1Department of Rehabilitation, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China. 2Med-X Research Institute and School of Biomedical Engineering, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China. 3Shenzhen Institutes of Advanced Technology Chinese Academy of Sciences, Shenzhen 518055, China. 4School of Information Engineering, Guangdong Medical University, Dongguan 523808, China
Transcranial focused ultrasound stimulation (tFUS) regulates neural activity in different brain regions in humans and animals. However, the role of ultrasound stimulation in modulating neural activity and promoting neurorehabilitation in the ischemic brain is largely unknown. In the present study, we explored the effect of tFUS on neurological rehabilitation and the underlying mechanism. Adult male ICR mice (n=42) underwent transient middle cerebral artery occlusion. One week after brain ischemia, low frequency (0.5 MHz) tFUS was applied to stimulate the ischemic hemisphere of mice for 7 consecutive days (10 minutes daily). Brain infarct volume, neurobehavioral tests, microglia activation, IL-10 and IL-10R levels were further assessed for up to 14 days. We found that the brain infarct volume was significantly reduced in the tFUS treated mice compared to that in the non-treated mice (p<0.05). Similarly, neurological severity scores, elevated body swing test, and corner test improved in the tFUS treated mice (p<0.05). We also demonstrated that tFUS resulted in increased M2 microglia in the ischemic brain region. The expression of IL-10R and IL-10 levels were also substantially upregulated (p<0.05). We concluded that tFUS served as a unique technique to promote neurorehabilitation after brain ischemia by promoting microglia polarization and further regulating IL-10 signaling in the ischemic brain.
Wang Jixian,Li Guofeng,Deng Lidong, et al. Transcranial Focused Ultrasound Stimulation Improves Neurorehabilitation after Middle Cerebral Artery Occlusion in Mice[J]. Aging and disease,
2021, 12(1): 50-60.
Figure 1. Flow diagram of the experimental design Animals underwent middle cerebral artery occlusion (MCAO). tFUS was applied for 7 days from day 7 of MCAO. The behavioral tests were performed following MCAO for 14 days. Brain atrophy volume and potential mechanisms were examined.
Figure 2. The setup used for low frequency transcranial ultrasound stimulation of the ischemic hemisphere of mice A) Photograph showed the experimental setup of ultrasound stimulation device. B) Photograph showed the tFUS process. The transducer was located on the ipsilateral hemisphere of the mouse brain. C) The photograph showed the ultrasound intensity distribution field relative to the spatial peak in the sanitation region. The Z axis represented the stimulus depth. The Y axis showed the stimulus width. Different colors represented different relative tFUS intensities. Red (high intensity); violet (low intensity). D) The photograph showed a general view of the tFUS sequences: 0.5 ms tone-burst-duration (TBD), 1000 Hz pulse repetition frequency (PRF), 300 ms sonication duration (SD), and 2.7 s inter-stimuli interval (ISI). Green, red and blue arrows indicate the portable tFUS system, the impedance matching device and the ultrasound transducer, respectively.
Figure 3. Ultrasound stimulation of the brain promoted neurofunctional recovery in mice that underwent MCAO A) Photomicrographs showed representative sets of cresyl violet-stained brain sections from mice treated with or without tFUS after 60 minutes of MCAO. The line illustrates the atrophy volume of the ipsilateral hemisphere following 7 days of tFUS treatment. Bar graph showed the semi-quantitative data from panel a. n=6 per group, p<0.05, tFUS treated mice after MCAO vs. MCAO alone. Line graphs showed the neurologic severity score (B), EBST (C) and corner test (D) results for the sham, MCAO alone, and MCAO mice with tFUS. n=6 per group, p<0.05, tFUS treated MCAO mice vs. MCAO alone group.
Figure 4. Ultrasound stimulation of the brain increased the number of M2 microglia after MCAO A) Co-immunostaining of the M1 microglia markers CD16/32 and IBA-1 (upper left), and the M2 microglia markers arginase and IBA-1 (upper right) in the mouse brain after one hour of MCAO in mice. After tFUS, there were fewer CD16/32+/IBA-1+ cells were detected while arginase+/IBA-1+ cells were increased in the ischemic perifocal region (below). Bar=20 μm. B) Bar graph showed the number of M1 and M2 microglia in the ischemic mouse brain treated with tFUS. Data are presented as mean ± SD. ***, p<0.001. C) CD16/32/CX3CR1+ (M1) and CD206/CX3CR1+ (M2) microglia, the number of CD16/32/CX3CR1+and CD206/CX3CR1+ microglia were increased. However, the number of CD206/CX3CR1+ cells was increased remarkably in the tFUS treated group. D) Bar graph showed the number of M1 and M2 microglia in the ischemic mouse brain treated with tFUS. Data are presented as mean ± SD. *, p<0.05.
Figure 5. Ultrasound stimulation of the brain upregulated IL-10R and IL-10 A) Photomicrographs showed IL-10R (green) and DAPI (blue) staining in the perifocal region of the ipsilateral hemisphere in sham, MCAO alone, and MCAO mice with tFUS. White arrowheads indicate that IL-10R is mainly expressed in the cell cytosol in the perifocal region in MCAO mice. IL-10R staining was greatly increased after tFUS. Scale bar= 25 μm. B) Western blot analysis showed that IL-10 expression (101 KD) was greatly increased after tFUS. The bar graph showed the quantitative data for the Western blot analysis. n=6 per group. *, p<0.05, tFUS treated mice after MCAO vs. MCAO alone. PCR showed the relative expression of IL-10 (C) and IL-10R (D) in the brain after tFUS treatment. n=6 per group. Data are presented as mean ± SD.*, p<0.05.
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