Induce Blood-brain Barrier Opening Research Articles

Multimodal evaluation of blood-brain barrier opening in mice in response to low-intensity ultrasound and a claudin-5 binder.

Background: The blood-brain barrier (BBB) is a major bottleneck in delivering therapeutics to the brain. Treatment strategies to transiently open this barrier include focused ultrasound combined with intravenously injected microbubbles (FUS+MB) and targeting of molecules that regulate BBB permeability. Methods: Here, we investigated BBB opening mediated by the claudin-5 binder cCPEm (a microorganismal toxin in a truncated form) and FUS+MB at a centre frequency of 1 MHz, assessing dextran uptake, broadband emission, and endogenous immunoglobulin G (IgG) extravasation. Results: FUS+MB-induced BBB opening was detectable at a pressure ≥0.35 MPa when assessed for leakage of 10 and 70 kDa dextran, and at ≥0.2 MPa for uptake of endogenous IgG. Treating mice with 20 mg/kg cCPEm failed to open the BBB, and pre-treatment with cCPEm followed by FUS+MB at 0.2 and 0.3 MPa did not overtly increase BBB opening compared to FUS+MB alone. Using passive cavitation detection (PCD), we found that broadband emission correlated with the peak negative pressure (PNP) and dextran leakage, indicating the possibility of using broadband emission for developing a feedback controller to monitor BBB opening. Conclusions: Together, our study highlights the challenges in developing combinatorial approaches to open the BBB and presents an additional IgG-based histological detection method for BBB opening.

Research and progress of focused ultrasound in the treatment of Alzheimer's disease.

Alzheimer's disease is one of the most common degenerative diseases of the central nervous system, with progressive cognitive and memory impairment and decreased ability of daily life as the cardinal symptoms, influencing the life quality of patients severely. There are currently approximately 46 million people living with Alzheimer's disease worldwide, and the number is expected to triple by 2050, which will pose a huge challenge for healthcare. At present, the Food and Drug Administration of the United States has approved five main drugs for the clinical treatment of Alzheimer's disease, which are cholinesterase inhibitors tacrine, galantamine, capalatine and donepezil, and N-methyl-d-aspartate receptor antagonist memantine, although these drugs have shown good efficacy in clinical trials, the actual clinical effect is less effective due to the existence of blood brain barrier. With the continuous development of ultrasound technology in recent years, focused ultrasound, as a non-invasive treatment technique, may target ultrasound energy to the deep brain for treatment without damaging the surrounding tissue. For the past few years, some studies could use focused ultrasound combined with microvesicles to induce blood brain barrier opening and targeted drug delivery to treat Alzheimer's disease, providing new opportunities for the treatment of Alzheimer's disease. This article reviews the application research and progress of focused ultrasound in the treatment of Alzheimer's disease, in order to provide new directions and ideas for the treatment of Alzheimer's disease.

Electromagnetic pulse induced blood-brain barrier breakdown through tight junction opening in rats.

The blood-brain barrier (BBB) is the main obstacle to hydrophilic and large molecules to enter the brain, maintaining the stability of the central nervous system (CNS). But many environmental factors may affect the permeability and structure of the BBB. Electromagnetic pulses (EMP) irradiation has been proven to enhance the permeability of the BBB, but the specific mechanism is still unclear. To explore the potential mechanism of EMP-induced BBB opening, this study investigated the permeability, fine structure and the proteins expression of the tight junction (TJ) of the BBB in the rats exposed to EMP. Using the leakage of fluorescein isothiocyanate-labeled dextran with different molecular mass under different field intensity of EMP exposure, we found that the tracer passing through the BBB is size-dependent in the rat exposed to EMP as field intensity increased. Transmission electron microscopy showed TJ of the endothelial cells in the EMP-exposed group was open, compared with the sham-irradiated group. But the levels of TJ proteins including ZO-1, claudin-5, or occludin were not changed as indicated by western blot. These data suggest that EMP induce BBB opening in a field intensity-dependent manner and probably through dysfunction of TJ proteins instead of their expression. Our findings increase the understanding of the mechanism for EMP working on the brain and are helpful for CNS protection against EMP.

Safety Profile of Low-Intensity Pulsed Ultrasound–Induced Blood–Brain Barrier Opening in Non-epileptic Mice and in a Mouse Model of Mesial Temporal Lobe Epilepsy

It is unknown whether ultrasound-induced blood-brain barrier (BBB) disruption can promote epileptogenesis and how BBB integrity changes over time after sonication. To gain more insight into the safety profile of ultrasound (US)-induced BBB opening, we determined BBB permeability as well as histological modifications in C57BL/6 adult control mice and in the kainate (KA) model for mesial temporal lobe epilepsy in mice after sonication with low-intensity pulsed ultrasound (LIPU). Microglial and astroglial changes in ipsilateral hippocampus were examined at different time points following BBB disruption by respectively analyzing Iba1 and glial fibrillary acidic protein immunoreactivity. Using intracerebral EEG recordings, we further studied the possible electrophysiological repercussions of a repeated disrupted BBB for seizure generation in nine non-epileptic mice. LIPU-induced BBB opening led to transient albumin extravasation and reversible mild astrogliosis, but not to microglial activation in the hippocampus of non-epileptic mice. In KA mice, the transient albumin extravasation into the hippocampus mediated by LIPU-induced BBB opening did not aggravate inflammatory processes and histologic changes that characterize the hippocampal sclerosis. Three LIPU-induced BBB opening did not induce epileptogenicity in non-epileptic mice implanted with depth EEG electrodes. Our experiments in mice provide persuasive evidence of the safety of LIPU-induced BBB opening as a therapeutic modality for neurological diseases.

Affordable stereotactic-guided focused ultrasound device for tunable drug delivery to the mouse brain

Focused ultrasound combined with microbubble (FUS+MB)-mediated blood brain barrier (BBB) opening is not only a promising technique for clinical applications but also a powerful tool for preclinical neuroscience and neuro-oncology research. However, existing FUS systems are expensive and lack the flexibility of modulating BBB opening volume, which prevents a broader research community from adopting the FUS+MB technique in preclinical studies. To address the challenge, we developed a low cost (∼$100), mini FUS transducer with the capability to modulate BBB opening size that can be readily integrated with a standard stereotaxic frame for mouse. Specifically, we manufactured in-house mini FUS transducers with three frequencies (1.5, 3.0, and 6.0 MHz), and quantified BBB opening and targeting accuracy at varying pressures (0.20 to 0.81 MPa). The volume of FUS+MB-induced BBB opening was evaluated using both contrast-enhanced MRI and Evans blue extravasation. Our results showed that we can achieve varying amount of Evans blue delivery and BBB opening size by modulating the transducer frequency and acoustic pressure. Additionally, the amount of Evans blue delivery and BBB opening had a significant linear correlation with the cavitation index (defined by the ratio between acoustic pressure and frequency). We believe that this stereotaxic-guided FUS system will lower the barrier for adopting the FUS technique in a broader research community.

Ternary Complexes of pDNA, Neuron-Binding Peptide, and PEGylated Polyethyleneimine for Brain Delivery with Nano-Bubbles and Ultrasound.

In brain-targeted delivery, the transport of drugs or genes across the blood−brain barrier (BBB) is a major obstacle. Recent reports found that focused ultrasound (FUS) with microbubbles enables transient BBB opening and improvement of drug or gene delivery. We previously developed nano-sized bubbles (NBs), which were prepared based on polyethylene glycol (PEG)-modified liposomes containing echo-contrast gas, and showed that our NBs with FUS could also induce BBB opening. The aim of this study was to enhance the efficiency of delivery of pDNA into neuronal cells following transportation across the BBB using neuron-binding peptides. This study used the RVG-R9 peptide, which is a chimeric peptide synthesized by peptides derived from rabies virus glycoprotein and nonamer arginine residues. The RVG peptide is known to interact specifically with the nicotinic acetylcholine receptor in neuronal cells. To enhance the stability of the RVG-R9/pDNA complex in vivo, PEGylated polyethyleneimine (PEG-PEI) was also used. The ternary complexes composed of RVG-R9, PEG-PEI, and pDNA could interact with mouse neuroblastoma cells and deliver pDNA into the cells. Furthermore, for the in vivo experiments using NBs and FUS, gene expression was observed in the FUS-exposed brain hemispheres. These results suggest that this systemic gene delivery system could be useful for gene delivery across the BBB.

Effect of ultra‑wide‑band electromagnetic pulses on blood‑brain barrier permeability in rats

The restrictive nature of the blood brain barrier (BBB) brings a particular challenge to the treatment of central nervous system (CNS) disorders. The effect of ultra-wide band electromagnetic pulses (UWB-EMPs) on BBB permeability was examined in the present study in order to develop a safe and effective technology that opens the BBB to improve treatment options for CNS diseases. Rats were exposed to a single UWB-EMP at various field strengths (50, 200 or 400 kV/m) and the BBB was examined using albumin immunohistochemistry and Evans blue staining at different time periods (0.5, 3, 6 and 24 h) after exposure. The expression and distribution of zonula occludens 1 (ZO-1) were evaluated using western blotting to identify a potential mechanism underlying BBB permeability. The results showed that the BBB permeability of rats exposed to UWB-EMP increased immediately following UWM-EMP treatment and peaked between 3 and 6 h after UWB-EMP exposure, returning to pre-exposure levels 24 h later. The data suggested that UWB-EMP at 200 and 400 kV/m could induce BBB opening, while 50 kV/m UWB-EMP could not. The levels of ZO-1 in the cerebral cortex were significantly decreased at 3 and 6 h after exposure; however, no change was observed in the distribution of ZO-1. The present study indicated that UWB-EMP-induced BBB opening was field strength-dependent and reversible. Decreased expression of ZO-1 may be involved in the effect of UWB-EMP on BBB permeability.

Characterization of Brain-Targeted Drug Delivery Enhanced by a Combination of Lipid-Based Microbubbles and Non-Focused Ultrasound

The combination of focused ultrasound (FUS) and microbubbles, an ultrasound (US) contrast agent, has attracted much attention for its ability to open the blood brain barrier (BBB) and deliver drugs to the brain parenchyma. FUS can concentrate US energy in a restricted space, whereas non-focused US can affect a wide area of tissue. Non-focused US is also promising for drug delivery to the brain and other tissues. We have previously developed lipid-based microbubbles (LBs), and demonstrated that non-focused US and LBs have potential for drug delivery to tumor tissues. In this study, to achieve efficient and safe brain-targeted drug delivery, we evaluated the characteristics of BBB opening using non-focused US and LBs. Our results indicated that LBs could induce BBB opening with non-focused US. US frequency and intensity affected the efficiency of BBB opening and brain damage, and showed that the dose of LBs was also related to the efficiency of BBB opening. Furthermore, the combination of non-focused US and LBs could deliver macromolecules at 2000 kDa to the brain, and the induction of BBB opening was found to be reversible. These results suggest that the combination of non-focused US and LBs has potential as a brain-targeted drug delivery system.

The neurovascular response is attenuated by focused ultrasound-mediated disruption of the blood-brain barrier

Focused ultrasound (FUS)-induced disruption of the blood-brain barrier (BBB) is a non-invasive method to target drug delivery to specific brain areas that is now entering into the clinic. Recent studies have shown that the method has several secondary effects on local physiology and brain function beyond making the vasculature permeable to normally non-BBB penetrant molecules. This study uses functional MRI methods to investigate how FUS BBB opening alters the neurovascular response in the rat brain. Nine rats underwent actual and sham FUS induced BBB opening targeted to the right somatosensory cortex (SI) followed by four runs of bilateral electrical hind paw stimulus-evoked fMRI. The neurovascular response was quantified using measurements of the blood oxygen level dependent (BOLD) signal and cerebral blood flow (CBF). An additional three rats underwent the same FUS-BBB opening followed by stimulus-evoked fMRI with high resolution BOLD imaging and BOLD imaging of a carbogen-breathing gas challenge. BOLD and CBF measurements at two different stimulus durations demonstrate that the neurovascular response to the stimulus is attenuated in both amplitude and duration in the region targeted for FUS-BBB opening. The carbogen results show that the attenuation in response amplitude, but not duration, is still present when the signaling mechanism originates from changes in blood oxygenation instead of stimulus-induced neuronal activity. There is some evidence of non-local effects, including a possible global decrease in baseline CBF. All effects are resolved by 24 h after FUS-BBB opening. Taken together, these results suggest that FUS-BBB opening alters that state of local brain neurovascular physiology in such a way that hinders its ability to respond to demands for increased blood flow to the region. The mechanisms for this effect need to be elucidated.

Focused ultrasound-induced blood brain-barrier opening enhanced vascular permeability for GDNF delivery in Huntington's disease mouse model

BackgroundHuntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the gene encoding the huntingtin (Htt) protein, which results in a protein containing an abnormally expanded polyglutamine (polyQ) sequence. The expanded polyQ in the Htt protein is toxic to brain cells. No therapy exists to delay disease progression. MethodsThis study describes a gene-liposome system that synergistically applied focused ultrasound (FUS)-blood-brain barrier (BBB) opening for rescuing motor and neuropathological impairments when administered from pre to post-symptomatic transgenic mouse models of HD. DPPC liposomes (LPs) are designed to carry glia cell line-derived neurotrophic factor (GDNF) plasmid DNA (GDNFp) to form a GDNFp-liposome (GDNFp-LPs) complex. Pulsed FUS exposure with microbubbles (MBs) was used to induce BBB opening for non-viral, non-invasive, and targeted gene delivery into the central nervous system (CNS) for therapeutic purposes. ResultsFUS-gene therapy significantly improved motor performance with GDNFp-LPs + FUS treated HD mice equilibrating longer periods in the animal behavior. Reflecting the improvements observed in motor function, GDNF overexpression results in significantly decreased formation of polyglutamine-expanded aggregates, reduced oxidative stress and apoptosis, promoted neurite outgrowth, and improved neuronal survival. Immunoblotting and histological staining further confirmed the neuroprotective effect from delivery of GDNF genes to neuronal cells. ConclusionsThis study suggests that the GDNFp-LPs plus FUS sonication can provide an effective gene therapy to achieve local extravasation and triggered gene delivery for non-invasive in vivo treatment of CNS diseases.

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.

Focused Ultrasound Induced-Blood-Brain Barrier Opening in Post-Radiosurgery Mouse Brain for Locally-Enhanced Systemic Therapy

Focused ultrasound (FUS) is a novel technique that is capable of non-invasively and transiently open the blood-brain barrier (BBB). It has been show that FUS can facilitate the delivery of systemically administered therapeutic agents. Our goal here is to investigate FUF induced BBB opening in a post stereotactic radiosurgery (SRS) mouse model for novel locally-enhanced systemic therapy. We hypothesized that microvascular effects of radiation could modulate the BBB-opening induced by FUS. In this study, normal brains of C57B5 mice were treated with a fractionated SRS dose, typically given to resected tumor cavities. A single beam radiation (6Gy x 5 daily fractions) was delivered through a 10×10 mm2 collimator to half of the mouse brain using the Small Animal Radiation Research Platform (SARRP). The dose delivered to the mice was verified via two mouse-like anthropomorphic phantoms in the axial and sagittal orientations. Upon the completion of SARRP treatments, mice were divided into two groups for FUS-induced BBB opening on day 7 (acute) and day 35 (chronic), respectively. A 1.5 MHz single element FUS transducer (-6dB focal zone was 1×1×7.5 mm3) was used for all sonications. The caudate-putamen (CPu) was the FUS target and four 30s sonications were delivered on each side of the brain. The BBB opening was confirmed with contrast-enhanced T1-weighted MRI scans, while dynamic MRI scans were also performed to evaluate the blood vessel permeability. The animals were sacrificed 2 hours post sonications and lectin was injected to label the vasculature. The isodose lines from the mouse-like phantom experiment agreed well with the projected values. BBB was successfully opened after FUS sonications on both sides of the brain (with or without radiation) as evidenced by contrast-enhanced MRI. In the acute group, higher gadolinium concentration (p = 0.072) was observed on the RT positive side. MR dynamic imaging revealed a similar trend in which RT positive side had a higher (p = 0.158) vascular permeability. Although the mean vessel area in the sonicated area remained similar between the two sides, significantly less lectin labeling (blood vessel density) was observed on the RT positive hemisphere (p = 0.003) in the acute group. These observations can be attributed to increased vasculature vulnerability immediately post SRS (within 2 days) resulting in more profound BBB opening. Interestingly, this difference diminished one month later in the chronic group, where FUS elicited comparable BBB openings and minimal vascular density changes in the CPu on both sides. Localized BBB opening can be successfully induced by FUS in previously stereotactically irradiated normal brains. BBB openings were evident in both acute and chronic settings with potentially more profound outcomes if FUS is performed within 2 days of RT treatment.

Abstract 025: Cellular Mechanisms of Blood-Brain Barrier Disruption in Ang II-Induced Hypertension

The blood-brain barrier (BBB) is critically important for brain health by regulating molecular exchanges between blood and brain. Hypertension (HTN) induces breakdown of the BBB which may contribute to its deleterious effect on the brain, but the cellular bases of the BBB opening remain to be established. We used a model of angiotensin II (AngII) HTN (600ng/kg/min x 2 weeks; n>5/group) to investigate the mechanisms of the BBB opening. BBB permeability was assessed spectrophotometrically in C57BL/6J mice using 3000 MW FITC-dextran as a tracer. HTN increased BBB permeability in Ang II HTN (29.8 ± 0.9 vs 18.1 ± 0.5 ng/g in control, p<0.01), an effect partially ameliorated by the angiotensin type 1 receptor (AT1R) antagonist losartan in the drinking water (22.9 ± 0.6 ng/g) but not by hydralazine + hydrochlorothiazide (27.4 ± 0.7 ng/g), suggesting involvement of AT1R and not elevated blood pressure. Next, we sought to identify the mechanisms of the breakdown of the BBB in HTN. First, we used electron microscopy to examine the ultrastructure of endothelial tight junctions (TJ) and assess vesicular transport, key components of the BBB. HTN reduced the length (-25%) and complexity (-11%) of TJ, and increased the number of endothelial vesicles (2.22 vs 1.42 vesicles/endothelial area, p<0.05). The TJ remodeling was associated with a reduction in occludin (-17%, p<0.01) and claudin-5 (-30%, p<0.05) mRNA in microvascular preparations. Additionally, the expression of Mfsd2a, a lipid transporter that also suppresses vesicular transcytosis, was markedly attenuated (-43%, p<0.01). Taken together, these data suggest a strong effect of Ang II on cerebral endothelial cells to induce BBB opening. Since perivascular macrophages (PVM) mediate cerebral endothelial dysfunction in HTN (J Clin Invest 2016;126:4674), we tested their involvement in the BBB opening. PVM depletion with icv clodronate or deletion of AT1 receptors in PVM partially attenuated the BBB opening in Ang II HTN (p<0.05). Thus, we conclude that AT1R in cerebral endothelial cells and PVM mediate the BBB opening by targeting both paracellular and vesicular transport. Such increase in BBB permeability to circulating agents may contribute to the cerebrovascular and cognitive dysfunction associated with HTN.

Quantitative assessment of the blood-brain barrier opening caused by Streptococcus agalactiae hyaluronidase in a BALB/c mouse model

Streptococcus agalactiae is a pathogen causing meningitis in animals and humans. However, little is known about the entry of S. agalactiae into brain tissue. In this study, we developed a BALB/c mouse model based on the intravenous injection of β-galactosidase-positive Escherichia coli M5 as an indicator of blood-brain barrier (BBB) opening. Under physiological conditions, the BBB is impermeable to E. coli M5. In pathological conditions caused by S. agalactiae, E. coli M5 is capable of penetrating the brain through a disrupted BBB. The level of BBB opening can be assessed by quantitative measurement of E. coli M5 loads per gram of brain tissue. Further, we used the model to evaluate the role of S. agalactiae hyaluronidase in BBB opening. The inactivation of hylB gene encoding a hyaluronidase, HylB, resulted in significantly decreased E. coli M5 colonization, and the intravenous injection of purified HylB protein induced BBB opening in a dose-dependent manner. This finding verified the direct role of HylB in BBB invasion and traversal, and further demonstrated the practicability of the in vivo mouse model established in this study. This model will help to understand the S. agalactiae–host interactions that are involved in this bacterial traversal of the BBB and to develop efficacious strategies to prevent central nervous system infections.

Integration of focused ultrasound with nanomedicine for brain drug delivery

Nanomedicine is the next-generation technology for cancer therapy. Liposomes are one of such nanomedicine that have well-established clinical applications. However, their large sizes make it challenging to deliver them to the brain due to the existence of the blood-brain barrier (BBB). Previous studies have demonstrated the potential of integrating focused ultrasound (FUS)-induced BBB opening with liposomes for brain drug delivery. However, no study has characterized the spatial distributions of the liposomes and released drugs in the brain parenchyma at a microscopic level after the BBB was disrupted by the FUS. In this study, we manufactured liposomes with a fluorescent dye embedded in the lipid layer and chemotherapeutic drug doxorubicin encapsulated in the core. These liposomes were intravenously injected into the mouse followed by FUS sonication of a targeted brain location in the presence of microbubbles. The mouse brains were coronally sectioned and imaged using fluorescence microscopy. Preliminary results confirmed the successful delivery of doxorubicin to the brain. However, the liposome distribution as indicated by the dye embedded in the lipid shell was not spatially correlated with the distribution of the doxorubicin. Further studies are needed to better understand the mechanism of FUS-enabled liposome delivery.

Characterization of Different Microbubbles in Assisting Focused Ultrasound-Induced Blood-Brain Barrier Opening

Microbubbles (MBs) serve as a critical catalyst to amplify local cavitation in CNS capillary lumen to facilitate focused ultrasound (FUS) to transiently open the blood-brain barrier (BBB). However, limited understanding is available regarding the effect of different microbubbles to induce BBB opening. The aim of this study is to characterize different MBs on their effect in FUS-induced BBB opening. Three MBs, SonoVue, Definity, and USphere, were tested, with 0.4-MHz FUS exposure at 0.62–1.38 of mechanical index (MI) on rats. Evans blue, dynamic contrast-enhanced (DCE) MRI and small-animal ultrasound imaging were used as surrogates to allow molecule-penetrated quantification, BBB-opened observation, and MBs circulation/persistence. Cavitation activity was measured via the passive cavitation detection (PCD) setup to correlate with the exposure level and the histological effect. Under given and identical MB concentrations, the three MBs induced similar and equivalent BBB-opening effects and persistence. In addition, a treatment paradigm by adapting exposure time is proposed to compensate MB decay to retain the persistence of BBB-opening efficiency in multiple FUS exposures. The results potentially improve understanding of the equivalence among MBs in focused ultrasound CNS drug delivery, and provide an effective strategy for securing persistence in this treatment modality.

PEGylated PLGA-based phase shift nanodroplets combined with focused ultrasound for blood brain barrier opening in rats

Previous studies have shown that focused ultrasound (FUS) combined with systematic administration of microbubbles (MBs) can open the blood brain barrier (BBB) locally, transiently and reversibly. However, because of the micro size diameters, MBs are restricted in the intravascular space and cannot extravasate into diseased sites through the opened BBB. In this study, we fabricated one kind of nanoscale droplets which consisted of encapsulated liquid perfluoropentane cores and poly (ethyleneglycol) - poly (lactide-co-glycolic acid) shells. The nanodroplets had the capacity to realize liquid to gas phase shift under FUS. Significant extravasation of Evan's blue appeared when acoustic pressure reached 1.0 MPa. Intracerebral hemorrhages and erythrocyte extravasations were observed when the pressure was increased to 1.5 MPa. Prolonged sonication duration could enhance the level of BBB opening and broaden the time window simultaneously. Furthermore, compared with MBs, the distribution of EB extravasation was firmly confined within narrow region in the center of focal zone, suggesting the site of FUS induced BBB opening could be controlled with high precision by this procedure. Our results show the feasibility of serving PEGylated PLGA-based phase shift nanodroplet as an effective alternative mediating agent for FUS induced BBB opening.

FOCUSED ULTRASOUND USING NEUROTROPHIC FACTORS FOR THE TREATMENT OF NEURODEGENERATIVE DISEASE

Focused Ultrasound (FUS) in conjunction with microbubbles has been shown to induce a safe, transient and localized opening of the blood-brain barrier (BBB). In this study, neurotrophic delivery through the opened BBB was studied in mice undergoing neurodegeneration. Wild-type mice received a sub-acute MPTP regimen, and the lesions were allowed to stabilize for 4 weeks. Mice then received sonication (FUS+) or not (FUS-), and NTN (NTN+) or saline (NTN-) IV injection, they were divided into 3 groups (FUS+/NTN+, FUS+/NTN-, FUS-/NTN-), and they were allowed to survive for 4 weeks. The caudate and the putamen were targeted using FUS (center frequency: 1.5 MHz, PRF: 10 Hz, acoustic pressure: 0.45 MPa; pulse length: 10,000 cycles;sonication duration: 60 s) immediately after the IV administration of monodisperse bubbles (diameter: 4-5 μm, 2.5*108#/kg). The mice were survived for 28 days before sacrifice for TH staining. Immunohistochemistry revealed statistically significant (p<.05) neurorestoration of the dopaminergic neurons only in the (FUS+/NTN+) group and only in the ipsilateral (FUS+) substantia nigra and caudate putamen. Since the MPTP lesions and the DA neuronal apoptosis preceded NTN delivery by 4 weeks, neuroregeneration was concluded for this group. Neurorestoration was shown to be feasible using neurotrophic factors through the induced BBB opening using focused ultrasound in a neurodegenerative mouse model.

Neuromodulation accompanying focused ultrasound-induced blood-brain barrier opening.

Burst-mode focused ultrasound (FUS) induces microbubble cavitation in the vasculature and temporarily disrupts the blood-brain barrier (BBB) to enable therapeutic agent delivery. However, it remains unclear whether FUS-induced BBB opening is accompanied by neuromodulation. Here we characterized the functional effects of FUS-induced BBB opening by measuring changes in somatosensory evoked potentials (SSEPs) and blood-oxygen-level dependent (BOLD) responses. Rats underwent burst-mode FUS (mechanical index (MI) of 0.3, 0.55 or 0.8) to the forelimb region in the left primary somatosensory cortex to induce BBB opening. Longitudinal measurements were followed for up to 1 week to characterize the temporal dynamics of neuromodulation. We observed that 0.8-MI FUS profoundly suppressed SSEP amplitude and prolonged latency, and this effect lasted 7 days. 0.55-MI FUS resulted in minimal and short-term suppression of SSEP for less than 60 minutes and didn’t affect latency. BOLD responses were also suppressed in an MI-dependent manner, mirroring the effect on SSEPs. Furthermore, repetitive delivery of 0.55-MI FUS every 3 days elicited no accumulative effects on SSEPs or tissue integrity. This is the first evidence that FUS-induced BBB opening is accompanied by reversible changes in neuron responses, and may provide valuable insight toward the development of FUS-induced BBB opening for clinical applications.

Chirp- and random-based coded ultrasonic excitation for localized blood-brain barrier opening

Chirp- and random-based coded excitation methods have been proposed to reduce standing wave formation and improve focusing of transcranial ultrasound. However, no clear evidence has been shown to support the benefits of these ultrasonic excitation sequences in vivo. This study evaluates the chirp and periodic selection of random frequency (PSRF) coded-excitation methods for opening the blood-brain barrier (BBB) in mice. Three groups of mice (n = 15) were injected with polydisperse microbubbles and sonicated in the caudate putamen using the chirp/PSRF coded (bandwidth: 1.5–1.9 MHz, peak negative pressure: 0.52 MPa, duration: 30 s) or standard ultrasound (frequency: 1.5 MHz, pressure: 0.52 MPa, burst duration: 20 ms, duration: 5 min) sequences. T1-weighted contrast-enhanced MRI scans were performed to quantitatively analyze focused ultrasound induced BBB opening. The mean opening volumes evaluated from the MRI were mm3, mm3and mm3 for the chirp, random and regular sonications, respectively. The mean cavitation levels were V.s, V.s and V.s for the chirp, random and regular sonications, respectively. The chirp and PSRF coded pulsing sequences improved the BBB opening localization by inducing lower cavitation levels and smaller opening volumes compared to results of the regular sonication technique. Larger bandwidths were associated with more focused targeting but were limited by the frequency response of the transducer, the skull attenuation and the microbubbles optimal frequency range. The coded methods could therefore facilitate highly localized drug delivery as well as benefit other transcranial ultrasound techniques that use higher pressure levels and higher precision to induce the necessary bioeffects in a brain region while avoiding damage to the surrounding healthy tissue.