Airy-beam holographic sonogenetics for advancing neuromodulation precision and flexibility.

Advancing our understanding of brain function and developing treatments for neurological diseases hinge on the ability to modulate neuronal groups in specific brain areas without invasive techniques. Here, we introduce Airy-beam holographic sonogenetics (AhSonogenetics) as an implant-free, cell type-specific, spatially precise, and flexible neuromodulation approach in freely moving mice. AhSonogenetics utilizes wearable ultrasound devices manufactured using 3D-printed Airy-beam holographic metasurfaces. These devices are designed to manipulate neurons genetically engineered to express ultrasound-sensitive ion channels, enabling precise modulation of specific neuronal populations. By dynamically steering the focus of Airy beams through ultrasound frequency tuning, AhSonogenetics is capable of modulating neuronal populations within specific subregions of the striatum. One notable feature of AhSonogenetics is its ability to flexibly stimulate either the left or right striatum in a single mouse. This flexibility is achieved by simply switching the acoustic metasurface in the wearable ultrasound device, eliminating the need for multiple implants or interventions. AhSonogentocs also integrates seamlessly with in vivo calcium recording via fiber photometry, showcasing its compatibility with optical modalities without cross talk. Moreover, AhSonogenetics can generate double foci for bilateral stimulation and alleviate motor deficits in Parkinson's disease mice. This advancement is significant since many neurological disorders, including Parkinson's disease, involve dysfunction in multiple brain regions. By enabling precise and flexible cell type-specific neuromodulation without invasive procedures, AhSonogenetics provides a powerful tool for investigating intact neural circuits and offers promising interventions for neurological disorders.

Static Magnetic Fields Dampen Focused Ultrasound-mediated Blood-Brain Barrier Opening.

Background Focused ultrasound combined with microbubbles has been used in clinical studies for blood-brain barrier (BBB) opening in conjunction with MRI. However, the impact of the static magnetic field generated by an MRI scanner on the BBB opening outcome has not been evaluated. Purpose To determine the relationship of the static magnetic field of an MRI scanner on focused ultrasound combined with microbubble-induced BBB opening. Materials and Methods Thirty wild-type mice were divided into four groups. Mice from different groups were sonicated with focused ultrasound in different static magnetic fields (approximately 0, 1.5, 3.0, and 4.7 T), with all other experimental parameters kept the same. Focused ultrasound sonication was performed after intravenous injection of microbubbles. Microbubble cavitation activity, the fundamental -physical mechanism underlying focused ultrasound BBB opening, was monitored with passive cavitation detection. After sonication, contrast-enhanced T1-weighted MRI was performed to assess BBB opening outcome. Intravenously injected Evans blue was used as a model agent to evaluate trans-BBB delivery efficiency. Results The microbubble cavitation dose decreased by an average of 2.1 dB at 1.5 T (P = .05), 2.9 dB at 3.0 T (P = .01), and 3.0 dB at 4.7 T (P = .01) compared with that outside the magnetic field (approximately 0 T). The static magnetic field of an MRI scanner decreased BBB opening volume in mice by 3.2-fold at 1.5 T (P < .001), 4.5-fold at 3.0 T (P < .001), and 11.6-fold at 4.7 T (P <.001) compared with mice treated outside the magnetic field. It also decreased Evans blue trans-BBB delivery 1.4-fold at 1.5 T (P = .009), 1.6-fold at 3.0 T (P < .001), and 1.9-fold at 4.7 T (P < .001). Conclusion Static magnetic fields dampened microbubble cavitation activity and decreased trans-blood-brain barrier (BBB) delivery by focused ultrasound combined with microbubble-induced BBB opening. © RSNA, 2021 An earlier incorrect version of this article appeared online. This article was corrected on July 8, 2021.

PINK1/Parkin-Mediated Mitophagy Promotes Resistance to Sonodynamic Therapy.

BACKGROUND/AIMS: Sonodynamic therapy (SDT), based on the synergistic effect of low-intensity ultrasound and sonosensitizer, is a potential approach for non-invasive treatment of cancers. In SDT, mitochondria played a crucial role in cell fate determination. However, mitochondrial activities and their response to SDT remain elusive. The purpose of this study was to examine the response of mitochondria to SDT in tumor cells. METHODS: A human breast adenocarcinoma cell line - MCF-7 cells were subjected to 5-aminolevulinic acid (ALA)-SDT, with an average ultrasonic intensity of 0.25W/cm2. Mitochondrial dynamics and redox balance were examined by confocal immunofluorescence microscopy and western blot. The occurrence of mitophagy was determined by confocal immunofluorescence microscopy. RESULTS: Our results showed that ALA-SDT could induce mitochondrial dysfunction through mitochondrial depolarization and fragmentation and lead to mitophagy. The Parkin-dependent signaling pathway was involved and promoted resistance to ALA-SDT induced cell death. Finally, excessive production of ROS was found to be necessary for the initiation of mitophagy. CONCLUSION: Taken together, we conclude that ROS produced by 5-ALA-SDT could initiate PINK1/Parkin-mediated mitophagy which may exert a protective effect against 5-ALA-SDT-induced cell death in MCF-7 cells.

Adeno-associated virus vector delivery to the brain: Technology advancements and clinical applications.

Adeno-associated virus (AAV) vectors have emerged as a promising tool in the development of gene therapies for various neurological diseases, including Alzheimer's disease and Parkinson's disease. However, the blood-brain barrier (BBB) poses a significant challenge to successfully delivering AAV vectors to the brain. Strategies that can overcome the BBB to improve the AAV delivery efficiency to the brain are essential to successful brain-targeted gene therapy. This review provides an overview of existing strategies employed for AAV delivery to the brain, including direct intraparenchymal injection, intra-cerebral spinal fluid injection, intranasal delivery, and intravenous injection of BBB-permeable AAVs. Focused ultrasound has emerged as a promising technology for the noninvasive and spatially targeted delivery of AAV administered by intravenous injection. This review also summarizes each strategy's current preclinical and clinical applications in treating neurological diseases. Moreover, this review includes a detailed discussion of the recent advances in the emerging focused ultrasound-mediated AAV delivery. Understanding the state-of-the-art of these gene delivery approaches is critical for future technology development to fulfill the great promise of AAV in neurological disease treatment.

Targeted delivery of therapeutic agents to the mouse brain using a stereotactic-guided focused ultrasound device.

Existing protocols of focused ultrasound (FUS) combined with microbubble-mediated blood-brain barrier (BBB) opening (FUS-BBBO) in preclinical research require expensive ultrasound equipment and complex operating procedures. We developed a low-cost, easy-to-use, and precise FUS device for small animal models in preclinical research. Here, we provide a detailed protocol for building the FUS transducer, attaching the transducer to a stereotactic frame for precise brain targeting, applying the integrated FUS device to perform FUS-BBBO in mice, and evaluating the FUS-BBBO outcome. For complete details on the use and execution of this protocol, please refer to Hu et al. (2022).1.

TRPV1-mediated sonogenetic neuromodulation of motor cortex in freely moving mice.

Background.Noninvasive and cell-type-specific neuromodulation tools are critically needed for probing intact brain function. Sonogenetics for noninvasive activation of neurons engineered to express thermosensitive transient receptor potential vanilloid 1 (TRPV1) by transcranial focused ultrasound (FUS) was recently developed to address this need. However, using TRPV1-mediated sonogenetics to evoke behavior by targeting the cortex is challenged by its proximity to the skull due to high skull absorption of ultrasound and increased risks of thermal-induced tissue damage.Objective.This study evaluated the feasibility and safety of TRPV1-mediated sonogenetics in targeting the motor cortex to modulate the locomotor behavior of freely moving mice.Approach.Adeno-associated viral vectors was delivered to the mouse motor cortex via intracranial injection to express TRPV1 in excitatory neurons. A wearable FUS device was installed on the mouse head after a month to control neuronal activity by activating virally expressed TRPV1 through FUS sonication at different acoustic pressures. Immunohistochemistry staining ofex vivobrain slices was performed to verify neuron activation and evaluate safety.Results.TRPV1-mediated sonogenetic stimulation at 0.7 MPa successfully evoked rotational behavior in the direction contralateral to the stimulation site, activated cortical neurons as indicated by the upregulation of c-Fos, and did not induce significant changes in inflammatory or apoptotic markers (GFAP, Iba1, and Caspase-3). Sonogenetic stimulation of TRPV1 mice at a higher acoustic pressure, 1.1 MPa, induced significant changes in motor behavior and upregulation of c-Fos compared with FUS sonication of naïve mice at 1.1 MPa. However, signs of damage at the meninges were observed at 1.1 MPa.Significance.TRPV1-mediated sonogenetics can achieve effective and safe neuromodulation at the cortex with carefully selected FUS parameters. These findings expand the application of this technique to include superficial brain targets.

First-in-human prospective trial of sonobiopsy in high-grade glioma patients using neuronavigation-guided focused ultrasound.

Sonobiopsy is an emerging technology that combines focused ultrasound (FUS) with microbubbles to enrich circulating brain disease-specific biomarkers for noninvasive molecular diagnosis of brain diseases. Here, we report the first-in-human prospective trial of sonobiopsy in high-grade glioma patients to evaluate its feasibility and safety in enriching plasma circulating tumor biomarkers. A nimble FUS device integrated with a clinical neuronavigation system was used to perform sonobiopsy following an established clinical workflow for neuronavigation. Analysis of blood samples collected before and after FUS sonication showed that sonobiopsy enriched plasma circulating tumor DNA (ctDNA), including a maximum increase of 1.6-fold for the mononucleosome cell-free DNA (cfDNA) fragments (120-280 bp), 1.9-fold for the patient-specific tumor variant ctDNA level, and 5.6-fold for the TERT mutation ctDNA level. Histological analysis of surgically resected tumors confirmed the safety of the procedure. Transcriptome analysis of sonicated and nonsonicated tumor tissues found that FUS sonication modulated cell physical structure-related genes. Only 2 out of 17,982 total detected genes related to the immune pathways were upregulated. These feasibility and safety data support the continued investigation of sonobiopsy for noninvasive molecular diagnosis of brain diseases.

Induction of a torpor-like hypothermic and hypometabolic state in rodents by ultrasound.

Torpor is an energy-conserving state in which animals dramatically decrease their metabolic rate and body temperature to survive harsh environmental conditions. Here, we report the noninvasive, precise and safe induction of a torpor-like hypothermic and hypometabolic state in rodents by remote transcranial ultrasound stimulation at the hypothalamus preoptic area (POA). We achieve a long-lasting (>24 h) torpor-like state in mice via closed-loop feedback control of ultrasound stimulation with automated detection of body temperature. Ultrasound-induced hypothermia and hypometabolism (UIH) is triggered by activation of POA neurons, involves the dorsomedial hypothalamus as a downstream brain region and subsequent inhibition of thermogenic brown adipose tissue. Single-nucleus RNA-sequencing of POA neurons reveals TRPM2 as an ultrasound-sensitive ion channel, the knockdown of which suppresses UIH. We also demonstrate that UIH is feasible in a non-torpid animal, the rat. Our findings establish UIH as a promising technology for the noninvasive and safe induction of a torpor-like state.

First-in-human prospective trial of sonobiopsy in glioblastoma patients using neuronavigation-guided focused ultrasound.

Sonobiopsy is an emerging technology that combines focused ultrasound (FUS) with microbubbles to enrich circulating brain disease-specific biomarkers for noninvasive molecular diagnosis of brain diseases. Here, we report the first-in-human prospective trial of sonobiopsy in glioblastoma patients to evaluate its feasibility and safety in enriching circulating tumor biomarkers. A nimble FUS device integrated with a clinical neuronavigation system was used to perform sonobiopsy following an established clinical workflow for neuronavigation. Analysis of blood samples collected before and after FUS sonication showed enhanced plasma circulating tumor biomarker levels. Histological analysis of surgically resected tumors confirmed the safety of the procedure. Transcriptome analysis of sonicated and unsonicated tumor tissues found that FUS sonication modulated cell physical structure-related genes but evoked minimal inflammatory response. These feasibility and safety data support the continued investigation of sonobiopsy for noninvasive molecular diagnosis of brain diseases.

An Affordable and Easy-to-Use Focused Ultrasound Device for Noninvasive and High Precision Drug Delivery to the Mouse Brain.

OBJECTIVE: Focused ultrasound (FUS) combined with microbubble-mediated blood-brain barrier (BBB) opening (FUS-BBBO) is not only a promising technique for clinical applications but also a powerful tool for preclinical research. However, existing FUS devices for preclinical research are expensive, bulky, and lack the precision needed for small animal research, which limits the broad adoption of this promising technique by the research community. Our objective was to design and fabricate an affordable, easy-to-use, high-precision FUS device for small animal research. METHODS: We designed and fabricated in-house mini-FUS transducers (∼$80 each in material cost) with three frequencies (1.5, 3.0, and 6.0 MHz) and integrated them with a stereotactic frame for precise mouse brain targeting using established stereotactic procedures. The BBB opening volume by FUS at different acoustic pressures (0.20-0.57 MPa) was quantified using T1-weighted contrast-enhanced magnetic resonance imaging of gadolinium leakage and fluorescence imaging of Evans blue extravasation. RESULTS: The targeting accuracy of the device as measured by the offset between the desired target location and the centroid of BBBO was 0.63 ± 0.19 mm. The spatial precision of the device in targeting individual brain structures was improved by the use of higher frequency FUS transducers. The BBB opening volume had high linear correlations with the cavitation index (defined by the ratio between acoustic pressure and frequency) and mechanical index (defined by the ratio between acoustic pressure and the square root of frequency). The correlation coefficient of the cavitation index was slightly higher than that of the mechanical index. CONCLUSION: This study demonstrated that spatially accurate and precise BBB opening was achievable using an affordable and easy-to-use FUS device. The BBB opening volume was tunable by modulating the cavitation index. This device is expected to decrease the barriers to the adoption of the FUS-BBBO technique by the broad research community.

Blood-Brain Barrier Opening by Individualized Closed-Loop Feedback Control of Focused Ultrasound.

Objective and Impact Statement. To develop an approach for individualized closed-loop feedback control of microbubble cavitation to achieve safe and effective focused ultrasound in combination with microbubble-induced blood-brain barrier opening (FUS-BBBO). Introduction. FUS-BBBO is a promising strategy for noninvasive and localized brain drug delivery with a growing number of clinical studies currently ongoing. Real-time cavitation monitoring and feedback control are critical to achieving safe and effective FUS-BBBO. However, feedback control algorithms used in the past were either open-loop or without consideration of baseline cavitation level difference among subjects. Methods. This study performed feedback-controlled FUS-BBBO by defining the target cavitation level based on the baseline stable cavitation level of an individual subject with "dummy" FUS sonication. The dummy FUS sonication applied FUS with a low acoustic pressure for a short duration in the presence of microbubbles to define the baseline stable cavitation level that took into consideration of individual differences in the detected cavitation emissions. FUS-BBBO was then achieved through two sonication phases: ramping-up phase to reach the target cavitation level and maintaining phase to control the stable cavitation level at the target cavitation level. Results. Evaluations performed in wild-type mice demonstrated that this approach achieved effective and safe trans-BBB delivery of a model drug. The drug delivery efficiency increased as the target cavitation level increased from 0.5 dB to 2 dB without causing vascular damage. Increasing the target cavitation level to 3 dB and 4 dB increased the probability of tissue damage. Conclusions. Safe and effective brain drug delivery was achieved using the individualized closed-loop feedback-controlled FUS-BBBO.

Airy Beam-enabled Binary Acoustic Metasurfaces for Underwater Ultrasound Beam Manipulation.

Airy beams are peculiar beams that are non-diffracting, self-accelerating, and self-healing, and they have offered great opportunities for ultrasound beam manipulation. However, one critical barrier that limits the broad applications of Airy beams in ultrasound is the lack of simply built device to generate Airy beams in water. This work presents a family of Airy beam-enabled binary acoustic metasurfaces (AB-BAMs) to generate Airy beams for underwater ultrasound beam manipulation. AB-BAMs are designed and fabricated by 3D printing with two coding bits: a polylactic acid (which is the commonly used 3D printing material) unit acting as a bit "1" and a water unit acting as a bit "0". The distribution of the binary units on the metasurface is determined by the pattern of Airy beam. To showcase the wavefront engineering capability of the AB-BAMs, several examples of AB-BAMs are designed, 3D printed, and coupled with a planar single-element ultrasound transducer for experimental validation. We demonstrate the capability of AB-BAMs in flexibly tuning the focal region size and beam focusing in 3D space by changing the design of the AB-BAMs. The focal depth of AB-BAMs can be continuous and electronical tuned by adjusting the operating frequency of the planar transducer without replacing the AB-BAMs. The superimposing method is leveraged to enable the generation of complex acoustic fields, e.g., multi-foci and letter patterns (e.g., "W" and "U"). The more complex focal patterns are shown to be also continuously steerable by simply adjusting the operating frequency. Furthermore, the proposed 3D-printed AB-BAMs are simple to design, easy to fabricate, and low-cost to produce with the capabilities to achieve tunable focal size, flexible 3D beam focusing, arbitrary multipoint focusing, and continuous steerability, which creates unprecedented potential for ultrasound beam manipulation.

Binary acoustic metasurfaces for dynamic focusing of transcranial ultrasound.

Transcranial focused ultrasound (tFUS) is a promising technique for non-invasive and spatially targeted neuromodulation and treatment of brain diseases. Acoustic lenses were designed to correct the skull-induced beam aberration, but these designs could only generate static focused ultrasound beams inside the brain. Here, we designed and 3D printed binary acoustic metasurfaces (BAMs) for skull aberration correction and dynamic ultrasound beam focusing. BAMs were designed by binarizing the phase distribution at the surface of the metasurfaces. The phase distribution was calculated based on time reversal to correct the skull-induced phase aberration. The binarization enabled the ultrasound beam to be dynamically steered along wave propagation direction by adjusting the operation frequency of the incident ultrasound wave. The designed BAMs were manufactured by 3D printing with two coding bits, a polylactic acid unit for bit "1" and a water unit for bit "0." BAMs for single- and multi-point focusing through the human skull were designed, 3D printed, and validated numerically and experimentally. The proposed BAMs with subwavelength scale in thickness are simple to design, easy to fabric, and capable of correcting skull aberration and achieving dynamic beam steering.

Incisionless targeted adeno-associated viral vector delivery to the brain by focused ultrasound-mediated intranasal administration.

BACKGROUND: Adeno-associated viral (AAV) vectors are currently the leading platform for gene therapy with the potential to treat a variety of central nervous system (CNS) diseases. There are numerous methods for delivering AAVs to the CNS, such as direct intracranial injection (DI), intranasal delivery (IN), and intravenous injection with focused ultrasound-induced blood-brain barrier disruption (FUS-BBBD). However, non-invasive and efficient delivery of AAVs to the brain with minimal systemic toxicity remain the major challenge. This study aims to investigate the potential of focused ultrasound-mediated intranasal delivery (FUSIN) in AAV delivery to brain. METHODS: Mice were intranasally administered with AAV5 encoding enhanced green fluorescence protein (AAV5-EGFP) followed by FUS sonication in the presence of systemically injected microbubbles. Mouse brains and other major organs were harvested for immunohistological staining, PCR quantification, and in situ hybridization. The AAV delivery outcomes were compared with those of DI, FUS-BBBD, and IN delivery. FINDINGS: FUSIN achieved safe and efficient delivery of AAV5-EGFP to spatially targeted brain locations, including a superficial brain site (cortex) and a deep brain region (brainstem). FUSIN achieved comparable delivery outcomes as the established DI, and displayed 414.9-fold and 2073.7-fold higher delivery efficiency than FUS-BBBD and IN. FUSIN was associated with minimal biodistribution in peripheral organs, which was comparable to that of DI. INTERPRETATION: Our results suggest that FUSIN is a promising technique for non-invasive, efficient, safe, and spatially targeted AAV delivery to the brain. FUNDING: National Institutes of Health (NIH) grants R01EB027223, R01EB030102, R01MH116981, and UG3MH126861.

A review of bioeffects induced by focused ultrasound combined with microbubbles on the neurovascular unit.

Focused ultrasound combined with circulating microbubbles (FUS+MB) can transiently enhance blood-brain barrier (BBB) permeability at targeted brain locations. Its great promise in improving drug delivery to the brain is reflected by a rapidly growing number of clinical trials using FUS+MB to treat various brain diseases. As the clinical applications of FUS+MB continue to expand, it is critical to have a better understanding of the molecular and cellular effects induced by FUS+MB to enhance the efficacy of current treatment and enable the discovery of new therapeutic strategies. Existing studies primarily focus on FUS+MB-induced effects on brain endothelial cells, the major cellular component of BBB. However, bioeffects induced by FUS+MB expand beyond the BBB to cells surrounding blood vessels, including astrocytes, microglia, and neurons. Together these cell types comprise the neurovascular unit (NVU). In this review, we examine cell-type-specific bioeffects of FUS+MB on different NVU components, including enhanced permeability in endothelial cells, activation of astrocytes and microglia, as well as increased intraneuron protein metabolism and neuronal activity. Finally, we discuss knowledge gaps that must be addressed to further advance clinical applications of FUS+MB.

Quantitative characterization of the colorectal cancer in a rabbit model using high-frequency endoscopic ultrasound.

PURPOSE: Colonoscopy accompanied with biopsy works as the routine endoscopic strategy for the diagnosis of colorectal cancer (CRC) in clinic; however, the colonoscopy is limited to the tissue surface. During the last decades, enabling technologies are emerging to complement with the colonoscopy for better administration of CRC. The conventional low-frequency (<12 MHz) endoscopic ultrasound (EUS) guided fine-needle aspiration (FNA) has been widely used to assess the lesion penetration. With the high-frequency ultrasound transducer (>20 MHz), EUS allows more precise visualization of the colorectal abnormalities. In order to achieve the accurate detection or in situ characterization of the colorectal lesions, the EUS diagnosis needs more patho-physiological related information in the micro-structural or molecular level. Quantitative ultrasound (QUS) technique, which could extract the micro-structural information from the ultrasound radio-frequency (RF) signal, is promising for the non-invasive tissue characterization. To date, the knowledge of the high-frequency endoscopic QUS for the CRC characterization has not been fully determined. METHODS: In this work, to our best knowledge, it is the first application of the QUS technique based on a customized high-frequency EUS system (30.5 MHz center frequency) to characterize the colorectal malignancies in a VX2 rabbit CRC model. To eliminate the response from the ultrasound electronic system and transducer, the ultrasound signals from colon tissue were calibrated. And, the resulting quasi-liner ultrasound spectra were fit by the linear regression test. As a result, three spectral parameters, including the slope (k), intercept (I) and Midband Fit (M), were obtained from the best-fit line. The three spectral parameters were compared between the malignant tissue regions and adjacent normal tissue regions of the colon tissue specimen ex vivo. The independent t-test was conducted between the three parameters from the normal and malignant group. The statistical method of Fisher Linear Discriminant (FLD) was used to explore the linear combinations of the three parameters, so as to provide more tissue micro-structural features than the single parameter alone. The three FLD values were derived from three different combinations among k, I and M. The threshold was selected from the statistical analysis to optimize the differentiation criteria between the malignant and the normal tissues. The color-coded images were used to display the local FLD values and combined with the EUS B-mode image. RESULTS AND CONCLUSIONS: The mean Midband Fit (M) and intercept (I) showed significant differences between the malignant and normal tissue regions. The statistical analysis showed that there were significant differences in all the mean FLD values of the spectral parameter combinations (kI, kM and IM) (t test, P < 0.05). And, the combined image result from the B-mode image and color-coded image could visually correlate with the histology result. In conclusion, the high-frequency endoscopic QUS technique was potential to be used as a complementary method to distinguish the colorectal malignancies by leveraging its morphological and micro-structural ultrasound information.

Mechanical and mechanothermal effects of focused ultrasound elicited distinct electromyographic responses in mice.

The objective of this study was to compare focused ultrasound (FUS) neuromodulation-induced motor responses under two physical mechanisms: mechanical and mechanothermal effects. Mice were divided into two groups. One group was subjected to short-duration FUS stimulation (0.3 s) that induced mechanical effects (mechanical group). The other group underwent long-duration FUS stimulation (15 s) that produced not only mechanical but also thermal effects (mechanothermal group). FUS was targeted at the deep cerebellar nucleus in the cerebellum to induce motor responses, which were evaluated by recording the evoked electromyographic (EMG) signals and tail movements. Brain tissue temperature rise associated with the FUS stimulation was quantified by noninvasive magnetic resonance thermometryin vivo. Temperature rise was negligible for the mechanical group (0.2 °C ± 0.1 °C) but did rise within the range of 0.6 °C ± 0.2 °C-3.3 °C ± 0.9 °C for the mechanothermal group. The elongated FUS beam also induced heating in the dorsal brain (below the top skull) and ventral brain (above the bottom skull) along the beam path for the mechanothermal group. Both mechanical and mechanothermal groups achieved successful FUS neuromodulation. EMG response latencies were within the range of 0.03-0.1 s at different intensity levels for the mechanical group. The mechanothermal effect of FUS could induce both short-latency EMG (0.2-1.4 s) and long-latency EMG (8.7-13.0 s) under the same intensity levels as the mechanical group. The different temporal dynamics of evoked EMG suggested that FUS-induced mechanical and mechanothermal effects could evoke different responses in the brain.

3-D Transcranial Microbubble Cavitation Localization by Four Sensors.

Cavitation is the fundamental physical mechanism of various focused ultrasound (FUS)-mediated therapies in the brain. Accurately knowing the three-dimensional (3-D) location of cavitation in real-time can improve the targeting accuracy and avoid off-target tissue damage. Existing techniques for 3-D passive transcranial cavitation detection require the use of expensive and complicated hemispherical phased arrays with 128 or 256 elements. The objective of this study was to investigate the feasibility of using four sensors for transcranial 3-D localization of cavitation. Differential microbubble cavitation detection combined with the time difference of arrival algorithm was developed for the localization using the four sensors. Numerical simulation using k-Wave toolbox was performed to validate the proposed method for transcranial cavitation source localization. The sensors with a center frequency of 2.25 MHz and a 6 dB bandwidth of 1.39 MHz were used to locate cavitation generated by FUS (500 kHz) sonication of microbubbles that were injected into a tube positioned inside an ex vivo human skullcap. Cavitation emissions from the microbubbles were detected transcranially using the four sensors. Both simulation and experimental studies found that the proposed method achieved accurate 3-D cavitation localization. When the cavitation source was located within 30 mm from the geometric center of the sensor network, the accuracy of the localization method with the skull was measured to be 1.9±1.0 mm, which was not significantly different from that without the skull (1.7 ± 0.5 mm). The accuracy decreased as the cavitation source was away from the geometric center of the sensor network. It also decreased as the pulse length increased. Its accuracy was not significantly affected by the sensor position relative to the skull. In summary, four sensors combined with the proposed localization algorithm offer a simple approach for 3-D transcranial cavitation localization.

Gas-filled protein nanostructures as cavitation nuclei for molecule-specific sonodynamic therapy.

Sonodynamic therapy (SDT) is a promising alternative for cancer therapy, understood to exert cytotoxicity through cavitation and subsequent production of large amounts of reactive oxygen species (ROS). Gas-filled protein nanostructures (gas vesicles or GVs) produced by cyanobacteria have a hollow structure similar to microbubbles and have demonstrated comparable enhancement of ultrasound imaging contrast. We thus hypothesized that GVs may act as stable nuclei for inertial cavitation to enhance SDT with improved enhanced permeability and retention (EPR) effects due to their nanometer scale. The function of GVs to mediate cavitation, ROS production, and cell-targeted toxicity under SDT was determined. In solution, we found that GVs successfully increased cavitation and enhanced ROS production in a dose- and time-dependent manner. Then, GV surfaces were modified (FGVs) to specifically target CD44+ cells and accumulate preferentially at the tumor site. In vitro sonodynamic therapy (SDT) showed ROS production and tumor cell toxicity substantially elevated in the presence of FGVs, and the addition of FGVs was found to enhance cavitation and subsequently inhibit tumor growth and exert greater damage to tumors under SDT in vivo. Our results thus demonstrate that FGVs can function as stable, nanosized, nuclei for spatially accurate and cell-targeted SDT. STATEMENT OF SIGNIFICANCE: The initiation of inertial cavitation is critical for ROS generation and subsequent cellular toxicity in SDT. Thus, precise control of the occurrence of cavitation is a key factor in increasing SDT's therapeutic efficacy. We explored nanometer-sized gas vesicles (GVs) as a new class of cavitation nuclei for molecule-specific sonodynamic therapy. Our results showed that GV-mediated SDT treatment enabled targeted disruption of specific cells expressing a known surface marker within the area of insonation, providing a spatially specific and targeted SDT treatment.