Transgenic mice with increased amyloid-β (Aβ) production show several aspects of Alzheimer’s disease, including Aβ deposition and memory impairment. By repeatedly treating these Aβ-forming mice with scanning ultrasound, Leinenga and Götz now demonstrate that Aβ is removed and memory is restored as revealed by improvement in three memory tasks. These improvements were achieved without the use of any therapeutic agent, and the scanning ultrasound treatment did not induce any apparent damage to the mouse brain. The authors then showed that scanning ultrasound activated resident microglial cells that took up Aβ into their lysosomes. These findings suggest that repeated scanning ultrasound may be a noninvasive method with potential for treating Alzheimer’s disease.

Amyloid-β (Aβ) peptide has been implicated in the pathogenesis of Alzheimer’s disease (AD). We present a nonpharmacological approach for removing Aβ and restoring memory function in a mouse model of AD in which Aβ is deposited in the brain. We used repeated scanning ultrasound (SUS) treatments of the mouse brain to remove Aβ, without the need for any additional therapeutic agent such as anti-Aβ antibody. Spinning disk confocal microscopy and high-resolution three-dimensional reconstruction revealed extensive internalization of Aβ into the lysosomes of activated microglia in mouse brains subjected to SUS, with no concomitant increase observed in the number of microglia. Plaque burden was reduced in SUS-treated AD mice compared to sham-treated animals, and cleared plaques were observed in 75% of SUS-treated mice. Treated AD mice also displayed improved performance on three memory tasks: the Y-maze, the novel object recognition test, and the active place avoidance task. Our findings suggest that repeated SUS is useful for removing Aβ in the mouse brain without causing overt damage, and should be explored further as a noninvasive method with therapeutic potential in AD.

Focused ultrasound allows for a transient opening of the BBB in the absence of tissue damage, as demonstrated in many experimental species, including rhesus macaques ( 13 ). In these primates, repeated opening of the BBB in the region of the visual cortex using focused ultrasound did not impair the ability of the animals to perform a complex visual acuity task in which they had been trained. Devices that emit ultrasound capable of penetrating the human brain are currently in clinical trials. Recently, a proof-of-concept study of using magnetic resonance–guided focused ultrasound to treat tremor and chronic pain has been successfully completed ( 15 ). Here, we investigate the use of SUS to remove Aβ from the AD mouse brain and to improve cognition and memory.

Here, we aim to establish whether a transient opening of the blood-brain barrier (BBB) using repeated scanning ultrasound (SUS) could assist in Aβ clearance. Only one method has been demonstrated to open the BBB noninvasively and repeatedly, that is, nonthermal focused ultrasound coupled with intravenous injection of microbubbles, which are used as ultrasound contrast agents ( 9 ). Ultrasound delivery is based on the principle that biologically inert and preformed microbubbles comprising either a lipid or polymer shell, a stabilized gas core, and a diameter of less than 10 μm are systemically administered and subsequently exposed to noninvasively delivered focused ultrasound pulses ( 10 ). Microbubbles within the target volume become “acoustically activated” by what is known as acoustic cavitation. In this process, the microbubbles expand and contract with acoustic pressure rarefaction and compression over several cycles ( 10 ). This activity has been associated with a range of effects, including the displacement of the vessel wall through dilation and contraction ( 11 , 12 ). More specifically, the mechanical interaction between ultrasound, microbubbles, and the vasculature transiently opens tight junctions and facilitates transport across the BBB ( 13 ). In assessing ultrasound-induced BBB opening, previous studies reported no difference in BBB opening or closing between Aβ plaque–forming APP/PS1 mice and nontransgenic (non-Tg) littermate controls ( 14 ).

Alzheimer’s disease (AD) is characterized by the presence of soluble oligomers of amyloid-β (Aβ) peptide that aggregate into extracellular fibrillar deposits known as amyloid plaques ( 1 – 3 ). Aβ is elevated in the AD brain because of the increased production of this peptide and its impaired removal ( 4 , 5 ). Recent therapeutic strategies have targeted both processes ( 6 ), including the inhibition of secretase enzymes to reduce Aβ production, as well as active and, in particular, passive immunization approaches for boosting Aβ clearance. These strategies, however, have side effects. Inhibition of secretases affects additional substrates with potential off-target effects ( 7 ), and passive immunization may be costly once effectiveness is demonstrated in clinical trials ( 8 ).

RESULTS

Scanning ultrasound is a safe method to transiently open the BBB We first established in C57BL/6 non-Tg wild-type mice that the BBB can be opened repeatedly by ultrasound, either by using single entry points (as is conventionally done) or by using SUS across the entire brain (Fig. 1, A to C). Mice were anesthetized, injected intravenously with microbubbles together with the indicator dye Evans blue, and then placed under the focus of a TIPS (therapy imaging probe system) ultrasound transducer (Philips Research) (16). Subsequent brain dissection revealed that a single ultrasound pulse resulted in a 1-mm-wide blue column of Evans blue dye, demonstrating focused opening of the BBB (Fig. 1B). When the focus of the ultrasound beam was moved in 1.5-mm increments until the entire forebrain of the mouse was sonicated with SUS, the BBB was opened throughout the brain, as evidenced by prevalent extravasation of Evans blue dye as early as 30 min after the treatment (fig. S1, A and B). This was also illustrated by fluorescence imaging 30 min to 1 hour after treatment (Fig. 1C). We optimized the ultrasound settings and established that a 0.7-MPa peak rarefactional pressure, 10-Hz pulse repetition frequency, 10% duty cycle, and 6-s sonication time per spot were optimal. These settings did not cause “dark” neurons, reflecting degeneration, as revealed by Nissl staining (fig. S1, C and D), or edema or erythrocyte extravasation as shown by hematoxylin and eosin (H&E) staining (fig. S1, E to H). To determine whether SUS caused immediate damage, we analyzed non-Tg mouse brain tissue 4 hours and 1 day after SUS treatment using acid fuchsin stain and found no evidence of ischemic damage (fig. S1, I and J). Fig. 1. Establishing SUS in an AD mouse model. (A) Setup of SUS equipment. (B and C) BBB opening by ultrasound was monitored by injecting wild-type mice with Evans blue dye that binds to albumin, a protein that is normally excluded from the brain. (B) A single entry point revealed a focal opening of the BBB in response to ultrasound treatment, with Evans blue dye able to enter the brain at this point. (C) Widespread opening of the BBB 1 hour after SUS was demonstrated with an Odyssey fluorescence LI-COR scanner of brain slices using nitrocellulose dotted with increasing concentrations of blue dye as a control. (D) Treatment scheme for the first cohort of hemizygous male Aβ plaque–forming APP23 mice (median age, 12.8 months). The mice received SUS or sham treatment for a total duration of the experiment of 6 weeks. Mice were randomly assigned to treatment groups. Using histological methods, Western blotting, enzyme-linked immunosorbent assay (ELISA), and confocal microscopy, we measured the effect of SUS treatment on amyloid pathology in mouse brain. Before the last SUS treatment, all mice were tested in the Y-maze. (E) The sequence of arm entries in the Y-maze was used to obtain a measure of alternation, reflecting spatial working memory. The percentage of alternation was calculated by the number of complete alternation sequences (that is, ABC, BCA, and CAB) divided by the number of alternation opportunities. Spontaneous alternation was restored in SUS-treated compared to sham-treated APP23 mice using non-Tg littermates as controls (n = 10 per group; one-way ANOVA followed by Dunnett’s posttest, P < 0.05). (F) Total number of arm entries did not differ between groups.

SUS reduces Aβ and amyloid plaque load in plaque-forming APP23 transgenic mice Having confirmed the viability of our protocol, we treated an initial cohort of 10 male Aβ plaque–forming APP23 transgenic mice five times with SUS over a period of 6 weeks (Fig. 1D, study design). At the age of 12 to 13 months, APP23 mice have a substantial plaque burden and spatial memory deficits (17). Age-matched APP23 mice in the control group (n = 10) received microbubble injections and were placed under the ultrasound transducer, but no ultrasound was emitted. After the 4-week sham or SUS treatment period, the mice underwent behavioral testing for a 2-week period in which they were not treated. We analyzed spatial working memory functions in the Y-maze. This test is based on the preference of mice to alternate between the arms of the maze. The analysis revealed that spontaneous alternation (calculated by the number of complete alternation sequences divided by the number of alternation opportunities) in APP23 mice treated with SUS, but not in sham-treated animals, was restored to wild-type levels [P < 0.05, one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison] (Fig. 1E). Total entries into the Y-maze arms did not differ between groups (Fig. 1F). The mice received one additional ultrasound treatment and were sacrificed 3 days later for histological and biochemical analysis. We next used Campbell-Switzer silver staining to distinguish the compact core of mature amyloid plaques from more dispersed Aβ deposits (Fig. 2, A and B). By analyzing every eighth section from −0.8 to −2.8 mm from bregma for each mouse (total of 8 to 10 sections per mouse), we found that the cortical area occupied by plaques was reduced by 56% (P = 0.014, unpaired t test) (Fig. 2C) and that the average number of plaques per section was reduced by 52% (P = 0.017, unpaired t test) (Fig. 2D) in SUS-treated compared to sham-treated mice. Thioflavin-S staining (Fig. 2E) and immunohistochemistry with the Aβ-specific antibody 4G8 (Fig. 2F) were used to confirm the specificity of the silver staining. We also plotted plaque load, as determined in Fig. 2C, as a function of age and included untreated mice to demonstrate the baseline of plaque load at the onset of treatment (Fig. 2G). It remains to be determined how our protocol would need to be modified to reveal efficacy in inducible models of AD, such as tetO-APPswe/ind mice (18). Fig. 2. SUS reduces Aβ plaques in an AD mouse model. (A and B) Representative images of free-floating coronal sections from APP23 transgenic mice (first cohort) with and without SUS treatment. Campbell-Switzer silver staining revealed compact, mature plaques (amber) and more diffuse Aβ deposits (black). A stained section at a higher magnification is shown in panel (B). (C and D) Quantification of amyloid plaques revealed a 56% reduction in the area of cortex occupied by plaques (unpaired t test, P = 0.017) and a 52% reduction in plaque number per section (t test, P = 0.014) in SUS-treated compared to sham-treated APP23 mice (n = 10 per group). (E and F) Representative sections of SUS-treated brains versus control brains stained with Thioflavin S (E) and 4G8 (F). (G) Plaque load plotted as a function of age confirmed that the SUS-treated group had significantly lower plaque load than the sham-treated group. Baseline plaque load at the onset of treatment is indicated by open circles. Scale bars, 1 mm (panel A) and 200 μm (panel B). We then extracted the right hemisphere from 10 SUS-treated and 10 sham-treated APP23 mice and used these tissues to obtain two lysates, one fraction enriched in extracellular proteins and a Triton-soluble fraction (19). By Western blotting with antibodies against Aβ, we were able to identify different species of the peptide (Fig. 3, A and B). The concentrations of these Aβ species were quantified, and reductions were found in the extracellular fraction for SUS-treated compared to sham-treated mice for high molecular weight species (HMW; 58% reduction), *56 oligomeric Aβ (Aβ*56; 38% reduction) and the trimeric Aβ/toxic APP C-terminal fragment (3-mer/CTFβ; 29% reduction) (Fig. 3C), and in the Triton-soluble fraction for *56 (50%) and trimeric Aβ/CTFβ (27%) (P < 0.05, unpaired t tests) (Fig. 3D). ELISA revealed a 17% reduction for Aβ 42 in the Triton-soluble fraction of SUS-treated compared to sham-treated mice (unpaired t test, P < 0.05; n = 10 per group) (Fig. 3E). Fig. 3. SUS treatment reduces different Aβ species. (A to D) Western blotting of extracellular-enriched (A) and Triton-soluble (B) fractions of the brains of the first cohort of APP23 mice with 6E10 and 4G8 anti-Aβ antibodies revealed a reduction in distinct Aβ species in both fractions in SUS-treated compared to sham-treated mice. These data are quantified in (C) and (D), respectively. The Western blots show significant reductions of HMW species, the 56-kD oligomeric Aβ*56 (*56) and trimeric Aβ (3-mer)/CTFβ, in the extracellular-enriched fraction and of *56 and 3-mer/CTFβ in the Triton-soluble fraction (unpaired t tests, P < 0.05). GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was used for normalization. MWM, molecular weight marker. (E) ELISA for Aβ42 in the Triton-soluble fraction revealed a significant reduction in SUS-treated compared to sham-treated mouse brains (unpaired t test, P < 0.05; n = 10 per group).

SUS treatment restores memory functions in AD mice To determine the functional outcome of our SUS treatment protocol in more robust behavioral tests, we next analyzed a second cohort of 20 gender-matched APP23 mice and non-Tg littermates (n = 10) in the active place avoidance (APA) task, a test of hippocampus-dependent spatial learning in which mice learn to avoid a shock zone in a rotating arena (Fig. 4A, study design). APP23 mice and non-Tg littermates underwent 4 days of training after habituation. There were significant effects of day of training (F 3,84 = 5.49, P = 0.002) and genotype (F 1,28 = 5.41, P = 0.028, two-way ANOVA), with day as the within-subjects factor (Fig. 4B). APP23 mice were divided into two groups with matching performance on the APA test and received weekly SUS or sham treatment for 7 weeks. Mice were retested in the APA test with the location of the shock zone in the opposite area of the arena (reversal learning). In the retest, there was a significant effect of day (F 3,84 = 2.809, P = 0.044) and treatment group (F 2,28 = 3.933, P = 0.0312). Multiple comparisons test for simple effects within rows showed that SUS-treated mice received fewer shocks on days 3 (P = 0.012) and 4 (P = 0.033) (Fig. 4C). SUS-treated mice also showed improvement when the first 5 min (long-term memory) and the last 5 min (short-term memory) of their performance were plotted separately (F 2,28 = 3.951, P = 0.0308) (Fig. 4D). We also performed an NOR test, which revealed improved performance after SUS treatment, with SUS-treated mice showing a preference for the novel object (labeled N, Fig. 4, E and F) [F 2,28 = 2.99, P = 0.066; t(20) = 2.33, P = 0.0356] compared to sham-treated control animals. Fig. 4. SUS treatment rescues memory deficits in an AD mouse model. (A) Treatment scheme of a second cohort of 20 gender-matched APP23 mice and 10 non-Tg littermates to determine the functional outcome of the SUS treatment protocol in more robust behavioral tests. The mice were analyzed in the APA task, a test of hippocampus-dependent spatial learning in which mice learned to avoid a shock zone in a rotating arena. After the APA test, the APP23 mice were divided into two groups with matching performance and received weekly SUS or sham treatment for 7 weeks. This was followed by an APA retest and a novel object recognition (NOR) test. One day after the final SUS treatment, mice were sacrificed and brain extracts were analyzed by Western blotting and ELISA. (B) Twenty APP23 mice and 10 non-Tg littermates tested in the APA test, with a habituation session (labeled H) followed by four training sessions (labeled D1 to D4). (C) In the APA retest, SUS-treated mice showed better learning than did sham-treated mice when tested for reversal learning (P = 0.031). (D) SUS-treated mice also showed improvement when the first 5 min (long-term memory) and last 5 min (short-term memory) were plotted separately (P = 0.031). (E) The APA retest was followed by the NOR test to determine the time spent with the novel object (labeled N) compared with the familiar object. (F) Analysis of the discrimination ratio that divides the above measure by the total time spent exploring both objects revealed that SUS-treated APP23 mice showed an increased preference for the novel object compared to sham-treated APP23 mice (P = 0.036). Upon sacrifice, we conducted a Western blot analysis using the Aβ-specific antibody W0-2, which showed a fivefold reduction of the monomer and a twofold reduction of the trimer in SUS-treated compared to sham-treated APP23 mice (unpaired t tests, P < 0.05) (Fig. 5, A and B). ELISA of the guanidine-insoluble brain fraction revealed a twofold reduction in Aβ 42 in SUS-treated samples (P < 0.008, unpaired t test) (Fig. 5C). Together, these data demonstrate that SUS has a robust effect on Aβ and memory function in AD mice. Fig. 5. SUS treatment reduces Aβ in a second cohort of AD mice. (A) A second cohort of APP23 mice was analyzed by Western blot with the anti-Aβ antibody W0-2; gel and transfer conditions were optimized to reveal the monomer and trimer specifically. The monomer was efficiently captured by using two sandwiched membranes. (B) The blots showed significant reduction of the monomer (fivefold reduction) and trimer (twofold reduction) in the extracellular fraction (unpaired t tests, P < 0.05). (C) ELISA for Aβ42 in the guanidine-insoluble fraction revealed a twofold reduction in SUS-treated compared to sham-treated mice (unpaired t test, P < 0.008; n = 10 per group).

SUS treatment causes uptake of Aβ into microglial lysosomes and clearance of plaques Our results revealed that the degree of Aβ reduction achieved by SUS treatment was comparable to that achieved by passive Aβ immunization (20, 21), but SUS treatment worked without the need for an additional therapeutic agent, such as antibodies, against Aβ. For passive vaccinations, different mechanisms have been proposed to remove Aβ from the brain (22, 23), with variable effects on microglial activation (20, 24). We therefore investigated whether microglial activation had an active mechanistic role in Aβ reduction caused by SUS treatment. On the basis of spinning disk confocal microscopy, an initial investigation of our first cohort of mice demonstrated that the microglia in SUS-treated brains fragmented and engulfed plaques (Fig. 6, A to D). We found that the microglia in SUS-treated APP23 mice contained twofold (P = 0.002, unpaired t test) more Aβ in lysosomal compartments than observed in sham-treated APP23 mice, as shown by costaining for Aβ and the microglial lysosomal marker CD68 (Fig. 6E). High-resolution three-dimensional (3D) reconstruction revealed extensive Aβ internalization in SUS-treated compared with sham-treated brains (Fig. 6, F to I, and movie S1). Confocal analysis of Aβ and CD68 further revealed cleared plaques in cortical areas in SUS-treated mice in which Aβ was almost completely contained in microglial lysosomes. This finding was observed in 75% of the SUS-treated mice but not in any of the sham-treated mice (Fisher’s exact test, P = 0.007; n = 8 per group), with four sections analyzed in each case) (Fig. 6J). Fig. 6. Microglial phagocytosis and lysosomal uptake of Aβ induced by SUS treatment. (A and B) Plaques in sham-treated animals were surrounded by lysosomal CD68-positive microglia that contained some Aβ. (C and D) In contrast, plaques in SUS-treated mouse brains were surrounded by microglia that contained significantly more Aβ in their lysosomal compartments, with some plaques appearing to be completely phagocytosed by microglia. (E) A twofold increase in microglia-internalized Aβ was observed in SUS-treated compared to sham-treated mouse brains (unpaired t test, P = 0.002). (F to I) Plaques imaged at high magnification in 3D. CD68 labeling revealed the extent of Aβ at the plaque site that was internalized by microglia into lysosomes. 4′,6-Diamidino-2-phenylindole (DAPI) was used to visualize nuclei. (J) Confocal analysis of Aβ and CD68 revealed that 6 of 8 SUS-treated mice and 0 of 8 sham-treated mice had “cleared plaques” in cortical areas, with Aβ being almost completely within microglial lysosomes (Fisher’s exact test, P = 0.007; n = 8 per group, with four sections analyzed in each case). Scale bars, 100 μm (A and C) and 10 μm (B, D, and F to I).

SUS treatment induces microglial activation We next sought to determine whether microglia in SUS-treated compared to sham-treated APP23 mice differed in other characteristics using sham-treated non-Tg littermates as control. Using the microglial cytoplasmic marker Iba1 (ionized calcium–binding adaptor molecule 1) (Fig. 7, A to C), we first determined the total microglial surface area, but we did not find differences between the three groups (t test) (Fig. 7D); there was also no difference in the size of microglial cell bodies (t test) (Fig. 7E). Resting microglia have highly branched extensions unlike activated phagocytic microglia. To quantify the extent of branching, after staining with the activated microglial marker Iba1, we converted the images to binary images that were then skeletonized (to obtain the most accurate tree geometry possible) (fig. S2, A to C). In this analysis, both the summed microglial process endpoints and the summed process length were normalized per cell using the Analyze Skeleton plugin in ImageJ (National Institutes of Health) (Fig. 7F). This showed that microglia in the SUS-treated group were more activated, a finding that was also reflected by a fivefold increase in the area of immunoreactivity for CD68 (t test, P = 0.001), a specific marker of microglial and macrophage lysosomes (Fig. 7, G to I). Fig. 7. Altered morphology after ultrasound but unaltered numbers of microglia in SUS-treated mice. (A to C) Sections of non-Tg mice (A) and sham-treated (B) and SUS-treated APP23 mice (C) stained with the microglial marker Iba1. (D) The microglial surface area did not differ between the three groups. (E) There was also no difference in the size of microglial cell bodies between the three groups. (F) A skeleton analysis in which both the summed microglial process endpoints and the summed process length were normalized per cell showing that microglia in the SUS-treated group were more activated (one-way ANOVA followed by Dunnett’s posttest, P < 0.05) (D to F: n = 4, non-Tg; n = 10, sham-treated and SUS-treated). (G to I) This is also reflected by the fivefold increase in the surface area of CD68 immuno-reactivity (G), a marker of microglial and macrophage lysosomes, in SUS-treated (I) compared with sham-treated (H) APP23 mice (n = 10, sham-treated and SUS-treated; t test, P = 0.001). Scale bars, 100 μm (A to C, H, and I).

Albumin may have a putative role in mediating Aβ uptake by microglia Phagocytosis of Aβ by microglia and perivascular macrophages has been shown to be assisted by blood-borne immune molecules, including Aβ-specific antibodies (20). Another Aβ-neutralizing molecule is albumin, which is present in the blood and may establish a ”peripheral sink” (25), although some reports argue against such a gradient (26). The fact that Evans blue dye–bound albumin can be detected in the brain after SUS treatment suggested to us that albumin may assist in Aβ engulfment not only in the periphery but also in the brain. After BBB disruption by ultrasound, albumin enters the brain where it is rapidly phagocytosed by glial cells but not by neurons (27). Albumin has also been demonstrated to bind to Aβ and inhibit its aggregation (28). To determine whether albumin may facilitate Aβ uptake by microglia, we incubated microglial BV-2 cells in culture with Aβ 42 with and without albumin (10 mg/ml; equivalent to 20% of the concentration in human serum) and found a 65% increase in Aβ 42 uptake in the presence of albumin (t test, P = 0.0188) (fig. S3). This result suggested that after SUS treatment, albumin may enter the brain and bind to Aβ, facilitating microglial phagocytosis. However, further work needs to be done to demonstrate a role for albumin in Aβ uptake by microglia in vivo.