Abstract
BackgroundMicroglial cells are tissue-resident macrophages of the central nervous system. They are extremely dynamic, sensitive to their microenvironment and present a characteristic complex and heterogeneous morphology and distribution within the brain tissue. Many experimental clues highlight a strong link between their morphology and their function in response to aggression. However, due to their complex “dendritic-like” aspect that constitutes the major pool of murine microglial cells and their dense network, precise and powerful morphological studies are not easy to realize and complicate correlation with molecular or clinical parameters.MethodsUsing the knock-in mouse model CX3CR1GFP/+, we developed a 3D automated confocal tissue imaging system coupled with morphological modelling of many thousands of microglial cells revealing precise and quantitative assessment of major cell features: cell density, cell body area, cytoplasm area and number of primary, secondary and tertiary processes. We determined two morphological criteria that are the complexity index (CI) and the covered environment area (CEA) allowing an innovative approach lying in (i) an accurate and objective study of morphological changes in healthy or pathological condition, (ii) an in situ mapping of the microglial distribution in different neuroanatomical regions and (iii) a study of the clustering of numerous cells, allowing us to discriminate different sub-populations.ResultsOur results on more than 20,000 cells by condition confirm at baseline a regional heterogeneity of the microglial distribution and phenotype that persists after induction of neuroinflammation by systemic injection of lipopolysaccharide (LPS). Using clustering analysis, we highlight that, at resting state, microglial cells are distributed in four microglial sub-populations defined by their CI and CEA with a regional pattern and a specific behaviour after challenge.ConclusionsOur results counteract the classical view of a homogenous regional resting state of the microglial cells within the brain. Microglial cells are distributed in different defined sub-populations that present specific behaviour after pathological challenge, allowing postulating for a cellular and functional specialization. Moreover, this new experimental approach will provide a support not only to neuropathological diagnosis but also to study microglial function in various disease models while reducing the number of animals needed to approach the international ethical statements.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0614-7) contains supplementary material, which is available to authorized users.
Highlights
Microglial cells are tissue-resident macrophages of the central nervous system
Hypotheses of an adaptation of their morphology to their specific function within the central nervous system (CNS) are formulated [11, 12] but neither could be substantiated due to two boundaries: (i) the difficulty-to-detect subtle morphological variations of microglia and probably intermediate forms between the categories of activation that are historically described [13, 14] and (ii) the need to couple a very precise morphological single cell approach with a proteomic or transcriptomic study using specific techniques [15] taking into account the regionalization that is specific to the CNS
No difference in the microglial cell green fluorescent protein (GFP) intensity between the two conditions, whatever the brain region, was found (Table 1 and Additional file 4)
Summary
Microglial cells are tissue-resident macrophages of the central nervous system. They are extremely dynamic, sensitive to their microenvironment and present a characteristic complex and heterogeneous morphology and distribution within the brain tissue. Microglia migrate to interact with other cell types (i.e. astrocyte and neurons) and produce a variety of factors required to induce neural progenitor differentiation [5] and neuronal apoptosis [6] Their activation is the main component of the neuroinflammatory process, which can result either from direct brain insult or systemic inflammation leading to either neuroprotective [7, 8] or neurotoxic [9, 10] responses. Since the expression of these markers depends on the intensity of microglial activation, immunostaining can be insufficient for accurately describing a “dendritic-like” or ramified phenotype [20, 21] that constitutes more than 90 % of the microglial cells in young mice [11] It required particular histological techniques such as paraffin embedding that may affect the precision of a morphological study. These limitations might be overcome by the use of transgenic mice, such as an Iba-1GFP/+ [22] or CX3CR1GFP/+ [23] mouse in which brain microglia express spontaneously green fluorescent protein (GFP) respectively under the control of the Iba-1 or the CX3CL1
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