Mouse monoclonal antibodies to human TPO (used as positive controls) were provided by Dr. P. Carayon and Dr. J. Ruf (Marseille, France). The authors thank all the participants in this study and the members of our research team. This work was in part supported by grants from the Postdoctoral Sustentation Fund of West China Hospital, Sichuan University, China (2020HXBH057) and the Sichuan Province Science and Technology Support Program (Project No. 2021YFS0166)

The protocol described below was approved by the care and use guidelines established by the Institutional Animal Care and Use Committee of Sichuan University.
1. Preparation
<ol>
	<li>House all mice in specific pathogen-free conditions under 12 h light-dark cycles (beginning at 07:00 a.m. and 07:00 p.m., respectively). Maintain the room temperature at 22 &#176;C. Change the bedding materials every week. Provide adequate quantities of standard rodent chow and water.</li>
	<li>When preparing the stock solution of NaI in water, weigh 2.5 g of the compound and dissolve it in 50 mL of sterile, pure water under vortexing to give a stock solution of 5%. Store this stock solution in a 4 &#176;C freezer, avoiding light, for up to 8 weeks.</li>
	<li>To prepare a working solution of NaI, draw 2.5 mL of stock solution and dissolve it in 250 mL of regular water as the daily drinking water for NOD.H-2h4 mice, to give a working solution of 0.05%.</li>
</ol>
2. Induction of thyroiditis
<ol>
	<li>SAT model
	<ol>
		<li>Prepare the NOD.H-2h4 mice (8 weeks of age) for the study by housing all mice of a single sex (no sexual dimorphism) in barrier cages (five animals per cage) with the feeding conditions described in step 1.1.</li>
		<li>To induce autoimmune thyroiditis in NOD.H-2h4 mice, feed all the mice with 0.05% NaI for 8 weeks. Change the NaI water every week and assess the status of the mice on a weekly basis, including appearance, weight, appetite, mental status, and mobility.</li>
	</ol>
	</li>
	<li>EAT model
	<ol>
		<li>Prepare the BALB/c mice (8 weeks of age) for the study by housing all mice of a single sex (no sexual dimorphism) in barrier cages (five animals per cage) with the feeding conditions described in step 1.1. Feed all mice with 0.05% NaI for 8 weeks.</li>
		<li>During the first 2 weeks, subcutaneously inject a mixture of CFA and Tg (200 &#181;L) once a week.</li>
		<li>From the third week on, subcutaneously inject a mixture of IFA and Tg (200 &#181;L) once a week for 3 weeks.</li>
	</ol>
	</li>
</ol>
3. Measurements
<ol>
	<li>Preparation of peripheral blood samples
	<ol>
		<li>After the induction, anesthetize the mice with a volume of 0.01 mL/g anesthetic by intraperitoneal injection. Prepare the anesthetic by mixing midazolam (40 &#181;g/100 &#181;L for sedation), medetomidine (7.5 &#181;g/100 &#181;L for sedation), and butorphanol tartrate (50 &#181;g/100 &#181;L for analgesia) in phosphate-buffered saline (PBS). 
		NOTE: The specific concentrations of each component in the anesthesia mixture are: midazolam 13.33&#181;g/100&#181;L, medetomidine 2.5&#181;g/100&#181;L, and butorphanol 16.7&#181;g/100&#181;L. For specific dosages used in mice, the doses are: midazolam 4&#181;g/g, medetomidine 0.75&#181;g/g, and butorphanol 1.67&#181;g/g.&#160;Anesthesia depth was confirmed when the mouse&#39;s limb muscles relaxed, the whiskers had no touch response, and there was loss of&#160; pedal reflex.</li>
		<li>After the mice are anesthetized, prepare the peripheral blood samples, by fixing the mouse with one hand and pressing the eye skin to protrude the eyeball. Then, insert the capillary tube into the inner corner of the eye and penetrate at a 30-45 degree angle to the plane of the nostril. Apply pressure while gently rotating the capillary tube. Blood will flow into the tube via capillary action.</li>
		<li>Put the blood into a 4 &#176;C refrigerator for 2 h and then centrifuge at 1,000 &#215; g for 10 min at 4 &#176;C to get the sample. For further analysis, store the rest of the sample at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Preparation of thyroid tissue samples
	<ol>
		<li>Humanely euthanize the animal according to the institutional policies. Then, dissect the chest wall to expose the heart, cut open the right atrium, and infuse saline into the left ventricle by an intravenous infusion needle attached to a 20 mL syringe until the tissue turns white.</li>
		<li>Fix the mouse on the dissection table with pins. Sterilize the neck with 75% ethanol. Cut the skin of the mouse along the median line of the neck from the top of the sternum to the lower jaw with tissue scissors to completely expose the neck tissue.</li>
		<li>Observe the pair of pink glands, the submandibular glands, below which is the anterior trachea muscle. Separate the glands and muscle with ophthalmic scissors and forceps to expose the trachea and thyroid cartilage.</li>
		<li>Separate the tissue under the cartilage, using curved forceps to pick up the cartilage and trachea together. Cut the distal and proximal ends of the cartilage and trachea, respectively, and remove the thyroid gland together with the trachea.</li>
		<li>Immerse the glands in 4% paraformaldehyde for 12-24 h. Then, transfer the tissue to 70% ethanol.</li>
		<li>Place the individual lobes of thyroid biopsy material in the processing cassettes and dehydrate through the following serial alcohol gradient: 70%, 80%, 90%, 95%, 100%, 100%, ethanol for 40 min each; 1/2 xylene + 1/2 absolute ethanol for 2 h; 100% xylene for 1.5 h; 100% xylene for 1.5 h; 1/2 xylene + 1/2 paraffin for 2 h; oven at 40 &#176; C for 40 min; paraffin I for 30 min; and paraffin II for 30 min. Embed them in paraffin wax blocks.</li>
		<li>Before the staining, dewax 5 &#181;m thick thyroid tissue sections in xylene (as follows) and rehydrate through the following decreasing concentrations of ethanol: xylene I for 10 min; xylene II for 10 min; xylene III for 10 min; absolute ethanol I for 5 min; absolute ethanol II for 5 min; 90% alcohol for 5 min; 80% alcohol for 5 min; 70% alcohol for 5 min; and 50% alcohol for 5 min.</li>
		<li>Stain the tissues with hematoxylin and eosin (H&#38;E).Stain with hematoxylin for 15 min, rinse with tap water for 15 min, add 1% hydrochloric acid ethanol for 10 s, rinse with running water, and then with 50%, 70%, and 80% ethanol, each for 3 min. Perform 0.5% eosin ethanol staining for 2 min and rinse with running water. Place the paraffin sections successively in 75% ethanol for 2 min, 85% ethanol for 2 min, absolute ethanol for 5 min, and xylene for 5 min. Fix the slices with neutral gum and glass slides.</li>
		<li>To evaluate the extent of lymphocytic thyroiditis, score the thyroid sections as follows: 0: little or no lymphocyte infiltration in the thyroid tissue; 1+: more than 1/8 of the gland is infiltrated in one or several foci; 2+: no more than 1/4 of the gland is infiltrated with lymphocytes; 3+: 1/4-1/2 of the gland is infiltrated with lymphocytes; 4+: more than 1/2 of the gland is destroyed. 
		NOTE: It is recommended to remove the thyroid cartilage to keep the whole structure of the mouse thyroid, which is too small to remove from the trachea.</li>
	</ol>
	</li>
	<li>Measurement of TPOAb 
	NOTE: Chinese hamster ovary (CHO) cells stably expressing mouse TPO were established in our lab. Mouse TPO cDNA was excised by XbaI and NheI. Then, the cDNA was transferred to pcDNA5/FRT. After the plasmid was constructed successfully, it was transfected into the CHO cells and selected with hygromycin B (100 g/mL).
	<ol>
		<li>After the construction of mTPO-CHO cells, dilute the mouse sera from step 3.1.3 (1:50) and incubate with the mTPO-CHO cells for 2 h. After incubation, exclude cell staining with propidium iodide (1 g/mL) and analyze the sample by flow cytometry using fluorescein isothiocyanate-conjugated, affinity-purified goat anti-mouse IgG. Screen surviving single-cell populations by FSC-A and SSC-A channels and select FITC- and PI-tagged cells from the single-cell populations21.</li>
		<li>Include the serum of unimmunized BALB/c or C57BL/6 mice bound to IgG as the negative control. Include mouse monoclonal antibodies to human TPO22 as positive controls that recognize mouse TPO23. Express TPO binding data as geometric means.</li>
	</ol>
	</li>
	<li>Measurement of TgAb 
	NOTE: Use an ELISA kit to detect thyroglobulin by following the manufacturer&#39;s instructions.
	<ol>
		<li>Dilute the standard samples in the microcentrifuge tube according to the instructions. Take the kit out from the refrigerator and keep it at room temperature for 30 min.</li>
		<li>Set the standard wells, blank wells, and testing wells. Blank wells only get buffer, while standard wells get standard solutions. Add 10 &#181;L of samples into the testing wells. When adding the samples, try not to touch the walls of the wells, and shake the plate gently to move the samples to the bottom of the wells.</li>
		<li>Include serum from BALB/c mice immunized with Tg, CFA, and IFA24 as the positive control and serum from 8-week-old NOD.H-2h4 mice on regular water as the negative control.</li>
		<li>Incubate the samples in a 37 &#176;C water bath for 30 min. Dilute the washing buffer with distilled water at a ratio of 1:30. Then, remove the sealing film, shake off the liquid inside the enzyme plate and pat dry on absorbent paper, and add 350 &#181;L of washing buffer for 30 s. Repeat this procedure five times and then pat the wells dry.</li>
		<li>Add 50 &#181;L of enzyme labeling reagent to each well, except for the blank wells, and incubate in a 37 &#176; water bath for 30 min. Remove the sealing film, shake off the liquid inside the enzyme plate, and pat dry on absorbent paper.</li>
		<li>Add 50 &#181;L of developer A to each well followed by 50 &#181;L of developer B and gently shake the wells to mix for 5 min. Add 50 &#181;L of the termination solution in each well for 15 min to stop the reaction. Measure the absorbance (OD) of each well at a wavelength of 450 nm using a microplate reader. Present the TgAb data as the optical density (OD) at 490 nm.</li>
	</ol>
	</li>
	<li>Measurement of T4 and thyroid stimulating hormone (TSH) 
	NOTE: Measure T4 and TSH levels (in 10 &#181;L aliquots) by ELISA by following the manufacturer&#39;s instructions.
	<ol>
		<li>Dilute the enzyme conjugate to 1:11 with the assay diluent; dilute the samples to be measured (1:100) using 0.01 M PBS.</li>
		<li>Add 10 &#181;L of the standard and the sample to the corresponding wells, add 100 &#181;L of enzyme conjugate to each well, and incubate at room temperature for 1 h.</li>
		<li>Discard the liquid in the wells and clean it with washing buffer three times.</li>
		<li>Add 100 &#181;L of 3,3&#39;,5,5&#39;-tetramethyl benzidine (TMB) and incubate at room temperature for 15 min.</li>
		<li>Add 50 &#181;L of stop solution and mix gently for 15-20 s.</li>
		<li>Determine the absorbance (OD) using a microplate reader at a wavelength of 450 nm and calculate the sample concentration.</li>
		<li>Use statistical software for performing t-test or rank sum test and plot the results.</li>
	</ol>
	</li>
</ol>

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<li>Carayanniotis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+parameters+linking+thyroglobulin+iodination+with+autoimmune+thyroiditis%5BTitle%5D)+AND+%22Hormones%22%5BJournal%5D)">Molecular parameters linking thyroglobulin iodination with autoimmune thyroiditis.</a> Hormones. 10 (1), 27-35 (2011).</li>
<li>Verginis, P., Li, H. S., Carayanniotis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tolerogenic+semimature+dendritic+cells+suppress+experimental+autoimmune+thyroiditis+by+activation+of+thyroglobulin-specific+CD4%2BCD25%2B+T+cells%5BTitle%5D)+AND+%22Journal+of+Immunology%22%5BJournal%5D)">Tolerogenic semimature dendritic cells suppress experimental autoimmune thyroiditis by activation of thyroglobulin-specific CD4+CD25+ T cells.</a> Journal of Immunology. 174 (11), 7433-7439 (2005).</li>
<li>Flynn, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Superiority+of+thyroid+peroxidase+DNA+over+protein+immunization+in+replicating+human+thyroid+autoimmunity+in+HLA-DRB1%2A0301+%28DR3%29+transgenic+mice%5BTitle%5D)+AND+%22Clinical+and+Experimental+Immunology%22%5BJournal%5D)">Superiority of thyroid peroxidase DNA over protein immunization in replicating human thyroid autoimmunity in HLA-DRB1*0301 (DR3) transgenic mice.</a> Clinical and Experimental Immunology. 137 (3), 503-512 (2004).</li>
<li>Akeno, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((IFN-%CE%B1+mediates+the+development+of+autoimmunity+both+by+direct+tissue+toxicity+and+through+immune+cell+recruitment+mechanisms%5BTitle%5D)+AND+%22Journal+of+Immunology%22%5BJournal%5D)">IFN-α mediates the development of autoimmunity both by direct tissue toxicity and through immune cell recruitment mechanisms.</a> Journal of Immunology. 186 (8), 4693-4706 (2011).</li>
</ol>

All the authors declare that they have no competing interests.

HT occurs due to an autoimmune system disorder caused by lymphocytes infiltrating the thyroid gland, further impairing thyroid function, while producing thyroid-specific antibodies. Serum TSH, TgAb, and TPOAb levels in HT patients are significantly elevated27. At present, two main kinds of murine models are widely used to study the etiology of autoimmune thyroiditis: EAT and SAT29. EAT mice are mostly immunized using proteins and adjuvants to create an abnormal immune envir...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/64609">Generation of a Mouse Spontaneous Autoimmune Thyroiditis Model</a>. The Protocol section was updated.
Step 3.1.1 of the Protocol was updated from:
After the induction, anesthetize the mice with a volume of 0.01 mL/g anesthetic by intraperitoneal injection. Prepare the anesthetic by mixing midazolam (40 &#181;g/100 &#181;L for sedation), medetomidine (7.5 &#181;g/100 &#181;L for sedation), and butorphanol tartrate (50 &#181;g/100 &#181;L for analgesia) in phosphate-buffered saline (PBS).
to
After the induction, anesthetize the mice with a volume of 0.01 mL/g anesthetic by intraperitoneal injection. Prepare the anesthetic by mixing midazolam (40 &#181;g/100 &#181;L for sedation), medetomidine (7.5 &#181;g/100 &#181;L for sedation), and butorphanol tartrate (50 &#181;g/100 &#181;L for analgesia) in phosphate-buffered saline (PBS). 
NOTE: The specific concentrations of each component in the anesthesia mixture are: midazolam 13.33&#181;g/100&#181;L, medetomidine 2.5&#181;g/100&#181;L, and butorphanol 16.7&#181;g/100&#181;L. For specific dosages used in mice, the doses are: midazolam 4&#181;g/g, medetomidine 0.75&#181;g/g, and butorphanol 1.67&#181;g/g.&#160;Anesthesia depth was confirmed when the mouse&#39;s limb muscles relaxed, the whiskers had no touch response, and there was loss of&#160; pedal reflex.
Step 3.1.2 of the Protocol was updated from:
After the mice are anesthetized, cut off their whiskers with ophthalmic scissors to prevent blood from flowing down the whiskers and causing hemolysis. Fix the mouse with one hand and press the skin of the eye to make the eyeball protrude. Quickly remove the eyeball and draw 1 mL of blood into the microcentrifuge tube via a capillary tube.
to
After the mice are anesthetized, prepare the peripheral blood samples, by fixing the mouse with one hand and pressing the eye skin to protrude the eyeball. Then, insert the capillary tube into the inner corner of the eye and penetrate at a 30-45 degree angle to the plane of the nostril. Apply pressure while gently rotating the capillary tube. Blood will flow into the tube via capillary action.
Step 3.2.1 of the Protocol was updated from:
Dissect the chest wall to expose the heart, cut open the right atrium, and infuse saline into the left ventricle by an intravenous infusion needle attached to a 20 mL syringe until the tissue turns white.
to
Humanely euthanize the animal according to the institutional policies. Then, dissect the chest wall to expose the heart, cut open the right atrium, and infuse saline into the left ventricle by an intravenous infusion needle attached to a 20 mL syringe until the tissue turns white.

An erratum was issued for:&#160;<a href="https://www.jove.com/t/64609">Generation of a Mouse Spontaneous Autoimmune Thyroiditis Model</a>. The Protocol section was updated.

Erratum: Generation of a Mouse Spontaneous Autoimmune Thyroiditis Model

Hashimoto&#39;s thyroiditis (HT), also known as chronic lymphocytic thyroiditis or autoimmune thyroiditis, was first reported in 19121. HT is characterized by lymphocyte infiltration and damage to thyroid follicular tissue. Laboratory tests are mainly manifested as increasing thyroid-specific antibodies, including anti-thyroglobulin antibody (TgAb) and anti-thyroid peroxidase antibody (TPOAb)2. The incidence of HT is in the range of 0.4%-1.5%, accounting for 20%-25% of all thyroid diseases, and this value has increased in recent years3. In addition, a large number of studies have reported that HT is associated with the oncogenesis and recurrence of papillary thyroid carcinoma (PTC)4,5; the potential mechanisms are still controversial. Autoimmune thyroiditis is also an important factor in female infertility6. Therefore, the pathogenesis of HT needs to be clear, for which a stable and simple animal model is essential.
To study the etiology of HT, two main kinds of murine models have been employed, including experimental autoimmune thyroiditis (EAT) and spontaneous autoimmune thyroiditis (SAT) in the present studies7,8. Susceptible mice were immunized with specific thyroid antigens (including the crude thyroid, purified thyroglobulin [TG], thyroid peroxidase [TPO], recombinant TPO ectodomain, and selected TPO peptides) to establish the EAT murine model. In addition, the adjuvants, including lipopolysaccharide (LPS), complete Freund&#39;s adjuvant (CFA), and other unusual adjuvants, are also used during the immunization to break down immune tolerance9,10,11,12,13,14,15,16,17.
The SAT model is an important model to study the spontaneous development of autoimmune thyroiditis, which is based on NOD.H-2h4 mice. The NOD.H-2h4 mouse is a new strain obtained from the cross of NOD and B10.A(4R) mice, followed by multiple backcrosses to NOD, with the autoimmune thyroiditis susceptibility gene IAk18,19. NOD.H-2h4 mice do not develop diabetes, but have a high incidence of autoimmune thyroiditis and Sjogren&#39;s syndrome (SS)19. Studies have found that intracellular adhesion molecule-1 (ICAM-1) is highly expressed in the thyroid tissue of NOD.H-2h4 mice at 3-4 weeks of age. Moreover, with the increase in iodine intake, the immunogenicity of the thyroglobulin molecule is enhanced, which further upregulates the expression of ICAM-1, which plays an important role in the process of monocyte infiltration21. This model simulates the autoimmune process while verifying the relationship between iodine dose and disease severity. The established method is stable, with a high probability of success. The SAT model has been applied to induce autoimmune thyroiditis for many years and continues to be an effective method to study the pathogenesis of autoimmune thyroiditis. However, the current construction method of the EAT model is more complicated and expensive; different laboratories use different immunization methods and injection sites. Furthermore, mice with different genetic backgrounds have different rates of induction, which need further study to reveal the potent mechanism.
However, the development of thyroiditis in the SAT model is associated with sodium iodide, sexual dimorphism, and the rearing conditions. To reveal the appropriate procedure of autoimmune thyroiditis in the SAT model, this article described the method of induction of autoimmune thyroiditis in different conditions. In addition, it allows for the study of the pathogenesis and immunological progress of autoimmune thyroiditis in different stages of this disease.

<table><tbody><tr><td>Butorphanol tartrate</td><td>Supelco</td><td>L-044&amp;nbsp;</td><td></td></tr><tr><td>Dexmedetomidine hydrochloride&amp;nbsp;</td><td>Sigma-Aldrich</td><td>145108-58-3</td><td></td></tr><tr><td>Enzyme-linked immunosorbent assay (ELISA) well</td><td>Sigma-Aldrich</td><td>M9410-1CS</td><td></td></tr><tr><td>Ethanol</td><td>macklin</td><td>64-17-5&amp;nbsp;</td><td></td></tr><tr><td>Freund&amp;rsquo;s Adjuvant, Complete&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F5881&amp;nbsp;</td><td></td></tr><tr><td>Freund&amp;rsquo;s Adjuvant, Incomplete&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F5506</td><td></td></tr><tr><td>Goat anti-Mouse IgG&amp;nbsp;</td><td>invitrogen</td><td>SA5-10275&amp;nbsp;</td><td></td></tr><tr><td>Midazolam solution&amp;nbsp;</td><td>Supelco</td><td>M-908&amp;nbsp;</td><td></td></tr><tr><td>Mouse/rat thyroxine (T4) ELISA</td><td>Calbiotech</td><td>DKO045</td><td></td></tr><tr><td>Paraformaldehyde</td><td>macklin</td><td>30525-89-4&amp;nbsp;</td><td></td></tr><tr><td>Propidium iodide</td><td>Sigma-Aldrich</td><td>P4864</td><td></td></tr><tr><td>Sodium Iodine</td><td>Sigma-Aldrich&amp;nbsp;</td><td>7681-82-5</td><td></td></tr><tr><td>Thyroglobulin</td><td>Sigma-Aldrich&amp;nbsp;</td><td>T1126</td><td></td></tr><tr><td>Thyroglobulin&amp;nbsp; ELISA Kit</td><td>Thermo Scientific</td><td>EHTGX5</td><td></td></tr><tr><td>TSH ELISA</td><td>Calbiotech</td><td>DKO200</td><td></td></tr><tr><td>Xylene</td><td>macklin</td><td>1330-20-7</td><td></td></tr></tbody></table>

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.

The histological changes were strikingly different in female and males, the duration of iodine intake, and the solution of NaI. As shown in Figure 1, ~10% of NOD.H-2h4 mice developed SAT even without iodine induction at the age of 24 weeks, and all the mice eventually developed thyroiditis. When given regular water, there was no significant difference in the histological changes between males and females. The addition of NaI to the drinking water accelerated the development of thyroiditis. I...

Watch this Scientific Journal Video about Generation of a Mouse Spontaneous Autoimmune Thyroiditis Model at JoVE.com

Generation of a Mouse Spontaneous Autoimmune Thyroiditis Model

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 ...

generation-of-a-mouse-spontaneous-autoimmune-thyroiditis-model

Research

JoVE Journal

Immunology and Infection

1.6K Views.  West China Hospital of Sichuan University. 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.

Video: Generation of a Mouse Spontaneous Autoimmune Thyroiditis Model

Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1. It has traditionally been considered a rare disease but reporting has increased markedly in recent years. Hypophysitis, in fact, develops not uncommonly as a "side effect" in cancer patients treated with antibodies that block inhibitory receptors expressed on T lymphocytes, such as CTLA-42 and PD-1 receptors. Autoimmune hypophysitis can be induced experimentally by injecting mice with pituitary proteins mixed with an adjuvant3. In this video article we demonstrate how to extract proteins from mouse pituitary glands and how to prepare them in a form suitable for inducing autoimmune hypophysitis in SJL mice.

Autoimmune hypophysitis can be reproduced in mice by injecting an extract of mouse pituitary proteins.

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1.  It has traditionally been considered a ...

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.

This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

The CYP2D6 Animal Model: How to Induce Autoimmune Hepatitis in Mice

Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2. In the past many attempts have been made to generate an animal model that reflects the characteristics of the human disease 3-5. However, in various models the induction of disease was rather complex and often hepatitis was only transient3-5. Therefore, we have developed a straightforward mouse model that uses the major human autoantigen in type 2 autoimmune hepatitis (AIH-2), namely hCYP2D6, as a trigger6. Type 1 liver-kidney microsomal antibodies (LKM-1) antibodies recognizing hCYP2D6 are the hallmark of AIH-27,8. Delivery of hCYP2D6 into wildtype FVB or C57BL/6 mice was by an Adenovirus construct (Ad-2D6) that ensures a direct delivery of the triggering antigen to the liver. Thus, the ensuing local inflammation generates a fertile field9 for the subsequent development of autoimmunity. A combination of intravenous and intraperitoneal injection of Ad-2D6 is the most effective route to induce a long-lasting autoimmune damage to the liver (section 1). Here we provide a detailed protocol on how autoimmune liver disease is induced in the CYP2D6 model and how the different aspects of liver damage can be assessed. First, the serum levels of markers indicating hepatocyte destruction, such as aminotransferases, as well as the titers of hCYP2D6 antibodies are determined by sampling blood retroorbitaly (section 2). Second, the hCYP2D6-specific T cell response is characterized by collecting lymphocytes from the spleen and the liver. In order to obtain pure liver lymphocytes, the livers are perfused by PBS via the portal vein (section 3), digested in collagen and purified over a Percoll gradient (section 4). The frequency of hCYP2D6-specific T cells is analyzed by stimulation with hCYP2D6 peptides and identification of IFN&gamma;-producing cells by flow cytometry (section 5). Third, cellular infiltration and fibrosis is determined by immunohistochemistry of liver sections (section 6). Such analysis regimen has to be conducted at several times after initiation of the disease in order to prove the chronic nature of the model. The magnitude of the immune response characterized by the frequency and activity of hCYP2D6-specific T and/or B cells and the degree of the liver damage and fibrosis have to be assessed for a subsequent evaluation of possible treatments to prevent, delay or abrogate the autodestructive process of the liver.

Infection of mice with an Adenovirus expressing the major human autoantigen cytochrome P450 2D6 (hCYP2D6) recognized by sera of patients suffering from type 2 autoimmune hepatitis results in a persistent form of autoimmune-mediated liver disease characterized by extensive hepatitis, fibrosis and generation of a CYP2D6-specific immune response.

Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2. In the past many attempts have been made to ...

Medicine

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.
Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.
Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Generation of an orthotopic mouse model of anaplastic thyroid carcinoma is described here. This technique employs surgical placement of human anaplastic thyroid cancer cells into the thyroid of immunodeficient mice, thus creating a more clinically relevant setting to study disease progression as well as screen innovative therapeutic interventions.

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer ...

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 ...

Cancer Research

The bm12 Inducible Model of Systemic Lupus Erythematosus (SLE) in C57BL/6 Mice

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible animal models mirror common components of human disease, including the bm12 transfer model. Upon transfer of bm12 splenocytes or purified CD4 T cells, C57BL/6 mice rapidly develop large frequencies of T follicular helper cells (Tfh), germinal center (GC) B cells, and plasma cells followed by high levels of circulating anti-nuclear antibodies. Since this model utilizes mice on a pure C57BL/6 background, researchers can quickly and easily study disease progression in transgenic or knockout mouse strains in a relatively short period of time. Here we describe protocols for the induction of the model and the quantitation Tfh, GC B cells, and plasma cells by multi-color flow cytometry. Importantly, these protocols can also be used to characterize disease in most mouse models of SLE and identify Tfh, GC B cells, and plasma cells in other disease models.

The bm12 Inducible Model of Systemic Lupus Erythematosus SLE in C57BL/6 Mice

The transfer of bm12 lymphocytes into a C57BL/6 recipient is an established model of systemic lupus erythematosus. Here we describe how to initiate disease using this model and how to characterize T follicular helper cells, germinal center B cells and plasma cells by flow cytometry.

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible ...

Imaging of Thyroid Hormone Action Indicator Mice to Assess Endocrine Disruption

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.

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.

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

Neuroscience

Induction and Evaluation of Experimental Autoimmune Encephalomyelitis in Mouse

Modeling Multiple Sclerosis in the Two Sexes: MOG35-55-Induced Experimental Autoimmune Encephalomyelitis

Multiple Sclerosis (MS) is a chronic autoimmune inflammatory disease affecting the central nervous system (CNS). It is characterized by different prevalence in the sexes, affecting more women than men, and different outcomes, showing more aggressive forms in men than in women. Furthermore, MS is highly heterogeneous in terms of clinical aspects, radiological, and pathological features. Thus, it is necessary to take advantage of experimental animal models that allow the investigation of as many aspects of the pathology as possible. Experimental autoimmune encephalomyelitis (EAE) represents one of the most used models of MS in mice, modeling different disease features, from the activation of the immune system to CNS damage. Here we describe a protocol for the induction of EAE in both male and female C57BL/6J mice using myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) immunization, which leads to the development of a chronic form of the disease. We also report the evaluation of the daily clinical score and motor performance of these mice for 28 days post immunization (28 dpi). Lastly, we illustrate some basic histological analysis at the CNS level, focusing on the spinal cord as the primary site of disease-induced damage.

Author Spotlight: Creating a Versatile Experimental Autoimmune Encephalomyelitis Model Relevant for Both Male and Female Mice 

Experimental autoimmune encephalomyelitis is one of the most widely used murine models of multiple sclerosis. In the current protocol, C57BL/6J mice of both sexes are immunized with myelin oligodendrocyte glycoprotein peptide, resulting mainly in ascending paresis of the tail and limbs. Here we discuss the protocol of EAE induction and evaluation.

Multiple Sclerosis (MS) is a chronic autoimmune inflammatory disease affecting the central nervous system (CNS). It is characterized by different ...

Orthotopic Inoculation of Thyroid Cancer Cells into Murine Thyroid: A Procedure to Establish Orthotopic Thyroid Cancer Mouse Model

Source: Julia Ramirez-Moya, et al. <a href="https://www.jove.com/video/59322" target="_blank">In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice.</a> J. Vis. Exp. (2019)
This video describes the procedure for orthotopic inoculation of thyroid cancer cells into the thyroid gland of mouse models, which may then serve as a valuable platform to assess carcinogenesis and discover potential treatment strategies.

Source: Julia Ramirez-Moya, et al. In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice. J. Vis. Exp. (2019)
This video describes the procedure ...

JoVE Encyclopedia of Experiments

Head and Neck Cancer

In vivo studies

Induction of Experimental Autoimmune Encephalomyelitis in a Mouse Model

Begin by subcutaneously injecting 10- to 13-week-old female mice with 200 micrograms of myelin oligodendrocyte glycoprotein, emulsified in 200 microliters of complete Freund&#39;s adjuvant containing 400 micrograms of Mycobacterium tuberculosis. Immediately, and 24 hours later, intraperitoneally inject the mice with 0.2 micrograms of pertussis toxin in 200 microliters of PBS.
It is essential that mice have been adapted both to handling and the experimental environment before induction to avoid inducing stress, which can prevent the development of clinical symptoms.
One week after the injection, examine the mice daily for clinical symptoms as described in the table.