Christopher S. Hall

High-resolution MRI characterization of human thrombus using a novel fibrin-targeted paramagnetic nanoparticle contrast agent

In this study, the sensitivity of a novel fibrin‐targeted contrast agent for fibrin detection was defined in vitro on human thrombus. The contrast agent was a lipid‐encapsulated perfluorocarbon nanoparticle with numerous Gd‐DTPA complexes incorporated into the outer surface. After binding to fibrin clots, scanning electron microscopy of treated clots revealed dense accumulation of nanoparticles on the clot surfaces. Fibrin clots with sizes ranging from 0.5–7.0 mm were imaged at 4.7 T with or without treatment with the targeted contrast agent. Regardless of sizes, untreated clots were not detectable by T1‐weighted MRI, while targeted contrast agent dramatically improved the detectability of all clots. Decreases in T1 and T2 relaxation times (20–40%) were measured relative to the surrounding media and the control clots. These results suggest the potential for sensitive and specific detection of microthrombi that form on the intimal surfaces of unstable atherosclerotic plaque. Magn Reson Med 44:867–872, 2000.

Blood Contrast Enhancement with a Novel, Non-Gaseous Nanoparticle Contrast Agent

Modern ultrasound contrast agents primarily comprise microbubble formulations that circulate in the intravascular compartment and are designed to enhance acoustic signals reflected from the blood pool. A variety of shell materials have been utilized to stabilize gas bubbles of the order of 1–10 microns in diameter. Reflectivity from microbubbles is enhanced by resonance and non-linear physical effects. However, the overall efficacy of bubbles as contrast agents must be considered in light of their marked instability to insonification pressures, marked attenuation artifacts, “blooming” effects, and their short circulatory half-life. Low molecular weight gaseous perfluorocarbon formulations have been utilized in vivo because they may offer advantages in formulation and reflectivity. In contrast, higher molecular weight perfluorocarbon emulsions that are liquid at body temperature have been formulated as nongaseous nanoparticle preparations (diameters 100– 300 nanometers), originally for use as blood substitutes. Unfortunately they exhibit low inherent echogenicity and are poor blood pool contrast agents under conditions of conventional 2-D echocardiography or harmonic imaging, or when imaged with color flow or spectral Doppler. Nevertheless, these nanoparticle formulations are chemically inert, manifest long circulatory half-lives, are not destroyed by ultrasonic imaging, and they possess low acoustic attenuation. Such features might still render them of interest as blood pool contrast agents if properly formulated and imaged. Recently, a new ultrasonic imaging modality, Power Doppler Harmonic Imaging (PDHI), has been introduced (4). This technique color-encodes changes in acoustic signal amplitude and motion of ultrasonic scatterers between insonifying pulses. PDHI has been used in a number of clinical studies to assess coronary artery bypass graft patency, tumor blood flow, and myocardial perfusion. In view of the exquisite sensitivity of Doppler for detecting the presence of small scatterers with limited scattering cross-sections as compared to microbubbles (e.g., red blood cells), and the enhanced ability of PDHI to register backscatter power, we hypothesized that certain liquid perfluorocarbon nanoparticle emulsions (5) might be more efficiently detected with this new imaging modality. Furthermore, although we have demonstrated previously that the liquid nanoparticle emulsions do not manifest any appreciable resonance behavior at clinically relevant imaging frequencies, they have performed well as targeted imaging agents in vitro and in vivo over a very broad range of frequencies (5–50 MHz)(6–8). Thus we anticipated that the PDHI method might permit imaging of these nanoparticles in the blood pool without reliance on any intrinsic resonance behavior.

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.

High-frequency ultrasound detection of the temporal evolution of protein cross linking in myocardial tissue

The progressive increase in stiffening of the myocardium associated with the aging process and abetted by comorbid conditions such as diabetes may be linked to an excessive number of collagen cross links within the myocardial extra-cellular matrix. To determine whether ultrasound can delineate changes in the physical properties of heart tissue undergoing cross linking, the authors employed a model in which increased cross linking was induced by treating rat myocardial tissue with specific chemical fixatives. Rat hearts (n=5 each group) were arrested at end-diastole, insonified (30 to 50 MHz) fresh within a few minutes of excision in a phosphate buffered solution, placed in a fixative (10% formalin or 2.5% glutaraldehyde) and insonified at 30-minute intervals thereafter for 24 hours. Ultrasonic attenuation increased in tissues cross linked with formalin (maximal change: 27.2/spl plusmn/3.4 dB/cm) and glutaraldehyde (maximal change: 40.2/spl plusmn/5.6 dB/cm) over a 24-hour period. The frequency dependence of the attenuation coefficient increased as a function of the extent of collagen cross links in formalin (maximal change: 0.8/spl plusmn/0.3 dB/cm-MHz) and glutaraldehyde (maximal change: 0.9/spl plusmn/0.6 dB/cm-MHz). This study represents the first time that the precise time course of myocardial protein cross linking in situ has been characterized by using real time monitoring, and the physiologic effect has been delineated on microscopic material properties.

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>>

Age-related alterations of cardiac tissue microstructure and material properties in Fischer 344 rats

The cardiac aging process is accompanied by global mechanical dysfunction that reflects increased myocardial stiffness. Accordingly, age-related changes in microscopic material properties of myocardium were delineated with high-frequency ultrasound (US) (30 to 44 MHz) tissue characterization methods for aging Fischer 344 rats at 6 (adult), 18 (aged), and 24 (senescent) months of age. The excised lateral wall of the left ventricle of rats (n = 10 per group) was insonified with a 50-MHz acoustic microscope for determination of integrated backscatter, backscatter coefficient and attenuation coefficient. Histological and biochemical analyses for collagen content and cardiac myocyte diameter were performed. Collagen concentration increased progressively with age, with the greatest increments occurring from 6 to 18 months (38.0 +/- 6.3 to 53.0 +/- 7.1 mg/g dry wt), and leveling off at 24 months (60.0 +/- 7.4 mg/g dry wt). Tissue microscopic material properties also changed progressively from 6 to 24 months of age, as determined by US methods: integrated backscatter increased (-44.7 +/- 1.8 vs. -40.8 +/- 1.9 dB, p < 0.05), attenuation increased (47.1 +/- 5.9 to 65.3 +/- 7.8 dB/cm, p < 0.05), and the backscatter coefficient increased (0.73 +/- 0.16 x 10(-5) to 3.76 +/- 1.6 x 10(-5) cm(-1), p < 0.05), from 6 to 24 months of age in each case. Age-related alterations in indices of cardiac microscopic material properties were closely correlated with the changes in cardiac microstructure. Ultrasonic tissue characterization may prove to be a sensitive tool to monitor changes in the cardiac microstructure, such as increased collagen deposition, that occur within age-related diastolic dysfunction.

HIGH-FREQUENCY ULTRASOUND FOR QUANTITATIVE CHARACTERIZATION OF MYOCARDIAL EDEMA

Myocardial edema has been associated with impaired ventricular compliance and diastolic filling. To determine the sensitivity of high-frequency (40 MHz) ultrasound to myocardial edema, we employed a model in which myocardial edema was induced by immersion of tissue in isotonic saline. The effect of freezing tissue on edema formation was also evaluated. Rat hearts were arrested at end-diastole and insonified fresh within 15 min of excision (n = 5) or following being frozen for 24 h and thawed (n = 4). Measurements of attenuation, backscatter, tissue thickness and speed of sound were performed at baseline and hourly for 4 h, and compared with direct measurements of myocardial edema. Fresh tissue demonstrated a greater propensity for the development of edema than frozen tissue. Integrated backscatter increased in both tissues, whereas the magnitude and slope of attenuation decreased as edema evolved. We conclude that high-frequency ultrasound sensitively detects myocardial edema, and we propose that the extension of these methods to clinical frequencies may prove useful for monitoring and treatment of cardiac edematous disease states.

Delineation of the extracellular determinants of ultrasonic scattering from elastic arteries

Elastic arteries consist of three primary components: elastin fibers, extracellular collagen matrix and smooth muscle cells. However, the relative contribution of elastin and collagen fibers to overall ultrasonic scattering from an intact arterial wall is poorly understood. To define the principal source of extracellular scattering from the medial layer of elastic arteries, canine ascending aortas (n = 10) were excised, fixed and sectioned for insonification. Subsequently, aortic specimens were restudied after treatment to dissolve all tissue components except extracellular collagen matrix (n = 5) and elastin fibers (n = 5). Histological staining revealed very few elastin fibers and sparse intact collagen in collagen-isolated and elastin-isolated tissues, respectively. Integrated backscatter, attenuation and backscatter coefficients differentiated these two treated tissues. The backscatter coefficient for elastin-isolated tissue demonstrated a fivefold increase over collagen-isolated tissue, suggesting that elastin fibers represent a primary scattering component within elastic arteries, and the collagen fibers may provide a secondary component of scattering.

In vivo ultrasonic detection of angiogenesis with site-targeted nanoparticle contrast agents using measure-theoretic signal receivers

Angiogenesis has been postulated as an important marker for the early detection of cancer. The proteins associated with new vessels are sub-resolution for ultrasonic imaging, necessitating the use of contrast agents. In this work we use a liquid, perfluorocarbon nanoparticle previously shown to enhance specific targets in in vitro and in situ settings. Previous studies focused on the use of conventional signal analysis techniques including signal amplitude, signal energy, and spectral analysis. To explore the possibility of further increasing contrast between targeted bio-markers and untargeted tissue, we applied concepts from measure-theoretic (e.g., information theory, thermodynamics) and topological dynamics. Specifically, Shannon entropy, H/sub S/, its continuous limit, H/sub C/, and three quantities obtained using analogies with thermodynamics: C/sub v/, E/sub th/, which have been described elsewhere. We describe the outcome of employing these types of dynamical quantities to ultrasonic data acquired in vivo using New Zealand white rabbits implanted with VX2-tumors and then exposed over the course of two hours to /spl alpha//sub v//spl beta//sub 3/ integrin-targeted liquid perfluorocarbon nanoparticles.

Angiotensin II Receptor Blockade in Syrian Hamster (T0-2) Cardiomyopathy Does Not Affect Microscopic Cardiac Material Properties: Implications for Mechanisms of Tissue Remodeling

This study delineates the role of angiotensin II type I (AT1) receptor in the remodeling of Syrian cardiomyopathic hamsters. Twelve cardiomyopathic (T0-2) hamsters received L-158,809 treatment ad libitum in their drinking water (27 µg/ml) and 9 cardiomyopathic and 9 normal F1-B hamsters received tap water from 1 to 4 months of age. Although pharmacologically effective with regard to complete suppression of the blood pressure response to angiotensin II infusion, L-158,809 did not diminish the progression or severity of cardiomyopathy. Heart weight/100 g body weight and left ventricular wall thickness adjusted for body weight of both L-158,809 and cardiomyopathic control hamsters did not differ and exceeded those of F1-B controls (p < 0.05). Myocardial material properties (e.g., stiffness and density) of cardiomyopathic hamsters treated with L-158,809 were not affected. Thus, the progression of fibrosis, calcification, and necrosis in T0-2 cardiomyopathic hamsters was not sensitive to AT1 receptor blockade.