Xing Hua

Myocardium-targeted transplantation of mesenchymal stem cells by diagnostic ultrasound-mediated microbubble destruction improves cardiac function in myocardial infarction of New Zealand rabbits.

BACKGROUND Therapeutic ultrasound-mediated microbubble destruction has been applied in the targeted delivery of genes, drugs and stem cells. We intended to study whether diagnostic US irradiating lipid-coated microbubble destruction combined with bone-marrow derived MSC infusion could enable the targeted delivery of MSCs into the myocardium and improve cardiac function of the myocardial infarction of New Zealand rabbits. METHODS Diagnostic ultrasound was applied to the anterior chest for 10 min after intravenous injection of lipid-coated microbubble followed by infusion of BM-MSCs. Echocardiography, histological examination, and western blotting were performed 4 weeks after cell transplantation. RESULTS The cardiac function (assessed by fractional shortening and ejection fraction) was markedly improved by US+Microbubble+MSC treatment. The number of capillaries stained by HE in US+Microbubble+MSC group (47+/-23) was much greater than that of the MSCs infusion group (26+/-7), US+Microbubble group(22+/-5) and PBS infusion group (19+/-10), P<0.01. US+Microbubble stimulation induced the expression of adhesion molecule (VCAM-1) in capillaries and enhanced the myocardial permeability of microvessels. US+Microbubble-mediated supply of MSCs increased the level of VEGF in ischemic myocardium. Area of cardiac fibrosis in the US+Microbubble+MSC group was significantly decreased by 25.6%,40.1% and 46.8% when compared with MSC infusion group, US+Microbubble group and PBS infusion group, respectively. CONCLUSIONS This non-invasive cell delivery system may be useful as a novel and efficient approach for angiogenic cell therapy to the infarcted myocardium.

Effects of a novel ultrasound contrast agent with long persistence on right ventricular pressure: Comparison with SonoVue.

This work investigated the effect of infusion of a self-made ultrasound contrast agent with long persistence (named ZHIFUXIAN) on rat right ventricular pressure and made a preliminary evaluation on the relative safety of the novel microbubbles. Normal saline, SonoVue and ZHIFUXIAN were injected through caudal vein at the total volume of 0.5ml for each injection. The right ventricular systolic pressure (RVSP) and end-diastolic pressure (RVEDP) were monitored and the changes of the pressure were compared with baseline readings. RVSP increased when saline, SonoVue or ZHIFUXIAN were injected, the greatest change being after SonoVue (about 2mmHg), but there was no statistical significance compared with baseline (P>0.05). There was no significant difference in RVSP between saline, SonoVue and ZHIFUXIAN at any time point. Also, there was no significant difference in RVEDP between groups at each time point and between different time points in each group. The results indicate that the self-made microbubbles effect on right ventricular hemodynamics is equivalent to that of normal saline at the same volume needed for effective enhanced imaging, demonstrating that it does not produce changes in right ventricular blood pressure under the study conditions. Pathological examination also showed it had no obvious influence on lung, liver and kidney.

Molecular imaging of vulnerable plaques in rabbits using contrast-enhanced ultrasound targeting to vascular endothelial growth factor receptor-2

Increased neovascularization has been identified as a feature of atherosclerotic plaque vulnerability and can be traced by microbubble ultrasound contrast agents (UCA). We investigated the relationship between retention of a vascular endothelial growth factor receptor 2 (VEGFR‐2) targeted UCA and VEGFR‐2 expression in a vulnerable plaque model in rabbits.

Effects of Diagnostic Contrast-Enhanced Ultrasound on Permeability of Placental Barrier: A Primary Study

OBJECTIVE To evaluate the effects of contrast-enhanced ultrasound (CEU) on the permeability of placental barrier primarily. METHODS A total of 60 pregnant Sprague Dawley (SD) rats were divided into 10 groups, including six groups of microbubbles-enhanced ultrasound (varied mechanical index (MI) of 0.13, 1.0 and 1.4 with continuous and intermittent insonation respectively) (US+MB), two groups of ultrasound insonation only (continuous and intermittent insonation respectively) (US), the group of microbubbles only (MB) and the control group. Evans blue (EB), as the tracer, was intravenously injected before treatment. The EB in placenta and fetus was observed under fluorescence microscope and analyzed quantitatively. The EB amount was compared between groups and between placenta and fetus. Lanthanum nitrate-tracing transmission electron microscope examination was performed to observe the distribution of lanthanum in the placenta and fetus. RESULTS Observed by naked eye, the plancenta was dyed into deep blue while there was no sign of dyeing to the fetus in all groups. Under fluorescence microscope, the red fluorescence radiated by EB was observed in placenta but not in fetus. The EB amount in placenta, insonated by microbubbles-enhanced ultrasound of varied MI, was higher significantly than that in MB, US and control group (all P<0.01) while there was no difference between the latter three groups. And in each group, EB amount was much higher in placenta than that in fetus (P<0.01). The lanthanum particles deposited in the intercellular space in the syntrophoblast while there was no lanthanum presented in the cytotrophoblast in all groups. CONCLUSION Our findings suggest that diagnostic CEU with SonoVue will not increase the permeability of placenta to the macromolecules larger than albumin, although it may affect placenta.

Vascular Effects of Microbubble-Enhanced, Pulsed, Focused Ultrasound on Liver Blood Perfusion

The purpose of this study was to investigate the vascular effects of microbubble-enhanced pulsed high-pressure ultrasound on liver blood perfusion. In the presence of circulating lipid-shell microbubbles, a focused ultrasound transducer was used to transcutaneously treat eight livers of healthy rabbits for perfusion analysis and to treat three livers with the abdomen open for histologic analysis. Twenty-two livers treated with the ultrasound only (n = 11) or microbubbles only (n = 11) served as the controls. The focused ultrasound was operated at a frequency of 1.22 MHz with a peak negative pressure of 4.6 MPa. The liver blood perfusion was estimated by performing contrast-enhanced ultrasound and gray-scale quantification on the livers before and after treatment. A temporary, nonenhanced region occurred in all of the experimental livers. The regional contrast gray-scale values of the experimental group dropped significantly from 88.4 before treatment to 2.7 after treatment. The liver perfusion also demonstrated a gradual recovery over a 60-min period. The liver perfusion of the control groups remained the same after treatment. We found microvascular rupture, hemorrhage and swelling hepatocytes upon histologic examination of the experimental group. Regional liver blood perfusion can be temporarily blocked by microbubble-enhanced focused ultrasound with high-pressure amplitude. These vascular effects can be explained as acute microvascular injury of the liver and may have clinical implications.

Ultrasound molecular imaging of acute cellular cardiac allograft rejection in rat with T-cell-specific nanobubbles.

Background Acute rejection (AR) is one of the main obstacles of cardiac transplantation; however, a noninvasive diagnostic method, which reflects its pathologic nature, has not been developed yet. In this study, we prepared a specific nanobubbles targeting to the activated T cells and applied it in the ultrasound molecular imaging of AR in heart transplantation by myocardial contrast echocardiography (MCE). Methods Nanobubbles loading anti-CD25 antibody (NBspecific) or isotype control antibody (NBnonspecific) were prepared and then applied in the ultrasound molecular imaging by MCE in a rat model. MCE was performed in 24 allografts and 18 isografts that were divided into three groups, including days 2, 4, and 6 after transplantation. Confocal laser scanning microscopy was used to evaluate the binding of nanobubbles and T cells in four allografts and four isografts. Results MCE with NBspecific in allograft showed a “delayed enhancement,” and the time-intensity curve presented a second peak. The intensity and time of second peak were both positively correlated with the transplant time (P<0.01) and the pathologic grade of AR (P<0.01). Confocal laser scanning microscopy demonstrated the binding of nanobubbles and lymphocytes in myocardium post-MCE with NBspecific. Conclusions Ultrasound molecular imaging of AR after heart transplantation can be achieved by using MCE with the nanobubbles targeted to T cells. The appearance of delayed enhancement indicates the occurrence of AR, and the intensity and time of the second peak in time-intensity curve provide potential quantitative indications for diagnosis and severity of AR.

Inhibition Effects of High Mechanical Index Ultrasound Contrast on Hepatic Metastasis of Cancer in a Rat Model

RATIONAL AND OBJECTIVES The liver is the most common organ for tumor metastasis. The development of new methods to depress hepatic metastasis is of great importance in improving survival. The aim of this study was to observe the effects of high-mechanical index ultrasound contrast on hepatic metastasis of colorectal cancer. MATERIALS AND METHODS Hepatic metastasis models were established by injecting human colon carcinoma LoVo cells into the spleens of Sprague-Dawley rats. The rats were divided into a control group, a microbubble plus ultrasound group, a simple ultrasound group, and a simple microbubble group. The ultrasound contrast agent SonoVue (1 mL/kg) was injected via the tail vein, and high-mechanical index ultrasound contrast (frequency, 1.5 MHz; mechanical index, 1.7) was performed on the spleen intermittently for 2 minutes. The animals were sacrificed after 10 days, and the sizes and number of hepatic metastases were measured and compared. Histologic pathology and splenic ultrastructure were observed. RESULTS The number and sizes of hepatic metastases patently decreased in rats in the microbubble plus ultrasound group (P < .01). There were no obvious differences among the control group, simple ultrasound group, and simple microbubble group in hepatic metastases (P > .05). Histologic pathology showed that the number of tumor cells in the spleens decreased considerably, and massive necroses, hemorrhages, and thrombi were observed in the tumor and spleen tissues of rats in the microbubble plus ultrasound group. Transmission electron microscopy showed that the mitochondria of tumor cells and endothelial cells were clearly swelled, and there were gaps among endothelial cells and platelets aggregated in capillary vessels. CONCLUSION This research shows that intermittent high-mechanical index ultrasound contrast may inhibit the hepatic metastasis of cancer in a rat model.

Renal interstitial permeability changes induced by microbubble-enhanced diagnostic ultrasound.

Abstract Ultrasound-targeted microbubble (MB) destruction (UTMD) has been shown to increase the glomerular permeability, providing a potential novel therapeutic approach in targeted drug release for kidney diseases. Therefore, we investigated the impact of UTMD on renal interstitial permeability using MB-mediated diagnostic ultrasound (DUS). The left kidney of Sprague–Dawley (SD) rat was insonated by UTMD with either continuous or intermittent mode for 5 min. Evans blue (EB) revealed that both modes induced renal vascular permeability increase after DUS but recovered after 24 h. Intermittent insonation caused more severe injury than continuous mode. Red blood cells leaked out of the capillaries into interstitium without glomerular capillary hemorrhage (GCH) by hematoxylin and eosin (HE) staining. Electronic microscopy revealed the disruption of focal capillary wall in interstitial tissues. Morphological results confirmed capillary wall recovered in 24 h post-treatment. Results from fluorescence-labeled MBs showed that MBs were mainly localized in the interstitial portion of the tubular region and retained at 24 h. Intriguingly, urinalysis showed no clinical proteinuria after treatment. Our results indicated that MB plus DUS specifically and reversibly enhanced the interstitial permeability without affecting glomerulus, which may be developed into a therapeutic approach for targeting drug release to individual renal compartments.

Unilateral Opening of Rat Blood-Brain Barrier Assisted by Diagnostic Ultrasound Targeted Microbubbles Destruction

Objective. Blood-brain barrier (BBB) is a key obstacle that prevents the medication from blood to the brain. Microbubble-enhanced cavitation by focused ultrasound can open the BBB and proves to be valuable in the brain drug delivery. The study aimed to explore the feasibility, efficacy, and safety of unilateral opening of BBB using diagnostic ultrasound targeted microbubbles destruction in rats. Methods. A transtemporal bone irradiation of diagnostic ultrasound and intravenous injection of lipid-coated microbubbles were performed at unilateral hemisphere. Pathological changes were monitored. Evans Blue extravasation grades, extraction from brain tissue, and fluorescence optical density were quantified. Lanthanum nitrate was traced by transmission electron microscopy. Results. After diagnostic ultrasound mediated microbubbles destruction, Evans Blue extravasation and fluorescence integrated optical density were significantly higher in the irradiated hemisphere than the contralateral side (all p < 0.01). Erythrocytes extravasations were demonstrated in the ultrasound-exposed hemisphere (4 ± 1, grade 2) while being invisible in the control side. Lanthanum nitrate tracers leaked through interendothelial cleft and spread to the nerve fiber existed in the irradiation side. Conclusions. Transtemporal bone irradiation under DUS mediated microbubble destruction provides us with a more accessible, safer, and higher selective BBB opening approach in rats, which is advantageous in brain targeted drugs delivery.

Anti-tumor effect of ultrasound-induced Nordy-loaded microbubbles destruction.

Abstract Background: Synthesized dl-Nordihydroguaiaretic acid (dl-NGDA or “Nordy”) can inhibit the growth of malignant human tumors, especially the tumor angiogenesis. However, its liposoluble nature limits its in vivo efficacy in the hydrosoluble circulation of human. Purpose: We tried to use the ultrasonic microbubble as the carrier and the ultrasound-induced destruction for the targeted release of Nordy and evaluate its in vitro and in vivo anti-tumor effect. Methods: Nordy-loaded lipid microbubbles were prepared by mechanical vibration. Effects of ultrasound-induced Nordy-loaded microbubbles destruction on proliferation of human umbilical vein endothelial cells (HUVECs), tumor derived endothelial cells (Td-ECs), and rabbit transplanted VX2 tumor models were evaluated. Results: The ultrasound-induced Nordy-loaded microbubbles destruction inhibited the proliferations of HUVECs and Td-ECs in vitro, and inhibited the tumor growth and the microvasculature in vivo. Its efficacy was higher than those of Nordy used only and Nordy with ultrasound exposure. Conclusion: Ultrasonic microbubbles can be used as the carrier of Nordy and achieve its targeted release with improved anti-tumor efficacy in the condition of ultrasound-induced microbubbles destruction.