Distributed Fault-Tolerant Containment Control for Linear Heterogeneous Multiagent Systems: A Hierarchical Design Approach.

In this article, the problem of distributed hierarchical fault-tolerant containment control for heterogeneous linear multiagent systems (MASs) is investigated. In most of the existing distributed methods for MASs with system failures, each agent broadcasts its state, or output, or the estimation of state to neighbors. Once an agent is subjected system failures, faults affect the dynamics of other agents over the network, that is, the influence of faults on the agent will propagate with the network. In order to overcome this drawback, a fault-tolerant hierarchical containment control protocol is developed, which includes two layers: 1) the upper layer and 2) the lower layer. The upper layer consists of a virtual system and a cooperative controller to achieve a virtual containment objective. The lower layer consists of an actual system and a fault-tolerant controller to track the upper layer virtual system. Compared with the existing results, the phenomenon of fault propagation can be avoided by introducing the hierarchical design approach, that is, the fault of agent i only affects the dynamics of itself, and does not affect the dynamics of other agents through the network. It is shown that each follower converges asymptotically to a convex hull spanned by leaders with external input. Finally, the developed method is demonstrated by simulation results.

Molecular photoacoustic imaging for early diagnosis and treatment monitoring of rheumatoid arthritis in a mouse model.

Rheumatoid arthritis (RA) is a progressive inflammatory joint disease. Early diagnosis is critical for timely therapeutic intervention. However, it lacks effective diagnostic methods capable of detecting disease progression in its early stage and evaluating treatment efficacy in clinics. Photoacoustic (PA) molecular imaging is a novel imaging modality that can detect in the early stage of disease and continuously monitor its progression. In this study, Evans blue (EB) was used as a PA contrast agent to detect the angiogenesis and microcirculation dysfunction in RA joint. In collagen-induced arthritis (CIA) mouse model, a distinct increase of PA signal was detected early at 2 weeks, with significant higher PA signal intensities from the RA joints compared to the normal joints. More importantly, we detected an increasing trend of PA signal intensity week by week post CIA induction, demonstrating the potential of EB-enhanced PA imaging in monitoring the development of RA. However, joint damage was silent in the X-ray at 2 weeks post CIA induction, which suggested the superiority of PA imaging in RA early detection. In addition, striking decrease of PA signal intensities in the RA joints was observed after administration with etanercept compared with the untreated RA joints. The signal changes exhibited by PA imaging were confirmed by clinical observation and histological examinations. This study demonstrated the promising use of EB-enhanced PA imaging for the early diagnosis and its feasibility for RA treatment monitoring.

Real-Time Quantification of Cartilage Degeneration by GAG-Targeted Cationic Nanoparticles for Efficient Therapeutic Monitoring in Living Mice.

One of the characterizations of degenerative cartilage disease is the progressive loss of glycosaminoglycans (GAGs). The real-time imaging method to quantify GAGs is of great significance for the biochemical analysis of cartilage and diagnosis and therapeutic monitoring of cartilage degeneration in vivo. To this end, a cationic photoacoustic (PA) contrast agent, poly-l-lysine melanin nanoparticles (PLL-MNPs), specifically targeting anionic GAGs was developed in this study to investigate whether it can image cartilage degeneration. PLL-MNP assessed GAG depletion by Chondroitinase ABC in vitro rat cartilage and intact ex vivo mouse knee joint. A papain-induced cartilage degenerative mice model was used for in vivo photoacoustic imaging (PAI). Oral cartilage supplement glucosamine sulfate was intragastrically administered for mice cartilage repair and the therapeutic efficacy was monitored by PLL-MNP-enhanced PAI. Histologic findings were used to further confirm PAI results. In vitro results revealed that the PLL-MNPs not only had a high binding ability with GAGs but also sensitively monitored GAG content changes by PAI. The PA signal was gradually weakened along with the depletion of GAGs in cartilage. Particularly, PLL-MNPs depicted the cartilage structure and the distribution of GAGs was demonstrated in PA images in ex vivo joints. Compared with the normal joint, a lower signal intensity was detected from degenerative joint at 3 weeks after papain injection, suggesting an early diagnosis of cartilage lesion by PLL-MNPs. Importantly, this PA-enhanced nanoprobe was suitable for monitoring in vivo efficacy of glucosamine sulfate, which effectively blocked cartilage degradation in a high dose manner. In vivo imaging findings correlated well with histological examinations. PLL-MNPs provided sensitive visualization of cartilage degeneration and promising monitoring of therapeutic response in living subjects.

The emerging landscape of nanotheranostic-based diagnosis and therapy for osteoarthritis.

Osteoarthritis (OA) is a common degenerative disease involving numerous joint tissues and cells, with a growing rate in prevalence that ultimately results in a negative social impact. Early diagnosis, OA progression monitoring and effective treatment are of significant importance in halting OA process. However, traditional imaging techniques lack sensitivity and specificity, which lead to a delay in timely clinical intervention. Additionally, current treatments only slow the progression of OA but have not meet the largely medical need for disease-modifying therapy. In order to overcome the above-mentioned problems and improve clinical efficacy, nanotheranostics has been proposed on OA remedy, which has confirmed success in animal models. In this review, different imaging targets-based nanoprobe for early and timely OA diagnosis is first discussed. Second, therapeutic strategies delivered by nanosystem are summarized as much as possible. Their advantages and the potential for clinical translation are detailed discussed. Third, nanomedicine simultaneously combined with the imaging for OA treatment is introduced. Nanotheranostics dynamically tracked the OA treatment outcomes to timely and individually adjust therapy. Finally, future prospects and challenges of nanotechnology-based OA diagnosis, imaging and treatment are concluded and predicted. It is believed that nanoprobe and nanomedicine will become prospective in OA therapeutic revolution.

Tracking Osteoarthritis Progress through Cationic Nanoprobe-Enhanced Photoacoustic Imaging of Cartilage.

A major obstacle in osteoarthritis (OA) theranostics is the lack of a timely and accurate monitoring method. It is hypothesized that the loss of anionic glycosaminoglycans (GAGs) in articular cartilage reflects the progression of OA. Thus, this study investigated the feasibility of photoacoustic imaging (PAI) applied for monitoring the in vivo course of OA progression via GAG-targeted cationic nanoprobes. The nanoprobes were synthesized through electrostatic attraction between poly-l-Lysine and melanin (PLL-MNPs). Cartilage explants with different concentrations of GAGs incubated with PLL-MNPs to test the relationship between GAGs content and PA signal intensity. GAG activity was then evaluated in vivo in destabilization of the medial meniscus (DMM) surgically-induced mouse model. To track OA progression over time, mice were imaged consistently for 10 weeks after OA-inducing surgery. X-ray was used to verify the superiority of PAI in detecting OA. The correlation between PAI data and histologic results was also analyzed. In vitro study demonstrated the ability of PLL-MNPs in sensitively detecting different GAGs concentrations. In vivo PAI exhibited significantly lower signal intensity from OA knees compared to normal knees. More importantly, PA signal intensity showed serial reduction over the course of OA, while X-ray showed visible joint destruction until 6 weeks. A decrease in GAGs content was confirmed by histologic examinations; moreover, histologic findings were well correlated with PAI results. Therefore, using cationic nanoprobe-enhanced PAI to detect the changes in GAG contents provides sensitive and consistent visualization of OA development. This approach will further facilitate OA theranostics and clinical translation. STATEMENT OF SIGNIFICANCE: The study of in vivo monitoring osteoarthritis (OA) is of high significance to tracking the trajectory of OA development and therapeutic monitoring. Here, we developed a cartilage-targeted cationic nanoprobe, poly-l-Lysine-melanin nanoparticles (PLL-MNPs), enhancing photoacoustic imaging (PAI) to monitor the progression of OA. The in vitro study demonstrated the ability of PLL-MNPs to detect different concentrations of GAGs with high sensitivity. We found that the contents of GAGs in vivo steadily decreased from the development of OA initial-stage to the end-point of our investigation via PAI; it reflected the course of OA in living subjects with high sensitivity. These results allow for further development in various aspects of OA research. It has potential for clinical translation and has a great impact on personalized medicine.

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.

OCTN2-targeted nanoparticles for oral delivery of paclitaxel: differential impact of the polyethylene glycol linker size on drug delivery in vitro, in situ, and in vivo.

Targeted nanocarriers have shown great promise in drug delivery because of optimized drug behavior and improved therapeutic efficacy. How to improve the targeting efficiency of nanocarriers for the maximum possible drug delivery is a critical issue. Here we developed L-carnitine-conjugated nanoparticles targeting the carnitine transporter OCTN2 on enterocytes for improved oral absorption. As a variable, we introduced various lengths of the polyethylene glycol linker (0, 500, 1000, and 2000) between the nanoparticle surface and the ligand (CNP, C5NP, C10NP and C20NP) to improve the ligand flexibility, and consequently for more efficient interaction with the transporter, to enhance the oral delivery of the cargo load into cells. An increased absorption was observed in cellular uptake in vitro and in intestinal perfusion assay in situ when the polyethylene glycol was introduced to link L-carnitine to the nanoparticles; the highest absorption was achieved with C10NP. In contrast, the linker decreased the absorption efficiency in vivo. As the presence or absence of the mucus layer was the primary difference between in vitro/in situ versus in vivo, the presence of this layer was the likely reason for this differential effect. In summary, the size of the polyethylene glycol linker improved the absorption in vitro and in situ, but interfered with the absorption in vivo. Even though this strategy of increasing the ligand flexibility with the variable size of the polyethylene glycol failed to increase oral absorption in vivo, this approach is likely to be useful for enhanced cellular uptake following intravenous administration of the nanocarriers.

UHPLC-MS/MS method for the quantification of aloin-A in rat plasma and its application to a pharmacokinetic study.

Aloin-A (also known as barbaloin), the main bioactive anthraquinone-C-glycoside of Aloe species, exhibits various beneficial pharmacological effects. However, the determination and pharmacokinetic study of aloin-A in rat plasma need to be improved and systematically demonstrated. In the present study, a simple, robust and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for rapid quantification of aloin-A in rat plasma was developed. Plasma preparation was conducted by a single step protein precipitation with obtusin serving as an internal standards (IS) followed by separation of the analytes using an Agilent C18 column with a gradient mobile phase comprised of acetonitrile and formic acid aqueous solution. Negative ion electrospray was used and multiple reaction monitoring transitions were m/z 417.1 → 297.0 for aloin-A and m/z 343.1 → 328.1 for IS, respectively. The developed method was validated with linear range of 1-1000 ng/mL. All validation parameters were well within the acceptance criteria based on the guidance of FDA. The validated approach was successfully applied to analyze samples from a pharmacokinetic study in healthy rats following intravenous and oral administration. Aloin-A was found to be quickly absorbed, extensively distributed and rapidly eliminated. The absolute bioavailability of aloin-A was 5.79%.

Therapeutic options and drug delivery strategies for the prevention of intrauterine adhesions.

Intrauterine adhesions (IUAs) are bands of fibrous tissue that form in the endometrial cavity and associated with the increased risk of abnormal menstruation, recurrent pregnancy loss, secondary infertility, and pregnancy complications. Physical barriers, including intrauterine device and hydrogel, were clinical available to prevent the post-operational IUAs. But physically separation of the injured endometrium relies on the own limited healing power and often ends with recurrence. In recent years, the mechanisms driving IUAs treatment has validated the application of hormones, and further stem cell therapy has also led to the development of novel therapeutic agents with promising efficacy in pre-clinical and initial clinical studies. Still, it is challenging to delivery the therpaeutic factors to the injured uterus. Herein, in this review, we discuss the traditional intervention methods for the prevention of IUAs, as well as novel therapeutics and delivery strategies that will most likely change the treatment paradigms for better clinical outcomes. The combination strategy that using physical barriers as the delivery carriers for therapeutics might provide new alternatives for the prevention of IUAs.

Nanomedicine - advantages for their use in rheumatoid arthritis theranostics.

Rheumatoid arthritis (RA) is an autoimmune disease accompanies with synovial inflammation and progressive bone destruction. Currently, anti-rheumatic drugs need high dose and frequent use for a long-term, which lead to serious side effect and low patient compliance. To overcome above problems and improve clinical efficacy, nano-technology with targeting ability, sustained release and so forth, has been proposed on RA treatment and already achieved success in RA animal models. In this review, authors summarize and illustrate representative nanomedicine targeting to RA states, which is achieved either through passive or active targeting with high affinity to the receptors that are over-expressed in macrophages or angiogenesis. In particular, authors highlight the new strategies to promote the efficacy of nanoscale treatments through phototherapy and the addition of contrast elements for theranostic application. The described advances may pave the way to better understanding and designing the novel nanomedicine and multifunctional nano-system on efficient RA treatment.

Biomaterial-engineered intra-articular drug delivery systems for osteoarthritis therapy.

Osteoarthritis (OA) is a progressive and degenerative disease, which is no longer confined to the elderly. So far, current treatments are limited to symptom relief, and no valid OA disease-modifying drugs are available. Additionally, OA relative joint is challenging for drug delivery, since the drugs experience rapid clearance in joint, showing a poor bioavailability. Existing therapeutic drugs, like non-steroidal anti-inflammatory drugs (NSAIDs) and corticosteroids, are not conducive for long-term use due to adverse effects. Though supplementations, including chondroitin sulfate and glucosamine, have shown beneficial effects on joint tissues in OA, their therapeutic use is still debatable. New emerging agents, like Kartogenin (KGN) and Interleukin-1 receptor antagonist (IL-1 ra), without a proper formulation, still will not work. Therefore, it is urgent to establish a suitable and efficient drug delivery system for OA therapy. In this review, we pay attention to various types of drug delivery systems and potential therapeutic drugs that may escalate OA treatments.

Effect of Ultrasound Combined With Microbubble Therapy on Interstitial Fluid Pressure and VX2 Tumor Structure in Rabbit.

Interstitial fluid pressure (IFP) in tumor tissue is significantly higher than that in normal tissue, which reduces the effectiveness of therapeutic drugs. There are several methods to decrease the IFP, such as normalizing blood vessel, decreasing hyaluronic acid and collagen fiber content in the extracellular matrix (ECM), and recovering lymphatic function. Reducing tumor IFP might be developed as a novel approach in cancer therapy. In this study, we aimed to elucidate the relationship between ultrasound combined with microbubble therapy and IFP, and the associated mechanism. VX2 tumor in rabbit was treated with ultrasound combined with microbubbles at different intensities. The IFP was measured using the wick-in-needle (WIN) method. The collagen and reticular fibers were stained by Masson and Gordon-Sweets, respectively. The results showed that low-frequency non-focus ultrasound combined with microbubbles therapy influences the IFP in tumor tissues; low-frequency non-focus ultrasound with low pressure increased the IFP, whereas middle-high pressure decreased the IFP. The results showed that the structure and content of collagen and reticular fibers in tumor tissue were rarely influenced by the treatment. Our study provides a novel approach of reduced IFP antitumor therapy.

Ambidextrous Approach To Disrupt Redox Balance in Tumor Cells with Increased ROS Production and Decreased GSH Synthesis for Cancer Therapy.

An effective steady-state redox balance is maintained in cancer cells, allowing for protection against oxidative stress and thereby enhancing cell proliferation and tumor growth. Disruption of this redox balance would increase the cellular content of reactive oxygen species (ROS) and potentiate oxidative stress-induced cell death in tumor cells, thus representing an effective strategy for cancer treatment. Glutathione (GSH) is a major reducing agent, and its cellular levels are determined at least partly by the availability of cysteine via xCT (SLC7A11)-mediated entry of cystine into cells. We developed a nanoplatform using ZnO nanoparticles (NPs) as a carrier, loaded with salicylazosulfapyridine (SASP), and stabilized with DSPE-PEG, to form ultra-small NPs (SASP/ZnO NPs). The goal of this NP strategy is to disrupt the redox balance in cells by two mechanisms: increased generation of ROS and decreased synthesis of GSH. Such an approach would be effective in killing tumor cells. As expected, the SASP/ZnO NPs enhanced ROS production because of ZnO and impaired GSH synthesis because of SASP-induced inhibition of xCT (SLC7A11) transport function. As a consequence, treatment of tumor cells with SASP/ZnO NPs in vitro and in vivo resulted in a synergistic disruptive effect on redox balance in tumor cells and induced cell death and decreased tumor growth. This ambidextrous approach has potential in cancer therapy by combining two complementary pathways to disrupt the redox balance in tumor cells.

Penetration of different molecule sizes upon ultrasound combined with microbubbles in a superficial tumour model.

Ultrasound combined with microbubbles (USMB) has been extensively applied to enhance drug and gene targeting delivery. However, the accumulation and distribution of particle size in the range of 5-30 nm (nano drug) to the tumour and the effects of intratumoral vascular density on permeability have been rarely reported. This study investigated Evans blue (EB) and fluorescein isothiocyanate-labelled dextran (FITC-dextran) distribution in tumour tissue upon USMB with various molecular sizes (3.7 nm and 30.6 nm). USMB increased the penetration of molecules with sizes of 5-20 nm in the whole tumour tissue, especially on the rim. For a molecule with sizes of 30.6 nm, USMB only increased penetration around the rim of the tumour with minimal improvement in the central of tumour. USMB enhanced the permeability of tumour tissue and increased tumour cells dose exposure without affecting tumour blood perfusion or microvessel density. The current study served as the foundation of parameter preference for therapeutic USMB drug delivery.

Correlation of virtual touch tissue quantification and liver biopsy in a rat liver fibrosis model.

Liver fibrosis assessment is very important to the treatment of chronic liver disease. In the present study, Virtual Touch Tissue Quantification (VTQ) and eSie Touch™ elasticity imaging techniques were used to examine the rat liver fibrosis model. Rat liver fibrosis was induced with thioacetamide and the degree of liver fibrosis was determined using pathological diagnosis as a gold standard. The right lobe of the liver was also examined with the VTQ and eSie Touch™ techniques. The VTQ and serological results were correlated and analyzed. The results were compared with those obtained from liver biopsies to investigate the accuracy and diagnostic value of eSie Touch™ and VTQ on the classification of liver fibrosis in rats. A total of 30 successful modeling cases were obtained, with a success rate of 86%. The mean acoustic radiation force impulse (ARFI) elastography­VTQ values were 1.08, 1.51, 1.88 and 2.50 m/sec for the normal and F1/F2, F3 and F4 fibrosis groups, respectively. A significant correlation (r = 0.969) was identified between the ARFI measurements and the degree of fibrosis assessed by pathological examination (P<0.001). The histological staging results correlated with those of the eSie Touch™ elasticity imaging of the biopsy site (r = 0.913, P<0.001). The predictive values of ARFI for various stages of fibrosis were as follows: F≥1 and 2 ­ cut­off >1.250 m/sec (when Vs >1.250 m/sec, the pathological grading was ≥F1/F2) [Area under receiver operating characteristic (AUROC) = 1.00], F≥3 ­ cut­off >1.685 m/sec (when Vs >1.685 m/sec, the pathological grading was ≥F3; AUROC = 1.00) and F≥4 ­ cut­off >2.166 m/sec (when Vs >2.166 m/sec, the pathological grading is cirrhosis; AUROC = 1.00). In conclusion, the eSie Touch™ elasticity imaging and VTQ techniques may be successfully adopted to assess the extent of liver stiffness. These techniques are expected to replace liver biopsy.

Hemostatic effects of microbubble-enhanced low-intensity ultrasound in a liver avulsion injury model.

OBJECTIVES: Microbubble-enhanced therapeutic ultrasound (MEUS) can block the blood flow in the organs. The aim of this study was to evaluate the hemostatic effect of microbubble-enhanced pulsed, low-intensity ultrasound in a New Zealand White rabbit model of avulsion trauma of the liver. The therapeutic ultrasound (TUS) transducer was operated with the frequency of 1.2 MHz and an acoustic pressure of 3.4 MPa. Microbubble-(MB) enhanced ultrasound (MEUS) (n = 6) was delivered to the distal part of the liver where the avulsion was created. Livers were treated by TUS only (n = 4) or MB only (n = 4) which served as controls. Bleeding rates were measured and contrast enhanced ultrasound (CEUS) was performed to assess the hemostatic effect, and liver hemoperfusion before and after treatment. Generally, bleeding rates decreased more than 10-fold after the treatment with MEUS compared with those of the control group (P<0.05). CEUS showed significant declines in perfusion. The peak intensity value and the area under the curve also decreased after insonation compared with those of the control group (P<0.05). Histological examination showed cloudy and swollen hepatocytes, dilated hepatic sinusoids, perisinusoidal spaces with erythrocyte accumulation in small blood vessels, obvious hemorrhage around portal areas and scattered necrosis in liver tissues within the insonation area of MEUS Group. In addition, necrosis was found in liver tissue 48 h after insonation. We conclude that MEUS might provide an effective hemostatic therapy for serious organ trauma such as liver avulsion injury.

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.