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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.

A single injection of neuraminidase (NA) within the cerebral ventricles (ICV) triggers an acute neuroinflammation. Neurological complications or behavioral alterations have been associated to neuroinflammation. While some of these symptoms decline with time along with inflammation, the possibility of long-term sequelae should be considered. Thus, we aimed to explore if NA-induced neuroinflammation provokes behavioral or neurological disturbances at medium (2 weeks) and long (10 weeks) term. Rats were ICV injected with NA or saline. First, neurological alterations of the sensorimotor reflexes were not found, suggesting that NA does not cause disturbances in major brain functions. While the open field test revealed normal locomotor capacity in the animals injected with NA, however the evaluation of specific behaviors (rearing and rearing with support) pointed out an increased anxiety state 2 weeks after NA administration, but not at long term (10 weeks). A histological study of brain areas related to emotions (amygdala) and stress response (hypothalamic PVN) revealed no significant differences in the number of microglia or astrocytes. Nevertheless, the morphological analysis of microglial cells demonstrated that, in the amygdala of NA injected rats, microglia presented a morphology consistent with a slightly activated state. Such morphological change, which was evident 2 weeks after NA injection, was virtually reverted 10 weeks post-ICV. These results point out that NA injected ICV may cause anxiety in the medium term (while not affecting other functions like sensorimotor functions or the locomotor capacity), a behavioral alteration that is transient and that concurs with a mild inflammation, evidenced by the overexpression of certain genes and, more notably, by the morphological bias of microglial cells located in the amygdala towards an activated profile. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.

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.

Tipo de presentación: Póster Here, 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).

Comunicación oral. El resumen no se publica por deseo de los autores NG2 cells are considered oligodendrocyte precursor cells (OPC). In pathological conditions, NG2 cells contribute to generate oligodendrocytes and reactive astrocytes. This study has been designed to uncover the role of NG2 cells in congenital hydrocephalus using the hyh mouse model. Materials and methods Brain sections and whole mount preparations were obtained from embryos and postnatal hyh and control mice. NG2 positive (NG2+) cells were co-labelled with different IHC markers to deepest identification. Additionally, trying to understand reproducibility of our results in different neurodegenerative conditions, same NG2 identification approach was performed using ventricular walls explants from control mice after mechanical induction of astrocyte reaction. Results A higher number of NG2+ cells were found in the hyh mice compared to the control mice. In addition, NG2+ cells in the hyh mice showed a higher NG2 antigen content compared to the control mice. In the hyh mouse, colocalization results showed that most of NG2+ cells were identified as OPC cells and pericytes, but never as reactive astrocytes o microglial cells. However, in the same approach performed in neurodegenerative conditions, NG2+ cells were mostly identified as reactive astrocytes. Conclusions NG2 progenitors appear to be affected in hyh mutant mice giving rise to a different NG2+population which role in hydrocephalus is still unkown. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.

Background: In many cases there seems to exist a tight relationship between the integrity of the ependymal barrier and the onset of hydrocephalus (see 1). We wanted to investigate this by means of a repetitive model that could be easily raised in the laboratory from normal rats. Material and methods: We observed that the, easily available, enzyme neuraminidase from Clostridum perfringens destroys the ependyma (2). So we proved different amounts of the drug at different times and explored by means of morphological techniques, at structural and ultra-structural levels, the events occurring after the intracerebroventricular administration of the drug into the normal rat brain Results: The administration provoked the detachment of a great part of the ciliated cubic ependyma but not other types of ependyma, particularly those attached by tight junctions, as is the notable case of the choroid plexus. The effect is dose-dependent and led to hydrocephalus when the detachment is massive and affects the brain aqueduct. The absence of ependyma marks a zone of great permeability where the immuno-responsible cells proceeding from the vessels, mainly from those venules located in the choroid plexus, easily penetrated the brain parenchyma. With time, these zones are covered by impermeable astrocytic glial scars (3, 4). We think that this could represent a good model for study the relationships between the ependymal detach and the onset of hydrocephalus. Conclusions: The microbial neuraminidase broke the intercellular junctions of the ependymal line, non sealed by tight junctions. The dissapearance of this barrier led to an increased permeability or/and a stenosis of the cerebral acueduct that in turn provokes hydrocephalus. References: 1. Jiménez AJ, et al. Structure and function of the ependymal barrier and diseases associated with ependyma disruption. Tissue Barriers 2014 Mar 19;2:e28426. 2. Grondona JM et al. Ependymal denudation, aqueductal obliteration and hydrocephalus after a single injection of neuraminidase into the lateral ventricle of adult rats. J Neuropathol Exp Neurol. 1996 55:999-1008.

Neurogenesis in the adult rat hippocampal subgranular zone and the subventricular zone of the lateral ventricles has been well documented. Increasing evidence suggests that neurogenesis also occurs in the adult hypothalamus. We report that, after intracerebroventricular treatment with insulin-like growth factor I (IGF-I), cell proliferation significantly increased in both the periventricular and the parenchymal zones of the whole hypothalamus. Proliferation was particularly abundant in a distinct periventricular zone of the caudal hypothalamus. This is an overlapping zone of transition, between the cubic ciliated ependymal, dorsally located, and the tanycytic ependyma, ventrally located, where three cell layers can be found: ciliated ependyma, subependyma and underlying tanycytes. After IGF-I treatment, proliferating cells were seen in the subependyma and in the layer of tanycytes. Neurons, astrocytes, tanycytes, microglia and endothelial cells of the local vessels were stained with the proliferative marker 5-bromo-2'-deoxyuridine (BrdU) in response to IGF-I. However, we never observed BrdU-positive ciliated cubic ependymal cells. In the subependyma, proliferating glial fibrillary acidic protein-positive astrocytes contacted the ventricle by an apical process bearing a single cilium and there were many labyrinthine extensions of the periventricular basement membranes. Both features are typical of neurogenic niches in other brain zones, suggesting that the central overlapping zone of the rat hypothalamic wall could be considered a neurogenic niche in response to IGF-I. These effects will be compared with those obtained for other factors. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.

Multiciliated ependymal cells form an epithelium lining most of the ventricular cavities of vertebrates brain. Although considered postmitotic and completely differentiated, ependymal cells maintain some phenotypic characteristics of neural stem cells. Thereby, under specific conditions they behave as neural stem cells, developing radial glia characteristics, and undergoing asymmetric division. Our group is searching for factors that promote dedifferentiation of ependymal cells in vitro. We developed a simple method to obtain pure cultures of non-adherent multiciliated ependymal cells from adult rats. These cultures were used to investigate the effect of FGF2 on the differentiation state and the aggregation of ependymal cells. Thus, FGF2 treated ependymal cells lose cilia and hence mobility, and after 7 days they aggregate to form irregular spheres (diameter ≥ 20-30 μm). Such changes were not observed when EFG was used instead of FGF2. To assess the specificity of FGF2 action on cell aggregation, the FGF receptor inhibitor PD166866 and an anti-FGF2 neutralizing antibody were used. In both conditions the aggregative effect of FGF2 was abolished. No cell proliferation was observed during sphere formation, at least in such experimental conditions. Spheres were analyzed by immunocytochemistry using radial glia markers. They were positive for GFAP, vimentin, BLBP and GLAST. These data suggest that FGF2 promotes the identity loss in multiciliated ependymal cells in vitro, which are transformed into cells with radial glia features. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.

Comunicación en forma de poster al congreso Neuraminidase from Clostridium perfringens, which cleaves terminal sialic acid from carbohydrate chains, was injected in the lateral ventricle of rats. It diffused in the ipsilateral ventricle, the third ventricle, and also towards the periventricular brain parenchyma. Soon after, the complement system activated, and some ependymal cells detached and died. In the affected zones, there was an increased expression of GFAP in astrocytes, IBA1 in microglia, and ICAM1 in the endothelial cells of blood vessels. Cytokines, such as IL1β secreted by activated macrophages and microglia, provoked the extravasation of leucocytes from about 4 h post-injection. The main sources of cells were large venules located in the choroid plexus, the meninges and the subependyma around the foramen interventricularis. Invading cells arrived orderly: first neutrophils, then macrophage-monocytes, and last lymphocytes (mainly CD8α-positive T-lymphocytes). Leucocytes invaded the ventricle and the meninges, and also penetrated the brain parenchyma, sometimes passing through the ependyma and the glia limitans. As a result, some myelinated tracts suffered vacuolar degeneration, being the stria medullaris consistently affected. Oligodendrocytes in the damaged tracts were not affected. Vacuolated myelin recovered with time. Thus, the intracerebroventricular injection of neuraminidase may represent a novel reversible animal model to study experimental neuroinflammation and myelin vacuolization. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Ministerio de Ciencia e Innovación, SAF2010-19087; Junta de Andalucía, Consejería de Sanidad, SAS 08-0029; Junta de Andalucía, Proyecto de excelencia P-11-CVI-07637.