Serum, uterine, and vaginal mucosal IgG antibody responses against Tritrichomonas foetus after administration of a commercial killed whole T foetus vaccine in beef cows.

The objective of this study was to determine the level and duration of IgG antibodies induced against killed whole Tritrichomonas foetus and T foetus-purified surface antigen (TF1.17) in serum, vaginal, and uterine secretions after systemic immunization of beef cows with a vaccine containing killed whole T foetus. Twenty nonpregnant beef cows were randomly assigned to vaccine or control groups as follows: Vaccine (n = 10): cows received 2 mL of a commercial vaccine containing killed whole T foetus subcutaneously and a 2-mL booster 2 weeks later. Control (n = 10): cows received 2 mL of sterile saline on the same schedule. Vaginal secretions and blood samples were collected on Days 0, 8, 15, 22, 29, 36, 43, 50, 60, 75, 89, 110, 146, and 182 relative to day of primary vaccination. Uterine flush fluid was collected on Days 0, 15, 29, and 43 after the day of primary vaccination. Samples were assayed for IgG antibodies to the killed whole T foetus and surface antigen TF1.17 using enzyme-linked immunosorbent assay. Serum whole T foetus-specific IgG levels were significantly increased (between Days 15 and 182) following vaccination with T foetus or with saline. No differences between vaccinates and controls in uterine responses to whole-cell antigen were detected. Serum anti-TF1.17 IgG responses to vaccination were significantly higher than Day 0 throughout the immunization period (P < 0.001) and were higher than responses in control animals on each day post immunization through Day 146 (P < 0.001). A significant rise in TF1.17-specific IgG levels was observed in vaginal and uterine fluids from Day 15 post vaccination compared to the Day 0 levels. These levels remained significantly elevated in vaginal and uterine fluids through Days 75 (P < 0.05) and 43 (P < 0.001) after primary vaccination, respectively. Antibody levels in serum, vaginal, and uterine secretions against TF1.17 remained low in the control group throughout the study. In conclusion, vaccination of beef cows with a commercial vaccine containing T foetus induced significant increase in the levels of IgG to the T foetus TF1.17 surface antigen in serum, vaginal secretions, and uterine fluid, which remained elevated through Days 43, 75, and 182 in uterine fluids, vaginal secretions, and serum, respectively. Since purified TF1.17 antigen has been shown to protect against experimental T foetus infection in heifers, the vaccine-induced TF1.17-specific IgG response is likely to be important in the prevention of trichomoniasis in beef cattle.

Instability development in heated human erthrocytes.

Heated human erythrocytes gradually lose their form-maintaining structure as the temperature is increased to 50 degrees C and can behave in some respects as a viscous fluid. We have developed a technique for heating and stressing these cells that is novel, simple and quantitatively precise. We have applied this technique to heated human erythrocytes and have measured instability development in cells. We have employed instability growth theory to calculate a value for an effective surface tension which, in contrast to other methods of membrane surface tension measurement sought to minimize the effects of membrane supporting structural elements. The value obtained for the surface tension of the heated erythrocyte membrane was 0.9 . 10(-6) N/m with a range of variation from 0.4 . 10(-6)N/m to 1.4 . 10(-6) N/m. The methods described may be useful for determining fundamental physical parameters such as internal viscosity and interfacial tension in other systems.

The influence of temperature and incubation time on deformability of human erythrocytes.

Human erythrocytes have been heated and stressed in a novel and controlled manner using rectangular microcapillaries. Heated cells attached to the capillary wall were stressed by liquid flow. Under particular conditions of stress, temperature and incubation time the body of the cell could be pulled in the flow, retaining a connection with the glass by means of a narrow process or tether. The tethers appear as: regularly beaded, irregularly beaded or without beads depending upon the incubation conditions. We have outlined the incubation regimes necessary to achieve these different responses in the temperature range 48--55 degrees C. The cells become less deformable as the incubation is continued beyond an optimum time. The behaviour of the tether is compared with that of a viscoelastic liquid. Circular dichroism studies of ghost membranes show that the denaturation of membrane proteins is partially reversible when incubation times are similar to those required to bring about a loss of deformability.

Correlation between sonoluminescence, sonochemistry and cavitation noise spectra.

The acoustic signal from the sonochemical production of H2O2 in water, as measured by the intensity and the width of the second harmonic, show a sensitive and correlated dependence to the presence of small amounts (millimolar range) of an anionic surfactant (SDS) in water. The graphic shows the link from the ultrasonic reaction to the measurable quantities. New possibilities to reliably control such processes is therefore opened.

Ultrasound accelerates functional recovery after peripheral nerve damage.

OBJECTIVE: Axonal injury in the peripheral nervous system is common, and often it is associated with severe long-term personal and societal costs. The objective of this study is to use an animal model to demonstrate that transcutaneous ultrasound can accelerate recovery from an axonotmetic injury. METHODS: The sciatic nerve of adult male Lewis rats was crushed in the right midthigh to cause complete distal degeneration of axons yet maintain continuity of the nerve. Beginning 3 days after surgery, various transcutaneous ultrasound treatments or sham treatments were applied 3 days per week for 30 days to the crush site of rats that were randomly assigned to two groups. In the preliminary experiments, there were three animals in each ultrasound group and two control animals. In the final experiment, there were 22 animals in the ultrasound group and 20 animals in the control group. Recovery was assessed by use of a toe spread assay to quantify a return to normal foot function in the injured leg. Equipment included a hand-held transducer that emitted continuous-wave ultrasound. The most successful ultrasound protocol had a spatial peak, time-averaged intensity of 0.25 W/cm2 operated at 2.25 MHz for 1 minute per application. RESULTS: Rats subjected to the most successful ultrasound protocol showed a statistically significant acceleration of foot function recovery starting 14 days after injury versus 18 days for the control group. Full recovery by the ultrasound group occurred before full recovery by the control group. CONCLUSION: Transcutaneous ultrasound applied to an animal model of axonotmetic injury accelerated recovery. Future studies should focus on identification of the mechanism(s) by which ultrasound creates this effect, as a prelude to optimization of the protocol, demonstration of its safety, and its eventual application to humans.

The significance of membrane changes in the safe and effective use of therapeutic and diagnostic ultrasound.

The cellular changes, such as alterations in motility and the stimulation of synthesis and secretion, induced by relatively low intensities of therapeutic ultrasound (e.g. 500 mW cm-2, SAPA; 100 mW cm-2 SATA) are primarily non-thermal in origin. They appear to be associated with changes in the permeability of the cell (plasma) membrane and in the transport of ions and molecules across it, effects which have been demonstrated in cells irradiated in suspension. In epithelial tissues, both in vitro and in vivo, it has been demonstrated that not only the cellular membrane transport pathways but also the paracellular or intercellular pathways are affected. Although membrane-mediated effects can be of value therapeutically, they could produce adverse effects if they were to occur during development, for the reception and transmission by the membrane of environmental signals are involved in determination of the fate of each cell. Determination is followed by selective gene expression and differentiation, that is, by the progressive increase in structural complexity brought about by the acquisition of specialised characteristics by various cell groups. Most cells of early embryos are ionically coupled via gap junctions which provide an intercellular pathway for electrochemical signalling and the maintenance of the concentration gradients which provide the cells with positional information. Differentiation of the cells varies according to their location with respect to these gradients. Increase in the intracellular concentration of calcium ions, which has been shown to occur after exposure to therapeutic levels of ultrasound, can decrease the permeability of gap junctions and uncouple cells, in the manner which occurs when they differentiate. Ultrasonically induced increases in calcium ion concentration are thus of considerable clinical significance, since they could affect differentiation and consequently histogenesis. Modification of plasma membrane permeability and transport properties, resulting in changes in the availability and activity of second messengers such as free calcium ions, can have profound effects on cell behaviour. Calcium channels appear to be the first channels to develop in the cell membranes of embryos, and internal calcium ion concentration is known to affect the synthesis of fetal proteins. Although generally reversible at intensities of less than 500 mW cm-2, changes in membrane permeability, particularly to calcium ions, could, if prolonged, have undesirable side effects not only on embryogenesis but on late prenatal and postnatal development. It is therefore recommended that the environmental conditions, thresholds, and mechanisms involved in the production of such changes be determined, so that they can be avoided when ultrasound is used diagnostically on sensitive targets such as embryos and fetuses.

The effect of therapeutic ultrasound on electrophysiological parameters of frog skin.

There are two groups of mechanisms through which ultrasound can affect biological systems, those of thermal origin and others of nonthermal origin. Since in almost every therapeutic application of ultrasound, movement of ions across cellular membranes is involved, it becomes important to study the effect of ultrasound on active and passive ionic conductance. In order to differentiate between thermal and nonthermal effects, a study was conducted on model systems in which the effect of temperature is known. The well-known sodium transporting epithelium, the epidermis of abdominal frog skin, was investigated and the effect of therapeutic ultrasound on its electrophysiological properties was determined. It was found that under open circuit conditions, irradiation of the skin with 1 MHz cw (60-480 mW/cm2) ultrasound caused a significant decrease (5-50%, depending on the applied power) in the transepithelial potential and resistance at room temperature (20-22 degrees C). Under short circuit conditions, also at room temperature, there was an increase in total ionic conductance (20-250%, depending on the applied power) and a decrease in the net actively transported current, measured as the short circuit current. These effects are reversible within the range of powers used. Furthermore, it was found that the magnitude of the observed changes was strongly dependent on the perfusion rate and the gas content of the bathing medium. The effect of ultrasound diminished in the presence of CO2 and was enhanced with faster perfusion rates. Pulsed ultrasound delivered at the same energy (Isata) as that of cw caused a significantly larger effect. At lower temperatures (12-14 degrees C) the effect of ultrasound was reduced. Analysis of the data reveals that the effects of ultrasound on ion transport reported here are not primarily of thermal origin but are probably due to cavitation and related effects, such as microsteaming.

Acoustic cavitation generated by an extracorporeal shockwave lithotripter.

Evidence is presented of acoustic cavitation generated by a Dornier extracorporeal shockwave lithotripter. Using x-ray film, thin aluminum sheets, and relatively thick metal plates as targets, evidence of liquid jet impacts associated with cavitation bubble collapse was observed. The jet impact was violent enough to puncture thin foils and deform metal plates. Furthermore, numerous jet impacts were generated over a volume of greater than 200 cm3. It is likely that such violent cavitation will also occur in tissue, and observed biological effects (e.g. renal calculus disintegration and tissue trauma) may be related to cavitation damage.

Ultrasonically induced gas bubble production in agar based gels: Part II. Theoretical analysis.

Visible size gas bubbles can be produced in an agar based gel when irradiated with either continuous wave (CW) or pulsed ultrasound. It is shown that many aspects of the production of these bubbles can be explained in a qualitative manner by a theoretical model based upon growth of a cavitation nucleus by rectified diffusion. Quantitative predictions for the number of bubbles produced as a function of various parameters tend to be different from measured values by less than an order of magnitude. The results given here provide a useful theoretical basis for the explanation of earlier measurements of ultrasonically induced bubbles in vivo.

Ultrasonically induced gas bubble production in agar based gels: Part I. Experimental investigation.

Macroscopically visible gas bubbles can be produced in an agar based gel by irradiation with either continuous or pulsed ultrasound at frequencies from 0.75 to 3.0 MHz. The variation in the number of bubbles formed with frequency, acoustic pressure, pulse length, duty cycle, and temperature closely resembles that seen in vivo. Furthermore, the acoustic pressure required to initiate bubble formation is also close to that required in vivo. It has been observed that alterations in the concentration and pH of the gels can have a profound effect on the nature and quantity of bubbles. This suggests that not only is this gel model suitable for the representation of the macroscopic features of bubble formation in vivo, but can be used to gain information about the preexisting bubble nuclei. Based on the experimental results obtained it can be suggested that for peak negative acoustic pressures of up 1 MPa (equivalent, for a plane travelling sinusoidal wave, to a time averaged intensity of 30 W/cm2) bubble formation can be avoided by the use of high frequencies, short pulse lengths and long duty cycles.

The effect of polypropylene vials on lithotripter shock waves.

In studies to understand the mechanisms responsible for shock wave lithotripsy (SWL) cell injury, we observed that shock waves (SWs) are influenced by the shape of the specimen vial. Lytic injury to kidney cells treated in a Dornier HM3 lithotripter was higher (p < 0.0001) when SWs entered the vial through the flat end (cap end) compared to the round end. Measurements of the acoustic field within polypropylene vials were carried out using both lithotripter SWs and pulsed ultrasound (US) in the megahertz frequency range. We compared pressure amplitudes inside the round and flat vials and found significant differences. When SWs entered through the round end, the average peak positive pressure was 40% of free-field pressure, due mostly to a dramatic reduction in pressure off axis. The average peak pressure inside the flat vial was twice that of the round vial. Experiments with US demonstrated that sound field focusing was induced by the curved interface of the round vial. Ray analysis for the round vial indicates the presence of "hot spots" on axis and "cold spots" off axis, in qualitative agreement with pressure profiles. We conclude that the shape of the specimen vial is an important factor that should be considered in model systems of SWL cell injury.

Color Doppler detection of acoustic streaming in a hematoma model.

Accurate differentiation between stagnant blood and soft tissue or clotted and unclotted blood has potential value in managing trauma patients with internal hemorrhage. Determination by regular ultrasound (US) imaging is sometimes difficult because the sonographic appearance of blood, clots and soft tissue may be similar. A hematoma model was developed to investigate the use of acoustic streaming for hematoma diagnosis in an in vivo environment. The results showed that a derated spatial peak temporal average (SPTA) intensity of 30 W/cm(2) was needed to generate color-Doppler-detectable streaming in stirred blood. The streaming velocity increased in proportion to the derated intensity. Streaming was also detected in stagnant blood, but at higher intensities. In clots, streaming was not detected even at high intensities. The streaming detection may be a valuable tool for improving the distinction between liquid blood and clots or soft tissue in hematoma diagnosis.

Calcium and the effects of ultrasound on frog skin.

Therapeutic ultrasound is used to enhance the repair of soft tissue, muscle, etc., and because many of the cellular reactions involved in these processes are dependent on the intracellular availability of free calcium ions, it becomes important to study the effects of ultrasound in the presence and the absence of calcium ions. Using frog skin as a biological model, the effect of therapeutic ultrasound (300 mW/cm2 1 MHz CW) was investigated. Sonication for two minutes caused a significantly larger increase in total ionic conductance (Gt) in the presence of calcium ions (140% vs. 27%). However, the time constant for Gt to return to steady state was significantly longer in calcium-free solutions (122 vs. 18 min.). This study demonstrates that the biological effects of ultrasound are influenced by calcium ions. Furthermore, the recovery time constants confirm recent findings regarding the function of calcium ions in the formation of tight junctions. The role of free radicals produced by cavitation and calcium potentiated lipid and protein peroxidation is discussed.

Activation, aggregation and adhesion of platelets exposed to high-intensity focused ultrasound.

Using platelet-rich plasma, we investigated the effect of 1.1-MHz continuous wave high-intensity focused ultrasound (HIFU) on platelet activation, aggregation and adhesion to a collagen-coated surface. Platelets were exposed for durations of 10-500 s at spatial average intensities of up to 4860 W/cm(2). To avoid heating effects, the average temperature in the HIFU tank was maintained at 33.8 +/- 4.0 degrees C during platelet experiments. Flow cytometry, laser aggregometry, environmental scanning electron microscopy and passive cavitation detection were used to observe and to quantify platelet activation, aggregation, adhesion to a collagen-coated surface and associated cavitation. It was determined that HIFU can activate platelets, stimulate them to aggregate and promote their adherence to a collagen-coated surface. In principle, HIFU can stimulate primary, or platelet-related, hemostasis. Cavitation was monitored by a passive cavitation detector during aggregation trials and was quantified to provide a relative measure of the amount of cavitation that occurred in each aggregation trial. Regression analysis shows a weak correlation (r(2) = 0.11) between aggregation and ultrasound intensity, but a substantial correlation (r(2) = 0.76) between aggregation and cavitation occurrence.

Hemostasis of punctured vessels using Doppler-guided high-intensity ultrasound.

The use of Doppler ultrasound was investigated to determine if it would aid in guiding the application of high-intensity focused ultrasound (HIFU) to stop bleeding from punctured vessels. Major vessels (abdominal aorta, illiac, carotid, common femoral and superficial femoral arteries and the jugular vein) were surgically exposed, punctured and treated in anesthetized pigs. Treatment was applied when the Doppler sounds indicated the focus coincided with the bleeding site. In 89 treatment trials, the average time to achieve major hemostasis (a point where bleeding was reduced to a level of only oozing) was 8 s, and for complete hemostasis was 13 s. These times were significantly shorter than those of an identical former study in which only visual guidance was used. In that study, the average times for major and complete hemostasis were 40 and 62 s, respectively. The advantage of Doppler guidance in applying HIFU in treating bleeding vessels was demonstrated.

In vivo pressure measurements of lithotripsy shock waves in pigs.

Stone comminution and tissue damage in lithotripsy are sensitive to the acoustic field within the kidney, yet knowledge of shock waves in vivo is limited. We have made measurements of lithotripsy shock waves inside pigs with small hydrophones constructed of a 25-microm PVDF membrane stretched over a 21-mm diameter ring. A thin layer of silicone rubber was used to isolate the membrane electrically from pig fluid. A hydrophone was positioned around the pig kidney following a flank incision. Hydrophones were placed on either the anterior (shock wave entrance) or the posterior (shock wave exit) surface of the left kidney. Fluoroscopic imaging was used to orient the hydrophone perpendicular to the shock wave. For each pig, the voltage settings (12-24 kV) and the position of the shock wave focus within the kidney were varied. Waveforms measured within the pig had a shape very similar to those measured in water, but the peak pressure was about 70% of that in water. The focal region in vivo was 82 mm x 20 mm, larger than that measured in vitro (57 mm x 12 mm). It appeared that a combination of nonlinear effects and inhomogeneities in the tissue broadened the focus of the lithotripter. The shock rise time was on the order of 100 ns, substantially more than the rise time measured in water, and was attributed to higher absorption in tissue.

Hemostasis of punctured blood vessels using high-intensity focused ultrasound.

The hemorrhagic complications of vascular injury can be significant. We report on the use of high-intensity focused ultrasound (HIFU) to stop the hemorrhage of punctured blood vessels in pigs. Two HIFU transducers with frequencies of 3.5 and 2.0 MHz, each equipped with a water-filled conical housing, were used. Major blood vessels (femoral artery and vein, axillary artery, carotid artery and jugular vein), 2-10 mm in diameter, of anesthetized pigs were exposed surgically and punctured with 14- and 18-gauge needles to produce moderate to profuse bleeding. Complete hemostasis was achieved in less than 3 min of HIFU treatment in most blood vessels, and all vessels were patent after the treatment. Both HIFU frequencies were effective in producing hemostasis. Gross examination of the HIFU-treated vessels showed a consistent hardening of the soft tissue surrounding the blood vessels, providing a seal for the puncture hole. Microscopic examination of the vessels showed a remarkably localized HIFU treatment, resulting in coagulation of the adventitia, and an extensive fibrin network around the vessels and in the puncture hole. The vessel walls exhibited focal swelling, without evidence of irreversible injury. HIFU may provide a useful method for achieving hemostasis of punctured and traumatized blood vessels in a variety of clinical settings.

Real-time visualization of high-intensity focused ultrasound treatment using ultrasound imaging.

High-intensity focused ultrasound (HIFU) and conventional B-mode ultrasound (US) imaging were synchronized to develop a system for real-time visualization of HIFU treatment. The system was tested in vivo in pig liver. The HIFU application resulted in the appearance of a hyperechoic spot at the focus that faded gradually after cessation of HIFU exposure. The duration of HIFU exposure needed for a hyperechoic spot to appear, was inversely related to the HIFU intensity. The threshold intensity required to produce a hyperechoic spot in liver in < 1 s was 970 W/cm(2), in situ. At this HIFU dose, no immediate cellular damage was observed, providing a potential for pretreatment targeting. The real-time visualization method was used in hemostasis of actively bleeding internal pelvic vessels, allowing targeting and monitoring of successful treatment. Real-time US imaging may provide a useful tool for image-guided HIFU therapy.

Use of overpressure to assess the role of bubbles in focused ultrasound lesion shape in vitro.

Overpressure--elevated hydrostatic pressure--was used to assess the role of gas or vapor bubbles in distorting the shape and position of a high-intensity focused ultrasound (HIFU) lesion in tissue. The shift from a cigar-shaped lesion to a tadpole-shaped lesion can mean that the wrong area is treated. Overpressure minimizes bubbles and bubble activity by dissolving gas bubbles, restricting bubble oscillation and raising the boiling temperature. Therefore, comparison with and without overpressure is a tool to assess the role of bubbles. Dissolution rates, bubble dynamics and boiling temperatures were determined as functions of pressure. Experiments were made first in a low-overpressure chamber (0.7 MPa maximum) that permitted imaging by B-mode ultrasound (US). Pieces of excised beef liver (8 cm thick) were treated in the chamber with 3.5 MHz for 1 to 7 s (50% duty cycle). In situ intensities (I(SP)) were 600 to 3000 W/cm(2). B-mode US imaging detected a hyperechoic region at the HIFU treatment site. The dissipation of this hyperechoic region following HIFU cessation corresponded well with calculated bubble dissolution rates; thus, suggesting that bubbles were present. Lesion shape was then tested in a high-pressure chamber. Intensities were 1300 and 1750 W/cm(2) ( +/- 20%) at 1 MHz for 30 s. Hydrostatic pressures were 0.1 or 5.6 MPa. At 1300 W/cm(2), lesions were cigar-shaped, and no difference was observed between lesions formed with or without overpressure. At 1750 W/cm(2), lesions formed with no overpressure were tadpole-shaped, but lesions formed with high overpressure (5.6 MPa) remained cigar-shaped. Data support the hypothesis that bubbles contribute to the lesion distortion.