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The effects of exposure of ischemic human microglial cells to a 50 Hz magnetic field on the cell survival rate, intracellular calcium and reactive oxygen species should be investigated.
Neurotoxic effects of hypoxic-ischemia are thought to be mainly caused by calcium overload and production of reactive oxygen species, ultimately resulting in cell death. It is suggested that microglia contribute to the protection of the brain against ischemic injury. Hence, their preservation in case of ischemia might be of therapeutic use. Ischemic conditions were experimentally induced via oxygen and glucose deprivation (OGD) in a special culture medium without glucose and in an incubator with little oxygen. Cells were divided into the following groups: 1) exposure to 10 Hz/1 mT, 2) exposure to 50 Hz/1 mT, 3) exposure to 100 Hz/1 mT, 4) exposure to 50 Hz/0.01 mT, 5) exposure to 50 Hz/0.1 mT, 6) OGD treatment alone, 7) OGD treatment and exposure to 50 Hz/1 mT, 8) no OGD treatment and no exposure (control group). Except for test on cell survival with all groups, only groups 6, 7 and 8 were used. Groups 1-5 were used to determine the optimum frequency and magnetic flux density of exposure for the most potent protection against OGD-induced cell death (remark EMF-Portal: however, there are no specifications on the use of OGD in these groups).
effects on microglia cells: survival, intracellular calcium and reactive oxygen species
cell culture dishes in gas incubator with 94% N2, 1% O2, and 5% CO2
exposure system consisted of two identical coils to generate the uniform vertical magnetic field; diameter of coils = 15 cm (inner) and 26 cm (outer); thickness of coils = 7.5 cm; distance between coils = 18 cm; wire diameter = 18 AWG; number of loops = 1000; the system was placed in the center of a gas addition incubator; cell culture dishes were maintained at the center of the uniform field area; a thermometric probe placed inside and outside the EMF generator revealed no significant temperature difference between culture media of exposed or unexposed cells
細胞生存力／細胞分裂/増殖: cell survival (with all groups, after exposure; also with ROS-generating enzyme inhibitors (see "others"), cell viability assays: 1. trypan blue stain, hemocytometer, light microscopy; 2. commercial kit, spectrophotometry)
intracellular calcium level (only with groups 6, 7 and 8, before and during exposure; commercial kit, spectrophotometry), level of reactive oxygen species (only with groups 6, 7 and 8, after exposure; with use of 15-100 µM of ROS-generating enzyme inhibitors apocynin (for NADPH oxidase), allopurinol (for xanthine oxidase) or TTFA (for mitochondrial respiration chain complex II); dichlorofluorescein stain, spectrophotometry and fluorescence microscopy)
In the pretests, group 2 (50 Hz/1 mT) showed the largest and significant increase in cell survival compared to the control group (group 8).Therefore, the authors suggested 50 Hz/1 mT as the optimum field for protection against cell death and used it in the subsequent tests. Ischemia evoked by OGD treatment with or without exposure to the 50 Hz/1 mT magnetic field (groups 6 and 7) significantly reduced the cell survival compared to the control group. However, OGD treatment and exposure to the magnetic field (group 7) showed a significantly higher cell survival compared to OGD treatment alone (group 6). The calcium level was significantly increased after 30 minutes of exposure in group 6 compared to the initial value and returned to the initial value after 60 minutes, while the calcium level in group 7 was significantly reduced compared to group 6 during the whole exposure. The level of reactive oxygen species was significantly increased in group 6 compared to group 7 and the control group and it was found that only xanthine oxidase was involved in OGD-induced cell death. The authors conclude that exposure to a 50 Hz magnetic field might protect human microglial cells from ischemia-induced cell death by reducing the intracellular calcium and ROS levels. The xanthine oxidase might be one of the main mediators of the ischemia-induced cell death. Magnetic field exposure could be clinically useful to attenuate hypoxic-ischemic brain injury.