Ultrasound Blood-brain Barrier Opening Research Articles

Ultrasound Blood–Brain Barrier Opening and Aducanumab in Alzheimer’s Disease

SummaryAntiamyloid antibodies have been used to reduce cerebral amyloid-beta (Aβ) load in patients with Alzheimer’s disease. We applied focused ultrasound with each of six monthly aducanumab infusions to temporarily open the blood–brain barrier with the goal of enhancing amyloid removal in selected brain regions in three participants over a period of 6 months. The reduction in the level of Aβ was numerically greater in regions treated with focused ultrasound than in the homologous regions in the contralateral hemisphere that were not treated with focused ultrasound, as measured by fluorine-18 florbetaben positron-emission tomography. Cognitive tests and safety evaluations were conducted over a period of 30 to 180 days after treatment. (Funded by the Harry T. Mangurian, Jr. Foundation and the West Virginia University Rockefeller Neuroscience Institute.)

Acoustically targeted noninvasive gene therapy in large brain volumes.

Focused Ultrasound Blood-Brain Barrier Opening (FUS-BBBO) can deliver adeno-associated viral vectors (AAVs) to treat genetic disorders of the brain. However, such disorders often affect large brain regions. Moreover, the applicability of FUS-BBBO in the treatment of brain-wide genetic disorders has not yet been evaluated. Herein, we evaluated the transduction efficiency and safety of opening up to 105 sites simultaneously. Increasing the number of targeted sites increased gene delivery efficiency at each site. We achieved transduction of up to 60% of brain cells with comparable efficiency in the majority of the brain regions. Furthermore, gene delivery with FUS-BBBO was safe even when all 105 sites were targeted simultaneously without negative effects on animal weight or neuronal loss. To evaluate the application of multi-site FUS-BBBO for gene therapy, we used it for gene editing using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system and found effective gene editing, but also a loss of neurons at the targeted sites. Overall, this study provides a brain-wide map of transduction efficiency, shows the synergistic effect of multi-site targeting on transduction efficiency, and is the first example of large brain volume gene editing after noninvasive gene delivery with FUS-BBBO.

Focused Ultrasound-Mediated Blood–Brain Barrier Opening Best Promotes Neuroimmunomodulation through Brain Macrophage Redistribution

Neuroimmunomodulation is a promising form of drug-free treatment for neurological diseases ranging from Alzheimer’s disease to depression. The evidence supporting the efficacy of focused ultrasound (FUS) neuroimmunomodulation is encouraging; however, the method has yet to be standardized, and its mechanism remains poorly understood. Methods of FUS neuroimmunomodulation can be categorized into three paradigms based on the parameters used. In the first paradigm, focused ultrasound blood–brain barrier opening (FUS-BBBO) combines FUS with microbubbles (MB) to transiently and safely induce BBB opening. In the second paradigm, focused ultrasound neuromodulation (FUS-N) harnesses the acoustic effects of FUS alone (without MB). In the third paradigm, focused ultrasound with microbubbles without BBBO (FUS + MB) combines MB with FUS below the BBBO pressure threshold—harnessing the mechanical effects of FUS without opening the barrier. Due to the recent evidence of brain macrophage modulation in response to FUS-BBBO, we provide the first direct comparison of brain macrophage modulation between all three paradigms both in the presence and absence of Alzheimer’s disease (AD) pathology. Flow cytometry and single-cell sequencing are employed to identify FUS-BBBO as the FUS paradigm, which maximizes brain macrophage modulation, including an increase in the population of neuroprotective, disease-associated microglia and direct correlation between treatment cavitation dose and brain macrophage phagocytosis. Next, we combine spatial and single-cell transcriptomics with immunohistochemical validation to provide the first characterization of brain macrophage distribution in response to FUS-BBBO. Given their relevance within neurodegeneration and perturbation response, we emphasize the analysis of three brain macrophage populations—disease- and interferon-associated microglia and central-nervous-system-associated macrophages. We find and validate the redistribution of each population with an overall trend toward increased interaction with the brain–cerebrospinal fluid barrier (BCSFB) after FUS-BBBO, an effect that is found to be more pronounced in the presence of disease pathology. This study addresses the prior lack of FUS neuroimmunomodulation paradigm optimization and mechanism characterization, identifying that FUS-BBBO best modulates brain macrophage response via complex redistribution.

874 Direct Visualization of Planned Resection Volumes with Microbubble-Enhanced MR-Guided Focused Ultrasound Blood-Brain Barrier Opening and Sodium Fluorescein in Infiltrating Gliomas

INTRODUCTION: Surgical resection of infiltrating gliomas (IG,Grade II-IV) can be challenging, as many do not consistently enhance on gadolinium-enhanced MRI. Evidence of survival benefit of supratotal resection is mounting. No method exists to preoperatively demarcate a planned surgical resection volume. Such physical demarcation could significantly improve the resection of IGs. METHODS: Four patients were treated (3M:1F), with a mean age of 32.5 (29-36). All were suspected of having or were previously diagnosed with a supratentorial IG. Patients underwent awake transcranial MB-FUS BBBO of the lesion bed and perilesional areas planned for resection at the discretion of the operating neurosurgeon (GFW). Subsequently, patients were transported directly to the operating room. For each patient, sodium fluorescein was administered, and then tumors were resected with standard craniotomies under general anesthesia, using neuronavigation and fluorescent microscopy. RESULTS: Patients tolerated the procedure well without adverse events with a median follow-up of 15.7 months. The mean tumor volume was 18.2 cm3 (6.8-32.9). The demarcated planned resection volume was visualized with fluorescent microscopy. Fluorescing tissue was completely resected. Gross-total or supratotal resection was achieved in all patients. Pathologies included diffuse astrocytoma, oligodendroglioma, and anaplastic oligodendroglioma. CONCLUSIONS: MB-FUS BBBO and sodium fluorescein present a safe and novel method of demarcating a preoperatively planned surgical resection volume with resultant intraoperative visualization in IGs. This method is resilient to brain shift, and is an addition to the current repertoire for visualizing non-contrast-enhancing gliomas. The same method can be investigated for use in epilepsy surgery.

MRI-guided focused ultrasound blood-brain barrier opening increases drug delivery and efficacy in a diffuse midline glioma mouse model.

Diffuse intrinsic pontine glioma (DIPG) is the most common and deadliest pediatric brainstem tumor and is difficult to treat with chemotherapy in part due to the blood-brain barrier (BBB). Focused ultrasound (FUS) and microbubbles (MBs) have been shown to cause BBB opening, allowing larger chemotherapeutics to enter the parenchyma. Panobinostat is an example of a promising in vitro agent in DIPG with poor clinical efficacy due to low BBB penetrance. In this study, we hypothesized that using FUS to disrupt the BBB allows higher concentrations of panobinostat to accumulate in the tumor, providing a therapeutic effect. Mice were orthotopically injected with a patient-derived diffuse midline glioma (DMG) cell line, BT245. MRI was used to guide FUS/MB (1.5 MHz, 0.615 MPa peak negative pressure, 1 Hz pulse repetition frequency, 10-ms pulse length, 3 min treatment time)/(25 µL/kg, i.v.) targeting to the tumor location. In animals receiving panobinostat (10 mg/kg, i.p.) in combination with FUS/MB, a 3-fold increase in tumor panobinostat concentration was observed, without significant increase of the drug in the forebrain. In mice receiving 3 weekly treatments, the combination of panobinostat and FUS/MB led to a 71% reduction of tumor volumes (P = .01). Furthermore, we showed the first survival benefit from FUS/MB improved delivery increasing the mean survival from 21 to 31 days (P < .0001). Our study demonstrates that FUS-mediated BBB disruption can increase the delivery of panobinostat to an orthotopic DMG tumor, providing a strong therapeutic effect and increased survival.

Cavitation Feedback Control of Focused Ultrasound Blood-Brain Barrier Opening for Drug Delivery in Patients with Parkinson's Disease.

Magnetic resonance-guided focused ultrasound (MRgFUS), in conjunction with circulating microbubbles, is an emerging technology that can transiently enhance the permeability of the blood-brain barrier (BBB) locally and non-invasively to facilitate targeted drug delivery to the brain. In this clinical trial, the feasibility and safety of BBB modulation in the putamen were evaluated for biweekly therapeutic agent delivery in patients with Parkinson's disease. The performance of the clinical MRgFUS system's cavitation feedback controller for active power modulation throughout the exposures was examined. The putamen was targeted unilaterally by an ExAblate Neuro MRgFUS system operating at 220 kHz. Definity microbubbles were infused via a saline bag gravity drip at a rate of 4 µL/kg per 5 min. A cavitation emissions-based feedback controller was employed to modulate the acoustic power automatically according to prescribed target cavitation dose levels. BBB opening was measured by Gadolinium (Gd)-enhanced T1-weighted MR imaging, and the presence of potential micro-hemorrhages induced by the exposures was assessed via T2*-weighted MR imaging. A total of 12 treatment sessions were carried out across four patients, with target cavitation dose levels ranging from 0.20-0.40. BBB permeability in the targeted putamen was elevated successfully in all treatments, with a 14% ± 6% mean increase in Gd-enhanced T1-weighted MRI signal intensity relative to the untreated contralateral side. No indications of red blood cell extravasations were observed on MR imaging scans acquired one day following each treatment session. The cavitation emissions-based feedback controller was effective in modulating acoustic power levels to ensure BBB permeability enhancement while avoiding micro-hemorrhages, however, further technical advancements are warranted to improve its performance for use across a wide variety of brain diseases.

Equivalent time active cavitation imaging

Rationale. Despite the development of a large number of neurologically active drugs, brain diseases are difficult to treat due to the inability of many drugs to penetrate the blood-brain barrier. High-intensity focused ultrasound (HIFU) blood-brain barrier opening in a site-specific manner could significantly expand the spectrum of available drug treatments. However, without monitoring, brain damage and off-target effects can occur during these treatments. While some methods can monitor inertial cavitation, temperature increase, or passively monitor cavitation events, to the best of our knowledge none of them can actively and spatiotemporally map the HIFU pressure field during treatment. Methods. Here we detail the development of a novel ultrasound imaging modality called equivalent time active cavitation imaging (ETACI) capable of characterizing the HIFU pressure field through stable cavitation events across the field of view with an ultrafast active imaging setup. This work introduces (1) a novel plane wave sequence whose transmit delays increase linearly with transmit events enabling the sampling of high-frequency cavitation events, and (2) an algorithm allowing the processing of the microbubble signal for pressure field mapping. The pressure measurements with our modality were first carried out in vitro for hydrophone comparison and then in vivo during blood-brain barrier opening treatment in mice. Results. This study demonstrates the capability of ETACI to spatiotemporally characterize a modulation pressure field with an active imaging setup. The resulting pressure field mapping reveals a good correlation with hydrophone measurements. Further results iareprovided experimentally in vivo with promising results. Conclusion. This proof of concept establishes the first steps towards a novel ultrasound modality for monitoring focused ultrasound blood-brain barrier opening, allowing new possibilities for a safe and precise monitoring method.

Cavitation-modulated inflammatory response following focused ultrasound blood-brain barrier opening

Focused ultrasound (FUS) in combination with systemically injected microbubbles can be used to non-invasively open the blood-brain barrier (BBB) in targeted regions for a variety of therapeutic applications. Over the past two decades, preclinical research into the safety and efficacy of FUS-induced BBB opening has proven this technique to be transient and efficacious, propelling FUS-induced BBB opening into several clinical trials in recent years. However, as clinical trials further progress, the neuroinflammatory response to FUS-induced BBB opening needs to be better understood. In this study, we provide further insight into the relationship of microbubble cavitation and the resulting innate immune response to FUS-induced BBB opening. By keeping ultrasound parameters fixed (i.e. frequency, pressure, pulse length, etc.), three groups of mice were sonicated using a real-time cavitation controller until a target cavitation dose was reached (1 x 107 V2•s, 5 x 107 V2•s, 1 x 108 V2•s). The change in relative gene expression of the mouse inflammatory cytokines and receptors were evaluated at three different time-points (6 h, 24 h, and 72 h) after FUS. At both 6 and 24 h time-points, significant changes in relative gene expression of inflammatory cytokines and receptors were observed across all cavitation groups. However, the degree of changes in relative expression levels and the number of genes with significant changes in expression varied across the cavitation groups. Groups with a higher cavitation dose exhibited both greater changes in relative expression levels and greater number of significant changes. By 72 h post-opening, the gene expression levels returned to baseline in all cavitation dose groups, signifying a transient inflammatory response to FUS-induced BBB opening at the targeted cavitation dose levels. Furthermore, the real-time cavitation controller was able to produce consistent and significantly different BBB permeability enhancement volumes across the three different cavitation dose groups. These results indicate that cavitation monitoring and controlling during FUS-induced BBB opening can be used to potentially modulate or limit the degree of neuroinflammation, further emphasizing the importance of implementing cavitation controllers as FUS-induced BBB opening is translated into the clinic.

Real-time cavitation control and monitoring for focused ultrasound blood-brain barrier opening via dB-based subject calibrated feedback controller

With proposed the dB-based subject calibrated feedback controller, we can achieve different levels of blood-brain barrier opening by adjusting the desired dB level.

Focused ultrasound blood brain barrier opening mediated delivery of MRI-visible albumin nanoclusters to the rat brain for localized drug delivery with temporal control

There is an ongoing need for noninvasive tools to manipulate brain activity with molecular, spatial and temporal specificity. Here we have investigated the use of MRI-visible, albumin-based nanoclusters for noninvasive, localized and temporally specific drug delivery to the rat brain. We demonstrated that IV injected nanoclusters could be deposited into target brain regions via focused ultrasound facilitated blood brain barrier opening. We showed that nanocluster location could be confirmed in vivo with MRI. Additionally, following confirmation of nanocluster delivery, release of the nanocluster payload into brain tissue can be triggered by a second focused ultrasound treatment performed without circulating microbubbles. Release of glutamate from nanoclusters in vivo caused enhanced c-Fos expression, indicating that the loading capacity of the nanoclusters is sufficient to induce neuronal activation. This novel technique for noninvasive stereotactic drug delivery to the brain with temporal specificity could provide a new way to study brain circuits in vivo preclinically with high relevance for clinical translation.

Blood-brain barrier opening with low intensity pulsed ultrasound for immune modulation and immune therapeutic delivery to CNS tumors.

Opening of the blood-brain barrier (BBB) by pulsed low intensity ultrasound has been developed during the last decade and is now recognized as a safe technique to transiently and repeatedly open the BBB. This non- or minimally invasive technique allows for a targeted and uniform dispersal of a wide range of therapeutic substances throughout the brain, including immune cells and antibodies. In this review article, we summarize pre-clinical studies that have used BBB-opening by pulsed low intensity ultrasound to enhance the delivery of immune therapeutics and effector cell populations, as well as several recent clinical studies that have been initiated. Based on this analysis, we propose immune therapeutic strategies that are most likely to benefit from this strategy. The literature review and trial data research were performed using Medline/Pubmed databases and clinical trial registry www.clinicaltrials.gov . The reference lists of all included articles were searched for additional studies. A wide range of immune therapeutic agents, including small molecular weight drugs, antibodies or NK cells, have been safely and efficiently delivered to the brain with pulsed low intensity ultrasound in preclinical models, and both tumor control and increased survival have been demonstrated in different types of brain tumor models in rodents. Ultrasound-induced BBB disruption may also stimulate innate and cellular immune responses. Ultrasound BBB opening has just recently entered clinical trials with encouraging results, and the association of this strategy with immune therapeutics creates a new field of brain tumor treatment.

The effect of ultrasound pulse length on microbubble cavitation induced antibody accumulation and distribution in a mouse model of breast cancer

In solid tumors, the limited diffusion of therapeutic molecules in the perivascular space is a known limitation impacting treatment efficacy. Ultrasound Targeted Microbubble Cavitation (UTMC) has been shown to increase vascular permeability and improve the delivery of therapeutic compounds including small molecules, antibodies (mAb), nanoparticles and even cells, notably across the blood-brain-barrier (BBB). In this study, we hypothesized that UTMC could improve the accumulation and biodistribution of mAb targeting the adenosinergic pathway (i.e. CD73) in mice bearing bilateral subcutaneous 4T1 mammary carcinoma. METHODS: A bolus of fluorescently labeled mAb was given intravenously, followed by a slow infusion of microbubbles. UTMC therapy (1 MHz, 850 kPa) was given under ultrasound image guidance for 5 minutes to the right side tumor only, using three different pulse lengths with identical ultrasound energy (5000cyc “long”, 125x40cyc “mid” and 500x10cyc “short”), and leaving the left tumor as a paired control. Longitudinal accumulation at 0 h, 4 h and 24 h was measured using whole-body biofluorescence and confocal microscopy. RESULTS: Our data support an increase in antibody accumulation and extravasation (# extravasated vessels and extravasated signal intensity) at 0 h for all pulses and at 4 h for the mid and short pulses when compared to the control non treated side. However, this difference was not found at 24 h post UTMC, indicative of the transient nature of UTMC. Interestingly, confocal data supported that the highest extravasation range was obtained at 0 h with the long pulse and that the short pulse caused no increase in the extravasation range. Overall, the mid pulse was the only pulse to increase all our metrics (biofluorescence, fraction of extravasated vessels, amount of extravasated Ab, and extravasation range) at 0 h and 4 h time points. CONCLUSIONS: Our results support that UTMC can enhance antibody accumulation in solid tumors at the macroscopic and microscopic levels. This preferential accumulation was evident at early time points (0 h and 4 h) but had started to fade by 24 h, a time dependence that is consistent with the ultrasound blood brain barrier opening literature. Further development and optimization of this theranostic platform, such as repeated UTMC, could help improve antibody based therapies against solid cancer.

Focused ultrasound in Parkinson's disease: A twofold path toward disease modification.

A major unmet need in Parkinson's disease (PD) is to slow the inexorable progression of neurodegeneration. Clinical trials that evaluated promising pharmacological strategies have repeatedly failed. Nonetheless, the advent of focused ultrasound provides new opportunities toward the goal of developing a safe and effective disease-modifying therapy for PD. Here we discuss the rationale, possible avenues, and challenges along this path, exploiting the potential of focused ultrasound for (1) performing focal thermal lesions to restore the basic basal ganglia abnormalities associated with dopamine depletion, and (2) transiently opening the blood-brain barrier for targeted delivery of therapeutic agents. First, the classic idea of excitotoxicity mediated by hyperactivity of the subthalamic nucleus suggests that focused ultrasound subthalamotomy may offer a clinically viable disease-modifying therapy in very-early PD. Second, the concept of retrograde nigrostriatal neurodegeneration, supported by our recent cortical pathogenic theory of PD, points toward the putamen as a principal site for focused ultrasound blood-brain barrier opening and targeted drug delivery. In principle, both therapeutic strategies-subthalamotomy and putaminal blood-brain barrier opening-could eventually be applied in the same patient. Clinical application is still a long road ahead; nevertheless, focused ultrasound may open a twofold path toward disease modification in PD. © 2019 International Parkinson and Movement Disorder Society.

Safety evaluation of frequent application of microbubble-enhanced focused ultrasound blood-brain-barrier opening

Focused ultrasound (FUS) with the presence of microbubbles induces blood brain barrier (BBB) opening in targeted areas and facilitates drug delivery. However, recent studies have indicated that FUS-BBB opening with excessive exposure levels may be associated with inflammatory response and cellular/tissue damage. Multiple weekly FUS exposures have been shown to be safe for human subjects. However the effect of more frequent FUS exposures is still unknown. This study examines whether frequent focused ultrasound blood brain barrier opening is associated with aggravated behavioral, histopathologic change or brain tissue damage. Two protocols of focused ultrasound blood brain barrier opening were devised using different microbubble doses (0.15 µl/kg and 0.4 µl/kg). Focused ultrasound exposure at a threshold level of BBB-opening, below-threshold level, or above level for intracerebral hemorrhage were delivered every 2 days. Animal behavioral and physiological changes were examined and recorded. Brain tissue was examined for hemorrhage and apoptosis. Results indicate that frequent exposure of excessive focused ultrasound (1.4 mechanical index) produced minor and short-term behavioral changes despite significant tissue damage, while frequent BBB opening with threshold or below-threshold FUS exposure (0.33–0.8 mechanical index) did not cause behavioral or histological change. Immunofluorescent examination of rat brain tissue indicated that excessive doses of microbubble administration induce an apparent cellular apoptotic response, which may be exacerbated by intracerebral hemorrhage. Experimental results suggest that frequent focused ultrasound blood brain barrier opening with sufficient ultrasound exposure level and a microbubble dose can be safe and pose minimal risk to brain tissue.

Real-time monitoring of focused ultrasound blood-brain barrier opening via subharmonic acoustic emission detection: implementation of confocal dual-frequency piezoelectric transducers

Burst-tone focused ultrasound exposure in the presence of microbubbles has been demonstrated to be effective at inducing temporal and local opening of the blood-brain barrier (BBB), which promises significant clinical potential to deliver therapeutic molecules into the central nervous system (CNS). Traditional contrast-enhanced imaging confirmation after focused ultrasound (FUS) exposure serves as a post-operative indicator of the effectiveness of FUS-BBB opening, however, an indicator that can concurrently report the BBB status and BBB-opening effectiveness is required to provide effective feedback to implement this treatment clinically. In this study, we demonstrate the use of subharmonic acoustic emission detection with implementation on a confocal dual-frequency piezoelectric ceramic structure to perform real-time monitoring of FUS-BBB opening.A confocal dual-frequency (0.55 MHz/1.1 MHz) focused ultrasound transducer was designed. The 1.1 MHz spherically-curved ceramic was employed to deliver FUS exposure to induce BBB-opening, whereas the outer-ring 0.55 MHz ceramic was employed to detect the subharmonic acoustic emissions originating from the target position. In stage-1 experiments, we employed spectral analysis and performed an energy spectrum density (ESD) calculation. An optimized 0.55 MHz ESD level change was shown to effectively discriminate the occurrence of BBB-opening. Wideband acoustic emissions received from 0.55 MHz ceramics were also analyzed to evaluate its correlations with erythrocyte extravasations. In stage-2 real-time monitoring experiments, we applied the predetermined ESD change as a detection threshold in PC-controlled algorithm to predict the FUS exposure intra-operatively.In stage-1 experiment, we showed that subharmonic ESD presents distinguishable dynamics between intact BBB and opened BBB, and therefore a threshold ESD change level (5.5 dB) can be identified for BBB-opening prediction. Using this ESD change threshold detection as a surrogate to on/off control the FUS exposure in stage-2 experiments, we demonstrated both excellent sensitivity (92%) and specificity (92.3%) in discriminating BBB-opening occurrence can be obtained in animal treatments, while concurrently achieving a high positive predicted value (95.8%). Wideband ESD was also highly correlated with the occurrence and level of erythrocyte extravasations (r 2 = 0.81).The proposed system configuration and corresponding analysis based on subharmonic acoustic emissions has the potential to be implemented as a real-time feedback control structure for reliable indication of intact FUS-BBB opening for CNS brain drug delivery.

Behavioral effects of targeted drug delivery via non-invasive microbubble enhanced focused ultrasound blood brain barrier opening in non-human primates

The Blood Brain Barrier (BBB) in Non-Human Primates (NHP) can be non-invasively opened through the use of Focused Ultrasound (FUS) in conjunction with microbubbles. This procedure allows for a targeted, transient opening in the BBB of the NHP which can be utilized to facilitate drug delivery. While FUS has been used to deliver various pharmacological compounds to promote neurogenesis or treat cancer, no group has investigated if drug delivery can affect behavioral responses. In this study, we show the effects of targeted ME-FUS drug delivery on the responses of NHP to a decision making task.

Reorganization of Gap Junctions after Focused Ultrasound Blood–Brain Barrier Opening in the Rat Brain

Ultrasound-induced opening of the blood-brain barrier (BBB) is an emerging technique for targeted drug delivery to the central nervous system. Gap junctions allow transfer of information between adjacent cells and are responsible for tissue homeostasis. We examined the effect of ultrasound-induced BBB opening on the structure of gap junctions in cortical neurons, expressing Connexin 36, and astrocytes, expressing Connexin 43, after focused 1-MHz ultrasound exposure at 1.25 MPa of one hemisphere together with intravenous microbubble (Optison, Oslo, Norway) application. Quantification of immunofluorescence signals revealed that, compared with non-insonicated hemispheres, small-sized Connexin 43 and 36 gap-junctional plaques were markedly reduced in areas with BBB breakdown after 3 to 6 hours (34.02+/-6.04% versus 66.49+/-2.16%, P=0.02 for Connexin 43; 33.80+/-1.24% versus 36.77+/-3.43%, P=0.07 for Connexin 36). Complementing this finding, we found significant increases in large-sized gap-junctional plaques (5.76+/-0.96% versus 1.02+/-0.84%, P=0.05 for Connexin 43; 5.62+/-0.22% versus 4.65+/-0.80%, P=0.02 for Connexin 36). This effect was reversible at 24 hours after ultrasound exposure. Western blot analyses did not show any change in the total connexin amount. These results indicate that ultrasound-induced BBB opening leads to a reorganization of gap-junctional plaques in both neurons and astrocytes. The plaque-size increase may be a cellular response to imbalances in extracellular homeostasis after BBB leakage.