Revascularization Surgery for Moyamoya Vasculopathy: An Australian Experience.

BACKGROUND: Moyamoya vasculopathy is a rare steno-occlusive cerebrovascular disorder presenting with ischemia or hemorrhage. There are racial and geographic differences in presentation and outcome. There is little information regarding moyamoya in Australia. METHODS: Moyamoya patients undergoing surgery from 2001 to 2022 were studied retrospectively. The outcomes of revascularization surgery in adult and pediatric patients, with ischemic and hemorrhagic disease were analyzed, including functional outcomes, postoperative complications, bypass patency, and long-term rates of ischemic and hemorrhagic events. RESULTS: A total of 68 patients with 122 revascularized hemispheres and 8 posterior circulation revascularizations were included in this study. Eighteen patients were of Asian descent and 46 were of Caucasian origin. Presentation was with ischemia in 124 hemispheres and hemorrhage in six hemispheres. There were 92 direct, 34 indirect, and 4 combined revascularization surgeries performed. Early postoperative complications occurred in 3.1% (n = 4) of operations and delayed complications (infection, subdural hematoma) occurred after 4.6% (n = 6) of operations. Mean follow-up was 6.5 years (3-252 months). There was 100% patency of direct grafts at last follow-up. There were no hemorrhagic events following surgery and 1 new ischemic event 2 years after surgery. There was significant improvement in physical health functional outcomes at most recent follow-up (P < 0.05); mental health outcomes were not different between preoperative and postoperative assessments. CONCLUSIONS: The majority of Australian moyamoya patients are Caucasian and the most common clinical presentation is ischemia. Revascularization surgery had excellent outcomes with very low rates of ischemia and hemorrhage, comparing favorably to the natural history of moyamoya vasculopathy.

Cerebellar Tonsillar Descent Mimicking Chiari Malformation.

Chiari I malformation has been defined as cerebellar tonsillar descent greater than 5 mm below the foramen magnum. Suboccipital decompression remains the mainstay of treatment for symptomatic patients. Other conditions sometimes have imaging features that mimic Chiari I malformation. These patients are at risk of misdiagnosis and mismanagement, including surgery that may be unnecessary or may even worsen the underlying condition. The aim of this study was to analyse a series of Chiari I malformation mimics and identify differentiating imaging features. The mimics are categorised as post-traumatic cranio-cervical junction arachnoiditis, dural band, spontaneous intracranial hypotension, idiopathic intracranial hypertension, and cysts. Better understanding of these conditions will assist with diagnosis and optimal management, including avoiding unnecessary surgery.

Preparation and Characterization of Targeted Microbubbles.

Targeting of microbubbles (ultrasound contrast agents for molecular imaging) has been researched for more than two decades. However, methods of microbubble preparation and targeting ligand attachment are cumbersome, complicated, and lengthy. Therefore, there is a need to simplify the targeted microbubble preparation procedure to bring it closer to clinical translation. The purpose of this publication is to provide a detailed description and explanation of the steps necessary for targeted microbubble preparation, functional characterization and testing. A sequence of the optimized and simplified procedures is presented for two systems: a biotin-streptavidin targeting pair model, and a cyclic RGD peptide targeting the recombinant αvß3 protein, which is overexpressed on the endothelial lining of the tumor neovasculature. Here, we show the following: covalent coupling of the targeting ligand to a lipid anchor, assessment of the reagent quality, and tests that confirm the successful completion of the reaction; preparation of the aqueous precursor medium containing microbubble shell components, followed by microbubble preparation via amalgamation; assessment of the efficacy of lipid transfer onto the microbubble stabilizer shell; adjustment of microbubble size distribution by flotation at normal gravity to remove larger microbubbles that might be detrimental for in vivo use; assessment of microbubble size distribution by electrozone sensing; evaluation of targeted binding of the microbubbles to receptor-coated surface in a static binding assay test (in an inverted dish); and evaluation of targeted binding of the microbubbles to receptor-coated surface in a parallel plate flow chamber test.

Emerging contrast-enhanced ultrasound applications in children.

Ultrasound contrast agent (UCA) use in radiology is expanding beyond traditional applications such as evaluation of liver lesions, vesicoureteral reflux and echocardiography. Among emerging techniques, 3-D and 4-D contrast-enhanced ultrasound (CEUS) imaging have demonstrated potential in enhancing the accuracy of voiding urosonography and are ready for wider clinical adoption. US contrast-based lymphatic imaging has been implemented for guiding needle placement in MR lymphangiography in children. In adults, intraoperative CEUS imaging has improved diagnosis and assisted surgical management in tumor resection, and its translation to pediatric brain tumor surgery is imminent. Because of growing interest in precision medicine, targeted US molecular imaging is a topic of active preclinical research and early stage clinical translation. Finally, an exciting new development in the application of UCA is in the field of localized drug delivery and release, with a particular emphasis on treating aggressive brain tumors. Under the appropriate acoustic settings, UCA can reversibly open the blood-brain barrier, allowing drug delivery into the brain. The aim of this article is to review the emerging CEUS applications and provide evidence regarding the feasibility of these applications for clinical implementation.

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.

Validation of Normalized Singular Spectrum Area as a Classifier for Molecularly Targeted Microbubble Adherence.

Ultrasound molecular imaging is a diagnostic technique wherein molecularly targeted microbubble contrast agents are imaged to reveal disease markers on the blood vessel endothelium. Currently, microbubble adhesion to affected tissue can be quantified using differential targeted enhancement (dTE), which measures the late enhancement of adherent microbubbles through administration of destructive ultrasound pressures. In this study, we investigated a statistical parameter called the normalized singular spectrum area (NSSA) as a means to detect microbubble adhesion without microbubble destruction. We compared the signal differentiation capability of NSSA with matched dTE measurements in a mouse hindlimb tumor model. Results indicated that NSSA-based signal classification performance matches dTE when differentiating adherent microbubble from non-adherent microbubble signals (receiver operating characteristic area under the curve = 0.95), and improves classification performance when differentiating microbubble from tissue signals (p < 0.005). NSSA-based signal classification eliminates the need for destruction of contrast, and may offer better sensitivity, specificity and the opportunity for real-time microbubble detection and classification.

Formation of Microbubbles for Targeted Ultrasound Contrast Imaging: Practical Translation Considerations.

For preparation of ligand-decorated microbubbles for targeted ultrasound contrast imaging, it is important to maximize the amount of ligand associated with the bubble shell. We describe optimization of the use of a biocompatible cosurfactant in the microbubble formulation media to maximize the incorporation of targeting ligand-lipid conjugate into the microbubble shell, and thus reduce the fraction of ligand not associated with microbubbles, following amalgamation preparation. The influence of the concentration of a helper cosurfactant propylene glycol (PG) on the efficacy of microbubble preparation by amalgamation and on the degree of association of fluorescent PEG-lipid with the microbubble shell was tested. Three sets of targeted bubbles were then prepared: with VCAM-1-targeting peptide VHPKQHRGGSK(FITC)GC-PEG-DSPE, cyclic RGDfK-PEG-DSPE, selective for αVß3, and control cRADfK-PEG-DSPE, without such affinity. Microbubbles were prepared by 45 s amalgamation, with DSPC and PEG stearate as the main components of the shell, with 15% PG in aqueous saline. In vitro microbubble targeting was assessed with a parallel plate flow chamber with a recombinant receptor coated surface. In vivo targeting was assessed in MC-38 tumor-bearing mice (subcutaneous tumor in hind leg), 10 min after intravenous bolus of microbubble contrast agent (20 million particles per injection). Ultrasound imaging of the tumor and control nontarget muscle tissue in a contralateral leg was performed with a clinical scanner. Amalgamation technique with PG cosurfactant produced microbubbles at concentrations exceeding 2 × 109 particles/mL, and ∼50-60% or more of the added fluorescein-PEG-DSPE or VCAM-1-targeted fluorescent peptide was associated with microbubbles, about 2 times higher than that in the absence of PG. After intravenous injection, peptide-targeted bubbles selectively accumulated in the tumor vasculature, with negligible accumulation in nontumor contralateral leg muscle, or with control nontargeted microbubbles (assessed by contrast ultrasound imaging). For comparison, administration of RGD-decorated microbubbles prepared by traditional sonication, and purified from free peptide-PEG-lipid by repeated centrifugation, resulted in the same accumulation pattern as for translatable amalgamated microbubbles. Following amalgamation in the presence of PG, efficient transfer of ligand-PEG-lipid to microbubble shell was achieved and quantified. Purification of microbubbles from free peptide-PEG-lipid was not necessary, as proven by in vitro and in vivo targeting studies, so PG cosurfactant amalgamation technique generated peptide-targeted microbubbles are amenable for bedside preparation and clinical translation. The pathway to clinical translation is simplified by the fact that most of the materials used in this study either are on the United States Food and Drug Administration GRAS list or can be procured as pharmaceutical grade substances.

Ultrasound Molecular Imaging of Inflammation in Mouse Abdominal Aorta.

OBJECTIVES: The aim of this study was to demonstrate a new clinically translatable ultrasound molecular imaging approach, modulated acoustic radiation force-based imaging, which is capable of rapid and reliable detection of inflammation as validated in mouse abdominal aorta. MATERIALS AND METHODS: Animal studies were approved by the Institutional Animal Care and Use Committee at the University of Virginia. C57BL/6 mice stimulated with tumor necrosis factor α, or fed with a high-fat diet, were used as inflammation (MInflammation) and diet-induced obesity (DIO) (MDIO) models, respectively. C57BL/6 mice, not exposed to tumor necrosis factor α or DIO, were used as controls (MNormal). P-selectin-targeted (MBP-selectin), vascular cell adhesion molecule (VCAM)-1-targeted (MBVCAM-1), and isotype control (MBControl) microbubbles were synthesized by conjugating anti-P-selectin, anti-VCAM-1, and isotype control antibodies to microbubbles, respectively. The abdominal aortas were imaged for 180 seconds during a constant infusion of microbubbles. A parameter, residual-to-saturation ratio (RSR), was used to assess P-selectin and VCAM-1. Statistical analysis was performed with the Student t test. RESULTS: For the inflammation model, RSR of the MInflammation + MBP-selectin group was significantly higher (40.9%, P < 0.0005) than other groups. For the DIO model, RSR of the MDIO + MBVCAM-1 group was significantly higher (60.0%, P < 0.0005) than other groups. Immunohistochemistry staining of the abdominal aorta confirmed the expression of P-selectin and VCAM-1. CONCLUSIONS: A statistically significant assessment of P-selectin and VCAM-1 in mouse abdominal aorta was achieved. This technique yields progress toward rapid targeted molecular imaging in large blood vessels and thus has the potential for early diagnosis, treatment selection, and risk stratification of atherosclerosis.

The Use of Acoustic Radiation Force Decorrelation-Weighted Pulse Inversion for Enhanced Ultrasound Contrast Imaging.

OBJECTIVES: The use of ultrasound imaging for cancer diagnosis and screening can be enhanced with the use of molecularly targeted microbubbles. Nonlinear imaging strategies such as pulse inversion (PI) and "contrast pulse sequences" (CPS) can be used to differentiate microbubble signal, but often fail to suppress highly echogenic tissue interfaces. This failure results in false-positive detection and potential misdiagnosis. In this study, a novel acoustic radiation force (ARF)-based approach was developed for superior microbubble signal detection. The feasibility of this technique, termed ARF decorrelation-weighted PI (ADW-PI), was demonstrated in vivo using a subcutaneous mouse tumor model. MATERIALS AND METHODS: Tumors were implanted in the hindlimb of C57BL/6 mice by subcutaneous injection of MC38 cells. Lipid-shelled microbubbles were conjugated to anti-VEGFR2 antibody and administered via bolus injection. An image sequence using ARF pulses to generate microbubble motion was combined with PI imaging on a Verasonics Vantage programmable scanner. ADW-PI images were generated by combining PI images with interframe signal decorrelation data. For comparison, CPS images of the same mouse tumor were acquired using a Siemens Sequoia clinical scanner. RESULTS: Microbubble-bound regions in the tumor interior exhibited significantly higher signal decorrelation than static tissue (n = 9, P < 0.001). The application of ARF significantly increased microbubble signal decorrelation (n = 9, P < 0.01). Using these decorrelation measurements, ADW-PI imaging demonstrated significantly improved microbubble contrast-to-tissue ratio when compared with corresponding CPS or PI images (n = 9, P < 0.001). Contrast-to-tissue ratio improved with ADW-PI by approximately 3 dB compared with PI images and 2 dB compared with CPS images. CONCLUSIONS: Acoustic radiation force can be used to generate adherent microbubble signal decorrelation without microbubble bursting. When combined with PI, measurements of the resulting microbubble signal decorrelation can be used to reconstruct images that exhibit superior suppression of highly echogenic tissue interfaces when compared with PI or CPS alone.

Optical Verification of Microbubble Response to Acoustic Radiation Force in Large Vessels With In Vivo Results.

OBJECTIVE: The objective of this study was to optically verify the dynamic behaviors of adherent microbubbles in large blood vessel environments in response to a new ultrasound technique using modulated acoustic radiation force. MATERIALS AND METHODS: Polydimethylsiloxane (PDMS) flow channels coated with streptavidin were used in targeted groups to mimic large blood vessels. The custom-modulated acoustic radiation force beam sequence was programmed on a Verasonics research scanner. In vitro experiments were performed by injecting a biotinylated lipid-perfluorobutane microbubble dispersion through flow channels. The dynamic response of adherent microbubbles was detected acoustically and simultaneously visualized using a video camera connected to a microscope. In vivo verification was performed in a large abdominal blood vessel of a murine model for inflammation with injection of biotinylated microbubbles conjugated with P-selectin antibody. RESULTS: Aggregates of adherent microbubbles were observed optically under the influence of acoustic radiation force. Large microbubble aggregates were observed solely in control groups without targeted adhesion. Additionally, the dispersion of microbubble aggregates were demonstrated to lead to a transient acoustic signal enhancement in control groups (a new phenomenon we refer to as "control peak"). In agreement with in vitro results, the control peak phenomenon was observed in vivo in a murine model. CONCLUSIONS: This study provides the first optical observation of microbubble-binding dynamics in large blood vessel environments with application of a modulated acoustic radiation force beam sequence. With targeted adhesion, secondary radiation forces were unable to produce large aggregates of adherent microbubbles. Additionally, the new phenomenon called control peak was observed both in vitro and in vivo in a murine model for the first time. The findings in this study provide us with a better understanding of microbubble behaviors in large blood vessel environments with application of acoustic radiation force and could potentially guide future beam sequence designs or signal processing routines for enhanced ultrasound molecular imaging.

Microbubbles as ultrasound contrast agents for molecular imaging: preparation and application.

OBJECTIVE: The purpose of this review is to describe trends in microbubble application in molecular imaging. CONCLUSION: Microbubbles are used for contrast ultrasound imaging as blood-pool agents in cardiology and radiology. Their promise as targeted agents for molecular imaging is now being recognized. Microbubbles can be functionalized with ligand molecules that bind to molecular markers of disease. Potential clinical applications of molecular imaging with microbubble-based ultrasound contrast agents are in the monitoring of the biomarker status of vascular endothelium, visualizing tumor vasculature, and imaging inflammation and ischemia-reperfusion injury zones and thrombi.

Nanobody-coupled microbubbles as novel molecular tracer.

Camelid-derived single-domain antibody-fragments (~15kDa), called nanobodies, are a new class of molecular tracers that are routinely identified with nanomolar affinity for their target and that are easily tailored for molecular imaging and drug delivery applications. We hypothesized that they are well-suited for the design of targeted microbubbles (µBs) and aimed to develop and characterize eGFP- and VCAM-1-targeted µBs. Anti-eGFP (cAbGFP4) and anti-VCAM-1 (cAbVCAM1-5) nanobodies were site-specifically biotinylated in bacteria. This metabolic biotinylation method yielded functional nanobodies with one biotin located at a distant site of the antigen-binding region of the molecule. The biotinylated nanobodies were coupled to biotinylated lipid µBs via streptavidin-biotin bridging. The ability of µB-cAbGFP4 to recognize eGFP was tested as proof-of-principle by fluorescent microscopy and confirmed the specific binding of eGFP to µB-cAbGFP4. Dynamic flow chamber studies demonstrated the ability of µB-cAbVCAM1-5 to bind VCAM-1 in fast flow (up to 5 dynes/cm(2)). In vivo targeting studies were performed in MC38 tumor-bearing mice (n=4). µB-cAbVCAM1-5 or control µB-cAbGFP4 were injected intravenously and imaged using a contrast-specific ultrasound imaging mode. The echo intensity in the tumor was measured 10min post-injection. µB-cAbVCAM1-5 showed an enhanced signal compared to control µBs (p<0.05). Using metabolic and site-specific biotinylation of nanobodies, a method to develop nanobody-coupled µBs was described. The application of VCAM-1-targeted µBs as novel molecular ultrasound contrast agent was demonstrated both in vitro and in vivo.

Analysis of leukocyte rolling in vivo and in vitro.

Leukocyte rolling is an important step for the successful recruitment of leukocytes from blood to tissues mediated by a specialized group of glycoproteins termed selectins. Because of the dynamic process of leukocyte rolling, binding of selectins to their respective counter-receptors (selectin ligands) needs to fulfill three major requirements: (1) rapid bond formation, (2) high tensile strength, and (3) fast dissociation rates. These criteria are perfectly met by selectins, which interact with specific carbohydrate determinants on selectin ligands. This chapter describes the theoretical background, technical requirements, and analytical tools needed to quantitatively assess leukocyte rolling in vivo and in vitro. For the in vivo setting, intravital microscopy allows the observation and recording of leukocyte rolling under different physiological and pathological conditions in almost every organ. Real-time and off-line analysis tools help to assess geometric, hemodynamic, and rolling parameters. Under in vitro conditions, flow chamber assays such as parallel plate flow chamber systems have been the mainstay to study interactions between leukocytes and adhesion molecules under flow. In this setting, adhesion molecules are immobilized on plastic, in a lipid monolayer, or presented on cultured endothelial cells on the chamber surface. Microflow chambers are available for studying leukocyte adhesion in the context of whole blood and without blood cell isolation. The microscopic observation of leukocyte rolling in different in vivo and in vitro settings has significantly contributed to our understanding of the molecular mechanisms responsible for the stepwise extravasation of leukocytes into inflamed tissues.

Turbulent flow evaluation of the venous needle during hemodialysis.

Arteriovenous (AV) grafts and fistulas used for hemodialysis frequently develop intimal hyperplasia (IH) at the venous anastomosis of the graft, leading to flow-limiting stenosis, and ultimately to graft failure due to thrombosis. Although the high AV access blood flow has been implicated in the pathogenesis of graft stenosis, the potential role of needle turbulence during hemodialysis is relatively unexplored. High turbulent stresses from the needle jet that reach the venous anastomosis may contribute to endothelial denudation and vessel wall injury. This may trigger the molecular and cellular cascade involving platelet activation and IH, leading to eventual graft failure. In an in-vitro graft/needle model dye injection flow visualization was used for qualitative study of flow patterns, whereas laser Doppler velocimetry was used to compare the levels of turbulence at the venous anastomosis in the presence and absence of a venous needle jet. Considerably higher turbulence was observed downstream of the venous needle, in comparison to graft flow alone without the needle. While turbulent RMS remained around 0.1 m/s for the graft flow alone, turbulent RMS fluctuations downstream of the needle soared to 0.4-0.7 m/s at 2 cm from the tip of the needle and maintained values higher than 0.1 m/s up to 7-8 cm downstream. Turbulent intensities were 5-6 times greater in the presence of the needle, in comparison with graft flow alone. Since hemodialysis patients are exposed to needle turbulence for four hours three times a week, the role of post-venous needle turbulence may be important in the pathogenesis of AV graft complications. A better understanding of the role of needle turbulence in the mechanisms of AV graft failure may lead to improved design of AV grafts and venous needles associated with reduced turbulence, and to pharmacological interventions that attenuate IH and graft failure resulting from turbulence.