Effects Of EMP And PMF With Different Parameters On BBB Permeability In Rats

BackgroundWith the aggravation of the aging degree of society,the incidence of CNS diseases is increasing year by year.By 2050,the number of people with Alzheimer’s disease worldwide will have increased from 46 million to 131.5 million.As one of the ten most common tumors at present,the incidence of glioma of China increased significantly(annual growth rate: 2.1%)from 2000 to 2011.In addition,the cost of other CNS diseases such as stroke,meningitis,Huntington’s disease,epilepsy,and mental illness also burden the society and health budget.Although technological tools and biomedical advances have led to a better understanding of the pathophysiological mechanisms of these CNS diseases,no significant progress has been made in drug therapy.Many CNS drugs have been discovered in recent years,but even when they worked well in the early stages of drug development,they often fail to pass subsequent clinical trials,in part because they cannot across the BBB.According to statistics of the world’s top prescription drugs in 2014,only one drug among the top ten molecules is a drug for the CNS diseases.In addition,there are only two CNS treatment areas in the top 10 treatment areas in the world by sales,namely,products related to pain and mental health.From these data,it can be seen that the CNS drug development is not satisfactory,the main reason is that the drug cannot pass BBB.To increase the delivery of CNS drugs,current methods include nanotechnology,drug modification,focused ultrasound combined with microbubbles.However,these methods have some disadvantages such as high cost,obvious side effects or slight effects.The previous results of our laboratory,it is found that EMP can increase the permeability of BBB to large molecules.Our laboratory has found that EMP of 200 kV/m field strength and 200 pulses could temporarily open BBB without causing thermal effect.But what is the parameters of EMP can achieve the best BBB opening effect? And what are the molecular mechanisms of EMP increasing BBB permeability? Can other frequency of pulsed EMR also lead to BBB opening? These questions have not yet been answered.In this study,SD rats were selected as object to study the effects of EMP with different parameters and PMF of different field strength on BBB permeability of rats and the changes of BBB related proteins,so as to provide experimental basis for the discovery of new CNS drug delivery strategy.Part Ⅰ: The effect of different parameters of EMP on BBB permeabilityObjective: To study the effects of different parameters of EMP on BBB permeability,so as to screen the optimal parameters of EMP to open BBB and explore the molecular mechanism of EMP increasing BBB permeability,providing experimental evidence for further application.Methods: Albumin immunohistochemistry and exogenous EB fluorescence tracer method were used to evaluate the BBB permeability when 3 hours after EMP exposure.1.Effects of different EMP field strength on BBB permeability: 48 SD rats were randomly divided into sham group and three different field strength groups(350 kV/m,480 kV/m,720 kV/m),with 12 rats in each group.3 hours after exposure,6 rats in each group were used for albumin immunohistochemistry and other 6 rats were used for EB fluorescence tracing.Other parameters of EMP : 800 pulses,pulse width was 40 ns,rising time was 19 ns,and repetition frequency was 1 pps.2.Effects of EMP of different pulses on BBB permeability: 48 SD rats were randomly divided into sham group and three different pulses groups(200,400 and 800 pulses),with 12 rats in each group.6 rats in each group were used for albumin immunohistochemistry and other 6 rats were used for EB fluorescence tracing.Other EMP parameters: field strength was 720 kV/m,pulse width was 40 ns,rising time was 19 ns,and repetition frequency was 1 pps.3.Effects of EMP of different repeat frequencies on BBB permeability: 48 SD rats were randomly divided into sham group and three different repeat frequency groups(1 pps,10 pps and 20 pps),with 12 rats in each group.6 rats in each group were used for albumin immunohistochemistry and other 6 rats were used for EB fluorescence tracing.Other EMP parameters: 720 kV/m field strength,40 ns pulse width,19 ns rising time,and 800 pulses.4.Changes of ultrastructure of TJ after EMP exposure: 3 hours after EMP exposure,using TEM to observe the effect of EMP on the ultrastructural changes of TJ.5.Changes of expression levels of BBB permeability related proteins after EMP exposure with different parameters: there were 5 rats in each group,3 hours after EMP exposure with different pulse(200,400 and 800 pulses)and different repeat frequency(1 pps and 10 pps),western blot was used to detect the changes of TJs related proteins,such as ZO-1,Occludin and Claudin-5,and expression of Caveolin-1,a structural protein of the caveolae,and MMP-9.Immunofluorescence method was used to detect the expression location and the difference of Caveolin-1 expression between exposure group and sham group.Results: the results of immunohistochemistry and EB fluorescence tracing method showed that EMP with different parameters could cause the extravasation of albumin and EB around microvessels,indicating the opening of BBB in rats.With the increasing of field strength and pulses,the degree of BBB permeability increased.Compared with sham group,groups of 10 pps and 20 pps repeat frequency had better BBB permeability,but there was no statistical difference between 10 pps and 20 pps.TEM results showed that,after EMP exposure,the ultrastructure of TJ became loose.Western blot results showed that compared with the sham group,the protein levels of TJ-related proteins ZO-1,Occludin and claudin-5 were significantly reduced after EMP exposure,with statistically significant differences(P < 0.05).The expression levels of Caveolin-1 and MMP-9 in the EMP group were significantly higher than those of the sham group(P < 0.05).The results of immunofluorescence showed that Caveolin-1 was mainly expressed around the microvessels of brain tissue and the intensity and quantity of fluorescence was higher in exposure groups.Part Ⅱ :The influence of different parameters of PMF on BBB permeabilityObjective: To investigate the effects of PMF of different magnetic field intensity on the BBB permeability in SD rats.Methods: 72 SD rats were randomly divided into sham group,positive control group and 4 groups with different magnetic field strength(100 mT、400 mT、800 mT、1.2 T),with 30 pulses per group.The frequency of PMF is 50 Hz,and the repetition frequency is 1 minute between pulses.3 hours after exposure,HE staining was used to evaluate the gross morphology of hippocampus.The extravasation of perivascular albumin was detected by immunohistochemistry.Exogenous EB fluorescence assay was used to observe EB extravasation around microvessels.Western blot analysis of the expression of TJ related proteins ZO-1 and Occludin after PMF exposure。Results: Compared with the sham group,there were no significant changes in the hippocampus histological structure of rats in the PMF group with different magnetic field strength,and no significant extravasation of albumin and EB around the cerebral microvessels.The results of semi-quantitative analysis showed that there were no statistical differences between the experimental groups and the sham group.The ZO-1 protein level in the 1.2 T exposure group was significantly lower than that in the sham group(P < 0.05),and there was no significant change in Occludin protein level in all magnetic field strength exposure groups.The above results suggested that although the low-frequency PMF exposure with high magnetic field strength did not significantly change the BBB permeability to albumin of SD rats under the experimental conditions,the decrease of ZO-1 protein level suggested that the integrity of BBB was damaged,the effect of higher magnetic field strength or more pulses of PMF on the BBB integrity should be discussed in the future.Conclusion 1.EMP can increase the permeability of BBB,and the experimental parameters that affect the opening of BBB by EMP include field strength,pulse number and repetition frequency.With the increase of EMP field strength and pulses,the effect of BBB opening also increased.In group of 10 pps or 20 pps repetition frequency,the increasing of BBB permeability of the rats was more than that of the 1pps group.2.After EMP exposure,the ultrastructure of TJ became loose,and the levels of TJ-related proteins ZO-1,Occludin and Claudin-5 decreased,in the same time,the expression levels of Caveolin-1 and MMP-9 increased,suggesting that these molecules may be involved in the mechanism of EMP-induced BBB permeability changes 3.The parameters of the PMF selected in this experiment could not lead to the increase of BBB permeability.However,the pulsed magnetic field with high magnetic field strength could lead to the decrease of TJ related protein ZO-1 protein level.