Time course of focused ultrasound effects on β-amyloid plaque pathology in the TgCRND8 mouse model of Alzheimer’s disease

Previous studies have demonstrated that temporarily increasing the permeability of the blood-brain barrier using focused ultrasound can reduce β-amyloid plaque load and improve cognitive function in animal models of Alzheimer’s disease. However, the underlying mechanism and duration for which the effects of one treatment persists for are unknown. Here, we used in vivo two-photon fluorescence microscopy to track changes in β-amyloid plaque sizes in the TgCRND8 mouse model of Alzheimer’s disease after one focused ultrasound treatment. We found that one treatment reduced plaques to 62 ± 16% (p ≤ 0.001) of their original volume two days post-sonication; this decrease in size persisted for two weeks. We then sought to evaluate the effectiveness of biweekly focused ultrasound treatments using magnetic resonance imaging-guided focused ultrasound treatments. Three to five biweekly treatments resulted in a 27 ± 7% (p ≤ 0.01) decrease in plaque number and 40 ± 10% (p ≤ 0.01) decrease in plaque surface area compared to untreated littermates. This study demonstrates that one focused ultrasound treatment reduces the size of existing β-amyloid plaques for two weeks, and that repeated biweekly focused ultrasound treatments is an effective method of reducing β-amyloid pathology in moderate-to-late stages of Alzheimer’s disease.


Supplementary
. Plaque chart. Maximum projection images of the raw XYZ image stacks of all the plaques collected for one animal are shown. Each column represents a different plaque, and each row represents a different imaging day.
Supplementary Figure S3. Changes in volume and cross-sectional area of larger and smaller plaques in Tg CTL (left) and Tg FUS (right) animals. The same data as that in Figure 3 is shown, but binned by size into larger (magenta) and smaller (blue) plaques based on the median of raw volume values measured on day 0. A t-test revealed that there is no difference in changes in volume or cross-sectional area between larger and smaller plaques (Tg CTL: p = 0.3 for volume, p = 0.2 for maximum cross-sectional area, Tg FUS: p = 0.8 for volume, p = 0.5 for maximum cross-sectional area,). Solid lines show mean  SD values, dotted lines show individual plaques.  Supplementary Figure S4. Survival curve in MRgFUS study. No difference in mortality of Tg and nTg animals in 10-week MRgFUS study.
Fisher's exact test shows that there is no significant difference in mortality between genotypes (Tg vs nTg, p > 0.99) or treatment (FUS vs CTL, p = 0.68). Supplementary Figure S7. Comparisons in BBB permeability between Tg and nTg groups. Increases in BBB permeability were evaluated by comparing the FUS-targeted regions with untargeted regions in the brain (baseline), in contrast-enhanced T1-weighted MRIs (nTg n = 7, Tg n = 5; mean ± SD; p = 0.9, unpaired t-test). Box-and-whiskers plots show mean and range of each group.

Time course of 1 FUS treatment Effects of repeated biweekly treatments Imaging modality
In vivo two photon fluorescence microscopy Treatments guided by MRI Aβ analyzed using IHC, stereology Aβ-specific labelling Methoxy X-04 6F3D antibody

Brain volume targeted
Up to 1 mm below the brain surface (limited by transducer design) Bilateral hippocampi

Attributes analyzed
Aβ plaque volume and maximum crosssectional area Aβ number, maximum cross-sectional area, and total surface area of brain section covered by Aβ Supplementary Table S8. Comparison of the two-photon fluorescence microscopy and MRguided FUS studies.

nTg Tg
Relative enhancement values 153 ± 24 146 ± 23 n 7 5 Supplementary Figure S9. Two photon fluorescence microscopy FUS system (left) and MRgFUS system (right). Left: In the two photon fluorescence microscopy FUS system, the ring transducer was positioned above the cranial window. A drop of degassed water was placed in the center of the transducer to accommodate the water-immersion objective lenses. Two photon fluorescence microscopy FUS experiments were operated at fixed pressures. Right: In the MRgFUS system, a spherically curved transducer and polyvinylidene difluoride hydrophone were positioned in a water tank below the subject's head. Acoustic signals from microbubble activity were measured using the hydrophone, and used to control acoustic pressures during the duration of sonication. Dotted red lines indicate the approximate focus of the transducers (not to scale).
Supplementary Figure S10. Image processing workflow of two-photon fluorescence microscopy image stacks of plaques. A maximum projection image of the raw image stack of a plaque is shown in (a). A nearby CAA-covered blood vessel can also be observed. XYZ image stacks collected with the two-photon microscope underwent the following image processing steps: importing image stack, identifying ROI (b; only step requiring user input), thresholding (c), defragmenting (d), computing volume (e). Scale bar = 20 μm.