Adult neurogenic sites are specialized microenvironments (niches) regulating neurogenesis in restricted brain regions. In the subventricular zone (SVZ), neurogenesis is supported by neural stem cells (NSCs). They are astrocytic cells which proliferate at low rate and contribute transit-amplifying progenitors which expand to generate neuroblasts (Doetsch, 2003). During the first three weeks after birth, radial glia transform into into SVZ astrocytes, but how this glial population changes remains to be understood (Bonfanti & Peretto, 2007). We are developing an ex vivo model of forebrain organotypic slices which contain the SVZ, to be employed as an alternative way of investigating the NSC niche. We adapted the original protocol for hippocampal cultures (Stoppini, 1991) to slices containing both neurogenic (SVZ) and non-neurogenic tissue (striatum, cortex). We extended this ex vivo approach to all ages involved in the SVZ postnatal modifications (0, 5, 13, 21 days from birth). The goal is a systematic study to define ultrastructural/antigenic features of SVZ niche at different time points in vitro (0, 12, 24, 48 hrs), focusing on the different cell types and their three-dimensional relationships. We show that conditions for these cultures change through the postnatal ages, due to modifications of the non-neurogenic tissue (Canalia, in preparation). Cell viability and behaviour are different in the SVZ and non-neurogenic tissue. Proliferation markers (BrdU, PH3) revealed that mitotic activity in the SVZ culture is not affected at any time point considered. We quantified apoptosis (casp3A1 cells) at the light and ultrastructural level. At early in vitro time points (t0-t6) SVZ cells are more healthy than surrounding tissue. With CD11 marker, we found both highly ramified (quiescent microglia) and fibroblastic (reactive microglia), maybe a possible gliosis resulting from technical procedures. We also started to compare the postnatal modification of glial populations in vivo and in culture. Our results show that it is possible to get viable organotypic cultures at different ages (first three postnatal weeks) that preserve SVZ cell types and relationships between neurogenic and non-neurogenic tissue, although changes in the SVZ three-dimensional arrangement start to occur after 24 hrs in colture.
Organotypic culture: an ex vivo approach to study postnatal modification
ARMENTANO, MARIA;AIMAR, Patrizia;BONFANTI, Luca
2009-01-01
Abstract
Adult neurogenic sites are specialized microenvironments (niches) regulating neurogenesis in restricted brain regions. In the subventricular zone (SVZ), neurogenesis is supported by neural stem cells (NSCs). They are astrocytic cells which proliferate at low rate and contribute transit-amplifying progenitors which expand to generate neuroblasts (Doetsch, 2003). During the first three weeks after birth, radial glia transform into into SVZ astrocytes, but how this glial population changes remains to be understood (Bonfanti & Peretto, 2007). We are developing an ex vivo model of forebrain organotypic slices which contain the SVZ, to be employed as an alternative way of investigating the NSC niche. We adapted the original protocol for hippocampal cultures (Stoppini, 1991) to slices containing both neurogenic (SVZ) and non-neurogenic tissue (striatum, cortex). We extended this ex vivo approach to all ages involved in the SVZ postnatal modifications (0, 5, 13, 21 days from birth). The goal is a systematic study to define ultrastructural/antigenic features of SVZ niche at different time points in vitro (0, 12, 24, 48 hrs), focusing on the different cell types and their three-dimensional relationships. We show that conditions for these cultures change through the postnatal ages, due to modifications of the non-neurogenic tissue (Canalia, in preparation). Cell viability and behaviour are different in the SVZ and non-neurogenic tissue. Proliferation markers (BrdU, PH3) revealed that mitotic activity in the SVZ culture is not affected at any time point considered. We quantified apoptosis (casp3A1 cells) at the light and ultrastructural level. At early in vitro time points (t0-t6) SVZ cells are more healthy than surrounding tissue. With CD11 marker, we found both highly ramified (quiescent microglia) and fibroblastic (reactive microglia), maybe a possible gliosis resulting from technical procedures. We also started to compare the postnatal modification of glial populations in vivo and in culture. Our results show that it is possible to get viable organotypic cultures at different ages (first three postnatal weeks) that preserve SVZ cell types and relationships between neurogenic and non-neurogenic tissue, although changes in the SVZ three-dimensional arrangement start to occur after 24 hrs in colture.
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