American Thyroid Association Guide to investigating thyroid hormone economy and action in rodent and cell models - PubMed (original) (raw)

Guideline

doi: 10.1089/thy.2013.0109. Epub 2013 Dec 12.

Grant Anderson, Douglas Forrest, Valerie Anne Galton, Balázs Gereben, Brian W Kim, Peter A Kopp, Xiao Hui Liao, Maria Jesus Obregon, Robin P Peeters, Samuel Refetoff, David S Sharlin, Warner S Simonides, Roy E Weiss, Graham R Williams; American Thyroid Association Task Force on Approaches and Strategies to Investigate Thyroid Hormone Economy and Action

Affiliations

Guideline

American Thyroid Association Guide to investigating thyroid hormone economy and action in rodent and cell models

Antonio C Bianco et al. Thyroid. 2014 Jan.

Abstract

Background: An in-depth understanding of the fundamental principles that regulate thyroid hormone homeostasis is critical for the development of new diagnostic and treatment approaches for patients with thyroid disease.

Summary: Important clinical practices in use today for the treatment of patients with hypothyroidism, hyperthyroidism, or thyroid cancer are the result of laboratory discoveries made by scientists investigating the most basic aspects of thyroid structure and molecular biology. In this document, a panel of experts commissioned by the American Thyroid Association makes a series of recommendations related to the study of thyroid hormone economy and action. These recommendations are intended to promote standardization of study design, which should in turn increase the comparability and reproducibility of experimental findings.

Conclusions: It is expected that adherence to these recommendations by investigators in the field will facilitate progress towards a better understanding of the thyroid gland and thyroid hormone dependent processes.

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Figures

<b>FIG. 1.</b>

**FIG. 1.

Microscopic structure of the mouse thyroid. (A) Hematoxylin and eosin (H&E) staining. (B) Periodic acid Schiff (PAS) staining. Mice were euthanized, and the thyroids dissected, fixed in buffered formalin, and embedded in paraffin. Thyroid sections (5 μm) were mounted on glass slides, de-paraffinated, and hydrated. For histological analysis, sections were stained with H&E, following a standard protocol. Glycoproteins were detected using PAS staining. Sections were stained with 0.5% periodic acid for 30 minutes and with Schiff's reagent for 20 minutes and then rinsed in running tap water for 5 minutes. Nuclei were counterstained with hematoxylin for 3 minutes. Sections were rinsed in running tap water, dehydrated, cleared, and mounted. Reproduced with permission from Senou et al. (20).

<b>FIG. 2.</b>

**FIG. 2.

Mouse thyroid transmission electron microscopy. Thyroid lobes were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 1.5 hours, post-fixed in 1% osmium tetroxide for 1 hour, and embedded in LX112 resin (Ladd Research Industries, Burlington, VT). (A) Thin sections (0.5 μm) were stained with toluidine blue and analyzed for morphology by light microscopy. (B) Ultrathin sections were prepared and stained with uranyl acetate and lead citrate and examined with an electron microscope Zeiss EM169 (Carl Zeiss, Oberkochen, Germany). (C) Ultrastructural distribution of 127I by secondary ion mass spectrometry (SIMS) imaging. Semi-thin sections were prepared, and the ultrastructural distribution of the iodide natural isotope (127I) was obtained through imaging by SIMS, using the NanoSIMS 50 system. Maps were acquired under standard analytic conditions: a Cs+ primary beam with impact energy of 16 keV and a probe with current intensity of 1 pA. The analyzed surface was 30×30 μm. Under these conditions, a lateral resolution of 100 nm is expected. All images were acquired in 256×256 pixels with a counting time of 20 milliseconds per pixel. White areas correspond to iodine detection. 127I is homogeneously distributed in the follicular lumina and in a few intracytoplasmic vesicles. Reproduced with permission from Senou et al. (20).

<b>FIG. 3.</b>

**FIG. 3.

Detection of thyroglobulin and iodinated thyroglobulin in the mouse thyroid by immunohistochemistry. (A) Thyroglobulin was detected on paraffin sections using anti-thyroglobulin rabbit polyclonal antibody (Dako) diluted 1/1500 and incubated overnight. (B) Iodinated thyroglobulin was detected using mouse monoclonal antibody (B1) diluted 1/3000 and incubated overnight. Negative controls included the replacement of primary antibody by the preimmune serum or absence of the primary antibody. Reproduced with permission from Senou et al. (20).

<b>FIG. 4.</b>

**FIG. 4.

Detection of dual oxidase (DUOX) and thyroperoxidase in the mouse thyroid by immunohistochemistry. (A) DUOX was detected on frozen sections with rabbit polyclonal antibody diluted 1/3000 and incubated overnight. Positivity is observed at the apical pole (arrows, inset). (B) thyroperoxidase was detected on paraffin sections with rabbit antibody Loαd TPO 821, 4 μg/mL and incubated for 3 hours. Reproduced with permission from Senou et al. (20).

<b>FIG. 5.</b>

**FIG. 5.

Detection of thyroglobulin in the mouse thyroid by immunogold electron microscopy. After wash with phosphate-buffered saline–bovine serum albumin (PBS-BSA 1%), ultrathin sections (0.1 μm) were incubated overnight with a rabbit polyclonal anti-thyroglobulin antibody (1/300, DAKO). Sections were then rinsed and incubated for 30 minutes with a 12-nm colloidal gold affinity pure goat anti-rabbit IgG (Jackson, 111-205-144, lot no. 71647). Sections were postfixed with 2.5% glutaraldehyde for 5 minutes and counterstained. They were examined with a Zeiss 109 transmission electron microscope. (A) Negative control obtained by omission of primary antibody. (B) Thyroglobulin was detected as small gold particles in the colloid limited by flat epithelial cells. Reproduced with permission from Senou et al. (20).

<b>FIG. 6.</b>

**FIG. 6.

Thyroid imaging using 124I-iodide in vivo. (A) Biodistribution of 124I-iodide in thyroid of genetically modified mice in which thyroid iodide uptake is suppressed by induction of a transgene; 1 week later suppression is relieved and iodide uptake is normalized. Top panels: representative images of uninduced mice, 1 week on doxycyclin to induce the transgene, followed by 1 week off doxycyclin. Positron emission tomography (PET) imaging was performed using an R4 microPET scanner (Concorde Microsystems) with Na124I produced on the MSKCC EBCO TR 19-9 (Advanced Cyclotron Systems Inc.) using 16 MeV protons on a tellurium-124 target. Mice were injected via tail vein with 1.7–2.0 MBq (45–55 μCi) of Na124I. Mice were imaged 24, 48, and 72 hours later under inhalational isoflurane anesthesia (Forane; Baxter Healthcare) at 1 L/min. List-mode data were acquired for 5 minutes using an energy window of 250–750 keV and a coincidence timing window of 6 nanoseconds, histogrammed into two-dimensional (2D) projected data by Fourier rebinning, and reconstructed by filter back-projection using a cut-off frequency equal to the Nyquist frequency. The image data were normalized to correct for nonuniformity of response of the PET, dead-time count losses, 124I positron branching ratio, and physical decay to the time of injection, but no attenuation, scatter, or partial-volume averaging correction was applied. (B) Quantification of thyroid 124I-iodide uptake in mice treated with the indicated conditions. ***p<0.001. An empirically determined system calibration factor (in units of [μCi/mL]/[cps/voxel]) was used to convert reconstructed voxel count rates to activity concentrations. The resulting image data were then normalized to the administered activity to parameterize images in terms of the percentage of the injected dose per gram of tissues (%ID/g). Manually drawn 2D regions of interest (ROIs) or three-dimensional (3D) volumes of interest (VOIs) were used to determined the %ID/g (decay corrected to the time of injection) in various tissues. Image visualization and analysis were performed using ASIPro VM software (Concorde Microsystems). (C) Representative gross appearance of thyroid glands at the indicated times. The boundaries of the thyroid are demarcated by dashed lines. Scale bar: 1 mm. ID/g, injected dose/gram Reproduced with permission from Chakravarty et al. (58).

<b>FIG. 7.</b>

**FIG. 7.

High-frequency ultrasonography (HFUS) of the mouse thyroid. (A) Representative image of mouse thyroid using HFUS and its anatomic correlation with (B) histological transversal images of the subhyoid and tracheal regions. Visible structures include: 1, tracheal cartilage ring; 2, salivary gland; 3, sternohyoideus and sternothyroideus muscles; 4, sternomastoideus muscle; 5, thyroid lobes; 6, common carotid arteries; 7, deep prevertebral muscles scalenus and longus colli; and 8, skin. A Vevo 770 microimaging system (Visualsonics, Toronto, Ontario, Canada) with a single element probe of center frequency of 40 MHz is used. The transducer has an active face of 3 mm, a lateral resolution of 68.2 μm, axial resolution of 38.5 μm, focal length of 6 mm, mechanical index 0.14, and a dynamic range 52 dB. A probe with lower frequency and more penetration depth can also be used (30 MHz center frequency single element with focal depth 12.7 mm, lateral resolution of 115 μm, axial resolution of 55 μm). HFUS is performed under general anesthesia. In this study, mice were anesthetized using 1.5%–2% isoflurane vaporized in oxygen on a heated stage, with constant monitoring of their body temperature. Area of interest was shaved (neck and the high thorax) with a depilatory cream to obtain a direct contact of the ultrasound gel to the skin of the animal minimizing ultrasound attenuation. To provide a coupling medium for the transducer warm gel was used. An outer ring of thick gel (Aquasonic 100; Parker Laboratories, Orange, NJ) was filled with a thinner gel (echo Gel 100; Eco-Med Pharmaceutical, Mississauga, Canada) over the region of interest. Reproduced with permission from Mancini et al. (61).

<b>FIG. 8.</b>

**FIG. 8.

Supply and metabolism of thyroid hormones affect negatively and positively T3-regulated genes in the brain. To construct this figure, the authors used individual reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) data from T3-regulated genes to calculate the fold change relative to the wild-type (WT) values, and plotted the Log2FC (fold change) to make the results quantitatively comparable. The data were represented in a box-and-whiskers (5%–95%) plot. Statistical significance between each group and the WT was calculated by one-way ANOVA. For the positive genes, _F_5,537=272, p<0.0001. For the negative genes, _F_5,400=145, p<0.0001. *p<0.05; **p<0.01; ***p<0.001. Reproduced with permission from Hernandez et al. (492).

<b>FIG. 9.</b>

**FIG. 9.

Strategies to measure individualized deiodinase activity in the presence of other deiodinases. (A) Two strategies to assess type II deiodinase (D2) activity in the presence of type I deiodinase (D1; e.g., in the human thyroid): (i) use 1 mM 6-n-propyl-2-thiouracil (PTU) to inhibit D1 or (ii) use 1–2 nM [125I]T4 in the presence or absence of 100 nM cold thyroxine (T4). (B) To measure D2 activity in D2/D3 co-expressing tissues (e.g., the brain), use 1000 nM cold T3 to saturate D3. (C) To measure D2 activity in D1/D2/D3 co-expressing tissues (e.g., rodent gonads, placenta, cerebrum, and skin), inhibit D1 with 1 mM PTU and saturate D3 with 1000 nM T3.

<b>FIG. 10.</b>

**FIG. 10.

In vitro modeling of thyroid hormone deiodination and transport in the brain. (A) Schematic representation of the Transwell System in which an insert is placed on a six-well plate and cells (D2-expressing H4 glial cells) are seeded inside the insert; D3-expressing neuroblastoma cells (SK-N-AS) are seeded at the bottom of the six-well plate. After cells are seeded, both cell types are kept separated overnight and then placed together in the same multiwell plate as indicated. (B) At the end of the incubation medium samples are collected, extracted and processed through a UPLC or HPLC connected to the flow gamma counter to separate and quantify the activity of each iodothyronine. The red arrow indicates the pathway completed by the column eluate through the gamma counter; courtesy Dr. Antonio Bianco. (C) Chromatograms of H4 cell medium at the indicated times after addition of 125I-T4. Typical peaks of 125-T3 and 125I are shown after 24 hours. (D) Same as in (C), except that 125I-T3 was added to cultures of SK-N-AS cells and 125I-T2 and 125I-T1 peaks are visualized. (E) Same as in (C), except that 125I-T4 was added to H4 and SK-N-AS cocultures and the indicated peaks are visualized. UPLC, ultrahigh performance liquid chromatography; HPLC, high performance liquid chromatography; T1, 5′-monoiodothyronine. Reproduced with permission from Freitas et al. (173).

<b>FIG. 11.</b>

**FIG. 11.

Dissection of the rodent thyroid gland. (A) Surgical incision, (B) isolate the salivary glands, (C) dissociate the muscles, (D) free the trachea, (E) section the lateral muscles, (F) slide a needle underneath the trachea, revealing the thyroid gland. Modified with permission from a web posting by Prof. Emeritus Jean-Pierre Herveg and Christian Regaert.

<b>FIG. 12.</b>

**FIG. 12.

Dio1 (type 1 deiodinase) is a representative T3-responsive gene in the liver. Northern blot analysis showing Dio1 mRNA levels in response to normal diet (ND), hypothyroid (LID, low-iodine diet and antithyroid agents), and hyperthyroid (0.5 T3) conditions. Dio1, upper panel, and control glyceraldehyde-3-phosphate dehyrogenase (G3pdh), lower panel. In WT mice (+/+), Dio1 mRNA is suppressed by hypothyroid conditions and induced by hyperthyroid conditions. Induction is defective in _Thrb_-deleted mice (−/−). Treatments: ND, normal diet; LID, hypothyroid groups (0.05% methimazole [MMI] and 1% potassium perchlorate in drinking water and low iodine chow, for 4 weeks); 0.5 T3, hyperthyroid groups (same as LID but with T3 added to drinking water at concentration of 0.5 mg/mL for an additional 8 days or more). Quantitation: Dio1 mRNA level in each condition is noted numerically below each lane relative to the level in +/+ mice on normal diet (assigned a value=1.0). Dio1 value is normalized to control G3pdh signal; UD, undetectable Dio1. Signals were quantified by phophorimager analysis of major bands.

<b>FIG. 13.</b>

**FIG. 13.

An example of using RT-qPCR to analyze a T3-responsive gene. The qPCR amplification plots indicate change in the mRNA level for the T3-responsive gene OPN1LW/MW (red/green opsin) in the human retinoblastoma cell line WERI following treatment with increasing T3 concentrations spanning the physiological range. The increased SYBER Green detection (ΔRn; SYBER Green fluorescence [reporter] normalized to background) following each PCR cycle demonstrates the accumulation of a PCR amplicon. (A) Amplification plots of the internal control (reference) gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Note that at the critical threshold (Ct, dotted line across graph) no difference is detected between the T3 doses for GAPDH indicating that this reference gene is not responsive to T3 treatment. (B) Amplification plots of the T3 target gene OPN1LW/MW. Note that as the T3 concentration increases, the PCR cycle needed to reach the threshold decreases indicating the presence of higher levels of mRNA induced by T3. A plateau in T3-induced expression of OPN1LW/MW is reached at 50 nM T3. (C) Melting curve analysis of OPN1LW/MW qPCR amplifications. Note that a single melt peak is observed in a plot of the first negative derivative (−R; fluorescence over time; i.e., the change rate) against temperature indicating that the increase in SYBER Green fluorescence detected is likely derived from a single PCR amplicon. Data are unpublished observations from D.S. Sharlin and D. Forrest and are consistent with previous reports (752).

<b>FIG. 14.</b>

**FIG. 14.

T3 signaling represented by β-galactosidase staining (blue) in coronal brain sections in postnatal day 5 mice carrying the FINDT3 reporter transgene. (A, D) FINDT3 reporter on wild-type (WT/FINDT3) mice; (B, E) FINDT3 reporter in mice lacking type 3 deiodinase, a thyroid hormone-inactivating enzyme (D3KO/FINDT3). (C, F) No β-galactosidase staining was detected in sections from control mice (WT) not carrying the transgene. S, septum; Pi, piriform cortex; MCx, motor cortex; SCx, sensory cortex. Note increased β-galactosidase activity in D3KO/FINDT3 mice in the piriform, motor, and sensory cortex, consistent with increased T3 exposure in mice lacking type 3 deiodinase. Cryosections (50 μm thick) were stained using 1 mg/mL of X-gal (5-bromo-4-chloro-3-indolyl-d-galactopyranoside), a colorigenic substrate for β-galactosidase. Reproduced and adapted from Hernandez et al. (278) with permission. © 2013, The Endocrine Society.

<b>FIG. 15.</b>

**FIG. 15.

In vivo transfection of rat cardiomyocytes by direct DNA injection. The animal is anesthetized with a mixture of N2O (0.2 L/min), O2 (0.2 L/min), and sevoflurane (2%–3%), and the heart is exposed through a right-lateral thoracotomy. The free wall of the right and/or the left ventricle is injected three to four times each, delivering a total of 20 μg of reporter plasmid(s)/ventricle in 100 μL of saline. In this example injection of the right ventricle is shown, with the 29-gauge needle bent at the tip at an almost right angle to allow easy injection of the thin right ventricle wall. The thorax is then closed and the animal is sacrificed 5 days later. Expression of luciferase reporter and normalization genes is determined in ventricle homogenates. Courtesy of Dr. Warner Simonides.

<b>FIG. 16.</b>

**FIG. 16.

Use of the Comprehensive Laboratory Animal Monitoring System to perform continuous indirect calorimetry in mice. (A) Two independent metabolic cages that are connected to a computer for recording and data analyses; (B) VO2 during a 24 hour time period showing the nocturnal (shaded area) increase in metabolism; (C) respiratory quotient (RQ) during the same period of time, depicting the nocturnal (shaded area) increase in RQ when animals are kept on a carbohydrate-enriched diet; (D) dramatic increase in VO2 during 48 hours cold exposure (shaded area); (E) decreased VO2 in hypothyroid mice that were kept on 0.1% MMI for 60 days; (F) RQ in the animals shown in (E). Courtesy of Drs. Antonio Bianco and Tatiana Fonseca.

<b>FIG. 17.</b>

**FIG. 17.

Measuring O2 consumption and the rate of medium acidification in cultured cells. Extracellular flux analysis is performed using XF Analyzers (Seahorse Bioscience, Billerica, MA). Cells are plated in monolayer 24- or 96-well format. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) are measured via chemiluminescent sensors applied to disposable cartridge-based probes that are lowered over the cells and a few microliters of media (lower picture), creating a transiently sealed micro-environment. As oxygen is depleted from the media and protons accumulate in the media, the device plots the concentration of O2 and pH in real time (upper picture). The OCR and ECAR are calculated as the slopes of the O2 and pH versus time curves. Modified with permission from a web posting by Seahorse Bioscience; courtesy of Dr. Brian Kim.

<b>FIG. 18.</b>

**FIG. 18.

Study of the interscapular brown adipose tissue (iBAT) thermal response to norepinephrine (NE) infusion. (A, B) A rat (or mouse) is anesthetized, and the iBAT pad is exposed through a surgical incision. A thermistor is placed under the iBAT pad and secured with a stitch; a rectal thermistor is inserted in the colon for measurement of core temperature (not shown). The right jugular vein is cannulated and connected to an infusion pump for infusion of catecholamines or other molecules. Temperatures are measured continuously before and during infusion. Courtesy of Dr. Miriam O. Ribeiro. (C) Plasma NE levels during infusion in intact and Tx rats; (D) iBAT temperature during infusion in intact and Tx rats. Infusion lasted for 60 minutes, and the temperature data points indicate the difference between baseline and maximum peak achieved during infusion. Reproduced with permission from Ribeiro et al. (627).

<b>FIG. 19.</b>

**FIG. 19.

Scheme for collection of bone samples, tissue fixation, and methods of analysis. The usage of this chart can maximize phenotype information obtained from juvenile and adult mice. qBSE-SEM, quantitative back-scattered electron-scanning electron microscopy. Courtesy of Dr. Graham Williams.

<b>FIG. 20.</b>

**FIG. 20.

Instron5534 load frame apparatus for destructive three-point bend testing of bone strength. Custom mounts incorporate rounded supports and loading pins that minimize cutting and shear forces, thus enabling biomechanical parameters of bone strength to be determined accurately. Courtesy of Dr. Graham Williams.

Comment in

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