Study On The Normal Or Pathological States Induced By Lipopolysaccharide Of Spiral Ganglion Neurons To Damage Caused By Mobile Phone Electromagnetic Radiation

Part Ⅰ primary culture and identification of cochlear spiral ganglion neuronsObjective:Newborn SD rat cochlea spiral ganglion cells (SGN) in vitro primary culture and identification.Methods:1-3days of ten neonatal SD rats were treated with75%ethanol after receiving ethyl ether inhaled anesthesia. The bilateral temporal bones were removed and the spiral ganglion tissue was dissected form the stria vascularis and basilar membrane, then the tissue digested with0.25%trypsin for30min at37℃. After the samples were centrifuged for8min at800rpm, the cells were resuspended and placed in12holes culture plates. Cells were maintained in a growth medium of with DMEM/F12, supplemented with10%fetal bovine serum,10%neurotrophic factors B27, and1%penicillin-streptomycin and placed in an incubator at37℃in5%CO2and95%air. We used5umol/ml cytarabine to purify the SGN for24,48and72h. Neuronal class Ⅲ anti-β-tubulin (TUJ1,1:200)(Santa Cruz, USA) and NeuN (abcam,USA) antibody was used to identify neurons.Results:We observed the SGN adherent growth after6hours in ordinary optical microscope. As shown in Fig.1-1, SGN can be distinguished from fibroblasts under an inverted phase contrast microscope based on their typical bipolar neurons. The SGN cell bodies are oval and smooth, and the axon extend out from cell body as slender projections. Fig.1-2and3show that an anti-β-Ⅲ tubulin (TUJ1) and NeuN fluorescent biomarker signal could be detected in the cultured cells.Conclusion:We used pancreatic enzyme digestion method to cultivate SD rats cochlear spiral ganglion cells in primary culture,5μmol/ml cytarabine was used to purify spiral ganglion cells, and after72h, we obtained high purity SGN. we used specific antibody NeuN and TUJ1antibdoy to identify SGN. Part Ⅱ the damage of mobile phone electromagnetic radiation on the normal cochlear spiral ganglion neuronsObjective:The aim of study is to investigate the normal spiral ganglion neurons cellular DNA damage, changes in ultrastructure, autophagy and ROS formation after exposure to the commonly cell phone frequency1800MHZ and intensity (SAR:2and4w/kg)Methods:In our experiment, we applied the SXc-1800MHz RF radiation exposure system that is characteristic of typical GSM "talk mode" signals to investigate whether the bio-effects might induce cellular DNA damage, autophagy, changes in ultrastructure and ROS formation at this frequency for the SAR value of2w/kg and4w/kg (during a24-h intermittent exposure pattern of5min on and10min off). DNA damage was examined by alkaline comet assay, ultrastructure changes were detected by transmission electron microscopy, and expression of the autopahgy markers LC3-Ⅱ and Beclinl were examined by immunofluorescence and confocal laser scanning microscopy. Reactive oxygen species (ROS) production was quantified by the dichlorofluorescin-diacetate assay.Results:The results of comet assay were negative in all of experimental groups. Transmission electron microscopy revealed mitochondrial, endoplasmic reticulum and nucleus structure were normal and had no obvious abnormalities. The ROS values significantly increased in4W/kg and H2O2exposure, compared with control and2W/kg groups (p<0.05,0.01).Conclusion:Electromagnetic radiation (EMR) could not induce DNA damage, autophagy, and changes in ultrastructure on1800MHz at specific absorption rate (2and4W/kg). Activating the ROS system is thought to be the main mechanism of electromagnetic radiation. PartⅢ the damage of mobile phone electromagnetic radiation on the Lipopolysaccharide-induced pathological states of cochlear spiral ganglion neurons Objective:To investigate whether the sensitivity of spiral ganglion neurons to damage caused by mobile phone electromagnetic radiation will increase in lipopolysaccharide-induced inflammation in vitro model.Methods:After the SGN were treated with different concentrations (0,20,40,50,100,200and400μg/ml) of LPS, the Cell Counting Kit-8(CCK-8) and alkaline comet assay were used to quantify cellular activity and DNA damage, respectively. The SGN were treated with the LPS40μg/ml before EMR exposure. Dishes were randomly divided into the following groups:(1) RF radiation control,(2)2W/kg (either sham exposure or exposure),(3)4W/kg (either sham exposure or exposure),(4)2W/kg combined with LPS (40μg/ml)(either sham exposure or exposure),(5)4W/kg combined with LPS (40μg/ml)(either sham exposure or exposure),(6). H2O2group. After24h intermittent exposure at an absorption rate of2and4W/kg, DNA damage was examined by alkaline comet assay, ultrastructure changes were detected by transmission electron microscopy, and expression of the autopahgy markers LC3-Ⅱ and Beclinl were examined by immunofluorescence and confocal laser scanning microscopy. Reactive oxygen species (ROS) production was quantified by the dichlorofluorescin-diacetate assay.Results:LPS (100μg/ml) induced DNA damage and suppressed cellular activity (p<0.05). LPS (40μg/ml) did not exhibit cellular activity changes or DNA damage (p>0.05), therefore,40μg/ml was used to pre-treatment concentration before exposed to RF-EMR. RF-EMR could not directly induce DNA damage. However, in4W/kg combined LPS (40ug/ml) group showed mitochondria vacuoles, karyopyknosis, presence of lysosomes and autophagosome, and increasing expression of LC3-Ⅱ and Beclinl. The ROS values significantly increased in4W/kg exposure,4W/kg combined LPS (40μg/ml) exposure and H2O2group (p<0.05,0.01).Conclusion:Short-term exposure to cell phone electromagnetic radiation (EMR) could not directly induce DNA damage in normal spiral ganglion neurons, but EMR could cause the changes of cell ultrastructure at special SAR4.0W/kg when cells in fragile or micro-damage condition. It seems that the sensitivity of SGN to damage caused by mobile phone electromagnetic radiation will increase in Lipopolysaccharide-induced inflammation vitro model. However, whether these alterations cause cochlear functions disorders still need further research. Activating the ROS system is thought to be the main mechanism of electromagnetic radiation biological effects.