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The kidney is an anisotropic organ, with higher elasticity along versus across nephrons. The degree of mechanical anisotropy in the kidney may be diagnostically relevant if properly exploited; however, if improperly controlled, anisotropy may confound stiffness measurements. The purpose of this study is to demonstrate the clinical feasibility of acoustic radiation force (ARF)-induced peak displacement (PD) measures for both exploiting and obviating mechanical anisotropy in the cortex of human kidney allografts, in vivo . Validation of the imaging methods is provided by preclinical studies in pig kidneys, in which ARF-induced PD values were significantly higher ( $p , Wilcoxon) when the transducer executing asymmetric ARF was oriented across versus along the nephrons. The ratio of these PD values obtained with the transducer oriented across versus along the nephrons strongly linearly correlated ( $R^{2} = 0.95$ ) to the ratio of shear moduli measured by shear wave elasticity imaging. On the contrary, when a symmetric ARF was implemented, no significant difference in PD was observed ( $p > 0.01$ ). Similar results were demonstrated in vivo in the kidney allografts of 14 patients. The symmetric ARF produced PD measures with no significant difference ( $p > 0.01$ ) between along versus across alignments, but the asymmetric ARF yielded PD ratios that remained constant over a six-month observation period post-transplantation, consistent with stable serum creatinine level and urine protein-to-creatinine ratio in the same patient population ( $p> 0.01$ ). The results of this pilot in vivo clinical study suggest the feasibility of 1) implementing symmetrical ARF to obviate mechanical anisotropy in the kidney cortex when anisotropy is a confounding factor and 2) implementing asymmetric ARF to exploit mechanical anisotropy when mechanical anisotropy is a potentially relevant biomarker.

Our group has previously found that in silico, mechanical anisotropy may be interrogated by exciting transversely isotropic materials with geometrically asymmetric acoustic radiation force excitations and then monitoring the associated induced displacements in the region of excitation. We now translate acoustic radiation force-based anisotropy assessment to human muscle in vivo and investigate its clinical relevance to monitoring muscle degeneration in Duchenne muscular dystrophy (DMD). Clinical anisotropy assessments were performed using Viscoelastic Response ultrasound, with a degree of anisotropy reflected by the ratios of Viscoelastic Response relative elasticity (RE) or relative viscosity (RV) measured with the asymmetric radiation force oriented parallel versus perpendicular to muscle fiber alignment. In vivo results from rectus femoris and gastrocnemius muscles of boys aged ∼7.9–10.4 y indicate that RE and RV anisotropy ratios in rectus femoris muscles of boys with DMD were significantly higher than those of healthy control boys (RE: DMD = 1.51 ± 0.87, control = 0.99 ± 0.69, p = 0.04, Wilcoxon rank sum test; RV: DMD = 1.04 ± 0.71, control = 0.74 ± 0.22, p = 0.02). In the gastrocnemius muscle, only the RV anisotropy ratio was significantly higher in dystrophic than control patients (DMD = 1.23 ± 0.35, control = 0.88 ± 0.31, p = 0.04). In the dystrophic rectus femoris muscle, the RE anisotropy ratio was inversely correlated (slope = –0.03/lbf, r = –0.43, p = 0.07, Pearson correlation) with quantitative muscle testing functional output measures but was not correlated with quantitative muscle testing in the dystrophic gastrocnemius. These results suggest that Viscoelastic Response RE and RV measures reflect differences in mechanical anisotropy associated with functional impairment with dystrophic degeneration that are relevant to monitoring DMD clinically.

Acoustic Radiation Force Impulse (ARFI) imaging employs sound impulses to induce and track tissue motion that conveys mechanical property. In application to carotid atherosclerotic plaque, ARFI noninvasively delineates features that convey risk for ischemic stroke: intraplaque hemorrhage, lipid-rich necrotic core, thin or ruptured fibrous cap, diffuse collagen, and calcium deposits. This talk will feature recent advances to in vivo ARFI atherosclerosis imaging technology, including the use of machine learning for automated feature classification, which are validated histologically in patients undergoing carotid endarterectomy. New developments, including incorporating vector blood flow estimation for combined shear strain assessment, will be highlighted.

Many tissues, such as muscle, kidney, and breast, are mechanically anisotropic. Appropriately exploited, mechanical anisotropy can be a clinically relevant target for noninvasive imaging. An imaging method's potential for interrogating mechanical anisotropy can be experimentally evaluated using tissue-mimicking materials, also known as phantoms. The objective of this work is to demonstrate the feasibility of constructing mechanically anisotropic phantoms using 3D-printed polylactic acid (PLA) fibers embedded in gelatin hydrogel or polyvinyl alcohol (PVA) cryogel. Four identical fiber sets were printed; two were embedded in gelatin and two in PVA. Acoustic Radiation Force Impulse (ARFI) imaging was performed on the constructed phantoms, with data acquisitions at 0°, 30°, 60°, and 90° concentric orientations, where 0° and 90° corresponded to the long-axis of the spatially asymmetric ARF excitation being aligned across and along the fibers, respectively. Degree of anisotropy (DoA) was calculated as the ratio of peak displacements achieved at 90° versus 0° orientations. While both gelatin and PVA embedded fibers demonstrated elastic anisotropy, DoA values were 32% higher in gelatin. These pilot experimental results demonstrate that phantoms constructed of 3D-printed PLA fibers embedded in gelatin or PVA exhibit mechanical anisotropy as assessed by ARFI ultrasound.

Viscoelastic Response (VisR) ultrasound is a noninvasive method for interrogating the viscoelastic properties of tissue by observing, in the region of excitation, tissue displacement in response to successive acoustic radiation force impulses. Recent VisR technology advancements now enable estimation of mechanical anisotropy and texture as well as, via machine learning approaches, quantitative evaluation of elastic and viscous moduli (as in Quantitative VisR (QVisR)). The diagnostic relevance of VisR outcome measures have been demonstrated for monitoring renal transplant status, differentiating malignant from benign breast masses, and delineating dystrophic muscle degeneration. For the latter two applications, method and pilot clinical results are reviewed herein. VisR-derived relative elasticity (RE) and relative viscosity (RV) mechanical anisotropy measures differentiated malignant from benign breast masses in women with sensitivities of 0.94 and specificities of 1.00, with accuracies of 0.96. In dystrophic muscle, QVisR-derived longitudinal shear elastic modulus in rectus femoris, vastus lateralus, sartorius, and gastrocnemius muscles statistically different between dystrophic and control for ages less than six years (Wilcoxon, p

Double-profile intersection (DoPIo) ultrasound is a new acoustic radiation force (ARF) imaging technique that exploits scatterer shearing under two different tracking point spread functions to estimate shear elastic moduli. Previous versions of DoPIo used an empirically derived model to relate the time where two displacement profiles, each measuring displacement in identical positions using different aperture sizes. We herein propose an alternative, machine learning-based approach that obviates the need for an empirically-derived model by replacing it with a support vector machine that uses paired displacement profiles in their entirety. In this work, we describe the implementation of such a model, validate its ability in shear modulus for simulated materials, and deploy it to image a calibrated phantom.

We present a quantitative extension of Viscoelastic Response (VisR) ultrasound that estimates shear elastic and viscous moduli from on-axis VisR displacement profiles in silico. Isotropic, homogeneous, linearly viscoelastic materials ranging from 1.57-33.33 kPa shear elasticity and 0.0033-2.34 Pa.s shear viscosity subject to a VisR beamsequence at 26 focal depths were simulated. Multi-target regression machine learning models were used to estimate shear elasticity and shear viscosity given the displacement profile, focal depth, and axial depth. The best performing models achieve a shear elasticity RMSE of 0.29 kPa and a shear viscosity RMSE of 0.13 Pa.s when predictions were made on the test set. These results suggest that machine learning methods can be used to quantitatively estimate viscoelasticity from VisR displacement profiles.

Differentiating malignant from benign breast lesions remains a challenge that may be met by a new imaging biomarker: lesion-to-background mechanical anisotropy assessed by VisR ultrasound. VisR is an ultrasound imaging method that uses 2 consecutive acoustic radiation force excitations, separated in time and delivered to the same region of excitation, to directionally interrogate peak displacement (PD), relative elasticity (RE), and relative viscosity (RV). From these directional measurements, mechanical degree of anisotropy is measured. In a pilot clinical study involving 29 women with BIRADS-4 or −5 breast masses, VisR PD, RE, and RV were measured in lesions and in surrounding tissue at 0°, 30°, 60°, and 90° concentric orientations. From these measurements, lesion DoA (LDoA), surrounding tissue DoA (SDoA), and log(LDoA/SDoA) were assessed per parameter and compared between biopsy-confirmed malignant versus benign masses. Across all patients, PD-, RE-, and RV-based LDoA or SDoA alone did not achieve statistical difference between malignant and benign masses (Wilcoxon, $\mathrm{p} > 0.05$ ). However, PD-, RE-, and RV-based log(LDoA/SDoA) statistically differentiated malignant from benign masses (Wilcoxon, $\mathrm{p} ), with AUC values of 0.96 for PD-based, and 0.94 for RE- and RV-based outcomes. These results demonstrate the relevance of lesion-to-background mechanical anisotropy assessed by VisR ultrasound for differentiating malignant from benign breast masses in women, in vivo.

Direct ultrasonic imaging of arterial and venous thrombi could aid in diagnosis and treatment planning by providing rapid and cost-effective measurements of thrombus volume and elastic modulus. Toward this end, it was demonstrated that open-air magnetomotive ultrasound (MMUS) provides specific contrast to superparamagnetic iron oxide-labeled model thrombi embedded in gelatin-based blood vessel-mimicking flow phantoms. MMUS was performed on model thrombi in the presence of pulsatile flow that mimics cardiac-induced motion found in real vasculature. The MMUS signal and contrast-to-noise ratio (CNR) were measured across a range of physiologically relevant thrombus volumes and elastic moduli. Model thrombus volumes as small as 0.5 ml were shown to be detectable (CNR > 1) over the entire range of elastic moduli tested (3.5–40 kPa). It was also found that MMUS signal and CNR are increased with increasing thrombus volume ( $r = 0.99$ ) and decreasing elastic modulus ( $r = -0.81$ ), while variations in pulsatile flow rate had little effect. These findings demonstrate that MMUS has promise as a direct in vivo thrombosis imaging modality for quantifying thrombus volume and stiffness.

Chronic kidney disease is most desirably and cost-effectively treated by renal transplantation, but graft survival is a major challenge. Although irreversible graft damage can be averted by timely treatment, intervention is delayed when early graft dysfunction goes undetected by standard clinical metrics. A more sensitive and specific parameter for delineating graft health could be the viscoelastic properties of the renal parenchyma, which are interrogated non-invasively by Viscoelastic Response (VisR) ultrasound, a new acoustic radiation force (ARF)-based imaging method. Assessing the performance of VisR imaging in delineating histologically confirmed renal transplant pathologies in vivo is the purpose of the study described here. VisR imaging was performed in patients with (n = 19) and without (n = 25) clinical indication for renal allograft biopsy. The median values of VisR outcome metrics (τ, relative elasticity [RE] and relative viscosity [RV]) were calculated in five regions of interest that were manually delineated in the parenchyma (outer, center and inner) and in the pelvis (outer and inner). The ratios of a given VisR metric for all possible region-of-interest combinations were calculated, and the corresponding ratios were statistically compared between biopsied patients subdivided by diagnostic categories versus non-biopsied, control allografts using the two-sample Wilcoxon test (p 0.05). Although τ ratios non-specifically differentiated allografts with vascular disease, tubular/interstitial scarring, chronic allograft nephropathy and glomerulonephritis from non-biopsied control allografts, RE distinguished only allografts with vascular disease and tubular/interstitial scarring, and RV distinguished only vascular disease. These results suggest that allografts with scarring and vascular disease can be identified using non-invasive VisR RE and RV metrics.

Degree of mechanical anisotropy in renal cortex can be assessed by evaluating the ratio of viscoelastic response (VisR) parameters achieved when the long axis of a spatially asymmetric ARF excitation PSF is aligned along versus across nephrons. In this work, intra-observer reproducibility and variability in VisR derived DoA assessment in renal cortex due to donor type, sex, race, and BMI is evaluated in healthy human kidney allografts, in vivo. Transcutaneous, in vivo VisR imaging was performed in 20 kidney allografts 2 months post-transplantation using a Siemens Acuson Antares™ imaging system and a VF7-3 linear array transducer. Ratios of VisR derived relative elasticity (RE), relative viscosity (RV), and peak displacement (PD) obtained with the ARF short-axis aligned along versus across nephrons were calculated. Patients were divided based on their sex (male: N=14 versus female: N=6), race (African American: N = 6 versus Caucasian: N=14), donor type (living: N=14 versus deceased: N =6), and BMI (BMI >30: N= 8 versus BMI 0.05) due to sex, race, donor types, or BMI. The median absolute percent difference in (PD, RE, RV) ratios were (23, 13, 23) due to sex, (5, 3, 3) due to race, (0.35, 14, 13) due to donor type, and 8.6, 6, 12) due to BMI. Ratios of PD, RE, and RV were not statistically different between 1st and 2nd imaging events with mean percent difference in (PD, RE, and RV) ratios of (-0.04, -0.11, and - 0.01). These findings suggest that VisR outcome measures are robust to potentially confounding demographic factors and reproducible by a trained practitioner.

Tissue elasticity can be measured by applying an acoustic radiation force (ARF) excitation and tracking mechanical responses like deformation or shear wave propagation via displacement tracking. However, tissue displacements tracked by ultrasound is underestimated due to the shearing of scatterers under the tracking point-spread function. While such displacement underestimation is a source of error in conventional elasticity approaches, we herein present an alternative method that exploits displacement underestimation: double-profile intersection (DoPIo) elastography. We hypothesize that DoPIo can quantify material elasticity in silico.

Mechanical property changes associated with inflammation, necrosis, fibrosis, and fatty deposition in dystrophic muscle may be quantitatively evaluated by Quantitative Viscoelastic Response (QVisR) ultrasound. QVisR uses a machine learning (ML) framework that takes as input tissue displacement in response to two consecutive and co-located acoustic radiation force (ARF) excitations and yields as output estimates of shear elastic and shear viscous moduli. QVisR imaging was performed in the vastus lateralis (VL) muscles of 11 boys with Duchenne muscular dystrophy (DMD) aged 5 to 12 years and of 8 age-matched boys with no known neuromuscular disorders, who served as controls. QVisR measures of elastic moduli differed between DMD and control VL in boys aged less than six years and six-to-seven years. Similarly, QVisR measures of viscous moduli differed between DMD and control VL in boys aged six-to-seven years. These results demonstrate that QVisR measures of elastic and viscous moduli differentiate dystrophic from control muscle. The findings suggest that QVisR may be relevant to monitoring dystrophic muscle degeneration and response to intervention, particularly in early stages when interventions are most likely to be impactful.

Chronic kidney disease is associated with progressive inflammation and fibrosis, which is conventionally assessed by invasive biopsy. A noninvasive alternative to biopsy may be interrogating elastic and viscous anisotropy. This work evaluates the feasibility of using in vivo Viscoelastic Response (VisR) ultrasound for detecting changes in the elastic and viscous degree of anisotropy (DoA) associated with fibrosis after chronic ischemia-reperfusion injury (IRI) in pig kidney. Histology was used to validate VisR DoA measurements, which were compared to those derived using Shear Wave Elasticity Imaging (SWEI) and Shearwave Dispersion Ultrasound Vibrometry (SDUV). In the injured and contralateral (control) kidneys of 8 pigs, VisR, SWEI, and SDUV-derived elastic DoA, evaluated as the ratio of longitudinal over transverse shear elasticity, was significantly (p

Quantitative Viscoelastic Response (QVisR) ultrasound is a new machine learning-based elasticity imaging method in which elastic and viscous moduli are estimated from tissue deformation in response to two consecutive acoustic radiation force (ARF) excitations. The estimated moduli are rendered into two-dimensional parametric images of elastic and viscous modulus. From QVisR images, the spatial distribution of elastic and viscous properties may be evaluated using established computational texture analysis tools. In this study, computational texture analysis by the omnidirectional gray-level run-length matrix (GLRLM) was applied to QVisR images. The images were obtained in the vastus lateralis (VL) muscles of 11 boys with Duchenne muscular dystrophy, aged 5-12 years, and 8 age-matched boys with no known neuromuscular disorders, who served as controls. GLRLM-derived elastic entropy in QVisR images was statistically higher (Wilcoxin, p

Chronic kidney disease is associated with progressive inflammation, which is conventionally assessed by invasive biopsy. A noninvasive alternative is to interrogate the mechanical property alterations that occur with inflammation. This work evaluates the feasibility of using in vivo Viscoelastic Response (VisR) ultrasound for detecting differences in mechanical degree of anisotropy (DoA) associated with inflammation after ischemia-reperfusion injury (IRI) in pig kidney. VisR measurements were validated by histology, Shear Wave Elasticity Imaging (SWEI), and Shearwave Dispersion Ultrasound Vibrometry (SDUV) derived DoA. In 6 pigs, data were acquired in vivo in the kidney following IRI and in the contralateral kidney, which did not undergo IRI and served as a control. VisR (relative elasticity (RE), relative viscosity (RV), and peak displacement (PD)), SWEI (shear elastic modulus $(\mu))$ , and SDUV (shear elastic $(\mu_{1})$ and viscous $(\mu_{2})$ moduli) outcomes were evaluated alone and as ratios to reflect DoA (longitudinal over transverse). VisR PD and RE, SWEI $\mu$ , and SDUV $\mu_{1}$ demonstrated significantly increased DoA (p $\mu$ in cortex). Notably, while elastic DoA increased in both medulla and cortex, the underlying causes differed (cortex: diminished transverse modulus v. medulla: elevated longitudinal modulus). VisR RV indicated decreased viscous DoA in the medulla after IRI. Spatially matched histology indicated mild inflammation in the injured, but not the control, kidneys. These results suggest that VisR noninvasively detects changes in DoA associated with inflammation induced by IRI in a pig model. VisR may be diagnostically relevant to delineating inflammation in human kidney.

Vulnerable atherosclerotic plaques have high risk for rupture, with rupture potential related to plaque composition and structure. We have previously shown that soft (intraplaque hemorrhage IPH, and lipid rich necrotic core LRNC) are differentiated from stiff (collagen COL, and calcium CAL) plaque elements in human carotid plaques by Acoustic Radiation Force Impulse (ARFI)-derived peak displacement (PD). However, PD had lower performance for differentiating between features with similar stiffness. Here we evaluate an alternative method to improve intraplaque feature delineation by using machine learning methods. From ARFI imaging data, SNR, cross-correlation coefficient, and displacement were used as inputs to random forests (RaF) and support vector machines (SVM) algorithms. The algorithms were trained to identify IPH, LRNC, COL and CAL by 5-fold cross-validation with ground truth identified from histology. From output likelihood matrices, CNR between plaque components were calculated and compared to the corresponding CNR achieved by ARFI PD and VoA. Results showed that both RaF and SVM achieved higher CNRs for distinguishing between features than ARFI outputs alone. These results suggest that, relative to PD, machine learning improves ARFI discrimination of carotid plaque components that are correlated to vulnerability for rupture.

In Golden Retriever Muscular Dystrophy (GRMD) dogs, a relevant animal model of human Duchenne muscular dystrophy (DMD), skeletal muscles progressively inflame, necrose, and undergo fibrous and fatty deposition. These degenerative changes alter the composition and structure of muscle, which affects the degree of directional variation, or anisotropy, in tissue mechanical properties. Further, such degenerative changes alter the spatial distribution of mechanical property across muscles. In this work we investigate the relevance of mechanical anisotropy and texture, estimated using Viscoelastic Response (VisR) ultrasound, to tracking progressive dystrophic muscle degeneration in GRMD dogs, in vivo. In a year-long study of 20 dogs, degree of shear elastic anisotropy (DoA), was significantly higher in the GRMD semitendinosus (ST) muscle compared to control ST (GRMD: 1.23 +- 0.25, Control 1.13 +- 0.27, p

In transversely isotropic (TI) materials, mechanical properties differ along versus across the axis of symmetry (AoS). The degree of these directional differences has been interrogated by ultrasound-based measures of shear wave velocity (SWV) and ARFI peak displacements (PD) acquired along versus across the AoS. When performed using a conventional linear array transducer, such measurements require a 90o transducer rotation, which can be cumbersome and prone to misalignment errors. Rather than 90o, we hypothesize that smaller rotation angles can be used to assess mechanical anisotropy. This hypothesis was tested in silico using finite-element method (FEM) models and Field II. ARFI and Shear Wave Elasticity Imaging (SWEI) were simulated in five TI elastic materials with longitudinal versus transverse shear elastic modulus ratios (SRs) of 1.22 to 5.44. The simulated rotation angles were 15o, 30o, and 90o. The ratio of PD was measured with the long-axis of the ARF PSF aligned along the AoS versus long-axis rotated by 90o, 30o, or 15o from the AoS. The ratio of SWV was measured along the AoS versus 90o, 30o, or 15o rotation from the AoS. For a 90o rotation angle, both SWV and PD ratios were statistically different (2-sample t-test, p

Previous work has shown that the degree of anisotropy in tissue can be assessed using Acoustic Radiation Force Impulse (ARFI) imaging performed with a geometrically asymmetric excitation. In this work, we investigate the clinical relevance of mechanical anisotropy in Duchenne muscular dystrophy (DMD). Anisotropy assessment was performed using relative elasticity (RE) and relative viscosity (RV) parameters measured by Viscoelastic Response (VisR) ultrasound. These parameters were evaluated as the ratio of their respective values obtained with two orthogonal transducer orientations, i.e. parallel and perpendicular to the muscle fiber direction, to represent the transverse over the longitudinal RE or RV. In a pilot clinical feasibility study performed in six, 7.9–10.4 year-old boys with (DMD) and five age-matched boys with no known neuromuscular disorders, VisR anisotropy imaging was performed in vivo in the rectus femoris (RF) serially for a total of 22 time points in DMD and 12 time points in control boys. Both the RE and RV anisotropy ratios in the RF muscles of boys with DMD were significantly higher (Wilcoxon, p

While skeletal muscle is often assumed to be structurally transversely isotropic (TI), its mechanical anisotropy is not well characterized in vivo. VisR ultrasound, a technique that provides information about the viscoelastic mechanical response of tissue, can be used for such in vivo characterization. Specifically, VisR-derived Relative Elasticity (RE) provides directionally dependent information about the shear modulus of materials. We hypothesize that VisR RE can be used in healthy older adults and in stroke patients to 1) assess the degree of anisotropy (DoA) of the biceps brachii (BB) and 2) characterize the effect of passive loading on the BB. VisR imaging was performed in the BB long head of 2 healthy controls and 1 stroke patient. The subjects' arms were imaged in full extension and approximately 90° flexion at rest. Data were acquired in the transverse and longitudinal imaging planes at the medial location of the BB. The ratio of RE obtained in the transverse to the longitudinal muscle views was found to reflect the DoA in shear modulus, where DoA = 1 indicates perfect isotropy. In control subjects, RE values for flexion and extension in the transverse imaging plane (previously shown to reflect the longitudinal shear modulus, μL) were higher than the RE values in the longitudinal imaging plane (which, primarily reflects the transverse shear modulus, μT). Additionally, both the longitudinal and transverse shear moduli increased in extension vs. flexion. Both the magnitude and direction of anisotropy appeared to change in the stroke patient compared to the healthy controls. This work indicates VisR can be used to assess DoA in skeletal muscle of older adults and examine effects of passive loading on mechanical properties, in vivo, which may be clinically relevant to studying impairment post stroke.

In Duchenne muscular dystrophy (DMD), muscle fiber necrosis and subsequent progressive replacement of muscle by fibrous tissue and fat impacts the degree of anisotropy (DoA) in mechanical properties. Mechanical DoA can be assessed in skeletal muscle using Viscoelastic Response (VisR) ultrasound. We hypothesize that in vivo VisR DoA measures in the rectus femoris (RF) muscles of boys with DMD statistically differ from those in control boys. Further, we hypothesize that VisR DoA is correlated to RF functional output.

Tissue mechanical anisotropy has been shown to be diagnostically relevant in numerous clinical applications. Anisotropy can be assessed using acoustic radiation force-based techniques, including Acoustic Radiation Force Impulse (ARFI) and Viscoelastic Response (VisR) ultrasound. In this work, ARFI peak displacement (PD), as well as VisR relative elasticity (RE) and relative viscosity (RV), were implemented to image mechanical anisotropy in ex vivo porcine posas major muscle. Two psoas major samples were imaged in this work. The first sample was imaged whole. The second sample was sectioned into a 1 cm-diameter cylinder (with cylinder height oriented along the muscle fibers) and embedded into an isotropic gelatin phantom for imaging. ARFI and VisR were performed on both muscle samples using a linear array transducer mounted to a translation and rotation stage. By rotating the transducer 90o as it stepped across a 2D imaging field of view, PD, RE, and RV were measured along and across muscle fibers. Then, the ratios of PD, RE, and RV measurements made along versus across the muscle fibers were calculated, and these ratios were parametrically rendered in to 2D images representing degree of anisotropy. In the whole psoas major, PD anisotropy ratio was 1.08 +− 0.11, the RE anisotropy ratio was 0.94 +− 0.09 and the RV ratio was 0.94 +− 0.08. These PD and RE anisotropy ratios suggest that the longitudinal shear elastic modulus was greater than the transverse shear elastic modulus, which agrees with previous studies on muscle anisotropy. The RV results suggest that the longitudinal viscous modulus was greater than the transverse viscous modulus. Similar PD, RE, and RV anisotropy ratios were measured in the embedded psoas major section, and contrast ratios were 0.21, 0.14, and 0.30 for PD, RE and RV 2D anisotropy images, respectively. CNR for PD, RE, and RV anisotropy images were 3.26, 2.09 and 2.41, respectively. These results demonstrate that ARFI and VisR anisotropy imaging are relevant for delineating the spatial distribution of anisotropic features in tissue.

Degree of anisotropy (DoA) has been evaluated for clinical significance in several organs. In skeletal muscle, mechanical DoA has been shown to increase with passive extension, i.e. the shear modulus parallel to muscle fibers (μ l ) increases relative to that perpendicular (μ t ). Imaging such variations in DoA could facilitate diagnosing and monitoring muscle pathologies. We have previously demonstrated the feasibility of imaging DoA with VisR ultrasound, in silico. The purpose of this work is to experimentally demonstrate VisR DoA imaging ex vivo in pig and in vivo in human muscles. We hypothesize that 2D VisR anisotropy images represent mechanical anisotropy in muscle.

The kidney is an anisotropic organ, with higher elasticity along versus across nephrons. The degree of elastic anisotropy in kidney may be diagnostically relevant if properly exploited; however, if improperly controlled, anisotropy may confound stiffness measurements. The purpose of this study is to demonstrate a novel method for selectively exploiting or obviating elastic anisotropy in kidney using Acoustic Radiation Force Impulse (ARFI)-induced peak displacement (PD). The kidneys of three pigs were imaged in vivo at baseline, with venous ligation, and with arterial ligation, and then kidneys were extracted and imaged ex vivo. Imaging was performed with ARF excitation impulses having F/1.5 or F/5.0 focal configurations, and data were acquired with the transducer oriented along and across nephrons alignment in the renal cortex. In addition to ARFI PD, shear wave velocity was measured along and across nephrons to estimate longitudinal and transverse shear elastic moduli. Elastic anisotropy was then assessed as the ratio of PD across versus along nephrons, and as the ratio of shear moduli along versus across nephrons. PD ratio using the F/1.5 ARF linearly correlated with shear moduli ratio (R2 = 0.95). However, PD ratio using the F/5.0 ARF was approximately 1.0 and had weak correlation to shear moduli ratio (R2= 0.56). Further, the average difference in PD measured along versus across nephrons was 2.91 μm for the F/1.5 ARF but was 0.2 μm for the F/5.0 ARF. These results suggest that the F/1.5 ARF excitation exploited elastic anisotropy in the renal cortex, while the F/5.0 ARF excitation obviated it.

Viscoelastic response (VisR) imaging is presented as a new acoustic radiation force (ARF)-based elastographic imaging method. Exploiting the Voigt model, VisR imaging estimates displacement in only the ARF region of excitation from one or two successive ARF impulses to estimate τσ, the relaxation time for constant stress. Double-push VisR τσ estimates were not statistically significantly different (p

Duchenne muscular dystrophy (DMD) is a genetic disorder that causes progressive muscle degeneration involving necrosis and inflammation, with subsequent replacement of muscle fibers by fibrosis and fatty tissue. These compositional changes underlie mechanical property alterations in affected muscles, which may be assessed using Viscoelastic Response (VisR) ultrasound. We hypothesize that VisR will delineate differences in the viscoelastic properties of lower limb skeletal muscles in boys with versus without DMD. VisR imaging was performed in the vastus intermedius (VI), rectus femoris (RF), sartorius (SM) and gastrocnemius (GM) muscles of seven boys (4 DMD, 3 control) aged 7.9 – 10.4 years. Parametric images of relative elasticity (RE) and relative viscosity (RV) were rendered. From the parametric images, percent muscle area with relatively high RE or RV value was calculated and compared (Wilcoxon rank-sum) between DMD and control on a per-muscle basis. In the VI, RF and SM, percent muscle with relatively high RV was larger (VI: 17.7% v. 13.1% RF: 98.9% v. 93.7%, SM: 43.2% v. 40.6% p < 0.05) in DMD than control muscles. In the VI, percent muscle with relatively high RE was larger (32.8% v. 29.5%, p < 0.05) in DMD muscles. No significant differences were observed in the GM between DMD and control. VisR results were consistent with temporally-matched functional testing using a hand-held dynamometer, which showed 40.5% to 70.0% lower force output in DMD RF, VL and SM - and only 21.8% lower force output in DMD GM - relative to the corresponding control muscles. These results suggest that VisR imaging is relevant to delineating viscoelastic property alterations that are associated with dystrophic muscle degeneration in boys with DMD, in vivo.

Acute cerebrovascular accidents are associated with the rupture of vulnerable atherosclerotic plaques in the carotid arteries. Fibrous cap thickness has been shown to be an important predictor of plaque rupture, but has been challenging to measure accurately with clinical noninvasive imaging modalities. The goals of this investigation were first, to evaluate the feasibility of using transcutaneous acoustic radiation force impulse (ARFI) ultrasound to quantify fibrous cap thickness and second, to optimize both imaging and motion tracking parameters to support such measurements. Fibrous caps with varying thickness (0.1 – 1.0 mm) were simulated using a simple layered geometry, and their mechanical response to an impulse of radiation force was solved using finite element method (FEM) modeling. Ultrasound tracking of FEM displacements was performed in Field II utilizing three center frequencies (6, 9, and 12 MHz) and eight motion tracking kernel lengths (0.5λ – 4λ). Additionally, fibrous cap thickness in two carotid plaques imaged in vivo was measured with ARFI and compared to matched histology. The results of this study demonstrate that 1) tracking pulse frequencies around 12 MHz are necessary to resolve caps around 0.2 mm, 2) large motion-tracking kernel sizes introduce bias into thickness measurements and overestimate the true cap thickness, and 3) color saturation settings on ARFI peak displacement images can impact thickness measurement accuracy substantially.

Objective Stroke is commonly caused by thromboembolic events originating from ruptured carotid plaque with vulnerable composition. This study assessed the performance of acoustic radiation force impulse (ARFI) imaging, a noninvasive ultrasound elasticity imaging method, for delineating the composition of human carotid plaque in vivo with histologic validation. Methods Carotid ARFI images were captured before surgery in 25 patients undergoing clinically indicated carotid endarterectomy. The surgical specimens were histologically processed with sectioning matched to the ultrasound imaging plane. Three radiologists, blinded to histology, evaluated parametric images of ARFI-induced peak displacement to identify plaque features such as necrotic core (NC), intraplaque hemorrhage (IPH), collagen (COL), calcium (CAL), and fibrous cap (FC) thickness. Reader performance was measured against the histologic standard using receiver operating characteristic curve analysis, linear regression, Spearman correlation (ρ), and Bland-Altman analysis. Results ARFI peak displacement was two-to-four-times larger in regions of NC and IPH relative to regions of COL or CAL. Readers detected soft plaque features (NC/IPH) with a median area under the curve of 0.887 (range, 0.867-0.924) and stiff plaque features (COL/CAL) with median area under the curve of 0.859 (range, 0.771-0.929). FC thickness measurements of two of the three readers correlated with histology (reader 1: R 2 = 0.64, ρ = 0.81; reader 2: R 2 = 0.89, ρ = 0.75). Conclusions This study suggests that ARFI is capable of distinguishing soft from stiff atherosclerotic plaque components and delineating FC thickness.

In this second of a two part series, we present pilot clinical data demonstrating Acoustic Radiation Force Impulse (ARFI) ultrasound for monitoring the onset of subcutaneous hemostasis at femoral artery puncture sites (arteriotomies), in vivo. We conducted a randomized, reader-blinded investigation of 20 patient volunteers who underwent diagnostic percutaneous coronary catheterization. After sheath removal (6 French), patients were randomized to treatment with either standard of care manual compression alone or, to expedite hemostasis, manual compression augmented with a p-GlcNAc fiber-based hemostatic dressing (Marine Polymer Technologies, Danvers MA). Concurrent with manual compression, serial ARFI imaging began at the time of sheath removal and continued every minute for 15 min. Serial data sets were processed with custom software to (1) estimate the time of hemostasis onset, and (2) render hybrid ARFI/B-Mode images to highlight displacements considered to correspond to extravasted blood. Images were read by an observer blinded to the treatment groups. Average estimated times to hemostasis in patient volunteers treated with manual compression alone ( n = 10) and manual compression augmented by hemostatic dressing ( n = 9) were, respectively, 13.00 ± 1.56 and 9.44 ± 3.09 min, which are statistically significantly different ( p = 0.0065, Wilcoxon two-sample test). Example images are shown for three selected patient volunteers. These pilot data suggest that ARFI ultrasound is relevant to monitoring subcutaneous bleeding from femoral arteriotomies clinically and that time to hemostasis was significantly reduced by use of the hemostatic dressing.

Acoustic radiation force impulse (ARFI) Surveillance of Subcutaneous Hemorrhage (ASSH) has been previously demonstrated to differentiate bleeding phenotype and responses to therapy in dogs and humans, but to date, the method has lacked experimental validation. This work explores experimental validation of ASSH in a poroelastic tissue-mimic and in vivo in dogs. The experimental design exploits calibrated flow rates and infusion durations of evaporated milk in tofu or heparinized autologous blood in dogs. The validation approach enables controlled comparisons of ASSH-derived bleeding rate (BR) and time to hemostasis (TTH) metrics. In tissue-mimicking experiments, halving the calibrated flow rate yielded ASSH-derived BRs that decreased by 44% to 48%. Furthermore, for calibrated flow durations of 5.0 minutes and 7.0 minutes, average ASSH-derived TTH was 5.2 minutes and 7.0 minutes, respectively, with ASSH predicting the correct TTH in 78% of trials. In dogs undergoing calibrated autologous blood infusion, ASSH measured a 3-minute increase in TTH, corresponding to the same increase in the calibrated flow duration. For a measured 5% decrease in autologous infusion flow rate, ASSH detected a 7% decrease in BR. These tissue-mimicking and in vivo preclinical experimental validation studies suggest the ASSH BR and TTH measures reflect bleeding dynamics.

Acoustic radiation force impulse (ARFI) ultrasound is an elasticity imaging technique that has the potential to identify vulnerable atherosclerotic plaque characteristics. Previous studies have shown that regions of high ARFI-induced displacement correspond to lipid/necrotic areas in plaques, whereas regions of low displacement correspond with fibrotic/calcified areas. In this work, we continue our analysis of ARFI images captured from patients undergoing carotid endarterectomy. ARFI imaging of carotid plaque was performed prior to surgery, and extracted specimens were histologically processed and matched to the ultrasound imaging plane. ARFI images and histological slides were annotated independently for various features including necrotic core, calcification, fibrosis and fibrous cap, and wide hemorrhage. Results are presented for a number of advanced plaque types (Va, Vb, and VI), and ARFI-derived fibrous cap thickness measurements are compared quantitatively to histology. Results suggest ARFI is capable of measuring fibrous cap thickness with a mean positive bias of 0.09 ± 0.12 mm, and may be capable of identifying ruptured plaque.

Viscoelastic Response (VisR) ultrasound is a new Acoustic Radiation Force (ARF)-based imaging method that uses two successive ARF excitations, delivered to the same region of excitation, to approximate a creep response in tissue and thereby estimate viscoelastic property. The viscoelasticity of dystrophic muscle is altered over time by ongoing necrotic, fatty, and fibrous degenerative changes. Evaluating such changes by VisR in the Golden Retriever Muscular Dystrophy (GRMD) canine model, with comparison to matched MRI and histology, is the purpose of this work. In a cross-sectional study, in vivo VisR imaging was performed on the vastus lateralis (VL) and cranial sartorius (CS) muscles in 20 dogs (n=10 control and GRMD age-matched pairs) aged 3, 6, 12, 24, or 60 months. Following VisR, T2-weighted MRI imaging was performed, and run percentage (RP) was calculated to reflect heterogeneity. Finally, muscle tissue samples were acquired by open surgical biopsy, sectioned, and stained, and percent collagen and fat were calculated from digital microscopy. In the VL, SD of VisR τ was consistently larger in GRMD versus age-matched control dogs (p=0.001, Wilcoxon two-sample test). This VisR result was consistent with higher MRI RP measures and higher histology percent collagen. Similarly in the CS, SD of VisR τ was larger in GRMD than control (p=0.001), consistent with higher MRI RP and with histology percent collagen. These results suggest that VisR measures of τ SD reflect heterogeneity due to collagen deposition in dystrophic muscles.

Cardiovascular disease (CVD) is the leading cause of death in the United States, with 70% of CVD mortalities the result of sequelae of atherosclerosis. An urgent need for enhanced delineation of vulnerable plaques has catalyzed the development of novel atherosclerosis imaging strategies that use X-ray computed tomography, magnetic resonance and ultrasound modalities. As suggested by the pathophysiology of plaque development and progression to vulnerability, insight to the focal material, i.e., mechanical, properties of arterial walls and plaques may enhance atherosclerosis characterization. We present acoustic radiation force impulse (ARFI) ultrasound in application to mechanically characterizing a raised focal atherosclerotic plaque in an iliac artery extracted from a relevant pig model. ARFI results are correlated to matched immunohistochemistry, indicating elastin and collagen composition. In regions of degraded elastin, slower recovery rates from peak ARFI-induced displacements were observed. In regions of collagen deposition, lower ARFI-induced displacements were achieved. This work demonstrates ARFI for characterizing the material nature of an atherosclerotic plaque.

Atherosclerotic plaque characterization with acoustic radiation force impulse (ARFI) imaging has recently been developed using phantoms and atherosclerotic pigs, but has yet to be validated in vivo in humans. We present initial results from an ongoing clinical trial investigating in vivo ARFI imaging of human carotid plaque with spatially-matched histopathology. Patients undergoing clinically-indicated carotid endarterectomy (CEA) were recruited from UNC Hospitals and imaged with ARFI prior to surgery. After surgery, the extracted specimen was sectioned according to noted arterial geometry for spatial registration to the ultrasound imaging plane and processed histologically. In two symptomatic patients, plaques were composed of a mixture of small and large necrotic cores, mild intra-plaque hemorrhage, and fibrosis. Mean ARFI peak displacements were observed to be two to three times higher in regions corresponding to either necrotic core or intra-plaque hemorrhage compared to regions of dense fibrosis or calcification. In an asymptomatic patient with predominantly calcified plaque, mean ARFI peak displacements were small (∼1–2 µm) suggesting stiff tissue. The results of this study indicate that areas of relatively large displacement by ARFI imaging correlate with lipid/necrotic cores and/or inflammation, which may confer an increased chance of plaque rupture and future ischemic event. This work demonstrates the feasibility of transcutaneous ARFI for characterizing the material and structural composition of carotid atherosclerotic plaques via mechanical properties, in humans, in vivo.

Viscoelastic Response (VisR) ultrasound is an acoustic radiation force (ARF)-based imaging method for noninvasively interrogating the viscoelastic properties of tissue. ViSR ultrasound uses two successive ARF impulses delivered to a single region of excitation (ROE) and tracks the micrometer-scale induced displacements. We have previously demonstrated that tracked displacements can be fit to the mass-spring-damper model in order to measure the relaxation time constant, τ, given by the ratio of viscosity to elasticity. Because it is a ratio, we cannot determine a change in τ is due to changes in elasticity, viscosity, or both. However, elasticity and viscosity may be evaluated uniquely by VisR if considered relative to the magnitude of the forcing function. It is hypothesized that their separation enhances discrimination of viscoelastic materials in VisR ultrasound. We demonstrate that VisR is able to isolate elasticity and viscosity from τ in FEM simulated data and experimentally in a gelatin phantom with a soft inclusion.

Acoustic Radiation Force Impulse (ARFI) ultrasound is used in ARFI Surveillance of Subcutaneous Hemorrhage (ASSH), a new in vivo hemostasis assay, to assess bleeding response in hemophilia A dogs. ASSH bleeding parameters were calculated from a small dose response study with 7 hemostatically normal dogs and 7 dogs exhibiting severe hemophilia A. Hemophilia A dogs were imaged in the naive state and/or when prophylactically treated with different levels of recombinant canine FVIII before bleeding was induced. A standardized injury of a 1–2mm vein by needle puncture induced subcutaneous bleeding in soft tissue. Bleeding was monitored by ASSH for 60 minutes. There was a significant difference (p

Viscoelastic Strain Response (ViSR) ultrasound is a novel acoustic radiation force (ARF)-based imaging method that noninvasively interrogates the viscoelastic properties of tissue by measuring the relaxation time constant for constant stress in the Voigt biomechanical model. The time constant is defined as the ratio of coefficient of viscosity to elastic modulus, so ViSR differentiates tissue with disparate viscosities and elasticities. ViSR ultrasound is performed by delivering two successive ARF impulses to a single region of exciation (ROE) and tracking the micrometer-scale displacements induced by the propagating longitudinal waves. ViSR does not rely on transverse wave propagation, which can be disrupted and difficult to track in heterogeneous and/or geometrically complex media. Another advantage to ViSR ultrasound is a large axial range relative to conventional ARF Impulse (ARFI) ultrasound. In this overview, ViSR methods are discussed and demonstrated in calibrated viscoelastic tissue mimicking materials. ViSR ultrasound is then applied to differentiating fatty and fibrous deposition in muscle in a golden retriever muscular dystrophy (GRMD) dog model and in boys with Duchenne muscular dystrophy (DMD) with correlation to standard physical testing. ViSR is also applied to delineating the structure and composition of atherosclerotic plaques in a hypercholesterolemic pig model with histochemical validation. ViSR’s key advantages and disadvantages are discussed in regard to its general clinical utility.

Acoustic radiation force (ARF) ultrasound is a method of elastographic imaging in which micron-scale tissue displacements, induced and tracked by ultrasound, reflect clinically relevant tissue mechanical properties. Our laboratory has recently shown that tissue viscoelasticity is assessed using the novel Multi-Push (MP) ARF method. MP ARF applies the Voigt model for viscoelastic materials and compares the displacements achieved by successive ARF excitations to qualitatively or quantitatively represent the relaxation time for constant stress, which is a direct descriptor of the viscoelastic response of the tissue. We have demonstrated MP ARF in custom viscoelastic tissue mimicking materials and implemented the method in vivo in canine muscle and human renal allografts, with strong spatial correlation between MP ARF findings and histochemical features and previously reported mechanical changes with renal disease. These data support that noninvasive MP ARF is capable of clinically relevant assessment of tissue viscoelastic properties.

Acoustic radiation force impulse (ARFI) imaging is an elastography technique that uses ultrasonic pulses to displace and track tissue motion. Previous modeling studies have shown that ARFI displacements are susceptible to underestimation due to lateral and elevational shearing that occurs within the tracking resolution cell. In this study, optical tracking was utilized to experimentally measure the displacement underestimation achieved by acoustic tracking using a clinical ultrasound system. Three optically translucent phantoms of varying stiffness were created, embedded with subwavelength diameter microspheres, and ARFI excitation pulses with F/1.5 or F/3 lateral focal configurations were transmitted from a standard linear array to induce phantom motion. Displacements were tracked using confocal optical and acoustic methods. As predicted by earlier finite element method studies, significant acoustic displacement underestimation was observed for both excitation focal configurations; the maximum underestimation error was 35% of the optically measured displacement for the F/1.5 excitation pulse in the softest phantom. Using higher F/#, less tightly focused beams in the lateral dimension improved accuracy of displacements by approximately 10 percentage points. This work experimentally demonstrates limitations of ARFI implemented on a clinical scanner using a standard linear array and sets up a framework for future displacement tracking validation studies.

The ability to image platelets in vivo can provide insight into blood clotting processes and coagulopathies, and aid in identifying sites of vascular endothelial damage related to trauma or cardiovascular disease. Toward this end, we have developed a magnetomotive ultrasound (MMUS) system that provides contrast-enhanced imaging of superparamagnetic iron oxide (SPIO) labeled platelets via magnetically-induced vibration. Platelets are a promising platform for functional imaging contrast because they readily take up SPIOs and are easily harvested from blood. Here we report a novel MMUS system that accommodates an arbitrarily thick sample while maintaining portability. We employed a frequency- and phase-locked motion detection algorithm based on bandpass filtering of the differential RF phase, which allows for the detection of sub-resolution vibration amplitudes on the order of several nanometers. We then demonstrated MMUS in homogenous tissue phantoms at SPIO concentrations as low as 0.09?mg ml?1?Fe (p < 0.0001, n = 6, t-test). Finally, we showed that our system is capable of three-dimensional imaging of a 185??L simulated clot containing SPIO-platelets. This highlights the potential utility for non-invasive imaging of platelet-rich clots, which would constitute a fundamental advance in technology for the study of hemostasis and detection of clinically relevant thrombi.

Acoustic radiation force impulse (ARFI) imaging is an elastography technique that uses ultrasonic pulses to both displace and track tissue motion. Previous modeling studies have shown that ARFI displacements are susceptible to underestimation due to lateral and elevational shearing that occurs within the tracking resolution cell. In this study, optical tracking was utilized to experimentally measure the displacement underestimation achieved by acoustic tracking using a clinical ultrasound system. Two optically translucent phantoms of varying stiffness with embedded microspheres were created, and ARFI excitation pulses with F/1.5 or F/3 lateral focal configurations were transmitted from a standard linear array to induce phantom motion. Displacements were tracked using confocal optical and acoustic methods, with an acoustic tracking focal configuration of F/1.5 laterally. Acoustic displacement underestimation was observed for both excitation focal configurations; the maximum underestimation error was 51% of the optically measured displacement for the F/1.5 excitation pulse. This work experimentally demonstrates limitations of ARFI implemented on a clinical scanner using a standard linear array and sets up a framework for future displacement tracking validation studies.

Researchers have begun to investigate novel meniscus graft construction with donor cell replacement through decellularization and host stem cell seeding. In these techniques, there is an unmet need for a nondestructive mechanical testing platform that can monitor changing mechanical properties of the seeded graft over time. In this study, Monitored Steady-State Excitation and Recovery (MSSER) ultrasound, which exploits multiple successive acoustic radiation force excitations interspersed with tracking lines to mimic a materials creep test, was used to nondestructively test the mechanical stiffness of meniscus grafts. Four human meniscus samples were evaluated ex vivo using MSSER: two chemically decellularized samples and two intact samples. MSSER testing measured a median stiffness difference of 37% between intact and decellularized samples, which agreed well with the 35% difference measured by destructive compression testing. Volumetric MSSER scanning showed highly heterogeneous stiffness throughout the samples. This demonstration substantiates the use of MSSER as a relevant nondestructive testing tool in tissue engineering applications.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder that is caused by a mutation in the gene for dystrophin leading to a loss of the dystrophin protein from the muscle cell (myofiber) membrane. Viscoelastic Strain Response (ViSR) ultrasound is a new, quantitative acoustic radiation force (ARF) based elastographic imaging method to calculate τ, the viscoelastic relaxation time constant for constant stress. We have investigated the use of ViSR imaging in human DMD. Imaging was performed in vivo on the right rectus femoris (RF), sartorius (SART), and gastrocnemius (GAST) muscles of two boys with DMD. The boys were 5- and 9-years-old at the time of imaging. ViSR results in the 5-year-old showed an average of 27.0%, 6.6%, and 18.2% fat/necrosis composition in the RF, SART, and GAST, respectively. In the 9-year old subject, ViSR showed an average fat/necrosis composition of 66.7% in the RF, 9.1% in the SART, and 37.2% in the GAST. These results are consistent with both the known phenotypic response of the three muscles and with functional testing results and point to ViSR's potential relevance as a novel outcome measure for diagnostics and clinical trials in DMD.

Background Studies published in the 1970s by Mostafa S. Fahim and colleagues showed that a short treatment with ultrasound caused the depletion of germ cells and infertility. The goal of the current study was to determine if a commercially available therapeutic ultrasound generator and transducer could be used as the basis for a male contraceptive. Methods Sprague-Dawley rats were anesthetized and their testes were treated with 1 MHz or 3 MHz ultrasound while varying power, duration and temperature of treatment. Results We found that 3 MHz ultrasound delivered with 2.2 Watt per square cm power for fifteen minutes was necessary to deplete spermatocytes and spermatids from the testis and that this treatment significantly reduced epididymal sperm reserves. 3 MHz ultrasound treatment reduced total epididymal sperm count 10-fold lower than the wet-heat control and decreased motile sperm counts 1,000-fold lower than wet-heat alone. The current treatment regimen provided nominally more energy to the treatment chamber than Fahim's originally reported conditions of 1 MHz ultrasound delivered at 1 Watt per square cm for ten minutes. However, the true spatial average intensity, effective radiating area and power output of the transducers used by Fahim were not reported, making a direct comparison impossible. We found that germ cell depletion was most uniform and effective when we rotated the therapeutic transducer to mitigate non-uniformity of the beam field. The lowest sperm count was achieved when the coupling medium (3% saline) was held at 37 degrees C and two consecutive 15-minute treatments of 3 MHz ultrasound at 2.2 Watt per square cm were separated by 2 days. Conclusions The non-invasive nature of ultrasound and its efficacy in reducing sperm count make therapeutic ultrasound a promising candidate for a male contraceptive. However, further studies must be conducted to confirm its efficacy in providing a contraceptive effect, to test the result of repeated use, to verify that the contraceptive effect is reversible and to demonstrate that there are no detrimental, long-term effects from using ultrasound as a method of male contraception.

A validated method for assessing hemostasis in vivo is critical for testing the hemostatic efficacy of therapeutic agents designed for patients with bleeding disorders such as von Willebrand disease (VWD) and hemophilia A. We hypothesize that rate of bleeding and time to hemostasis can be monitored in vivo by acoustic radiation force impulse (ARFI) ultrasound. We performed ARFI imaging following 12-gauge needle puncture of hind limb muscle encompassing an ∼2 mm vein in six normal, eight naive hemophilia A before and after infusing canine factor VIII, three hemophilia A expressing canine factor VIIa following gene transfer, and two naive VWD dogs. Serial data sets were processed with custom software to (1) estimate the rate of hemorrhage and (2) estimate the time of hemostasis onset. The rate of hemorrhage during the first 30 min following puncture was markedly increased in the VWD dogs relative to normal but was not significantly different between normal, naive hemophilia A or hemophilia A expressing cFVIIa. ARFI-derived times to hemostasis were significantly longer in naive hemophilia A dogs than in normal dogs and were shortened by canine coagulation factors VIII and VIIa. These data support our hypothesis that rate of hemorrhage and time to hemostasis in vivo in response to a standardized hemostatic challenge can be detected by ARFI ultrasound in canine models of VWD and hemophilia. These data also suggest that the ARFI-monitored hemostatic challenge is relevant for in vivo testing of the hemostatic efficacy of therapeutic clotting factor replacement products used to treat inherited bleeding disorders.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder that is caused by a mutation in the gene for dystrophin leading to a loss of the dystrophin protein from the muscle cell (myofiber) membrane. The golden retriever muscular dystrophy (GRMD) model also has a naturally-occurring dystrophin gene mutation and progressive phenotypic features analogous to those seen in DMD. We have investigated the use of Acoustic Radiation Force Impulse (ARFI) imaging in the GRMD canine model. Imaging was performed in vivo on the right rectus femoris (RF) and cranial sartorius (CS) muscles in both transverse and parallel planes. A cohort of 6 normal dogs and 4 GRMD dogs were imaged at 3, 6, and 12 months of age. Results showed significant differences in peak displacement in the RF for GRMD vs normal dog, but similar ARFI results in the CS. These results are consistent with known variations in disease progression in RF versus CS muscles in the dog model and suggest that ARFI could be relevant to monitoring therapy in affected individuals.