| Objective:With the development of scientific technology, progress of society, electromagnetic radiation has been utilized in almost every fields such as broadcasting, communicating, industry, research, national defense, medicine and health and so on, even in the daily life. In the other hand, the electromagnetic radiation has also impacted the human health as the forth biggest environmental pollution source. Thus, the research on biological effects of different kinds of electromagnetic fields 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 pulsed magnetic fields on neurons in order to further explore the impact of pulsed magnetic fields on nervous system. The effects of extremely low-frequency pulsed magnetic fields(ELFPMF) on neurons depends on two aspects: one, the parameters of ELFPMF such as frequency, intensity, action time; the other, the parameters of neurons such as resource of neurons, location, growth condition and so on.Aim to explore the effects of ELFPMF(15Hz) on learning, memory, EEG, Morphology of brain tissue and cultured cortical neurons and analyze the possible mechanism, the ELFPMF was acted on the head of rat and the cultured cortical neurons.Methods:1. Thirty-six adult male Wistar rats were divided into control group (Group A) and experimental group (Group B) randomly. Rats in Group B were exposed to 0.1mT intensity ELFPMF of 15Hz for 45 minutes per day for 30 days, the irradiation source was positioned 2mm from the surface of conscious rats' parietal bone. The influence of ELFPMF on the rats was detected by the following methods:(1). Morris maze test was used to test the changes in learning and memory. Shotthe behavior of rats in two groups with video camera in a real-time way.(2). Morphology observation:①. Light Microscopy: Small pieces were dissected from the cortex of frontallobe, the section was prepared in routine, HE stain, observed and recorded(×200).②. Transmission Electron Microscopy(TEM): Small pieces dissected fromthe cortex of frontal lobe were cut into several smaller pieces which had avolume of 1 mm3, then were quickly transferred to flaskets containing the 2.5%glutaral, ultra-thin section was prepared in routine, observed and recorded withJEM-1200EX electron microscope.(3). Electroencephalography:The RM-600 Eight-channel Electroencephalograph was used to get the EEG2. Primary cortical neurons were prepared from embryonic day 15.5～16.5 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 ELFPMF(frequency: 15Hz, average magnetic field intensity: 0.1mT) for 2 days, 3times/d, 30minutes/time. 12h afterthe last irradiation, the influence of ELFPMF on the neurons was detected bythe following methods:(1). Morphological changes: Observed the changes in neuron ultra-structureusing TEM.(2). The relative viability of cells after irradiation was measured by MTT assay.(3). Intracellular free calcium level of neurons was determined by FlowCytometry: Collected cells, added Fluo3/AM to a final concentration of10μmol/L, incubated for 1h, protected from light. Flow Cytometry was appliedto check the difference of intracellular calcium level of neurons.(4). Mitochondrial membrane potential: Collected cells, added Rhodamine123to a final concentration of 10μg/ml, incubated for 30min, Flow Cytometry wasapplied to check the changes in mitochondrial membrane potential of neurons.Results:1. Effects of extremely low-frequency pulsed magnetic fields on learning,memory, brain tissue Morphology and EEG in rats:(1). Effects of ELFPMF on behavior of rats:Compared with group A, 24h after the first irradiation, the rats' swimmingactions in group B were not coordinative in the water maze, the reflection ofshaking hair got weaker, etc. But this phenomenon disappeared gradually.(2). Effects of ELFPMF on learning and memory of rats:The value of Escape Latency of rats in two groups at 1d, 2d, 3d, 4d, 5d, 9d, 20d,29d were 19.4±10.0, 14.1±10.0, 12.7±10.1, 6.6±3.5, 6.5±3.0, 6.1±2.8, 7.1±2.2,6.1±16.8 and 55.2±32.2, 17.3±9.9, 13.3±9.0, 8.9±3.1, 7.7±2.6, 6.5±1.4,7.8±10.5, 7.1±7.3 respectively.(3). Effects of ELFPMF on brain Morphology of rats:①. Observation with light microscope: The neuron axons of the cortex ofthe rats in Group A were evident. The amount of apoptotic cells in the cortex ofrats in Group B was larger than that of Group A. ②. Observation with TEM: The shape of the neuron and neuroglia cells ofrats in group A was natural. The absence of the mitochondria cristae in theneuroglia cells, the perinuclear cisternae expansion and the swelling and evendeath of the endoplasmic reticulum were common in group B. There wasevident twisting in some neuron karyotheca, different amounts of small bubblesencircled by the monolayer were distributed in the nuclear matrix. Theapoptotic neuron was visible, the cell pycnosed and deformed, although everyorganelle was visible, the electron intensity wais abnormal, the nucleus wasirregular in shape, and the chromatin agglutinated in the mass shape(4). Effects of ELFPMF on the EEG of rats:The frequency and amplitude of EEG of rats in Group A were 9.67±1.37(Hz)and 76.7±10.8(μm) respectively. The frequency and amplitude of EEG of rats inGroup B were 7.71±1.12(Hz) and 58.9±8.6(μm) respectively.2. Damaging effects of extremely low-frequency pulsed magnetic fields oncultured cortical neurons:(1). Morphological observation:After initial plating, the embryonic cortical neurons were spherical andsuspended in medium. 24h later, most cells spreaded around and adhered to thedishes with a triangular or polygonal plump cyton. There was axon or dendritespreading from the neuron. The surface of cyton and axon or dendrite wassmooth. Under TEM, the double layer nuclear membrane of neuron was smooth,there was abundant euchromatin in the nucleus, there were more mitochondriain the perikaryon, their double layer membrane and crista were clear. Inexperimental group, the surface of cyton and axon or dendrite was rough. UnderTEM, there were more myelin body in the intracytoplasm, several mitochondriaaggregated and were enveloped by monolayer membrane.(2). The relative viability:The values of A570 of neurons in two groups were 0.494±0.031 and 0.401±0.023respectively. (3).Intracellular free calcium level of neurons:The values of mean fluorescence intensity of intracellular calcium were66.69±2.24 and 78.22±1.59 respectively.(4).Mitochondrial membrane potential:The values of mean fluorescence intensity of mitochondrial membrane potentialwere 69.49±1.94 and 41.66±2.01 respectively.Conclusions:1. Under this experiment condition, long-term irradiation of ELFPMF(15Hz) could impact EEG of rats, resulted in apoptosis of cortical neurons and other ultra-structural changes, could inhibit the growth of cultured cortical neurons, resulted in metabolic abnormality, elevated intracellular free calcium level and decreased mitochondrial membrane potential of neurons.2. When long-term radiation of ELFPMF(frequency: 15Hz, average magnetic field intensity: 0.1mT) is adopted, the rats in Group B showed cinesipathy, sensory disturbance and dysmnesia at first and recovered gradually, but both the brain Morphology detection and the EEG demonstrated abnormalities, so to explore the biological effects of ELFPMF, it is essential to aggregate analysis index of all aspects, in addition, the detrimental effects brought by the long-term radiation of the ELFPMF should be taken into full consideration.Significance:This work chose similar frequency with consciousness(15Hz) to act on the head of adult rats and cultured cortical neurons, uncovered the detrimental effects brought by the long-term radiation of the ELFPMF on rats and cultured neurons, could provide experimental data for further research on the biological effects of ELFPMF.
|
PDF Full Text Request