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Table 3 Summary of the selected transcranial ultrasound stimulation studies

From: Multi-mechanical waves against Alzheimer’s disease pathology: a systematic review

Publication

Center frequency

Duration and periodicity

Model/subjects under study

Delivery mode

Primary outcomes of transcranial ultrasound stimulation (TUSS)

Jordão et al. [59]

0.5 MHz (pulsing at 1 Hz)

Single session of 10-ms bursts for 2 min/spot (4 spots)

TgCRND8 mice (4 months old)

Transcranial (unilateral target)

Cellular and molecular readouts

 Reduced Aβ plaque size in the treated cortex at the 4-days follow-up (IHC)

 Tendency to reduction of Aβ plaques number in the treated cortex at the 4-day follow-up (IHC)

 Enhanced interaction between endogenous antibodies and Aβ plaques in the treated cortex at the 4-day follow-up (IHC and confocal microscopy)

 Improved bioavailability of endogenous antibodies in the treated hemisphere at the 4-day follow-up (IHC and immunoblotting)

 Increased glial cell activity in the treated hemisphere at the 4-h, 4-day and 15-day follow-ups (IHC and WB)

 Increased Aβ internalization by glial cells at the 4-day follow-up in both treated and non-treated cortices, more marked in the treated hemisphere (IHC and confocal microscopy)

Safety and feasibility

 Safe US-mediated BBB opening

 No adverse effects

Burgess et al. 2014 [38]

1.68 MHz (pulsing at 1 Hz)

10-ms bursts for 2 min (2 spots/ hemisphere), once a week over 3 weeks

TgCRND8 mice

(7 months old)

Transcranial (bilateral hippocampi)

Cellular and molecular readouts

 Reduced Aβ plaque size and number in the HPC (IHC)

 Increased number of immature hippocampal neurons, increased dendrite length and branching in pre-existing neurons of HPC (IHC)

Behavioral readouts

 Improved spontaneous alternation (short-term memory) and exploratory skills (Y-maze test)

Safety and feasibility

 Safe US-mediated BBB opening

 Well-tolerated by mice

Leinenga and Götz 2015 [36]

1 MHz (pulsing at 10 Hz)

6 s per spot (spots 1.5-mm apart through the entire brain)

 Over 4 weeks (sessions with 2-weeks interval), followed by a final US session (5 sonications in total)

 Weekly sessions over 7 weeks

 Aged APP23 mice (in vivo)

 Microglial cells exposed to Aβ42 (in vitro)

Transcranial (whole brain)

Cellular and molecular readouts

 Reduced Aβ plaque area and number in mice after the 4-weeks + 1 session regimen (IHC and HC)

 Reduced soluble and oligomeric Aβ species in the right hemisphere of mice after the 4-weeks + 1 session regimen (WB and ELISA)

 Reduced Aβ1-42 levels (ELISA andWB) in the brains of mice after the 7-week regimen

 Activation of microglia in the brain after the 7-week regimen (IHC)

 Increased internalization of Aβ by microglia in mice after the 4-weeks + 1 session regimen (spinning disk confocal microscopy and high-resolution 3D reconstruction)

 Increased Aβ42 uptake by microglial cells in the presence of albumin after the 7-weeks regimen (IHC and confocal microscopy)

 No significant differences in Tau hyperphosphorylation load after the 7-week regimen (WB)

 No differences in astrocyte immunoreactivity after the 7-week regimen (IHC)

Behavioral readouts

 Improved spontaneous alternation (short-term memory) after the 4-week regimen (Y-maze)

 Enhanced spatial learning, long- and short-term memory after the 7-week regimen (active avoidance test)

 Improved recognition memory after the 7-week regimen (novel object recognition)

Safety and feasibility

 No adverse effects

O’Reilly et al. 2017 [62]

0.28 MHz (pulsing at 1 Hz)

- Single session of 60 10-ms bursts/spot for 3 min;

- 120 10-ms bursts/spot for 2 min (5-min interval), weekly over 4 weeks

Aged beagle dogs (Aβ-positive)

(9–11 years old)

Transcranial (whole-left hemisphere)

Cellular and molecular readouts

 No significant differences in Aβ load between hemispheres after both regimens (IHC)

 Tendency to reduced Aβ load in the left cortex compared to the ipsilateral area after both regimens (IHC)

 No significant differences in microglia activity between hemispheres after both regimens (IHC)

Behavioral readouts

 No changes in gait, postural reaction or cranial nerve test after both regimens

Safety and feasibility

 Intact BBB and no observed brain damage in all animals one week after both treatments (MRI)

 One dog suffered from thermal damage due to too high-pressure exposures, with no other adverse events

Leinenga and Götz [60]

1 MHz (pulsing at 10 Hz)

4 sessions of 2.4 min each (over 24 spots)

Aged APP23 mice

(21–22 months old)

Transcranial (whole brain)

Cellular and molecular readout

 No significant differences in Aβ plaque area and number in the forebrain after sonication (IHC)

 Reduced Aβ plaque area in the forebrain (IHC)

 Increased Aβ-associated microglia, mainly in larger plaques (IHC)

Safety and feasibility

 Safe US-mediated BBB opening

Eguchi et al. 2018 [57]

1.875 MHz

(pulsing at 6 kHz)

20 min/spot (3 spots), 3 sessions on days 1, 3, 5, 28, 30, 32, 56, 58, 60, 84 and 86

(over 12 weeks)

5xFAD mice

(14–16 weeks old)

Transcranial (whole brain)

Cellular and molecular readout

 Regulation of immune-related genes (RNA-sequencing)

 Reduced microgliosis in the cortex,  despite increased microglia phagocytosis towards Aβ deposits (IHC)

 Upregulation of endothelial nitric oxide synthase, neurotrophins, and heat-shock protein 90 (WB)

 Downregulation of amyloid precursor protein and BACE-1 (WB)

 Reduced Aβ load and plaque burden throughout the brain (IHC and ELISA)

 Enhanced cerebral blood flow for up to 84 days after sonication (laser speckle blood flow imager)

Behavioral readouts

 Improved memory performance in the spontaneous alternation task (Y-maze test)

Safety and feasibility

 No adverse effects

Poon et al. 2018 [37]

- 1.1 MHz (pulsing at 1 Hz)

- 1.68 MHz (pulsing at 1 Hz)

10-ms bursts for 2 min:

 - in a single session

 - once every 2 weeks, over 10 weeks

TgCRND8 mice

(6 months old)

Transcranial (bilateral hippocampi)

Cellular and molecular readouts

 Reduced Aβ plaque volume after one session of US until 14-day follow-up (two-photon fluorescence microscopy)

 Reduced Aβ plaque maximum cross-sectional area after one session of US until 7-day follow-up (two-photon fluorescence microscopy)

 Decreased Aβ plaque number and surface area in the HPC after 10-week treatment (IHC)

Safety and feasibility

 Safe US-mediated BBB opening after both regimens (two-photon fluorescence microscopy and MRI)

Pandit, Leinenga & Götz 2019 [66]

1 MHz (pulsing at 10 Hz)

2.4 min (over 24 spots), once a week over 15 weeks

K3 mice

(6 weeks old)

Transcranial (whole brain)

Cellular and molecular readouts

 Tendency for reduced p-Tau levels in hippocampal slices

 Reduced p-Tau levels in the HPC, but not in the cortex (IHC)

 Reduced neurofibrillary tangles in the HPC and cortex (HC and IHC)

 Activation of microglia

 Increased autophagy in the neurons (IHC, WB, and proximity ligation assays)

Behavioral readouts

 Improved motor ability and coordination, but no increased grip strength (repeated Rotarod paradigm)

 Enhanced spatial working memory (Y-maze test)

 Tendency for improved short-term memory (novel object recognition test)

Safety and feasibility

 Safe US-mediated BBB opening, as no differences in opening volume occurred (MRI)

 Well-tolerated by mice

Karakatsani et al. 2019 [45]

1.5 MHz (pulsing at 10 Hz)

single session of 60 s

rTg4510 mice (Tau pathology model)

(3.5–4.5 months old)

Transcranial (unilateral target)

Cellular and molecular readouts

 Reduced p-Tau in the HPC of both hemispheres mainly in the treated one (IHC)

 Increased immune cells activation in both hemispheres compared to untreated brains (IHC)

 Fragments of p-Tau were found within microglia in both hemispheres (IHC)

 Increased immune cell activation was correlated with p-Tau reduction

Safety and feasibility

 Safe US-mediated BBB opening, as no differences in opening volume occurred (MRI)

Bobola et al. [61]

2 MHz (pulsing at 40 Hz)

- Single session of 1 h (acute stimulation)

- 1 h/day over 5 days (chronic stimulation)

5xFAD mice

(6 months old)

Transcranial (left hippocampus for acute stimulation and bilateral hippocampi for chronic stimulation)

Cellular and molecular readouts

 No significant differences in endothelial nitric oxide synthase production after chronic stimulation (histological analysis)

 Activation of microglia that co-localized with Aβ plaques after acute stimulation in the treated hemisphere (histological analysis)

 No significant changes in Aβ plaque load after acute stimulation in both hemispheres (IHC)

 Activation of microglia that co-localized with Aβ plaques after chronic stimulation in the treated brains

 Reduction in Aβ plaque burden after chronic stimulation (histological analysis)

Brain function readouts

 Strong and temporally non-uniform signal entrainment at 40 Hz during acute stimulation (continuous wavelet transforms analysis of EEG signal)

Safety and feasibility

 No side effects after treatment

Shen et al. [58]

0.996 MHz (pulsing at 1 Hz)

60-s sessions, twice a week over 6 weeks

3xTg-AD mice

(8 months old)

Transcranial (unilateral target)

Cellular and molecular readouts

 Reduced Aβ pathology in the cortex, amygdala, CA1 and CA3 hippocampal regions of the treated hemisphere (IHC)

 Decreased p-Tau levels in the cortex, HPC, and amygdala (IHC)

Improvement of axonal neurofilament degeneration in the HPC of treated mice (confocal microscopy)

 Activation of microglial phagocytosis and extensive internalization of Aβ aggregates (confocal microscopy)

 Reduced microglia branching in the stratum radiatum layer in the treated hemisphere near Aβ deposits (confocal microscopy)

 Modulation of the expression of synaptic, microtubule, mitochondrial, glycolytic, and ubiquitin proteins (2D fluorescence difference gel electrophoresis combined with mass spectroscopy)

Behavioral readouts

 Improved spatial learning  as well as short- and long-term memory skills (Y maze, MWM, and step-down passive avoidance test)

Safety and feasibility

 Well-tolerated by mice

 No neuron or tissue damage in the brain

 Safe and reversible US-mediated BBB opening (fluorescence imaging)

Lee et al. [56]

715 kHz (pulsing at 1 Hz)

Single session with 20-ms bursts delivered over 60 s

5xFAD mice

(4 months old)

Transcranial (unilateral target)

Cellular and molecular readouts

 Reduced total area of Aβ deposits, but not Aβ plaques, in both treated and non-treated hemispheres (IHC)

 Reduced gliosis in the HPC and entorhinal cortex in both treated and non-treated hemispheres (IHC)

 Increased Aβ species around meningeal vessels (IHC)

 Increased microglia around Aβ plaques (IHC)

 Increased drainage of soluble Aβ to the cerebrospinal fluid space in TUSS-treated mice in which ligation of lymphatics to the deep cervical lymph nodes was performed (IHC)

 Prevention of neuronal loss and glial cell reactivity/activation in the entorhinal cortex, but not in other brain areas (H&E and TUNEL staining)

Behavioral readouts

 Improved working memory, with no alterations in mobility (Y maze)

Safety and feasibility

 Well-tolerated by mice

 No neuron or tissue damage in the brain

 Safe and reversible US-mediated BBB opening (fluorescence imaging)

Lipsman et al. [63]

220 kHz (continuous mode)

7.5 min s/session, along 3.6 sessions (stage 1), followed by 7.5 sessions (stage 2) over 3 months, with 1-month interval between stages

AD patients

(mild to moderate AD)

(age: 66.2 ± 6.6)

Transcranial

(whole brain)

Cellular and molecular readouts

 No significant differences in Aβ levels ([18F]-florbetaben PET scans)

Behavioral readouts

 No changes in cognition and daily functioning at the 3-month follow-up (MMSE, ADAS-cog, ADCS-ADL, GDS, and NPI-Q)

 One of the patients showed a transient cognitive improvement in the 1-month follow-up after stage 2 (NPI-Q)

Safety and feasibility:

 Safe, temporary and repeatable USS-mediated opening, even in Aβ-rich brain regions (MRI)

 No serious adverse events

Meng et al. [68]

220 kHz (continuous mode)

Two sessions,  one month apart, doubling the target volume in the second intervention

AD patients

(mild to moderate AD)

(age: 66.8 ± 6.1)

Transcranial (right frontal lobe)

Brain function readouts

 Decreased functional connectivity in the right frontoparietal networks during BBB opening, which was reversed at 1-day and 1-week follow-up (resting-state BOLD fMRI signal)

 Tendency for decreased functional connectivity of the right frontoparietal network at the 3-month follow-up compared to baseline, although the US inhibited the marked decrease that occurred in the control group (resting-state BOLD fMRI signal)

 Tendency for increased functional connectivity of the default mode network at the 3-month follow-up in US-treated patients compared to controls (resting-state BOLD fMRI signal)

Safety and feasibility

 Safe,  effective, and reversible US-mediated BBB opening (MRI)

Beisteiner et al. [69]

-

(pulsing at 5 Hz)

2 spots per session, 3 sessions/week over 2–4 weeks

AD patients

Transcranial (bilateral

target)

 Behavioral and brain function readouts

 Improved cognitive status after treatment and at the 3-month follow-up improved memory and verbal processing and decline in visuospatial processing (CERAD scores and SEG scale)

 Upregulation of memory network after treatment (fMRI)

 Increased functional connectivity in HPC, parahippocampal cortex, parietal cortex and precuneus (resting-state fMRI) and bilateral HPC (task-based fMRI)

 Improved brain functional connectivity that was correlated with cognitive improvements (CERAD scores)

Safety and feasibility

 No side effects after treatment and at the 3-month follow-up (clinical assessments, patients reports, and MRI)

Rezai et al. [82]

220 kHz (continuous mode)

2–5 sonications over 3 sessions separated by 2 weeks

AD patients

(early AD)

(age: 55–75)

Transcranial (bilateral hippocampi/ entorhinal cortex)

Behavioral readouts

 No significant changes in cognitive function at the 30-day follow-up (formal cognitive assessments)

Safety and feasibility

 Well-tolerated by the patients

 No side effects after treatment

 Safe, feasible, reversible, reproducible, focused, and noninvasive US-mediated BBB opening in the HPC and entorhinal cortex (MRI)

D’Haese et al. [65]

220 kHz (continuous mode)

3 sessions separated by 2 weeks

AD patients

(early AD)

(age: 55–73)

Transcranial (unilateral hippocampi/ entorhinal cortex)

Cellular and molecular readouts

 Reduced Aβ plaques in the stratum radiatum layer in the HPC and entorhinal cortex of the treated hemisphere ([18F]-florbetaben PET scans)

Behavioral readouts

 No significant changes in cognitive function at the 1-week follow-up (formal cognitive assessments)

Safety and feasibility

 Safe, feasible and reversible US-mediated BBB opening in the HPC and entorhinal cortex (MRI)

  1. Tg: transgenic; Aβ: β-amyloid; IHC: immunohistochemistry; WB: Western blot; US: ultrasound; ; BBB: blood–brain barrier; AlCl3: aluminum chloride; MWM: Morris water maze; HPC: hippocampus; ELISA: enzyme-linked immunosorbent assay; p-Tau: phosphorylated Tau; MRI: magnetic resonance imaging; EGR1: early growth response protein 1; CA1, CA3: Cornu Ammonis hippocampal areas; DG: dentate gyrus; AChE: acetylcholinesterase; BrdU: bromodeoxyuridine; BDNF: brain-derived neurotrophic factor; RT-PCR: real-time polymerase chain reaction; PET: positron emission tomography; MMSE: mini-mental state examination, ADAS-cog: Alzheimer’s disease assessment scale – cognitive; ADCS-ADL: Alzheimer’s disease cooperative study group—activities of daily living; GDS: geriatric depression scale; NPI-Q: neuropsychiatric inventory questionnaire; BOLD: blood-oxygenation-level-dependent; fMRI: functional MRI; CERAD: consortium to establish a registry for Alzheimer's disease; SEG: scale for subjective evaluation of memory performance (translated from German)