WO2024069059A1

WO2024069059A1 - Method for ultrasound imaging of a microvascular structure

Info

Publication number: WO2024069059A1
Application number: PCT/FR2022/051843
Authority: WO
Inventors: Olivier Couture; Vincent HINGOT; Louise DENIS; Abderrahmane AISSANI; Sylvain BODARD; Jean-Michel CORREAS
Original assignee: Sorbonne Universite; Centre National De La Recherche Scientifique; Institut National De La Sante Et De La Recherche Medicale; Assistance Publique Hopitaux De Paris; Universite Paris Cite
Priority date: 2022-09-29
Filing date: 2022-09-29
Publication date: 2024-04-04

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

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

[0001] The subject of the present invention is a method for ultrasonic 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 microcirculation. The method makes it possible in particular to obtain imaging of the glomeruli of the kidney. The invention also relates to a device enabling the method to be implemented.

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) which provides its filtration capacity. The glomerulus is impacted by several chronic diseases such as hypertension, diabetes, autoimmune diseases and cancer which can lead to its complete destruction and therefore to the loss of its filtration functions. Imaging of 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 indirectly assessed in the clinic. , by blood or urine tests which provide access to the overall glomerular filtration rate and not to the observation of individual glomeruli. Renal biopsy is the only current way to observe and analyze these structures.

[0003] In the same way, numerous 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, islets of Langherans 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 provide an understanding of the processes involved, nor good diagnostic tools. [0004] Until recently, imaging technologies for microvascular anatomy and physiology were limited due to two contradictory aspects: resolution and penetration. Indeed, medical imaging techniques such as MRI, X-ray scanner (CT) or ultrasound cannot visualize the microcirculation directly, their resolution being generally limited to the scale of millimeters or millimeters. submillimeter (a few hundred microns or more). Indirect techniques, for example using organ perfusion by contrast agents, can be used to enable characterization of organ perfusion. At the opposite end of the spectrum, methods that can image the microcirculation directly such as confocal microscopy, two-photon microscopy, optical coherence tomography are limited by their penetration or high invasiveness. They can only image microstructures at a depth of a few hundred microns, at best, while keeping the animal alive.

[0005] Ultrasound localization microscopy (ULM) technology has made it possible to overcome the compromise between resolution and penetration by making it possible to visualize the microcirculation in depth. It relies on tracking isolated clinical contrast agents, namely microbubbles, moving through blood vessels and has since been implemented in humans. It is carried out 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 thanks to filters which can highlight microbubbles going at high speed thanks to 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 single objects are located, their position can be determined with precision much finer than the wavelength depending on the signal-to-noise ratio,

- monitoring of microbubbles to determine their path, and

- accumulation of paths for super-resolved visualization.

The ULM technique is an acoustic super-resolution technique making it possible to map the microcirculation with unprecedented resolution on the scale of the organ of a living animal, which typically allows a gain of a factor of ten.

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

The present invention aims to remedy these drawbacks.

[0009] The subject of the invention is thus a method of ultrasonic 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 as to that the contrast agents are sufficiently distant to prevent interference between their echoes,

- a step of detection/filtering and localization of the contrast agents,

- an individual monitoring step for the contrast agents,

- a step of classifying the behaviors of the contrast agents followed individually, according to 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, depending on the classification thus carried out.

The injection step is typically carried out intravenously and it is the circulation which distributes the contrast agents.

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

[0013] By way of example, we know that the contrast agents (the microbubbles) visualized travel through the glomeruli by swirling through the capillary balls which 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 classification of unique contrast agents circulating in glomeruli. We can add filters to categorize the contrast agents between those behaving this way and the others. An example of predetermined behavior representative of glomeruli is stationary behavior of contrast agents over a certain period of time.

[0014] Said predetermined behavior characteristic of the circulation of the contrast agents in the microvascular structure may be a predetermined movement (for example a whirling 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 may also be a variation in the amplitude of the linear or non-linear echo of the contrast agent.

[0016] Other predetermined behaviors can for example be a swirling movement of the contrast agents, or other types of movement, modifications of their speed, a residence time in a restricted zone, their average free path, or again their dispersity. [0017] By using this classification, it is possible to generate a specific image of these microvascular structures, from trajectories compatible with the predetermined behavior.

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

The microvascular structure can be a functional unit of the zone, said zone being an organ.

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

The contrast agents may be microbubbles.

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 movement, a predetermined passage time and/or a predetermined speed of the contrast agents.

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

[0025] The process may include:

- an ultrasound imaging step of the kidney, using a device emitting and receiving ultrasounds and reconstructing an image on the basis of the ultrasound flight times,

- a step of injecting ultrasound contrast agents into blood vessels of the kidney,

- a step of detection and localization of the contrast agents,

- an individual monitoring step for the 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 lower 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 carrying out imaging of the glomeruli, by selecting contrast agents presenting a movement characteristic of a glomerulus.

The step of performing imaging of the glomeruli can be carried out by selecting contrast agents exhibiting a movement characteristic of a glomerulus and contrast agents having a speed greater than the first predetermined speed.

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 enabling the method to be implemented.

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 in said zone including the microvascular structure,

- advantageously a contrast agent filtering system which does not irreversibly exclude the contrast agents depending on their speed,

- a system for detecting and locating contrast agents,

- an individual monitoring system for contrast agents,

- a system capable of imaging the microvascular structure, by ultrasound, and by selecting contrast agents monitored individually and having a predetermined behavior characteristic of the circulation of the contrast agents in the microvascular structure.

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 process 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 detailed view of the image in Figure 4,

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

[0037] [Fig. 7] is a diagram illustrating the speed 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 micro-tomography,

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

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

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

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

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

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

[0048] [Fig. 18] is a detailed 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 speed 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 lomeruli

[0054] Firstly, the ULM is applied to observe the microvessels at low blood flow in a kidney 3 of rat 1, using a probe 2 (Figures 1 and 2). In general, ULM consists of imaging the microbubbles in the microvasculature with an ultrasound scanner at a frame rate of 500 Hz and a contrast agent perfusion of 13 μl/min which allows the localization and subwavelength monitoring of the individual gas particles. Eleven rats were examined using a research ultrasound machine and a 15 MHz high-frequency linear 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 sequence of amplitude modulation of long sets (LEAM in English or AMLE in French) in order to obtain classic linear data, allowing the observation of fast microbubbles, and non-linear data which reinforce the signal from slow or stopped microbubbles. The ULM post-processing steps were then performed twice in order to obtain a tracking map of fast microbubbles, from the linear acquisition filtered with singular value decomposition (SVD), and a map of slow microbubbles to from non-linear acquisition. Conventional “Power Doppler in English” ultrasound, that is to say carried out using linear acquisition and SVD, is presented in Figure 3.

[0055] Thanks to this double tracking, it is possible to obtain a composite density map of the ULMs with the slow runways in light gray and the fast runways 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, we could observe a particular behavior of microbubbles in the cortex: traces of microbubbles that enter or exit an area where they rotate at speeds corresponding to capillary flow. A zoom 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, traces 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.

The segmentation of the different regions of the kidney, namely the medulla, the large arteries (interlobar and arcuate) and the cortex, was carried out manually in order to compare the behavior of the microbubbles in the different zones. A glomerular mask was also constructed by selecting cortical tracks with a high mean free path value, i.e. stagnant microbubbles. Tracks that have at least one point in the region were included in the region analysis.

[0057] Specific metrics to highlight capillary entanglements, including normalized speeds, persistence time (i.e. the duration of the trace within a radius of 50 microns from the center of the trace) , and the 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 carried out on every 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 the traces in the medullary region and in the main arteries. Student's t tests (at a margin of 5%) show that these differences are still significant from the main vessels to the glomeruli (P<0.001 for normalized speed, P<0.001 for afterglow, P<0.001 for dispersion), and from the main vessels to the medulla (P<0.001 for normalized velocity, P<0.001 for afterglow, P<0.002 for dispersity).

[0058] The micro CT indicates that the capillary tangle detected in the ULM corresponds to the glomeruli Among the 11 rats examined by ULM in vivo, 6 were also observed post-mortem by micro CT (X-ray micro-tomography) after injection of barium contrast product at the end of the ultrasound acquisition. The manual registration of the ULM (figure 10) and the micro CT section (figure 11) was carried out on a 160 pm section, that is to say corresponding to the elevation resolution of the ultrasound probe at level of the focal point. Glomerular counting was performed using local regional high intensity 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] Manual segmentation of the renal capsule was carried out in order to compare the density of the glomeruli as a function of their distance relative to it, in the two imaging modalities. Thus, we can see 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 the 6 rats with the two modalities in order to know if the distributions of the two sets come from the same continuous distribution. The null hypothesis, i.e., h, is accepted in all rats, meaning that the glomeruli are equally distributed in both modalities at the 5% significance level.

The calculation of the size of the glomeruli was also carried out in both modalities. For ULM, in 11 rats, we plotted the glomerular density curve as a function of the distance from the center of the glomeruli and for micro CT, the intensity curve as a function of the distance from the central pixel. The full width at half maximum of these normalized data was used to estimate 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 kidney glomeruli in humans

[0063] ULM was performed on 5 transplant patients who underwent a routine clinical ultrasound examination at the hospital. We used sequences conventional contrast-enhanced ultrasound (CEUS), integrated into a clinical ultrasound machine and a convex ultrasound probe (2MHz) 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 was split with a band-pass filter to obtain the fast microbubbles, and with specific ULM parameters to highlight stagnant microbubbles.

This composite ULM density map, with the fast microbubbles in dark gray (zones B) and the slow microbubbles in light gray (zones A), presents the same glomerular shapes as rats in the distribution of the cortex (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 their spatial distribution were studied with the same technical tools. It was observed that the glomerular diameters estimated by ULM are slightly greater than those found in the literature, i.e. 200 pm in diameter, but remain in the same order of magnitude.

[0066] Manual segmentations of the areas of the kidney were carried out, namely the medulla, the cortex and the main vessels (interlobar and arcuate arteries), and the glomerular mask was calculated as previously. Thus, we were able to compare the behavior of the tracks in each region. Student's t tests were performed with a margin of 5% 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 (P < 0.001) vessels; they are more persistent, that is to say stagnant, than the medullary (P < 0.01) and main (P < 0.001) vessels; and they are also less dispersed than the medullary (P < 0.001) and main (P < 0.001) vessels.

[0067] Conclusion [0068] The invention implements a new form of ULM which exploits the kinetics of individual microbubbles, including those circulating in the capillaries, to map the microarchitecture of the vessels. By classifying their behavior according to known anatomical characteristics, we seek in this case the precise complexion of the microcirculation of the glomeruli, including the afferent and efferent arterioles. The rotating microbubbles were tracked specifically 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 through a comparison with micro CT: the spatial distribution of glomeruli by ULM and by 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 results previously found in the literature in rats and humans. Finally, glomeruli are similarly distributed between ULM and micro-CT around the intervening blood vessels.

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 indirect estimation of the glomerular filtration rate, and not their distinct observation. In addition to being able to observe a new category of slow microbubbles whose behavior and localization are comparable to glomerular organization, the process reduces the gap between anatomical imaging and functional imaging, since we are able to image the very structures responsible for the function of the organ. Thanks to ULM and the use of information given 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, acquisitions in humans were performed with a clinical ultrasound system widely available and used worldwide.

Claims

[Claim 1] Ultrasound imaging method of a microvascular structure, characterized in that it comprises:

- a step of detection/filtering and localization of the contrast agents,

- an individual monitoring step for the contrast agents,

- a step of classifying the behaviors of the contrast agents followed individually, according to 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, depending on the classification thus carried out.

[Claim 2] Method according to claim 1, characterized in that the step of carrying out the imaging is carried out by selecting and displaying the contrast agents followed individually and having a predetermined behavior characteristic of the circulation of the contrast agents in the microvascular structure.

[Claim 3] 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 predetermined movement, a predetermined passage time and/or a predetermined speed of the contrast agents. contrast.

[Claim 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.

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

[Claim 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.

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

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

[Claim 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.

[Claim 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.

[Claim 11] Method according to one of claims 8 to 10, characterized in that it comprises:

- a step of detection/filtering and localization of the contrast agents,

- an individual monitoring step for the contrast agents,

- 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 whirling movement, - a step of carrying out imaging of the glomeruli, by selecting contrast agents presenting a movement characteristic of a glomerulus.

[Claim 12] Method according to claim 11, characterized in that the step of carrying out imaging of the glomeruli is carried out by selecting contrast agents having a movement characteristic of a glomerulus and contrast agents having a higher speed at the first predetermined speed.

[Claim 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.

[Claim 14] Device making it possible to implement the method according to claim 1, characterized in that it comprises:

- a contrast agent filtering system which does not irreversibly exclude contrast agents depending on their speed,

- a system for detecting and locating contrast agents,

- an individual monitoring system for contrast agents,

- a system capable of imaging the microvascular structure, by ultrasound, and by selecting contrast agents monitored individually and having a predetermined behavior characteristic of the circulation of the contrast agents in said microvascular structure.