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Physical Factors Involved in Ultrasonically Induced Changes in Living Systems: I. Identification of Non-Temperature Effects

W. J. Fry, V. J. Wulff, D. Tucker, F. J. Fry

The Journal of the Acoustical Society of America 1950, 22, 867-876 · 10.1121/1.1906707

other vertebrateinvertebratehealthyinvasive electrophysiologybehaviourhistology molecular

Abstract

The results of the first step in a systematic investigation of the mechanism of the action of ultrasound on tissue are reported. The temperature changes resulting from absorption of acoustic energy were determined while irradiation was in progress. Experimental evidence is presented which demonstrates the existence of non-temperature effects in various nerve tissue preparations.

Abstract via crossref.

SpeciesRana pipiens (frog) and crayfish (species not stated)
Subjects7, 12, 50, 12, 6, 5, 5swept animals
Sessions per subjectnot reported
Randomisednot reported
Blindingnot reported
Sham / controlnone
Auditory controlnot reported
Readout timingboth
Anaesthesianot reported
Readoutsinvasive electrophysiology, behaviour, histology molecularextracellular spike-potential recording from the crayfish ventral nerve cord and frog sciatic nerve; hind-limb paralysis as a behavioural/motor endpoint; osmic-acid and thionine histology of the sciatic nerve and spinal cord.
Direction of effectmixed or unclearUltrasound reversibly depressed spontaneous spike activity in the excised crayfish ventral nerve cord (inhibitory), had no detectable effect on excitability or spike waveform in excised frog sciatic nerve, and produced irreversible paralysis with axonal and motor-neuron degeneration when applied to the intact frog spinal cord at sufficient dose (a damaging/lesioning effect not attributable to bulk heating).
Adverse eventsobservedSufficient ultrasound dose to the frog spinal cord produced permanent hind-limb paralysis and histological degeneration of sciatic-nerve axons and spinal ventral-horn motor neurons; the authors argue this is not a simple thermal effect.

Exposures

Exposure 1: Crayfish ventral abdominal nerve cord (ganglia) and walking leg nerves

Target: crayfish neurons — “ventral abdominal nerve cord (ganglia 1 and 2) and walking leg nerves of crayfish
Device: custom-built

Pulse timing
Waveformcontinuous
Fundamental frequency (kHz)975, 980swept✓✓
Pulse duration (ms)not applicable
Pulse repetition frequency (Hz)not applicable
Duty cycle (%)not applicable
Sonication duration (s)43.5✓✓
Pressure and intensity, by domain
Free-field pressure (kPa)1,013✓✓
Free-field Isppa (W/cm²)35✓✓
Free-field Ispta (W/cm²)not reported
In-situ estimatenot applicable
In-situ pressure (kPa)not applicable
In-situ Isppa (W/cm²)not applicable
In-situ Ispta (W/cm²)not applicable
Protocol, in the paper’s words

Continuous-wave ultrasound (~35 W/cm2) was applied to two adjacent ganglia and the connecting commissure of the excised ventral nerve cord; spontaneous spike frequency first increased, then decreased, then disappeared entirely after 43.5 s of exposure, recovering over about 25-30 s once the sound was switched off. Similar observations were made on six other preparations, and similar exposures of the excised walking-leg nerve gave negative results.

Exposure 2: Excised frog sciatic nerve

Target: sciatic nerve — “excised frog sciatic nerve suspended in Ringer's solution
Device: custom-built

Pulse timing
Waveformnot reported
Fundamental frequency (kHz)975, 980swept✓✓
Pulse duration (ms)not reported
Pulse repetition frequency (Hz)not reported
Duty cycle (%)not reported
Sonication duration (s)not reported
Pressure and intensity, by domain
Free-field pressure (kPa)1,013✓✓
Free-field Isppa (W/cm²)35✓✓
Free-field Ispta (W/cm²)not reported
In-situ estimatenot applicable
In-situ pressure (kPa)not applicable
In-situ Isppa (W/cm²)not applicable
In-situ Ispta (W/cm²)not applicable
Protocol, in the paper’s words

Prolonged application of ultrasound to excised frog sciatic nerves suspended in Ringer's solution and in contact with salt bridges produced no detectable change in wave form, spike magnitude or excitability, whether the sound was incident on the stimulating electrodes, the recording electrodes, or the intermediate region; no intensity or duration is given for this specific test.

Exposure 3: Intact frog, lumbar enlargement of the spinal cord sonicated through the back

Target: lumbar spinal cord — “lumbar enlargement of the spinal cord, intact frog, sound beam incident on the dorsal surface
Device: custom-built

Pulse timing
Waveformcontinuous, pulsed
Fundamental frequency (kHz)975, 980swept✓✓
Pulse duration (ms)10, 80swept✓✓
Pulse repetition frequency (Hz)2, 20swept✓✓
Duty cycle (%)not reported
Sonication duration (s)2.8, 3.3, 4.3, 7.3swept✓✓
Pressure and intensity, by domain
Free-field pressure (kPa)1,013✓✓
Free-field Isppa (W/cm²)35✓✓
Free-field Ispta (W/cm²)not reported
In-situ estimatenot reported
In-situ pressure (kPa)not reported
In-situ Isppa (W/cm²)not reported
In-situ Ispta (W/cm²)not reported
Protocol, in the paper’s words

Frogs suspended under water were exposed to a single continuous sonication of the lumbar spinal cord region: complete, permanent paralysis of the hind legs followed exposures of 4.3 s at room temperature (21-25C) or 7.3 s at 1-2C, while shorter exposures gave no or only temporary paralysis. In a separate comparison at room temperature, repetitive short pulses (0.08 s at 2/s, or 0.01 s at 20/s) were used to vary the temperature-rise time course; the slower/longer pulse train (0.08 s, 2/s) paralysed frogs despite reaching a lower peak cord temperature than the faster/shorter train (0.01 s, 20/s), which did not paralyse.

Flags from extraction

  • exposures[0].free_field.isppa_w_cm2Paper calls this the approximate intensity used ('~35 w/cm2'); domain is inferred as free-field from the paper's separate statement that 'the measured free field sound intensity at the positions used in the experiments ... is between 30 and 40 w/cm2', not stated explicitly for this experiment.
  • exposures[2].timing.waveformThe lumbar spinal cord exposure combines a continuous single-burst paralysis-threshold protocol (4.3 s / 7.3 s) with a separate pulsed temperature-matching sub-experiment (0.08 s at 2/s vs 0.01 s at 20/s); only the pulsed condition that produced paralysis (0.08 s, 2 Hz) is captured in the numeric pulse fields. Duty cycle is not stated and not computed here.
  • n_subjectsThe paper reports many different sub-group sizes across sub-experiments (e.g. 12 frogs at room temperature, 50 frogs cooled, 6 frogs for histology, 5+5 frogs for the two pulsed-duration comparisons, 7 crayfish nerve-cord preparations) without ever stating an aggregate total across the whole study.
  • subject_unitStudy mixes intact frogs (animal), excised crayfish nerve-cord/leg-nerve preparations, and excised frog sciatic nerve (preparation); 'animal' is used as the majority category.