Low-intensity pulsed ultrasound ameliorates glia-mediated inflammation and neuronal damage in experimental intracerebral hemorrhage conditions
Wei-Shen Su, Chun-Hu Wu, Wen-Shin Song, Szu-Fu Chen, Feng-Yi Yang
Journal of Translational Medicine 2023 · 10.1186/s12967-023-04377-z
Abstract
Background Intracerebral hemorrhage (ICH) is a condition associated with high morbidity and mortality, and glia-mediated inflammation is a major contributor to neurological deficits. However, there is currently no proven effective treatment for clinical ICH. Recently, low-intensity pulsed ultrasound (LIPUS), a non-invasive method, has shown potential for neuroprotection in neurodegenerative diseases. This study aimed to investigate the neuroprotective effects and potential mechanisms of LIPUS on glia-mediated inflammation in ICH. Methods This study used 289 mice to investigate the effects of LIPUS on ICH. ICH was induced by injecting bacterial collagenase (type VII-S; 0.0375 U) into the striatum of the mice. LIPUS was applied noninvasively for 3 days, including a 2-h-delayed intervention to mimic clinical usage. The study evaluated neurological function, histology, brain water content, hemoglobin content, MRI, and protein expression of neurotrophic factors, inflammatory molecules, and apoptosis. In vitro studies investigated glia-mediated inflammation by adding thrombin (10 U/mL) or conditioned media to primary and cell line cultures. The PI3K inhibitor LY294002 was used to confirm the effects of PI3K/Akt signaling after LIPUS treatment. Results LIPUS treatment improved neurological deficits and reduced tissue loss, edema, and neurodegeneration after ICH. The protective effects of LIPUS resulted from decreased glia-mediated inflammation by inhibiting PI3K/Akt-NF-κB signaling, which reduced cytokine expression and attenuated microglial activation-induced neuronal damage in vitro. Conclusions LIPUS treatment improved neurological outcomes and reduced glia-mediated inflammation by inhibiting PI3K/Akt-NF-κB signaling after ICH. LIPUS may provide a non-invasive potential management strategy for ICH.
Abstract via europepmc.
Exposures
Exposure 1: LIPUS to striatal ICH lesion (in vivo mouse)
Target: striatum — “striatum (collagenase injection/needle entry site, ICH lesion)”
Device: Mettler · Mettler Electronics · ME740 ✓
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 1,000 | ✓✓✓ |
| Pulse duration (ms) | 2 | ✓✓✓ |
| Pulse repetition frequency (Hz) | 100 | ✓✓✓ |
| Duty cycle (%) | 20pulse duration × PRF gives 20% | ✓✓✓ |
| Sonication duration (s) | 300 | ✓✓✓ |
| 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 | |
| Ispta, domain unspecified (W/cm²) | 0.528 | ✓✓✓ |
LIPUS treatment was administered either as a single 5-min sonication session or as three 5-min sonication sessions with two 5-min rest intervals in between. The transducer was applied with a 2-ms burst length, a duty cycle of 20%, and a repetition frequency of 100 Hz ± 5%. LIPUS treatment (red dash) was performed daily from D0 to D2 in the brain, and mice were sacrificed on D3 (Experiment 2); a separate cohort received a 2-h-delayed LIPUS intervention after ICH to mimic clinical usage (Experiment 3).
Exposure 2: LIPUS applied to glial/neuronal cell line and primary cultures (in vitro)
Target: cultured glia, cultured neurons — “BV2 microglia, CTX TNA2 astrocytes, Neuro-2A neurons, and primary rat microglia/astrocyte cultures”
Device: Mettler · Mettler Electronics · ME740 ✓
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 1,000 | ✓✓✓ |
| Pulse duration (ms) | 2 | ✓✓✓⚑ |
| Pulse repetition frequency (Hz) | 100 | ✓✓✓ |
| Duty cycle (%) | 20pulse duration × PRF gives 20% | ✓✓✓ |
| Sonication duration (s) | 900 | ✓✓✓⚑ |
| Free-field pressure (kPa) | not reported | |
|---|---|---|
| Free-field Isppa (W/cm²) | not reported | |
| Free-field Ispta (W/cm²) | not reported | |
| 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 |
During the in vitro study, LIPUS was applied by sonication from the bottom of the culture plate to stimulate the cells. After thrombin/conditioned-media induction, BV2/TNA2/N2A cell-line cultures received 15-min LIPUS treatment and were measured at 24 h or 48 h; primary microglia/astrocyte cultures were stimulated with thrombin in the absence or presence of LIPUS treatment and harvested at 1, 3, or 24 h (durations for the primary-culture experiments are not separately restated). Ultrasound coupling gel was used between the transducer and the culture plate.
Flags from extraction
n_subjects— Paper states a study-wide total of 289 mice across sham/ICH/ICH+LIPUS arms and multiple sub-experiments with varying group sizes (e.g. n=7-9/group, n=8/group, n=7/group, n=6-8/group) but never gives a single total specific to LIPUS-exposed animals, so n_subjects is left not_reported.subject_unit— Study combines in vivo mouse (animal) work with in vitro cell-line and primary rat culture (culture) work; subject_unit/n_sessions_per_subject given here reflect only the in vivo animal arm.exposures[1].timing.pulse_duration_ms— In vitro sonication parameters (burst length, duty cycle, PRF) are not explicitly restated for the cell-culture experiments; assumed but not confirmed to use the same ME740 apparatus settings as the in vivo work, so left not_reported.exposures[1].unspecified_domain.ispta_w_cm2— The only in vitro intensity value given (ISPTA of 30 mW/cm2) is attributed to 'our previous study' as background safety context, not stated as the intensity actually used in this paper's in vitro experiments, so was not recorded as this exposure's value.exposures[1].timing.sonication_duration_s— 15-min duration explicitly stated only for the cell-line (BV2/TNA2/N2A) experiment; primary microglia/astrocyte culture experiments do not restate a duration.