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The development of animal models of mental disorders is an important task since such models are useful for studying the neurobiological mechanisms of psychopathologies and for trial of new therapeutic drugs. One way to model pathologies of the nervous system is to impair fetal neurodevelopment through stress of the pregnant future mother, or prenatal stress (PS). The use of variable frequency ultrasound (US) in rodents is a promising method of imitating psychological stress, to which women in modern society are most often subjected. The aim of our study was to investigate the effect of PS induced by exposure to variable frequency ultrasound (US PS) throughout the gestational period on the adult rat offspring, namely, to identify features of behavioral alterations and neurochemical brain parameters that can be associated with certain mental disorders in humans, to determine the possibility of creating a new model of psychopathology. Our study included a study of some behavioral characteristics of male and female rats in the elevated plus maze, open-field test, object recognition test, social interaction test, sucrose preference test, latent inhibition test, Morris water maze, forced swimming test, acoustic startle reflex, and prepulse inhibition tests. We also determined the activity of the serotonergic, dopaminergic, and noradrenergic neurotransmitter systems in the hippocampus and frontal cortex by HPLC-ED. Concentration of norepinephrine, dopamine, DOPAC, serotonin, and HIAA, as well as DOPAC/dopamine and HIAA/serotonin ratios were determined. A correlation analysis of behavioral and neurochemical parameters in male and female rats was performed based on the data obtained. The results of the study showed that US PS altered the behavioral phenotype of the rat offspring. US PS increased the level of anxious behavior, impaired orientation-research behavior, increased grooming activity, decreased the desire for social contacts, shifted behavioral reactions from social interaction to interaction with inanimate objects, impaired latent inhibition, and decreased the startle reflex. US PS activated the serotonergic, dopaminergic, and noradrenergic neurotransmitter systems of the rat frontal cortex and hippocampus. A correlation between neurochemical and behavioral parameters was revealed. Our study showed that US PS leads to a certain dysfunction on behavioral and neurochemical levels in rats that is most closely associated with symptoms of schizophrenia or autism. We hypothesize that this could potentially be an indicator of face validity for a model of psychopathology based on neurodevelopmental impairment. [ABSTRACT FROM AUTHOR]

The aim of the present study was to investigate the possibility of encapsulating the plant antioxidant taxifolin in the living cells of the yeast Saccharomyces cerevisiae. Taxifolin is an unstable substance prone to oxidative degradation and actively enters into chemical reactions with a decrease or loss of bioactive properties. To minimize these problems, the use of encapsulation technology has been proposed. The cells of the yeast Saccharomyces cerevisiae have been chosen as a protective material for taxifolin. The encapsulation process was carried out using simple diffusion methods in living Saccharomyces cerevisiae cells in a thermostatically controlled shaker for 24 h. The aim of the study was to evaluate the effect of preliminary microstructuring of taxifolin on the efficiency of its encapsulation in yeast cells. The microstructuring process was carried out using low-frequency ultrasonic cavitation exposure for 7 min with a frequency of 22 ± 1.6 kHz and a power of 600 W/100 mL. The studies confirmed the feasibility of the proposed approach. It was found that microstructuring changes the dispersed composition of taxifolin particles and their morphology in solution and also increases the value of the antioxidant activity. Preliminary microstructuring of taxifolin increases the efficiency of its encapsulation in Saccharomyces cerevisiae yeast cells by 1.42 times compared to the initial form. A positive dependence of the growth of the encapsulation efficiency on the duration of the process was also established. Thus, the conducted studies confirmed the advantage of encapsulation of taxifolin in living cells of the yeast Saccharomyces cerevisiae in microstructured form. [ABSTRACT FROM AUTHOR]

The aim of the present study was to investigate the possibility of encapsulating the plant antioxidant taxifolin in the living cells of the yeast Saccharomyces cerevisiae. Taxifolin is an unstable substance prone to oxidative degradation and actively enters into chemical reactions with a decrease or loss of bioactive properties. To minimize these problems, the use of encapsulation technology has been proposed. The cells of the yeast Saccharomyces cerevisiae have been chosen as a protective material for taxifolin. The encapsulation process was carried out using simple diffusion methods in living Saccharomyces cerevisiae cells in a thermostatically controlled shaker for 24 h. The aim of the study was to evaluate the effect of preliminary microstructuring of taxifolin on the efficiency of its encapsulation in yeast cells. The microstructuring process was carried out using low-frequency ultrasonic cavitation exposure for 7 min with a frequency of 22 ± 1.6 kHz and a power of 600 W/100 mL. The studies confirmed the feasibility of the proposed approach. It was found that microstructuring changes the dispersed composition of taxifolin particles and their morphology in solution and also increases the value of the antioxidant activity. Preliminary microstructuring of taxifolin increases the efficiency of its encapsulation in Saccharomyces cerevisiae yeast cells by 1.42 times compared to the initial form. A positive dependence of the growth of the encapsulation efficiency on the duration of the process was also established. Thus, the conducted studies confirmed the advantage of encapsulation of taxifolin in living cells of the yeast Saccharomyces cerevisiae in microstructured form.

Abstract: Combining liposomally encapsulated cytotoxic drugs with ultrasound exposure has improved the therapeutic response to cancer in animal models; however, little is known about the underlying mechanisms. This study focused on investigating the effect of ultrasound exposures (1 MHz and 300 kHz) on the delivery and distribution of liposomal doxorubicin in mice with prostate cancer xenografts. The mice were exposed to ultrasound 24 h after liposome administration to study the effect on release of doxorubicin and its penetration through the extracellular matrix. Optical imaging methods were used to examine the effects at both microscopic subcellular and macroscopic tissue levels. Confocal laser scanning microscopy revealed that ultrasound-exposed tumors had increased levels of released doxorubicin compared with unexposed control tumors and that the distribution of liposomes and doxorubicin through the tumor tissue was improved. Whole-animal optical imaging revealed that liposomes were taken up by both abdominal organs and tumors. [Copyright &y& Elsevier]

Abstract: Development of a gene delivery system to transfer the gene of interest selectively and efficiently into targeted cells is essential for achievement of sufficient therapeutic effects by gene therapy. Here, we succeeded in developing the gene transfection method using ultrasound (US)-responsive and mannose-modified gene carriers, named Man-PEG2000 bubble lipoplexes. Compared with the conventional lipofection method using mannose-modified carriers, this transfection method using Man-PEG2000 bubble lipoplexes and US exposure enabled approximately 500∼800-fold higher gene expressions in the antigen presenting cells (APCs) selectively in vivo. This enhanced gene expression was contributed by the improvement of delivering efficiency of nucleic acids to the targeted organs, and by the increase of introducing efficiency of nucleic acids into the cytoplasm followed by US exposure. Moreover, high anti-tumor effects were demonstrated by applying this method to DNA vaccine therapy using ovalbumin (OVA)-expressing plasmid DNA (pDNA). This US-responsive and cell-specific gene delivery system can be widely applied to medical treatments such as vaccine therapy and anti-inflammation therapy, which its targeted cells are APCs, and our findings may help in establishing innovative methods for in-vivo gene delivery to overcome the poor introducing efficiency of carriers into cytoplasm which the major obstacle associated with gene delivery by non-viral carriers. [ABSTRACT FROM AUTHOR]

Small interfering RNA (siRNA) has been used to knock down the expression of specific genes. However, its delivery remains a challenge. Recently, ultrasound combined with microbubbles has been used to deliver plasmid into cells and has shown much superiority. Whether a survivin siRNA transfected with a microbubble contrast agent combined with ultrasound exposure could inhibit survivin expression and induce ovarian cancer cell apoptosis was investigated.The survivin gene was amplified and inserted into vector pEGFP-N1, resulting in psurvivin-EGFP. Three siRNA expression cassettes (SECs) targeting survivin were obtained by 2-step polymerase chain reaction. The SECs and psurvivin-EGFP were cotransfected into 293T cells. A functional SEC was selected and inserted into pMD18T, resulting in the survivin-targeting siRNA expression plasmid, which was transfected into SKOV-3 ovarian carcinoma cells using SonoVue, a microbubble contrast agent, and ultrasound exposure. Survivin expression was monitored by Western blot, and apoptosis was determined by fluorescence-activated cell sorting.One siRNA effectively inhibited survivin expression. SonoVue with ultrasound effectively delivered survivin siRNA to SKOV-3 cells, inhibited survivin expression, and induced apoptosis.Delivery of survivin siRNA using a microbubble contrast agent combined with ultrasound exposure can effectively inhibit survivin expression and induce apoptosis, providing a new promising approach for siRNA delivery in vivo. [ABSTRACT FROM AUTHOR]

AbstractAim:To determine the effects of ultrasound exposure in combination with a microbubble contrast agent (SonoVue) on the cellular uptake and delivery of drugs/genes into human umbilical vein endothelial cells (HUVECs) as well as their biological effects on migration.Methods:HUVECs in suspension were exposed to pulsed ultrasound with a 10% duty cycle in combination with various concentrations of a microbubble contrast agent (SonoVue) using a digital sonifier at a frequency of 20 kHz and an intensity of 3.77 W/cm2 on the surface of a horn tip. Cell culture inserts were used to determine the cell migration ability.Results:Exposure to pulsed ultrasound resulted in enhanced green fluorescent protein (EGFP) gene transfection efficiencies ranging from 0.2% to 2%. The transfection efficiency of HUVECs was approximately 3-fold higher in the presence of SonoVue than in its absence at the effective exposure time of 6 s. For drug delivery to HUVECs using ultrasound, the delivery efficiencies of a low-molecular-weight model drug (TO-PRO®−1, MW 645.38) were significantly higher when compared to drug delivery without ultrasound, with a maximum efficiency of approximately 34%. However, the delivery efficiencies of a high-molecular-weight model drug (Dextran-Rhodamine B, MW 70 000) were low, with a maximum delivery efficiency of nearly 0.5%, and gene transfection results were similarly poor. The migration ability of HUVECs exposed to ultrasound was also lower than that of the control (no exposure).Conclusion:The use of low-frequency and low-energy ultrasound in combination with microbubbles could be a potent physical method of increasing drug/gene delivery efficiency. This technique is a promising nonviral approach that can be used in cardiovascular disease therapy. [ABSTRACT FROM AUTHOR]

Abtract: This work investigates the effect of ultrasound exposure parameters on the sonoporation of KHT-C cells in suspension by perflutren microbubbles. Variations in insonating acoustic pressure (0.05 to 3.5 MPa), pulse frequency (0.5 to 5.0 MHz), pulse repetition frequency (10 to 3000 Hz), pulse duration (4 to 32 μs) and insonation time (0.1 to 900 s) were studied. The number of cells permeabilised to a fluorescent tracer molecule (70 kDa FITC-dextran) and the number of viable cells were measured using flow cytometry. The effect of exposure on the microbubble population was measured using a Coulter counter. Cell viability and membrane permeability were found to depend strongly on the acoustic exposure conditions. Cell permeability increased and viability decreased with increasing peak negative pressure, pulse repetition frequency, pulse duration and insonation time and with decreasing pulse centre frequency. The highest therapeutic ratio (defined as the ratio of permeabilised to nonviable cells) achieved was 8.8 with 32 ± 4% permeabilisation and 96 ± 1% viability at 570 kPa peak negative pressure, 8 μs pulse duration, 3 kHz pulse repetition frequency, 500 kHz centre frequency and 12 s insonation time with microbubbles at 3.3% volume concentration. These settings correspond to an acoustic energy density (ESPPA) of 3.12 J/cm2. Cell permeability and viability did not correlate with bubble disruption. The results indicate that ultrasound exposure parameters can be optimized for therapeutic sonoporation and that bubble disruption is a necessary but insufficient indicator of ultrasound-induced permeabilisation. (E-mail: raffik@sri.utoronto.ca) [Copyright &y& Elsevier]

The effects of low-intensity pulsed ultrasound (30 mW/cm2) were investigated in experimental cancellous bone fracture healing in bioabsorbable self-reinforced poly-l-lactide (SR-PLLA) rod fixed distal femur osteotomy in rats. A transverse transcondylar osteotomy was fixed with one SR-PLLA rod in 32 male Wistar rats of the age of 20 weeks. Half of the rats had a daily 20-min ultrasound exposure for three weeks. The follow-up times were three, six, and 12 weeks. Radiographical, histological, microradiographical, oxytetracycline labeling, and histomorphometrical analyses were performed. No foreign-body reactions were noted. The biocompatibility of SR-PLLA and ultrasound was found to be good. In the radiological and histological assessments there was a slight tendency for enhanced healing in the ultrasound group at three weeks, but at six and 12 weeks no differences were observed. The histomorphometrical and oxytetracycline labeling analyses showed that ultrasound exposure had no significant effects on bone healing. The present study shows that there were no obvious findings to support the hypothesis that low-intensity pulsed ultrasound enhances bone healing in self-reinforced poly-l-lactide (SR-PLLA) rod fixed experimental metaphyseal distal femur osteotomy in rats. The observed good biocompatibility provides a safe starting-point for clinical trials on bioabsorbable fixation combined with low-intensity ultrasound. [ABSTRACT FROM AUTHOR]

Abstract: 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” (1MHz and 1.6MHz) therapeutic ultrasound range and low (20kHz) 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 5mol% PEG were prepared with sizes ranging from 100nm to 1μm. 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 1.6MHz (focused) and 1.0MHz (unfocused) were used, the I spta of which were 46.9W/cm2 and 3.0W/cm2, respectively. A commercial 20kHz, point-source, continuous wave transducer with an I spta of 0.13W/cm2 was also used for comparative purposes. Whereas complete leakage was obtained for all vesicle sizes at 20kHz, no leakage was observed for vesicles smaller than 100nm in diameter at 1.6 or 1.0MHz. However, introducing leakage at the higher frequencies became feasible when larger (greater than 300nm) vesicles were used, and the extent of leakage correlated well with vesicle sizes between 100nm and 1μm. 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” (1MHz) 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. [Copyright &y& Elsevier]

Abstract: We investigated the effect of ultrasound on plasma membrane (PM) Ca2+-ATPase activity of Aloe arborescens callus cells in solid culture. The calluses were exposed by a 20kHz digital sonifier at the powers of 2 and 10W from the effective exposure times of 2–10s. PM Ca2+-ATPase activity was almost significantly higher at 2W both in continuous wave and 10% duty cycle than that of the control (no ultrasound) at effective exposure times of 5 and 10s. However, its activity decreased at 10W in continuous wave exposure. It is possible that the PM Ca2+-ATPase configuration or structure may be partly damaged by high-energy ultrasound at 10W. Our results showed that low-energy ultrasound exposure was a useful physical field to stimulate A. arborescens callus cells to adapt environmental stress through PM Ca2+-ATPase activity increase. [Copyright &y& Elsevier]

Effects of ultrasound on the growth and vacuolar H+-ATPase (V-ATPase) activity of aloe arborescens callus cells were investigated. The callus cells cultured in suspension were agitated by an ultrasonic bath at 28 kHz from the exposure time ts=2–60 s, and the cell growth was the highest at ts=5 s as measured by the fresh weight of the callus. The callus cells cultured in solid were exposed by a digital sonifier at 20 kHz in 2 s

• Current in vitro sonication experiments show big variability in experimental methods. • Many factors influence reproducibility and outcome of sonication experiments in vitro. • Reproducibility and validity of in vitro sonication experiments can be altered. Current in vitro sonication experiments show immense variability in experimental set-ups and methods used. As a result, there is uncertainty in the ultrasound field parameters experienced by sonicated samples, poor reproducibility of these experiments and thus reduced scientific value of the results obtained. The scope of this narrative review is to briefly describe mechanisms of action of ultrasound, list the most frequently used experimental set-ups and focus on a description of factors influencing the outcomes and reproducibility of these experiments. The factors assessed include: proper reporting of ultrasound exposure parameters, experimental geometry, coupling medium quality, influence of culture vessels, formation of standing waves, motion/rotation of the sonicated sample and the characteristics of the sample itself. In the discussion we describe pros and cons of particular exposure geometries and factors, and make a few recommendations as to how to increase the reproducibility and validity of the experiments performed. [ABSTRACT FROM AUTHOR]