Name: in vivo characterization of endocrine disrupting chemical effects via thyroid hormone action indicator mouse
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Description: Watch this Scientific Journal Video about In vivo Characterization of Endocrine Disrupting Chemical Effects via Thyroid Hormone Action Indicator Mouse at JoVE.com

This work was supported by Project no. RRF-2.3.1-21-2022-00011, titled National Laboratory of Translational Neuroscience has been implemented with the support provided by the Recovery and Resilience Facility of the European Union within the framework of Programme Sz&#233;chenyi Plan Plus.

The present protocol was reviewed and approved by the Animal Welfare Committee at the Institute of Experimental Medicine (PE/EA/1490-7/2017, PE/EA/106-2/2021). The presented data is from FVB/Ant background14, 3-month-old male THAI mice (n = 3-6/group). FVB/Ant background THAI animals tend to have highly pigmented spots on their skin that may distort measurements. Hence, search for pigmented spots on the skin of the imaged area after fur removal. Animals do not require special housing conditions unless the experiment specifically requires so (e.g., a special diet).
1. Hyperthyroid treatment
NOTE: A general protocol for inducing hyperthyroidism in mice is provided here. The ATA guide19 offers detailed explanations on the background of the methods with alternatives mentioned.
<ol>
	<li>Dissolve T3 (3,5,3&#39;-triiodothyronine, see Table of Materials) in 40 mM NaOH to create a stock solution with a concentration between 5-10 mg/mL.</li>
	<li>Dilute the stock solution with saline to a final concentration of 0.1 &#181;g/&#181;L.</li>
	<li>Inject the diluted T3 solution intraperitoneally (i.p.) into awake animals at a volume of 10 &#181;L per gram of body weight (bwg). After 24 h, the animals will be considered hyperthyroid. 
	NOTE: T3 treatment can be replaced by any other kind of treatment. The treatment does not affect the protocol of in vivo imaging.</li>
</ol>
2. Hypothyroid treatment
NOTE: Here, only a general protocol for inducing hypothyroidism in mice is provided. ATA guide19 describes detailed explanations on the background of the methods with alternatives mentioned.
<ol>
	<li>Switch the diet to an iodine-free chow diet and add KClO4 and methimazole to the drinking water (0.01 % methimazole, 0.05 % KClO4) (see Table of Materials).</li>
	<li>Replace the drinking solution regularly (every 2-3 days) with a fresh drinking solution because methimazole is light-sensitive and degrades quickly.</li>
	<li>Keep up the treatment regime for at least 2 weeks, no more than 4 weeks. Animals will lose weight and will show discomfort. If animals are hardly moving around, have lost much of their hair, or are barely conscious, use a humane endpoint and terminate them by any method (following institutionally approved protocols).</li>
	<li>Continue hypothyroid treatment when combined with other treatments to prevent potential recovery from hypothyroidism.</li>
</ol>

3. In vivo imaging
<ol>
	<li>Start the software compatible with the in vivo imaging system (see Table of Materials).</li>
	<li>Log in and wait for the &#34;Imaging Wizard&#34; panel to load. It is a smaller panel on the bottom left of the window.</li>
	<li>On the &#34;Imaging Wizard&#34; initiate camera cooling by clicking on Initialize in the &#34;Image Wizard panel&#34;. This makes the instrument run a setup protocol, wait for it to complete. The &#34;Imaging Wizard panel&#34; turns blue, and a green light will turn on in the panel when the camera temperature is low enough and the instrument is ready.</li>
	<li>Set the temperature of the heating pad to 30-37 &#176;C to keep the measured animals warm. 
	NOTE: Continue with the protocol while waiting for the camera temperature to be optimal.</li>
	<li>Do not keep or treat animals near the instrument. Avoid excess amounts of hair circulating in the air around the instrument.</li>
	<li>Anesthetize 1-3 animals with ketamine-xylazine i.p. injection (ketamine 50 mg/kg body weight, xylazine 10 mg/kg body weight, see Table of Materials). Alternatively, if an isoflurane anesthetic system is installed, follow institutionally approved protocols to use isoflurane anesthesia, replacing the ketamine-xylazine mixture. 
	NOTE: Hypothyroid mice are more sensitive to ketamine-xylazine; use half dose.</li>
	<li>Use eye protection gel during anesthesia.</li>
	<li>Check pedal reflex by pinching the footpads. No pedal reflex confirms the state of surgical plane anesthesia.</li>
	<li>After the anesthesia takes effect, remove fur from the imaged body parts using the most suitable fur removal method (epilator, shaving, cream, etc.). Ensure no fur is left on the imaged body parts to prevent the scattering of luminescent light.</li>
	<li>Dissolve Na-luciferin (see Table of Materials) in 1x phosphate-buffered saline (PBS) at a concentration of 15 mg/mL. Luciferin is light-sensitive; avoid direct light exposure. Store the solution in amber tubes or wrap it in aluminum foil.</li>
	<li>Treat shaven animals with luciferin solution 10 &#181;L/bwg i.p.</li>
	<li>Place the animals into the instrument with the camera&#39;s center point marked as a &#39;+&#39; on the pad. Ensure proper placement by checking the gridlines and confirming with a single &#39;Photo&#39; capture if unsure.</li>
	<li>Wait for 15 min after substrate administration before taking the first measurement. During this time, set the imaging time in the &#34;Image Wizard&#34; panel to 3 min for luminescence and check the boxes for Photo and Luminescence. The &#39;Photo&#39; is necessary to coincide with luminescence to identify the source of the measured signal. 
	NOTE: 15 min is required for optimal substrate uptake and tissue distribution. The luminescent signal plateaus 15-20 min after luciferin administration. The signal starts to slowly decrease after the plateau.</li>
	<li>Take the first measurement by clicking on Measure in the &#34;Imaging Wizard&#34; panel.</li>
	<li>If both ventral and dorsal imaging are performed, rearrange animals for the second body part to be imaged immediately after concluding the first.</li>
	<li>After imaging is done, return the animals to their cages and continue the experiment with the next set of animals.</li>
	<li>Allow the animals to recover, which typically takes 1-2 h at most. Place a tube filled with warm water near the animals to facilitate recovery, and monitor vital signs such as breathing and perfusion.</li>
	<li>Decide the fate of measured animals. In the data presented in this article, the measured animals were euthanized following institutionally approved protocols for ex vivo measurements. However, this is not necessary. Consider whether euthanasia or follow-up experiments are ethical.</li>
</ol>

4. Data analysis
<ol>
	<li>Open the &#34;ClickInfo&#34; file in the software. A panel named &#34;Tool Palette&#34; for image analysis and editing will open on the right side of the window.</li>
	<li>Convert scale to radiance in the top left corner of the image.</li>
	<li>Click on&#160;&#34;Image Adjust&#34;.</li>
	<li>Decide on the optimal binning and color scale of the images. Make all images utilize the same settings.</li>
	<li>Click on &#34;ROI tools&#34; on the &#34;Tool palette&#34;.</li>
	<li>Select areas of interest by clicking on &#34;Place ROI&#34; in the &#34;ROI tools&#34;. Using the same sized ROIs or different sized ROIs can also be meaningful depending on the experimental design.</li>
	<li>Click on &#34;Measure ROIs&#34;. A new window will open with the data of placed ROIs. Export the data with ctr + c-ctrl + v Windows command into an organizing or statistical software of choice.</li>
	<li>Data can be exported as total flux or average radiance. Choose which variable is the most relevant in the current experimental setting.</li>
	<li>Continue data analysis in accordance with the experimental design. Individual calculation of (treated-background) values as &#34;measured effect in one animal&#34; is recommended.</li>
</ol>

Imaging of Thyroid Hormone Action Indicator Mice to Assess Endocrine Disruption

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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=American+thyroid+association+guide+to+investigating+thyroid+hormone+economy+and+action+in+rodent+and+cell+models.">American thyroid association guide to investigating thyroid hormone economy and action in rodent and cell models.</a> Thyroid. 24 (1), 88-168 (2014).</li><li>Silva, J. E., Larsen, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adrenergic+activation+of+triiodothyronine+production+in+brown+adipose+tissue.">Adrenergic activation of triiodothyronine production in brown adipose tissue.</a> Nature. 305 (5936), 712-713 (1983).</li><li>Bianco, A. C., Silva, J. 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The authors have nothing to disclose.

The threats posed by Endocrine-Disrupting Chemicals (EDCs) to human health are well recognized; however, research on EDCs faces formidable challenges. These challenges are partially a consequence of the complexity of the endocrine system. Many EDCs have been identified to simultaneously disrupt multiple endocrine systems22. Additionally, in the context of Thyroid Hormone (TH) economy, there exists an additional layer of complexity due to tissue-specific differences in regulating TH action. This complexity provides a novel perspective on expanding the assessment of TH signaling by characterizing TH action in various tissues. The challenge is further exacerbated by the metabolism of compounds, which can either enhance or attenuate their effects on the endocrine system. It&#39;s important to note that current screening methods for identifying compounds are well established and function with high performance8,11. However, there is still a lack of test systems to characterize tissue-specific effects and consequences of identified compounds.
The THAI mouse model was developed to tackle the challenges of characterizing tissue-specific thyroid hormone economy. Its potential has been demonstrated under various circumstances14,15,16,18. The THAI model offers an advantage in characterizing chemicals that disrupt thyroid hormones. It&#39;s important to note that the model isn&#39;t intended for rapid compound screening, but to provide insights into disruption mechanisms in an in vivo mammalian model.
In this article, a protocol is presented outlining how the THAI mouse can be used for in vivo imaging studies. This method allows testing of treatments affecting the Hypothalamus-Pituitary-Thyroid (HPT) axis and/or thyroid hormone action in animals. The protocol supports self-controlled studies and follow-up designs. Additionally, the protocol can be used to generate control animals with various thyroid hormone states, serving as references in experimental settings. The presented hypo- and hyperthyroid treatments can be replaced, extended, and combined with other treatments as needed. This versatility is valuable for assessing tissue thyroid hormone economy, especially for endocrine-disrupting chemical (EDC) characterization.
In vivo imaging signals on the dorsal side come from the Brown Adipose Tissue (BAT), while ventral signals originate from the small intestines14. Thus, the method primarily characterizes these tissues, and measuring other body parts might be challenging. Overcoming these technical limitations through organ exposure requires careful consideration of ethical and technical implications.
Combining in vivo imaging with ex vivo studies on the THAI model allows for assessing thyroid hormone signaling in various tissues and brain regions14. For instance, qPCR can extend and specify the effects seen in in vivo imaging. However, this sacrifices follow-up and self-controlled designs, so costs and benefits should be evaluated. Combining in vivo imaging with ex vivo measurements is recommended for a comprehensive investigation.

Thyroid hormone (TH) signaling is a fundamental regulator of cellular metabolism, essential for normal development and optimal tissue function in adulthood1. Within tissues, TH action is finely controlled by a complex molecular machinery, allowing for tissue-specific maintenance of local TH levels. This autonomy of different tissues from circulating TH levels is of great importance2,3,4.
Numerous chemicals have the potential to disrupt endocrine functions and are found in the environment as pollutants. It is a growing concern that these molecules can enter the food chain through wastewater and agricultural production, thereby impacting the health of livestock and humans5,6,7.
One of the significant challenges in addressing this issue is the sheer number of compounds involved, including both authorized and already banned, but still persistently present, molecules. In recent years, substantial efforts have been made to develop test systems for screening and identifying the disruptive potential of various chemicals8,9,10,11. While these methods excel in high-throughput screening of thousands of compounds and identifying potential threats, a detailed analysis of specific in vivo effects of these molecules is essential to establish the hazards of human exposure. Thus, a multifaceted approach is necessary when studying and characterizing endocrine-disrupting chemicals (EDCs).
In the context of TH regulation, understanding the tissue-specific consequences of EDC exposure requires quantifying local TH action. Although several in vivo models have been developed for this purpose, most rely on endogenous markers as their output measure. Despite being physiological, these markers are subject to numerous regulatory mechanisms, both direct and indirect, making their interpretation more challenging. Therefore, characterizing EDC effects on TH regulation at the tissue level remains a significant challenge12,13.
To address the challenges of measuring tissue-specific TH signaling, the Thyroid Hormone Action Indicator (THAI) mouse model was recently developed. This model allows for specific quantification of changes in local TH action under endogenous conditions. A luciferase transgene was introduced into the mouse genome, which is highly sensitive to regulation by TH action14. This model has demonstrated effectiveness in answering various research questions that require quantifying changes in local tissue TH signaling14,15,16,17,18.
Recognition of one potential use of the THAI model is characterizing tissue-specific effects of EDCs on TH signaling. The model has recently been employed successfully to investigate the tissue-specific effects of tetrabromobisphenol A and diclazuril on TH signaling15. Here, baseline protocols are presented for utilizing in vivo imaging techniques on the THAI model as a test system for characterizing EDCs that disrupt TH function. This method leverages the bioluminescent nature of the luciferin-luciferase reaction. Essentially, the transgenically expressed luciferase enzyme catalyzes the oxidation of administered luciferin, generating luminescent light proportional to the amount of luciferase in the tissue (Figure 1). Consequently, the biological response measured is luciferase activity, which has been validated as a suitable measure of local TH action14. While the THAI model is applicable for quantifying TH action in virtually all tissues, in vivo imaging primarily focuses on TH action in the small intestine (ventral imaging) and the interscapular brown adipose tissue (BAT, dorsal imaging)14.
A significant advantage of the in vivo imaging technique is that it eliminates the need to sacrifice animals for measurements. This allows investigators to design longitudinal and follow-up experiments as self-controlled studies, reducing between-subjects bias and the number of animals used. This aspect is particularly crucial in EDC characterization, and the method&#39;s strength and versatility for this purpose have been previously demonstrated14,15.

<table><tbody><tr><td>3,5,3&#039;-triiodothyronine (T3)</td><td>Merck</td><td>T2877</td><td></td></tr><tr><td>Animals, mice</td><td></td><td></td><td>THAI mouse</td></tr><tr><td>Eye protection gel</td><td>Oculotect</td><td></td><td>1000 IU/g</td></tr><tr><td>Falcon tube</td><td>Thermo Fisher Scientific</td><td></td><td>50 mL volume</td></tr><tr><td>Iodine-free chow diet</td><td>Research Diets</td><td>custom</td><td></td></tr><tr><td>IVIS Lumina II in vivo imaging system</td><td>Perkin Elmer</td><td>-</td><td></td></tr><tr><td>Ketamine</td><td>Vetcentre</td><td>E1857</td><td></td></tr><tr><td>Living Image software 4.5</td><td>Perkin Elmer</td><td>-</td><td>provided with the instrument</td></tr><tr><td>Measuring cylinder</td><td></td><td></td><td>250 mL</td></tr><tr><td>methimazole</td><td>Merck</td><td>M8506</td><td></td></tr><tr><td>Microfuge tubes</td><td>Eppendorf</td><td></td><td>For diluting treatment materials</td></tr><tr><td>NaClO&lt;sub&gt;4&lt;/sub&gt;</td><td>Merck</td><td>71852</td><td></td></tr><tr><td>Na-luciferin, substrate</td><td>Goldbio</td><td>103404-75-7</td><td></td></tr><tr><td>NaOH</td><td>Merck</td><td>101052833</td><td></td></tr><tr><td>Phoshphate buffer saline</td><td>Chem Cruz</td><td>sc-362302</td><td></td></tr><tr><td>Pipette</td><td>Gilson</td><td></td><td>For diluting treatment materials</td></tr><tr><td>Pipette tips</td><td>Axygen</td><td></td><td>For diluting treatment materials</td></tr><tr><td>Shaving cream/epilator/shaver</td><td></td><td></td><td>Personal preference</td></tr><tr><td>Syringe</td><td>B Braun</td><td></td><td>1 mL volume</td></tr><tr><td>Syringe needle</td><td>B Braun</td><td></td><td>0.3 x 12 mm</td></tr><tr><td>Xylazine</td><td>Vetcentre</td><td>E1852</td><td></td></tr></tbody></table>

in vivo characterization of endocrine disrupting chemical effects via thyroid hormone action indicator mouse

Thyroid hormones (TH) play a critical role in cell metabolism and tissue function. TH economy is susceptible to endocrine disrupting chemicals (EDCs) that can disturb hormone production or action. Many environmental pollutants are EDCs, representing an emerging threat to both human health and agricultural production. This has led to an increased demand for proper test systems to examine the effects of potential EDCs. However, current methodologies face challenges. Most test systems use endogenous markers regulated by multiple, often complex regulatory processes, making it difficult to distinguish direct and indirect effects. Moreover, in vitro test systems lack the physiological complexity of EDC metabolism and pharmacokinetics in mammals. Additionally, exposure to environmental EDCs usually involves a mixture of multiple compounds, including in vivo generated metabolites, so the possibility of interactions cannot be ignored. This complexity makes EDC characterization difficult. The Thyroid Hormone Action Indicator (THAI) mouse is a transgenic model that carries a TH-responsive luciferase reporter system, enabling the assessment of tissue-specific TH action. One can evaluate the tissue-specific effects of chemicals on local TH action by quantifying luciferase reporter expression in tissue samples. Furthermore, with in vivo imaging, the THAI mouse model allows for longitudinal studies on the effects of potential EDCs in live animals. This approach provides a powerful tool for testing long-term exposure, complex treatment structures, or withdrawal, as it enables the assessment of changes in local TH action over time in the same animal. This report describes the process of in vivo imaging measurements on THAI mice. The protocol discussed here focuses on developing and imaging hyper- and hypothyroid mice, which can serve as controls. Researchers can adapt or expand the treatments presented to meet their specific needs, offering a foundational approach for further investigation.

Generally, the measured radiance ranges from magnitudes of 105 to 1010 p/s/cm2/sr. However, exact values can vary among animals within the same image and across different images. Therefore, comparing raw data might be misleading. It&#39;s crucial to establish control and background signals in all experiments, making self-controlled designs highly recommended.
Figure 2 presents representative images and data from ventral and dorsal views in an experimental setup involving hypo-, eu-, and hyperthyroid mice. The lowest signals are expected in hypothyroid mice, often falling below the lower limit of the color scale.
Areas lacking fur, such as footpads, tail, and nose, exhibit relatively high basal signals. Importantly, the luciferase signal is influenced by the thyroid hormone (TH) status, as observed in Figure 2. Intriguingly, Figure 3 demonstrates that TH action is significantly heightened in the brown adipose tissue (BAT) of cold-stressed THAI mice14. However, this treatment does not impact the luciferase signal in the footpads and tail. This disparity underscores the potential for markedly distinct TH actions in tissues of the same organism. Cold exposure triggers BAT activation, necessitating localized upregulation of type 2 deiodinase-mediated TH activation20,21. Following 24 h of cold exposure, circulating TH levels remain unaltered, resulting in no TH-dependent signal change in the footpads and tail. In contrast, in the scenario presented in Figure 2, where elevated blood TH levels correspondingly increase TH action in the footpad, tail, and BAT.
Both Figure 2 and Figure 3 reveal robust signals in the testicular region. This is attributed to the TH-independent high basal expression of the luciferase transgene in the testicles, a characteristic of the THAI model. In this organ, the luciferase signal remains unaffected by changes in circulating TH levels.
As previously mentioned, hypo- and hyperthyroid treatments can be substituted with other interventions, such as testing endocrine-disrupting compounds (EDCs). Figure 4 displays BAT imaging in a three-week-long follow-up experiment involving diclazuril, a veterinary drug with EDC potential16. Signals between time points are easily distinguishable, and the method effectively captures the accumulation and clearance of diclazuril.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65657/65657fig01.jpg" /> 
Figure 1: Concept and working principles of the THAI Construct. (A) The Recombinant THAI construct. This figure is adapted from Moh&#225;csik et al.14. (B) Schematic illustration of luciferase-catalyzed luciferin oxidation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65657/65657fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65657/65657fig02.jpg" /> 
Figure 2: Representative dorsal and ventral images of hypo-, eu-, and hyperthyroid THAI Mice, along with an intensity diagram of luciferase activity. (A) Representative Images of hypo-, eu-, and hyperthyroid THAI mice. (B) Quantification of signals in (A). Mean photon/s &#177; SEM (n = 3). *P &#60; 0.05; ***P &#60; 0.001, determined by one-way ANOVA, followed by Newman-Keuls post hoc test. This figure is adapted from Moh&#225;csik et al.14. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65657/65657fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65657/65657fig03.jpg" /> 
Figure 3: Paw, tail, and BAT signals before and after cold stress, along with light intensity diagram of luciferase activity. (A) Representative dorsal images of control and cold-stressed THAI mice. (B) Quantification of signals in (A). Mean photon/s &#177; SEM (n = 4). **P &#60; 0.001, determined by Student&#39;s t-test. This figure is adapted from Moh&#225;csik et al.14. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65657/65657fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65657/65657fig04.jpg" /> 
Figure 4: Representative BAT images of a three-week long diclazuril follow-up study, accompanied by light intensity diagram of luciferase activity. THAI mice were orally treated for 2 weeks with 10 mg/bwkg/day of diclazuril as a saline suspension, followed by one week of recovery. (A) Quantification of BAT bioluminescent signals in (B) before, during, and after diclazuril treatment. (B) Representative dorsal images of THAI mice before, during and after diclazuril treatment. n = 4-6 mice/group; the figure displays a Tukey Box Plot of photon/s, &#945; = 0.05; ***: p &#60; 0.001. This figure is adapted from Sinko et al.15.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65657/65657fig04large.jpg" style="font-size: 14px;" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about In vivo Characterization of Endocrine Disrupting Chemical Effects via Thyroid Hormone Action Indicator Mouse at JoVE.com

Author Spotlight: In Vivo Assessment of Thyroid Hormone Disruption Using the THAI Mouse Model

The Thyroid Hormone Action Indicator mouse model was developed to enable tissue-specific quantification of local thyroid hormone action using its endogenous regulatory machinery. Recently, it has been shown that the model is suitable for characterizing endocrine-disrupting chemicals interacting with thyroid hormone economy, both by ex vivo and in vivo methodologies.

In vivo Characterization of Endocrine Disrupting Chemical Effects via Thyroid Hormone Action Indicator Mouse

Thyroid hormones (TH) play a critical role in cell metabolism and tissue function. TH economy is susceptible to endocrine disrupting chemicals (EDCs) that ...

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Nanyang Technological University

Research

JoVE Journal

Neuroscience

749 Views.  Institute of Experimental Medicine. The Thyroid Hormone Action Indicator mouse model was developed to enable tissue-specific quantification of local thyroid hormone action using its endogenous regulatory machinery. Recently, it has been shown that the model is suitable for characterizing endocrine-disrupting chemicals interacting with thyroid hormone economy, both by ex vivo and in vivo methodologies.

Video: Author Spotlight: In Vivo Assessment of Thyroid Hormone Disruption Using the THAI Mouse Model

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been hindered partly due to a lack of standardized methods and availability of robust, user-friendly technology. In this study, commercially available, division-arrested cell lines were employed to quantitatively screen for endocrine activity of chemicals present in water samples of interest to environmental quality professionals. A single, standardized protocol that included comprehensive quality assurance/quality control (QA/QC) checks was developed for Estrogen and Glucocorticoid Receptor activity (ER and GR, respectively) using a cell-based Fluorescence Resonance Energy Transfer (FRET) assay. Samples of treated municipal wastewater effluent and surface water from freshwater systems in California (USA), were extracted using solid phase extraction and analyzed for endocrine activity using the standardized protocol. Background and dose-response for endpoint-specific reference chemicals met QA/QC guidelines deemed necessary for reliable measurement. The bioassay screening response for surface water samples was largely not detectable. In contrast, effluent samples from secondary treatment plants had the highest measurable activity, with estimated bioassay equivalent concentrations (BEQs) up to 392 ng dexamethasone/L for GR and 17 ng 17&#946;-estradiol/L for ER. The bioassay response for a tertiary effluent sample was lower than that measured for secondary effluents, indicating a lower residual of endocrine active chemicals after advanced treatment. This protocol showed that in vitro transactivation bioassays that utilize commercially available, division-arrested cell &#34;kits&#34;, can be adapted to screen for endocrine activity in water.

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

A protocol to screen for endocrine activity in organic extracts of water samples, including treated wastewater effluent and surface (receiving) water, was adapted using commercially available division-arrested ("freeze and thaw") in vitro transactivation bioassays.

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been ...

Environment

In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ever-increasing number of studies have identified miRNAs as potential biomarkers for cancer diagnosis and prognosis, and also as therapeutic targets, adding an extra dimension to cancer evaluation and treatment. In the context of thyroid cancer, tumorigenesis results not only from mutations in important genes, but also from the overexpression of many miRNAs. Accordingly, the role of miRNAs in the control of thyroid gene expression is evolving as an important mechanism in cancer. Herein, we present a protocol to examine the effects of miRNA-inhibitor delivery as a therapeutic modality in thyroid cancer using human tumor xenograft and orthotopic mouse models. After engineering stable thyroid tumoral cells expressing GFP and luciferase, cells are injected into nude mice to develop tumors, which can be followed by bioluminescence. The in vivo inhibition of a miRNA can reduce tumor growth and upregulate miRNA gene targets. This method can be used to assess the importance of a determined miRNA in vivo, in addition to identifying new therapeutic targets.

This protocol describes xenograft and orthotopic mouse models of human thyroid tumorigenesis as a platform to test microRNA-based inhibitor treatments. This approach is ideal to study the function of non-coding RNAs and their potential as new therapeutic targets.

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ...

Cancer Research

An In Vivo Estrogen Deficiency Mouse Model for Screening Exogenous Estrogen Treatments of Cardiovascular Dysfunction After Menopause

Postmenopausal women are at greater risk of developing cardiovascular diseases than premenopausal women. Female mice ovariectomized (OVX) at weaning display increased atherosclerotic lesions in the aorta compared with female mice with intact ovarian function. However, laboratory models involving estrogen-deficient mice with atherosclerosis-prone status are lacking. This deficit is crucial because clinical estrogen deficiency in menopausal women may aggravate the incidence of pre-existing or ongoing lipid disruption and atherosclerosis. In this study, we establish an in vivo estrogen-deficient mouse model by bilateral ovariectomy via a double dorsal-lateral incision in apolipoprotein E (apoE)-/- mice. We then compare the effects of 17&#946;-estradiol and pseudoprotodioscin (PPD) (a phytoestrogen) perorally administered via hazelnut spread. We find that although PPD exerts some effect on reducing final body weight and plasma TG in OVX apoE-/- mice, it has anti-atherosclerotic and cardiac-protective capacities comparable with its 17&#946;-estradiol counterpart. PPD is a phytoestrogen that has been reported to exert anti-tumor properties. Thus, the proposed method is applicable for screening phytoestrogens via peroral administration to substitute for traditional hormone replacement therapy in postmenopausal women, which has been reported to have potentially deleterious tumorigenetic capacity. Peroral administration via hazelnut spread is noninvasive, rendering it widely applicable to many patients. This article contains step-by-step demonstrations of bilateral ovariectomy via the double dorsal-lateral incision in apoE-/- mice and peroral 17&#946;-estradiol or phytoestrogen hormone replacement via hazelnut spread. Plasma lipid and cardiovascular function analyses using echocardiography follow.

Clinically, estrogen deficiency in menopausal women may aggravate the incidence of lipid disruption and atherosclerosis. We established an in vivo estrogen deficiency model by bilateral ovariectomy via a double dorsal-lateral incision in apoE-/- mice. The mouse model is applicable for screening exogenous estrogen treatments of cardiovascular dysfunction after menopause.

Postmenopausal women are at greater risk of developing cardiovascular diseases than premenopausal women. Female mice ovariectomized (OVX) at weaning ...

Biochemistry

A Hyperandrogenic Mouse Model to Study Polycystic Ovary Syndrome

Hyperandrogenemia plays a critical role in reproductive and metabolic function in females and is the hallmark of polycystic ovary syndrome. Developing a lean PCOS-like mouse model that mimics women with PCOS is clinically meaningful. In this protocol, we describe such a model. By inserting a 4 mm length of DHT (dihydrotestosterone) crystal powder pellet (total length of pellet is 8 mm), and replacing it monthly, we are able to produce a PCOS-like mouse model with serum DHT levels 2 fold higher than mice not implanted with DHT (no-DHT). We observed reproductive and metabolic dysfunction without changing body weight and body composition. While exhibiting a high degree of infertility, a small subset of these PCOS-like female mice can get pregnant and their offspring show delayed puberty and increased testosterone as adults. This PCOS-like lean mouse model is a useful tool to study the pathophysiology of PCOS and the offspring from these PCOS-like dams.

We describe the development of a lean PCOS-like mouse model with&#160;dihydrotestosterone pellet to study the pathophysiology of PCOS and the offspring from these PCOS-like dams.

Hyperandrogenemia plays a critical role in reproductive and metabolic function in females and is the hallmark of polycystic ovary syndrome. Developing a ...

Developmental Biology

Spontaneous Murine Model of Anaplastic Thyroid Cancer

Anaplastic thyroid cancer (ATC) is a rare but lethal malignancy with a dismal prognosis. There is an urgent need for more in-depth research on the carcinogenesis and development of ATC, as well as therapeutic methods, since standard treatments are essentially depleted in ATC patients. However, low prevalence has hampered thorough clinical studies and the collection of tissue samples, so little progress has been achieved in creating effective treatments. We used genetic engineering to create a conditionally inducible ATC murine model (mATC) in a C57BL/6 background. The ATC murine model was genotyped by TPO-cre/ERT2; BrafCA/wt; Trp53ex2-10/ex2-10 and induced by intraperitoneal injection with tamoxifen. With the murine model, we investigated the tumor dynamics (tumor size ranged from 12.4 mm2 to 32.5 mm2 after 4 months of induction), survival (the median survival period was 130 days), and metastasis (lung metastases occurred in 91.6% of mice) curves and pathological features (characterized by Cd8, Foxp3, F4/80, Cd206, Ki67, and Caspase-3 immunohistochemical staining). The results indicated that spontaneous mATC possesses highly similar tumor dynamics and immunological microenvironment to human ATC tumors. In conclusion, with high similarity in pathophysiological features and unified genotypes, the mATC model resolved the shortage of clinical ATC tissue and sample heterogeneity to some extent. Therefore, it would facilitate the mechanism and translational studies of ATC and provide an approach to investigate the treatment potential of small molecular drugs and immunotherapy agents for ATC.

Here, we present a standard pipeline to obtain murine ATC tumors by spontaneous genetically engineered mouse models. Further, we present tumor dynamics and pathological information about the primary and metastasized lesions. This model will help researchers to understand tumorigenesis and facilitate drug discoveries.

Anaplastic thyroid cancer (ATC) is a rare but lethal malignancy with a dismal prognosis. There is an urgent need for more in-depth research on the ...

Assessing Motor Performance in TH-Related Murine Models Using the Ladder Beam Test

A Versatile, Behavioral Method to Investigate Thyroid Hormone Effects on Cerebellar Function

Thyroid hormone (TH) action is essential during the development of the central nervous system, including the cerebellum. In case of TH deficiency in early life such as congenital hypothyroidism, patients display neurological disorders such as cognitive retardation and motor deficits. There are various studies using mouse models with tissue- or cell-specific TH deficiency to investigate the role of TH in the cerebellum. Compared to generalized congenital hypothyroid mice, cerebellar cell-specific TH-deficient mice display milder and subtler ataxic features, making the assessment of motor function difficult when using conventional tests such as the rotarod test. 
Due to the need for an alternative tool to assess motor function in TH-related animal models, we developed a versatile behavioral method called the "ladder beam test," in which we can design the various ladder tests depending on the severity of ataxia in model mice. We utilized transgenic mice expressing a dominant-negative TH receptor specifically in the cerebellar Purkinje cell, a sole output neuron in the cerebellar cortex modulating motor performance. The newly-built ladder beam test successfully detected robust impairments in motor performance in the transgenic mice at a greater level compared to the rotarod test. Disruption of motor learning was also detected in the ladder beam test but not in the rotarod test. The protocol with this novel behavioral apparatus can be applied to other animal models that may show mild ataxic phenotype to examine subtle changes in cerebellar function.

Author Spotlight: Accurately Assessing Thyroid Hormone-Driven Motor Alterations in Mouse

Here we present a protocol for a versatile behavior test developed recently, the ladder beam test. This test has the advantage of detecting subtle cerebellar ataxia caused by a defect of thyroid hormone action in the central nervous system over the conventional behavior tests assessing motor performance.

Thyroid hormone (TH) action is essential during the development of the central nervous system, including the cerebellum. In case of TH deficiency in early ...

Behavior

Generation of a Mouse Spontaneous Autoimmune Thyroiditis Model

In recent years, Hashimoto's thyroiditis (HT) has become the most common autoimmune thyroid disease. It is characterized by lymphocyte infiltration and the detection of specific serum autoantibodies. Although the potential mechanism is still not clear, the risk of Hashimoto's thyroiditis is related to genetic and environmental factors. At present, there are several types of models of autoimmune thyroiditis, including experimental autoimmune thyroiditis (EAT) and spontaneous autoimmune thyroiditis (SAT).
EAT in mice is a common model for HT, which is immunized with lipopolysaccharide (LPS) combined with thyroglobulin (Tg) or supplemented with complete Freund's adjuvant (CFA). The EAT mouse model is widely established in many types of mice. However, the disease progression is more likely associated with the Tg antibody response, which may vary in different experiments.
SAT is also widely used in the study of HT in the NOD.H-2h4 mouse. The NOD.H2h4 mouse is a new strain obtained from the cross of the nonobese diabetic (NOD) mouse with the B10.A(4R), which is significantly induced for HT with or without feeding iodine. During the induction, the NOD.H-2h4 mouse has a high level of TgAb accompanied by lymphocyte infiltration in the thyroid follicular tissue. However, for this type of mouse model, there are few studies to comprehensively evaluate the pathological process during the induction of iodine.
A SAT mouse model for HT research is established in this study, and the pathologic changing process is evaluated after a long period of iodine induction. Through this model, researchers can better understand the pathological development of HT and screen new treatment methods for HT.

Several types of animal models of Hashimoto's thyroiditis have been established, as has spontaneous autoimmune thyroiditis in the NOD mouse. H-2h4 mice are a simple and reliable model for HT induction. This article describes this approach and evaluates the pathological process for a better understanding of the SAT murine model.

In recent years, Hashimoto's thyroiditis (HT) has become the most common autoimmune thyroid disease. It is characterized by lymphocyte infiltration and ...

Immunology and Infection

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

Biology

Developing an Ovariectomized Mouse Model with Elevated FSH Level

Exploring Independent Effects of Follicle-Stimulating Hormone In Vivo in a Mouse Model

During the transition from a reproductive to a nonreproductive phase (menopause), many women experience significant physiological and pathological changes, including decreased bone mass, increased blood lipids, and increased visceral adiposity. Levels of follicle-stimulating hormone (FSH) rise during the menopausal transition. Many studies have shown that FSH in various extragonadal tissues and organs is associated with the pathogenesis of multiple diseases. Thus, building an animal model that can help study the independent effects of FSH in vivo is particularly important. In this study, C57BL/6 female mice were ovariectomized and supplemented with estradiol valerate (OVX + E2) to eliminate the effect of the hypothalamic-pituitary-gonadal axis. The OVX + E2 mice received solvent (N.S.) or different doses of recombinant FSH via intraperitoneal injection to create a mouse model (OVF) characterized by relatively stable estrogen and rising FSH levels. Thus, we successfully generated an experimental mouse model to mimic the early stage of menopause transition, characterized by elevated serum FSH levels. The OVF model has the advantages of being stable, low cost, and easy to operate, which is suitable for studies to explore the extragonadal actions of FSH. Here, we describe detailed protocols for the mouse OVF model.

Author Spotlight: Investigating the Relationship Between FSH and Pathophysiological Changes in Perimenopausal Women - Insights from a Mouse Model 

Follicle-stimulating hormone (FSH) in various extragonadal tissues and organs is associated with the pathogenesis of multiple diseases. The ovariectomized and FSH-treated mouse model (OVF) can be used to explore the extragonadal actions of FSH.

During the transition from a reproductive to a nonreproductive phase (menopause), many women experience significant physiological and pathological ...

Establishment of Rat Models Mimicking Gender-affirming Hormone Therapies

Transgender (TG) people are individuals whose gender identity and sex assigned at birth do not match. They often undergo gender-affirming hormone therapy (GAHT), a medical intervention that allows the acquisition of secondary sex characteristics more aligned with their individual gender identity, providing consistent results in the improvement of numerous socio-psychological variables. However, GAHT targets different body systems, and some side effects are recorded, although not yet fully identified and characterized. Therefore, TG people undergoing GAHT may be considered as a susceptible sub-group of population and specific attention should be paid in the frame of risk assessment, e.g., through the use of targeted animal models. The present work describes the procedures set to implement two rat models mimicking GAHT: the demasculinizing-feminizing model (dMF) mimicking the GAHT for TG women and the defeminizing-masculinizing model (dFM) mimicking the GAHT for TG men. The models have&#160;been implemented through the administration of the same hormones used for human GAHT, namely, &#946;-estradiol plus cyproterone acetate for dMF and testosterone for dFM, by the same routes of exposure for a 2 week period. Rats are checked daily during the treatment to evaluate health status and potentially aggressive behaviors. At sacrifice, blood and target tissues have been sampled and stored for biochemical, molecular, and histopathological analysis. Sex-specific parameters, namely, sperm count and clitoral dimensions, have also been evaluated. In addition, CYP450 isoforms, exclusively and/or preferentially expressed in male and female rat liver, are identified and characterized as novel biomarkers to verify the success of GAHT and to set the model. Thyroid involvement has also been explored as a key target in the endocrine system.

The protocol describes the development of two rodent models mimicking gender-affirming hormone therapies through subcutaneous administration of testosterone or estradiol plus cyproterone acetate (used in human therapies for transgender people): setting of the doses, identification of relevant biomarkers, and evaluation of the effects.

Transgender (TG) people are individuals whose gender identity and sex assigned at birth do not match. They often undergo gender-affirming hormone therapy ...

In vivo Characterization of Endocrine Disrupting Chemical Effects via Thyroid Hormone Action Indicator Mouse

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Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice

An In Vivo Estrogen Deficiency Mouse Model for Screening Exogenous Estrogen Treatments of Cardiovascular Dysfunction After Menopause

A Hyperandrogenic Mouse Model to Study Polycystic Ovary Syndrome

Spontaneous Murine Model of Anaplastic Thyroid Cancer

A Versatile, Behavioral Method to Investigate Thyroid Hormone Effects on Cerebellar Function

Generation of a Mouse Spontaneous Autoimmune Thyroiditis Model

An Ex vivo Culture System to Study Thyroid Development

Exploring Independent Effects of Follicle-Stimulating Hormone In Vivo in a Mouse Model

Establishment of Rat Models Mimicking Gender-affirming Hormone Therapies