Super-Resolution Imaging with Ultrasound for Visualization of the Renal Microvasculature in Rats Before and After Renal Ischemia: A Pilot Study

Sofie Bech Andersen, Iman Taghavi, Carlos Armando Villagómez Hoyos, Stinne Byrholdt Søgaard, Fredrik Gran, Lars Lönn, Kristoffer Lindskov Hansen, Jørgen Arendt Jensen, Michael Bachmann Nielsen, Charlotte Mehlin Sørensen, Sofie Bech Andersen, Iman Taghavi, Carlos Armando Villagómez Hoyos, Stinne Byrholdt Søgaard, Fredrik Gran, Lars Lönn, Kristoffer Lindskov Hansen, Jørgen Arendt Jensen, Michael Bachmann Nielsen, Charlotte Mehlin Sørensen

Abstract

In vivo monitoring of the microvasculature is relevant since diseases such as diabetes, ischemia, or cancer cause microvascular impairment. Super-resolution ultrasound imaging allows in vivo examination of the microvasculature by detecting and tracking sparsely distributed intravascular microbubbles over a minute-long period. The ability to create detailed images of the renal vasculature of Sprague-Dawley rats using a modified clinical ultrasound platform was investigated in this study. Additionally, we hypothesized that early ischemic damage to the renal microcirculation could be visualized. After a baseline scan of the exposed kidney, 10 rats underwent clamping of the renal vein (n = 5) or artery (n = 5) for 45 min. The kidneys were rescanned at the onset of clamp release and after 60 min of reperfusion. Using a processing pipeline for tissue motion compensation and microbubble tracking, super-resolution images with a very high level of detail were constructed. Image filtration allowed further characterization of the vasculature by isolating specific vessels such as the ascending vasa recta with a 15-20 μm diameter. Using the super-resolution images alone, it was only possible for six assessors to consistently distinguish the healthy renal microvasculature from the microvasculature at the onset of vein clamp release. Future studies will aim at attaining quantitative estimations of alterations in the renal microvascular blood flow using super-resolution ultrasound imaging.

Keywords: Sprague-Dawley; kidney; microcirculation; rats; reperfusion injury; super-resolution ultrasound imaging; vasa recta.

Conflict of interest statement

C.A.V.H. and F.G. are employed at BK Medical ApS, Herlev, Denmark. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Experiment overview of the ischemia-reperfusion intervention. AM = amplitude modulation, f0 = center frequency of the probe, fr = frame rate, MI = mechanical index, SR = super-resolution, MB = microbubble.
Figure 2
Figure 2
(A–C) Super-resolution images (log-scaled, i.e., color bar shows the value of intensity after logarithmic compression, ranging from 0~30 dB, and intensity corresponds to the number of microbubbles) of the healthy rat renal microvasculature. (A) There is a clear distinction between the dense cortical (CO) microvascular network and the vasa recta of the outer medulla (OM) and inner medulla (IM), leading down to the papilla (PA). Even though the scans were performed under the same conditions, the images show how the number of microbubbles varied between the rats, indicated by the higher intensity on image (B) (average of 117 detected microbubbles/frame) compared with image (C) (average of 70 detected microbubbles/frame). (D) Ex vivo magnetic resonance T1 contrast-enhanced image of another rat kidney for comparison. The kidneys measure approximately 2 cm in craniocaudal length and 1 cm in the medial-lateral direction. Scale bar: 2 mm.
Figure 3
Figure 3
Super-resolution microbubble track maps. (A) Unfiltered microbubble track map. The color of the wheel indicates microbubble flow direction, and the color brightness indicates MB flow velocity. The image shows the opposite flow of the paired arteries and veins of the renal vascular tree. (B) Without filtering, this velocity map (from another rat) contains a high number of tracks (343,939). (C) The microbubble track map from (B) filtered to increase the robustness of the included tracks and highlight the long straight vasa recta, thereby allowing a distinction between the descending (green/blue) and ascending (red) vasa recta (insert). (D) Another way to filter these maps is by direction. This image is filtered to show microbubbles with a direction going from left to right, leaving the descending vasa recta visible on the left side of the medulla, and the ascending visible on the right side. Scale bar: 2 mm.
Figure 4
Figure 4
(A) Super-resolution images (log-scaled, i.e., color bar shows the value of intensity after logarithmic compression, ranging from 0~30 dB, and intensity corresponds to the number of microbubbles) of three rat kidneys before and after clamping of the renal vein. (B) Super-resolution images of three rat kidneys before and after clamping of the renal artery. Scale bar: 10 mm.
Figure 5
Figure 5
(A) Super-resolution (log-scaled) and power Doppler images of two rat kidneys before and after clamping of the renal vein. (B) Super-resolution (log-scaled) and power Doppler images of two rat kidneys before and after clamping of the renal artery. In these examples, all the super-resolution images showed a complete refilling of the microvascular bed after 60 min of reperfusion. The power Doppler scans from all animals showed a decreased signal in the cortex after 60 min of reperfusion compared with baseline. Scale bar: 10 mm.
Figure 6
Figure 6
Hematoxylin and eosin staining of the renal cortex from three rats. (A) Control. (B) After renal artery clamping. (C) After renal vein clamping. The black arrows show intratubular cast formation in the tubules. The yellow arrow shows erythrocytes accumulated in the renal interstitial space around a glomerulus. Scale bar: 100 μm.

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