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Preliminary Study Of Diagnostic Ultrasound Associated With Microbubbles To Open The Blood-brain Barrier: Mechanism And Effects On Permeability

Posted on:2009-12-28Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y N YangFull Text:PDF
GTID:1114360278976920Subject:Medical imaging and nuclear medicine
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BackgroundVykhodtseva has been demonstrated that the ultrasound pulses can temporarily disrupt the blood brain barrier (BBB) with negligible associated effects to the brain firstly, this phenomenon which could be exploited for a non-invasive means for ultrasound technology on transcranial therapy, broaden the research at lowest risk. Ultrasound could potentially disrupt the barrier in a volume that conforms to the desired anatomical site. Ultrasonic effects totally different from its former used in the fields of (Extracorporeal shock wave lithotripsy, High intensity focused ultrasound, Sonodynamic therapy), when choose some diagnostic ultrasound combined with an ultrasound contrast agent (also equal to microbubble), could temporarily open the BBB and reversablely. This method challenges the risk limit on BBB function. Ultrasound combined with microbubbles (UCM) is well-recognized as a powerful tool, widely applied in several series of research on BBB.The blood brain barrier serves to protect the central nervous system from blood-borne infections and toxic agents. Its unique features, such as tight junctions, low vesicular transport, and high metabolic activity, accomplish the barrier function and maintain the homeostasis of the brain parenchymal microenvironment. While beneficial, the physiological characteristics of BBB make the treatment of systemic agents into brain particularly difficult. A variety of approaches have been undertaken to open the BBB for facilitating drug delivery into brain without highly invasive procedures such as opening the cranium. For example, the BBB has been opened with intra-arterial injection of hyperosmotic solutions such as mannitol. This action of mannitol causes the endothelial cells to shrink resulting in an opening of the tight junctions for lasting a few hours. However, both osmotic and chemical methods require invasive intra-arterial catheterization and produce diffuse, transient BBB opening within the entire tissue volume supplied by the arterial branch that is injected. Likewise, lipid soluble solvents (such as high-dose ethanol or DMSO), alkylating agents (like etoposide and melphalan), immune adjuvants, and cytokines, have been used to disrupt the BBB. Localized drug delivery with disruption of the BBB can be accomplished only by injecting through a needle or catheter directly into the targeted brain area. Such direct injections not only are invasive and require opening the skull but cause non-targeted penetration of brain tissue, even to carry the risk of brain damage, bleeding, and infection. There are ways to enhance propagation through the barrier: Chemical modification of the drugs or the use of other carriers such as amino acid and peptide carriers can increase transport through the BBB. This type of localized BBB disruption hardly can be accomplished clinically until further application. Previous studies have demonstrated that many ultrasound techniques can be used to increase BBB permeability. The way of ultrasound to open BBB makes it a promising tool for targeting drug delivery.A number of animal studies have demonstrated that local BBB disrupt is possible under burst ultrasound exposures and intravascular micro-bubbles. While the exact mechanisms for the disruption are not known, it is presumably related to the interaction between the ultrasound fields, the interaction between microbubbles and the ultrasound field is strongly affected by the ultrasound frequency. For example, the frequency has a large effect on the inertial cavitation threshold and by the growth of microbubbles within the ultrasound field during sonication. This could be caused by bubble collapse with associated jet formation that punctured the vessel wall.Several hypotheses on the mechanism of BBB disruption with MBs and ultrasound have been proposed. This interaction creates a change of the pressure in the capillary to evoke biochemical reactions that trigger the opening of the BBB. Therefore, we conducted the present experiment with a non-invasive way to increase BBB permeability, and to further estimate the relationship between UCM enhanced BBB permeability, also including security studies. Hence, we utilized mouse brain microvessel endothelial cells isolated and grown in monolayer culture on porous support as a BBB model in vitro, associate with animal research data to support a useful reference on clinical therapy.Methods:1. Endothelial cells were isolated from BALB/c mice brain capillaries enzyme digest, mechanical separation and density centrifugal. In brief, the meninges of mice brain were removed and the cerebra were transferred to a pre-chilled glass homogenizer. Supernatant was carefully separated from the vasculature-enriched pellet, after 2–3 washes with the mixture of homogenization buffer and 30% dextran–70 solution (3:4), resuspend the pellet very gently and plate the cells at density of one brain equivalent. BMVECs monolayers grown in collagen-coated, inner cell structure were scanned by electron microscope. VIII factor detected by the methods of Strept Avidin Biotin-peroxidase Complex (SABC); BMVECs were plated on the Lab-Tek chambered slides and grown until confluence (2–3 days); Growth line described by the depicted of Methyl thiazolyl tetrazolium (MTT).2. Brain endothelial cells are grown on cell inserts; many porous membranes for cell culture are available in different materials and pore sizes. The pore size of these membranes is 8.0μm. The permeable membranes divide the transwell device into two compartments: the inner and outer chamber. The distributions of typical tight junction proteins Zonula occludens protein 1 (ZO-1) in mouse brain microvascular endothelial cells were studied using confocal microscopy, tight junction between cells scanned by the transmission electron microscope; A constant TEER value across the cell layer will obtained and to estimate the function of BBB in vitro. The effect of the human skull on the ultrasound beam propagation was using a single-element transducer what investigated through a piece of excised human skulls and held stationary in the transwell. There's an ultrathin acoustically and optically transparent plastic layer between the skull and transwell.3. Probe conditions will be detected by the hydrophone and choose the optimizing acoustic parameter, support the quantum data during analysing. Safety estimate will undergoing by the TEER detect and BMVECs re-culture after ultrasound exposure. Assessment of BBB permeability: was used for the HRP permeability study to investigate the paracellular diffusion across the cell monolayers.4. Mechanism research: BBB in vitro model will be devided into four groups, control group, ultrasound group, microbubble group, UCM group. The ultrasound contrast agent (self-made) contained microbubbles (approximately 2.0×107). Ultrasound waves were generated by transcranial Doppler transducer, cell ultrastructure, ZO-1 protein, TEER and HRP peameability will be detected to estimate the feasible mechanism on BBB open.5. Twenty-four transwells were divided into four groups randomly: UCM group, ultrasound group, microubbbles group and control group. Each group added 50μl colloidal gold (500ng/μl) and 9μg/cm2 Diamminedichloroplatinum (DDP). The DDP permeability was detected by the High Performance Liquid Chromatogram (HPLC) technique; The ultrastructureof brain microvascular endothelial cells (BMVEC) and colloidal gold distribution were observed under transmission electron microscope (TEM); Spectrum analysis were used to detect the two important trace elements of Carbon and Zinc on the BBB.6. Evans blue tracer experiment: the human skull covered on the rat, which original temporal bone removed. Quantitative evaluation of Evans blue dye was performed after exposure process and cardiac open, using saline to filling the heart before the atrium run off clear liquid. Each brain tissue sample was weighed, homogenized in a three-fold volume of 50% trichloroacetic acid solution, and centrifuged. The supernatants were diluted with ethanol (1:3), and fluorescence was quantified by using Ultraviolet light (UVL) reader Sample value calculations were based on Evans blue dye standards mixed with the same solvent. Results were expressed in optical density (OD) value of Evans blue dye per-milligram of tissue.7. DDP in vivo permeability: same experiment as Evans blue tracer set up described, quantitative evaluation of DDP detected by HPLC.8. Lanthanum nitrate tracing was employed to observe the mechanism which BBB permeability increased after ultrasound exposures on the presence of microbubbles. Transmission electron microscope (TEM) was used to assess the microstructure of the brain.Result:1. BBB model in vitro: the tested BALB/c mouse brain microvascular endothelial cells (BMVECs) grown on collagen-coated and fibronectin-treated culture dishes retained the morphological characteristic of microvascular endothelial cells. Typical BBB character formed a continuous monolayer with an elongated, spindle-shaped morphology, a useful tool on trans-BBB research. Electron microscopic examination, the confluent BMVECs grew into a confluent monolayer on top of the collagen fibronectin-coated transwell inserts with apparent intercellular unique W-P body.2. Tight junction transform to bridge junction. After exposure to ultrasound with the absence of microbubble, the tight junction between the endothelial cells transformed to bridge junction. Tight-junction formation decreased in the group solo-ultrasound exposure, but not apparently separation between cells. HRP permeability on group ultrasound combined with microbubble display a fluctuation wave after exposure and could recover in 18 h. TEER decreased at the lowest (179±8) ?/cm2, follows back at the same time while HRP decreased. Indicates: this technique could successfully use on transient BBB open and separate the tight junction between cells.3. Group UCM and group ultrasound sonicated by TCD, with 2MHz,0.6w/cm2 and 10 min exposure. We found that the ultrastructure of BMVEC in group UCM changed greatly from pinocytosis increased to Colloidal gold distribution in the cells. Minor Colloidal gold exist betweent the cells in group ultrasound, none in the cells, neither found in the group control and microbbles; Spectrum analysis indicates the group UCM and group ultrasound could effect the spectrum distribution of these trace elements Carbon and Zinc, but not significant difference in the group control and group microbubbles. BBB permeability of DDP increased greatly in the group UCM and group ultrasound, significantly difference among the four groups. Indicates: our results show that transcranial ultrasound associated with MBs effectively increases the permeability of DDP on the BBB in vitro, which may open the BBB reversibility.4. Lanthanum nitrate trace: In the group control and group microbbles, lanthanum only stays in the blood vessel, none in the cells and exist along the cell basal membrane, but not break through it. In group UCM, we could find the Lanthanum distribution along the BMVEC basal membrane and through the break of tight junction outside into the brain tissue.5. BBB permeability of DDP increased greatly in the group UCM, group ultrasound also increased the permeability in some extent, but only group UCM has significantly difference among the four groups (P<0.01).
Keywords/Search Tags:Blood brain barrier, Ultrasound, Microbubble, Brain microvascular endothelial cell
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