Impact of Therapeutic Ultrasound on Metabolic Hormone Release From Thyroid Gland

Abstract

Objectives

This study focused on determining if ultrasound therapy could stimulate and control the function of the thyroid gland, specifically the synthesis and release of metabolic hormones thyroxine (T4) and triiodothyronine (T3) in a physiologically relevant ex vivo model.

Methods

Excised thyroid glands obtained from healthy adult Sprague–Dawley rats were diced into sections and placed in wells containing 1 mL of Krebs Ringer Buffer with 5 mmol glucose. Continuous, unfocused ultrasound was applied at 1 W/cm² for 5 minutes at frequencies of 600 kHz, 800 kHz, and 1 MHz using a circular planar transducer positioned at the near field–far field transition distance. Samples were collected at time points 0, 5, and 35 minutes after ultrasound sonication. T3 and T4 concentrations were analyzed using competitive enzyme‐linked immunosorbent assays, and histological examination was performed using hematoxylin and eosin staining.

Results

There was no statistically significant difference in T4 or T3 release between the ultrasound treated and sham treated samples at any combination of frequency and time point tested (p > .05 for all comparisons). Histological analysis revealed no structural abnormalities or gross microscopic changes in ultrasound‐treated samples.

Conclusions

Unlike previous studies demonstrating ultrasound‐stimulated insulin release from pancreatic beta cells, therapeutic ultrasound at the tested parameters did not stimulate T3 or T4 release from ex vivo thyroid tissue.

Abbreviation

ELISA

enzyme‐linked immunosorbent assay

The thyroid gland is a critical part of the endocrine system and is responsible for development, activation, and regulation of thyroid hormones and iodine homeostasis. ¹ Thyroid hormones occur primarily in 2 forms, thyroxine (T4) as the inactive thyroid hormone and triiodothyronine (T3) as the active thyroid hormone. Thyroid hormone production is approximately 90% T4 production and 10% T3 production. ¹ These thyroid hormones affect nearly all body systems and act to regulate metabolism, stimulate cellular respiration, and maintain cardiovascular function. ² , ³ , ⁴

Thyroid disease is the second most common disease state in the endocrine system, after diabetes mellitus. ⁵ Dysfunction of the thyroid and thyroid hormone release has an impact on most organ systems of the body. ⁶ The 2 major thyroid disorders are hyperthyroidism and hypothyroidism. Hyperthyroidism, a condition where there is too much thyroid hormone production, can cause increased metabolism, heart rate, and cellular respiration, and affects approximately 1.2% of the US population. ⁷ , ⁸ , ⁹ Hypothyroidism, abnormally low thyroid gland activity and hormone production, affects approximately 5% of the US population and is one of the most prevalent endocrine diseases worldwide. ¹⁰ , ¹¹

The use of ultrasound for therapeutic medical applications has a long and varied history. In physical therapy applications, ultrasound can be used to reduce soft tissue inflammation, augment scar tissue remodeling, and reduce pain. ¹² Therapeutic ultrasound has also been extensively studied for the treatment of kidney stones and there are ongoing clinical trials to investigate ultrasound‐based propulsion of kidney stones. ¹³ Applications of therapeutic ultrasound continue to expand beyond traditional physical therapy uses, with applications now including the removal of cataracts, ¹⁴ fracture healing, ¹⁵ and drug delivery. ¹⁶

More recently, therapeutic ultrasound has been explored as a modality for neuromodulation, demonstrating its ability to influence both neural activity and downstream physiological processes. ¹⁷ Emerging evidence also suggests that ultrasound‐mediated neuromodulation may impact endocrine function, either directly through tissue interactions or indirectly via neural and neuroendocrine pathways. ¹⁸ Ultrasound stimulation has been shown to influence pancreatic endocrine function through mechanical stimulation of beta cells, suggesting a potential noninvasive method for hormone secretion modulation in endocrine tissues.

Previous studies in our laboratory have demonstrated that ultrasound therapy can trigger insulin release from rodent pancreatic beta cells. ¹⁹ In vitro studies were conducted to determine the effects of ultrasound on rat INS 823/13 beta cells to determine insulin release via an insulin enzyme‐linked immunosorbent assay (ELISA), and a significant release of insulin was observed after cell exposure to 800 kHz ultrasound application and 1 MHz at an intensity of 1 W/cm². ²⁰ These studies allowed for investigation of the translational potential of therapeutic ultrasound application for treatment of early‐stage type 2 diabetes.

Given the successful stimulation of insulin release from pancreatic beta cells, we hypothesized that ultrasound therapy might similarly stimulate hormone secretion from other endocrine tissues. The thyroid gland was selected due to its similar characteristics as a hormone‐secreting endocrine organ, its superficial anatomical location that facilitates ultrasound delivery, and the clinical relevance of modulating thyroid hormone levels in disease states. The goal of this study was to determine whether ultrasound stimulation affects metabolic hormone release from thyroid tissue.

Thyroid hormone regulation differs fundamentally from pancreatic insulin secretion. Thyroid hormones are synthesized and stored extracellularly within the follicular colloid and are released though a multistep process that involves endocytosis, lysosomal processing, and membrane transportation. ²¹ As a result, thyroid hormone release typically occurs on longer time scales than insulin secretion from pancreatic beta cells.

Accordingly, the present study tests a constrained hypothesis that therapeutic ultrasound stimulation can induce acute changes in thyroid hormones T3 and T4 release from excised thyroid tissue over short post‐stimulation time scales. This experimental design isolates direct ultrasound‐tissue interactions in an ex vivo model, excluding systemic regulatory feedback such as hypothalamic–pituitary thyroid signaling. ²²

Materials and Methods

Tissue Preparation

Excised thyroid glands were obtained from healthy adult Sprague–Dawley rats aged 8–10 weeks of both sexes from Charles River Laboratories (Germantown, MD) and delivered overnight in ice‐cold Dulbecco's modified solution. As the excised thyroid glands were obtained from a commercial supplier, this experimentation did not require ethical approval or informed consent processes. The average thyroid gland mass varied by sex, with female glands averaging 17 mg and male glands averaging 22 mg.

The thyroid glands were maintained at 4°C after delivery and were processed within 3 hours of delivery. Tissue preparation was performed under sterile conditions, with each whole thyroid gland first rinsed 3 times in ice‐cold phosphate buffered saline (pH 7.4) to remove residual transport solution. Using a sterile surgical scalpel, glands were then diced into quarter sections of approximately 0.5 mm³ on sterile dental wax with a thin layer of PBS to maintain tissue viability. The 4 diced sections from each gland were randomly allocated into 2 groups: ultrasound‐treated and sham‐treated.

The prepared sections were placed into wells containing 1 mL of Krebs Ringer Buffer supplemented with 5 mmol/L glucose, and all samples were plated in 12‐well flat‐bottom cell culture plates (Corning, Corning, NY).

Experimental Setup

The experimental setup for the ultrasound application utilized a water tank, a water heater, a waveform generator, a radiofrequency amplifier, and a series of circular planar ultrasound transducers at the desired frequencies. A schematic of the experimental setup is shown in Figure 1.

Figure 1.

Depiction of experimental set‐up.

The circular planar transducers utilized in this study had an active diameter of 1.5 cm and center frequencies of 600 kHz, 800 kHz, and 1 MHZ (Sonic Concepts, Inc., Bothell, WA). Each transducer was positioned at the near field–far field transition distance from the cell culture plate and was oriented with the acoustic axis perpendicular to the bottom of the plate for optimal sonication of tissue samples. The water bath was kept at the physiologically relevant temperature of 37°C throughout experimentation, and temperature measurements were taken within the ultrasound‐treated and sham‐treated wells using a calibrated digital thermometer which confirmed a well temperature of 35°C ± 1°C throughout the studies conducted.

An Agilent 33220A arbitrary waveform generator (Agilent Technologies, Santa Clara, CA) and a 150A100B radiofrequency amplified (Amplifier Research, Souderton, PA) were used to generate ultrasound waveforms. Ultrasound experiments were performed in the continuous wave mode at a spatial‐average temporal‐average intensity of 1 W/cm² for 5 minutes, with frequencies of 600 kHz, 800 kHz, and 1 MHz. For sham‐control experiments, tissue preparations were placed in wells with the same preparation and maintained in the water bath for the same duration as experimentation. Samples of 150 μL were collected immediately after ultrasound exposure (t = 5 min) and 30 minutes after ultrasound exposure (t = 35 min).

Cell Viability Experiments

To confirm tissue viability during the experimental protocol, thyroid sections were exposed to rat thyroid‐stimulating hormone (rTSH). Thyroid sections were incubated with 10 mU/mL rTSH for 30 minutes at 37°C, and the T3 and T4 release was measured using the established sample collection and ELISA protocols.

Quantification of Thyroid Hormones

The metabolic hormone concentrations were quantified using competitive enzyme‐linked immunosorbent assay kits specific for thyroxine (T4) and triiodothyronine (T3) (ThermoFisher Scientific Invitrogen, Waltham, MA). ²³ , ²⁴ These assays allow quantification of thyroid hormones in serum, plasma, or cell culture media. All samples were processed according to manufacturer protocols, and optical density was measured at 450 nm using a SpectraMax iD5 Multi‐Mode Microplate Reader (Molecular Devices, San Jose, CA). ²⁵

Statistical Analysis

Hormone release values are reported as the difference between ultrasound‐treated and sham‐treated thyroid hormone concentrations. For each experimental condition, the mean hormone concentration of the sham‐treated sample concentration was subtracted from the corresponding ultrasound‐treated sample concentration. This approach accounts for baseline hormone release and any non‐specific leakage that results from the tissue preparations. The standard deviation of the difference values was normalized and calibrated using the following formula:

$$\begin{gathered} {\mathrm{SD}}_{E,\text{Calibrated}}=\sqrt{{\mathrm{SD}}_{E,\text{Measured}}^2+{\mathrm{SD}}_{\mathrm{E},\text{Background}}^2 \\ -\left(2\times \text{Corr}\times {\mathrm{SD}}_{E,\text{Measured}}^2\times {\mathrm{SD}}_{E,\text{Background}}^2\right)} \end{gathered}$$

where SD _E,Measured is the ultrasound‐treated sample concentration and SD _E,Background is the sham‐treated sample concentration. Corr is the correlation between the ultrasound‐treated and sham‐treated hormone release, which is assumed to be negligible as the samples were prepared in separate wells.

Welch's unequal variance t‐test was selected to address differing variability between ultrasound‐treated and sham‐treated samples across experimental conditions, with this test providing a robust comparison in the presence of heterogeneous variability. ²⁶ Separate analysis was conducted for each frequency and time point combination and statistical significance was defined as p < .05. Statistical analyses were performed using MATLAB.

Power Analysis and Detectable Effect Size

To aid interpretation of the results of this study, a post hoc power analysis was performed to estimate the minimum detectable effect size under the experimental conditions. This study consisted of matched experimental conditions derived from the same thyroid gland divided into ultrasound‐treated and sham‐treated samples.

Power analysis was conducted assuming a 2‐sample t‐test with unequal variance, consistent with the statistical approach utilized in the hypothesis testing. Statistical significance was defined as $\alpha =0.05$, the sample size was $n=45$ paired experiments, and the statistical power was assumed to be 80%. Under these conditions, the corresponding standardized effect size detectable is approximately Cohen's d = 0.42. This standardized effect size corresponds to a minimum detectable difference of approximately 0.001–0.002 ng/mL for T3 and 0.015–0.020 ng/mL for T4 dependent on frequency and time point. Any effects smaller than these minimums are thus unlikely to reach statistical significance given the variability in experimental measurements.

These calculations indicate that the study was adequately powered to detect moderate ultrasound‐induced changes in hormone release, but may not have been sufficiently sensitive to detect sub‐threshold effects. Therefore, the absence of statistically significant changes in hormone concentrations is unlikely to be explained solely by insufficient statistical power.

Histological Analysis

Thyroid tissue samples from ultrasound‐treated, sham treated, and untreated control groups were immediately fixed in 10% neutral buffered formalin after experimentation and then were sent to HistoServ, Inc. (Germantown, MD) for processing. Samples were stained with hematoxylin and eosin (H&E) according to standard protocols allowing differentiation of cellular and tissue structures. ²⁷

Light microscopy images were obtained at 10× and 40× magnification using a ZEISS Axio Imager.A2 microscope and AxioCam MRc camera (Carl Zeiss, Oberkochen, Germany). Images were analyzed for the structural integrity of thyroid follicles and colloid, follicular epithelial cell morphology, and the presence of any tissue damage. A minimum of 5 representative fields were examined for each sample.

Results

A total of 45 experiments were conducted, and each experiment included thyroid tissue from 1 animal, divided between ultrasound‐treated and sham‐treated conditions.

T3 Hormone Release

Triiodothyronine (T3) concentrations measured at 5 and 35 minutes after ultrasound exposure are presented in Figure 2, with the average difference in release across all frequencies at 5 minutes being −0.001 ng/mL and at 35 minutes being 0.001 ng/mL.

Figure 2.

T3 ELISA results for all experiments.

At 5 minutes post‐exposure, the difference in T3 concentration between the ultrasound‐treated and sham‐treated samples was 600 kHz: −0.001 ± 0.002 ng/mL; 800 kHz: 0.001 ± 0.003 ng/mL; 1 MHz: 0.002 ± 0.005 ng/mL. No statistically significant differences were observed between ultrasound‐treated and sham‐treated groups at any frequency (600 kHz: p = .990; 800 kHz: p = .990; 1 MHz: p = .781).

At 35 minutes post‐exposure, the difference in T3 concentration between the ultrasound‐treated and sham‐treated samples was 600 kHz: 0.001 ± 0.001 ng/mL; 800 kHz: 0.001 ± 0.001 ng/mL; 1 MHz: 0.002 ± 0.013 ng/mL. Again, no statistically significant differences were detected between treatment groups at any frequency (600 kHz: p = .991; 800 kHz: p = .990; 1 MHz: p = .982).

T4 Hormone Release

Thyroxine (T4) concentrations measured at 5 and 35 minutes after ultrasound exposure are presented in Figure 3, with the average difference in release across all frequencies at 5 minutes being 0.031 ng/mL and at 35 minutes being 0.003 ng/mL.

Figure 3.

T4 ELISA results for all experiments.

At 5 minutes post‐exposure, the difference in T4 concentration between the ultrasound‐treated and sham‐treated samples was 600 kHz: 0.037 ± 0.041 ng/mL; 800 kHz: 0.037 ± 0.038 ng/mL; 1 MHz: 0.020 ± 0.035 ng/mL. No statistically significant differences were observed between ultrasound‐treated and sham‐treated groups (600 kHz: p = .735; 800 kHz: p = .676; 1 MHz: p = .987). At 35 minutes post‐exposure, the difference in T4 concentration between the ultrasound‐treated and sham‐treated samples was 600 kHz: 0.003 ± 0.031 ng/mL; 800 kHz: 0.002 ± 0.064 ng/mL; 1 MHz: 0.004 ± 0.024 ng/mL. Statistical analysis revealed no significant differences between treatment groups at any frequency tested (600 kHz: p = .980; 800 kHz: p = .985; 1 MHz: p = .937).

Histological Findings

Histological examination of H&E‐stained thyroid sections revealed normal thyroid architecture in all experimental groups. Representative images are shown in Figures 4, 5, 6, 7. The thyroid follicles in both the untreated control samples (Figure 4) and the sham treated samples (Figures 5, 6, 7) displayed the expected round to oval shapes with uniform colloid content and a single layer of cuboidal follicular epithelial cells.

Figure 4.

Untreated microscopy images.

Figure 5.

600 kHz applied ultrasound and sham microscopy images.

Figure 6.

800 kHz applied ultrasound and sham microscopy images.

Figure 7.

1 MHz applied ultrasound and sham microscopy images.

Thyroid tissue exposed to 600 kHz (Figure 5), 800 kHz (Figure 6), and 1 MHz (Figure 7) ultrasound similarly demonstrated preserved thyroid architecture.

Follicular cells maintained normal morphology with basally located nuclei and the colloid content appeared unchanged compared to the controls. No evidence of hemorrhage, necrosis, cellular disruption, or structural abnormalities was observed in any ultrasound‐treated samples.

Discussion

The aim of this study was to determine whether therapeutic ultrasound can stimulate thyroid hormone release from ex vivo rat thyroid tissue preparations. Contrary to our hypothesis, the ultrasound parameters tested did not produce statistically significant changes in T3 or T4 hormone release when compared to sham treated controls. Histological examination revealed no structural changes in ultrasound‐treated thyroid tissue when compared to sham‐treated tissue, indicating that the experimental parameters tested did not cause tissue damage while also failing to trigger hormone secretion. Negative results remain critical for defining biological boundaries of emerging therapeutic modalities and help refine future experimental design.

Importantly, the absence of detectable hormone release under these conditions should not be interpreted as evidence that thyroid tissue is entirely unresponsive to therapeutic ultrasound, but rather that acute stimulation within the tested parameters is unlikely to induce measurable T3 or T4 release. While the present study does not support a measurable stimulatory effect of ultrasound stimulation on thyroid hormone release under the experimental conditions, we cannot exclude the possibility of smaller sub‐threshold biological effects. These findings help define tissue‐specific limits of ultrasound‐induced endocrine modulation.

Comparison with Pancreatic Beta Cell Studies

The contrasting outcomes between these thyroid experiments and previously conducted pancreatic studies provide an interesting avenue for investigation. Several fundamental differences in tissue structure and hormone secretion mechanisms may explain these differing results.

Firstly, the cellular organization and hormone storage differ substantially between pancreatic islets and thyroid follicles. Pancreatic beta cells store insulin in cytoplasmic secretory granules that readily undergo exocytosis in response to various stimuli, including glucose, membrane depolarization, and calcium influx. ²⁸ In contrast, thyroid hormones are stored extracellularly within the colloid of thyroid follicles, bound to thyroglobulin. Thyroid hormone release requires a multi‐step process involving endocytosis of colloid, proteolytic cleavage of thyroglobulin within lysosomes, and subsequent hormone secretion, ²⁹ a fundamentally different mechanism than the rapid exocytosis observed in beta cells. This may imply that thyroid hormone release via ultrasound will need to employ different parameters to trigger hormone release. Unlike excitable secretory cells such as neurons and pancreatic beta cells, thyroid follicular cells are non‐excitable epithelial cells whose hormone secretion is governed by TSH‐mediated endocytosis rather than membrane depolarization, ³⁰ potentially reducing susceptibility to acute mechanically driven secretion.

Secondly, the time course of hormone secretion differs markedly. Insulin secretion from beta cells occurs within minutes of stimulation, consistent with our experimental observation period. Thyroid hormone synthesis and release under physiological TSH stimulation typically requires hours to days for maximal effect, although acute release of stored hormone can occur more rapidly. ³¹ Our sampling at 5 and 35 minutes may have been appropriate for detecting preformed hormone release from pancreatic beta cells but insufficient for capturing synthesis‐dependent secretion in the thyroid gland.

Additionally, the sensitivity to mechanical stimulation may differ between cell types. Pancreatic beta cells express numerous mechanosensitive ion channels, including TRPV and Piezo channels, which can be activated by membrane deformation or shear stress. ³² While thyroid follicular cells also express some mechanosensitive channels, their role in regulating hormone secretion is less well characterized. The acoustic radiation force and associated mechanical stress generated by our ultrasound parameters may be insufficient to activate secretory pathways in thyroid tissue.

Potential Mechanisms and Theoretical Considerations

Therapeutic ultrasound can influence biological tissues through several mechanisms, including thermal effects, mechanical effects (acoustic radiation force, acoustic streaming, shear stress), and cavitation. At the intensity used in this study (1 W/cm²), thermal effects are expected to be minimal, particularly given the small tissue volume and efficient heat dissipation. Our temperature measurements confirmed that well temperature remained within physiological range throughout exposure.

Mechanical effects represent the most likely mechanism for any potential ultrasound‐induced hormone secretion. Acoustic radiation force can generate localized tissue displacement and cellular deformation. In mechanosensitive cells, such deformation can activate stretch‐activated ion channels, alter membrane potential, trigger calcium influx, and ultimately stimulate secretory pathways. However, the magnitude of mechanical stress required to activate these pathways likely varies among cell types. The threshold for activating secretory mechanisms in thyroid follicular cells may exceed that generated by our ultrasound parameters. To better contextualize the mechanical stimulation generated in this study, we performed a first‐order theoretical estimate of the acoustic radiation pressure. The time‐averaged radiation pressure was approximated using the equation. ³³

$$P_{\mathrm{rad}}=\frac{2I}{c}$$

where $I$ is the spatial‐average acoustic intensity and $c$ is the speed of sound in soft tissue. In this study, ultrasound was applied at an intensity of 1 W/cm² for all frequencies tested (600 kHz, 800 kHz, 1 MHz) and we assume the speed of sound in soft tissue is approximately 1540 m/s. ³⁴ This yields an estimated rational pressure, $P_{\mathrm{rad}}$, of approximately 13 Pa, which is an approximate order‐of‐magnitude estimate of the mechanical stress exerted on the tissue surface.

This estimated radiation pressure of approximately 13 Pa is comparable to values reported in other ultrasound bioeffect studies ³⁵ and falls within a range consistent with the activation of mechanosensitive ion channels such as Piezo1, TRAAK, and TRPC, which have been shown to respond to ultrasound‐induced mechanical forces including acoustic radiation force and membrane tension. ³⁶

Cavitation effects, including stable and inertial cavitation, can generate more substantial mechanical perturbations but require specific conditions including the presence of gas nuclei and sufficient acoustic pressure. ³⁷ At 1 W/cm² continuous wave ultrasound, significant cavitation is unlikely in tissue preparations, though localized microbubble activity cannot be entirely excluded.

The follicular structure of the thyroid gland may provide mechanical protection against ultrasound‐induced stimulation. The thyroid follicle consists of a monolayer of epithelial cells surrounding a proteinaceous colloid core, encased by a basement membrane. ³⁸ This architecture may dampen acoustic energy transmission to the follicular cells or distribute mechanical stress in a manner that fails to reach stimulatory thresholds. The enclosed colloid matrix may act as a mechanical buffer, redistributing acoustic energy and reducing localized membrane deformation at the follicular epithelium, limiting activation of mechanotransducive signaling pathways. ³⁹

Limitations and Clinical/Translational Implications

Several limitations of this study should be acknowledged. First, the ex vivo tissue model preparation lacks the intact vascular supply, innervation, and humoral regulation present in vivo, which limits our ability to mimic physiologically relevant conditions that could influence the response to ultrasound stimulation.

Second, we tested a limited range of ultrasound parameters. The frequencies, intensity, and duration were selected based on our successful pancreatic studies, but optimal parameters for thyroid stimulation may differ substantially.

Third, our sampling time points may not have captured the full temporal dynamics of thyroid hormone release. While we observed no changes at 5 or 35 minutes post‐exposure, delayed effects occurring over time could have been missed. This sampling may have been appropriate for detecting preformed hormone release from pancreatic beta cells but may be insufficient for capturing the synthesis dependent secretion of thyroid hormones. Given the kinetics of thyroid hormone mobilization, longer observation windows may be required to detect delayed or indirect effects of ultrasound stimulation. Thus, the negative findings of this study may reflect intrinsic physiological time constraints rather than suboptimal ultrasound delivery alone.

Finally, we cannot exclude the possibility that ultrasound did influence thyroid cells in ways not measured by our assays. A post hoc power analysis indicates that the present study was sufficiently powered to detect moderate ultrasound‐induced changes in hormone secretion, but may not detect small concentration differences. More comprehensive investigations including gene expression analysis, enzyme activity assays, and longer‐term culture studies would be needed to fully characterize ultrasound effects on thyroid physiology.

Although the present study did not demonstrate acute ultrasound‐mediated thyroid hormone release under the tested parameters, these negative findings represent important clinical and translational implications for ultrasound‐based thyroid applications.

From a safety perspective, the absence of stimulated T3 or T4 hormone secretion at the tested parameters 600 kHz–1 MHz and 1 W/cm² suggests that therapeutic ultrasound delivered within this parameter range is unlikely to inadvertently disrupt thyroid hormone homeostasis. This has relevance for patients receiving therapeutic ultrasound treatment in anatomically adjacent regions, such as carotid vasculature, cervical spine, or regional lymphatics, and provides reassurance for individuals with pre‐existing thyroid conditions of unlikely off‐target effects.

More broadly, the contrast between the present findings and prior observations in pancreatic beta cells underscores the tissue‐specific nature of ultrasound responsiveness. These results suggest that mechanosensitive pathways capable of driving hormone secretion are not uniformly accessible across endocrine tissues. For thyroid‐directed applications, the parameter space explored here is insufficient to engage follicular release mechanisms, and future approaches may require alternative strategies, including modified acoustic parameters, longer stimulation timescales consistent with thyroid hormone kinetics, or indirect targeting of the hypothalamic–pituitary–thyroid axis.

Taken together, this study helps define a boundary condition for ultrasound–thyroid interactions, providing a foundation for the rational design of future therapeutic strategies targeting thyroid function.

Conclusions

Therapeutic ultrasound application at frequencies of 600 kHz, 800 kHz, and 1 MHz with corresponding intensity of 1 W/cm² applied continuously for 5 minutes did not stimulate a statistically significant difference in T3 or T4 hormone release from ex vivo rat thyroid tissue preparations. These negative results contrast with our laboratory's previous findings of significant ultrasound‐stimulated insulin secretion from pancreatic beta cells, which demonstrates the importance of tissue‐specific differences relating to ultrasound responsiveness. Examination of histology confirmed that while ultrasound exposure was ineffective for stimulating hormone secretion, it was not structurally damaging. These findings suggest that the thyroid gland may represent a boundary case for direct ultrasound mediated endocrine modulation, underscoring the need for physiologically informed parameter selection and experimental design. Importantly, these findings suggest that therapeutic ultrasound delivered within the tested parameter range is unlikely to inadvertently alter thyroid hormone release, providing reassurance for its safe use in anatomically adjacent clinical applications while also informing the design of future thyroid‐directed ultrasound therapies. Future investigations into ultrasound stimulation of the thyroid gland should incorporate alternative ultrasound parameter combinations, differing endpoints, and in vivo experimentation which may reveal experimental conditions under which ultrasound can modulate thyroid function.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.