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In this work, the Kramers-Kronig (K-K) relations are applied to experimental data of resonant nature by limiting the interval of integration to the measurement spectrum. The data are from suspensions of encapsulated microbubbles (Albunex) and have the characteristics of an ultrasonic notch filter. The goal is to test the consistency of this dispersion and attenuation data with the Kramers-Kronig relations in a strict manner, without any parameters from outside the experimental bandwidth entering in to the calculations. In the course of reaching the goal, the artifacts associated with the truncation of the integrals are identified and it is shown how their impacts on the results can be minimized. The problem is first approached analytically by performing the Kramers-Kronig calculations over a restricted spectral band on a specific Hilbert transform pair (Lorentzian curves). The resulting closed-form solutions illustrate the type of artifacts that can occur due to truncation and also show that accurate results can be achieved. Next, both twice-subtracted and lower-order Kramers-Kronig relations are applied directly to the attenuation and dispersion data from the encapsulated microbubbles. Only parameters from within the experimental attenuation coefficient and phase velocity data sets are used. The twice-subtracted K-K relations produced accurate estimates for both the attenuation coefficient and dispersion across all 12 data sets. Lower-order Kramers-Kronig relations also produced good results over the finite spectrum for most of the data. In 2 of the 12 cases, the twice-subtracted relations tracked the data markedly better than the lower-order predictions. These calculations demonstrate that truncation artifacts do not overwhelm the causal link between the phase velocity and the attenuation coefficient for finite bandwidth calculations. This work provides experimental evidence supporting the validity of the subtracted forms of the acoustic K-K relations between the phase velocity and attenuation coefficient.

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.

RATIONALE AND OBJECTIVES Molecular imaging with targeted contrast agents enables tissues to be distinguished by detecting specific cell-surface receptors. In the present study, a ligand-targeted acoustic nanoparticle system is used to identify angioplasty-induced expression of tissue factor by smooth muscle cells within carotid arteries. METHODS Pig carotid arteries were overstretched with balloon catheters, treated with tissue factor-targeted or a control nanoparticle system, and imaged with intravascular ultrasound before and after treatment. RESULTS Tissue factor-targeted emulsions bound and increased the echogenicity and gray-scale levels of overstretched smooth muscle cells within the tunica media, versus no change in contralateral control arteries. Expression of stretch-induced tissue factor in carotid artery media was confirmed by immunohistochemistry. CONCLUSIONS The potential for abnormal thrombogenicity of balloon-injured arteries, as reflected by smooth muscle expression of tissue factor, was imaged using a novel, targeted, nanoparticulate ultrasonic contrast agent.

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.

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. © 2000 Wiley-Liss, Inc.

Ultrasound tissue characterization with measurement of backscatter has been employed in numerous experimental and clinical studies of cardiac pathology, yet the cellular components responsible for scattering from cardiac tissues have not been unequivocally identified. This laboratory has proposed a mathematical model for myocardial backscatter that postulates the fibrous extracellular matrix (ECM) as a significant determinant of backscatter. To demonstrate the importance of ECM, this group sought to determine whether measurements of backscatter from the isolated ECM could reproduce the known directional dependence, or anisotropy of backscatter, from intact cardiac tissues in vitro. Segments of left ventricular free wall from ten formalin fixed porcine hearts were insonified at 50 MHz, traversing the heart wall from endo- to epicardium to measure the anisotropy of myocardial backscatter, defined as the difference between peak (perpendicular to fibers) and trough (parallel to fibers) backscatter amplitude. The tissue segments were then treated with 10% NaOH to dissolve all of the cellular components, leaving only the intact ECM. Scanning electron micrographs (SEM) were obtained of tissue sections to reveal complete digestion of the cellular elements. The dimensions of the residual voids resulting from cell digestion were approximately the diameter of the intact myocytes (10-30 microm). These samples were reinsonified after seven days of treatment to compare the anisotropy of integrated backscatter. The magnitude of anisotropy of backscatter changed from 15.4 +/- 0.8 to 12.6 +/- 1.1dB for intact as compared with digested specimens. Because digestion of the myocardium leaves only extracellular sources of ultrasonic scattering, and because the isolated ECM exhibits similar ultrasonic anisotropy as does the intact myocardium, it is concluded that there is a direct association between the ECM and the anisotropy of backscatter within intact tissue. Thus, it is suggested that ultrasonic tissue characterization represents a potentially clinically applicable method for delineating the structure and function of the ECM.

The phase velocities and attenuation coefficients for suspensions of narrowly sized polymer microspheres are reported over a broadband spectrum from 3 to 30 MHz. The six suspensions used in this work contain microspheres with respective average diameters near 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, and 100 μm. The results of these measurements are compared with theoretical expressions for the phase velocity and attenuation coefficient derived from the scattering properties of an elastic sphere in water using the weak scattering limit of the Waterman and Truell dispersion relation [J. Math. Phys. 2, 512–537 (1961)]. This single-scattering limit of the theory is found to be sufficient for predicting the ultrasonic transport properties of these suspensions to a considerable degree of accuracy.

Objective: To assess the efficacy of salvage therapy containing ritonavir and saquinavir after failure of indinavir- or nelfinavir-containing regimens, and to determine correlates of success or failure. Design: Retrospective chart review. Setting. The Moore Clinic - the HIV clinic of Johns Hopkins Hospital. Patients: Forty-one HIV-infected patients were identified through physician contacts, referrals from other providers, and review of a comprehensive clinical database. Main outcome measures: To determine response to salvage therapy, HIV-1 viral RNA (absolute and log 10 -transformed) was measured using the Roche Amplicor quantitative HIV-1 RNA assay after initiation of the salvage regimen. Potential correlates of response included: viral RNA at the time of switch; viral RNA at the time of switch as a percentage of baseline viral RNA; magnitude of decline in viral RNA; and the interval between virologic failure of single protease inhibitor therapy and switch to the salvage regimen. Results: Thirteen (56.5%) of 23 patients failing indinavir responded to salvage therapy (HIV RNA < 400 copies/ml) with persistence throughout the follow-up period (median of 37 weeks; range 18-67 weeks). Mean absolute viral RNA at the time of switch was 20 238 copies/ml (median, 9281) compared with 42 953 copies/ml (median, 24 650) for the 10 non-responders. Mean log 10 viral RNA at switch was 3.804 for responders versus 4.405 for non-responders (P = 0.040). Among four responders who had failed nelfinavir, mean viral RNA was 9634 copies/ml and mean log 10 viral RNA was 3.749 at the time of switch. Two non-responders had a mean viral RNA of 21 551 and a mean log 10 viral RNA of 4.037 at switch. Conclusions: In contrast with previous reports, salvage regimens containing ritonavir and/or saquinavir can be effective and durable following the failure of combination regimens containing either indinavir or nelfinavir. Salvage therapy may be more likely to succeed when it is initiated early in failure at low viral loads.

To the Editor: Streptococcus suis, commensal and opportunistic pathogens of swine, and prevalent zoonotic agents worldwide, are α-hemolytic gram-positive cocci with 35 different serotypes (1). In humans, S. suis infection has been associated with bacterial meningitis, septic shock, arthritis, pneumonia, endocarditis, endophthalmitis, and spontaneous bacterial peritonitis (2,3). Most at risk are those who handle or eat undercooked pork, e.g., farm workers, butchers, and slaughterhouse workers (4). Most cases have been reported in Europe or Southeast Asia (2,3). Meningitis, first recognized in 1968 in Denmark (1), is the most common clinical manifestation of human infection with S. suis. A case of S. suis meningitis in a pig farmer was reported in the United States (5). Here, we describe another case in a 60-year-old man from San Francisco who had consumed raw pork while traveling in the Philippines. In June 2003, this man became ill with fever, diaphoresis, headache, nausea, and anorexia. He had just returned from a 7-month vacation in the Philippines. Three days after symptoms onset, his physician prescribed doxycycline. Symptoms continued and he was admitted to a local hospital 5 days later with a fever of 38.9°C, nuchal rigidity, headache, and general malaise. The patient described no recent contact with sick persons; past medical history was unremarkable. On physical examination, he was somnolent but fully oriented, with no focal findings on neurologic examination and only slight nuchal rigidity. He had a leukocyte count of 21,000/mm3, including 16,400/mm3 neutrophils. Cerebrospinal fluid (CSF) showed leukocyte count of 487/μL with 80% polymorphonuclear cells and 18% lymphocytes, and glucose and protein levels

Targeted ultrasonic contrast systems are designed to enhance the reflectivity of selected tissues in vivo [Lanza et al., Circulation 94, 3334 (1996)]. In particular, these agents hold promise for the minimally invasive diagnosis and treatment of a wide array of pathologies, most notably tumors, thromboses, and inflamed tissues. In the present study, acoustic microscopy was used to assess the efficacy of a novel, perfluorocarbon based contrast agent to enhance the inherent acoustic reflectivity of biological and synthetic substrates. Data from these experiments were used to postulate a simple model describing the observed enhancements. Frequency averaged reflectivity (30-55 MHz) was shown to increase 7.0 +/- 1.1 dB for nitrocellulose membranes with targeted contrast. Enhancements of 36.0 +/- 2.3 dB and 8.5 +/- 0.9 dB for plasma and whole blood clots, respectively, were measured between 20 and 35 MHz. A proposed acoustic transmission line model predicted the targeted contrast system would increase the acoustic reflectivity of the nitrocellulose membrane, whole blood clot, and fibrin plasma clot by 2.6, 8.0, and 31.8 dB, respectively. These predictions were in reasonable agreement with the experimental results of this paper. In conclusion, acoustic microscopy provides a rapid and sensitive approach for in vitro chracterization, development, and testing of mathematical models of targeted contrast systems. Given the current demand for targeted contrast systems for medical diagnostic and therapeutic use, the use of acoustic microscopy may provide a useful tool in the development of these agents.

A broadband ultrasonic measurement system has been utilized to characterize the concentration and frequency dependence of in vitro suspensions of Albunex® microspheres at concentrations ranging from 1.7×105 to 2.1×107 microspheres/mL and over a bandwidth of 1–16 MHz. The apparent backscattered power (not compensated for effects due to attenuation) was shown to increase with dose for lower concentrations of microspheres, but then to decrease rapidly with increasing concentration where attenuation effects become significant. Measurements of signal loss demonstrated that the attenuation grew exponentially with increasing concentration, so that a doubling of the number of microspheres led to a doubling of the value of the attenuation coefficient measured in dB/cm. This relationship was demonstrated over the entire system bandwidth. Compensation of the apparent backscattered power for the attenuation yielded the backscatter transfer function. This quantity was shown to be linearly proportional to concentration, so that a doubling of the number of microspheres led to a 3-dB increase in the backscatter transfer function. A broadband data reduction technique was used to further reduce the data to backscatter coefficient, an absolute parameter describing the intrinsic scattering efficiency of the Albunex® microsphere suspensions. The backscatter coefficient was shown to be linearly proportional to microsphere concentration at all concentrations investigated and over all the usable bandwidth. This suggests that, with appropriate compensation for attenuation and equipment parameters, perfusion or flow quantification techniques which assume a linear dependence of backscatter with contrast agent concentration should be applicable. The backscatter coefficient exhibits a rapid rise with frequency below 3 MHz, and appears to approach a frequency independent limit above 3 MHz. The relationships of the attenuation coefficient and backscatter transfer function to concentration were generally consistent with predictions from a simple scattering model. These relationships appear to be valid within the usable bandwidth of our measurement system for all concentrations investigated.

In this paper, we report the effects of inherent tissue anisotropy on the spectral properties of backscattered ultrasound when measured with a commercially-available imaging system. We insonified five specimens of bovine tendon immersed in a water tank and rotated in 10° increments while being imaged with a Hewlett-Packard Sonos 1500 system. The backscattered RF signals corresponding to each angle of insonification were digitized and the spectral characteristics of the backscattered ultrasound were determined. The mean anisotropy, defined as the average difference between values at perpendicular and parallel insonification, for band-limited estimates of backscattered power, centroid frequency, upper-band to lower-band power ratio, and upper-band to total-band power ratio were found to be 24.6 ± 1.1 dB, 142 ± 27 kHz, 32 ± 13%, and 22 ± 5%, respectively (mean ± SE). The magnitude of each of these backscatter spectral parameters was larger at perpendicular insonification compared with the corresponding values at parallel insonification, consistent with previous measurements of the inherent anisotropy of ultrasonic attenuation and backscatter in tissue.

The phase velocities in suspensions of Albunex, an ultrasound contrast agent, are determined using the technique of broadband phase spectroscopy. In these suspensions, phase velocities were measured as a function of Albunex concentration in Isoton II (buffered saline). The dispersion over the measurement bandwidth (1-15 MHz) grew with increasing Albunex concentration, exhibiting a 221-m/s change (17% increase) in phase velocity between 1.0 and 3.8 MHz at the highest concentration reported (32 microL Albunex/8 mL Isoton). The dispersion behavior of the Albunex suspensions is described using a model of encapsulated gas bubbles. The influence of the dispersion in Albunex on broadband pulses is discussed, as well as the potential impact this dispersion may have on the formation of ultrasonic images of the heart.

Diagnostic ultrasonic characterization of the heart is designed to assess the state of the myocardium with ultrasonic parameters that relate to structural or functional components of cardiac muscle. The potential cross fertilization of ideas and methods between those borne out of research in quantitative nondestructive evaluation of materials and those borne out of research in diagnostic medical applications in the area of ultrasonics represents an attractive goal. In this paper we describe our approach to tissue characterization of the heart based on quantitative ultrasonic imaging. We discuss methods of ultrasonic tissue characterization applied in laboratory and clinical investigations. Specific examples of the application of clinical quantitative tissue characterization include measurements of the hearts of patients with remote myocardial infarction, dilated cardiomyopathy, acute myocardial ischemia, and hearts of patients with diabetes. The role of anisotropy in quantitative tissue characterization a...

Tissue characterization would be facilitated by improved methods of calibration with which to make absolute measurements of the backscatter and attenuation of tissue or contrast agents. In this paper, the use of polystyrene microspheres is examined as a potential broadband in vitro calibration tool by employing an experimental system designed to characterize ultrasonic contrast agents. The frequency-dependent attenuation coefficient and backscatter coefficient were measured for a series of microsphere size distributions with a broadband ultrasound system using a custom-built specimen chamber and a novel suspension mixing technique. The measurements were used in a broadband reduction method to yield the backscatter coefficient. The range of ka spanned in this study is from ka=0.5 to ka=12. The broadband nature of the pulses permitted simultaneous measurement of different regimes of scattering of the microspheres (specifically, the transition region from Rayleigh to short-wavelength asymptotic scattering). ...

Broadband normalized signal loss of commercially available Albunex®, an ultrasonic contrast agent, was measured in vitro at concentrations approaching those which may be found in vivo for clinical doses. The measurements were made using a novel specimen chamber, careful material handling procedures, and a broadband square wave pulser system. Results were obtained over the full bandwidth of the experimental system (1 to 20 MHz) at concentrations up to 1.9×106 microspheres/mL. Further results were obtained over a partial bandwidth of the experimental system at concentrations up to 1.5×107 microspheres/mL. The frequency-dependent signal loss exhibited a peak for all concentrations investigated. In the meaningful bandwidth of the system, the signal loss (expressed in dB) was directly proportional to microsphere concentration. The experimental results for normalized signal loss were compared with predictions from a linear single-scattering model for encapsulated bubbles. The experimental data was used to estimate values for the two adjustable parameters in the model: microsphere shell elasticity (4200±1000 dyn/cm) and friction (0.0054±0.0015 dyn s/cm).

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.

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. >

The physical structures responsible for ultrasonic scattering from myocardial tissue have not yet been conclusively defined. It is hypothesized in this paper that the backscatter from myocardium is primarily due to inhomogeneities approximately the size of the myocytes. In particular, it is proposed that the acoustic contrast responsible for the scattering is that between the extracellular collagen network that surrounds each myocyte (or myocyte bundle) and the rest of the tissue (the myocytes' intracellular contents). To test this hypothesis, a simple elastic wave scattering model for myocardium was developed. An elementary scatterer is modeled as an ellipsoidal shell, having the material properties of wet collagen, imbedded in a host medium having the average properties of myocardium. The first Born approximation to elastic scattering is used to calculate the frequency-dependent scattering from a single scatterer. To scale up from a single scatterer to a distribution of scatterers, it is assumed that the power received at the transducer is simply the sum of the power scattered in the direction of the transducer by each individual scatterer located in the active volume of the beam (an independent-scatterer approximation). Calculations are restricted to the backscattering direction (pulse-echo), although the theory can accommodate pitch-catch scattering at all angles. With the aid of a computer program, the acoustic backscatter coefficient is calculated using the Born formalism and then measurement effects (frequency-dependent beam width and attenuation correction factors) are incorporated to arrive at calculated integrated (frequency-averaged) backscatter. Both the backscatter coefficient and integrated backscatter are calculated for angles of incidence that range from parallel to the long axis of the scatterer to perpendicular to this fiber direction. For the low MHz frequencies typically used in clinical echocardiography, the calculated absolute magnitude of the acoustic backscatter coefficient lies within a range from 0.0001 to 0.001 cm-1 sr-1. For selected fiber geometries, the anisotropy in integrated backscatter as the angle of incidence is varied with respect to the fiber orientation is about 10 dB. The predicted frequency dependence of the acoustic backscatter coefficient is calculated to be about f3.9 in the low MHz frequency range. These calculated results are reasonably consistent with published experimental measurements and provide a successful preliminary test of the hypothesis.

BACKGROUND Although 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 RESULTS We 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 high-resolution 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. CONCLUSIONS The 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.

Ultrasound-mediated delivery (USMD) of drugs and genes, especially with the enhancement of microbubble or nanoparticle agents, has been demonstrated in various settings for some years. Translation to pre-clinical disease models faces many challenges. This paper reports on the development and characteristics of a device for preclinical translational research in USMD. Feasibility of short-haripin RNA (shRNA) delivery in vivo using this device was demonstrated.

Ultrasound-mediated delivery (USMD) is an active research topic, as researchers develop applications for therapeutic ultrasound in addition to thermal ablation. In USMD, ultrasound is used in conjunction with microbubbles and drugs, nanoparticles, siRNA, pDNA, stem cells, etc., to facilitate their cellular delivery and uptake using pressure and temperature-mediated mechanisms to bring about a desired therapeutic effect. To investigate the potential of targeted USMD of nanoparticles, pDNA, and stem cells for cardiovascular and other applications, a general-purpose preclinical research tool, therapy imaging probe system (TIPS) was designed. It consists of a wideband annular array, a small-animal acoustic coupler, a motorized positioning system, integrated control software for ultrasound image-guided treatment planning and execution, and triggering electronics that allow ECG and respiration-gated ultrasound exposures. TIPS was then used to enhance delivery of nanoparticles into the murine myocardium and heart vessel walls to demonstrate the feasibility of the technology, pave the way for additional basic research in cardiovascular USMD, and begin to explore the requirements that USMD devices will have to meet to be useful in a clinical setting.

The integration of therapeutic interventions with diagnostic imaging has been recognized as one of the next technological developments that will have a major impact on medical treatments. Important advances in this field are based on a combination of progress in guiding and monitoring ultrasound energy, novel drug classes becoming available, the development of smart delivery vehicles, and more in depth understanding of the mechanisms of the cellular and molecular basis of diseases. Recent research demonstrates that both pressure sensitive and temperature sensitive delivery systems hold promise for local treatment. The use of ultrasound for the delivery of drugs has been demonstrated in particular the field of cardiology and oncology for a variety of therapeutics ranging from small drug molecules to biologics and nucleic acids.

Localized therapeutic effects of an experimental therapeutic agent on a mouse MC38 tumor model were evaluated using a combined ultrasound therapy and imaging probe system (TIPS). With ultrasound activated paclitaxel delivery, the tumor growth is substantially retarded for at least 3 days. The therapeutic effect of ultrasound is statistically significant (p=0.03) within one day after treatment but gradually decreases with time. Therefore, it is suggested that multiple treatments over a long period are required for cancer treatment. It is also demonstrated in this preclinical study that TIPS is an effective tool for imaging-guided, ultrasound-activated drug delivery.

A new set of multiple-pulse contrast modalities with combinations of power modulation (PM), pulse inversion (PI) and contrast resonance (CR) are proposed in order for contrast imaging sensitivity improvement. For the CR mode, an imaging pulse is ldquoembeddedrdquo in a long ldquoresonancerdquo pulse so that the dynamic ldquoresonantrdquo state of a contrast bubble encountered by the embedded imaging pulse is very different from the static state of the bubble for the imaging pulse alone. Numerical and experimental evaluations of the mode combinations indicate great potential for contrast imaging sensitivity improvement (without resolution degradation) over the standard PM or PI&PM mode.

Novel polymeric microcapsules, filled with a mixture of gas and oil, were produced and their potential as ultrasound contrast agent-based drug delivery system for lipophilic drugs was investigated. Microcapsules were synthesized that contained either no oil, were almost half-filled with oil, or were almost completely filled with oil. Mean number weighted diameters were between 1.22 and 1.31 μm. At a low MI (1 MHz, Place of 0.24 MPa), microcapsules typically compressed without cracking. At a high MI (1 MHz, Place of 0.51 MPa), microcapsules cracke, thereby releasing their content. Guidance and monitoring of therapy will be possible because the microcapsules were echogenic and stable at low MI. These microcapsules therefore have great potential as local drug delivery system for lipophilic drugs.

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.

Targeted acoustic contrast agents are designed to enhance the sensitivity and specificity of ultrasonic diagnoses. The authors have previously developed a ligand targeted ultrasonic contrast system which is a liquid, lipid encapsulated 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. The authors have recently proposed a simple acoustic transmission line model that treats the emulsion particles as a thin layer over the targeted surface. The acoustic reflectivity of the contrast layer is related to the longitudinal velocity and density of the perfluorocarbon. Improvement of the contrast effect may be achieved by using perfluorocarbons with slower longitudinal velocities and/or lower densities. The authors report the longitudinal velocities as a function of frequency of 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/sec (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. The authors' 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 the acoustic scattering.

A novel class of acoustically active microcapsules that partially contain drug-bearing oil with gas offers great potential for localized drug release. The acoustical activation threshold and rate of such microcapsules with different oil-to-gas ratios were measured in vitro. Drug loaded microcapsules were prepared from which the anti-cancer drug paclitaxel was delivered in murine tumors. For recovered mice each with two tumors on the left and right hind legs, the growth rate of the acoustically treated tumors was substantially reduced in comparison to that of the untreated tumor on the same mouse. These initial results showed that lipophilic drugs can be packaged in microcapsules and released locally using focused ultrasound.

Although infection with Hepatitis C Virus (HCV) and Human Immunodeficiency Virus (HIV) frequently co-exist, there has been little research to determine the effects of HIV/HCV co-infection on health-related quality of life (HRQOL). We performed a cross-sectional analysis of baseline data from 216 participants enrolled in a community based study of HIV-infected homeless and marginally housed individuals, using multivariable linear regression analysis to determine if co-infection with HCV was independently associated with lower short-form 36 (SF-36) questionnaire scores. We found that individuals with HCV had significantly lower mean SF-36 scores in the domains of physical functioning, bodily pain, social functioning and role limitation due to emotional health, and that HIV/HCV co-infection was independently associated with a lower physical component score but not a lower mental component score after controlling for numerous covariates. These results suggest that co-infection with HCV may have an adverse effect on HRQOL among homeless and marginally housed individuals with HIV.

Molecular imaging employs the use of site-targeted contrast agents that, once bound, indicate the expression of a molecular signal uniquely associated with a specific disease state. Ultrasonic contrast agents including microbubbles, nanoparticles, and liposomes have been successfully targeted towards angiogenesis associated with tumor growth; inflammation associated with ischemic events; formation of thrombi associated with heart attack and stroke; atherosclerosis; and many other pathologies. Ultrasound has the unique property of being able to interact with tissue while simultaneously visualizing anatomical features and function. This ability makes ultrasound a unique tool for both visualization of pathology and for the potential delivery of drugs to specific locations within the body.

Ultrasonically-triggered drug delivery may be ac- complished through the attachment of drug-enriched liposomes to microbubble contrast agent. Understanding of the dynamics and mechanisms of drug release is needed to ensure robust and reproducible delivery of pharmaceutical payloads. Fluorescent microbeads were attached to stabilized perfluorobutane mi- crobubbles with avidin-biotin bonding. The resulting bubble-bead complex is a model for a drug-loaded ultrasound contrast agent. In a preliminary experiment using fluorescent microscopy, the destruction of microbubbles and release of beads were observed during exposure to diagnostic ultrasound. Detailed observations were made using the Brandaris-128 camera operated at 13 million frames per second (Mfps) speed. The bead-bubble complexes were exposed to 2.0 MHz, 10-cycle ultrasonic pulses at up to 0.65 MPa peak negative amplitude. Release and displacement of fluorescent microbeads from their original position was observed during insonation. Detached beads track the fluid flow induced by the bubble compression and expansion, at velocities greater than 10 m/sec. The translational and oscillatory movement of detached beads diminishes as the distance between the bubble and the bead increases. The results did not establish the capability of ultrasound to propel microbeads to a distance greater than a few cell layers. In short, this study exposed the process of the ultrasound-mediated release and subsequent movement of shell- associated microbeads with high temporal resolution.

Thermal therapies such as radio frequency, heated saline, and high-intensity focused ultrasound ablations are often performed sub-optimally due to the inability to monitor the spatial and temporal distribution of delivered heat and the extent of tissue necrosis. Ultrasound-based temperature imaging has been recently proposed as a means to measure non-invasively the deposition of heat by tracking the echo arrival time shifts in the ultrasound backscatter caused by changes in speed of sound and tissue thermal expansion. However, the clinical applicability of these techniques has been hampered by the two-dimensional nature of traditional ultrasound imaging. In this study, we present methodology, results, and validation of a three-dimensional spatial and temporal ultrasound temperature estimation technique to track the evolution of heat deposition over a treatment volume. Ultrasonic backscattered radio-frequency data were captured using a commercial clinical imaging system to monitor heat generated by a complex, three-dimensional geometrical construct of resistive wires in an ultrasonic phantom. Thermocouples were embedded at various locations in the phantom to independently measure the temperature evolution during 90 seconds of heating (peak temperature increase of 8.5 degC) and the subsequent cool-down. X-ray computed tomography was used to ensure accurate placement of thermocouples. Good agreement was observed between the ultrasound derived temperature estimates and the invasive thermocouple measurements throughout the heating and cool-down phase. The mean difference between the ultrasound and thermocouple data was calculated to be 0.06 degC, RMS difference: 0.12 degC, and maximum difference: 0.24 degC. Time varying, three-dimensional ultrasonic measurements of temperature yielded parametric maps of thermal deposition within the phantom, and illustrated the complex three-dimensional geometry of the heat source. The results demonstrate potential for the applicability of the technique in monitoring and guidance of thermal therapy. To the best of the knowledge of the authors, this work represents the first demonstration of three-dimensional spatial and temporal ultrasound thermometry

Localized drug delivery should significantly improve the therapeutic efficacy for treatment of pathological lesions and concurrently reduce toxic exposure to the surrounding healthy tissue. A novel contrast agent, consisting of mono-dispersed gas-filled microcapsules, offers great potential for localized drug release. The destruction threshold and rate of various microcapsules and their dependence on the microcapsule size and shell thickness have been investigated via optical and acoustical measurements. The results presented in this paper will increase understanding of specific physical attributes needed to improve ultrasonically-activated drug delivery particles

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.

The ability to enhance specific molecular markers of pathology with ultrasound has been previously demonstrated by our group employing a nanoparticle contrast agent [Lanza et al., Invest. Radiol. 35, 227–234 (2000); Ultrasound Med. Biol. 23, 863–870 (1997)]. One of the advantages of this agent is very low echogenicity in the blood pool that allows increased contrast between the blood pool and the bound, site-targeted agent. We measured acoustic backscatter and attenuation coefficient as a function of the contrast agent concentration, ambient pressure, peak acoustic pressure, and as an effect of duty cycle and wave form shape. Measurements were performed while the nanoparticles were suspended in either whole porcine blood or plasma. The nanoparticles were only detectable when insonified within plasma devoid of red blood cells and were shown to exhibit backscatter levels more than 30 dB below the backscatter from whole blood. Attenuation of nanoparticles in whole porcine blood was not measurably different from that of whole blood alone over a range of concentrations up to eight times the maximum in vivo dose. The resulting data provide upper bounds on blood pool attenuation coefficient and backscatter and will be needed to more precisely define levels of molecular contrast enhancement that may be obtained in vivo.

Advances in molecular biology and cellular biochemistry are providing new opportunities for diagnostic medical imaging. Molecular imaging provides an opportunity to "see" the earliest biochemical signatures of disease in vivo analogous to microscopic detection of epitopes with immunohistochemistry techniques. Previously the province of nuclear medicine, today numerous, highly active research programs can be found for all clinically relevant imaging modalities. In many instances, these emerging site-directed imaging agents also incorporate therapeutic agents for drug delivery, allowing noninvasive confirmation and quantification of the targeted therapeutic dose. We have developed a novel multi-modal site-targeted contrast agent for sensitive and specific imaging of molecular epitopes and local therapy, which illustrates the key features of these emerging platform technologies. Targeted nanoparticles are applicable to ultrasound, nuclear, CT, and magnetic resonance imaging and have been used to detect a variety of epitopes expressed on angiogenic vessels, thrombus, and within vascular walls. Moreover, this particular targeted agent can deposit a therapeutic payload through a unique mechanism termed "contact-facilitated delivery". The combined benefits of molecular imaging and therapeutic systems are merging and will potentially alter many current clinical paradigms in the next decade.

The ability to enhance specifically molecular markers of pathology with ultrasound has been previously demonstrated by our group employing a nanoparticle contrast agent. One of the advantages of this agent is it's relative non-echogenicity in the blood pool that allows increased contrast-to-noise between the blood pool and the bound, site-targeted agent. We sought to define the contrast agent concentration and acoustic parameters necessary to detect contrast enhancement in the blood so that molecular contrast enhancement could be more precisely defined. This study addresses two potential mechanisms that have been proposed for backscatter from the nanoparticle contrast agent in the blood pool - concentration-related scattering and phase conversion from liquid to gaseous perfluorocarbon. The nanoparticles were produced by methods currently standard in our laboratory using perfluorooctyl bromide (PFOB: b.p. 142/spl deg/C) as the major component. Particle size was measured at 200/spl plusmn/30nm. Attenuation coefficient and backscatter of the agent were measured in whole porcine blood (hct 40%) and porcine plasma maintained at 37/spl deg/C. Specimens were insonified using a broadband, single element transducer (5 MHz, 2.54 cm diameter, 5.08 cm focal length). Acoustic pulses with usable bandwidth of 1.5 to 10 MHz, a repetition rate of 1kHz, and peak negative pressure of 3.9, 2.7, 1.5, and 0.8MPa (equivalent to M.I. of: 1.7, 1.2, 0.67, 0.36) were used to measure of attenuation coefficient and backscatter of nanoparticles at concentrations of 0.26, 0.51, 1.02, 2.04, 4.08/spl times/10/sup 14/ particles/mL while suspended in either whole porcine blood or porcine plasma. The attenuation coefficient was linear at all concentrations and power levels and shows no evidence of a resonant peak characteristic of liquid-to-gas phase conversion. The back-scatter coefficient in plasma increased with concentration. However, in blood, backscatter was only significantly different from baseline at 2.04/spl times/l0/sup 14/ particles/mL and above (8x the maximum anticipated dose). These data indicate that phase conversion of PFOB to gas is not the source of the contrast in molecular imaging with site targeted nanoparticles.

Myocardial edema is associated with impaired ventricular compliance and diastolic dysfunction. To determine the sensitivity of high-frequency ultrasound to myocardial edema, we employed a model in which edema was induced by immersion of heart tissue in isotonic saline. Formation of edema after rapid-freezing and thawing of myocardium was also evaluated. Rat hearts were arrested at end-diastole and insonified fresh within 15 minutes of excision (n=5) or after being frozen for 24 hours and thawed (n=4). A high-frequency, acoustic microscope was employed to perform ultrasonic measurements of attenuation, backscatter, speed of sound, and tissue thickness at baseline and hourly for 4 hours while immersed in solution. Parametric images of these ultrasonic indices were constructed to monitor changes in microscopic material properties due to edema and to allow identification of localized regions of interest. Fresh tissue demonstrated a greater propensity for the development of edema than frozen tissue. Integrated backscatter increased in both tissues while the magnitude and slope of attenuation decreased as edema evolved. We conclude that high-frequency ultrasound sensitively detects myocardial edema and may prove useful for monitoring and treatment of cardiac edematous disease states.

The authors have developed a nongaseous, ligand-targeted perfluorocarbon nanoparticle emulsion which can acoustically enhance the presence of molecular epitopes on tissue surfaces. In a two-part study, they first demonstrate how perfluorocarbons with varying phase velocities impact the acoustic reflectivity of plasma clots targeted with an anti-fibrin perfluorocarbon nanoparticle in vitro. The targeted perfluorocarbon contrast agents increased backscattered power from 18.2 to 23.4 dB with the greatest enhancement noted for formulations with low acoustic impedance. Porcine carotid arteries were injured by percutaneous balloon angioplasty and treated in situ with a tissue factor-targeted or control nanoparticulate immunoemulsion. Intravascular ultrasound images (30 MHz) of the arteries before and after application of contrast agent demonstrated the acoustic localization and enhancement of stretch-induced tissue factor expression within the carotid media, which was not apparent in the control vessels.

Specific ultrasonic tissue characterization parameters were correlated with the three-dimensional architecture and material properties (density, compressibility, size, and orientation) of aortic elastic elements at the microscopic level. The medial layer of 10 samples of normal canine aorta were insonified in vitro utilizing acoustic microscopy from 30 to 44 MHz. The following quantitative indexes exhibited substantial anisotropic elastic behavior in radial (R), circumferential (C), and longitudinal (L) directions: backscatter coefficient (R:0.9/spl plusmn/0.2; C:0.008/spl plusmn/0.0008; L:0.0077/spl plusmn/0.0008 sr/sup -1/ cm/sup -1/); frequency dependence of backscatter (R:3.3; C:1.4; L:1.5); attenuation coefficients (R:105/spl plusmn/22; L:135/spl plusmn/13; C:131/spl plusmn/14 dB/cm). Thus, the ultrasonic indexes are anisotropic: equivalent in the C and L directions, but markedly different in the R direction. These data are indicative of an aortic microstructure that interacts with ultrasonic waves as thin sheet-like elastic layers instead of independent elastin fibers. This specific sheet-like organization of elastin microfibers may function to limit shear injury to concentric aortic lamellae and prevent aortic dissection. The marked anisotropic behavior of normal aortas suggests that ultrasound may be useful for nondestructive characterization of vascular integrity.

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. The time-evolution of the crosslinking of proteins was monitored with high frequency ultrasound (30-50 MHz). Rats hearts (n=5 each group) were arrested at end-diastole and then insonified fresh within a few minutes of excision in a phosphate buffered solution. The solution was then replaced with a fixative and the tissue was insonified at 30-minute intervals thereafter for 24 hours. A parametric image of the magnitude and frequency dependence of ultrasonic attenuation in heart tissue was constructed at each time point to yield a time-lapse visualization of the process of protein crosslinking.

The goal of myocardial ultrasonic tissue characterization is to complement two-dimensional and Doppler echocardiography by providing information (such as assessment of regional viability based on localized values of backscatter) beyond that derived from an assessment of myocardial dimensions and motion. Quantitative backscatter imaging can be subdivided into three broad areas: (1) direct applications, in which specific pathologies are identified and monitored, (2) indirect applications, in which quantitative techniques designed for use in tissue characterization serve to expand the role of echocardiography, and (3) contributions to the understanding of cardiac structure and function.

The authors' goal is to apply the techniques of quantitative ultrasonic tissue characterization to intravascular ultrasound. They have developed a system to acquire radio frequency ultrasonic signals from a modified commercially available intravascular ultrasound scanner. This system incorporates a novel method of acoustic signal calibration. Experiments have been completed demonstrating the ability to make repeatable measurements of frequency-averaged backscattered power. In order to test and evaluate this system, intravascular ultrasound was used to characterize the enhancement of backscattered power from clot specifically targeted with an acoustic contrast agent recently developed in the authors' laboratory. The agent has the unique physical attribute of low inherent echogenicity in suspension, while attaining high acoustic reflectivity when bound to a biologic surface or to itself. Clots from canine plasma were formed in vitro on nitrocellulose membranes. Clots were incubated with either a clot-specific (n=2) or a clot-nonspecific (n=2) contrast emulsion. Specimens were imaged with a 30 MHz intravascular ultrasonic catheter. Radio frequency signals backscattered from the clots were acquired, gated, and their power spectra obtained by FFT. Frequency-averaged power backscattered from the clots exposed to clot-specific contrast exceeded that from clots exposed to clot-nonspecific contrast by 5.6/spl plusmn/0.7 dB (/spl plusmn/SD,p

Ventricular remodeling after myocardial infarction elicits structural and functional changes that can compromise global cardiac function. To determine whether age influences this process, we characterized remodeling after myocardial infarction in Fischer 344 rats that undergo natural age-related changes in heart structure and function that parallel those observed in human hearts. Young (3 month, n=7) and old (18 month, n=7) rats underwent myocardial infarction by coronary occlusion. Sham-infarct procedures were performed in both groups as controls. Twelve weeks after infarction, hearts were excised, fixed, and sliced into 2 mm thick cross-sections. Ultrasonic RF signals were acquired with a 50 MHz transducer and integrated backscatter was calculated from 30 to 50 MHz. Heart slices were then stained and their images digitized to permit histology with computer assisted planimetry. Infarct sizes were similar in young and old rats (12.1/spl plusmn/2.4% and 11.6/spl plusmn/5.4%; p=NS). Integrated backscatter from the infarct scar tissue in both the young and old rats were similar, (-41.4/spl plusmn/1.2 dB vs. -42.7/spl plusmn/1.1 dB, p=NS). Integrated backscatter from the remodeled but non-infarcted viable tissue was -48.4/spl plusmn/0.8 dB and -45.4/spl plusmn/1.0 dB in young and old rats, respectively, both statistically different from integrated backscatter from scar tissue (p

Progressive collagen cross-linking is a feature of many cardiovascular conditions (e.g., aging, diabetes, remodeling) and may contribute to impaired ventricular relaxation. However, it is not known whether cross-linking affects anisotropic tissue material properties more in one direction than another. We hypothesized that biological tissue comprising a highly ordered collagen matrix (porcine tendon) would manifest a directional dependence of the physical effects of cross-links that could be quantitatively delineated with ultrasound. Fresh porcine flexor tendons were sliced in 2-mm planes parallel (n=7) and perpendicular (n=7) to the tissue fiber axis for attenuation measurements (1-7 MHz). Tendons subjected to cross-linking treatment were immersed in 10% formalin, and control tendons in normal saline solution. Attenuation measurements were made immediately after immersion for both tendon groups at numerous intervals for 1 hr. The attenuation coefficient of the samples insonified perpendicular to the collagen fibers increased after formalin fixation over 1-hr period, while attenuation for parallel measurements decreased at selected frequencies during the first 20 min and subsequently increased modestly by 1 hr. Although the molecular mechanism responsible for the unexpected decrease in attenuation in the parallel direction as compared with the increase in the perpendicular direction is not clear, it may reflect rapid alterations in directionally dependent viscous and/or elastic components that contribute to material properties of well-ordered biological tissues.

The emerging field of "molecular imaging" refers to in vivo diagnosis of complex pathological processes by specific detection of unique molecular signatures of disease states either at the cellular, subcellular, or gene level. Identification of specific molecular epitopes causally associated with pathologies can be accomplished with the use of various novel targeted contrast agents, which permit conjunctive delivery and local deposition of therapeutic agents directly at the site of disease. Combining imaging with drug delivery provides verification of drug delivery and quantification of treatment, serving in effect as a new paradigm for rational drug dosing. Important potential clinical applications for targeted contrast agents include: (1) characterization of vulnerable plaques by detection of microscopic deposits of fibrin in plaque fissures prior to rupture. (2) early detection and treatment of solid tumors and metastases (colon, breast, prostate, etc.) by localization of angiogenesis. (3) detection of early atherosclerosis by assessment of epitopes related to macrophage trafficking (e.g., VCAM), and (4) diagnosis and treatment of inflammatory pathologies (infection, arthritis, myocarditis, transplant rejection, etc.) by delineation of epitopes that are markers for inflammatory cell recruitment (adhesion molecules, selectins, etc.) We provide a brief review of the field and discuss specific applications.

Molecular imaging with targeted contrast agents is emerging as an important technological advance for diagnostic ultrasound. In vivo, these agents must bind to and acoustically enhance their targets before the agents are cleared or destroyed. In this study, we present results using a system that was designed and constructed for visualization of the evolution of acoustic contrast enhancement. To evaluate the system, two targets were examined: avidin-complexed nitrocellulose membrane and human plasma clots. The time evolution of enhancement was visualized in 10-minute increments for one hour. A monotonic increase was observed in ultrasonic reflection enhancement from specially treated nitrocellulose membranes for targeted emulsions containing perfluorooctylbromide (1.3/spl plusmn/0.3 dB) and perfluorooctane (2.6/spl plusmn/0.5 dB) within the first 60 minutes of imaging. In comparison, the inherently non-echogenic plasma clots showed a substantial increase of 12.0/spl plusmn/0.9 dB when targeted with a perfluorooctane emulsion. This study demonstrates the concept of molecular imaging and provides the first quantifiable time evolution report of the binding of a site-targeted ultrasonic contrast agent.