Generation of a Mouse Spontaneous Autoimmune Thyroiditis Model

Yuanfan Qian; Linye He; Anping Su; Yiguo Hu; Jingqiang Zhu

doi:10.3791/64609

JoVE Journal > Immunology and Infection

Immunology and Infection

Generation of a Mouse Spontaneous Autoimmune Thyroiditis Model

Published: March 17, 2023

doi:

10.3791/64609

Yuanfan Qian^1,2, Linye He^1,2, Anping Su^1,2, Yiguo Hu³, Jingqiang Zhu¹

¹Thyroid and Parathyroid Surgery Center,West China Hospital of Sichuan University, ²The Laboratory of Thyroid and Parathyroid Disease,West China Hospital of Sichuan University, ³State Key Laboratory of Biotherapy and Cancer Center, West China Hospital,Sichuan University and Collaborative Innovation Center

Summary

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.

Abstract

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.

Introduction

Hashimoto'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'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'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.

Protocol

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

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 °C. Change the bedding materials every week. Provide adequate quantities of standard rodent chow and water.
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 °C freezer, avoiding light, for up to 8 weeks.
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%.

2. Induction of thyroiditis

SAT model
1. 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.
2. 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.
EAT model
1. 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.
2. During the first 2 weeks, subcutaneously inject a mixture of CFA and Tg (200 µL) once a week.
3. From the third week on, subcutaneously inject a mixture of IFA and Tg (200 µL) once a week for 3 weeks.

3. Measurements

Preparation of peripheral blood samples
1. 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 µg/100 µL for sedation), medetomidine (7.5 µg/100 µL for sedation), and butorphanol tartrate (50 µg/100 µL for analgesia) in phosphate-buffered saline (PBS).
  NOTE: The specific concentrations of each component in the anesthesia mixture are: midazolam 13.33µg/100µL, medetomidine 2.5µg/100µL, and butorphanol 16.7µg/100µL. For specific dosages used in mice, the doses are: midazolam 4µg/g, medetomidine 0.75µg/g, and butorphanol 1.67µg/g. Anesthesia depth was confirmed when the mouse's limb muscles relaxed, the whiskers had no touch response, and there was loss of pedal reflex.
2. 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.
3. Put the blood into a 4 °C refrigerator for 2 h and then centrifuge at 1,000 × g for 10 min at 4 °C to get the sample. For further analysis, store the rest of the sample at -80 °C.
Preparation of thyroid tissue samples
1. 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.
2. 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.
3. 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.
4. 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.
5. Immerse the glands in 4% paraformaldehyde for 12-24 h. Then, transfer the tissue to 70% ethanol.
6. 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 ° C for 40 min; paraffin I for 30 min; and paraffin II for 30 min. Embed them in paraffin wax blocks.
7. Before the staining, dewax 5 µm 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.
8. Stain the tissues with hematoxylin and eosin (H&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.
9. 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.
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).
1. 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.
2. 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.
Measurement of TgAb
NOTE: Use an ELISA kit to detect thyroglobulin by following the manufacturer's instructions.
1. 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.
2. Set the standard wells, blank wells, and testing wells. Blank wells only get buffer, while standard wells get standard solutions. Add 10 µL 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.
3. 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.
4. Incubate the samples in a 37 °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 µL of washing buffer for 30 s. Repeat this procedure five times and then pat the wells dry.
5. Add 50 µL of enzyme labeling reagent to each well, except for the blank wells, and incubate in a 37 ° water bath for 30 min. Remove the sealing film, shake off the liquid inside the enzyme plate, and pat dry on absorbent paper.
6. Add 50 µL of developer A to each well followed by 50 µL of developer B and gently shake the wells to mix for 5 min. Add 50 µL 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.
Measurement of T4 and thyroid stimulating hormone (TSH)
NOTE: Measure T4 and TSH levels (in 10 µL aliquots) by ELISA by following the manufacturer's instructions.
1. Dilute the enzyme conjugate to 1:11 with the assay diluent; dilute the samples to be measured (1:100) using 0.01 M PBS.
2. Add 10 µL of the standard and the sample to the corresponding wells, add 100 µL of enzyme conjugate to each well, and incubate at room temperature for 1 h.
3. Discard the liquid in the wells and clean it with washing buffer three times.
4. Add 100 µL of 3,3',5,5'-tetramethyl benzidine (TMB) and incubate at room temperature for 15 min.
5. Add 50 µL of stop solution and mix gently for 15-20 s.
6. Determine the absorbance (OD) using a microplate reader at a wavelength of 450 nm and calculate the sample concentration.
7. Use statistical software for performing t-test or rank sum test and plot the results.

Representative Results

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. In the solutions of 0.005%, 0.05%, and 0.5%, SAT reached maximal severity in weeks 16, 8, and 8, respectively, after giving the NaI water. Once thyroiditis was induced, lymphocyte infiltration continued throughout the mouse's life. During the induction, female mice seemed to have a trend in developing more severe thyroiditis than males under the same condition, but there were no significant differences in the severity of lymphocytic infiltration, which might be limited by the number of samples.

When given regular water without additional iodine, the level of TgAb did not rise significantly at the age of 16 weeks, and no significant difference was found between males and females. TgAb levels in NOD.H-2h4 mice began to rise at 24 weeks, with no difference between sexes, and TgAb levels continued to rise until 72 weeks (Figure 2A). With the addition of NaI to drinking water, the levels of TgAb began to rise at weeks 16, 8, and 8 with solutions of 0.005%, 0.05%, and 0.5% NaI, respectively. However, the levels of TgAb showed no difference between males and females in any of the solutions (Figure 2B–D). Regardless of whether NaI water was given, the levels of TgAb reached their highest at the age of 72 weeks (or 64 weeks after NaI water was first given).

There was a longer delay in the detectability of TPOAb levels compared to TgAb. These antibodies were rarely detected in NOD.H-2h4 mice with regular water at the age of 24 weeks. When fed with regular water, females exhibited much higher TPOAb levels than males at 72 weeks (Figure 2E). The same trend of intersex difference was also emerging in NOD.H-2h4 mice during the duration of 16, 8, and 8 weeks when given NaI water of 0.005%, 0.05%, and 0.5%, respectively (Figure 2F–H) Remarkably, however, when the concentration of NaI was over 0.05% during the induction, the levels of TPOAb reached their highest at 64 weeks after NaI water was first given, and there was no difference between males and females (Figure 2G,H).

In addition, serum TSH levels in male NOD.H-2h4 mice were significantly higher than those of females at different feeding stages, regardless of whether NAI water was given or not. When given regular water, the TSH levels of NOD.H-2h4 mice began to increase at the age of 24 weeks, and a similar tendency was observed in the iodine diet groups with the concentrations of 0.005%, 0.05%, and 0.5% at 16, 8, and 8 weeks, respectively, after iodine was given separately. TSH levels continued to rise throughout the induction period, peaking at 64 weeks. Furthermore, there was a significant difference in TSH levels between males and females in the rest of the NOD.H-2h4 mice (Figure 3A–D). NOD.H-2h4 mice did not experience goiter or hypothyroidism after iodine ingestion. Regardless of whether iodine agents were administered, there was no sex-related difference in T4 levels in NOD.H-2h4 mice. However, with the duration of induction increasing, the levels of T4 had a tendency to decrease in both sexes in the iodine water groups (no significant difference) (Figure 3E–H).

Sexual dimorphism in the pathological process and TPOAb levels were found in the progeny of NOD.H-2h4 mice, but not TgAb. Similar to other studies²⁵^,²⁶, TPOAb levels were significantly increased in model mice after receiving dietary iodide for 2-4 months, and there was a sex-related difference. In addition, TSH levels were higher in male than female NOD.H-2h4 mice with or without dietary iodide supplementation, which is similar to other mouse strains. This difference remained throughout the lives of the animals, which may not have beeb influenced by the levels of TPOAb or TgAb.When exposed to 0.05% NaI, most of the mice developed thyroiditis in 8 weeks, which had a similar effect with 0.5% NaI. While 0.005% NaI had a similar effect on NOD.H-2h4 mice fed with regular water, the concentration of 0.05% NaI (after 8 weeks of induction) might be the preferable condition to induce autoimmune thyroiditis. T4 levels in NOD.H-2h4 mice had no sex-related differences and were not affected by iodine agents, which is consistent with other studies²⁷^,²⁸. This may be due to the relatively short onset time of the disease and the compensatory thyroid function of the mice, resulting in a phenomenon similar to subclinical hypothyroidism in the mice. We speculate that the TSH axis of the mice would eventually become decompensated and produce differences over time.

Figure 1: Pathological changes and lymphocyte infiltration inflammatory scores of thyroid glands in NOD.H-2h4 mice with/without the iodine diet. (A,C,E,G) Pathological changes of the thyroid gland under the iodine diet of 0%, 0.005%, 0.05%, and 0.5% by HE staining; 200x magnification, N = 10/group. (B,D,F,G) Thyroid inflammation was determined according to the lymphocyte infiltration area; grading of the degree of lymphocyte infiltration: 0: almost no lymphocyte infiltration; 1+: more than one-eighth of the gland is invaded; 2+: one-quarter of the gland is invaded; 3+: one-quarter to one-half of the gland is invaded; 4+: more than one-half of the gland is destroyed. N = 10/group. Significant differences: *p < 0.05; **p < 0.01. Abbreviations: F = female; M = male; NW = N weeks. Please click here to view a larger version of this figure.

Figure 2: Autoantibodies in NOD.H-2h4 mice on regular and iodide-containing water. (A–D) Autoantibodies to Tg in NOD.H-2h4 mice on regular water (at the age of 8, 16, 24, and 72 weeks separately) and iodide water-0.005%, 0.05%, and 0.5% (at 0, 8, 16, and 64 weeks after the iodine water was begun). (E–H) Autoantibodies to TPO in NOD.H-2h4 mice on regular water (at the age of 8, 16, 24, and 72 weeks separately) and iodide water-0.005%, 0.05%, and 0.5% (at 0, 8, 16, and 64 weeks after the iodine water was begun). Values for TgAb are reported as ELISA optical density (OD) 490 nm (mean ± standard error of the mean SEM) and values for TPOAb are reported as the geometric mean in flow cytometry. N = 8/group. Significant differences: *p < 0.05; **p < 0.01; ***p < 0.001. Abbreviations: Tg = thyroglobulin; TgAb = autoantibodies to Tg; TPO = thyroid peroxidase; TPOAb = autoantibodies to TPO; F = female; M = male; NW = N weeks. Please click here to view a larger version of this figure.

Figure 3: TSH and T4 levels in NOD.H-2h4 mice on regular and iodide-containing water. (A–D) TSH and (E–H) T4 levels on regular water (at the age of 8, 16, 24, and 72 weeks separately) and iodide water-0.005%, 0.05%, and 0.5% (at 0, 8, 16, and 64 weeks after the iodine water was begun). TSH values were measured by radioimmunoassay (mU/L, mean ± standard error of the mean SEM). N = 8/group. Significant differences: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Abbreviations: TSH = thyroid stimulating hormone; F = female; M = male; NW = N weeks. Please click here to view a larger version of this figure.

Discussion

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 environment in vivo. This approach has been used for many years and continues to be effective³⁰^,³¹. In addition, novel approaches to induce thyroiditis include injecting dendric cells (DCs) pulsed with Tg³², the expression of Tg or TPO in vivo by adenoviral vectors or plasmids³¹, transplanting the thyroid gland of allogeneic mice, and injecting LPS. However, the successful construction of the thyroiditis model is usually more restricted by the genetic background of the experimental murine model, syngeneic antigens, and cell biology technologies. Furthermore, specific adjuvants may induce a more complex immune environment, making it difficult to investigate the immunological process of autoimmune thyroiditis. Therefore, we believe that the SAT of NOD.H-2h4 mice can better illustrate the pathogenesis of Hashimoto’s thyroiditis compared with the EAT model. Specifically, immunization with potent adjuvants and thyroglobulin is not necessary for SAT³³. To speed up the progress of SAT, iodine is added to the drinking water of NOD.H-2h4 mice. Although the resulting thyroiditis does not share the same mechanism by which thyroiditis occurs in humans, iodine is indeed an important influencing factor of thyroiditis. Furthermore, in this model, iodine agents can quickly induce thyroiditis in NOD.H-2h4 mice, and NOD.H-2h4 mice that do not receive iodine may eventually develop thyroiditis as well. As the experimental protocol in most laboratories is different, we performed a more comprehensive evaluation to the NOD.H-2h4 mice during the induction of thyroiditis.

In the process of model construction, some points need careful attention. Adding drinking water regularly and evaluating the general condition of the mice avoids accidental mouse deaths. It is recommended to keep no more than five mice per cage during the induction to provide enough NaI feeding to each mouse. However, the number of mice can also change following the specific institution’s rules and regulations for animal. As sexual dimorphism is found in our laboratory, single-sex mice are suggested for the experiment (for experiments with special requirements for the detection of thyroid-specific antibodies, female are recommended). It is suggested to select 8-week-old mice, as 10% of the mice develop spontaneous thyroiditis by the age of 16 weeks and all the mice develop thyroiditis eventually without NaI water. The thyroid lesions persist throughout the lifetimes of the mice and the next generation develop thyroiditis when born. Therefore, for the preservation of mice, it is recommended to use 8-week-old mice.It is important to note that TPOAb is more suitable as a detection indicator than TgAb when using NOD.H-2h4 mice to mimic thyroiditis. However, when measuring the levels of TPOAb, the duration of induction needs to be more delayed than TgAb.

In conclusion, this paper describes the establishment of a SAT model with NOD.H-2h4 mice and the exploration of the influence of different factors on this model. Although it does not perfectly simulate the pathogenesis and mechanism of autoimmune thyroid disease, the NOD.H-2h4 mouse is a stable animal model with great potential in the field of autoimmune thyroid disease. Given that this is an easy animal model to construct and is highly reproducible, it is hoped that this method will help improve applications of SAT murine models in different research institutions.

Disclosures

The authors have nothing to disclose.

Acknowledgements

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)

Materials

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

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Qian, Y., He, L., Su, A., Hu, Y., Zhu, J. Generation of a Mouse Spontaneous Autoimmune Thyroiditis Model. J. Vis. Exp. (193), e64609, doi:10.3791/64609 (2023).

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