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

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. Important clinical practices in use today
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  American Thyroid Association Guide to InvestigatingThyroid Hormone Economy and Actionin Rodent and Cell Models Report of the American Thyroid Association Task Forceon Approaches and Strategies to Investigate Thyroid Hormone Economy and Action Antonio C. Bianco, 1, * Grant Anderson, 2 Douglas Forrest, 3 Valerie Anne Galton, 4 Bala´zs Gereben, 5 Brian W. Kim, 1 Peter A. Kopp, 6 Xiao Hui Liao, 7 Maria Jesus Obregon, 8 Robin P. Peeters, 9 Samuel Refetoff, 7 David S. Sharlin, 10 Warner S. Simonides, 11 Roy E. Weiss, 7 and Graham R. Williams 12 Background:  An in-depth understanding of the fundamental principles that regulate thyroid hormone homeostasisis 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, hyper-thyroidism, 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 theAmerican Thyroid Association makes a series of recommendations related to the study of thyroid hormoneeconomy and action. These recommendations are intended to promote standardization of study design, whichshould 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 facilitateprogress towards a better understanding of the thyroid gland and thyroid hormone dependent processes. INTRODUCTION O ver the past  150  years,  investigators utilizing animaland cell culture–based experimental models haveachieved landmark discoveries that have shaped our under-standing of thyroid physiology and disease. From the iden-tification of the long-acting thyroid stimulator to thediscovery of antithyroid drugs, basic research studies haveprovided the fundamentals upon which our clinical diag-nostic and therapeutic tools are based. Tens of thousands of publications indexed on PubMed ( fea-ture cells or small animals made hypothyroid or thyrotoxic.The great similarities in multiple aspects of thyroid physi-ology between humans and small rodents have facilitated therapid translation of experimental findings to the clinicalrealm. At the same time, fundamental interspecies differencesdo exist and must be carefully accounted for if the experi-mental findings are to have clinical relevance. 1 Division of Endocrinology, Diabetes and Metabolism, University of Miami Miller School of Medicine, Miami, Florida. 2 Department of Pharmacy Practice and Pharmaceutical Sciences, College of Pharmacy, University of Minnesota Duluth, Duluth, Minnesota. 3 Laboratory of Endocrinology and Receptor Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland. 4 Department of Physiology and Neurobiology, Dartmouth Medical School, Lebanon, New Hampshire. 5 Department of Endocrine Neurobiology, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary. 6 Division of Endocrinology, Metabolism, and Molecular Medicine, and Center for Genetic Medicine, Feinberg School of Medicine,Northwestern University, Chicago, Illinois. 7 Section of Adult and Pediatric Endocrinology, Diabetes, and Metabolism, The University of Chicago, Chicago, Illinois. 8 Institute of Biomedical Investigation (IIB), Spanish National Research Council (CSIC) and Autonomous University of Madrid, Madrid,Spain. 9 Division of Endocrinology, Department of Internal Medicine, Erasmus Medical Center, Rotterdam, The Netherlands. 10 Department of Biological Sciences, Minnesota State University, Mankato, Minnesota. 11 Laboratory for Physiology, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, The Netherlands. 12 Department of Medicine, Imperial College London, Hammersmith Campus, London, United Kingdom.*Chair; all other authors are listed in alphabetical order. THYROIDVolume 24, Number 1, 2014 ª  American Thyroid AssociationDOI: 10.1089/thy.2013.0109 88  While certain experimental techniques have been widelyaccepted and adapted following their use in papers gener-ated by influential labs, lack of standardization has un-doubtedly promoted heterogeneity of results. Becausecertain experimental variables may have unknown biologi-calthresholdlevels,lackofstandardizationmayleadtohavehighly discordant results in different studies examining thesame issue.To address this lack of standardization, the AmericanThyroid Association (ATA) convened a panel of specialists inthe field of basic thyroid research to define consensus strate-gies and approaches for thyroid studies in rodents and in cellmodels. This task force was charged with reviewing the lit-erature first to determine which experimental practices could benefit from standardization and second to identify criticalexperimental variables that demand consideration whenthyroid studies are being designed. The conclusions of thetask force are presented in this document as ‘‘AmericanThyroid Association Guide to Investigating Thyroid Hor-mone Economy and Action in Rodent and Cell Models.’’ The70 recommendations and their accompanying commentariesexamine topics ranging from ‘‘making cells hypothyroid’’ to‘‘how to study the thyrotoxic bone.’’ While far from exhaus-tive, these recommendations touch on certain fundamentalaspects of thyroid research relevant for all investigators inthe field.Each recommendation in this guide promotes a particularexperimental approach based on criteria including theprevalence of the approach, with widely used techniques being given precedence, and in particular whether the ap-proach has been shown to lead to reproducible results instudies by independent investigators. Because head-to-headscientific comparisons of experimental methods in this fieldare virtually nonexistent, these recommendations cannot begraded on the basis of strength of evidence in the fashion of clinical guidelines; indeed, all would be graded as ‘‘expertopinion.’’ At the same time, unlike clinical guidelines, themain goal of these recommendations and their accompany-ing commentaries is not to identify the single best practice  per se , but instead to encourage investigators to choosestandard approaches; for example, avoiding random treat-ment doses or methods of thyroid hormone administration,which would only serve to limit comparison with previousstudies.The practical nature of recommendations should becomereadily apparent to the reader. This document is intendedto serve as a reference for investigators, assisting them inmaking design choices that avoid well-known pitfallswhile increasing standardization in the field. As part of this practical approach, reference credit is often given tomanuscripts in which the technical details are most clearlyor comprehensively explained, rather than the first publi-cation to use a technique. In addition, emphasis wasplaced on contemporary approaches, rather than historicalstrategies, such that the document illustrates what is cur-rently available for the contemporary study of thyroidhormone homeostasis, metabolism, and action. It is theposition of the ATA that animal studies should be per-formed in accordance with all applicable ethical standardsand research protocols approved by local institutionalanimal committees. METHODS OF DEVELOPMENTOF RECOMMENDATIONS Administration  TheATAExecutiveCouncilselectedachairpersontoleadthetask force, and this individual (A.C.B.) identified the other 14members of the panel in consultation with the ATA board of directors.Membershiponthepanelwasbasedonexpertiseandprevious contributions to the thyroid field. Panel members de-claredwhethertheyhadanypotentialconflictofinterestduringthe course of deliberations. Funding for the guide was derivedfrom the ATA and thus the task force functioned withoutcommercial support.To develop a useful document, the task force first devel-oped a list of the topics that would be most helpful and themost important questions that scientists working in the thy-roid field might pose when planning an experiment or inter-preting experimental data. Each of the 10 topics wasdistributed to a primary writer who used his or her knowl-edge of the subject as well as a systematic PubMed andGoogle Scholar search for primary references, reviews, andother materials publicly available before December 2012, todevelop a set of recommendations. All drafts were reviewedand edited by the chair for consistency and sent back to theprimary writers for review; in some cases multiple iterationstook place until the recommendation was finalized. A pre-liminarydraftof eachrecommendation wasthen reviewed bysecondary and tertiary reviewers within the group who thenprepared additional critiques. These were addressed by theprimary writer and sent back to the chair. All drafts weremerged and posted at a protected web address available onlyto the task force members and ATA office. This documentremained available for periodic review by the task force atlarge, with critiques and suggestions sent back to the chairthat updated the document. In a few cases the chair asked foroutside experts to critically review specific recommendationsgiven their expertise in a focused area. Their comments andsuggestions werethen worked intothe master document, andthe contributions are acknowledged at the end of this article.The panel agreed that recommendations would be basedon consensus of the panel. Task force deliberations took placelargely through electronic communication. There were also afew meetings of the authors and telephone conference calls. Presentation, Approval, and Endorsement of Recommendations  ThestructureofourrecommendationsispresentedinTable1. Specific recommendations are presented within the main body of the text and in many cases broken down in subitemsidentified by letters. The page numbers and the locationkey can be used to quickly navigate to specific topics andrecommendations.Priortotheinitialsubmissionoftheseguidelines,theywereapproved by the board and executive committee of the ATAand afterwards submitted to the membership of the ATA inearly 2013 for comments and suggestions. This feedback wasconsidered in the further preparation of the document thatwas submitted for publication. Subsequent to the document being accepted for publication in  Thyroid , it was approved bythe board and executive committee of the ATA. INVESTIGATING THYROID HORMONE ECONOMY AND ACTION 89  Table  1.  Organization of the Task Force’s Recommendations Locationkey Sections and subsections PageLocationkey Sections and subsections Page T 3 , 3,3 ¢ ,5-triiodothyronine; TR, thyroid hormone receptor; PCR, polymerase chain reaction. [A] Assessing the Thyroid Gland  91[A.1] Structure–function relationships 91 Recommendation 1  91[A.2] Thyroid iodide kinetics 93 Recommendation 2  94 Recommendation 3  95[A.3] Thyroid imaging 95 Recommendation 4  95 [B] Assessing Circulating and TissueThyroid Hormone Levels 97[B.1] Serum 97 Recommendation 5  98 Recommendation 6  99 Recommendation 7  100[B.2] Tissue 100 Recommendation 8  100[B.3] Sources of tissue T 3  and TR saturation 100 Recommendation 9  101 [C] Assessing Thyroid HormoneTransport Into Cells 101[C.1] Thyroid hormone transport in vitro 102 Recommendation 10  102 Recommendation 11  103[C.2] Thyroid hormone transport  in vivo  103 Recommendation 12  103 [D] Assessing Thyroid Hormone Deiodination  104[D.1] Identification, expression, andquantification of deiodinases104 Recommendation 13  104 Recommendation 14  105[D.2] Deiodination in intact cells 106 Recommendation 15  106[D.3] Deiodination in perfused organs 106 Recommendation 16  106[D.4] Deiodination in whole animals 107 Recommendation 17  107[D.5] Non-deiodination pathways of thyroid hormone metabolism108 Recommendation 18  109 [E] Inducing Hypothyroidism andThyroid Hormone Replacement 109[E.1] Hypothyroidism in animals 109 Recommendation 19  109 Recommendation 20  110 Recommendation 21  111 Recommendation 22  111 Recommendation 23  112[E.2] Thyroid hormone replacementin animals113 Recommendation 24  113[E.3] Hypothyroidism in cultured cells 114 Recommendation 25  114 [F] Increasing Thyroid HormoneSignaling 114[F.1] Thyrotoxicosis in animals 114 Recommendation 26  115 Recommendation 27  115[F.2] Thyrotoxicosis in cultured cells 115 Recommendation 28  115[F.3] Use of thyroid hormone analogues 116 Recommendation 29  116 [G] Iodine Deficiency and Maternal–FetalTransfer of Thyroid Hormone 117[G.1] Iodine deficiency in rodents 117 Recommendation 30  117 Recommendation 31  118 Recommendation 32  118[G.2] Placental transfer of thyroid hormone 118 Recommendation 33  118 [H] Models of Nonthyroidal Illness  118 Recommendation 34  119 Recommendation 35  119 [I] Assessing Thyroid Hormone Signalingat Tissue and Cellular Levels 119[I.1] Gene expression as a marker of thyroid hormone status120 Recommendation 36  120[I.2] PCR analysis of mRNA expression levels 120 Recommendation 37  120[I.3] Genome-wide analysis of thyroidhormone-responsive mRNA122 Recommendation 38  122[I.4] Mechanisms of gene regulation bythyroid hormone122 Recommendation 39  122 Recommendation 40  123[I.5] Mouse models for indicating thyroidhormone and TR signaling in tissues123 Recommendation 41  124 [J] Assessing Thyroid Hormone Signalingby Way of Systemic Biological Parameters 124[J.1] Central nervous system 125 Recommendation 42  126 Recommendation 43  126 Recommendation 44  127 Recommendation 45  127 Recommendation 46  127 Recommendation 47  128 Recommendation 48  128[J.2] Heart and cardiovascular system 129 Recommendation 49  129 Recommendation 50  129 Recommendation 51  130 Recommendation 52  130 Recommendation 53  131 Recommendation 54  132[J.3] Intermediary metabolism andenergy homeostasis132 Recommendation 55  132 Recommendation 56  135 Recommendation 57  135 Recommendation 58  136 Recommendation 59  137[J.4] Skeletal muscle 137 Recommendation 60  138 Recommendation 61  138 Recommendation 62  138 Recommendation 63  139 Recommendation 64  139 Recommendation 65  139[J.5] Skeleton 140 Recommendation 66  140 Recommendation 67  140 Recommendation 68  140 Recommendation 69  140 Recommendation 70  142 90  ThefinaldocumentwasofficiallyendorsedbytheAmericanAcademy of Otolaryngology–Head and Neck Surgery (AAO-HNS), American Association of Endocrine Surgeons (AAES),American College of Nuclear Medicine (ACNM), Asia andOceania Thyroid Association (AOTA), British Nuclear Medi-cine Society (BNMS), British Thyroid Association (BTA),European Thyroid Association (ETA), International Associa-tion of Endocrine Surgeons (IAES), Italian Endocrine Society(SIE), Japan Thyroid Association (JTA), Korean Society of Head and Neck Surgery (KSHNS), Latin American ThyroidSociety (LATS), Korean Society of Nuclear Medicine (KSNM)and The Endocrine Society (TES). RESULTS[A]  Assessing the Thyroid Gland  Overview.  Studies of function–structure relationship of the thyroid gland, as well as studies of thyroid iodide kineticsand imaging are traditionally employed to assess the thyroidgland. Structural characterization is important to assessfunctional changes such as hypo- and hyperthyroidism andforevaluatingtransformationofthyroidcellsintoamalignantphenotype (1–3). At the same time, the study of thyroidaliodide economy and thyroid imaging are relevant not only tostudies of thyroid hormone synthesis but also to under-standing the effects of environmental toxins such as perchlo-rate or thiocyanate on thyroid economy (4–7). [A.1]  Structure–function relationships  Background.  While the human thyroid consists of a leftand a right lobe that are connected by an isthmus, rodentshave two independent thyroid lobes. The thyroid gland isdivided by connective tissue septa into lobules, each one of these containing from 20 to 40 follicles, the basic functionalunit of the thyroid gland. The follicle is a round or elongatedhollow structure lined by a single layer of polarized cuboidalor flattened follicular cells that is filled with thyroglobulin-containing colloid. It is surrounded by a basal membrane anda rich capillary network with high blood flow (8). The folliclesnormally vary considerable in size, and the follicular cellmorphology is usually monotonous. The height of the cellsvaries according to the functional status of the gland. & RECOMMENDATION 1a Morphometryofthyroidfolliclescanbeusedasanindexof thyroidal activity. Commentary.  The entire gland should always be dis-sected while attached to the trachea and immediately fixedwith 10% neutral buffered formalin for histological and im-munohistochemical analysis. Hematoxylin and eosin (H&E)staining is widely used to assess the thyrocytes, whereasperiodic-acidSchiffstainingstainsthyroglobulinavidlyandiswell suited to highlight follicular protein content and follic-ular structure (Fig. 1) (8). Structural modifications reflectchanges in secretory activity resulting from iodine deficiency(9), chronic cold exposure (10), or treatment with antithyroiddrugs (11). Some follicular cell parameters such as height can be measured under light microscopy using an ocular mi-crometer grid (e.g., in a 1-month-old rat, the epithelial cellheight is about 10 l m) (12). A flat epithelium is hypoactive,while a heightened epithelium is observed in glands in whichthe thyrotropin (TSH) pathway is stimulated (10). The use of computerizedsemiautomaticimageanalysisismoreobjectiveand used widely (13). Such morphometric analysis should befocusedononeofthecentralsectionsofthethyroid(13)thatisrepresentative of the whole lobe (14). The data obtained arereduced by predefined mathematical models that assumethyroid follicles have a spherical shape and follicular cells areoctagons with a square base. This data reduction yields thefollowing parameters: mean follicle circumference; surfacearea and volume; total volume of epithelium and colloid;number of epithelial cell nuclei visible in each follicle; and theheight, surface area, and volume of thyroid epithelial cell,which can be used to estimate the functional state of thethyroid gland. Thus, the activation index, expressed by theepithelial volume/colloid volume ratio, increases as the thy-roid becomes more active, reflecting an increase in the epi-thelial volume and a decrease in the colloid volume (13).Measurement of total cell volume in cultures of primarythyrocytes or cell lines cultured  in vitro  can be performedusing confocal laser-scanning microscopy after cells are loa-ded with octadecylrhodamine B (15,16). FIG. 1.  Microscopic structure of the mouse thyroid.  (A) Hematoxylin and eosin (H&E) staining.  (B)  Periodic acidSchiff (PAS) staining. Mice were euthanized, and the thy-roids dissected, fixed in buffered formalin, and embedded inparaffin. Thyroid sections (5 l m) were mounted on glassslides, de-paraffinated, and hydrated. For histological anal-ysis, sections were stained with H&E, following a standardprotocol. Glycoproteins were detected using PAS staining.Sections were stained with 0.5% periodic acid for 30 minutesand with Schiff’s reagent for 20 minutes and then rinsed inrunning tap water for 5 minutes. Nuclei were counterstainedwith hematoxylin for 3 minutes. Sections were rinsed inrunning tap water, dehydrated, cleared, and mounted. Re-produced with permission from Senou  et al.  (20). INVESTIGATING THYROID HORMONE ECONOMY AND ACTION 91  & RECOMMENDATION 1b Autoradiography can be used to quantify the overall ac-tivity of thyroid follicles and to determine the location of iodide within follicles. Commentary.  Thyroid follicular cells concentrate iodideaccording to their activity. Although the activity of the thou-sands of follicular cells should be similar within a given thy-roidgland,thereisagreatdealofvariationamongcellswithinthe same follicle and between follicles. Thus autoradiographyprovides unique insights into the activity of individual thy-roid follicular cells. 125 Iisinjectedintravenously,typically48to72hourspriortokilling the animal. Thyroid glands are dissected and processedfor autoradiography using standard techniques (17,18). Orga-nification of iodide can be blocked by treatment of the animalswith methimazole (MMI). Autoradiography experiments withhuman, rodent, and feline goiter tissue have also been per-formed after xenotransplantation of thyroid tissue into nudemice. Subcutaneously implanted fragments are maintained inrecipient mice for several weeks before further analysis (19). & RECOMMENDATION 1c The ultrastructural distribution of iodide within thyroidfollicles can be defined with secondary ion mass spec-trometry (SIMS). Commentary.  SIMS is a technique used to analyze thecomposition of thin films by sputtering the surface of thespecimenwithafocusedprimaryionbeamandcollectingandanalyzing ejected secondary ions (Fig. 2). The mass/chargeratios of these secondary ions are measured with a massspectrometer to determine the elemental, isotopic, or molec-ular composition of the surface to a depth of 1–2nm. SIMS isthe most sensitive surface analysis technique, with elementaldetection limits ranging from parts per million to parts per billion. It is uniquely suited for the study of trace ions distri- bution at the ultrastructural level (20).Ionic images show that the early distribution of iodine isheterogeneous from one follicle to another, from one thyr-ocyte to another inside the same follicle, and that this dis-tribution varies as a function of time (21). In normal thyroidsthe natural  127 I isotope is found predominantly in the fol-licular lumina. The identification of lumina devoid of   127 Iand/or the demonstration of significant amounts of   127 I inthe cytoplasm of the epithelial cells or on the apical mem- brane indicates impairment of the iodination pathway. Todefine the ultrastructural distribution of iodide using SIMS,thyroid lobes are processed in a similar way as for electronmicroscopy, including fixation with glutaraldehyde andpreparation of semithin sections (20). & RECOMMENDATION 1d Confocal microscopy in conjunction with immunohisto-chemistry (IHC) can be used for two- or three-dimensional(2D or 3D) image reconstruction to study protein expres-sionin thyroid follicles, thesurrounding capillarynetwork,and the stroma. Commentary.  Antibodies are available against most keyproteins in thyrocyte biology (22,23). Thus, standard IHCtechniques are commonly used in thyroid studies (Figs. 3 and4) (24,25). Visualization can be performed with conventionallight microscopy, immunofluorescence microscopy, or con-focal microscopy for higher resolution and 2D or 3D imagereconstruction (26). Cell surface proteins and processes are best investigated using scanning electron microscopy (10).Endogenous peroxidase activity is very high in thyroidcells and is detected by reacting fixed tissue sections with 3,3 ¢ -diaminobenzidine substrate; pretreatment with hydrogen FIG. 2.  Mouse thyroid transmission electron microscopy. Thyroid lobes were fixed in 2.5% glutaraldehyde in 0.1M 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 l m) 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 microscopeZeiss EM169 (Carl Zeiss, Oberkochen, Germany).  (C)  Ultrastructural distribution of   127 I by secondary ion mass spectrometry(SIMS) imaging. Semi-thin sections were prepared, and the ultrastructural distribution of the iodide natural isotope ( 127 I) wasobtained through imaging by SIMS, using the NanoSIMS 50 system. Maps were acquired under standard analytic conditions: aCs +  primary beam with impact energy of 16keV and a probe with current intensity of 1pA. The analyzed surface was30 · 30 l m. Under these conditions, a lateral resolution of 100nm is expected. All images were acquired in 256 · 256 pixels with acounting time of 20 milliseconds per pixel. White areas correspond to iodine detection.  127 I is homogeneously distributed in thefollicular lumina and in a few intracytoplasmic vesicles. Reproduced with permission from Senou  et al.  (20). 92 BIANCO ET AL.
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