Research ArticleAlzheimer’s Disease

Scanning ultrasound removes amyloid-β and restores memory in an Alzheimer’s disease mouse model

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Science Translational Medicine  11 Mar 2015:
Vol. 7, Issue 278, pp. 278ra33
DOI: 10.1126/scitranslmed.aaa2512
  • 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.

  • 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).

  • 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).

  • 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).

  • 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).

  • 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).

  • 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).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/278/278ra33/DC1

    Methods

    Fig. S1. Absence of brain damage after either repeated or short-term SUS treatment.

    Fig. S2. Skeleton analysis of microglia.

    Fig. S3. Increased Aβ uptake by microglial cells in the presence of albumin.

    Fig. S4. Analysis of IDE and tau phosphorylation after SUS treatment in AD mice.

    Fig. S5. Analysis of SUS-treated mice for inflammatory markers.

    Fig. S6. Absence of astrogliosis but activation of microglia after acute ultrasound treatment in wild-type mice.

    Movie S1 (mp4 format). High-resolution 3D reconstruction of a plaque imaged in a 40-μm section of a SUS-treated mouse.

  • Supplementary Material for:

    Scanning ultrasound removes amyloid-β and restores memory in an Alzheimer's disease mouse model

    Gerhard Leinenga and Jürgen Götz*

    *Corresponding author. E-mail: j.goetz@uq.edu.au

    Published 11 March 2015, Sci. Transl. Med. 7, 278ra33 (2015)
    DOI: 10.1126/scitranslmed.aaa2512

    This PDF file includes:

    • Methods
    • Fig. S1. Absence of brain damage after either repeated or short-term SUS treatment.
    • Fig. S2. Skeleton analysis of microglia.
    • Fig. S3. Increased Aβ uptake by microglial cells in the presence of albumin.
    • Fig. S4. Analysis of IDE and tau phosphorylation after SUS treatment in AD mice.
    • Fig. S5. Analysis of SUS-treated mice for inflammatory markers.
    • Fig. S6. Absence of astrogliosis but activation of microglia after acute ultrasound treatment in wild-type mice.
    • Legend for movie S1

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). High-resolution 3D reconstruction of a plaque imaged in a 40-μm section of a SUS-treated mouse.

    [Download Movie S1]

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