Effects Of Power-frequency Magnetic Field On Cortical Neurons

Objective:With the development of electricity technology, application of electrical equipment, we contact a lot of Power-frequency magnetic field in the daily life. Many researches indicated that Power-frequency magnetic field has also impacted the human health as the biggest environmental pollution source. Thus, the research on biological effects of Power-frequency magnetic field becomes an focus in Bio-electromagnetism.Central nervous system is the most sensitive system of human, neuron is the fundamental unit of the nervous system structure and function, so it is necessary to study the effects of Power-frequency magnetic field on neurons in order to further explore the impact of Power-frequency magnetic field on nervous system. The effects of Power-frequency magnetic field on neurons depends on two aspects: one, the parameters of Power-frequency magnetic field such as intensity, action time; the other, the parameters of neurons such as resource of neurons, location, growth condition and so on.Aim to explore the damaging effects of Power-frequency magnetic field (50-60Hz) on cortical neurons of rats and analyze the possible mechanism, the Power-frequency magnetic field was acted on the cultured cortical neurons.Methods:Primary cortical neurons were prepared from embryonic day 13.5-14 Wistar rats. The cultured neurons were divided into control group and experimental group randomly when they were cultured for 48h. Then the experimental group was exposed to the Power-frequency magnetic field (frequency: 50Hz, average magnetic field intensity: 0.3mT) for 2 days, 3times/d, 30minutes/time. 12h after the last irradiation, the influence of Power-frequency magnetic field on the neurons was detected by the following methods:1. Morphology observation:(1) Light Microscopy: Observed the growth of neuron using Light Microscopy.(2) Transmission Electron Microscope: Observed the changes in neuron ultra-structure using TEM.(3) Scanning Electron Microscope: Observed the changes in neuron surface structure using SEM.2. The detection of cell death: Cell death was determined by Ho33342 and PI fluorescence dual-stain.3. The relative viability of cells after irradiation was measured by MTT assay.4. Intracellular free calcium level of neurons was determined by Flow Cytometry: Collected cells, added Fluo3/AM to a final concentration of 10μmol/L, incubated for 1h, protected from light. Flow Cytometry was applied to check the difference of intracellular calcium level of neurons.5. Mitochondrial membrane potential: Collected cells, added Rhodamine 123 to a final concentration of 10μg/ml, incubated for 30min, Flow Cytometry was applied to check the changes in mitochondrial membrane potential of neurons.6. lipid peroxidation: Collected cells, broke the cell with ultrasonic waves, got the supernatant after 15min centrifuge (12000rpm/m) with 4℃super centrifuge, undertook the following reaction on the description of kit, determined the contents of malondialdehyde(MDA),the activities of superoxide dismutase (SOD) using 721 ultraviolet spectrophotometer.Results:1. Morphological observation:(1) Light Microscopy: In control group, after initial plating, the embryonic cortical neurons were spherical and suspended in medium. 24h later, some cells spreaded around and adhered to the dishes. There was axon or dendrite spreading from the neuron. 48h later, most cells spreaded around. 96h later, most cells adhered to the 1dishes with a plump cyton, the surface of cyton and axon or dendrite was smooth. In experiment group, the condition of neurons were not good, some cells spreaded around but the surface of cyton and axon or dendrite was rough. There were obvious differences between the control group and the experiment group.(2) Transmission Electron Microscope: In control group, there was abundant euchromatin in the nucleus, there were more mitochondria, endoplasmic reticulum and Golgi complex in the endochylema, there were more microbule and microfilament in the endochylema and the ecphyma. In experiment group, some mitochondria cristae were chaotic and there were huge mitochondria in the endochylema, autophagosomen and remnant cytolysosome which were circled by single layer membrane or double layer membrane were obviously increased in most neurons, endoplasmic reticulum dilated which were filled up retention, and Golgi complex were not good.(3) Scanning Electron Microscope: In control group, most cells spreaded around, there was axon or dendrite spreading from the neuron, the surface of cyton and axon or dendrite was smooth. In experiment group, obvious morphological changes were observed, the ecphyma of cell were contracted and broken, the surface of cyton and axon or dendrite was not smooth.2. The detection of cell death:Ho33342 and PI fluorescence dual-stain: In control group, caryoplasm were uniform and presented blue fluorescence. In experiment group, there were necrotic cell which presented red fluorescence.3. The relative viability:The values of A₅₇₀ of neurons in two groups were 0.515±0.029 and 0.398±0.016 respectively.4. Intracellular free calcium level of neurons:The values of mean fluorescence intensity of intracellular calcium were 91.23±1.16 and 88.42±2.08 respectively.5. Mitochondrial membrane potential: The values of mean fluorescence intensity of mitochondrial membrane potential were63.34±1.55 and 44.36±1.73 respectively.6. lipid peroxidation:The contents of MDA were 13.24±0.36 and 18.67±0.45 respectively, the activities ofSOD were 32.98±1.35 and 17.74±0.96 respectively.Conclusions:Under this experiment condition, Power-frequency magnetic field (repetitive frequency was 15Hz,average strength was 0.3mT ) could result in morphologia change of cortical neurons of rat, could inhibit the growth of cultured cortical neurons, resulted in metabolic abnormality, decreased mitochondrial membrane potential of neurons, elevated the contents of malondialdehyde(MDA) and degraded the activities of superoxide dismutase (SOD) in the neurons. Intracellular free calcium basicly was not to change, probably related to the persistence time of irradiation, number of times of irradiation and the time cell between finished irradiation and collected cells. So to explore the biological effects of Power-frequency magnetic field, it is essential to aggregate analysis index of all aspects and make full consideration.Significance:This work chose Power-frequency magnetic field (50Hz) to act on cultured cortical neurons of rat, uncovered the detrimental effects brought by the Power-frequency magnetic field on cultured neurons, could provide experimental data for further research on the biological effects of Power-frequency magnetic field.