Effects of ultrasound on placental transfer during the last third of gestation in the rat

The effect of ultrasound on placental transfer in the rat has been measured in vivo. At 15, 18, 21 and 22 days of gestation one uterine horn of each anaesthetized rat was exposed, after laparatomy, to ultrasound. The control horn was effectively shielded. Radio-labelled tracers were then injected into the maternal circulation via a jugular vein cannula and fetuses removed simultaneously from both experimental and control horns at times predetermined to ensure minimal backflux from fetus to mother. Samples of maternal plasma were also taken at suitable intervals. The radioactive content of fetuses and plasma were measured and a unidirectional materno-fetal clearance value (Kmf) for each fetus calculated. It was shown that in rats whose body temperature was accurately maintained at 37 degrees C, ultrasound at the level used in the present study had no effect on placental clearance. However, in rats whose body temperature was allowed to fall to 32 degrees C, a 3 min exposure to 1.1 MHz continuous wave ultrasound at 1 W cm-2 caused a significant (p less than 0.001) decrease in the placental clearance of 22Na (by 55%) and 51Cr-EDTA (by 45%) in rats at 15 days of gestation. These effects showed no dose response and were not observed in rats at any of the other stages of gestation which we investigated. However, in rats at 21 days of gestation the clearance of 45Ca2+ was significantly (p less than 0.01) reduced (by 30%) by a similar exposure to ultrasound. It is concluded that although an interesting biological effect of ultrasound at low body temperatures has been observed, it has little relevance for the normal use of ultrasound in obstetrics.     

Ultrasonic stimulation of the brain to enhance the release of dopamine – A potential novel treatment for Parkinson’s disease

Parkinson’s disease (PD) is characterized by the decrease of dopamine (DA) production and release in the substantia nigra and striatum regions of the brain. Transcranial ultrasound has been exploited recently for neuromodulation of the brain in a number of fields. We have stimulated DA release in PC12 cells using low-intensity continuous ultrasound (0.1 W/cm² − 0.3 W/cm², 1 MHz), 12 h after exposure at 0.2 W/cm², 40 s, the amount of DA content eventually increased 78.5% (p = 0.004). After 10-day ultrasonic treatment (0.3 W/cm², 5 min/d), the DA content in the striatum of PD mice model restored to 81.07% of the control (vs 43.42% in the untreated PD mice model). In addition to this the locomotion activity was restored to the normal level after treatment. We suggest that the low intensity ultrasound-induced DA release can be attributed to a combination of neuron regeneration and improved membrane permeability produced by the mechanical force of ultrasound. Our study indicates that the application of transcranial ultrasound applied below FDA limits, could provide a candidate for relatively safe and noninvasive PD therapy through an amplification of DA levels and the stimulation of dopaminergic neuron regeneration without contrast agents.     

Calcium permeation property and firmness change of cherry tomatoes under ultrasound combined with calcium lactate treatment

This study aimed to investigate the effect of ultrasound combined with calcium lactate (2%, w/v) treatment (U + Ca) on calcium permeation and firmness of cherry tomatoes. Calcium distribution and fruit pectin nanostructure were also analysed by transmission electron microscope (TEM) and atomic force microscopy (AFM), respectively. The firmness (31.45 N) was maintained when ultrasound energy density was 20 W/L for 15 min at 15 °C. The Ca content increased in U + Ca treated fruit. Meanwhile, the Peleg’s model could be used to express the change of solid gain in cherry tomatoes under ultrasound treatment at 15, 20, and 25 °C. According to the AFM results, the width (≥40 nm) and length (≥2 μm) of chelate-soluble pectin (CSP) and sodium carbonate-soluble pectin (SSP) chains with large frequency was observed in U + Ca treated fruit. Under desirable conditions (15 °C, 15 min, 20 W/L), ultrasound combined with calcium lactate could maintain the quality of cherry tomatoes.     

Postpartum survival of mice exposed in utero to ultrasound

We found no credible enhancement of neonatal mortality in mice exposed in utero on Day 8 of gestation to ultrasound at 0.44 W/cm2 (+/- 20%) spatial average intensity, 2 MHz, continuous wave, for 60--180 s in a 37 degrees C water bath. This result is compared with the adverse effect on neonatal survival reported by Curto [in Ultrasound in Medicine (Plenum, New York, 1976), Vol. 2, pp. 535--536] for exposure on Day 14 of gestation at about 0.125 to 0.5 W/cm2, 1MHz, continuous wave, for 180 s in a 30 degrees C bath. It is concluded that a temporary reduction in fetal viability, lasting from 4 days to less than 10 days after exposure, could account for both sets of data. It is also evident from these data that improved methods of determining intensities within mammalian tissues are highly desirable.     

Biological effects of combined ultrasound and cisplatin treatment on ovarian carcinoma cells

The effects of low-power ultrasound, the anti-cancer drug cisplatin, and their combined application were studied in two lines of human ovarian carcinoma cells, A2780 and A2780cis. Four modes of treatment were used: exposure to ultrasonic field, application of cisplatin, exposure to ultrasound followed by cisplatin, and presence of cisplatin followed by exposure to application ultrasound. Ultrasound was used at intensities of 0.5 W/cm² and 1.0 W/cm² for 10 min, cisplatin was applied at concentrations of 1 μM and 6 μM per cell suspension treated in A2780 and cisplatin-resistant A2780cis cells, respectively. The results of each experimental treatment were assessed by the resultant cell viability related to the viability of control cells, using a standard MTT test. It was shown that a combined effect of ultrasound and cisplatin was more effective than that of ultrasound or cisplatin alone. It also appeared that the order of application played a role, with the cisplatin-ultrasound treatment lowering cell viability more than the ultrasound-cisplatin treatment. It can be assumed that the exposure of cells to a low-power ultrasonic field has an immediate effect on the structure of cell surfaces and, consequently, on entry of cisplatin into the cell.The study also included observations on changes in the cell cycle associated with the treatments used in both cell lines and their evaluation by flow cytometry.     

Effects of ultrasound on apoptosis induced by anti-CD20 antibody in CD20-positive B lymphoma cells

AIM: The present study was conducted to examine the thermal and non-thermal effects of ultrasound on apoptosis induced by anti-CD20 monoclonal antibody (rituximab). MATERIALS AND METHODS: SU-DHL-4 cells, a CD20-positive cell line derived from B cell lymphomas with a BCL2 gene rearrangement, were exposed to continuous 1 MHz ultrasound for therapeutic use under an air- or CO(2)-saturated condition to control cavitation. Early apoptosis (EA) and secondary necrosis (SN) were examined by flow cytometry. Cavitation was determined by detecting the hydroxyl radicals derived from pyrolysis of water molecules using electron paramagnetic resonance-spin trapping. To assess thermal effects, cells were treated in a temperature-controlled water bath. RESULTS: There was a significant additive increase in EA and EA+SN observed in cells treated with rituximab combined with heat at 42 degrees C or non-thermal ultrasound at 0.5 W/cm(2) under an air-saturated condition, where heat or ultrasound induced some cell death. A significant synergistic increase in EA and EA+SN was observed in cells treated with rituximab and ultrasound at 2.5 W/cm(2) under CO(2)-saturated conditions, where inertial cavitations were completely suppressed. No enhancement was observed at a temperature less than 40 degrees C or ultrasound at 0.5 W/cm(2) under CO(2)-saturated conditions. CONCLUSION: These results suggest that the immuno-therapeutic application of ultrasound at relatively high-intensities combined with rituximab thus produces synergistic effects under conditions where the non-thermal and non-cavitational effects are predominant.     

Intrapulpar temperature during continuous CO2 laser irradiation in human molars: an in vitro study

To establish safety parameters, we in vitro studied the increase in intrapulpal temperature caused by the use of a cw CO2 laser. A thermistor was implanted in the inner part of the pulpal chamber of 25 human lower third molars to measure the intrapulpal temperature produced by laser powers between 2-10 W and exposure times of 0.5-25.0 s. The Pearson linear correlation factor applied to the measured values showed there is a direct relationship between the independent variable and the applied power. A variance analysis produced the linear regression equation: T = 1.10 + (0.127)E where T is the temperature and E the energy. The results showed that, with a power of 4 W and maximum exposure time of 2:5 s (10 J) and a power density of 12,738.85 W cm-2, there will be no damaging reactions affecting the pulpal tissues.     

Lack of effect of continuous wave ultrasound exposure on in vivo Chinese hamster cheek pouch epithelial mitotic index

Chinese hamster cheek pouches were everted and the epithelia exposed in vivo for 1 min to continuous wave 1.07 MHz ultrasound at intensities ranging from 0.5 to 10.0 W cm-2. Since the mitotic index of the hamster epithelium exhibited a circadian rhythm, the times of sonication and tissue sampling were arranged to allow for detection of a division delay as evidenced by a change in mitotic index in cells insonated in the early S or late S/early G2 phases of the cell cycle. There was no demonstrated effect of the ultrasound exposures on mitotic index.     

红外激光对可见光CCD成像系统的干扰

采用连续波红外激光对可见光面阵CCD成像系统进行了干扰实验,观察到饱和串扰、全屏饱和等干扰现象,并测到串扰阈值小于2.0×102W/cm2,全屏饱和阈值为1.2×104W/cm2。通过分析CCD输出电压、视频信号,提出基于有效干扰面积的干扰效果评估方法,并利用该方法对干扰面积与激光功率密度、辐照时间之间的关系进行了半定量分析。结果显示:激光功率密度对干扰效果的影响较大,而辐照时间在0.5~2.0 s之间时,辐照时间对CCD干扰效果的影响不明显。     

Increased accumulation of paclitaxel and doxorubicin in proliferating capillary cells and prostate cancer cells following ultrasound exposure

Introduction

We have previously reported enhanced cytotoxic effects of both doxorubicin and antisense oligonucleotides using an optimized ultrasound regime of a single 10 s exposure in burst-mode (4 MHz, 32 W/cm²(SaTa), 50 ms burst period) in both PC3 (prostate cancer) cells and angiogenic Huvec (human umbilical cord endothelial cells). The objective of this study was to investigate the effect of ultrasound on the cellular uptake of both hydrophilic agents (rhodamine R123, doxorubicin hydrochloride and mannitol) and hydrophobic agents (rhodamine R6G and paclitaxel) using the same 4 MHz ultrasound exposure system.

Methods

PC3 cells and Huvec were incubated with solutions of radioactive or fluorescent compounds for 1 h and ultrasound was then applied to cells. Following washing and lysis of cells, the degree of drug uptake was measured using liquid scintillation counting or fluorescence spectroscopy.

Results

Ultrasound exposure resulted in the enhanced uptake of both hydrophilic and hydrophobic compounds into cells. For paclitaxel, approximately 100% increased uptake was observed when the drug was encapsulated in a nanoparticulate micellar formulation compared to approximately 50% for free drug.

Conclusions

The 4 MHz, 32 W/cm² ultrasound exposure regime (using burst-mode with 50 ms burst period) allows for the enhanced uptake of both water soluble and insoluble compounds into proliferating cancer and angiogenic cells.     

Effect of exposure parameters on cavitation induced by low-level dual-frequency ultrasound

In order to quantify the effects of exposure parameters under therapeutic conditions such as sonodynamic therapy, it is necessary initially to evaluate the inertial cavitation activity in vitro. In this study, the dependence of cavitation activity induced by the low-level dual-frequency ultrasound irradiation on exposure parameters has been studied. Experiments were performed in the near 150 kHz and 1 MHz fields in the progressive wave mode. It has been shown that at constant ultrasound energy the fluorescence intensity for continuous sonication is higher than for pulsed mode. With increasing the duty cycle of pulsed field, the inertial cavitation activity is increased. The activity of cavitation produced by simultaneous combined sonication by two ultrasound fields is remarkably higher than the algebraic sum of effects produced by fields separately (p-value < 0.05). This study shows that simultaneous combined dual-frequency ultrasound sonication in continuous mode is more effective in producing inertial cavitation activity at low-level intensity. Therefore, it is concluded that investigations in this combined ultrasound sonication can be useful in sonodynamic therapy for superficial tumors.     

连续波HF激光泵浦Fe2+: ZnSe激光器的可行性

针对目前Fe2+: ZnSe激光器缺乏有效的高功率泵浦源这一关键瓶颈,提出了采用连续波HF化学激光器泵浦Fe2+: ZnSe来实现4 μm波段激光输出的技术路线,结合实验和理论手段考察了此技术路线的可行性。首次获得了由连续波HF化学激光泵浦的Fe2+: ZnSe激光器的W级激光输出,输出功率约为1.7 W,谱线峰值波长为4.18 μm,出光时间约2 s。     

Instant magnetic labeling of tumor cells by ultrasound in vitro

Magnetic labeling of living cells creates opportunities for numerous biomedical applications. Here we describe an instantly cell magnetic labeling method based on ultrasound. We present a detailed study on the ultrasound performance of a simple and efficient labeling protocol for H-22 cells in vitro. High frequency focus ultrasound was investigated as an alternative method to achieve instant cell labeling with the magnetic particles without the need for adjunct agents or initiating cell cultures. Mean diameter of 168 nm dextran-T40 coated superparamagnetic iron oxide (SPIO) nanoparticles were prepared by means of classical coprecipitation in solution in our laboratory. H-22 tumor cells suspended in phosphate-buffered saline (PBS, pH=7.2) were exposed to ultrasound at 1.37 MHz for up to 120 s in the presence of SPIOs. The cellular uptake of iron oxide nanoparticles was detected by prussion blue staining. The viability of cells was determined by a trypan blue exclusion test. At 2 W power and 60 s ultrasound exposure in presence of 410 μg/ml SPIOs, H-22 cell labeling efficiency reached 69.4±6.3% and the labeled cells exhibited an iron content of 10.38±2.43 pg per cell. Furthermore, 95.2±3.2% cells remained viable. The results indicated that the ultrasound protocol could be potentially applied to label cells with large-sized magnetic particles. We also calculated the shear stress at the 2 W power and 1.37 MHz used in experiments. The results showed that the shear stress threshold for ultrasonically induced H-22 cell reparable sonoporation was 697 Pa. These findings provide a quantitative guidance in designing ultrasound protocols for cell labeling.     

Impact of thermal effects induced by ultrasound on viability of rat C6 glioma cells

In order to have consistent and repeatable effects of sonodynamic therapy (SDT) on various cancer cells or tissue lesions we should be able to control a delivered ultrasound energy and thermal effects induced. The objective of this study was to investigate viability of rat C6 glioma cells in vitro depending on the intensity of ultrasound in the region of cells and to determine the exposure time inducing temperature rise above 43 °C, which is known to be toxic for cells. For measurements a planar piezoelectric transducer with a diameter of 20 mm and a resonance frequency of 1.06 MHz was used. The transducer generated tone bursts with 94 μs duration, 0.4 duty-cycle and initial intensity I_SATA (spatial averaged, temporal averaged) varied from 0.33 W/cm² to 8 W/cm² (average acoustic power varied from 1 W to 24 W). The rat C6 glioma cells were cultured on a bottom of wells in 12-well plates, incubated for 24 h and then exposed to ultrasound with measured acoustic properties, inducing or causing no thermal effects leading to cell death. Cell viability rate was determined by MTT assay (a standard colorimetric assay for assessing cell viability) as the ratio of the optical densities of the group treated by ultrasound to the control group. Structural cellular changes and apoptosis estimation were observed under a microscope. Quantitative analysis of the obtained results allowed to determine the maximal exposure time that does not lead to the thermal effects above 43 °C in the region of cells for each initial intensity of the tone bursts used as well as the threshold intensity causing cell death after 3 min exposure to ultrasound due to thermal effects. The averaged threshold intensity was found to be about 5.7 W/cm².     

Early iNOS impairment and late eNOS enhancement during reperfusion following 2.49 MHz continuous ultrasound exposure after ischemia

ObjectiveIschemia reperfusion (IR) injury, occurring during heart attacks, hemorrhagic shock, and bypass and transplant surgeries, impairs microcirculatory function and nitric oxide (NO) synthesis. We report the regulation of endothelial and inducible NO synthase (eNOS and iNOS) proteins as a consequence of the application of continuous mode diagnostic frequency ultrasound application following IR injury.MethodsAnimals were assigned to one of five groups for microcirculatory assessment or Western blot analysis (WB) as follows: (1) IR+iNOS inhibition (1400W); and (2) IR+1400W+ultrasound for microcirculatory assessment, (3) Control; (4) IR; and (5) IR+ultrasound for WB. Functional capillary density and microvascular diameter, flow velocity, and flow were monitored for microcirculatory assessment. Skin tissue samples were harvested for WB. 2.49 MHz continuous ultrasound was used for application.ResultsBoth the inhibition of iNOS alone and iNOS inhibition with ultrasound irradiation positively influenced the microcirculation of observed animals relative to baseline values. Ultrasound exposure resulted in a significant production of eNOS protein in skin tissue harvested 24 h into reperfusion (p < 0.01). iNOS levels from the same tissue of ultrasound exposed animals were found to be significantly decreased 0.5 h into reperfusion (p < 0.05).ConclusionProtection from lasting IR injury effects in the microcirculation, with continuous mode diagnostic frequency ultrasound, results from augmented eNOS protein levels during late reperfusion. Ultrasound inhibited iNOS protein production during early reperfusion may also confer protection from IR injury.     

Preparation and in vitro evaluation of an ultrasound-triggered drug delivery system: 10-hydroxycamptothecin loaded PLA microbubbles

10-Hydroxycamptothecin (HCPT) loaded PLA microbubbles, used as an ultrasound-triggered drug delivery system, were fabricated by a double emulsion-solvent evaporation method. The obtained microbubbles were characterized by scanning electron microscope (SEM), transmission electron microscope (TEM), X-ray diffraction (XRD), differential scanning calorimetry (DSC) and confocal laser scanning microscope (CLSM). In addition, the effect of diagnostic ultrasound exposure on BEL-7402 cells combined with HCPT-loaded PLA microbubbles was evaluated using cytotoxicity assay, CLSM and flow cytometry (FCM). It was found that the HCPT-loaded PLA microbubbles showed smooth surface and spherical shape, and the drug was amorphously dispersed within the shell and the drug loading content reached up to 1.69%. Nearly 20% of HCPT was released upon exposure to diagnostic ultrasound at frequency of 3.5 MHz for 10 min. Moreover, HCPT fluorescence in the cells treated only with the HCPT-loaded PLA microbubbles was discernible, but less intense, while those treated with the microbubbles in conjunction with ultrasound exposure was evident and intense, indicating an increased cellular uptake of HCPT by ultrasound exposure. Cytotoxicity test on BEL-7402 cells indicated that the HCPT-loaded PLA microbubbles combined with ultrasound exposure were more cytotoxic than the microbubbles alone. The results suggest that the combination of drug loaded PLA microbubbles and diagnostic ultrasound exposure exhibit an effective intracellular drug uptake by tumor cells, indicating their great potential for antitumor therapy.     

Sonodynamically induced antitumor effect of hematoporphyrin on Hepatoma 22

The ultrasonically induced cytotoxic effects of hematoporphyrin (Hp) on Hepatoma 22 (H22) cells in vitro and vivo were investigated. Tumor cells were suspended in saline and exposed to ultrasound at 1.43 MHz for up to 60s in the presence and absence of Hp. The viability of cells was evaluated by trypan blue exclusion test. The ultra-structure changes of H22 cells induced by ultrasonic irradiation were evaluated by scanning electron microscope (SEM) and transmission electron microscope (TEM). Lipid peroxidation in cell was estimated by the thiobarbicturic acid (TBA) method. Our experiments indicated that the ultrasonic intensity of 2 W/cm(2), the Hp concentration of 100 microg/ml and the ultrasound exposure time of 60s were the best conditions for sonodynamic treatment in vitro. The tumor volume and weight after the combination of Hp with ultrasound were remarkably inhibited. SEM and TEM observation found the cell ultra-structure was significantly damaged, and lipid peroxidation level remarkably increased after sonodynamic treatment. This study suggested the ultra-structural changes may play a key role in cell destruction induced by sonodynamic treatment and the biological mechanism might be involved in mediating the killing effect on H22 cells in our experiment mode.     

Modulation of luciferase activity using high intensity focused ultrasound in combination with bioluminescence imaging, magnetic resonance imaging and histological analysis in muscle tissue

This study investigates the effect of high intensity focused ultrasound (HIFU) to muscle tissue transfected with a luciferase reporter gene under the control of a CMV-promoter. HIFU was applied to the transfected muscle tissue using a dual HIFU system. In a first group four different intensities (802 W/cm², 1401 W/cm², 2117 W/cm², 3067 W/cm²) of continuous HIFU were applied 20 s every other week for four times. In a second group two different intensities (802 W/cm², 1401 W/cm²) were applied 20 s every fourth day for 20 times. The luciferase activity was determined by bioluminescence imaging. The effect of HIFU to the muscle tissue was assessed by T1-weighted ± Gd-DTPA, T2-weighted and a diffusion-weighted STEAM sequence obtained on a 1.5-T GE-MRI scanner. Histology of the treated tissue was done at the end. In the first group the photon emission was at 3067.6 W/cm² 1.28 × 10⁷ ± 3.1 × 10⁶ photon/s (5.5 ± 1.2-fold), of 2157.9 W/cm² 8.1 ± 2.7 × 10⁶ photon/s (3.2 ± 1.1-fold), of 1401.9 W/cm² 9.3 ± 1.3 × 10⁶ photon/s (4.9 ± 0.4-fold) and of 802.0 W/cm² 8.6x ± 1.2 × 10⁶ photon/s (4.5 ± 0.6-fold) compared to baseline. In the second group the photon emission was at 1401.9 W/cm² and 802.0 W/cm² 14.1 ± 3.6 × 10⁶ photon/s (6.1 ± 1.5-fold), respectively, 5.1 ± 4.7 × 10⁶ photon/s (6.5 ± 2.0-fold). HIFU can enhance the luciferase activity controlled by a CMV-promoter.     

In vitro ultrasound-mediated leakage from phospholipid vesicles

Interest in using ultrasound energy in wound management and intracellular drug delivery has been growing rapidly. Development and treatment optimization of such non-diagnostic applications requires a fundamental understanding of interactions between the acoustic wave and phospholipid membranes, be they cell membranes or liposome bilayers. This work investigates the changes in membrane permeation (leakage mimicking drug release) in vitro during exposure to ultrasound applied in two frequency ranges: “conventional” (1 MHz and 1.6 MHz) therapeutic ultrasound range and low (20 kHz) frequency range. Phospholipids vesicles were used as controllable biological membrane models. The membrane properties were modified by changes in vesicle dimensions and incorporation of poly(ethylene glycol) i.e. PEGylated lipids. Egg phosphatidylcholine vesicles with 5 mol% PEG were prepared with sizes ranging from 100 nm to 1 μm. Leakage was quantified in terms of temporal fluorescence intensity changes observed during carefully controlled ultrasound ON/OFF time intervals. Custom-built transducers operating at frequencies of 1.6 MHz (focused) and 1.0 MHz (unfocused) were used, the I_spta of which were 46.9 W/cm² and 3.0 W/cm², respectively. A commercial 20 kHz, point-source, continuous wave transducer with an I_spta of 0.13 W/cm² was also used for comparative purposes. Whereas complete leakage was obtained for all vesicle sizes at 20 kHz, no leakage was observed for vesicles smaller than 100 nm in diameter at 1.6 or 1.0 MHz. However, introducing leakage at the higher frequencies became feasible when larger (greater than 300 nm) vesicles were used, and the extent of leakage correlated well with vesicle sizes between 100 nm and 1 μm. This observation suggests that physico-chemical membrane properties play a crucial role in ultrasound mediated membrane permeation and that low frequency (tens of kilohertz) ultrasound exposure is more effective in introducing permeability change than the “conventional” (1 MHz) therapeutic one. The experimental data also indicate that the leakage level is controlled by the exposure time. The results of this work might be helpful to optimize acoustic field and membrane parameters for gene or drug delivery. The outcome of this work might also be useful in wound management.     

H-22细胞声孔效应的应力阈值

理论和实验研究了超声空化场中的H-22型肝癌细胞产生可逆声孔效应的剪应力阈值.本文用1.37 MHz的聚焦声场,当超顺磁性纳米氧化铁在细胞悬液中的终浓度为410 μg/mL,换能器负载电功率为2 W,超声辐照60 s,细胞存活率90%以上时,有45.9±13.5%的细胞显示普鲁士蓝染阳性,暗示超声作用下,这些细胞表面曾出现可逆性微孔而使磁性微粒由此进入细胞内.利用无界自由空间微泡运动方程的球对称稳态解对实验条件下细胞膜表面的切变应力进行数值估算,结果表明,使H-22细胞产生可逆性声孔效应的微流剪应力阈值为697 Pa. 关键词： 声孔效应 磁性标记 微流 剪应力