Ultrasonic Characterization of Ibidi µ-Slide I Luer Channel Slides for Studies With Ultrasound Contrast Agents.

Understanding and controlling the ultrasound contrast agent (UCA)'s response to an applied ultrasound pressure field are crucial when investigating ultrasound imaging sequences and therapeutic applications. The magnitude and frequency of the applied ultrasonic pressure waves affect the oscillatory response of the UCA. Therefore, it is important to have an ultrasound compatible and optically transparent chamber in which the acoustic response of the UCA can be studied. The aim of our study was to determine the in situ ultrasound pressure amplitude in the ibidi µ -slide I Luer channel, an optically transparent chamber suitable for cell culture, including culture under flow, for all microchannel heights (200, 400, 600, and [Formula: see text]). First, the in situ pressure field in the 800- [Formula: see text] high channel was experimentally characterized using Brandaris 128 ultrahigh-speed camera recordings of microbubbles (MBs) and a subsequent iterative processing method, upon insonification at 2 MHz, 45° incident angle, and 50-kPa peak negative pressure (PNP). Control studies in another cell culture chamber, the CLINIcell, were compared with the obtained results. The pressure amplitude was -3.7 dB with respect to the pressure field without the ibidi µ -slide. Second, using finite-element analysis, we determined the in situ pressure amplitude in the ibidi with the 800- [Formula: see text] channel (33.1 kPa), which was comparable to the experimental value (34 kPa). The simulations were extended to the other ibidi channel heights (200, 400, and [Formula: see text]) with either 35° or 45° incident angle, and at 1 and 2 MHz. The predicted in situ ultrasound pressure fields were between -8.7 and -1.1 dB of the incident pressure field depending on the listed configurations of ibidi slides with different channel heights, applied ultrasound frequencies, and incident angles. In conclusion, the determined ultrasound in situ pressures demonstrate the acoustic compatibility of the ibidi µ -slide I Luer for different channel heights, thereby showing its potential for studying the acoustic behavior of UCAs for imaging and therapy.

Intra-renal microcirculatory alterations on non-traumatic hemorrhagic shock induced acute kidney injury in pigs.

Acute kidney injury (AKI) is frequently seen in patients with hemorrhagic shock due to hypotension, tissue hypoxia, and inflammation despite adequate resuscitation. There is a lack of information concerning the alteration of renal microcirculation and perfusion during shock and resuscitation. The aim of this study was to investigate the possible role of renal microcirculatory alterations on development of renal dysfunction in a pig model of non-traumatic hemorrhagic shock (HS) induced AKI.Fully instrumented female pigs were divided into the two groups as Control (n = 6) and HS (n = 11). HS was achieved by withdrawing blood until mean arterial pressure (MAP) reached around 50 mmHg. After an hour cessation period, fluid resuscitation with balanced crystalloid was started for the duration of 1 h. The systemic and renal hemodynamics, renal microcirculatory perfusion (contrast-enhanced ultrasound (CEUS)) and the sublingual microcirculation were measured.CEUS peak enhancement was significantly increased in HS during shock, early-, and late resuscitation indicating perfusion defects in the renal cortex (p < 0.05 vs. baseline, BL) despite a stable renal blood flow (RBF) and urine output. Following normalization of systemic hemodynamics, we observed persistent hypoxia (high lactate) and high red blood cell (RBC) velocity just after initiation of resuscitation resulting in further endothelial and renal damage as shown by increased plasma sialic acid (p < 0.05 vs. BL) and NGAL levels. We also showed that total vessel density (TVD) and functional capillary density (FCD) were depleted during resuscitation (p < 0.05).In this study, we showed that the correction of systemic hemodynamic variables may not be accompanied with the improvement of renal cortical perfusion, intra-renal blood volume and renal damage following fluid resuscitation. We suggest that the measurement of renal injury biomarkers, systemic and renal microcirculation can be used for guiding to the optimization of fluid therapies.

Coupling Two Ultra-high-Speed Cameras to Elucidate Ultrasound Contrast-Mediated Imaging and Therapy.

Ultrasound contrast-mediated medical imaging and therapy both rely on the dynamics of micron- and nanometer-sized ultrasound cavitation nuclei, such as phospholipid-coated microbubbles and phase-change droplets. Ultrasound cavitation nuclei respond non-linearly to ultrasound on a nanosecond time scale that necessitates the use of ultra-high-speed imaging to fully visualize these dynamics in detail. In this study, we developed an ultra-high-speed optical imaging system that can record up to 20 million frames per second (Mfps) by coupling two small-sized, commercially available, 10-Mfps cameras. The timing and reliability of the interleaved cameras needed to achieve 20 Mfps was validated using two synchronized light-emitting diode strobe lights. Once verified, ultrasound-activated microbubble responses were recorded and analyzed. A unique characteristic of this coupled system is its ability to be reconfigured to provide orthogonal observations at 10 Mfps. Acoustic droplet vaporization was imaged from two orthogonal views, by which the 3-D dynamics of the phase transition could be visualized. This optical imaging system provides the temporal resolution and experimental flexibility needed to further elucidate the dynamics of ultrasound cavitation nuclei to potentiate the clinical translation of ultrasound-mediated imaging and therapy developments.

Dispersing and Sonoporating Biofilm-Associated Bacteria with Sonobactericide.

Bacteria encased in a biofilm poses significant challenges to successful treatment, since both the immune system and antibiotics are ineffective. Sonobactericide, which uses ultrasound and microbubbles, is a potential new strategy for increasing antimicrobial effectiveness or directly killing bacteria. Several studies suggest that sonobactericide can lead to bacterial dispersion or sonoporation (i.e., cell membrane permeabilization); however, real-time observations distinguishing individual bacteria during and directly after insonification are missing. Therefore, in this study, we investigated, in real-time and at high-resolution, the effects of ultrasound-induced microbubble oscillation on Staphylococcus aureus biofilms, without or with an antibiotic (oxacillin, 1 µg/mL). Biofilms were exposed to ultrasound (2 MHz, 100-400 kPa, 100-1000 cycles, every second for 30 s) during time-lapse confocal microscopy recordings of 10 min. Bacterial responses were quantified using post hoc image analysis with particle counting. Bacterial dispersion was observed as the dominant effect over sonoporation, resulting from oscillating microbubbles. Increasing pressure and cycles both led to significantly more dispersion, with the highest pressure leading to the most biofilm removal (up to 83.7%). Antibiotic presence led to more variable treatment responses, yet did not significantly impact the therapeutic efficacy of sonobactericide, suggesting synergism is not an immediate effect. These findings elucidate the direct effects induced by sonobactericide to best utilize its potential as a biofilm treatment strategy.

Internalization of targeted microbubbles by endothelial cells and drug delivery by pores and tunnels.

Ultrasound insonification of microbubbles can locally enhance drug delivery by increasing the cell membrane permeability. To aid development of a safe and effective therapeutic microbubble, more insight into the microbubble-cell interaction is needed. In this in vitro study we aimed to investigate the initial 3D morphology of the endothelial cell membrane adjacent to individual microbubbles (n = 301), determine whether this morphology was affected upon binding and by the type of ligand on the microbubble, and study its influence on microbubble oscillation and the drug delivery outcome. High-resolution 3D confocal microscopy revealed that targeted microbubbles were internalized by endothelial cells, while this was not the case for non-targeted or IgG1-κ control microbubbles. The extent of internalization was ligand-dependent, since αvß3-targeted microbubbles were significantly more internalized than CD31-targeted microbubbles. Ultra-high-speed imaging (~17 Mfps) in combination with high-resolution confocal microscopy (n = 246) showed that microbubble internalization resulted in a damped microbubble oscillation upon ultrasound insonification (2 MHz, 200 kPa peak negative pressure, 10 cycles). Despite damped oscillation, the cell's susceptibility to sonoporation (as indicated by PI uptake) was increased for internalized microbubbles. Monitoring cell membrane integrity (n = 230) showed the formation of either a pore, for intracellular delivery, or a tunnel (i.e. transcellular perforation), for transcellular delivery. Internalized microbubbles caused fewer transcellular perforations and smaller pore areas than non-internalized microbubbles. In conclusion, studying microbubble-mediated drug delivery using a state-of-the-art imaging system revealed receptor-mediated microbubble internalization and its effect on microbubble oscillation and resulting membrane perforation by pores and tunnels.

Theranostic Microbubbles with Homogeneous Ligand Distribution for Higher Binding Efficacy.

Phospholipid-coated targeted microbubbles are used for ultrasound molecular imaging and locally enhanced drug delivery, with the binding efficacy being an important trait. The use of organic solvent in microbubble production makes the difference between a heterogeneous or homogeneous ligand distribution. This study demonstrates the effect of ligand distribution on the binding efficacy of phospholipid-coated ανß3-targeted microbubbles in vitro using a monolayer of human umbilical-vein endothelial cells and in vivo using chicken embryos. Microbubbles with a homogeneous ligand distribution had a higher binding efficacy than those with a heterogeneous ligand distribution both in vitro and in vivo. In vitro, 1.55× more microbubbles with a homogeneous ligand distribution bound under static conditions, while this was 1.49× more under flow with 1.25 dyn/cm2, 1.56× more under flow with 2.22 dyn/cm2, and 1.25× more in vivo. The in vitro dissociation rate of bound microbubbles with homogeneous ligand distribution was lower at low shear stresses (1-5 dyn/cm2). The internalized depth of bound microbubbles was influenced by microbubble size, not by ligand distribution. In conclusion, for optimal binding the use of organic solvent in targeted microbubble production is preferable over directly dispersing phospholipids in aqueous medium.

Hydroxyl Ethyl Starch (HES) Preserves Intrarenal Microcirculatory Perfusion Shown by Contrast-Enhanced Ultrasound (Ceus), and Renal Function in a Severe Hemodilution Model in Pigs.

ABSTRACT: Acute normovolemic hemodilution (ANH) is associated with low oxygen carrying capacity of blood and purposed to cause renal injury in perioperative setting. It is best accomplished in a perioperative setting by a colloid such as hydroxyl ethyl starch (HES) due its capacity to fill the vascular compartment and maintain colloidal pressure. However, alterations of intra renal microvascular perfusion, flow and its effects on renal function and damage during ANH has not been sufficiently clarified. Based on the extensive use of HES in the perioperative setting we tested the hypothesis that the use of HES during ANH is able to perfuse the kidney microcirculation adequately without causing renal dysfunction and injury in pigs. Hemodilution (n = 8) was performed by stepwise replacing blood with HES to hematocrit (Hct) levels of 20% (T1), 15% (T2), and 10% (T3). Seven control animals were investigated. Systemic and renal hemodynamics were monitored. Renal microcirculatory perfusion was visualized and quantified using contrast-enhanced ultrasound (CEUS) and laser speckle imaging (LSI). In addition, sublingual microcirculation was measured by handheld vital microscopy (HVM). Intrarenal mean transit time of ultrasound contrast agent (IRMTT-CEUS) was reduced in the renal cortex at Hct 10% in comparison to control at T3 (1.4 ±â€Š0.6 vs. 2.2 ±â€Š0.7 seconds, respectively, P < 0.05). Although renal function was preserved, the serum neutrophil gelatinase-associated lipocalin (NGAL) levels was higher at Hct 10% (0.033 ±â€Š0.004 pg/µg protein) in comparison to control at T3 (0.021 ±â€Š0.002 pg/µg protein. A mild correlation between CO and IRMTT (renal RBC velocity) (r -0.53; P = 0.001) and CO and NGAL levels (r 0.66; P = 0.001) was also found. Our results show that HES induced ANH is associated with a preserved intra renal blood volume, perfusion, and function in the clinical range of Hct (<15%). However, at severely low Hct (10%) ANH was associated with renal injury as indicated by increased NGAL levels. Changes in renal microcirculatory flow (CEUS and LSI) followed those seen in the sublingual microcirculation measured with HVM.

Vancomycin-decorated microbubbles as a theranostic agent for Staphylococcus aureus biofilms.

Bacterial biofilms are a huge burden on our healthcare systems worldwide. The lack of specificity in diagnostic and treatment possibilities result in difficult-to-treat and persistent infections. The aim of this in vitro study was to investigate if microbubbles targeted specifically to bacteria in biofilms could be used both for diagnosis as well for sonobactericide treatment and demonstrate their theranostic potential for biofilm infection management. The antibiotic vancomycin was chemically coupled to the lipid shell of microbubbles and validated using mass spectrometry and high-axial resolution 4Pi confocal microscopy. Theranostic proof-of-principle was investigated by demonstrating the specific binding of vancomycin-decorated microbubbles (vMB) to statically and flow grown Staphylococcus aureus (S. aureus) biofilms under increasing shear stress flow conditions (0-12 dyn/cm2), as well as confirmation of microbubble oscillation and biofilm disruption upon ultrasound exposure (2 MHz, 250 kPa, and 5,000 or 10,000 cycles) during flow shear stress of 5 dyn/cm2 using time-lapse confocal microscopy combined with the Brandaris 128 ultra-high-speed camera. Vancomycin was successfully incorporated into the microbubble lipid shell. vMB bound significantly more often than control microbubbles to biofilms, also in the presence of free vancomycin (up to 1000 µg/mL) and remained bound under increasing shear stress flow conditions (up to 12 dyn/cm2). Upon ultrasound insonification biofilm area was reduced of up to 28%, as confirmed by confocal microscopy. Our results confirm the successful production of vMB and support their potential as a new theranostic tool for S. aureus biofilm infections by allowing for specific bacterial detection and biofilm disruption.

Phospholipid-coated targeted microbubbles for ultrasound molecular imaging and therapy.

Phospholipid-coated microbubbles are ultrasound contrast agents that, when functionalized, adhere to specific biomarkers on cells. In this concise review, we highlight recent developments in strategies for targeting the microbubbles and their use for ultrasound molecular imaging (UMI) and therapy. Recently developed novel targeting strategies include magnetic functionalization, triple targeting, and the use of several new ligands. UMI is a powerful technique for studying disease progression, diagnostic imaging, and monitoring of therapeutic responses. Targeted microbubbles (tMBs) have been used for the treatment of cardiovascular diseases and cancer, with therapeutics either coadministered or loaded onto the tMBs. Regardless of which disease was treated, the use of tMBs always resulted in a better therapeutic outcome than non-tMBs when compared in vitro or in vivo.

High Frame Rate Volumetric Imaging of Microbubbles Using a Sparse Array and Spatial Coherence Beamforming.

Volumetric ultrasound imaging of blood flow with microbubbles enables a more complete visualization of the microvasculature. Sparse arrays are ideal candidates to perform volumetric imaging at reduced manufacturing complexity and cable count. However, due to the small number of transducer elements, sparse arrays often come with high clutter levels, especially when wide beams are transmitted to increase the frame rate. In this study, we demonstrate with a prototype sparse array probe and a diverging wave transmission strategy, that a uniform transmission field can be achieved. With the implementation of a spatial coherence beamformer, the background clutter signal can be effectively suppressed, leading to a signal to background ratio improvement of 25 dB. With this approach, we demonstrate the volumetric visualization of single microbubbles in a tissue-mimicking phantom as well as vasculature mapping in a live chicken embryo chorioallantoic membrane.

Photoacoustic flow velocity imaging based on complex field decorrelation.

Photoacoustic (PA) imaging can be used to monitor flowing blood inside the microvascular and capillary bed. Ultrasound speckle decorrelation based velocimetry imaging was previously shown to accurately estimate blood flow velocity in mouse brain (micro-)vasculature. Translating this method to photoacoustic imaging will allow simultaneous imaging of flow velocity and extracting functional parameters like blood oxygenation. In this study, we use a pulsed laser diode and a quantitative method based on normalized first order field autocorrelation function of PA field fluctuations to estimate flow velocities in an ink tube phantom and in the microvasculature of the chorioallantoic membrane of a chicken embryo. We demonstrate how the decorrelation time of signals acquired over frames are related to the flow speed and show that the PA flow analysis based on this approach is an angle independent flow velocity imaging method.

Paracetamol modulates biofilm formation in Staphylococcus aureus clonal complex 8 strains.

Staphylococcus aureus biofilms are a major problem in modern healthcare due to their resistance to immune system defenses and antibiotic treatments. Certain analgesic agents are able to modulate S. aureus biofilm formation, but currently no evidence exists if paracetamol, often combined with antibiotic treatment, also has this effect. Therefore, we aimed to investigate if paracetamol can modulate S. aureus biofilm formation. Considering that certain regulatory pathways for biofilm formation and virulence factor production by S. aureus are linked, we further investigated the effect of paracetamol on immune modulator production. The in vitro biofilm mass of 21 S. aureus strains from 9 genetic backgrounds was measured in the presence of paracetamol. Based on biofilm mass quantity, we further investigated paracetamol-induced biofilm alterations using a bacterial viability assay combined with N-Acetylglucosamine staining. Isothermal microcalorimetry was used to monitor the effect of paracetamol on bacterial metabolism within biofilms and green fluorescent protein (GFP) promoter fusion technology for transcription of staphylococcal complement inhibitor (SCIN). Clinically relevant concentrations of paracetamol enhanced biofilm formation particularly among strains belonging to clonal complex 8 (CC8), but had minimal effect on S. aureus planktonic growth. The increase of biofilm mass can be attributed to the marked increase of N-Acetylglucosamine containing components of the extracellular matrix, presumably polysaccharide intercellular adhesion. Biofilms of RN6390A (CC8) showed a significant increase in the immune modulator SCIN transcription during co-incubation with low concentrations of paracetamol. Our data indicate that paracetamol can enhance biofilm formation. The clinical relevance needs to be further investigated.

Corrections to "Microbubble Composition and Preparation for High-Frequency Contrast-Enhanced Ultrasound Imaging: In Vitro and In Vivo Evaluation".

In the above article [1], the authors regret that there was a mistake in calculating the mol% of the microbubble coating composition used. For all experiments, the unit in mg/mL was utilized and the conversion mistake only came when converting to mol% in order to define the ratio between the coating formulation components. The correct molecular weight of PEG-40 stearate is 2046.54 g/mol [2], [3], not 328.53 g/mol. On page 556, Table I should read as shown here.

The Preparation of Chicken Ex Ovo Embryos and Chorioallantoic Membrane Vessels as In Vivo Model for Contrast-Enhanced Ultrasound Imaging and Microbubble-Mediated Drug Delivery Studies.

The chicken embryo and the blood-vessel rich chorioallantoic membrane (CAM) is a valuable in vivo model to investigate biomedical processes, new ultrasound pulsing schemes, or novel transducers for contrast-enhanced ultrasound imaging and microbubble-mediated drug delivery. The reasons for this are the accessibility of the embryo and vessel network of the CAM as well as the low costs of the model. An important step to get access to the embryo and CAM vessels is to take the egg content out of the eggshell. In this protocol, three methods for taking the content out of the eggshell between day 5 and 8 of incubation are described thus allowing the embryos to develop inside the eggshell up to these days. The described methods only require simple tools and equipment and yield a higher survival success rate of 90% for 5-day, 75% for 6-day, 50% for 7-day, and 60% for 8-day old incubated eggs in comparison to ex ovo cultured embryos (~50%). The protocol also describes how to inject cavitation nuclei, such as microbubbles, into the CAM vascular system, how to separate the membrane containing the embryo and CAM from the rest of the egg content for optically transparent studies, and how to use the chicken embryo and CAM in a variety of short-term ultrasound experiments. The in vivo chicken embryo and CAM model is extremely relevant to investigate novel imaging protocols, ultrasound contrast agents, and ultrasound pulsing schemes for contrast-enhanced ultrasound imaging, and to unravel the mechanisms of ultrasound-mediated drug delivery.

Corrections to "Vibrational Responses of Bound and Nonbound Targeted Lipid-Coated Single Microbubbles".

In the above article [1], the authors regret that there was a mistake in calculating the mol% of the microbubble coating composition. For all experiments, the unit in mg/mL was utilized and the conversion mistake only came when converting to mol% in order to define the ratio between the coating formulation components. The correct molecular weight of PEG-40 stearate is 2046.54 g/mol [2], [3], not 328.53 g/mol. On page 786, paragraph II-A, it should read "The coating was composed of 84.8 mol% DSPC (P6517, Sigma-Aldrich, Zwijndrecht, The Netherlands) or DPPC (850355, Avanti Polar Lipids, Alabaster, AL, USA); 8.2 mol% polyoxyethylene-40-stearate (PEG-40 stearate, P3440, Sigma-Aldrich); 5.9 mol% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG(2000), 880125, Avanti Polar Lipids); and 1.1 mol% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (DSPE-PEG(2000)-biotin, 880129, Avanti Polar Lipids)."

Corrections to "Targeted Microbubble Mediated Sonoporation of Endothelial Cells In Vivo".

In the above article [1], the authors regret that there was a mistake in calculating the mol% of the microbubble coating composition. For all experiments, the unit in mg/mL was utilized and the conversion mistake only came when converting to mol% in order to define the ratio between the coating formulation components. The correct molecular weight of PEG-40 stearate is 2046.54 g/mol [2], [3], not 328.53 g/mol. On page 1661, paragraph II-A, it should read "The coating was composed of DSPC (84.8 mol%; P 6517; Sigma-Aldrich, Zwijndrecht, The Netherlands);PEG-40 stearate (8.2 mol%; P 3440; Sigma-Aldrich); DSPE-PEG(2000) (5.9 mol%; 880125 P; Avanti Polar Lipids, Alabaster, AL, USA); and DSPE-PEG(2000)-biotin (1.1 mol%; 880129 C; Avanti Polar Lipids)".