Method for ultrasound imaging of a microvascular structure

Abstract

The present invention relates to a method for ultrasound imaging of a microvascular structure, which method characterised in that it comprises: - a step of injecting ultrasound contrast agents into blood vessels in a region comprising a microvascular structure, - a step of ultrasound imaging the region comprising the microvascular structure while the contrast agents flow through the blood vessels, the concentration of the contrast agents and / or the ultrasound frequency of the imaging being chosen such that the contrast agents are sufficiently separated to prevent feedback, - a step of detecting / filtering and locating the contrast agents, - a step of individually monitoring the contrast agents, - a step of classifying the behaviours of the individually monitored contrast agents according to at least one predetermined behaviour characteristic of contrast agents flowing through the microvascular structure, and - a step of imaging the microvascular structure according to the thus-performed classification.

Description

Description Title of the invention: METHOD FOR ULTRASOUND IMAGING OF A MICROVASCULAR STRUCTURE

[0001] The present invention relates to a method for ultrasound imaging of a microvascular structure, and in particular of a functional unit of an organ of the human or animal body or a physiological or pathological process affecting the microcirculation. The method makes it possible in particular to obtain imaging of the glomeruli of the kidney. The invention also relates to a device for implementing the method.

[0002] One of the basic functional units of the kidney is the glomerulus. This component of the nephron is a tangle of capillaries located between two resistance vessels (afferent arteriole and efferent arteriole) that provides its filtration capacity. The glomerulus is impacted by several chronic diseases such as hypertension, diabetes, autoimmune diseases and cancer that can lead to its complete destruction and therefore to the loss of its filtration functions. Imaging of the glomeruli is necessary for the exploration, diagnosis and understanding of these pathologies. However, the diameter of glomeruli in humans is approximately 200 microns, which is well below the resolution limit of most medical imaging techniques, and their function is only assessed indirectly in the clinic, by blood or urine tests that provide access to the global glomerular filtration rate and not by the observation of individual glomeruli.Renal biopsy is the only current way to observe and analyze these structures.

[0003] Similarly, many functional structures are thus listed throughout the human body, in which the architecture of the microvasculature is very deeply linked to the function of the organ. For example, the islets of Langerhans and insulin secretion in the pancreas, Kupffer cells and filtration in the liver. For all these structures, medical imaging is limited to the observation of macroscopic processes with poor resolution, which does not allow for an understanding of the processes involved, nor good diagnostic tools.

[0004] Until recently, imaging technologies for microvascular anatomy and physiology have been limited by two conflicting aspects: resolution and penetration. Indeed, medical imaging techniques such as MRI, X-ray computed tomography (CT), or ultrasound cannot directly visualize the microcirculation, their resolution being generally limited to the millimeter or submillimeter scale (a few hundred microns or more). Indirect techniques, such as organ perfusion with contrast agents, can be used to characterize organ perfusion. At the other end of the spectrum, methods that can image the microcirculation directly, such as confocal microscopy, two-photon microscopy, and optical coherence tomography, are limited by their penetration or high invasiveness.They can only image microstructures to a depth of a few hundred microns, at best, while keeping the animal alive.

[0005] Ultrasound Localization Microscopy (ULM) technology has overcome the trade-off between resolution and penetration by enabling the deep visualization of microcirculation. It relies on tracking isolated clinical contrast agents, namely microbubbles, moving through blood vessels and has since been implemented in humans. It is achieved through the following steps: - injection of contrast agents, often microbubbles, at an optimized concentration, - ultrasound imaging of the area of ​​interest including the blood vessels in which the contrast agents circulate, - isolation of contrast agents on ultrasound images using filters that can highlight high-speed microbubbles using spatio-temporal or temporal filters, - sub-wavelength localization of contrast agents. The principle of superresolution here is based on the fact that the microbubbles are distant from each other, preventing interference between their echoes. It is this interference that limits the resolution to about half a wavelength. When isolated objects are located, their position can be determined with precision much finer than the wavelength as a function of the signal-to-noise ratio, - tracking microbubbles to determine their path, and - accumulation of paths for super-resolution visualization.

[0006] The ULM technique is an acoustic super-resolution technique that allows mapping of microcirculation with unprecedented resolution at the organ scale of a living animal, typically allowing a gain of a factor of ten.

[0007] Although it allows for rich microvascular mapping, it remains unsuitable for imaging the functional units of organs. This limitation stems from a lack of contrast for these structures. Unlike large vessels, few contrast agents pass through individual microstructures, and they circulate very slowly; they therefore do not appear on state-of-the-art ULM images.

[0008] The present invention aims to remedy these drawbacks.

[0009] The invention thus relates to a method of ultrasound imaging of a microvascular structure.

[0010] The method according to the invention comprises: - a step of injecting ultrasound contrast agents into blood vessels in an area including a microvascular structure, - a step of ultrasound imaging of said area including the microvascular structure, during the circulation of the contrast agents in the blood vessels, the concentration of the contrast agents and / or the ultrasound frequency of the imaging being chosen so that the contrast agents are sufficiently distant to prevent interference between their echoes, - a step of detection / filtering and localization of contrast agents, - a step of individual monitoring of contrast agents, - a step of classifying the behaviors of the contrast agents monitored individually, based on at least one predetermined behavior characteristic of the circulation of the contrast agents in the microvascular structure, and - a step of carrying out imaging of the microvascular structure, based on the classification thus carried out.

[0011] The injection step is typically performed intravenously and it is the circulation that distributes the contrast agents.

[0012] The imaging step can thus be carried out by selecting and displaying individually tracked contrast agents having a predetermined behavior characteristic of the circulation of contrast agents in the microvascular structure.

[0013] For example, it is known that the contrast agents (microbubbles) visualized travel through the glomeruli by swirling through the capillary balls that form them. The invention consists of translating this very precise behavior, distinct from what is expected in the other vessels of the kidney, to allow a classification of unique contrast agents circulating in glomeruli. Filters can be added to categorize the contrast agents into those behaving in this way and the others. An example of a predetermined behavior representative of the glomeruli is a stationary behavior of the contrast agents over a certain period of time.

[0014] Said predetermined behavior characteristic of the circulation of contrast agents in the microvascular structure may be a predetermined displacement (for example, swirling in the case of glomeruli), a predetermined passage time and / or a predetermined speed of the contrast agents.

[0015] Said predetermined behavior characteristic of the circulation of contrast agents in the microvascular structure can also be a variation in the amplitude of the linear or non-linear echo of the contrast agent.

[0016] Other predetermined behaviors may include, for example, swirling motion of the contrast agents, or other types of motion, changes in their speed, residence time in a restricted area, their mean free path, or their dispersity.

[0017] Using this classification, it is possible to generate a specific image of these microvascular structures, from the trajectories compatible with the predetermined behavior.

[0018] This image can then be used to derive fundamental information about these structures, such as their number, size, location, and spatial distribution.

[0019] The microvascular structure may be a functional unit of the area, said area being an organ.

[0020] The contrast agents are advantageously separated by a distance at least equal to the wavelength of the ultrasound.

[0021] Contrast agents can be microbubbles.

[0022] The microvascular structure may be a glomerulus of a kidney.

[0023] In this case, the predetermined behavior characteristic of the circulation of contrast agents in a glomerulus may be a predetermined displacement, a predetermined passage time and / or a predetermined speed of the contrast agents.

[0024] Said predetermined behavior characteristic of the circulation of contrast agents in a glomerulus may be a predetermined swirling of the contrast agents.

[0025] The method may include: - a stage of ultrasound imaging of the kidney, using a device emitting and receiving ultrasounds and reconstructing an image based on the flight times of the ultrasounds, - a step of injecting ultrasound contrast agents into the blood vessels of the kidney, - a step of detection and localization of contrast agents, - a step of individual monitoring of contrast agents, - a step of detecting contrast agents having a speed greater than a first predetermined speed and of detecting contrast agents having a speed lower than a second predetermined speed, - a detection step, among the contrast agents having a speed less than a second predetermined speed, of contrast agents exhibiting a movement characteristic of the circulation of contrast agents in a glomerulus, and in particular a swirling movement, - a step of performing imaging of the glomeruli, by selecting contrast agents presenting a movement characteristic of a glomerulus.

[0026] The step of performing glomeruli imaging can be performed by selecting contrast agents exhibiting a motion characteristic of a glomerulus and contrast agents having a speed greater than the first predetermined speed.

[0027] The method may further comprise a step of analyzing the number, position, and / or variability of the contrast agents in the glomeruli.

[0028] The invention also relates to a device for implementing the method.

[0029] The device according to the invention comprises: - an ultrasound imaging system of an area including the microvascular structure, - a system for injecting ultrasound contrast agents into blood vessels of said area including the microvascular structure, - advantageously a contrast agent filtering system which does not irreversibly exclude contrast agents according to their speed, - a system for detecting and localizing contrast agents, - an individual monitoring system for contrast agents, - a system capable of performing imaging of the microvascular structure, by ultrasound, and by selecting individually monitored contrast agents having a predetermined behavior characteristic of the circulation of contrast agents in the microvascular structure.

[0030] Other advantages and particularities of the present invention will result from the description which follows, given by way of non-limiting example and made with reference to the appended figures:

[0031] [Fig. 1] illustrates a rat subjected to an imaging method according to the invention,

[0032] [Fig. 2] illustrates a kidney of the rat of Figure 1,

[0033] [Fig. 3] is a Doppler ultrasound of the kidney in Figure 2,

[0034] [Fig. 4] is an image of the kidney, obtained by an imaging method according to the invention,

[0035] [Fig. 5] is a detail view of the image in Figure 4,

[0036] [Fig. 6] is a detail view of the image of Figure 5,

[0037] [Fig. 7] is a diagram illustrating the velocity of contrast agents in different areas of the rat kidney,

[0038] [Fig. 8] is a diagram illustrating the persistence of contrast agents in different areas of the rat kidney,

[0039] [Fig. 9] is a diagram illustrating the dispersity of contrast agents in different areas of the rat kidney,

[0040] [Fig. 10] is an image of the kidney obtained by ultrasound localization microscopy,

[0041] [Fig. 11] is an image of the kidney obtained by X-ray microtomography,

[0042] [Fig. 12] is a detail view of the image in Figure 10 illustrating the counting of glomeruli,

[0043] [Fig. 13] is a detail view of the image in Figure 11 illustrating the counting of glomeruli,

[0044] [Fig. 14] is a diagram illustrating the probability of density of glomeruli,

[0045] [Fig. 15] is an image of a human kidney obtained by an imaging method according to the invention,

[0046] [Fig. 16] is a detail view of the image of Figure 15,

[0047] [Fig. 17] is a detail view of the image of Figure 16 illustrating a first trajectory of a contrast agent in a glomerulus,

[0048] [Fig. 18] is a detail view of the image of Figure 16 illustrating a second trajectory of a contrast agent in a glomerulus,

[0049] [Fig. 19] is a diagram illustrating the velocity of contrast agents in different areas of the human kidney,

[0050] [Fig. 20] is a diagram illustrating the persistence of contrast agents in different areas of the human kidney, and

[0051] [Fig. 21] is a diagram illustrating the dispersion of contrast agents in different areas of the human kidney.

[0052] DETAILED DESCRIPTION

[0053] Ultrasound imaging of rat kidney globules

[0054] First, ULM is applied to observe low-flow microvessels in a rat kidney 3 using a probe 2 (Figures 1 and 2). Typically, ULM involves imaging microbubbles in the microvasculature with an ultrasound scanner at a frame rate of 500 Hz and a contrast agent infusion of 13 pl / min, which allows subwavelength localization and tracking of individual gas particles. Eleven rats were examined using a research ultrasound scanner and a 15 MHz high-frequency linear array probe. The animals were placed in a supine position, and the left kidney was externalized to acquire ultrasound data in a P-plane longitudinal to the kidney (Figure 2).The ultrasound acquisitions consisted of a Long Ensemble Amplitude Modulation (LEAM) sequence to obtain conventional linear data, allowing the observation of fast microbubbles, and nonlinear data that enhance the signal of slow or stopped microbubbles. The ULM post-processing steps were then performed twice to obtain a fast microbubble tracking map, from the linear acquisition filtered with a singular value decomposition (SVD), and a slow microbubble map from the nonlinear acquisition. The conventional “Power Doppler” ultrasound, i.e., performed using linear acquisition and SVD, is shown in Figure 3.

[0055] Thanks to this double tracking, it is possible to obtain a composite density map of the ULMs with the slow tracks in light gray and the fast tracks in dark gray (Figure 4). Two examples of zones A (inside the circles) of slow tracks and zones B (inside the rectangles) of fast tracks have been illustrated in Figures 4 and 15. On this map, a particular behavior of the microbubbles in the cortex could be observed: traces of microbubbles that enter or exit an area where they rotate at speeds corresponding to capillary flow. A zoom in on the density map of ULMs in the cortex (Figure 5) confirms the presence of these ultrasound diffusers slowly rotating in circles. As an example, the tracks 4 of two independent microbubbles were followed along this path leading to a glomerular shape (enlarged in Figure 6): the microbubbles in the cortex appear to rise, then swirl before leaving again. Such behavior of microbubbles is observable along the intermediate vessels of the cortex.

[0056] Segmentation of the different kidney regions, namely the medulla, large arteries (interlobar and arcuate), and cortex, was performed manually to compare microbubble behavior in the different areas. A glomerular mask was also constructed by selecting cortical tracks with a high mean free path value, i.e., stagnant microbubbles. Tracks with at least one point in the region were included in the region analysis.

[0057] Specific metrics to highlight capillary tangles, including normalized velocities, afterglow time (i.e., the duration of the trace within a 50-micron radius from the center of the trace), and dispersity (i.e., the number of times a trace goes in the same direction, with a tolerance of plus or minus 20°, divided by the number of points constituting the trace) were performed on each trace of the 11 rats. These measurements highlight slower (Figure 7), more stagnant (Figure 8), and less dispersed (Figure 9) microbubbles in the glomerular structures compared to traces in the medullary region and in the main arteries.Student's t-tests (at a margin of 5%) show that these differences are always significant from the main vessels to the glomeruli (P<0.001 for normalized velocity, P<0.001 for remanence, P<0.001 for dispersion), and from the main vessels to the medulla (P<0.001 for normalized velocity, P<0.001 for remanence, P<0.002 for dispersity).

[0058] Micro CT indicates that the capillary tangle detected in the ULM corresponds to the glomeruli

[0059] Among the 11 rats examined by ULM in vivo, 6 were also observed post mortem by micro CT (micro X-ray tomography) after injection of barium contrast agent at the end of the ultrasound acquisition. Manual registration of the ULM (Figure 10) and the micro CT slice (Figure 11) was performed on a 160 μm slice, i.e. corresponding to the elevation resolution of the ultrasound probe at the focal point. Glomerular counting was performed using the high-intensity local regional maxima on the micro CT slice (Figure 13). On the ULM, capillary tangles were counted by selecting cortical tracks with a high mean free path value in the ULM (Figure 12).

[0060] A manual segmentation of the renal capsule was performed to compare the density of glomeruli as a function of their distance from it, in both imaging modalities. Thus, it can be seen that the spatial distribution is similar between the glomeruli detected by a known approach with micro CT and the capillary tangles observed by ULM in rat number 7 (Figure 14). A two-sample Kolmogorov-Smirnov statistical test was performed in all 6 rats with both modalities to find out if the distributions of both sets come from the same continuous distribution. The null hypothesis, i.e., h, is accepted in all rats, meaning that the glomeruli are distributed equally in both modalities at the 5% significance level.

[0061] Calculation of glomerular size was also performed in both modalities. For ULM, in 11 rats, the glomerular density curve was plotted as a function of the distance to the center of the glomeruli and for micro CT, the intensity curve as a function of the distance to the central pixel. The full width at half maximum of these normalized data was used to estimate the glomerular diameter in all rats. The glomeruli appear to have a similar diameter in both modalities, i.e., 60 pm in diameter, and smaller than those observed in the literature, i.e., 50 pm in radius.

[0062] Ultrasound imaging of human kidney glomeruli

[0063] ULM was performed in 5 transplant patients who underwent routine clinical ultrasound examination at the hospital. Sequences were used conventional contrast-enhanced ultrasound (CEUS) integrated into a clinical ultrasound system and a convex ultrasound probe (2 MHz) to acquire clinical data and perform ULM. The ULM post-processing steps are similar to those performed in rats, except that, since the CEUS sequence is a non-linear sequence, the data were split with a band-pass filter to obtain fast microbubbles, and with specific ULM parameters to highlight stagnant microbubbles.

[0064] This composite ULM density map, with fast microbubbles in dark gray (areas B) and slow microbubbles in light gray (areas A), shows the same glomerular shapes as rats in the cortex distribution (Figure 15). A specific zoom in this area confirms the presence of afferent and efferent arterioles (in green) with a rotating bubble in the middle of the path (in pink) (Figure 16). Tracks 5 and 6 are examples of this particular path (highlighted in Figure 16, and zoomed in Figures 17 and 18).

[0065] In the same way as the treatment carried out in rats, the glomeruli were counted and then their diameter and spatial distribution were studied with the same technical tools. It was observed that the glomerular diameters estimated by ULM are slightly higher than those found in the literature, i.e. 200pm in diameter, but remain in the same order of magnitude.

[0066] Manual segmentations of the kidney areas were performed, namely the medulla, cortex, and main vessels (interlobar and arcuate arteries), and the glomerular mask was calculated as before. Thus, the behavior of the tracks in each region could be compared. Student's t-tests were performed with a 5% margin in 8 patients. In the same way as in rats, and even more pronounced than in humans, the glomeruli remain slower than the medullary (P < 0.05) and main vessels (P < 0.001); they are more remanent, i.e., stagnant, than the medullary (P < 0.01) and main vessels (P < 0.001); and they are also less dispersed than the medullary (P < 0.001) and main vessels (P < 0.001).

[0067] Conclusion

[0068] The invention implements a new form of ULM that exploits the kinetics of individual microbubbles, including those circulating in capillaries, to map the microarchitecture of the vessels. By classifying their behavior according to known anatomical characteristics, in this case the precise complexion of the microcirculation of the glomeruli, including the afferent and efferent arterioles, is sought. The rotating microbubbles were specifically tracked in the cortex and further analysis of the traces confirms the presence of these particular shapes in this area, i.e. slower, more stagnant and less dispersed. It could also be confirmed that what was observed by ULM were glomeruli thanks to a comparison with micro CT: the spatial distribution of glomeruli by ULM and micro CT statistically belong to the same continuous distribution.Furthermore, the size of glomeruli measured by micro-CT and ULM is similar with both techniques in all rats, and close to the results previously found in the literature in rats and humans. Finally, glomeruli are distributed similarly between ULM and micro-CT around intermediate blood vessels.

[0069] The method according to the invention therefore makes it possible to observe individual glomeruli in humans for the first time with a clinical imaging device. Current imaging techniques only allow an indirect estimation of the glomerular filtration rate, and not their separate observation. In addition to being able to observe a new category of slow microbubbles whose behavior and location are comparable to the glomerular organization, the method reduces the gap between anatomical imaging and functional imaging, since it is possible to image the very structures responsible for the function of the organ. Thanks to ULM and the use of the information provided by the microbubble itself, it is possible to image functional microvascular units. This type of imaging makes it possible to study many organs and diseases such as diabetes or cancer.Additionally, human acquisitions were performed with a clinical ultrasound system that is widely available and used worldwide.

Claims

Claims

1. Method for ultrasonic imaging of a microvascular structure, characterized in that it comprises: - a step of injecting ultrasound contrast agents into blood vessels in an area including a microvascular structure, - a step of ultrasound imaging of said area including the microvascular structure, during the circulation of the contrast agents in the blood vessels, the concentration of the contrast agents and / or the ultrasound frequency of the imaging being chosen so that the contrast agents are sufficiently distant to prevent interference between their echoes, - a step of detection / filtering and localization of contrast agents, - a step of individual monitoring of contrast agents, - a step of classifying the behaviors of the contrast agents monitored individually, based on at least one predetermined behavior characteristic of the circulation of the contrast agents in the microvascular structure, and - a step of carrying out imaging of the microvascular structure, based on the classification thus carried out.

2. A method according to claim 1, characterized in that the step of performing the imaging is performed by selecting and displaying the individually tracked contrast agents having a predetermined behavior characteristic of the circulation of the contrast agents in the microvascular structure.

3. Method according to claim 1 or 2, characterized in that said predetermined behavior characteristic of the circulation of the contrast agents in the microvascular structure is a predetermined displacement, a predetermined passage time and / or a predetermined speed of the contrast agents.

4. Method according to claim 1 or 2, characterized in that said predetermined behavior characteristic of the circulation of contrast agents in the microvascular structure is a variation of the amplitude of the linear or non-linear echo of the contrast agent.

5. Method according to one of claims 1 to 4, characterized in that the microvascular structure is a functional unit of the area, said area being an organ.

6. Method according to one of claims 1 to 5, characterized in that the contrast agents are separated by a distance at least equal to the wavelength of the ultrasound.

7. Method according to one of claims 1 to 6, characterized in that the contrast agents are microbubbles.

8. Method according to one of claims 1 to 7, characterized in that the microvascular structure is a glomerulus of a kidney

9. Method according to claim 8, characterized in that said predetermined behavior characteristic of the circulation of contrast agents in a glomerulus is a predetermined displacement, a predetermined passage time and / or a predetermined speed of the contrast agents.

10. Method according to claim 9, characterized in that said predetermined behavior characteristic of the circulation of contrast agents in a glomerulus is a predetermined swirling of the contrast agents.

11. Method according to one of claims 8 to 10, characterized in that it comprises: - a stage of ultrasound imaging of the kidney, using a device emitting and receiving ultrasounds and reconstructing an image based on the flight times of the ultrasounds, - a step of injecting ultrasound contrast agents into the blood vessels of the kidney, - a step of detection / filtering and localization of contrast agents, - a step of individual monitoring of contrast agents, - a step of detecting contrast agents having a speed greater than a first predetermined speed and of detecting contrast agents having a speed lower than a second predetermined speed, - a step of detecting, among the contrast agents having a speed lower than a second predetermined speed, contrast agents exhibiting a movement characteristic of a glomerulus, and in particular a swirling movement, - a step of performing imaging of the glomeruli, by selecting contrast agents presenting a movement characteristic of a glomerulus.

12. A method according to claim 11, characterized in that the step of performing the imaging of the glomeruli is carried out by selecting contrast agents having a movement characteristic of a glomerulus and contrast agents having a speed greater than the first predetermined speed.

13. Method according to claim 11 or 12, characterized in that it further comprises a step of analyzing the number, position, and / or variability of the contrast agents in the glomeruli.

14. Device for implementing the method according to claim 1, characterized in that it comprises: - an ultrasound imaging system of an area including the microvascular structure, - a system for injecting ultrasound contrast agents into blood vessels of said area including the microvascular structure, - a contrast agent filtering system that does not irreversibly exclude contrast agents based on their speed, - a system for detecting and localizing contrast agents, - an individual monitoring system for contrast agents, - a system capable of performing imaging of the microvascular structure, by ultrasound, and by selecting individually monitored contrast agents having a predetermined behavior characteristic of the circulation of the contrast agents in said microvascular structure.

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