Enhancement of Neurotrophic Factors in Astrocyte for Neuroprotective Effects in Brain Disorders Using Low-intensity Pulsed Ultrasound Stimulation
Feng-Yi Yang, Wen-Wei Lu, Wei-Ting Lin, Chi-Wei Chang, Sin-Luo Huang
Brain Stimulation 2015, 8, 465-473 · 10.1016/j.brs.2014.11.017
Abstract
Background Astrocytes play an important role in the growth and survival of developing neurons by secreting neurotrophic factors. Objective The goal of this study was to investigate how low-intensity pulsed ultrasound (LIPUS) stimulation directly affects brain astrocyte function. Methods Here, we report that LIPUS stimulation increased protein levels of BDNF, GDNF, VEGF, and GLUT1 in rat brain astrocytes as measured by western blot analysis. Histological outcomes including demyelination and apoptosis were examined in rats after administration of aluminum chloride (AlCl3). Results At the mechanistic level, integrin inhibitor (RGD peptide) attenuated the LIPUS-induced neurotrophic factor expression. The data suggest that neurotrophic factor protein levels may be promoted by LIPUS through activation of integrin receptor signaling. In addition, LIPUS stimulation protected cells against aluminum toxicity as demonstrated by an increase in the median lethal dose for AlCl3 from 3.77 to 6.25 mM. In in vivo histological evaluations, LIPUS significantly reduced cerebral damages in terms of myelin loss and apoptosis induced by AlCl3. Conclusions The results of this study demonstrate that transcranial LIPUS is capable of enhancing the protein levels of neurotrophic factors, which could have neuroprotective effects against neurodegenerative diseases.
Abstract via europepmc.
Exposures
Exposure 1: LIPUS sonication of cultured rat brain astrocytes (RBAC)
Target: cultured glia — “rat brain astrocyte cells (RBAC, CTX TNA2)”
Device: Olympus / Panametrics · Panametrics · A394S-SU ✓
| 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 | ✓✓✓ |
| 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 |
LIPUS was generated by a 1-MHz plane piezoelectric transducer with 50 ms burst lengths at a 50% duty cycle and a repetition frequency of 10 Hz, transmitted from the plane transducer to the bottom of the cell culture plate. Astrocyte cells were treated with multiple LIPUS stimulations by triple sonications; the duration of each sonication was 5 min with a 5 min interval between sonications, for a total sonication time of 15 min. A duty-cycle range from 0-100% was also tested for cell growth (Fig. 1A), but 50% duty cycle was used for the reported neurotrophic-factor and mechanistic experiments.
Exposure 2: LIPUS sonication of rat brain (in vivo)
Target: other — “rat brain hemisphere (3.5 mm posterior, 2.5 mm lateral to bregma)”
Device: Olympus / Panametrics · Panametrics · A392S ✓
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 1,000 | ✓✓✓ |
| Pulse duration (ms) | 50 | ✓✓✓ |
| Pulse repetition frequency (Hz) | 1 | ✓✓✓ |
| Duty cycle (%) | 5pulse duration × PRF gives 5% | ✓✓✓ |
| Sonication duration (s) | 300 | ✓✓✓ |
| Free-field pressure (kPa) | not reported | |
|---|---|---|
| Free-field Isppa (W/cm²) | not reported | |
| Free-field Ispta (W/cm²) | 0.528 | ✓✓✓ |
| 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 |
LIPUS was generated by a 1-MHz focused piezoelectric transducer with 50 ms burst lengths at a 5% duty cycle and a repetition frequency of 1 Hz, directed at a region 3.5 mm posterior and 2.5 mm lateral to bregma. Each rat hemisphere was treated with multiple LIPUS stimulations by triple sonications; the duration of each sonication was 5 min with a 5 min interval between sonications, for a total sonication time of 15 min. LIPUS was applied daily for 49 days, starting 7 days before AlCl3 administration and continuing through the 42 days of AlCl3 administration.
Flags from extraction
n_subjects— Total ultrasound-exposed animal count across sub-experiments is not stated; group sizes reported are the main AlCl3 study's two LIPUS-exposed arms (LIPUS n=6, LIPUS+AlCl3 n=6) plus a separate normal-rat western-blot pilot (Fig. 5, n=4); in vitro astrocyte culture replicate counts (typically n=4 per assay) are excluded from this animal-only count because model_system includes both rodent and in_vitro_cell.exposures[1].target— The in vivo sonicated site is described only by stereotaxic coordinates (3.5 mm posterior, 2.5 mm lateral to bregma) and as 'rat hemisphere'; no specific named brain structure (e.g., hippocampus) is stated for the sonication target itself in this paper.exposures[0].timing.duty_cycle_pct— A duty-cycle dose-response from 0-100% was tested for cell growth optimisation (Fig. 1A) but only readable from the figure; the single value (50%) used for the reported neurotrophic-factor experiments is the one taken from the text.direction_of_effect— This study reports biochemical/neuroprotective and imaging outcomes (neurotrophic factor expression, demyelination, apoptosis) rather than a direct measure of neuronal excitation or inhibition.exposures[0].in_situ— This exposure is an in vitro cell-culture (bath/dish) preparation with no skull or tissue path; in_situ is recorded as not_reported (rather than null) because the record also includes an in vivo exposure and model_system is not tissue/cell-only.