Microbubbles and Ultrasound Accelerated Thrombolysis for Peripheral Arterial Occlusions: The Outcomes of a Single Arm Phase II Trial.

OBJECTIVE: Acute peripheral arterial occlusions can be treated by catheter directed thrombolysis (CDT). However, CDT is time consuming and accompanied by the risk of bleeding complications. The addition of contrast enhanced ultrasound and microbubbles could improve thrombus susceptibility to thrombolytic agents and potentially shorten treatment time with a lowered risk of bleeding complications. This article reports the outcomes of the safety and feasibility of this novel technique. METHODS: In this single arm phase II trial, 20 patients with acute lower limb ischaemia received CDT combined with an intravenous infusion of microbubbles and locally applied ultrasound during the first hour of standard intra-arterial thrombolytic therapy. The primary endpoint was safety, i.e., occurrence of serious adverse events (haemorrhagic complications and/or amputation) and death within one year. Secondary endpoints included angiographic and clinical success, thrombolysis duration, additional interventions, conversion, and quality of life. RESULTS: The study included 20 patients (16 men; median age 68.0 years; range, 50.0 - 83.0; and 40% native artery and 60% bypass graft). In all patients, the use of microbubble contrast enhanced sonothrombolysis could be applied successfully. There were no serious adverse events related to the experimental treatment. Duplex examination showed flow distal from the occlusion after 23.1 hours (range 3.1 - 46.5) with a median thrombolysis time of 47.5 hours (range 6.0 - 81.0). The short term ABI and pain scores significantly improved; however, no changes were observed before or after thrombolysis in the microcirculation. Overall mortality and amputation rates were both 2% within one year. The one year patency rate was 55%. CONCLUSION: Treatment of patients with acute peripheral arterial occlusions with contrast enhanced sonothrombolysis is feasible and safe to perform in patients. Further research is necessary to investigate the superiority of this new treatment over standard treatment.

Non-invasive, neurotoxic surgery reduces seizures in a rat model of temporal lobe epilepsy.

Surgery can be highly effective for treating certain cases of drug resistant epilepsy. The current study tested a novel, non-invasive, surgical strategy for treating seizures in a rat model of temporal lobe epilepsy. The surgical approach uses magnetic resonance-guided, low-intensity focused ultrasound (MRgFUS) in combination with intravenous microbubbles to open the blood-brain barrier (BBB) in a transient and focal manner. During the period of BBB opening, a systemically administered neurotoxin (Quinolinic Acid: QA) that is normally impermeable to the BBB gains access to a targeted area in the brain, destroying neurons where the BBB has been opened. This strategy is termed Precise Intracerebral Non-invasive Guided Surgery (PING). Spontaneous recurrent seizures induced by pilocarpine were monitored behaviorally prior to and after PING or under control conditions. Seizure frequency in untreated animals or animals treated with MRgFUS without QA exhibited expected seizure rate fluctuations frequencies between the monitoring periods. In contrast, animals treated with PING targeting the intermediate-temporal aspect of the hippocampus exhibited substantial reductions in seizure frequency, with convulsive seizures being eliminated entirely in two animals. These findings suggest that PING could provide a useful alternative to invasive surgical interventions for treating drug resistant epilepsy, and perhaps for treating other neurological disorders in which aberrant neural circuitries play a role.

Silicone microbubbles after anti-vascular endothelial growth factor injections in patients with wet age-related macular degeneration: incidence, quantification and secondary optical coherence tomography artfacts.

PURPOSE: To report the incidence and quantity of silicone oil microbubbles and the relationship with the number of intravitreal anti-vascular endothelial growth factor (VEGF) injections and evaluate if microbubbles induce artefacts on optical coherence tomography (OCT) images. METHODS: Observational, descriptive, cross-sectional study. Patients with wet age-related macular degeneration were included who had been treated for 1 year minimally with anti-VEGF injections repackaged in the hospital pharmacy. Detection and quantification of silicone microbubbles by mydriatic biomicroscopic examination were conducted 1 month after the last injection. The numbers of microbubbles were quantified on a scale of 0-3: 0, none; 1 scarce (1-10 microbubbles); 2 moderate (10-30); or 3 numerous (>30). Shadowing on OCT images was classified as 0-3: 0, none; 1 obscuring some retinal layers; 2 obscuring all retinal layers; or 3 obscuring the retinal thickness. RESULTS: The study included 142 eyes of 98 patients (mean age, 82.4 years + 7.3; range, 65-97) treated with 2377 injections. Microbubbles were detected in 127 (89.4%) eyes, 62 (43.6%) with numerous microbubbles and 36 (25.4%) and 29 (20.4%), respectively, with scarce and moderate numbers. A positive correlation was found between the numbers of injections and intravitreal silicone (rho, 0.7). Optical coherence tomography (OCT) artefacts were detected in 11 eyes; the artefacts obscured all retinal layers in three eyes. No significant relationship could be established between the appearance of floaters and the microbubbles. CONCLUSION: The presence and number of silicone microbubbles were correlated with the number of intravitreal injections. Microbubbles can produce OCT artefacts, which can hinder the treatment decision.

Neural network-based modeling of the number of microbubbles generated with four circulation factors in cardiopulmonary bypass.

The need for the estimation of the number of microbubbles (MBs) in cardiopulmonary bypass surgery has been recognized among surgeons to avoid postoperative neurological complications. MBs that exceed the diameter of human capillaries may cause endothelial disruption as well as microvascular obstructions that block posterior capillary blood flow. In this paper, we analyzed the relationship between the number of microbubbles generated and four circulation factors, i.e., intraoperative suction flow rate, venous reservoir level, continuous blood viscosity and perfusion flow rate in cardiopulmonary bypass, and proposed a neural-networked model to estimate the number of microbubbles with the factors. Model parameters were determined in a machine-learning manner using experimental data with bovine blood as the perfusate. The estimation accuracy of the model, assessed by tenfold cross-validation, demonstrated that the number of MBs can be estimated with a determinant coefficient R2 = 0.9328 (p < 0.001). A significant increase in the residual error was found when each of four factors was excluded from the contributory variables. The study demonstrated the importance of four circulation factors in the prediction of the number of MBs and its capacity to eliminate potential postsurgical complication risks.

Molecular Ultrasound Imaging.

Contrast-enhanced ultrasound (CEUS) imaging is a valuable tool for preclinical and clinical diagnostics. The most frequently used ultrasound contrast agents are microbubbles. Besides them, novel nano-sized materials are under investigation, which are briefly discussed in this chapter. For molecular CEUS, the ultrasound contrast agents are modified to actively target disease-associated molecular markers with a site-specific ligand. The most common markers for tumor imaging are related to neoangiogenesis, like the vascular endothelial growth factor receptor-2 (VEGFR2) and αvß3 integrin. In this chapter, applications of molecular ultrasound to longitudinally monitor receptor expression during tumor growth, to detect neovascularization, and to evaluate therapy responses are described. Furthermore, we report on first clinical trials of molecular CEUS with VEGFR2-targeted phospholipid microbubbles showing promising results regarding patient safety and its ability to detect tumors of prostate, breast, and ovary. The chapter closes with an outlook on ultrasound theranostics, where (targeted) ultrasound contrast agents are used to increase the permeability of tumor tissues and to support drug delivery.

Observed Effects of Whole-Brain Radiation Therapy on Focused Ultrasound Blood-Brain Barrier Disruption.

As focused ultrasound for blood-brain barrier disruption (FUS-BBBD) has progressed to human application, it has become necessary to consider the potential effects of prior irradiation treatments. Using a murine model, we examined the effects of whole-brain irradiation on FUS-BBBD. We first subjected half of the experimental cohort to daily 3-Gy whole-brain irradiation for 10 consecutive days. Then, microbubble-assisted FUS-BBBD was performed unilaterally while the contralateral sides served as unsonicated controls. FUS-BBBD, as evident by measuring the fluorescence yield of extravasated trypan blue dye, was identified at all sites with minimal or no apparent pathology. The peak fluorescence intensity caused by extravasated dye in the sonicated region was 17.5 ± 12.1% higher after radiation and FUS-BBBD than after FUS-BBBD alone, suggesting that prior radiation of the brain may be a sensitizing factor for FUS-BBBD. Radiation alone-without FUS-BBBD-resulted in mild BBB disruption. Hemorrhagic petechiae were observed in 9 of 12 radiated brains, with 77% of them clearly located outside the sonicated area; no petechiae were found in non-irradiated animals. This radiation protocol did not appear to increase the risk for vascular damage associated with FUS-BBBD.

Precision pulse capsulotomy complicated by radial tear of the anterior capsule: a proposed mechanism.

Zepto precision pulsed capsulotomy is an emerging technology aimed at providing a safer and more reproducible anterior capsulorhexis, with potential advantages in challenging cases. Initial reports suggest high safety, and thus far to our knowledge, no complications have been reported. Herein we report an unexpected complication. After the pulse delivery phase on a routine cataract case, a radial tear of the anterior capsule was observed. Upon careful review of the surgery video, an air bubble was noted, at the precise clock hour of the radial tear, trapped between the device wire and capsule. This air bubble presumably prevented the transfer of rapid phase transition at this site, interfering with capsule cleavage, and resulting in incomplete capsulotomy. Based on this observation, if a trapped air bubble is observed after the vacuum phase, we recommend applying more vacuum or disengaging and reattaching, before proceeding to the pulse delivery stage.

Improved delivery of doxorubicin by altering the tumor microenvironment using ultrasound combined with microbubbles and chemotherapy.

PURPOSE: To determine whether low-intensity pulsed ultrasound (US) using microbubbles (MB) can temporarily promote regional blood flow in the tumor and increase the delivery of doxorubicin (ad). METHODS: We randomly divided 66 tumor-bearing rabbits into 6 groups (n=11/group). The 6 groups were as follows: doxorubicin and ultrasound combined with microbubble treatment group (Ad-US-MB treatment group), US-MB treatment group, US treatment group, MB treatment group, doxorubicin treatment group (Ad-treatment group), and control group. The animals were intravenously injected with doxorubicin hydrochloride; next, the tumors in the Ad-US-MB treatment group were subjected to low-intensity ultrasound with microbubbles for 10 min. Contrast-enhanced ultrasound (CEUS) imaging of tumor tissues was performed before and after the intervention. Next, we randomly selected 8 rabbits/group, which were euthanized immediately after treatment. The remaining rabbits were reared and underwent the intervention every 7 days. RESULTS: Tumor perfusion increased immediately in the Ad-US-MB treatment group (p<0.01). Unlike the Ad treatment group, the Ad-US-MB treatment group showed high levels of doxorubicin in the tumor samples (p<0.05). Immunofluorescent staining showed high levels of doxorubicin mainly around the blood vessels; in addition, doxorubicin was observed in other areas in the Ad-US-MB treatment group. Inhibition of tumor growth was observed in the Ad-US-MB treatment group. CONCLUSIONS: Low-intensity ultrasound combined with microbubbles and chemotherapy can alter the tumor microenvironment and temporarily increase the regional blood flow to the tumor.

A combination of ultrasound-targeted microbubble destruction with transplantation of bone marrow mesenchymal stem cells promotes recovery of acute liver injury.

BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) can provide an additional source of therapeutic stem cells for regeneration of liver cells during acute liver injury (ALI). However, the insufficient hepatic homing by the transplanted BMSCs limits their applications. Ultrasound-targeted microbubble destruction (UTMD) has been reported to promote the homing of transplanted stem cells into the ischemic myocardium. In this study, we investigated whether UTMD promotes the hepatic homing of BMSCs in ALI rats and evaluated the therapeutic effect. METHODS: BMSCs were isolated from the femurs and tibias of Sprague-Dawley (SD) rats. The isolated BMSCs were stably transfected with a lentivirus expressing enhanced green fluorescent protein (EGFP) that can be visualized and quantified in vivo after transplantation. Both tumor necrosis factor α (TNF-α) and stromal cell-derived factor 1 (SDF-1) were used to verify the appropriate ultrasound parameters. The ALI rats were divided into four groups: control, BMSCs, UTMD, and UTMD + BMSCs. The protein and mRNA expression levels of SDF-1, intercellular cell adhesion molecule (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), hepatocyte growth factor (HGF), and monocyte chemotactic protein 1 (MCP-1) in the exposed livers were analyzed at 48 h after treatment. ALI recovery was determined by serum biochemical parameters and histology. RESULTS: The isolated rat BMSCs demonstrated a good proliferation potential that was both osteogenic and adipogenic in differentiation and expressed cluster of differentiation (CD) 29 and CD90, but not CD45 or CD11b/c. After BMSC and/or UTMD treatment, the number of GFP-labeled BMSCs in the UTMD + BMSCs group was significantly higher than that of the BMSCs group (9.8 ± 2.3 vs. 5.2 ± 1.1/per high-power field). Furthermore, the expression of GFP mRNA was performed for evaluation of the homing rate of BMSCs in injury sites as well. In addition, the expression levels of SDF-1, ICAM-1, VCAM-1, HGF, and MCP-1 were higher (p < 0.01) in UTMD+BMSCs group. The serum levels of biomarkers were significantly lower in the UTMD + BMSCs group, and the apoptotic rate of hepatocytes in the UTMD + BMSCs group was markedly lower than that of the BMSCs group (all p < 0.05). The hepatic pathology was significantly alleviated in the UTMD + BMSCs group. CONCLUSIONS: UTMD treatment efficiently induced a favorable microenvironment for cell engraftment, resulting in improvement of hepatic homing of BMSCs, which was probably mediated through upregulation of the expression of adhesion molecules and cytokines. UTMD treatment appeared to be an effective and noninvasive approach to achieve better efficacy of BMSC-based therapy for repairing a severely injured liver.

Frequency bands for ultrasound, suitable for the consideration of its health effects.

It is proposed that the ultrasound frequency spectrum should be divided into three bands in order to facilitate a more rational assessment of its health effects. Whilst statement of the frequencies at the borders of these bands facilitates their definition, it is recognized that these observables vary continuously with frequency and consequently these border frequencies should not be used to rule out the possibility of a given effect occurring. The lowest band, US(A), lies between 17.8 and 500 kHz. In this band acoustic cavitation and its associated forces form the dominant process resulting in biological effects in liquids and soft tissues, whereas health effects from airborne ultrasound have been reported but are far less researched. In the middle band, US(B), between 500 kHz and 100 MHz, temperature rise in tissues becomes the most important biological effect of exposure. The highest band, US(C), covers frequencies above 100 MHz, for which the radiation force becomes an increasingly important biophysical mechanism. A justification for the selection of 17.8 kHz in preference to any other threshold for the lower frequency limit for ultrasound is given.

A microbubble of gas as an early indication of macular hole formation after vitrectomy surgery for retinal detachment repair.

We present a novel case of a 67-year-old man who developed a full-thickness macular hole in the presence of a microbubble after repair of rhegmatogenous retinal detachment.

Influences of Flap Shape and Hinge Angle on Opaque Bubble Layer Formation in Femtosecond Laser-Assisted LASIK Surgery.

PURPOSE: To evaluate the effects of different flap shapes and hinge angles on opaque bubble layer (OBL) formation using a femtosecond laser for flap creation in LASIK surgery. METHODS: This retrospective study evaluated 138 eyes of 73 patients who underwent femtosecond laser-assisted LASIK with a 150-kHz IntraLase femtosecond laser (Abbott Medical Optics, Inc., Santa Ana, CA) for myopic astigmatism and compared differences between different flap shapes and hinge angles on OBL formation. The surgical procedures were videotaped, and the patterns and sizes of the OBLs seen during surgery were analyzed. Preoperative and postoperative data including patient demographics, refractive status, keratometry, central corneal thickness, and intraoperative data (flap size and pocket parameters) were recorded. The eyes were divided into four groups based on the corneal flap shape (elliptical versus round) and hinge angle (50° versus 60°). RESULTS: The preoperative demographic data, mean spherical errors, cylindrical power, and central corneal thickness were not significantly different among the groups. Of the 138 eyes, 107 (77%) developed an OBL covering a mean area of 13.8% ± 12.6% in each case. This area was significantly smaller in the elliptical flap with 60° hinge angle group (P < .05). CONCLUSIONS: An oval-shaped flap with a larger hinge angle tended to result in less OBL formation in femtosecond laser-assisted LASIK. [J Refract Surg. 2017;33(3):178-182.].

New horizons for focused ultrasound (FUS) - therapeutic applications in neurodegenerative diseases.

Access to the CNS and delivery of therapeutics across the blood-brain barrier remains a challenge for most treatments of major neurological diseases such as AD or PD. Focused ultrasound represents a potential approach for overcoming these barriers to treating AD and PD and perhaps other neurological diseases. Ultrasound (US) is best known for its imaging capabilities of organs in the periphery, but various arrangements of the transducers producing the acoustic signal allow the energy to be precisely focused (F) within the skull. Using FUS in combination with MRI and contrast agents further enhances accuracy by providing clear information on location. Varying the acoustic power allows FUS to be used in applications ranging from imaging, stimulation of brain circuits, to ablation of tissue. In several transgenic mouse models of AD, the use of FUS with microbubbles reduces plaque load and improves cognition and suggests the need to investigate this technology for plaque removal in AD. In PD, FUS is being explored as a way to non-invasively ablate the brain areas responsible for the tremor and dyskinesia associated with the disease, but has yet to be utilized for non-invasive delivery of putative therapeutics. The FUS approach also greatly increases the range of possible CNS therapeutics as it overcomes the issues of BBB penetration. In this review we discuss how the characteristics and various applications of FUS may advance the therapeutics available for treating or preventing neurodegenerative disorders with an emphasis on treating AD and PD.

Microvascular Injury and Perfusion Changes Induced by Ultrasound and Microbubbles in a Machine-Perfused Pig Liver.

Localized drug delivery and uptake can benefit from the combined action of ultrasound and microbubbles at a specific site. Some of the possible mechanisms suggested are vessel poration and/or cell poration, but the exact acoustic parameters that trigger those phenomena remain unknown. Ex vivo machine perfusion of human-sized organs is a technique that provides an ideal environment for pre-clinical investigations with high physiologic relevance not possible with in vitro experiments. In this work, ex vivo machine-perfused pig livers were combined with an image-guided therapy system to investigate microvascular flow changes caused by the interaction of ultrasound-driven microbubbles with the vasculature. The effects of acoustic pressure (1.7-4 MPa peak negative pressures) and number of cycles (1000 or 20 cycles) were examined. Perfusion changes caused by the action of ultrasound on microbubbles in the microcirculation were qualitatively and quantitatively assessed with contrast-enhanced ultrasound and used as a metric of the extent of vessel perforation, thus, extravasation. Areas that were exposed to peak negative pressures above 1.7 MPa underwent a detectable and irreversible perfusion change. Complete devascularization of the area exposed to ultrasound was observed at much larger acoustic pressures (∼4 MPa). Shorter acoustic pulses (20 cycles) produced markedly fewer perfusion changes than longer pulses (1000 cycles) under the same acoustic amplitude exposure.

Controllable permeability of blood-brain barrier and reduced brain injury through low-intensity pulsed ultrasound stimulation.

It has been shown that the blood-brain barrier (BBB) can be locally disrupted by focused ultrasound (FUS) in the presence of microbubbles (MB) while sustaining little damage to the brain tissue. Thus, the safety issue associated with FUS-induced BBB disruption (BBBD) needs to be investigated for future clinical applications. This study demonstrated the neuroprotective effects induced by low-intensity pulsed ultrasound (LIPUS) against brain injury in the sonicated brain. Rats subjected to a BBB disruption injury received LIPUS exposure for 5 min after FUS/MB application. Measurements of BBB permeability, brain water content, and histological analysis were then carried out to evaluate the effects of LIPUS. The permeability and time window of FUS-induced BBBD can be effectively modulated with LIPUS. LIPUS also significantly reduced brain edema, neuronal death, and apoptosis in the sonicated brain. Our results show that brain injury in the FUS-induced BBBD model could be ameliorated by LIPUS and that LIPUS may be proposed as a novel treatment modality for controllable release of drugs into the brain.

Role of US Contrast Agents in the Assessment of Indeterminate Solid and Cystic Lesions in Native and Transplant Kidneys.

Ultrasonography (US) is often the initial imaging modality employed in the evaluation of renal diseases. Despite improvements in B-mode and Doppler imaging, US still faces limitations in the assessment of focal renal masses and complex cysts as well as the microcirculation. The applications of contrast-enhanced US (CEUS) in the kidneys have dramatically increased to overcome these shortcomings with guidelines underlining their importance. This article describes microbubble contrast agents and their role in renal imaging. Microbubble contrast agents consist of a low solubility complex gas surrounded by a phospholipid shell. Microbubbles are extremely safe and well-tolerated pure intravascular agents that can be used in renal failure and obstruction, where computed tomographic (CT) and magnetic resonance (MR) imaging contrast agents may have deleterious effects. Their intravascular distribution allows for quantitative perfusion analysis of the microcirculation, diagnosis of vascular problems, and qualitative assessment of tumor vascularity and enhancement patterns. Low acoustic power real-time prolonged imaging can be performed without exposure to ionizing radiation and at lower cost than CT or MR imaging. CEUS can accurately distinguish pseudotumors from true tumors. CEUS has been shown to be more accurate than unenhanced US and rivals contrast material-enhanced CT in the diagnosis of malignancy in complex cystic renal lesions and can upstage the Bosniak category. CEUS can demonstrate specific enhancement patterns allowing the differentiation of benign and malignant solid tumors as well as focal inflammatory lesions. In conclusion, CEUS is useful in the characterization of indeterminate renal masses and cysts.

Silicone oil microbubble found in failed full-thickness macular hole closure.

PURPOSE: To describe a case of failed macular hole closure after vitrectomy with silicone oil tamponade because of a silicone oil microbubble. METHODS: This is a retrospective case review of a single patient's clinical course. RESULTS: Because of the inability of the patient to assume a prone position after vitrectomy for full-thickness macular hole, 1000-centistoke silicone oil tamponade was used at the initial repair. Optical coherence tomography showed persistent full-thickness macular hole with evidence of a silicone oil microbubble within the macular hole. Subsequent vitrectomy with the removal of silicone oil and exchange for 25% sulfur hexafluoride (SF6) gas with attempted prone positioning failed to provide hole closure. Successful hole closure was accomplished after tamponade with 5000-centistoke silicone oil, without prone positioning. No recurrence of the full-thickness defect was seen after the eventual removal of 5000-centistoke silicone oil. CONCLUSION: It is possible that the silicone oil microbubble formation and migration within a full-thickness macular hole defect may contribute to surgical failure.

New-onset seizure after perflutren microbubble injection during dobutamine stress echocardiography.

Intravenous microbubble contrast agents are frequently used during ultrasound imaging to improve endocardial border detection, enhance Doppler signals, differentiate thrombi from tumors or define vascular anatomy. Dobutamine stress echocardiography (DSE) with or without addition of atropine is a standard technique for evaluation of coronary artery disease. Noncontrast or contrast-enhanced DSE is generally considered a safe procedure. We report what appears to be the first case of new-onset seizure activity following perflutren microbubble contrast injection during dobutamine-atropine stress echocardiography. On the basis of this single occurrence, we are only able to demonstrate a temporal, but not a causal relationship between the administration of microbubble echo contrast and onset of seizure. We do not suggest withholding administration of microbubble contrast when clinically indicated. However, increased vigilance in monitoring for seizure development in patients receiving microbubble contrast seems warranted.

Preparation and antitumor activity of bFGF-mediated active targeting doxorubicin microbubbles.

Characterization and antitumor activity of basic fibroblast growth factor-mediated active targeting doxorubicin microbubbles (bFGF-DOX-MB) were investigated. Pluronic F68 with chemical conjugation of doxorubicin (DOX-P) and peptide KRTGQYKLC-conjugated DSPE-PEG2000 were prepared. bFGF-DOX-MB had a normal distribution of particle size, with average particle size of 2.7 µm. Using A549 mouse model, bFGF-DOX-MB combined ultrasound showed the best inhibition effect on tumor volume growth among all the test groups. Similar conclusion was obtained from experimental measurements of tumor weight change and blood cell count. From the results, chemotherapeutic drug inhibition on tumor growth could be enhanced by local ultrasound combined with active targeting bFGF-DOX-MB, which might provide a potential application for ultrasound-mediated chemotherapy.