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-1β positive, i.e., M1-state) compared to those from control animals (none of them IL-1β 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.
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.
Neuraminidase (NA) is a sialidase present, among various locations, in the envelope/membrane of some bacteria/viruses (e.g., influenza virus), and is involved in infectiveness and/or dispersion. The ...administration 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.
Mouse strains deficient in either TLR2 (TLR2
) or TLR4 (TLR4
) were used. NA was injected in the lateral ventricle, and the inflammatory reaction was studied by immunohistochemistry (IBA1 and IL-1β) and qPCR (cytokine response). Also, microglia was isolated from those strains and in vitro stimulated with NA, or with TLR2/TLR4 agonists as positive controls (P3C and LPS respectively). The relevance of the sialidase activity of NA was investigated by stimulating microglia with heat-inactivated NA, or with native NA in the presence of sialidase inhibitors (oseltamivir phosphate and N-acetyl-2,3-dehydro-2-deoxyneuraminic acid).
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 were only slightly diminished in TLR2
mice. Similarly, the NA-induced expression of IL-1β, TNFα, and IL-6 was completely blocked in TLR4
mice, and only partially reduced in TLR2
mice. In isolated cultured microglia, 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 microglial response. When WT microglia was exposed in vitro to heat-inactivated NA or to native NA along with sialidase inhibitors, the NA-induced microglia activation was almost completely abrogated.
NA is able to directly activate microglial cells, and it does so mostly acting through the TLR4 receptor, while TLR2 has a secondary role. Accordingly, the inflammatory reaction induced by NA in vivo is partially dependent on TLR2, while TLR4 plays a crucial role. Also, the sialidase activity of NA is critical for microglial activation. These results highlight the relevance of microbial NA in the neuroinflammation provoked by NA-bearing pathogens and the possibility of targeting its sialidase activity to ameliorate its impact.
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-1β, which highlight features of the cell´s shape and inflammatory activation, respectively. Activated (IL-1β 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-1β (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-1β 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-1β 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.
Abstract
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.
The administration of microbial neuraminidase into the brain ventricular cavities of rodents represents a model of acute aseptic neuroinflammation. Ependymal cell death and hydrocephalus are unique ...features of this model. Here we demonstrate that activated microglia participates in ependymal cell death. Co-cultures of pure microglia with ependymal cells (both obtained from rats) were performed, and neuraminidase or lipopolysaccharide were used to activate microglia. Ependymal cell viability was unaltered in the absence of microglia or inflammatory stimulus (neuraminidase or lipopolysaccharide). The constitutive expression by ependymal cells of receptors for cytokines released by activated microglia, such as IL-1β, was demonstrated by qPCR. Besides, neuraminidase induced the overexpression of both receptors in ventricular wall explants. Finally, ependymal viability was evaluated in the presence of functional blocking antibodies against IL-1β and TNFα. In the co-culture setting, an IL-1β blocking antibody prevented ependymal cell death, while TNFα antibody did not. These results suggest that activated microglia are involved in the ependymal damage that occurs after the administration of neuraminidase in the ventricular cavities, and points to IL-1β as possible mediator of such effect. The relevance of these results lies in the fact that brain infections caused by neuraminidase-bearing pathogens are frequently associated to ependymal death and hydrocephalus.
Soy extracts have been claimed to be neuroprotective against brain insults, an effect related to the estrogenic properties of isoflavones. However, the effects of individual isoflavones on ...obesity-induced disruption of adult neurogenesis have not yet been analyzed. In the present study we explore the effects of pharmacological administration of daidzein, a main soy isoflavone, in cell proliferation, cell apoptosis and gliosis in the adult hippocampus of animals exposed to a very high-fat diet. Rats made obese after 12-week exposure to a standard or high-fat (HFD, 60%) diets were treated with daidzein (50 mg kg(-1)) for 13 days. Then, plasma levels of metabolites and metabolic hormones, cell proliferation in the subgranular zone of the dentate gyrus (SGZ), and immunohistochemical markers of hippocampal cell apoptosis (caspase-3), gliosis (GFAP and Iba-1), food reward factor FosB and estrogen receptor alpha (ERα) were analyzed. Treatment with daidzein reduced food/caloric intake and body weight gain in obese rats. This was associated with glucose tolerance, low levels of HDL-cholesterol, insulin, adiponectin and testosterone, and high levels of leptin and 17β-estradiol. Daidzein increased the number of phospho-histone H3 and 5-bromo-2-deoxyuridine (BrdU)-ir cells detected in the SGZ of standard diet and HFD-fed rats. Daidzein reversed the HFD-associated enhanced immunohistochemical expression of caspase-3, FosB, GFAP, Iba-1 and ERα in the hippocampus, being more prominent in the dentate gyrus. These results suggest that pharmacological treatment with isoflavones regulates metabolic alterations associated with enhancement of cell proliferation and reduction of apoptosis and gliosis in response to high-fat diet.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
Main Points
Ventricular inflammation drives long‐lasting priming of hypothalamic microglia.
Response of primed hypothalamic microglia to challenges such as HDF and obesity is increased.
Past inflammatory events alter hypothalamus and energy balance regulation.
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Changes in endogenous cannabinoid homeostasis are associated with both ethanol-related neuroinflammation and memory decline. Extensive research is still required to unveil the role of ...endocannabinoid signaling activation on hippocampal microglial cells after ethanol exposure. Either microglial morphology, phenotype and recruitment may become notably altered after chronic alcohol-related neurodegeneration. 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 subchronic (2 weeks) ethanol diet (11% v/v) exposure. URB597 turned to be the most effective treatment. URB597 increased microglial (IBA-1+) cell population, and changed morphometric features (cell area and perimeter, roughness, fractal dimension, lacunarity) associated with activated microglia in the hippocampus of ethanol-exposed rats. Regarding innate immune activity, URB597 specifically increased mRNA levels of toll-like receptor 4 (TLR4), glial fibrillary acidic protein (Gfap) and the chemokine stromal cell-derived factor 1 (SDF-1α/CXCL12), and elevated the cell population expressing the chemokine receptors CX3CR1, CCR2 and CCR4 in the ethanol-exposed rat hippocampus. Contrary to ethanol effect, URB597 reduced mRNA levels of Iba-1, Tnfα, IL-6 and the monocyte chemoattractant protein-1 (MCP-1/CCL2), as well as cell population expressing iNOS. URB597 effects on hippocampal immune system were accompanied by changes in short and long-term visual recognition memory. These results suggest that FAAH inhibition may modulates hippocampal microglial recruitment and activation that can be associated with improved hippocampal-dependent memory despite ethanol exposure.
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.
Diet‐induced obesity produces changes in endocannabinoid signaling (ECS), influencing the regulation of energy homeostasis. Recently, we demonstrated that, in high‐fat‐diet‐fed rats, blockade of CB1 ...receptor by AM251 not only reduced body weight but also increased adult neurogenesis in the hippocampus, suggesting an influence of diet on hippocampal cannabinoid function. To further explore the role of hippocampal ECS in high‐fat‐diet‐induced obesity, we investigated whether the immunohistochemical expression of the enzymes that produce (diacylglycerol lipase alpha and N‐acyl phosphatidylethanolamine phospholipase D) and degrade (monoacylglycerol lipase and fatty acid amino hydrolase) endocannabinoids may be altered in the hippocampus of AM251 (3 mg/kg)‐treated rats fed three different diets: standard diet (normal chow), high‐carbohydrate diet (70% carbohydrate) and high‐fat diet (60% fat). Results indicated that AM251 reduced caloric intake and body weight gain, and induced a modulation of the expression of ECS‐related proteins in the hippocampus of animals exposed to hypercaloric diets. These effects were differentially restricted to either the 2‐arachinodoyl glycerol or anandamide signaling pathways, in a diet‐dependent manner. AM251‐treated rats fed the high‐carbohydrate diet showed a reduction of the diacylglycerol lipase alpha : monoacylglycerol lipase ratio, whereas AM251‐treated rats fed the high‐fat diet showed a decrease of the N‐acyl phosphatidylethanolamine phospholipase D : fatty acid amino hydrolase ratio. These results are consistent with the reduced levels of hippocampal endocannabinoids found after food restriction. Regarding the CB1 expression, AM251 induced specific changes focused in the CA1 stratum pyramidale of high‐fat‐diet‐fed rats. These findings indicated that the cannabinoid antagonist AM251 modulates ECS‐related proteins in the rat hippocampus in a diet‐specific manner. Overall, these results suggest that the hippocampal ECS participates in the physiological adaptations to different caloric diets.
Diet‐induced obesity produces changes in endocannabinoid signaling (ECS), influencing the regulation of energy homeostasis. Recently, we demonstrated that in high‐fat diet‐fed rats, blockade of CB1 receptor by AM251 not only reduced body weight but also increased adult neurogenesis in the hippocampus, suggesting an influence of diet on hippocampal cannabinoid function.
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