It is known that microglia morphology and function are closely related, but only few studies have subjectively 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 within the lateral ventricle 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 hours). 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. This method pointed out the classification of microglia population in four clusters. Furthermore, a linear discriminant analysis suggested three specific parameters to objectively classify any microglia by a decision tree. In addition, a principal components analysis 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 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.

Background: 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.Methods: 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).Results: 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.Conclusions: 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.

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.

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.