Abstract

Recent advances in transcranial ultrasound stimulation (TUS) pulsed at 40 Hz have demonstrated the potential to ameliorate cognitive deficits in mouse models of Alzheimer’s disease. However, technical barriers remain as general anesthesia is required for mice, which restricts the accurate elucidation of biological mechanisms and behavioral effects under awake physiological conditions. Here, we report a wearable, free-moving ultrasound stimulation system that delivers TUS pulsed at 40 Hz to female 5xFAD transgenic mice to systematically evaluate the behavioral outcomes and underlying mechanistic pathways. Among the treatment groups, a 14-d regimen at an acoustic intensity of 2.14 W/cm 2 yielded the optimal cognitive outcome in Alzheimer’s disease mice, which was consistently verified across Y-maze and Morris water maze tests. Additionally, this group showed reduced Aβ plaque deposition and increased plaque-associated microglial activity. Furthermore, enhanced gamma oscillations in the hippocampus were detected following treatment. RNA sequencing revealed modulation of innate immune and inflammatory pathways. Corresponding molecular analysis demonstrated a marked down-regulation in RIPK1 , phosphorylated NF-κB , and necroptosis markers, alongside reductions in key pro-inflammatory cytokines ( IL-6 , IL-1β , and TNF-α ). Collectively, our findings suggest that the cognitive improvement observed after treatment with TUS pulsed at 40 Hz may be linked to the modulation of neuroinflammatory and necroptotic pathways, possibly involving RIPK1 / NF-κB signaling.