Pulmonary Artery Proportional Pulse Pressure (PAPP) Index Identifies Patients With Improved Survival From the CardioMEMS Implantable Pulmonary Artery Pressure Monitor.

BACKGROUND: Pulmonary artery proportional pulse pressure (PAPP) was recently shown to have prognostic value in heart failure (HF) with reduced ejection fraction (HFrEF) and pulmonary hypertension. We tested the hypothesis that PAPP would be predictive of adverse outcomes in patients with implantable pulmonary artery pressure monitor (CardioMEMS™ HF System, St. Jude Medical [now Abbott], Atlanta, GA, USA). METHODS: Survival analysis with Cox proportional hazards regression was used to evaluate all-cause deaths and HF hospitalisation (HFH) in CHAMPION trial1 patients who received treatment with the CardioMEMS device based on the PAPP. RESULTS: Among 550 randomised patients, 274 had PAPP ≤ the median value of 0.583 while 276 had PAPP>0.583. Patients with PAPP≤0.583 (versus PAPP>0.583) had an increased risk of HFH (HR 1.40, 95% CI 1.16-1.68, p=0.0004) and experienced a significant 46% reduction in annualised risk of death with CardioMEMS treatment (HR 0.54, 95% CI 0.31-0.92) during 2-3 years of follow-up. This survival benefit was attributable to the treatment benefit in patients with HFrEF and PAPP≤0.583 (HR 0.50, 95% CI 0.28-0.90, p<0.05). Patients with PAPP>0.583 or HF with preserved EF (HFpEF) had no significant survival benefit with treatment (p>0.05). CONCLUSION: Lower PAPP in HFrEF patients with CardioMEMS constitutes a higher mortality risk status. More studies are needed to understand clinical applications of PAPP in implantable pulmonary artery pressure monitors.

Ultrasound Modulates the Splenic Neuroimmune Axis in Attenuating AKI.

We showed previously that prior exposure to a modified ultrasound regimen prevents kidney ischemia-reperfusion injury (IRI) likely via the splenic cholinergic anti-inflammatory pathway (CAP) and α7 nicotinic acetylcholine receptors (α7nAChR). However, it is unclear how ultrasound stimulates the splenic CAP. Further investigating the role of the spleen in ischemic injury, we found that prior splenectomy (-7d) or chemical sympathectomy of the spleen with 6-hydroxydopamine (6OHDA; -14d) exacerbated injury after subthreshold (24-minute ischemia) IRI. 6-OHDA-induced splenic denervation also prevented ultrasound-induced protection of kidneys from moderate (26-minute ischemia) IRI. Ultrasound-induced protection required hematopoietic but not parenchymal α7nAChRs, as shown by experiments in bone marrow chimeras generated with wild-type and α7nAChR(-/-) mice. Ultrasound protection was associated with reduced expression of circulating and kidney-derived cytokines. However, splenocytes isolated from mice 24 hours after ultrasound treatment released more IL-6 ex vivo in response to LPS than splenocytes from sham mice. Adoptive transfer of splenocytes from ultrasound-treated (but not sham) mice to naïve mice was sufficient to protect kidneys of recipient mice from IRI. Ultrasound treatment 24 hours before cecal ligation puncture-induced sepsis was effective in reducing plasma creatinine in this model of AKI. Thus, splenocytes of ultrasound-treated mice are capable of modulating IRI in vivo, supporting our ongoing hypothesis that a modified ultrasound regimen has therapeutic potential for AKI and other inflammatory conditions.

Oscillatory Dynamics and In Vivo Photoacoustic Imaging Performance of Plasmonic Nanoparticle-Coated Microbubbles.

Microbubbles bearing plasmonic nanoparticles on their surface provide contrast enhancement for both photoacoustic and ultrasound imaging. In this work, the responses of microbubbles with surface-bound gold nanorods-termed AuMBs-to nanosecond pulsed laser excitation are studied using high-speed microscopy, photoacoustic imaging, and numerical modeling. In response to laser fluences below 5 mJ cm(-2) , AuMBs produce weak photoacoustic emissions and exhibit negligible microbubble wall motion. However, in reponse to fluences above 5 mJ cm(-2) , AuMBs undergo dramatically increased thermal expansion and emit nonlinear photoacoustic waves of over 10-fold greater amplitude than would be expected from freely dispersed gold nanorods. Numerical modeling suggests that AuMB photoacoustic responses to low laser fluences result from conductive heat transfer from the surface-bound nanorods to the microbubble gas core, whereas at higher fluences, explosive boiling may occur at the nanorod surface, producing vapor nanobubbles that contribute to rapid AuMB expansion. The results of this study indicate that AuMBs are capable of producing acoustic emissions of significantly higher amplitude than those produced by conventional sources of photoacoustic contrast. In vivo imaging performance of AuMBs in a murine kidney model suggests that AuMBs may be an effective alternative to existing contrast agents for noninvasive photoacoustic and ultrasound imaging applications.

Simultaneous photoacoustic microscopy of microvascular anatomy, oxygen saturation, and blood flow.

Capitalizing on the optical absorption of hemoglobin, photoacoustic microscopy (PAM) is uniquely capable of anatomical and functional characterization of the intact microcirculation in vivo. However, PAM of the metabolic rate of oxygen (MRO2) at the microscopic level remains an unmet challenge, mainly due to the inability to simultaneously quantify microvascular diameter, oxygen saturation of hemoglobin (sO2), and blood flow at the same spatial scale. To fill this technical gap, we have developed a multi-parametric PAM platform. By analyzing both the sO2-encoded spectral dependence and the flow-induced temporal decorrelation of photoacoustic signals generated by the raster-scanned mouse ear vasculature, we demonstrated-for the first time-simultaneous wide-field PAM of all three parameters down to the capillary level in vivo.

In vivo imaging of microfluidic-produced microbubbles.

Microfluidics-based production of stable microbubbles for ultrasound contrast enhancement or drug/gene delivery allows for precise control over microbubble diameter but at the cost of a low production rate. In situ microfluidic production of microbubbles directly in the vasculature may eliminate the necessity for high microbubble production rates, long stability, or small diameters. Towards this goal, we investigated whether microfluidic-produced microbubbles directly administered into a mouse tail vein could provide sufficient ultrasound contrast. Microbubbles composed of nitrogen gas and stabilized with 3 % bovine serum albumin and 10 % dextrose were injected for 10 seconds into wild type C57BL/6 mice, via a tail-vein catheter. Short-axis images of the right and left ventricle were acquired at 12.5 MHz and image intensity over time was analyzed. Microbubbles were produced on the order of 10(5) microbubbles/s and were observed in both the right and left ventricles. The median rise time, duration, and decay time within the right ventricle were 2.9, 21.3, and 14.3 s, respectively. All mice survived the procedure with no observable respiratory or heart rate distress despite microbubble diameters as large as 19 µm.

A Quantitative Method for the Evaluation of Deep Vein Thrombosis in a Murine Model Using Three-Dimensional Ultrasound Imaging.

Deep vein thrombosis (DVT) is a life-threatening condition that can lead to its sequelae pulmonary embolism (PE) or post-thrombotic syndrome (PTS). Murine models of DVT are frequently used in early-stage disease research and to assess potential therapies. This creates the need for the reliable and easy quantification of blood clots. In this paper, we present a novel high-frequency 3D ultrasound approach for the quantitative evaluation of the volume of DVT in an in vitro model and an in vivo murine model. The proposed method involves the use of a high-resolution ultrasound acquisition system and semiautomatic segmentation of the clot. The measured 3D volume of blood clots was validated to be correlated with in vitro blood clot weights with an R2 of 0.89. Additionally, the method was confirmed with an R2 of 0.91 in the in vivo mouse model with a cylindrical volume from macroscopic measurement. We anticipate that the proposed method will be useful in pharmacological or therapeutic studies in murine models of DVT.

Shear forces from flow are responsible for a distinct statistical signature of adherent microbubbles in large vessels.

Real-time ultrasound-based targeted molecular imaging in large blood vessels holds promise for early detection and diagnosis of stroke risk by identifying early markers for atherosclerosis prior to plaque formation. Singular spectrum-based targeted molecular (SiSTM) imaging is a recently proposed method that uses changes in statistical dimensionality-quantified by a normalized singular spectrum area (NSSA)-to image receptor-ligand-bound adherent microbubbles. However, the precise physical mechanism responsible for the distinct statistical signature was previously unknown. In this study, in vitro flow phantom experiments were performed to elucidate the physical mechanism in large blood vessel environments. In the absence of flow, an increase in the NSSA of adherent microbubbles with respect to tissue was not observed with increased microbubble concentration or pulse length (p > .23; n  =  5) but was observed with increased flow rate (p < .01; n  =  10). When observing the dynamics of the adherent microbubble statistics, a good correlation was observed between the NSSA and the derivative of image intensity (R2 > .97). In addition, a monotonic relationship between the NSSA and decorrelation was demonstrated. These findings confirm the hypothesis that the statistical signature of adherent microbubbles is derived from frame-to-frame decorrelation, which is induced by flow shear forces.

In Vivo Validation of Modulated Acoustic Radiation Force-Based Imaging in Murine Model of Abdominal Aortic Aneurysm Using VEGFR-2-Targeted Microbubbles.

OBJECTIVES: The objective of this study is to validate the modulated acoustic radiation force (mARF)-based imaging method in the detection of abdominal aortic aneurysm (AAA) in murine models using vascular endothelial growth factor receptor 2 (VEGFR-2)-targeted microbubbles (MBs). MATERIALS AND METHODS: The mouse AAA model was prepared using the subcutaneous angiotensin II (Ang II) infusion combined with the ß-aminopropionitrile monofumarate solution dissolved in drinking water. The ultrasound imaging session was performed at 7 days, 14 days, 21 days, and 28 days after the osmotic pump implantation. For each imaging session, 10 C57BL/6 mice were implanted with Ang II-filled osmotic pumps, and 5 C57BL/6 mice received saline infusion only as the control group. Biotinylated lipid MBs conjugated to either anti-mouse VEGFR-2 antibody (targeted MBs) or isotype control antibody (control MBs) were prepared before each imaging session and were injected into mice via tail vein catheter. Two separate transducers were colocalized to image the AAA and apply ARF to translate MBs simultaneously. After each imaging session, tissue was harvested and the aortas were used for VEGFR-2 immunostaining analysis. From the collected ultrasound image data, the signal magnitude response of the adherent targeted MBs was analyzed, and a parameter, residual-to-saturation ratio ( Rres - sat ), was defined to measure the enhancement in the adherent targeted MBs signal after the cessation of ARF compared with the initial signal intensity. Statistical analysis was performed with the Welch t test and analysis of variance test. RESULTS: The Rres - sat of abdominal aortic segments from Ang II-challenged mice was significantly higher compared with that in the saline-infused control group ( P < 0.001) at all 4 time points after osmotic pump implantation (1 week to 4 weeks). In control mice, the Rres - sat values were 2.13%, 1.85%, 3.26%, and 4.85% at 1, 2, 3, and 4 weeks postimplantation, respectively. In stark contrast, the Rres - sat values for the mice with Ang II-induced AAA lesions were 9.20%, 20.6%, 22.7%, and 31.8%, respectively. It is worth noting that there was a significant difference between the Rres - sat for Ang II-infused mice at all 4 time points ( P < 0.005), a finding not present in the saline-infused mice. Immunostaining results revealed the VEGFR-2 expression was increased in the abdominal aortic segments of Ang II-infused mice compared with the control group. CONCLUSIONS: The mARF-based imaging technique was validated in vivo using a murine model of AAA and VEGFR-2-targeted MBs. Results in this study indicated that the mARF-based imaging technique has the ability to detect and assess AAA growth at early stages based on the signal intensity of adherent targeted MBs, which is correlated with the expression level of the desired molecular biomarker. The results may suggest, in very long term, a pathway toward eventual clinical implementation for an ultrasound molecular imaging-based approach to AAA risk assessment in asymptomatic patients.

Targeted PERK inhibition with biomimetic nanoclusters confers preventative and interventional benefits to elastase-induced abdominal aortic aneurysms.

Abdominal aortic aneurysm (AAA) is a progressive aortic dilatation, causing ∼80% mortality upon rupture. Currently, there is no approved drug therapy for AAA. Surgical repairs are invasive and risky and thus not recommended to patients with small AAAs which, however, account for ∼90% of the newly diagnosed cases. It is therefore a compelling unmet clinical need to discover effective non-invasive strategies to prevent or slow down AAA progression. We contend that the first AAA drug therapy will only arise through discoveries of both effective drug targets and innovative delivery methods. There is substantial evidence that degenerative smooth muscle cells (SMCs) orchestrate AAA pathogenesis and progression. In this study, we made an exciting finding that PERK, the endoplasmic reticulum (ER) stress Protein Kinase R-like ER Kinase, is a potent driver of SMC degeneration and hence a potential therapeutic target. Indeed, local knockdown of PERK in elastase-challenged aorta significantly attenuated AAA lesions in vivo. In parallel, we also conceived a biomimetic nanocluster (NC) design uniquely tailored to AAA-targeting drug delivery. This NC demonstrated excellent AAA homing via a platelet-derived biomembrane coating; and when loaded with a selective PERK inhibitor (PERKi, GSK2656157), the NC therapy conferred remarkable benefits in both preventing aneurysm development and halting the progression of pre-existing aneurysmal lesions in two distinct rodent models of AAA. In summary, our current study not only establishes a new intervention target for mitigating SMC degeneration and aneurysmal pathogenesis, but also provides a powerful tool to facilitate the development of effective drug therapy of AAA.

Adeno-associated virus serotype 9 administered systemically after reperfusion preferentially targets cardiomyocytes in the infarct border zone with pharmacodynamics suitable for the attenuation of left ventricular remodeling.

BACKGROUND: Adeno-associated virus serotype 9 (AAV9) vectors provide efficient and uniform gene expression to normal myocardium following systemic administration, with kinetics that approach steady-state within 2-3 weeks. However, as a result of the delayed onset of gene expression, AAV vectors have not previously been administered intravenously after reperfusion for post-infarct gene therapy applications. The present study evaluated the therapeutic potential of post-myocardial infarction gene delivery using intravenous AAV9. METHODS: AAV9 vectors expressing firefly luciferase, enhanced green fluorescent protein (eGFP) or extracellular superoxide dismutase genes from the cardiac troponin-T (cTnT) promoter (AcTnTLuc, AcTnTeGFP, AcTnTEcSOD) were employed. AcTnTLuc was administered intravenously at 10 min and at 1, 2 and 3 days post-ischemia/reperfusion (IR), and the kinetics of luciferase expression were assessed with bioluminescence imaging. AcTnTeGFP was used to evaluate the distribution of eGFP expression. High-resolution echocardiography was used to evaluate the effects of AcTnTEcSOD on left ventricular (LV) remodeling when injected 10 min post-IR. RESULTS: Compared to sham animals, luciferase expression at 2 days after vector administration was elevated by four-, 24-, 210- and 213-fold in groups injected at 10 min, 1 day, 2 days and 3 days post-IR, respectively. The expression of cTnT-driven eGFP was strongest in cardiomyocytes bordering the infarct zone. In the efficacy study of EcSOD, post-infarct LV end-systolic and end-diastolic volumes at days 14 and 28 were significantly smaller in the EcSOD group compared to the control. CONCLUSIONS: Systemic administration of AAV9 vectors after IR both elevates and accelerates gene expression that preferentially targets cardiomyocytes in the border zone with pharmacodynamics suitable for the attenuation of LV remodeling.

Focused ultrasound-mediated drug delivery from microbubbles reduces drug dose necessary for therapeutic effect on neointima formation--brief report.

OBJECTIVE: We hypothesized that (1) neointima formation in a rat carotid balloon injury model could be reduced in vivo following targeted ultrasound delivery of rapamycin microbubbles (RMBs), and (2) the addition of dual-mode ultrasound decreases the total amount of drug needed to reduce neointima formation. METHODS AND RESULTS: Balloon injury was performed in rat carotids to induce neointima formation. High or low doses of RMBs were injected intravenously and ruptured at the site of injury with ultrasound. Compared with nontreated injured arteries, neointima formation was reduced by 0% and 35.9% with 10(8) RMBs and by 28.7% and 34.9% in arteries treated with 10(9) RMBs with and without ultrasound, respectively. CONCLUSIONS: Without ultrasound, 10-fold higher concentrations of RMBs were needed to reduce neointima formation by at least 28%, whereas 10(8) RMBs combined with ultrasound were sufficient to achieve the same therapeutic effect, demonstrating that this technology may have promise for localized potent drug therapy.

Recovery of Blood Flow From Undersampled Photoacoustic Microscopy Data Using Sparse Modeling.

Photoacoustic microscopy (PAM) leverages the optical absorption contrast of blood hemoglobin for high-resolution, multi-parametric imaging of the microvasculature in vivo. However, to quantify the blood flow speed, dense spatial sampling is required to assess blood flow-induced loss of correlation of sequentially acquired A-line signals, resulting in increased laser pulse repetition rate and consequently optical fluence. To address this issue, we have developed a sparse modeling approach for blood flow quantification based on downsampled PAM data. Evaluation of its performance both in vitro and in vivo shows that this sparse modeling method can accurately recover the substantially downsampled data (up to 8 times) for correlation-based blood flow analysis, with a relative error of 12.7 ± 6.1 % across 10 datasets in vitro and 12.7 ± 12.1 % in vivo for data downsampled 8 times. Reconstruction with the proposed method is on par with recovery using compressive sensing, which exhibits an error of 12.0 ± 7.9 % in vitro and 33.86 ± 26.18 % in vivo for data downsampled 8 times. Both methods outperform bicubic interpolation, which shows an error of 15.95 ± 9.85 % in vitro and 110.7 ± 87.1 % in vivo for data downsampled 8 times.

Contrast-Enhanced Ultrasound Reveals Partial Perfusion Recovery After Hindlimb Ischemia as Opposed to Full Recovery by Laser Doppler Perfusion Imaging.

Mouse models are critical in developing new therapeutic approaches to treat peripheral arterial disease (PAD). Despite decades of research and numerous clinical trials, the efficacy of available therapies is limited. This may suggest shortcomings in our current animal models and/or methods of assessment. We evaluated perfusion measurement methods in a mouse model of PAD by comparing laser Doppler perfusion imaging (LDPI, the most common technique), contrast-enhanced ultrasound (CEUS, an emerging technique) and fluorescent microspheres (conventional standard). Mice undergoing a femoral artery ligation were assessed by LDPI and CEUS at baseline and 1, 4, 7, 14, 28, 60, 90 and 150 d post-surgery to evaluate perfusion recovery in the ischemic hindlimb. Fourteen days after surgery, additional mice were measured with fluorescent microspheres, LDPI, and CEUS. LDPI and CEUS resulted in broadly similar trends of perfusion recovery until 7 d post-surgery. However, by day 14, LDPI indicated full recovery of perfusion, whereas CEUS indicated ∼50% recovery, which failed to improve even after 5 mo. In agreement with the CEUS results, fluorescent microspheres at day 14 post-surgery confirmed that perfusion recovery was incomplete. Histopathology and photoacoustic microscopy provided further evidence of sustained vascular abnormalities.

Real-time technique for improving molecular imaging and guiding drug delivery in large blood vessels: in vitro and ex vivo results.

Ultrasound-based molecular imaging employs targeted microbubbles to image vascular pathology. This approach also has the potential to monitor molecularly targeted microbubble-based drug delivery. We present an image-guided drug delivery technique that uses multiple pulses to translate, image, and cavitate microbubbles in real time. This technique can be applied to both imaging of pathology in large arteries (sizes and flow comparable to those in humans) and guiding localized drug delivery in blood vessels. The microbubble translation (or pushing) efficacy of this technique was compared in a variety of flow media: saline, viscous saline (4 cp), and bovine blood. It was observed that the performance of this approach was marginally better (by 6, 4, and 2 dB) in viscous saline than in bovine blood with varying levels of hematocrit (40%, 30%, and 10%). The drug delivery efficacy of this technique was evaluated by in vitro and ex vivo experiments. High-intensity pulses mediated fluorophore (DiI) deposition on endothelial cells (in vitro) without causing cell destruction. Ex vivo fluorophore delivery experiments conducted on swine carotids of 2 and 5 mm cross-section diameter demonstrated a high degree of correspondence in spatial localization of the fluorophore delivery between the ultrasound and composite fluorescence microscopy images of the arterial cross sections.

Closed-Loop Low-Rank Echocardiographic Artifact Removal.

Echocardiographic image sequences are frequently corrupted by quasi-static artifacts ("clutter") superimposed on the moving myocardium. Conventionally, localized blind source separation methods exploiting local correlation in the clutter have proven effective in the suppression of these artifacts. These methods use the spectral characteristics to distinguish the clutter from tissue and background noise and are applied exhaustively over the data set. The exhaustive application results in high computational complexity and a loss of useful tissue signal. In this article, we develop a closed-loop algorithm in which the clutter is first detected using an adaptively determined weighting function and then removed using low-rank estimation methods. We show that our method is adaptable to different low-rank estimators, by presenting two such estimators: sparse coding in the principal component domain and nuclear norm minimization. We compare the performance of our proposed method (CLEAR) with two methods: singular value filtering (SVF) and morphological component analysis (MCA). The performance was quantified in silico by measuring the error with respect to a known "ground truth" data set with no clutter for different combinations of moving clutter and tissue. Our method retains more tissue with a lower error of 3.88 ± 0.093 dB (sparse coding) and 3.47 ± 0.78 (nuclear norm) compared with the benchmark methods 8.5 ± 0.7 dB (SVF) and 9.3 ± 0.5 dB (MCA) particularly in instances where the rate of tissue motion and artifact motion is small (≤0.25 periods of center frequency per frame) while producing comparable clutter reduction performance. CLEAR was also validated in vivo by quantifying the tracking error over the cardiac cycle on five mouse heart data sets with synthetic clutter. CLEAR reduced the error by approximately 50%, compared with 25% for the SVF.

Dynamic Filtering of Adherent and Non-adherent Microbubble Signals Using Singular Value Thresholding and Normalized Singular Spectrum Area Techniques.

Ultrasound molecular imaging techniques rely on the separation and identification of three types of signals: static tissue, adherent microbubbles and non-adherent microbubbles. In this study, the image filtering techniques of singular value thresholding (SVT) and normalized singular spectrum area (NSSA) were combined to isolate and identify vascular endothelial growth factor receptor 2-targeted microbubbles in a mouse hindlimb tumor model (n = 24). By use of a Verasonics Vantage 256 imaging system with an L12-5 transducer, a custom-programmed pulse inversion sequence employing synthetic aperture virtual source element imaging was used to collect contrast images of mouse tumors perfused with microbubbles. SVT was used to suppress static tissue signals by 9.6 dB while retaining adherent and non-adherent microbubble signals. NSSA was used to classify microbubble signals as adherent or non-adherent with high accuracy (receiver operating characteristic area under the curve [ROC AUC] = 0.97), matching the classification performance of differential targeted enhancement. The combined SVT + NSSA filtering method also outperformed differential targeted enhancement in differentiating MB signals from all other signals (ROC AUC = 0.89) without necessitating destruction of the contrast agent. The results from this study indicate that SVT and NSSA can be used to automatically segment and classify contrast signals. This filtering method with potential real-time capability could be used in future diagnostic settings to improve workflow and speed the clinical uptake of ultrasound molecular imaging techniques.

Quantitative analysis of in-vivo microbubble distribution in the human brain.

Microbubbles (MB) are widely used as contrast agents to perform contrast-enhanced ultrasound (CEUS) imaging and as acoustic amplifiers of mechanical bioeffects incited by therapeutic-level ultrasound. The distribution of MBs in the brain is not yet fully understood, thereby limiting intra-operative CEUS guidance or MB-based FUS treatments. In this paper we describe a robust platform for quantification of MB distribution in the human brain, allowing to quantitatively discriminate between tumoral and normal brain tissues and we provide new information regarding real-time cerebral MBs distribution. Intraoperative CEUS imaging was performed during surgical tumor resection using an ultrasound machine (MyLab Twice, Esaote, Italy) equipped with a multifrequency (3-11 MHz) linear array probe (LA332) and a specific low mechanical index (MI < 0.4) CEUS algorithm (CnTi, Esaote, Italy; section thickness, 0.245 cm) for non-destructive continuous MBs imaging. CEUS acquisition is started by enabling the CnTI PEN-M algorithm automatically setting the MI at 0.4 with a center frequency of 2.94 MHz-10 Hz frame rate at 80 mm-allowing for continuous non-destructive MBs imaging. 19 ultrasound image sets of adequate length were selected and retrospectively analyzed using a custom image processing software for quantitative analysis of echo power. Regions of interest (ROIs) were drawn on key structures (artery-tumor-white matter) by a blinded neurosurgeon, following which peak enhancement and time intensity curves (TICs) were quantified. CEUS images revealed clear qualitative differences in MB distribution: arteries showed the earliest and highest enhancement among all structures, followed by tumor and white matter regions, respectively. The custom software built for quantitative analysis effectively captured these differences. Quantified peak intensities showed regions containing artery, tumor or white matter structures having an average MB intensity of 0.584, 0.436 and 0.175 units, respectively. Moreover, the normalized area under TICs revealed the time of flight for MB to be significantly lower in brain tissue as compared with tumor tissue. Significant heterogeneities in TICs were also observed within different regions of the same brain lesion. In this study, we provide the most comprehensive strategy for accurate quantitative analysis of MBs distribution in the human brain by means of CEUS intraoperative imaging. Furthermore our results demonstrate that CEUS imaging quantitative analysis enables discernment between different types of brain tumors as well as regions and structures within the brain. Similar considerations will be important for the planning and implementation of MB-based imaging or treatments in the future.

A Targeted Molecular Localization Imaging Method Applied to Tumor Microvasculature.

OBJECTIVES: Ultrasound contrast agents, consisting of gas-filled microbubbles (MBs), have been imaged using several techniques that include ultrasound localization microscopy and targeted molecular imaging. Each of these techniques aims to provide indicators of the disease state but has traditionally been performed independently without co-localization of molecular markers and super-resolved vessels. In this article, we present a new imaging technology: a targeted molecular localization (TML) approach, which uses a single imaging sequence and reconstruction approach to co-localize super-resolved vasculature with molecular imaging signature to provide simultaneous anatomic and biological information for potential multiscale disease evaluation. MATERIALS AND METHODS: The feasibility of the proposed TML technique was validated in a murine hindlimb tumor model. Targeted molecular localization imaging was performed on 3 groups, which included control tissue (leg), tumor tissue, and tumor tissue after sunitinib an-tivascular treatment. Quantitative measures for vascular index (VI) and molecular index (MITML) were calculated from the microvasculature and TML images, respectively. In addition to these conventional metrics, a new metric unique to the TML technique, reporting the ratio of targeted molecular index to vessel surface, was assessed. RESULTS: The quantitative resolution results of the TML approach showed resolved resolution of the microvasculature down to 28.8 µm. Vascular index increased in tumors with and without sunitinib compared with the control leg, but the trend was not statistically significant. A decrease in MITML was observed for the tumor after treatment (P < 0.0005) and for the control leg (P < 0.005) compared with the tumor before treatment. Statistical differences in the ratio of molecular index to vessel surface were found between all groups: the control leg and tumor (P < 0.05), the control leg and tumor after sunitinib treatment (P < 0.05), and between tumors with and without sunitinib treatment (P < 0.001). CONCLUSIONS: These findings validated the technical feasibility of the TML method and pre-clinical feasibility for differentiating between the normal and diseased tissue states.

Focused in vivo delivery of plasmid DNA to the porcine vascular wall via intravascular ultrasound destruction of microbubbles.

BACKGROUND: Safety concerns associated with drug-eluting stents have spurred interest in alternative vessel therapeutics following angioplasty. Microbubble contrast agents have been shown to increase gene transfection in vivo in the presence of ultrasound. OBJECTIVES/METHODS: The purpose of this study was to determine whether an intravascular ultrasound (IVUS) catheter could mediate plasmid DNA transfection from microbubble carriers to the porcine coronary artery wall following balloon angioplasty. RESULTS: In the presence of plasmid-coupled microbubbles in vitro only cells exposed to ultrasound from the modified IVUS catheter significantly expressed the transgene. A porcine left anterior descending coronary artery underwent balloon angioplasty followed by injection and insonation of microbubbles from the IVUS catheter at the site of angioplasty. After 3 days, an approximately 6.5-fold increase in transgene expression was observed in arteries that received microbubbles and IVUS compared to those that received microbubbles with no IVUS. CONCLUSIONS: The results of this study demonstrate for the first time that IVUS is required to enhance gene transfection from microbubble carriers to the vessel wall in vivo. This technology may be applied to both drug and gene therapy to reduce vessel restenosis.

A non-linear three-dimensional model for quantifying microbubble dynamics.

A three-dimensional non-linear model for simulating microbubble response to acoustic insonation is presented. A 1 mum radius microbubble stimulated using positive and inverted 2.4 MHz pulses produced radius-time curves that matched (error <10%) with the experimental observation. A bound 2.3 mum radius microbubble insonated using 2.25 MHz 6 cycle pulse was observed to oscillate with max/min oscillations 45% lower than that of the free microbubble, this correlated ( approximately 10% error) with the observations of Garbin et al. [Appl. Phys. Lett. 90, 114103 (2007)]. The adherent microbubble oscillated asymmetrically in the plan view and symmetrically in the elevation view, consistent with the previous experimental results.