Microbubble-enhanced Ultrasound Research Articles

Inhibition of Walker‐256 Tumor Growth by Combining Microbubble‐Enhanced Ultrasound and Endostar

This research is to investigate the anti-tumor effects by combining anti-vascular effect of microbubble enhanced ultrasound (MEUS) mechanical destruction and anti-angiogenic effect of Endostar. Rats bearing Walker-256 tumor were randomly divided into 4 groups treated by Endostar + MEUS combination, Endostar, MEUS or Sham ultrasound (US), respectively. MEUS was induced by Sonazoid microbubble and a focused therapeutic US device. Contrast-enhanced ultrasound (CEUS) was used to assess tumor perfusion before and after treatment. Microvessel density (MVD) was evaluated with immunohistochemical staining of CD31, CD34, and VEGFA. TUNEL assay was used to determine the apoptosis rate of tumor cells. Endostar + MEUS combined group induced the most reduced blood perfusion and most retarded tumor growth compared with other 3 groups. Decreased MVD was shown in Endostar + MEUS, Endostar and MEUS group, but the lowest MVD value was presented in the combined treatment group. Significant increase was observed in the combined therapy group and MEUS group. This study showed an improved anti-vascular and anti-angiogenic effect achieved by combining Endostar and MEUS, and may provide a new method potential for anti-tumor therapy.

Promoting the effect of microbubble-enhanced ultrasound on hyperthermia in rabbit liver

PurposeThe heat-sink effect is one reason for the insufficient temperature increase in hyperthermia (HT) treatment for cancer. Microbubbles (MBs) nucleate inertial cavitation under therapeutic ultrasound (TUS) exposure, which form microbubble-enhanced ultrasound (MEUS), which results in blocking blood perfusion in the targeted liver tissues. This study aimed to determine if synergistic effects exist during HT in the liver when combined with MEUS.MethodsForty rabbits with surgically exposed livers were randomly divided into TUS + MB + HT, MB + HT, normal saline + HT, and MB + sham groups (n = 10 in each group). Liver perfusion was evaluated using contrast-enhanced ultrasound. The temperatures of the liver tissues were monitored using thermocouples. Pathological changes were determined by hematoxylin and eosin (H&E) staining. Serum hepatic transaminases were evaluated.ResultsMEUS pretreatment almost completely blocked the perfusion of targeted areas. The TUS + MB + HT and MB + HT groups showed significantly higher temperatures in treated areas than those in the other groups. However, the TUS + MB + HT group exhibited a more stable and regular increase in temperatures in the fitting curves compared with the MB + HT group. H&E staining revealed swelling hepatocytes, hemorrhage, and thrombosis in the portal area in the TUS + MB + HT group.ConclusionMEUS reduced the blood perfusion in the targeted liver tissues, and, therefore, overcame the heat-sink effect during the HT procedure in rabbits. MEUS pretreatment might have the potential to enhance the therapeutic effect of HT.

In vivo Nakagami-m parametric imaging of microbubble-enhanced ultrasound regulated by RF and VF processing techniques.

Application of the Nakagami statistical model and associated m parameter has the potential to suppress artifacts from adjustable system parameters and operator selections typical in echo amplitude-coded microbubble-enhanced ultrasound (MEUS). However, the feasibility of applying m estimation and determination of the associated Nakagami distribution features for in vivo MEUS remain to be investigated. Sensitivity and discriminability of m-coded MEUS are often limited since raw envelopes are regulated by complex radiofrequency (RF) and video-frequency (VF) processing. This study aims to develop an improved imaging approach for the m parameter estimation which can overcome the above limitations in in vivo condition. The regulation effects of RF processing of pulse-inversion (PI) harmonic detection techniques and VF processing of logarithmic compression in Nakagami distributions were investigated in MEUS. A window-modulated compounding moment estimator was developed to estimate the MEUS m values. The sensitivity and discriminability of m-coded MEUS were quantified with contrast-to-tissue ratio (CTR), contrast-to-noise ratio (CNR), and axial and lateral resolutions, which were validated through in vivo perfusion experiments on rabbit kidneys. Regulated by RF and VF processing, the distributions of MEUS obeyed the Nakagami statistical model. The Nakagami-fitted correlation coefficient was 0.996±0.003 (P<0.05 in the t test and P<0.001 in the Kolmogorov-Smirnov test). Among each of the m-coded MEUS methods, the logarithmic m-coded PI-MEUS scheme effectively characterized the peripheral rim perfusion features and details within the renal cortex. The CTR and CNR in this region reached 7.9±1.5dB and 34.4±1.7dB, respectively, which were higher than those of standard amplitude-coded MEUS; and the axial and lateral resolutions were 1.02±0.02 and 0.91±0.02mm, respectively, which were slightly longer than those of amplitude-coded MEUS. The Nakagami statistical model could characterize MEUS even when the envelope distributions were regulated by RF and VF processing. The logarithmic m-coded PI-MEUS scheme significantly improved the sensitivity, discriminability, and robustness of m estimation in MEUS. The scheme provides an option to remove artifacts in echo amplitude-coded MEUS and to distinctly characterize the inherent microvasculature enhanced by microbubbles, with potential to improve and expand the role of MEUS in diagnostic ultrasound.

Characteristic Blood-Perfusion Reduction of Walker 256 Tumor Induced by Diagnostic Ultrasound and Microbubbles

Tumor angiogenesis is characterized by a defective, leaky and fragile microvascular construction, and microbubble-enhanced ultrasound (MEUS) with high-pressure amplitude is capable of disrupting tumor microvasculature and arresting blood perfusion. In this study, we tried to investigate whether the blood perfusion of a malignant tumor can be characteristically interrupted by combining microbubbles and diagnostic ultrasound (US). Twenty-nine Sprague-Dawley (SD) rats with subcutaneous Walker 256 tumors and seven healthy SD rats were included. Fifteen tumors were treated by MEUS, which combined constant microbubble injection and 20 episodes of irradiation by diagnostic US (i.e., acoustic radiation force impulse [ARFI] imaging). The other 14 tumors were treated by ARFI or sham US only. Seven skeleton muscles from healthy SD rats were also treated with MEUS, serving as the control. Contrast-enhanced ultrasound (CEUS) was performed before and after all treatments. The blood perfusion of the tumor MEUS group showed a significant drop immediately after treatment, followed by a quick, incomplete perfusion recovery within 10–20 min. The visual perfusion scoring result was consistent with the quantitative analysis by CEUS peak intensity. However, there were no significant perfusion changes in the tumor control groups or the muscle control group. Histologic examination found severe microvascular disruption and hemorrhage in the MEUS-treated tumors but not in the control groups. Therefore, the treatment combining diagnostic US and microbubbles can specifically decrease or interrupt the blood perfusion of Walker 256 tumors, which could be a potential new imaging method for diagnosing malignant tumors.

Enhancement Effect of Microbubble-Enhanced Ultrasound in Microwave Ablation in Rabbit VX2 Liver Tumors.

Objectives One reason for the high recurrence and metastatic rates of tumors such as hepatocellular carcinoma (HCC) treated by microwave ablation (MWA) is the presence of residual foci in the tumor due to heat sink effect. Microbubble-enhanced ultrasound (MEUS) can noninvasively disrupt and block the tumor blood perfusion and has the potential to overcome the heat sink effect and enhance the therapeutic effect of MWA. The study aimed at evaluating the potential additional benefit of microbubble-enhanced ultrasound (MEUS) in hepatocellular carcinoma (HCC) treated by microwave ablation (MWA). Methods In this study, a new strategy of combining MWA with MEUS for treating HCC was proposed. Twenty-four rabbits with VX2 tumors in livers were randomly divided into MEUS + MWA, MEUS alone, MWA alone, and blank control groups, respectively (n = 6). In the MEUS group, the tumors were directly exposed to therapeutic ultrasound for 5 min with a concurrent intravenous injection of microbubbles (0.1 ml/kg diluted into 5 ml saline). In the MWA group, the tumors were treated by MWA for 1 min. In the MEUS + MWA group, tumors were ablated by MWA for 1 min after ultrasound cavitation enhanced by microbubbles as in the MEUS group. In the blank control group, the tumors received probe sham and intravenous saline. Contrast-enhanced ultrasound (CEUS) was performed before treatment and immediately after treatment to display the size, shape, and contour of the tumors. Throughout the treatment process, the local temperature of the treatment area was detected by a temperature needle punctured into the tumor. The blood samples of animals were obtained after treatment for evaluating the liver function. Tumor cell necrosis and apoptotic rates were observed after treatment by histological examination. Results CEUS showed that although perfusion defects appeared in all the treatment groups, especially in the MEUS + MWA group, there was no significant difference between the two groups on the volumes of perfusion defects, which were 1.78 ± 0.31 (cm3) in the MWA group and 1.84 ± 0.20 (cm3) in the combined group (P < 0.01). The time to reach the peak temperature of the treatment area was 21.7 ± 5.0 (s) in the MWA group and 10.3 ± 5.0 (s) in the MEUS + MWA group (P < 0.01). The time to reach the peak temperature of the treatment area was 21.7 ± 5.0 (s) in the MWA group and 10.3 ± 5.0 (s) in the MEUS + MWA group (P < 0.01). The time to reach the peak temperature of the treatment area was 21.7 ± 5.0 (s) in the MWA group and 10.3 ± 5.0 (s) in the MEUS + MWA group (Conclusions These results suggested MEUS treatment alone may significantly reduce tumor blood perfusion and led to a sharp rise in the local temperature of the treatment area to a higher PT using MEUS + MWA with higher rates of necrosis and apoptosis of cancer cells without severe liver function damage, which might be a safe strategy for treating HCC.

The outcomes and bioactivities of microbubble-enhanced ultrasound combined with prothrombin on microwave ablation in rabbit VX2 liver tumors

Objective To investigate the sequential biological effects and outcomes of microbubble-enhanced ultrasound (MEUS) combined with prothrombin on microwave ablation (MWA) in rabbit VX2 liver tumors using examination of transmission electron microscopy, histopathology and contrast-enhanced ultrasound(CEUS). Methods Eighty New Zealand rabbits with VX2 liver tumors were randomly divided into 4 groups(20 per group), including physiological saline group, prothrombin group, cavitation of microbubbles group (cavitation group) and cavitation of microbubbles combined prothrombin group (combined group). After treatment, the targeted liver tumors in all groups were ablated with MWA. On the 0, 3, 7 and 14 d, the volumes of coagulated areas of 5 rabbits of each group randomly were measured using CEUS. Tissues in ablated areas, transition areas and surrounding areas were examined under light microscopy with histopathology and transmission electron microscopy to observe the differences among 4 groups. Tumor metastasis was graded using visual method. Results On the 0, 3, 7 and 14 d, the coagulated volumes in combined group were larger than those in remaining 3 groups (all P<0.001). Under light microscopy with HE stain, in transition area, the fibra band in combined group was wider than those in remaining 3 groups on the 7 and 14 d (all P<0.05). The observations under transmission electron microscopy showed that the cellular ultrastructure disorder in ablated area on the 0 d and mitochondrial injury in transition area on the 7 d were more severe in combined group than those in remaining 3 groups. Compared with the remaining 3 groups, the degree of tumor metastasis in combined group was less, and the time of ocurrence was later. Conclusions MEUS combined with prothrombin can significantly expand ablation volume, enhance the necrosis of ablated tissues, and inhibit tumor metastasis on rabbit VX2 liver tumors. The fibra bands in the transition areas can promote the organization and repair the surrounding tissue in the ablation areas. Key words: Cavitation; Microwave ablation; Prothrombin; VX2 liver tumors

Effect of Microbubble-Enhanced Ultrasound on Radiofrequency Ablation of Rabbit Liver

Microbubble-enhanced ultrasound (MEUS) can non-invasively disrupt and block liver blood perfusion. It may potentially overcome the heat sink effect during a thermal ablation and consequently enhance radiofrequency ablation (RFA) of the liver. We propose a new strategy combining RFA with MEUS. For ultrasound treatment, an 831-kHz air-backed focused transducer directed 400-cycle bursts at 4.3 MPa to the liver at a 9-Hz rate. The treatment was nucleated by a lipids microbubble forming MEUS. Eighteen surgically exposed rabbit livers were treated using MEUS combined with RFA; the other 32 livers were treated using MEUS (n = 14) or RFA (n = 18) alone and served as the controls. Contrast ultrasound imaging confirmed that MEUS treatment significantly reduced liver blood perfusion by cutting contrast peak intensities in half (44.7%–54.1%) without severe liver function damage. The ablated liver volume treated using MEUS combined with RFA was 2.8 times greater than that treated using RFA alone. In conclusion, RFA of the liver can be safely and greatly enhanced by combination with MEUS pre-treatment.

The impact of microbubble-enhanced therapeutic ultrasound combined with prothrombin on microwave ablation in the rabbit liver.

This study aimed to investigate the effect of microbubble-enhanced ultrasound (MEUS) combined with prothrombinon regional hepatic circulation and microwave ablation (MWA) in rabbit livers. High-pressureamplitude therapeutic ultrasound (TUS) was used to treat 52 surgically exposed livers of healthy New Zealand rabbits: 13 liverswere treated with MEUS alone, 13 with MEUS and prothrombin (PMEUS), 13 with ultrasound plus normal saline and 13with ultrasound plus prothrombin as controls. Contrast-enhanced ultrasound (CEUS) imaging was performed on the exposedlivers before and after treatment, and acoustic quantification was done to assess liver perfusion. Then, the liver was dividedinto two parts, one was used for pathologic examination and the other was ablated with microwave (MWA) and then processedfor pathologic examination. The CEUS images and Peak value after treatment in the PMEUS group were significantlyreduced as compared to the remaining 3 groups (p<0.05). Occasional piecemeal hemorrhage was evidenced in the pathologicalexamination in the MEUS group. Obvious cellular degeneration and necrosis with thrombosis were observed in the PMEUSgroup. Electron microscopy showed endothelial damage in both the MEUS group and PMEUS group. After MWA, coagulatedvolumes (V) in the PMEUS group were larger than in the remaining 3 groups (p<0.05). The cell ultrastructure disorder wasmore severe in the PMEUS group than in remaining 3 groups. PMEUS promotes endothelial injury and producesmore obvious thrombotic occlusion, improving the therapeutic effect of MWA on the rabbit liver.

Disruption of Prostate Microvasculature by Combining Microbubble-Enhanced Ultrasound and Prothrombin.

Previous studies have shown a unique method to disrupt tumor vasculature using pulsed, high-pressure amplitude therapeutic ultrasound combined with microbubbles. In this study, we attempted to destroy the prostate vasculature of canine prostates using microbubble-enhanced ultrasound (MEUS) and prothrombin. The prostates of 43 male mongrel canines were surgically exposed. Twenty-two prostates were treated using MEUS (n = 11) or MEUS and prothrombin (PMEUS, n = 11). The other 21 prostates, which were treated using microbubbles (n = 7), ultrasound (n = 7) or prothrombin (n = 7) only, served as the controls. Prothrombin was intravenously infused at 20 IU/kg. MEUS was induced using a therapeutic ultrasound device at a peak negative pressure of 4.47 MPa and a microbubble injection. Contrast-enhanced ultrasound was performed to assess the blood perfusion of the prostates. Then, the prostate tissue was harvested immediately after treatment and at 48 hours later for pathological examination. The contrast-enhanced ultrasound peak value of the prostate decreased significantly from 36.2 ± 5.6 to 27.1 ± 6.3 after treatment in the PMEUS group, but it remained unchanged in the other groups. Histological examination found severe microvascular rupture, hemorrhage and thrombosis in both MEUS- and PMEUS-treated prostates immediately after treatment, while disruption in the PMEUS group was more severe than in the MEUS group. Forty-eight hours after treatment, massive necrosis and infiltration of white blood cells occurred in the PMEUS group. This study demonstrated that PMEUS disrupted the normal microvasculature of canine prostates and induced massive necrosis. PMEUS could potentially become a new noninvasive method used for the treatment of benign prostatic hyperplasia.

Effect of microbubble-enhanced ultrasound on percutaneous ethanol ablation of rat walker-256 tumour.

Percutaneous ethanol ablation (PEA) is an effective method for treating small liver cancer. Microbubble-enhanced ultrasound (MEUS) can potentially promote PEA by disrupting the tumour's circulation. In this study, treatment combining MEUS and PEA was performed to find any synergistic effects in tumour ablation. Ten rats bearing subcutaneous Walker-256 tumours were treated by MEUS combined with PEA. The other 18 tumour-bearing rats that were treated by MEUS or PEA served as the controls. MEUS was conducted by therapeutic ultrasound (TUS) and microbubble injection. TUS was operated at a frequency of 831 KHz with a pressure amplitude of 4.3MPa. Tumour blood perfusion was assessed by contrast-enhanced ultrasound (CEUS), and the tumour necrosis rate was determined by histological examination. CEUS showed that the tumour blood perfusion almost vanished in all of the MEUS-treated tumours. The contrast peak intensity dropped 84.8% in the MEUS + PEA-treated tumours when compared to 46.3% (p < 0.05) in the PEA-treated tumours 24h after treatment. The tumour necrosis rate of the combination therapy was 97.50%, which is much higher than that of the MEUS- (66.2%) and PEA-treated (81.0%) tumours. PEA combined with MEUS can induce a much more complete tumour necrosis. • This experiment demonstrated a novel method for enhancing percutaneous ethanol ablation. • Microbubble-enhanced therapeutic ultrasound is capable of disrupting tumour circulation. • Combined therapy of MEUS and PEA can induce more complete necrosis of tumours.

2076859 Effect of Microbubble-Enhanced Ultrasound on Percutaneous Ethanol Ablation of Rat Walker-256 Tumor

2076859 Effect of Microbubble-Enhanced Ultrasound on Percutaneous Ethanol Ablation of Rat Walker-256 Tumor

Application of microbublle-enhanced ultrasound in preoperative mapping of perforators in supraclavicular artery based flaps

To investigate the effectiveness of microbubble-enhanced ultrasound (MEUS) for detecting perforators preoperatively in supraclavicular flap surgery. From May 2009 to October 2013, there were 20 patients (26 flaps were involved) who planned to undergo supraclavicular artery based flap surgeries to recover the large-area defects in head and neck. The MEUS together with regular color Doppler ultrasound ( CDUS) were conducted preoperatively to determine the anatomical features of perforators branching from supraclavicular arteries (SCA). The perforator with wider caliber, faster flow speed, longer pedicles and closer pivot point was selected and the flap was designed according to the observed results. There were 37 perforators of SCA detected by CDUS, whose calibers were ranging from 0.5 to 0.8 mm [Mean: (0.6 ± 0.1) mm]. There were 48 perforators of SCA detected by MEUS. Compared to CDUS, the caliber obtained from MEUS for same vessel is significantly increased [(0.7 ± 0.3) mm vs (0.6 ± 0.1) mm, P < 0.05]. According to the results of MEUS and three-dimensional reconstructive techniques, in at least 65.4% (17/26) of the flaps, thoracic branch of SCA (TBSA) has large caliber and good flow velocity which can be regarded as the predominant vessel and used as the pedicle of flap. The results of the operations confirmed the existences of all the marked vessels. 25 flaps were obtained according to the preoperative plans and one case used perforators of internal mammary artery as free flaps since the perforator of SCA was found improper. The contrast-related complication occurred in one patient which was manifested by gastrointestinal adverse effect like nausea and anorexia. The patient recovered 1 day later without treatment. All the patients have been followed up for 3 to 16 months (Mean: 8 months) with well-survived flaps. The perforators of SCA demonstrated significant variations and preoperative mapping was vital for the success of surgery. MEUS is a valuable imaging modality for the preoperative assessment of the vascular supply for supraclavicular artery based flap.

Comparing Efficiency of micro-RNA and mRNA Biomarker Liberation with Microbubble-Enhanced Ultrasound Exposure

Blood biomarkers are potentially powerful diagnostic tools that are limited clinically by low concentrations, the inability to determine biomarker origin and unknown patient baseline. Recently, ultrasound has been shown to liberate proteins and large mRNA biomarkers, overcoming many of these limitations. We have since demonstrated that adding lipid-stabilized microbubbles elevates mRNA concentration an order of magnitude compared with ultrasound without microbubbles, in vitro. Unfortunately the large size of some mRNA molecules may limit efficiency of release and hinder efficacy as an ultrasound-liberated biomarker. We hypothesize that smaller molecules will be released more efficiently with ultrasound than larger molecules. Although investigation of large libraries of biomarkers should be performed to fully validate this hypothesis, we focus on a small subset of mRNA and micro-RNAs. Specifically, we focus on miR-21 (22 base pairs [bp]), which is upregulated in certain forms of cancer, compared with previously investigated mammaglobin mRNA (502 bp). We also report release of micro-RNA miR-155 (22 bp) and housekeeping rRNA S18 (1869 bp). More than 10 million additional miR-21 copies per 100,000 cells are released with ultrasound-microbubble exposure. The low- molecular-weight miR-21 proved to be liberated 50 times more efficiently than high-molecular-weight mammaglobin mRNA, releasing orders of magnitude more miR-21 than mammaglobin mRNA under comparable conditions.

Impact of Microbubble-Enhanced Ultrasound on Liver Ethanol Ablation

Ethanol ablation (EA) is a safe and effective method for treating small liver cancer. However, the ethanol is rapidly washed out by blood perfusion, preventing its accumulation within tumors. Microbubble-enhanced ultrasound (MEUS) is capable of disrupting tumor and liver circulation. We hypothesized that this disruption could be used to enhance EA of normal liver tissue. We treated surgically exposed rabbit liver with a combination of MEUS and EA. The controls were treated with only MEUS or 0.05 mL EA. MEUS treatment was administered with a high-pressure-amplitude, pulsed therapeutic ultrasound device and intra-venous injection of microbubbles. Therapeutic ultrasound was delivered at an acoustic pressure of 4.3 MPa and a duty cycle of 0.22%. Contrast-enhanced ultrasound was performed to estimate liver blood perfusion. Livers were harvested for necrotic volume measurements 48 h after treatment. Contrast-enhanced ultrasound demonstrated that liver perfusion was temporally arrested, with a significant peak intensity decline from −46.9 ± 3.8 to −64.0 ± 3.3 dB, after MEUS treatment. The mean volume ablated in MEUS + EA-treated livers (3.3 ± 2.3 cm3) was more than 10 times larger than that in livers treated only with EA (0.3 ± 0.2 cm3). The volume of liver ablated by MEUS treatment alone was minor, scattered and immeasurable. These results indicate that MEUS disruption of the liver circulation can greatly promote EA of liver.

Disruption of Splenic Circulation Using Microbubble-Enhanced Ultrasound and Prothrombin: A Preliminary Study

The spleen is a solid organ in which splenomegaly frequently develops and to which abdominal blunt trauma occurs. In this study, we demonstrated the potential therapeutic effect of microbubble-enhanced ultrasound (MEUS) combined with prothrombin to disrupt splenic circulation. A high-pressure-amplitude therapeutic ultrasound (TUS) device was used to treat 36 surgically exposed spleens in healthy New Zealand rabbits. Eighteen spleens were treated with either MEUS (n = 9) or MEUS combined with prothrombin (n = 9). The other 18 spleens were treated with TUS only or sham ultrasound exposure and served as the controls. The TUS was operated at a frequency of 831 kHz and a peak negative pressure of 4.8 MPa. Prothrombin was administered intravenously at 20 IU/kg. Contrast-enhanced ultrasound (CEUS) and acoustic quantification were performed to assess splenic blood perfusion. We found significant blood perfusion slowdown and drop-off in the MEUS-treated spleens. The peak intensity dropped from 20.2 ± 2.70 dB to 11.6 ± 4.58 dB immediately after treatment. The spleens treated with the combination of MEUS and prothrombin showed consistently poor perfusion within 1 h. In histologic examination of the MEUS-treated spleens, we found significant dilatation of splenic sinuses, hemorrhage, interstitial edema and thrombosis. This study demonstrated that the vascular effects induced by microbubble-enhanced, high-pressure ultrasound can slow down or block blood perfusion in the rabbit spleen. Prothrombin helps to enhance and extend the effects for up to 1 h.

Disruption of Tumor Neovasculature by Microbubble Enhanced Ultrasound: A Potential New Physical Therapy of Anti-Angiogenesis

Tumor angiogenesis is of vital importance to the growth and metastasis of solid tumors. The angiogenesis is featured with a defective, leaky and fragile vascular construction. Microbubble enhanced ultrasound (MEUS) cavitation is capable of mechanical disruption of small blood vessels depending on effective acoustic pressure amplitude. We hypothesized that acoustic cavitation combining high-pressure amplitude pulsed ultrasound (US) and circulating microbubble could potentially disrupt tumor vasculature. A high-pressure amplitude, pulsed ultrasound device was developed to induce inertial cavitation of circulating microbubbles. The tumor vasculature of rat Walker 256 was insonated percutaneously with two acoustic pressures, 2.6 MPa and 4.8 MPa, both with intravenous injection of a lipid microbubble. The controls were treated by the ultrasound only or sham ultrasound exposure. Contrast enhanced ultrasound (CEUS) and histology were performed to assess tumor circulation and pathological changes. The CEUS results showed that the circulation of Walker 256 tumors could be completely blocked off for 24 hours in 4.8 MPa treated tumors. The CEUS gray scale value (GSV) indicated that there was significant GSV drop-off in both of the two experimental groups but none in the controls. Histology showed that the tumor microvasculature was disrupted into diffuse hematomas accompanied by thrombosis, intercellular edema and multiple cysts formation. The 24 hours of tumor circulation blockage resulted in massive necrosis of the tumor. MEUS provides a new, simple physical method for anti-angiogenic therapy and may have great potential for clinical applications.

Liver haemostasis using microbubble-enhanced ultrasound at a low acoustic intensity

To explore the haemostatic effects of microbubble-enhanced ultrasound (MEUS) at a very low acoustic intensity on the bleeding liver of rabbits. Liver incisions made on 20 rabbits were treated with a pulsed therapeutic ultrasound transducer. The transducer was operated at 831 KHz with an acoustic intensity of 0.4 W/cm(2). The treatment was coordinated with intravenous injection of microbubbles. Ultrasound only and sham treatment served as the controls. Visual bleeding score and 10-min bleeding volume were evaluated for haemostatic efficacy. Contrast-enhanced ultrasound (CEUS) was performed to assess the liver perfusion. Nine treated livers were harvested for acute histological examination. Regarding the bleeding incisions made on rabbit livers, the haemorrhage stopped immediately after 2 min of MEUS treatment but bleeding continued in the controls treated by ultrasound or microbubble injection alone. The bleeding scores and the 10-min haemorrhagic volumes dropped significantly in the MEUS group compared with those of the controls (p < 0.01). The mechanism of MEUS haemostasis appears to involve the extensive swelling of hepatocytes and the haemorrhage of the portal area, which formed a joint compression on the regional liver circulation. Low acoustic intensity MEUS might provide a novel method for liver haemostasis. • This animal experiment demonstrates a novel method of controlling hepatic haemorrhage • The treatment uses therapeutic ultrasound during enhancement with intravenous microbubbles • This combined therapy was more effective than ultrasound or intravenous microbubbles alone • More work is required with larger animals before potential human trials.

Era of diagnostic and interventional ultrasound

It is an era of diagnostic and interventional ultrasound (US). Various new techniques such as three-dimensional US (3D US), interventional US, and contrast-enhanced US (CEUS) have been introduced into clinical practice. Dr. Xu and his colleagues have taken advantage of these techniques and carried out a series of relevant studies. Their use of 3D US in the liver, gallbladder, liver tumor volumetry, guidance for ablation, and 3D CEUS has widened the application of 3D US in the clinic. They found that prognosis in patients with hepatocellular carcinoma (HCC) after thermal ablation with curative intent was determined by treatment response to ablation, pretreatment serum AFP, and liver function reserve. Tumor response to treatment was the most predictive factor for long-term survival. They compared the use of percutaneous microwave ablation and radiofrequency ablation for the treatment of HCC and found that both are effective methods in treating HCCs. The local tumor control, complications related to treatment, and long-term survival were equivalent for the two modalities. They first compared the enhancement patterns of HCC and intrahepatic cholangiocarcinoma (ICC) and proposed the diagnostic clues for ICC, liver angiomyolipoma (AML), gallbladder cancer, renal carcinoma, and renal AML, which have greatly enhanced the role of CEUS in the clinic. They also evaluated the diagnostic performance of CEUS in characterizing complex cystic focal liver lesions and the agreement between two investigators with different experience levels; and found that CEUS is especially useful for the young investigator. They assessed the effect of anti-angiogenic gene therapy for HCC treated by microbubble-enhanced US exposure and concluded that gene therapy mediated by US exposure enhanced by a microbubble contrast agent may become a new treatment option for HCC.

Efficacy of Perflubutane Microbubble-Enhanced Ultrasound in the Characterization and Detection of Focal Liver Lesions: Phase 3 Multicenter Clinical Trial

The purpose of this study was to assess the efficacy and safety of contrast-enhanced ultrasound performed with perflubutane microbubbles in comparison with unenhanced ultrasound and dynamic CT in the characterization of focal liver lesions during the vascular phase of imaging and in the detection of lesions during the Kupffer phase. A total of 196 patients were enrolled at 15 centers in Japan. Vascular phase images were obtained before contrast injection until 1 minute after injection. Kupffer phase images were obtained 10 minutes after injection. Dual-phase CT was performed as determined by standard clinical practice at each center. Unenhanced ultrasound, contrast-enhanced ultrasound, and CT images were read by blinded reviewers, and the results they reached regarding characterization and detection were compared with reference standard findings made by onsite investigators. The safety observation period was 72 hours after contrast administration. Among the 190 patients included in the characterization analysis, the accuracy of contrast-enhanced ultrasound (88.9%) was significantly greater than that of unenhanced ultrasound (68.4%) and dynamic CT (80.5%) (p < 0.001 and p = 0.008). Among the 191 patients in the detection analysis, the efficacy of contrast-enhanced ultrasound in detection of lesions was significantly higher than that of unenhanced ultrasound and dynamic CT (p < 0.001 and p = 0.008), predominantly because more metastatic lesions were detected (both p < 0.001). In particular, contrast-enhanced ultrasound was superior to dynamic CT in the detection of metastatic lesions measuring 1 cm or smaller. The incidence of adverse events was 49.2% and that of adverse drug reactions was 10.4%. All adverse drug reactions were mild. Compared with unenhanced ultrasound and dynamic CT, contrast-enhanced ultrasound with perflubutane microbubbles improved diagnostic efficacy in both characterization and detection of focal liver lesions with no serious adverse drug reactions.

Sustained Transgene Expression in Intervertebral Disc Cells In Vivo Mediated by Microbubble-Enhanced Ultrasound Gene Therapy

In vivo studies using a rat model were performed to determine the feasibility of microbubble-enhanced ultrasound gene transfer technique to the intervertebral disc. 1) To establish this microbubble-enhanced ultrasound gene therapy technique for intervertebral disc cells in vivo without using viral vectors and 2) to estimate the duration of transgene expression in vivo. Intervertebral disc degeneration and associated spinal disorders remain a formidable problem. Although gene therapy approaches have been reported as having promising therapeutic potential to regenerate the disc, concerns over safety issues using recombinant viral vectors limits its application. Successful gene transfer using ultrasound has been reported in muscle and cardiovascular systems in vivo. Two different reporter plasmid DNA encoding green fluorescent protein (GFP) and firefly luciferase were used. Plasmid DNA was mixed with ultrasonography contrast agent (microbubbles) and injected into coccygeal intervertebral discs of Sprague-Dawley rats. The therapeutic ultrasound was irradiated on the surface of injected discs. Rats were killed 1, 3, 6, 12, and 24 weeks after gene transduction. Harvested nucleus pulposus tissues were used for evaluation of transgene expression. The intact discs were used as a control. Seven days after gene transfection, considerable numbers of GFP-positive cells were observed in nucleus pulposus from the GFP-transfected group. Luciferase assay revealed that the ultrasound group demonstrated approximately an 11-fold increase in luciferase activity over the plasmid DNA-only group. Furthermore, transgene expression mediated by this method was observed, at least up to 24 weeks. Our study indicated that ultrasound transfection method with microbubbles significantly enhanced transfection efficiency of plasmid DNA into the nucleus pulposus cells in vivo. Furthermore, the sustained transgene expression in vivo was possible up to 24 weeks. The long-term gene expression mediated by simple and safe procedure has important clinical applications, including the treatment of chronic types of disease such as degenerative disc diseases.