Form Of Microglia Research Articles

A new method for obtaining bankable and expandable adult-like microglia in mice

BackgroundThe emerging role of microglia in neurological disorders requires a novel method for obtaining massive amounts of adult microglia. We aim to develop a new method for obtaining bankable and expandable adult-like microglia in mice.MethodsThe head neuroepithelial layer (NEL) that composed of microglial progenitor and neuroepithelial cells at mouse E13.5 was dissected and then cultured or banked. Microglia (MG) isolated from the cultured NEL by magnetic-activated cell sorting system were obtained and named NEL-MG.ResultsThe NEL included microglia progenitors that proliferate and ramify over time with neuroepithelial cells as feeder. In functional analysis, NEL-MG exhibited microglial functions, such as phagocytosis (microbeads, amyloid β, synaptosome), migration, and inflammatory response following lipopolysaccharide (LPS) stimulation. NEL was passage cultured and the NEL-MG exhibited a higher expression of microglia signature genes than the neonatal microglia, a widely used in vitro surrogate. Banking or long-term passage culture of NEL did not affect NEL-MG characteristics. Transcriptome analysis revealed that NEL-MG exhibited better conservation of microglia signature genes with a closer fidelity to freshly isolated adult microglia than neonatal microglia. NEL-MG could be re-expandable when they were plated again on neuroepithelial cells.ConclusionsThis new method effectively contributes to obtaining sufficient matured form of microglia (adult-like microglia), even when only a small number of experimental animals are available, leading to a broad application in the field of neuroscience.

Microglial Morphometric Parameters Correlate With the Expression Level of IL-1β, 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-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.

Alerted microglia and the sympathetic nervous system: A novel form of microglia in the development of hypertension

Microglia, commonly known as the tissue resident macrophages of the central nervous system (CNS), are ubiquitously expressed in the CNS. Microglia, in their resting, or surveilling, stage, play a critical role in the maintenance of normal neuronal physiology and homeostasis. On activation, microglia can acquire either a neurotoxic (M1) or a neuroprotective (M2) phenotype. Prior to development of the M1 or M2 phenotype, little was known about changes in microglial activity, when subjected to stimuli. It is postulated, that an inability of microglia to maintain neuronal physiology within a normal working range can contribute to the development of cardiovascular disorders (CVDs) such as hypertension, but clear evidence supporting this hypothesis is missing. Even though our understanding of microglial function in a state of CNS injury/inflammation is extensive, the literature concerning role of microglia in the healthy CNS, is limited. Involvement of microglia in the pathophysiology of CVDs, in a neuroprotective/neurotoxic manner, is a key area that requires further investigation.

Microglial changes accompanying the promotion of retinal ganglion cell axonal regeneration into peripheral nerve grafts.

Intravitreal injection of the microglia inhibitor tuftsin 1-3 leads to an increase in retinal ganglion cell axonal regeneration into peripheral nerve grafts and a decrease in phagocytic cells in the retina. However, the relation of phagocytic cells and particularly microglia towards axonal regeneration remains unclear. Initially, to assess this, tuftsin 1-3's effect on axonal regeneration was reexamined by doing a dose-response study. Optimal doses were found to be 2.5 microg/ml and 250 microg/ml in rats and hamsters respectively. We then studied retinal phagocytic cells in rats. Microglial cells were classified as resting or activated based on their morphology following OX42 immunolabelling. In controls, most microglial cells were in the resting state. Optic nerve cut led to an increase in the total number of microglia and a ten-fold elevation in the proportion of activated cells; changes were more pronounced at the optic nerve stump. Anastomosis of an autologous segment of sciatic nerve to the stump of the freshly cut optic nerve minimized the overall increase in microglia, and combined with 2.5 microg/ml tuftsin 1-3, lead to a marked blunting of activation. Preservation within the retina of a higher proportion of resting over active form of microglia, and not the prevention of microglial proliferation per se, may be a crucial factor in allowing additional retinal ganglion cell axons to regenerate into peripheral nerve grafts.

Bacterial Endotoxin Induces Changes in the Organization and Expression of Actin and Modulation of the Cell Membrane of Microglia

Background and Objectives: In situ there are at least three morphologically and functionally different forms of microglia: the resting, the activated, and the phagocytic microglia. The signals promoting the morphological changes which adapt microglia to specific functions are still unknown. In this study the effect of bacterial wall lipopolysaccharide (LPS) on the morphology and organization and expression of actin in microglia was investigated. In addition, the changes in the appearance of the cell membrane were investigated. Methods: Microglia cultures were prepared from neopallia of newborn mice and treated with LPS. Scanning electron microscopy, labeling with phalloidin, and immunoblotting were used. Results: The majority of nontreated microglia were ameboid in shape with many short processes that extended into lamellipodia. When microglia were treated with LPS most of the microglia acquired a large, round, and flat shape. The rest of the ameboid microglia became larger in size. Fluorescent labeling with phalloidin showed that the F-actin network appeared diffusely arranged throughout the cytoplasm of nontreated microglia. In LPS-treated microglia the F-actin network was reorganized into filamentous bundles extending into microspike-like projections. Using scanning electron microscopy, the nontreated microglia had large membrane folds and few large blebs. In LPS-treated microglia most of the membrane folds and blebs at the cell periphery disappeared with the appearance of many microspike-like projections. Immunoblotting showed that LPS-treated microglia upregulated their actin protein. Conclusions: These changes in the organization of F-actin and the cell membrane may reflect adaptation of activated microglia to specific functional activity, such as increases in their phagocytic activity.

Identification of microglia in primary cultures of mixed cerebral cortical cells

Microglia were investigated in tissue cultures of dissociated cerebral cortex from embryonic rat. Three different forms of microglia were identified in relatively mature cultures on the basis of morphological criteria, esterase histochemistry and immunohistochemical staining for Fc receptors. Two of these forms corresponded to the amoeboid and ramified microglia; the amoeboid form stained intensely for esterase activity and ramified cells possessed Fc receptors. The third microglial cell form was distinguished by its morphological properties and was characterized by the presence of numerous filopodial processes. This morphological form occasionally possessed esterase staining and was the predominate type of microglia observed in the cultures. The identification of these cell types supports the potential use of this culture system to investigate functional aspects of microglia and their interaction with other brain cells.