- Email: [email protected]
0359: Enhanced Effects of Liposome Microbubble on Ultrasound Mediated Gene Transfection
S48
Ultrasound in Medicine and Biology
pathology of some of the more important ligaments and retinacula. In the upper limb, particular attention will be paid to the rotator interval, ulnar collateral ligament of the elbow, and the pulley and sagittal band system of the fingers. In the lower limb the important ligaments include the collateral ligaments of the knee and ankle as well as the intertarsal ligaments. The presentation will demonstrate the anatomy, technical tips on ultrasound scanning and pathological findings in these structures. 0353 Validity of Carotid Duplex Scan Performed with the Patient Seated Lucia B Pemble, Griffith University, Australia Carotid duplex ultrasound scanning is widely used in clinical practice for diagnosis of patients with symptoms of cerebrovascular disease. It has been extensively validated and is often the only diagnostic imaging performed prior to intervention. Carotid duplex ultrasound examination of the cerebrovasculature and angiographical studies used for correlation have been performed with subjects in the supine position. Clinically patients can present for carotid duplex ultrasound evaluation who may be unable to assume a supine position. They may be quadriplegic, suffer severe vertigo, spinal abnormalities or shortness of breath and are restricted in their ability to assume a supine position for the scanning procedure. Objective: The purpose of this study is to compare pulsed wave (PW) Doppler velocity measurements obtained from duplex ultrasound of the carotid arteries with subjects in the seated and supine positions. It will then postulate if this would change the category of stenosis interpreted using the current velocity criteria for the classification of the severity of disease as recommended by the Australasian Society of Ultrasound in Medicine. Conclusions: Duplex ultrasound PW Doppler derived velocity criteria are used to classify the severity of stenosis in turn impacting on patient treatment options. The carotid duplex scan can be adequately performed on patients in the seated position but velocities measured will be reduced and modification to the existing velocity criteria is needed. 0354 Measuring Whole Body Skeletal Muscle Mass - is Ultrasound an Alternative Technique Kerry Thoirs, University of South Australia Objective: This paper will review the increasing evidence that ultrasound techniques may be suitable to determine whole body skeletal muscle mass. Background: Whole body skeletal muscle mass constitutes approximately 30-40% of the body mass in healthy individuals. Skeletal muscle mass plays an important role in many physiologic and disease processes such as the growth and development of children, catabolic disease, exercise science and gerontology. Measurements of total skeletal muscle mass are required in clinical and research fields to establish normative and baseline data, and to monitor the progress of physiologic processes, disease and interventions. Although there are a number of techniques which are available to measure whole body skeletal muscle mass, the imaging techniques of Magnetic Resonance Imaging, Computed tomography and Dual energy X-ray absorptiometry have been established as the most precise and accurate techniques. These techniques however are not always suitable due to safety concerns including exposure to ionising radiation, expense, limited availability and non-portability. There is a need for accurate and precise measurement techniques for whole body skeletal muscle mass which are not only precise and accurate but also safe, convenient and portable.
Volume 35, Number 8S, 2009 Conclusion: Ultrasound is emerging as a technique that potentially can be used to accurately predict whole body skeletal muscle mass without the limitations of other imaging modalities. 0357 Conjoined Twins - First Trimester Case Study Karen Waalwyk, Monash Ultrasound for Women, Australia The advent of high-resolution transvaginal ultrasound allows for improved early diagnosis of conjoined twins. This is a rare phenomenon with a high prenatal mortality rate and differentiation from ‘normal’ monoamniotic twins can be difficult. This presentation will provide an example of first trimester diagnosis by 2D imaging in an IVF pregnancy. Further confirmation of the diagnosis was obtained through the use of 3D/4D technology. The ultrasound findings leading us to this diagnosis will be illustrated and discussed. 0358 Methods for Sonochemical Manufacture of Proteinaceous Ultrasound Contrast Agents Brian G Lee, Primary Healthcare/Monash University, Australia Ben Aldham, CSIRO, Australia Judy Scoble, CSIRO, Australia Anna Raicevic, CSIRO, Australia Ilija Sutalo, CSIRO, Australia Robert Stewart, CSIRO, Australia Richard Manasseh, CSIRO, Australia Morry Silberstein, Monash Univeristy, Australia Laboratory procedures for the sonochemical manufacture of proteinaceous contrast agents are reviewed and documented. The objective was to develop a standard protocol to enable ultrasound contrast researchers to develop consistent agents. Proteinaceous agents continue to be in clincal use and are the subject of renewed interest owing to their potential as a platform for antibody-targeted diagnostics. The agents are prepared by sonication. Protocols for agent preparation are scattered throughout literature with methodologies varying between laboratories. While new imaging technologies improve agent visualisation, there are limited correlations between contrast agent manufacturing methods and in vitro performance. The present experimental study has determined that successful production of a stable shell coating the bubble is a result of precise attention to denaturation of protein prior to sonication. The resulting shell coats the bubble and greatly slows down gas dissolution that would otherwise result in rapid disappearance of the agent or poor shelf life. The fluid-dynamical process by which the agent is formed is discussed. Issues of technique such as the location of the sonotrode tip, sonication time and power, and and the container size are detailed. Finally, procedures for quantitative characterisation of the agent by a range of instruments are discussed. In conclusion, a complete and consistent protocol from the initial biochemical source to the agent characterisation can be produced. 0359 Enhanced Effects of Liposome Microbubble on Ultrasound Mediated Gene Transfection Yi Zhi Chen, Department of Medical Ultrasound, The Third Affiliated Hospital of Guangzhou Medical College, Guangzhou, China, China Hua Jian Liu, Department of Function Imaging and Ultrasonography, Guangzhou First Municipal People’s Hospital, Guangzhou Medical College, Gua, China Kun Liang, Department of Obstetrics and Gynecology, Guangzhou Research Institute of Obstetrics and Gynecology, The Third Affiliated Hospit, China
Abstracts Objectives: Noninvasive, efficient and tissue-specific technologies of gene transfection would be valuable in clinical gene therapy. Although non-viral carriers may be safer and cheaper, they have much lower transfection efficiency than viral gene carriers. This present study was designed to test the transfection efficiency and safety of liposome microbubble (LM) on red fluorescent protein (RFP) in Hela cells in vitro and transplanted tumors of nude mice in vivo under ultrasound mediated gene transfection (UMGT) conditions. Methods: Plasmids containing RFP were gently mixed with LM. The complexes were added to Hela cells or injected into BALB/c mice by tail vein under different ultrasound and LM conditions, and then the transfection and expression efficiency were examined. Results: The results both in vivo and in vitro demonstrated that LM and UMGT could significantly increase the gene expression as compared with plasmid group, ultrasound ⫹ plasmid group, LM ⫹ plasmid group (P⬍0.01). These results also indicated that the optimal irradiation parameters would obtain higher transfection efficiency. Moreover, targeted delivery by UMGT and LM In Vivo results in tissue specificity and histological analyses showed that tissue damage was not seen in tumors. Conclusions: From the study, we concluded that LM could enhance UMGT effectively without causing any apparently detrimental effect. The combination of UMGT and LM could enhance the gene expression in vivo and in vitro, and this noninvasive technique might be a promising candidate for non-viral gene therapy. 0360 SonoVue Enhances in Vivo Goat Liver Ablation Induced by High Intensity Focused Ultrasound: The Safe Dosage of SonoVue Under Different Acoustic Intensity Faqi Li, Department of Biomedical Engineering, Chongqing Medical University, China Ting Zhang, Department of Biomedical Engineering, Chongqing Medical University, China Liyuan Fu, Department of Biomedical Engineering, Chongqing Medical University, China Shouming Chen, Department of Biomedical Engineering, Chongqing Medical University, China Xiaobo Gong, Department of Biomedical Engineering, Chongqing Medical University, China Zhibiao Wang, China Objective: To investigate the safe dosage of SonoVue under different acoustic intensity in enhancing goat liver ablation in vivo by high intensity focused ultrasound(HIFU). Methods: The acoustic intensity used were 8960W/cm2, 14900 W/cm2 and 20900W/cm2 respectively, the dose of SonoVue used were 0.01ml/kg, 0.03ml/kg and 0.05ml/kg respectively. Forty-five goats were randomly divided into nine groups of five. They injected with SonoVue intravenously 20s before HIFU exposure. Using a clinical HIFU device, the liver of all goats were ablated under the following protocol: a single exposure, ultrasound frequency 0.8MHz, the distance from skin to the target liver tissue was 30mm and the exposure time was set at 15s. All animals were euthanized 7 days later, the tissues in HIFU pathway were observed and the volumes of coagulated necrosis were measured. Pathological examinations for exposed region were performed. Results: Using the same acoustic intensity, the coagulated volumes increased significantly with increasing dose of SonoVue (p ⬍0.05). The tissues in HIFU pathway were damaged in the groups of 14900W/ cm2, 0.05ml/kg; 20900W/cm2, 0.03ml/kg and 20900W/cm2, 0.05ml/ kg. For acoustic intensity of 8960W/cm2, 14900 W/cm2 and 20900W/ cm2, the safe dosage of SonoVue were 0.05ml/kg, 0.03ml/kg and 0.01ml/kg respectively. Pathological examinations confirmed that there were no residual normal tissues within the exposed volume. There was demarcation between the targeted tissue and normal tissue.
S49
Conclusion: The safe dose of SonoVue under the lower acoustic intensity in enhancing goat liver ablation in vivo by HIFU was biger than that under higher acoustic intensity. 0361 Study on Suppression of Tissue Signal in Ultrasound Contrast Imaging Tetsuya Yoshida, Ultrasound Division, Toshiba Medical Systems Corp., Japan Hiroki Yoshiara, Ultrasound Division, Toshiba Medical Systems Corp., Japan Tomohisa Imamura, Ultrasound Division, Toshiba Medical Systems Corp., Japan Takeshi Sato, Ultrasound Division, Toshiba Medical Systems Corp., Japan Naohisa Kamiyama, Ultrasound Division, Toshiba Medical Systems Corp., Japan Background: In ultrasound contrast imaging, it is important to distinguish bubble signal from tissue signal. However, when we use second harmonic component for imaging, not only bubble but also tissue is visualized. Especially, this nonlinear propagation is not negligible when higher acoustic power is transmitted. In this report, we conducted feasibility study on suppression of tissue signal in contrast imaging. Methods: We used an agar-based graphite phantom, which has a groove for filling diluted contrast agents, SonazoidR. RF signals were acquired by an ultrasound equipment AplioXGTM(SSA-790A) with a convex probe (3MHz). Then, RF signals were transferred to an off-line system where analysis was performed. Two pulses of differing phase by 180 degrees were transmitted into the phantom. We extracted fundamental component (Sf) from one of two received pulses, and extracted second harmonic component (S2) by summing two received pulses. After detecting envelope of each component, we visualized two kinds of B-mode images by calculating the equations as follows: (a) logS2-logSf⫹␣ (b) logS2-logSf2⫹ where ␣ and  are appropriate gain values. It is expected that uniformity in tissue signal is obtained by normalizing S2 with Sf. Results and Conclusion: In comparison with conventional second harmonic imaging, signal intensity ratio between bubble and tissue was improved by about 6dB in equation (a). Furthermore, tissue signal became fairly flat spatially, and bubble signal became uniform near to deep area in equation both (a) and (b). These results show that the proposed method has a possibility to suppress tissue signal in contrast imaging. 0362 The Effect of a Surface on the Response of Microbubbles to Ultrasound Edward Payne, CSIRO, Australia Richard Manasseh, CSIRO, Australia Andrew Ooi Experimental data on the response frequencies of microbubbles are compared with a range of theoretical and numerical models. The objective was to determine how the surface affected the bubble resonant frequency. Recent proposals for ultrasound molecular imaging (targeted diagnostics) and targeted therapeutics require contrast agent microbubbles to be bound to a target surface, usually vascular endothelium expressing pathological markers to which the bubble shell has been made specific. Continuous-wave ultrasound was applied to microbubbles attached to surfaces both as individuals and in pairs. An optical imaging system acquired data on the bubble radius as a function of the transmitted
Ultrasound in Medicine and Biology
pathology of some of the more important ligaments and retinacula. In the upper limb, particular attention will be paid to the rotator interval, ulnar collateral ligament of the elbow, and the pulley and sagittal band system of the fingers. In the lower limb the important ligaments include the collateral ligaments of the knee and ankle as well as the intertarsal ligaments. The presentation will demonstrate the anatomy, technical tips on ultrasound scanning and pathological findings in these structures. 0353 Validity of Carotid Duplex Scan Performed with the Patient Seated Lucia B Pemble, Griffith University, Australia Carotid duplex ultrasound scanning is widely used in clinical practice for diagnosis of patients with symptoms of cerebrovascular disease. It has been extensively validated and is often the only diagnostic imaging performed prior to intervention. Carotid duplex ultrasound examination of the cerebrovasculature and angiographical studies used for correlation have been performed with subjects in the supine position. Clinically patients can present for carotid duplex ultrasound evaluation who may be unable to assume a supine position. They may be quadriplegic, suffer severe vertigo, spinal abnormalities or shortness of breath and are restricted in their ability to assume a supine position for the scanning procedure. Objective: The purpose of this study is to compare pulsed wave (PW) Doppler velocity measurements obtained from duplex ultrasound of the carotid arteries with subjects in the seated and supine positions. It will then postulate if this would change the category of stenosis interpreted using the current velocity criteria for the classification of the severity of disease as recommended by the Australasian Society of Ultrasound in Medicine. Conclusions: Duplex ultrasound PW Doppler derived velocity criteria are used to classify the severity of stenosis in turn impacting on patient treatment options. The carotid duplex scan can be adequately performed on patients in the seated position but velocities measured will be reduced and modification to the existing velocity criteria is needed. 0354 Measuring Whole Body Skeletal Muscle Mass - is Ultrasound an Alternative Technique Kerry Thoirs, University of South Australia Objective: This paper will review the increasing evidence that ultrasound techniques may be suitable to determine whole body skeletal muscle mass. Background: Whole body skeletal muscle mass constitutes approximately 30-40% of the body mass in healthy individuals. Skeletal muscle mass plays an important role in many physiologic and disease processes such as the growth and development of children, catabolic disease, exercise science and gerontology. Measurements of total skeletal muscle mass are required in clinical and research fields to establish normative and baseline data, and to monitor the progress of physiologic processes, disease and interventions. Although there are a number of techniques which are available to measure whole body skeletal muscle mass, the imaging techniques of Magnetic Resonance Imaging, Computed tomography and Dual energy X-ray absorptiometry have been established as the most precise and accurate techniques. These techniques however are not always suitable due to safety concerns including exposure to ionising radiation, expense, limited availability and non-portability. There is a need for accurate and precise measurement techniques for whole body skeletal muscle mass which are not only precise and accurate but also safe, convenient and portable.
Volume 35, Number 8S, 2009 Conclusion: Ultrasound is emerging as a technique that potentially can be used to accurately predict whole body skeletal muscle mass without the limitations of other imaging modalities. 0357 Conjoined Twins - First Trimester Case Study Karen Waalwyk, Monash Ultrasound for Women, Australia The advent of high-resolution transvaginal ultrasound allows for improved early diagnosis of conjoined twins. This is a rare phenomenon with a high prenatal mortality rate and differentiation from ‘normal’ monoamniotic twins can be difficult. This presentation will provide an example of first trimester diagnosis by 2D imaging in an IVF pregnancy. Further confirmation of the diagnosis was obtained through the use of 3D/4D technology. The ultrasound findings leading us to this diagnosis will be illustrated and discussed. 0358 Methods for Sonochemical Manufacture of Proteinaceous Ultrasound Contrast Agents Brian G Lee, Primary Healthcare/Monash University, Australia Ben Aldham, CSIRO, Australia Judy Scoble, CSIRO, Australia Anna Raicevic, CSIRO, Australia Ilija Sutalo, CSIRO, Australia Robert Stewart, CSIRO, Australia Richard Manasseh, CSIRO, Australia Morry Silberstein, Monash Univeristy, Australia Laboratory procedures for the sonochemical manufacture of proteinaceous contrast agents are reviewed and documented. The objective was to develop a standard protocol to enable ultrasound contrast researchers to develop consistent agents. Proteinaceous agents continue to be in clincal use and are the subject of renewed interest owing to their potential as a platform for antibody-targeted diagnostics. The agents are prepared by sonication. Protocols for agent preparation are scattered throughout literature with methodologies varying between laboratories. While new imaging technologies improve agent visualisation, there are limited correlations between contrast agent manufacturing methods and in vitro performance. The present experimental study has determined that successful production of a stable shell coating the bubble is a result of precise attention to denaturation of protein prior to sonication. The resulting shell coats the bubble and greatly slows down gas dissolution that would otherwise result in rapid disappearance of the agent or poor shelf life. The fluid-dynamical process by which the agent is formed is discussed. Issues of technique such as the location of the sonotrode tip, sonication time and power, and and the container size are detailed. Finally, procedures for quantitative characterisation of the agent by a range of instruments are discussed. In conclusion, a complete and consistent protocol from the initial biochemical source to the agent characterisation can be produced. 0359 Enhanced Effects of Liposome Microbubble on Ultrasound Mediated Gene Transfection Yi Zhi Chen, Department of Medical Ultrasound, The Third Affiliated Hospital of Guangzhou Medical College, Guangzhou, China, China Hua Jian Liu, Department of Function Imaging and Ultrasonography, Guangzhou First Municipal People’s Hospital, Guangzhou Medical College, Gua, China Kun Liang, Department of Obstetrics and Gynecology, Guangzhou Research Institute of Obstetrics and Gynecology, The Third Affiliated Hospit, China
Abstracts Objectives: Noninvasive, efficient and tissue-specific technologies of gene transfection would be valuable in clinical gene therapy. Although non-viral carriers may be safer and cheaper, they have much lower transfection efficiency than viral gene carriers. This present study was designed to test the transfection efficiency and safety of liposome microbubble (LM) on red fluorescent protein (RFP) in Hela cells in vitro and transplanted tumors of nude mice in vivo under ultrasound mediated gene transfection (UMGT) conditions. Methods: Plasmids containing RFP were gently mixed with LM. The complexes were added to Hela cells or injected into BALB/c mice by tail vein under different ultrasound and LM conditions, and then the transfection and expression efficiency were examined. Results: The results both in vivo and in vitro demonstrated that LM and UMGT could significantly increase the gene expression as compared with plasmid group, ultrasound ⫹ plasmid group, LM ⫹ plasmid group (P⬍0.01). These results also indicated that the optimal irradiation parameters would obtain higher transfection efficiency. Moreover, targeted delivery by UMGT and LM In Vivo results in tissue specificity and histological analyses showed that tissue damage was not seen in tumors. Conclusions: From the study, we concluded that LM could enhance UMGT effectively without causing any apparently detrimental effect. The combination of UMGT and LM could enhance the gene expression in vivo and in vitro, and this noninvasive technique might be a promising candidate for non-viral gene therapy. 0360 SonoVue Enhances in Vivo Goat Liver Ablation Induced by High Intensity Focused Ultrasound: The Safe Dosage of SonoVue Under Different Acoustic Intensity Faqi Li, Department of Biomedical Engineering, Chongqing Medical University, China Ting Zhang, Department of Biomedical Engineering, Chongqing Medical University, China Liyuan Fu, Department of Biomedical Engineering, Chongqing Medical University, China Shouming Chen, Department of Biomedical Engineering, Chongqing Medical University, China Xiaobo Gong, Department of Biomedical Engineering, Chongqing Medical University, China Zhibiao Wang, China Objective: To investigate the safe dosage of SonoVue under different acoustic intensity in enhancing goat liver ablation in vivo by high intensity focused ultrasound(HIFU). Methods: The acoustic intensity used were 8960W/cm2, 14900 W/cm2 and 20900W/cm2 respectively, the dose of SonoVue used were 0.01ml/kg, 0.03ml/kg and 0.05ml/kg respectively. Forty-five goats were randomly divided into nine groups of five. They injected with SonoVue intravenously 20s before HIFU exposure. Using a clinical HIFU device, the liver of all goats were ablated under the following protocol: a single exposure, ultrasound frequency 0.8MHz, the distance from skin to the target liver tissue was 30mm and the exposure time was set at 15s. All animals were euthanized 7 days later, the tissues in HIFU pathway were observed and the volumes of coagulated necrosis were measured. Pathological examinations for exposed region were performed. Results: Using the same acoustic intensity, the coagulated volumes increased significantly with increasing dose of SonoVue (p ⬍0.05). The tissues in HIFU pathway were damaged in the groups of 14900W/ cm2, 0.05ml/kg; 20900W/cm2, 0.03ml/kg and 20900W/cm2, 0.05ml/ kg. For acoustic intensity of 8960W/cm2, 14900 W/cm2 and 20900W/ cm2, the safe dosage of SonoVue were 0.05ml/kg, 0.03ml/kg and 0.01ml/kg respectively. Pathological examinations confirmed that there were no residual normal tissues within the exposed volume. There was demarcation between the targeted tissue and normal tissue.
S49
Conclusion: The safe dose of SonoVue under the lower acoustic intensity in enhancing goat liver ablation in vivo by HIFU was biger than that under higher acoustic intensity. 0361 Study on Suppression of Tissue Signal in Ultrasound Contrast Imaging Tetsuya Yoshida, Ultrasound Division, Toshiba Medical Systems Corp., Japan Hiroki Yoshiara, Ultrasound Division, Toshiba Medical Systems Corp., Japan Tomohisa Imamura, Ultrasound Division, Toshiba Medical Systems Corp., Japan Takeshi Sato, Ultrasound Division, Toshiba Medical Systems Corp., Japan Naohisa Kamiyama, Ultrasound Division, Toshiba Medical Systems Corp., Japan Background: In ultrasound contrast imaging, it is important to distinguish bubble signal from tissue signal. However, when we use second harmonic component for imaging, not only bubble but also tissue is visualized. Especially, this nonlinear propagation is not negligible when higher acoustic power is transmitted. In this report, we conducted feasibility study on suppression of tissue signal in contrast imaging. Methods: We used an agar-based graphite phantom, which has a groove for filling diluted contrast agents, SonazoidR. RF signals were acquired by an ultrasound equipment AplioXGTM(SSA-790A) with a convex probe (3MHz). Then, RF signals were transferred to an off-line system where analysis was performed. Two pulses of differing phase by 180 degrees were transmitted into the phantom. We extracted fundamental component (Sf) from one of two received pulses, and extracted second harmonic component (S2) by summing two received pulses. After detecting envelope of each component, we visualized two kinds of B-mode images by calculating the equations as follows: (a) logS2-logSf⫹␣ (b) logS2-logSf2⫹ where ␣ and  are appropriate gain values. It is expected that uniformity in tissue signal is obtained by normalizing S2 with Sf. Results and Conclusion: In comparison with conventional second harmonic imaging, signal intensity ratio between bubble and tissue was improved by about 6dB in equation (a). Furthermore, tissue signal became fairly flat spatially, and bubble signal became uniform near to deep area in equation both (a) and (b). These results show that the proposed method has a possibility to suppress tissue signal in contrast imaging. 0362 The Effect of a Surface on the Response of Microbubbles to Ultrasound Edward Payne, CSIRO, Australia Richard Manasseh, CSIRO, Australia Andrew Ooi Experimental data on the response frequencies of microbubbles are compared with a range of theoretical and numerical models. The objective was to determine how the surface affected the bubble resonant frequency. Recent proposals for ultrasound molecular imaging (targeted diagnostics) and targeted therapeutics require contrast agent microbubbles to be bound to a target surface, usually vascular endothelium expressing pathological markers to which the bubble shell has been made specific. Continuous-wave ultrasound was applied to microbubbles attached to surfaces both as individuals and in pairs. An optical imaging system acquired data on the bubble radius as a function of the transmitted