| BackgroundWith the aggravation of environmental pollution and population aging,the incidences of central nervous system(CNS)diseases have increased a lot.The treatment of these CNS diseases is facing with a common bottleneck—blood brain barrier(BBB).BBB is formed by the tight junction(TJ)between the endothelial cells,which maintains the delicate environment of the brain to ensure the normal CNS function.Since BBB keeps most of CNS drugs in the systemic circulation from entering the brain because of its strict selective permeability,the efficacy of these drugs to treat CNS diseases is very poor.How to open BBB safely and effectively has become one of the hot topics in this field?Previous studies have demonstrated that electromagnetic radiation(EMR),especially electromagnetic plus(EMP)can increase the permeability of BBB reversibly.However,the optimal physical parameters of EMP to induce BBB opening are unclear.In addition,lots of questions have been still unresolved,such as whether EMR with other parameters increases the permeability of BBB and what the dose-effect and time-effect are? In order to answer the questions above,SD rats were used in this study to investigate the effects of different EMR on BBB permeability,including EMP,ultra-wide band EMP(UWB-EMP)and radiofrequency radiation(RFR).ObjectiveTo explore the effects of different EMR(EMP,UWB-EMP,RFR)on BBB permeability so as to provide theoretical and experimental basis for the clinical application of EMRinduced BBB opening.Part I: The effects of EMP on BBB permeabilityMaterials and Methods 1 EMP equipment: the EMP signal was produced by SPG200-EMP equipment with a uniform electromagnetic field in its exposure chamber(350k V/m).The pulse width was 7ns or 50 ns.The number of pulses could be set from 0 to 6000.The repetition frequency was 1Hz.2 Animal groups and parameters: 108 healthy male SD rats were divided into exposure groups(84)and sham groups(24)randomly.The exposure groups were set as follows: 2.1 EMP with different numbers of pulses: in this part,the exposure groups were diviede into 5 subgroups according to the number of pulses(100,200,400,800,and 1600 pulses).All rats were exposed to EMP with a field strength of 350 k V/m at the pulse width of 50 ns.Animals were sacrificed at 3h after whole-body exposure.2.2 EMP with different pulse width: in this part,the exposure groups were divided into 2 subgroups according to the pulse width(7 and 50ns).All rats were exposed to EMP with a field strength of 350 k V/m for 200 pulses.Animals were sacrificed 3h after whole-body exposure.3 Detection methods: albumin and Evans blue(EB)were used as vascular tracers,which were measured by immunohistochemistry and fluorescence microscope respectively.Results 1 The effects of EMP with different numbers of pulses on BBB permeability: after exposure to EMP with a field strength of 350 k V/m and a pulse width of 50 ns,the strongest albumin immunoreactivity was found in 200 pulses group,followed by 100 pulses group and 400 pulse group.The albumin immunohistochemistry results of 800 pulses group and 1600 pulses group showed no difference compared with that of sham group.EB fluorescence showed the similar results to albumin immunohistochemistry.2 The effects of EMP with different pulse width on BBB permeability: after exposure to EMP with a field strength of 350 k V/m for 200 pulses,an increasing albumin immunoreactivity was found in both 50 ns group and 7ns group compared to sham group.And 50 ns group had a higher density.While EB results showed only in 50ns-EMP group,the BBB permeability significantly increased.Part II: The effects of UWB-EMP on BBB permeabilityMaterials and Methods 1 UWB-EMP equipment: the UWB-EMP signal was produced by UWB-EMP equipment made of generator and horn antenna.The field strength could be adjusted by changing the distance from animals to horn mouth.The pulse width was fixed while the number of pulses was set as necessary.The repetition frequency was 10 Hz.2 Animal groups and parameters: 180 healthy male SD rats were divided into exposure groups(132),positive control group(12)and sham groups(36)randomly.The exposure groups were set as follows: 2.1 UWB-EMP with different field strengths: in this part the exposure groups were divided into 3 subgroups according to the field strength(50,200 and 400 k V/m).All rats were exposed to EMP for 20000 pulses and sacrificed at 3h after wholebody exposure.2.2 The time-effect of UWB-EMP on BBB permeability: in this part,exposure groups were devided into 4 subgroups according to the sampling time(0.5,3,6 and 24 h after exposure).All animals were whole-body exposed to UWB-EMP at 400 k V/m for 20000 pulses.3 Detection methods: albumin,S100? and EB were used as vascular tracers,which were measured by immunohistochemistry,enzyme linked immunosorbent assay(ELISA) and fluorescence microscope.The expression and distribution of ZO-1 were analyzed by Western blotting(WB)and immunofluorescence staining respectively.Results 1 The effects of UWB-EMP with different field strength on BBB permeability: after exposure to UWB-EMP at 400 k V/m,enhanced albumin extravasation around the vessels in frontal cortex was found in rats.However after exposure to UWB-EMP at 50 or 200 k V/m,the albumin extravasation showed no significant difference between exposure groups and sham group.These results were in accordance with EB results.2 The time-effect of UWB-EMP on BBB permeability: after exposure to UWB-EMP at 400 k V/m for 20000 pulses,albumin extravasation began to increase at 0.5h after exposure and reached to the peak at 3~6h after exposure.The results of 24 h group were the same as those of sham group.Moreover,ELISA results showed the content of S100? significantly increased at 6h after exposure.3 The protein level and distribution of ZO-1 after exposure to UWB-EMP: after exposure to UWB-EMP at 400 k V/m for 20000 pulses,the protein level of ZO-1 in cerebral cortex decreased significantly at 3h and 6h.ZO-1 immunofluorescence staining showed that the intensity of ZO-1 decreased at 3h after exposure and there was no difference in the distribution of ZO-1 in each group.Part III: The effects of RFR on BBB permeabilityMaterials and Methods 1 RFR equipment: the frequency was 1840 MHz.The output power and exposure time could be adjusted as necessary.2 Animal groups and parameters: 36 healthy male SD rats were divided into exposure group(18)and sham group(18)randomly.Rats in exposure group were whole-body exposed to 1840 MHz RFR at a specific absorption rate(SAR)of 2W/kg for 1h/d over 7d and sacrificed immediately after exposure 3 Detection methods: albumin and lanthanum nitrate were used as vascular tracers,which were measured by immunohistochemistry and transmission electron microscopy(TEM)respectively.The expression of ZO-1 was analyzed by WB.Results After exposure to 1840 MHz RFR at 2W/kg for 7d,the albumin extravasation in frontal cortex in rats increased significantly compared to the sham group.TEM showed that lanthanum particles deposited in the basal membrane of some microvessels and a small amount of lanthanum particles penetrated into the brain parenchyma.WB results showed that the protein level of ZO-1 decreased significantly after RFR exposure.Conclusions 1 EMP exposure at 350 k V/m for 200 pulses with a pulse width of 50 ns could induce BBB opening obviously.2 UWB-EMP exposure at 400 k V/m for 20000 pulses could reversiblely increase BBB permeability significantly by reducing the protein level of ZO-1.3 1840 MHz RFR at a SAR of 2W/kg could induce BBB opening significantly.The reduction of ZO-1 may be involved in it.4 The increase in BBB permeability was not depended on the numbers of EMP pulses.This finding should be verified by more experiments and its mechanism also needed further studies. |
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