Survival of amphibian embryos after continuous ultrasound treatment

The influence of continuous ultrasound on the embryonic development of grass frog Rana temporaria has been investigated. Intact embryos at the blastula stage were treated by ultrasound of different frequency (0.88 and 2.64 MHz), intensity (0.05-1.0 W/cm2), and duration (1-15 min). The treatment with ultrasound of frequency 0.88 MHz and intensity 0.05 W/cm2 for 1-5 min tended to increase the proportion of normally developing embryos up to hatch (10-25% of control). Increasing the intensity of ultrasound (0.88 MHz) to 0.7-1.0 W/cm2 and the duration of its action to 5-15 min induced the death of almost all of treated embryos. No significant differences were found between the development of control embryos and embryos treated with ultrasound of middle intensity (0.2-0.7 W/cm2) for 1-5 min. The exposure of amphibian embryos to ultrasound of frequency 2.64 MHz and intensity 0.05-0.7 W/cm2 for 1-5 min did not change their survival. Increasing the intensity of ultrasound (2.64 MHz) to 1.0 W/cm2 and the duration of its action to 5 min decreased the number of normal developing embryos (by 35%).     

Production of thymine base damage in ultrasound-exposed EMT6 mouse mammary sarcoma cells

Mouse mammary sarcoma cells, line EMT6/Ro, were exposed for 1 min to 1 MHz continuous wave ultrasound over a range of intensities from 0.5 to 30 W/cm2 (spatial peak). The presence of thymine base damage (TBD) products of the 5,6-dihydroxydihydrothymine type was determined by an alkali degradation assay. Production of damage was found to be greatest (approximately 2.7 X 10(-3%) t'/T) at an intensity of 10 W/cm2 and fell off rapidly above and below this intensity. The amount of base damage produced at 10 W/cm2 ultrasound was approximately equivalent to the damage produced by a gamma-ray absorbed dose of 12 krad. Assay of cells immediately after sonication at 10 W/cm2 showed that approximately 14% of the cells had been lysed. Tests showed that it was the DNA of the intact cells, however, which sustained all of the TBD. Survival data demonstrated that of the remaining unlysed cell population approximately 5% were viable, whereas cells exposed to 12 krad showed no survival. Additionally, cells were exposed for up to 5 min at 5 W/cm2. An increase in TBD was demonstrated with increasing time of exposure such that the rate of production at 5 min was approximately three times greater than that of a 1-min exposure. TBD was found to be completely suppressed when cells were sonicated at 10 W/cm2 for 2 min under 4 bar of hydrostatic pressure. Addition of the radical scavengers beta-MEA and cystamine eliminated TBD but had minimal effect on survival. The pressure and scavenger experiments demonstrate that TBD results from cavitation-induced free radicals. Based on the values for both the half-life and diffusion distance of such radicals, our results indicate that at least part of the bubble collapse occurs intracellularly.     

Effect of 2 MHz ultrasound on DNA, RNA and protein synthesis in Pisum sativum root meristem cells.

The amounts of DNA, RNA and protein synthesis were determined in Pisum sativum root meristem cells at various times after a 1 min exposure to 1 MHz ultrasound at a power density of 30 W/cm2. Immediate depressions in all three macromolecular syntheses occurred after sonication, followed by an apparent recovery several hours later. These events appear to correlate in time with the subsequent reduction and recovery in mitotic index in Pisum sativum root meristem cells exposed to 2 MHz ultrasound.     

Effect of ultrasound on a bilayer lipid membrane.

The effects of continuous wave ultrasound at a frequency of 1 MHz in the intensity range of 0-1.4 W/cm2 on an oxidized cholesterol bilayer lipid membrane (BLM) were observed. Ultrasound at 1.5 W/cm2 broke the membrane; in the range from 0.5 to 1.4 W/cm2, it accelerated the draining of the bulk lipid solution from the annulus to the Teflon support. At all intensities it has no effect on the conductance, the capacitance, or the dependence of each on the voltage applied across the membrane. Electrical parameters were measured in the presence of aqueous solutions of NaCl, KCl, and distilled water. The motivation and results of this project are explained in relation to an overall objective of determining the specific effects of ultrasound on biological membranes.     

声化学诱导艾氏腹水瘤细胞凋亡机制初探

本研究采用频率1.43MHz,声强3W/cm2的高频聚焦超声处理艾氏腹水肿瘤细胞,研究超声激活血卟啉诱导艾氏腹水肿瘤细胞凋亡的途径及其与癌细胞内的氧自由基之间的关系。通过细胞免疫组织化学方法检测与癌细胞凋亡相关的Bax,细胞色素c和caspase-3蛋白的动态表达,黄嘌呤氧化酶法检测超氧化物歧化酶活性变化,硫代巴比妥酸法检测膜脂质过氧化物的含量。结果发现超声加血卟啉处理1h,癌细胞胞浆中的三种促凋亡蛋白表达增多,3h时表现为高表达;处理1h的癌细胞,超氧化物歧化酶活性下降,膜脂质过氧化物增多。研究结果表明超声激活血卟啉诱导艾氏腹水肿瘤细胞凋亡可能通过线粒体途径,且与癌细胞受损后产生的氧自由基有关。     

Bioeffect of ultrasound on endothelial cells in vitro

The effects of low-intensity ultrasound (US) on biological systems have been investigated extensively; however, the effects of ultrasound stimulation on endothelial cells were rarely studied. In this study, 1 MHz, pulsed 1:4, and four different spatial-average temporal-peak intensities (0.5, 1.0, 1.6, and 2W/cm2) of ultrasound were used to stimulate endothelial cells for 10 min per day. The results showed that ultrasound (intensity 1.6-2.0W/cm2) treatment after 6 days enhanced the nitric oxide (NO) and Ca2+ release from the endothelial cells but did not promote cell growth. In addition, ultrasound stimulation changed the cellular morphology and orientation, and increased extracellular matrix secretion from endothelial cells.     

声化学激活血卟啉诱导艾氏腹水肿瘤细胞凋亡

本实验采用频率为2．0MHz，声强分别为1．0w／cm^2、1．5w／cm^2、2．0w／cm^2等不同参数，研究超声激活血卟啉对艾氏腹水肿瘤细胞的杀伤作用和诱导肿瘤细胞凋亡现象。通过扫描电镜、透射电镜以及荧光显微镜观察受损后细胞形态结构的变化，主要表现为细胞微绒毛的减少，胞膜结构和通透性的改变，细胞器的受损以及核物质的分解、丢失；同时发现处理后的肿瘤细胞有核物质凝集、趋边排列以及凋亡小体的形成等细胞凋亡特征。研究中首次发现声化学激活血卟啉在对艾氏腹水肿瘤细胞杀伤的同时，也能诱导艾氏腹水肿瘤细胞发生凋亡，提示在声动力疗法中并存着对癌细胞的直接杀伤和通过诱导癌细胞凋亡的两种抗癌途径。     

The effect of frequency and grounding on whole-body absorption of humans in E-polarized radiofrequency fields

The radiofrequency absorption rates of five male human volunteers have been measured from 3 to 41 MHz. The subjects were exposed at about 10 microW /cm2 inside a very large transverse electromagnetic (TEM) cell and never absorbed more than 1 W. Both the EKH and EHK orientations were employed under both free-space and grounded conditions. Absorption rates for the EKH orientation exceed those of the EHK orientation by 40% in free space, but only by 6% when grounded. The absorption rates for the grounded men vary with frequency, f, as f1.9 from 3 to 25 MHz and then level off at peak. The free-space absorption rates vary as f1.7 from 3 to 18 MHz and as f2.9 from 18 to 41 MHz. The average measured absorption rates at 10 MHz exceed the average of the standard model calculations by a factor of three (for free space) or four (grounded). The average man, when exposed grounded in an EKH orientation to the maximum permitted exposure levels under ANSI standard C95 .1-1982, will absorb 0.58 +/- 0.14 W/kg over most of the 3 to 41-MHz frequency range. This slightly exceeds the whole-body maximum of 0.40 W/kg underlying the standard.     

Ultrasound stimulates proteoglycan synthesis in bovine primary chondrocytes

Mechanical forces can stimulate the production of extracellular matrix molecules. We tested the efficacy of ultrasound to increase proteoglycan synthesis in bovine primary chondrocytes. The ultrasound-induced temperature rise was measured and its contribution to the synthesis was investigated using bare heat stimulus. Chondrocytes from five cellular isolations were exposed in triplicate to ultrasound (1 MHz, duty cycle 20%, pulse repetition frequency 1 kHz) at average intensity of 580 mW/cm2 for 10 minutes daily for 1-5 days. Temperature evolution was recorded during the sonication and corresponding temperature history was created using a controllable water bath. This exposure profile was used in 10-minute-long heat treatments of chondrocytes. Heat shock protein 70 (Hsp70) levels after one-time treatment to ultrasound and heat was analyzed by Western blotting, and proteoglycan synthesis was evaluated by 35S-sulfate incorporation. Ultrasound treatment did not induce Hsp70, while heat treatment caused a slight heat stress response. Proteoglycan synthesis was increased approximately 2-fold after 3-4 daily ultrasound stimulations, and remained at that level until day 5 in responsive cell isolates. However, chondrocytes from one donor cell isolation out of five remained non-responsive. Heat treatment alone did not increase proteoglycan synthesis. In conclusion, our study confirms that pulsed ultrasound stimulation can induce proteoglycan synthesis in chondrocytes.     

The survival of amphibian embryos after continuous ultrasonic treatment

The effect of continuous ultrasonic treatment on the development of early embryos of common frog Rana temporaria was studied. Intact embryos at the blastula stage were exposed to ultrasound of various frequencies (0.88 and 2.64 MHz) and intensities (0.05 to 1.0 W/cm²) for various periods (1 to 15 min). The increase in ultrasound intensity to 0.7–1.0 W/cm² and exposure time to 5–15 min resulted in nearly complete mortality. Development of the embryos exposed to ultrasound of medium intensity (0.2–0.7 W/cm²) for 1–5 min was virtually similar to the control. Treatment at a frequency of 2.64 MHz and intensity of 0.05–0.7 W/cm² for 1–5 min had no effect on the development of amphibian embryos and their survival rate. The increase in intensity of the ultrasound of this frequency to 1 W/cm² and the exposure time to 5 min decreased the number of normally developing embryos by 35%.     

Cell inactivation by ultrasound

Cell survival curves have been obtained for Escherichia coli B (E. coli B) after the sonication of suspensions of the bacteria with continuous wave ultrasound at a fixed frequency of 2 MHz between peak intensities of 8.7 and 2.25 W cm^?2. It was found that under suitable conditions the survival curves were reproducible and it also was found that there was a clear relationship between the rate of inactivation and the peak acoustic intensity of the ultrasound. There appeared to be a lower threshold of peak intensity below which no inactivation was observed.     

Absence of synergistic enhancement of non-thermal effects of ultrasound on cell killing induced by ionizing radiation

The present study was performed to elucidate the role of non-thermal effects (cavitation and direct effects) of ultrasound, in simultaneous combination with X-irradiation on the cytotoxicity of mouse L cells. Firstly, mouse L cells were exposed to X-rays and ultrasound (1 MHz continuous wave, spatial peak temporal average intensity; 3.7 W/cm2) simultaneously at 37 degrees C under O2 or Ar saturated conditions to examine the cavitational effect of ultrasound. Secondly, cells were exposed to X-rays and ultrasound at 37 degrees C under N2O saturated conditions, which suppresses the cavitation, to examine the direct effects of ultrasound. The cavitational effect under O2 and Ar saturated conditions induced an exponential decrease in cell survival, and resulted in an additive effect on cell killing with the combination of X-rays and ultrasound. The direct effect in the N2O conditions induced no cell killing and did not modify the cell killing induced by X-rays. These results suggested that the non-thermal effects of ultrasound did not interact synergistically with X-rays for cell killing.     

Changes in the permeability of amphibian embryo envelope for fluorochromes under the action of ultrasound

It was determined whether high-frequency ultra sound can change the permeability of gray toad Bufo bufo and grass frog Rana temporaria under the action of high-frequency ultrasound. The changes in the permeability of embryonic envelope were assessed by using slowly penetrating fluorochromes ANS, FDA, and fluorescein. It was found that the ultrasound of 0.88 MHz and 0.4 - 0.7 W/cm2 intensity increased the permeability of amphibian embryonic envelope for ANS and FDA, whereas the ultrasound of 2.64 MHz and the same intensity increased that for fluorescein with the retention of low permeability for FDA. Embryos continued the normal development after treatment with ultrasound under these conditions.     

The effect of ultrasound on the antigenic activity of human erythrocytes

The effect of ultrasound (frequency 0.88 MHz, intensity from 0.05 to 1 W/cm2) on alterations in antigenic activity has been investigated in vitro using ABO antigens of human erythrocytes. The existence of threshold doses of ultrasound influence has been found. These doses are shown to be independent of ultrasound intensity. The dependence of the effect on erythrocyte concentration has been established. Individual and group differences in the antigenic resistance to ultrasonic exposure in donors of groups A and B have been revealed. A drop in antigenic activity equal to 97% has been obtained.     

Reactive oxygen species and DNA damage after ultrasound exposure

The aim of this work was to detect the formation of hydrogen peroxide and hydroxyl radicals after ultrasound (US) exposure and test the hypothesis that reactive oxygen species induced by ultrasound can contribute to DNA damage. Formation of reactive oxygen species was observed in incubated medium after sonication with 1 MHz continuous ultrasound at the intensities of 0.61-2.44 W/cm2. Free radicals and hydrogen peroxide produced by ultrasound exposure of cells can lead to DNA damage. Comet assay was used to assess the effect of ultrasound on the level of nuclear DNA damage. The nucleated erythrocytes from fish were exposed in vitro to ultrasound at the same intensities and frequency. It was noticed that ultrasound in all used intensities induced DNA damage. The effect was not eliminated by the addition of catalase, which indicates that DNA damage was not caused by hydrogen peroxide only. The results showed that the DNA damage can be repair and this mechanism was the most effective after 30 and 60 min after sonication. Furthermore, the ultrasound-induced DNA damage in the presence of sonosensitizer (Zn- and AlCl-phthalocyanine) was studied. It was noticed that phthalocyaniens (Pcs) alone or with ultrasound did not induce significant changes in the level of DNA damage.     

Energy-efficient sludge sonication: power and sludge characteristics

This paper improves the energy efficiency for sludge treatment using ultrasound. The sludge organic content, solid concentration, ultrasonic intensity, and time were optimized. A new index, kWh/kgSCOD-increase, was used to evaluate the energy efficiency that covers both sludge characteristics and lysis effectiveness. The results showed that the energy efficiency of sludge sonication was in the range of 102-347kWh/kgSCOD-increase. Higher VS/TS ratio gave higher energy efficiency. Optimal solid concentration (TS) was found to be 20g/L; low TS (5.0g/L) consumed 137% more energy; while higher TS might assimilate the sound energy and decrease the efficiency. The cavitation threshold for sludge was 20-30W/cm(2), below which no sludge lysis was observed. The lowest effective sound intensity (31.4W/cm(2)) was best and saved 20-35% energy compared to higher intensities. The initial first minutes of sonication were most effective; hence intensive-short treatment was preferred.     

The embryotoxic effects of ultrasound exposure in pregnant ICR mice

The embryotoxicity of ultrasound exposure during pregnancy was investigated in DUB:(ICR) mice. On day 0 of gestation (day of plug), pregnant mice were assigned to one of five groups: cage control, sham exposed (0 W/cm2), 0.05 W/cm2, 0.50 W/cm2. or 1.00 W/cm2. Females were anesthetized on day 8 of gestation and their abdomens were shaved to assure good acoustic coupling. The animals were strapped on a lucite board and placed vertically into a distilled degassed water bath (30 degrees C) so that the abdomen was fully submerged and centered in the axis of the ultrasonic beam. Insonation was carried out using a PZT transducer with a radius of 1.27 cm and a frequency of 1 MHz under continuous wave conditions. Each animal was placed at a distance of 25 cm from the transducer and exposed to the appropriate intensity for 120 seconds. On day 17 of gestation, the maternal animals were killed, the uterine contents were examined, and live fetuses were weighed and then shipped in cold lactated Ringer's solution from Maryland to Arkansas. Fetuses were examined on the day following maternal sacrifice for external and visceral defects and skeletons were prepared and examined subsequently. Slight but significant differences were detected between the cage control and sham-exposed groups. No statistically significant changes were seen that could be attributed to ultrasound exposure, although there was a slight increase in the incidence of malformed fetuses and the occurrence of multiple malformations in individual fetuses as intensity of the ultrasonic exposure increased.     

Chromosome aberrations induced in the lymphocytes of rabbits by ultrasounds of 1MHz alone or followed by treatment with 5-bromo-2'-deoxyuridine

Rabbit lymphocytes were treated in Go with therapeutical ultrasound waves (1 W/cm2; 1 MHz; continuous wave mode) alone or followed by an additional exposure to 5-bromo-2'-deoxyuridine (BrdU). Doses in the Fresnel zone ranged from 120 to 960 J/cm2; no refrigeration was provided, and temperature increased from 37 degrees C to 45 degrees C. In the Fraunhofer zone, only an ultrasound dose of 960 J/cm2 was used, and the temperature of the insonated blood sample was maintained at 37 degrees C. For the sequential treatment, BrdU (10 micrograms/ml medium) was added immediately after the ultrasound treatment and was left in contact with the lymphocytes for 48 hours. Ultrasound alone did not induce chromosome aberrations, but ultrasound followed by the BrdU exposure resulted in an increased number of abnormal cells.     

Acoustically active liposomes for drug encapsulation and ultrasound-triggered release

Acoustically active liposomes (AAL), previously developed as ultrasound contrast agents, contain small amounts of air. These AAL have potential to carry pharmaceutics and their acoustic activity could enable them to respond to ultrasound stimulation by releasing their contents. Since liposomes can entrap many kinds of drugs, if such entrapment did not affect their echogenicity, then the release of contents could potentially be controlled by ultrasound stimulation. The aim of this research was to investigate the capacity of acoustically active liposomes for hydrophilic molecule encapsulation and to determine their sensitivity to ultrasound-triggered release. Liposomes, composed of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and cholesterol, were made acoustically active by hydrating a lipid film, sonication, freezing in the presence of mannitol, lyophilization, and rehydration. As a test molecule, calcein was added in the hydration step. The procedure for generating acoustically active liposomes was compatible with an encapsulation efficiency of 15% or more. The presence of mannitol during freeze-drying was essential not only for generation of acoustic activity but also for efficient encapsulation. Ultrasound-triggered release was achieved by applying 1 MHz ultrasound at 2 W/cm2 for 10 s. The inclusion of 4% diheptanolyphosphatidylcholine (DHPC) increased the sensitivity of liposomes to ultrasound stimulation and resulted in very efficient stimulated release of contents (1/3 released in 10 s, 2/3 released in six such applications). Release of contents was highly correlated with the loss of air induced either by ultrasound or rapid pressure reduction. These encapsulation and triggered release techniques are highly efficient, and hence may be applicable to drug delivery.     

Assessment of fibroblast cells submitted to ultrasonic irradiation

Physiotherapists consider ultrasound an indispensable tool, which is commonly employed in clinical practice as a treatment aid for musculoskeletal dysfunctions. The aim of our study has been to analyze fibroblast cell structures following low-intensity pulsed ultrasonic irradiation. Fibroblast cell cultures irradiated with ultrasound were analyzed through electron microscopy to determine an ideal irradiation beam that preserved cell morphology and integrity. Analysis by fluorescence microscopy and transmission electron microscopy was used to follow morphological changes of the nucleus and cytoskeleton following different ultrasound irradiation intensities. According to the parameters used in the pulsed irradiation of fibroblast cultures, control over the intensity employed is fundamental to the optimal use of therapeutic ultrasound. Cell cultures submitted to low-intensity pulsed ultrasonic irradiation (0.2-0.6 W/cm2) at 10% (1:9 duty cycle) and 20% (2:8 duty cycle) maintained shape and cellular integrity, with little damage. In the group irradiated with an intensity of 0.8 W/cm2, a loss of adhesion was observed along with an alteration in the morphology of some cells at an intensity of 1.0 W/cm2, which resulted in the presence of cellular fragments and a decrease of adhering cells. In cells irradiated at 2.0 W/cm2, there was a complete loss of adhesion and aggregation of cellular fragments. The present study confirms that biophysical properties of pulsed ultrasound may accelerate proliferation processes in different biological tissues.