Effects of Metformin on Spontaneous Ca2+ Signals in Cultured Microglia Cells under Normoxicand Hypoxic Conditions

Microglia are the main resident immune cells that are among the first responders to hypoxic/ischemic brain damages. Because of their sensitivity to blood flow fluctuations, microglia become activated and undergo morphological changes under hypoxic/ischemic conditions. However, intracellular mechanisms mediating microglial activation under hypoxic conditions are not well understood. When activated, microglia can cause a cascade of inflammatory processes and initiate cytokine release, activate reactive oxygen species production. It is known that intracellular Ca2+ signaling is linked with pathophysiological functions of microglia and its signaling changes, arising in response to brain damage. Previous studies found that calcium signaling is important for microglial immune function— cytokine release, P2X receptor trafficking and diffusion. Imaging of calcium-dependent fluorescence in vivo has demonstrated that microglial cells have spontaneous Ca2+ signaling which is important for microglial function. Spontaneous signals and their rate were changed by lipopolysaccharide and by local neuronal tissue injury. Also, changes in neuronal activity triggered increased microglial process Ca2+ signaling, which was related to process extension. However, there is still little knowledge on how Ca2+ spontaneous signals change in response to hypoxia. Metformin is widely used as anti-hyperglycemic agent for non-insulin-dependent (type 2) diabetes therapy. It has been suggested that metformin decreases hepatic glucose production mostly via inhibition of complex I of the mitochondrialrespiratory chain and activation of AMP-kinase signaling pathway. Recent studies suggest that metformin can also act as a neuroprotective agent during hypoxia/ischemia by suppressing mitochondrial complex I activity in neuronal cultures, by reducing ischemic stroke-induced oxidative stress, inhibiting neuronal apoptosis and suppressing neuroinflammation. Metformin has also been suggested to inhibit mitochondrial permeability transition pore (mPTP) opening and to exert neuroprotective effects by this mechanism. However, the link between mitochondrial dysfunction and inflammatory responses mediated by the activated microglia are not clear yet, and the role of metformin in these processes remains largely unknown. It has been demonstrated that metformin and phenformin at pharmacologically relevant concentrations differently improve Ca2+ homeostasis in hypoxia-affected primary cortical neuronal cell cultures, but little is known about their role in microglial cultures. In this study, we investigated the effect of metformin on spontaneous calcium signals in cultured microglia cells grown under normoxic and mild hypoxic conditions in order to elucidate the mechanism by which ischemichypoxic injury induce spontaneous calcium signaling changes in microglia. By using Ca2+ sensitive fluorescence dye, we studied how inhibition of mitochondrial respiration changed spontaneous Ca2+ signals in soma of microglial cells from 5–7-day-old rats grown under normoxic and mild-hypoxic conditions. In microglia under normoxic conditions, metformin or rotenone elevated the rate and the amplitude of Ca2+ signals 10–15 min after drug application. Addition of cyclosporin A, a blocker of mPTP, antioxidant trolox, or inositol 1,4,5-trisphosphate receptor (IP3R) blocker caffeine in the presence of rotenone reduced the elevated rate and the amplitude of the signals implying sensitivity to reactive oxygen species, and involvement of mitochondrial mPTP together with IP3R. Microglial cells exposed to mild hypoxic conditions for 24 h showed elevated rate and increased amplitude of Ca2+ signals. Application of metformin or rotenone but not phenformin before mild hypoxia reduced this elevated rate. Thus, metformin and rotenone had the opposing fast action in normoxia after 10–15 min and the slow action during 24 h mild-hypoxia implying activation of different signaling pathways. The slow action of metformin through inhibition of complex I could stabilize Ca2+ homeostasis after mild hypoxia and could be important for reduction of ischemia-induced microglial activation.