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.

Anxiety-like behavior and microglial activation in the amygdala after acute neuroinflammation induced by microbial neuraminidase.

Short-term behavioral alterations are associated with infection and aid the recovery from sickness. However, concerns have raised that sustained behavioral disturbances after acute neuroinflammation could relate to neurological diseases in the long run. We aimed to explore medium- and long-term behavioral disturbances after acute neuroinflammation in rats, using a model based on the intracerebroventricular administration of the enzyme neuraminidase (NA), which is part of some pathogenic bacteria and viruses. Neurological and behavioral assessments were performed 2 and 10 weeks after the injection of NA, and neuroinflammation was evaluated by gene expression and histology. No alterations were observed regarding basic neurological functions or locomotor capacity in NA-injected rats. However, they showed a reduction in unsupported rearing, and increased grooming and freezing behaviors, which indicate anxiety-like behavior. A principal component analysis including a larger set of parameters further supported such anxiety-like behavior. The anxiety profile was observed 2 weeks after NA-injection, but not after 10 weeks. Concomitantly, the amygdala presented increased number of microglial cells showing a morphologic bias towards an activated state. A similar but subtler tendency was observed in hypothalamic microglia located in the paraventricular nucleus. Also, in the hypothalamus the pattern recognition receptor toll-like receptor 4 (TLR4) was slightly overexpressed 2 weeks after NA injection. These results demonstrate that NA-induced neuroinflammation provokes anxiety-like behavior in the medium term, which disappears with time. Concurrent microgliosis in the amygdala could explain such behavior. Further experiments should aim to explore subtle but long-lasting alterations observed 10 weeks after NA injection, both in amygdala and hypothalamus, as well as mild behavioral changes.

Microglial Morphometric Parameters Correlate With the Expression Level of IL-1ß, and Allow Identifying Different Activated Morphotypes.

Microglia are the resident macrophages in the brain. Traditionally, two forms of microglia have been described: one considered as a resting/surveillant state in which cells have a highly branched morphology, and another considered as an activated state in which they acquire a de-ramified or amoeboid form. However, many studies describe intermediate microglial morphologies which emerge during pathological processes. Since microglial form and function are closely related, it is of interest to correlate microglial morphology with the extent of its activation. To address this issue, we used a rat model of neuroinflammation consisting in a single injection of the enzyme neuraminidase (NA) within the lateral ventricle. Sections from NA-injected animals were co-immunolabeled with the microglial marker IBA1 and the cytokine IL-1ß, which highlight features of the cell's shape and inflammatory activation, respectively. Activated (IL-1ß positive) microglial cells were sampled from the dorsal hypothalamus nearby the third ventricle. Images of single microglial cells were processed in two different ways to obtain (1) an accurate measure of the level of expression of IL-1ß (indicating the degree of activation), and (2) a set of 15 morphological parameters to quantitatively and objectively describe the cell's shape. A simple regression analysis revealed a dependence of most of the morphometric parameters on IL-1ß expression, demonstrating that the morphology of microglial cells changes progressively with the degree of activation. Moreover, a hierarchical cluster analysis pointed out four different morphotypes of activated microglia, which are characterized not only by morphological parameters values, but also by specific IL-1ß expression levels. Thus, these results demonstrate in an objective manner that the activation of microglial cells is a gradual process, and correlates with their morphological change. Even so, it is still possible to categorize activated cells according to their morphometric parameters, each category presenting a different activation degree. The physiological relevance of those activated morphotypes is an issue worth to be assessed in the future.

Evidence for a role of the ubiquitin-proteasome pathway in pancreatic islets.

The ubiquitin-proteasome pathway is crucial for protein turnover. Part of the pathway involves deubiquitination, which is carried out by cystein proteases known as ubiquitin COOH-terminal hydrolases. The isoform Uch-L1 was found to be present in large amounts in rat islets by immunostaining, Western blot analysis, and RT-PCR. Culturing islets in high glucose concentrations (16.7 mmol/l) for 24 h led to decreased gene expression. Exposure to chronic hyperglycemia following 90% partial pancreatectomy also led to reduced Uch-L1 expression. Expression of other members of the ubiquitin-proteasome pathway studied after culturing islets at high glucose concentrations revealed little change except for modest declines in parkin, human ubiquitin-conjugating enzyme 5 (UbcH5), and beta-TRCP (transducin repeat-containing protein). With the pancreatectomy model, expression of polyubiquitin-B and c-Cbl were increased and E6-associated protein was reduced. Further insight about the proteasome pathway was obtained with the proteasome inhibitor lactacystin, which in short-term 2-h experiments enhanced glucose-induced insulin secretion. An important role for the ubiquitin-proteasome pathways in beta-cells is suggested by the findings that changes in glucose concentration influence expression of genes in the pathway and that blockade of the proteasome degradation machinery enhances glucose-stimulated insulin secretion.

Microglia Morphological Categorization in a Rat Model of Neuroinflammation by Hierarchical Cluster and Principal Components Analysis.

It is known that microglia morphology and function are closely related, but only few studies have objectively described different morphological subtypes. To address this issue, morphological parameters of microglial cells were analyzed in a rat model of aseptic neuroinflammation. After the injection of a single dose of the enzyme neuraminidase (NA) within the lateral ventricle (LV) an acute inflammatory process occurs. Sections from NA-injected animals and sham controls were immunolabeled with the microglial marker IBA1, which highlights ramifications and features of the cell shape. Using images obtained by section scanning, individual microglial cells were sampled from various regions (septofimbrial nucleus, hippocampus and hypothalamus) at different times post-injection (2, 4 and 12 h). Each cell yielded a set of 15 morphological parameters by means of image analysis software. Five initial parameters (including fractal measures) were statistically different in cells from NA-injected rats (most of them IL-1ß positive, i.e., M1-state) compared to those from control animals (none of them IL-1ß positive, i.e., surveillant state). However, additional multimodal parameters were revealed more suitable for hierarchical cluster analysis (HCA). This method pointed out the classification of microglia population in four clusters. Furthermore, a linear discriminant analysis (LDA) suggested three specific parameters to objectively classify any microglia by a decision tree. In addition, a principal components analysis (PCA) revealed two extra valuable variables that allowed to further classifying microglia in a total of eight sub-clusters or types. The spatio-temporal distribution of these different morphotypes in our rat inflammation model allowed to relate specific morphotypes with microglial activation status and brain location. An objective method for microglia classification based on morphological parameters is proposed. Main points Microglia undergo a quantifiable morphological change upon neuraminidase induced inflammation.Hierarchical cluster and principal components analysis allow morphological classification of microglia.Brain location of microglia is a relevant factor.

Neuroinflammation induced by intracerebroventricular injection of microbial neuraminidase.

In the present paper, we describe the facts that took place in the rat brain after a single injection of the enzyme neuraminidase from Clostridium perfringens into the right lateral ventricle. After injection, it diffused through the cerebrospinal fluid of the ipsilateral ventricle and the third ventricle, and about 400 µm into the periventricular brain parenchyma. The expression of ICAM1 in the endothelial cells of the periventricular vessels, IBA1 in microglia, and GFAP in astrocytes notably increased in the regions reached by the injected neuraminidase. The subependymal microglia and the ventricular macrophages begun to express IL1ß and some appeared to cross the ependymal layer. After about 4 h of the injection, leukocytes migrated from large venules of the affected choroid plexus, the meninges and the local subependyma, and infiltrated the brain. The invading cells arrived orderly: first neutrophils, then macrophage-monocytes, and last CD8α-positive T-lymphocytes and B-lymphocytes. Leukocytes in the ventricles and the perivascular zones penetrated the brain parenchyma passing through the ependyma and the glia limitans. Thus, it is likely that a great part of the damage produced by microorganism invading the brain may be due to their neuraminidase content.

Survival of microencapsulated adult pig islets in mice in spite of an antibody response.

The aim of this study was to assess the capacity of simple alginate capsules to protect adult pig islets in a model of xenotransplantation. Adult pig islets were microencapsulated in alginate, with either single alginate coats (SAC) or double alginate coats (DAC), and transplanted into the streptozotocin-induced diabetic B6AF1 mice. Normalization of glucose levels was associated with an improvement of the glucose clearance during intravenous glucose tolerance tests. After explantation, all mice became hyperglycemic, demonstrating the efficacy of the encapsulated pig islets. Explanted capsules were mainly free of fibrotic reaction and encapsulated islets were still functional, responding to glucose stimulation with a 10-fold increase in insulin secretion. However, a significant decrease in the insulin content and insulin responses to glucose was observed for encapsulated islets explanted from hyperglycemic mice. An immune response of both IgG and IgM subtypes was detectable after transplantation. Interestingly, there were more newly formed antibodies in the serum of mice transplanted with SAC capsules than in the serum of mice transplanted with DAC capsules. In conclusion, alginate capsules can prolong the survival of adult pig islets transplanted into diabetic mice for up to 190 days, even in the presence of an antibody response.

Development and retroviral transduction of porcine neonatal pancreatic islet cells in monolayer culture.

To learn more about the potential of neonatal porcine pancreatic duct and islet cells for xenotransplantation, the development of these cells when cultured as monolayers was evaluated. Immunostaining for islet hormones and cytokeratin-7 revealed that day eight monolayers consisted of approximately 70% duct cells and less than 10% beta cells. Using Ki-67 immunostaining as a proliferation marker, the fraction of beta cells in the cell cycle was shown to decrease from 20% at day three to 10% at day eight, and for duct cells from 36 to 19%. Insulin secretion increased 2.4-fold upon glucose stimulation, and 38-fold when 10 mm theophylline was added, showing the responsiveness of the neonatal beta cells. Reaggregated monolayers consisted mostly of duct cells, but 4 weeks after transplantation, grafts contained predominantly endocrine cells, with duct cells being almost absent, suggesting in vivo differentiation of duct cells to endocrine cells. Monolayer susceptibility to retroviral transduction was also investigated using a Moloney Murine Leukemia Virus-based vector. Approximately 60% of duct cells but less than 5% of beta cells expressed the transgene, indicating that precursor duct cells are better targets for transgene expression. These results show that porcine neonatal pancreatic cells can be cultured as monolayers in preparation for transplantation. Furthermore, in such a culture setting, precursor duct cells have a high rate of proliferation and are more efficiently transduced with a retrovirus-based reporter gene than are beta cells.