Effects of metformin, cyclosporine and rotenone on activation of developing brain microglia under normoxic and hypoxic conditions

Abstract

Hypoxia may affect neural tissue via microglia, however, mechanisms and consequences of microglial activation during hypoxia are not well understood. We aimed to investigate effects of inhibitors of mitochondrial complex I – metformin and rotenone, and classical inhibitor of permeability transition pore (mPTP) on microglial cells. Primary rat microglial cultures at 7–11 DIV were treated with metformin (Met, 3 mM), cyclosporin (CsA, 10 μM) and rotenone (Ro, 5 nM) under normoxic or hypoxic (2% O2) conditions for 24 h. We show that 24 h hypoxia reduced number of microglia and had a cell viability- -reducing effect compared to normoxia. Met had no effect on viability and number of cells, CsA under hypoxic conditions tended to decrease both –viability and cell number, while Ro decreased cell viability but had no effect on microglia cell numbers. Hypoxia increased glutamate in microglia culture media, and pre-treatment with Met but not Ro or CsA tended to reduce glutamate levels after hypoxia. We also found that none of the compounds effectively blocked mPTP opening in intact cells. Calcium dependent fluorescence measurements showed spontaneous calcium spikes; their generation was suppressed by CsA or trolox (0.1 mM), and enhanced by Ro, suggesting that Ca2+ spikes were mediated by mPTP opening. Hypoxia increased the frequency of Ca2+ spikes, while Met reduced the effect of hypoxia. In conclusion, our results suggest that hypoxia facilitates opening of mPTP, slightly reduces microglial viability in monotypic cell cultures and causes release of glutamate into culture medium which may be reduced by Met.