Sensing ultrasound localization microscopy for the visualization of glomeruli in living rats and humans.

BACKGROUND: Estimation of glomerular function is necessary to diagnose kidney diseases. However, the study of glomeruli in the clinic is currently done indirectly through urine and blood tests. A recent imaging technique called Ultrasound Localization Microscopy (ULM) has appeared. It is based on the ability to record continuous movements of individual microbubbles in the bloodstream. Although ULM improved the resolution of vascular imaging up to tenfold, the imaging of the smallest vessels had yet to be reported. METHODS: We acquired ultrasound sequences from living humans and rats and then applied filters to divide the data set into slow-moving and fast-moving microbubbles. We performed a double tracking to highlight and characterize populations of microbubbles with singular behaviors. We decided to call this technique "sensing ULM" (sULM). We used post-mortem micro-CT for side-by-side confirmation in rats. FINDINGS: In this study, we report the observation of microbubbles flowing in the glomeruli in living humans and rats. We present a set of analysis tools to extract quantitative information from individual microbubbles, such as remanence time or normalized distance. INTERPRETATION: As glomeruli play a key role in kidney function, it would be possible that their observation yields a deeper understanding of the kidney. It could also be a tool to diagnose kidney diseases in patients. More generally, it will bring imaging capabilities closer to the functional units of organs, which is a key to understand most diseases, such as cancer, diabetes, or kidney failures. FUNDING: This study was funded by the European Research Council under the European Union Horizon H2020 program (ERC Consolidator grant agreement No 772786-ResolveStroke).

Ultrasound localization microscopy of the human kidney allograft on a clinical ultrasound scanner.

Chronic kidney disease is a major medical problem, causing more than a million deaths each year worldwide. Peripheral kidney microvascular damage characterizes most chronic kidney diseases, yet noninvasive and quantitative diagnostic tools to measure this are lacking. Ultrasound Localization Microscopy (ULM) can assess tissue microvasculature with unprecedented resolution. Here, we optimized methods on 35 kidney transplants and studied the feasibility of ULM in seven human kidney allografts with a standard low frame rate ultrasound scanner to access microvascular damage. Interlobar, arcuate, cortical radial vessels, and part of the medullary organization were visible on ULM density maps. The medullary vasa recta can be seen but are not as clear as the cortical vessels. Acquisition parameters were derived from Contrast-Enhanced Ultrasound examinations by increasing the duration of the recorded clip at the same plane. ULM images were compared with Color Doppler, Advanced Dynamic Flow, and Superb Microvascular Imaging with a contrast agent. Despite some additional limitations due to movement and saturation artifacts, ULM identified vessels two to four times thinner compared with Doppler modes. The mean ULM smallest analyzable vessel cross section was 0.3 ± 0.2 mm in the seven patients. Additionally, ULM was able to provide quantitative information on blood velocities in the cortical area. Thus, this proof-of-concept study has shown ULM to be a promising imaging technique for qualitative and quantitative microvascular assessment. Imaging native kidneys in patients with kidney diseases will be needed to identify their ULM biomarkers.

3D transcranial ultrasound localization microscopy for discrimination between ischemic and hemorrhagic stroke in early phase.

Early diagnosis is a critical part of the emergency care of cerebral hemorrhages and ischemia. A rapid and accurate diagnosis of strokes reduces the delays to appropriate treatments and a better functional recovery. Currently, CTscan and MRI are the gold standards with constraints of accessibility, availability, and possibly some contraindications. The development of Ultrasound Localization Microscopy (ULM) has enabled new perspectives to conventional transcranial ultrasound imaging with increased sensitivity, penetration depth, and resolution. The possibility of volumetric imaging has increased the field-of-view and provided a more precise description of the microvascularisation. In this study, rats (n = 9) were subjected to thromboembolic ischemic stroke or intracerebral hemorrhages prior to volumetric ULM at the early phases after onsets. Although the volumetric ULM performed in the early phase of ischemic stroke revealed a large hypoperfused area in the cortical area of the occluded artery, it showed a more diffused hypoperfusion in the hemorrhagic model. Respective computations of a Microvascular Diffusion Index highlighted different patterns of perfusion loss during the first 24 h of these two strokes' subtypes. Our study provides the first proof that this methodology should allow early discrimination between ischemic and hemorrhagic stroke with a potential toward diagnosis and monitoring in clinic.

Performance benchmarking of microbubble-localization algorithms for ultrasound localization microscopy.

Ultrafast ultrasound localization microscopy can be used to detect the subwavelength acoustic scattering of intravenously injected microbubbles to obtain haemodynamic maps of the vasculature of animals and humans. The quality of the haemodynamic maps depends on signal-to-noise ratios and on the algorithms used for the localization of the microbubbles and the rendering of their trajectories. Here we report the results of benchmarking of the performance of seven microbubble-localization algorithms. We used metrics for localization errors, localization success rates, processing times and a measure of the reprojection of the localization of the microbubbles on the original beamformed grid. We combined eleven metrics into an overall score and tested the algorithms in three simulated microcirculation datasets, and in angiography datasets of the brain of a live rat after craniotomy, an excised rat kidney and a mammary tumour in a live mouse. The algorithms, metrics and datasets, which we have made openly available at https://github.com/AChavignon/PALA and https://doi.org/10.5281/zenodo.4343435 , will facilitate the identification or generation of optimal microbubble-localization algorithms for specific applications.

3D Transcranial Ultrasound Localization Microscopy in the Rat Brain With a Multiplexed Matrix Probe.

OBJECTIVE: Ultrasound Localization Microscopy (ULM) provides images of the microcirculation in-depth in living tissue. However, its implementation in two-dimension is limited by the elevation projection and tedious plane-by-plane acquisition. Volumetric ULM alleviates these issues and can map the vasculature of entire organs in one acquisition with isotropic resolution. However, its optimal implementation requires many independent acquisition channels, leading to complex custom hardware. METHODS: In this article, we implemented volumetric ultrasound imaging with a multiplexed 32 × 32 probe driven by a single commercial ultrasound scanner. We propose and compare three different sub-aperture multiplexing combinations for localization microscopy in silico and in vitro with a flow of microbubbles in a canal. Finally, we evaluate the approach for micro-angiography of the rat brain. The "light" combination allows a higher maximal volume rate than the "full" combination while maintaining the field of view and resolution. RESULTS: In the rat brain, 100,000 volumes were acquired within 7 min with a dedicated ultrasound sequence and revealed vessels down to 31 µm in diameter with flows from 4.3 mm/s to 28.4 mm/s. CONCLUSION: This work demonstrates the ability to perform a complete angiography with unprecedented resolution in the living rat's brain with a simple and light setup through the intact skull. SIGNIFICANCE: We foresee that it might contribute to democratize 3D ULM for both preclinical and clinical studies.

Megalencephalic leukoencephalopathy with subcortical cysts is a developmental disorder of the gliovascular unit.

Absence of the astrocyte-specific membrane protein MLC1 is responsible for megalencephalic leukoencephalopathy with subcortical cysts (MLC), a rare type of leukodystrophy characterized by early-onset macrocephaly and progressive white matter vacuolation that lead to ataxia, spasticity, and cognitive decline. During postnatal development (from P5 to P15 in the mouse), MLC1 forms a membrane complex with GlialCAM (another astrocytic transmembrane protein) at the junctions between perivascular astrocytic processes. Perivascular astrocytic processes along with blood vessels form the gliovascular unit. It was not previously known how MLC1 influences the physiology of the gliovascular unit. Here, using the Mlc1 knock-out mouse model of MLC, we demonstrated that MLC1 controls the postnatal development and organization of perivascular astrocytic processes, vascular smooth muscle cell contractility, neurovascular coupling, and intraparenchymal interstitial fluid clearance. Our data suggest that MLC is a developmental disorder of the gliovascular unit, and perivascular astrocytic processes and vascular smooth muscle cell maturation defects are primary events in the pathogenesis of MLC and therapeutic targets for this disease.

Large-scale functional ultrasound imaging of the spinal cord reveals in-depth spatiotemporal responses of spinal nociceptive circuits in both normal and inflammatory states.

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 (Aß, 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.

Early Ultrafast Ultrasound Imaging of Cerebral Perfusion correlates with Ischemic Stroke outcomes and responses to treatment in Mice.

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. Conclusion: We thus provide the proof of concept that in a mouse model of thromboembolic stroke with an intact skull, early ultrafast ultrasound can be indicative of responses to treatment and cerebral tissue fates following stroke. It brings new tools to study ischemic stroke in preclinical models and is the first step prior translation to the clinical settings.

Circulating tPA contributes to neurovascular coupling by a mechanism involving the endothelial NMDA receptors.

The increase of cerebral blood flow evoked by neuronal activity is essential to ensure enough energy supply to the brain. In the neurovascular unit, endothelial cells are ideally placed to regulate key neurovascular functions of the brain. Nevertheless, some outstanding questions remain about their exact role neurovascular coupling (NVC). Here, we postulated that the tissue-type plasminogen activator (tPA) present in the circulation might contribute to NVC by a mechanism dependent of its interaction with endothelial N-Methyl-D-Aspartate Receptor (NMDAR). To address this question, we used pharmacological and genetic approaches to interfere with vascular tPA-dependent NMDAR signaling, combined with laser speckle flowmetry, intravital microscopy and ultrafast functional ultrasound in vivo imaging. We found that the tPA present in the blood circulation is capable of potentiating the cerebral blood flow increase induced by the activation of the mouse somatosensorial cortex, and that this effect is mediated by a tPA-dependent activation of NMDAR expressed at the luminal part of endothelial cells of arteries. Although blood molecules, such as acetylcholine, bradykinin or ATP are known to regulate vascular tone and induce vessel dilation, our present data provide the first evidence that circulating tPA is capable of influencing neurovascular coupling (NVC).

Ultrafast 3D Ultrasound Localization Microscopy Using a 32 × 32 Matrix Array.

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.

Microvascular flow dictates the compromise between spatial resolution and acquisition time in Ultrasound Localization Microscopy.

Medical ultrasound is a widely used diagnostic imaging technique for tissues and blood vessels. However, its spatial resolution is limited to a sub-millimeter scale. Ultrasound Localization Microscopy was recently introduced to overcome this limit and relies on subwavelength localization and tracking of microbubbles injected in the blood circulation. Yet, as microbubbles follow blood flow, long acquisition time are required to detect them in the smallest vessels, leading to long reconstruction of the microvasculature. The objective of this work is to understand how blood flow limits acquisition time. We studied the reconstruction of a coronal slice of a rat's brain during a continuous microbubble injection close to clinical concentrations. After acquiring 192000 frames over 4 minutes, we find that the biggest vessels can be reconstructed in seconds but that it would take tens of minutes to map the entire capillary network. Moreover, the appropriate characterization of flow profiles based on microbubble velocity within vessels is bound by even more stringent temporal limitations. As we use simple blood flow models to characterize its impact on reconstruction time, we foresee that these results and methods can be adapted to determine adequate microbubble injections and acquisition times in clinical and preclinical practice.

Ultrasound Localization Microscopy and Super-Resolution: A State of the Art.

Because it drives the compromise between resolution and penetration, the diffraction limit has long represented an unreachable summit to conquer in ultrasound imaging. Within a few years after the introduction of optical localization microscopy, we proposed its acoustic alter ego that exploits the micrometric localization of microbubble contrast agents to reconstruct the finest vessels in the body in-depth. Various groups now working on the subject are optimizing the localization precision, microbubble separation, acquisition time, tracking, and velocimetry to improve the capacity of ultrasound localization microscopy (ULM) to detect and distinguish vessels much smaller than the wavelength. It has since been used in vivo in the brain, the kidney, and tumors. In the clinic, ULM is bound to improve drastically our vision of the microvasculature, which could revolutionize the diagnosis of cancer, arteriosclerosis, stroke, and diabetes.

Subwavelength motion-correction for ultrafast ultrasound localization microscopy.

Ultrafast Ultrasound Localization Microscopy uses microbubbles that are individually localized with a resolution below 10µm. Positions of the microbubbles are accumulated to create a super resolution image, which bypass the diffraction-limit of spatial resolution. However, microbubbles localization is affected by physiological motions at the micrometric scale. Here, we demonstrate a phase correlation method for rigid motion correction. Spatiotemporal filters extract tissue dominated images, which are tracked to correct linear motions and improve the precision of microbubbles' localization, improving the quality of the image. It is the first proof of concept towards a full motion correction strategy and super-resolution imaging in moving tissues.