# Effects of blood-brain barrier opening using ultrasound on tauopathies: A systematic review

Amandine Géraudie, Pierre de Rossi, Michael Canney, Alexandre Carpentier, Benoît Delatour

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Amandine Géraudie, Pierre de Rossi, Michael Canney, Alexandre Carpentier, Benoît Delatour. Effects of blood-brain barrier opening using ultrasound on tauopathies: A systematic review. Journal of Controlled Release, 2025, 379, pp.1029 - 1044. 10.1016/j.jconrel.2025.01.056. hal-05012221

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# Effects of blood-brain barrier opening using ultrasound on tauopathies: A systematic review

Amandine Géraudie<sup>a, *,</sup> Pierre De Rossi<sup>b</sup>, Michael Canney<sup>b</sup>, Alexandre Carpentier<sup>c,d,e</sup>, Benoît Delatour<sup>a</sup>

<figure><img src="image_3.png" /></figure>

## ARTICLE INFO

**Keywords:**

Tauopathies

Blood-brain barrier

Alzheimer's disease

Ultrasound

Microbubbles

## ABSTRACT

Blood-brain barrier opening with ultrasound can potentiate drug efficacy in the treatment of brain pathologies and also provides therapeutic effects on its own. It is an innovative tool to transiently, repeatedly and safely open the barrier, with studies showing beneficial effects in both preclinical models for Alzheimer's disease and recent clinical studies. The first preclinical and clinical work has mainly shown a decrease in amyloid burden in mice models and in patients. However, Alzheimer's disease pathology also encompasses tauopathy, which is closely related to cognitive decline, making it a crucial therapeutic target. The effects of blood-brain barrier opening with ultrasound have been rarely assessed on tau and are still unclear.

**Methods:** This systematic review, conducted through searches using Pubmed, Embase, Web of Science and Cochrane Central databases, extracted results of 15 studies reporting effects of blood-brain barrier opening using ultrasound on tau proteins.

**Results:** This review of the literature indicates that blood-brain barrier opening using ultrasound can decrease the extent of the tau pathology or potentialize the effect of a therapeutic drug. However, selected studies report paradoxically that blood-brain barrier opening can increase tau pathology burden and induce brain damage.

**Discussion:** Apparent discrepancies between reports could originate from the variability in protocols or analytical methods that may impact the effects of blood-brain barrier opening with ultrasound on tauopathies, glial populations, tissue integrity and functional outcomes.

**Conclusion:** This calls for a better standardization effort combined with improved methodologies allowing between-studies comparisons, and for further understanding of the effects of blood-brain barrier opening on tau pathology as an essential prerequisite before translation to clinic.

## 1. Introduction

Alzheimer's disease (AD) represents both the most frequent neurodegenerative disease and the leading cause of dementia in the world. In 2015, 46.8 million people worldwide lived with dementia, a number still increasing with the ageing population and generating major public health and socio-economic repercussions [1]. The amyloid cascade hypothesis [2] points at the extracellular accumulation of β-amyloid peptides (Aβ), the so-called amyloid plaques, as the main cause for the disease, triggering large research efforts and the development of anti-Aβ

therapies. To date, three anti-Aβ immunotherapies have been FDA-approved (aducanumab [3], lecanemab [4], donanemab [5]) based on positive signs on fluid and neuroimaging biomarkers (reduction of amyloid loads) but still with a questionable risk-benefit profile due to mild or disputable clinical improvements and worrying side effects observed in clinical trials. Along with extracellular Aβ deposits, AD is also characterized by intracellular aggregates of abnormally phosphorylated tau proteins making it a secondary tauopathy [6]. AD shares tau pathological hallmark with different primary tauopathies, each disease being characterized by a specific type and topographical/cellular distribution

<footnote><sup>a</sup> Paris Brain Institute, ICM, Inserm U1127, CNRS UMR 7225, Sorbonne University, 75013 Paris, France</footnote>

<footnote><sup>b</sup> Carthera, 1 Pl. Vaclav Havel, 69007 Lyon, France</footnote>

<footnote><sup>c</sup> Department of Neurosurgery, Sorbonne University, APHP, La Pitié-Salpetrière Hospital, 75013 Paris, France</footnote>

<footnote><sup>d</sup> Faculty of Medicine, Sorbonne University, GRC 23, Brain Machine Interface, APHP, La Pitié-Salpetrière Hospital, 75013 Paris, France</footnote>

<footnote><sup>e</sup> Advanced Surgical Research Technology Lab, Sorbonne University, 75013 Paris, France</footnote>

<footnote>* Corresponding author at: Brain Institute, 47, boulevard de l'Hôpital, 75013 Paris, France.</footnote>

<footnote>E-mail address: amandine.géraudie@icm-institute.org (A. Géraudie).</footnote>
of abnormally phosphorylated tau proteins prone to aggregation. These aggregates cause neuroinflammation, synaptic loss and, in the end, neurodegeneration [7]. In AD, tauopathies are better correlated with cognitive decline than Aβ deposits [8,9], justifying the development of therapies against abnormal tau deposition. However, despite encouraging pre-clinical results [10–15], most clinical trials targeting tau in Alzheimer's disease and other tauopathies have failed [16].

When developing brain therapeutics, one major obstacle is the crossing of the blood-brain barrier (BBB). Indeed, while protecting the brain from pathogens and tightly maintaining homeostasis between blood and brain parenchyma compartments, the BBB limits bioavailability of drugs in the brain. It has been estimated that less than 0.1 % of the systemic concentration of a peripherally administered antibody reaches the cerebrospinal fluid (CSF) [17,18] and only 0.01–0.02 % the brain parenchyma [19]. Multiple methods have been developed to overcome the BBB, with significant invasiveness (i.e. intracerebral or intraventricular injection) or systemic side effects (i.e. intra-arterial infusion of mannitol or bivalent molecules hijacking transferrin or insulin receptor) [17,20–22]. Back in the early 2000’s, it was shown that the BBB can be transiently and safely disrupted using ultrasound associated with microbubbles [23–26]. Microbubbles are made of a lipidic or polymeric shell and filled with gas, yielding a diameter of 1–10 µm. When systemically administered concomitantly with the use of low intensity ultrasound, these bubbles oscillate, with biological effects such as disruption of the tight junctions and increased transcytosis [27–30], allowing larger molecules to cross the BBB. Mechanistic studies have explored the effects of ultrasound parameters [31,32] as well as microbubble dosage modulation [33,34], progressively improving the technique and making the association of ultrasound and systemic microbubbles a safe technique to transiently and repeatedly disrupt the BBB.

This technique has been applied to numerous brain diseases, including AD. In AD, BBB opening mediated by sonication has been used to increase the penetration of antibodies in the brain parenchyma, initially in amyloid animal models. These studies showed that ultrasound allowed both an increased penetration of antibodies into the brain and an improvement of neuropathological phenotypes (e.g. decreased amyloid plaques [35,36], increased neurogenesis [36]). The first clinical trial combining BBB opening and peripherally administered anti-Aβ antibody recently provided exciting results [37]. Surprisingly, it was also reported that BBB opening alone, without any adjunct therapeutic, is able to promote therapeutic effects on its own (decreased amyloid plaques in number and size [38–40], improved cognitive performances especially in tasks involving memory [39,40], increased neurogenesis [36]). The main mechanism of action evoked in these studies involves the modulation of neuroinflammation stimulating microglia phagocytosis of extracellular amyloid plaques [38,39].

Since then, and after demonstrating very encouraging results when associated to brain tumor chemotherapy [41–44], the technology of BBB opening with sonication has rapidly translated to patients with AD. Safety endpoints were reached and BBB opening was achieved in AD patients [37,45–47]. In some reports, sonication was associated with discrete reductions of brain amyloid burden [37,45,47–49]. However, the clinical efficacy of US-mediated BBB opening still needs to be assessed in dedicated clinical trials. In addition, the translation of this technique to patients with AD needs to demonstrate its effects on both Aβ and tau pathologies that co-exist in the brain of patients with AD. Indeed, the amyloid cascade, suggesting that tau is secondary to amyloid pathology, remains debated and clearing amyloid from patients’ brains does not appear to be sufficient to have meaningful clinical effects [50,51]. Tau pathology is, on the other side, a strong clinicopathological marker of cognitive symptoms and might be considered as a key therapeutic target. Furthermore, abnormal tau aggregates are also characteristic of other neurodegenerative diseases gathered under the umbrella term of tauopathies [7,52]. However, to date, the effects of BBB opening using sonication on tau pathologies as well as the underlying

mechanisms of action remain unclear.

This systematic review aims to compile and summarize data from the literature on the effects of BBB opening using ultrasound associated with microbubbles on tau pathology, either in animal models or in patients with tauopathies and also in other animal models in which effects of sonication on tau were analyzed. In addition, we listed additional effects of sonication on other neuropathological outcomes (e.g. neuroinflammation) and behavior in animal models of tauopathies and assessed safety concerns in all the included studies. This review finally underlines current limitations and challenges and suggests implications for future studies.

## 2. Methods

This systematic review follows Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [53,54].

### 2.1. Literature search

A systematic review of the literature was performed using the following information sources: Pubmed, Embase, Web of Science, Cochrane Central, with the help of a university librarian. Literature search with Pubmed used the following search terms: "tauopathies" (as a MeSH term) or "Alzheimer's disease" (as a MeSH term) or "tau" or "Alzheimer*" or "progressive supranuclear palsy" or ("frontotemporal" or "corticobasal") and ("dementia" or "degeneration" or "syndrome")) AND ("ultrasound*" or "ultrasonic waves" (as a MeSH term)). The same terms were used for the search on other databases and adapted for each database (use of Entree terms in place of MeSH terms for Embase, for instance). The search was conducted on November 8th, 2023. Additionally, references of selected reviews and included papers were manually reviewed in search of supplementary relevant articles that would not have been detected by the search equation but this manual search did not find any additional publications. Duplicates were removed.

### 2.2. Study inclusion

Inclusion criteria were determined following the Population, Intervention, Comparison, Outcomes and Study (PICOS) framework. Two independent raters (AG, BD) selected the included studies based on the following criteria: i) Population (P): animal models or patients with tauopathies or in which tau protein levels have been measured; in particular studies investigating tau or phosphorylated tau (ptau) dosages in mixed Aβxtau models, in Aβ models, in patients with AD and even in non-transgenic animals were considered; ii) Intervention (I): BBB opening with ultrasound associated with microbubbles; iii) Comparison (C): comparison to at least one group without BBB opening or between before and after BBB opening or comparison of different antibodies used as adjunct sonication; iv) Outcome (O): at least one outcome evaluating BBB opening, drug delivery, tau pathology, neuroinflammation, behavioral, cognitive or motor changes, side effects in brain parenchyma (e.g. hemorrhages, neuronal loss) in mouse models of tauopathies or at least one outcome evaluating tau (in the brain or in peripheral fluids (plasma, CSF)) in other models (mixed Aβ x tau models, Aβ models) and in AD patients; v) Study design (S): original studies in animal models or clinical trials, published in English. Exclusion criteria were as follows: i) not an experimental study (review articles, editorials, theses, expert opinions, or commentaries); ii) conference materials or abstracts. To identify the included studies, two authors (AG, BD) separately screened first the titles and abstracts and then assessed the full texts of selected papers for eligibility. In case of disagreement, consensus was reached through discussion between the two authors.
## 2.3. Data extraction

After the screening procedure, each selected article was used to extract the following data: i) authors, journal and publication year; ii) types of animal models or clinical data for each patient, including their

sex and age; iii) type of ultrasound and sonication parameters; iv) type of anesthesia; v) type and dose of microbubbles; vi) target of BBB opening; vii) type and dose of associated drug if any; viii) type of sham treatment; viii) assessment of BBB opening; ix) main findings on the different outcomes; x) supposed mechanism of action.

<figure><img src="image_4.png" /><figcaption>Fig. 1. PRISMA flowchart of literature search.</figcaption></figure>
Importantly, due to the heterogeneity of the different animal models screened in this review we applied the following rules for data extraction of the outcomes:

- In models specifically designed to develop a brain tauopathy as seen in AD or frontotemporal dementia patients (i.e. mutant tau transgenic mice), all outcomes were considered (modulation of tau as a primary outcome but also consequences of sonication on glial populations and on behavioral-cognitive phenotypes).

- In other models and in patients with AD, in which tau anomalies can be observed in association with other hallmarks (e.g. Aβ-positive lesions), data extraction only focused on reported changes in the tau burden after BBB opening. We do indeed consider that other phenotypic traits (e.g. gliosis or functional impairments) could have several origins and cannot be strictly related to sonication-induced tau modulations that are the focus of the present review.

- In wild-type animals that, by definition, do not present any AD-like neuropathological features, all post-sonication outcomes, in addition to the tau assessment, were considered and extracted.

- Relationships between the change of magnitude/nature of the tau burden after BBB opening and safety issues were analyzed in all studies.

## 2.4. Risk of bias assessment

In all included studies, risk of bias was assessed using the Systematic Review Center for Laboratory Animal Experimentation risk of bias (SYRCLE's RoB) tool for animal studies [55]. This tool precises 10 items related to selection bias, performance bias, detection bias, reporting bias and other biases. As for clinical trials, we used PEDro, a tool recommended for interventional studies in patients. It evaluated eligibility criteria, random allocation, concealed allocation, similitude of characteristics at baseline, blinding of subjects, therapists and assessors, completeness of results on the different outcomes, intention to treat design, between groups statistical comparisons and reports of points measures as well as estimates for variability [56].

# 3. Results

## 3.1. Included studies

The reviewing process is shown step by step in the PRISMA flowchart (Fig. 1). Out of the 2538 manuscripts retrieved, 871 duplicates were removed. 1667 titles and abstracts were then screened and 1474 of them were excluded because judged as irrelevant to the topic. 193 articles were fully reviewed for eligibility. 178 of them were excluded (wrong population, no BBB opening, no outcome of interest, not a research article, not in English language) and 15 were finally selected for this systematic review. Fourteen of them concerned animal models (7 studies involving animal models for tauopathies [57–63], 3 involving mouse models for both amyloid and tau neuropathologies [64–66], 3 using mouse models for amyloidosis [39,67,68] and 1 using a Sprague Dawley rat model to explore the effects on tau [69]). One study was conducted in patients with AD [70]. Of note, over 50 % of the publications (8/15) were attributed to two research groups (Queensland Brain Institute and Colombia University). The main characteristics of the included studies are presented in Table 1. Risk of bias assessment of the 14 included animal studies showed that over 65 % of the items indicated a possible risk of bias, according to criteria explicit in [55]. The only study involving patients had a global score of 4/11 but with a lot of items “not applicable” due to the open-labelled single-arm design of the study. All the details of the risk of bias assessment can be found in Tables S1-S2.

## 3.2. Ultrasound for BBB opening: methodologies

Different types of ultrasound can be used to transiently open the BBB. Most of the included studies used focused ultrasound (FUS) that have the ability to induce a BBB opening from a precise brain location [59,63,64,67,68] or at several focused brain loci [62,65,66,69,70]. Two of the included studies used FUS under magnetic resonance imaging (MRI) guidance [69,70]. To provide a better coverage of the diffuse distribution of the pathology, five studies used scanning ultrasound (SUS) which open the BBB in several sequential focal points distributed over the brain surface [39,57,58,60,63]. Lastly one study used low-intensity pulsed ultrasound (LIPU), an unfocused ultrasound system

<table><caption>**Table 1**
Main characteristics of included studies.</caption><thead><tr><th>First author, [...] Last author</th><th>Reference</th><th>Year of publication</th><th>Journal</th><th>Center, Country</th><th>Animal model / Patients with pathology</th></tr></thead><tbody><tr><td>Leinenga, [...] Götz</td><td>39</td><td>2016</td><td>Science translational medicine</td><td>Queensland Brain Institute, Australia</td><td>APP23 mice</td></tr><tr><td>Nisbet, [...] Götz</td><td>57</td><td>2017</td><td>Brain</td><td>Queensland Brain Institute, Australia</td><td>pR5 mice</td></tr><tr><td>Janowicz, [...] Nisbet</td><td>63</td><td>2019</td><td>Scientific reports</td><td>Queensland Brain Institute, Australia</td><td>pR5 mice</td></tr><tr><td>Pandit, [...] Götz</td><td>58</td><td>2019</td><td>Theranostics</td><td>Queensland Brain Institute, Australia</td><td>K3 mice</td></tr><tr><td>Bajracharya, [...] Nisbet Karakatsani, [...] Konofagou</td><td>60</td><td>2022</td><td>Journal of Controlled Release</td><td>Queensland Brain Institute, Australia</td><td>K3 mice</td></tr><tr><td>Karakatsani, [...] Konofagou</td><td>59</td><td>2019</td><td>Theranostics</td><td>Columbia University, USA</td><td>rTg4510</td></tr><tr><td>Noel, [...] Konofagou</td><td>66</td><td>2023</td><td>Theranostics</td><td>Columbia University, USA</td><td>3xTg</td></tr><tr><td>Noel, [...] Konofagou</td><td>65</td><td>2023</td><td>Frontiers in neuroscience</td><td>Columbia University, USA National Institutes of Health, USA</td><td>3xTg mice</td></tr><tr><td>Kovacs, [...] Frank</td><td>69</td><td>2018</td><td>Theranostics</td><td>Shenzhen University, China</td><td>Sprague Dawley rats</td></tr><tr><td>Shen, [...] Yang</td><td>64</td><td>2020</td><td>Theranostics</td><td>Kunming Medical University, China</td><td>3xTg</td></tr><tr><td>Luo, [...] Ao</td><td>67</td><td>2022</td><td>Neural plasticity</td><td>Nankai University, China</td><td>Mice with ICV injection of Aβ</td></tr><tr><td>Mi, [...] Tan</td><td>68</td><td>2022</td><td>Acta Biomaterialia</td><td>Nankai University, China</td><td>APP/PS1</td></tr><tr><td>Géraudie, [...] Delatour</td><td>61</td><td>2023</td><td>International Journal of Molecular Sciences</td><td>Paris Brain Institute, France</td><td>P301S (PS19) mice</td></tr><tr><td>Meng, [...] Lipsman</td><td>70</td><td>2023</td><td>Brain</td><td>University of Toronto, Canada</td><td>Patients with AD</td></tr><tr><td>Pacia, [...] Chen</td><td>62</td><td>2023</td><td>Radiology</td><td>Washington University, USA</td><td>P301S (PS19) mice</td></tr></tbody></table>

Studies are classified first by research group and then by year of publication. AD: Alzheimer's disease; ICV: intracerebroventricular.
[61]. Importantly, due to the small brain size in mice, one may consider that both focused and unfocused ultrasounds induce the same BBB opening (in terms of volume).

Across all the included studies, different ultrasound parameters have been used (Table 2). Peak negative pressures varied from 0.3 to 0.7 MPa and the ultrasound frequency varied from 0.5 MHz to 1.5 MHz. We computed the mechanical index (MI) (peak negative pressure amplitude divided by the square root of the center frequency), a standardized measure which has been reported to correlate with the probability of both BBB opening and tissue damage [71], for all the studies reporting sufficient information (12/15 studies). MI varied between studies (from 0.3 to 0.7). Pulse length (6,7–25 ms) and pulse repetition frequency (0,5–10 Hz) also varied largely between studies. Most studies used in-house manufactured microbubbles but commercial microbubbles (Sonovue, Optison, Definity) were also used in some studies [61,62,67,69,70]. The type and dosage of microbubbles varied largely between reports. To account for these variations, we computed the estimated number of microbubbles administered as well as the estimated microbubbles gas volume (for papers with data available) which has been shown to be correlated to the extent of BBB opening [34]. This latter varied up to x7000 between reports. Most studies used repeated sonication sessions with different repetition patterns and timelines. The delay between BBB opening and post-sonication analysis of brain tissues varied largely between studies (from 30 min to 13 weeks). Finally, concerning anesthesia, 7/15 studies used isoflurane [59,63–67], 3/15 a mix of ketamine and xylazine [58,60,61] and 1/15 a mix of toletamine, zolazepam and xylazine [39]. No anesthesia modality was documented in 4/15 studies.

### 3.3. Ultrasound for BBB opening: assessment of the procedure

Six of the included studies used intravenous injections of gadolinium to assess the efficiency of BBB opening [59,62,65,66,69,70]. This method aims to visualize in vivo the disruption of the BBB with gadolinium-enhanced MRI contrast at the site of permeabilization (an intact BBB precluding intraparenchymal gadolinium extravasation). BBB opening can also be confirmed once the brain is extracted using Evans blue dye visualization in brain tissue [39,60,61,64,67]. The Evans blue method consists of intravenously injecting mice with the dye immediately after the sonication session and analyzing the coloration of parenchyma after brain extraction. Indeed, under physiological conditions, Evans blue, of molecular weight around 1 kDa, links to albumin, reaching a molecular weight of ~67 kDa and does not cross the BBB. Once the BBB is opened, Evans blue can pass through the BBB at the site of opening and stains brain parenchyma with a blue coloration. Other studies used immunohistochemistry to evaluate the intra-parenchymal penetration of albumin [63] or fibrinogen [64], both present in blood while not physiologically entering the brain. In addition, studies which used BBB opening to maximize brain penetration of antibodies also confirmed enhanced BBB passage by measuring intracerebral antibodies concentrations after sonication [57,60,63,68]. Some studies assessed BBB opening longitudinally after a sonication session, showing that the BBB remained open for 3 to 4 h and was closed 6 h after sonication [64,67]. However, in one study implicating AD patients, 15 % of targeted sites presented with persistent hypersignal due to gadolinium, one day post-treatment, suggesting incomplete closure of BBB [70]. Of note, Karakatsani et al. compared the volume of BBB opening (assessed by MRI) between tau transgenic and wild-type animals and did not report differences between genotypes [59].

### 3.4. Effects of BBB opening with sonication

Main results of the included studies on the effects of BBB opening using sonication are synthesized in Table 3.

### 3.5. Effects on tau pathology

A total of 13 studies assessed tau levels after sonication with no adjunct drugs: 12 animal studies (6 in models for tauopathies, 3 in mixed models for amyloid and tau, 2 in models for amyloid and 1 in wild-type (Sprague Dawley) rats) and 1 study in patients with AD.

As a main result, six studies performed on mouse models displaying tau anomalies (listed as A, B, C in Table 3) reported significant decreased ptau or tau levels after sonication [57–60,64,66]. In the first published study, Nisbet et al. showed that BBB opening was able to reduce hyperphosphorylated tau (AT8 and AT180 immunostaining) [57]. This protective effect was confirmed by subsequent studies in different transgenic models of tauopathies [58–60] and in mouse models combining both amyloid and tau pathologies [64,66]. Sonication-induced reduction of tau seemed to be more consistent in brain regions with high levels of tau pathology (i.e. amygdala in pR5 mouse model [57], hippocampus and cortex in K3 mouse model [58,60], amygdala and hippocampus in 3xTg mouse model [64]). One study [59] showed reduced tau densities in rTg4510 mouse model after BBB opening with FUS but the overall number of tau-positive cells was unaffected by treatment which might suggest a main effect of sonication on dendritic tau versus somatic tau (see also [66]).

Mitigation of tauopathies by sonication was reported to be consistent using AT8 and AT180 immunostained-material and was also evidenced for aggregated, mature forms of tau (RN235 antibody [58,60,72], Bielschowsky-silver stain [58]). On the other side, phospho-epitopes at tau C-terminus appeared to be unaffected by sonication (12E8, pS404 antibodies [57]). This may suggest that abnormally phosphorylated tau species are differentially impacted by transient BBB opening using ultrasound.

Beneficial effects of sonication on tau were confirmed using biochemical dosages (HT7 immunoblots) in one study [66], while other reports failed to report decreased ptau dosages by WB or ELISA after sonication [39,57,58,60,65,67], presumably because of a lack of sensitivity or specificity, especially in models with low intrinsic tau levels (e. g. models primarily designed to develop amyloid pathology [39]).

More concerning, within the set of studies that claimed a therapeutic effect of sonication on tauopathies, conflicting results were sometimes reported. For instance, the studies of Karakatsani and colleagues, using both a tau model (rTg4510 line) and a mixed model for amyloid and tau pathologies (3xTg line) [59,66], showed an unclear therapeutic effect. They found decreased phosphorylated tau density [59] as well as reduced length of phosphorylated tau-stained neuronal processes [59,66] in sonicated animals which might be interpreted, as stated above, as a therapeutic effect on dendritic tau. However, the same histological changes (decreased ptau densities) were concurrently observed in aged non-sonicated transgenics ([59]; fig. S1B) casting doubt on the therapeutic nature of the reported effects of sonication on tau lesions. In a similar vein, Noel et al. [65] claimed for a decrease of total tau (as measured by ELISA) in sonicated 3xTg transgenics but statistical analysis did not confirm this trend (Fig. 6C in their report). Microscopic illustration of reduced tau immunofluorescent staining was also provided in this study but without quantification and no visual difference was observed between sonicated and non-sonicated female transgenics (Fig. 6D in their report).

While several reports showed a positive effect of sonifications on tau/ptau accumulations, other studies reported paradoxical deleterious impacts of BBB opening with ultrasound on tau levels in both transgenic and wild-type animals as well as in patients with AD [61,62,69,70]. Géraudie et al. quantified for instance a slight increased tau pathology in piriform cortex of sonicated P301S tau transgenics, using a semi-quantitative approach (H-scores from AT8 immunostained sections) [61].

Pacia et al. performed focused ultrasound-mediated blood-based liquid biopsy (sonobiopsy) in P301S mouse model with the aim to improve detection of peripheral biomarkers. They showed that
<table><caption>**Table 2**
Summary of the type and parameters of ultrasound and microbubbles used for BBB opening in the studies included in this systematic review.</caption><thead><tr><th rowspan="2">Reference</th><th rowspan="2">US type</th><th rowspan="2">Associated drug</th><th rowspan="2">Pressure (Mpa)</th><th rowspan="2">Center frequency (MHz)</th><th rowspan="2">Mechanical index</th><th rowspan="2">Pulse length (ms)</th><th rowspan="2">PRF (Hz)</th><th rowspan="2">Duration (s)</th><th rowspan="2">Anesthesia</th><th colspan="4">MB</th><th rowspan="2">Sonication regimen</th><th rowspan="2">Brain target</th></tr><tr><th>type</th><th>Average volume</th><th>injected volume</th><th>estimated amount</th><th>gas volume dose</th></tr></thead><tbody><tr><td colspan="14" style="text-align: left; font-weight: bold;">A-TAU MODELS</td></tr><tr><td>Nisbet, 2017 [57]</td><td>SUS</td><td>RN2N (scFv)</td><td>0,7</td><td>1</td><td>0,7</td><td>10</td><td>10</td><td>6 s/spot</td><td>-</td><td>in-house; lipid shell, octafluoropropane</td><td>-</td><td>30 µL</td><td>-</td><td>-</td><td>once per week for 4 weeks</td><td>whole brain</td></tr><tr><td>Pandit, 2019 [58]</td><td>SUS</td><td>x</td><td>0,65</td><td>1</td><td>0,65</td><td>10</td><td>10</td><td>6 s/spot (24 spots)</td><td>ketamine (90 mg/kg) and xylazine (6 mg/kg)</td><td>in-house; phospholipid shell, octafluoropropane; 1-5 × 10<sup>8</sup>/mL</td><td>-</td><td>1 µL/g</td><td>3 × 10<sup>6</sup></td><td>-</td><td>once per week for 15 weeks</td><td>whole brain</td></tr><tr><td>Karakatsani, 2019 [59]</td><td>FUS</td><td>x</td><td>0,45</td><td>1,5</td><td>0,37</td><td>6,7</td><td>10</td><td>60</td><td>isoflurane 1-2 %</td><td>in-house; 8 × 10<sup>8</sup>/mL</td><td>1,4 µm</td><td>0,1 µL/g</td><td>3 × 10<sup>5</sup></td><td>4,2 × 10<sup>5</sup></td><td>once per week for 4 weeks</td><td>left hippocampus</td></tr><tr><td>Bajracharya, 2022 [60]</td><td>SUS</td><td>RNF5 (IgG)</td><td>0,5</td><td>1</td><td>0,5</td><td>10</td><td>10</td><td>6 s/spot (48 spots then 72 spots)</td><td>ketamine (90 mg/kg) and xylazine (6 mg/kg)</td><td>in-house; phospholipid shell, octafluoropropane; 1-5 × 10<sup>9</sup>/mL</td><td>1 µm</td><td>1 µL/g</td><td>1,5 × 10<sup>8</sup></td><td>1,5 × 10<sup>8</sup></td><td>once per week for 12 weeks</td><td>whole brain</td></tr><tr><td>Geraudie, 2023 [61]</td><td>LIPU</td><td>x</td><td>0,3</td><td>1</td><td>0,3</td><td>25</td><td>1</td><td>120</td><td>ketamine (100 mg/kg) and xylazine (10 mg/kg)</td><td>Sonovue: 2 × 10<sup>8</sup>/mL</td><td>2,5 µm</td><td>100-120 µL</td><td>2 × 10<sup>7</sup></td><td>5 × 10<sup>7</sup></td><td>once per week for 5 weeks</td><td>right hemisphere</td></tr><tr><td>Pacia, 2023 [62]</td><td>FUS</td><td>x</td><td>0,68</td><td>1,5</td><td>0,56</td><td></td><td>6,7</td><td>5</td><td>32</td><td>-</td><td>Definity; 4 × 10<sup>6</sup>/mL</td><td>1,4 µm</td><td>25 µL</td><td>1 × 10<sup>5</sup></td><td>1,4 × 10<sup>5</sup></td><td>once</td><td>right hemisphere right cortex and hippocampus</td></tr><tr><td>Janowicz, 2019 [63]</td><td>SUS</td><td>RN2N (scFv, Fab, IgG format)</td><td>0,65</td><td>1</td><td>0,65</td><td>10</td><td>10</td><td>6 s/spot</td><td>isoflurane 1-2 %</td><td>in-house; phospholipid shell, octafluoropropane diluted in saline; 1-5 × 10<sup>7</sup>/mL</td><td>4 µm</td><td>40 µL</td><td>4 × 10<sup>5</sup></td><td>1,6 × 10<sup>6</sup></td><td>once</td><td>whole brain</td></tr><tr><td>FUS</td><td>RN2N (scFv, Fab, IgG format)</td><td>0,6</td><td>1</td><td>0,6</td><td>10</td><td>10</td><td>60</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>once</td><td>left hippocampus</td></tr><tr><td colspan="14" style="text-align: left; font-weight: bold;">B-MIXED MODELS</td></tr><tr><td>Shen, 2020 [64]</td><td>FUS</td><td>x</td><td>0,64</td><td>0,996</td><td>0,64</td><td>10</td><td>1</td><td>60</td><td>isoflurane 1.5 %</td><td>in-house; lipid shell, perfluoropropane; 1-3 × 10<sup>9</sup>/mL</td><td>1,5 µm</td><td>0.2 µL/g</td><td>6 × 10<sup>6</sup></td><td>1,8 × 10<sup>7</sup></td><td>twice per week for 6 weeks</td><td>right hippocampus</td></tr><tr><td>Noel, 2023 [65]</td><td>FUS</td><td>x</td><td>0,45</td><td>1,5</td><td>0,37</td><td>-</td><td>4</td><td>60 s (4 spots)</td><td>isoflurane 1.5-2 %</td><td>in-house; lipid-shelled, perfluorobutane; 8 × 10<sup>8</sup>/mL</td><td>-</td><td>50 µL</td><td>4 × 10<sup>7</sup></td><td>-</td><td>once per month for 5 months</td><td>bilateral hippocampus</td></tr><tr><td>Karakatsani, 2023 [66]</td><td>FUS</td><td>x</td><td>0,4</td><td>1,5</td><td>0,33</td><td>10</td><td>5</td><td>8-360</td><td>isoflurane 1-2 %</td><td>in-house; 8 × 10<sup>8</sup>/mL</td><td>1,4 µm</td><td>0,1 µL/g</td><td>2,4 × 10<sup>6</sup></td><td>3,4 × 10<sup>6</sup></td><td>once per week for 4 weeks</td><td>bilateral hippocampus</td></tr><tr><td colspan="14" style="text-align: left; font-weight: bold;">C-Aβ MODELS</td></tr><tr><td>Leinenga, 2016 [39]</td><td>SUS</td><td>x</td><td>0,7</td><td>1</td><td>0,7</td><td>10</td><td>10</td><td>6 s/spot</td><td>Zoletil (tolétamine, zolazépam) (20 mg/kg) and xylazine (10 mg/kg)</td><td>in-house; phospholipid shell, octafluoropropane diluted in saline; 1-5 × 10<sup>7</sup>/mL</td><td>4 µm</td><td>1 µL/g</td><td>3 × 10<sup>5</sup></td><td>4,2 × 10<sup>5</sup></td><td>once per week for 4 weeks and one 2 weeks after</td><td>whole brain</td></tr></tbody></table>

(continued on next page)
<table><caption>Table 2 (continued)</caption><thead><tr><th rowspan="2">Reference</th><th rowspan="2">US type</th><th rowspan="2">Associated drug</th><th rowspan="2">Pressure (Mpa)</th><th rowspan="2">Center frequency (MHz)</th><th rowspan="2">Mechanical index</th><th rowspan="2">Pulse length (ms)</th><th rowspan="2">PRF (Hz)</th><th rowspan="2">Duration (s)</th><th rowspan="2">Anesthesia</th><th colspan="4">MB</th><th rowspan="2">Sonication regimen</th><th rowspan="2">Brain target</th></tr><tr><th>type</th><th>Average injected volume</th><th>estimated amount</th><th>gas volume dose</th></tr></thead><tbody><tr><td>Luo, 2022 [67]</td><td>FUS</td><td>Gastrodin</td><td>—</td><td>1</td><td>—</td><td>10</td><td>1</td><td>120</td><td>isoflurane 1.5 %</td><td>Sonovue: 2 × 10<sup>8</sup>/mL</td><td>2,5 µm</td><td>2,5 µl/g</td><td>—</td><td>—</td><td>5 times every 3 days</td><td>left hippocampus</td></tr><tr><td>Mi, 2022 [68]</td><td>FUS</td><td>asparagine endopeptidase inhibitor</td><td>—</td><td>1</td><td>—</td><td>—</td><td>—</td><td>120</td><td>in-house; nanobubbles loaded with the drug</td><td>50 nm</td><td>—</td><td>—</td><td>—</td><td>—</td><td>once per week for 5 weeks</td><td>hippocampus</td></tr><tr><td>Kovacs, 2018 [69]</td><td>FUS (MRI-guided)</td><td>x</td><td>0,3</td><td>0,548</td><td>0,41</td><td>—</td><td>—</td><td>—</td><td>D-WILD-TYPE MICE</td><td>Optison; 5-8 × 10<sup>8</sup>/mL</td><td>3 µm</td><td>460 µL/kg repeated 4-6 times</td><td>4,5 × 10<sup>7</sup></td><td>1,4 × 10<sup>8</sup></td><td>once then once per week for 6 weeks</td><td>left cortex and right hippocampus</td></tr><tr><td>Meng, 2023 [70]</td><td>FUS (MRI-guided)</td><td>x</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>E-AD patients</td><td>Definity; 4 × 10<sup>6</sup></td><td>1,4 µm</td><td>4µL/g</td><td>5 × 10<sup>8</sup></td><td>7 × 10<sup>8</sup></td><td>every two weeks for 6 weeks (1 to 4 in practice)</td><td>default mode network</td></tr></tbody></table>

Fab: Fragment antigen-binding; FUS: Focused ultrasound; IgG: Immunoglobulin G; LIPU: Low-intensity pulsed ultrasound; MB: Microbubbles; MRI: Magnetic resonance imaging; PRF: Pulse repetition frequency; scFv: single-chain fragment variable; SUS: Scanning ultrasound; US: ultrasound. x indicates sonication treatment with no adjunct therapies. — indicates when data are missing. Studies are organized given the type of used ultrasound and publication date.

plasmatic tau (ptau-231 and ptau-181) was indeed significantly increased in the hour following BBB opening with ultrasound. This result may be interpreted as an increased release of tau from the brain to the blood circulation but could concurrently indicate a sonication-induced increase of central brain tau pathology as captured in peripheral fluids [62]. Similar results came from a study involving patients with AD who received sonication targeting the default mode network (composed of different brain regions, activated when a subject is at rest, not engaged in a specific activity) and that showed increased total tau in CSF one week after treatment [70]. These results might be interpreted as an increased release (clearance) of brain tau in the CSF after BBB opening or alternatively as evidence of neuronal/axonal injury increasing tau CSF dosage after sonication [73]. Finally, Kovacs et al. reported the emergence of hyperphosphorylated tau immunoreactivity and of neurons with neurofibrillary tangles-like morphologies in the sonicated hemisphere of WT rats after repeated sonications; these results were confirmed by increased biochemical dosage (Western blot) of phosphorylated tau in the same areas.

A final point concerns the topography and spatial extent of tau-induced modifications (when observed, either as an up- or down-regulation) after sonication. Four studies made use of unilateral targets and assessed whether the effects of BBB opening were different between the sonicated and the non-sonicated brain areas. A lateralization effect was clear in two of these studies [64,69] with effects limited to the ipsilateral (sonicated) hemisphere but was not confirmed in two other studies using lower ultrasound intensities [59,61] that showed, in contrary, tau modulations similarly distributed across ipsi- and contralateral hemispheres.

### 3.6. Effects on glial cells

Five studies included in this systematic review explored effects of sonication on glial cells (in mouse models for tau pathology [59–61] and in WT rats [69]). Two studies in the tau transgenic K3 model reported an increased number of cells as well as of area occupied by Iba1-positive microglia [60] subsequent to BBB opening by sonication. CD68 immunofluorescence signal (activated microglia and macrophages) was also reported to be increased in sonicated Tg4510 transgenics [59]. On the contrary, microglial cell density and area of occupancy (Iba1 immunostaining) were found to be decreased in sonicated P301S model after repeated sonication sessions [61], suggesting that the effects of BBB opening with sonication might be model-dependent or related to sonication protocols (FUS/SUS versus LIPU) and variable levels of BBB opening between studies. To our knowledge, no study has further explored, besides density and occupancy metrics, the impact of sonication on fine morphological modifications of microglia in tau models. One report quantified the astrocyte population in P301S mice and found no differences between sonicated and control groups [61].

Kovacs and colleagues analyzed the effects of sonication in wild-type rats and showed upregulation of both astrocytes and microglia populations (surface of occupancy), 1 and 7 weeks after treatment [69]. Glial brain coverage returned to baseline 13 weeks after sonication underlining possible reversibility of the effects of BBB opening. However, an increase of CD68+ microglia was still observed in sonicated areas (rostral cortex, hippocampus) at 1, 7 weeks post-sonication and remained persistent at 13 weeks. The increase of CD68+ cells was particularly important in rats having received multiple rounds of sonication. These results indicate that the different glial cells (and their activation status) may undergo changes with specific kinetics after sonication. The delay between sonication and subsequent analysis of brain tissue and repeated sonications may also modulate the effects of BBB opening on glial cells.

Interestingly, the two studies showing distributed (bilateral) effects of unilateral sonication on tau lesions [59,61] also reported wide modulation of microglial populations, in both hemispheres, after BBB opening.
<table><caption>**Table 3**
Synthesis of the main results of studies reporting effects of sonication (+/- adjunct drugs) on tau levels and associated phenotypes.</caption><thead><tr><th rowspan="2">Reference</th><th rowspan="2">Animal model or patients population (n group)</th><th rowspan="2">Sex</th><th rowspan="2">Age at onset of treatment</th><th rowspan="2">Tau pathology (plaques groups)</th><th rowspan="2">Ultrasmall type</th><th rowspan="2">Associated drug</th><th rowspan="2">Delay after last sonication</th><th rowspan="2">Sonication regimen</th><th colspan="2">Modulation of tau</th><th colspan="2">Modulation of neuroinflammation</th><th colspan="4">Modulation of functional phenotypes</th><th rowspan="2">Safety</th></tr><tr><th>IHC</th><th>WB/ELISA</th><th>Biomarkers (Blood or CSF)</th><th>Potentiation drug+US</th><th>Microglia</th><th>Astrocyes</th><th>Potentiation drug+US</th><th>Motor</th><th>Behaviour</th><th>Cognition</th><th>Potentiation drug+US</th></tr></thead><tbody><tr><td colspan="17">A-TAU MODELS</td></tr><tr><td>Nisbet, 2017 [57]</td><td>pR5 mice (5)</td><td>F</td><td>4.5 months</td><td>++</td><td>SUS</td><td>RN2N (scFv)</td><td>1 week ?</td><td>Uweek for 4 weeks</td><td>▼ AT8, AT180</td><td>-</td><td>-</td><td>▼ AT8, AT180, 128</td><td>-</td><td>n/a</td><td>-</td><td>Elevated plus mice</td><td>-</td><td>Elevated plus mice</td><td>-</td></tr><tr><td>Pandini, 2019 [58]</td><td>K3 mice (10)</td><td>M-F</td><td>1.5 month</td><td>++</td><td>SUS</td><td>x</td><td>3 days</td><td>Uweek for 15 weeks</td><td>▼ AT8, RN235, Bielschowsky</td><td>++ AT8</td><td>-</td><td>-</td><td>n/a</td><td>-</td><td>n/a</td><td>▲ Rotarod</td><td>-</td><td>Y-maze Novel object</td><td>n/a</td><td>** weight, HE (neuronal integrity, hemorrhages)</td></tr><tr><td>Karatsanasi, 2019 [59]</td><td>τε4510 mice (5)</td><td>F</td><td>3.5 months</td><td>++</td><td>FUS</td><td>x</td><td>1 day</td><td>Uweek for 4 weeks</td><td>▼ AT8</td><td>-</td><td>-</td><td>n/a</td><td>▲</td><td>-</td><td>n/a</td><td>-</td><td>-</td><td>-</td><td>n/a</td><td>** MRI (tissue damage); NonN, β-tubulin III (neuronal integrity)</td></tr><tr><td>Bujracharya, 2022 [60]</td><td>K3 mice (7)</td><td>M</td><td>1 month</td><td>++</td><td>SUS</td><td>RNF5 (IgG)</td><td>A few days (unclear)</td><td>Uweek for 12 weeks</td><td>▼ AT180, RN235, AT8</td><td>** Tau 5, AT8, AT180</td><td>-</td><td>** Tau 5, AT8, AT180, RN235</td><td>▲</td><td>-</td><td>++ Rotarod</td><td>-</td><td>-</td><td>** Rotarod</td><td>** weight</td><td>** weight</td></tr><tr><td>Gerardic, 2023 [61]</td><td>P301S (P519) mice (7)</td><td>M</td><td>4.5 months</td><td>+</td><td>LIPU</td><td>x</td><td>7 days</td><td>Uweek for 5 weeks</td><td>▲ AT8</td><td>-</td><td>-</td><td>n/a</td><td>▼</td><td>++</td><td>n/a</td><td>-</td><td>-</td><td>-</td><td>n/a</td><td>▲ 1/26 mice died after 1st sonication</td></tr><tr><td></td><td></td><td></td><td></td><td>+</td><td></td><td></td><td>1 day</td><td></td><td>▲ AT8</td><td>-</td><td>-</td><td>n/a</td><td>-</td><td>-</td><td>n/a</td><td>-</td><td>-</td><td>-</td><td>n/a</td><td>▲ 1/26 mice died after 1st sonication</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>30 min</td><td>ance</td><td>-</td><td>▲ pTau-181 pTau-231</td><td>-</td><td>-</td><td>-</td><td>n/a</td><td>-</td><td>-</td><td>-</td><td>n/a</td><td>▲ HE (micro-hemorrhages)</td><td>▲ HE (micro-hemorrhages)</td></tr><tr><td colspan="17">B-MIXED MODELS</td></tr><tr><td>Shin, 2020 [64]</td><td>3xTg mice (10)</td><td>F</td><td>8 months</td><td>+</td><td>FUS</td><td>x</td><td>6 weeks</td><td>2week for 6 weeks</td><td>▼ AT8, AT180</td><td>-</td><td>-</td><td>n/a</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>** HE (hemorrhage, vaculation)</td><td>** HE (hemorrhage, vaculation)</td></tr><tr><td>Noel, 2023 [65]</td><td>3xTg mice (10)</td><td>F</td><td>2 months</td><td>+</td><td>FUS</td><td>x</td><td>8 days</td><td>1/month for 5 months</td><td>-</td><td>▼ total tau</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>** weight, HE (neuronal integrity, hemorrhages)</td><td>** weight, HE (neuronal integrity, hemorrhages)</td></tr><tr><td>Karatsanasi, 2023 [66]</td><td>3xTg mice (5)</td><td>F</td><td>14 months</td><td>++</td><td>FUS</td><td>x</td><td>approx. 1 week (unclear)</td><td>Uweek for 4 weeks</td><td>▼ HT7</td><td>▼ HT7</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>** MRI (oedema, persistence of BBB opening)</td><td>** MRI (oedema, persistence of BBB opening)</td></tr><tr><td colspan="17">C-AB MODELS</td></tr><tr><td>Leinenga, 2016 [39]</td><td>APP23 mice (10)</td><td>M-F</td><td>14 months</td><td>-</td><td>SUS</td><td>x</td><td>3 days</td><td>1/week for 4 weeks and 1-2 weeks after</td><td>-</td><td>** AT8</td><td>-</td><td>-</td><td>n/a</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>** Nsul, HE (oedema, hemorrhage); fuchin acid (sulbemia)</td><td>** Nsul, HE (oedema, hemorrhage); fuchin acid (sulbemia)</td></tr><tr><td>Luo, 2022 [67]</td><td>Kunming mice with IVC Aβ (5)</td><td>M</td><td>-</td><td>-</td><td>FUS</td><td>Gastrodin</td><td>2 days</td><td>1/3 days for 15 days</td><td>-</td><td>** tau, pTau</td><td>▼ pTau</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>** HE, TUNEL (neuronal integrity apoptosis)</td><td>** HE, TUNEL (neuronal integrity apoptosis)</td></tr><tr><td>Mi, 2022 [68]</td><td>APP/PS mice (6)</td><td>-</td><td>6.5 months</td><td>-</td><td>FUS</td><td>asparagine oxidoreductase inhibitor</td><td>1 week</td><td>1/week for 5 weeks</td><td>-</td><td>-</td><td>▼ clear tau, pTau</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>▲ plasma NfL, CSF total tau (neuronal integrity, MRI (micro-hemorrhages))</td><td>▲ plasma NfL, CSF total tau (neuronal integrity, MRI (micro-hemorrhages))</td></tr><tr><td colspan="17">D-WILD-TYPE MICE</td></tr><tr><td>Kovacs, 2018 [69]</td><td>Sprague Dawley rats (6)</td><td>F</td><td>1.5 months</td><td>-</td><td>FUS</td><td>x</td><td>13 weeks</td><td>once</td><td>▲ AT270</td><td>** AT180</td><td>-</td><td>n/a</td><td>▲</td><td>▲</td><td>n/a</td><td>-</td><td>-</td><td>-</td><td>n/a</td><td>▲ histology, MRI (micro-hemorrhages, atrophy)</td><td>▲ histology, MRI (micro-hemorrhages, atrophy)</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>1 week</td><td>1/week for 6 weeks</td><td>▲ AT270</td><td>▲ AT180</td><td>-</td><td>n/a</td><td>▲</td><td>▲</td><td>n/a</td><td>-</td><td>-</td><td>-</td><td>n/a</td><td>▲ histology, MRI (micro-hemorrhages, atrophy)</td><td>▲ histology, MRI (micro-hemorrhages, atrophy)</td></tr><tr><td colspan="17">E-AD patients</td></tr><tr><td>Meng, 2023 [70]</td><td>AD patients (9)</td><td>M-F</td><td>70.2±7.2 years</td><td>+</td><td>FUS</td><td>x</td><td>follow up to 6 months</td><td>1/2 weeks for 6 weeks</td><td>-</td><td>-</td><td>▲ total tau (CSF)</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>▲ plasma NfL, CSF total tau (neuronal integrity, MRI (micro-hemorrhages))</td><td>▲ plasma NfL, CSF total tau (neuronal integrity, MRI (micro-hemorrhages))</td></tr></tbody></table>

CSF: Cerebrospinal fluid; F: female; FUS: Focused ultrasound; HE: hematoxylin eosin coloration; ICV: intracerebroventricular; IHC: Immunohistochemistry; IgG: Immunoglobulin G; LIPU: Low-intensity pulsed ultrasound; M: male; MRI: Magnetic resonance imaging; NfL: neurofilament light chain; ptau: phosphor tau; scFv: single-chain fragment variable; SUS: Scanning ultrasound.

**A- Animal models for tauopathies** are the most adequate models to evaluate the effects of sonication-induced BBB opening on tau proteinopathies and associated phenotypes (neuroinflammation status, neurological symptoms)

**B- Animal models for tauopathies + amyloidosis (mixed).** While not being pure models for tauopathies, mixed lines for Aβ/tau can still be considered for evaluating specific effects of BBB opening on tau lesions. However, due to the composite nature of these models, it is not possible to decipher if modulations of neuroinflammatory and neurological (motor-behavioral-cognitive) phenotypes are linked to sonication impacts on tau and/or Aβ proteinopathies. Therefore, these outcomes will not be considered and discussed in the present review (dark grey).

**C- Animal models for amyloidosis.** Mice engineered to harbor Aβ brain lesions can also develop subtle ptau accumulations. For the same reasons as for mixed models (see above), modulations of inflammatory/neurological phenotypes in these models will not be considered and discussed in the present review (dark grey).

**D-Wild-type animals.** A single report on sonication in non-transgenic animals is indicated (presenting an evaluation of ptau levels in sonicated animals).

**E-Patients with AD.** For the same reasons as exposed above, the table focused on post-sonication tau/ptau changes in patients reported in this single study. Other readouts will not be considered (dark grey) as their association with tau modulations cannot be strictly assessed.

Modulation of tauopathies, markers of neuroinflammation and of functional phenotypes are depicted for each listed paper.

▼ indicates a decrement in the studied variable (i.e. biological marker, behavioral performance).

▲ indicates an increase of the studied variable.

(▼/▲) indicate minimal effects (trends, non-significant results).

↔ underlines no significant variation of the studied variable.

? points to results with disputable interpretation.

– indicates when data are missing (information omission in reports or variable not investigated in the study).

x indicates sonication treatment with no adjunct therapies.

n/a non-applicable

Green color indicates beneficial effects of treatments (e.g. reduction of tauopathies, rescue of behavioral phenotypes) while red color pinpoints deleterious effects (e.g. increase in pathological markers, demonstration of adverse effects).

For each variable investigated, in addition to the direction and magnitude of changes post-sonication, the different markers used (e.g. antibodies for IHC, behavioral tests etc.), when reported in papers, are also indicated.
## 3.7. Effects on behavior and cognition

Within the 15 selected studies, only 3 studies have assessed the effects of sonication on behavior or cognition in mice modelling pure tauopathies (i.e. without co-induced amyloid pathologies) [57,58,60]. Studies exploring impacts of BBB opening on motor functions (coordination in the rotarod test) showed divergent results: while Pandit et al. described evidence of recovery of motor coordination (rotarod test) in the K3 model post sonication, Bajracharya et al., using the same mouse model and the same motor evaluation test, did not find any effects of sonication [58,60]. Besides using different ultrasound parameters, these two studies differed in the number of sonication sessions (15 in [58] and 12 in [60]) and the positive effects were seen only after 10 sessions suggesting that the functional improvement may depend on the number of treatment sessions.

No effect of sonication were reported on anxiety-related behaviors (elevated plus maze task) that remained altered in both control and treated pR5 model for tau pathology [57].

The effects of BBB opening on cognitive functions were assessed in a single study [58]. No rescuing effects were noted for recognition memory (novel object recognition task). The same study claimed for a recovery of function assessed in the Y-maze (spontaneous alternation task) as sonicated tau transgenics performed better than untreated mice. However, untreated transgenic mice did not display any impairment at baseline (no difference with wild-type littermates) making results difficult to interpret [58].

## 3.8. Underlying mechanisms of action

Several mechanisms have been proposed and explored in the included studies to explain the modulation of tau and associated phenotypes (inflammation, neurological functions) following BBB opening with sonication.

As previously evoked to explain clearance of amyloid under the action of sonication [38,39,74], microglia phagocytosis may play a mechanistic role in the effects of sonication on tau pathologies. Indeed, Karakatsani et al. [59] found that microglia activation (quantified with CD68 staining) correlated with a decrease in phosphorylated tau densities and that CD68 and AT8 markers colocalized more frequently in sonicated mice than in non-sonicated mice. Using confocal microscopy, hyperphosphorylated tau was observed inside microglia of sonicated mice, evocative of phagocytosis. Pandit et al., on the contrary, failed to observe colocalization of microglia markers and tau aggregates [58]. Bajracharya et al. reported, in sonicated tau transgenics, an increased microglia occupancy and a concomitant decrease of tau burden [60]. Géraudie et al. observed an opposite pattern of sonication-induced effects: reduction in microglia densities paralleled by a moderate increase in tau pathology, but without a significant correlation between these modulations [61]. In summary, a clear demonstration that increased or decreased microgliosis in sonicated animals impacts directly on tau pathology is still lacking.

Other mechanisms have been explored to decipher the effects of BBB opening with sonication on tau lesions. First, glycogen synthase kinase 3β (GSK3β), a serine and threonine kinase [75], which is closely implicated in tau aggregation and in regulating tau under physiological and pathological conditions [76,77], was hypothesized as participating to the molecular effects of sonication on tau. GSK3β activity was reported as decreased with sonication but with inconsistent results (decreased in [57] but not in [58]). Second, ubiquitination, which has been described to favor the degradation of aggregated tau through the proteasomal pathway [78], has been quantified but did not show any significant difference between sonicated and non-sonicated groups [58]. Third, exploring the autophagosomal pathway, Pandit et al. showed an increased neuronal autophagy of hyperphosphorylated tau in sonicated mice which may mediate the overall decrease in tau pathology observed in this study [58].

## 3.9. Safety considerations

A total of 12 of the reviewed studies provided feedback on the safety of BBB opening using sonication. Most of them found that sonication did not create oedema or hemorrhage as assessed by MRI examination [59,66] and/or histological analysis [39,58,59,64,65,67]. Consistently, in most of the studies, there were no reports of neuronal or tissular alteration post-sonication [39,58,59,64–67]. The body weights of mice also remained stable after the multiple rounds of sonication [58,60,65].

However, some reports underlined significant side effects. Géraudie et al. reported that 1/26 mouse with P301S background died after the first sonication session [61]. Additionally, micro-hemorrhages and an increase in plasmatic dosage of light neurofilaments was observed in one pre-clinical and one clinical studies, along with an increase of CSF total tau in the clinical study [62,70]. The levels of light neurofilaments in plasma returned to baseline after 6 months in the clinical study. Kovacs et al. also reported microhemorrhages stigma with MRI (T2* hypointensities) in all animals (WT rats) after multiple sonication sessions, especially in the sonicated hemisphere, along with cortical atrophy [69].

### 3.9.1. BBB opening with sonication combined to adjunct drug

The main results of the included studies on the potentiation of therapeutic drugs with BBB opening using sonication are included in Table 3.

### 3.10. Potentiation of brain pharmacokinetics of drugs acting on tau

In most of the studies combining BBB opening with a therapeutic drug (3/4), sonication greatly potentiated the entry of the drug into the brain parenchyma [57,60,68]. The drugs used were a scFv antibody (RN2N) directed against tau [57], an anti-tau monoclonal IgG (RNF5) [60], gastrodin, a phenolic glycoside extracted from the traditional Chinese herb [67] and an asparaginase endopeptidase inhibitor [68]. Interestingly, Nisbet et al. showed that concentration of the RN2N scFv gained an 11-fold increase (quantified by fluorescence intensity) and that RN2N was efficiently taken up by neurons under the action of sonication [57]. On the contrary, despite an increased penetration into the brain parenchyma, Bajracharya et al. failed to show neuronal uptake of their anti-tau IgG [60]. Janowicz et al. explored further the ability of different types of anti-tau antibodies (scFv, Fab, IgG formats) to enter the brain after sonication [63]. They showed that sonication increased the brain concentrations of the three antibody formats, but these effects were mitigated by serum biodisponibility. Indeed, despite advantages in terms of diffusion, reduced immunogenicity, and increased binding, small-sized scFv antibodies were rapidly eliminated from the blood by the kidneys, hampering their brain penetration. On the contrary, the IgG format, with more limited plasmatic clearance, displayed an overall increased brain concentration after sonication.

### 3.11. Potentiated effects on tau pathology

Two studies found an effect of combined treatment (therapeutic compound + sonication) on tau pathology (assessed by Western blots) while single treatments (i.e. drug without sonication) had no effect [67,68].

Considering passive immunotherapy, Nisbet et al. also showed augmented efficacy (reduction of ptau aggregates evaluated by immunohistochemistry) of scFv anti-tau antibody injection associated with BBB opening when compared to drug or sonication alone [57]. However, Bajracharya et al. [60] failed to show a similar potentiated effect of combined treatments (immunotherapy + sonication) on tau pathology assessed by immunohistochemistry or biochemical methods (WB). These last negative results might be related to the properties of the monoclonal tau-specific IgG antibody (RNF5) used in the latter study: RNF5 was detected in the brain parenchyma after sonication but in unidentified cells of the hippocampal pyramidal cells layer that did not contain
hyperphosphorylated tau. Moreover, the same unspecific cell-targeting of RNF5 was observed in wild-type mice devoid of tau lesions. This suggests a particular in vivo cell tropism of injected RNF5 IgGs and a lack of specificity of this anti-tau antibody [60].

### 3.12. Potentiated effects on other phenotypes

Only one study included in our systematic review assessed, using a tau transgenic model, the effects on glial cells of sonication associated with a monoclonal anti-tau antibody. In this study, there was no cumulative effect of the combined treatment compared to sonication alone: microglia densities and loads were similarly increased in the two groups of mice compared with mice receiving sham procedures [60].

Functional effects of combined treatments (drug + sonication) were assessed in two studies. Rescuing effects were depicted on anxiety-related behaviors (elevated plus maze) of pR5 mice treated with a combination of sonication and injection of an anti-tau scFv antibody [57]. However, it was not clear from the results that treatment with the antibody alone was less efficient than combined treatment. Another study failed to show beneficial effect of sonication combined to the RNF5 anti-tau IgG on motor capacities (rotarod) but, as discussed above, this antibody lacked efficacy (no impact on tau burden) and specificity (uncontrolled cell targeting) [60].

## 4. Discussion

This review collected studies exploring the effects of BBB opening using ultrasound associated with microbubbles on tau protein, either in animals or patients with tauopathies or in other preclinical models in which the effects of sonication on endogeneous tau/ptau levels were assessed. An overview of the results and of the major elements of discussion are presented in Fig. 2.

### 4.1. Summary of the results

A first critical point concerns the paucity of experimental reports (n = 15) that were identified in this systematic review, underlining that the field of research on the modulation of tau proteins by ultrasound BBB opening is still in its infancy, which may explain number of divergent results and unbalanced interpretations. Bibliographic analysis and data extraction was performed at the end of 2023 but to our knowledge no new experimental work was published in 2024 that may modify the overall findings and conclusions of the present systematic review.

Mirroring the beneficial effects observed on amyloid pathology [38–40], several studies highlighted that ultrasound-mediated BBB opening is able to decrease levels of (hyper)phosphorylated tau in mouse models overexpressing mutant human tau [57–60] as well as in mixed models combining Aβ and tau pathologies [64,66]. However, other

<figure><img src="image_5.png" /><figcaption>Fig. 2. Overview of the main findings of this systematic review concerning the effects of BBB opening with ultrasound in models of tauopathies.</figcaption></figure>

❶ The sonication procedure is schematized: once anesthetized, the animals received ultrasound through the skull. Under the action of ultrasound, systemic circulating microbubbles oscillate, contract and expand widening the tight junctions, increasing transcytosis of vesicles and inhibiting efflux pumps of the blood-brain barrier.

❷ The ultrasound are applied with several varying parameters, among which the peak negative pressure, the center frequency, the pulse length and pulse repetition frequency (PRF). When these parameters or the microbubbles dosage are not appropriate, stable cavitation may turn into inertial cavitation causing brain damage such as hemorrhages or oedema.

❸ Concerning results, several studies showed that BBB opening using ultrasound is safe and may have beneficial effects (mitigation) on tauopathies. However, other reports underlined side effects affecting brain integrity as well as an increase of tau pathology in sonicated animals. Studies evaluating the effects on neuroimmune cells and behavior are scarce. In the schematic representation of the results, green color is used for beneficial and/or safe effects and red color for detrimental effects; numbers refer to numbers of studies reporting positive/negative outcomes. The outcomes on neuroimmune cells are presented in black and grey because of the difficulty of determining if increase or decrease in cell population is beneficial versus detrimental.

❹ Divergent results between studies may be the result of several accumulating and interacting sources of variability concerning a) the ultrasound procedure and the resulting magnitude of BBB opening, b) the used mouse models and c) the assessment of the different outcomes.
studies showed that BBB opening with ultrasound can lead to paradoxical increase in tau/ptau levels. These latter contradictory results were reported in association with safety issues [61,62,69,70] leading to the hypothesis that adverse effects of sonication (e.g. induction of brain hemorrhages or neuronal injuries), when observed, can be paralleled by increased neurodegeneration markers levels including abnormally high levels of tau proteins in brain and peripheral fluids (see Table 3). Considering the effects of sonication on glial cell populations several reports underlined a “boosting” effect on microglia and astrocytes (see however [61]). Modulation of behavioral/cognitive phenotypes in tau transgenic models following sonication remains poorly explored and with, for now, divergent results [57,58,60]. Similarly, several mechanisms of action have been explored to account for the effects of BBB opening in animal models of tauopathies and did not allow emergence of a unified model hypothesis.

The added value of BBB sonication to increase brain penetration of drugs acting on tau pathology has only been assessed in a reduced number of studies with, again, varying results. A reduction of the tau burden with combined treatment was shown in the study of Nisbet et al. [57] but not in the one from Bajracharya et al. [60], possibly related to the type of antibody used. Two other reports showed that drugs combined with sonication can decrease tau and ptau species in brain tissue, but the effects of sonication alone were not assessed [67,68] precluding clear evaluation of beneficial effects of combined treatments. To our knowledge no modified behavioral performance was reported when combining drugs with sonication in mice with tauopathies [57,60].

## 4.2. Limitations of included studies

The identified studies evaluating the effects of BBB opening on tau were carried out using different animal models that underwent heterogeneous sonication protocols using inconstant control (sham) procedures. Also, the assessment methods for the principal readouts (measurement of tau/ptau) and associated phenotypes (glial response, behavioral changes etc.) varied largely between studies. Methodological diversity is common to young growing fields of research and must be taken into consideration to explain discrepant findings and afford refinement and optimization of subsequent research effort. We will first list here the different bottlenecks identified from existing literature that limit data comparison and integration and will propose possible directions for the future in the next section (“Implications for future studies”).

## 4.3. Animal models

In the 14 animal studies included in this review, 9 different models were used. In additions, some reports relied on models that are not widely used in preclinical research (e.g. K3, pR5 lines) [79,80] or that do present very aggressive tau pathology (rTg4510 model) [81]. Only 2 studies [61,62] were performed on PS19-P301S mice [82], one of the most frequently used model for tauopathy for preclinical research. Across all the models mimicking tauopathies, mice develop neuropathological lesions and especially tau accumulation with various topographical distributions, magnitudes, and kinetics. Moreover, some models display other neuropathological phenotypes such as early microglial activation in the P301S model [82] which may modulate the effects of sonication. The age and sex of mice used in the included studies is also variable and sexual dimorphism has been demonstrated in several mouse models mimicking AD pathologies [83–87]. Additionally, it has been found that both age and genotype variables impact BBB permeability and sensitivity to BBB disruption [88]. It seems therefore predictable that characteristics of the model used (line, sex, age, phenotypes, BBB vulnerability) are active contributors to the diverse effects of ultrasound BBB opening reported in the literature.

A second point concerns the design of control groups. Some FUS studies compared the sonicated hemisphere with the “control” non-

sonicated contralateral hemisphere [69] but most of the analyzed reports included a separate group of animals receiving a sham procedure. However, the management of the sham procedure varied between studies: injection of microbubbles without applying ultrasound [39,57–59,61,63,64], ultrasound without microbubbles [64] or no ultrasound and no microbubbles [60,62,65,68]. Only one study involved two sham groups: a control group receiving ultrasound without microbubbles and another group receiving microbubbles without ultrasound [64]. Indeed, one may consider that the effects of sonication (ultrasound + microbubbles) can partly be mediated by the sole action of ultrasound without requirements of BBB opening. Several studies showed beneficial effects of neuromodulation (application of low-intensity ultrasound) on memory along with an increase in neurotrophic factors in mice [89,90] and in patients with AD [91]. A subset of these studies used comparable ultrasound parameters as in standard BBB sonication protocols and reported positive effects on cognition in both wild-type [92] and amyloid transgenic mice [93]. The application of low-intensity pulsed ultrasound stimulation, with no BBB opening, was also found to be sufficient to induce significant modulation of microglial recruitment and activation [89,94]. All these data suggest that ultrasound alone may exert specific effects through mechanical forces applied to the brain. These “ultrasound alone” effects may contribute to the overall brain modulations observed after sonication and need to be taken into consideration (requirement for supplementary control groups) to carefully study the effects of BBB opening in tau models while minimizing or controlling the impact of confounding variables.

## 4.4. Sonication protocols

Induction of BBB opening by combined ultrasound and microbubbles involve different procedural steps that greatly vary between studies extracted for the present systematic review.

1) **Anesthesia modality** diverged between reports and anesthetics could have a differential impact on BBB permeability [95]. Spieth et al. found that isoflurane, the most commonly used anesthetics, can by itself enhance BBB permeability [96]. It has also been shown that using medical air (21 % O2) rather than 100 % oxygen as carrier gas extends in-vivo circulation time of microbubbles [97] and increases the amount of wideband emissions and BBB opening [98]. McDan-nold et al. reported however an increased BBB opening using keta-mine/xylazine compared to isoflurane and suggested that it could be accounted by the vasodilator effect of isoflurane that may decrease the action of microbubbles on the endothelium [99]. Finally, it is known that isoflurane promotes tau phosphorylation through cas-pase activation [100]. Altogether, this underlines that the variability in the anesthetics used can modulate the effects on BBB opening and the outcomes of interest, in particular tau dosages.

2) **Wide variability of sonication protocols** (FUS, SUS, LIPU) may affect the outcomes reported. Both SUS and FUS showed some congruent effects [57–60,64,66] on tau/ptau and glial cell populations but sometimes associated with side effects [62,69,70]. Only one study relied on LIPU precluding any firm conclusions [61]. Different ultrasound parameters were also used in the extracted studies. It is known that both acoustic pressure and center frequency modulate BBB opening [71,101] and influence the likelihood of inertial cavitation, which may increase the occurrence of inflammation and tissue damage [32,33,101–103]. However, from the present review it does not seem that specific parameters of ultrasound (mechanical index, pulse length/frequency, total duration of sonication etc.) could account neither for specific biological effects (tau/ptau, microglia, behavior) nor for the occurrence of adverse effects (hemorrhages, tissue damage). **Microbubbles** used in the included studies also differed widely between studies (in terms of volume, concentration, timing and duration of infusion), which may participate in discrepant outcomes such as BBB opening [34,101,104],
neuroinflammation and microhemorrhages [34,102,105]. The estimated microbubbles gas volume considered to be a good proxy of BBB opening extent [34] varied largely in the different collected reports. Importantly, more than the type/parameter of ultrasound/microbubbles, the overall amount of sonicated tissue and the magnitude of BBB opening (rarely quantified in studies) may influence the induced effects on the primary outcomes that were analyzed in this review (tau/ptau). This is certainly a critical aspect to be evaluated and controlled in future studies.

3) **Sonication regimen** could also be a contributive variable to the discrepant experimental results we listed. The frequency of sonication sessions (usually one per week but varying from once every 3 days [67] to once per month [65]) as well as their total number (varying from 1 to 15) differed largely between studies. This may impact the different outcomes. Kovacs et al. showed that the repetition of sonication sessions induce sterile inflammation and tissue damage as well as tau phosphorylation while these histopathological changes were not observed after a single sonication session [69]. Conversely, Pandit et al. reported motor improvement in the K3 mouse model only after 12 sessions of sonication [58]. The effect of the delay between the last session of sonication and analysis of brain tissue may also be an important variable. In a previous study on amyloid models, Jordao et al. showed an effect of this delay on microglia and astrocytes populations, depending on whether the mice were sacrificed 4 h or 15 days after sonication [38]. Consistently, it was found that increased transcription of proinflammatory cytokine genes increased at 6 h post-sonication and went back to baseline 24 h after treatment [106]. Variation in delays between sonication and outcomes assessment could therefore impact on measures of tau pathology, glial cells, neuronal health, and behavior and explain, in part, discrepancies between results.

To summarize, it appears important to consider all these protocol steps to better understand the effects of sonication on tau/ptau dosages and associated phenotypes. As detailed later (§ “Implications for future studies”), we will propose several options for assessing and controlling methodological diversity.

## 4.5. Data measurement and analysis

Besides experimental models and protocols multiplicity that may lead to heterogeneity in reported results, it is also important to pinpoint issues concerning analytical approaches. For instance, the evaluation of sonication-induced modification of tau burden underlined some divergent results when obtained through immunohistochemical quantifications versus biochemical analyses [57,58,60]. This might be explained by non-detectable changes of tau/ptau using biochemical assays [57] but it remains a limit to decipher the precise effects of BBB opening on tau lesions as standard efficacy should be confirmed by concurrent methods. A more standardized and unbiased approach may be required to precisely evaluate the different post-sonication readouts (tau/ptau, glial cells, functional outcomes). For future studies, it would be important, in particular, to use directly quantitative methods and established clear immunostaining procedures to limit the specificity/sensitivity issues of routinely used antibodies, and finally, to use appropriate imaging techniques to eliminate artifacts and generate unbiased data that could be compared between studies.

Concerning safety outcomes of sonication, different measures were also used, but often limited to H&E staining or identification of damaged neurons, and only few articles provided quantitative evaluation of tissue alterations (e.g. hemorrhage, oedema) which would be a prerequisite to firmly assess the possible deleterious effects of sonication in tau models.

In addition, it appears crucial to complete experimental findings by an upstream precise and reproducible assessment of sonication (e.g. feedback on cavitation) and of resulting BBB opening (volume estimation, location, time-duration of the opening etc.) which is lacking in

most of the examined reports while being potentially crucial in driving biological effects.

## 4.6. Implications for future studies

### 4.6.1. Improving comparability of results between studies

#### 4.6.1.1. Animal models and experimental groups.
The variety of models used in the cited studies should not be viewed as an obstacle to research but more as reflecting the polymorphism of existing human tauopathies (displaying lesions with various nature, density, topography and kinetics). Still, due to the reduced number of published reports, there is a clear need for extended studies both in models of tauopathies and in non-transgenic mice as genotype (and presumably genetic background) is linked to intrinsic BBB frailty characteristics [107]. Whatever the model used, it seems to be necessary, as a prerequisite for interventional action (sonication treatment), to identify and document i) the characteristics of induced BBB opening that may vary between models, ii) the amount of pathology (e.g. tau burden) displayed at initiation of treatment and that relies on sex/age variables. This will allow precise evaluation of tau sonication-induced changes as a function of intrinsic neuropathological staging and quantitation of BBB permeation.

Control groups in sonication paradigms should also be better defined. In particular, the addition of a “ultrasound-only” group seems important to clearly disentangle the biological effects of BBB opening vs. the possible action of ultrasound. Finally, while rarely performed, the inclusion of non-transgenic animals undergoing the same sonication regimen would allow to decipher specific effects (disease-modifier) vs. non-specific effects (observed in both transgenics and wild-type littermates) of sonication. Indeed, it remains possible that sonication could impact similarly both genotypes, at the level of behavioral-cognitive performances (e.g. increased memories), glial cell populations (e.g. increased number of microglia) and even tau/ptau brain concentrations (e.g. increased brain ptau dosage [108]).

#### 4.6.1.2. Evaluation of BBB opening and standardization of analytical procedures.
As for the diversity of animal models used, the variability of sonication procedure previously highlighted could offer precious information for the optimization of the different techniques and innovation in this field. However, to be able to take advantage of this variability, the evaluation of biological effects of sonication in tau mice should be better standardized.

Firstly, BBB opening induced by sonication, while poorly documented in most of reported studies, is crucial to be assessed and controlled as it is supposed to drive the main biological outcomes. A first option would be to measure in real time the mechanical effects of ultrasound in experimental setups. It is hence possible to use cavitation monitoring through real-time passive cavitation detection and feedback control of peak negative pressure given the ultra-harmonics and broadband emissions detected. This monitoring would help to evaluate related risk of inertial cavitation [109,110]. Also, in postmortem studies it is possible to quantify extravasation of peripheral markers (mouse IgG, albumin, intravenously injected dye) from serial brain sections. A more quantitative and precise quantification of Evans Blue is also available by spectrophotometry [111] but without topographical information on BBB opening. The assessment of BBB opening magnitude could also participate in the evaluation of the potential BBB opening by the anesthesia itself and a change of anesthetic protocol might be necessary if found to promote, per se, large or uncontrolled BBB opening in sham groups.

Secondly, evaluation of changes induced by sonication need also some degree of standardization. For instance, assessment of tau burden by immunohistochemistry should primarily rely on automated immunofluorescence staining. Applying H-score calculations that consider both labeling intensity and spatial coverage of staining is a good and validated option [112] that may help to quantify tau immunostaining
that often present both diffuse and focal distributions. The selection of quantitative and highly sensitive biochemical methods (e.g. multiplex ELISA, ©MSD) could minimize discrepant results (intra- and inter-studies) in the future. Behavioral-cognitive modifications should similarly be assessed with standard protocols to measure the functional outcomes of sonication in tau models. Quick and robust neurological battery exist [113] and can be complemented by highly standardized test for cognitive evaluation. For instance, Pandit et al. relied on short-term memory test (Y-maze alternation, object recognition) to measure effects of sonication in tau transgenics but the evaluation of spatial long-term memories (Morris Water Maze, Barnes maze) may provide a more robust readout, highly sensitive to therapeutic intervention [58]. Alternatively, the use of user-independent automated behavioral protocols could be an option to generate unbiased data and facilitate inter-studies comparison.

### 4.6.1.3. Refining mechanisms of action of ultrasound in tau models.
The mechanism of action (MOA) of potential effects of sonication in tau models remains elusive. As described above, enhanced microgliosis (including possible phagocytic activity) is still debated. Other signaling or molecular pathways (eg GSK3β modulation, stimulation of autophagy) rely on isolated reports and require additional experimental support. Other MOA need also to be explored as they have already been described in several publications on the general effects of BBB opening by ultrasound. In particular, modulation of neurogenesis [114,115] and of brain vasculature [106,116,117] may constitute potential mechanism to trigger (indirectly or directly) sonication-induced effects in tau mice.

More generally, studies will also need to assess neuropathology at different time points post-sonication. Indeed, the delay between sonication and tissue analysis differs largely between studies and this delay might have an impact on the nature and magnitude of histological changes. Only one study evaluated tauopathy in a mouse model at different time points (one day and seven days) after sonication and did not find differences in the profile of induced tau changes [61]. In addition, it will be necessary to understand the long-term effect of sonication on tau pathology and whether these effects are sustained over time or if the pathology might show a rebound. Indeed, Karakatsani et al. found decreased amyloid burden in brain of one patient with AD 3 weeks after sonication, but this beneficial effect did not persist after 3 months of treatment cessation [66] suggesting a therapeutic effect within a limited time window.

Considering tau lesions, it would be of great interest to assess if the effects of BBB opening impacts on different species or conformations of tau, for instance on aggregated tau (AT100, AT180 antibodies) or oligomeric forms (T22, TOC1 antibodies), on top of immunohistological quantitation using gold-standard antibodies (eg AT8) that was performed in most of the reported studies. Also, in relation with the prion-like spreading hypothesis and propagation of tau [118], it would be interesting to investigate the effects of sonication on the progression of tau lesions from brain to brain areas in addition to standard lesion load assessment.

Several studies included in this systematic review analyzed the effects of sonication on tau/ptau and concurrently on microglia [59–61]. A more comprehensive analysis of morphological modifications in microglial cells caused by BBB opening should be performed in the future (e.g. assessment of microglial typologies, immunohistochemical profiling using dedicated markers such as Clec7a or P2Y12, spatial transcriptomics analysis etc). In addition, other cells involved in neuroinflammation (astrocytes, infiltrating T-cells etc.) and dosages of pro-and anti-inflammatory cytokines should be explored to draw a clearer portrait of the glial/immune modulations occasioned by sonication in mouse models for tauopathies. Understanding further the effects of sonication on neuroinflammation and the effects of these modulations on the underlying proteinopathies could also be informative about the complex links between inflammation and tau burden. Indeed, if a

controlled increase in neuroinflammation might be beneficial to combat amyloid pathology [38,39,64,119], the same assertion remains poorly documented for tau lesions.

### 4.6.1.4. Other developments.
Finally, studies combining BBB opening with sonication and a therapeutic molecule targeting tau/ptau are very scarce [57,60]. The format of therapeutic antibodies seems to play a role in the efficiency to target phosphorylated tau into neurons (beneficial effects with scFv antibody [57] but not with IgG antibody [60]); this needs to be further investigated and confirmed, especially because the short-sized antibodies, while presenting obvious therapeutic advantages, have also a payload in terms of plasmatic clearance [63].

## 5. Conclusions

Ultrasound combined with microbubbles is an innovative, non-invasive, technique allowing to transiently open the BBB, with a potential to enhance brain pharmacokinetics of CNS-directed drugs. First applied in patients to treat brain tumors, sonication also showed beneficial effects in mouse models of neurodegenerative diseases such as Alzheimer's disease with observed decrease of amyloid burden after sonication, with or even without combined anti-amyloid therapeutics [74,120]. Several safety assays were performed in patients with AD to demonstrate the relative innocuity of the method [45–49]. The first clinical study in patients combining focused ultrasound mediated BBB opening and aducanumab (a monoclonal FDA-approved anti-Αβ antibody) has been recently published with enhanced clearance of Αβ pathology [37], increasing the clinicians' attention to this technique. Despite encouraging results in several preclinical studies, the effects of BBB opening with ultrasound are less clear for tau pathologies. Some studies reported warning signs of worsening tau pathology and safety issues in animal models. To our knowledge, only one study evaluated the effect of sonication on tau in patients with AD; an increase in CSF total tau was reported in this study [70] allowing no clear conclusion on the effects of treatment. For now, preclinical studies evaluating the potential effect of BBB opening combined with a therapeutic drug acting on tau are scarce and show divergent or unclear results.

A better characterization of the effects of ultrasound on all the neuropathological hallmarks (mostly tau pathology but also glial/immune cells and neuroinflammatory markers) as well as behavioral-cognitive outcomes in various animal models and a deeper understanding of underlying mechanisms remain critical before any translation to the patients.

## Fundings

The present work was also supported by funding from the program "Investissements d'avenir" ANR-10IAIHU-06, ANR-11-INBS-0011-NeurATRIS: Translational Research Infrastructure for Biotherapies in Neurosciences. A.G. received a PhD grant from the 'Institut Universitaire d'Ingénierie en Santé' at Sorbonne University.

## CRediT authorship contribution statement

**Amandine Géraudie:** Conceptualization, Methodology, Investigation, Writing – original draft, Visualization. **Pierre De Rossi:** Writing – review & editing. **Michael Canney:** Writing – review & editing. **Alexandre Carpentier:** Writing – review & editing, Supervision, Funding acquisition. **Benoît Delatour:** Conceptualization, Methodology, Investigation, Writing – review & editing, Visualization, Supervision, Project administration, Funding acquisition.

## Declaration of competing interest

MC and PDR are employees of Carthera. MC and AC are inventors of
patents related to the technology and have stock ownership in Carthera. AC has received funding support from Horizon 2020 European Innovation Council, is a paid consultant of Carthera, and is part of the Board of Directors of Carthera.

## Acknowledgments

The authors wish to thank the librarians of Paris Cité (Bernadette Brizion) and Sorbonne University (Agnès Kermin) for their help in retrieving references.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jconrel.2025.01.056.

## Data availability

The data is available from the corresponding author on reasonable request.

## References

[1] World Alzheimer Report, The Global Impact of Dementia: An Analysis of Prevalence, Incidence, Cost and Trends, Disponible sur, https://www.alzint.org/resource/world-alzheimer-report-2015/, 2015.

[2] J.A. Hardy, G.A. Higgins, Alzheimer's disease: the amyloid cascade hypothesis, Science 256 (5054) (1992) 184–185, 10 avr.

[3] J. Sevigny, P. Chiao, T. Bussière, P.H. Weinreb, L. Williams, M. Maier, et al., The antibody aducanumab reduces β plaques in Alzheimer's disease, Nature 537 (7618) (2016) 50–56, 1 sept.

[4] C.H. van Dyck, C.J. Swanson, P. Aisen, R.J. Bateman, C. Chen, M. Gee, et al., Lecanemab in early Alzheimer's disease, N. Engl. J. Med. 388 (1) (2023) 9–21, 5 janv.

[5] J.R. Sims, J.A. Zimmer, C.D. Evans, M. Lu, P. Ardayfio, J. Sparks, et al., Donanemab in early symptomatic Alzheimer disease: the TRAILBLAZER-ALZ 2 randomized clinical trial, JAMA 330 (6) (2023) 512, 8 août.

[6] M.A. DeTure, D.W. Dickson, The neuropathological diagnosis of Alzheimer's disease, Mol. Neurodegener. 14 (1) (2019) 32, déc.

[7] J. Götz, G. Halliday, R.M. Nisbet, Molecular pathogenesis of the Tauopathies, Annu. Rev. Pathol. Mech. Dis. 14 (1) (2019) 239–261, 24 janv.

[8] P.T. Nelson, I. Alafuzoff, E.H. Bigio, C. Bouras, H. Braak, N.J. Cairns, et al., Correlation of Alzheimer disease neuropathologic changes with cognitive status: a review of the literature, J. Neuropathol. Exp. Neurol. 71 (5) (2012) 362–381, mai.

[9] J.A. Tanner, L. Iaccarino, L. Edwards, B.M. Asken, M.L. Gorno-Tempini, J. H. Kramer, et al., Amyloid, tau and metabolic PET correlates of cognition in early and late-onset Alzheimer's disease, Brain 145 (12) (2022) 4489–4505, 19 déc.

[10] A. Boutajangout, J. Ingadottir, P. Davies, E.M. Sigurdsson, Passive immunization targeting pathological phospho-tau protein in a mouse model reduces functional decline and clears tau aggregates from the brain, J. Neurochem. 118 (4) (2011) 658–667, août.

[11] K. Yanamandra, N. Kfoury, H. Jiang, T.E. Mahan, S. Ma, S.E. Maloney, et al., Anti-tau antibodies that block tau aggregate seeding in vitro markedly decrease pathology and improve cognition in vivo, Neuron 80 (2) (2013) 402–414, oct.

[12] K. Yanamandra, H. Jiang, T.E. Mahan, S.E. Maloney, D.F. Wozniak, M. I. Diamond, et al., Anti-tau antibody reduces insoluble tau and decreases brain atrophy, Ann. Clin. Transl. Neurol. 2 (3) (2015) 278–288, mars.

[13] A. Ittner, J. Bertz, L.S. Suh, C.H. Stevens, J. Götz, L.M. Ittner, Tau-targeting passive immunization modulates aspects of pathology in tau transgenic mice, J. Neurochem. 132 (1) (2015) 135–145, janv.

[14] X. Chai, S. Wu, T.K. Murray, R. Kinley, C.V. Cella, H. Sims, et al., Passive immunization with anti-tau antibodies in two transgenic models, J. Biol. Chem. 286 (39) (2011) 34457–34467, sept.

[15] M. Albert, G. Mairet-Coello, C. Danis, S. Lieger, R. Caillierez, S. Carrier, et al., Prevention of tau seeding and propagation by immunotherapy with a central tau epitope antibody, Brain 142 (6) (2019) 1736–1750, 1 juin.

[16] J. Cummings, Y. Zhou, G. Lee, K. Zhong, J. Fonseca, F. Cheng, Alzheimer's disease drug development pipeline: 2024, Alzheimers Dement. Transl. Res. Clin. Interv. 10 (2) (2024) avr. (e12465).

[17] W.M. Pardridge, Blood-brain barrier and delivery of protein and gene therapeutics to brain, Front. Aging Neurosci. 11 (2019) 373.

[18] B. Bohrmann, K. Baumann, J. Benz, F. Gerber, W. Huber, F. Knoflach, et al., Gantenerumab: a novel human anti-β antibody demonstrates sustained cerebral amyloid-β binding and elicits cell-mediated removal of human amyloid-β, J. Alzheimers Dis. 28 (1) (2012) 49–69, 12 janv.

[19] D.B. Yadav, J.A. Maloney, K.R. Wildsmith, R.N. Fuji, W.J. Meilandt, H. Solanoy, et al., Widespread brain distribution and activity following i.c.v. infusion of anti-β-secretase (BACE1) in nonhuman primates, Br. J. Pharmacol. 174 (22) (2017) 4173–4185, nov.

[20] W.M. Pardridge, R.J. Boado, R. Giugliani, M. Schmidt, Plasma pharmacokinetics of Valanafusp alpha, a human insulin receptor antibody-Iduronidase fusion protein, in patients with Mucopolysaccharidosis type I, BioDrugs Clin. Immunother. Biopharm. Gene Ther. 32 (2) (2018) 169–176, avr.

[21] C. Chu, A. Jablonska, Y. Gao, X. Lan, W.G. Lesniak, Y. Liang, et al., Hyperosmolar blood-brain barrier opening using intra-arterial injection of hyperosmotic mannitol in mice under real-time MRI guidance, Nat. Protoc. 17 (1) (2022) 76–94, janv.

[22] C. Warren Olanow, R.T. Bartus, T.L. Baumann, S. Factor, N. Boulis, M. Stacy, et al., Gene delivery of neuritin to putamen and substantia nigra in Parkinson disease: a double-blind, randomized, controlled trial, Ann. Neurol. 78 (2) (2015) 248–257, août.

[23] K. Hynynen, F.A. Jolesz, Demonstration of potential noninvasive ultrasound brain therapy through an intact skull, Ultrasound Med. Biol. 24 (2) (1998) 275–283, févr.

[24] K. Hynynen, N. McDannold, N. Vykhodtseva, F.A. Jolesz, Noninvasive MR imaging-guided focal opening of the blood-brain barrier in rabbits, Radiology 220 (3) (2001) 640–646, sept.

[25] N.I. Vykhodtseva, K. Hynynen, C. Damianou, Histologic effects of high intensity pulsed ultrasound exposure with subharmonic emission in rabbit brain in vivo, Ultrasound Med. Biol. 21 (7) (1995) 969–979.

[26] K. Hynynen, N. McDannold, H. Martin, F.A. Jolesz, N. Vykhodtseva, The threshold for brain damage in rabbits induced by bursts of ultrasound in the presence of an ultrasound contrast agent (Optison®), Ultrasound Med. Biol. 29 (3) (2003) 473–481, mars.

[27] N. Sheikov, N. McDannold, N. Vykhodtseva, F. Jolesz, K. Hynynen, Cellular mechanisms of the blood-brain barrier opening induced by ultrasound in presence of microbubbles, Ultrasound Med. Biol. 30 (7) (2004) 979–989, juill.

[28] N. Sheikov, N. McDannold, F. Jolesz, Y.Z. Zhang, K. Tam, K. Hynynen, Brain arterioles show more active vesicular transport of blood-borne tracer molecules than capillaries and venules after focused ultrasound-evoked opening of the blood-brain barrier, Ultrasound Med. Biol. 32 (9) (2006) 1399–1409, sept.

[29] N. Sheikov, N. McDannold, S. Sharma, K. Hynynen, Effect of focused ultrasound applied with an ultrasound contrast agent on the tight junctional integrity of the brain microvascular endothelium, Ultrasound Med. Biol. 34 (7) (2008) 1093–1104, juill.

[30] S.B. Raymond, J. Skoch, K. Hynynen, B.J. Bakskai, Multiphoton imaging of ultrasound/Optison mediated cerebrovascular effects in vivo, J. Cereb. Blood Flow Metab. 27 (2) (2007) 393–403, févr.

[31] K. Hynynen, N. McDannold, N.A. Sheikov, F.A. Jolesz, N. Vykhodtseva, Local and reversible blood-brain barrier disruption by noninvasive focused ultrasound at frequencies suitable for trans-skull sonifications, NeuroImage 24 (1) (2005) 12–20, janv.

[32] H. Chen, E.E. Konofagou, The size of blood-brain barrier opening induced by focused ultrasound is dictated by the acoustic pressure, J. Cereb. Blood Flow Metab. 34 (7) (2014) 1197–1204, juill.

[33] N. McDannold, N. Vykhodtseva, K. Hynynen, Effects of acoustic parameters and ultrasound contrast agent dose on focused-ultrasound induced blood-brain barrier disruption, Ultrasound Med. Biol. 34 (6) (2008) 930–937, juin.

[34] K.H. Song, A.C. Fan, J.J. Hinkle, J. Newman, M.A. Borden, B.K. Harvey, Microbubble gas volume: a unifying dose parameter in blood-brain barrier opening by focused ultrasound, Theranostics 7 (1) (2017) 144–152.

[35] J.F. Jordão, C.A. Ayala-Grosso, K. Markham, Y. Huang, R. Chopra, J. McLaurin, et al., Antibodies targeted to the brain with image-guided focused ultrasound reduces amyloid-β plaque load in the TgCRND8 mouse model of Alzheimer's disease. El Khoury J, éditeur, PLoS One 5 (5) (2010), 11 mai. (e10549).

[36] S. Dubey, S. Heinen, S. Krantic, J. McLaurin, D.R. Branch, K. Hynnen, et al., Clinically approved IVIg delivered to the hippocampus with focused ultrasound promotes neurogenesis in a model of Alzheimer's disease, Proc. Natl. Acad. Sci. USA 117 (51) (2020) 32691–32700, 22 déc.

[37] A.R. Rezai, P.F. D'Haese, V. Finomore, J. Carpenter, M. Ranjan, K. Wilhelmsen, et al., Ultrasound blood-brain barrier opening and aducanumab in Alzheimer's Disease, N. Engl. J. Med. 390 (1) (2024) 55–62, 4 janv.

[38] J.F. Jordão, E. Thévenot, K. Markham-Coultes, T. Scarcelli, Y.Q. Weng, K. Xhima, et al., Amyloid-β plaque reduction, endogenous antibody delivery and glial activation by brain-targeted, transcranial focused ultrasound, Exp. Neurol. 248 (2013) 16–29, oct.

[39] G. Leinenga, J. Götz, Scanning ultrasound removes amyloid-β and restores memory in an Alzheimer's disease mouse model, Sci. Transl. Med. 7 (278) (2015), 11 mars. (278ra33).

[40] A. Burgess, S. Dubey, S. Yeung, O. Hough, N. Eterman, I. Aubert, et al., Alzheimer disease in a mouse model: MR imaging-guided focused ultrasound targeted to the Hippocampus opens the blood-brain barrier and improves pathologic abnormalities and behavior, Radiology 273 (3) (2014) 736–745.

[41] A. Carpentier, M. Canney, A. Vignot, V. Reina, K. Beccaria, C. Horodyckid, et al., Clinical trial of blood-brain barrier disruption by pulsed ultrasound, Sci. Transl. Med. [Internet] 8 (343) (2016), https://doi.org/10.1126/scitranslmed.aaf6086, 15 juin. Disponible sur. cité 15 avr 2024.

[42] A. Carpentier, R. Stupp, A.M. Sonabend, H. Dufour, O. Chinot, B. Mathon, et al., Repeated blood-brain barrier opening with a nine-emitter implantable ultrasound device in combination with carboplatin in recurrent glioblastoma: a phase I/II clinical trial, Nat. Commun. 15 (1) (2024) 1650, 23 févr.

[43] D.Y. Zhang, C. Dmello, L. Chen, V.A. Arrieta, E. Gonzalez-Buendia, J.R. Kane, et al., Ultrasound-mediated delivery of paclitaxel for glioma: a comparative study of
distribution, toxicity, and efficacy of albumin-bound versus Cremophor formulations, Clin. Cancer Res. 26 (2) (2020) 477–486, 15 janv.

[44] A. Idbaih, M. Canney, L. Belin, C. Desseaux, A. Vignot, G. Bouchoux, et al., Safety and feasibility of repeated and transient blood-brain barrier disruption by pulsed ultrasound in patients with recurrent glioblastoma, Clin. Cancer Res. 25 (13) (2019) 3793–3801, 1 juill.

[45] P.F. D’Haese, M. Ranjan, A. Song, M.W. Haut, J. Carpenter, G. Dieb, et al., β-Amyloid plaque reduction in the Hippocampus after focused ultrasound-induced blood-brain barrier opening in Alzheimer’s disease, Front. Hum. Neurosci. 14 (2020) 593672.

[46] N. Lipsman, Y. Meng, A.J. Bethune, Y. Huang, B. Lam, M. Masellis, et al., Blood-brain barrier opening in Alzheimer’s disease using MR-guided focused ultrasound, Nat. Commun. 9 (1) (2018) 2336, 25 juill.

[47] S. Epelbaum, N. Burgos, M. Canney, D. Matthews, M. Houot, M.D. Santin, et al., Pilot study of repeated blood-brain barrier disruption in patients with mild Alzheimer’s disease with an implantable ultrasound device, Alzheimers Res. Ther. 14 (1) (2022) 40, 8 mars.

[48] S.H. Park, K. Baik, S. Jeon, W.S. Chang, B.S. Ye, J.W. Chang, Extensive frontal focused ultrasound mediated blood-brain barrier opening for the treatment of Alzheimer’s disease: a proof-of-concept study, Transl. Neurodegener. 10 (1) (2021) 44, 5 nov.

[49] A.R. Rezai, M. Ranjan, P.F. D’Haese, M.W. Haut, J. Carpenter, U. Najib, et al., Noninvasive hippocampal blood-brain barrier opening in Alzheimer’s disease with focused ultrasound, Proc. Natl. Acad. Sci. USA 117 (17) (2020) 9180–9182, 28 avr.

[50] S.E. Nadeau, Lecanemab questions, Neurology 102 (7) (2024), 9 avr. (e209320).

[51] M.H. Ebell, H.C. Barry, K. Baduni, G. Grasso, Clinically important benefits and harms of monoclonal antibodies targeting amyloid for the treatment of Alzheimer disease: a systematic review and meta-analysis, Ann. Fam. Med. 22 (1) (2024) 50–62.

[52] G. Devi, The tauopathies, Handb. Clin. Neurol. 196 (2023) 251–265.

[53] M.J. Page, J.E. McKenzie, P.M. Bossuyt, I. Boutron, T.C. Hoffmann, C.D. Mulrow, et al., The PRISMA 2020 statement: an updated guideline for reporting systematic reviews, BMJ n71 (2021), 29 mars.

[54] D. Moher, L. Shamseer, M. Clarke, D. Ghersi, A. Liberati, M. Petticrew, et al., Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015 statement, Syst. Rev. 4 (1) (2015) 1.

[55] C.R. Hooijmans, M.M. Rovers, R.B. de Vries, M. Leenaars, M. Ritskes-Hoitinga, M. W. Langendam, SYRCLE’s risk of bias tool for animal studies, BMC Med. Res. Methodol. 14 (1) (2014) 43.

[56] A.G. Cashin, J.H. McAuley, Clinimetrics: physiotherapy evidence database (PEDro) scale, J. Phys. 66 (1) (2020) 59.

[57] R.M. Nisbet, A. Van der Jeugd, G. Leinenga, H.T. Evans, P.W. Janowicz, J. Götz, Combined effects of scanning ultrasound and a tau-specific single cell antibody in a tau transgenic mouse model, Brain J. Neurol. 140 (5) (2017) 1220–1230, 1 mai.

[58] R. Pandit, G. Leinenga, J. Götz, Repeated ultrasound treatment of tau transgenic mice clears neuronal tau by autophagy and improves behavioral functions, Theranostics 9 (13) (2019) 3754–3767.

[59] M.E. Karakatsani, T. Kugelman, R. Ji, M. Murillo, S. Wang, Y. Niimi, et al., Unilateral focused ultrasound-induced blood-brain barrier opening reduces phosphorylated tau from the rTg4510 mouse model, Theranostics 9 (18) (2019) 5396–5411.

[60] R. Bajracharya, E. Cruz, J. Götz, R.M. Nisbet, Ultrasound-mediated delivery of novel tau-specific monoclonal antibody enhances brain uptake but not therapeutic efficacy, J. Control. Release 349 (2022) 634–648.

[61] A. Géraudie, M. Riche, T. Lestra, A. Trotier, L. Dupuis, B. Mathon, et al., Effects of low-intensity pulsed ultrasound-induced blood-brain barrier opening in P301S mice modeling Alzheimer’s disease Tauopathies, Int. J. Mol. Sci. 24 (15) (2023) 12411, 3 août.

[62] C.P. Pacia, J. Yuan, Y. Yue, E.C. Leuthardt, T.L.S. Benzinger, A. Nazeri, et al., Focused ultrasound-mediated liquid biopsy in a Tauopathy mouse model, Radiology 307 (2) (2023), 1 avr. (e220869).

[63] P.W. Janowicz, G. Leinenga, J. Götz, R.M. Nisbet, Ultrasound-mediated blood-brain barrier opening enhances delivery of therapeutically relevant formats of a tau-specific antibody, Sci. Rep. 9 (1) (2019) 9255, 25 juin.

[64] Y. Shen, L. Hua, C.K. Yeh, L. Shen, M. Ying, Z. Zhang, et al., Ultrasound with microbubbles improves memory, ameliorates pathology and modulates hippocampal proteomic changes in a triple transgenic mouse model of Alzheimer’s disease, Theranostics 10 (25) (2020) 11794–11819.

[65] R.L. Noel, S.L. Gorman, A.J. Batts, E.E. Konofagou, Getting ahead of Alzheimer’s disease: early intervention with focused ultrasound, Front. Neurosci. 17 (2023) 1229683, 27 juill.

[66] M.E. Karakatsani, R. Ji, M.F. Murillo, T. Kugelman, N. Kwon, Y.H. Lao, et al., Focused ultrasound mitigates pathology and improves spatial memory in Alzheimer’s mice and patients, Theranostics 13 (12) (2023) 4102–4120.

[67] K. Luo, Y. Wang, W.S. Chen, X. Feng, Y. Liao, S. Chen, et al., Treatment combining focused ultrasound with Gastrodin alleviates memory deficit and neuropathology in an Alzheimer’s disease-like experimental mouse model. Lu R, éditeur, Neural Plast. (2022) 1–13, 13 janv.

[68] X. Mi, H. Du, X. Guo, Y. Wu, L. Shen, Y. Luo, et al., Asparagine endopeptidase-targeted ultrasound-responsive nanobubbles alleviate tau cleavage and amyloid-β deposition in an Alzheimer’s disease model, Acta Biomater. 141 (2022) 388–397, 15 mars.

[69] Z.I. Kovacs, T.W. Tu, M. Sundby, F. Qureshi, B.K. Lewis, N. Jikaria, et al., MRI and histological evaluation of pulsed focused ultrasound and microbubbles treatment effects in the brain, Theranostics 8 (17) (2018) 4837–4855.

[70] Y. Meng, M. Goubran, J.S. Rabin, M. McSweeney, J. Ottoy, C.B. Pople, et al., Blood-brain barrier opening of the default mode network in Alzheimer’s disease with magnetic resonance-guided focused ultrasound, Brain 146 (3) (2023) 865–872, 1 mars.

[71] N. McDannold, N. Vykhodtseva, K. Hynynen, Blood-brain barrier disruption induced by focused ultrasound and circulating preformed microbubbles appears to be characterized by the mechanical index, Ultrasound Med. Biol. 34 (5) (2008) 834–840.

[72] D. Brici, J. Götz, R.M. Nisbet, A novel antibody targeting tau phosphorylated at serine 235 detects neurofibrillary tangles. Kayed R, éditeur, J. Alzheimers Dis. 61 (3) (2018) 899–905, 9 janv.

[73] G.M. Giuffrè, D. Quaranta, E.M. Costantini, S. Citro, N. Martellacci, G. De Ninno, et al., Cerebrospinal fluid neurofilament light chain and total-tau as biomarkers of neurodegeneration in Alzheimer’s disease and frontotemporal dementia, Neurobiol. Dis. 186 (2023) 106267.

[74] C. Kong, E.J. Yang, J. Shin, J. Park, S.H. Kim, S.W. Park, et al., Enhanced delivery of a low dose of aducanumab via F5 × FAD mice, an AD model, Transl. Neurodegener. 11 (1) (2022) 57, 27 déc.

[75] F. Hernandez, J.J. Lucas, J. Avila, GSK3 and tau: two convergence points in Alzheimer’s disease, J. Alzheimers Dis. 33 (Suppl. 1) (2013) S141–S144.

[76] J.H. Cho, G.V.W. Johnson, Glycogen synthase kinase 3β induces caspase-cleaved tau aggregation in situ, J. Biol. Chem. 279 (52) (2004) 54716–54723.

[77] D.P. Hanger, W. Noble, Functional implications of glycogen synthase Kinase-3-mediated tau phosphorylation, Int. J. Alzheimers Dis. 2011 (2011) 1–11.

[78] M.J. Lee, J.H. Lee, D.C. Rubinsstein, Tau degradation: the ubiquitin–proteasome system versus the autophagy-lysosome system, Prog. Neurobiol. 105 (2013) 49–59.

[79] L.M. Ittner, T. Fath, Y.D. Ke, M. Bi, J. van Eersel, K.M. Li, et al., Parkinsonism and impaired axonal transport in a mouse model of frontotemporal dementia, Proc. Natl. Acad. Sci. 105 (41) (2008) 15997–16002, 14 oct.

[80] J. Götz, F. Chen, R. Barmettler, R.M. Nitsch, Tau filament formation in transgenic mice expressing P301L tau, J. Biol. Chem. 276 (1) (2001) 529–534.

[81] M. Ramsden, L. Kotilinek, C. Forster, J. Paulson, E. McGowan, K. SantaCruz, et al., Age-dependent neurofibrillary tangle formation, neuron loss, and memory impairment in a mouse model of human Tauopathy (P301L), J. Neurosci. 25 (46) (2005) 10637–10647, 16 nov.

[82] Y. Yoshiyama, M. Higuchi, B. Zhang, S.M. Huang, N. Iwata, T.C. Saido, et al., Synapse loss and microglial activation precede tangles in a P301S tauopathy mouse model, Neuron 53 (3) (2007) 337–351, 1 févr.

[83] Y. Sun, Y. Guo, X. Feng, M. Jia, N. Ai, Y. Dong, et al., The behavioural and neuropathologic sexual dimorphism and absence of MIP-3α in tau P301S mouse model of Alzheimer’s disease, J. Neuroinflammation 17 (1) (2020) 72.

[84] C.H. Poon, S.T.N. Wong, J. Roy, Y. Wang, H.W.H. Chan, H. Steinbusch, et al., Sex differences between neuronal loss and the early onset of amyloid deposits and behavioral consequences in 5xFAD transgenic mouse as a model for Alzheimer’s disease, Cells 12 (5) (2023) 780, 1 mars.

[85] H.M. Edwards, C.E. Wallace, W.D. Gardiner, B.M. Doherty, R.T. Harrigan, K. M. Yuede, et al., Sex-dependent effects of acute stress on amyloid-β in male and female mice, Brain 146 (6) (2023) 2268–2274, 1 juin.

[86] G. Blázquez, T. Cañete, A. Tobéla, L. Giménez-Llort, A. Fernández-Teruel, Cognitive and emotional profiles of aged Alzheimer’s disease (3×TgAD) mice: effects of environmental enrichment and sexual dimorphism, Behav. Brain Res. 268 (2014) 185–201.

[87] J.L. Dennison, N.R. Ricciardi, I. Lohse, C.H. Volmar, C. Wahlestedt, Sexual dimorphism in the 3xTg-AD mouse model and its impact on pre-clinical research, J. Alzheimers Dis. 80 (1) (2021) 41–52, 9 mars.

[88] R.L. Noel, A.J. Batts, R. Ji, A.N. Pouliopoulos, S. Bae, A.R. Kline-Schoder, et al., Natural aging and Alzheimer’s disease pathology increase susceptibility to focused ultrasound-induced blood-brain barrier opening, Sci. Rep. 13 (1) (2023) 6757, 25 avr.

[89] C. Eguchi, T. Shindo, K. Ito, T. Ogata, R. Kurosawa, Y. Kagaya, et al., Whole-brain low-intensity pulsed ultrasound therapy markedly improves cognitive dysfunctions in mouse models of dementia - crucial roles of endothelial nitric oxide synthase, Brain Stimulat. 11 (5) (2018) 959–973.

[90] W.T. Lin, R.C. Chen, W.W. Lu, S.H. Liu, F.Y. Yang, Protective effects of low-intensity pulsed ultrasound on aluminum-induced cerebral damage in Alzheimer’s disease rat model, Sci. Rep. 5 (1) (2015) 9671, 15 avr.

[91] H. Shimokawa, T. Shindo, A. Ishiki, N. Tomita, S. Ichijyo, T. Watanabe, et al., A pilot study of whole-brain low-intensity pulsed ultrasound therapy for early stage of Alzheimer’s disease (LIPUS-AD): a randomized, double-blind, Placebo-Controlled Trial. Tohoku J. Exp. Med. 258 (3) (2022) 167–175.

[92] D.G. Blackmore, F. Turpin, T. Palliyaguru, H.T. Evans, A. Chicoteau, W. Lee, et al., Low-intensity ultrasound restores long-term potentiation and memory in senescent mice through pleiotropic mechanisms including NMDAR signaling, Mol. Psychiatry 26 (11) (2021) 6975–6991, nov.

[93] G. Leinenga, XV To, L.G. Bodea, J. Yousef, G. Richter-Stretton, T. Palliyaguru, et al., Scanning ultrasound-mediated memory and functional improvements do not require amyloid-β reduction, Mol. Psychiatry. 29 (8) (2024) 2408–2423.

[94] F. Li, J. Gallego, N.N. Tirko, J. Greaser, D. Bashe, R. Patel, et al., Low-intensity pulsed ultrasound stimulation (LIPUS) modulates microglial activation following intracortical microelectrode implantation, Nat. Commun. 15 (1) (2024) 5512, 29 juin.
[95] A.S. Mathew, C.M. Gorick, E.A. Thim, W.J. Garrison, A.L. Klibanov, G.W. Miller, et al., Transcriptomic response of brain tissue to focused ultrasound-mediated blood-brain barrier disruption depends strongly on anesthesia, Bioeng. Transl. Med. 6 (2) (2021) e10198 nov.

[96] L. Spieth, S.A. Berghoff, S.K. Stumpf, J. Winchenbach, T. Michaelis, T. Watanabe, et al., Anesthesia triggers drug delivery to experimental glioma in mice by hijacking caveolar transport, Neuro-Oncol. Adv. 3 (1) (2021), 1 janv. (vdab140).

[97] L. Mullin, R. Gessner, J. Kwan, M. Kaya, M.A. Borden, P.A. Dayton, Effect of anesthesia carrier gas on *in vivo* circulation times of ultrasound microbubble contrast agents in rats, Contrast Media Mol. Imag. 6 (3) (2011) 126–131.

[98] N. McDannold, Y. Zhang, N. Vykhodtseva, The effects of oxygen on ultrasound-induced blood-brain barrier disruption in mice, Ultrasound Med. Biol. 43 (2) (2017) 469–475.

[99] N. McDannold, Y. Zhang, N. Vykhodtseva, Blood-brain barrier disruption and vascular damage induced by ultrasound bursts combined with microbubbles can be influenced by choice of anesthesia protocol, Ultrasound Med. Biol. 37 (8) (2011) 1259–1270.

[100] Y. Dong, X. Wu, Z. Xu, Y. Zhang, Z. Xie, Anesthetic isoflurane increases phosphorylated tau levels mediated by caspase activation and Aβ generation. Zheng JC, éditeur, PLoS One 7 (6) (2012), 20 juin. e39386.

[101] J. Shin, C. Kong, J.S. Cho, J. Lee, C.S. Koh, M.S. Yoon, et al., Focused ultrasound-mediated noninvasive blood-brain barrier modulation: preclinical examination of efficacy and safety in various sonication parameters, Neurosurg. Focus. 44 (2) (2018) E15, févr.

[102] A. Pascal, N. Li, K.J. Lechtenberg, J. Rosenberg, R.D. Airan, M.L. James, et al., Histologic evaluation of activation of acute inflammatory response in a mouse model following ultrasound-mediated blood-brain barrier using different acoustic pressures and microbubble doses, Nanotheranostics 4 (4) (2020) 210–223.

[103] B. Baseri, J.J. Choi, Y.S. Tung, E.E. Konofagou, Multi-modality safety assessment of blood-brain barrier opening using focused ultrasound and definity microbubbles: a short-term study, Ultrasound Med. Biol. 36 (9) (2010) 1445–1459, sept.

[104] G. Samiotaki, F. Vlachos, Y. Tung, E.E. Konofagou, A quantitative pressure and microbubble-size dependence study of focused ultrasound-induced blood-brain barrier opening reversibility in vivo using MRI, Magn. Reson. Med. 67 (3) (2012) 769–777, mars.

[105] D. McMahon, K. Hynynen, Acute inflammatory response following increased blood-brain barrier permeability induced by focused ultrasound is dependent on microbubble dose, Theranostics 7 (16) (2017) 3989–4000.

[106] D. McMahon, R. Bendayan, K. Hynynen, Acute effects of focused ultrasound-induced increases in blood-brain barrier permeability on rat microvascular transcriptome, Sci. Rep. 7 (1) (2017) 45657, 4 avr.

[107] L.J. Blair, H.D. Frauen, B. Zhang, B.A. Nordhues, S. Bijan, Y.C. Lin, et al., Tau depletion prevents progressive blood-brain barrier damage in a mouse model of tauopathy, Acta Neuropathol. Commun. 3 (1) (2015) 8, déc.

[108] E. Planel, K.E.G. Richter, C.E. Nolan, J.E. Finley, L. Liu, Y. Wen, et al., Anesthesia leads to tau hyperphosphorylation through inhibition of phosphatase activity by hypothermia, J. Neurosci. 27 (12) (2007) 3090–3097, 21 mars.

[109] T. Sun, Y. Zhang, C. Power, P.M. Alexander, J.T. Sutton, M. Aryal, et al., Closed-loop control of targeted ultrasound drug delivery across the blood-brain/tumor barriers in a rat glioma model, Proc. Natl. Acad. Sci. [Internet]. (2017), https://doi.org/10.1073/pnas.1713328114, 28 nov. [cité 15 avr 2024];114(48). Disponible sur.

[110] C.Y. Chien, Y. Yang, Y. Gong, Y. Yue, H. Chen, Blood-brain barrier opening by individualized closed-loop feedback control of focused ultrasound, BME Front. 2022 (2022) 9867230, janv.

[111] A.S. Montero, I. Aliouat, M. Ribon, M. Canney, L. Goldwirt, S. Mourah, et al., Effect of ultrasound-mediated blood-spinal cord barrier opening on survival and motor function in females in an amyotrophic lateral sclerosis mouse model, eBioMedicine 106 (2024) août. (105235).

[112] D.K. Meyerholz, A.P. Beck, Principles and approaches for reproducible scoring of tissue stains in research, Lab. Investig. 98 (7) (2018) 844–855, juill.

[113] R. Lalonde, M. Filali, C. Strazielle, SHIRPA as a neurological screening battery in mice, Curr. Protoc. 1 (5) (2021) mai. (e135).

[114] S.J. Mooney, K. Shah, S. Yeung, A. Burgess, I. Aubert, K. Hynynen, Focused ultrasound-induced neurogenesis requires an increase in blood-brain barrier permeability. Deli MA, éditeur, PLoS One 11 (7) (2016), 26 juill. (e0159892).

[115] J. Shin, C. Kong, J. Lee, B.Y. Choi, J. Sim, C.S. Koh, et al., Focused ultrasound-induced blood-brain barrier opening improves adult hippocampal neurogenesis and cognitive function in a cholinergic degeneration dementia rat model, Alzheimers Res. Ther. 11 (1) (2019) 110, déc.

[116] D. McMahon, E. Mah, K. Hynynen, Angiogenic response of rat hippocampal vasculature to focused ultrasound-mediated increases in blood-brain barrier permeability, Sci. Rep. 8 (1) (2018) 12178, 15 août.

[117] E.E. Cho, J. Drazic, M. Ganguly, B. Stefanovic, K. Hynynen, Two-photon fluorescence microscopy study of cerebrovascular dynamics in ultrasound-induced blood-brain barrier opening, J. Cereb. Blood Flow Metab. 31 (9) (2011) 1852–1862, sept.

[118] A. Mudher, M. Colin, S. Dujardin, M. Medina, I. Dewachter, S.M. Alavi Naini, et al., What is the evidence that tau pathology spreads through prion-like propagation? Acta Neuropathol. Commun. 5 (1) (2017) 99, déc.

[119] G. Leinenga, J. Götz, Safety and efficacy of scanning ultrasound treatment of aged APP23 mice, Front. Neurosci. 12 (2018) 55, 7 févr.

[120] G. Leinenga, W.K. Koh, J. Götz, A comparative study of the effects of Aducanumab and scanning ultrasound on amyloid plaques and behavior in the APP23 mouse model of Alzheimer disease, Alzheimers Res. Ther. 13 (1) (2021) 76.