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Intravital correlated microscopy reveals differential macrophage and microglial dynamics during resolution of neuroinflammation.

ABSTRACT: Many brain diseases involve activation of resident and peripheral immune cells to clear damaged and dying neurons. Which immune cells respond in what way to cues related to brain disease, however, remains poorly understood. To elucidate these in vivo immunological events in response to brain cell death we used genetically targeted cell ablation in zebrafish. Using intravital microscopy and large-scale electron microscopy, we defined the kinetics and nature of immune responses immediately following injury. Initially, clearance of dead cells occurs by mononuclear phagocytes, including resident microglia and macrophages of peripheral origin, whereas amoeboid microglia are exclusively involved at a later stage. Granulocytes, on the other hand, do not migrate towards the injury. Remarkably, following clearance, phagocyte numbers decrease, partly by phagocyte cell death and subsequent engulfment of phagocyte corpses by microglia. Here, we identify differential temporal involvement of microglia and peripheral macrophages in clearance of dead cells in the brain, revealing the chronological sequence of events in neuroinflammatory resolution. Remarkably, recruited phagocytes undergo cell death and are engulfed by microglia. Because adult zebrafish treated at the larval stage lack signs of pathology, it is likely that this mode of resolving immune responses in brain contributes to full tissue recovery. Therefore, these findings suggest that control of such immune cell behavior could benefit recovery from neuronal damage.

SUBMITTER: van Ham TJ

PROVIDER: S-EPMC4073275 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Intravital correlated microscopy reveals differential macrophage and microglial dynamics during resolution of neuroinflammation.

van Ham Tjakko J TJ Brady Colleen A CA Kalicharan Ruby D RD Oosterhof Nynke N Kuipers Jeroen J Veenstra-Algra Anneke A Sjollema Klaas A KA Peterson Randall T RT Kampinga Harm H HH Giepmans Ben N G BN

Disease models & mechanisms 20140701 7

Many brain diseases involve activation of resident and peripheral immune cells to clear damaged and dying neurons. Which immune cells respond in what way to cues related to brain disease, however, remains poorly understood. To elucidate these in vivo immunological events in response to brain cell death we used genetically targeted cell ablation in zebrafish. Using intravital microscopy and large-scale electron microscopy, we defined the kinetics and nature of immune responses immediately followi ...[more]

PMID: 24973753

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Project description:Cellular communication constitutes a fundamental mechanism of life, for instance by permitting transfer of information through synapses in the nervous system and by leading to activation of cells during the course of immune responses. Monitoring cell-cell interactions within living adult organisms is crucial in order to draw conclusions on their behavior with respect to the fate of cells, tissues and organs. Until now, there is no technology available that enables dynamic imaging deep within the tissue of living adult organisms at sub-cellular resolution, i.e. detection at the level of few protein molecules. Here we present a novel approach called multi-beam striped-illumination which applies for the first time the principle and advantages of structured-illumination, spatial modulation of the excitation pattern, to laser-scanning-microscopy. We use this approach in two-photon-microscopy--the most adequate optical deep-tissue imaging-technique. As compared to standard two-photon-microscopy, it achieves significant contrast enhancement and up to 3-fold improved axial resolution (optical sectioning) while photobleaching, photodamage and acquisition speed are similar. Its imaging depth is comparable to multifocal two-photon-microscopy and only slightly less than in standard single-beam two-photon-microscopy. Precisely, our studies within mouse lymph nodes demonstrated 216% improved axial and 23% improved lateral resolutions at a depth of 80 µm below the surface. Thus, we are for the first time able to visualize the dynamic interactions between B cells and immune complex deposits on follicular dendritic cells within germinal centers (GCs) of live mice. These interactions play a decisive role in the process of clonal selection, leading to affinity maturation of the humoral immune response. This novel high-resolution intravital microscopy method has a huge potential for numerous applications in neurosciences, immunology, cancer research and developmental biology. Moreover, our striped-illumination approach is able to improve the resolution of any laser-scanning-microscope, including confocal microscopes, by simply choosing an appropriate detector.

Project description:BackgroundIn the healthy central nervous system (CNS), microglia are found in a homeostatic state and peripheral macrophages are absent from the brain. Microglia play key roles in maintaining CNS homeostasis and acting as first responders to infection and inflammation, and peripheral macrophages infiltrate the CNS during neuroinflammation. Due to their distinct origins and functions, discrimination between these cell populations is essential to the comprehension of neuroinflammatory disorders. Studies comparing the gene profiles of microglia and peripheral macrophages, or macrophages in vitro-derived from bone marrow, under non-infectious conditions of the CNS, have revealed valuable microglial-specific genes. However, studies comparing gene profiles between CNS-infiltrating macrophages and microglia, when both are isolated from the CNS during viral-induced neuroinflammation, are lacking.MethodsWe isolated, via flow cytometry, microglia and infiltrating macrophages from the brains of Theiler's murine encephalomyelitis virus-infected C57BL/6 J mice and used RNA-Seq, followed by validation with qPCR, to examine the differential transcriptional profiles of these cells. We utilized primary literature defining subcellular localization to determine whether or not particular proteins extracted from the transcriptional profiles were expressed at the cell surface. The surface expression and cellular specificity of triggering receptor expressed on myeloid cells 1 (TREM-1) protein were examined via flow cytometry. We also examined the immune response gene profile within the transcriptional profiles of these isolated microglia and infiltrating macrophages.ResultsWe have identified and validated new microglial- and macrophage-specific genes, encoding cell surface proteins, expressed at the peak of neuroinflammation. TREM-1 protein was confirmed to be expressed by infiltrating macrophages, not microglia, at the peak of neuroinflammation. We also identified both unique and redundant immune functions, through examination of the immune response gene profiles, of microglia and infiltrating macrophages during neurotropic viral infection.ConclusionsThe differential expression of cell surface-specific genes during neuroinflammation can potentially be used to discriminate between microglia and macrophages as well as provide a resource that can be further utilized to target and manipulate specific cell responses during neuroinflammation.

Dataset Information

Intravital correlated microscopy reveals differential macrophage and microglial dynamics during resolution of neuroinflammation.

Publications

Intravital correlated microscopy reveals differential macrophage and microglial dynamics during resolution of neuroinflammation.

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