Study Of Flash Technique Of Low-frequency Focused Ultrasound Associated With Microbubbles Opening The Blood Brain Barrier Of Mice

In recent years, the neuron-protective treatment such as stem celltransplantation, gene therapy and neuron-protective drugs have developed rapidlyand successed in some animal models. The application prospects are broad. However,since the gene fragment and neural therapeutic drugs can’t pass through the bloodbrain barrier, they have to be injected directly or by intra-ventricular catheter. Thislimits their clinical application because they are all invasive. Thus, how to makethese therapeutic substances pass through the blood brain barrier safely、effectively、 and targetedly is one of the hotpots in clinical researches. A number ofstudies show that low frequency focused ultrasound combined with ultrasoundcontrast agent is the most promising approach to open the blood brain barriernon-invasively and reversibly. Most of these studies use rats as experimental subjectsand few with mice. But a lot of nervous system diseases use mice as animal models.Therefore, our study intends to explore the applied value using low-frequencyfocused ultrasound combined with Sonovue to open the mice’ blood brain barrier,via the skin and skull.This study includes two parts as follow： Part1. Effectiveness and safety of Flash technique oflow-frequency focused ultrasound associated with microbubblesopening the blood brain barrier of mice【Objective】 To explore the effectiveness and safety of Flash technique oflow-frequency focused ultrasound associated with Sonovue opening the blood brainbarrier(BBB) of mice【Methods】 Forty eight mice were randomly divided into four groups： thecontrol group、 the MB(microbubbles) group、 the Flash group、 the MB andFlash group. The right basal ganglia of the mice was sonicated by the flashprocedures in the low frequency focused ultrasound, through their intact skin andskull, after continuous intravenous injection of Sonovue and Evans blue(EB). Thedegree of BBB opening was evaluated quantitatively based on the extravasation ofEB and qualitatively under the fluorescence microscope． The safety was inspectedby observation of cell morphology under hematoxylin eosin(HE) staining andelectron microscopy．【Results】 Only in the MB and Flash group, EB extravasation in the sonicatedregion and significantly red EB fluorescence under fluorescence microscope wereshown, which can’t be seen in other three groups. The EB concentration in thesonicated side in this group was also much higher than other groups (P <0.01). In allgroups, there were no obvious neuronal damage and erythrocyte extravasationshowed by HE staining. Perivascular edema and vesicle within the cytoplasm ofendothelial cells can be seen in the Flash and microbubbles group.【Conclusions】 The BBB can be opened targetedly, safely and noninvasively byFlash procedure of low-frequency focused ultrasound with the microbubbles in mice. Part2. Optimization of Flash technique of low-frequencyfocused ultrasound associated with microbubbles opening theblood brain barrier of mice【Objective】 Preliminary studies have confirmed the effectiveness of Flashprocedure of low-frequency focused ultrasound associated with Sonovue to open theblood brain barrier(BBB)． Now we shall explore what are the optimal conditions ofFlash procedure combined with Sonovue. And then applying “the optimalconditions”, the permeability of the BBB and safety will be assessed as a function oftime after focused ultrasound exposure for the sonicated location.【Methods】 The right basal ganglia of the mice was sonicated by Flash procedureof low frequency focused ultrasound, through their intact skull, after continuousintravenous injection of Sonovue and Evans blue(EB). The permeability of the BBBwas evaluated quantitatively based on the extravasation of EB and qualitatively underthe fluorescence microscope． The relationship between the permeability of the BBBand different irradiation duration、 different Flash interval、 different doses ofcontrast agent would be discussed. The safety was inspected by observation of cellmorphology under hematoxylin eosin(HE) staining and electron microscopy．【Results】BBB disruption, as quantified by EB extraction, was measured in thetargeted region of the brain.①The EB extravasation in the sonicated regionincreased with the irradiation time within3min(3min>2min>1min,p<0.05), yet notincreased significantly from3min to5min(p>0.05); So, the irradiation time of3minwas selected.②Under the condition of irradiation time3min, the EBextravasation increased with doses of Sonovue from0.1ml/25g to0.2ml/25g(p<0.05),yet not increased significantly from0.2ml/25g to0.3ml/25g(p>0.05); So, the dose of Sonovue0.2ml/25g was selected.③Under the conditions of irradiation time3min、dose of Sonovue0.2ml/25g, the EB extravasation increased with the shortening ofthe Flash interval(2s>5s>10s, p<0.05). In short, The EB extravasation in thesonicated region was most significant when the irradiation time was3min、 theFlash procedure interval was2s、 the dose of Sonovue was0.2ml/25g. Further, usingthe optimal conditions, the EB extravasation decreased gradually with the extensionof time after irradiation. The time group comparison between groups werestatistically significant, except followings:(0min and1min group)、（60min and180min group). There were no obvious neuronal damage and erythrocyte extravasationshowed by HE staining4h after sonication.【Conclusions】 The optimal conditions is as following: irradiation time3min、Flash procedure interval2s、 the dose of Sonovue0.2ml/25g, using Flashprocedures of low-frequency focused ultrasound combined with continuousintravenous injection of Sonovue. After the sonication, the permeability of BBBdecreased gradually, and recovery completely after4hours. In short, the BBB can beopened noninsavely、directionally、safely and reversibly using the method mentionedabove.