Assessing vesicoureteral reflux in live inbred mice via ultrasound with a microbubble contrast agent

Vesicoureteral reflux (VUR) is a common pediatric anomaly linked to renal scarring and hypertension. Although there are many mouse VUR models, cystograms have previously only been performed in euthanized animals, thus preventing serial assessments for VUR in the same animal and not delineating "live" physiology. Our purpose was to develop a live murine cystogram assay that could be used serially to track reflux. We injected microbubbles via transurethral catheters into bladders of C57BL6/J and C3H/HeJ inbred mouse strains that are known to have low and high VUR rates, respectively. We performed ultrasound to visualize microbubbles in the renal pelvis to determine feasibility of the procedure. We then repeated the microbubble ultrasound using a transducer allowing for visualization of both kidneys and ureters simultaneously and for 3 dimensional (3D) reconstruction. We then performed "euthanized" cystograms on all mice for comparison. C3H/HeJ mice had a strong and persistent microbubble signal in the renal pelvis and ureters bilaterally with low-contrast infusion volumes (<100 μl) and similarly showed bilateral reflux by euthanized cystograms. With larger infused volumes (≥150 μl), C57BL6/J mice had small volumes of microbubbles in the renal pelvis that cleared quickly and did not show reflux on euthanized cystograms. Thus, using animal models of known VUR, we demonstrate the utility of contrast-enhanced ultrasound to visualize reflux in live mice.     

Soft trapping and manipulation of cells using a disposable nanoliter biochamber

Low-power continuous-wave laser radiation is used to form a very stable microbubble at the end of a specially etched and metalized optical fiber probe. We demonstrate that the microbubble, which is firmly attached to the fiber probe, can be used to benignly trap and manipulate living swine sperm cells as well as human embryonic kidney cells. The lifetime of the microbubble has been prolonged and the gaseous environment inside the bubble controlled using micropipette gas injection. The controlled fusion of two microbubbles is demonstrated as a means of transferring microparticles from one bubble to another. These experiments lay the foundation for the use of the microbubble as a mobile, nanoliter-volume disposable biochamber for cellular studies.     

Ultrasound／Microbubble Enhances Foreign Gene Expression in ECV304 Cells and Murine Myocardium

The success of gene therapy is largely dependent onthe development of vectors or vehicles that can selectivelyand efficiently deliver a therapeutic gene to cells or targetissues with minimal toxicity. Viruses are efficient transducing vectors. However, the safety concerns regardingthe use of virus vector in human make nonviral deliverysystem an attractive focus. Nonviral vectors are particularly suitable with respect to the simplicity of use, possibility of large-scale production and lack of s…     

A mathematical model of tumor oxygen and glucose mass transport and metabolism with complex reaction kinetics

Hypoxia imparts radioresistance to tumors, and various approaches have been developed to enhance oxygenation, thereby improving radiosensitivity. This study explores the influence of kinetic and physical factors on substrate metabolism in a tumor model, based on a Krogh cylinder. In tissue, aerobic metabolism is assumed to depend on glucose and oxygen, represented by the product of Michaelis-Menten expressions. For the base case, an inlet pO(2) of 40 mmHg, a hypoxic limit of 5 mmHg, and a tissue/capillary radius ratio of 10 are used. For purely aerobic metabolism, a hypoxic fraction of 0.16 and volume-average pO(2) of 8 mmHg are calculated. Reducing the maximum oxygen rate constant by 9%, decreasing the tissue cylinder radius by 5%, or increasing the capillary radius by 8% abolishes the hypoxic fraction. When a glycolytic term is added, concentration profiles are similar to the base case. Using a distribution of tissue/capillary radius ratios increases the hypoxic fraction and reduces sensitivity to the oxygen consumption rate, compared to the case with a single tissue/capillary radius ratio. This model demonstrates that hypoxia is quite sensitive to metabolic rate and geometric factors. It also predicts quantitatively the effects of inhibited oxygen metabolism and enhanced mass transfer on tumor oxygenation.     

Improvement of tumor oxygenation by mild hyperthermia

There is now abundant evidence that oxygenation in rodent, canine and human tumors is improved during and for up to 1-2 days after heating at mild temperatures. An increase in tumor blood perfusion along with a decline in the oxygen consumption rate appears to account for the improvement of tumor oxygenation by mild hyperthermia. The magnitude of the increase in tumor pO(2), determined with oxygen-sensitive microelectrodes, caused by mild hyperthermia is less than that caused by carbogen breathing. However, mild hyperthermia is far more effective than carbogen breathing in increasing the radiation response of experimental tumors, probably because mild hyperthermia oxygenates both (diffusion-limited) chronically hypoxic and (perfusion-limited) acutely hypoxic cells, whereas carbogen breathing oxygenates only the chronically hypoxic cells. Mild hyperthermia is also more effective than nicotinamide, which is known to oxygenate acutely hypoxic cells, in enhancing the radiation response of experimental tumors. The combination of mild hyperthermia with carbogen or nicotinamide is highly effective in reducing the hypoxic cell fraction in tumors and increasing the radiation response of experimental tumors. A primary rationale for the use of hyperthermia in combination with radiotherapy has been that hyperthermia is equally cytotoxic toward fully oxygenated and hypoxic cells and that it directly sensitizes both fully oxygenated and hypoxic cells to radiation. Such cytotoxicity and such a radiosensitizing effect may be expected to be significant when the tumor temperature is elevated to at least 42-43 degrees C. Unfortunately, it is often impossible to uniformly raise the temperature of human tumors to this level using the hyperthermia devices currently available. However, it is relatively easy to raise the temperature of human tumors into the range of 39-42 degrees C, which is a temperature that can improve tumor oxygenation for up to 1-2 days. The potential usefulness of mild hyperthermia to enhance the response of human tumors to radiotherapy by improving tumor oxygenation merits continued investigation.     

The powerful microbubble: from bench to bedside, from intravascular indicator to therapeutic delivery system, and beyond

This review discusses the development, current applications, and therapeutic potential of ultrasound contrast agents. Microbubbles containing gases act as true, intravascular indicators, permitting a noninvasive, quantitative analysis of the spatial and temporal heterogeneity of blood flow and volumes within the microvasculature. These shelled microbubbles are near-perfect reflectors of acoustic ultrasound energy and, when injected intravenously into the bloodstream, reflect ultrasound waves within the capillaries without disrupting the local environment. Accordingly, microbubble ultrasound contrast agents are clinically useful in enhancing ultrasound images and improving the accuracy of diagnoses. More recently, ultrasound contrast agents have been used to directly visualize the vasa vasorum and neovascularization of atherosclerotic carotid artery plaques, thus suggesting a new paradigm for diagnosis and treatment of atherosclerosis. Future applications of these microscopic agents include the deliver of site-specific therapy to targeted organs in the body. Medical therapies may use these microbubbles as carriers for newer therapeutic options.     

载氢-纳米氧化铈微泡对小鼠造血系统抗辐射作用的分析

为探讨载氢-纳米氧化铈微泡对小鼠辐射损伤的防护作用。本研究检测载氢-纳米氧化铈微泡的表征,并将60只BALB/c小鼠随机分为正常对照组、照射对照组、载氢-纳米氧化铈微泡组。小鼠经6Gy x射线一次性全身照射(剂量率2 Gy/min)。于照射后3 d和8 d处死小鼠,检测其外周血细胞数、脾脏和胸腺指数、骨髓和脾脏组织病理学变化。结果显示,照射后3 d和8 d,与正常对照组相比,载氢-纳米氧化铈微泡组和照射对照组的白细胞均明显下降,相比照射对照组,载氢-纳米氧化铈微泡组有改善(p<0.05或p<0.01);而载氢-纳米氧化铈微泡组和照射对照组的红细胞数和血红蛋白均略有下降,但差异无统计学意义。与正常对照组相比,微泡组的胸腺指数、脾脏指数均有下降,和照射对照组相比,载氢-纳米氧化铈微泡组的胸腺指数明显改善(p<0.05或p<0.01)。照射后3 d,与正常对照组相比,照射对照组的骨髓细胞较少,存在细胞碎片,载氢-纳米氧化铈微泡组骨髓细胞数量略有减少,存在细胞核松散现象。而照射后8 d,与正常对照组相比,照射对照组的骨髓细胞几乎找不到,载氢-纳米氧化铈微泡组骨髓细胞有一定数量,存在细胞凋亡现象。本研究表明,载氢-纳米氧化铈微泡通过保护造血组织、改善造血功能,对机体起到一定的辐射防护作用。     

Differences in the strategies and potentials of human adaptation to hypoxia

The general patterns and individual specific features of human adaptation to acute hypoxic hypoxia caused by breathing a hypoxic oxygen-nitrogen gas mixture containing 8.0% oxygen have been studied. It was found that, at the initial stage of hypoxia, all examined subjects demonstrated a reduced oxygen consumption as compared to normoxia; then, this parameter increased and, beginning from a certain moment (after 5–15 min of exposure), exceeded the baseline level by 10–40%. Hypotheses explaining the mechanisms of this growth in oxygen consumption during hypoxia are considered. It has been found that the roles of the cardiovascular system and mechanisms of the tissue and cellular utilization of oxygen in the growth of the rate of oxygen consumption caused by hypoxia vary in different subjects. The hypothesis is put forward that the relatively low potential for rearrangement of the biological oxidation system at the cellular level, aimed at increasing the rate of oxygen consumption, predetermines a need to increase the rate of oxygen supply by the blood and, therefore, a greater strain of the cardiovascular system. In many cases, this strain can cause failure of adaptation to hypoxia. Other parameters that can serve as characteristics of a subject’s resistance to hypoxia, such as the intensity of EEG slow waves and the level of blood oxygenation, are also considered.     

Manufacture of Concentrated,Lipid-based Oxygen Microbubble Emulsions by High Shear Homogenization and Serial Concentration

Gas-filled microbubbles have been developed as ultrasound contrast and drug delivery agents. Microbubbles can be produced by processing surfactants using sonication, mechanical agitation, microfluidic devices, or homogenization. Recently, lipid-based oxygen microbubbles (LOMs) have been designed to deliver oxygen intravenously during medical emergencies, reversing life-threatening hypoxemia, and preventing subsequent organ injury, cardiac arrest, and death. We present methods for scaled-up production of highly oxygenated microbubbles using a closed-loop high-shear homogenizer. The process can produce 2 L of concentrated LOMs (90% by volume) in 90 min. Resulting bubbles have a mean diameter of ~2 μm, and a rheologic profile consistent with that of blood when diluted to 60 volume %. This technique produces LOMs in high capacity and with high oxygen purity, suggesting that this technique may be useful for translational research labs.     

Molecular imaging with targeted contrast ultrasound

Molecular imaging with contrast ultrasound relies on the detection of targeted microbubbles or other acoustically active nanoparticles. These microbubbles are retained in diseased tissue where they produce an acoustic signal because of their resonant properties in the ultrasound field. Targeting is accomplished either through manipulating the chemical properties of the microbubble shell or through conjugation of disease-specific ligands for the targeted molecule to the microbubble surface. As microbubbles cannot leave the intravascular space, the disease process must be characterized by molecular changes in the vascular compartment to be imaged. Inflammation, angiogenesis and thrombus formation are central pathophysiologic processes in many disease states and produce phenotypic changes in the vascular compartment. Thus, targeted contrast ultrasound in the future could aid in the diagnosis of such diverse diseases as atherosclerosis, transplant rejection and tumor-related angiogenesis.     

Microultrasound molecular imaging of vascular endothelial growth factor receptor 2 in a mouse model of tumor angiogenesis

High-frequency microultrasound imaging of tumor progression in mice enables noninvasive anatomic and functional imaging at excellent spatial and temporal resolution, although microultrasonography alone does not offer molecular scale data. In the current study, we investigated the use of microbubble ultrasound contrast agents bearing targeting ligands specific for molecular markers of tumor angiogenesis using high-frequency microultrasound imaging. A xenograft tumor model in the mouse was used to image vascular endothelial growth factor receptor 2 (VEGFR-2) expression with microbubbles conjugated to an anti-VEGFR-2 monoclonal antibody or an isotype control. Microultrasound imaging was accomplished at a center frequency of 40 MHz, which provided lateral and axial resolutions of 40 and 90 Im, respectively. The B-mode (two-dimensional mode) acoustic signal from microbubbles bound to the molecular target was determined by an ultrasound-based destruction-subtraction scheme. Quantification of the adherent microbubble fraction in nine tumor-bearing mice revealed significant retention of VEGFR-2-targeted microbubbles relative to control-targeted microbubbles. These data demonstrate that contrast-enhanced microultrasound imaging is a useful method for assessing molecular expression of tumor angiogenesis in mice at high resolution.     

The influence of environmental salinity on hemocyanin function in the blue crab, Callinectes sapidus.

The effects of inorganic ions and of the hydrogen ion on oxygen-binding properties of most respiratory pigments are opposite. The addition of salt to the medium increases oxygen affinities, and the addition of H+ decreases oxygen affinities of crustacean hemocyanins. These oxygenation properties, as observed in vitro, suggest that the oxygen-transport system must adapt to ionic changes in the blood. In fact, decreases in the salt concentration of the blood of estaurine blue crabs are accompanied by increases in pH, probably resulting from the input of ammonia produced in deamination of the intracellular pool of free amino acids as the cells conform to osmotic changes in body fluids. The result is a stability of hemocyanin function until the blood becomes very dilute. As the acclimation salinity is reduced from 35 to 15 o/ooo, the ionic effects on respiratory transport are balanced and there is no change in total oxygen uptake. At 5 o/ooo salinity, however, the higher blood pH is manifested in an elevation of the total oxygen concentration of prebranchial blood, probably because the Bohr shift is no longer opposed by a critical level of salt in the blood. Under these conditions the role of hemocyanin in aerobic respiration is reduced at high environmental oxygen levels, but it may be enhanced in hypoxic uaters.     

Cerebral hypoperfusion during hypoxic exercise following two different hypoxic exposures: independence from changes in dynamic autoregulation and reactivity

We examined the effects of exposure to 10-12 days intermittent hypercapnia [IHC: 5:5-min hypercapnia (inspired fraction of CO(2) 0.05)-to-normoxia for 90 min (n = 10)], intermittent hypoxia [IH: 5:5-min hypoxia-to-normoxia for 90 min (n = 11)] or 12 days of continuous hypoxia [CH: 1,560 m (n = 7)], or both IH followed by CH on cardiorespiratory and cerebrovascular function during steady-state cycling exercise with and without hypoxia (inspired fraction of oxygen, 0.14). Cerebrovascular reactivity to CO(2) was also monitored. During all procedures, ventilation, end-tidal gases, blood pressure, muscle and cerebral oxygenation (near-infrared spectroscopy), and middle cerebral artery blood flow velocity (MCAv) were measured continuously. Dynamic cerebral autoregulation (CA) was assessed using transfer-function analysis. Hypoxic exercise resulted in increases in ventilation, hypocapnia, heart rate, and cardiac output when compared with normoxic exercise (P < 0.05); these responses were unchanged following IHC but were elevated following the IH and CH exposure (P < 0.05) with no between-intervention differences. Following IH and/or CH exposure, the greater hypocapnia during hypoxic exercise provoked a decrease in MCAv (P < 0.05 vs. preexposure) that was related to lowered cerebral oxygenation (r = 0.54; P < 0.05). Following any intervention, during hypoxic exercise, the apparent impairment in CA, reflected in lowered low-frequency phase between MCAv and BP, and MCAv-CO(2) reactivity, were unaltered. Conversely, during hypoxic exercise following both IH and/or CH, there was less of a decrease in muscle oxygenation (P < 0.05 vs. preexposure). Thus IH or CH induces some adaptation at the muscle level and lowers MCAv and cerebral oxygenation during hypoxic exercise, potentially mediated by the greater hypocapnia, rather than a compromise in CA or MCAv reactivity.     

Effects of L-carnitine L-tartrate supplementation on muscle oxygenation responses to resistance exercise

Previous research has shown that L-carnitine L-tartrate (LCLT) supplementation beneficially affects markers of hypoxic stress following resistance exercise. However, the mechanism of this response is unclear. Therefore, the primary purpose of this study was to determine the effects of LCLT supplementation on muscle tissue oxygenation during and after multiple sets of squat exercise. Nine healthy, previously resistance-trained men (25.2 +/- 6.years, 91.2 +/- 10.2 kg, 180.2 +/- 6.3 cm) ingested 2 g.d of LCLT or an identical placebo for 23 days in a randomized, balanced, crossover, double-blind, placebo-controlled, repeated-measures study design. On day 21, forearm muscle oxygenation was measured during and after an upper arm occlusion protocol using near infrared spectroscopy (NIRS), which measures the balance of oxygen delivery in relation to oxygen consumption. On day 22, subjects performed 5 sets of 15 to 20 repetitions of squat exercise with corresponding measures of thigh muscle oxygenation, via NIRS, and serial blood draws. Compared to the placebo trial, muscle oxygenation was reduced in the LCLT trial during upper arm occlusion and following each set of resistance exercise. Despite reduced oxygenation, plasma malondealdehyde, a marker of membrane damage, was attenuated during the LCLT trial. There were no differences between trials in the vasoactive substance prostacyclin. In conclusion, because oxygen delivery was occluded during the forearm protocol, it is proposed that enhanced oxygen consumption mediated the reduced muscle oxygenation during the LCLT trial. Enhanced oxygen consumption would explain why hypoxic stress was attenuated with LCLT supplementation.     

Expression of endostatin mediated by a novel non-viral delivery system inhibits human umbilical vein endothelial cells in vitro

Ultrasound (US)-mediated microbubble destruction is recognized to have considerable potential for gene delivery, whereas, there is few report of its effect on enhancing liposomal transfection. In this study, we used pIRES2-EGFP/hES containing human endostatin (hES) cDNA as target gene to test the hypothesis that US exposure with microbubbles could improve liposomal transfection, and to investigate the possibility of intracellular delivery of ES gene using this method. Under the controlled US exposure condition with microbubbles, the plasmid:liposome was transferred into COS-7 cells. The transfection rate, the expression of endostatin and the inhibition effect of transfection-endostatin on endothelial cells were assessed. The results revealed that US-mediated microbubble destruction together with liposome could significantly enhance gene transfection without obvious cell damage. By this means, endostatin gene could be efficiently transferred into COS-7 cells and expressed. The transfection-endostatin could inhibit endothelial proliferation and migration, which suggests that the non-viral method might be useful in anti-angiogenesis therapy in the future.     

Re-oxygenation causes hypoxic tumor regression through restoration of p53 wild-type conformation and post-translational modifications

Hypoxic tumors are resistant to conventional therapies through indirect mechanisms such as the selection of resistant phenotype under chronic hypoxia. Hyperbaric oxygen (HBO) therapy has been shown to increase oxygen level and induce apoptosis in hypoxic tumor. However, it could produce significant adverse effects including oxygen toxic seizures and severe radiation tissue injury due to high pressure. We have shown that repeated oxygenation at 30% O₂ (1 atmospheres absolute) results in significant regression of MCF-7 tumor xenografts without any adverse effect. In MCF-7 cells, re-oxygenation showed an eightfold increase in cellular apoptosis. Both in hypoxic tumor and in hypoxic cells, that exclusively favor p53 to exist in mutant conformation, re-oxygenation restores p53 wild-type conformation. The oxygen-mediated rescue of mutant p53 followed by its trans-activation is responsible for the induction of p53-downstream apoptotic, cell-cycle arrest and DNA-repair genes. Further, p53 trans-activation may thus be due to its post-translational modifications as a result of re-oxygenation. We have thus concluded that oxygen therapy without pressure, as opposed to HBO therapy, may be ideal for hypoxic tumor regression, which functions through oxygen-mediated rescue of mutant p53 followed by induction of apoptosis.     

The range of oxygenation in SiHa tumor xenografts

Tumor cells at very low oxygen tensions are known to be about three times more resistant to killing by ionizing radiation. Since cells at intermediate oxygen tensions (defined here as greater than 0.1% and less than 2% O(2)) show partial radioresistance, they should be a consideration in tumor treatment. In an effort to estimate the extent and range of oxygenation in SiHa human cervical carcinoma xenografts, patterns of cell killing and DNA damage by radiation and two bioreductive drugs, PD-144872 and RSU-1069, were compared to those seen in SiHa cells grown as spheroids. These drugs produce DNA interstrand crosslinks that are largely responsible for cell killing, and the degree of crosslinking increases as the oxygenation is reduced. About 60% of the cells in SiHa xenografts exhibited drug-induced crosslinks, but only about 35% showed extensive crosslinking indicative of hypoxia below 0.1% oxygen. Patterns of toxicity and DNA damage in xenografts were comparable to those of spheroids equilibrated with about 2% oxygen, indicating that most cells in the xenografts exhibit some radioresistance due to lack of oxygen. Similarly, pimonidazole binding indicated that about 60% of the cells in SiHa xenografts were either intermediate in oxygenation or hypoxic, but only about half of those were consistent with extreme oxygen depletion. The apparent size of the population of "intermediately hypoxic" cells has implications for the use of ionizing radiation, hypoxic cell cytotoxins, and other antitumor agents whose cytotoxicity is dependent on cellular oxygen content.     

Ultrasound-targeted microbubble enhances migration and therapeutic efficacy of marrow mesenchymal stem cell on rat middle cerebral artery occlusion stroke model

To investigate the role of ultrasound-targeted microbubbles in the homing effect of bone marrow-derived mesenchymal stem cells (BMSCs) and in the therapeutic efficacy of BMSCs on the ischemic stroke. A middle cerebral artery occlusion (MCAO) model was induced by plug wire preparation. Seventy-two hours after MCAO, the treatment of BMSCs with ultrasound-targeted microbubble was assessed via modified neurological severity score (mNSS), infarct volumes, and cerebral edema. In addition, immunofluorescence was performed to analyze the homing effect of BMSCs with ultrasound-targeted microbubble. We find that BMSCs with ultrasound-targeted microbubble (BMMSCs with ultrasound-targeted microbubble [USMM] group) could significantly ameliorate mNSS, infarct volumes, and cerebral edema of MCAO compared with phosphate buffer saline group, BMSCs alone group (BMSC group), and BMSCs with Ultrasound group (Ultrasound group). Immunofluorescence analysis demonstrated that ultrasound-targeted microbubbles promoted the accumulation of BMSCs in rat MCAO brains. Our findings demonstrated that ultrasound-targeted microbubble could be an effective approach for the accumulation of BMSCs on ischemic stroke, and further improved the therapeutic efficacy of BMSCs on MCAO.     

Inhibitory effect of 5-FU loaded ultrasound microbubbles on tumor growth and angiogenesis

The anti-neovascularization treatment is one of the effective strategies for tumor molecular target therapy. At present, the target and effect of the anti-neovascularization treatment is limited, and it is urgent to establish a new vascular targeting strategy to effectively treat tumors. In this work, we used high intensity focused ultrasound (HIFU) combined with targeted microbubbles to establish a molecular targeted ultrasound response microbubble for neovascular cells. Furthermore, the effects of drug loaded microbubbles on neovascularization and tumor cells were studied. The tumor vascular targeted and ultrasound-responsive microbubbles of 5-FU@DLL4-MBs were prepared by the thin-film dispersion method. The size and zeta potential of 5-FU@DLL4-MBs was about 1248 nm and −9.1 mV. 5-FU@DLL4-MBs released 5-FU showed an ultrasound-responsive manner, and had better vascular-targeting ability. Furthermore, the 5-FU@DLL4-MBs showed the strongest cytotoxic effect on HUVECs or HepG-2 cells and can be effectively internalized into the HUVECs cells. Thus, 5-FU@DLL4-MBs combined with HIFU can be considered as a potential method for antitumor angiogenesis in the future.