Low-Intensity Ultrasound Reduces Brain Infarct Size by Upregulating Phosphorylated Endothelial Nitric Oxide in Mouse Model of Middle Cerebral Artery Occlusion
Catherine M. Davis, Azzdine Y. Ammi, Wenbin Zhu, Carmen Methner, Zhiping Cao, David Giraud, Nabil J. Alkayed, Randy L. Woltjer, Sanjiv Kaul
Ultrasound in Medicine & Biology 2023 · 10.1016/j.ultrasmedbio.2022.12.008
Abstract
Objective There have been attempts to use therapeutic ultrasound (US) for the treatment of both experimental and clinical stroke. We hypothesized that low-intensity US has direct beneficial effects on the brain independent of cerebral blood flow (CBF) during middle cerebral artery occlusion (MCAO). Methods Three groups of mice were studied. Group I included 84 mice with MCAO undergoing US treatment/no treatment at two US frequencies (0.25 and 1.05 MHz) with three different acoustic pressures at each frequency in which infarct size (IS) was measured 24 h later. Group II included 11 mice undergoing treatment based on best US results from group I animals in which the IS/risk area (RA) ratio was measured 24 h later. Group III included 38 normal mice undergoing US treatment/no treatment for assessment of CBF, tissue metabolite and protein expression and histopathology. Discussion Ultrasound at both frequencies and most acoustic pressures resulted in reduction in IS in group I animals, with the best results obtained with 0.25 MHz at 2.0 MPa: IS was reduced 4-fold in the cerebral cortex, 1.5-fold in the caudate putamen and 3.5-fold in the cerebral hemisphere compared with control. US application in group III animals elicited only a marginal increase in CBF despite a 2.6-fold increase in phosphorylated endothelial nitric oxide synthase (p-eNOS)-S1177 and a corresponding decrease in p-eNOS-T494. Histopathology revealed no evidence of hemorrhage, inflammation or necrosis. Conclusion Low-intensity US at specific frequencies and acoustic pressures results in marked neuroprotection in a mouse model of stroke by modulation of p-eNOS independent of its effect on CBF.
Abstract via europepmc.
Exposures
Exposure 1: 250 kHz US during MCAO
Target: brain — “region of the mouse brain covered by the beam (right side of head, frontal and parietal acoustic window)”
Device: Sonic Concepts · Sonic Concepts · H-117 ✓
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 250 | ✓✓✓ |
| Pulse duration (ms) | 0.008 | ✓✓✓⚑ |
| Pulse repetition frequency (Hz) | 25 | ✓✓✓ |
| Duty cycle (%) | 0.02pulse duration × PRF gives 0.02% | ✓✓✓ |
| Sonication duration (s) | 1,800 | ✓✓✓ |
| Free-field pressure (kPa) | 1,200, 2,000, 2,600swept | ✓✓✓ |
|---|---|---|
| Free-field Isppa (W/cm²) | 18.6, 52.3, 84.8swept | ✓✓✓ |
| Free-field Ispta (W/cm²) | 0.0074, 0.0209, 0.0339swept | ✓✓✓ |
| 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 |
Single 30 min exposure started 15 min after MCAO onset; 2-cycle pulses at 25 Hz PRF; three peak rarefactional pressures per frequency in separate animal groups.
Exposure 2: 1050 kHz US during MCAO
Target: brain — “region of the mouse brain covered by the beam (right side of head, frontal and parietal acoustic window)”
Device: Sonic Concepts · Sonic Concepts · H-102 ✓
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 1,050 | ✓✓✓ |
| Pulse duration (ms) | 0.00476 | ✓✓✓ |
| Pulse repetition frequency (Hz) | 50 | ✓✓✓ |
| Duty cycle (%) | 0.02pulse duration × PRF gives 0.024%, which disagrees with the stated value | ✓✓✓ |
| Sonication duration (s) | 1,800 | ✓✓✓ |
| Free-field pressure (kPa) | 1,000, 2,000, 4,750swept | ✓✓✓ |
|---|---|---|
| Free-field Isppa (W/cm²) | 23.1, 102.7, 914.1swept | ✓✓✓ |
| Free-field Ispta (W/cm²) | 0.0073, 0.0327, 0.291swept | ✓✓✓ |
| 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 |
Single 30 min exposure started 15 min after MCAO onset; 5-cycle pulses at 50 Hz PRF; three peak rarefactional pressures per frequency in separate animal groups.
Flags from extraction
n_subjects— 133 is the total of completed mice including untreated controls; the number exposed to US is not stated as a total.exposures[0].unspecified_domain.pressure_kpa— Peak negative pressure (PRAPA); only the 2 MPa beam is said to be measured in water (Figure 2), so all values kept as unspecified domain.exposures[1].unspecified_domain.pressure_kpa— Table 1 gives 4.75 MPa, Table 2 and text give 4.7/4.75 MPa; Table 1 value recorded.exposures[0].timing.pulse_duration_ms— Pulse given in cycles (2 at 0.25 MHz, 5 at 1.05 MHz), not in ms; left not_reported.exposures[0].target— Target is the brain region covered by the beam (7 mm at 0.25 MHz, 2 mm at 1.05 MHz); infarct assessed in cortex, caudate putamen and hemisphere.sham_type— Group III normal mice had 'sham' treatment; how sham was produced is not described. Group I/II controls were untreated.exposures[0].unspecified_domain.ispta_w_cm2— Table gives mW/cm2; converted to W/cm2.