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    Transcranial focused ultrasound stimulation reduces vasogenic edema after middle cerebral artery occlusion in mice
  • Figure 1|The low-frequency tFUS setup on the mouse ischemic brain. 

    Seventy-six mice were divided into the following 6 groups (12–13 mice per group) using the random number table method: sham, sham/tFUS, tMCAO alone, tMCAO/tFUS at 2 hours, tMCAO/tFUS at 4 hours, and tMCAO/tFUS at 8 hours. Mice were anesthetized with 1.5% isoflurane (RWD Life Science, Shenzhen, China) in air under spontaneous breathing conditions. After the mice were fixed on the stereotaxic apparatus, tFUS was applied to the left hemisphere (ipsilateral to the side of occlusion) for 10 minutes with the probe attached to the overlying skull (Figure 1A). The pulsed wave bursts were delivered from a single-element focused ultrasound transducer (diameter, 2.5 cm; geometrical focal length, 12.44 mm) (Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, China). Ultrasound was induced using the following parameters: fundamental frequency, 500 kHz; spatial peak temporal average intensity, 39 mW/cm2; pulse width, 500 μs; pulse interval,1 ms; stimulation duration, 300 ms; and stimulation interval, 3 seconds (Figure 1B and C).

    Figure 2| tFUS reduces brain edema and neurological deficit.  

    The detailed tMCAO procedure has been described previously with minor modification (Lu et al., 2017). Animals were anesthetized with 1.5% isoflurane in air under spontaneous breathing conditions. Body temperature was maintained at 37.0 ± 0.5°C during surgery using a heating pad (RWD Life Science). The entire surgical procedure was performed under a surgical microscope (Leica, Wetzlar, Germany). A midline incision was made in the neck to expose and separate the left common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). A round head 6-0 suture (Dermalon, 1741-11, Covidien, OH) coated with silicone was gently inserted into the ICA from the ECA stump to induce MCA occlusion. The suture insertion distance from the bifurcation to the left MCA opening was 9.5 ± 0.5 mm. Successful occlusion was defined as a 20% reduction in cerebral blood flow (CBF) as determined by laser speckle imaging (LSI, Figure 2A). After 90 minutes of occlusion, the suture was withdrawn, the CCA restored, and blood flow returned to at least 70% of baseline blood. Sham surgery mice underwent the same procedure without suture insertion.  

    We first examined HE-stained brain tissue to assess the effect of tFUS and found that tFUS-treated mouse brain had no detectable pathological changes, the same as normal control mouse brain (Figure 2B). To explore the effect of tFUS on mouse neurobehavioral outcomes, mice were treated with tFUS for 10 minutes 2, 4, and 8 hours after tMCAO. We found that mNSS on days 1 and 3 after tMCAO did not differ between mice treated with tFUS 2 hours after tMCAO (tMCAO/tFUS at 2-hour group) and tMCAO alone mice. The tMCAO/tFUS at 4-hour group performed significantly better than the tMCAO alone group on day 1 (P < 0.05) but not on day 3. mNSS score was significantly lower in the tMCAO/tFUS at 8-hour group than the tMCAO alone group on days 1 and 3 (P < 0.01; Figure 2C). These results demonstrated that tFUS attenuated neurological deficit in tMCAO mice compared to control mice. 
    We also examined animal body weight before surgery and on days 1 and 3 following tMCAO. Before tMCAO and 1 day after, body weight did not differ among the tMCAO alone and the three tMCAO/tFUS groups. However, on day 3 after tMCAO, body weight was significantly lower in the tMCAO alone group than the tMCAO/tFUS at 8-hour group (P < 0.05; Figure 2D), suggesting that tFUS can improve mouse general condition after ischemic brain injury. 
    To determine if better neurological outcome in the tMCAO/tFUS at 8-hour group was associated with less brain tissue injury, we examined brain infarct volume and edema formation (Figure 2E). Edema volume was smaller in the tMCAO/tFUS at 8-hour group than the tMCAO alone group on day 3 after tMCAO (P < 0.05; Figure 2F). However, edema volume did not differ among the tMCAO/tFUS at 2 hours, tMCAO/tFUS at 4 hours, and tMCAO alone groups. Infarct volume did not differ among groups (P > 0.05; Figure 2G). 

    Figure 3| tFUS improves CBF.

    To investigate the effect of tFUS on CBF, we measured CBF in the normal mouse brain using LSI 10 minutes before and 10 minutes after tFUS stimulation. tFUS caused an approximately 20% increase in CBF (Figure 3A), indicating a direct effect on CBF. We then measured CBF in tMCAO mice before and after tFUS. CBF consistently changed in tMCAO mice. Compared to the tMCAO alone group, CBF increased in the tMCAO/tFUS at 8 hours group (Figure 3B), indicating that tFUS stimulation applied 8 hours after occlusion improved CBF.

    Figure 4|  tFUS treatment reduces IgG leakage and enhances ZO-1 expression in tMCAO mice. 

    To determine whether tFUS treatment reduced tMCAO-induced brain edema by protecting the BBB, we examined BBB leakage using IgG staining. We did not detect IgG leakage in the tFUS-treated normal mice, indicating that tFUS itself did not affect BBB permeability. However, degree of IgG leakage was smaller in the tMCAO/tFUS at 8-hour group compared to the tMCAO alone group on day 3 after tMCAO (P < 0.05; Figure 4A). This suggests that tFUS treatment reduced brain edema by attenuating disruption of the BBB in tMCAO mice.
    We then further examined whether tFUS treatment protected the structural integrity of the BBB. Expression of the tight junction proteins ZO-1, occludin, and claudin-5 was examined in tMCAO mice using immunostaining and Western blotting 3 days after occlusion. Expression of ZO-1, but not occludin or claudin-5, was significantly higher in the tMCAO/tFUS at 8-hour group than the tMCAO alone group (P < 0.05; Figure 4B and C). These results suggest that tFUS attenuated BBB disruption by reducing tight junction protein ZO-1 degradation.

    Figure 5| MMP-9 activity and TNF-α expression decrease in ischemic brain after tFUS treatment.

    To demonstrate whether the protective effects of tFUS on the BBB in ischemic mice were associated with decreased MMP-9 activity, we examined MMP-9 enzyme activity in tMCAO mice 3 days after occlusion using the gelatin zymography assay. MMP-9 activity was lower in the tMCAO/tFUS at 8-hour group than the tMCAO alone group (P < 0.05, Figure 5A), suggesting that the decrease in tight junction degradation induced by tFUS may be related to reduction in MMP-9 activity.
    We also examined mRNA expression of the cytokines IL-1β, TNF-α, and TGF-β on day 3 after occlusion and found that expression of TNF-α, but not IL-1β or TGF-β, was significantly lower in the tMCAO/tFUS at 8-hour group than the tMCAO alone group (P < 0.05; Figure 5B). This suggests that tFUS can attenuate focal inflammation.


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  • 发布日期: 2022-03-10  浏览: 341
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