Auditory independent low-intensity ultrasound stimulation of mouse brain is associated with neuronal ERK phosphorylation and an increase of Tbr2 marked neuroprogenitors
Jormay Lim, Ya-Cherng Chu, Hsiao-Hsin Tai, Andy Chien, Shao-Shiang Huang, Chih-Cheng Chen, Jaw-Lin Wang
Biochemical and Biophysical Research Communications 2022, 613, 113-119 · 10.1016/j.bbrc.2022.04.123
Abstract
Transcranial ultrasound stimulation is an emerging technique for the development of a non-invasive neuromodulation device for the treatment of various types of neurodegenerations and brain damages. However, there are very few studies that have quantified the optimal ultrasound dosage and the long-term associated effects of transcranial ultrasound treatments of brain diseases. In this study, we used a simple ex vivo hippocampal tissues stimulated by different dosages of ultrasound in combination with different chemical treatments to quantify the required energy for a measurable effect. After determining the most desirable ex vivo stimulation conditions, it was then replicated for the in vivo mouse brains. It was discovered that transcranial ultrasound promoted the increase of Tbr2-expressing neural progenitors in an ASIC1a-dependent manner. Furthermore, such effect was observable at least a week after the initial ultrasound treatments and was not abolished by auditory toxicity.
Abstract via europepmc.
Exposures
Exposure 1: Ex vivo hippocampal tissue and cultured primary neurons sonicated in a water chamber (dose-response for p-ERK)
Target: hippocampus, cultured neurons — “freshly isolated neonatal mouse hippocampus and primary cultured neurons, sonicated via a water chamber”
Device: not reported · 1 MHz disc-type transducer driven via a function generator (Tektronix AFG1022) and power amplifier (E&I 210L) ✓
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 1,000 | ✓✓✓ |
| Pulse duration (ms) | not reported | |
| Pulse repetition frequency (Hz) | not reported | |
| Duty cycle (%) | 1, 0.5, 0.1swept | ✓✓✓ |
| Sonication duration (s) | 60, 300swept | ✓✓✓ |
| Free-field pressure (kPa) | 7.2, 50swept | ✓✓✓ |
|---|---|---|
| Free-field Isppa (W/cm²) | not reported | |
| Free-field Ispta (W/cm²) | 0.0001, 0.0069swept | ✓✓✓ |
| 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 |
Input voltage was varied from 100 to 900 mVpp with different duty cycles to assess dose-dependence. A 1-min stimulation at 900 mVpp/1% DC and a 5-min stimulation at 100 mVpp/0.5% DC were the minimal settings found to give a reproducible p-ERK response; duty cycle was also lowered stepwise from 1% to 0.5% and 0.1% at fixed voltage to find the minimal effective dose. Hippocampus tissue was either sham-treated, PMA-treated (positive control for p-ERK), or ultrasound-treated; pharmacological blockers (EGTA, Gadolinium, PcTx-1, Xestospongin C) were applied prior to sonication in separate experiments to probe the mechanotransduction pathway.
Exposure 2: Transcranial ultrasound stimulation of mouse brain in vivo (dentate gyrus neuroprogenitor and trigeminal ganglion effects)
Target: whole brain or unfocused — “mouse head (transcranial, non-focused disc transducer); downstream effects assessed in hippocampal dentate gyrus, cortex, and trigeminal ganglion”
Device: Olympus / Panametrics · Olympus · C539-SM ✓
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 1,000 | ✓✓✓ |
| Pulse duration (ms) | not reported | |
| Pulse repetition frequency (Hz) | not reported | |
| Duty cycle (%) | 1 | ✓✓✓ |
| Sonication duration (s) | not reported |
| Free-field pressure (kPa) | not reported | |
|---|---|---|
| Free-field Isppa (W/cm²) | not reported | |
| 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 |
In vivo mice were stimulated with a commercial 1 MHz transducer using an input parameter of 900 mVpp, 1% duty cycle (estimated from the ex vivo dose-response study). For the DCX/Tbr2 neurogenesis experiment, 5-6 week-old wild-type, Asic1a-/-, or Asic3-/- mice were randomly allocated to sham or ultrasound-treated groups and given 3 days of treatment, then perfusion-fixed at day 7. In a separate ototoxicity experiment, mice were given auditory toxins (kanamycin then furosemide) or saline 2 h before a single ultrasound stimulation session, followed by p-ERK immunohistochemistry; trigeminal ganglia were also collected from stimulated and control wild-type and Asic3-/- mice to test involvement of meningeal/dural nerve pathways.
Flags from extraction
n_subjects— The paper reports several separate cohorts (ex vivo hippocampus pups, cultured neurons, in vivo WT/Asic1a-/-/Asic3-/- mice for DCX/Tbr2, a 3-mouse ototoxicity experiment, and trigeminal ganglion collection) without ever stating a single total N.n_sessions_per_subject— The 3-day treatment schedule applies to the DCX/Tbr2 in vivo cohort; other sub-experiments (e.g. the ototoxicity/p-ERK experiment) describe a single ultrasound session, so this value does not apply uniformly to all animals in the paper.exposures[0].unspecified_domain.pressure_kpa— Domain is ambiguous: pressure/intensity were measured 'inside the culture dish' containing the ex vivo/in vitro tissue itself (no skull), which could be read as either a free-field water measurement or the dose delivered at the target; placed in unspecified_domain rather than guessing.sham_type— Paper states tissue/animals were 'sham treated' or allocated to 'sham control' without describing the sham mechanism (e.g. transducer off, blocked, or aimed away).exposures[1].target— The in vivo transducer is described only as being used 'for mouse brain stimulation' without a stated focal target or skull location; classified as whole_brain_or_unfocused since a non-focused disc transducer was used and effects were assessed in hippocampus, cortex, and (via meningeal innervation) trigeminal ganglion.