Acta Neuropathol (2010) 119:7–35 9 Fig. 2 Schematic representations that summarize, a astrocyte func- tions in healthy CNS, and b triggers and molecular regulators of reactive astrogliosis Nevertheless, it is important to recognize the appropriate uses and limitations of GFAP as an astrocyte marker. GFAP was ﬁrst isolated as a protein highly concentrated in old demyelinated plaques from multiple sclerosis patients and was then found to be associated immunohistochemically with reactive astrocytes in such plaques and in other patho- logical contexts [62, 63]. In line with this original mode of identiﬁcation, GFAP expression can be regarded as a sensi- tive and reliable marker that labels most, if not all, reactive astrocytes that are responding to CNS injuries (Fig. 3). However, GFAP is not an absolute marker of all non-reactive astrocytes and is often not immunohistochemically detectable in astrocytes in healthy CNS tissue or remote from CNS lesions (Figs. 3a, 4a). Although GFAP is immunohistochemically detectable in many astrocytes throughout the healthy CNS, it is clear from double staining with multiple markers (including transgenic reporter pro- teins) that many mature astrocytes in healthy CNS tissue do not express detectable levels of GFAP and that GFAP expression by astrocytes exhibits both regional and local variability that is dynamically regulated by a large number of inter- and intra-cellular signaling molecules [226]. GFAP has been studied extensively. GFAP is one of a family of intermediate ﬁlament proteins, including vimentin, nestin, and others, that serve largely cyto-archi- tectural functions [179]. Studies in transgenic mice indicated that the expression of GFAP is not essential for the normal appearance and function of most astrocytes in healthy CNS of transgenic mice, but is essential for the process of reactive astrogliosis and glial scar formation [95, 179, 180]. There are different isoforms and splice variants of GFAP including GFAP a, b, c, d, and j, and these may be expressed in a heterogeneous manner in both healthy CNS and in pathological specimens including glioma, but the differential distribution and roles of GFAP isoforms are only beginning to be studied [4, 20, 195]. At the single-cell level, in line with its structural role, GFAP is not present throughout astrocyte cytoplasm, and GFAP immunohistochemistry does not label all portions of the astrocyte but only in the main stem branches (Fig. 1b). GFAP is entirely absent from the ﬁnely branching astrocyte processes and is often not detectably present in the cell body (Figs. 1b, 3a). Consequently, GFAP immunohisto- chemistry can markedly underestimate the extent of astrocyte branching and territory in comparison with other means of detection such as Golgi staining (Fig. 1a) or expression of reporter proteins such as GFP or b-galacto- sidase (Fig. 1b), or ﬁlling with ﬂuorescent dyes [34]. In addition, as regards the use of GFAP as an astrocyte marker, it is important to note that GFAP expression is not exclusive to protoplasmic and ﬁbrous astrocytes. Within the CNS, GFAP is also expressed by a number of cells that can be considered part of an extended astroglial family that is described in more detail below. Outside of the CNS, GFAP is expressed widely in many tissues by a variety of cell types, also discussed below. Other molecular markers that have been used for immunohistochemical identiﬁcation of astrocytes and reactive astrocytes include glutamine synthetase and S100b [85, 165], but these molecules are not entirely exclusive to astrocytes. Several recent studies have conducted large- scale genetic analysis of the astrocyte transcriptome in rodents and humans, and have identiﬁed large numbers of molecules enriched in astrocytes as compared with other neural cells such as neurons and oligodendrocytes [35, 129]. Such studies will provide a rich resource for identi- fying molecules for mechanistic analyses of functions and 123