Abstract
Extracellular zinc, which is released from hippocampal neurons in response to brain ischaemia, triggers morphological changes in microglia. Under ischaemic conditions, microglia exhibit two opposite activation states (M1 and M2 activation), which may be further regulated by the microenvironment. We examined the role of extracellular zinc on M1 activation of microglia. Pre-treatment of microglia with 30–60 μM ZnCl2 resulted in dose-dependent increases in interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumour necrosis factor-alpha (TNFα) secretion when M1 activation was induced by lipopolysaccharide administration. In contrast, the cell-permeable zinc chelator TPEN, the radical scavenger Trolox, and the P2X7 receptor antagonist A438079 suppressed the effects of zinc pre-treatment on microglia. Furthermore, endogenous zinc release was induced by cerebral ischaemia–reperfusion, resulting in increased expression of IL-1β, IL-6, TNFα, and the microglial M1 surface marker CD16/32, without hippocampal neuronal cell loss, in addition to impairments in object recognition memory. However, these effects were suppressed by the zinc chelator CaEDTA. These findings suggest that extracellular zinc may prime microglia to enhance production of pro-inflammatory cytokines via P2X7 receptor activation followed by reactive oxygen species generation in response to stimuli that trigger M1 activation, and that these inflammatory processes may result in deficits in object recognition memory.
Highlights
Ischaemia results in the immediate release of zinc into the hippocampal extracellular space and is followed by a second release of zinc at the onset of reperfusion16
The present study demonstrated the following: (1) Extracellular zinc enhanced the LPS-induced secretion of pro-inflammatory cytokines from microglia in a dose-dependent manner; (2) this zinc-induced enhancement was mediated by microglial zinc uptake, P2X7 receptor activation, and reactive oxygen species (ROS) generation; (3) microglial uptake of extracellular zinc enhanced LPS-induced expression of the M1 marker inducible nitric oxide synthase (iNOS); (4) CaEDTA, but not ZnEDTA, suppressed ischaemia-induced increases in the expression of pro-inflammatory cytokines and the M1 microglial cell-surface maker CD16/32 in the hippocampus; and (5) CaEDTA protected mice from ischaemia-induced deficits in object recognition memory
These findings suggest that extracellular zinc may be an endogenous factor involved in the promotion of the inflammatory M1 phenotype of microglia in response to M1 stimuli
Summary
Ischaemia results in the immediate release of zinc into the hippocampal extracellular space and is followed by a second release of zinc at the onset of reperfusion. We demonstrated that extracellular chelatable zinc triggers morphological changes in cultured microglia and the brain following cerebral ischaemia, and that these morphological changes are mediated by zinc uptake, P2X7 receptor activation, and reactive oxygen species (ROS) generation. Toll-like receptor 4 (TLR4), which is predominantly expressed in brain microglia, has been observed to participate in such inflammatory responses. Recent research has demonstrated that the activation of TLR4 by LPS induces the M1 phenotype of microglia, which is characterised by an increase in the expression of pro-inflammatory cytokines and M1 cell-surface markers such as CD16/3223. We hypothesised that endogenous extracellular chelatable zinc would promote inflammatory activity of microglia with the M1 phenotype in the hippocampus following cerebral ischaemia, and that these pro-inflammatory functions would be further mediated by zinc uptake, P2X7 receptor activation, and ROS generation
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