tsa trichostatin We hypothesized that type B astrocytes migh

We hypothesized that type B astrocytes might be among the other SVZ-derived cell types able to migrate in response to a damage insult. In this study we used Nilo1, a previously characterized mAb that identifies early neural progenitors in the SVZ niche (Del Valle et al., 2010), demonstrating that Nilo1+ cells showed an immunophenotype and subependymal localization compatible with B astrocytes. After coupling Nilo1 mAb to magnetic nanoparticles, we followed by MRI and confirmed by immunohistochemistry the fast mobilization of B astrocytes towards the lesion site, as a trait shared by different tsa trichostatin injuries in adult mice.
Materials and methods
Results
Discussion Although there are evidences that immature neurons present in brain lesion sites, such as peri-infarcted tissue, come from GFAP+ SVZ-derived neural stem cells (Garcia et al., 2004; Ohab and Carmichael, 2008; Ohab et al., 2006), a direct mobilization of type B astrocytes towards a lesion site has not yet been reported. To study the migration of early neural progenitors towards a brain injury, we used Nilo1 mAb. Here we demonstrate that Nilo1 identified type B astrocytes (or adult neural stem cells) since i) it recognized GFAP+, Sox2+ and EGFR+ cells, being negative for the neuroblast markers DCX and Nilo2; ii) Nilo1 did not stain ependymal CD24+ cells, having instead subependymal localization on the SVZ; iii) transient amplifying cells (or type C cells, which are Pax6highBrdU+ on tissue sections from in vivo labeled animals with a short BrdU pulse) (Aguirre and Gallo, 2004; Kim et al., 2009; Parras et al., 2004) and Nilo1+ cells represented two distinct populations; iv) Nilo1+ cells were GFAP+, excluding the possibility that this mAb identified intermediate progenitors in the differentiation process, like NG2-precursors which are GFAP− (Dawson et al., 2003; Nishiyama et al., 2005); v) double staining of E10 embryo brains with Nilo1 and either vimentin or nestin suggested that Nilo1 recognizes embryonic radial glia. Nilo1 mAb, coupled to functionalized magnetic nanoparticles in combination with magnetic resonance imaging allowed us to identify the Nilo1+ cells at their niche in the SVZ and, in animals carrying a lesion, at intermediate positions between the niche and the lesion site, where they accumulated over time. The migratory response was very fast since MRI hypointense signals at the lesion site were detected by MRI 3h after injection of the Nilo1–mGNP complexes in animals bearing tumors, or 3.5h after injection of the CT-2A cells on animals that had previously been injected with Nilo1–mGNP complexes. The presence of Nilo1+ cells at the lesion site was corroborated by immunohistochemistry analyses on fixed brain sections from these mice, incubating with a fluorescent secondary antibody, since the Nilo1+ cells were already labeled in vivo with the Nilo1–mGNPs. Immunohistochemistry confirmed not only that the hypointense signals corresponded to Nilo1+ cells surrounding the tumor, but also that these cells retained their type B astrocyte phenotype (GFAP+, EGFR+, Sox2+) following their migration. The migration of adult neural stem cells (type B astrocytes) towards a tumor site, rather than a tumor-specific response, turned out to be a more generalized response to brain insults since we detected Nilo1+ cells at the sites where other types of lesions were produced (i.e. cryolesion, demyelination, mechanical injury). These lesions were chosen because they are very different from each other and from the tumor implantation model described above. In all of them, Nilo1+ cells were detected surrounding the lesions, although we cannot formally exclude at this time that some of the Nilo1+ cells surrounding the lesion represent cells around the injury site de novo expressing Nilo1 upon injury. In addition to Nilo1+ cells migrating to the lesion sites, both in a mechanical lesion model and after any stereotactic injection, we detected, one day after the lesion, coalescing structures filling the wound-lesion that expressed high levels of Nilo1 antigen. These Nilo1+ processes were GFAP+, vimentin− and CD11b−, forming structures associated to PSA-NCAM. On the one hand, the observation that these structures did not express vimentin or CD11b allowed us to exclude that they represented proximal reactive astrocytes (Holmin et al., 1997; Raedt et al., 2009; Ridet et al., 1997; Robel et al., 2011). On the other hand, their GFAP+ staining was compatible with the presence of adult radial glia fibers or glial tubes. In physiological conditions they have been described to support the migration of neuroblasts either towards the olfactory bulb (Doetsch and Alvarez-Buylla, 1996; Ohab and Carmichael, 2008; Yang et al., 2005), acting as substrate for the migrating progenitor cells after focal apoptosis in the adult brain (Leavitt et al., 1999), or from their proliferation zones to the lesion sites in adult brain in fish (Clint and Zupanc, 2001; Zupanc and Clint, 2003). The appearance of fibers either from astrocytes or adult radial glia emanating from their SVZ subependymal position has also been described not only under physiological conditions (Kriegstein and Alvarez-Buylla, 2009; Thored et al., 2006; Teramoto et al., 2003), but also in rodent or fish brain lesions (Thored et al., 2006; Holmin et al., 1997; Ohab and Carmichael, 2008; Clint and Zupanc, 2001; Zupanc and Clint, 2003; Szele and Chesselet, 1996). Radial glia cells have been defined as neuronal precursors (Robel et al., 2011; Noctor et al., 2001; Malatesta et al., 2000) and it has been suggested that they could represent a specific subpopulation of astrocytes in adult mammals (Kriegstein and Alvarez-Buylla, 2009; Robel et al., 2011; Noctor et al., 2001; Malatesta et al., 2000; Gubert et al., 2009). Furthermore, the presence of radial glia in adult hippocampus where neurogenesis occurs throughout life, or in non-mammalian vertebrates where neurogenesis persists in a rather wide-spread fashion in the adult brain (Clint and Zupanc, 2001; Pinto and Gotz, 2007), raise the possibility that the neurogenic potential of radial glia may extend into adulthood in some brain regions (Noctor et al., 2001) or even as an acute response to brain lesions, were neurogenesis is necessary and commonly associated to brain tissue repairing processes.