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Methods to assess neuroinflammation.
Title of the book
Current protocols in Toxicology
Neuroinflammation is observed in many brain pathologies: in neurodegenerative diseases and multiple sclerosis as well as in chemically induced lesions. It is characterized by the reactivity of microglial cells and astrocytes, activation of inducible NO-synthase (i-NOS), and increased expression and/or release of cytokines and chemokines. Clearly, cell-to-cell signaling between the different brain cell types plays an important role in the initiation and propagation of neuroinflammation, but despite the growing list of known molecular actors, the underlying pathways and the sequence of events remain to be fully elucidated. The present chapter presents an example of how to assess neuroinflammation in complex brain tissues, using aggregating brain cell cultures as an in vitro model. This three-dimensional cell culture system provides optimal cell-to-cell interactions crucial for histotypic cellular maturation and control of neuroinflammatory processes. The techniques described here comprise immunocytochemistry to assess the reactivity of microglia and astrocytes and the expression of cytokines; quantitative RT-PCR to measure the mRNA expression of cytokines (TNF-α, IL-1β, IL-6, IL-1ra, TGF-β, IL-15, IFN-γ), chemokines (ccl5, cxcl1, cxcl2), and i-NOS; and immunoblotting to assess MAP kinase pathway activation (phosphorylation of p38 and p44/42 MAP kinases).
Animals, Astrocytes/cytology, Astrocytes/immunology, Biological Markers/analysis, Cell Culture Techniques/methods, Cytokines/biosynthesis, Cytokines/genetics, Encephalitis/immunology, Encephalitis/metabolism, Humans, Immunoblotting, Immunohistochemistry, Microglia/cytology, Microglia/immunology, Mitogen-Activated Protein Kinases/metabolism, RNA, Messenger/genetics, Reverse Transcriptase Polymerase Chain Reaction
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