The Responses Of Microglia To Infrasound And Its Effects On Primary Cultured Neuron Cell

Infrasound(IS)is a kind of sound wave,whose frequency of mechanical vibration is below 20 Hz.It is acoustic energy with physical characters of strong penetration and less attenuation in long distance propagating.Infrasound arises from a large variety of natural and man-made phenomena,including avalanches, ocean waves,tornadoes,earthquakes,rocket launch and so on.Though it is usually inaudible,humans can perceive infrasound if its intensity is sufficiently high.Experimental studies had reported that infrasound can injure the central nervous system(CNS)of humans and various species of animals,such as the cortex,hypothalamus and other subcortical structures et al.With the development of modern industry and transportation,infrasound plays a more and more important role in noise pollution.Although there have been some studies indicate that one of mechanisms of infrasound injure the CNS is to cause inflammatory reaction,little is known about the adverse effects of infrasound on inflammation of CNS.MI,as the most important immunological cell in the inflammation process,become a hot topic in the researchers on the CNS inflammation process.MI can migrate to the sites of damaged tissue when the CNS injuries.On the one hand,microglia protect the CNS,restrict and remove the foreign microorganism,phagocytose and eliminate noxious substances and dead cerebrum chips;On the other,MI produce lots of cytokines and participate the antigen presentation process and actively construct immune system of the CNS.MI actively participate in the process of the nosogenesis, development and turnover of many diseases.Sometimes it provides neuronprotective effects against the diseases and sometimes it causes the diseases to protract and aggravate the diseases.In the prophase study of our laboratory,we found that exposure to infrasound could induce microglia activation in the brain.Since the activated microglia are the double-edge sword for the neuron cells,they can exert not only benefit but also harmful effects on neurons.So far,the effects of infrasound-activated microglia on neurons are largely unknown.In the present study,we exposed cultured rat microglia to high-intensity infrasound,and try to explore the effects and functions of microglia on neurons.Experiments were carried out in cultured microglia cells and neuron cells in vitro.Experiment 1:The responses of the cultured microglia to infrasound. Experiment 2:The influences on cultured neuron cells induced by the supernatant of the microglia exposed to infrasound.Experiment 3:The simulation injury of TNF-αand IL-1βon the primary cultured neurons secreted by infrasound-activated microglia and the protective effects produced by neutralization antibodies of TNF-αand IL-1β.Experiment 4:The influences of infrasound on the process that microglia and astrocyte secrete some kinds of cytokines.The results were showed as the following:Experiment 1:The responses of the cultured microglia to infrasound.Cell morphology:Twenty-four hours after isolation,microglia showed a little cytoplasm and circular shape.Microglia enlarged at once after IS exposed.4h after IS exposed,ameboid cells thrived and binucleated ameboid cells were often observed with occasional telophase pairs and mitotic figures present general. Microglia,12h after IS exposed,is significant swelling and changes anomality. 24h after IS exposed,microglia cell has more vacuoles and phagocytic vesicles.Cytokine analysis:IL-10 was released as early as 0h after exposure and concentration reached the higlhest at 12h after exposure.While the significant upregulation of TNF-αand IL-1βwas detected at 12h and then degraded.After 1h exposure to IS,the levels of TNF-αand IL-1βelevated gradually until the end point of observation,but the secretion of the two cytokines show derangement. The level of IL-1βin the supernatent of infrasound-activated MI upregulated instantly after 1h exposure to IS;TNF-αdid not upregulate until 12h after 1h exposure to IS and secretion of TNF-αwas significantly hysteresis compared with IL-1β.IL-10,previously known as a protective factor,showed two peak levels at 4h and 24h and the levels were higher at all observing points compared to control.Experiment 2:The influences on cultured neuron cells induced by the supernatant of the microglia exposed to infrasound.The influences on cultured neurons of the supernatant of the microglia exposeded to infrosound.(a)The result of MTT:The gathered supernatant of cultured microglia at different time point after 30min IS exposed did not injure primary cultured neuron cells;the gathered supernatant of cultured microglia at different time point after lh IS exposed could all injure primary cultured neuron cells,(b)Appoptosis of neuron detected by TUNEL:The gathered supernatant of cultured microglia at different time point after 30min IS exposed did not promote primary cultured neuron cells appoptosis;the gathered supernatant of cultured microglia at different time point after 1h IS exposed all could all promote primary cultured neuron cells appoptosis,furthermore the inducing apoptosis effects of the supernatant gathered at 24h was most significant.Experiment 3:The simulation injury of TNF-αand IL-1βon the neuron secreted by infrasound-activated microglia and the protective effects produced by neutralization antibodies of TNF-αand IL-1β.(l)The simulation injury of TNF-αand IL-1βon the primary cultured neurons secreted by the microglia exposed to 1h infrasound and the protective effects produced by neutralization antibodies of TNF-αand IL-1β.Flow cytometry result: The apoptosis ratio on the primary cultured neuron cells,caused by TNF-αor IL-1βthat secreted by microglia when it exposed to infrasound after 24h,was 44±3.2%or 57±4.8%.The apoptosis ratio was 67.5±6.4%that caused by TNF-αand IL-1β.Then 5μg/ml concentration of the neutralization antibody of TNF-αor IL-1βcould completely neutralize the bioactivity of TNF-αor IL-1β.5μg/ml concentration of the neutralization antibody of TNF-αor IL-1βdid not influence the primary cultured neuron cells.(2)Admoving the neutralization antibody of TNF-αand IL-1βinto the gathered supernatant of microglia at 24h after 1h IS exposed and using the supernatant to the cultured neuron cell then detecting the apoptosis.(a)Flow cytometry result: The apoptosis ratio of the two groups admoved IL-1βand TNF-α/IL-1βantibody was obviously lessened compared with the groups that were not admoved the antibody and admoved TNF-αantibody;Compared with the group was not admove the antibody,the apoptosis ratio of TNF-αantibody group had not obvious difference.Experiment 4:The influences of infrasound on the process that microglia and astrocyte secret some kinds of cytokines.Cell morphology:Normal astrocyte show anomalism of appearances and abundant kytoplasm.The cell body has more branches.The nucleus is oval-shap and in the lateralization of the cell body.In 24h after 1h exposure to IS,astrocyte did not show significant changes compareded with the control observed by microscopy. The responses of MI to infrasound had been described in experiment 1.Cytokine analysis:At every observering point after IS exposed,the cytokines in supernatant including TNF-α,IL-1βand IL-10 and so on had not diversity in 24h after exposed to infrasound compared to the control.But many kinds of cytokines in the supernatant of infrasound-activated MI increased in 4h after exposure.The following is the conclusion of experiments:(1)Microglia could be activated by exposing to infrasound of 16 Hz,130 dB. The microglial may be the first cell that reacted to infrasound in the CNS.(2)The supernatant gathered after 30min IS exposed dose not cause neuron cells injuried.The supernatant gathered at 4h,12h and 24h point after 1h IS exposed can all cause the primary cultured neuron cells apoptosis.The apoptosis caused by 24h time point supernatant is the best obvious than the others.(3)The microglia after 1h IS exposed through secreting cytokines to cause neuron apoptosis;IL-1βmay play an important role in the process of neuron apoptosis caused by the microglia exposed to infrasound.