Effect Of Ultrasound-targeted Microbubble Destruction Research Articles

Ultrasound-Targeted Microbubble Destruction Accelerates Angiogenesis and Ameliorates Left Ventricular Dysfunction after Myocardial Infarction in Mice

Failure of coronary recanalization within 12 h or no flow in the myocardium after percutaneous coronary intervention is associated with high mortality from myocardial infarction, and insufficient angiogenesis in the border zone results in the expansion of infarct area. In this study, we examined the effects of ultrasound-targeted microbubble destruction (UTMD) on angiogenesis and left ventricular dysfunction in a mouse model of myocardial infarction. Fifty-four mice with MI were treated with no UTMD, ultrasound (US) alone or UTMD four times (days 1, 3, 5 and 7), and another 18 mice underwent sham operation and therapy. Therapeutic US was generated with a linear transducer connected to a commercial diagnostic US system (VINNO70). UTMD was performed with the VINNO70 at a peak negative pressure of 0.8 MPa and lipid microbubbles. Transthoracic echocardiography was performed on the first and seventh days. The results indicated that UTMD decreased the infarct size ratio from 78.1 ± 5.3% (untreated) to 43.3 ± 6.4%, accelerated angiogenesis and ameliorated left ventricular dysfunction. The ejection fraction increased from 25.05 ± 8.52% (untreated) to 42.83 ± 9.44% (UTMD). Compared with that in other groups, expression of vascular endothelial growth factor and endothelial nitric oxide synthase and release of nitric oxide were significantly upregulated after UTMD treatment, indicating angiogenesis. Therefore, UTMD is a potential physical approach in the treatment of myocardial infarction.

Using ultrasound-targeted microbubble destruction to enhance radiotherapy of glioblastoma

ObjectiveTo investigate the efficacy and mechanism of ultrasound-targeted microbubble destruction (UTMD) combined with radiotherapy (XRT) on glioblastoma.MethodsThe enhanced radiosensitization by UTMD was assessed through colony formation and cell apoptosis in Human glioblastoma cells (U87MG). Subcutaneous transplantation tumors in 24 nude mice implanted with U87MG cells were randomly assigned to 4 different treatment groups (Control, UTMD, XRT, and UTMD + XRT) based on tumor sizes (100–300 mm3). Tumor growth was observed for 10 days after treatment, and then histopathology stains (HE, CD34, and γH2AX) were applied to the tumor samples. A TUNEL staining experiment was applied to detect the apoptosis rate of mice tumor samples. Meanwhile, tissue proteins were extracted from animal specimens, and the expressions of dsDNA break repair-related proteins from animal specimens were examined by the western blot.ResultsWhen the radiotherapy dose was 4 Gy, the colony formation rate of U87MG cells in the UTMD + XRT group was 32 ± 8%, lower than the XRT group (54 ± 14%, p < 0.01). The early apoptotic rate of the UTMD + XRT group was 21.1 ± 3% at 48 h, higher than that of the XRT group (15.2 ± 4%). The tumor growth curve indicated that the tumor growth was inhibited in the UTMD + XRT group compared with other groups during 10 days of observation. In TUNEL experiment, the apoptotic cells of the UTMD + XRT group were higher than that of the XRT group (p < 0.05). The UTMD + XRT group had the lowest MVD value, but was not significantly different from other groups (p > 0.05). In addition, γH2AX increased due to the addition of UTMD to radiotherapy compared to XRT in immunohistochemistry (p < 0.05).ConclusionsOur study clearly demonstrated the enhanced destructive effect of UTMD combined with 4 Gy radiotherapy on glioblastoma. This could be partly achieved by the increased ability of DNA damage of tumor cells.

Ultrasound-Targeted Microbubble Destruction Mediated si-CyclinD1 Inhibits the Development of Hepatocellular Carcinoma via Suppression of PI3K/AKT Signaling Pathway.

Background and AimIn our study, we aimed to investigate the effect of ultrasound-targeted microbubble destruction (UTMD)mediated si-CyclinD1 (CCND1) on the growth of hepatocellular carcinoma (HCC) cells.Patients and MethodsBioinformatics analysis was performed to detect the difference of CCND1 expression of HCC and normal liver tissues. After treatment with UTMDmediated si-CCND1, the growth and apoptosis of HepG2 cells were detected by flow cytometry, MTT, EdU staining, colony formation assay, Hoechst 33,258 staining and Western blot analysis. The growth of HepG2 cells in vivo was also studied via xenograft tumor in nude mice.ResultsCCND1 was highly expressed in HCC tissues and HCC cell lines. UTMDmediated si-CCND1 could inhibit the growth of HepG2 cells and promote apoptosis via suppression of the PI3K/AKT signaling pathway. UTMDmediated si-CCND1 could also suppress the growth of HepG2 cells in vivo.ConclusionOur study provided evidence that UTMDmediated si-CCND1 could inhibit the growth of HepG2 cells and promote apoptosis via suppression of the PI3K/AKT signaling pathway.

The effects of ultrasound-targeted microbubble destruction (UTMD) carrying IL-8 monoclonal antibody on the inflammatory responses and stability of atherosclerotic plaques

This study investigated the value of using ultrasound-targeted microbubble destruction (UTMD) to deliver an IL-8 monoclonal antibody to inhibit the inflammatory response and increase plaque stability in a rabbit model of atherosclerosis (AS). An abdominal aortic atherosclerotic plaque model was established in sixty 4-week-old male New Zealand rabbits. The rabbits were fed a high-fat diet for 12 weeks. On the 12th week, the abdominal aorta was subjected to both balloon-induced mechanical injury and bovine serum albumin-induced immunological injury. After these injuries were established, the rabbits were fed a high-fat diet for 8 additional weeks. On the 20th week, the rabbits were divided into three groups: the pretreatment (PT) group, the control group, and the IL-8 group. The ultrasonic parameters and histological data associated with the plaques from the PT group were acquired on the 20th week after targeted contrast-enhanced ultrasonography (CEUS) was performed. The rabbits in the IL-8 and control groups received targeted CEUS and UTMD every 2 weeks. A targeted contrast agent carrying IL-8 monoclonal antibody was used for the IL-8 group, whereas normal saline was used for the control group. The rabbits in these two groups underwent the same procedure four times beginning during the 20th week. On the 26th week after UTMD, ultrasonic parameters and histological data were collected. The peak intensity (PI), microvessel density (MVD), and macrophage count of the PT group were significantly higher than those of both the control and IL-8 groups (P < 0.05). Additionally, these three parameters were higher in the control group than in the IL-8 group (P < 0.05). The two-dimensional (2D) ultrasonic parameters, including the maximum thickness of the plaque and the intima-media thickness (IMT), did not differ significantly among the three groups (P > 0.05). PI, MVD, and macrophage count were positively correlated with each other (r=0.564, r=0.6034, and r=0.536, respectively; P<0.05). UTMD-delivered IL-8 monoclonal antibodies alleviate inflammation within atherosclerotic plaques. UTMD is a novel and effective method for plaque stabilization.

Enhancement of gene transfection efficiency and therapeutic effect of ultrasound-targeted microbubble destruction in vivo with cationic microbubble

To construct a cationic microbubble (CMB), and investigate the enhancement of gene transfection efficiency and therapeutic effect of ultrasound-targeted microbubble destruction (UTMD) in vivo with CMB compared to definity MB (DMB). In vitro, the CMB was prepared by the method of thin film hydration. The morphology, size, zeta potential, and gene-carrying capacity of CMB were compared with the DMB. In vivo, the firefly luciferase gene which was used as a reporter gene was targeted transfected into myocardium of 16 rats with CMB and DMB, respectively. The gene transfection efficiency and targeting were observed dynamically. Then, ischemia-reperfusion (I/R) model was performed on 64 rats. The models of 60 rats were successfully confirmed by using ultrasonography at 5 days after I/R. The rats were divided into 3 groups ( n=20) randomly. The control group received DMB carrying empty plasmid for transfection; DMB group received DMB carrying AKT plasmid for transfection; and CMB group received CMB carrying AKT plasmid for transfection. The cardiac perfusion, cardiac function, infarct size, and infarct thickness were measured by ultrasonography and histological observations after treatment. In addition, the capillary and arteriolar densities were measured with immunohistochemical staining. The myocyte apoptosis was measured with TUNEL staining. The protein expressions of AKT, phospho-AKT (P-AKT), Survivin, and phospho-BAD (P-BAD) were measured by Western blot. The size of CMB was uniformly. The zeta potential of CMB was significantly higher than that of DMB ( t=28.680, P=0.000). The CMB bound more plasmid DNA than the DMB ( P<0.05). The luciferase activity of myocardium were higher in CMB group than in DMB group both in vitro and in vivo measurements ( P<0.05). There was no significant difference between groups in the ratio of signal intensity in anterior wall to posterior wall, ejection fraction (EF), and fractional shortening (FS) at 5 days after I/R ( P>0.05), but the above indexes were significant higher in CMB and DMB groups than in control group at 21 days after I/R ( P<0.05). Besides, the above indexes were significant higher in CMB group than in DMB group at 21 days after I/R ( P<0.05). The infarct size was the smallest and infarct thickness was the thickest in the CMB group, followed by DMB group, control group at 21 days after I/R. The capillary and arteriolar densities of CMB and DMB groups were significant higher than those of control group at 21 days after I/R ( P<0.05). Besides, the capillary and arteriolar densities of CMB group were significant higher than those of DMB group ( P<0.05). The apoptotic cells were the most in the control group, followed by DMB group, CMB group at 3 days after gene transfection, showing significant differences between groups ( P<0.05). The protein expressions of AKT, P-AKT, Survivin, and P-BAD were significant higher in CMB and DMB groups than those in control group at 3 days after gene transfection ( P<0.05). Besides, these protein expressions were significant higher in CMB group than those in DMB group ( P<0.05). The DNA-carrying capacity and gene transfection efficiency are elevated by CMB, although its physicochemical property is the same as DMB. When ultrasound-targeted AKT gene transfection is used to treat myocardial I/R injury in rats, delivery of AKT with the CMB can result in higher transfection efficiency and greater cardiac functional improvements compared to the DMB.

Ultrasound-targeted microbubble destruction enhances polyethylenimine-mediated gene transfection in vitro in human retinal pigment epithelial cells and in vivo in rat retina.

The aim of the present study was to investigate the efficacy and safety of ultrasound-targeted microbubble destruction (UTMD)-mediated polyethylenimine (PEI) transfection in cultured human retinal pigment epithelial (RPE) cells in vitro and rat retinas in vivo. An enhanced green fluorescent protein plasmid (pEGFP) was incubated with PEI to prepare a cationic complex (PEI/pEGFP), which was confirmed using a gel retardation assay. In the in vitro study, cultured human RPE cells were subjected to US waves under different conditions with or without microbubbles. The effect of UTMD on the viability of the cells was evaluated. In the in vivo study, gene transfer was examined by injecting PEI/pEGFP into the subretinal space of the rats. The rats treated with PEI/pEGFP and UTMD served as the experimental group, while rats treated with PEI/pEGFP alone served as the control group. The transfected tissue was visualized using an inverted fluorescence microscope. The expression of EGFP was classified into three groups, negative, weak positive and strong positive. Hematoxylin and eosin staining of frozen sections was used to observe tissue damage and the location of the EGFP gene expression. The electrophoresis experiment revealed that PEI treatment was able to condense DNA efficiently. In the in vitro study, the gene transfer efficiency under the optimal UTMD condition was enhanced and significantly higher than control groups. In the in vivo study, UTMD was able to enhance transgene expression in the retina without marked tissue damage. Frozen sections of the optic cups exhibited pEGFP‑positive cells, predominantly distributed in the retina. This noninvasive novel combination of UTMD with PEI was able to enhance targeted gene delivery and gene expression in the rat retina without causing any apparent tissue damage, and may be a safe method to transfer genes and drug treatments directly to the retina, therefore being of potential therapeutic value.

Recombinant adeno-associated virus-, polyethylenimine/plasmid- and lipofectamine/carboxyfluorescein-labeled small interfering RNA-based transfection in retinal pigment epithelial cells with ultrasound and/or SonoVue

The present study was conducted to investigate the efficacy and safety of ultrasound (US)‑mediated transfection of the type 2 recombinant adeno‑associated virus (AAV) vectors encoding the enhanced green fluorescent protein (EGFP) gene (rAAV), polyethylenimine (PEI)/plasmid EGFP‑N1 (pDNA) or lipofectamine (L)/carboxyfluorescein (FAM)‑labeled small interfering RNA (siRNA) in the human ARPE‑19 retinal pigment epithelial (RPE) cell line, with or without the addition of SonoVue. Cultured RPE cells were exposed to US, with or without SonoVue under different conditions, including variation in the intensity and duration of treatment, and the dose of microbubbles. The effects of ultrasound‑targeted microbubble destruction (UTMD) on the structure of pDNA and the transfection ability of rAAV, PEI/pDNA and L/siRNA were also evaluated. Furthermore, the effect of UTMD on RPE cells was evaluated at 0 and 24 h following UTMD. US‑mediated transfection (USMT) significantly increased L/siRNA transfection efficiency, as measured by the transgene expression per cell and the percentage of transfected cells. UTMD significantly increased rAAV and PEI/pDNA transfer to RPE cells. UTMD‑mediated rAAV or PEI/pDNA delivery was more effective than USMT‑mediated delivery of siRNA. Evaluating cell viability at 24 h post‑UTMD provided more valuable information than immediate evaluation following UTMD. Furthermore, there was minimal cytotoxicity and minimal change to the structure of pDNA under the optimal parameters. UTMD/US may be of use in enhancing rAAV, PEI/pDNA and L/siRNA transgene expression of ARPE‑19 cells in vitro. Studies on the transfection of different nucleotides (such as pDNA and siRNA) and different types of vectors (chemical and biological) mediated by UTMD may provide useful information to guide future in vivo and transfection studies.

A targeted ultrasound contrast agent carrying gene and cell-penetrating peptide: Preparation and gene transfection in vitro

Targeted and high efficient gene delivery is a main issue in gene treatment. Taking advantage of ischemic memory target P-selectin and our previous study-synergistic effects of ultrasound-targeted microbubble destruction (UTMD) and TAT peptide on gene transfection, which were characterized by targeted aggregation and high efficient gene transfection, we set up a ‘smart’ gene delivery system-targeted ultrasound contrast agent (UCA) carrying gene and cell-permeable peptides (CPP). Such UCA had a strong binding force with DNA which was protected from being hydrolysed by nuclease. Moreover, synergistic effects of UTMD and TAT peptide increased gene transfection. Specifically, the UCA were reacted with an ischemic memory target P-selectin overexpressed by ischemic issues (including ischemic heart disease) and loaded with gene and CPP, which enabled targeted localization and gene delivery to ischemic cells overexpressing P-selectin. We demonstrated their targeting affinity for hypoxia human umbilical vein endothelial cell (HUVEC) and gene transfection in vitro. The results of confocal laser scanning microscopy (CLSM) showed that gene and CPP were distributed on the shell of UCA. Red fluorescence was observed on the surface of targeted UCA using immunofluorescent microscopy, which demonstrated that the antibody was successfully connected to the UCA. The targeted UCA was specifically and tightly binded to hypoxia HUVEC, while there were no or little non-targeted UCA binding around hypoxia HUVEC. 24h after transfection, gene transfection efficiency detected by FCM was higher in targeted group than non-targeted group. Overall, the targeted UCA carrying gene and CPP was prepared successfully. It had a strong target binding capacity to hypoxia HUVEC and high efficient gene transfection, which maybe provide a novel strategy for gene therapy.

The dual effect of ultrasound-targeted microbubble destruction in mediating recombinant adeno-associated virus delivery in renal cell carcinoma: transfection enhancement and tumor inhibition

Recombinant adeno-associated virus (rAAV) is recognized as a promising vector for cancer gene therapy, although its low transfer efficiency in less permissive cells limits extensive application. Our previous studies reported that ultrasound-targeted microbubble (MB) destruction (UTMD) enhanced rAAV transfer in its permissive retinal cells. In the present study, we investigated whether UTMD increased rAAV transfer in less permissive human renal cell carcinoma (hRCC) cells and tumors. hRCC cells were treated with rAAV2 under different conditions of UTMD, and the viral transfer efficiency and cell viability were analyzed. Fifty-two male nude mice (BALB/c) implanted with hRCC cells were randomly assigned to four groups consisting of rAAV, rAAV + ultrasound and rAAV + UTMD (20 µl and 40 µl of MBs). UTMD was initiated immediately after intratumoral viral injection, and viral transfer efficiency and tumor volumes were analyzed at 12 weeks after infection. The efficiency of non-augmented transfer of rAAV2 into hRCC cells was low (17.28 ± 2.44%). The use of UTMD enhanced viral transfer efficiency by two- to three-fold, and enhanced viral genomic DNA by more than nine-fold, without decreasing cell viability. In vivo studies also showed that UTMD increased rAAV2 transfer in tumor. The enhancements were maintained for a period of 12 weeks. Tumor growth in mice was inhibited by UTMD treatment, and UTMD treatment augmented by MBs (40 µl) produced an even stronger effect. UTMD enhanced rAAV2 transfer into less permissive RCC cells and tumors, resulting in inhibition of tumor growth, which suggests that UTMD may be a useful delivery tool for cancer gene therapy.

Synergistic effects of ultrasound-targeted microbubble destruction and TAT peptide on gene transfection: An experimental study in vitro and in vivo

Cell-permeable peptides (CPPs) and ultrasound-targeted microbubble destruction (UTMD) have tremendous potential for gene delivery. However, their applications are limited due to nonspecificity of CPPs and low transfection efficiency of UTMD. Here, we developed a ‘smart’ gene delivery system by encapsulating TAT peptide (TATp) and hepatocyte growth factor (HGF) gene within lipid microbubbles, in which TATp was protected from being enzymatically cleaved and HGF gene was protected from degradation. This new strategy had synergistic effects of UTMD and TATp on gene transfection. We investigated the efficacy and safety of HGF gene transfection mediated by the combination of UTMD and TATp in vitro and in vivo. The results from MTT assay and flow cytometry analyses indicated that the combination of UTMD and TATp could enhance HGF gene expression in HUVECs without any significant side effect on cell viability. In rat myocardial infarction models, we demonstrated that the protein and mRNA expressions of HGF in myocardium caused by the combination of UTMD and TATp were the highest.Histopathological findings demonstrated that the combination of UTMD and TATp enhanced myocardial microvasculature and ameliorated myocardial fibrosis. In conclusion, the combination of UTMD and TATp might be a safe and efficient technique for gene delivery.

Inhibitory effects of ultrasound-targeted microbubble destruction mediated HSV-TK/GCV system on ovarian cancer in BALB/c-nu mice

Objective To investigate the anti-tumor effect of ultrasound-targeted microbubble destruction (UTMD) mediated herpes simplex virus thymedine kinase (HSV-TK) on mice ovarian cancer.Methods Forty female BALB/c-nu mice were randomly divided into four groups after the models of subcutaneous transplantation tumors were established:(A)HSV-TK + Microbubbles + Ultrasound (HSV-TK+ MBs + US);(B) HSV-TK+ Ultrasound (HSV-TK + US);(C) HSV-TK;(D) PBS.TheTK protein and mRNA expression were separately detected by western-blot and real time RT-PCR.TUNEL staining was used to detect the tumor cell apoptosis.The inhibition rates and survival time of the animals were compared among all groups.Results The HSV-TK gene transfection efficacy and tumor inhibitory effect of HSV-TK on mice transplantable tumor in group A (HSV-TK + MBs + US) were significantly improved compared with group B (HSV-TK + US),group C (HSV-TK) and group D (PBS) (P ＜0.05).However,group A has no significant difference (P ＞0.05) with other groups in improving the survival time of tumor-bearing mice.Conclusions Ultrasound-targeted microbubble destruction can effectively transfect HSV-TK gene into target tissues and play a significant inhibition effect on ovarian cancer in mice.However,this new method is not able to improve the survival time of mice for a short-term observation. Key words: Ultrasonography; Microbubbles; Gene therapy; Ovarian neoplasms

Ultrasound-targeted microbubble destruction promotes proliferative vitreoretinopathy induced by RPE-J cells and platelet-rich plasma

Ultrasound-targeted microbubble destruction (UTMD) has been considered as a new approach to the gene therapy of proliferative vitreoretinopathy (PVR), but whether it affect the in vivo PVR model induction remains unknown. In this study, 90 Wistar rats were averagely divided into three groups according to the content of intravitreal injection: normal saline (Group 1), retinal pigmented epithelial -J cells and PRP (Groups 2 and 3). In Group 3, a condition of UTMD was used additionally on days 3 and 7 after injection. On days 14 and 28, the pathological changes of eye grounds were assessed, and the expression levels of transforming growth factor (TGF)-β2 and platelet-derived growth factor (PDGF)-BB were tested. In Group 3, proliferation in the eyes was significantly stronger and faster than that of Group 2 and the ratio of PVR was significantly higher than that of Group 2. The expression levels of TGF-β2 and PDGF-BB were significantly higher in Group 3 than in Group 2. These data suggested that UTMD promotes PVR induced by RPE-J cells and PRP which provide a new method for the development of rat PVR model. This also reminds us that the effects of UTMD should be taken into consideration when using UTMD as an approach to attenuate PVR. Key words: Proliferative vitreoretinopathy, rat model, ultrasound-targeted microbubble destruction, retinal pigmented epithelial -J cells, platelet-rich plasma.

Ultrasound-targeted microbubble destruction mediated herpes simplex virus-thymidine kinase gene treats hepatoma in mice

ObjectiveThe purpose of the study was to explore the anti-tumor effect of ultrasound -targeted microbubble destruction mediated herpes simplex virus thymidine kinase (HSV-TK) suicide gene system on mice hepatoma.MethodsForty mice were randomly divided into four groups after the models of subcutaneous transplantation tumors were estabilished: (1) PBS; (2) HSV-TK (3) HSV-TK+ ultrasound (HSV-TK+US); (4) HSV-TK+ultrasound+microbubbles (HSV-TK+US+MB). The TK protein expression in liver cancer was detected by western-blot. Applying TUNEL staining detected tumor cell apoptosis. At last, the inhibition rates and survival time of the animals were compared among all groups.ResultsThe TK protein expression of HSV-TK+MB+US group in tumor-bearing mice tissues were significantly higher than those in other groups. The tumor inhibitory effect of ultrasound-targeted microbubble destruction mediated HSV-TK on mice transplantable tumor was significantly higher than those in other groups (p < 0.05), and can significantly improve the survival time of tumor-bearing mice.ConclusionUltrasound-targeted microbubble destruction can effectively transfect HSV-TK gene into target tissues and play a significant inhibition effect on tumors, which provides a new strategy for gene therapy in liver cancer.

Comparative analysis of the effects of ultrasound-targeted microbubble destruction on recombinant adeno-associated virus- and plasmid-mediated transgene expression in human retinal pigment epithelium cells

Ultrasound-targeted microbubble destruction (UTMD) has been utilized to deliver a drug/gene into cells in both in vitro and in vivo studies. This study was performed to investigate the feasibility of UTMD-enhanced recombinant adeno-associated virus- (rAAV) and plasmid-mediated transfection into the human retinal pigment epithelium (RPE) cell line ARPE-19. Additionally, the transfection efficiency of rAAV and plasmid was compared in order to choose the appropriate gene vector or strategy to be used with UTMD for RPE-based gene modification. rAAV or plasmid encoding an enhanced green fluorescent protein gene was administered to ARPE-19 cells under various UTMD conditions. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl-tetrazolium bromide (MTT) assay. Transfection efficiency was determined by fluorescence microscopy and flow cytometry. UTMD significantly enhanced the transfection efficiency of rAAV and plasmid in ARPE-19 cells without adverse effects on cell viability. The transfection efficiency of rAAV was higher than that of plasmid. rAAV is therefore an appropriate gene vector for use with UTMD in retinal gene modification.

Effects of ultrasound-targeted microbubble destruction on cardiac gene expression

Ultrasound (US) contrast agents are increasingly used in diagnostic echocardiography. Recent studies have suggested unanticipated effects of microbubble destruction. This study was designed to evaluate gene regulation caused by US-mediated destruction of microbubbles in the heart. During IV infusion of Optison™, triggered US was applied to rat hearts to destroy microbubbles. A control group received only saline and US. RNA was isolated from hearts 24 and 72 h after treatment. Analysis with a deeply representative murine cardiac-specific microarray was used to identify regulated genes. Real-time polymerase chain reaction (PCR) was then applied to verify regulated genes. Microarray analysis revealed only 5 regulated genes in the 24-h group and 4 in the 72-h group. Of these genes, only carbonic anhydrase was significantly upregulated in the 24-h Optison™ group (4.3 fold; p = 0.0005) when examined in individual animals by real-time PCR. By this very sensitive technique, the bioeffects of microbubble destruction are negligible. (E-mail: ralph.shohet@utsouthwestern.edu)