Brain ischemia resembles other brain injuries in producing enhanced neurogenesis in neuroproliferative regions of the rodent brain, including subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the dentate gyrus (DG) of the hippocampus. Newly-generated neurons would be incorporated in the hippocampal local circuitry and involved in brain repair. Organotypic culture of hippocampal slices (OHC) provides an alternative model of hippocampus in vivo. Moreover, exposure of the organotypic slices to oxygen and glucose deprivation (OGD) mimics cerebral ischemia. The aim of the present study was to investigate whether deprivation of oxygen and glucose might stimulate cell proliferation and neurogenesis in organotypic hippocampal slice culture. Furthermore, we evaluate whether the activity of matrix metalloproteinases (MMPs) in the OHC parallels the rate of cell proliferation and/or further differentiation. Cell death in the organotypic hippocampal slices was determined with propidium iodide (IP) staining. Stem cells proliferation was detected by using DNA replication marker – 5-Bromo 2-Deoxyuridine (BrdU) followed by immunoreaction with specific antibodies. Newly generated BrdU(+) cells were identified by an analysis of neural, glial and microglial markers expression – NF-200 NeuN, GFAP, ED1, respectively. In order to check the activity and localization of metalloproteinases, MMP-2 and MMP-9, we conducted in situ zymography in conjunction with immunohistochemistry. Exposing rat OHC for 40 min OGD followed by 24h of reoxygenation induces cell death in CA1 area with only negligible damage in DG. At 1 week cell death appears all over the slice in control conditions as well as after OGD. The stimulation of cell proliferation was observed 7 days after OGD exclusively in CA1. At the same time the number of BrdU(+) cells in DG remained on the level characteristic for control cultures. The majority of BrdU positive cells presents expression of microglial specific stain (ED1) pronounced particularly in CA1 at 3 days after OGD. However, some BrdU labeled nuclei were encapsulated by GFAP positive processes especially in CA1 region of the hippocampus (3 and 7 days after OGD). We do not notice coexpression of BrdU-positive cells with NeuN(+) mature neurons. The study suggests that slice cultures do not show neurogenesis for chosen cultivation period. Activation of MMPs was localized mainly in microglial cells and may be associated with their proliferation in situ. Supported by MSHE grant no 0154/B/P01/2010/38
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