| Background Blood-brain barrier(BBB)is a hightly specialized barrier,which strictly limits potentially neurotoxic components from plasma entering into the brain to stabilize homeostasis of the CNS.However,BBB also keeps most of drugs from entering the brain,and seriously affects the treatment efficiency of CNS diseases.How to overcome BBB remains to be one of the big challenges in modern medicine.Electromagnetic pulse(EMP)is a kind of transient electromagnetic field,which is characterized by rapid generation,short duration,high peak intensity and wide spectrum range.It is now widely used in communication engineering and electronic countermeasures.Previous studies have shown that EMP can increase BBB permeability and its effect was related to its electric field strengths,pulse width,pulse number and repetition frequency.However,which parameter plays the key role,the specific dose-effect and time-effect relationships between these physical parameters and BBB permeability,and the effect of EMP on different brain regions are still not very clear.Therefore,this study focuses on the regulation and mechanism of EMP-induced BBB opening.On the one hand,the results lay a foundation for EMP clinical application to assist drug delivery in the treatment of CNS diseases.On the other hand,it is of great significance to establish EMP protection standard and put forward prevention strategy in special working environment.Objective1.To identify the key EMP parameter that plays a major role in the EMP-induced BBB opening,the dose-effect relationship of the effect of EMP with different electric field strengths on BBB permeability,and time-effect relationship of EMP-induced BBB opening2.To reveal the sensitive brain area to EMP.3.Comprehensive analysis of possible molecular mechanisms in the EMP-induced BBB opening to provide theoretical and experimental evidence for EMP protection and clinical applications.Materials and Methods1.Healthy male SD rats were used as research subjects.The Evans blue(EB)leakage were quantitatively caculated to detect the BBB permeability of rats.Referring to the EMP parameters reported in the previous literature that can lead to BBB opening,the effects of EMP exposure with different electric field strength(400,800 k V/m),pulse width(40 ns、1.3 μs)and pulse numbers(100,800 pulses)on the BBB permeability were compared according to the factorial design.2.Selecting endogenous albumin and exogenous EB as BBB permeability tracers,albumin immunohistochemistry and EB fluorescence imaging were applied respectively to observe the changes in BBB permeability of rats after exposure to EMP with the electric field intensity of 600,800,1000,1200 or 1500 k V/m.Apoptosis of cells in rat brain was detected by Td T-mediated d UTP nick end labeling(TUNEL).Other EMP parameters: pulse width 40 ns,800 pulses,repetition frequency 10 Hz.3.EB fluorescence imaging was selected to determin the BBB permeability of rats at different time(1,3,6,9,12,24 hours)after EMP exposure.EMP parameters: electric field intensity 1200 k V/m,pulse width 40 ns,800 pulses,repetition frequency 10 Hz.4.The electric field strengths distribution in the brain was calculated by simulation to predict the sensitive brain area where BBB would be disrupted.To verify the simulation results,EB fluorescence oberservation and in vitro fluorescent imaging were applied to detect and quantitatively analyze BBB permeability in different brain regions,including prefrontal cortex,parietal cortex,hippocampus,striatum,and thalamus.EMP parameters: electric field intensity 1200 k V/m,pulse width 40 ns,800 pulses,repetition frequency 10 Hz.5.Tandem Mass Tag(TMT)quantitative proteomics technology was used to comprehensively observe and analyze the function of significantly differently expressed proteins in the cerebral cortex of rats after EMP exposure,and searched for key molecules related to the structure and function of BBB.6.Transmission electron microscopy(TEM)was used to observe the ultrastructure of brain microvessels.Western blot and q RT-PCR was used to detect the expression of Glyceraldehyde-3-phosphate dehydrogenase(GFAP),Aquaporin 4(AQP4),Platelet derived growth factor receptor β(PDGFRβ)and α-smooth muscle actin(α-SMA)in cerebral cortex.Immunofluorescence staining was used to observe the distribution of GFAP,AQP4 and α-SMA.Results1.Electric field intensity was the main factor influencing EMP-induced BBB opening in rats.The analysis of variance by factorial design showed that among the three physical parameters of EMP including electric field intensity,pulse width and pulse number,only electric field intensity had an effect on EMP-induced BBB opening in rats,and there was no interaction between electric field intensity,pulse width and pulse times.2.Electric field intensity was positively correlated with EMP-induced BBB opening.Three hours after exposure EMP with different electric field strengths(600,800,1000,1200,and 1500 k V/m),albumin and EB extravasation were observed around microvessels in rat brain tissue.By quantitative analysis of integral optical density and average optical density,it was found that when the EMP electric field intensity was higher than 800 k V/m,the total extravasation of albumin and EB increased significantly,and increased with the rising of the electric field strength.When the EMP electric field strength exceeded 1200 k V/m,the proportion of individuals with BBB opening in same EMP group was the largest(8/10).When the EMP electric field strength reached 1500 k V/m,the apoptosis level in cerebral cortex increased significantly.These results implied that the effect of EMP-induced BBB opening was positively related to its electric field intensity,and EMP with electric field intensity of 1200 k V/m can maximize BBB permeability of rat without causing obvious damage on rat brain.3.EMP-induced BBB opening was reversible.After exposure to EMP with 1200 k V/m,40 ns,10 Hz for 800 pulses,the analysis of EB integral optical density,positive area,and average optical density in rat brain showed that compared with sham group,EB extravasation increased at 1 h after exposure,but there was no statistical significance;3 h after exposure,EB exuded the most and the difference was statistically significant;and then EB extravasation decreased significantly at 6 h after exposure;the leakage increased slightly 9-12 h after exposure;and no obvious exudation of EB was observed till 24 h after exposure.4.The sensitivity of BBB to EMP was different in different brain regions.After exposure to EMP with 1200 k V/m,40 ns,10 Hz for 800 pulses,the internal electric field strengths of different parts of rat brain were different.Assessment by simulation calculations,it was found that the average electric field strenght was the largest at the cerebral coronal plane where was 6 mm behind the olfactory bulb,and the electric field intensity was larger near the outer cortex in the same plane.According to EB extravasation,the most sensitive brain region to EMP was cortex around the parietal cortex(cerebral cortex within 3-9 mm behind the olfactory bulb),followed by the prefrontal cortex and hippocampus.However,no significant EB exudation was observed at the striatum and thalamus.The results indicated that BBB in different brain regions had different sensitivity to EMP exposure;with the current parameters,the BBB permeability increased most around the parietal cortex of rats after EMP exposure;the spatial distribution of EMP-induced BBB opening had a certain correlation with the electric field strengths distribution in the brain.5.EMP caused significant differences in the expression of 515 proteins in cerebral cortex of rats,among which 246 were up-regulated and 269 were down-regulated.GO analysis showed that these proteins were mainly located in the cytoplasm and cell membrane.The proteins mainly played protein binding functions,including enzyme binding,cytoskeleton protein binding,ion channel binding,protein macromolecule and nucleoside binding.KEGG pathway analysis indicated that the adhesion plaques pathway and tight junction pathway which are associated with BBB permeability were also significantly enriched.6.Analysis of protein-protein interaction network revealed that after EMP exposure,the key differentially expressed proteins closely related to the structure and function of BBB were Caveolin-1,GFAP and AQP4.7.EMP could affect the distribution and function of astrocytes and pericytes.After EMP exposure,astrocytes around brain capillaries were slightly swollen after EMP exposure.The expression level of AQP4 in the cerebral cortex was significantly reduced,however,the GFAP expression was not.GFAP and AQP4 distributions in BBB opening areas were abnormal to a certain extent.The expression of pericyte-associated protein α-SMA increased,and it was mainly distributed in micro and small blood vessels.ConclusionsThe current study identified the electric field intensity of EMP is the key physical parameter that affects its effect on opening BBB.The optimal EMP parameters to open BBB in rats were as follows: electric field intensity 1200 k V/m,pulse width 40 ns,pulse frequency 800 times and repetition frequency 10 Hz.EMP could increase the BBB permeability reversibly.The study revealed that BBB in different brain regions has different sensitivity to EMP exposure,and BBB in the parietal cortex of rats is most sensitive to EMP with current parameters.After EMP exposure,the protein expression profile in the brain tissue of rats was significantly changed.The results of proteomics and animal experiments suggested that after EMP exposure,astrocyte terminal foot swelling resulted in disruption of BBB integrity,which may be involved in the process of EMP-induced BBB opening.However,the mechanism of how EMP induced astrocyte swelling needs further study.In this paper,interdisciplinary technology was applied to reveal the sensitivity of BBB in different parts of the brain to EMP.It is the first time to demonstrate the effect of EMP on the protein expression profile of rat cortical tissue.The relevant results will provide theoretical and experimental basis for the application of EMP-induced BBB opening and the study of its mechanism. |
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