Microglia in Culture

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Culture experiments have allowed the study of basic functional capacities of microglia. Most of these studies have been performed with microglia that were isolated from mixed glial cell cultures based on newborn rodent brain as starting material. In these isolated culture systems, microglia lose their characteristic ramified shape and round up to an ameboid morphology reminescent of activated microglia in areas of tissue remodeling during development and after brain injury (Fig. 3A). Compared to the normal in vivo situation, production of inflammatory cytokines is markedly enhanced in cultured microglia and likely further increased by the presence of serum that is routinely added to the culture medium but normally excluded from the brain parenchyma.

Treatment of cultured microglia with proinflammatory substances such as interferon-g (IFN-g), which is

Ameboid Microglia Tissue

Figure 2 Perivascular microglia in the rat brain. Immunolabeling with ED2 antibody reveals a unique cell population of microglia also designated perivascular cells (A) that also express CD4 (B). Note the exclusive localization of the ED2 + (A) and CD4 + (B) microglia in close vicinity to cerebral blood vessels. (C-E) Perivascular microglia at higher magnification and their expression of ED2 (C), CD4 (D), and MHC class II antigens (E). Scale bars = 40 mm in A and B and 20 mm in (C-E).

Figure 2 Perivascular microglia in the rat brain. Immunolabeling with ED2 antibody reveals a unique cell population of microglia also designated perivascular cells (A) that also express CD4 (B). Note the exclusive localization of the ED2 + (A) and CD4 + (B) microglia in close vicinity to cerebral blood vessels. (C-E) Perivascular microglia at higher magnification and their expression of ED2 (C), CD4 (D), and MHC class II antigens (E). Scale bars = 40 mm in A and B and 20 mm in (C-E).

secreted by T cells during inflammation, leads to induction of MHC class II antigens and a number of adhesion and costimulatory molecules, such as intercellular adhesion molecule-1 (ICAM-1), B7-1, and CD40, that are essential participants in the process of antigen-specific T cell stimulation. Accordingly, activated microglia derived from newborn rodent brain efficiently stimulated the activation and proliferation of antigen-specific T cells in vitro, supporting a role for microglia as the major antigen-presenting and immune-stimulatory cell type in the mammalian brain. However, this concept has been challenged by recent studies using ramified microglia isolated from the adult brain that have shown downregulatory rather than immune-stimulatory interactions of parenchymal mi-croglia with T cells.

When microglial cells are grown on an astrocytic monolayer they develop processes similar to those of

Figure 3 Rat microglia in culture dishes: immunostaining for complement receptor 3 by monoclonal antibody Ox42, a microglial marker. (A) Note the round (ameboid) morphology of isolated microglia resembling monocytes/macrophages. In the presence of astrocytes in primary mixed glial cultures (barely visible in the background), microglia develop multiple processes and acquire their distinctive morphological features (B). Scale bar = 20 mm.

Figure 3 Rat microglia in culture dishes: immunostaining for complement receptor 3 by monoclonal antibody Ox42, a microglial marker. (A) Note the round (ameboid) morphology of isolated microglia resembling monocytes/macrophages. In the presence of astrocytes in primary mixed glial cultures (barely visible in the background), microglia develop multiple processes and acquire their distinctive morphological features (B). Scale bar = 20 mm.

ramified microglia in brain parenchyma (Fig. 3B). This points to an important influence of astroglia on microglial properties that is further substantiated by the fact that coculturing microglia with astrocytes markedly suppressed phagocytosis. Nondiffusible matrix components as well as astrocyte-derived cytokines such as the macrophage colony-stimulating factor (M-CSF) have been implicated in the induction of microglial ramification in these coculture models. In support of the proposed hematogenous origin of ramified microglia, monocytes or spleen macrophages brought into coculture with astrocytes likewise displayed a ramified morphology that was indistinguishable from that of CNS-derived microglia. Functionally, ramification was accompanied by the downregulation of immunomoleucles such as MHC class II, leukocyte function-associated antigen-1, and ICAM-1. Microglia bear a variety of receptor molecules on their surface, facilitating signaling through complement components, growth factors, cytokines, and chemokines. Antiinflammatory cytokines, such as interleukin-(IL-10) and transforming growth factor-b (TGF-b), can effectively suppress the expression of MHC class II antigens on microglia as well as neuronally produced factors such as brain-derived neurotrophic factor. In vivo, the cellular microenvir-

onment of the CNS thus provides multiple autocrine as well as paracrine antiinflammatory feedback loops that tightly control the potentially harmful activation of microglia in disease states.

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Responses

  • Diamante
    How do you do gene expression on microglia cells in culture?
    11 months ago

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