Multiparametric ultrasound imaging for early-stage steatosis: Comparison with magnetic resonance imaging-based proton density fat fraction.

BACKGROUND: The prevalence of liver diseases, especially steatosis, requires a more convenient and noninvasive tool for liver diagnosis, which can be a surrogate for the gold standard biopsy. Magnetic resonance (MR) measurement offers potential, however ultrasound (US) has better accessibility than MR. PURPOSE: This study aims to suggest a multiparametric US approach which demonstrates better quantification and imaging performance than MR imaging-based proton density fat fraction (MRI-PDFF) for hepatic steatosis assessment. METHODS: We investigated early-stage steatosis to evaluate our approach. An in vivo (within the living) animal study was performed. Fat inclusions were accumulated in the animal livers by feeding a methionine and choline deficient (MCD) diet for 2 weeks. The animals (n = 19) underwent US and MR imaging, and then their livers were excised for histological staining. From the US, MR, and histology images, fat accumulation levels were measured and compared: multiple US parameters; MRI-PDFF; histology fat percentages. Seven individual US parameters were extracted using B-mode measurement, Burr distribution estimation, attenuation estimation, H-scan analysis, and shear wave elastography. Feature selection was performed, and the selected US features were combined, providing quantification of fat accumulation. The combined parameter was used for visualizing the localized probability of fat accumulation level in the liver; This procedure is known as disease-specific imaging (DSI). RESULTS: The combined US parameter can sensitively assess fat accumulation levels, which is highly correlated with histology fat percentage (R = 0.93, p-value < 0.05) and outperforms the correlation between MRI-PDFF and histology (R = 0.89, p-value < 0.05). Although the seven individual US parameters showed lower correlation with histology compared to MRI-PDFF, the multiparametric analysis enabled US to outperform MR. Furthermore, this approach allowed DSI to detect and display gradual increases in fat accumulation. From the imaging output, we measured the color-highlighted area representing fatty tissues, and the fat fraction obtained from DSI and histology showed strong agreement (R = 0.93, p-value < 0.05). CONCLUSIONS: We demonstrated that fat quantification utilizing a combination of multiple US parameters achieved higher performance than MRI-PDFF; therefore, our multiparametric analysis successfully combined selected features for hepatic steatosis characterization. We anticipate clinical use of our proposed multiparametric US analysis, which could be beneficial in assessing steatosis in humans.

Simultaneous imaging of ultrasonic relative backscatter and attenuation coefficients for quantitative liver steatosis assessment.

Prevalence of liver disease is continuously increasing and nonalcoholic fatty liver disease (NAFLD) is the most common etiology. We present an approach to detect the progression of liver steatosis based on quantitative ultrasound (QUS) imaging. This study was performed on a group of 55 rats that were subjected to a control or methionine and choline deficient (MCD) diet known to induce NAFLD. Ultrasound (US) measurements were performed at 2 and 6 weeks. Thereafter, animals were humanely euthanized and livers excised for histological analysis. Relative backscatter and attenuation coefficients were simultaneously estimated from the US data and envelope signal-to-noise ratio was calculated to train a regression model for: (1) fat fraction percentage estimation and (2) performing classification according to Brunt's criteria in grades (0 <5%; 1, 5-33%; 2, >33-66%; 3, >66%) of liver steatosis. The trained regression model achieved an [Formula: see text] of 0.97 (p-value < 0.01) and a RMSE of 3.64. Moreover, the classification task reached an accuracy of 94.55%. Our results suggest that in vivo QUS is a promising noninvasive imaging modality for the early assessment of NAFLD.

Image-guided focused ultrasound-mediated molecular delivery to breast cancer in an animal model.

Tumors become inoperable due to their size or location, making neoadjuvant chemotherapy the primary treatment. However, target tissue accumulation of anticancer agents is limited by the physical barriers of the tumor microenvironment. Low-intensity focused ultrasound (FUS) in combination with microbubble (MB) contrast agents can increase microvascular permeability and improve drug delivery to the target tissue after systemic administration. The goal of this research was to investigate image-guided FUS-mediated molecular delivery in volume space. Three-dimensional (3-D) FUS therapy functionality was implemented on a programmable ultrasound scanner (Vantage 256, Verasonics Inc.) equipped with a linear array for image guidance and a 128-element therapy transducer (HIFUPlex-06, Sonic Concepts). FUS treatment was performed on breast cancer-bearing female mice (N= 25). Animals were randomly divided into three groups, namely, 3-D FUS therapy, two-dimensional (2-D) FUS therapy, or sham (control) therapy. Immediately prior to the application of FUS therapy, animals received a slow bolus injection of MBs (Definity, Lantheus Medical Imaging Inc.) and near-infrared dye (IR-780, surrogate drug) for optical reporting and quantification of molecular delivery. Dye accumulation was monitored viain vivooptical imaging at 0, 1, 24, and 48 h (Pearl Trilogy, LI-COR). Following the 48 h time point, animals were humanely euthanized and tumors excised forex vivoanalyzes. Optical imaging results revealed that 3-D FUS therapy improved delivery of the IR-780 dye by 66.4% and 168.1% at 48 h compared to 2-D FUS (p= 0.18) and sham (p= 0.047) therapeutic strategies, respectively.Ex vivoanalysis revealed similar trends. Overall, 3-D FUS therapy can improve accumulation of a surrogate drug throughout the entire target tumor burden after systemic administration.

3-D H-scan ultrasound imaging of relative scatterer size using a matrix array transducer and sparse random aperture compounding.

H-scan ultrasound (US) is a high-resolution imaging technique for soft tissue characterization. By acquiring data in volume space, H-scan US can provide insight into subtle tissue changes or heterogenous patterns that might be missed using traditional cross-sectional US imaging approaches. In this study, we introduce a 3-dimensional (3-D) H-scan US imaging technology for voxel-level tissue characterization in simulation and experimentation. Using a matrix array transducer, H-scan US imaging was developed to evaluate the relative size of US scattering aggregates in volume space. Experimental data was acquired using a programmable US system (Vantage 256, Verasonics Inc, Kirkland, WA) equipped with a 1024-element (32 × 32) matrix array transducer (Vermon Inc, Tours, France). Imaging was performed using the full array in transmission. Radiofrequency (RF) data sequences were collected using a sparse random aperture compounding technique with 6 different data compounding approaches. Plane wave imaging at five angles was performed at a center frequency of 8 MHz. Scan conversion and attenuation correction were applied. To generate the 3-D H-scan US images, a convolution filter bank (N = 256) was then used to process the RF data sequences and measure the spectral content of the backscattered US signals before volume reconstruction. Preliminary experimental studies were conducted using homogeneous phantom materials embedded with spherical US scatterers of varying diameter, i.e., 27 to 45, 63 to 75, or 106-126 µm. Both simulated and experimental results revealed that 3-D H-scan US images have a low spatial variance when tested with homogeneous phantom materials. Furthermore, H-scan US is considerably more sensitive than traditional B-mode US imaging for differentiating US scatterers of varying size (p = 0.001 and p = 0.93, respectively). Overall, this study demonstrates the feasibility of 3-D H-scan US imaging using a matrix array transducer for tissue characterization in volume space.

Photoacoustic graphic equalization and application in characterization of red blood cell aggregates.

A photoacoustic (PA) graphic equalization (PAGE) algorithm was developed to characterize the relative size of optical absorbing aggregates. This technique divides the PA signal into frequency bands related to different-sized optical absorbers. Simulations of a material containing optical absorbing microparticles of varying size were used to assess PAGE performance. Experiments were performed on phantom materials containing microspheres of varying size and concentration. Additional experiments were performed using tubes with fresh clotting blood. PA data was obtained using a Vevo LAZR-X system (FUJIFILM VisualSonics Inc). PAGE imaging of phantoms with varying-sized optical absorbers found a 1.5-fold difference in mean image intensity (p < 0.001). Conversely, PA images from these same materials exhibited no intensity changes (p = 0.68). PAGE imaging results from clotting blood exhibited differences for clot sizes in the range 0.30-0.64 mm (p < 0.001). In summary, PAGE imaging can distinguish optical absorbing aggregates of varying size.

Disease-Specific Imaging Utilizing Support Vector Machine Classification of H-Scan Parameters: Assessment of Steatosis in a Rat Model.

In medical imaging, quantitative measurements have shown promise in identifying diseases by classifying normal versus pathological parameters from tissues. The support vector machine (SVM) has shown promise as a supervised classification algorithm and has been widely used. However, the classification results typically identify a category of abnormal tissues but do not necessarily differentiate progressive stages of a disease. Moreover, the classification result is typically provided independently as a supplement to medical images, which contributes to an overload of information sources in the clinic. Hence, we propose a new imaging method utilizing the SVM to integrate classification results into medical images. This framework is called disease-specific imaging (DSI) that produces a color overlaid highlight on B-mode ultrasound images indicating the type, location, and severity of pathology from different conditions. In this article, the SVM training was performed to construct hyperplanes that can differentiate normal, fibrosis, steatosis, and pancreatic ductal adenocarcinoma (PDAC) metastases in livers based on ultrasound echoes. Also, cluster centroids for specific diseases define unique disease axes, and the inner product between measured features and any disease axis selected by the SVM quantifies the disease progression. The features were measured from 2794 ultrasound frames using the H-scan analysis, attenuation estimation, and B-mode image analysis. The performance of our proposed DSI method was evaluated for a preclinical model of steatosis ( n = 400 frames). The contribution of each feature was assessed, and the results were compared with ground truth from histology. Moreover, the images generated by our DSI were compared with earlier imaging methods of B-mode, H-scan, and histology. The comparisons demonstrate that DSI images yield higher sensitivity to monitor progressive steatosis than B-mode and H-scan and provide a comparable performance with the histology. For the parameter comparison, DSI and H-scan resulted in similar correlation with histology ( rs = 0.83 ) but higher than attenuation ( rs = 0.73 ) and B-mode ( rs = 0.47 ). Therefore, we conclude that DSI utilizing the SVM applied to steatosis can visually represent the classification results with color highlighting, which can simplify the interpretation of classification compared to the traditional SVM result. We expect that the proposed DSI can be used for any medical imaging modality that can estimate multiple quantitative parameters at high resolution.

Clusters of Ultrasound Scattering Parameters for the Classification of Steatotic and Normal Livers.

The study of ultrasound tissue interactions in fatty livers has a long history with strong clinical potential for assessing steatosis. Recently we proposed alternative measures of first- and second-order statistics of echoes from soft tissues, namely, the H-scan, which is based on a matched filter approach, to quantify scattering transfer functions and the Burr distribution to model speckle patterns. Taken together, these approaches produce a multiparameter set that is directly related to the fundamentals of ultrasound propagation in tissue. To apply this approach to the problem of assessing steatotic livers, these analyses were applied to in vivo rat livers (N=21) under normal feeding conditions or after receiving a methionine- and choline-deficient diet that produces steatosis within a few weeks. Ultrasound data were acquired at baseline and again at weeks 2 and 6 before applying the H-scan and Burr analyses. Furthermore, a classification technique known as the support vector machine was then used to find clusters of the five parameters that are characteristic of the different steatotic liver conditions as confirmed by histologic processing of excised liver tissue samples. With the in vivo multiparametric ultrasound measurement approach and determination of clusters, steatotic can be discriminated from normal livers with 100% accuracy in a rat animal model.

Intercellular Arc Signaling Regulates Vasodilation.

Injury responses require communication between different cell types in the skin. Sensory neurons contribute to inflammation and can secrete signaling molecules that affect non-neuronal cells. Despite the pervasive role of translational regulation in nociception, the contribution of activity-dependent protein synthesis to inflammation is not well understood. To address this problem, we examined the landscape of nascent translation in murine dorsal root ganglion (DRG) neurons treated with inflammatory mediators using ribosome profiling. We identified the activity-dependent gene, Arc, as a target of translation in vitro and in vivo Inflammatory cues promote local translation of Arc in the skin. Arc-deficient male mice display exaggerated paw temperatures and vasodilation in response to an inflammatory challenge. Since Arc has recently been shown to be released from neurons in extracellular vesicles (EVs), we hypothesized that intercellular Arc signaling regulates the inflammatory response in skin. We found that the excessive thermal responses and vasodilation observed in Arc defective mice are rescued by injection of Arc-containing EVs into the skin. Our findings suggest that activity-dependent production of Arc in afferent fibers regulates neurogenic inflammation potentially through intercellular signaling.SIGNIFICANCE STATEMENT Nociceptors play prominent roles in pain and inflammation. We examined rapid changes in the landscape of nascent translation in cultured dorsal root ganglia (DRGs) treated with a combination of inflammatory mediators using ribosome profiling. We identified several hundred transcripts subject to rapid preferential translation. Among them is the immediate early gene (IEG) Arc. We provide evidence that Arc is translated in afferent fibers in the skin. Arc-deficient mice display several signs of exaggerated inflammation which is normalized on injection of Arc containing extracellular vesicles (EVs). Our work suggests that noxious cues can trigger Arc production by nociceptors which in turn constrains neurogenic inflammation in the skin.

Multiparametric ultrasound imaging for the assessment of normal versus steatotic livers.

Liver disease is increasing in prevalence across the globe. We present here a multiparametric ultrasound (mpUS) imaging approach for assessing nonalcoholic fatty liver disease (NALFD). This study was performed using rats (N = 21) that were fed either a control or methionine and choline deficient (MCD) diet. A mpUS imaging approach that includes H-scan ultrasound (US), shear wave elastography, and contrast-enhanced US measurements were then performed at 0 (baseline), 2, and 6 weeks. Thereafter, animals were euthanized and livers excised for histological processing. A support vector machine (SVM) was used to find a decision plane that classifies normal and fatty liver conditions. In vivo mpUS results from control and MCD diet fed animals reveal that all mpUS measures were different at week 6 (P < 0.05). Principal component analysis (PCA) showed that the H-scan US data contributed the highest percentage to the classification among the mpUS measurements. The SVM resulted in 100% accuracy for classification of normal and high fat livers and 92% accuracy for classification of normal, low fat, and high fat livers. Histology findings found considerable steatosis in the MCD diet fed animals. This study suggests that mpUS examinations have the potential to provide a comprehensive estimation of the main components of early stage NAFLD.

Multifocused Ultrasound Therapy for Controlled Microvascular Permeabilization and Improved Drug Delivery.

Focused ultrasound (FUS) exposure of micro-bubble (MB) contrast agents can transiently increase microvascular permeability allowing anticancer drugs to extravasate into a targeted tumor tissue. Either fixed or mechanically steered in space, most studies to date have used a single element focused transducer to deliver the ultrasound (US) energy. The goal of this study was to investigate various multi-FUS strategies implemented on a programmable US scanner (Vantage 256, Verasonics Inc.) equipped with a linear array for image guidance and a 128-element therapy transducer (HIFUPlex-06, Sonic Concepts). The multi-FUS strategies include multi-FUS with sequential excitation (multi-FUS-SE) and multi-FUS with temporal sequential excitation (multi-FUS-TSE) and were compared to single-FUS and sham treatment. This study was performed using athymic mice implanted with breast cancer cells ( N = 20 ). FUS therapy experiments were performed for 10 min after a solution containing MBs (Definity, Lantheus Medical Imaging Inc.) and near-infrared (NIR, surrogate drug) dye were injected via the tail vein. The fluorescent signal was monitored using an in vivo optical imaging system (Pearl Trilogy, LI-COR) to quantify intratumoral dye accumulation at baseline and again at 0.1, 24, and 48 h after receiving US therapy. Animals were then euthanized for ex vivo dye extraction analysis. At 48 h, fluorescent tracer accumulation within the tumor space for the multi-FUS-TSE therapy group animals was found to be 67.3%, 50.3%, and 36.2% higher when compared to sham, single-FUS, and multi-FUS-SE therapy group measures, respectively. Also, dye extraction and fluorescence measurements from excised tumor tissue found increases of 243.2%, 163.1%, and 68.1% for the multi-FUS-TSE group compared to sham, single-FUS, and multi-FUS-SE therapy group measures, respectively. In summary, experimental results revealed that for a multi-FUS sequence, increased microvascular permeability was considerably influenced by both the spatial and temporal aspects of the applied US therapy.