Transcranial Low-Intensity Pulsed Ultrasound Stimulation Induces Neuronal Autophagy

Autophagy, or cellular self-digestion, is an essential process for eliminating abnormal protein in mammalian cells. Accumulating evidence indicates that increased neuronal autophagy has a protective effect on neurodegenerative disorders. It has been reported that low-intensity pulsed ultrasound (LIPUS) can noninvasively modulate neural activity in the brain. Yet, the effect of LIPUS on neuronal autophagy is still unclear. The objective of this study was to examine whether LIPUS stimulation could induce neuronal autophagy. Primary neurons were treated by LIPUS with a frequency of 0.68 MHz, a pulse repetition frequency (PRF) of 500 Hz, a spatial peak temporal-average intensities (I _SPTA ) of 70 and 165 mW/cm ² . Then, the immunofluorescent analysis of LC3B was carried out for evaluating neuronal autophagy. Furthermore, 0.5-MHz LIPUS was noninvasively delivered to the cortex and hippocampus of adult mice (n = 16) with PRF of 500 Hz and I _SPTA of 235 mW/cm ² . The LC3BII/LC3BI ratio and p62 (autophagic markers) were measured by western blot analysis. In the in vitro study, the expression of LC3B in primary neurons was statistically improved after LIPUS stimulation was implemented for 4 h (p <; 0.01). With the increase in the irradiation duration or acoustic intensity of LIPUS stimulation, the expression of LC3B in primary neurons was increased. Furthermore, transcranial LIPUS stimulation increased the LC3BII/LC3BI ratio (p <; 0.05) and decreased the expression of p62 (p <; 0.05) in the cortex and hippocampus.We concluded that LIPUS provides a safe and capable tool for activating neuronal autophagy in vitro and in vivo.vivo.