Rett syndrome (RTT) is a severe X-linked neurodevelopmental disorder due to pathogenic variants in the methyl CpG binding protein 2 gene (MECP2). The discovery that deficits resulting from Mecp2 loss are reversible in mice has increased interest in gene therapy as a potential cure for RTT. We have previously evaluated the efficacy of a self-complementary AAV9 vector expressing a codon-optimized version of Mecp2 (scAAV9-MCO) delivered via a systemic approach in early symptomatic Mecp2-knock-out male (KO) mice. In the present study, focused ultrasound (FUS) was used to transiently disrupt the blood-brain barrier (BBB) in a RTT mouse model, thereby facilitating enhanced AAV delivery to the central nervous system (CNS). Our findings demonstrate that scAAV9-MCO administration, when combined with FUS, significantly improves survival, body weight, respiratory function, and locomotor activity, while restoring the excitatory-inhibitory synaptic balance in hippocampal neurons in treated KO mice relative to untreated animals. Quantification of the brain infection level revealed that 20-40% of cells are Mecp2-positive in the brain of KO mice following the treatment with scAAV9-MCO and FUS. This is a significant improvement compared to prior results without FUS. The evaluation of the protein levels indicates a possible overdose of Mecp2 protein in the brain cells. Nevertheless, these results demonstrate that using FUS following systemic administration of an AAV9 vector represents a significant improvement over classical gene therapy protocol for RTT.

Introduction:Glioblastoma (GBM) remains the most aggressive primary brain tumor, characterized by a high recurrence rate and a poor prognosis, particularly due to the blood-brain/tumor barrier, which severely limits the intracerebral drug delivery. This study evaluates a novel strategy combining a nanomedicine-based drug delivery approach with focused ultrasound-mediated blood-brain barrier disruption (FUS-BBBD) to achieve localized therapeutic drug concentrations to the tumor. Methods:Paclitaxel and docetaxel were encapsulated in perfluorooctyl bromide nanodroplets, stabilized with fluorinated surfactants. The formulation was optimized and characterized in terms of drug loading, encapsulation efficiency, size, size distribution and stability. In vivo pharmacokinetics (PK) and safety were assessed in C57BL/6 mice. Therapeutic efficacy was evaluated using an orthotopic syngeneic GL261 glioma model combined with hemispheric 1.5 MHz FUS-BBBD. Results:Docetaxel-loaded nanodroplets (DTX-NDs) emerge as the most promising candidates. Optimized DTX-NDs exhibited a mean diameter of 62 ± 4 nm with an encapsulation efficiency exceeding 90%, a good stability achieved by freeze-drying, and a sustained release profile. PK analysis demonstrated a 28-fold reduction in systemic clearance and a significantly prolonged terminal half-life compared to free docetaxel. Quantitative LC-MS confirmed that FUS-BBBD enhanced docetaxel accumulation 9-fold in healthy brain tissue (p < 0.05) and 6-fold in GL261 glioma-bearing mice (p < 0.05) when using the nanodroplet formulation. An optimized treatment plan with DTX-NDs (20 mg kg-1 every 72 hours) successfully balanced efficacy and safety, extending median survival to 36 days versus 20 days for free DTX (p < 0.05), while achieving a 33% long-term survival rate. Toxicity was limited to transient and reversible hepatotoxicity. Conclusion:This study demonstrates that the repeated combination of DTX-NDs and FUS-BBBD is a biocompatible and effective strategy for enhancing brain drug delivery while minimizing peripheral toxicity, thereby offering a promising translational approach for the treatment of GBM.

Focused ultrasound (FUS) offers reversible disruption of the blood-brain barrier (BBB), which enables drug delivery to the brain. However, the impact of FUS on the blood-tumor barrier (BTB) remains largely misunderstood. The reversibility of FUS-induced BTB opening was monitored using PET imaging in a glioblastoma model. C57Bl/6 mice with bilateral GL261-GFP tumors received FUS specifically targeting the right hemisphere, followed by injections of the BBB permeability marker [18F]fluoro-deoxysorbitol (183 Da) or the radiolabeled antibody [18F]avelumab (150 kDa). PET acquisitions were performed at 1 h, 24 h and 72 h post-FUS. The uptake of [18F]avelumab and [18F]fluoro-deoxysorbitol increased immediately after FUS. At 24 h post-FUS, BTB permeability returned to the baseline, as evidenced by consistent [18F]avelumab distribution volumes (VT) between tumors. By 72 h, increased radiotracer uptake indicated tumor progression. These findings highlight the potential of FUS to enhance the delivery of therapeutics to the brain while preserving BTB integrity over time.

Background Noise correlation tomography has revolutionized seismology by enabling constant monitoring of our planet. Considering the brain as a system full of noise, the same algorithm might be adapted to monitor the brain using MRI.Purpose To apply the MR passive elastography sequence to patients diagnosed with brain tumors.Study Type Prospective.Population Four healthy volunteers and 18 patients (11 women and 7 men) with histopathology-confirmed brain tumors. There were 12 malignant tumors (10 high-grade gliomas, 2 metastases) and 6 had benign tumors (2 low-grade gliomas, 4 meningiomas).Field Strength/Sequence A modified T2-weighted spin-echo sequence with motion encoded in one spatial direction at 3.0 T.Assessment Shear wavelength estimated from the passive elastography sequence was compared to the shear modulus measured with intraoperative ultrasound.Statistical Tests Agreement of tumor mechanical properties measurements was assessed using Pearson correlation and Bland-Altman plots. The ability to distinguish malignant from benign tumors was evaluated using receiver operating characteristic curve analysis. Significance was considered at a p value of 0.05.Results Tumor wavelength values obtained with passive elastography were significantly correlated with ultrasound elastography (r = 0.59). Moreover, by calculating the ratio between normal brain and tumor, passive elastography was able to distinguish malignant tumors (shear wavelength of 0.26 m) from benign tumors (shear wavelength of 0.29 m) (AUC = 0.93).Data Conclusions Intracranial passive elastography is a feasible technique that does not require an external vibrating driver and has the potential to provide valuable information about tumors.Evidence Level 2.Technical Efficacy Stage 2.

Objective. Temporary, non-invasive, and localized permeabilization of the blood-brain barrier (BBB) can be achieved through focused ultrasound and microbubbles (MB). This technique has been extensively employed in rodent and non-human primate (NHP) studies for testing various drugs but requires precise control of ultrasonic pressure. However, controlling cavitation in NHP is challenging due to their thicker skull inducing strong ultrasonic attenuation. Furthermore, extra-cranial cavitation may occur masking the cavitation signal at the focal region (cerebral cavitation). Particularly in larger male NHP, temporal muscles are highly perfused and filled with MB.Approach. This study proposes a feedback loop control strategy to distinguish between intra- and extra-cerebral cavitation by analyzing broadband noise recorded by passive cavitation detection sensors.Main results. The frequency-dependent low-pass filtering effect by the skull allows differentiation of distinct frequency components, providing insights into cavitation origin. The present study involved 17 BBB opening experiments in NHP.Significance. Although successful BBB disruption can be achieved in NHP with thin temporal muscles (<5 mm) using a regular feedback loop algorithm, NHP having thicker muscles (>15 mm) require the use of an optimized algorithm able to specifically extract the signature of intra-cerebral cavitation.

Organophosphates (OP) found in pesticides and chemical weapons irreversibly inhibit acetylcholinesterases (AChE) and cause toxic accumulation of acetylcholine throughout the organism. Due to their lipophilicity, OP easily cross the blood-brain barrier (BBB) and affect the central nervous system (CNS), resulting in epileptic seizures and long-term cognitive impairment. The antidote includes oximes which reactivate inhibited AChE. Unfortunately, oximes have limited BBB penetration and therefore fail to prevent neurological damage. Improving the penetration of oximes through the CNS and their therapeutic effect on the brain, is a major challenge. Recent studies have demonstrated the efficacy of transcranial focused ultrasound (FUS), in combination to intravenously injected microbubbles, to transiently disrupt the BBB for drug delivery. We assessed the efficacy of FUS to deliver two known oximes (2-PAM, HI-6) into the brain and reactivate AChE following an exposure to VX in a mouse model. After both sub-lethal and supra-lethal exposure, HI-6 + FUS treatment reactivated nearly 30 % more AChE in the hippocampus than HI-6 alone. In contrast, 2-PAM+FUS was not effective. Furthermore, animals treated with HI-6 + FUS following an exposure to a supra-lethal dose of VX exhibited enhanced short-term recovery and an increased 24 hours survival rate. Finally, up to 7 days after exposure to a supra-lethal dose of VX, HI-6 + FUS showed a significant reduction of pro-inflammatory cytokines IL-6 and MIP-1α expression levels in the hippocampus. Thus, the use of FUS is very promising for improving the medical care of OP exposure because it enables antidotes to treat central symptoms and it may reduce brain damage.

Focused-ultrasound (FUS) in conjunction with microbubbles contrast agent is a non-invasive methods to induce transient blood brain barrier (BBB) disruption. This review aims to provide an overview of the imaging strategies (magnetic resonance imaging, positron emission tomography, and ultrasound imaging) used in parallel with FUS-induced BBB opening in pre-clinical and clinical studies to ensure the safety and efficacy of the technique.

Microbubble-mediated focused ultrasound is a promising strategy for transient and localized blood-brain barrier (BBB) permeabilization, enabling drug delivery to the brain. Optimizing microbubble stability and acoustic response is essential to maximize treatment efficiency and minimize potential damage. This study introduces an innovative microbubble formulation with a phospholipid-fluoropolymer shell (LIP-POL), designed to enhance circulation persistence while maintaining a low cavitation threshold. The physicochemical and acoustic properties of LIP-POL microbubbles were systematically compared with phospholipid-shell microbubbles (LIP) and the commercial agent SonoVue®. Both SonoVue and LIP-POL microbubbles have similar concentrations and sizes (approximately 5 × 108 bubbles/mL, mean size of 2.5-2.7 µm), whereas LIP microbubbles are around 100 times more concentrated (7.3 × 1010 bubbles/mL) and slightly smaller (1.9 µm). In vitro, ultra-harmonics appeared at 120 kPa for LIP, 150 kPa for LIP-POL, and 200 kPa for SonoVue (fc = 1 MHz, PRF = 10 kHz, 40 cycles). Consistent with microbubbles' acoustic signature, the BBB opening threshold (fc = 1.5 MHz) occurred at lower Mechanical Indices (MI) for LIP and LIP-POL microbubbles (MI = 0.16) compared to SonoVue (MI = 0.20). Stability of circulating microbubbles was assessed using BBB permeabilization protocol at various time points post microbubble-injection. LIP-POL microbubbles remained effective for up to 15 min post-injection, compared to 7.5 min for LIP and 5 min for SonoVue (2 × 107 microbubbles injected). The prolonged efficacy of LIP-POL microbubbles (three times longer than SonoVue) opens the possibility for extended ultrasound treatments, particularly for BBB permeabilization across larger areas in large animal models or humans.

Numerous studies suggest that blood-brain barrier (BBB) dysfunction may contribute to the progression of Alzheimer’s disease (AD). Clinically available neuroimaging methods are needed for quantitative “scoring” of BBB permeability in AD patients. [18F]2-fluoro-2-deoxy-sorbitol ([18F]FDS), which can be easily obtained from simple chemical reduction of commercial [18F]2-fluoro-2-deoxy-glucose ([18F]FDG), was investigated as a small-molecule marker of BBB permeability, in a preclinical model of AD using in vivo PET imaging.Chemical reduction of [18F]FDG to [18F]FDS was obtained with a 100% conversion yield. Dynamic PET acquisitions were performed in the APP/PS1 rat model of AD (TgF344-AD, n=3) compared with age-matched littermates (WT, n=4). The brain uptake of [18F]FDS was determined in selected brain regions, delineated from a coregistered rat brain template. The brain uptake of [18F]FDS in the brain regions of AD rats versus WT rats was compared using a 2-way ANOVA.The uptake of [18F]FDS was significantly higher in the whole-brain of AD rats, as compared with WT rats (p<0.001), suggesting increased BBB permeability. Enhanced brain uptake of [18F]FDS in AD rats was significantly different across brain regions (p<0.001). Minimum difference was observed in the amygdala (+89.0±7.6%, p<0.001) and maximum difference was observed in the midbrain (+177.8±29.2%, p<0.001).[18F]FDS, initially proposed as radiopharmaceutical to estimate renal filtration using PET imaging, can be repurposed for non-invasive and quantitative determination of BBB permeability in vivo. Making the best with the quantitative properties of PET imaging, it was possible to estimate the extent of enhanced BBB permeability in a rat model of AD.

Rationale: Glioblastoma (GBM) poses significant challenges regarding complete tumor removal due to its heterogeneity and invasiveness, emphasizing the need for effective therapeutic options. In the last two decades, fluorescence-guided surgery (FGS), employing fluorophores such as 5-aminolevulinic acid (5-ALA) to enhance tumor delineation, has gained attraction among neurosurgeons. However, some low-grade tumors do not show any accumulation of the tracers, and the lack of patient stratification represents an important limitation. Since 2000, endothelin axis has been extensively investigated for its role in cancer progression. More specifically, our team has identified endothelin A receptors (ETA), overexpressed in glioblastoma cancer stem cells, as a target of interest for GBM imaging. This study aims to evaluate the efficacy of a novel preclinical bimodal imaging agent, [89Zr]Zr-axiRA63-MOMIP, as a theranostic approach to: i) detect ETA + cells in an orthotopic model of human GBM, ii) achieve complete tumoral resection. Methods: Monomolecular multimodal imaging platform (MOMIP) - containing both a fluorophore (IRDye800CW) and a chelator for a positron-emitting radiometal (desferroxamine B, DFO) - was conjugated to the axiRA63 antibody targeting ETA receptors, overexpressed on the surface of GBM stem cells. Mice bearing orthotopic human GBM were imaged 48 h post injection of [89Zr]Zr-axiRA63-MOMIP via positron emission tomography (PET) and optical imaging. Subsequently, post-mortem proof-of-concept FGS was implemented as well as ex vivo analyses (H&E staining, autoradiography, serial block face imaging) on brains with resected or unresected tumor to assess the correlation between PET and fluorescence signals. Results: PET imaging of [89Zr]Zr-axiRA63-MOMIP enabled a clear detection of ETA + cells in an orthotopic model of human GBM. Intraoperative optical imaging allowed a near-complete tumor resection together with the visualization of a weak fluorescence signal, after a prolonged exposure time, that was attributed to residual tumor cells via H&E staining. Besides, a qualitative correlation between the signals of both modalities was observed. Conclusions: The use of [89Zr]Zr-axiRA63-MOMIP provides an effective theranostic approach to detect and treat GBM by surgery in a preclinical mouse model. Thanks to the high correlation between PET and fluorescence signal allowing patients stratification, this bimodal agent should have a great potential for clinical translation and should present a significant advantage over non-targeted fluorophores already used in the clinic.

Objective. To optimize and ensure the safety of ultrasound brain therapy, personalized transcranial ultrasound simulations are very useful. They allow to predict the pressure field, depending on the patient skull and probe position. Most transcranial ultrasound simulations are based on numerical methods which have a long computation time and a high memory usage. The goal of this study is to develop a new semi-analytical field computation method that combines realism and computation speed. Approach. Instead of the classic ray tracing, the ultrasonic paths are computed by time of flight minimization. Then the pressure field is computed using the pencil method. This method requires a smooth and homogeneous skull model. The simulation algorithm, so-called SplineBeam, was numerically validated, by comparison with existing solvers, and experimentally validated by comparison with hydrophone measured pressure fields through an ex vivo human skull. Main results. SplineBeam simulated pressure fields were close to the experimentally measured ones, with a focus position difference of the order of the positioning error and a maximum pressure difference lower than 6.02%. In addition, for those configurations, SplineBeam computation time was lower than another simulation software, k-Wave's, by two orders of magnitude, thanks to its capacity to compute the field only at the focal spot. Significance. These results show the potential of this new method to compute fast and realistic transcranial pressure fields. The combination of this two assets makes it a promising tool for real time transcranial pressure field prediction during ultrasound brain therapy interventions.

Low-boiling point perfluorocarbon nanodroplets (NDs) are valued as effective sonosensitive agents, encapsulating a liquid perfluorocarbon that would instantaneously vaporize at body temperature without the NDs shell. Those NDs have been explored for both therapeutic and diagnostic purposes. Here, phospholipid-shelled nanodroplets containing octafluoropropane (C3F8) or decafluorobutane (C4F10) formed by condensation of microbubbles were thoroughly characterized before blood-brain (BBB) permeabilization. Transmission electron microscopy (TEM) and cryo-TEM were employed to confirm droplet formation while providing high-resolution insights into the droplet surface and lipid arrangement assessed from electron density observation after condensation. The vaporization threshold of NDs was determined with a high-speed camera, and the frequency signal emitted by the freshly vaporized bubbles was analyzed using cavitation detection. C3F8 NDs exhibited vaporization at 0.3 MPa (f0 = 1.5 MHz, 50 cycles), and emitted signals at 2 f0 and 1.5 f0 from 0.45 MPa onwards (f0 = 1.5 MHz, 50 cycles), while broadband noise was measured starting from 0.55 MPa. NDs with the higher boiling point C4F10 vaporized at 1.15 MPa and emitted signals at 2 f0 from 0.65 MPa and 1.5 f0 from 0.9 MPa, while broadband noise was detected starting from 0.95 MPa. Both ND formulations were used to permeabilize the BBB in healthy mice using tailored ultrasound sequences, allowing for the identification of optimal applications for each NDs type. C3F8 NDs proved suitable and safe for permeabilizing a large area, potentially the entire brain, at low acoustic pressure. Meanwhile, C4F10 droplets facilitated very localized (400 μm isotropic) permeabilization at higher pressure. This study prompts a closer examination of the structural rearrangements occurring during the condensation of microbubbles into NDs and highlights the potential to tailor solutions for different brain pathologies by choosing the composition of the NDs and adjusting the ultrasound sequence.

Objective.Focused ultrasound (FUS) is a promising non-invasive therapeutic approach that can be used to generate thermal and non-thermal bioeffects. Several non-thermal FUS therapies rely on FUS-induced oscillations of microbubbles (MBs), a phenomenon referred to as cavitation. Cavitation monitoring in real time is essential to ensure both the efficacy and the safety of FUS therapies. This study aims to introduce a new magnetic resonance (MR) method for cavitation monitoring during FUS therapies.Approach.By finely synchronizing the FUS pulse with an accelerated turbo spin-echo MR sequence, the cavitation effect could be quantitatively estimated on the acquired images at 1-Hz refresh rate. The proposed method was assessed in vitro in a water bath. A series of FUS pulses were generated on a silicone tube filled with MBs at different acoustic pressures (0.07-2.07 MPa) and pulse durations (20-2000μs). MR images and passive cavitation detection (PCD) signals were simultaneously acquired for each FUS pulse.Main results.Inertial cavitation was found to induce a quantitatively interpretable signal loss on the MR image. The transition from stable to inertial cavitation was identified on MR cavitation maps with high repeatability. These results were found to be in good agreement with PCD measurements in terms of pressure thresholds between stable and inertial cavitation. MR cavitation imaging was shown to be sensitive to short and even ultrashort FUS pulses, from 2 ms down to 20μs. The presented theoretical model suggests that the signal loss in MR cavitation imaging relies on susceptibility changes related to the diameter of the oscillating MBs.Significance.The proposed MR cavitation imaging method can both locate and characterize cavitation activity. It has therefore the potential to improve the efficacy and safety of FUS therapies, particularly for localized drug delivery applications.

Objective.Cavitation dose monitoring plays a key role in ultrasound drug delivery to the brain. The use of capacitive micromachined ultrasonic transducer (CMUT) technology has a great potential for passive cavitation detection (PCD).Approach.Here, a circular (diameter 7 mm) CMUT centered at 5 MHz was designed to be inserted into a therapeutic transducer (1.5 MHz) used for ultrasound-induced blood-brain barrier (BBB) disruption on mice. CMUT-based real-time cavitation detection was performed during the ultrasound procedure (50μl intravenous injection of SonoVue microbubbles, frequency 1.5 MHz, PNP 480 kPa, duty Cycle 10%, PRF 10 Hz, duration 60 s). BBB disruption were confirmed by contrast-enhanced 7T-MRI.Main results.The CMUT device has a fractional bandwidth of 140%, almost twice a conventional piezocomposite PCD transducer. As expected, the CMUT device was able to detect the occurrence of harmonic, subharmonic and ultraharmonic frequencies as well as the increase of broadband signal indicating inertial cavitation in a wide frequency range (from 0.75 to 6 MHz). Signal-to-noise ratio was high enough (>40 dB) to perform ultrafast monitoring and follow the subtle intrapulse variations of frequency components at a rate of 10 kHz.Significance. This firstin vivoproof of concept demonstrates the interest of CMUT for PCD and encourages us to develop devices for PCD in larger animals by integrating an amplifier directly to the CMUT front-end to considerably increase the signal-to-noise ratio.

Transcranial ultrasound simulations are crucial to optimize and secure ultrasound interventions in brain therapy, depending on the patient skull. When performing such simulations, accurate modeling of the skull is essential, although very challenging, because of the inter/intra sample property variability. Simulations based on semi-analytical methods require a homogeneous description of the skull. Averaging the acoustic property maps derived from the CT scan does not modify the focus shift, but it leads to an overestimation of the pressure field amplitude. The purpose of this work is to provide a homogenization method that compensates for this amplitude overestimation. First, the skull acoustic property maps are segmented into a three-layer medium to represent the different types of skull bone (cortical - trabecular - cortical). Then, equivalent properties are computed so as to minimize the time of flight and transmission coefficient errors between the three-layer medium and the one-layer equivalent medium. This method was validated using 3D simulations with CIVA Healthcare and k-Wave and has proven to be very efficient.

Alzheimer′s disease (AD), the most common cause of dementia, represents one of the main clinical challenges of the century as the number of patients is predicted to triple by 2050. Despite the recent approval of three monoclonal antibodies targeting Amyloid β (Aβ) aggregates by the Food and Drug Administration (FDA), immunotherapies still face challenges due to the difficulty of antibodies crossing the blood-brain barrier (BBB). This necessitates administering large doses of drugs to achieve their therapeutic effects, which is associated with significant side effects. In this context, low-intensity focused ultrasound (LiFUS) appears as an innovative and non-invasive method which, in association with intravenous injection of microbubbles (MB), leads to a transient BBB opening. This innovative strategy has been extensively studied in different preclinical models and more recently in human clinical trials, particularly in the context of AD. LiFUS+MB seems to increase the inflammatory response at short term, but the time course of this response is not consistent between studies, certainly due to the discrepancy between LiFUS protocols used. Moreover, the impact at longer term is understudied and the mechanisms underlying this effect are still not well understood. In our study, we therefore used the TgF344-AD rat model of AD, to investigate the effect of a single or multiple exposures to LiFUS+MB in the entire brain, on inflammatory response and amyloid load. The ultrasound attenuation through the skull was corrected to apply a peak negative acoustic pressure of 450 kHz in all treated animals. Single LiFUS+MB exposure induces a slight astrocyte and microglial response 24 hours post-treatment whereas repeated LiFUS treatment seems to induce microglial reprogramming, leading to the adaptation of gene expression related to key functions such as inflammatory response, mitochondrial and energetic metabolism. In our rat model and LiFUS+MB protocol conditions, multiple exposures did not modulate soluble/poorly aggregated forms nor the highly aggregated forms of Aβ40 and Aβ42. For therapeutic AD management, LiFUS+MB could be combined with drugs such as immunotherapies. In a proof-of-concept experiment, we validated that LiFUS was also efficient to improve the brain entry of the anti-Aβ antibody, Aducanumab. ### Competing Interest Statement The authors have declared no competing interest.