Neurogenesis in organotypic hippocampal slice cultures inhibited by inflammation early after oxygen-glucose deprivation is restored at later time point

Abstract

The neurogenesis in the dentate gyrus (DG) of the hippocampus is regulated by many factors including the pathological conditions like stroke, epilepsy, traumatic injury or inflammation. Increase of neurogenesis occurring in response to injury has been reported and considered as a mechanism of regeneration after neuronal loss. We studied the neurogenesis in organotypic hippocampal slice cultures (OHCs) after oxygen and glucose deprivation (OGD). 14 days old OHCs from P7-9 rats were exposed to 40 min of OGD. OGD caused the neuronal damage that reached the significant level at 6h as revealed by propidium iodide uptake. At the same time point activation and increase in number of microglial cells was observed that resulted in the development of inflammatory reaction in the system. Application of anti-inflammatory drug indomethacine (10M) prevented neuronal degeneration. Upregulation of pro-inflammatory cytokines was found 8h after OGD. qPCR showed 4 fold increase in the IL-1b and 2 fold increase in TNF-a mRNA. To label proliferating cells the OHCs were treated with bromodeoxyuridine (BrdU) and increase in cell proliferation was revealed by BrdU immunohistochemistry at 16h after OGD. Newly generated neurons were detected with early neuronal markers doublecortin (DCX) and -III Tubulin. The neurogenesis (DCX immunostaining) was restricted in the dentate gyrus where the BrdU/DCX+ neurons could be found already at day 3 after first BrdU application with further increase to day 6. At early time point (3d) after OGD very few BrdU/DCX+ cells might be observed in DG however at 6d the number of BrdU/DCX+ neurons increased and some of the newly generated neurons expressed -III Tubulin. Our data indicate that OGD results in the neuronal degeneration and activation of inflammatory reactions that inhibit the basal neurogenesis in the DG at early time point. However neurogenesis seems to restore later on.