The Effect of metformin on cultured microglia cells under normoxic and mildhypoxic conditions

Abstract

Hypoxic brain injury may affect neural tissue via microglia activation, however, mechanisms and consequences of these processes during hypoxia are not fully elucidated yet. We aimed to investigate effects of anti-hyperglycemic agent – metformin on developing brain microglia cells under normoxic or mild- hypoxic conditions. In this study primary rat microglial cultures (≥85 % microglia) at 7–11 DIV were treated with metformin (Met), cyclosporin (CsA) and rotenone (Ro). Cell cultures were incubated with or without pharmacological agents under normoxic and mild-hypoxic (93% N2, 5% CO2, 2% O2; 37°C) conditions for 24 h. It was shown that mild hypoxia (2% oxygen) had no effect on microglial cell viability which remained above 90%. None of Met concentrations (0,1mM; 0,5mM and 3mM) had effect on viability and number of microglial cells. CsA under hypoxic conditions tended to decrease both – cell number and viability, while Ro has no effect on number and viability of cells. Mild-hypoxia increases glutamate in microglia culture media, and pretreatment with Met but not Ro or CsA tend to reduce glutamate levels. 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. These results suggest that hypoxia facilitates opening of mPTP in monotypic cell cultures and may cause release of glutamate into culture medium which may be reduced by Met.