Subsequent Ultrasound Vascular Targeting Therapy of Hepatocellular Carcinoma Improves the Treatment Efficacy.

The response of hepatocellular carcinoma (HCC) to anti-vascular ultrasound therapy (AVUS) can be affected by the inherent differences in tumor vascular structure, and the functionality of tumor vessels at the time of treatment. In this study, we evaluate the hypothesis that repeated subsequent AVUS therapies are a possible approach to overcome these factors and improve the therapeutic efficacy of AVUS. HCC was induced in 30 Wistar rats by oral ingestion of diethylnitrosamine (DEN) for 12 weeks. A total of 24 rats received treatment with low intensity, 2.8 MHz ultrasound with an intravenous injection of microbubbles. Treated rats were divided into three groups: single therapy group (ST), 2-days subsequent therapy group (2DST), and 7-days subsequent therapy group (7DST). A sham control group did not receive ultrasound therapy. Tumor perfusion was measured by quantitative contrast-enhanced ultrasound (CEUS) nonlinear and power-Doppler imaging. Tumors were harvested for histologic evaluation of ultrasound-induced vascular changes. ANOVA was used to compare the percent change of perfusion parameters between the four treatment arms. HCC tumors treated with 2DST showed the largest reduction in tumor perfusion, with 75.3% reduction on average for all perfusion parameters. The ST group showed an average decrease in perfusion of 54.3%. The difference between the two groups was significant p < 0.001. The 7DST group showed a reduction in tumor perfusion of 45.3%, which was significant compared to the 2DST group (p < 0.001) but not different from the ST group (p = 0.2). The use of subsequent targeted AVUS therapies applied shortly (two days) after the first treatment enhanced the anti-vascular effect of ultrasound. This gain, however, was lost for a longer interval (1 week) between the therapies, possibly due to tumor necrosis and loss of tumor viability. These findings suggest that complex interplay between neovascularization and tumor viability plays a critical role in treatment and, therefore, must be actively monitored following treatment by CEUS for optimizing sequential treatment.

Photoacoustic Imaging for Assessing Tissue Oxygenation Changes in Rat Hepatic Fibrosis.

Chronic liver inflammation progressively evokes fibrosis and cirrhosis resulting in compromised liver function, and often leading to cancer. Early diagnosis and staging of fibrosis is crucial because the five-year survival rate of early-stage liver cancer is high. This study investigates the progression of hepatic fibrosis induced in rats following ingestion of diethylnitrosamine (DEN). Changes in oxygen saturation and hemoglobin concentration resulting from chronic inflammation were assayed longitudinally during DEN ingestion by photoacoustic imaging (PAI). Accompanying liver tissue changes were monitored simultaneously by B-mode sonographic imaging. Oxygen saturation and hemoglobin levels in the liver increased over 5 weeks and peaked at 10 weeks before decreasing at 13 weeks of DEN ingestion. The oxygenation changes were accompanied by an increase in hepatic echogenicity and coarseness in the ultrasound image. Histology at 13 weeks confirmed the development of severe fibrosis and cirrhosis. The observed increase in PA signal representing enhanced blood oxygenation is likely an inflammatory physiological response to the dietary DEN insult that increases blood flow by the development of neovasculature to supply oxygen to a fibrotic liver during the progression of hepatic fibrosis. Assessment of oxygenation by PAI may play an important role in the future assessment of hepatic fibrosis.

Microbubble-enhanced ultrasound for the antivascular treatment and monitoring of hepatocellular carcinoma.

Background and Objective: Hepatocellular carcinoma (HCC) is the most common primary liver malignancy, and its current management relies heavily on locoregional therapy for curative therapy, bridge to transplant, and palliative therapy. Locoregional therapies include ablation and hepatic artery therapies such as embolization and radioembolization. In this study we evaluate the feasibility of using novel antivascular ultrasound (AVUS) as a noninvasive locoregional therapy to reduce perfusion in HCC lesions in a rat model and, monitor the effect with contrast-enhanced ultrasound imaging. Methods: HCC was induced in 36 Wistar rats by the ingestion of 0.01% diethylnitrosamine (DEN) for 12 weeks. Two therapy regimens of AVUS were evaluated. A primary regimen (n = 19) utilized 2-W/cm2, 3-MHz ultrasound (US) for 6 minutes insonation with 0.7 ml of microbubbles administered as an intravenous bolus. An alternate dose at half the primary intensity, sonication time, and contrast concentration was evaluated in 11 rats to assess the efficacy of a reduced dose. A control group (n = 6) received a sham therapy. Tumor perfusion was measured before and after AVUS with nonlinear contrast ultrasound (NLC) and power Doppler (PD). The quantitative perfusion measures included perfusion index (PI), peak enhancement (PE), time to peak (TTP), and perfusion area from NLC and PD scans. Total tumor area perfused during the scan was measured by a postprocessing algorithm called delta projection. Tumor histology was evaluated for signs of tissue injury and for vascular changes using CD31 immunohistochemistry. Results: DEN exposure induced autochthonous hepatocellular carcinoma lesions in all rats. Across all groups prior to therapy, there were no significant differences in the nonlinear contrast observations of peak enhancement and perfusion index. In the control group, there were no significant differences in any of the parameters after sham treatment. After the primary AVUS regimen, there were significant changes in all parameters (p ≤ 0.05) indicating substantial decreases in tumor perfusion. Peak enhancement in nonlinear contrast scans showed a 37.9% ± 10.1% decrease in tumor perfusion. Following reduced-dose AVUS, there were no significant changes in perfusion parameters, although there was a trend for the nonlinear contrast observations of peak enhancement and perfusion index to increase. Conclusion: This study translated low-intensity AVUS therapy into a realistic in vivo model of HCC and evaluated its effects on the tumor vasculature. The primary dose of AVUS tested resulted in significant vascular disruption and a corresponding reduction in tumor perfusion. A reduced dose of AVUS, on the other hand, was ineffective at disrupting perfusion but demonstrated the potential for enhancing tumor blood flow. Theranostic ultrasound, where acoustic energy and microbubbles are used to monitor the tumor neovasculature as well as disrupt the vasculature and treat lesions, could serve as a potent tool for delivering noninvasive, locoregional therapy for hepatocellular carcinoma.

B-mode ultrasound for the assessment of hepatic fibrosis: a quantitative multiparametric analysis for a radiomics approach.

Hepatic fibrosis and cirrhosis are a growing global health problem with increasing mortality rates. Early diagnosis and staging of hepatic fibrosis represent a major challenge. Currently liver biopsy is the gold standard for fibrosis assessment; however, biopsy requires an invasive procedure and is prone to sampling error and reader variability. In the current study we investigate using quantitative analysis of computer-extracted features of B-mode ultrasound as a non-invasive tool to characterize hepatic fibrosis. Twenty-two rats were administered diethylnitrosamine (DEN) orally for 12 weeks to induce hepatic fibrosis. Four control rats did not receive DEN. B-mode ultrasound scans sampling throughout the liver were acquired at baseline, 10, and 13 weeks. Computer extracted quantitative parameters representing brightness (echointensity, hepatorenal index) and variance (heterogeneity, anisotropy) of the liver were studied. DEN rats showed an increase in echointensity from 37.1 ± SD 7.8 to 53.5 ± 5.7 (10 w) to 57.5 ± 6.1 (13 w), while the control group remained unchanged at an average of 34.5 ± 4.5. The three other features studied increased similarly over time in the DEN group. Histologic analysis showed METAVIR fibrosis grades of F2-F4 in DEN rats and F0-F1 in controls. Increasing imaging parameters correlated with increasing METAVIR grades, and anisotropy showed the strongest correlation (ρ = 0.58). Sonographic parameters combined using multiparametric logistic regression were able to differentiate between clinically significant and insignificant fibrosis. Quantitative B-mode ultrasound imaging can be implemented in clinical settings as an accurate non-invasive tool for fibrosis assessment.

A review of low-intensity ultrasound for cancer therapy.

The literature describing the use of low-intensity ultrasound in four major areas of cancer therapy-sonodynamic therapy, ultrasound-mediated chemotherapy, ultrasound-mediated gene delivery and anti-vascular ultrasound therapy-was reviewed. Each technique consistently resulted in the death of cancer cells, and the bio-effects of ultrasound were attributed primarily to thermal actions and inertial cavitation. In each therapeutic modality, theranostic contrast agents composed of microbubbles played a role in both therapy and vascular imaging. The development of these agents is important as it establishes a therapeutic-diagnostic platform that can monitor the success of anti-cancer therapy. Little attention, however, has been given either to the direct assessment of the mechanisms underlying the observed bio-effects or to the viability of these therapies in naturally occurring cancers in larger mammals; if such investigations provided encouraging data, there could be prompt application of a therapy technique in the treatment of cancer patients.

Modeling of thermal effects in antivascular ultrasound therapy.

Antivascular ultrasound consisting of low-intensity sonication in the presence of circulating microbubbles of an ultrasound contrast agent has been demonstrated to disrupt blood flow in solid cancers. In this study a mathematical framework is described for the microbubble-induced heating that occurs during antivascular ultrasound. Biological tissues are modeled as a continuum of microbubble-filled vasculature, cells, and interstitial fluids with compressibility equal to the sum of the compressibility of each component. The mathematical simulations show that the absorption of ultrasound waves by viscous damping of the microbubble oscillations induced significant local heating of the tissue vasculature. The extent and the rate of temperature increase not only depends on the properties of the microbubbles and the sonication parameters but is also influenced markedly by the blood flow. Slow flow conditions lead to higher tissue temperatures due to a stronger interaction between microbubbles and ultrasound and reduced heat dissipation. Because tumors have slower blood flow than healthy tissue, the microbubble-induced ultrasound antivascular therapy is likely to affect cancerous tissue more extensively than healthy tissue, providing a way to selectively target the vasculature of cancers.

Antivascular ultrasound therapy extends survival of mice with implanted melanomas.

The goal of this murine investigation was to evaluate the effect of an antivascular ultrasound treatment on the growth of an implanted melanoma and the consequent survival rate. After the intravenous injection of 0.2 mL ultrasound contrast agent (Definity), therapy (n = 15) was performed on 1-mL tumors for 3 min with low-intensity continuous ultrasound (3 MHz; 2.4 +/- 0.1 W cm(-2) [I(SATA)]); control mice (n = 17) received a sham treatment. Mice were euthanized once the tumor had reached 3 mL, and then survival percentage vs. time curves were plotted. The median survival time (time for tumor to reach 3 mL) for the treated group was 23 d and for the control group was 18 d; the difference was statistically significant (p

Acute increases in murine tumor echogenicity after antivascular ultrasound therapy: a pilot preclinical study.

OBJECTIVE: This study was designed to determine whether the echogenicity of neoplastic tissues changed as a result of low-intensity insonation and whether such alterations were related to an anti-vascular effect. METHODS: In 21 mice, implanted melanomas were insonated at either 1, 2, or 3 MHz using low-intensity ultrasound (spatial-average temporal-average intensity, 2.1 W/cm(2)). B-mode (mean gray scale) and contrast-enhanced power Doppler (percentage area of flow) measurements were made on each tumor before and after therapy. RESULTS: There was an increase in the echogenicity of the tumors with the increase in the frequency of the therapy beam and an accompanying decrease in tumor vascularity. CONCLUSIONS: Although the mechanisms responsible for the echogenicity change are not fully understood, it appears that an increase in the tumor mean gray scale was, at least in part, related to tissue inhomogeneities formed after disruption of the tumor neovasculature.

Delta-projection imaging on contrast-enhanced ultrasound to quantify tumor microvasculature and perfusion.

RATIONALE AND OBJECTIVES: The aim of this study was to assess the Delta-projection image processing technique for visualizing tumor microvessels and for quantifying the area of tissue perfused by them on contrast-enhanced ultrasound images. MATERIALS AND METHODS: The Delta-projection algorithm was implemented to quantify perfusion by tracking the running maximum of the difference (Delta) between the contrast-enhanced ultrasound image sequence and a baseline image. Twenty-five mice with subcutaneous K1735 melanomas were first imaged with contrast-enhanced grayscale and then with minimum-exposure contrast-enhanced power Doppler (minexCPD) ultrasound. Delta-projection images were reconstructed from the grayscale images and then used to evaluate the evolution of tumor vascularity during the course of contrast enhancement. The extent of vascularity (ratio of the perfused area to the tumor area) for each tumor was determined quantitatively from Delta-projection images and compared to the extent of vascularity determined from contrast-enhanced power Doppler images. Delta-projection and minexCPD measurements were compared using linear regression analysis. RESULTS: Delta-projection was successfully performed in all 25 cases. The technique allowed the dynamic visualization of individual blood vessels as they filled in real time. Individual tumor blood vessels were distinctly visible during early image enhancement. Later, as an increasing number of blood vessels were filled with the contrast agent, clusters of vessels appeared as regions of perfusion, and the identification of individual vessels became difficult. Comparisons were made between the perfused area of tumors in Delta-projections and in minexCPD images. The Delta-projection perfusion measurements were correlated linearly with minexCPD. CONCLUSION: Delta-projection visualized tumor vessels and enabled the quantitative assessment of the tumor area perfused by the contrast agent.

The disruption of murine tumor neovasculature by low-intensity ultrasound-comparison between 1- and 3-MHz sonication frequencies.

RATIONALE AND OBJECTIVES: The goal was to determine whether the tumor vascular disrupting actions of low-intensity ultrasound were frequency dependent. MATERIALS AND METHODS: The effect of the frequency (1 MHz at 2.2 W/cm2 or 3 MHz at 2.4 W/cm2) of low-intensity ultrasound as a neovascular disrupting modality was investigated in 15 murine melanomas (K1735(22)) insonated for 3 minutes after the intravenous injection of a microbubble contrast agent (Definity). In contrast-enhanced power Doppler observations of each tumor (before and after treatment), measurements were made of the size of the area of the tumor that was perfused with blood containing the ultrasound contrast agent (percentage area of flow [PAF]), and the volume of contrast agent flowing through the unit volume of the tumor (color-weighted fractional area [CWFA]). During insonation of the tumor, the temperature was measured with a fine wire thermocouple in an additional eight mice. RESULTS: The antivascular action of low-intensity ultrasound was significantly enhanced (PAF by 64%; CWFA by 106%) when the tumor was treated with 3-MHz ultrasound rather than 1 MHz (analysis of variance: PAF, P=.02; CWFA, P=.04). The average rate of tumor temperature increase was 2.6+/-1.3 degrees C/min for 1 MHz and 5.0+/-1.7 degrees C/min for 3 MHz; these increases were significantly different (P=.04). CONCLUSIONS: Insonation of the tumor at a higher frequency amplified the heating of the neoplasm and led to greater disruption of the tumor vasculature; 3-MHz ultrasound was more efficacious than 1 MHz for antivascular cancer therapy.

The antivascular action of physiotherapy ultrasound on a murine tumor: role of a microbubble contrast agent.

This study investigated whether a microbubble-containing ultrasound contrast agent had a role in the antivascular action of physiotherapy ultrasound on tumor neovasculature. Ultrasound images (B-mode and contrast-enhanced power Doppler [0.02 mL Definity]) were made of 22 murine melanomas (K1735(22)). The tumor was insonated (I(SATA) = 1.7 W cm(-2), 1 MHz, continuous output) for 3 min and the power Doppler observations of the pre- and postinsonation tumor vascularities were analyzed. Significant reductions (p = 0.005 for analyses of color-weighted fractional area) in vascularity occurred when a contrast-enhanced power Doppler study occurred before insonation. Vascularity was unchanged in tumors without a pretherapy Doppler study. Histologic studies revealed tissue structural changes that correlated with the ultrasound findings. The underlying etiology of the interaction between the physiotherapy ultrasound beam, the microbubble-containing contrast agent and the tumor neovasculature is unknown. It was concluded that contrast agents play an important role in the antivascular effects induced by physiotherapy ultrasound.

Histopathological observations of the antivascular effects of physiotherapy ultrasound on a murine neoplasm.

This study evaluates the histopathological changes that follow insonation of a neoplasm with physiotherapy ultrasound. In 27 mice (C3HV/HeN strain), a subcutaneous melanoma (K1735(22)) was insonated with continuous physiotherapy ultrasound (1 MHz; spatial-average-temporal-average = 2.3 W cm(-2)). Analyses of contrast enhanced (0.1 mL Optison) power Doppler observations showed that insonation significantly (p < 0.05) increased the avascular area in the neoplasm. The predominant acute effect of insonating the neoplasm was an apparently irreparable dilation of the tumor capillaries with associated intercellular oedema; other immediate effects were haemorrage and increased intercellular fluid. Liquefactive necrosis of neoplastic cells was a delayed effect. There was a high correlation (R2 = 0.91) between the percent area affected on histologic examination and the percent increase in avascularity of the neoplasm in the Doppler study. In conclusion, physiotherapy ultrasound produced histologic changes in the tumor vasculature that were consistent with observations made by contrast enhanced power Doppler ultrasound.

The antivascular action of physiotherapy ultrasound on murine tumors.

This study was aimed at determining if physiotherapy ultrasound (US) affected the fragile and leaky angiogenic blood vessels in a tumor. In 22 C3HV/HeN mice, a subcutaneous melanoma (K1735(22)) was insonated (1, 2 or 3 min) with continuous 1-MHz low-intensity (spatial-average temporal-average = 2.28 W cm(-2)), physiotherapy US. Contrast-enhanced (0.1 mL Optison) power Doppler US observations were made and histogram analyses of the images were performed. Before insonation, all but 7% of the tumor was perfused. The avascular area in tumors receiving 3-min treatment increased to 82% (p < 0.001). A linear regression analysis showed that each min of insonation led to a 25% reduction in tumor vascularity; the antivascular activity persisted for 24 h. Histology demonstrated disruption of vascular walls and tumor cell death in areas of vascular congestion and thrombosis. Physiotherapy US particularly targeted the vascular structures, and the effects on tumor cells appeared to be secondary to the resultant ischemia.

Gadodiamide T1 relaxivity in brain tissue in vivo is lower than in saline.

In vivo measurements of gadodiamide (Gd-DTPA-BMA) T(1) relaxivity were performed at 4.7 T in injured and normal rat brains. Cerebral lesions were induced in nine rats by a localized freezing method. T(1) maps of the lesions were generated before and after injection of Gd-DTPA-BMA (0.1-0.6 mmol/kg). Samples of normal and necrotic brain were collected postmortem; the wet and dry weights were determined, and Gd content was measured by inductively coupled plasma mass spectroscopy. The in vivo relaxivity was determined by a linear fit of a plot of the change in relaxation rate following injection of the contrast agent as a function of Gd content. This analysis yielded a relaxivity in the injured brain of 2.8 sec(-1) mmol(-1) kg tissue water at 36 degrees C. The water weight fraction was 0.90 +/- SD 0.02 wt/wt in injured brain and 0.79 +/- 0.02 in normal brain. Relaxivity measurements were also performed on solutions of Gd-DTPA-BMA (0.0-0.6 mmol) and albumin (0-30% wt/wt) in normal saline at room and physiologic temperatures. The relaxivity in the albumin/saline increased with increasing solids content with values of 4.0-4.9 sec(-1) mmol(-1)kg at 21 degrees C and 3.4-4.5 sec(-1) mmol(-1) kg at 37 degrees C. The relaxivity of the tissues differed significantly from that of the saline solutions of comparable solids content, suggesting that the solids content of a tissue is not the only factor that determines in vivo relaxivity.

A novel method for analysis of TOMROP data.

PURPOSE: To develop an efficient method for extracting maps of the corrected T1 from images generated using the T One by Multiple Read Out Pulses (TOMROP) sequence. MATERIALS AND METHODS: An expression is developed for the true T1 in terms of the parameters from a three-parameter fit of the TOMROP data. Solutions of gadodiamide in normal saline with concentrations of 0.0, 0.06, 0.11, 0.23, 0.46, and 0.91 mM were prepared and T1 measurements were performed using both the inversion recovery (IR) and the TOMROP methods. The TOMROP data were analyzed using the proposed technique and the results compared to those from the IR measurements. RESULTS: The T1 estimates generated from the TOMROP data using the proposed method were consistent with the IR results. However, systematic errors were observed in the T1 estimates when the repetition time was not sufficient for full recovery of axial magnetization. Relaxation times determined using the proposed method were within 1% of the spectroscopiclly determined values for T1 values in the range of 0.28-2.8 seconds when a suitable delay was employed. CONCLUSION: The proposed method of analysis was found to yield accurate T1 estimates when the assumptions used in the analysis were not violated.

Correlation of ultrasonographic observations with anatomic features and radiography of the elbow joint in dogs.

OBJECTIVE: To define the ultrasonographic appearance of the elbow joint of dogs and to develop an ultrasonographic imaging protocol to reliably accomplish complete evaluation of that joint. ANIMALS: 11 clinically normal mixed-breed dogs. PROCEDURE: Ultrasonographic observations (by use of a 5 to 10 MHz linear array probe) were made of 22 elbow joints in cadaveric forelimbs from clinically normal dogs. Images in standard anatomic planes were recorded with a multi-image camera, on videotape, or onto a computer. The anatomic plane of the ultrasonographic beam and position of the ultrasonographic probe were also recorded. Dissection of each elbow joint was performed, and anatomic features were correlated with ultrasonographic images. RESULTS: Structures clearly identified ultrasonographically included the lateral and medial humeral epicondyles, the humeroradial and humeroulnar joints, anconeal process, medial coronoid process, hyaline cartilage covering the proximal articular surface of the radius, collateral ligaments, tendons of triceps brachii and supinator muscles, and the supinator tendon and sesamoid in the supinator cartilage. An ultrasonographic imaging protocol for examination of the elbow joint was developed. CONCLUSIONS AND CLINICAL RELEVANCE: Precise correlations were established between the gross anatomic appearance of the elbow joint and the ultrasonographic images of its component structures. The ultrasonographic imaging protocol will enable complete examination of all regions of the joint for detection of pathologic lesions.