Abstract
Microglial functioning depends on Ca2+ signaling. 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 mitochondrial permeability transition pore (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 (ROS), 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.
Highlights
Microglia are the main resident immune cells that are among the first responders to hypoxic/ischemic brain damages [1,2]
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 ischemic-hypoxic injury induce spontaneous calcium signaling changes in microglia
When cells were exposed to mild hypoxia (2% oxygen) for 24 h a tendency of increased cell death was observed, though viability of microglia still remained above 90% (Figure 1a,b)
Summary
Microglia are the main resident immune cells that are among the first responders to hypoxic/ischemic brain damages [1,2]. Because of their sensitivity to blood flow fluctuations, microglia become activated and undergo morphological changes under hypoxic/ischemic conditions [3]. It is known that intracellular Ca2+ signaling is linked with pathophysiological functions of microglia and its signaling changes, arising in response to brain damage, may be activation-associated [7,8,9,10,11]. Previous studies found that calcium signaling is important for microglial immune function—cytokine release [12,13], P2X receptor trafficking and diffusion [14]. The rate of signal generation was increased in activated microglia in Alzheimer’s disease mouse models [17], the rate was elevated by local neuronal tissue injury [18] and aging modified microglial spontaneous Ca2+ signaling [19]
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