Neuraminidase-induced neuroinflammation is largely dependent on microglial TLR4 receptor

The sialidase neuraminidase (NA) cleaves terminal sialic acid from glycoproteins and glycolipids. Among its various locations, it is present in the envelope/membrane of some bacteria/viruses (e.g. influenza virus), where it is involved in infectiveness and dispersion. The injection of NA within the brain lateral ventricle represents a model of acute sterile inflammation. The relevance of the toll-like receptors TLR2 and TLR4 (particularly those in microglial cells) in such process was investigated using mouse strains deficient in these receptors. In septofimbria and hypothalamus, IBA1-positive and IL-1β-positive cell counts increased after NA injection in wild type (WT) mice. In TLR4-/- mice such increases were largely abolished, while only slightly affected in TLR2-/- mice. Similarly, the NA-induced expression of IL-1β, TNFα and IL-6 (evaluated by qPCR) was completely blocked in TLR4-/- mice, and only partially reduced in TLR2-/- mice. Microglia was isolated from the three mouse strains and exposed to NA or to specific TLR2 and TLR4 agonists (Pam3CSK4 and LPS respectively) in vitro. NA induced a cytokine response (IL-1β, TNFα and IL-6) in WT microglia, but was unable to do so in TLR4-/- microglia; TLR2 deficiency partially affected the NA-induced microglia response. To investigate if such response of microglial cells to NA was dependent on the sialidase activity of the enzyme, WT microglia was exposed in vitro to NA previously inactivated with heat, or inhibited with two different sialidase inhibitors (oseltamivir phosphate and N-acetyl-2,3-dehydro-2-deoxyneuraminic acid). In all cases, NA- induced microglia activation was dependent on the intact sialidase activity of NA. Therefore, we conclude that NA is able to directly activate microglial cells, mostly through TLR4 receptor and due to its sialidase activity. Accordingly, the inflammatory reaction induced by NA in vivo is partially dependent on TLR2, while TLR4 plays a crucial role.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Morphological traits of surveillant/activated microglia during an experimentally induced acute neuroinflammatory process

Poster en CongresoSeveral studies show that morphological changes of microglia over the course of inflammation are tightly coupled to function. However the progressive transformation into activated microglia is poorly characterized. AIMS: This study aimed to establish a spatiotemporal correlation between quantifiable morphological parameters of microglia and the spread of an acute ventricular inflammatory process. METHODS: Inflammation was induced by a single injection of the enzyme neuraminidase within the lateral ventricle of rats. Animals were sacrificed 2, 4 and 12 hours after injection. Coronal slices were immunostained with Iba1 to label microglia and with IL1β to delimit the spread of inflammation. Digital images were obtained by scanning the labelled sections. Single microglia images were randomly selected from periventricular areas of caudate putamen, hippocampus and hypothalamus. FracLac for ImageJ software was used to measure the following morphological parameters: fractal dimension, lacunarity, area, perimeter and density. RESULTS: Significant differences were found in fractal dimension, lacunarity, perimeter and density of microglia cells of neuraminidase injected rats compared to sham animals. However no differences were found in the parameter “area”. In hipoccampus there was a delay in the significant change of the measured parameters. These morphological changes correlated with IL1β-expression in the same areas. CONCLUSIONS: Ventricular inflammation induced by neuraminidase provokes quantifiable morphological changes in microglia restricted to areas labelled with IL1β. Morphological parameters of microglia such as fractal dimension, lacunarity, perimeter and density are sensitive and valuable tools to quantify activation. However, the extensively used parameter “area” did not change upon microglia activation.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Grant: Junta de Andalucía (Spain) P11-CVI-07637 Ibima, Andalucía Tech, UM

Long-term priming of hypothalamic microglia is associated with energy balance disturbances under diet-induced obesity

Exposure of microglia to an inflammatory environment may lead to their priming and exacerbated response to future inflammatory stimuli. Here we aimed to explore hypothalamic microglia priming and its consequences on energy balance regulation. A model of intracerebroventricular administration of neuraminidase (NA, which is present in various pathogens such as influenza virus) was used to induce acute neuroinflammation. Evidences of primed microglia were observed 3 months after NA injection, namely (1) a heightened response of microglia located in the hypothalamic arcuate nucleus after an in vivo inflammatory challenge (high fat diet [HFD] feeding for 10 days), and (2) an enhanced response of microglia isolated from NA-treated mice and challenged in vitro to LPS. On the other hand, the consequences of a previous NA-induced neuroinflammation were further evaluated in an alternative inflammatory and hypercaloric scenario, such as the obesity generated by continued HDF feeding. Compared with sham-injected mice, NA-treated mice showed increased food intake and, surprisingly, reduced body weight. Besides, NA-treated mice had enhanced microgliosis (evidenced by increased number and reactive morphology of microglia) and a reduced population of POMC neurons in the basal hypothalamus. Thus, a single acute neuroinflammatory event may elicit a sustained state of priming in microglial cells, and in particular those located in the hypothalamus, with consequences in hypothalamic cytoarchitecture and its regulatory function upon nutritional challenges.Ministerio de Economía, Industria yCompetitividad, Gobierno de España, Grant/Award Number: SAF2017-83645; Ministeriode Educación y Formación Profesional; Funding for open access charge: Universidadde Málaga/CBU

Primed microglia after acute neuroinflammation may drive an enhanced stress response.

Microglial cells become activated during acute neuroinflammation and usually they return to their basal surveillant state in a few days. However, sometimes microglia evolve towards a primed state characterized by an exacerbated response to new stimuli, which may jeopardize brain functions. Here we aimed to explore microglial priming in the hypothalamus and its consequences on the neuroendocrine regulation of the stress response. To induce priming we used a model of acute ventricular neuroinflammation by intracerebroventricular (ICV) injection of the enzyme neuraminidase (NA). Three months later, an acute stressor (consisting in forced swimming) was applied to investigate the activation of the hypothalamic-pituitary-adrenal axis and the stress response elicited, as well as the inflammatory activation of hypothalamic microglial cells. Stressed rats previously injected with NA had increased plasma levels of corticosterone compared to control rats that were equally stressed but had been ICV injected with saline. Also, qPCR studies revealed that NA-treated rats presented an increased expression of the microglial marker IBA1 and of the inflammasome protein NLRP3. Concomitantly, the morphological analysis of hypothalamic microglial cells showed a morphological bias towards a slightly activated state in microglia of NA injected rats compared to those of saline injected controls. Furthermore, in the open field test NA-treated rats displayed increased locomotor activity. These results suggest that prior neuroinflammatory episodes might result in subtle but persistent changes in microglial cells that could determine the response to future challenges such as stressful events.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Microbial neuraminidase induces TLR4-dependent long-term immune priming in the brain

Innate immune memory explains the plasticity of immune responses after repeated immune stimulation, leading to either enhanced or suppressed immune responses. This process has been extensively reported in peripheral immune cells and also, although modestly, in the brain. Here we explored two relevant aspects of brain immune priming: its persistence over time and its dependence on TLR receptors. For this purpose, we used an experimental paradigm consisting in applying two inflammatory stimuli three months apart. Wild type, toll-like receptor (TLR) 4 and TLR2 mutant strains were used. The priming stimulus was the intracerebroventricular injection of neuraminidase (an enzyme that is present in various pathogens able to provoke brain infections), which triggers an acute inflammatory process in the brain. The second stimulus was the intraperitoneal injection of lipopolysaccharide (a TLR4 ligand) or Pam3CSK4 (a TLR2 ligand). One day after the second inflammatory challenge the immune response in the brain was examined. In wild type mice, microglial and astroglial density, as well as the expression of 4 out of 5 pro-inflammatory genes studied (TNFα, IL1β, Gal-3, and NLRP3), were increased in mice that received the double stimulus compared to those exposed only to the second one, which were initially injected with saline instead of neuraminidase. Such enhanced response suggests immune training in the brain, which lasts at least 3 months. On the other hand, TLR2 mutants under the same experimental design displayed an enhanced immune response quite similar to that of wild type mice. However, in TLR4 mutant mice the response after the second immune challenge was largely dampened, indicating the pivotal role of this receptor in the establishment of immune priming. Our results demonstrate that neuraminidase-induced inflammation primes an enhanced immune response in the brain to a subsequent immune challenge, immune training that endures and that is largely dependent on TLR4 receptor

Neuraminidase-induced neuroinflammation causes anxiety and microgliosis in the amygdala

An intracerebroventricular (ICV) injection of neuraminidase (NA) within the lateral ventricles originates an acute event of neuroinflammation, which is solved to a great extent after two weeks. Recently, neurological problems or behavioral alterations have been associated with neuroinflammation. Although the majority of them fade along with inflammation resolution, the possibility of long-term sequelae should be taken into consideration. Thus, we aimed to explore if NA-induced neuroinflammation provokes behavioral or neurological disturbances at medium (2 weeks) and long (10 weeks) term. Initially, rats were ICV injected with NA or saline. Two or 10 weeks later they were made to perform a series of neurological tests and behavioral evaluations (open field test). The neuroinflammation status of the brain was studied by immunohistochemistry and qPCR. While no neurological alterations were found, the open field test revealed an increased anxiety state 2 weeks after NA administration, which was not observed after 10 weeks. In accordance with this behavioral findings, an overexpression of the molecular pattern receptor TLR4 was revealed by qPCR in hypothalamic tissue in NA treated animals after 2 weeks of ICV, but not after 10 weeks. Moreover, histological studies showed a microgliosis in the amygdala of NA injected rats 2 weeks post-ICV, as well as a slightly activated state evidenced by morphometric parameters of these cells. These histological findings were not present 10 weeks after the ICV injection. These results suggest that NA-induced neuroinflammation might cause anxiety, with no neurological manifestations, in the medium term, along with a mild microglial activation in amygdala. Such symptoms seem to revert, as they were not detected 10 weeks after NA administration.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Enhanced stress response in rats that suffered acute neuroinflammation induced by neuraminidase three months before

Microglial cells are protagonists in neuroinflammatory processes and their activation is a notorious feature of such events. In acute inflammation, microglial cells return to their basal surveillant state in few days. However, sometimes they evolve towards a primed state, characterized by hypersensitivity to new stimuli and an exacerbated response which may jeopardize brain functions. Because the hypothalamus is a pivotal hub for neuroendocrine and autonomic functions, we have been exploring evidences of microglial priming in this region and its consequences. We used a model of acute ventricular neuroinflammation consisting in the intracerebroventricular (ICV) injection of neuraminidase (NA). This enzyme is found in the cover of neurotropic bacteria and viruses, e.g. influenza, mumps or measles viruses, thus mimicking a brain infection. Three months after inducing neuroinflammation with NA to rats, an acute stressor was applied to investigate the activation of the hypothalamic-pituitary-adrenal (HPA) axis and the stress response elicited, as well as the inflammatory activation of hypothalamic microglial cells. The acute stressor was forced swimming for 6 minutes. Afterwards, blood samples were retrieved to determine corticosterone levels by ELISA, and the brains extracted to analyze microglial cells in histological sections by immunohistochemistry with IBA1 and inflammatory markers by qPCR. Stressed rats previously injected with NA had increased levels of corticosterone compared with control rats that were equally stressed but had been ICV injected with saline. Also, qPCR studies in hypothalamic tissue revealed that NA treated rats presented an increased expression of the genes for the inflammasome protein NLR family pyring domain containing 3 (NLRP3) and the microglial marker IBA1. Concomitantly, the morphological analysis of microglial cells located in the paraventricular nucleus (PVN) showed a morphological bias towards a slightly activated state in microglia...Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Sudden cessation of fluoxetine before alcohol drinking reinstatement alters microglial morphology and TLR4/inflammatory neuroadaptation in the rat brain

Preclinical studies on the efects of abrupt cessation of selective serotonin reuptake inhibitors (SSRIs), a medication often prescribed in alcohol use disorder (AUD) patients with depression, results in alcohol consumption escalation after resuming drinking. However, a potential neuroinfammatory component on this escalation remains unexplored despite the immunomodulatory role of serotonin. Here, we utilized a rat model of 14-daily administration of the SSRI fuoxetine (10 mg/kg/day) along alcohol self-administration deprivation to study the efects of fuoxetine cessation on neuroinfammation after resuming alcohol drinking. Microglial morphology and infammatory gene expression were analyzed in prelimbic cortex, striatum, basolateral amygdala and dorsal hippocampus. Results indicated that alcohol drinking reinstatement increased microglial IBA1 immunoreactivity and altered morphometric features of activated microglia (fractal dimension, lacunarity, density, roughness, and cell area, perimeter and circularity). Despite alcohol reinstatement, fuoxetine cessation modifed microglial morphology in a brain region-specifc manner, resulting in hyper-ramifed (spatial complexity of branching), reactive (lower heterogeneity and circularity)-like microglia. We also found that microglial cell area correlated with changes in mRNA expression of chemokines (Cx3cl1/fractalkine, Cxcl12/SDF1α, Ccl2/MCP1), cytokines (IL1β, IL6, IL10) and the innate immune toll-like receptor 4 (TLR4) in dorsal hippocampus. Specifcally, TLR4 correlated with microglial spatial complexity assessed by fractal dimension in striatum, suggesting a role in process branching. (...)Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. Funding for open access charge: Universidad de Málaga/CBUA. RETICS Red de Trastornos Adictivos, Instituto de Salud Carlos III (ISCIII), Ministerio de Ciencia e Innovación and European Regional Development Funds-European Union (ERDF-EU) (Grant No. RD16/0017/0001); ISCIII, ERDF-EU (Grant No. PI17/02026, Grant No. PI19/01577); Ministerio de Sanidad, Delegación de Gobierno para el Plan Nacional sobre Drogas (Grant No. PND 2020/048, Grant No. PND 2019/040, Grant No. PND 2018/044, Grant No. PND 2018/033); and Consejería de Salud y Familia, Junta de Andalucía (Neuro-RECA, Grant No. RIC-0111–2019). JS (Grant No. CPII17/00024), FJP (Grant No. CPII19/00022) and AS (Grant No. CPII19/00031) hold “Miguel Servet II” research contracts from the National System of Health, ISCIII, ERDF-EU. FJP also holds a “Nicolas Monardes” contract from Servicio Andaluz de Salud, Consejería de Salud y Familia, Junta de Andalucía (Grant No. C1-0049–2019). PR (Grant No. CP19/00068) hold “Miguel Servet I” research contracts from the National System of Health, ISCIII, ERDF-EU. The funding sources had no further role in study design; in the collection, analysis and interpretation of data; in writing of the report; and in the decision to submit the paper for publication

Chronic ethanol induces morpohological changes on hippocampal microglia, which are reverted by pharmacological blockade of faah with urb597

Tipo de presentación: PósterHere, we evaluated the pharmacological effects of fatty-acid amide-hydrolase (FAAH) inhibitor URB597 (0.3 mg/kg), oleoylethanolamide (OEA, 10 mg/kg), arachidonoylethanolamide (AEA, 10 mg/kg), the CB1 receptor agonist ACEA (3 mg/kg) and the CB2 receptor agonist JWH133 (0.2 mg/kg) administered for 5 days in a rat model of sub-chronic (2 weeks) ethanol diet (11% v/v) exposure. As a result of these trials, URB597 turned to be the most effective treatment. Contrary to ethanol, URB597 reduced the mRNA levels of Iba-1, Tnfα, IL-6 and monocyte chemoattractant protein-1 (MCP-1/CCL2), as well as the number of cells expressing GFAP or iNOS. Moreover, URB597 effects on hippocampal immune system were accompanied by changes in short and long-term visual recognition memory. Microglial morphometric analysis pointed out significant changes after ethanol exposure, suggesting that microglial cell morphology is closely related to ethanol-induced neuroinflammation. Ethanol provoked changes in fractal dimension, lacunarity, density, roughness, cell area and cell perimeter, which explain a decreased complexity of branches and increased cell surface irregularities. Such changes may represent a chronic activation state of microglia. In addition, ethanol effects on the microglial morphological parameters density and fractal dimension were reverted by URB597. Thus, this FAAH inhibitor was able to counteract the sub-chronic ethanol-induced morphological changes of microglia, resulting in a more compact and increased branch complexity, which apparently relate to a less activated state. Therefore, these morphometric parameters are sensitive and valuable tools to evaluate the chronic activation of microglia by ethanol and its pharmacological blockade.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. RETICS Red de Trastornos Adictivos, ISCIII, MINECO, ERDF-EU (RD16/0017/0001; PI17/02026; SAF2017-83645R). Plan Nacional sobre Drogas, MSCBS (PNSD2015/047; PND2017/043). Proyectos de investigación de excelencia, Junta de Andalucía (P11-CVI-07637)

Despertando la curiosidad científica en infantil a través de la colaboración de familia, escuela y centro universitario

The experience presented is an activity for preschool students. The aim is to arouse their scientific curiosity and encourage collaboration among members of the educational community by participating students' parents, and students and teachers from the Teacher Training College ‘Sagrada Familia’ in Úbeda. The experience consisted of a day of workshops for preschool students from the state school "San Ginés de la Jara" in Sabiote (Jaén). In those workshops the students discovered scientific phenomena through experiments. A total of four workshops were carried out. They were based on scientific methodology in which children first observe, then formulate hypotheses, check the phenomenon and, finally, draw a conclusion. To study the initiation to scientific curiosity in the group of children we used the qualitative technique of text production. Also, as the experience was designed as a training project through the collaborative work of several sectors of the community, we decided to study the evolution in the correlation between theory and practice that trainee teachers had by means of a pretest-posttest research design with a single group, on the basis of a questionnaire on solving problematic scientific situations. In the same way, we used a pretest-posttest design based on a questionnaire with open-ended questions to analyze whether the family context was a collaborative environment to foster scientific curiosity and to contribute to its development in a setting other than the school. The results show that, after the intervention, a significant group of children remind one or more of the experiments of the workshops and are able to give a scientific explanation of them. The information drawn from the data collected in the family setting expand the results mentioned before, since it shows that, in some homes, children have applied scientific knowledge and, thus, the family context has become a collaborative environment to encourage scientific curiosity. Besides, the analysis of the data collected from trainee teachers show that most of them are capable of linking theory and practice after the experience. Key words: Scientific methodology, collaborative work, scientific curiosity, initial training, permanent training.La experiencia se plantea como una actividad para el alumnado de Educación Infantil. El fin es despertar la curiosidad científica y fomentar la colaboración entre los miembros de la comunidad educativa al participar padres y madres del alumnado, estudiantes de Educación Infantil y profesores de la Escuela Universitaria. El desarrollo de la experiencia se basó en la organización de una jornada de talleres para el alumnado de segundo ciclo de Educación Infantil del CEIP “San Ginés de la Jara”, de Sabiote (Jaén), en los que se presentaba el descubrimiento de fenómenos científicos a través de experimentos, con la colaboración de los padres y madres interesados en el proyecto y del alumnado de Magisterio de la Escuela Universitaria de Magisterio “Sagrada Familia”, de Úbeda, cercana al centro en el que se desarrolló la experiencia. Se realizaron un total de cuatro talleres basados en una metodología científica, dejando a los niños que observaran en primer lugar, lanzaran hipótesis, comprobaran el fenómeno sobre el que estaban haciendo las conjeturas y, finalmente, extrajeran una conclusión. Para estudiar la iniciación a la curiosidad científica en el grupo de niños y niñas de infantil utilizamos la técnica cualitativa de producción de textos. Asimismo, como la experiencia se preparó como un proyecto de formación a través del trabajo colaborativo de varios sectores de la comunidad, nos propusimos estudiar la evolución en la interrelación de teoría y práctica entre el alumnado de magisterio a través de un diseño de investigación de un sólo grupo con pretest-postest y a partir de un cuestionario de resolución de situaciones científicas problemáticas. De manera similar, para analizar si el contexto familiar constituía un entorno de colaboración para alentar la curiosidad científica y contribuir desde otro ambiente diferente al escolar a su desarrollo, utilizamos un diseño pretest-postest a partir de un cuestionario de preguntas abiertas