Transcranial focused ultrasound stimulation alleviates NLRP3-related neuroinflammation induced by ischemic stroke via regulation of the Nespas/miR-383-3p/SHP2 pathway

Zhongqiu Hong, Zejie Zuo, Yun Zhao, Yinan Ai, Liying Zhang, Lili Li, Xiaofei He, Jing Luo, Jinghui Xu, Xiaofeng Yang, Shasha Yi, Haiqing Zheng, Changjun Tie, Lili Niu, Xiquan Hu

International Immunopharmacology 2025, 144, 113680 · 10.1016/j.intimp.2024.113680

rodentin vitro cellstrokebehaviourother mrihistology molecular

Abstract

Transcranial focused ultrasound stimulation (tFUS) has emerged as a promising therapeutic strategy for mitigating brain injury in animal models. In this study, the effects and mechanisms of tFUS on ischemic stroke were explored in a transient middle cerebral artery occlusion (MCAO) rat model. Low-intensity tFUS was administered to the ischemic hemisphere 24 h post-MCAO for seven consecutive days. Neurological function was evaluated through neurobehavioral assessments following tFUS treatment. Western blotting, immunofluorescence staining, and quantitative real-time PCR were performed to examine the impact of tFUS on NLRP3-related neuroinflammation using brain tissues from MCAO rats and BV2 cells subjected to oxygen glucose deprivation/reperfusion (OGD/R). Additionally, RNA sequencing and cell transient transfection were employed to elucidate the underlying mechanisms. The findings revealed that tFUS improved neurobehavioral performance, reduced infarct size, and suppressed NLRP3 inflammasome activation seven days post-MCAO. Notably, Nespas expression was significantly elevated in tFUS-treated rats, whereas Nespas silencing exacerbated neurological deficits and enhanced NLRP3 activation. Moreover, Nespas positively regulated src homology 2 domain-containing tyrosine phosphatase-2 (SHP2), and SHP2 inhibition significantly amplified NLRP3 activation. Mechanistic in vitro studies further demonstrated that Nespas attenuated microglial NLRP3 activation via the Nespas/miR-383-3p/SHP2 pathway. These results suggest that the neuroprotective effects of tFUS are likely mediated through the upregulation of Nespas and suppression of NLRP3 via the Nespas/miR-383-3p/SHP2 axis, offering new insights into the molecular mechanisms supporting tFUS as a potential therapeutic approach for stroke-induced brain injury.

Abstract via europepmc.

Speciesrat (Sprague-Dawley); BV2 murine microglial cell line

Subjectsnot reported animals

Sessions per subject7

Randomisednot reported

Blindingnot reported

Sham / controlinactive transducer

Auditory controlnot reported

Readout timingoffline

Anaesthesiaanaesthetised

Readoutsbehaviour, other mri, histology molecular — Modified neurological severity score (mNSS), elevated body swing test, rotarod test, open field test; T2-weighted MRI infarct volume; Western blot, immunofluorescence, qRT-PCR, TUNEL staining, fluorescence in situ hybridization, dual-luciferase reporter assay

Direction of effectinhibitorytFUS suppressed microglial NLRP3 inflammasome activation and pro-inflammatory cytokine release (NLRP3, ASC, Caspase1, IL-1β, TNF-α, IL-6) while increasing anti-inflammatory IL-10, and improved neurobehavioral recovery and reduced infarct volume after MCAO.

Adverse eventsnot reported

Exposures

Exposure 1: tFUS to the ischemic cortex after MCAO

Target: cerebral cortex — “the cortex (ischemic hemisphere)”
Device: custom-built · ndtXducer (transducer element); mobile system integrated by Shenzhen Institutes of Advanced Technology · 1905055 ✓

Pulse timing
Waveform	pulsed
Fundamental frequency (kHz)	500	✓✓✓
Pulse duration (ms)	1	✓✓✓
Pulse repetition frequency (Hz)	500	✓✓✓
Duty cycle (%)	not reportedpulse duration × PRF gives 50%
Sonication duration (s)	0.4	✓✓✓

Pressure and intensity, by domain
Free-field pressure (kPa)	not reported
Free-field Isppa (W/cm²)	5.1	✓✓✓⚑
Free-field Ispta (W/cm²)	not reported
In-situ estimate	not reported
In-situ pressure (kPa)	not reported
In-situ Isppa (W/cm²)	not reported
In-situ Ispta (W/cm²)	not reported

Protocol, in the paper’s words

Ultrasound stimulation was administered for 15 min daily for seven consecutive days, starting 24 h post-MCAO, using a 400 ms sonication-duration train (1 ms tone bursts at 500 Hz PRF) with a 2.6 s inter-stimulus interval; the sham-tFUS (MCAO) group underwent the identical procedure with the power amplifier deactivated.

Flags from extraction

n_subjects — Paper reports a total pool of 135 rats obtained ('A total of 135 male Sprague-Dawley (SD) rats ... were obtained'; 'A total of 17 rats were excluded due to post-ischemia death, and 8 were excluded for unsuccessful ischemia induction'), but animals were then split across many sub-experiments/figures with individually reported group sizes of n=4-10 each (sham, MCAO, MCAO+tFUS, plus shRNA/vehicle arms), so the number of animals that actually received active tFUS cannot be reduced to one total from the text.
exposures[0].free_field.isppa_w_cm2 — The 5.1 W/cm2 Isppa is assumed to be a free-field (water tank) value because the Methods state acoustic parameters were 'precisely measured using an ultrasonic parameter water tank system ... and hydrophones', but the paper does not explicitly label this value as free-field or in-situ.
device.manufacturer — The transducer element is credited to ndtXducer (model 1905055), but it was integrated by the Shenzhen Institutes of Advanced Technology into a custom 'mobile ultrasound stimulation system' along with a separate signal generator (RIGOL) and power amplifier (MiniCircuits); coded as a custom/research system overall.
direction_of_effect — This study measures tFUS's anti-inflammatory/neuroprotective effect on microglial NLRP3 signaling and post-stroke behavioural recovery, not neuronal excitability directly; 'inhibitory' here refers to suppression of NLRP3-driven neuroinflammation/microglial activation rather than a measured decrease in neural firing.