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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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superiority+of+thyroid+peroxidase+DNA+over+protein+immunization+in+replicating+human+thyroid+autoimmunity+in+HLA-DRB1*0301+(DR3)+transgenic+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IFN-&#945;+mediates+the+development+of+autoimmunity+both+by+direct+tissue+toxicity+and+through+immune+cell+recruitment+mechanisms.">IFN-&#945; 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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Butorphanol tartrate</td>
			<td>Supelco</td>
			<td>L-044&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexmedetomidine hydrochloride&#160;</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&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Freund&#8217;s Adjuvant, Complete&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>F5881&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Freund&#8217;s Adjuvant, Incomplete&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>F5506</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG&#160;</td>
			<td>invitrogen</td>
			<td>SA5-10275&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Midazolam solution&#160;</td>
			<td>Supelco</td>
			<td>M-908&#160;</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&#160;</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&#160;</td>
			<td>7681-82-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Thyroglobulin</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>T1126</td>
			<td></td>
		</tr>
		<tr>
			<td>Thyroglobulin&#160; 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

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JoVE Journal

Immunology and Infection

1.5K 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 ...

Parasite Induced Genetically Driven Autoimmune Chagas Heart Disease in the Chicken Model

The Trypanosoma cruzi acute infections acquired in infancy and childhood seem asymptomatic, but approximately one third of the chronically infected cases show Chagas disease up to three decades or later. Autoimmunity and parasite persistence are competing theories to explain the pathogenesis of Chagas disease 1, 2. To separate roles played by parasite persistence and autoimmunity in Chagas disease we inoculate the T. cruzi in the air chamber of fertilized eggs. The mature chicken immune system is a tight biological barrier against T. cruzi and the infection is eradicated upon development of its immune system by the end of the first week of growth 3. The chicks are parasite-free at hatching, but they retain integrated parasite mitochondrial kinetoplast DNA (kDNA) minicircle within their genome that are transferred to their progeny. Documentation of the kDNA minicircle integration in the chicken genome was obtained by a targeted prime TAIL-PCR, Southern hybridizations, cloning, and sequencing 3, 4. The kDNA minicircle integrations rupture open reading frames for transcription and immune system factors, phosphatase (GTPase), adenylate cyclase and phosphorylases (PKC, NF-Kappa B activator, PI-3K) associated with cell physiology, growth, and differentiation 3, 5-7, and other gene functions. Severe myocarditis due to rejection of target heart fibers by effectors cytotoxic lymphocytes is seen in the kDNA mutated chickens, showing an inflammatory cardiomyopathy similar to that seen in human Chagas disease. Notably, heart failure and skeletal muscle weakness are present in adult chickens with kDNA rupture of the dystrophin gene in chromosome 1 8. Similar genotipic alterations are associated with tissue destruction carried out by effectors CD45+, CD8&gamma;&delta;+, CD8&alpha; lymphocytes. Thus this protozoan infection can induce genetically driven autoimmune disease.

The inoculation of Trypanosoma cruzi in fertile eggs prior to incubation renders the parasite kDNA minicircle integration in embryo cells genome. Crossbreeding reveals the vertical transfer of the mutations to progeny. The kDNA integrates into coding regions at several chromosomes and the chickens die with an inflammatory autoimmune heart disease.

The Trypanosoma cruzi acute infections acquired in infancy and childhood seem asymptomatic, but approximately one third of the chronically infected cases ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Antibody Binding Specificity for Kappa (V&#954;) Light Chain-containing Human (IgM) Antibodies: Polysialic Acid (PSA) Attached to NCAM as a Case Study

Antibodies of the IgM isotype are often neglected as potential therapeutics in human trials, animal models of human diseases as well as detecting agents in standard laboratory techniques. In contrast, several human IgMs demonstrated proof of efficacy in cancer models and models of CNS disorders including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). Reasons for their lack of consideration include difficulties to express, purify and stabilize IgM antibodies, challenge to identify (non-protein) antigens, low affinity binding and fundamental knowledge gaps in carbohydrate and lipid research. 
This manuscript uses HIgM12 as an example to provide a detailed protocol to detect antigens by Western blotting, immunoprecipitations and immunocytochemistry. HIgM12 targets polysialic acid (PSA) attached to the neural cell adhesion molecule (NCAM). Early postnatal mouse brain tissue from wild type (WT) and NCAM knockout (KO) mice lacking the three major central nervous system (CNS) splice variants NCAM180, 140 and 120 was used to evaluate the importance of NCAM for binding to HIgM12. Further enzymatic digestion of CNS tissue and cultured CNS cells using endoneuraminidases led us to identify PSA as the specific binding epitope for HIgM12.

We present a protocol to identify protein moieties containing antigens for human and mouse IgM antibodies with specific kappa (V&#954;) light chains. This protocol is not limited to IgM class antibodies but applies to all immunoglobulin isotypes that target their antigen with sufficiently high affinity during immunoprecipitations.

Antibodies of the IgM isotype are often neglected as potential therapeutics in human trials, animal models of human diseases as well as detecting agents ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and consequently poses an urgent threat to public health. Recurrence of a C. difficile infection (CDI) after successful treatment with antibiotics is high, occurring in 20-30% of patients, thus necessitating the discovery of novel therapeutics against this pathogen. Current animal models of CDI result in high mortality rates and thus do not approximate the chronic, insidious disease manifestations seen in humans with CDI. To evaluate therapeutics against C. difficile, a mouse model approximating human disease utilizing a clinically-relevant strain is needed. This protocol outlines the cefoperazone mouse model of CDI using a clinically-relevant and genetically-tractable strain, R20291. Techniques for clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol. Compared to other mouse models of CDI, this model is not uniformly lethal at the dose administered, allowing for the observation of a prolonged clinical course of infection concordant with the human disease. Therefore, this cefoperazone mouse model of CDI proves a valuable experimental platform to assess the effects of novel therapeutics on the amelioration of clinical disease and on the restoration of colonization resistance against C. difficile.

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

This protocol outlines the cefoperazone mouse model of Clostridium difficile infection (CDI) using a clinically-relevant and genetically-tractable strain, R20291. Emphasis on clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol.

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and ...

A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue

Dry eye disease is a very common condition that causes morbidity and healthcare burden and decreases the quality of life. There is a need for a suitable dry eye animal model to test novel therapeutics to treat autoimmune dry eye conditions. This protocol describes a chronic autoimmune dry eye rat model. Lewis rats were immunized with an emulsion containing lacrimal gland extract, ovalbumin, and complete Freund&#39;s adjuvant. A second immunization with the same antigens in incomplete Freund&#39;s adjuvant was administered two weeks later. These immunizations were administered subcutaneously at the base of the tail. To boost the immune response at the ocular surface and lacrimal glands, lacrimal gland extract and ovalbumin were injected into the forniceal subconjunctiva and lacrimal glands 6 weeks after the first immunization. The rats developed dry eye features, including reduced tear production, decreased tear stability, and increased corneal damage. Immune profiling by flow cytometry showed a preponderance of CD3+ effector memory T cells in the eyeball.

This report describes a method to induce chronic experimental autoimmune dry eye in Lewis rats through immunization with an emulsion of rat lacrimal gland extract, ovalbumin, and complete Freund's adjuvant, followed by the injection of lacrimal gland extract and ovalbumin into the forniceal subconjunctiva and lacrimal glands six weeks later.

Dry eye disease is a very common condition that causes morbidity and healthcare burden and decreases the quality of life. There is a need for a suitable ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse polymicrobial sepsis can be induced by injecting cecal slurry intraperitoneally into day of life 7 mice and monitoring them for the following week. Presented here are the detailed steps necessary for the implementation of this neonatal sepsis model. This includes making a homogeneous cecal slurry stock, diluting it to a weight- and litter-adjusted dose, an outline of the monitoring schedule, and a definition of observed health categories used to define humane endpoints. The generation of a homogeneous cecal slurry stock from pooled donors allows for the administration into many litters over time, reducing the variation between donors, and preventing the use of potentially toxic glycerol. The monitoring strategy used allows for the anticipation of survival outcome and the identification of mice that would later progress to death, allowing for an earlier identification of the humane endpoint. Two main behavioral features are used to define the health scores, namely, the ability of the neonatal mice to right themselves when placed on their back and their level of mobility. These criteria could potentially be applied to address humane endpoints in other studies of neonatal disease in mice, as long as a pilot study is performed to confirm accuracy. In conclusion, this approach provides a standardized method to model newborn sepsis in mice, while providing resources to assess animal welfare used to define early humane endpoints for challenged animals.

This protocol provides the necessary steps to establish and evaluate neonatal sepsis in 7-day-old mice.

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse ...