Ultrasound-mediated Blood-brain Barrier Research Articles

Closed-loop cavitation control for focused ultrasound-mediated blood–brain barrier opening by long-circulating microbubbles

Focused ultrasound (FUS) exposure in the presence of microbubbles (MBs) has been successfully used in the delivery of various sizes of therapeutic molecules across the blood–brain barrier (BBB). While acoustic pressure is correlated with the BBB opening size, real-time control of BBB opening to avoid vascular and neural damage is still a challenge. This arises mainly from the variability of FUS-MB interactions due to the variations of animal-specific metabolic environment and specific experimental setup. In this study, we demonstrate a closed-loop cavitation control framework to induce BBB opening for delivering large therapeutic molecules without causing macro tissue damages. To this end, we performed in mice long-term (5 min) cavitation monitoring facilitated by using long-circulating MBs. Monitoring the long-term temporal kinetics of the MBs under varying level of FUS pressure allowed to identify in situ, animal specific activity regimes forming pressure-dependent activity bands. This enables to determine the boundaries of each activity band (i.e. steady oscillation, transition, inertial cavitation) independent from the physical and physiological dynamics of the experiment. However, such a calibration approach is time consuming and to speed up characterization of the in situ, animal specific FUS-MB dynamics, we tested a novel method called ‘pre-calibration’ that closely reproduces the results of long-term monitoring but with a much shorter duration. Once the activity bands are determined from the pre-calibration method, an operation band can be selected around the desired cavitation dose. To drive cavitation in the selected operation band, we developed an adaptive, closed-loop controller that updates the acoustic pressure between each sonication based on measured cavitation dose. Finally, we quantitatively assessed the safety of different activity bands and validated the proposed methods and controller framework. The proposed framework serves to optimize the FUS pressure instantly to maintain the targeted cavitation level while improving safety control.

Abstract #14: Resting State Functional Connectivity Changes After MR-Guided Focused Ultrasound Mediated Blood-Brain Barrier Opening

Abstract #14: Resting State Functional Connectivity Changes After MR-Guided Focused Ultrasound Mediated Blood-Brain Barrier Opening

Repeated ultrasound treatment of tau transgenic mice clears neuronal tau by autophagy and improves behavioral functions.

Intracellular deposits of pathological tau are the hallmark of a broad spectrum of neurodegenerative disorders collectively known as tauopathies, with Alzheimer's disease, a secondary tauopathy, being further characterized by extracellular amyloid plaques. A major obstacle in developing effective treatments for tauopathies is the presence of the blood-brain barrier, which restricts the access of therapeutic agents to the brain. An emerging technology to overcome this limitation is the application of low-intensity ultrasound which, together with intravenously injected microbubbles, transiently opens the blood-brain barrier, thereby facilitating the delivery of therapeutic agents into the brain. Interestingly, even in the absence of therapeutic agents, ultrasound has previously been shown to reduce amyloid plaques and improve cognitive functions in amyloid-depositing mice through microglial clearance. Ultrasound has also been shown to facilitate the delivery of antibody fragments against pathological tau in P301L tau transgenic mice; however, the effect of ultrasound alone has not been thoroughly investigated in a tauopathy mouse model.Methods: Here, we performed repeated scanning ultrasound treatments over a period of 15 weeks in K369I tau transgenic mice with an early-onset tau-related motor and memory phenotype. We used immunohistochemical and biochemical methods to analyze the effect of ultrasound on the mice and determine the underlying mechanism of action, together with an analysis of their motor and memory functions following repeated ultrasound treatments.Results: Repeated ultrasound treatments significantly reduced tau pathology in the absence of histological damage. Associated impaired motor functions showed improvement towards the end of the treatment regime, with memory functions showing a trend towards improvement. In assessing potential clearance mechanisms, we ruled out a role for ubiquitination of tau, a prerequisite for proteasomal clearance. However, the treatment regime induced the autophagy pathway in neurons as reflected by an increase in the autophagosome membrane marker LC3II and a reduction in the autophagic flux marker p62, along with a decrease of mTOR activity and an increase in beclin 1 levels. Moreover, there was a significant increase in the interaction of tau and p62 in the ultrasound-treated mice, suggesting removal of tau by autophagosomes.Conclusions: Our findings indicate that a neuronal protein aggregate clearance mechanism induced by ultrasound-mediated blood-brain barrier opening operates for tau, further supporting the potential of low-intensity ultrasound to treat neurodegenerative disorders.

Therapeutic Effect of Cabazitaxel and Blood-Brain Barrier opening in a Patient-Derived Glioblastoma Model.

Treatment of glioblastoma and other diseases in the brain is especially challenging due to the blood-brain barrier, which effectively protects the brain parenchyma. In this study we show for the first time that cabazitaxel, a semi-synthetic derivative of docetaxel can cross the blood-brain barrier and give a significant therapeutic effect in a patient-derived orthotopic model of glioblastoma. We show that the drug crosses the blood-brain barrier more effectively in the tumor than in the healthy brain due to reduced expression of p-glycoprotein efflux pumps in the vasculature of the tumor. Surprisingly, neither ultrasound-mediated blood-brain barrier opening (sonopermeation) nor drug formulation in polymeric nanoparticles could increase either accumulation of the drug in the brain or therapeutic effect. This indicates that for hydrophobic drugs, sonopermeation of the blood brain barrier might not be sufficient to achieve improved drug delivery. Nonetheless, our study shows that cabazitaxel is a promising drug for the treatment of brain tumors.

Low-Intensity MR-Guided Focused Ultrasound Mediated Disruption of the Blood-Brain Barrier for Intracranial Metastatic Diseases.

Low-intensity MR-guided focused ultrasound in combination with intravenously injected microbubbles is a promising platform for drug delivery to the central nervous system past the blood-brain barrier. The blood-brain barrier is a key bottleneck for cancer therapeutics via limited inter- and intracellular transport. Further, drugs that cross the blood-brain barrier when delivered in a spatially nonspecific way, result in adverse effects on normal brain tissue, or at high concentrations, result in increasing risks to peripheral organs. As such, various anti-cancer drugs that have been developed or to be developed in the future would benefit from a noninvasive, temporary, and repeatable method of targeted opening of the blood-brain barrier to treat metastatic brain diseases. MR-guided focused ultrasound is a potential solution to these design requirements. The safety, feasibility and preliminary efficacy of MRgFUS aided delivery have been demonstrated in various animal models. In this review, we discuss this preclinical evidence, mechanisms of focused ultrasound mediated blood-brain barrier opening, and translational efforts to neuro-oncology patients.

Feedback control of microbubble cavitation for ultrasound-mediated blood–brain barrier disruption in non-human primates under magnetic resonance guidance

Focused ultrasound (FUS) in combination with microbubbles is capable of noninvasive, site-targeted delivery of drugs through the blood–brain barrier (BBB). Although acoustic parameters are reproducible in small animals, their control remains challenging in primates due to skull heterogeneity. This study describes a 7-T magnetic resonance (MR)-guided FUS system designed for BBB disruption in non-human primates (NHP) with a robust feedback control based on passive cavitation detection (PCD). Contrast enhanced T1-weighted MR images confirmed the BBB opening in NHP sonicated during 2 min with 500-kHz frequency, pulse length of 10 ms, and pulse repetition frequency of 5 Hz. The safe acoustic pressure range from 185 ± 22 kPa to 266 ± 4 kPa in one representative case was estimated from combining data from the acoustic beam profile with the BBB opening and hemorrhage profiles obtained from MR images. A maximum amount of MR contrast agent at focus was observed at 30 min after sonication with a relative contrast enhancement of 67% ± 15% (in comparison to that found in muscles). The feedback control based on PCD using relative spectra was shown to be robust, allowing comparisons across animals and experimental sessions. Finally, we also demonstrated that PCD can test acoustic coupling conditions, which improves the efficacy and safety of ultrasound transmission into the brain.

Establishing sheep as an experimental species to validate ultrasound-mediated blood-brain barrier opening for potential therapeutic interventions

Rationale: Treating diseases of the brain such as Alzheimer's disease (AD) is challenging as the blood-brain barrier (BBB) effectively restricts access of a large number of potentially useful drugs. A potential solution to this problem is presented by therapeutic ultrasound, a novel treatment modality that can achieve transient BBB opening in species including rodents, facilitated by biologically inert microbubbles that are routinely used in a clinical setting for contrast enhancement. However, in translating rodent studies to the human brain, the presence of a thick cancellous skull that both absorbs and distorts ultrasound presents a challenge. A larger animal model that is more similar to humans is therefore required in order to establish a suitable protocol and to test devices. Here we investigated whether sheep provide such a model.Methods: In a stepwise manner, we used a total of 12 sheep to establish a sonication protocol using a spherically focused transducer. This was assisted by ex vivo simulations based on CT scans to establish suitable sonication parameters. BBB opening was assessed by Evans blue staining and a range of histological tests.Results: Here we demonstrate noninvasive microbubble-mediated BBB opening through the intact sheep skull. Our non-recovery protocol allowed for BBB opening at the base of the brain, and in areas relevant for AD, including the cortex and hippocampus. Linear time-shift invariant analysis and finite element analysis simulations were used to optimize the position of the transducer and to predict the acoustic pressure and location of the focus.Conclusion: Our study establishes sheep as a novel animal model for ultrasound-mediated BBB opening and highlights opportunities and challenges in using this model. Moreover, as sheep develop an AD-like pathology with aging, they represent a large animal model that could potentially complement the use of non-human primates.

Three-dimensional transcranial microbubble imaging for guiding volumetric ultrasound-mediated blood-brain barrier opening.

Focused ultrasound (FUS)-mediated blood-brain barrier (BBB) opening recently entered clinical testing for targeted drug delivery to the brain. Sources of variability exist in the current procedures, motivating the development of real-time monitoring and control techniques to improve treatment safety and efficacy. Here we used three-dimensional (3D) transcranial microbubble imaging to calibrate FUS exposure levels for volumetric BBB opening.Methods: Using a sparse hemispherical transmit/receive ultrasound phased array, pulsed ultrasound was focused transcranially into the thalamus of rabbits during microbubble infusion and multi-channel 3D beamforming was performed online with receiver signals captured at the subharmonic frequency. Pressures were increased pulse-by-pulse until subharmonic activity was detected on acoustic imaging (psub), and tissue volumes surrounding the calibration point were exposed at 50-100%psub via rapid electronic beam steering.Results: Spatially-coherent subharmonic microbubble activity was successfully reconstructed transcranially in vivo during calibration sonications. Multi-point exposures induced volumetric regions of elevated BBB permeability assessed via contrast-enhanced magnetic resonance imaging (MRI). At exposure levels ≥75%psub, MRI and histological examination occasionally revealed tissue damage, whereas sonications at 50%psub were performed safely. Substantial intra-grid variability of FUS-induced bioeffects was observed via MRI, prompting future development of multi-point calibration schemes for improved treatment consistency. Receiver array sparsity and sensor configuration had substantial impacts on subharmonic detection sensitivity, and are factors that should be considered when designing next-generation clinical FUS brain therapy systems.Conclusion: Our findings suggest that 3D subharmonic imaging can be used to calibrate exposure levels for safe FUS-induced volumetric BBB opening, and should be explored further as a method for cavitation-mediated treatment guidance.

Ultrasound-mediated blood-brain barrier disruption: Correlation with acoustic emissions

Blood-brain barrier (BBB) disruption mediated by ultrasound and microbubbles (US-BBBD) is a promising strategy for non-invasive and targeted delivery of therapeutics to the brain. In US-BBBD, treatment control is achieved by externally monitoring acoustic emissions (AE) and adjusting ultrasound parameters in real-time to avoid AE associated with damage. Recent work suggests that AE may also provide insight regarding the extent of BBB opening and BBB recovery time. The mechanisms underlying BBB opening and recovery, however, are largely not understood. To investigate US-BBBD mechanisms with regard to AE, we developed an in vitro platform for monitoring both BBB integrity and AE during US-BBBD. Temporally resolved BBB integrity monitoring was achieved using a microfluidic BBB-on-a-chip device with integrated trans-endothelial electrical resistance (TEER) measurements. Well-characterized ultrasound exposure and AE monitoring were achieved using a focally aligned high-intensity focused ultrasound transducer and passive cavitation detector. In addition to recording TEER and AE data, our platform is compatible with fluorescence microscopy during ultrasound exposure, providing further insight into US-BBBD mechanisms. This work further demonstrates potential for in vitro screening of cavitation agents and/or therapeutics for novel US-BBBD applications and strategies.

Transcranial acoustic imaging for real-time control of ultrasound-mediated blood-brain barrier opening using a clinical-scale prototype system

Multichannel beamforming of passively detected ultrasound (US)-stimulated acoustic emissions is a promising method for guiding cavitation-mediated therapies. In the context of brain applications, our group and others have previously demonstrated the use of conventional beamforming techniques to transcranially map cavitation activity during microbubble (MB)-mediated blood-brain barrier (BBB) opening. MB activity can be mapped at pressure levels below the BBB opening threshold, allowing target confirmation prior to therapy delivery. By including skull-specific phase and amplitude corrections in the reconstruction process, the aberrating effects of the cranial bone can be compensated for to improve image quality. Recently, we have designed, fabricated, and characterized multi-frequency, transmit/receive, sparse hemispherical phased arrays for MB-mediated brain therapy and simultaneous cavitation mapping [Deng et al. , Phys. Med. Biol. 61, 8476-8501 (2016)]. This talk will review our progress to date in using these prototype systems to exploit the spatial information obtained from receive beamforming to actively modulate the therapeutic exposures during US-induced BBB opening, following our previously developed single-element internal calibration approach [O’Reilly & Hynynen, Radiology 263, 96-106 (2012)]. We anticipate that this technique will improve the safety and efficacy of MB-mediated BBB opening, as well as other future non-thermal US brain treatments such as cavitation-enhanced ablation, sonothrombolysis, and histotripsy.

The breakup of intravascular microbubbles and its impact on the endothelium.

Encapsulated microbubbles (MBs) serve as endovascular agents in a wide range of medical ultrasound applications. The oscillatory response of these agents to ultrasonic excitation is determined by MB size, gas content, viscoelastic shell properties and geometrical constraints. The viscoelastic parameters of the MB capsule vary during an oscillation cycle and change irreversibly upon shell rupture. The latter results in marked stress changes on the endothelium of capillary blood vessels due to altered MB dynamics. Mechanical effects on microvessels are crucial for safety and efficacy in applications such as focused ultrasound-mediated blood-brain barrier (BBB) opening. Since direct in vivo quantification of vascular stresses is currently not achievable, computational modelling has established itself as an alternative. We have developed a novel computational framework combining fluid-structure coupling and interface tracking to model the nonlinear dynamics of an encapsulated MB in constrained environments. This framework is used to investigate the mechanical stresses at the endothelium resulting from MB shell rupture in three microvessel setups of increasing levels of geometric detail. All configurations predict substantial elevation of up to 150% for peak wall shear stress upon MB breakup, whereas global peak transmural pressure levels remain unaltered. The presence of red blood cells causes confinement of pressure and shear gradients to the proximity of the MB, and the introduction of endothelial texture creates local modulations of shear stress levels. With regard to safety assessments, the mechanical impact of MB breakup is shown to be more important than taking into account individual red blood cells and endothelial texture. The latter two may prove to be relevant to the actual, complex process of BBB opening induced by MB oscillations.

Growth inhibition in a brain metastasis model by antibody delivery using focused ultrasound-mediated blood-brain barrier disruption

HER2-targeting antibodies (i.e. trastuzumab and pertuzumab) prolong survival in HER2-positive breast cancer patients with extracranial metastases. However, the response of brain metastases to these drugs is poor, and it is hypothesized that the blood-brain barrier (BBB) limits drug delivery to the brain. We investigated whether we could improve the response by temporary disruption of the BBB using focused ultrasound in combination with microbubbles. To study this, we inoculated 30 nude rats with HER2-positive cells derived from a brain metastasis of a breast cancer patient (MDA-MB-361). The animals were divided into three groups: a control-group that received no treatment; an antibody-only group that received six weekly treatments of trastuzumab and pertuzumab; and an ultrasound+antibody group that received trastuzumab and pertuzumab in combination with six weekly sessions of BBB disruption using focused ultrasound. In two animals, the leakiness of the tumors before disruption was evaluated using contrast-enhanced T1-weighted magnetic resonance imaging and found that the tumors were not leaky. The same technique was used to evaluate the effectiveness of BBB disruption, which was successful in all sessions.The tumor in the control animals grew exponentially with a growth constant of 0.042±0.011mm3/day. None of the antibody-only animals responded to the treatment and the growth constant was 0.033±0.009mm3/day during the treatment period. Four of the ten animals in the ultrasound+antibody-group showed a response to the treatment with an average growth constant of 0.010±0.007mm3/day, compared to a growth constant 0.043±0.013mm3/day for the six non-responders. After the treatment period, the tumors in all groups grew at similar rates. As the tumors were not leaky before BBB disruption and there were no responders in the antibody-only group, these results show that at least in some cases disruption of the BBB is necessary for a response to the antibodies in these brain metastases. Interestingly, only some of the rats responded to the treatment. We did not observe a difference in tumor volume at the start of the treatment, nor in HER2 expression or in contrast-enhancement on MRI between the responders and non-responders to explain this. Better understanding of why certain animals respond is needed and will help in translating this technique to the clinic. In conclusion, we demonstrate that BBB disruption using focused ultrasound in combination with antibody therapy can inhibit growth of breast cancer brain metastasis.

LIFESPAN OF ONE FOCUSED ULTRASOUND MEDIATED BLOOD-BRAIN BARRIER TREATMENT IN A MOUSE MODEL OF ALZHEIMER'S DISEASE

LIFESPAN OF ONE FOCUSED ULTRASOUND MEDIATED BLOOD-BRAIN BARRIER TREATMENT IN A MOUSE MODEL OF ALZHEIMER'S DISEASE

Multiple sessions of liposomal doxorubicin and focused ultrasound mediated blood-brain barrier disruption: safety study

Transcranial MRI-guided focused ultrasound is a rapidly advancing field for delivering therapeutic and imaging agents to the brain. It has the ability to facilitate the passage of therapeutics from the vasculature to the brain parenchyma, which is normally protected by the blood-brain barrier (BBB). Its main advantages are that it is targeted, noninvasive, and readily repeatable in nature. Studies have shown that it can deliver liposomal doxorubicin (DOX), a chemotherapy agent with promise for tumors in the central nervous system, into the brain across BBB. However, prior studies have suggested that this agent may be significantly neurotoxic, even at small concentrations. Here, we studied whether multiple sessions of DOX administered after FUS-induced BBB disruption (FUS-BBBD) caused adverse events in the normal brain. First, we used fluorometry to measure the doxorubicin concentrations in the brain after FUS-BBBD. Next, we performed three weekly sessions with FUS-BBBD ± DOX administration.

Localized delivery of non-viral gene-bearing nanoparticles into the rat brain following focused ultrasound-mediated BBB opening

By preventing more than 98% of currently used pharamaceutical agents from entering the brain, the blood-brain barrier critically reduces the ability of therapeutics to treat a variety of central nervous system (CNS) disorders including glioblastoma and neurodegenerative diseases. Focused ultrasound (FUS) mediated microbubble oscillation and subsequent blood brain barrier (BBB) permeabilization has been explored as a powerful non-invasive strategy for the delivery of circulating therapeutic agents into the CNS. FUS in conjunction with microbubbles has been shown to facilitate non-damaging, reversible and localized disruption of the BBB, leading to substantial increases in nanoparticle (NP) concentrations in ultrasound-treated tissue. Once beyond the BBB, the extracellular matrix acts as a steric and adhesive barrier and limits NP distribution. Coating sub-100 nm nanoparticles with a dense brush layer of polyethylene glycol (PEG) to limit the interactions with the ECM leads to a significant improvement of their diffusivity in brain tissue. The current study investigates the ability of FUS to deliver densely PEGylated brain penetrating cationic polymer-based gene vectors across the BBB to mediate robust and long-term transgene expression.

Therapeutic effects of focused ultrasound-mediated blood-brain barrier opening in a mouse model of Alzheimer’s disease

Focused ultrasound (FUS)-mediated opening of the blood-brain barrier (BBB) has been used to promote drug delivery to the brain. However, in a mouse model of Alzheimer’s disease, FUS has proven to be effective for reducing pathology and improving cognition, even in the absence of exogenous drug application. We have explored single and repeated FUS treatments in mice at both early and late stages of plaque pathology using a transgenic (Tg) mouse model of Alzheimer’s disease.

Multiple sessions of liposomal doxorubicin delivery via focused ultrasound mediated blood–brain barrier disruption: A safety study

Transcranial MRI-guided focused ultrasound is a rapidly advancing method for delivering therapeutic and imaging agents to the brain. It has the ability to facilitate the passage of therapeutics from the vasculature to the brain parenchyma, which is normally protected by the blood–brain barrier (BBB). The method's main advantages are that it is both targeted and noninvasive, and that it can be easily repeated. Studies have shown that liposomal doxorubicin (Lipo-DOX), a chemotherapy agent with promise for tumors in the central nervous system, can be delivered into the brain across BBB. However, prior studies have suggested that doxorubicin can be significantly neurotoxic, even at small concentrations. Here, we studied whether multiple sessions of Lipo-DOX administered after FUS-induced BBB disruption (FUS-BBBD) induces severe adverse events in the normal brain tissues. First, we used fluorometry to measure the doxorubicin concentrations in the brain after FUS-BBBD to ensure that a clinically relevant doxorubicin concentration was achieved in the brain. Next, we performed three weekly sessions with FUS-BBBD±Lipo-DOX administration. Five to twelve targets were sonicated each week, following a schedule described previously in a survival study in glioma-bearing rats (Aryal et al., 2013). Five rats received three weekly sessions where i.v. injected Lipo-DOX was combined with FUS-BBBD; an additional four rats received FUS-BBBD only. Animals were euthanized 70days from the first session and brains were examined in histology. We found that clinically-relevant concentrations of doxorubicin (4.8±0.5μg/g) were delivered to the brain with the sonication parameters (0.69MHz; 0.55–0.81MPa; 10ms bursts; 1Hz PRF; 60s duration), microbubble concentration (Definity, 10μl/kg), and the administered Lipo-DOX dose (5.67mg/kg) used. The resulting concentration of Lipo-DOX was reduced by 32% when it was injected 10min after the last sonication compared to cases where the agent was delivered before sonication. In histology, the severe neurotoxicity observed in some previous studies with doxorubicin by other investigators was not observed here. However, four of the five rats who received FUS-BBBD and Lipo-DOX had regions (dimensions: 0.5–2mm) at the focal targets with evidence of minor prior damage, either a small scar (n=4) or a small cyst (n=1). The focal targets were unaffected in rats who received FUS-BBBD alone. The result indicates that while delivery of Lipo-DOX to the rat brain might result in minor damage, the severe neurotoxicity seen in earlier works does not appear to occur with delivery via FUS-BBB disruption. The damage may be related to capillary damage produced by inertial cavitation, which might have resulted in excessive doxorubicin concentrations in some areas.

Improved Anti-Tumor Effect of Liposomal Doxorubicin After Targeted Blood-Brain Barrier Disruption by MRI-Guided Focused Ultrasound in Rat Glioma

The blood-brain barrier (BBB) inhibits the entry of the majority of chemotherapeutic agents into the brain. Previous studies have illustrated the feasibility of drug delivery across the BBB using focused ultrasound (FUS) and microbubbles. Here, we investigated the effect of FUS-enhanced delivery of doxorubicin on survival in rats with and 9L gliosarcoma cells inoculated in the brain. Each rat received either: (1) no treatment (control; N = 11), (2) FUS only (N = 9), (3) IV liposomal doxorubicin (DOX only; N = 17), or (4) FUS with concurrent IV injections of liposomal doxorubicin (FUS+DOX; N = 20). Post-treatment by magnetic resonance imaging (MRI) showed that FUS+DOX reduced tumor growth compared with DOX only. Further, we observed a modest but significant increase in median survival time after a single treatment FUS+DOX treatment (p = 0.0007), whereas neither DOX nor FUS had any significant impact on survival on its own. These results suggest that combined ultrasound-mediated BBB disruption may significantly increase the antineoplastic efficacy of liposomal doxorubicin in the brain.

Clearance of albumin following ultrasound-induced blood–brain barrier opening is mediated by glial but not neuronal cells

Ultrasound-mediated opening of the blood–brain barrier (BBB) in the presence of gas-filled microbubbles is a potential strategy for drug delivery across the blood–brain barrier to promote regeneration after ischemic stroke. However, related bioeffects and potential side-effects that could limit a translation into clinical application are poorly understood so far. We therefore examined the clearance of extravasated albumin following ultrasound-mediated BBB opening. Autofluorescence of albumin-bound Evans Blue dye indicated cellular albumin uptake as soon as 30 min after insonation (2 ± 0.72 cells/optical field). Cellular albumin uptake increased constantly over 24 h (22 ± 3.33 cells/optical field, p < 0.05). Initially, the majority of albumin-positive cells were located in the periphery of brain capillaries. Most albumin phagocyting cells stained positive for CD163 and Iba-1, identifying them as activated microglia. Further, a small fraction of albumin-positive cells stained positive for the astroglial markers GFAP/S100B. Some perivascular cells with intracellular albumin were shown to express the endothelial marker protein EN4. Albumin uptaking cells stained negative for the neuronal TubulinIII. Thus, ultrasound-induced BBB opening leads to albumin extravasation which is phagocytized predominantly by activated microglia, astrocytes and endothelial cells. As albumin uptake into neurons has been shown to be neurotoxic, rapid albumin clearance by microglia might prevent neuronal cell death.

Focused ultrasound-mediated bbb disruption is associated with an increase in activation of AKT: experimental study in rats

BackgroundThe Blood Brain Barrier (BBB) maintains the homeostasis of central nervous system by preventing the free passage of macromolecules from the systemic circulation into the brain. This normal physiological function of the BBB presents a challenge for delivery of therapeutic compounds into the brain. Recent studies have shown that the application of focused ultrasound together with ultrasound contrast agent (microbubbles) temporarily increases the permeability of the BBB. This effect is associated with breakdown of tight junctions, the structures that regulate the paracellular permeability of the endothelial cell layer. The influence of this ultrasound effect on the activation of intracellular signaling proteins is currently not well understood. Therefore, the aim of this study was to investigate the activation of cell survival signaling molecules in response to ultrasound-mediated BBB opening;MethodsThe BBB was disrupted in two four-spot lines (1-1.5 mm spacing) along the right hemisphere of rat brain with ultrasound beams (0.3 MPa, 120 s, 10 ms bursts, repetition frequency = 1 Hz) in the presence Definity microbubbles. Contrast-enhanced MRI images were acquired to assess the extent of BBB opening upon which the animals were sacrificed and the brains removed and processed for biochemical and immunohistochemical analyses;ResultsImmunoblotting of sonicated brain lysates resolved by SDS-PAGE demonstrated an increase in phosphorylation of Akt and its downstream signaling molecule, GSK3β, while the phosphorylation of MAPK remained unchanged. The elevated levels of pAkt and pGSK3β are still evident after 24 hours post-sonication, a time point where the integrity of the BBB is known to be re-established. Furthermore, immunofluoresence staining localized this increase in pAkt and pGSK3β levels to neuronal cells flanking the region of the disrupted BBB;ConclusionsOur data demonstrates that ultrasound-mediated BBB disruption causes an activation of the Akt signaling pathway in neuronal cells surrounding the disrupted BBB.