Aging-related cerebral microvascular changes visualized using ultrasound localization microscopy in the living mouse

Aging-related cognitive decline is an emerging health crisis; however, no established unifying mechanism has been identified for the cognitive impairments seen in an aging population. A vascular hypothesis of cognitive decline has been proposed but is difficult to test given the requirement of high-fidelity microvascular imaging resolution with a broad and deep brain imaging field of view, which is restricted by the fundamental trade-off of imaging penetration depth and resolution. Super-resolution ultrasound localization microscopy (ULM) offers a potential solution by exploiting circulating microbubbles to achieve a vascular resolution approaching the capillary scale without sacrificing imaging depth. In this report, we apply ULM imaging to a mouse model of aging and quantify differences in cerebral vascularity, blood velocity, and vessel tortuosity across several brain regions. We found significant decreases in blood velocity, and significant increases in vascular tortuosity, across all brain regions in the aged cohort, and significant decreases in blood volume in the cerebral cortex. These data provide the first-ever ULM measurements of subcortical microvascular dynamics in vivo within the context of the aging brain and reveal that aging has a major impact on these measurements.

Ultrafast Doppler imaging and Ultrasound Localization Microscopy reveal the complexity of vascular rearrangement in chronic spinal lesion

Acute spinal cord injury (SCI) leads to severe damage to the microvascular network. The process of spontaneous repair is accompanied by formation of new blood vessels; their functionality, however, presumably very important for functional recovery, has never been clearly established, as most studies so far used fixed tissues. Here, combining ultrafast Doppler imaging and Ultrasound Localization Microscopy (ULM) on the same animals, we proceeded at a detailed analysis of structural and functional vascular alterations associated with the establishment of chronic SCI, both at macroscopic and microscopic scales. Using a standardized animal model of SCI, our results demonstrate striking hemodynamic alterations in several subparts of the spinal cord: a reduced blood velocity in the lesion site, and an asymmetrical hypoperfusion caudal but not rostral to the lesion. In addition, the worsening of many evaluated parameters at later time points suggests that the neoformed vascular network is not yet fully operational, and reveals ULM as an efficient in vivo readout for spinal cord vascular alterations. Finally, we show statistical correlations between the diverse biomarkers of vascular dysfunction and SCI severity. The imaging modality developed here will allow evaluating recovery of vascular function over time in pre-clinical models of SCI. Also, used on SCI patients in combination with other quantitative markers of neural tissue damage, it may help classifying lesion severity and predict possible treatment outcomes in patients.

Ultrafast Doppler imaging and Ultrasound Localization Microscopy reveal the complexity of vascular rearrangement in chronic spinal lesion

Acute spinal cord injury (SCI) leads to severe damage to the microvascular network. The process of spontaneous repair is accompanied by formation of new blood vessels; their functionality, however, presumably very important for functional recovery, has never been clearly established, as most studies so far used fixed tissues. Here, combining ultrafast Doppler imaging and Ultrasound Localization Microscopy (ULM) on the same animals, we proceeded at a detailed analysis of structural and functional vascular alterations associated with the establishment of chronic SCI, both at macroscopic and microscopic scales. Using a standardized animal model of SCI, our results demonstrate striking hemodynamic alterations in several subparts of the spinal cord: a reduced blood velocity in the lesion site, and an asymmetrical hypoperfusion caudal but not rostral to the lesion. In addition, the worsening of many evaluated parameters at later time points suggests that the neoformed vascular network is not yet fully operational, and reveals ULM as an efficient in vivo readout for spinal cord vascular alterations. Finally, we show statistical correlations between the diverse biomarkers of vascular dysfunction and SCI severity. The imaging modality developed here will allow evaluating recovery of vascular function over time in pre-clinical models of SCI. Also, used on SCI patients in combination with other quantitative markers of neural tissue damage, it may help classifying lesion severity and predict possible treatment outcomes in patients.

An Anatomically and Hemodynamically Realistic Simulation Framework for 3D Ultrasound Localization Microscopy

The resolution of 3D Ultrasound Localization Microscopy (ULM) is determined by acquisition parameters such as frequency and transducer geometry but also by microbubble (MB) concentration, which is also linked to the total acquisition time needed to sample the vascular tree at different scales. In this study, we introduce a novel 3D anatomically- and physiologically-realistic ULM simulation framework based on two-photon microscopy (2PM) and in-vivo MB perfusion dynamics. As a proof of concept, using metrics such as MB localization error, MB count and network filling, we could quantify the effect of MB concentration and PSF volume by varying probe transmit frequency (3-15 MHz). We find that while low frequencies can achieve sub-wavelength resolution as predicted by theory, they are also associated with prolonged acquisition times to map smaller vessels, thus limiting effective resolution. A linear relationship was found between maximal MB concentration and inverse point spread function (PSF) volume. Since inverse PSF volume roughly scales cubically with frequency, the reconstruction of the equivalent of 10 minutes at 15 MHz would require hours at 3 MHz. We expect that these findings can be leveraged to achieve effective reconstruction and serve as a guide for choosing optimal MB concentrations in ULM.

Improved Ultrasound Localization Microscopy based on Microbubble Uncoupling via Transmit Excitation (MUTE)

Ultrasound localization microscopy (ULM) demonstrates great potential for visualization of tissue microvasculature at depth with high spatial resolution. The success of ULM heavily depends on the robust localization of isolated microbubbles (MBs), which can be challenging in vivo especially within larger vessels where MBs can overlap and cluster close together. While MB dilution alleviates the issue of MB overlap to a certain extent, it drastically increases the data acquisition time needed for MBs to populate the microvasculature, which is already on the order of several minutes using recommended MB concentrations. Inspired by optical super-resolution imaging based on stimulated emission depletion (STED), here we propose a novel ULM imaging sequence based on microbubble uncoupling via transmit excitation (MUTE). MUTE “silences” MB signals by creating acoustic nulls to facilitate MB separation, which leads to robust localization of MBs especially under high concentrations. The efficiency of localization accomplished via the proposed technique was first evaluated in simulation studies with conventional ULM as a benchmark. Then an in vivo study based on the chorioallantoic membrane (CAM) of chicken embryos showed that MUTE could reduce the data acquisition time by half thanks to the enhanced MB separation and localization. Finally, the performance of MUTE was validated in an in vivo mouse brain study. These results demonstrate the high MB localization efficacy of MUTE-ULM, which contributes to a reduced data acquisition time and improved temporal resolution for ULM.

Simulation-Based Resilience Quantification of an Indoor Ultrasound Localization System in the Presence of Disruptions

Time difference of arrival (TDOA) based indoor ultrasound localization systems are prone to multiple disruptions and demand reliable, and resilient position accuracy during operation. In this challenging context, a missing link to evaluate the performance of such systems is a simulation approach to test their robustness in the presence of disruptions. This approach cannot only replace experiments in early phases of development but could also be used to study susceptibility, robustness, response, and recovery in case of disruptions. The paper presents a simulation framework for a TDOA-based indoor ultrasound localization system and ways to introduce different types of disruptions. This framework can be used to test the performance of TDOA-based localization algorithms in the presence of disruptions. Resilience quantification results are presented for representative disruptions. Based on these quantities, it is found that localization with arc-tangent cost function is approximately 30% more resilient than the linear cost function. The simulation approach is shown to apply to resilience engineering and can be used to increase the efficiency and quality of indoor localization methods.

Volumetric ultrasound localization microscopy of the whole brain microvasculature

Technologies to visualize whole organs across scales in vivo are essential for our understanding of biology in health and disease. To date, only post-mortem techniques such as perfused computed tomography scanning or optical microscopy of cleared tissues achieve cellular resolution across entire organs and imaging methods with equal performance in living mammalian organs have yet to be developed. Recently, 2D ultrasound localization microscopy has successfully mapped the fine-scale vasculature of various organs down to a 10 μm precision. However, reprojection issues and out-of-plane motion prevent complex blood flow quantification and fast volumetric imaging of whole organs. Here, we demonstrate for the first time in vivo volumetric ultrasound localization microscopy mapping of the rodent brain vasculature. We developed a complete methodological pipeline that includes specific surgery, a dedicated 3D ultrasound acquisition sequence, localization and tracking algorithms, motion correction and realignment, as well as the post-processing quantification of cerebral blood flow. We illustrate the power of this approach, by mapping the whole rat brain vasculature at a resolution of 12 μm, revealing mesoscopic to macroscopic vascular architectures and cerebral blood flows ranging from 1 to 100 mm/s. Our results pave the way to the investigation of in vivo vascular processes across the mammalian brain in health and disease, in a wide range of contexts and models.