Physique pour la médecine Paris

To understand brain circuits it is necessary both to record and manipulate their activity. Transcranial ultrasound stimulation (TUS) is a promising non-invasive brain stimulation technique. To date, investigations report short-lived neuromodulatory effects, but to deliver on its full potential for research and therapy, ultrasound protocols are required that induce longer-lasting 'offline' changes. Here, we present a TUS protocol that modulates brain activation in macaques for more than one hour after 40 s of stimulation, while circumventing auditory confounds. Normally activity in brain areas reflects activity in interconnected regions but TUS caused stimulated areas to interact more selectively with the rest of the brain. In a within-subject design, we observe regionally specific TUS effects for two medial frontal brain regions - supplementary motor area and frontal polar cortex. Independently of these site-specific effects, TUS also induced signal changes in the meningeal compartment. TUS effects were temporary and not associated with microstructural changes.

Singular value decomposition of ultrafast imaging ultrasonic data sets has recently been shown to build a vector basis far more adapted to the discrimination of tissue and blood flow than the classical Fourier basis, improving by large factor clutter filtering and blood flow estimation. However, the question of optimally estimating the boundary between the tissue subspace and the blood flow subspace remained unanswered. Here, we introduce an efficient estimator for automatic thresholding of subspaces and compare it to an exhaustive list of thirteen estimators that could achieve this task based on the main characteristics of the singular components, namely the singular values, the temporal singular vectors, and the spatial singular vectors. The performance of those fourteen estimators was tested in vitro in a large set of controlled experimental conditions with different tissue motion and flow speeds on a phantom. The estimator based on the degree of resemblance of spatial singular vectors outperformed all others. Apart from solving the thresholding problem, the additional benefit with this estimator was its denoising capabilities, strongly increasing the contrast to noise ratio and lowering the noise floor by at least 5 dB. This confirms that, contrary to conventional clutter filtering techniques that are almost exclusively based on temporal characteristics, efficient clutter filtering of ultrafast Doppler imaging cannot overlook space. Finally, this estimator was applied in vivo on various organs (human brain, kidney, carotid, and thyroid) and showed efficient clutter filtering and noise suppression, improving largely the dynamic range of the obtained ultrafast power Doppler images.

This manuscript describes the use of ultrasound elastography, with the exception of liver applications, and represents an update of the 2013 EFSUMB (European Federation of Societies for Ultrasound in Medicine and Biology) Guidelines and Recommendations on the clinical use of elastography.

PURPOSE: Low-intensity focused ultrasound has been shown to stimulate the brain noninvasively and without noticeable tissue damage. Such a noninvasive and localized neurostimulation is expected to have a major impact in neuroscience in the coming years. This emerging field will require many animal experiments to fully understand the link between ultrasound and stimulation. The primary goal of this paper is to investigate transcranial ultrasonic neurostimulation at low frequency (320 kHz) on anesthetized rats for different acoustic pressures and estimate the in situ pressure field distribution and the corresponding motor threshold, if any. The corresponding acoustic pressure distribution inside the brain, which cannot be measured in vivo, is investigated based on numerical simulations of the ultrasound propagation inside the head cavity, reproducing at best the experiments conducted in the first part, both in terms of transducer and head geometry and in terms of acoustic parameters. METHODS: In this study, 37 ultrasonic neurostimulation sessions were achieved in rats (N=8) using a 320 kHz transducer. The corresponding beam profile in the entire head was simulated in order to investigate the in situ pressure and intensity level as well as the spatial pressure distribution, thanks to a rat microcomputed tomography scan (CT)-based 3D finite differences time domain solver. RESULTS: Ultrasound pulse evoked a motor response in more than 60% of the experimental sessions. In those sessions, the stimulation was always present, repeatable with a pressure threshold under which no motor response occurred. This average acoustic pressure threshold was found to be 0.68±0.1 MPa (corresponding mechanical index, MI=1.2 and spatial peak, pulse averaged intensity, Isppa=7.5 W cm(-2)), as calibrated in free water. A slight variation was observed between deep anesthesia stage (0.77±0.04 MPa) and light anesthesia stage (0.61±0.03 MPa), assessed from the pedal reflex. Several kinds of motor responses were observed: movements of the tail, the hind legs, the forelimbs, the eye, and even a single whisker were induced separately. Numerical simulations of an equivalent experiment with identical acoustic parameters showed that the acoustic field was spread over the whole rat brain with the presence of several secondary pressure peaks. Due to reverberations, a 1.8-fold increase of the spatial peak, temporal peak acoustic pressure (Psptp) (±0.4 standard deviation), a 3.6-fold increase (±1.8) for the spatial peak, temporal peak acoustic intensity (Isptp), and 2.3 for the spatial peak, pulse averaged acoustic intensity (Isppa), were found compared to simulations of the beam in free water. Applying such corrections due to reverberations on the experimental results would yield a higher estimation for the average acoustic pressure threshold for motor neurostimulation at 320 KHz at 1.2±0.3 MPa (MI=2.2±0.5 and Isppa=17.5±7.5 W cm(-2)). CONCLUSIONS: Transcranial ultrasonic stimulation is pressure- and anesthesia-dependent in the rat model. Numerical simulations have shown that the acoustic pattern can be complex inside the rat head and that special care must be taken for small animal studies relating acoustic parameters to neurostimulation effects, especially at a low frequency.

The advent of neuroimaging has increased our understanding of brain function. While most brain-wide functional imaging modalities exploit neurovascular coupling to map brain activity at millimeter resolutions, the recording of functional responses at microscopic scale in mammals remains the privilege of invasive electrophysiological or optical approaches, but is mostly restricted to either the cortical surface or the vicinity of implanted sensors. Ultrasound localization microscopy (ULM) has achieved transcranial imaging of cerebrovascular flow, up to micrometre scales, by localizing intravenously injected microbubbles; however, the long acquisition time required to detect microbubbles within microscopic vessels has so far restricted ULM application mainly to microvasculature structural imaging. Here we show how ULM can be modified to quantify functional hyperemia dynamically during brain activation reaching a 6.5-µm spatial and 1-s temporal resolution in deep regions of the rat brain.

Ultrasound localization microscopy can map blood vessels with a resolution much smaller than the wavelength by localizing microbubbles. The current implementations of the technique are limited to 2-D planes or small fields of view in 3-D. These suffer from minute-long acquisitions, out-of-plane microbubbles, and tissue motion. In this paper, we exploit the recent development of 4D ultrafast ultrasound imaging to insonify an isotropic volume up to 20 000 times per second and perform localization microscopy in the three dimensions. Specifically, a 32 ×32 elements, 9-MHz matrix-array probe connected to a 1024-channel programmable ultrasound scanner was used to achieve sub-wavelength volumetric imaging of both the structure and vector flow of a complex 3D structure (a main canal branching out into two side canals). To cope with the large volumes and the need to localize the bubbles in the three dimensions, novel algorithms were developed based on deconvolution of the beamformed microbubble signal. For tracking, individual particles were paired following a Munkres assignment method, and velocimetry was done following a Lagrangian approach. ULM was able to clearly represent the 3-D shape of the structure with a sharp delineation of canal edges (as small as [Formula: see text]) and separate them with a spacing as low as [Formula: see text]. The compounded volume rate of 500 Hz was sufficient to describe velocities in 2.5-150-mm/s range and to reduce the maximum acquisition time to 12 s. This paper demonstrates the feasibility of in vitro 3-D ultrafast ultrasound localization microscopy and opens up the way toward in vivo volumetric ULM.

Ultrasound sensitivity to slow blood flow motion gained two orders of magnitude in the last decade thanks to the advent of ultrafast ultrasound imaging at thousands of frames per second. In neuroscience, this access to small cerebral vessels flow led to the introduction of ultrasound as a new and full-fledged neuroimaging modality. Much as functional MRI or functional optical imaging, functional Ultrasound (fUS) takes benefit of the neurovascular coupling. Its ease of use, portability, spatial and temporal resolution makes it an attractive tool for functional imaging of brain activity in preclinical imaging. A large and fast-growing number of studies in a wide variety of small to large animal models have demonstrated its potential for neuroscience research. Beyond preclinical imaging, first proof of concept applications in humans are promising and proved a clear clinical interest in particular in human neonates, per-operative surgery, or even for the development of non-invasive brain machine interfaces.

Neuroimaging modalities such as MRI and EEG are able to record from the whole brain, but this comes at the price of either limited spatiotemporal resolution or limited sensitivity. Here, we show that functional ultrasound imaging (fUS) of the brain is able to assess local changes in cerebral blood volume during cognitive tasks, with sufficient temporal resolution to measure the directional propagation of signals. In two macaques, we observed an abrupt transient change in supplementary eye field (SEF) activity when animals were required to modify their behaviour associated with a change of saccade tasks. SEF activation could be observed in a single trial, without averaging. Simultaneous imaging of anterior cingulate cortex and SEF revealed a time delay in the directional functional connectivity of 0.27 ± 0.07 s and 0.9 ± 0.2 s for both animals. Cerebral hemodynamics of large brain areas can be measured at high spatiotemporal resolution using fUS.

BACKGROUND: Non-invasive high-resolution imaging of the cerebral vascular anatomy and function is key for the study of intracranial aneurysms, stenosis, arteriovenous malformations, and stroke, but also neurological pathologies, such as degenerative diseases. Direct visualization of the microvascular networks in the whole brain remains however challenging in vivo. METHODS: In this work, we performed 3D ultrafast ultrasound localization microscopy (ULM) using a 2D ultrasound matrix array and mapped the whole-brain microvasculature and flow at microscopic resolution in C57Bl6 mice in vivo. FINDINGS: in large vessels. The vascular maps were registered automatically with the Allen atlas in order to extract quantitative vascular parameters such as local flow rates and velocities in regions of interest. INTERPRETATION: We show the potential of 3D ULM to provide new insights into whole-brain vascular flow in mice models at unprecedented vascular scale for an in vivo technique. This technology is highly translational and has the potential to become a major tool for the clinical investigation of the cerebral microcirculation. FUNDING: This study was supported by the European Research Council under the European Union's Seventh Framework Program (FP/2007-2013) / ERC Grant Agreement n° 311025 and by the Fondation Bettencourt-Schueller under the program "Physics for Medicine". We acknowledge the ART (Technological Research Accelerator) biomedical ultrasound program of INSERM.

As transcranial ultrasound stimulation (TUS) advances as a precise, non-invasive neuromodulatory method, there is a need for consistent reporting standards to enable comparison and reproducibility across studies. To this end, the International Transcranial Ultrasonic Stimulation Safety and Standards Consortium (ITRUSST) formed a subcommittee of experts across several domains to review and suggest standardised reporting parameters for low intensity TUS, resulting in the guide presented here. The scope of the guide is limited to reporting the ultrasound aspects of a study. The guide and supplementary material provide a simple checklist covering the reporting of: (1) the transducer and drive system, (2) the drive system settings, (3) the free field acoustic parameters, (4) the pulse timing parameters, (5) in situ estimates of exposure parameters in the brain, and (6) intensity parameters. Detailed explanations for each of the parameters, including discussions on assumptions, measurements, and calculations, are also provided.

Abstract Clinicians have long been interested in functional brain monitoring, as reversible functional losses often precedes observable irreversible structural insults. By characterizing neonatal functional cerebral networks, resting-state functional connectivity is envisioned to provide early markers of cognitive impairments. Here we present a pioneering bedside deep brain resting-state functional connectivity imaging at 250-μm resolution on human neonates using functional ultrasound. Signal correlations between cerebral regions unveil interhemispheric connectivity in very preterm newborns. Furthermore, fine-grain correlations between homologous pixels are consistent with white/grey matter organization. Finally, dynamic resting-state connectivity reveals a significant occurrence decrease of thalamo-cortical networks for very preterm neonates as compared to control term newborns. The same method also shows abnormal patterns in a congenital seizure disorder case compared with the control group. These results pave the way to infants’ brain continuous monitoring and may enable the identification of abnormal brain development at the bedside.

Functional ultrasound imaging (fUS) recently emerged as a promising neuroimaging modality to image and monitor brain activity based on cerebral blood volume response (CBV) and neurovascular coupling. fUS offers very good spatial and temporal resolutions compared to fMRI gold standard as well as simplicity and portability. It was recently extended to 4D fUS imaging in preclinical settings although this approach remains limited and complex. Indeed 4D fUS requires a 2D matrix probe and specific hardware able to drive the N <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> elements of the probe with thousands of electronic channels. Several under-sampling approaches are currently investigated to limit the channel count and spread ultrasound 4D modalities. Among them, the Row Column Addressing (RCA) approach combined with ultrafast imaging is a compelling alternative using only N + N channels. We present a large field of view RCA probe prototype of 128 + 128 channels and 15 MHz central frequency adapted for preclinical imaging. Based on the Orthogonal Plane Wave compounding scheme, we were able to perform 4D vascular brain acquisitions at high volume rate. Doppler volumes of the whole rat brain were obtained in vivo at high rates (23 dB CNR at 156 Hz and 19 dB CNR at 313 Hz). Visual and whiskers stimulations were performed and the corresponding CBV increases were reconstructed in 3D with successful functional activation detected in the superior colliculus and somato-sensorial cortex respectively. This proof of concept study demonstrates for the first time the use of a low-channel count RCA array for in vivo 4D fUS imaging in the whole rat brain.

Brain perturbation studies allow detailed causal inferences of behavioral and neural processes. Because the combination of brain perturbation methods and neural measurement techniques is inherently challenging, research in humans has predominantly focused on non-invasive, indirect brain perturbations, or neurological lesion studies. Non-human primates have been indispensable as a neurobiological system that is highly similar to humans while simultaneously being more experimentally tractable, allowing visualization of the functional and structural impact of systematic brain perturbation. This review considers the state of the art in non-human primate brain perturbation with a focus on approaches that can be combined with neuroimaging. We consider both non-reversible (lesions) and reversible or temporary perturbations such as electrical, pharmacological, optical, optogenetic, chemogenetic, pathway-selective, and ultrasound based interference methods. Method-specific considerations from the research and development community are offered to facilitate research in this field and support further innovations. We conclude by identifying novel avenues for further research and innovation and by highlighting the clinical translational potential of the methods.

Elucidating organismal developmental processes requires a comprehensive understanding of cellular lineages in the spatial, temporal, and molecular domains. In this study, we introduce Zebrahub, a dynamic atlas of zebrafish embryonic development that integrates single-cell sequencing time course data with lineage reconstructions facilitated by light-sheet microscopy. This atlas offers high-resolution and in-depth molecular insights into zebrafish development, achieved through the sequencing of individual embryos across ten developmental stages, complemented by reconstructions of cellular trajectories. Zebrahub also incorporates an interactive tool to navigate the complex cellular flows and lineages derived from light-sheet microscopy data, enabling in silico fate-mapping experiments. To demonstrate the versatility of our multimodal resource, we utilize Zebrahub to provide fresh insights into the pluripotency of neuro-mesodermal progenitors (NMPs) and the origins of a joint kidney-hemangioblast progenitor population.

Deep regions of the brain are not easily accessible to investigation at the mesoscale level in awake animals or humans. We have recently developed a functional ultrasound (fUS) technique that enables imaging hemodynamic responses to visual tasks. Using fUS imaging on two awake nonhuman primates performing a passive fixation task, we constructed retinotopic maps at depth in the visual cortex (V1, V2, and V3) in the calcarine and lunate sulci. The maps could be acquired in a single-hour session with relatively few presentations of the stimuli. The spatial resolution of the technology is illustrated by mapping patterns similar to ocular dominance (OD) columns within superficial and deep layers of the primary visual cortex. These acquisitions using fUS suggested that OD selectivity is mostly present in layer IV but with extensions into layers II/III and V. This imaging technology provides a new mesoscale approach to the mapping of brain activity at high spatiotemporal resolution in awake subjects within the whole depth of the cortex.

ABSTRACT Elucidating the developmental processes of organisms requires a comprehensive understanding of cellular lineages in the spatial, temporal, and molecular domains. In this study, we introduce Zebrahub, a dynamic atlas of zebrafish embryonic development that integrates single-cell sequencing time course data with lineage reconstructions facilitated by light-sheet microscopy. This atlas offers high-resolution and in-depth molecular insights into zebrafish development, achieved through the sequencing of individual embryos across ten developmental stages, complemented by trajectory reconstructions. Zebrahub also incorporates an interactive tool to navigate the complex cellular flows and lineages derived from light-sheet microscopy data, enabling in silico fate mapping experiments. To demonstrate the versatility of our multi-modal resource, we utilize Zebrahub to provide fresh insights into the pluripotency of Neuro-Mesodermal Progenitors (NMPs). Our publicly accessible web-based platform, Zebrahub, is a foundational resource for studying developmental processes at both transcriptional and spatiotemporal levels, providing researchers with an integrated approach to exploring and analyzing the complexities of cellular lineages during zebrafish embryogenesis.

Despite a century of research on the physiology/pathophysiology of the spinal cord in chronic pain condition, the properties of the spinal cord were rarely studied at the large-scale level from a neurovascular point of view. This is mostly due to the limited spatial and/or temporal resolution of the available techniques. Functional ultrasound imaging (fUS) is an emerging neuroimaging approach that allows, through the measurement of cerebral blood volume, the study of brain functional connectivity or functional activations with excellent spatial (100 μm) and temporal (1 msec) resolutions and a high sensitivity. The aim of this study was to increase our understanding of the spinal cord physiology through the study of the properties of spinal hemodynamic response to the natural or electrical stimulation of afferent fibers. Using a combination of fUS and ultrasound localization microscopy, the first step of this study was the fine description of the vascular structures in the rat spinal cord. Then, using either natural or electrical stimulations of different categories of afferent fibers (Aβ, Aδ, and C fibers), we could define the characteristics of the typical hemodynamic response of the rat spinal cord experimentally. We showed that the responses are fiber-specific, located ipsilaterally in the dorsal horn, and that they follow the somatotopy of afferent fiber entries in the dorsal horn and that the C-fiber response is an N-methyl-D-aspartate receptor-dependent mechanism. Finally, fUS imaging of the mesoscopic hemodynamic response induced by natural tactile stimulations revealed a potentiated response in inflammatory condition, suggesting an enhanced response to allodynic stimulations.

A shift of paradigm is currently underway in biomedical ultrasound thanks to\nplane and diverging waves for ultrafast imaging. One remaining challenge\nconsists in the correction of phase and amplitude aberrations induced during\npropagation through complex layers. Unlike conventional line-per-line imaging,\nultrafast ultrasound provides for each transmission, backscattering information\nfrom the whole imaged area. Here, we take benefit from this feature and propose\nan efficient approach to perform fast aberration correction based on the\nSingular Value Decomposition of an ultrafast compound matrix built from\nbackscattered data for several plane wave transmissions. First, we explain the\nphysical signification of SVD and associated singular vectors within the\nultrafast matrix formalism. We theoretically demonstrate that the spatial and\nangular variables separation, rendered by SVD on ultrafast data, provides an\nelegant and straightforward way to optimize angular coherence of backscattered\ndata. In heterogeneous media with an aberrating phase screen approximation, we\ndemonstrate that the first spatial and angular singular vectors retrieve on one\nside the non-aberrated image, and on the other, the phase and amplitude of the\naberration law. In vitro results prove the efficiency of the image correction,\nbut also the accuracy of the aberrator determination. Based on spatial and\nangular coherence, we introduce a complete methodology for adaptive beamforming\nof ultrafast data, performed on successive isoplanatism patches undergoing SVD\nbeamforming. The simplicity of this method paves the way to real-time adaptive\nultrafast ultrasound imaging and provides a theoretical framework for future\nquantitative ultrasound applications.\n

Remote and precisely controlled activation of the brain is a fundamental challenge in the development of brain-machine interfaces for neurological treatments. Low-frequency ultrasound stimulation can be used to modulate neuronal activity deep in the brain, especially after expressing ultrasound-sensitive proteins. But so far, no study has described an ultrasound-mediated activation strategy whose spatiotemporal resolution and acoustic intensity are compatible with the mandatory needs of brain-machine interfaces, particularly for visual restoration. Here we combined the expression of large-conductance mechanosensitive ion channels with uncustomary high-frequency ultrasonic stimulation to activate retinal or cortical neurons over millisecond durations at a spatiotemporal resolution and acoustic energy deposit compatible with vision restoration. The in vivo sonogenetic activation of the visual cortex generated a behaviour associated with light perception. Our findings demonstrate that sonogenetics can deliver millisecond pattern presentations via an approach less invasive than current brain-machine interfaces for visual restoration.

In the field of ischemic cerebral injury, precise characterization of neurovascular hemodynamic is required to select candidates for reperfusion treatments. It is thus admitted that advanced imaging-based approaches would be able to better diagnose and prognose those patients and would contribute to better clinical care. Current imaging modalities like MRI allow a precise diagnostic of cerebral injury but suffer from limited availability and transportability. The recently developed ultrafast ultrasound could be a powerful tool to perform emergency imaging and long term follow-up of cerebral perfusion, which could, in combination with MRI, improve imaging solutions for neuroradiologists. Methods: In this study, in a model of in situ thromboembolic stroke in mice, we compared a control group of non-treated mice (N=10) with a group receiving the gold standard pharmacological stroke therapy (N=9). We combined the established tool of magnetic resonance imaging (7T MRI) with two innovative ultrafast ultrasound methods, ultrafast Doppler and Ultrasound Localization Microscopy, to image the cerebral blood volumes at early and late times after stroke onset and compare with the formation of ischemic lesions. Results: Our study shows that ultrafast ultrasound can be used through the mouse skull to monitor cerebral perfusion during ischemic stroke. In our data, the monitoring of the reperfusion following thrombolytic within the first 2 h post stroke onset matches ischemic lesions measured 24 h. Moreover, similar results can be made with Ultrasound Localization Microscopy which could make it applicable to human patients in the future.