Absence of COVID-19-associated changes in plasma coagulation proteins and pulmonary thrombosis in the ferret model.

BACKGROUND: Many patients who are diagnosed with coronavirus disease 2019 (COVID-19) suffer from venous thromboembolic complications despite the use of stringent anticoagulant prophylaxis. Studies on the exact mechanism(s) underlying thrombosis in COVID-19 are limited as animal models commonly used to study venous thrombosis pathophysiology (i.e. rats and mice) are naturally not susceptible to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Ferrets are susceptible to SARS-CoV-2 infection, successfully used to study virus transmission, and have been previously used to study activation of coagulation and thrombosis during influenza virus infection. OBJECTIVES: This study aimed to explore the use of (heat-inactivated) plasma and lung material from SARS-CoV-2-inoculated ferrets studying COVID-19-associated changes in coagulation and thrombosis. MATERIAL AND METHODS: Histology and longitudinal plasma profiling using mass spectrometry-based proteomics approach was performed. RESULTS: Lungs of ferrets inoculated intranasally with SARS-CoV-2 demonstrated alveolar septa that were mildly expanded by macrophages, and diffuse interstitial histiocytic pneumonia. However, no macroscopical or microscopical evidence of vascular thrombosis in the lungs of SARS-CoV-2-inoculated ferrets was found. Longitudinal plasma profiling revealed minor differences in plasma protein profiles in SARS-CoV-2-inoculated ferrets up to 2 weeks post-infection. The majority of plasma coagulation factors were stable and demonstrated a low coefficient of variation. CONCLUSIONS: We conclude that while ferrets are an essential and well-suited animal model to study SARS-CoV-2 transmission, their use to study SARS-CoV-2-related changes relevant to thrombotic disease is limited.

Measurements of bubble-enhanced heating from focused, MHz-frequency ultrasound in a tissue-mimicking material.

Time-resolved measurements of the temperature field in an agar-based tissue-mimicking phantom insonated with a large aperture 1-MHz focused acoustic transducer are reported. The acoustic pressure amplitude and insonation duration were varied. Above a critical threshold acoustic pressure, a large increase in the temperature rise during insonation was observed. Evidence for the hypothesis that cavitation bubble activity in the focal zone is the cause of enhanced heating is presented and discussed. Mechanisms for bubble-assisted heating are presented and modeled, and quantitative estimates for the thermal power generated by viscous dissipation and bubble acoustic radiation are given.

Comparisons of sonoluminescence from single-bubbles and cavitation fields: bridging the gap.

Sonoluminescence (SL) refers to the generation of light through the energetic pulsations of acoustic cavitation bubbles in a liquid. For years, SL was observed primarily in cavitation fields. These bubbles are believed by many to undergo near-adiabatic compression, resulting in the heating of the bubble contents and the subsequent emission of light. Recently, researchers have discovered a 'new' form of sonoluminescence in which light is observed to emanate from a single bubble undergoing very large volume excursions. The mechanism for light production is unknown, but many believe it is due to a rapid heating of the central core by an imploding shock wave. Based in part on the emission time scales, there is a common belief that the two forms of SL are quite distinct. We address this issue by comparing the two phenomena with regards to their light-flash durations and emission spectra--leading to some surprising differences and similarities.

In vitro detection of cavitation induced by a diagnostic ultrasound system.

Experiments were performed to determine whether a clinical diagnostic scanner, a Hewlett-Packard (HP) 77020A, could produce cavitation in water containing suspensions of either 0.245-mum polystyrene spheres or Albunex, 1-10 mum albumin-coated microbubbles. Two calibrated, phased-array HP imaging transducers with 2.5- and 5.0-MHz operating frequencies were driven in M-mode (single cycle) and Doppler mode 4 cycles by the HP imaging system. Cavitation was detected in the water with polystyrene spheres at 2.5 MHz in both M-mode and Doppler mode at a peak negative acoustic pressure of 1.1 MPa or greater. Insonification at 5.0 MHz in either mode did not produce a detectable amount of cavitation, even with peak negative pressures as high as 1.2 MPa. Cavitation was not detected in water with the Albunex spheres at either frequency.

Amplitude degradation of time-reversed pulses in nonlinear absorbing thermoviscous fluids.

The linear wave equation in a lossless medium is time reversible, i.e., every solution p(x, t) has a temporal mirror solution p(x, -t). Analysis shows that time reversal also holds for the lossless nonlinear wave equation. In both cases, time-reversal invariance is violated when losses are present. For nonlinear propagation loses cannot normally be ignored; they are necessary to prevent the occurrence of multivalued waveforms. Further analysis of the nonlinear wave equation shows that amplification of a time-reversed pulse at the array elements also leads to a violation of time reversal even for lossless nonlinear acoustics. Numerical simulations are used to illustrate the effect of nonlinearity on the ability of a time-reversal system to effectively focus on a target in an absorbing fluid medium. We consider both the amplitude and arrival time of retrodirected pulses. The numerical results confirm that both shock generation (with the accompanying absorption) and amplification at the array, adversely affect the ability of a time-reversal system to form strong retrodirective sound fields.

Thresholds for cavitation produced in water by pulsed ultrasound.

The threshold for transient cavitation produced in water by pulsed ultrasound was measured as a function of pulse duration and pulse repetition frequency at both 0.98 and 2.30 MHz. The cavitation events were detected with a passive acoustic technique which relies upon the scattering of the irradiation field by the bubble clouds associated with the events. The results indicate that the threshold is independent of pulse duration and acoustic frequency for pulses longer than approximately 10 acoustic cycles. The threshold increases for shorter pulses. The cavitation events are likely to be associated with bubble clouds rather than single bubbles.

Role of acoustic cavitation in the delivery and monitoring of cancer treatment by high-intensity focused ultrasound (HIFU).

Acoustic cavitation has been shown to play a key role in a wide array of novel therapeutic ultrasound applications. This paper presents a brief discussion of the physics of thermally relevant acoustic cavitation in the context of high-intensity focussed ultrasound (HIFU). Models for how different types of cavitation activity can serve to accelerate tissue heating are presented, and results suggest that the bulk of the enhanced heating effect can be attributed to the absorption of broadband acoustic emissions generated by inertial cavitation. Such emissions can be readily monitored using a passive cavitation detection (PCD) scheme and could provide a means for real-time treatment monitoring. It is also shown that the appearance of hyperechoic regions (or bright-ups) on B-mode ultrasound images constitutes neither a necessary nor a sufficient condition for inertial cavitation activity to have occurred during HIFU exposure. Once instigated at relatively large HIFU excitation amplitudes, bubble activity tends to grow unstable and to migrate toward the source transducer, causing potentially undesirable pre-focal damage. Potential means of controlling inertial cavitation activity using pulsed excitation so as to confine it to the focal region are presented, with the intention of harnessing cavitation-enhanced heating for optimal HIFU treatment delivery. The role of temperature elevation in mitigating bubble-enhanced heating effects is also discussed, along with other bubble-field effects such as multiple scattering and shielding.

Mechanical characterization of microparticles by scattered ultrasound.

A technique for determining the compressibility and density of individual microparticles in suspension is described. The particles have diameters on the order of 10 microns Ultrasonic tone bursts of 2-microseconds duration and 30-MHz center frequency scatter from individual particles as they traverse the confocal zone of two transducers. The resulting scattered tone bursts are detected at 90 degrees and 180 degrees (backscattering). The received rf signals are demodulated, peak detected, digitized, and stored in computer memory. Using Rayleigh scattering theory, the compressibility and density of a particle can be computed given knowledge of the particle size and host fluid properties. Results of experiments with latex microspheres are presented and compared with calculations based on long-wavelength (Rayleigh) and elastic scattering theory.

Some observations on the breakage of ultrasonic files driven piezoelectrically.

The incidence of breakage of Piezon-Master ultrasonic K files were evaluated. Three groups of unused files were subjected to three treatments, namely; free vibration in air without irrigation, free vibration in root canal while minimizing contact with the wall of canal in the presence of irrigation and light filing in root canal with free flow of irrigation. Cavitation produced by files in contact and free of contact with a glass surface was examined in order to observe the relationship between cavitation defects and breakage. In addition, the fractured and unfractured files were examined under a scanning electron microscope for the presence of cavitation pits. The results indicated that more files broke in air. In water, a higher incidence of breakage occurred when files were allowed to freely vibrate while no breakage occurred when the files were used in filing. All files generated cavitation which resulted in pitting of their surfaces. However, it was considered unlikely that the pits contributed to fracture. Fatigue cracks which could be the result of the manufacturing process were observed at some of the corners of the cross sections of the fractured files and could be the main contributory factor to fracture.

Dynamics of gas bubbles in viscoelastic fluids. II. Nonlinear viscoelasticity.

The nonlinear oscillations of a spherical, acoustically forced gas bubble in nonlinear viscoelastic media are examined. The constitutive equation [Upper-Convective Maxwell (UCM)] used for the fluid is suitable for study of large-amplitude excursions of the bubble, in contrast to the previous work of the authors which focused on the smaller amplitude oscillations within a linear viscoelastic fluid [J. S. Allen and R. A. Roy, J. Acoust. Soc. Am. 107, 3167-3178 (2000)]. Assumptions concerning the trace of the stress tensor are addressed in light of the incorporation of viscoelastic constitutive equations into bubble dynamics equations. The numerical method used to solve the governing system of equations (one integrodifferential equation and two partial differential equations) is outlined. An energy balance relation is used to monitor the accuracy of the calculations and the formulation is compared with the previously developed linear viscoelastic model. Results are found to agree in the limit of small deformations; however, significant divergence for larger radial oscillations is noted. Furthermore, the inherent limitations of the linear viscoelastic approach are explored in light of the more complete nonlinear formulation. The relevance and importance of this approach to biomedical ultrasound applications are highlighted. Preliminary results indicate that tissue viscoelasticity may be an important consideration for the risk assessment of potential cavitation bioeffects.

Observations of acoustic streaming fields around an oscillating ultrasonic file.

The steady acoustic streaming generated around straight and precurved oscillating ultrasonic files driven by the Piezon-Master 400 unit was examined in the free field and in small channels using a stereomicroscope. In addition, the effect of file-wall contact on streaming production was also investigated. The results indicated that the ultrasonic files can generate acoustic streaming both in the free field and in the small channel. Higher velocity streaming was observed when smaller size files were employed and when the file was precurved. Light file-wall contact did not totally inhibit streaming while severe file-wall contact inhibited movement of the file and, as a result, no streaming was observed. The positions and length scales of the streaming vortices appeared to be influenced by the presence of boundaries. In the free field, two rows of vortices were situated along the sides of the file while in the small channel, the vortices were positioned above the surface of the file. These results indicated that it is possible for acoustic streaming to occur in a confined space as in a root canal provided that severe file-wall contact is avoided. It is therefore recommended that light filing or allowing the file to freely vibrate during some stage of treatment should be carried out in order to generate streaming in the root canal.

Physical mechanisms governing the hydrodynamic response of an oscillating ultrasonic file.

Ultrasonically driven vibrating files are known to enhance the efficiency of root canal debridement. This paper presents a phenomenological view of the hydrodynamic response of an oscillating ultrasonic file and the relationship between the file response and various physical factors such as file size and curvature, file surface properties, file velocity amplitude, root canal geometry, and the type of irrigant. Relevant hydrodynamic properties include the propensity of a file to produce stable and transient cavitation, steady streaming, and cavitation microstreaming. These relationships were explored by experiment. Sonoluminescence was employed as an indicator of transient cavitation activity and photographic analysis was utilized as a means for detecting steady streaming, microstreaming, and stable cavitation. Measurements failed to indicate any strong correlation between registered driving power and the propensity to produce transient cavitation. Files that were pitted or possessed salient edges were very effective at generating transient cavitation. When observed, transient cavitation activity generally occurred near the tip of the straight file, provided the wall-loading did not inhibit file motion. In all cases studied, steady streaming and stable cavitation were observed to varying degrees, depending on the amount of file to wall contact. Stable cavitation was probably enhanced by the addition of moderate amounts of dissolved gas into the irrigant. Although the imposition of file-wall contact served to inhibit the production of transient cavitation, this action had relatively little effect on the ability of a file to produce a nominal level of streaming, microstreaming, and stable cavitation. The relationship between these hydrodynamic properties and the process of root canal debridement is addressed. Observations suggest that it is not prudent to ascribe enhanced cleaning effects to any one phenomenon, for it is likely that several factors are involved to varying degrees depending on the local conditions of application.

Variations in power output of the Piezon-Master 400 ultrasonic endodontic unit.

The present study was undertaken to see if there was any variability in the power output of Piezon-Master 400 ultrasonic files when driven using different generators, tranducers and file holders. The displacement amplitude of the oscillating tip of the file in air was used as a measure of the power output. The results showed that there was considerable variability in the power output of Piezon-Master 400 ultrasonic files of similar size and length when driven using different generators, transducers and file holders. In consideration of this, it is recommended that a calibration device be incorporated in the ultrasonic unit so that the operator will have some knowledge of when the unit is working at its maximum efficiency.

An acoustic backscattering technique for the detection of transient cavitation produced by microsecond pulses of ultrasound.

An acoustic backscattering technique for detecting transient cavitation produced by 10-microseconds-long pulses of 757-kHz ultrasound is described. The system employs 10-microseconds-long, 30-MHz center frequency tone bursts that scatter from cavitation microbubbles. Experiments were performed with suspensions of hydrophobic polystyrene spheres in ultraclean water. Transient cavitation threshold pressures measured with the active cavitation detector (ACD) were always less than or equal to those measured using a passive acoustic detection scheme. The measured cavitation thresholds decreased with increasing dissolved gas content and increasing suspended particle concentration. Results also show that ultrasonic irradiation of the polystyrene sphere suspensions by the ACD lowered the threshold pressure measured with the passive detector. A possible mechanism through which suspensions of hydrophobic particles might nucleate bubbles is presented.

Acoustic microcavitation: its active and passive acoustic detection.

In this work acoustic microcavitation in water is studied primarily at 0.75 MHz and 1% duty cycle. To detect cavitation, two kinds of acoustic detectors are used. The first one is an unfocused, untuned 1-MHz receiver transducer that serves as a passive detector. The other one is a focused 30-MHz transducer that is used in pulse-echo mode and is called the active detector. Cavitation itself is brought about by a focused PZT-8 crystal driven in pulse mode. The active detector is arranged confocally with respect to the cavitation transducer. Both the interrogating pulse and the cavitation pulse arrive simultaneously at the common focus, which is the region of cavitation. With the test chamber filled with clean water, no cavitation is observed, even when the cavitation transducer is driven to give its peak output of 22 bar peak negative. Cavitation is, however, observed when polystyrene microparticles are added to the host water. Our view of how these smooth, spherical, monodispersed microparticles give rise to cavitation is described with some estimates. An attempt has been made to understand whether the presence of "streaming" affects the thresholds, and it has been found that the active detector field affects the cavitation process.

The vibratory pattern of ultrasonic files driven piezoelectrically.

The pattern of oscillation of a Piezon-Master 400 ultrasonic file driven by a piezoelectric transducer was studied in air and on water. In addition, the displacement amplitudes of the files were measured. The findings were compared with those observed with the Cavi-Endo unit reported in another study (Ahmad 1969). It was observed that the file vibrated such that a standing wave was formed on the file and it exhibited points of maximum deflection (antinode) and points of minimum deflection (node) with the largest deflection occurring at the apical end. This pattern of oscillation was similar to that exhibited by the Cavi-Endo file which employed a magnetostrictive transducer. However, the displacement amplitudes were very much higher than those exhibited by the Cavi-Endo. It is considered that the 120 degrees angle of the file holder inherent in the Piezon-Master 400 unit and the more effective power transmission with the piezoelectric transducer may have contributed to the large amplitudes.

A precise technique for the measurement of acoustic cavitation thresholds and some preliminary results.

A description is given of a precise technique for measuring the threshold for acoustic cavitation inception. The system, which is automated so as to remove operator involvement, utilizes a slow ramping of the acoustic pressure amplitude until cavitation occurs. The detection criterion is the generation of a sufficiently intense sonoluminescent signal. Measurements made in filtered water show a well-defined, reproducible, and stable cavitation threshold. Measurements of the dependence of the threshold on filter size, on time, and on the concentration of dissolved ions for various salts are also presented. Many of these results appear anomalous.

Acoustic cavitation produced by microsecond pulses of ultrasound: a discussion of some selected results.

Because of its extensive utilization in clinical practice, and because the subjects examined are often fragile and sensitive to trauma, the safety of diagnostic ultrasound has always been of concern. Of the various mechanisms through which ultrasound could act in a manner deleterious to a patient, acoustic cavitation, should it occur, appears to possess significant potential for biological damage. This paper reviews several recent reports of progress by our two groups and demonstrates the conditions under which cavitation has been observed by microsecond pulses of ultrasound. Although these results give no indications that diagnostic ultrasound may pose a true risk to a patient, they do indicate that in vivo cavitation may occur under certain conditions.