Lok, U‐Wai

Abstract Background Spinal cord injury (SCI) induces complex secondary pathophysiological cascades, including microvascular disruption, glial activation, and chronic neuroinflammation. These vascular changes are dynamic and spatially heterogeneous across acute, subacute, and chronic phases. However, limitations in in vivo imaging resolution have impeded precise, longitudinal evaluation of spinal microcirculation. Objective To quantify post-SCI alterations in spinal cord vessel density, flow speed, and morphology over time using ultrasound localization microscopy (ULM), and to correlate these changes with chronic astrocytic reactivity assessed by GFAP expression. Methods Eleven Sprague-Dawley rats underwent T9–T11 laminectomy; nine received a standardized 200 kdyn contusion using the Infinite Horizons Impactor and were assigned to acute (24h), subacute (7d), and chronic (21d) groups (n = 3/group), while two served as uninjured controls. ULM was performed using a Vevo 3100 high-frequency ultrasound system with a 40 MHz transducer following intravenous Definity® microbubble injection. Vessel density (VD) and flow speed (FS) were quantified at the lesion epicenter and adjacent rostral/caudal regions. Arterial and venous compartments were distinguished by flow direction. Histological sections were stained for glial fibrillary acidic protein (GFAP), and %Area was quantified across phases. One-way ANOVA and Tukey’s test evaluated between-group differences; Pearson correlation identified phase-specific relationships between vascular metrics and chronic GFAP expression. Results ULM provided super-resolved imaging of spinal microvasculature at 5–10 µm resolution. Venous VD significantly declined from 0.92 ± 0.18 (acute) to 0.42 ± 0.23 (subacute) and 0.11 ± 0.07 (chronic, p < 0.001), particularly in dorsal regions. Arterial VD remained stable across timepoints (p > 0.05), while FS demonstrated transient reductions in both arterial and venous systems during the subacute phase. Chronic venous FS partially recovered by Day 21. GFAP-positive area increased significantly in the chronic group (3.3 ± 0.87%) compared to acute (1.52 ± 0.42%) and subacute (1.40 ± 0.05%) (p < 0.05), indicating delayed astrocyte reactivity. Correlation analysis revealed significant inverse associations between chronic GFAP %Area and early ventral venous density (r = − 1.0, p < 0.05), as well as positive correlations with chronic ventral arterial FS (r = 1.0, p < 0.01), suggesting early microvascular deficits may predict long-term gliosis. Conclusion This study demonstrates the feasibility and power of ULM to quantify dynamic microvascular remodeling in SCI with unprecedented resolution. Venous rarefaction precedes chronic astrocytosis, while arterial flow speed partially recovers over time. These findings highlight key spatiotemporal biomarkers of injury progression and support the use of ULM as a noninvasive platform for monitoring SCI pathophysiology and guiding therapeutic timing.

Objective: This study aims to develop a contrast-free, high-sensitivity ultrasound method, denoted as Radon transform-based flow measurement (R-Flow), for in vivo mapping of microvascular flow vectors and for establishing R-Flow-derived vector-field metrics to noninvasively quantify microcirculatory patterns in liver cirrhosis. Impact Statement: R-Flow enables robust, contrast-free imaging of microvascular dynamics and demonstrates translational feasibility in the human liver. Its direction-aware indices offer pilot in vivo quantification of flow redistribution and remodeling, providing unique insights into hepatic flow dynamics. Introduction: Microvascular dysfunction is a hallmark of many diseases, yet noninvasive visualization and quantitative assessment of abnormal microcirculation remain limited. Methods: R-Flow leverages Radon transform to decode red blood cell dynamics from the spatiotemporal domain of ultrasound flow signals, reconstructing velocity vectors at microvascular scale. From these vector maps, unique direction-aware indices are further derived to characterize flow distribution and heterogeneity. Results: Validated across simulations, phantoms, and in vivo studies, R-Flow provides robust velocity estimation across a wide range (1 to 60 mm/s). Notably, it enables high-sensitivity microvascular flow vector mapping of human liver, showing strong agreement with references ( r > 0.9). In a rat model, these direction-aware indices revealed a shift from healthy multipath perfusion in control livers to directionally biased vascular pattern in cirrhotic livers, demonstrating significant correlation with pathological indicators. Conclusion: R-Flow enables noninvasive, contrast-free mapping of microvascular blood flow velocity and offers a promising approach for high-resolution assessment of microvascular flow characteristics.

Ultrasound localization microscopy (ULM) enables imaging of the microvasculature at spatial resolutions surpassing the limits of conventional ultrasound, while maintaining comparable penetration depth. This super-resolution technique relies on the localization and tracking of individual microbubbles (MBs) administered into the bloodstream, providing detailed visualization of microvascular structures and functions. However, clinical implementation of ULM faces significant challenges, such as low signal-to-noise ratio (SNR), tissue motion artifacts, and prolonged acquisition times. In this presentation, we will discuss advancements aimed at achieving robust and reproducible clinical implementation of ULM through a series of technical innovations and optimizations. These include signal enhancement techniques to boost MB SNR, advanced algorithms for accurate MB localization and tracking at clinically relevant dosages, optimized data acquisition strategies tailored to clinical workflows, and the development of 3-D imaging capabilities. Through pilot preclinical and clinical studies, we demonstrate the feasibility and efficacy of ULM in various applications, including chronic kidney disease (CKD), kidney transplant, liver disease evaluation, and beyond, in both animal models and human subjects under clinical scanning conditions. These findings underscore the transformative potential of super-resolution ultrasound imaging as a diagnostic tool, paving the way for widespread clinical adoption.

ObjectivesFetal growth restriction (FGR) is commonly associated with placental dysfunction, increasing perinatal morbidity and mortality. Visualizing placental vessels in utero would be advantageous for identifying functional FGR cause and determining proper management strategies. We aimed to utilize high‐sensitivity ultrasound microvessel imaging (HUMI) for quantifying placental vessel density (VD) in pregnancies diagnosed with FGR.MethodsThis pilot case–control study enrolled subjects in the third trimester with a diagnosis of FGR (n = 40) and gestational age‐matched controls with normal fetal growth (n = 20) at a 2:1 ratio, respectively. The Verasonics Vantage ultrasound system was used to perform HUMI on each participant at one timepoint. Scanning involved randomized singular value decomposition‐based clutter filtering to identify the villous tree, followed by step‐by‐step scanning to acquire 3‐dimensional‐like data. Mean VD was calculated from three ultrasound measurements per subject. Additional clinical and pathology data were also collected and compared.ResultsSixteen participants were utilized to establish the scanning protocol and 2 met exclusion criteria at delivery. Thus, VD was successfully measured on 42 pregnancies scanned at 35 weeks 5 days on average. In FGR (n = 24), placental VD was significantly reduced compared to controls (P < .01). VD measures were as good at predicting FGR as systolic/diastolic (S/D) ratios (area under the curve 0.86 versus 0.80). In a smaller cohort, VD in placentas with a diagnosis of inflammatory villitis (n = 10) by histology showed an increase in VD compared to those without inflammation (P = .01). Low VD was correlated with increased S/D ratios (P = .03).ConclusionsHUMI is useful for identifying altered placental vascularization in utero for FGR. VD may be a valuable indicator for placental health and could lead to improved risk stratification methods considering underlying biology.

ObjectivesMetabolic dysfunction‐associated steatotic liver disease (MASLD) is the most prevalent liver disorder in Western countries, with approximately 20%–30% of the MASLD patients progressing to severe stages. There is an urgent need for noninvasive, cost‐effective, widely accessible, and precise biomarkers to evaluate liver steatosis. This study aims to assess and compare the diagnostic performance of a novel reference frequency method‐based ultrasound attenuation coefficient estimation (ACE) in both fundamental (RFM‐ACE‐FI) and harmonic (RFM‐ACE‐HI) imaging for detecting and grading liver steatosis.MethodsAn Institutional Review Board‐approved prospective study was carried out between December 2018 and October 2022. A total number of 130 subjects were enrolled in the study. The correlation between RFM‐ACE‐HI values and magnetic resonance imaging proton density fat fraction (MRI‐PDFF), as well as between RFM‐ACE‐FI values and MRI‐PDFF were calculated. The diagnostic performance of RFM‐ACE‐FI and RFM‐ACE‐HI was evaluated using receiver operating characteristic (ROC) curve analysis, as compared to MRI‐PDFF. The reproducibility of RFM‐ACE‐HI was assessed by interobserver agreement between two sonographers.ResultsA strong correlation was observed between RFM‐ACE‐HI and MRI‐PDFF, with R = 0.88 (95% confidence interval [CI]: 0.83–0.92; P < .001), while the correlation between RFM‐ACE‐FI and MRI‐PDFF was R = 0.65 (95% CI: 0.50–0.76; P < .001). The area under the ROC (AUROC) curve for RFM‐ACE‐HI in staging liver steatosis grades of S ≥ 1 and S ≥ 2 was 0.97 (95% CI: 0.91–0.99; P < .001) and 0.98 (95% CI: 0.93–1.00; P < .001), respectively, and 0.76 (95% CI: 0.65–0.85) and 0.80 (95% CI: 0.70–0.88) for RFM‐ACE‐FI, respectively. Great reproducibility was achieved for RFM‐ACE‐HI, with an interobserver agreement of R = 0.97 (95% CI: 0.94–0.99; P < .001).ConclusionsThe novel RFM‐ACE‐HI method offered high liver steatosis diagnostic accuracy and reproducibility, which has important clinical implications for early disease intervention and treatment evaluation.