Ultrasound Used for Diagnostic Imaging Facilitates Dendritic Branching of Developing Neurons in the Mouse Cortex
Tamas Papp, Zsuzsanna Ferenczi, Bernadette Szilagyi, Matyas Petro, Angelika Varga, Eva Kókai, Ervin Berenyi, Gabor Olah, Gabor Halmos, Peter Szucs, Zoltan Meszar
Frontiers in Neuroscience 2022, 16 · 10.3389/fnins.2022.803356
Abstract
Neuronal differentiation and synaptogenesis are regulated by precise orchestration of intrinsic and extrinsic chemical and mechanical factors throughout all developmental steps critical for the assembly of neurons into functional circuits. While ultrasound is known to alter neuronal migration and activity acutely, its chronic effect on neuronal behavior or morphology is not well characterized. Furthermore, higher-frequency (3-5 MHz) ultrasound (HFU) is extensively used in gynecological practice for imaging, and while it has not been shown harmful for the developing brain, it might be associated with mild alterations that may have functional consequences. To shed light on the neurobiological effects of HFU on the developing brain, we examined cortical pyramidal cell morphology in a transgenic mouse model, following a single and short dose of high-frequency ultrasound. Layer V neurons in the retrosplenial cortex of mouse embryos were labeled with green and red fluorescent proteins by in utero electroporation at the time of their appearance (E14.5). At the time of their presumptive arrival to layer V (E18.5), HFU stimulation was performed with parameters matched to those used in human prenatal examinations. On the third postnatal day (P3), basic morphometric analyses were performed on labeled neurons reconstructed with Neurolucida. Low-intensity HFU-treated cells showed significantly increased dendritic branching compared to control (non-stimulated) neurons and showed elevated c-fos immunoreactivity. Labeled neurons were immunopositive for the mechanosensitive receptor TRPC4 at E18.5, suggesting the role of this receptor and the associated signaling pathways in the effects of HFU stimulation.
Abstract via europepmc.
Exposures
Exposure 1: Diagnostic ultrasound exposure of pregnant mice / pups (retrosplenial cortex neurons analysed)
Target: retrosplenial cortex — “retrosplenial cortex”
Device: other named manufacturer · GE · Logiq V2 (4C-RS convex probe) ✓
| Waveform | not reported | |
|---|---|---|
| Fundamental frequency (kHz) | 3,000 | ✓✓✓ |
| Pulse duration (ms) | not reported | |
| Pulse repetition frequency (Hz) | not reported | |
| Duty cycle (%) | not reported | |
| Sonication duration (s) | 600 | ✓✓✓ |
| 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 | |
| Pressure, domain unspecified (kPa) | 1,557 | ✓✓✓⚑ |
| Ispta, domain unspecified (W/cm²) | 0.056 | ✓✓✓⚑ |
Mice previously subjected to in utero electroporation were deeply anesthetized and received a single 10-minute abdominal diagnostic ultrasound exposure (GE Logiq V2, 4C-RS convex probe, 3 MHz, MI=0.9, TIS=0.8) at embryonic day 18.5 (E18.5); the probe covered the whole abdominal region so all embryos received the same dose. After birth, unfocused ultrasound was applied directly to the skull. In a separate cohort, the E18.5 exposure was followed by 4 further weekly ultrasound stimuli during the first 4 postnatal weeks (5 exposures total). Sham/control animals underwent identical handling with the transducer pressed to the abdomen but without actual ultrasound output.
Flags from extraction
exposures[0].target.terms— Ultrasound was applied to the whole maternal abdomen (unfocused with respect to the fetal brain); retrosplenial cortex was the region subsequently analysed for morphological effects, not a spatially targeted stimulation site.exposures[0].unspecified_domain.ispta_w_cm2— Paper states a single 'calculated average intensity' (56 mW/cm2) without specifying whether this is spatial-peak pulse-average (Isppa) or spatial-peak temporal-average (Ispta) intensity, or the domain (water/free-field vs in situ); recorded as Ispta by convention and flagged.exposures[0].unspecified_domain.pressure_kpa— Domain (free-field vs in situ) not stated by the paper for this 'calculated peak negative acoustic pressure'; placed in unspecified_domain per the three-way rule.n_subjects— Paper gives separate group sizes for the electroporation/morphometry cohort (24 embryos enrolled, real US) and a separate qRT-PCR cohort (9 pups, real US); recorded as a list per SCHEMA.md guidance. An additional 'five times repetitive' subgroup exists but its own N is not stated, so it is not included in this list.n_sessions_per_subject— Most animals received a single 10-min exposure at E18.5, but a separate repetitive-exposure subgroup received 5 total exposures (E18.5 plus 4 further weekly doses); no single value applies to the whole study.adverse_events— Paper reports no gross morphological changes in most dendritic parameters and intact cortical layering, but does not make an explicit overall safety/adverse-event statement for the animals or fetuses.