A functional ultrasound brain GPS for automatic vascular-based neuronavigation.

Recent advances in ultrasound imaging triggered by transmission of ultrafast plane waves have rendered functional ultrasound (fUS) imaging a valuable neuroimaging modality capable of mapping cerebral vascular networks, but also for the indirect capture of neuronal activity with high sensitivity thanks to the neurovascular coupling. However, the expansion of fUS imaging is still limited by the difficulty to identify cerebral structures during experiments based solely on the Doppler images and the shape of the vessels. In order to tackle this challenge, this study introduces the vascular brain positioning system (BPS), a GPS of the brain. The BPS is a whole-brain neuronavigation system based on the on-the-fly automatic alignment of ultrafast ultrasensitive transcranial Power Doppler volumic images to common templates such as the Allen Mouse Brain Common Coordinates Framework. This method relies on the online registration of the complex cerebral vascular fingerprint of the studied animal to a pre-aligned reference vascular atlas, thus allowing rapid matching and identification of brain structures. We quantified the accuracy of the automatic registration using super-resolution vascular images obtained at the microscopic scale using Ultrasound Localization Microscopy and found a positioning error of 44 µm and 96 µm for intra-animal and inter-animal vascular registration, respectively. The proposed BPS approach outperforms the manual vascular landmark recognition performed by expert neuroscientists (inter-annotator errors of 215 µm and 259 µm). Using the online BPS approach coupled with the Allen Atlas, we demonstrated the capability of the system to position itself automatically over chosen anatomical structures and to obtain corresponding functional activation maps even in complex oblique planes. Finally, we show that the system can be used to acquire and estimate functional connectivity matrices automatically. The proposed functional ultrasound on-the-fly neuronavigation approach allows automatic brain navigation and could become a key asset to ensure standardized experiments and protocols for non-expert and expert researchers.

Functional ultrasound imaging of the spreading activity following optogenetic stimulation of the rat visual cortex.

Optogenetics has revolutionized neurosciences by allowing fine control of neuronal activity. An important aspect for this control is assessing the activation and/or adjusting the stimulation, which requires imaging the entire volume of optogenetically-induced neuronal activity. An ideal technique for this aim is fUS imaging, which allows one to generate brain-wide activation maps with submesoscopic spatial resolution. However, optical stimulation of the brain with blue light might lead to non-specific activations at high irradiances. fUS imaging of optogenetic activations can be obtained at these wavelengths using lower light power (< 2mW) but it limits the depth of directly activatable neurons from the cortical surface. Our main goal was to report that we can detect specific optogenetic activations in V1 even in deep layers following stimulation at the cortical surface. Here, we show the possibility to detect deep optogenetic activations in anesthetized rats expressing the red-shifted opsin ChrimsonR in V1 using fUS imaging. We demonstrate the optogenetic specificity of these activations and their neuronal origin with electrophysiological recordings. Finally, we show that the optogenetic response initiated in V1 spreads to downstream (LGN) and upstream (V2) visual areas.

A simple novel approach for detecting blood-brain barrier permeability using GPCR internalization.

AIMS: Impairment of blood-brain barrier (BBB) is involved in numerous neurological diseases from developmental to aging stages. Reliable imaging of increased BBB permeability is therefore crucial for basic research and preclinical studies. Today, the analysis of extravasation of exogenous dyes is the principal method to study BBB leakage. However, these procedures are challenging to apply in pups and embryos and may appear difficult to interpret. Here we introduce a novel approach based on agonist-induced internalization of a neuronal G protein-coupled receptor widely distributed in the mammalian brain, the somatostatin receptor type 2 (SST2). METHODS: The clinically approved SST2 agonist octreotide (1 kDa), when injected intraperitoneally does not cross an intact BBB. At sites of BBB permeability, however, OCT extravasates and induces SST2 internalization from the neuronal membrane into perinuclear compartments. This allows an unambiguous localization of increased BBB permeability by classical immunohistochemical procedures using specific antibodies against the receptor. RESULTS: We first validated our approach in sensory circumventricular organs which display permissive vascular permeability. Through SST2 internalization, we next monitored BBB opening induced by magnetic resonance imaging-guided focused ultrasound in murine cerebral cortex. Finally, we proved that after intraperitoneal agonist injection in pregnant mice, SST2 receptor internalization permits analysis of BBB integrity in embryos during brain development. CONCLUSIONS: This approach provides an alternative and simple manner to assess BBB dysfunction and development in different physiological and pathological conditions.

Quantitative imaging of coronary flows using 3D ultrafast Doppler coronary angiography.

Coronary flow rate remains complex to assess in clinical practice using non-invasive, non-ionizing imaging tools. In this study, we introduce 3D ultrafast Doppler coronary angiography (3D UDCA), an ultrasound-based method to assess coronary blood flows in three-dimensions at high volume-rate and in one single heartbeat. We demonstrate that 3D UDCA can visualize the coronary vasculature with high temporal and spatial resolution and quantify the absolute flow. The feasibility of the technique was demonstrated in an open-chest swine model. The flow rate of the left-anterior descending artery (LAD) assessed by 3D UDCA was reconstructed successfully at the early diastolic and late diastolic phases and was in good agreement with an invasive gold-standard flowmeter during baseline, reactive hyperemia and coronary stenosis (r2 = 0.84). Finally, we demonstrate that a coronary stenosis on the LAD can be visualized as well as its associated flow acceleration.

4D simultaneous tissue and blood flow Doppler imaging: revisiting cardiac Doppler index with single heart beat 4D ultrafast echocardiography.

The goal of this study was to demonstrate the feasibility of semi-automatic evaluation of cardiac Doppler indices in a single heartbeat in human hearts by performing 4D ultrafast echocardiography with a dedicated sequence of 4D simultaneous tissue and blood flow Doppler imaging. 4D echocardiography has the potential to improve the quantification of major cardiac indices by providing more reproducible and less user dependent measurements such as the quantification of left ventricle (LV) volume. The evaluation of Doppler indices, however, did not benefit yet from 4D echocardiography because of limited volume rates achieved in conventional volumetric color Doppler imaging but also because spectral Doppler estimation is still restricted to a single location. High volume rate (5200 volume s-1) transthoracic simultaneous tissue and blood flow Doppler acquisitions of three human LV were performed using a 4D ultrafast echocardiography scanner prototype during a single heartbeat. 4D color flow, 4D tissue Doppler cineloops and spectral Doppler at each voxel were computed. LV outflow tract, mitral inflow and basal inferoseptal locations were automatically detected. Doppler indices were derived at these locations and were compared against clinical 2D echocardiography. Blood flow Doppler indices E (early filling), A (atrial filling), E/A ratio, S (systolic ejection) and cardiac output were assessed on the three volunteers. Simultaneous tissue Doppler indices e' (mitral annular velocity peak), a' (late velocity peak), e'/a' ratio, s' (systolic annular velocity peak), E/e' ratio were also estimated. Standard deviations on three independent acquisitions were averaged over the indices and was found to be inferior to 4% and 8.5% for Doppler flow and tissue Doppler indices, respectively. Comparison against clinical 2D echocardiography gave a p  value larger than 0.05 in average indicating no significant differences. 4D ultrafast echocardiography can quantify the major cardiac Doppler indices in a single heart beat acquisition.

A versatile and robust microfluidic device for capillary-sized simple or multiple emulsions production.

Ultrasound-vaporizable microdroplets can be exploited for targeted drug delivery. However, it requires customized microfluidic techniques able to produce monodisperse, capillary-sized and biocompatible multiple emulsions. Recent development of microfluidic devices led to the optimization of microdroplet production with high yields, low polydispersity and well-defined diameters. So far, only few were shown to be efficient for simple droplets or multiple emulsions production below 5 µm in diameter, which is required to prevent microembolism after intravenous injection. Here, we present a versatile microchip for both simple and multiple emulsion production. This parallelized system based on microchannel emulsification was designed to produce perfluorocarbon in water or water within perfluorocarbon in water emulsions with capillary sizes (<5 µm) and polydispersity index down to 5% for in vivo applications such as spatiotemporally-triggered drug delivery using Ultrasound. We show that droplet production at this scale is mainly controlled by interfacial tension forces, how capillary and viscosity ratios influence droplet characteristics and how different production regimes may take place. The better understanding of droplet formation and its relation to applied pressures is supported by observations with a high-speed camera. Compared to previous microchips, this device opens perspectives to produce injectable and biocompatible droplets with a reasonable yield in order to realize preclinical studies in mice.

A large aperture row column addressed probe for in vivo 4D ultrafast doppler ultrasound imaging.

Four-dimensional (4D) Ultrafast ultrasound imaging was recently proposed to image and quantify blood flow with high sensitivity in 3D as well as anatomical, mechanical or functional information. In 4D Ultrafast imaging, coherent compounding of tilted planes waves emitted by a 2D matrix array were used to image the medium at high volume rate. 4D ultrafast imaging, however, requires a high channel count (>1000) to drive those probes. Alternative approaches have been proposed and investigated to efficiently reduce the density of elements, such as sparse or under-sampled arrays while maintaining a decent image quality and high volume rate. The row-columns configuration presents the advantage of keeping a large active surface with a low amount of elements and a simple geometry. In this study, we investigate the row and column addressed (RCA) approach with the orthogonal plane wave (OPW) compounding strategy using real hardware limitations. We designed and built a large 7 MHz 128 + 128 probe dedicated to vascular imaging and connected to a 256-channel scanner to implement the OPW imaging scheme. Using this strategy, we demonstrate that 4D ultrafast Power Doppler imaging of a large volume of [Formula: see text] up to [Formula: see text] depth, both in vitro on flow phantoms and in vivo on the carotid artery of a healthy volunteer at a volume rate of 834 Hz.

Self-adaptive ultrasonic beam amplifiers: application to transcostal shock wave therapy.

Ultrasound shock wave therapy is increasingly used for non-invasive surgery. It requires the focusing of very high pressure amplitude in precisely controlled focal spots. In transcostal therapy of the heart or the liver, the high impedance mismatch between the bones and surrounding tissues gives rise to strong aberrations and attenuation of the therapeutic wavefront, with potential risks of injury at the tissue-bone interface. An adaptive propagation of the ultrasonic beam through the intercostal spaces would be required. Several solutions have been developed so far, but they require a prior knowledge of the patient's anatomy or an invasive calibration process, not applicable in clinic. Here, we develop a non-invasive adaptive focusing method for ultrasound therapy through the ribcage using a time reversal cavity (TRC) acting as an ultrasonic beam amplifier. This method is based on ribcage imaging through the TRC and a projection orthogonally to the strongest identified reflectors. The focal pressure of our device was improved by up to 30% using such self-adaptive processing, without degrading the focal spots size and shape. This improvement allowed lesion formation in an Ultracal® phantom through a ribcage without invasive calibration of the device. This adaptive method could be particularly interesting to improve the efficiency and the safety of pulsed cavitational therapy of the heart or the liver.

3D elastic tensor imaging in weakly transversely isotropic soft tissues.

We present herein 3D elastic tensor imaging (3D ETI), an ultrasound-based volumetric imaging technique to provide quantitative volumetric mapping of tissue elastic properties in weakly elastic anistropic media. The technique relies on (1) 4D ultrafast shear wave elastography (SWE) at very high volume rate (e.g. > 8000 Hz, depending only on the imaging depth), (2) a volumetric estimation of shear wave velocity using the eikonal equation and (3) a generalized 3D elastic tensor-based approach. 3D ETI was first evaluated using numerical simulations in homogeneous isotropic and transverse isotropic media. Results showed that 3D ETI can accurately assess tissue stiffness and tissue anisotropy in weakly transversely isotropic media (elastic fractional anisotropy coefficient < 0.34). Experimental feasibility was shown in vitro in a transverse isotropic phantom. Quantification of the elastic properties by 3D ETI was in good agreement with 2D SWE results performed at different orientations using a clinical ultrafast ultrasound scanner. 3D ETI has the potential to provide a volumetric quantitative map of tissue elastic properties in weakly transversely isotropic soft tissues within less than 20 ms of acquisition for the entire imaged volume.

Pulsed cavitational therapy using high-frequency ultrasound for the treatment of deep vein thrombosis in an in vitro model of human blood clot.

Post-thrombotic syndrome, a frequent complication of deep venous thrombosis, can be reduced with early vein recanalization. Pulsed cavitational therapy (PCT) using ultrasound is a recent non-invasive approach. We propose to test the efficacy and safety of high-frequency focused PCT for drug-free thrombolysis (thrombotripsy) in a realistic in vitro model of venous thrombosis. To reproduce venous thrombosis conditions, human whole blood was allowed to clot by stasis in silicone tubes (6 mm internal diameter) at a 30 cm H2O pressure, maintained during the whole experiment. We engineered an ultrasound device composed of dual 2.25 MHz transducers centered around a 6 MHz imaging probe. A therapeutic focus was generated at a 3.2 cm depth from the probe. Thrombotripsy was performed by longitudinally scanning the thrombus at three different speeds: 1 mm s-1 (n = 6); 2 mm s-1 (n = 6); 3 mm s-1 (n = 12). Restored outflow was measured every three passages. Filters were placed to evaluate the debris size. Twenty-four occlusive thrombi, of 2.5 cm mean length and 4.4 kPa mean stiffness, were studied. Flow restoration was systematically obtained by nine subsequent passages (4.5 min maximum). By varying the device's speed, we found an optimal speed of 1 mm s-1 to be efficient for effective recanalization with 90 s (three passages). Within 90 s, flow restoration was of 80, 62 and 74% at respectively 1, 2 and 3 mm s-1. For all groups, cavitation cloud drilled a 1.7 mm mean diameter channel throughout the clot. Debris analysis showed 92% of debris <10 µm, with no fragment > 200 µm.

A semi-analytical model of a time reversal cavity for high-amplitude focused ultrasound applications.

Time reversal cavities (TRC) have been proposed as an efficient approach for 3D ultrasound therapy. They allow the precise spatio-temporal focusing of high-power ultrasound pulses within a large region of interest with a low number of transducers. Leaky TRCs are usually built by placing a multiple scattering medium, such as a random rod forest, in a reverberating cavity, and the final peak pressure gain of the device only depends on the temporal length of its impulse response. Such multiple scattering in a reverberating cavity is a complex phenomenon, and optimisation of the device's gain is usually a cumbersome process, mostly empirical, and requiring numerical simulations with extremely long computation times. In this paper, we present a semi-analytical model for the fast optimisation of a TRC. This model decouples ultrasound propagation in an empty cavity and multiple scattering in a multiple scattering medium. It was validated numerically and experimentally using a 2D-TRC and numerically using a 3D-TRC. Finally, the model was used to determine rapidly the optimal parameters of the 3D-TRC which had been confirmed by numerical simulations.

Ex vivo optimisation of a heterogeneous speed of sound model of the human skull for non-invasive transcranial focused ultrasound at 1 MHz.

Transcranial brain therapy has recently emerged as a non-invasive strategy for the treatment of various neurological diseases, such as essential tremor or neurogenic pain. However, treatments require millimetre-scale accuracy. The use of high frequencies (typically ≥1 MHz) decreases the ultrasonic wavelength to the millimetre scale, thereby increasing the clinical accuracy and lowering the probability of cavitation, which improves the safety of the technique compared with the use of low-frequency devices that operate at 220 kHz. Nevertheless, the skull produces greater distortions of high-frequency waves relative to low-frequency waves. High-frequency waves require high-performance adaptive focusing techniques, based on modelling the wave propagation through the skull. This study sought to optimise the acoustical modelling of the skull based on computed tomography (CT) for a 1 MHz clinical brain therapy system. The best model tested in this article corresponded to a maximum speed of sound of 4000 m.s-1 in the skull bone, and it restored 86% of the optimal pressure amplitude on average in a collection of six human skulls. Compared with uncorrected focusing, the optimised non-invasive correction led to an average increase of 99% in the maximum pressure amplitude around the target and an average decrease of 48% in the distance between the peak pressure and the selected target. The attenuation through the skulls was also assessed within the bandwidth of the transducers, and it was found to vary in the range of 10 ± 3 dB at 800 kHz and 16 ± 3 dB at 1.3 MHz.

4D in vivo ultrafast ultrasound imaging using a row-column addressed matrix and coherently-compounded orthogonal plane waves.

4D ultrafast ultrasound imaging was recently shown using a 2D matrix (i.e. fully populated) connected to a 1024-channel ultrafast ultrasound scanner. In this study, we investigate the row-column addressing (RCA) matrix approach, which allows a reduction of independent channels from N × N to N + N, with a dedicated beamforming strategy for ultrafast ultrasound imaging based on the coherent compounding of orthogonal plane wave (OPW). OPW is based on coherent compounding of plane wave transmissions in one direction with receive beamforming along the orthogonal direction and its orthogonal companion sequence. Such coherent recombination of complementary orthogonal sequences leads to the virtual transmit focusing in both directions which results into a final isotropic point spread function (PSF). In this study, a 32 × 32 2D matrix array probe (1024 channels), centered at 5 MHz was considered. An RCA array, of same footprint with 32 + 32 elements (64 channels), was emulated by summing the elements either along a line or a column in software prior to beamforming. This approach allowed for the direct comparison of the 32 + 32 RCA scheme to the optimal fully sampled 32 × 32 2D matrix configuration, which served as the gold standard. This approach was first studied through PSF simulations and then validated experimentally on a phantom consisting of anechoic cysts and echogenic wires. The contrast-to-noise ratio and the lateral resolution of the RCA approach were found to be approximately equal to half (in decibel) and twice the values, respectively, obtained when using the 2D matrix approach. Results in a Doppler phantom and the human humeral artery in vivo confirmed that ultrafast Doppler imaging can be achieved with reduced performances when compared against the equivalent 2D matrix. Volumetric anatomic Doppler rendering and voxel-based pulsed Doppler quantification are presented as well. OPW compound imaging using emulated RCA matrix can achieve a power Doppler with sufficient contrast to recover the vein shape and provides an accurate Doppler spectrum.

A 3D time reversal cavity for the focusing of high-intensity ultrasound pulses over a large volume.

Shock wave ultrasound therapy techniques, increasingly used for non-invasive surgery, require extremely high pressure amplitudes in precise focal spots, and large high-power transducers arranged on a spherical shell are usually used to achieve that. This solution allows limited steering of the beam around the geometrical focus of the device at the cost of a large number of transducer elements, and the treatment of large and moving organs like the heart is challenging or impossible. This paper validates numerically and experimentally the possibility of using a time reversal cavity (TRC) for the same purpose. A 128-element, 1 MHz power transducer combined with different multiple scattering media in a TRC was used. We were able to focus high-power ultrasound pulses over a large volume in a controlled manner, with a limited number of transducer elements. We reached sufficiently high pressure amplitudes to erode an Ultracal® target over a 10 cm2 area.

In vivo real-time cavitation imaging in moving organs.

The stochastic nature of cavitation implies visualization of the cavitation cloud in real-time and in a discriminative manner for the safe use of focused ultrasound therapy. This visualization is sometimes possible with standard echography, but it strongly depends on the quality of the scanner, and is hindered by difficulty in discriminating from highly reflecting tissue signals in different organs. A specific approach would then permit clear validation of the cavitation position and activity. Detecting signals from a specific source with high sensitivity is a major problem in ultrasound imaging. Based on plane or diverging wave sonications, ultrafast ultrasonic imaging dramatically increases temporal resolution, and the larger amount of acquired data permits increased sensitivity in Doppler imaging. Here, we investigate a spatiotemporal singular value decomposition of ultrafast radiofrequency data to discriminate bubble clouds from tissue based on their different spatiotemporal motion and echogenicity during histotripsy. We introduce an automation to determine the parameters of this filtering. This method clearly outperforms standard temporal filtering techniques with a bubble to tissue contrast of at least 20 dB in vitro in a moving phantom and in vivo in porcine liver.

In vivo quantification of the shear modulus of the human Achilles tendon during passive loading using shear wave dispersion analysis.

The shear wave velocity dispersion was analyzed in the Achilles tendon (AT) during passive dorsiflexion using a phase velocity method in order to obtain the tendon shear modulus (C 55). Based on this analysis, the aims of the present study were (i) to assess the reproducibility of the shear modulus for different ankle angles, (ii) to assess the effect of the probe locations, and (iii) to compare results with elasticity values obtained with the supersonic shear imaging (SSI) technique. The AT shear modulus (C 55) consistently increased with the ankle dorsiflexion (N = 10, p < 0.05). Furthermore, the technique showed a very good reproducibility (all standard error of the mean values <10.7 kPa and all coefficient of variation (CV) values ⩽ 0.05%). In addition, independently from the ankle dorsiflexion, the shear modulus was significantly higher in the proximal location compared to the more distal one. The shear modulus provided by SSI was always lower than C55 and the difference increased with the ankle dorsiflexion. However, shear modulus values provided by both methods were highly correlated (R = 0.84), indicating that the conventional shear wave elastography technique (SSI technique) can be used to compare tendon mechanical properties across populations. Future studies should determine the clinical relevance of the shear wave dispersion analysis, for instance in the case of tendinopathy or tendon tear.

In Vivo Measurement of Brain Tumor Elasticity Using Intraoperative Shear Wave Elastography.

Purpose: Objective Shear wave elastography (SWE) enabled living tissue assessment of stiffness. This is routinely used for breast, thyroid and liver diseases, but there is currently no data for the brain. We aim to characterize elasticity of normal brain parenchyma and brain tumors using SWE. Materials and Methods: Patients with scheduled brain tumor removal were included in this study. In addition to standard ultrasonography, intraoperative SWE using an ultrafast ultrasonic device was used to measure the elasticity of each tumor and its surrounding normal brain. Data were collected by an investigator blinded to the diagnosis. Descriptive statistics, box plot analysis as well as intraoperator and interoperator reproducibility analysis were also performed. Results: 63 patients were included and classified into four main types of tumor: meningiomas, low-grade gliomas, high-grade gliomas and metastasis. Young's Modulus measured by SWE has given new insight to differentiate brain tumors: 33.1 ±â€Š5.9 kPa, 23.7 ±â€Š4.9 kPa, 11.4 ±â€Š3.6 kPa and 16.7 ±â€Š2.5 kPa, respectively, for the four subgroups. Normal brain tissue has been characterized by a reproducible mean stiffness of 7.3 ±â€Š2.1 kPa. Moreover, low-grade glioma stiffness is different from high-grade glioma stiffness (p = 0.01) and normal brain stiffness is very different from low-grade gliomas stiffness (p < 0.01). Conclusion: This study demonstrates that there are significant differences in elasticity among the most common types of brain tumors. With intraoperative SWE, neurosurgeons may have innovative information to predict diagnosis and guide their resection.

Observation of the internal response of the kidney during compressive loading using ultrafast ultrasonography.

A protocol based on ultrafast ultrasonography was developed to study the internal response of isolated perfused human (n=3) and porcine (n=11) kidneys subjected to loading at 0.003 m/s and 0.3m/s respectively. Regional uniaxial strains were calculated based on natural target tracking. The effect of loading speed and regional differences could be statistically detected on the porcine specimens. However, despite the inhomogeneity of their anatomical structures, strains' responses appeared relatively homogeneous at 0.3m/s in both porcine and human kidneys. Failure, identified as a sudden change on the ultrasonography movie, also appeared at similar compression levels for both species (38.3% of applied strain in average for human and 35.8% of applied strain in average for porcine).

Effects of pressure on the shear modulus, mass and thickness of the perfused porcine kidney.

Eleven fresh ex vivo porcine kidneys were perfused in the artery, vein and ureter with degassed Dulbecco׳s Modified Eagle Medium (DMEM). The effect of perfusion pressure was evaluated using ten different pressures combinations. The shear modulus of the tissues was estimated during perfusion using shear wave elastography. The organ weight change was measured by a digital scale and cameras were used to follow the changes of the dimensions after each pressure combination. The effect of perfusion on the weight and the thickness was non-reversible, whereas the effect on the shear modulus was reversible. Pressure was found to increase the average shear modulus in the cortex by as much as 73%. A pressure of 80 mmHg was needed to observe tissues shear modulus in the same range as in vivo tests (Gcortex=9.1 kPa, Gmedulla=8.5 kPa ex vivo versus Gcortex=9.1 kPa, Gmedulla=8.7 kPa in vivo in Gennisson et al., 2012).