Ultrasound Enhances the Expression of Brain-Derived Neurotrophic Factor in Astrocyte Through Activation of TrkB-Akt and Calcium-CaMK Signaling Pathways

Shing-Hwa Liu, Yi-Long Lai, Bo-Lin Chen, Feng-Yi Yang

Cerebral Cortex 2016, bhw169 · 10.1093/cercor/bhw169

in vitro cellhealthyhistology molecular

Abstract

Low-intensity pulsed ultrasound (LIPUS) stimulation has been shown to increase the expression of brain-derived neurotrophic factor (BDNF) in astrocytes of an in vitro model and rat brains of an in vivo model; however, their molecular mechanisms are still not well clarified. Here, we investigated the underlying mechanisms of BDNF enhancement by LIPUS in rat cerebral cortex astrocytes. After LIPUS stimulation in astrocytes, the protein and mRNA expressions were measured by western blot and RT-PCR, respectively. The concentration of intracellular calcium was determined spectrophotometrically. The results showed that LIPUS enhanced the phosphorylation of tropomyosin-related kinase B (TrkB) and Akt but had no effect on Erk1/2 phosphorylation. Additionally, LIPUS increased the intracellular concentration of calcium and enhanced the protein levels of calmodulin-dependent kinase (CaMK) II and CaMKIV. LIPUS also activated the phosphorylation of NF-κB-p65 but did not promote the activation of cAMP response element-binding protein (CREB). Taken together, our results suggest that LIPUS stimulation upregulates BDNF production in astrocytes through the activation of NF-κB via the TrkB/PI3K/Akt and calcium/CaMK signaling pathways. BDNF has emerged as a major molecular player in the regulation of neural circuit development and function. Therefore, LIPUS stimulation may play a crucial and beneficial role in neurodegenerative diseases.

Abstract via europepmc.

Speciesrat

Subjects4 cultures

Sessions per subjectnot applicable

Randomisednot applicable

Blindingnot applicable

Sham / controlnone

Auditory controlnot reported

Readout timingoffline

Anaesthesianot applicable

Readoutshistology molecular — Western blotting (p-TrkB, p-Akt, p-Erk1/2, PLCγ, CaMKII, CaMKIV, p-CREB, p-NF-κB p65, BDNF), quantitative real-time RT-PCR (BDNF, c-Fos mRNA), spectrophotometric intracellular calcium assay, MTT cell-growth assay

Direction of effectnot assessedLIPUS increased phosphorylation of TrkB and Akt, PLCγ protein, intracellular calcium, CaMKII/CaMKIV protein, and NF-κB p65 phosphorylation, leading to increased BDNF mRNA/protein in cultured astrocytes; no change in Erk1/2 or CREB phosphorylation. Calcium chelation (BAPTA-AM), Akt inhibition (MK2206) and NF-κB inhibition (QNZ) each blocked the BDNF increase.

Adverse eventsnot applicable

Exposures

Exposure 1: LIPUS stimulation of cultured rat cerebral cortex astrocytes

Target: cultured glia — “rat cerebral cortex astrocytes”
Device: Olympus / Panametrics · Panametrics · A394S-SU ✓

Pulse timing
Waveform	pulsed
Fundamental frequency (kHz)	1,000	✓✓✓
Pulse duration (ms)	50	✓✓✓
Pulse repetition frequency (Hz)	10	✓✓✓
Duty cycle (%)	50pulse duration × PRF gives 50%	✓✓✓
Sonication duration (s)	300	✓✓✓

Pressure and intensity, by domain
Free-field pressure (kPa)	not reported
Free-field Isppa (W/cm²)	not reported
Free-field Ispta (W/cm²)	0.11	✓✓✓
In-situ estimate	not applicable
In-situ pressure (kPa)	not applicable
In-situ Isppa (W/cm²)	not applicable
In-situ Ispta (W/cm²)	not applicable

Protocol, in the paper’s words

Each sample of astrocyte cells was treated with multiple LIPUS stimulations by triple sonication. The duration of each sonication was 5 min, and there was an interval of 5 min between the first and second and between the second and third sonications. The total sonication time of LIPUS stimulation was 15 min.

Flags from extraction

n_subjects — n=4 is the number of independent culture replicates reported consistently across figures (means ± SEM, n=4); paper does not separately state a total number of biological replicates.