Brain Stimulation 11 (2018) 959e973

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Brain Stimulation
journal homepage: http://www.journals.elsevier.com/brain-stimulation

Whole-brain low-intensity pulsed ultrasound therapy markedly
improves cognitive dysfunctions in mouse models of
dementia - Crucial roles of endothelial nitric oxide synthase
Kumiko Eguchi a, Tomohiko Shindo a, Kenta Ito a, Tsuyoshi Ogata a, Ryo Kurosawa a,
Yuta Kagaya a, Yuto Monma a, Sadamitsu Ichijo a, Sachie Kasukabe a, Satoshi Miyata a,
Takeo Yoshikawa b, Kazuhiko Yanai b, Hirofumi Taki c, Hiroshi Kanai d, Noriko Osumi e,
Hiroaki Shimokawa a, *a

Department of Cardiovascular Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan
Department of Pharmacology, Tohoku University School of Medicine Sendai, Japanc
Biomedical Engineering for Health and Welfare, Graduate School of Biomedical Engineering, Tohoku University, Sendai, Japand
Department of Electronic Engineering, Tohoku University Graduate School of Engineering, Sendai, Japane
Department of Developmental Neuroscience, Tohoku University, Sendai, Japanba r t i c l e i n f o

a b s t r a c t

Article history:
Received 22 January 2018
Received in revised form
1 May 2018
Accepted 20 May 2018
Available online 22 May 2018

Background: Therapeutic focused-ultrasound to the hippocampus has been reported to exert neuroprotective effects on dementia. In the present study, we examined whether the whole-brain LIPUS (lowintensity pulsed ultrasound) therapy is effective and safe in 2 mouse models of dementia (vasculardementia, VaD and Alzheimer's disease, AD), and if so, to elucidate the common underlying mechanism(s) involved.
Methods: We used bilateral carotid artery stenosis (BCAS) model with micro-coils in male C57BL/6 miceas a VaD model and 5XFAD transgenic mice as an AD model. We applied the LIPUS therapy (1.875 MHz,
6.0 kHz, 32cycles) to the whole brain.
Results: In both models, the LIPUS therapy markedly ameliorated cognitive impairments (Y-maze test
and/or passive avoidance test) associated with improved cerebral blood ﬂow (CBF). Mechanistically, the
LIPUS therapy signiﬁcantly increased CD31-positive endothelial cells and Olig2-positive oligodendrocyte
precursor cells (OPCs) in the VaD model, while it reduced Iba-1-positive microglias and amyloid-b (Ab)plaque in the AD model. In both models, endothelium-related genes were signiﬁcantly upregulated in
RNA-sequencing, and expressions of endothelial nitric oxide synthase (eNOS) and neurotrophins wereupregulated in Western blotting. Interestingly, the increases in glia cells and neurotrophin expressions
showed signiﬁcant correlations with eNOS expression. Importantly, these beneﬁcial effects of LIPUS wereabsent in eNOS-knockout mice.
Conclusions: These results indicate that the whole-brain LIPUS is an effective and non-invasive therapy
for dementia by activating speciﬁc cells corresponding to each pathology, for which eNOS activation
plays an important role as a common mechanism.
© 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords:
LIPUS
Dementia
eNOS

1. Introduction
Novel, effective strategies for treating dementia are urgently
needed, particularly given alarming increases in the global* Corresponding author. Department of Cardiovascular Medicine, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai, 980-8574,
Japan.
E-mail address: shimo@cardio.med.tohoku.ac.jp (H. Shimokawa).

prevalence of dementia. According to the current estimates, nearly
47 million patients worldwide had dementia in 2015, and thisnumber is estimated to exceed 131 million by 2050 [1]. However,no curative treatment is yet available for vascular dementia (VaD)
or Alzheimer's disease (AD) [2,3], both of which comprise the mostcommon causes of dementia. VaD and AD share risk factors, such as
hypertension, hypercholesterolemia, and diabetes mellitus [2,3].
Long-term exposure to these risk factors results in common
outcome, i.e., impairment of vascular endothelial functions [4].

https://doi.org/10.1016/j.brs.2018.05.012
1935-861X/© 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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K. Eguchi et al. / Brain Stimulation 11 (2018) 959e973

Abbreviations
Ab
AD
APP
BACE-1
BCAS
BDNF
CBF
CREB
CXCR4
DCX
eNOS
FGF-2

amyloid-beta
Alzheimer's diseaseamyloid precursor proteinb-site APP-cleaving enzyme-1
bilateral carotid artery stenosis
brain-derived neurotrophic factor
cerebral blood ﬂow
cAMP response element-binding protein
CXC chemokine receptor 4
doublecortin
endothelial nitric oxide synthaseﬁbroblast growth factor-2

Thus, the endothelium has been recognized as an important andcommon therapeutic target for prevention and treatment of dementia [4].
Among several reports on therapeutic applications of ultrasound technology, low-intensity pulsed ultrasound (LIPUS) has
emerged as a non-invasive therapy for several diseases. We havepreviously demonstrated that vascular endothelial cells substantially contribute to the therapeutic effect of LIPUS, inducing therapeutic angiogenesis in myocardial ischemia [5,6]. Meanwhile, LIPUS
has also been reported to increase the production of brain-derived
neurotrophic factor (BDNF) in astrocytes [7] and nerve growth
factor (NGF) in PC12 cells [8], and to promote nerve regeneration[9]. Also, focused LIPUS to the hippocampus could ameliorate dementia (VaD and AD) in mice via increasing endogenous neurotrophins and vascular endothelial growth factor (VEGF) [10,11].
However, it is still unknown what kind of cell type contributes tothe LIPUS-induced cognitive improvement, and whether there is acommon factor in the two disorders. Moreover, from clinical pointof view, we hypothesized that whole-brain LIPUS, rather thanfocused ultrasound, is effective and feasible since VaD is characterized by the widespread white matter lesions (WML) and AD by
the widespread deposition of amyloid-b (Ab) in the brain [2,3].
Thus, in the present study, we aimed to examine the effectivenessand safety of whole-brain LIPUS therapy that has never been used
before. We ﬁrst examined whether the whole-brain LIPUS iseffective in different mouse models of dementia (VaD and AD), andif so, to elucidate the common mechanism underlying the beneﬁcial effects of LIPUS.
2. Methods
See Supplementary Methods.
3. Results
3.1. LIPUS therapy improves cognitive dysfunctions in the VaD
model
In the VaD model, the LIPUS therapy (Fig. 1A and B,
Supplementary Figs. 1A and B) had no effects on body weight orsystolic blood pressure (Supplementary Fig. 1C). LIPUS-treatedmice showed no signs of cramps, paralysis, cerebral hemorrhage,
hypothermia, hyperthermia, or increased mortality compared with
control mice, which underwent the same procedure without LIPUS
treatment (data not shown). In the Y-maze test, the number of
entries was comparable between the LIPUS and control groups,

GFAP
GO
Iba-1
iNOS
LIPUS
MAPK
NGF
nNOS
OPC
Hsp 90RNA-seq
VaD
VEGF
WML

glial ﬁbrillary acidic protein
gene ontology
ionized calcium-binding adapter molecule-1
inducible nitric oxide synthaselow-intensity pulsed ultrasoundmitogen-activated protein kinase
nerve growth factor
neuronal nitric oxide synthaseoligodendrocyte precursor cellheat shock protein 90
RNA-sequencing
vascular dementia
vascular endothelial growth factor
white matter lesions

suggesting that LIPUS causes no hyperactivity (Fig. 1C). Next, the
LIPUS-treated BCAS group had signiﬁcantly improved performancefor cognitive functions, including spontaneous alternation task in
the Y-maze and retention in the passive avoidance test (Fig. 1C). In
the novel object recognition test, the LIPUS-treated BCAS grouptended to have improved cognitive function compared with thecontrol group (Fig. 1C). Similar results were also obtained at 56
and 84 days after the LIPUS therapy (Supplementary Fig. 1D), andthere were no side effects of LIPUS for 3 months (data not shown).
The LIPUS therapy signiﬁcantly improved cerebral blood ﬂow(CBF) in the BCAS group for up to 28 days after the therapy,
whereas it had no effect in the control groups (Fig. 1D). Taken
together, these results suggest that the cognitive improvement by
the LIPUS therapy is associated with increased CBF and can last for
at least 3 months.

3.2. LIPUS therapy improves WML in the VaD model
Next, we examined brain histology to determine the mechanism of the beneﬁcial effects of the LIPUS therapy in the VaD
model. We focused on the constituent cells of neurovascular unit
possibly contributing to multifaceted actions of LIPUS. The results showed that the number of cells in the corpus callosum thatwere positive for GST-p, a marker of mature oligodendrocytes,
was signiﬁcantly higher in the LIPUS-treated group comparedwith the control group (Fig. 2A). In addition, Klüver-Barrera
staining showed that the LIPUS therapy reduced the severity of
WML following BCAS (Fig. 2A). Although there was no differencein CD31-positive cells in the corpus callosum (for assessment ofcapillary density), signiﬁcantly more CD31-positive cells were
present in the hippocampus of LIPUS-treated group comparedwith control group (Fig. 2A). Compared with control group,
LIPUS-treated group showed no difference in the number of cells
positive for GFAP (glial ﬁbrillary acidic protein; for astrocytes) or
Iba-1 (ionized calcium-binding adapter molecule-1; for microglias) (Supplementary Fig. 2). Moreover, doublecortin (DCX)
staining of the hippocampus showed signiﬁcantly more newly
formed neurons in the dentate gyrus of LIPUS-treated groupcompared with control group (Fig. 2B, Supplementary Fig. 3).
These results suggest that the LIPUS-induced angiogenesis
ameliorates cognitive dysfunctions through neurogenesis andreduction of WML. Since we found no signiﬁcant difference between the LIPUS-treated sham group and the control group, we
focused on the BCAS group in the subsequent biochemical
analyses.

Fig. 1. Whole-brain LIPUS therapy improves cognitive dysfunctions in the VaD model.
(A) The timeline of the BCAS experiments. (B) Schematic drawing showing the application of whole-brain LIPUS in mice. (C) Behavioral testing was performed on day 28 following
BCAS or sham surgery; n ¼ 12e17 (Y-maze and Passive avoidance) and n ¼ 5e12 (Novel object recognition).
(D) Representative images of cerebral blood ﬂow (CBF) measured using laser speckle and time-course of CBF relative to baseline; n ¼ 9e10 (sham) and n ¼ 20e25 (BCAS). *P<0.05,**P<0.005, and ***P<0.0005 (two-way ANOVA). LIPUS, low-intensity pulsed ultrasound.
Sham/Control; sham operation without the LIPUS treatment.
Sham/LIPUS; sham operation with the LIPUS treatment.
BCAS/Control; BCAS operation without the LIPUS treatment.
BCAS/LIPUS; BCAS operation with the LIPUS treatment.

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Fig. 2. LIPUS therapy enhances angiogenesis and reduces the severity of WML in the VaD model.
(A) Representative images of corpus callosum and hippocampal sections stained for GST-p, Klüver-Barrera (KB), and CD31 on day 28 following sham surgery or BCAS. The scale bar
represents 100 mm. Quantitative analysis of GST-p-positive cells, white matter lesion severity, and CD31-positive cells in the corpus callosum and hippocampus; n ¼ 3 (sham) andn ¼ 12e13 (BCAS). (B) Representative images of hippocampal sections stained for DCX on day 28. The scale bar represents 50 mm. Quantitative analysis of DCX-positive cells in the
hippocampus (n ¼ 12e13). *P<0.05 and ***P<0.0005 (A; two-way ANOVA, B; Student's t-test).

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3.3. LIPUS therapy upregulates genes related to OPC andangiogenesis in the VaD model
To examine the effects of the LIPUS therapy at the molecularlevel, we performed RNA-sequencing (RNA-seq) using total brain
samples. The results showed that 3 days after BCAS, the expressionsof ~2000 genes differed signiﬁcantly between the LIPUS-treatedand control groups; speciﬁcally, 981 and 1239 genes were signiﬁcantly upregulated and downregulated, respectively (Fig. 3A). Gene
ontology (GO) term analysis showed that the genes differentiallyregulated by the LIPUS therapy were enriched for genes that play arole in angiogenesis and proliferation (Fig. 3B). Regarding the cell
type responding to the LIPUS therapy, the highest number of LIPUSenhanced genes was annotated to astrocytes, followed by genes
annotated to endothelial cells and oligodendrocytes (Fig. 3C). Mostof the genes listed as oligodendrocyte-related genes were annotated as playing a role in cell differentiation and development;
however, since mature oligodendrocytes do not proliferate, theseoligodendrocyte-related genes were likely to be oligodendrocyte
precursor cells (OPCs). A heat map of upregulated and downregulated genes conﬁrmed that the differentially expressed genes
in response to LIPUS were related to astrocytes, OPCs, myelination,
and angiogenesis (Fig. 3D).
To conﬁrm our RNA-seq data, we performed RT-qPCR analyses
for the following genes; GFAP (for astrocytes), Olig2 (for OPCs),eNOS (for angiogenesis and vasodilatation), and FGF-2 (ﬁbroblast
growth factor-2) and CXCR4 (CXC chemokine receptor 4) (forangiogenesis [12,13] and OPC differentiation [14,15]). mRNA levelsof all 5 genes were higher in the LIPUS group compared with thecontrol group (Fig. 3E).
In contrast, regarding the trend for improved histological and
CBF state in the sham-operation group in response to LIPUS, we
assumed the following hypothesis; since the sham-operation/LIPUS
group underwent the same procedure with BCAS except theplacement of micro-coils, those mice were under temporaryischemic burden. In fact, CBF in the sham-operated groups
decreased to ~90% compared with baseline CBF at 2 h to 7 days after
operation. We do not consider that this mild decrease in CBF led tocerebral dysfunction including cognitive function. We rather hypothesize that LIPUS treatment has a potential to upregulate
angiogenesis-related genes. To conﬁrm this hypothesis, we performed RNA-seq analysis with sham group animals and found that
LIPUS upregulated angiogenesis-related genes including eNOS(Supplementary Fig. 4).
3.4. LIPUS increases the protein levels related to angiogenesis, OPCgenesis, and neurotrophins in the VaD model
Next, we examined the effect of the LIPUS therapy at the protein
level. The results of multiplex analysis using Bio-Plex showed that
LIPUS increased proteins in the MAPK (mitogen-activated protein
kinase) pathway, including CREB (cAMP response element-binding
protein), ERK1/2, MEK, p38 MAPK, and proteins downstream of thePI3K/Akt pathway, including p70 S6 kinase, whereas GSK-3ab wasslightly but signiﬁcantly decreased (Supplementary Fig. 5A). To
conﬁrm this ﬁnding, we measured the extent of phosphorylation of
ERK1/2 and Akt, which are main mediators of the MAPK and PI3KAkt pathways for angiogenesis and cell proliferation, respectively.
We found that p-ERK1/2 was signiﬁcantly increased in the LIPUStreated group on day 3, but not on day 7 (Supplementary Fig. 5B).
In contrast, p-Akt did not differ between the LIPUS-treated andcontrol groups at either time point (Supplementary Fig. 5B).
We thus measured the protein levels of GFAP, Olig2, eNOS,CXCR4, and FGF2 as mRNA expressions of those factors wereincreased by LIPUS, and nerve growth factor (NGF), pro-BDNF, and963

VEGF (vascular endothelial growth factor) as those were reported
to be increased by LIPUS [7,8]. As shown in Fig. 3F and
Supplementary Fig. 5D, the protein levels of GFAP, p-eNOS, t-eNOS,
FGF2, NGF, and pro-BDNF were signiﬁcantly higher in the LIPUS
group on day 3, whereas those of Olig2, CXCR4, and VEGF were
unchanged at this time point. In contrast, t-eNOS, CXCR4, FGF2,
VEGF, and NGF, but not GFAP, Olig2, p-eNOS or pro-BDNF, weresigniﬁcantly increased in the LIPUS group on day 7 (SupplementaryFigs. 5C and 5D). Interestingly, we found a positive correlation between eNOS expression and NGF, pro-BDNF, and VEGF (Fig. 3G),
indicating that the expression of neurotrophins is correlated witheNOS expression. Besides, LIPUS had no signiﬁcant effect onneuronal NO synthase (nNOS) or inducible NO synthase (iNOS) at
either time point (Supplementary Fig. 6A). Thus, the upregulationof endogenous growth factors in the LIPUS-treated group may
contribute to improved cognitive functions, for which eNOS plays
an important role.
3.5. LIPUS enhances OPC proliferation in the VaD model
Mechanistically, we examined histological changes in the
corpus callosum and hippocampus on day 3 after BCAS surgery. In
the corpus callosum, the LIPUS therapy signiﬁcantly increased thenumber of Olig2-positive cells in the BCAS group, but had no signiﬁcant effect on GFAP-positive or Ki67-positive cells (Fig. 4A). In
contrast, in the hippocampus, LIPUS signiﬁcantly increased thenumber of Ki67-positive cells in the BCAS group, but had no signiﬁcant effect on GFAP-positive or Olig2-positive cells (Fig. 4A). We
also examined which cell types are involved in LIPUS-induced
proliferation by co-immunostaining for Ki67 and Olig2, and foundthat the LIPUS therapy increases OPC proliferation (Fig. 4B). In
contrast, by staining both eNOS and CD31, we found that eNOS was
expressed speciﬁcally in the vascular endothelium of the corpus
callosum following the LIPUS therapy (Supplementary Fig. 6B).
Furthermore, consistent with our Western blot analysis, immunostaining showed that eNOS expression was increased following the
LIPUS therapy in the BCAS group (Supplementary Fig. 6C). We also
found a positive correlation between eNOS and Olig2 and between
eNOS and GFAP (Fig. 4C). Taken together, these results suggest thatthe whole-brain LIPUS therapy in mice with BCAS activated OPCs
and astrocytes associated with eNOS upregulation.
3.6. Essential role of eNOS in the beneﬁcial effects of the LIPUS
therapy in the VaD model
We next examined the role of eNOS in the LIPUS-induced
improvement in cognitive function using eNOS-knockout (eNOSe/e) mice. The LIPUS therapy had no signiﬁcant effect on BCASinduced changes in CBF (Fig. 5A). Importantly, in eNOSe/e mice,
LIPUS had no beneﬁcial effects on BCAS-induced behavior outcome(Fig. 5B), the number of GST-p-positive cells, the extent of WML, or
the number of CD31-positive cells (Fig. 5C). Moreover, the expression levels of VEGF and neurotrophins, both of which were significantly increased in LIPUS-treated wild-type mice with BCAS, were
unaffected by the LIPUS therapy in eNOSe/e mice (Fig. 5D). Thus, weconclude that eNOS is crucial for mediating the beneﬁcial effects of
the LIPUS therapy in the VaD model.
3.7. LIPUS therapy ameliorates cognitive dysfunctions in the AD
model
Next, we examined the effect of another common form of dementia, Alzheimer's disease (AD), using 5XFAD mice (Fig. 6A), an
established mouse model of AD pathology [16]. We ﬁrst conﬁrmedthat whole-brain LIPUS had no effects on body weight or systolicFig. 3. LIPUS therapy enhances angio-, OPC-genesis-related molecules, and neurotophins in correlation with eNOS.
(A) Scatter plot depicting differential gene expression between the LIPUS-treated BCAS group and the control BCAS group on day 3. Signiﬁcantly upregulated or downregulated
genes are colored in red and blue, respectively. (B) Gene ontology (GO) analyses of the signiﬁcantly upregulated and downregulated genes between the LIPUS-treated and control
BCAS groups. The top 10 GO terms are shown in descending order of P-value. (C) The percentage of differentially regulated genes for each cell type annotated by GO terms. (D) Heatmap depicting the relative expressions of the genes related to angiogenesis, oligodendrocytes, myelination, and astrocytes in the control and LIPUS-treated BCAS groups (n ¼ 1
pooled sample obtained from 4 separate animals per group). (E) RT-qPCR analysis of the indicated genes in the control and LIPUS-treated BCAS groups; each gene is plotted relative
to the respective control group (n ¼ 18 per group). (F) Western blot analysis of GFAP, Olig2, p-eNOS, t-eNOS, CXCR4, and FGF2 on days 3 (n ¼ 10 per group). (G) NGF and pro-BDNF
plotted against eNOS measured on days 3 and 7 (n ¼ 7e10); the correlation coefﬁcients and P-value are indicated. *P < 0.05, and **P < 0.005 (Student's t-test).

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Fig. 4. LIPUS therapy enhances OPC proliferation and activates glia cells associated with eNOS upregulation.
(A) Representative images of corpus callosum and hippocampal sections immunostained for GFAP, Olig2, and Ki67 in control and LIPUS-treated sham mice and in control and LIPUStreated BCAS mice on day 3. The scale bar represents 100 mm. Quantitative analysis of GFAP-, Olig2-, and Ki67-positive cells in the corpus callosum and hippocampus of mice treatedas indicated; n ¼ 3 (sham) and n ¼ 9e10 (BCAS). (B) Representative images of corpus callosum section co-immunostained for Olig2 and Ki67 in control and LIPUS-treated mice on
day 3. The scale bar indicates 50 mm. (C) Olig2 and GFAP mRNA plotted against eNOS mRNA(n ¼ 16 per group); the correlation coefﬁcients and P-value are indicated. *P<0.05 (twoway ANOVA).

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Fig. 5. eNOS is required for the beneﬁcial effects of LIPUS in the VaD model.
(A) Representative laser speckle images showing cerebral blood ﬂow (CBF) measured at the indicated times in control and LIPUS-treated eNOSe/e mice subjected to BCAS surgery.
Time-course of CBF in the indicated groups (n ¼ 6 per group). (B) Behavioral tests were performed on day 28 in control and LIPUS-treated eNOSe/e mice subjected to BCAS surgery(n ¼ 6 per group). (C) Representative images of corpus callosum and hippocampal sections immunostained for GST-p or CD31 or stained with Klüver-Barrera on day 28. The scale
bar represents 100 mm. Quantitative analysis of GST-p-positive cells, white matter lesion severity score, and CD31-positive cells in the corpus callosum and hippocampus (n ¼ 6 pergroup). (D) Western blot analysis of NGF, pro-BDNF, and VEGF on day 3 and/or on day 7 (n ¼ 5 per group).

Fig. 6. Whole-brain LIPUS therapy ameliorates cognitive dysfunctions in the AD model.
(A) Timeline of the experiments with 5XFAD mice. (B) Summary of Y-maze test results measured on day 86 (n ¼ 18 per group). (C) Representative laser speckle images of CBF andthe time-course of CBF relative to baseline; n ¼ 3 (WT) and n ¼ 14 (5XFAD). (D) Scatter plot depicting differential gene expressions between the LIPUS-treated and control 5XFAD
groups, on day 86. Signiﬁcantly upregulated or downregulated genes are colored in red and blue, respectively. (E) Gene ontology (GO) analyses of the signiﬁcantly upregulated or
downregulated genes measured between the LIPUS-treated and control groups. The top ten GO terms are shown in descending order of P-value. (F) The percentage of differentiallyregulated genes for each cell type annotated by GO terms. (G) Heat map depicting the relative expression of angiogenesis and microglia-related genes in the control and LIPUStreated groups (n ¼ 1 pooled sample obtained from 4 separate animals per group). *P<0.05, **P<0.005 and ***P<0.0005 (two-way ANOVA). LIPUS, low-intensity pulsed ultrasound.

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blood pressure in 5XFAD mice (Supplementary Fig. 7A). Moreover,
the LIPUS therapy did not cause cramps, paralysis, cerebral hemorrhage, hypothermia, hyperthermia, or death (data not shown) or
hyperactivity (Fig. 6B). Importantly, the LIPUS therapy signiﬁcantly
improved cognitive functions in 5XFAD mice in the spontaneousalternation task (Fig. 6B). The LIPUS therapy had no signiﬁcant effect in the passive avoidance test (Supplementary Fig. 7B). Moreover, consistent with previous ﬁnding [17], we found an agingdependent decrease in CBF in 5XFAD (Fig. 6C). LIPUS had no effect on CBF in wild-type mice for 84 days after the treatment(Fig. 6C). Interestingly, however, this decline in CBF in 5XFAD mice
was ameliorated by the LIPUS therapy, resulting in signiﬁcantly
higher CBF in the LIPUS-treated group at 84 days after the treatment (Fig. 6C).
Next, we performed RNA-seq analysis using whole-brain samples to compare the global gene expression patterns between
LIPUS-treated and control 5XFAD mice on day 86. We found thatnearly 2500 genes were differentially expressed in the LIPUS andthe control groups, including 1354 upregulated and 1126 downregulated genes (Fig. 6D). GO term analysis showed that the genes
regulated by the LIPUS therapy in the 5XFAD mice encoded key
regulators of immune function and angiogenesis (Fig. 6E), similar to
the genes identiﬁed in the VaD model. We next calculated thenumber of genes annotated to each type of cells as a percentage ofthe total number of genes, demonstrating that the majority of genes
were annotated to microglia. Interestingly, endothelium-related
genes were the second most frequently enhanced genes in thismodel as well as in the BCAS model (Fig. 6F). The heat map
expression conﬁrmed that the LIPUS therapy signiﬁcantly upregulated and/or downregulated genes related to amyloid, microglia,
inﬂammation, and angiogenesis (Fig. 6G). These results suggest that
the LIPUS therapy suppressed chronic inﬂammatory response of
microglias, and that endothelium-related genes were alsoenhanced in this model.
3.8. LIPUS therapy reduces microgliosis along with eNOS
upregulation in the AD model
Based on the results from RNA-seq, we examined microgliosis
and angiogenesis in those mice (Fig. 7A). Histological analysis
showed a signiﬁcant reduction in microgliosis in the cortex of
5XFAD mice following the LIPUS therapy, reﬂecting a decrease in
Iba-1-positive cells (Fig. 7A). The number of CD31-positive cells
tended to increase in the hippocampus of LIPUS-treated 5XFAD
mice compared with the control group (Fig. 7A). Since we found no
signiﬁcant difference between the LIPUS-treated and the control
groups, we focused on the 5XFAD group in the subsequentbiochemical analyses.
Next, we examined whether the LIPUS therapy affects eNOS
expression in the 5XFAD mice since the signiﬁcance of eNOS as atherapeutic target in AD has been recently reported [4]. Western
blot analysis showed that the LIPUS therapy signiﬁcantly increased
t-eNOS protein levels in 5XFAD mice (Fig. 7B), and this increase wascorrelated with a reduction in Iba-1-positive cells (Fig. 7C).
Furthermore, the expression of APP (amyloid precursor protein)
and BACE-1 (b-site amyloid precursor protein cleaving enzyme-1),
both of which are known to be suppressed by eNOS [18], weresigniﬁcantly decreased by the LIPUS therapy (Supplementary
Fig. 7C). Hsp 90 (heat shock protein 90), an activator of eNOS[19], was signiﬁcantly increased by the LIPUS therapy(Supplementary Fig. 7C). In contrast, the protein levels of both NGF
and pro-BDNF were signiﬁcantly increased in LIPUS-treated micecompared with control mice, whereas VEGF levels were unchanged(Supplementary Fig. 7D). Importantly, we found a positive correlation between eNOS and pro-BDNF as well as in the VaD model(Fig. 7D). Taken together, these results indicate that the wholebrain LIPUS therapy in the AD mice had potential to suppress
neuroinﬂammation through suppression of microglia and amyloidb (Ab) accumulation associated with eNOS upregulation.
3.9. LIPUS therapy reduces Ab plaque in the AD model
Mechanistically, we performed histological and biochemical
analyses to examine the effect of the LIPUS therapy on accumulation of Ab in the brain of 5XFAD mice. Interestingly, we found that
LIPUS signiﬁcantly decreased the Ab-42 plaque throughout thebrain in 5XFAD mice (Fig. 8A). Enzyme-linked immunosorbent
assay (ELISA) showed that the amount of Ab-42 in the tritonsoluble fraction (which contains primarily oligomeric Ab [20])was decreased in the LIPUS-treated group (Fig. 8B). In contrast,
LIPUS had no effect on Ab-42 levels in the TBS-soluble fraction,
which contains primarily monomeric Ab (Fig. 8B) [20]. Importantly,
recruitment of microglia cells to Ab deposits were enhanced in
LIPUS-treated mice compared with control mice (Fig. 8C). These
results suggest that the LIPUS therapy enhances the phagocytosis of
Ab plaque.
4. Discussion
In the present study, we report the ﬁrst experimental evidence
that the whole-brain LIPUS therapy markedly improves cognitive
dysfunctions without serious side effects in the 2 major mousemodels of dementia (VaD and AD) by enhancing the speciﬁc cells
corresponding to each pathology, and that eNOS plays importantroles in mediating the beneﬁcial effects of LIPUS (see Fig. 9). Since
large molecules cannot cross the blood-brain barrier [21,22], thestrategy to increase the local expression of endogenous neurotrophins is important for dementia. Furthermore, the present study
provides 3 important advances in this ﬁeld. First, our study showsthat the whole-brain LIPUS therapy is effective and safe whenapplied in vivo. Second, we identiﬁed the main processes by which
LIPUS improves cognitive dysfunctions, namely increased OPC
production in the VaD model and reduced microgliosis in the AD
model. Third, we were able to demonstrate that eNOS activation
plays crucial and common roles in the beneﬁcial effects of LIPUS
therapy in both models of dementia.
4.1. LIPUS ameliorates cognitive dysfunctions by promoting OPC-,
angiogenesis in the VaD model
The VaD model with BCAS is characterized by WML due tochronic cerebral ischemia, similar to patients with the disorder[23]. OPC proliferation is enhanced in WML in patients with multiple cerebral infarctions, suggesting that white matter has thecapacity to regenerate via OPC proliferation following ischemic
injury [24]. In the present study, the LIPUS therapy signiﬁcantly
increased the number of both OPCs and oligodendrocytes, suggesting the maturation and differentiation of oligodendrocytes
from OPCs, ultimately leading to reduced WML. Moreover, OPCs
themselves can contribute to angiogenesis following white matterinjury [25]. Thus, LIPUS-induced OPC proliferation and angiogenesis may be a novel strategy that can improve cognitive dysfunction
for VaD patients.
4.2. LIPUS ameliorates cognitive dysfunctions by reducing Ab andmicrogliosis in the AD model
The primary phenotype in 5XFAD mice includes the progressive
accumulation of Ab and neuroinﬂammation, thereby mimicking
the main pathological features of AD patients [16]. In the present

Fig. 7. LIPUS therapy reduces microgliosis in relation to the eNOS expression in the AD model.
(A) Representative images of cortical and hippocampal sections immunostained for Iba-1 (a marker of microglia) and CD31 on day 86. Low-magniﬁcation images are shown in theupper panels, and high-magniﬁcations in the lower panels. The scale bars represent 100 mm. Quantitative analysis of Iba-1-positive cells and CD31-positive cells measured in thecortex and/or hippocampus; n ¼ 3 (WT) and n ¼ 11e15 (5XFAD). (B) Western blot analysis of p-eNOS and t-eNOS in whole-brain homogenates (n ¼ 9 per group). (C) The number of
Iba-1-positive cells is plotted against eNOS mRNA level; the correlation coefﬁcient and P-value are indicated. The scale bar indicates 50 mm. (D) NGF and pro-BDNF plotted against
eNOS; the correlation coefﬁcients and P-values are indicated. *P<0.05 (Panel A; two-way ANOVA, B; Student's t-test).

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K. Eguchi et al. / Brain Stimulation 11 (2018) 959e973

Fig. 8. LIPUS therapy reduces Ab plaques in the AD model.
(A) Representative images of coronal sections immunostained for Ab on day 86; the lower images show magniﬁed views of the cerebral cortex, hippocampus, and thalamus. The
scale bars represent 500 mm (whole brain) or 50 mm (other rows). Quantitative analysis of Ab plaques area measured in whole-brain sections (n ¼ 11e15 per group). (B) Summary of
Ab-42 measured in whole-brain samples using ELISA (n ¼ 9 per group). (C) Representative images of co-immunostaining for Ab and Iba-1 in control and LIPUS-treated mice on day
86 (n ¼ 6 per group). *P<0.05 (Student's t-test).

K. Eguchi et al. / Brain Stimulation 11 (2018) 959e973

971

Fig. 9. Schematic diagram showing the proposed mechanisms underlying the beneﬁcial effects of whole-brain LIPUS in the 2 mouse models of dementia.
Whole-brain LIPUS treatment can activate a variety of cell types corresponding to each pathology in the neurovascular unit (i.e., neurons, glial cells, and vascular cells). The present
study indicates that activation of endothelial NO synthase (eNOS) is a key component underlying the beneﬁcial effects of the LIPUS therapy in both models of dementia. eNOS
increases the proliferation of oligodendrocyte precursor cells (OPCs) in the VaD mouse model and decreases microglias in the AD mouse model. Proliferation of OPCs leads to anincrease in mature oligodendrocytes, ultimately leading to re-myelination. Moreover, eNOS may also have signiﬁcant effects on astrocytes, reported to release neurotrophins. In
addition to suppressing microgliosis, eNOS also inhibits the production and accumulation of Ab. eNOS-induced angiogenesis plays an important role in neurogenesis via a change in
cerebral blood ﬂow. Finally, the cognitive dysfunction can be inhibited by re-myelination, angiogenesis, and neurogenesis in the VaD mouse model, and by reduced microgliosis andAb plaques, increased angiogenesis in the AD mouse model.

study, the expression of APP and BACE-1 were signiﬁcantly
decreased by the LIPUS therapy, and Ab was signiﬁcantly reduced intriton-soluble fraction and histological assessment. Regarding themechanism of unchanged TBS soluble Ab levels, Ab is known todissolve in a monomer form at concentrations lower than 3 mM,
whereas it aggregates at concentrations higher than 3 mM [29]. In
the present study, although the total amount of Ab was actually
decreased by LIPUS treatment, more amount of Ab was produced
than the amount that could dissolve in extracellular ﬂuid. Thus, weconsider that the amount of Ab in the extracellular ﬂuid continued
to be saturated, resulting in no difference in the TBS soluble fraction. On the other hand, it is generally accepted that microgliamediated neuroinﬂammatory responses can promote neurodegeneration in AD [26]. Thus, a strategy to suppress this microglial
response could be a therapeutic approach for AD [26,27]. However,since microglia acts in both protective and toxic manners, its
function in AD is still controversial. In the present study, the ratio of
recruited microglia to phagocytosis for amyloid plaque was significantly higher in the LIPUS-treated group (Fig. 7B). Although thetotal number of Iba-1-positive microglia decreased by LIPUS,
phagocytosis was rather enhanced in the LIPUS-treated group.
Besides, Hsp 90, which is known to inhibit aggregation of Ab [28],was enhanced by LIPUS in the present study, a consistent ﬁnding of
the previous study [29]. Hsp 90 is promptly recruited by endothelial
stimulation of shear stress induced by ultrasonic waves, and enhances eNOS activation [30]. Thus, we consider that LIPUS-induced
reduction in Ab triton fraction and plaque load can be explained, atleast in part, by the following mechanisms; (i) reduction in Ab
production, (ii) the changes in characteristics of microglia, and (iii)
refolding of Ab by Hsp 90. Multiple factors may be involved in themechanisms of LIPUS-induced reduction in Ab triton fraction andplaque load. Further studies are needed to clarify the mechanisms
of Ab plaque reduction by the LIPUS therapy.
4.3. Important roles of eNOS as a common mechanism in the twomouse models of dementia
Our RNA-seq analyses with 2 different models of dementia
revealed that the LIPUS therapy signiﬁcantly upregulated genes
related to vascular endothelial cells. In the VaD model, LIPUS
improved CBF promptly after the LIPUS therapy, suggesting that theacute effect of LIPUS on CBF is likely to be mediated by a vasodilating factor rather than angiogenesis. eNOS produces relatively
low concentrations of NO, which are essential for maintaining
endothelial function and integrity [31,32]. NO exerts a variety ofphysiological effects, including regulation of vascular tone andangiogenesis [32,33]. Thus, eNOS activation is an effective therapeutic strategy for a variety of cardiovascular diseases [31,32].
Moreover, in the hippocampus, eNOS plays a crucial role in longterm potentiation [34]. In endothelial cells, eNOS localizes in the
caveolae, and its activity is controlled by binding with caveolin [30].
Cyclic strain induced by ultrasonic pulse activates mechanotransduction via mechanosensors [35], such as caveolin, leading toeNOS activation [30]. Indeed, in the present study, eNOS was
upregulated by LIPUS at both mRNA and protein levels. We also
found a signiﬁcant correlation between eNOS and activated glial
cells/molecules in both models; eNOS and Olig2 expression/neurotrophins in the BCAS model and eNOS and Iba-1-positive cells/
neurotrophin in the AD model. eNOSe/e mice have activatedmicroglial cells and spontaneously develop cognitive impairment[18]. Moreover, the factors contributing to Ab reduction, such as
microglias, APP, BACE-1, and Hsp 90, are known to be linked with

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K. Eguchi et al. / Brain Stimulation 11 (2018) 959e973

eNOS [18,30]. Our ﬁnding that the beneﬁcial effects of LIPUS areabsent in eNOS/- mice provides further evidence that eNOS plays
key roles in mediating the effects of LIPUS.
4.4. Important role of astrocytes in the LIPUS-induced beneﬁcial
effects on cognitive functions
Astrocytes play an important role in maintaining neural functions through releasing neurotrophins (e.g. BDNF and NGF), which
are increased in response to LIPUS [7, 39]. Since LIPUS-induced
increase in neurotrophins was absent in eNOS/- mice, activationof endothelial cells may have signiﬁcant effects on astrocytes,
resulting in improvement of cognitive functions in the present
study. The enhancement of GFAP expression also supports theinvolvement of astrocytes in the LIPUS-induced beneﬁcial effects
on cognitive functions.
4.5. Interpretation of the behavioral improvements
We consider that the behavioral improvements observed in thepresent study reﬂect improved cognition. In the present study, Ymaze and novel object recognition test were analyzed independently for motor functions because they were corrected by calculating as ratio. In contrast, in passive avoidance test, theimprovement of motor performance shortens the latency. In general, BCAS mice show motor dysfunction 3 month after operation[36], while 5XFAD after 9 months of age [37]. In the present study,
we performed behavioral tests at 1 month after operation in BCAS
mice, and at 6e7 months in 5XFAD mice. In the Y maze test, wefound no difference in the number of entries between LIPUS andcontrol groups in both models.
4.6. Study limitations
Several limitations should be mentioned for the present study.
First, in the present study, we were unable to examine the contribution of eNOS in 5XFAD mice because this model was technically
difﬁcult to generate. Second, the use of 5XFAD mice has some
limitations. With this model, we were obviously unable to evaluate
tau protein. Furthermore, 5XFAD mouse is a model of forced
amyloidosis but not an actual human AD model. Third, we did notuse endothelium-speciﬁc eNOS/- mice. However, eNOS is mainly
localized in endothelial cells, including the brain, which was also
conﬁrmed in the present study. Finally, we did not directly compare
the effectiveness between our whole-brain LIPUS therapy and thefocused LIPUS therapy to the hippocampus by others [10,11].
5. Conclusions
We were able to demonstrate that the whole-brain LIPUS is aneffective and safe therapy for ameliorating cognitive dysfunction
induced by either ischemia or overexpression of amyloid-related
genes in mice, for which eNOS plays crucial roles to improve cerebral circulatory dysfunction. These ﬁndings suggest that wholebrain LIPUS therapy may be beneﬁcial in vascular dementia and
AD in humans.
Conﬂicts of interest
None declared.
Appendix A. Supplementary data
Supplementary data related to this article can be found athttps://doi.org/10.1016/j.brs.2018.05.012.

References[1] Prince AC-H M, Knapp M, Guerchet M, Karagiannidou M. World Alzheimer
report 2016. London: Alzheimer's Disease International; 2016. 2016.
[2] Scheltens P, Blennow K, Breteler MM, de Strooper B, Frisoni GB, Salloway S,
et al. Alzheimer's disease. Lancet 2016;388:505e17.
[3] O'Brien JT, Thomas A. Vascular dementia. Lancet 2015;386:1698e706.
[4] Katusic ZS, Austin SA. Neurovascular protective function of endothelial nitricoxide- recent advances. Circ J 2016;80:1499e503.
[5] Hanawa K, Ito K, Aizawa K, Shindo T, Nishimiya K, Hasebe Y, et al. Low-intensity pulsed ultrasound induces angiogenesis and ameliorates left ventricular dysfunction in a porcine model of chronic myocardial ischemia. PLoS One
2014;9:e104863.
[6] Shindo T, Ito K, Ogata T, Hatanaka K, Kurosawa R, Eguchi K, et al. Low-intensity
pulsed ultrasound enhances angiogenesis and ameliorates left ventricular
dysfunction in a mouse model of acute myocardial infarction. Arterioscler
Thromb Vasc Biol 2016;36:1220e9.
[7] Liu SH, Lai YL, Chen BL, Yang FY. Ultrasound enhances the expression of brainderived neurotrophic factor in astrocyte through activation of TrkB-Akt andCalcium-CaMK signaling pathways. Cerebr Cortex 2017;27:3152e60.
[8] Zhao L, Feng Y, Hu H, Shi A, Zhang L, Wan M. Low-intensity pulsed ultrasoundenhances nerve growth factor-induced neurite outgrowth throughmechanotransduction-mediated ERK1/2-CREB-Trx-1 signaling. Ultrasound
Med Biol 2016;42:2914e25.
[9] Chang CJ, Hsu SH, Lin FT, Chang H, Chang CS. Low-intensity-ultrasoundaccelerated nerve regeneration using cell-seeded poly(D,L-lactic acid-coglycolic acid) conduits: an in vivo and in vitro study. J Biomed Mater Res B
Appl Biomater 2005;75:99e107.
[10] Lin WT, Chen RC, Lu WW, Liu SH, Yang FY. Protective effects of low-intensity
pulsed ultrasound on aluminum-induced cerebral damage in Alzheimer's
disease rat model. Sci Rep 2015;5:9671.
[11] Huang SL, Chang CW, Lee YH, Yang FY. Protective effect of low-intensity
pulsed ultrasound on memory impairment and brain damage in a rat modelof vascular dementia. Radiology 2017;282:113e22.
[12] Salcedo R, Oppenheim JJ. Role of chemokines in angiogenesis: CXCL12/SDF-1
and CXCR4 interaction, a key regulator of endothelial cell responses. Microcirculation 2003;10:359e70.
[13] Shing Y, Folkman J, Sullivan R, Butterﬁeld C, Murray J, Klagsbrun M. Heparin
afﬁnity: puriﬁcation of a tumor-derived capillary endothelial cell growth
factor. Science 1984;223:1296e9.
[14] McKinnon RD, Matsui T, Dubois-Dalcq M, Aaronson SA. FGF modulates the
PDGF-driven pathway of oligodendrocyte development. Neuron 1990;5:603e14.
[15] Patel JR, McCandless EE, Dorsey D, Klein RS. CXCR4 promotes differentiation ofoligodendrocyte progenitors and remyelination. Proc Natl Acad Sci U S A
2010;107:11062e7.
[16] Oakley H, Cole SL, Logan S, Maus E, Shao P, Craft J, et al. Intraneuronal betaamyloid aggregates, neurodegeneration, and neuron loss in transgenic micewith ﬁve familial Alzheimer's disease mutations: potential factors in amyloid
plaque formation. J Neurosci 2006;26:10129e40.
[17] Wiesmann M, Zerbi V, Jansen D, Lutjohann D, Veltien A, Heerschap A, et al.
Hypertension, cerebrovascular impairment, and cognitive decline in aged
AbetaPP/PS1 mice. Theranostics 2017;7:1277e89.
[18] Katusic ZS, Austin SA. Endothelial nitric oxide: protector of a healthy mind.
Eur Heart J 2014;35:888e94.
[19] Garcia-Cardena G, Fan R, Shah V, Sorrentino R, Cirino G, Papapetropoulos A,
et al. Dynamic activation of endothelial nitric oxide synthase by Hsp90. Nature 1998;392:821e4.
[20] Hao W, Liu Y, Liu S, Walter S, Grimm MO, Kiliaan AJ, et al. Myeloid differentiation factor 88-deﬁcient bone marrow cells improve Alzheimer's diseaserelated symptoms and pathology. Brain 2011;134:278e92.
[21] Pardridge WM. Drug and gene delivery to the brain: the vascular route.
Neuron 2002;36:555e8.
[22] Patel MM, Patel BM. Crossing the blood-brain barrier: recent advances in drugdelivery to the brain. CNS Drugs 2017;31:109e33.
[23] Shibata M, Ohtani R, Ihara M, Tomimoto H. White matter lesions and glial
activation in a novel mouse model of chronic cerebral hypoperfusion. Stroke
2004;35:2598e603.
[24] Miyamoto N, Tanaka R, Shimura H, Watanabe T, Mori H, Onodera M, et al.
Phosphodiesterase III inhibition promotes differentiation and survival ofoligodendrocyte progenitors and enhances regeneration of ischemic whitematter lesions in the adult mammalian brain. J Cerebr Blood Flow Metabol
2010;30:299e310.
[25] Pham LD, Hayakawa K, Seo JH, Nguyen MN, Som AT, Lee BJ, et al. Crosstalk
between oligodendrocytes and cerebral endothelium contributes to vascularremodeling after white matter injury. Glia 2012;60:875e81.
[26] Block ML, Zecca L, Hong JS. Microglia-mediated neurotoxicity: uncovering themolecular mechanisms. Nat Rev Neurosci 2007;8:57e69.
[27] Zenaro E, Pietronigro E, Della Bianca V, Piacentino G, Marongiu L, Budui S,
et al. Neutrophils promote Alzheimer's disease-like pathology and cognitive
decline via LFA-1 integrin. Nat Med 2015;21:880e6.
[28] Evans CG, Wisen S, Gestwicki JE. Heat shock proteins 70 and 90 inhibit earlystages of amyloid beta-(1-42) aggregation in vitro. J Biol Chem 2006;281:
33182e91.

K. Eguchi et al. / Brain Stimulation 11 (2018) 959e973[29] Miyasaka M, Nakata H, Hao J, Kim YK, Kasugai S, Kuroda S. Low-intensity
pulsed ultrasound stimulation enhances Heat-shock protein 90 and mineralized nodule formation in mouse calvaria-derived osteoblasts. Tissue Eng
2015;21:2829e39.
[30] Bredt DS. Nitric oxide signaling speciﬁcityethe heart of the problem. J Cell Sci
2003;116:9e15.
[31] Vanhoutte PM, Zhao Y, Xu A, Leung SW. Thirty years of saying NO: sources,
fate, actions, and misfortunes of the endothelium-derived vasodilator mediator. Circ Res 2016;119:375e96.
[32] Shimokawa H. 2014 Williams Harvey Lecture: importance of coronary vasomotion abnormalities-from bench to bedside. Eur Heart J 2014;35:3180e93.
[33] Albrecht EW, Stegeman CA, Heeringa P, Henning RH, van Goor H. Protectiverole of endothelial nitric oxide synthase. J Pathol 2003;199:8e17.

973[34] Kantor DB, Lanzrein M, Stary SJ, Sandoval GM, Smith WB, Sullivan BM, et al.
A role for endothelial NO synthase in LTP revealed by adenovirus-mediatedinhibition and rescue. Science 1996;274:1744e8.
[35] McCormick SM, Saini V, Yazicioglu Y, Demou ZN, Royston TJ. Interdependenceof pulsed ultrasound and shear stress effects on cell morphology and gene
expression. Ann Biomed Eng 2006;34:436e45.
[36] Nishio K, Ihara M, Yamasaki N, Kalaria RN, Maki T, Fujita Y, et al. A mousemodel characterizing features of vascular dementia with hippocampal atrophy. Stroke 2010;41:1278e84.
[37] Jawhar S, Trawicka A, Jenneckens C, Bayer TA, Wirths O. Motor deﬁcits,
neuron loss, and reduced anxiety coinciding with axonal degeneration andintraneuronal Abeta aggregation in the 5XFAD mouse model of Alzheimer's
disease. Neurobiol Aging 2012;33. 196.e129-140.