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Transcranial focused ultrasound stimulation reduces vasogenic edema after middle cerebral artery occlusion in mice

Guo-Yuan Yang, Ji-Xian Wang, Li-Dong Deng, Lin Qi, Qian Suo, Sheng-Ju Wu, Muyassar Mamtilahun, Ru-Bing Shi, Ze Liu, Jun-Feng Sun, Yao-Hui Tang, Zhi-Jun Zhang

Neural Regeneration Research 2022, 17, 2058 · 10.4103/1673-5374.335158

rodentstrokebehaviourhistology molecularcerebral haemodynamics

Abstract

Blood-brain barrier (BBB) disruption underlies the vasogenic edema and neuronal cell death induced by acute ischemic stroke. Reducing this disruption has therapeutic potential. Transcranial focused ultrasound stimulation has shown neuromodulatory and neuroprotective effects in various brain diseases including ischemic stroke. Ultrasound stimulation can reduce inflammation and promote angiogenesis and neural circuit remodeling. However, its effect on the BBB in the acute phase of ischemic stroke is unknown. In this study of mice subjected to middle cerebral artery occlusion for 90 minutes, low-intensity low-frequency (0.5 MHz) transcranial focused ultrasound stimulation was applied 2, 4, and 8 hours after occlusion. Ultrasound stimulation reduced edema volume, improved neurobehavioral outcomes, improved BBB integrity (enhanced tight junction protein ZO-1 expression and reduced IgG leakage), and reduced secretion of the inflammatory factors tumor necrosis factor-α and activation of matrix metalloproteinase-9 in the ischemic brain. Our results show that low-intensity ultrasound stimulation attenuated BBB disruption and edema formation, which suggests it may have therapeutic use in ischemic brain disease as a protector of BBB integrity.

Abstract via europepmc.

Speciesmouse (C57BL/6J)
Subjects76 animals
Sessions per subject1
Randomisedyes
Blindingdouble
Sham / controlnone
Auditory controlnot reported
Readout timingoffline
Anaesthesiaanaesthetised
Readoutsbehaviour, histology molecular, cerebral haemodynamicsmodified neurological severity score (mNSS); cresyl violet staining for infarct/edema volume; Western blot and immunostaining for tight junction proteins (ZO-1, occludin, claudin-5); IgG leakage staining; gelatin zymography (MMP-9/MMP-2 activity); RT-qPCR (IL-1beta, TNF-alpha, TGF-beta); laser speckle imaging of cerebral blood flow
Direction of effectnot assessedThis study assessed neuroprotective and vascular outcomes (edema volume, blood-brain barrier integrity, inflammation, cerebral blood flow) rather than direct neuronal excitability, so excitatory/inhibitory direction of neuromodulatory effect was not assessed.
Adverse eventsnone observedNo detectable pathological changes were seen on hematoxylin and eosin staining of tFUS-treated brain compared to normal controls, and tFUS itself did not cause IgG (BBB) leakage in normal (sham) mice.

Exposures

Exposure 1: tFUS to left (ipsilateral) hemisphere after transient MCA occlusion

Target: hemisphere unspecified — “left hemisphere (ipsilateral to MCA occlusion), 2 mm beneath the cortical surface
Device: custom-built · Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences

Pulse timing
Waveformpulsed
Fundamental frequency (kHz)500✓✓
Pulse duration (ms)0.5✓✓
Pulse repetition frequency (Hz)1,000✓✓
Duty cycle (%)not reportedpulse duration × PRF gives 50%
Sonication duration (s)0.3✓✓
Pressure and intensity, by domain
Free-field pressure (kPa)not reported
Free-field Isppa (W/cm²)not reported
Free-field Ispta (W/cm²)not reported
In-situ estimatenot reported
In-situ pressure (kPa)not reported
In-situ Isppa (W/cm²)not reported
In-situ Ispta (W/cm²)not reported
Ispta, domain unspecified (W/cm²)0.039✓✓
Protocol, in the paper’s words

tFUS was applied to the left hemisphere for 10 minutes with the probe attached to the overlying skull, using a single-element focused ultrasound transducer. Parameters: fundamental frequency 500 kHz; spatial peak temporal average intensity 39 mW/cm2; pulse width 500 microseconds; pulse interval 1 ms; stimulation (train) duration 300 ms; stimulation interval 3 seconds. Treatment was given once, 2, 4, or 8 hours after occlusion depending on group.

Flags from extraction

  • n_subjects76 mice were enrolled in total across all 6 groups (12-13 per group), but only 4 of the 6 groups (sham/tFUS, tMCAO/tFUS at 2h/4h/8h) actually received active tFUS; the paper does not give an exact count of ultrasound-exposed animals, only the per-group range, so the total N=76 enrolled is recorded here.
  • exposures[0].timing.pulse_repetition_frequency_hzPaper reports pulse width (500 microseconds) and pulse interval (1 ms) rather than PRF directly; PRF was not computed here.
  • exposures[0].timing.duty_cycle_pctDuty cycle is not explicitly stated; it would require computing pulse width / (pulse width + pulse interval), which was not done here.
  • exposures[0].unspecified_domain.ispta_w_cm2The paper states a single 'spatial peak temporal average intensity' without specifying whether it was measured/derived in free field or in situ.
  • direction_of_effectStudy measured edema, BBB integrity, inflammation and cerebral blood flow rather than neuronal excitability; classified as not_assessed.