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OPEN

Low‑intensity pulsed ultrasoundtherapy promotes recovery
from stroke by enhancing
angio‑neurogenesis in mice in vivo
Sadamitsu Ichijo1, Tomohiko Shindo1, Kumiko Eguchi1, Yuto Monma1, Takashi Nakata1,
Yoshihiko Morisue2, Hiroshi Kanai3,4, Noriko Osumi2, Satoshi Yasuda1 &Hiroaki Shimokawa1,5*
Since the treatment window of thrombolytic therapy for stroke is limited, new therapy remains to be
developed. We have recently developed low-intensity pulsed ultrasound (LIPUS) therapy to improve
cognitive dysfunction in mouse models of vascular dementia and Alzheimer’s disease. Here, we
further aimed to examine whether our LIPUS therapy improves neurological recovery from ischemic
stroke, and if so, to elucidate the mechanisms involved. In a mouse model of middle cerebral artery
occlusion (MCAO), we applied LIPUS (32 cycles, 193 mW/cm2) to the whole brain 3 times in the firstweek (days 1, 3, and 5) after MCAO. We evaluated neurological functions using behavioral tests andperformed histological analyses. Furthermore, to elucidate how LIPUS works within the injured
brain, we also tested the effects of LIPUS in endothelial nitric oxide synthase (eNOS)-deficient­(eNOS−/−) mice. In wild-type mice, the LIPUS therapy markedly improved neurological functions in
the tightrope and rotarod tests at 28 days after MCAO. Histological analyses showed that the LIPUS
therapy significantly increased the numbers of CD31-positive blood vessels in the perifocal lesion
and doublecortin (DCX)-positive neurons in the ischemic striatum, indicating the angio-neurogenesis
effects of the therapy. Importantly, these beneficial effects of the LIPUS therapy were totally absent
in ­eNOS−/− mice. No adverse effects of the LIPUS therapy were noted. These results indicate that the
LIPUS therapy improves neurological functions after stroke through enhanced neuro-angiogenesis
in mice in vivo in an eNOS-dependent manner, suggesting that it could a novel and non-invasive
therapeutic option for stroke.
Stroke is the leading cause of death and disability associated with a limited degree of functional ­recovery1. With
the progress of medical technology in the past decades, increasing number of stroke patients survived from theinitial injury. Consequently, 60% of survivors have disabilities in the arm or leg, and up to one third of themneed to stay in a nursing home or to use assistant devices for independent ­living2,3. Thus, it is crucial to develop
effective treatment for stroke patients. Brain neuro-restoration leads to considerable post-stroke functional
recovery. The brain attempts to repair itself after ischemic stroke by neurogenesis and a­ ngiogenesis4. Neural
stem/progenitor cells (NPCs) and endothelial progenitor cells (EPCs) play important roles in neurogenesis andangiogenesis, ­respectively5. After stroke, NPCs migrate to the ischemic boundary where angiogenesis takes place,which is closely associated with cerebral vessels in neuro-vascular unit. Indeed, suppression of angiogenesis
substantially reduces migration of newly formed NPCs to the ischemic r­ egion5, and activated endothelial cells(ECs) secrete vascular endothelial growth factor (VEGF) to increase n­ eurogenesis5.
Among several ultrasound technologies, low-intensity pulsed ultrasound (LIPUS) has emerged as a noninvasive therapy for several diseases. We have previously demonstrated that the LIPUS therapy with specificpulsed conditions has therapeutic effects for ischemic and pressure-overloaded heart disease in animal ­models6–8.
1

Department of Cardiovascular Medicine, Tohoku University Graduate School of Medicine, 1‑1 Seiryo‑machi,
Aoba‑ku, Sendai 980‑8574, Japan. 2Department of Developmental Neuroscience, Tohoku University, Sendai,
Japan. 3Biomedical Engineering for Health and Welfare, Graduate School of Biomedical Engineering, Tohoku
University, Sendai, Japan. 4Department of Electronic Engineering, Tohoku University Graduate School ofEngineering, Sendai, Japan. 5International University of Health and Welfare, Narita, Japan. *email: shimo@
cardio.med.tohoku.ac.jp
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Figure 1.  Whole-brain LIPUS therapy improves functional recovery after MCAO. (a) The timeline of theexperiments. (b) Schematic drawing showing the application of whole-brain LIPUS in mice. (c) Tightrope androtarod tests were performed on days 7, 14, 21, and 28 following MCAO or sham surgery; n = 10. *P < 0.05 (C;two-way ANOVA, D; Student’s t-test), †P < 0.05 (two-way ANOVA). (d) Cresyl violet staining was performedon day 28 following MCAO or sham surgery; n = 3 (sham), n = 8 (MCAO). The scale bar represents 100 μm.
*P < 0.05 (Student’s t-test).
We found that the LIPUS therapy upregulated neurotrophins, including VEGF and endothelial nitric oxide
synthase (eNOS)6–8. Since endothelium-derived NO exerts a variety of beneficial cardiovascular effects, activation of eNOS could be an effective therapeutic strategy for cardiovascular d­ isease9,10. Furthermore, we also haverecently demonstrated that the LIPUS therapy improves cognitive dysfunction in mouse models of vasculardementia and Alzheimer’s disease through enhancement of eNOS ­upregulation11. However, the effects of the
LIPUS therapy on stroke remain to be examined.
In the present study, we thus examined whether the LIPUS therapy can promote post-stroke functional
recovery through angiogenesis and neurogenesis, and if so, to elucidate the mechanisms involved.

Results

LIPUS therapy improves functional recovery following MCAO.

In the middle cerebral artery occlusion (MCAO) model in mice, the LIPUS therapy (Fig. 1a,b) had no effects on body weight or systolic bloodpressure (P = 0.454, P = 0.971, Fig. S1). The LIPUS group showed no increased events of cramps or cerebral hem-

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Figure 2.  The LIPUS therapy promotes angiogenesis and upregulates angiogenesis- related molecules. (a)Schematic drawing showing the ischemic hemisphere section and 4 regions related with the lesion at (i)40–250 μm, (ii) 250–500 μm, (iii) 500–750 μm, and (iv) 750–1000 μm from the SVZ. Representative imagesof ischemic hemisphere sections at 500–750 μm from the SVZ immunostained for CD31 (endothelial cellmarker) on days 7, 14, and 28. The scale bar represents 50 μm. (b) Schematic drawing showing 4 lesions ofinterest at different distances from the SVZ. The numbers of CD31-positive vessels in the indicated lesions atdays 7, 14, and 28 (n = 8 per group). (c) RT-qPCR analysis of eNOS and VEGF at day 3 (n = 14 per group). (d)Representative Western blot and quantification of phosphorylated eNOS at Ser1177 and VEGF at day 5 (n = 10
per group). *P < 0.05 (Student’s t-test). Uncropped blots are shown in Fig. S10.
orrhage compared with the Non-LIPUS group, which underwent the same procedure but without the LIPUS
therapy (data not shown). The LIPUS therapy markedly improved neurological functions compared with the
Non-LIPUS group in the rotarod and in the tightrope tests (rotarod test, P = 0.012; tightrope test, P < 0.001,
Fig. 1c, Movie S1, Movie S2). There were no significant differences in the corner or the Barnes circular mazetest between the 2 groups (Fig. S3). Moreover, the LIPUS therapy significantly reduced the infarct size at day 28
compared with the Non-LIPUS group (P = 0.045, Fig. 1d).

LIPUS therapy promotes angiogenesis around ischemic lesion. Next, to examine whether the
LIPUS therapy increases vascular density in the MCAO model, we examined CD31-positive vessel density in the
injured striatum (Fig. 2a). In the normal striatum areas, including the subventricular zone (SVZ), there was no
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difference in CD31-positive vessels between the 2 groups. However, in the ischemic lesion, the number of CD31positive vessels, especially at 250 ~ 750 μm from the lateral ventricle, was significantly increased in the LIPUS
group compared with the non-LIPUS group at day 7 (250–500 μm, P = 0.019; 500–750 μm, P = 0.039) (Fig. 2b).
Moreover, the number of CD31/Ki67 double positive cells at 500–750 μm from the lateral ventricle at day 7 wassignificantly increased in the LIPUS group compared with the Non-LIPUS group (P = 0.029, Fig. S4c), suggestingthat the LIPUS therapy induced angiogenesis in the MCAO model. This angiogenic effect of the LIPUS therapy
was noted until day 28 (Fig. 2b).

LIPUS therapy upregulates angiogenic factors.

In order to elucidate the molecular mechanisms of
the beneficial effects of the LIPUS therapy on angiogenesis, we examined the expressions of mRNA and proteinsthat are related to angiogenesis. mRNA expression levels of eNOS, VEGF, and fibroblast growth factor (FGF)
were significantly higher in the LIPUS group compared with the Non-LIPUS group (eNOS, P = 0.047; VEGF,
P = 0.041; FGF, P = 0.021, Fig. 2c, Fig. S5a). The protein levels of eNOS phosphorylated at Ser1177, VEGF, andextracellular signal-regulated kinase (ERK) phosphorylated at Thr202/Tyr204 were higher in the LIPUS groupcompared with the Non-LIPUS group (eNOS, P = 0.031; VEGF, P = 0.034; ERK, P = 0.030, Fig. 2d, Fig. S5b). In
contrast, mRNA expression level of angiopoietin-1 was comparable between the 2 groups (Fig. S5a).

LIPUS therapy promotes neurogenesis. To examine whether the LIPUS therapy promotes neurogenesis after MCAO, we examined the number of cells expressing the immature neuronal marker, doublecortin(DCX), in the striatum. Although there was no significant difference in DCX-positive cells at day 7, more DCXpositive cells were noted in the ischemic striatum of the LIPUS-treated group compared with the Non-LIPUS
group at days 14 and 28 (500–750 μm at day 14, P = 0.033; 500–750 μm at day 28, P = 0.042, Fig. 3a,b).
LIPUS therapy increases protein levels of factors related to neuronal migration. In order toelucidate the molecular mechanisms of the beneficial effects of the LIPUS therapy on neurogenesis, we thenexamined mRNA and protein expressions of neurogenesis-related factors. We found that mRNA expression levels of stromal cell-derived factor (SDF)-1α and C-X-C motif chemokine receptor 4 (CXCR4) were significantly
higher in the LIPUS group compared with the Non-LIPUS group (SDF-1α, P = 0.012; CXCR4, P = 0.013; Fig. 3c),which also was the case for protein level of CXCR4 (P = 0.044, Fig. 3d). In contrast, mRNA and protein levelsof brain-derived neurotrophic factor (BDNF) were comparable between the Non-LIPUS and LIPUS groups(Fig. 3c,d). The protein levels of SDF-1 and phosphorylated Akt at Ser 473 were also unchanged between the twogroups (Fig. 3d, Fig. S5b).
To examine cellular source of SDF1 and CXCR4, we performed double immunofluorescence staining forboth CD31-positive and DCX-positive cells. SDF-1 and CXCR4 were expressed in CD31-positive cells at day 7(Fig. S6a,b). In contrast, few DCX-positive cells expressed SDF-1 at day 7 (Fig. S7a), and CXCR4 was expressed
only in a few DCX-positive cells (Fig. S7b). These results suggest that CD31-positive cells contributed to the
production of SDF-1 and CXCR4 and migration of DCX positive cells.

Essential role of eNOS in the beneficial effects of the LIPUS therapy. Our previous study showedthat up-regulation of eNOS is crucial for the angiogenic effects of the LIPUS therapy. Indeed, in the present
study, the protein level of phosphorylated eNOS was significantly higher in the LIPUS group compared with the
Non-LIPUS group. To evaluate the role of eNOS in the LIPUS-induced effects, we next examined the influenceof eNOS ablation in the MCAO model using eNOS-deficient ­(eNOS−/−) ­mice12. Importantly, in e­ NOS−/− mice,the LIPUS therapy had no effect on MCAO-induced behavior outcome, infarct size, CD31-positive vessel density, or the number of DCX-positive neuroblasts (Fig. 4a–f). Furthermore, the expression levels of neurotrophins
related to angiogenesis (VEGF) and neurogenesis (CXCR4 and ERK) after MCAO were not increased in LIPUStreated ­eNOS−/− mice (Fig. 4g, Fig. S8). Thus, eNOS is crucial for the beneficial effects of the LIPUS therapy in
the MCAO model.
LIPUS therapy does not improve functional recovery following MCAO when used in the chronic
phase. Finally, we assessed whether the LIPUS therapy improves neurological functions after MCAO whenused in the chronic phase. In this experiment, the LIPUS therapy was started 28 days after MCAO (Fig. S9a). We
performed behavioral tests at days 28, 56, 84 and 112. However, there were no significant differences between
the LIPUS and the Non-LIPUS groups in the rotarod, tightrope, or corner test (Fig. S9b). This may be because
maturation of blood vessels induced by angiogenic stimuli is completed within 28 days in m
­ ice13, and even if theangiogenesis is stimulated thereafter, the tissues are unable to respond.

Discussion

Cerebral infarction is a disease with poor prognosis that significantly impairs activities of daily living (ADL)
due to paralysis of the extremities of the patients. We have demonstrated that the LIPUS exerts angiogenic andanti-inflammatory effects and have developed the LIPUS therapy for the treatment of ischemic heart disease­first6,7 followed by that for d­ ementia11. Thus, in the present study, we aimed to examine whether the whole-brain
LIPUS therapy is also effective for the treatment of ischemic stroke in mice in vivo, and if so, to elucidate themechanisms involved.

LIPUS promotes angiogenesis and subsequent neurogenesis. We have previously reported that
the LIPUS therapy mainly acts on vascular endothelial cells, inducing angiogenesis in ischemic tissue throughScientific Reports |Vol:.(1234567890)(2021) 11:4958 |https://doi.org/10.1038/s41598-021-84473-6

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Figure 3.  The LIPUS therapy promotes neurogenesis and upregulates the molecules related to neuronalmigration. (a) Schematic drawing showing the coronal section of the mouse brain and lesion areas of interest.
Representative images of ischemic hemisphere sections immunostained for DCX on days 7, 14, and 28. The
scale bar represents 100 μm. (b) Schematic drawing showing the ischemic hemisphere section and 4 regions
related with the lesion at (i) 40–250 μm, (ii) 250–500 μm, (iii) 500–750 μm, and (iv) 750–1000 μm from the
SVZ. The numbers of DCX-positive cells in the indicated regions at day 7, 14, and 28 (n = 8 per group). (c)RT-qPCR analysis of SDF-1, CXCR4, and BDNF at day 3 (n = 14 per group). (d) Representative Western blot andquantification of SDF-1, CXCR4 and BDNF at day 5 (n = 10 per group). *P < 0.05 (Student’s t-test). Uncropped
blots are shown in Fig. S10.
upregulation of ­eNOS6–8,11. In the present study, we consistently observed increased vessel density in the periinfarct lesion associated with upregulation of VEGF and eNOS in the LIPUS group as we observed in the mousemodel of vascular ­dementia11. During the recovery process, the LIPUS therapy induced more CD31-positive
blood vessels, followed by DCX-positive neurons. This implies that neovessels generated by angiogenic effectsof LIPUS induced neurogenesis and neuronal migration to the infarct lesion. Furthermore, we also confirmed
a significant increase in the expressions of SDF-1α and CXCR4, which are known as molecules that regulate
cell proliferation and cell ­migration14. It has been reported that CXCR4 is expressed in NPCs and the actions of
SDF-1/CXCR4 complex are crucial for NPCs migration to SDF-1-riched injury sites after cerebral ­ischemia15–17.
Thus, it is highly possible that the LIPUS therapy can enhance proliferation of neural stem cells and support theirmigration by creating suitable vasculature as a scaffold (Fig. 5).
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Figure 4.  Crucial role of eNOS for the beneficial effects of LIPUS in the MCAO model. (a) Behavioral tests
were performed on days 7, 14, 21, and 28 in the Non-LIPUS and LIPUS-treated e­ NOS−/− mice subjected to
MCAO (n = 6 per group). (b) Cresyl violet staining was performed on day 28 following MCAO (n = 6 pergroup). (c) Representative images in the peri-infarct lesion immunostained for CD31 on day 28. The scale bar
represents 50 μm. (d) The number of CD31-positive vessels at (i) 40–250 μm, (ii) 250–500 μm, (iii) 500–750 μm,
and (iv) 750–1000 μm from the SVZ on day 28 (n = 6 per group). (e) Representative images of the ischemic
striatum immunostained for DCX on day 28. The scale bar represents 100 μm. (f) The numbers of DCX-positive
cells at (i) 40–250 μm, (ii) 250–500 μm, (iii) 500–750 μm, and (iv) 750–1000 μm from the SVZ at day 28 (n = 6
per group). (g) Western blot analysis of VEGF and CXCR4 on day 5 (n = 6 per group). Uncropped blots areshown in Fig. S10.

First evidence of the LIPUS treatment for clinical medicine. A few studies have previously attempted

to apply focused LIPUS for the treatment of stroke in rodent ­models18–20. Although it has been reported that
focused LIPUS applied to the ischemic site of brain directly induces ­angiogenesis19, our whole brain irradiation
may be advantageous as it stimulates not only the ischemic border zone but also the SVZ known as neurogenic
niche. Additional advantage of our whole-brain LIPUS therapy over the focused LIPUS therapies is that it does
not require an expensive MRI machine and is easy to implement in clinical practice. Actually, our whole-brain
LIPUS using convenient equipment is already in the phase 2 of the dementia trial (UMIN000033071). Moreover,

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Figure 5.  Schematic diagram showing the proposed mechanisms underlying the beneficial effects of wholebrain LIPUS in the MCAO model in mice. The present study demonstrates that the LIPUS therapy promotes
functional recovery in MCAO mouse model, and that the beneficial effects of the LIPUS therapy depend on
endothelial NO synthase (eNOS). Both angiogenesis and neurogenesis were promoted by the LIPUS therapy
sequentially in this order, indicating that VEGF and eNOS-induced angiogenesis subsequently upregulates
neurotrophins, including SDF-1, and CXCR4, and these molecules mediate neuronal proliferation andmigration. Additionally, increased vessel density supports neuronal migration structurally. Finally, neuronal
functional recovery can be improved by differentiated neural stem cells.
the focused LIPUS has been applied either before or immediately after the ­injury18–20, whereas our whole-brain
LIPUS was applied 1, 3, and 5 days after the injury, which may be more suitable for future clinical application.

Study limitations. Several limitations should be mentioned for the present study. First, we did not analyze
the function of individual brain cells, such as astrocytes, microglia, and other types of cells, for neuronal recovery after stroke. For example, LIPUS stimulates astrocytes and upregulates their BDNF ­production21. Other
types of cells (e.g. oligodendrocyte precursor cells) may also have important roles in the beneficial effects of the
LIPUS therapy on post-stroke neuronal recovery. Second, to confirm that newborn neurons are incorporated
into the surrounding tissue and work functionally, differentiation, axonal outgrowth, and synaptogenesis should
be analyzed. Further studies are needed to elucidate the mechanism of the beneficial effects of the LIPUS therapy
on post-stroke functional recovery.

Conclusions

The LIPUS therapy could be a novel treatment with minimal invasiveness, which may potentially be applied toa huge number of patients with stroke in an aging society. In the current clinical medicine, treatment options
for ischemic brain injury are not sufficient, and there is no treatment for promoting neurogenesis after stroke.
Thus, our whole-brain LIPUS therapy could be a novel treatment option not only for ischemic stroke but also
for other neurodegenerative diseases, such as Parkinson disease, amyotrophic lateral sclerosis, spinocerebellar
degeneration in future.

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Methods

All procedures were performed in accordance with the protocols approved by the Institutional Committee for the
Use and Care of Laboratory Animals of Tohoku University (2017Ikumikae-174, 2017Idou-266), and the Guide
for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication
No. 85-23, revised 1985). The experimental timeline is shown in Fig. 1a.

Mice. Male C57BL/6 mice (10–12 weeks-old, weight 22 to 28 g) and eNOS-deficient ­(eNOS−/−) mice (10–12 weeks-old, on a C57BL/6 background; weight 22 to 28 g) were used. ­eNOS−/− mice were originally provided
by P. Huang (Harvard Medical School, Boston, MA)12 and were backcrossed to C57BL/6 mice more than 10
generations. All mice were housed in a temperature-controlled (20 ± 2 °C) and humidity-controlled (60%) room.
The number of animals used in each experiment is shown in Table S1.

Induction of transient focal cerebral ischemia. Cerebral ischemia was induced using the intraluminal
middle cerebral artery occlusion (MCAO) model as described ­previously22,23. Mice were anesthetized with 1.1%
isoflurane (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and were placed on a heating pad to prevent
hypothermia; body temperature was maintained at 36.5 to 37.5 °C. MCAO was induced using a commerciallyavailable suture (602312PK10Re, Doccol Corp., MA, USA), which was withdrawn after 45 min of ischemia toallow reperfusion of the middle cerebral artery. Regional cerebral blood flow was measured with a laser-Doppler
flowmetry (Omegawave, Tokyo, Japan) to confirm the induction of ischemia and reperfusion (Fig. S2). Animals
that did not show less than 30% reduction of regional cerebral blood flow from pre-ischemia baseline levels during MCAO were excluded from further experimentation. All animals were randomly assigned to each treatmentgroups.
Whole‑brain LIPUS therapy. The experimental timelines are shown in Fig. 1a. The LIPUS therapy was
performed as previously described, with slight ­modifications11. The LIPUS group was subjected to the LIPUS
therapy 3 times in the first week (1, 3, and 5 days after MCAO), while the Non-LIPUS group underwent the sameprocedures including anesthesia but without the LIPUS treatment (Fig. 1a). For the LIPUS therapy, we used aplaner ultrasound probe (HONDA ELECTRONICS, Co., Aichi, Japan). The LIPUS therapy was performed using
the following conditions; center frequency = 0.5 MHz, pulse repetition frequency = 780 Hz, number of cycles = 32(64-μsec burst length), and spatial peak temporal average intensity = 193 mW/cm2 11. While in the previous
study, the center frequency was 1.875 M­ Hz11, we used in the present study 0.5 MHz for the purpose of effectively
penetration through the skull. Additionally, in order to apply the LIPUS therapy for humans in future clinical trials, we have changed the shape of the transducer from the sector-type to the convex-type, which has already beenused in our clinical trial for patients with Alzheimer’s disease (UMIN000033071). As in the previous s­ tudy11, weused whole brain irradiation, and we applied LIPUS three times in the same position. Whole-brain irradiation isused to cover a wider range of lesions than the focused-ultrasound. The mouse’s head was shaved, and the LIPUS
was applied to the head via an agar phantom gel under inhalation anesthesia with 0.5–1.0% isoflurane (Fig. 1b).
The treatment was performed three times a day with each duration of 20 min.
Behavioral tests. Behavioral tests were conducted on days 7, 14, 21, 28 following MCAO or sham surgery(Fig. 1a). The tests were conducted by researchers who were blinded with respect to whether the mouse received
the LIPUS therapy or not.
Rotarod test. The Rotarod test was performed with MK-630B (Muromachi Kikai, Co., Tokyo, Japan) as previously ­described24. The cylinder speed was slowly increased from 4 to 40 rpm. Maximum speed was achieved after
260 s, and maximum testing time was 300 s. Mice were put on the cylinder, and the time spent on the device wasdetermined. Mice were trained for 5 days before MCAO. The mean duration on the device was recorded with 2
measurements.
Tightrope test. Animals were placed on a 60 cm-long tightrope grasping the string with their forepaws according to a modified protocol from Doeppner et al.24. All animals were tested twice per time point. Maximum test
time was 60 s, and results were scored from 0 (minimum) to 20 (maximum) according to a validated score(Table S2), depending on the time that animals spent on the rope and whether or not they reached to the platform.
Corner test. The corner test was used to evaluate sensorimotor asymmetry, as described ­previously25. The mice
were placed in a corner with an angle of 30°. To exit the corner, the mouse could turn to the left or right and thischoice was recorded. Although healthy animals leave the corner without side preference, mice suffering fromstroke preferentially turn to the left, non-impaired body s­ ide25. The test was repeated 10 times, and the percentage of left turnings was calculated. Only turns involving full rearing along either wall were included.
Barnes circular maze test. We performed the Barnes task on “dry land,” a gray acrylic platform (122 cm indiameter), with 16 equidistant holes around the perimeter (O’Hara & Co., Tokyo, Japan). An overhead camera
assessed the performance of mice as previously ­reported26. In the 3-day training period, 3 trials were performed
per day, with a maximum trial length of 300 s. Each trial began with the start box positioned at the center ofthe maze and a mouse placed inside it for 30 s; the mouse was then permitted to explore the maze freely. Afterreaching the target and before being returned to its home cage, the mouse was left in the escape box for 30 s. If
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the mouse did not enter the escape box within 300 s, the researcher gently picked it up and placed it inside thetarget box for 30 s before returning it to its home cage. We assessed the latency (time to reach the target box) andcompared the results between sham animals and the MCAO groups with or without the LIPUS therapy.

Histological analyses. To assess the acute and chronic phase effects of the LIPUS therapy in the MCAO

model in mice, histological analyses were performed on days 7, 14, and 28 after the procedure (Fig. 1a). Under
deep anesthesia with isoflurane, the mouse was perfused with 4% paraformaldehyde, the brain was removed,
fixed in 4% paraformaldehyde for 48 h, cryoprotected in 30% sucrose, and 10-μm coronial sections were cut
using a cryostat (LEICA, Wetzlar, Germany). Cresyl violet staining was performed to evaluate infarct ­area27–29.
The tissue defect in each experimental group was defined as the lesion, and the area of this defect was calculated
by subtracting the area of the ipsilateral hemisphere from the area of the contralateral hemisphere. Each area
was estimated by the ImageJ software (ImageJ version 1.52, National Institutes of Health, MD, USA; available
at: http://image​j.nih.gov/ij/). The lesion volume was calculated from the sum of lesion areas of the 4 serial slices,
spaced 2-mm apart. The percentage of the lesion volume presented in this study was calculated by dividing lesion
volume by the volume of the contralateral hemisphere.
For immunofluorostaining, the sections were blocked with PBS containing 5% goat serum (Invitrogen, CA,
USA) and 0.3% Triton X-100, and then incubated with primary antibodies at 4 °C overnight. After washingwith PBS, sections were incubated with secondary antibodies conjugated with Alexa Fluor 488 (1:500, ThermoFisher Scientific, Waltham, MA, USA), Alexa Fluor 564 (1:500, Thermo-Fisher Scientific, Waltham, MA, USA)
or Cy3 (1:500, Jackson Immuno Research, West Grove, PA, USA) for 1 h at room temperature. After washingwith PBS, the cells were covered with VECTASHIELD mounting medium (Vector Laboratories, CA, USA) with40, 6-diamidino-2-phenylindole (DAPI). The sections were observed using a BZ-X 800 (KEYENCE, Osaka,
Japan). The numbers of DCX-positive cells, CD31-positive microvessels, and CD31/Ki67 double positive cells
in the indicated region were counted and quantified by an investigator who was blinded. Four serial sections
spaced 400-μm apart (1.10 to − 0.1 mm from the bregma) were selected from each a­ nimal30. The numbers of
DCX-positive cells, CD31-positive microvessels and CD31/Ki67 double positive cells were counted and averaged
from 4 sections of each animal. Sections were incubated with the same primary antibodies and imaged under
the same conditions by a blinded investigator. Negative control staining without primary antibodies resulted inno signal. The antibodies used in this study are listed in Table S3.

Quantitative real‑time PCR. To assess the acute effects of the LIPUS therapy in the MCAO model inmice, real-time PCR analysis was performed on day 3 after MCAO. Total RNA was extracted from the ipsilateral striatum and isolated using the RNeasy universal kit (Qiagen, Hilden, Germany) in accordance with the
manufacturer’s protocol. After reverse transcription, real-time PCR was performed using SYBR Premix Ex Taq
II (Takara-Bio Inc.) and a CFX96TM Real-Time system C1000TM Thermal Cycler (Bio-Rad Laboratories Inc.,
California, USA). The number of transcripts was normalized to GAPDH as a housekeeping gene. Melting curves
were run to ensure amplification of a single product. The primers used in this study are listed in Table S4.
Western blot analysis. To assess the acute effects of the LIPUS therapy in the MCAO model in mice, Western blot analysis was performed on day 5 after the procedure. The brain was perfused with PBS and cut into 4
sections by 3 blades that are 2-mm apart; the second rostral section, including the ischemic core, was collected.
The protein extracted from ipsilateral striatum was used for further Western blot analysis. Western blot analysis
was performed on day 5 after MCAO. The tissue was homogenized using Tissue Protein Extraction Reagent(Thermo-Fisher Scientific, Waltham, MA, USA) containing a protease inhibitor cocktail (Sigma-Aldrich, MO,
USA). The samples were then centrifuged at 15,000 g for 20 min at 4 °C, and the supernatants were collected andnormalized for protein concentration, which was measured using the BCA Protein Assay Kit (Biotechnology
Inc., Rockford, IL, USA). The samples were then separated by SDS-PAGE and transferred to a polyvinylidene
difluoride (PVDF) membrane (GE Healthcare, IL, USA). After blocking with Tris-buffered saline (TBS) containing 5% (w/v) skim milk or BSA and 0.05% Tween 20, the membranes were incubated with primary antibodies
overnight at 4 °C. The antibodies used in this study are listed in Table S3. The membrane was then incubated in
the appropriate secondary antibody (1:5,000 dilution) for 1 h at room temperature. The proteins were visualizedusing enhanced chemiluminescence (ECL Western Blotting Detection Kit; GE Healthcare, IL, USA) and quantified using ImageJ Software (NIH).
Statistical analysis. All summary data are presented as the mean ± s.e.m. Although no statistical methods
were used to predetermine sample sizes for the studies, our sample sizes were similar to those generally used in
the field. Differences between 2 groups were analyzed using an unpaired two-tailed Student’s t-test. Data frommultiple groups were analyzed using analysis of variance (ANOVA) followed by Tukey’s multiple comparison(GraphPad Prism Software Inc.). Differences with a P-value < 0.05 were considered to be statistically significant.

Data availability

Data are available on request by contacting the corresponding author.
Received: 28 October 2020; Accepted: 14 January 2021

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Acknowledgements

The authors thank the collaborators at the Department of Neurosurgery, Tohoku University Graduate School of
Medicine (Drs. Naoya Iwabuchi, Takatsugu Abe, Natsumi Konno, Marisa Ohno, Kuniyasu Niizuma, and Teiji
Tominaga), and the experiment assistants (Mss. Ai Nishihara, Misako Suzuki, Hiromi Yamashita, and Yumi
Watanabe).

Author contributions

S.I., T.S., N.O., H.K, and H.S. designed research. S.I., K.E., Y.Mon., T.N., Y.Mor. performed research. S.I. T.N.,
and Y.Mor. analyzed data. S.I., T.S., N.O., S.Y., and H.S. wrote the manuscript.

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Funding

This work was supported in part by the Japan Agency for Medical Research and Development Grants(16lm0103007j0005 and 17lk1403011h0001).

Competing interests

The authors declare no competing interests.

Additional information

Supplementary Information The online version contains supplementary material available at https​://doi.
org/10.1038/s4159​8-021-84473​-6.
Correspondence and requests for materials should be addressed to H.S.
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