S.A. Wickline

Ultrasonic tissue characterization with integrated backscatter. Acute myocardial ischemia, reperfusion, and stunned myocardium in patients.

We have previously shown in studies of experimental animals that myocardium exhibits a cardiac cycle-dependent variation of integrated backscatter that reflects regional myocardial contractile performance and that is blunted promptly after arterial occlusion and recovers after reperfusion. To define the clinical utility of ultrasonic tissue characterization with integrated backscatter for detection of acute myocardial infarction and reperfusion, 21 patients (14 men and seven women) were studied in the cardiac care unit within the first 24 hours (mean time, 11.3 hours; range, 3.5-23.8 hours) after the onset of symptoms indicative of acute myocardial infarction with conventional two-dimensional and M-mode echocardiography and with analysis of integrated backscatter. The magnitude of cyclic variation of integrated backscatter was measured from several sites within acute infarct regions and normal regions remote from the infarct zone for each patient. The average magnitude of cyclic variation among all patients (n = 21) was 4.8 +/- 0.5 dB in normal regions compared with 0.8 +/- 0.3 dB in infarct regions (p less than 0.05) within the first 24 hours after the onset of symptoms. Among the patients who had two studies, 15 (mean, 7.1 days; range, 2-31 days for second study) underwent coronary arteriography to define vessel patency. In patients with vessels with documented patency (n = 10), the magnitude of cyclic variation in infarct regions increased over time from 1.3 +/- 0.6 to 2.5 +/- 0.5 dB from the initial to final study (p less than 0.05). Patients with occluded infarct-related arteries (n = 5) exhibited no significant recovery of cyclic variation (0.3 +/- 0.3-0.6 +/- 0.3 dB). A blinded analysis of standard two-dimensional echocardiographic images revealed no significant recovery of wall thickening in either group over the same time intervals. Ultrasonic tissue characterization promptly detects acute myocardial infarction and may delineate potential beneficial effects of coronary artery reperfusion manifest by restoration of cyclic variation of integrated backscatter in the presence of severe wall motion abnormalities.

Sensitive detection of the effects of reperfusion on myocardium by ultrasonic tissue characterization with integrated backscatter.

We have shown recently that tissue characterization of myocardium with ultrasound reflects changes associated with contractile function throughout the cardiac cycle. To determine whether ultrasonic tissue characterization can sensitively detect the impact of ischemic injury and reperfusion on contractile properties of the heart, we studied the time course of change of backscatter after 5, 20, and 60 min of coronary occlusion followed by reperfusion in 15 dogs. The time-averaged integrated backscatter (IB) and the amplitude and phase of cyclic variation of IB (phase relative to the left ventricular pressure waveform) were measured. A novel ultrasonic index of acute injury was identified, the phase-weighted amplitude of cyclic variation, and calculated by weighting the amplitude of cyclic variation of IB with respect to the phase. We hypothesized that backscatter variables would change dramatically after occlusion and that their restitution after reperfusion would sensitively reflect the extent and time course of reversibility of ischemic injury. After coronary occlusion, segmental wall thickening decreased from approximately 55% to 5% regardless of the duration of ischemia. Changes in backscatter associated with this decrease included an increase in time-averaged IB of approximately 5 dB, a 5 dB decrease in cyclic variation, an 80 degree phase shift, and a 7 dB decrease in phase-weighted amplitude. Wall thickening after reperfusion immediately after the 5, 20, or 60 min occlusions recovered to 45%, 27%, and 12% of baseline values, respectively. Within 3 hr it recovered to 53%, 44%, and 22%. Time-averaged IB recovered initially by 89%, 61%, and 44% (all p less than .05) and continued to recover subsequently although more slowly. Ultimate recovery was virtually complete. In contrast to the rapid recovery of time-averaged IB, phase-weighted amplitude recovered initially to only 72%, 41%, and -7% of baseline (all p less than .05) and manifested slower and incomplete recovery when ischemia had been present for 20 or 60 min. After reperfusion, the time course of both cyclic variation and phase were reflected by changes in the phase-weighted amplitude. The backscatter variables assessed appear to sensitively delineate the duration, time course of recovery, and reversibility of ischemic injury in response to reperfusion. The results suggest that early recovery of time-averaged IB corresponds in part to the restoration of tissue ultrastructural integrity.(ABSTRACT TRUNCATED AT 400 WORDS)

Structural remodeling of human myocardial tissue after infarction. Quantification with ultrasonic backscatter.

BackgroundRemodeling of myocardial tissue after infarction may culminate in the development of either a well-healed scar or a thin, expanded heart wall segment that predisposes to ventricular aneurysm formation, congestive heart failure, or ventricular tachycardia. The three-dimensional architecture of mature human infarct tissue and the mechanisms that determine it have not been elucidated. We have previously shown that quantitative ultrasonic backscatter can be used to define the transmural organization of human myofibers in the normal ventricular wall by measuring the dependence of backscatter on the angle of insonification, or ultrasonic anisotropy. We propose that measurement of ultrasonic anisotropy of backscatter may permit quantitative characterization of the transmural architecture of tissue from areas of myocardial infarction and facilitate identification of fundamental mechanisms of remodeling of the ventricular wall. Methods and ResultsWe measured integrated backscatter in 33 transmural sections from 12 cylindrical biopsy specimens (1.4-cm diameter) sampled from central regions of mature infarction in six explanted fixed human hearts. Tissue samples were insonified in two-degree steps around their entire circumference at successive transmural levels with a 5-MHz broad-band piezoelectric transducer. Backscatter radio frequency data were gated from the center of each specimen, and spectral analysis was performed on the gated radio frequency for the computation of integrated backscatter. Histological morphometric analysis was performed on each specimen for determination of the predominant fiber orientation and the percentage of tissue infarcted at consecutive transmural levels. The average percentage of tissue infarcted for all transmural levels was 49±3% (range, 13-80%). Histological attributes varied from patchy fibrosis to extensive confluent zones of scar tissue. The angle-averaged integrated backscatter for all transmural levels in infarct tissue was approximately 5 dB greater than that previously measured in normal tissue in our laboratory (−48.3±0.5 versus −53.4±0.4 dB, infarct versus normal). Marked anisotropy of backscatter was observed in tissue from areas of infarction and was characterized by a sinusoid-like dependence on the angle of insonification at each transmural level. Insonification perpendicular to infarct fibers yielded values for integrated backscatter 14.8±0.5 dB greater than those for insonification parallel to these fibers. Juxtaposition of the sinusoid-like anisotropy functions from all consecutive transmural levels demonstrated a progressive shift in the orientation of scar tissue elements from epicardial to endocardial levels of 14.6±1.5°/mm of tissue. The transmural shift in fiber orientation per millimeter of tissue from the area of infarction exceeded that previously measured for normal tissue (9.2±0.7°/mm) by 59%. This marked augmentation in angular shift per millimeter of tissue results from a generalized structural rearrangement (or reorientation) of fibers across the entire ventricular wall in the infarct zone that we hypothesize is determined in part by dynamic mechanical forces, imposed by the surrounding functional normal tissue, that tether the infarcted tissue ConclusionsMyocardial tissue from areas of myocardial infarction manifests substantial anisotropy of ultrasonic scattering that may be useful for quantitative characterization of the alignment and overall three-dimensional anatomic organization of mature infarct scars.

Quantitative real-time imaging of myocardium based on ultrasonic integrated backscatter

The integrated backscatter calculation over the full, two-dimensional echocardiographic sector is implemented to produce images from closed-chest dogs. This new real-time integrated backscatter measurement system allows a continuous determination of integrated backscatter from all myocardial regions in the ultrasonic view. By replacing the conventional video processor in a commercial two-dimensional echocardiographic imager with this new real-time backscatter measurement system, it is possible to produce real-time two-dimensional images based on integrated backscatter.<<ETX>>

Detection of unique transmural architecture of human idiopathic cardiomyopathy by ultrasonic tissue characterization.

BackgroundNoninvasive approaches to the evaluation of idiopathic cardiomyopathy are limited. Recent work from our laboratory has used quantitative ultrasound to define the three-dimensional structure of normal human myocardium and the myocardial remodeling associated with infarction. Our goal was to define the role of ultrasonic tissue characterization for detection of specific alterations in the threedimensional transmural architecture of idiopathic dilated cardiomyopathy Methods and ResultsWe measured frequency-dependent backscatter from 22 cylindrical biopsy specimens from nine explanted fixed hearts of patients who underwent heart transplantation for idiopathic cardiomyopathy, seven specimens from normal portions, and 12 specimens of infarcted tissue from six explanted fixed human hearts. Consecutive transmural levels from each specimen were insonified with a 5-MHz broadband transducer. The dependence of apparent (uncompensated for attenuation) backscatter, B(f), on frequency (f) was computed from radiofrequency (rf) data as: IB(f)I2=afn, where n is an index that reflects in part the size of the dominant scatterers in myocardial tissue. Myofiber diameter and percentage fibrosis were determined at each transmural level for each specimen. For cardiomyopathic tissue, the frequency dependence of backscatter (n) increased progressively from epicardial to endocardial (0.02±0.37 to 1.01±0.12, p < 0.05) levels in conjunction with a progressive decrease in myofiber diameter (29.5±0.9 to 21.4±0.6,um, p < 0.0001). In contrast, in tissue from areas of infarction, the frequency dependence decreased progressively from epicardium to endocardium (0.91±0.20 to 0.23±0.21, p < 0.05) in conjunction with a progressive increase in the percentage of fibrosis (23.5±9.4% to 54.5±4.9%, p < 0.005). Normal tissue exhibited no significant transmural trend for frequency dependence, myofiber diameter, or percentage fibrosis ConclusionsThese data indicate the presence of a heterogenous transmural distribution of scattering structures associated with human idiopathic cardiomyopathy and myocardial infarction that may be detected by ultrasonic tissue characterization. The divergence of these transmural trends for frequency dependence of backscatter reflects distinct mechanisms of structural heterogeneity for different pathological processes that comprise a transmural gradation of cell size and fibrosis for idiopathic cardiomyopathy and infarction, respectively.

Experimental determination of phase velocity of perfluorocarbons: Applications to targeted contrast agents

Targeted acoustic contrast agents are designed to enhance the sensitivity and specificity of ultrasonic diagnoses. We have previously developed a ligand targeted ultrasonic contrast system that is a lipid-encapsulated, liquid-perfluorocarbon emulsion. The emulsion particles are small (250 nm) and have inherently low echogenicity unless bound to a surface by a pretargeted ligand through avidin-biotin interactions. We have recently proposed a simple acoustic transmission line model that treats the emulsion particles as a thin layer over the targeted surface. In this model, the acoustic reflectivity of the sample increases for perfluorocarbons with smaller velocities of longitudinal sound or lower densities. In this study, we measure and report the velocity of longitudinal sound for 20 perfluorocarbons using a broadband phase spectroscopic approach for estimating phase velocities. Experimentally determined velocities ranged from 520/spl plusmn/2 m/sec (perfluorohexane) to 705/spl plusmn/5 m/s (perfluorodecalin). No measurable dispersion was observed over the useful bandwidth of 2 to 22 MHz. Increasing carbon backbone chain length and fluorine substitution with halogens of greater atomic weight increased the measured speed of sound. Our experimental data were consistent (R=0.87) with a published empirical model that predicts velocity as a function of molecular structure. These data provide a rational basis for optimizing targeted perfluorocarbon-based contrast agents and offer further insight into the physical mechanisms responsible for the observed enhancement of surface acoustic reflectivity.

Quantification of atherosclerotic plaque composition in cholesterol-fed rabbits with 50-MHz acoustic microscopy.

To determine whether high-frequency ultrasound could distinguish normal from pathological vascular structure and to elucidate the determinants of ultrasonic backscatter in different layers of normal and atherosclerotic arteries, high-resolution acoustic microscopy at 50 MHz was used to characterize aortic plaque in six New Zealand White rabbits fed a 2% cholesterol diet for 3.5 months. Four rabbits were fed a standard diet for 3.5 months to provide normal control data. Segments of aortas were excised, fixed in formalin, opened longitudinally, and mounted flat for insonification. For each specimen, backscattered radio frequency (rf) data were acquired from 30 to 100 independent sites separated by 500 microns. Portions of rf data were gated from discrete layers of the vessel wall for computation of integrated backscatter. Results of histological and immunocytochemical analyses of vessel wall thickness and composition were compared with those of ultrasonic analysis. Normal aortas manifested prominent but homogeneous backscatter (average integrated backscatter, -28.5 +/- 2.9 dB) throughout the vessel wall, with no clear distinction between intimal and medial layers. The atherosclerotic aortas manifested substantially reduced integrated backscatter from the thickened intima (-47.5 +/- 3.2 dB, p < 0.0001) but relatively normal integrated backscatter from the media (-31.2 +/- 1.6 dB; p = NS versus normal aortas). The thickness of the media for both normal and atherosclerotic rabbits was approximately 300 microns. Histological characteristics of atherosclerotic aortas confirmed the presence of substantial intimal thickening, with prominent foam cell and lipid infiltration abutting a more normal medial layer.(ABSTRACT TRUNCATED AT 250 WORDS)

Cellular mechanisms of captopril-induced matrix remodeling in Syrian hamster cardiomyopathy.

BackgroundAlthough angiotensin-converting enzyme (ACE) inhibitors have become a mainstay of treatment for chronic congestive heart failure (CHF), it is not known whether the cardiac remodeling effects are a secondary phenomenon, resulting from ACE inhibitors hemodynamic actions of afterload reduction, or occur through an independent mechanism. Methods and ResultsWe used ultrasonic tissue characterization to define potentially salutary effects of treatment with ACE inhibitors on the material properties of the heart and its potential influence on cardiac remodeling at the cellular level. Ten 1-month-old, cardiomyopathic (CM) Syrian hamsters and 6 normal (NL) hamsters were treated with captopril (2 g/L water ad libitum), and 10 CM hamsters and 10 NL hamsters were maintained untreated for 3 months. Hearts were excised, and backscattered radiofrequency data were acquired from 1200 independent sites from each specimen with a highresolution 50-MHz acoustic microscope for calculation of integrated backscatter (IB). Treatment with captopril reduced left ventricular mass, calcium concentration, and IB in CM hearts without affecting myofiber size or collagen concentration. The IB from grossly normal regions of myocardium in NL hamsters, treated CM hamsters, and untreated CM hamsters was not significantly different. The IB from the microscopic regions of scar tissue in treated CM hamsters was significantly less (P=.0004) than that from scar tissue in untreated CM hamsters. ConclusionsThe reduced IB from treated scar tissue components reflects specific alterations in the material properties (elastic stiffness, density) of fibrous regions in CM hearts induced by captopril. This is the first report that defines specific cellular effects of ACE inhibitors on the material properties of isolated components of cardiac tissue in experimental cardiomyopathy. These alterations in material properties of scar tissue components represent a potential mechanism for the salutary actions of ACE inhibitors in heart failure.

Mechanism of the view-dependence of ultrasonic backscatter from normal myocardium

Anisotropy of ultrasonic scattering and attenuation in heart tissue depends on the specific orientation of myofibers with respect to angle of insonification. The present study was designed to delineate the effect of the angle of insonification with respect to the alignment of myofibers on measurements of integrated backscatter. A transmural cube of myocardium was cut from the anterior wall of the left ventricles from 5 normal canine hearts and their transmural scattering behavior was studied with the use of reflection acoustic microscopy at 50 MHz. A theoretical model for scattering based on the Born approximation (weak scattering) was employed to predict the relationship between backscatter and angle of insonification. Insonification of the basal face (basal view) demonstrated a wide transmural variation in integrated backscatter (/spl sim/15 dB), while insonification of the lateral face (lateral view) had much reduced variation (/spl sim/4 dB), despite an equivalent overall shift in transmural fiber angle of /spl sim/85/spl deg/ across the ventricular wall. Integrated backscatter was greatest in the midmyocardium when the basal face was viewed and least in the midmyocardium when the lateral face was viewed. The backscatter in the subepicardial and subendocardial regions was similar for both views. The maximum difference in backscatter from basal and lateral views at the midmyocardial level was approximately 18 dB, which represents a 64-fold change in the intensity of ultrasonic backscatter. The mathematical model developed for scattering based on the Born approximation (weak scattering) predicted the observed relationship between backscatter and angle of insonification. The rapid angular variation of integrated backscatter perpendicular to the fiber direction and the slow variation at parallel incidence observed experimentally were predicted by the model. This angular variation is due to the specific shape and elastic properties assumed for the predominant myocardial scatterer. There was a strong relationship between backscatter and fiber orientation, indicating that the view chosen for insonification of myocardium in clinical imaging may influence the estimation of scattering behavior. The mathematical model utilized here predicts the anisotropic behavior of scattering and suggests that the principal scattering structure in normal myocardium may be a stiff collagen shell surrounding a more compliant myocyte. This model might provide a valid approach for the study of material properties of the heart with the use of ultrasound.<<ETX>>

New mechanisms for non-porative ultrasound stimulation of cargo delivery to cell cytosol with targeted perfluorocarbon nanoparticles

The cell membrane constitutes a major barrier for non-endocytotic intracellular delivery of therapeutic molecules from drug delivery vehicles. Existing approaches to breaching the cell membrane include cavitational ultrasound (with microbubbles), electroporation and cell-penetrating peptides. We report the use of diagnostic ultrasound for intracellular delivery of therapeutic bulky cargo with the use of molecularly targeted liquid perfluorocarbon (PFC) nanoparticles. To demonstrate the concept, we used a lipid with a surrogate polar head group, nanogold-DPPE, incorporated into the nanoparticle lipid monolayer. Melanoma cells were incubated with nanogold particles and this was followed by insonication with continuous wave ultrasound (2.25 MHz, 5 min, 0.6 MPa). Cells not exposed to ultrasound showed gold particles partitioned only in the outer bilayer of the cell membrane with no evidence of the intracellular transit of nanogold. However, the cells exposed to ultrasound exhibited numerous nanogold-DPPE components inside the cell that appeared polarized inside intracellular vesicles demonstrating cellular uptake and trafficking. Further, ultrasound-exposed cells manifested no incorporation of calcein or the release of lactate dehydrogenase. These observations are consistent with a mechanism that suggests that ultrasound is capable of stimulating the intracellular delivery of therapeutic molecules via non-porative mechanisms. Therefore, non-cavitational adjunctive ultrasound offers a novel paradigm in intracellular cargo delivery from PFC nanoparticles.