| Popis: |
As resident central nervous system (CNS) immune cells, microglia play an important role of active sensor and adaptable effector cells both in healthy brain and pathologic conditions, one of them being cerebral ischemia. Cerebral ischemia is followed by neuroinflammatory response initiated in ischemic penumbra – a metabolically active but neurophysiologically silent region around the infarct core. Part of this response includes the activation of microglia that along with macrophages first infiltrate penumbra where, depending on the surrounding conditions, this activation can lead to protection or exhibit detrimental effects. Direct effect of ischemia on microglia response can be examined in oxygen- glucose deprivation (OGD) model in vitro. What triggers the transformation of microglia from resting to activated state is still unclear. Therefore, we used BV-2 microglial cell line to examine the effect of dimethyoxalylglycine (DMOG), a prolyl hydroxylase inhibitor, and Necrostatin 1 (Nec-1), a RIP1 kinase inhibitor, on the response of ramified i.e. resting BV-2 cells to OGD followed by reoxygenation. 24h prior and during the cells’ exposure to OGD, they were grown in complete RPMI supplemented with 0, 5% FBS, to support ramified microglial phenotype. Either DMOG (10M) or Nec-1 (40M) were present during 8h of oxygen deprivation. Cell viability as well as the presence of apoptotic cells were followed after 16hr of reoxygenation period by MTS and Annexin V/Propidium iodide assay, respectively. Furthermore, changes in the activation of caspase 3 and RIP1K protein level in OGD exposed cells were examined by Western blot. While DMOG, protected BV-2 cells from dying as revealed by MTS assay, Nec-1 exhibited the opposite effect. Flow cytometric analysis has shown that DMOG protective effect is based primarily on reduction in the number of early apoptotic cells while Nec-1 increases their number. Also, DMOG reduced caspase 3 activity but did not affect RIP1 kinase protein level in OGD exposed microglia. The obtained data indicate that these two important enzymatic regulators of cell’s death could have a role in regulation of microglia’s response to OGD. |
