In-field use of I-VED electrical impedance sensor for assessing post-dive decompression stress in humans.

Purpose: Ultrasound imaging is commonly used in decompression research to assess venous gas emboli (VGE) post-dive, with higher loads associated with increased decompression sickness risk. This work examines, for the first time in humans, the performance of a novel electrical impedance spectroscopy technology (I-VED), on possible detection of post-dive bubbles presence and arterial endothelial dysfunction that may be used as markers of decompression stress. Methods: I-VED signals were recorded in scuba divers who performed standardized pool dives before and at set time points after their dives at 35-minute intervals for about two hours. Two distinct frequency components of the obtained signals, Low-Pass Frequency-LPF: 0-0.5 Hz and Band-Pass Frequency-BPF: 0.5-10 Hz, are extracted and respectively compared to VGE presence and known flow-mediated dilation trends for the same dive profile for endothelial dysfunction. Results: Subjects with VGE counts above the median for all subjects were found to have an elevated average LPF compared to subjects with lower VGE counts, although this was not statistically significant (p=0.06), as well as significantly decreased BPF standard deviation post-dive compared to pre-dive (p=0.008). Conclusions: I-VED was used for the first time in humans and operated to provide qualitative in-vivo electrical impedance measurements that may contribute to the assessment of decompression stress. Compared to ultrasound imaging, the proposed method is less expensive, not operator-dependent and compatible with continuous monitoring and application of multiple probes. This study provided preliminary insights; further calibration and validation are necessary to determine I-VED sensitivity and specificity.

Development of a graphical user interface for automatic separation of human voice from Doppler ultrasound audio in diving experiments.

Doppler ultrasound (DU) is used in decompression research to detect venous gas emboli in the precordium or subclavian vein, as a marker of decompression stress. This is of relevance to scuba divers, compressed air workers and astronauts to prevent decompression sickness (DCS) that can be caused by these bubbles upon or after a sudden reduction in ambient pressure. Doppler ultrasound data is graded by expert raters on the Kisman-Masurel or Spencer scales that are associated to DCS risk. Meta-analyses, as well as efforts to computer-automate DU grading, both necessitate access to large databases of well-curated and graded data. Leveraging previously collected data is especially important due to the difficulty of repeating large-scale extreme military pressure exposures that were conducted in the 70-90s in austere environments. Historically, DU data (Non-speech) were often captured on cassettes in one-channel audio with superimposed human speech describing the experiment (Speech). Digitizing and separating these audio files is currently a lengthy, manual task. In this paper, we develop a graphical user interface (GUI) to perform automatic speech recognition and aid in Non-speech and Speech separation. This constitutes the first study incorporating speech processing technology in the field of diving research. If successful, it has the potential to significantly accelerate the reuse of previously-acquired datasets. The recognition task incorporates the Google speech recognizer to detect the presence of human voice activity together with corresponding timestamps. The detected human speech is then separated from the audio Doppler ultrasound within the developed GUI. Several experiments were conducted on recently digitized audio Doppler recordings to corroborate the effectiveness of the developed GUI in recognition and separations tasks, and these are compared to manual labels for Speech timestamps. The following metrics are used to evaluate performance: the average absolute differences between the reference and detected Speech starting points, as well as the percentage of detected Speech over the total duration of the reference Speech. Results have shown the efficacy of the developed GUI in Speech/Non-speech component separation.

Effect of Anesthetic Carrier Gas on In Vivo Circulation Times of Intravenously Administered Phospholipid Oxygen Microbubbles in Rats.

OBJECTIVE: For the treatment of tumor hypoxia, microbubbles comprising oxygen as a majority component of the gas core with a stabilizing shell may be used to deliver and release oxygen locally at the tumor site through ultrasound destruction. Previous work has revealed differences in circulation half-life in vivo for perfluorocarbon-filled microbubbles, typically used as ultrasound imaging contrast agents, as a function of anesthetic carrier gas. These differences in circulation time in vivo were likely due to gas diffusion as a function of anesthetic carrier gas, among other variables. This work has motivated studies to evaluate the effect of anesthetic carrier gas on oxygen microbubble circulation dynamics. METHODS: Circulation time for oxygen microbubbles was derived from ultrasound image intensity obtained during longitudinal kidney imaging. Studies were constructed for rats anesthetized on inhaled isoflurane with either pure oxygen or medical air as the anesthetic carrier gas. RESULTS: Results indicated that oxygen microbubbles were highly visible via contrast-specific imaging. Marked signal enhancement and duration differences were observed between animals breathing air and oxygen. Perhaps counterintuitively, oxygen microbubbles disappeared from circulation significantly faster when the animals were breathing pure oxygen compared with medical air. This may be explained by nitrogen counterdiffusion from blood into the bubble, effectively changing the gas composition of the core, as has been observed in perfluorocarbon core microbubbles. CONCLUSION: Our findings suggest that the apparent longevity and persistence of oxygen microbubbles in circulation may not be reflective of oxygen delivery when the animal is anesthetized breathing air.

Overcoming biological barriers to improve treatment of a Staphylococcus aureus wound infection.

Chronic wounds frequently become infected with bacterial biofilms which respond poorly to antibiotic therapy. Aminoglycoside antibiotics are ineffective at treating deep-seated wound infections due to poor drug penetration, poor drug uptake into persister cells, and widespread antibiotic resistance. In this study, we combat the two major barriers to successful aminoglycoside treatment against a biofilm-infected wound: limited antibiotic uptake and limited biofilm penetration. To combat the limited antibiotic uptake, we employ palmitoleic acid, a host-produced monounsaturated fatty acid that perturbs the membrane of gram-positive pathogens and induces gentamicin uptake. This novel drug combination overcomes gentamicin tolerance and resistance in multiple gram-positive wound pathogens. To combat biofilm penetration, we examined the ability of sonobactericide, a non-invasive ultrasound-mediated-drug delivery technology to improve antibiotic efficacy using an in vivo biofilm model. This dual approach dramatically improved antibiotic efficacy against a methicillin-resistant Staphylococcus aureus (MRSA) wound infection in diabetic mice.

An open-source framework for synthetic post-dive Doppler ultrasound audio generation.

Doppler ultrasound (DU) measurements are used to detect and evaluate venous gas emboli (VGE) formed after decompression. Automated methodologies for assessing VGE presence using signal processing have been developed on varying real-world datasets of limited size and without ground truth values preventing objective evaluation. We develop and report a method to generate synthetic post-dive data using DU signals collected in both precordium and subclavian vein with varying degrees of bubbling matching field-standard grading metrics. This method is adaptable, modifiable, and reproducible, allowing for researchers to tune the produced dataset for their desired purpose. We provide the baseline Doppler recordings and code required to generate synthetic data for researchers to reproduce our work and improve upon it. We also provide a set of pre-made synthetic post-dive DU data spanning six scenarios representing the Spencer and Kisman-Masurel (KM) grading scales as well as precordial and subclavian DU recordings. By providing a method for synthetic post-dive DU data generation, we aim to improve and accelerate the development of signal processing techniques for VGE analysis in Doppler ultrasound.

A fully automated algorithm for heart rate detection in post-dive precordial Doppler ultrasound.

Background: Doppler ultrasound is used currently in decompression research for the evaluation of venous gas emboli (VGE). Estimation of heart rate from post-dive Doppler ultrasound recordings can provide a tool for the evaluation of physiological changes from decompression stress, as well as aid in the development of automated VGE detection algorithms that relate VGE presence to cardiac activity. Method: An algorithm based on short-term autocorrelation was developed in MATLAB to estimate the heart rate in post-dive precordial Doppler ultrasound. The algorithm was evaluated on 21 previously acquired and labeled precordial recordings spanning Kisman-Masurel (KM) codes of 111-444 (KM I-IV) with manually derived instantaneous heart rates. Results: A window size of at least two seconds was necessary for robust and accurate instantaneous heart rate estimation with a mean error of 1.56 ± 7.10 bpm. Larger window sizes improved the algorithm performance, at the cost of beat-to-beat heart rate estimates. We also found that our algorithm provides good results for low KM grade Doppler recordings with and without flexion, and high KM grades without flexion. High KM grades observed after movement produced the greatest mean absolute error of 6.12 ± 8.40 bpm. Conclusion: We have developed a fully automated algorithm for the estimation of heart rate in post-dive precordial Doppler ultrasound recordings.

Deep Learning-Based Venous Gas Emboli Grade Classification in Doppler Ultrasound Audio Recordings.

OBJECTIVE: Doppler ultrasound (DU) is used to detect venous gas emboli (VGE) post dive as a marker of decompression stress for diving physiology research as well as new decompression procedure validation to minimize decompression sickness risk. In this article, we propose the first deep learning model for VGE grading in DU audio recordings. METHODS: A database of real-world data was assembled and labeled for the purpose of developing the algorithm, totaling 274 recordings comprising both subclavian and precordial measurements. Synthetic data was also generated by acquiring baseline DU signals from human volunteers and superimposing laboratory-acquired DU signals of bubbles flowing in a tissue mimicking material. A novel squeeze-and-excitation deep learning model was designed to effectively classify recordings on the 5-class Spencer scoring system used by trained human raters. RESULTS: On the real-data test set, we show that synthetic data pretraining achieves average ordinal accuracy of 84.9% for precordial and 90.4% for subclavian DU which is a 24.6% and 26.2% increase over training with real-data and time-series augmentation only. The weighted kappa coefficients of agreement between the model and human ground truth were 0.74 and 0.69 for precordial and subclavian respectively, indicating substantial agreement similar to human inter-rater agreement for this type of data. CONCLUSION: The present work demonstrates the first application of deep-learning for DU VGE grading using a combination of synthetic and real-world data. SIGNIFICANCE: The proposed method can contribute to accelerating DU analysis for decompression research.

Hyperbaric exposure in rodents with non-invasive imaging assessment of decompression bubbles: A scoping review protocol.

Hyperbaric pressure experiments have provided researchers with valuable insights into the effects of pressure changes, using various species as subjects. Notably, extensive work has been done to observe rodents subjected to hyperbaric pressure, with differing imaging modalities used as an analytical tool. Decompression puts subjects at a greater risk for injury, which often justifies conducting such experiments using animal models. Therefore, it is important to provide a broad view of previously utilized methods for decompression research to describe imaging tools available for researchers to conduct rodent decompression experiments, to prevent duplicate experimentation, and to identify significant gaps in the literature for future researchers. Through a scoping review of published literature, we will provide an overview of decompression bubble information collected from rodent experiments using various non-invasive methods of ultrasound for decompression bubble assessment. This review will adhere to methods outlined by the Joanna Briggs Institute Manual for Evidence Synthesis and be reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses for Scoping Reviews (PRISMA-ScR). Literature will be obtained from the PubMed, Embase, and Scopus databases. Extracted sources will first be sorted to a list for inclusion based on title and abstract. Two independent researchers will then conduct full-text screening to further refine included papers to those relevant to the scope. The final review manuscript will cover methods, data, and findings for each included publication relevant to non-invasive in vivo bubble imaging.

Comparison of Newer Hand-Held Ultrasound Devices for Post-Dive Venous gas Emboli Quantification to Standard Echocardiography.

Decompression sickness (DCS) can result from the growth of bubbles in tissues and blood during or after a reduction in ambient pressure, for example in scuba divers, compressed air workers or astronauts. In scuba diving research, post-dive bubbles are detectable in the venous circulation using ultrasound. These venous gas emboli (VGE) are a marker of decompression stress, and larger amounts of VGE are associated with an increased probability of DCS. VGE are often observed for hours post-dive and differences in their evolution over time have been reported between individuals, but also for the same individual, undergoing a same controlled exposure. Thus, there is a need for small, portable devices with long battery lives to obtain more ultrasonic data in the field to better assess this inter- and intra-subject variability. We compared two new handheld ultrasound devices against a standard device that is currently used to monitor post-dive VGE in the field. We conclude that neither device is currently an adequate replacement for research studies where precise VGE grading is necessary.

Perspective on ultrasound bioeffects and possible implications for continuous post-dive monitoring safety.

Ultrasound monitoring, both in the form of Doppler and 2D echocardiography, has been used post-dive to detect decompression bubbles circulating in the bloodstream. With large variability in both bubble time course and loads, it has been hypothesised that shorter periods between imaging, or even continuous imaging, could provide more accurate post-dive assessments. However, while considering applications of ultrasound imaging post-decompression, it may also be prudent to consider the possibility of ultrasound-induced bioeffects. Clinical ultrasound studies using microbubble contrast agents have shown bioeffect generation with acoustic powers much lower than those used in post-dive monitoring. However, to date no studies have specifically investigated potential bioeffect generation from continuous post-dive echocardiography. This review discusses what can be drawn from the current ultrasound and diving literature on the safety of bubble sonication and highlights areas where more studies are needed. An overview of the ultrasound-bubble mechanisms that lead to bioeffects and analyses of ultrasound contrast agent studies on bioeffect generation in the pulmonary and cardiovascular systems are provided to illustrate how bubbles under ultrasound can cause damage within the body. Along with clinical ultrasound studies, studies investigating the effects of decompression bubbles under ultrasound are analysed and open questions regarding continuous post-dive monitoring safety are discussed.

Effect of Acoustic Parameters and Microbubble Concentration on the Likelihood of Encapsulated Microbubble Coalescence.

Microbubble contrast agents are commonly used for therapeutic and diagnostic imaging applications. Under certain conditions, these contrast agents can coalesce on ultrasound application and form larger bubbles than the initial population. The formation of large microbubbles potentially influences therapeutic outcomes and imaging quality. We studied clinically relevant ultrasound parameters related to low-pressure therapy and contrast-enhanced ultrasound imaging to determine their effect on microbubble coalescence and subsequent changes in microbubble size distributions in vitro. Results indicate that therapeutic ultrasound at low frequencies, moderate pressures and high duty cycles are capable of forming bubbles greater than two times larger than the initial bubble distribution. Furthermore, acoustic parameters related to contrast-enhanced ultrasound imaging that are at higher frequency, low-pressure and low-duty cycle exhibit no statistically significant changes in bubble diameter, suggesting that standard contrast ultrasound imaging does not cause coalescence. Overall, this work suggests that the microbubble coalescence phenomenon can readily occur at acoustic parameters used in therapeutic ultrasound, generating bubbles much larger than those found in commercial contrast agents, although coalescence is unlikely to be significant in diagnostic contrast-enhanced ultrasound imaging. This observation warrants further expansion of parameter ranges and investigation of resulting effects.

Harnessing ultrasound-stimulated phase change contrast agents to improve antibiotic efficacy against methicillin-resistant Staphylococcus aureus biofilms.

Bacterial biofilms, often associated with chronic infections, respond poorly to antibiotic therapy and frequently require surgical intervention. Biofilms harbor persister cells, metabolically indolent cells, which are tolerant to most conventional antibiotics. In addition, the biofilm matrix can act as a physical barrier, impeding diffusion of antibiotics. Novel therapeutic approaches frequently improve biofilm killing, but usually fail to achieve eradication. Failure to eradicate the biofilm leads to chronic and relapsing infection, is associated with major financial healthcare costs and significant morbidity and mortality. We address this problem with a two-pronged strategy using 1) antibiotics that target persister cells and 2) ultrasound-stimulated phase-change contrast agents (US-PCCA), which improve antibiotic penetration. We previously demonstrated that rhamnolipids, produced by Pseudomonas aeruginosa, could induce aminoglycoside uptake in gram-positive organisms, leading to persister cell death. We have also shown that US-PCCA can transiently disrupt biological barriers to improve penetration of therapeutic macromolecules. We hypothesized that combining antibiotics which target persister cells with US-PCCA to improve drug penetration could improve treatment of methicillin resistant S. aureus (MRSA) biofilms. Aminoglycosides alone or in combination with US-PCCA displayed limited efficacy against MRSA biofilms. In contrast, the anti-persister combination of rhamnolipids and aminoglycosides combined with US-PCCA dramatically improved biofilm killing. This novel treatment strategy has the potential for rapid clinical translation as the PCCA formulation is a variant of FDA-approved ultrasound contrast agents that are already in clinical practice and the low-pressure ultrasound settings used in our study can be achieved with existing ultrasound hardware at pressures below the FDA set limits for diagnostic imaging.

An echo from the past: Building a Doppler repository for big data in diving research.

Decompression sickness (DCS) remains a major operational concern for diving operations, submarine escape and high-altitude jumps. Aside from DCS symptoms, venous gas emboli (VGE) detected with ultrasound post-dive are often used as a marker of decompression stress in humans, with a specificity of 100% even though the sensitivity is poor [1]. Being non-invasive, portable and non-ionizing, ultrasound is particularly suited to regular and repeated monitoring. It could help elucidate inter- and intra-subject variability in VGE and DCS susceptibility, but analyzing these recordings remains a cumbersome task [2].

Ultrasound in decompression research: fundamentals, considerations, and future technologies.

It is widely accepted that bubbles are a necessary but insufficient condition for the development of decompression sickness. However, open questions remain regarding the precise formation and behavior of these bubbles after an ambient pressure reduction (decompression), primarily due to the inherent difficulty of directly observing this phenomenon in vivo. In decompression research, information about these bubbles after a decompression is gathered via means of ultrasound acquisitions. The ability to draw conclusions regarding decompression research using ultrasound is highly influenced by the variability of the methodologies and equipment utilized by different research groups. These differences play a significant role in the quality of the data and thus the interpretation of the results. The purpose of this review is to provide a technical overview of the use of ultrasound in decompression research, particularly Doppler and brightness (B)-mode ultrasound. Further, we will discuss the strengths and limitations of these technologies and how new advancements are improving our ability to understand bubble behavior post-decompression.

A fully automated method for late ventricular diastole frame selection in post-dive echocardiography without ECG gating.

Venous gas emboli (VGE) are often quantified as a marker of decompression stress on echocardiograms. Bubble-counting has been proposed as an easy to learn method, but remains time-consuming, rendering large dataset analysis impractical. Computer automation of VGE counting following this method has therefore been suggested as a means to eliminate rater bias and save time. A necessary step for this automation relies on the selection of a frame during late ventricular diastole (LVD) for each cardiac cycle of the recording. Since electrocardiograms (ECG) are not always recorded in field experiments, here we propose a fully automated method for LVD frame selection based on regional intensity minimization. The algorithm is tested on 20 previously acquired echocardiography recordings (from the original bubble-counting publication), half of which were acquired at rest (Rest) and the other half after leg flexions (Flex). From the 7,140 frames analyzed, sensitivity was found to be 0.913 [95% CI: 0.875-0.940] and specificity 0.997 [95% CI: 0.996-0.998]. The method's performance is also compared to that of random chance selection and found to perform significantly better (p≺0.0001). No trend in algorithm performance was found with respect to VGE counts, and no significant difference was found between Flex and Rest (p>0.05). In conclusion, full automation of LVD frame selection for the purpose of bubble counting in post-dive echocardiography has been established with excellent accuracy, although we caution that high quality acquisitions remain paramount in retaining high reliability.

Contrast-Enhanced Sonography with Biomimetic Lung Surfactant Nanodrops.

Nanodrops comprising a perfluorocarbon liquid core can be acoustically vaporized into echogenic microbubbles for ultrasound imaging. Packaging the microbubble in its condensed liquid state provides some advantages, including in situ activation of the acoustic signal, longer circulation persistence, and the advent of expanded diagnostic and therapeutic applications in pathologies which exhibit compromised vasculature. One obstacle to clinical translation is the inability of the limited surfactant present on the nanodrop to encapsulate the greatly expanded microbubble interface, resulting in ephemeral microbubbles with limited utility. In this study, we examine a biomimetic approach to stabilize an expanding gas surface by employing the lung surfactant replacement, beractant. Lung surfactant contains a suite of lipids and proteins that provide efficient shuttling of material from bilayer folds to the monolayer surface. We hypothesized that beractant would improve stability of acoustically vaporized microbubbles. To test this hypothesis, we characterized beractant surface dilation mechanics and revealed a novel biophysical phenomenon of rapid interfacial melting, spreading, and resolidification. We then harnessed this unique functionality to increase the stability and echogenicity of microbubbles produced after acoustic droplet vaporization for in vivo ultrasound imaging. Such biomimetic lung surfactant-stabilized nanodrops may be useful for applications in ultrasound imaging and therapy.

Phospholipid Oxygen Microbubbles for Image-Guided Therapy.

In recent work, oxygen microbubbles (OMB) have been shown to oxygenate hypoxic tumors, increase radio-sensitivity and improve tumor control by radiation therapy. Compared to intra-tumoral injection, intravenous delivery of adjuvant agents such as OMBs for radiotherapy offers an attractive means of achieving true theranostic function in a minimally invasive manner via contrast-enhanced ultrasound (CEUS), while reducing the risk of injury, infection or displacing tumor cells. However, short intravascular circulation times with conventional DSPC-lipid OMBs may lead to premature off-target dissolution of OMBs with an associated reduction in tumoral oxygen delivery. Prior work on microbubble stability and gas exchange suggests that increasing phospholipid acyl-chain length of the encapsulating shell and OMB size may increase circulation persistence, delivery and dissolved oxygen content. In the following studies, we investigate the effect of two phospholipid shell compositions, DSPC (C18:0) and DBPC (C22:0), as well as three size distributions (0.5-2 µm, 2-10 µm and polydisperse) on OMB circulation persistence utilizing CEUS in the kidneys of live C57B1/6 male and female mice, six weeks of age. DBPC OMB formulations demonstrated increased circulation half-lives versus DSPC formulations (2.4 ± 1.0 vs. 0.6 ± 0.5 s, p<0.01 for 2-10 µm), as well as an increased maximum intensity by over tenfold (p<0.01). Size-dependent effects remained consistent across both formulations with larger 2-10 µm microbubbles demonstrating significantly increased half-lives (2.4 ± 1.0 vs. 0.3 ± 0.2 s, p < 0.01) compared to smaller 0.5-2 µm formulations of DBPC. These studies indicate that DBPC 2-10 µm OMBs may be improved adjuvant agents for radiotherapy with significant potential for CEUS interrogation.

Ultrasound-Stimulated Phase-Change Contrast Agents for Transepithelial Delivery of Macromolecules, Toward Gastrointestinal Drug Delivery.

The gastrointestinal (GI) tract presents a notoriously difficult barrier for macromolecular drug delivery, especially for biologics. Herein, we demonstrate that ultrasound-stimulated phase change contrast agents (PCCAs) can transiently disrupt confluent colorectal adenocarcinoma monolayers and improve the transepithelial transport of a macromolecular model drug. With ultrasound treatment in the presence of PCCAs, we achieved a maximum of 44 ± 15% transepithelial delivery of 70-kDa fluorescein isothiocyanate-dextran, compared with negligible delivery through sham control monolayers. Among all tested rarefactional pressures (300-600 kPa), dextran delivery efficiency was consistently greatest at 300 kPa. To explore this unexpected finding, we quantified stable and inertial cavitation energy generated by various ultrasound exposure conditions. In general, lower pressures resulted in more persistent cavitation activity during the 30-s ultrasound exposures, which may explain the enhanced dextran delivery efficiency. Thus, a unique advantage of using low boiling point PCCAs for this application is that the same low-pressure pulses can be used to induce vaporization and provide maximal delivery.

Ultrasound multiple scattering with microbubbles can differentiate between tumor and healthy tissue in vivo.

Most solid tumors are characterized by highly dense, isotropic vessel networks. Characterization of such features has shown promise for early cancer diagnosis. Ultrasound diffusion has been used to characterize the micro-architecture of complex media, such as bone and the lungs. In this work, we examine a non-invasive diffusion-based ultrasound technique to assess neo-vascularization. Because the diffusion constant reflects the density of scatterers in heterogeneous media, we hypothesize that by injecting microbubbles into the vasculature, ultrasound diffusivity can reflect vascular density (VD), thus differentiating the microvascular patterns between tumors and healthy tissue. The diffusion constant and its anisotropy are shown to be significantly different between fibrosarcoma tumors (n = 16) and control tissue (n = 18) in a rat animal model in vivo. The diffusion constant values for control and tumor were found to be 1.38 ± 0.51 mm2 µs-1 and 0.65 ± 0.27 mm2 µs-1, respectively. These results are corroborated with VD from acoustic angiography (AA) data, confirming increased vessel density in tumors compared to controls. The diffusion constant offers a promising way to quantitatively assess vascular networks when combined with contrast agents, which may allow early tumor detection and characterization.