, we found that the proneurogenic effect of 1 ng/ml TNF- is mediated via tumor necrosis factor receptor 1 activation. Accordingly, the percentage of neuronal nuclear protein-positive neurons was increased following exposure to mouse TNF-. Interestingly, exposure of SVZ cultures to 1 ng/ml TNF- induced cell proliferation, whereas 10 and 100 ng/ml TNF- induced apoptotic cell death. Moreover, we found that exposure of SVZ cells to TNF- for 15 minutes or 6 hours caused an increase in the phospho-stress-activated protein kinase/c-Jun N-terminal kinase immunoreactivity initially in the nucleus and then in growing axons, colocalizing with tau, consistent with axonogenesis. Taken together, these results show that TNF- induces neurogenesis in neonatal SVZ cell cultures of mice. TNF-, a proinflammatory cytokine and a proneurogenic factor, may play a central role in promoting neurogenesis and brain repair in response to brain injury and infectio

Itens cirúrgicos individualizados e packs cirúrgicos personalizados na gestão do bloco opertaório

Background: in the operation room (OR), ideal efficiency is achieved by the professional’s ability to offer high quality healthcare and simultaneously find new and creative ideas to lower costs and improve productivity. Using Customized Surgical Packs (CSP) instead of Individualized Surgical Items (ISI) could be a way to work more efficiently. Objective: to evaluate the costs and time spent while using ISI and compare them with CSP in trochanteric fracture surgery. Methodology: qualitative study, done in OR, between november 2017 and april 2018, through participative observation and cost documentary analysis. Data analysis was performed with Statistical Package for the Social Sciences (SPSS), version 25. Results: costs associated with ISI, per surgery, are in average 42,20€ while using CSP has an average cost of 33,33€. Setting up the operating table with the use of ICI takes an average time of 6 minutes and 4 seconds, whereas using CSP takes that value down 49 seconds. Conclusion: OR efficiency and financial sustainability are guaranteed when comparing the use of CSP instead of ISI, contributing for less wasting by optimizing the available resources.Marco contextual: en el quirófano, la capacidad de los profesionales para ofrecer atención médica de alta calidad y, al mismo tiempo, encontrar ideas nuevas y creativas para reducir costos y mejorar la productividad, logran la eficiencia ideal. Usar Paquetes Quirúrgicos Personalizados (PQP) en lugar de Artículos Quirúrgicos Individuales (AQI) podría ser una forma de trabajar de manera más eficiente. Objetivo: evaluar los costos y el tiempo empleado en el uso de AQI y compararlos con la PQP en la cirugía de fractura trocantérica. Metodología: estudio cuantitativo, realizado en el quirófano, entre noviembre de 2017 y abril de 2018, mediante observación participativa y análisis documentado de costos. El análisis de los datos se realizó con Statistical Package for the Social Sciences (SPSS), versión 25. Resultados: los costos asociados con AQI, por cirugía, son en promedio 42,20 € mientras que el uso de PQP tiene un costo promedio de 33,33 €. La configuración de la mesa de operaciones con el uso de AQI requiere un tiempo promedio de 6 minutos e 4 segundos, mientras que el uso de PQP reduce ese valor a 49 segundos. Conclusión: La eficiencia del quirófano y la sostenibilidad financiera están garantizadas cuando se compara el uso de PQP en lugar de AQI, lo que contribuye a una menor pérdida mediante la optimización de los recursos disponibles.Enquadramento: no Bloco Operatório (BO) a eficiência é alcançada pela capacidade de os profissionais oferecerem cuidados de saúde de alta qualidade e simultaneamente encontrarem ideias inovadoras e criativas para diminuir os custos e aumentar a produtividade. Usar Packs Cirúrgicos Personalizados (PCP) em vez dos Itens Cirúrgicos Individuais (ICI) descartáveis poderá ser uma forma de alcançar essa eficiência. Objetivos: avaliar os custos e tempo dispensado na utilização dos ICI comparando-os com a utilização dos PCP, nas cirurgias de fratura trocantérica. Metodologia: estudo de natureza qualitativa, realizado num bloco operatório, entre novembro de 2017 e abril de 2018, com observação participante e análise documental de custos, tratamento de dados com recurso ao Statistical Package for the Social Sciences (SPSS), versão 25. Resultados: os custos associados aos ICI, por cirurgia, são em média 42,20€ enquanto que nos PCP o custo médio é de 33,33€. O tempo utilizado na colocação da mesa operatória com a utilização dos ICI é em média 6 minutos com 4 segundos e na utilização dos PCP decresce para 49 segundos. Conclusão: eficiência em BO e sustentabilidade financeira são garantidas com a utilização de PCP ao invés de ICI, contribuindo para a eliminação do desperdício com recurso a uma estratégia de otimização dos recursos disponíveis

Neuropeptide Y inhibits interleukin-1β-induced phagocytosis by microglial cells

<p>Abstract</p> <p>Background</p> <p>Neuropeptide Y (NPY) is emerging as a modulator of communication between the brain and the immune system. However, in spite of increasing evidence that supports a role for NPY in the modulation of microglial cell responses to inflammatory conditions, there is no consistent information regarding the action of NPY on microglial phagocytic activity, a vital component of the inflammatory response in brain injury. Taking this into consideration, we sought to assess a potential new role for NPY as a modulator of phagocytosis by microglial cells.</p> <p>Methods</p> <p>The N9 murine microglial cell line was used to evaluate the role of NPY in phagocytosis. For that purpose, an IgG-opsonized latex bead assay was performed in the presence of lipopolysaccharide (LPS) and an interleukin-1β (IL-1β) challenge, and upon NPY treatment. A pharmacological approach using NPY receptor agonists and antagonists followed to uncover which NPY receptor was involved. Moreover, western blotting and immunocytochemical studies were performed to evaluate expression of p38 mitogen-activated protein kinase (MAPK) and heat shock protein 27 (HSP27), in an inflammatory context, upon NPY treatment.</p> <p>Results</p> <p>Here, we show that NPY inhibits phagocytosis of opsonized latex beads and inhibits actin cytoskeleton reorganization triggered by LPS stimulation. Co-stimulation of microglia with LPS and adenosine triphosphate also resulted in increased phagocytosis, an effect inhibited by an interleukin-1 receptor antagonist, suggesting involvement of IL-1β signaling. Furthermore, direct application of LPS or IL-1β activated downstream signaling molecules, including p38 MAPK and HSP27, and these effects were inhibited by NPY. Moreover, we also observed that the inhibitory effect of NPY on phagocytosis was mediated <it>via </it>Y₁receptor activation.</p> <p>Conclusions</p> <p>Altogether, we have identified a novel role for NPY in the regulation of microglial phagocytic properties, in an inflammatory context.</p

Determination of catecholamines and endogenous related compounds in rat brain tissue exploring their native fluorescence and liquid chromatography

The profiling analysis of catecholamines and their metabolites in brain tissue offers a crucial key to understand their functions in the body and the opportunity to follow up neural diseases. A rapid and simple liquid chromatography-fluorescence detection (LC-FLD) method was developed and validated for simultaneously measuring several catecholamines and endogenous related compounds in the rat brain tissue samples. The target analytes measured in this bioanalytical assay were levodopa (L-DOPA), dopamine (DA), norepinephrine (NE), epinephrine (E), 3-O-methyldopa (3-O-MD), and homovanillic acid (HVA), being the 3,4-dihydroxybenzylamine (DHBA) used as internal standard (IS). The six analytes (L-DOPA, DA, NE, E, 3-O-MD and HVA) can be determined in a single chromatographic run of less than 12min, and all the compounds (analytes and IS) were detected using their native fluorescence and monitored at excitation/emission wavelengths of 279nm/320nm, respectively. The chromatographic and detection conditions were experimentally optimized and then several validation parameters (linearity, limits of quantification and detection, precision and accuracy, recovery, stability and selectivity) were examined. In accordance with the international guidelines of the Food and Drug Administration and European Medicines Agency the method described herein exhibited limits of quantification in the range of 2-25ngmL-1, linearity in wide concentration ranges (r2≥0.994), and acceptable precision (coefficient variation ≤8.76%) and accuracy (bias ±14.65%) levels. Since the bioanalytical procedure does not involve pre-purification or derivatization of the sample, the absolute recovery was found to be around 100%. Moreover, the developed LC-FLD method was successfully applied for the determination of the compounds of interest in tissue samples of different rat brain regions (cerebellum, amygdala, cortex, hippocampus, striatum, mesencephalon, medulla oblongata, substantia nigra and ventral tegmental area). Hence, this assay represents a valuable bioanalytical tool to support several pre(non)clinical studies in the broad field of neurosciences, requiring the quantitative analysis of these bioamines and their metabolites

From Expression Pattern to Dopaminergic Survival

Funding Information: Open access funding provided by FCT|FCCN (b-on). This work was supported by “Programa Operacional do Centro, Centro 2020” through the funding of the ICON project (Interdisciplinary Challenges On Neurodegeneration; CENTRO-01–0145-FEDER-000013), and by the following national funds: Foundation for Science and Technology (doctoral grant SFRH/BD/121822/2016), UBI-Santander/Totta (BID/ICI-FCS/CICS/Santander Universidades-UBI/2017) and the PPBI-Portuguese Platform of BioImaging: POCI-01–0145-FEDER-022122. Publisher Copyright: © 2023, The Author(s).C-terminal binding proteins (CtBP) are transcriptional co-repressors regulating gene expression. CtBP promote neuronal survival through repression of pro-apoptotic genes, and may represent relevant targets for neurodegenerative disorders, such as Parkinson’s disease (PD). Nevertheless, evidence of the role of CtBP1 and CtBP2 in neurodegeneration are scarce. Herein, we showed that CtBP1 and CtBP2 are expressed in neurons, dopaminergic neurons, astrocytes, and microglia in the substantia nigra (SN) and striatum of adult mice. Old mice showed a lower expression of CtBP1 in the SN and higher expression of CtPB2 in the SN and striatum compared with adult mice. In vivo models for PD (paraquat, MPTP, 6-OHDA) showed increased expression of CtBP1 in the SN and striatum while CtBP2 expression was increased in the striatum of paraquat-treated rats only. Moreover, an increased expression of both CtBP was found in a dopaminergic cell line (N27) exposed to 6-OHDA. In the 6-OHDA PD model, we found a dual effect using an unspecific ligand of CtBP, the 4-methylthio 2-oxobutyric acid (MTOB): higher concentrations (e.g. 2500 µM, 1000 µM) inhibited dopaminergic survival, while at 250 μM it counteracted cell death. In vitro, this latter protective role was absent after the siRNA silencing of CtBP1 or CtBP2. Altogether, this is the first report exploring the cellular and regional expression pattern of CtBP in the nigrostriatal pathway and the neuroprotective role in PD toxin-based models. CtBP could counteract dopaminergic cell death in the 6-OHDA PD model and, therefore, CtBP function and therapeutic potential in PD should be further explored.publishersversionepub_ahead_of_prin

Anti-Inflammatory Strategy for M2 Microglial Polarization Using Retinoic Acid-Loaded Nanoparticles

Inflammatory mechanisms triggered by microglial cells are involved in the pathophysiology of several brain disorders, hindering repair. Herein, we propose the use of retinoic acid-loaded polymeric nanoparticles (RA-NP) as a means to modulate microglia response towards an anti-inflammatory and neuroprotective phenotype (M2). RA-NP were first confirmed to be internalized by N9 microglial cells; nanoparticles did not affect cell survival at concentrations below 100 μg/mL. Then, immunocytochemical studies were performed to assess the expression of pro- and anti-inflammatory mediators. Our results show that RA-NP inhibited LPS-induced release of nitric oxide and the expression of inducible nitric oxide synthase and promoted arginase-1 and interleukin-4 production. Additionally, RA-NP induced a ramified microglia morphology (indicative of M2 state), promoting tissue viability, particularly neuronal survival, and restored the expression of postsynaptic protein-95 in organotypic hippocampal slice cultures exposed to an inflammatory challenge. RA-NP also proved to be more efficient than the free equivalent RA concentration. Altogether, our data indicate that RA-NP may be envisioned as a promising therapeutic agent for brain inflammatory diseases

Heterocellular Contacts with Mouse Brain Endothelial Cells Via Laminin and alpha 6 beta 1 Integrin Sustain Subventricular Zone (SVZ) Stem/Progenitor Cells Properties

Neurogenesis in the subventricular zone (SVZ) is regulated by diffusible factors and cell-cell contacts. In vivo, SVZ stem cells are associated with the abluminal surface of blood vessels and such interactions are thought to regulate their neurogenic capacity. SVZ neural stem cells (NSCs) have been described to contact endothelial-derived laminin via (01 integrin. To elucidate whether heterocellular contacts with brain endothelial cells (BEG) regulate SVZ cells neurogenic capacities, cocultures of SVZ neurospheres and primary BEG, both obtained from C57BL/6 mice, were performed. The involvement of laminin integrin interactions in SVZ homeostasis was tested in three ways. Firstly, SVZ cells were analyzed following incubation of BEC with the protein synthesis inhibitor cycloheximide (GHX) prior to coculture, a treatment expected to decrease membrane proteins. Secondly, SVZ cells were cocultured with BEG in the presence of an anti-alpha 6 integrin neutralizing antibody. Thirdly, BEC were cultured with beta 1(-/-) SVZ cells. We showed that contact with BEC supports, at least in part, proliferation and stemness of SVZ cells, as evaluated by the number of BrdU positive (+) and Sox2+ cells in contact with BEG. These effects are dependent on BEG-derived laminin binding to alpha 6 beta 1 integrin and are decreased in cocultures incubated with anti-alpha 6 integrin neutralizing antibody and in cocultures with SVZ beta 1(-/-) cells. Moreover, BEG-derived laminin sustains sternness in SVZ cell cultures via activation of the Notch and mTOR signaling pathways. Our results show that BEC/SVZ interactions involving alpha 6 beta 1 integrin binding to laminin, contribute to SVZ cell proliferation and stemness