Induction of a torpor-like hypothermic and hypometabolic state in rodents by ultrasound
Yaoheng Yang, Jinyun Yuan, Rachael L. Field, Dezhuang Ye, Zhongtao Hu, Kevin Xu, Lu Xu, Yan Gong, Yimei Yue, Alexxai V. Kravitz, Michael R. Bruchas, Jianmin Cui, Jonathan R. Brestoff, Hong Chen
Nature Metabolism 2023, 5, 789-803 · 10.1038/s42255-023-00804-z
Abstract
Torpor is an energy-conserving state in which animals dramatically decrease their metabolic rate and body temperature to survive harsh environmental conditions. Here, we report the noninvasive, precise and safe induction of a torpor-like hypothermic and hypometabolic state in rodents by remote transcranial ultrasound stimulation at the hypothalamus preoptic area (POA). We achieve a long-lasting (>24 h) torpor-like state in mice via closed-loop feedback control of ultrasound stimulation with automated detection of body temperature. Ultrasound-induced hypothermia and hypometabolism (UIH) is triggered by activation of POA neurons, involves the dorsomedial hypothalamus as a downstream brain region and subsequent inhibition of thermogenic brown adipose tissue. Single-nucleus RNA-sequencing of POA neurons reveals TRPM2 as an ultrasound-sensitive ion channel, the knockdown of which suppresses UIH. We also demonstrate that UIH is feasible in a non-torpid animal, the rat. Our findings establish UIH as a promising technology for the noninvasive and safe induction of a torpor-like state.
Abstract via europepmc.
Exposures
Exposure 1: Ultrasound-induced hypothermia/hypometabolism (UIH), mouse POA, 3.2 MHz
Target: hypothalamus — “hypothalamus preoptic area (POA)”
Device: custom-built
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 3,200 | ✓✓✓ |
| Pulse duration (ms) | 50 | ✓✓✓ |
| Pulse repetition frequency (Hz) | 10 | ✓✓✓ |
| Duty cycle (%) | 50pulse duration × PRF gives 50% | ✓✓✓ |
| Sonication duration (s) | 2, 10swept | ✓✓✓ |
| Free-field pressure (kPa) | not reported | |
|---|---|---|
| Free-field Isppa (W/cm²) | not reported | |
| Free-field Ispta (W/cm²) | not reported | |
| In-situ estimate | measurementsingle value | |
| In-situ pressure (kPa) | 400, 800, 1,200, 1,600swept | ✓✓✓ |
| In-situ Isppa (W/cm²) | not reported | |
| In-situ Ispta (W/cm²) | not reported |
Default protocol: peak negative in-situ pressure 1.6 MPa, duty cycle 50%, PRF 10 Hz, stimulus duration 10 s, inter-stimulus interval 30 s, 6 stimuli total, via a wearable transducer glued to the mouse skull over the POA. A gaussian envelope was applied to pulse onset/offset to reduce broadband auditory activation. In a parameter study, stimulus duration (2-10 s) and acoustic pressure (0.4, 0.8, 1.2, 1.6 MPa) were varied independently while other parameters were held constant. A closed-loop feedback controller turned ultrasound on/off (same parameters, 20 s inter-stimulus interval) to hold core temperature below 34 degC for approximately 24 h continuously, with pressure increased 10% and stimulus number increased to 8 after 12 h to compensate for gel attenuation.
Exposure 2: Ultrasound-induced hypothermia (UIH), rat POA, 1.5 MHz
Target: hypothalamus — “hypothalamus preoptic area (POA)”
Device: custom-built
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 1,500 | ✓✓✓ |
| Pulse duration (ms) | 50 | ✓✓✓⚑ |
| Pulse repetition frequency (Hz) | 10 | ✓✓✓ |
| Duty cycle (%) | 50pulse duration × PRF gives 50% | ✓✓✓ |
| Sonication duration (s) | 10 | ✓✓✓ |
| Free-field pressure (kPa) | not reported | |
|---|---|---|
| Free-field Isppa (W/cm²) | not reported | |
| Free-field Ispta (W/cm²) | not reported | |
| In-situ estimate | measurementsingle value | |
| In-situ pressure (kPa) | 1,400 | ✓✓✓ |
| In-situ Isppa (W/cm²) | not reported | |
| In-situ Ispta (W/cm²) | not reported |
Same wearable-transducer protocol as in mice (duty cycle 50%, PRF 10 Hz, stimulus duration 10 s, inter-stimulus interval 30 s), scaled to a 1.5 MHz transducer and a peak negative in-situ pressure of 1.4 MPa, with 20 stimuli delivered to freely-moving rats.
Exposure 3: In vitro TRPM2 activation by ultrasound in HEK293T cells
Target: other — “HEK293T cells overexpressing TRPM2 (in vitro)”
Device: custom-built
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 1,700 | ✓✓✓ |
| Pulse duration (ms) | not reportedimplied by duty cycle ÷ PRF: 40 ms (not stated by the paper) | ⚑ |
| Pulse repetition frequency (Hz) | 10 | ✓✓✓ |
| Duty cycle (%) | 40 | ✓✓✓ |
| Sonication duration (s) | 60 | ✓✓✓ |
| 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 | |
| Pressure, domain unspecified (kPa) | 1,000 | ✓✓✓ |
Cultured HEK293T cells overexpressing TRPM2 were sonicated with a customized in vitro US setup (different transducer from the in vivo experiments) 30 s after the start of Ca2+ imaging, to test direct ultrasound sensitivity of the TRPM2 channel; a subset of wells received the TRPM2 blocker 2-APB before sonication.
Flags from extraction
n_subjects— Dozens of separate cohorts (mice, UCP1-KO mice, rats, in vitro cells) with different, non-overlapping n reported per assay across the paper; no single study-wide total of subjects exposed to ultrasound is given.n_sessions_per_subject— Most animals received a single acute stimulation session, but the closed-loop feedback experiment delivered many intermittent stimuli continuously over ~24 h; no single value applies across the study.exposures[1].timing.pulse_duration_ms— The 50 ms pulse duration is explicitly stated only in the context of the mouse experiment (Fig. 1c); it is assumed to also apply to rats because duty cycle (50%) and PRF (10 Hz) are stated to be identical for mice and rats, but this is not separately confirmed in the text for rats.exposures[2].timing.pulse_duration_ms— Pulse duration for the in vitro TRPM2 experiment is not stated directly; only duty cycle (40%) and PRF (10 Hz) are given, and pulse duration was not computed from these per instructions.anaesthesia— Ultrasound stimulation itself was delivered to awake, freely-moving animals; brief isoflurane anaesthesia (1-1.5%) was used only for surgery and daily transducer attachment (<=15 min), not during stimulation/testing.sham_type— The exact mechanism of the 'US-' (no ultrasound) control condition (e.g., whether a transducer was worn but inactive) is not explicitly described.