Isoflurane Preconditioning Attenuates The Brain Injury Induced By Electromagnetic Pulse Via TLR4/NFκB Signaling Pathway

With the development of science and technology,electromagnetic wave exists widely in the daily life,and the chances of people exposed to electromagnetic radiation are greatly increased.Electromagnetic pulse(EMP)is a special type of electromagnetic radiation.EMP exposure will cause a series of biological effects.We have a lot of research on the physical protection of EMP exposure,but the prevention and treatment of drugs has rarely been studied.This experiment focuses on the effects of EMP exposure on the cerebral cortex of rats,and the neuroprotection of isoflurane preconditioning on cerebral cortex injury induced by electromagnetic pulse exposure.Part1 Effects of EMP on neurons,proinflammatory cytokines and Caspase3 in cerebral cortex of SD ratsObjective: Study the effects of electromagnetic pulse on neurons,proinflammatory cytokines and Caspase3 in frontal cortex of rats.Methods:Healthy adult male SD rats were randomly divided into 4 groups(n = 8): Control group,1 h after EMP group,6 h after EMP group and 24 h after EMP group.The animals were exposed or not to 200 pulses of EMP at 200 kv/m for 3days.HE staining was used to observe the effects of EMP exposure on neurons in frontal cortex of rats.ELISA was used to detect the level of proinflammatory cytokines TNF-α and IL-1β in the frontal cortex of rats.Western Blotting was used to detect the expression of Caspase3 in the frontal cortex of rats.Results: After EMP exposure,The number of abnormal neurons in the EMP1 h group,EMP6 h group,EMP24 h group was increased compared with the Control group,which in EMP6 h group and EMP24 h group was increased significantly(P < 0.01).After EMP exposure,the level of TNF-α gradually was increased with time,which in the EMP6 h group is more obvious,and compared with the Control group the difference was statistically significant(P<0.01);the level of IL-1β of each experimental group compared with the Control group was increased,which in the EMP1 h group was increased most obviously(P<0.01).Western Blotting showed that the expression of Caspase3 of the experimental group was significantly increased compared with the Control group,and the difference was statistically significant(P<0.05),which in the EMP 6h group was increased most obviously(P<0.01).Conclusion:Electromagnetic pulse exposure can cause neuronal damage in frontal cortex of rats,the inflammatory reaction and cells apoptosis.Part2 Effects of microglial and TLR4 / NFκBp65 signaling pathway induced by EMP in rat cerebral cortexObjective: Study the effect of microglial and the expression of TLR4 and NFκBp65 induced by EMP,and determine the time point after EMP exposure for the next experiment.Methods: The group of experimental animals was same as the first part.Western Blotting was used to detect the expression of CD11 b,TLR4,NFκBp65 in the frontal cortex of rats after EMP exposure.Immunofluorescence was used to observe the effects of EMP exposure on the expression of TLR4 and NFκBp65 in the frontal cortex of rats.Results: Western Blotting showed that at 6h and 24 h after EMP,the expression of CD11 b in frontal cortex of rats was increased compared with the Control group,which in the EMP6 h group was increased most obviously(P<0.01).Western Blotting showed that the expression of TLR4,NFκBp65 in the EMP6 h group and EMP24 h group was significantly increased compared with the Control group,which in the EMP 6h group was increased most obviously(P<0.01).Immunofluorescence showed that the expression of TLR4 and NFκBp65 in frontal cortex neurons of rats was increased after EMP exposure,which in the EMP6 h group and EMP24 h group was increased obviously.Conclusion: EMP exposure can cause microglial activation.EMP exposure can increase the expression of TLR4 and NFκBp65,which indicates that the brain damage induced by EMP may be via the TLR4/NFκB signaling pathway.Part3 Isoflurane preconditioning alleviate the brain injure induced by EMP via TLR4 / NFκB signaling pathwayObjective: Study the neuroprotective of isoflurane preconditioning from the EMP exposure and the mechanism of isoflurane preconditioning neuroprotective is via inhibiting the microglia activation and TLR4 / NFκB signaling pathway induced by EMP.Method: The healthy adult male SD rats were randomly divided into 4 groups: the Control group,the EMP exposure group(EMP group),the EMP exposure after isoflurane preconditioning group(EMP+Iso group),the isoflurane preconditioning group(Iso grop).the time point after EMP exposure was 6h after EMP,which was selected according to the results of Part2.HE staining was used to observe the effects of frontal cortex neurons.ELISA was used to observe the level of TNF-α and IL-1β.Western Blotting was used to detect the expression of Caspase3,CD11 b,TLR4,NFκBp65 on the frontal cortex of rat.Result: HE staining showed that the number of abnormal neurons after EMP exposure was increased(P<0.05),which in the EMP+Iso group was significantly reduced compared with the EMP exposure group(P<0.05).Isoflurane preconditioning had no effect on normal brain cortex neurons.The results of ELISA showed that the level of TNF-α and IL-1β was increased after EMP exposure(P<0.05),which in the EMP+Iso group were reduced compared with the EMP group(P<0.05).Isoflurane preconditioning had no effect on it.Western Blotting showed that the expression of Caspase3,CD11 b,TLR4,NFκBp65 was increased after EMP exposure(P<0.05),which in the EMP+Iso group was reduced compared with the EMP group(P<0.05).Isoflurane preconditioning had no effect on it.Immunofluorescence staining showed that after EMP exposure,a large number of TLR4 and NFκBp65 in frontal cortex activated,and after isoflurane pretreatment,the expression of TLR4 and NFκBp65 was significantly reduced.Isoflurane preconditioning had no effect on it.Conclusion: Isoflurane preconditioning can protect neurons in the cerebral cortex from EMP exposure,and allivate the inflammatory reaction and cells apotosis.It may be through down-regulation of TLR4 / NFκB signaling pathway and inhibition of microglial activation.Part4 The effect of isoflurane preconditioning on microglia activation and TLR4-NFκB signaling pathway induced by EMP esposureObjective: In vitro study,the effects of EMP on microglia and the role of TLR4/NFκB pathway in the protection of isoflurane preconditioning from EMP exposure.Method: N9 microglial cells were cultured and divided into the Control group,electromagnetic pulse exposure group(EMP group),electromagnetic pulse exposure after isoflurane preconditioning group(ISO+EMP group)and isoflurane preconditioning group(Iso group).Western Blotting was used to detect the expression of CD11 b,TLR4,NFκBp65 in microglia.Result: Western Blotting showed that the expression of CD11 b,TLR4 and NFκBp65 in the EMP group was significantly increased(P<0.05),which in the EMP+Iso group was reduced compared with the EMP group(P<0.05).Isoflurane had no significant effect on the microglia.Conclusion: In vitro experiment proved that isoflurane preconditioning could decrease the activation of microglia and down regulate the expression of TLR4/NFκB signaling pathway.